You are on page 1of 55

Progress in Neurobiology Vol. 54, pp.

193 to 247, 1998


# 1997 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
0301-0082/98/$19.00

PII: S0301-0082(97)00068-3

THE VESTIBULAR HAIR CELLS:


POST-TRANSDUCTIONAL SIGNAL PROCESSING

P. S. GUTH*%, P. PERIN$, C. H. NORRIS* and P. VALLI$


*Departments of Pharmacology and Otolaryngology, Tulane University School of Medicine, New Orleans
LA 70112, U.S.A. and $Istituto di Fisiologia Generale, Universita di Pavia, Pavia 27100, Italy

(Received 4 August 1997)

AbstractÐHair cells in mechanosensory systems transduce mechanical stimuli into biological signals to
be presented to and analyzed by the brain. Vestibular hair cells transduce stimuli primarily associated
with the organism's orientation and motion in space. When examined super®cially it may appear that
the hair cells act as passive transducers whereby mechanical stimulation of their hair bundle results in
transmitter release at their a€erent synapses. In fact, hair cell functions are more complicated, and the
mechanical signals are heavily processed even before being encoded in a€erent nerve activity. Hair cells
are di€erent from one another in morphology, biophysics, transmitter and transmitter receptor comp-
lements, not only across di€erent organs (as one might expect), but even in the same organ. This review
focuses on hair cell morpho-physiological properties, ionic conductances, neurotransmitters/modulators
and their receptors, second messengers and e€ectors. Special features of hair cell neurotransmission, as
the synaptic body and the presence of autoreceptors and local circuits, are also discussed, as is the possi-
bility of a di€erential modulation of hair cell transmitter release in the resting and mechanically-stimu-
lated states. # 1997 Elsevier Science Ltd

CONTENTS
1. Introduction 194
1.1. Accessory structures 196
1.2. Transduction 196
2. Morpho-physiological di€erences among hair cells 197
2.1. Hair cell morphology 197
2.1.1. Hair bundle 198
2.1.2. Hair cell body 198
3. Hair cell conductances 201
3.1. Potassium channels 201
3.2. Calcium channels 202
3.3. Functional roles 204
4. Vestibular hair cell motility 205
5. Neurotransmission 207
5.1. A€erent innervation 207
5.1.1. The role of the synaptic body 207
5.2. E€erent innervation 209
5.2.1. Physiological e€ects of the vestibular e€erents 209
5.3. Reciprocal synapses 211
5.4. Synaptic transmission 211
5.4.1. Generalities 211
5.4.2. The activity of the a€erent neurons depends completely on transmitter release from hair cells 212
5.4.3. Evoked and resting activities of the a€erents are di€erentially modulated 212
5.4.4. Calcium sources in the resting and evoked modes of transmitter release 213
5.5. Adaptation 213
5.6. Potassium ¯uxes in vestibular organs 214
5.7. Neurotransmitters and neuromodulators 214
5.7.1. Acetylcholine 214
5.7.1.1. Generalities 214
5.7.1.2. The pharmacology of ACh 215
5.7.1.3. The hair cell ACh receptor 215
5.7.1.4. Evidence for two ACh receptors in VHC 216
5.7.1.5. The nicotinic-like receptor(s) 216
5.7.1.6. The muscarinic-like receptor(s) 217
5.7.1.7. The putative role of chloride 219
5.7.2. The hair cell a€erent transmitter 219
5.7.2.1. Generalities 219
5.7.2.2. The presence of Glu 220

% Author for correspondence.

193
194 P. S. Guth et al.

CONTENTS (continued)
5.7.2.3. The pharmacology of Glu 220
5.7.2.4. The release of Glu 222
5.7.2.5. Molecular biological evidence of Glu as hair cell transmitter 223
5.7.2.6. Hair cell autoreceptors 224
5.7.2.7. The Sewell hair cell transmitter search 224
5.7.3. Calcitonin gene-related peptide 225
5.7.4. Enkephalins and other opioids 226
5.7.5. g-Amino butyric acid 226
5.7.6. Substance P 227
5.7.7. ATP 228
5.7.8. Adenosine 229
5.7.9. Histamine 229
5.7.10. N-Acetylhistidine 230
6. Second messengers and e€ectors of hair cells 230
6.1. Cyclic nucleotides 231
6.2. The phospholipase C cascade 231
6.3. Regulation of cytoplasmic calcium 232
7. Summary 233
8. Pertinent review articles 233
Acknowledgements 234
References 234

ABBEVIATIONS
ATP adenosine 5'-triphosphate EPSP excitatory postsynaptic potential
AMP adenosine 5'-monophosphate PLA phospholipase A2
GTP guanosine 5'-triphosphate PLD2 phospholipase D
EGTA ethylene glycol-bis(b-aminoethylether)N,N,N',N' VHC vestibular hair cells
tetraacetic acid MEPSP miniature excitatory postsynaptic potential
TEA tetraethylammonium ER endoplamic reticulum.

1. INTRODUCTION have a hair-like appearance. Hudspeth and Jacobs


(1979) showed that the kinocilia are not essential for
Gravitationally-sensitive receptors appear in vir- signal transduction, while the stereocilia are.
tually all motile multicellular organisms from coe- However, in all hair cells equipped with a kinocilium
lenterates onward (Bullock and Horridge, 1966). mechanical stimuli are transferred exclusively from
They are surprisingly constant in principle and in it to the stereocilia (Hillman and Lewis, 1971).
anatomy and function throughout the animal king- Among mammals the vestibular organs consist of
dom. This universality and constancy of structure the three semicircular canals (SCC) (anterior vertical,
and function underscores the fundamental import- posterior vertical and horizontal), the utricle and the
ance to the survival of the organism of sensing its saccule. The sensory epithelium of the SCC is
orientation (and motion) in space. Mechanoreceptor arranged along a crista (crest) and those of the oto-
organs are thought to have developed several times lithic organs (saccule and utricle) on a surface called
independently in evolution (Garcia-Anoveros and macula (spot). These otolithic organs are largely re-
Corey, 1997). However, the vertebrate vestibular sponsible for sensing linear acceleration as well as
system appears to be more complex than most ana- static tilt (Precht, 1976).
logous systems in invertebrates, and its functions Signals from the three orthogonally-arranged
[integrated with information coming from the visual SCC are presented to the central nervous system
and proprioceptive systems (Diener and Dichgons, (CNS) so as to generate an appreciation of angular
1988)] are re®ned enough to allow the amazing con- acceleration, angular velocity and position of the
trol of movements required for the preying birds' head and/or body in three-dimensional space. The
swoops and for the proverbial cats always landing hair cells of each of the SCCs have only a single
on their feet. functional directionality (Hillman, 1976) and thus
All vertebrate forms have inner ear organs pri- the decoding task of the CNS (of signals from the
marily responsible for sensing linear and angular SCC) is already simpli®ed.
acceleration and orientation of the head with regard In the otolithic organs the hair cells are generally
to gravity. These are the vestibular organs. The ves- polarized about a central line called striola
tibular organs transduce mechanical signals, such as (Hillman, 1976). In the utricle the functional polar-
are produced by active or passive head or body ization of the stereocilia of the hair cells is toward
movements, into biological signals ultimately carried and in the saccule it is away from the striola. Their
to the brain and spinal cord (Goldberg and arrangement may be summarized in this way: the
Fernandez, 1984). Some of these organs (e.g. sac- functional polarization vectors of utricular signals
cule) also transduce vibratory signals (Precht, 1976). lie near the horizontal plane while those of saccular
The central sensory elements in this transduction are signals lie near the sagittal plane of the head. Within
the hair cells, so-called because they are invested at the horizontal plane (Baird, 1992), the sensory rep-
their apical ends with villi, generally called stereoci- resentation of linear force from utricular units is
lia, and in vestibular organs a kinocilium, which two-dimensional. Signals from the saccule give in-
The Vestibular Hair Cells 195

formation in the third or dorso-ventral dimension Kinetics of response may also account for di€er-
(Ashcroft and Hallpike, 1934). Thus, together these ences among hair cells. How long will a particular
two otolithic organs send signals that are based cell output last in the presence of a constant input?
upon linear force in three dimensions much as the Phasic cells display rapid and transient responses
SCCs provide information about angular accelera- while tonic cells show a slower and longer lasting re-
tion in three dimensions. sponse. Thus, during a sustained input, individual
The vast majority of nonmammalian vertebrates vestibular hair cells are turning on and o€ at di€er-
have a third otolithic organ, the lagena. The lagena's ent rates. In high-frequency-responding saccular
function is primarily gravity sensing and its morpho- hair cells, receptor currents adapt in >100 msec in
logical polarization is similar to that of the saccule. the presence of a constant mechanical stimulus
That is, hair cells are arranged on either side of the (Eatock et al., 1987; Howard and Hudspeth, 1987);
striola with their kinocilia away from it. Some ver- however, after complete adaptation during a con-
tebrates also have a non-otolithic structure called stant stimulus, a second superimposed stimulus can
the papilla neglecta (Popper et al., 1990) whose func- elicit a new partial-to-full response. This has been
tion is not clear. explained by hypothesizing that, during a constant
The main task of sensory systems is optimizing step stimulus, the zero-input set point for mechanoe-
the response to stimuli in regards to sensitivity, sig- lectrical transduction (MET) current moves, restor-
nal-to-noise ratio, dynamic range and kinetics ing full sensitivity to subsequent stimulation. This
(Torre et al., 1995). Vestibular organs accomplish apical adaptation has been reviewed in Hudspeth
these tasks with a remarkable precision. As regards and Gillespie (1994). A di€erent adaptation system
sensitivity, hair cells are able to detect stereociliary may be working in SCCs, involving more complex
displacement of only a few atomic diameters metabolic processes [e.g. autoreceptor-mediated
(Hudspeth, 1983). The plot of membrane voltage vs feedback (Prigioni et al., 1990)], whose mechanisms
stereociliary de¯ection amplitude is asymmetrical are at present only partially understood (Masetto et
about the resting position of the stereociliary bundle al., 1995a,b). Utricular cells appear to use both sys-
(Howard et al., 1988). Inhibitory movements induce tems even on the same time scale (Baird, 1994b),
hyperpolarizations that saturate for very small suggesting that speed of response is not the only
angles of stereociliary de¯ection, while in the de- determinant of a double adaptation mechanism.
polarizing direction saturation occurs for much lar- All of the vestibular organs respond, in varying
ger de¯ection angles.
degree, to vibratory stimulation. In some instances,
Signal-to-noise ratio is optimized di€erently for
the vestibular organs respond to vibratory signals in
each hair cell. First, the accessory structures
air or substrate (Koyama et al., 1982). In certain
attached to the hair bundles reduce their thermal
vertebrates (e.g. ostariophysean ®sh) the saccule
motion (Torre et al., 1995). In addition, in saccular
does this so well that it is the major organ of hear-
and auditory hair cells from lower vertebrates, an
ing. The response of vestibular organs and a€erent
electrical tuning (Ashmore, 1983) [reviewed in
vestibular neurons to auditory stimuli has been stu-
(Fettiplace, 1987)] enhances the responses to a par-
ticular frequency, while in SCC hair cells, which are died by several laboratories (Cazals et al., 1980,
not sharply tuned, high frequency noise generated 1982; Didier and Cazals, 1989; McCue and Guinan,
by random channel openings is probably reduced by 1995; Murofushi et al., 1995; Young et al., 1977).
an inactivating K current (IA) (Norris et al., 1992; And, in fact, these investigators have all reported re-
Masetto et al., 1995b). Another noise reduction sponses to intense auditory stimuli in vestibular
strategy is the presence of the low-conductance a€erents. The threshold for activation of vestibular
delayed recti®ers instead of the high-conductance a€erents with clicks is ca 60 dB above the threshold
BK channels in hair cells tuned to low frequencies for auditory ®bers (Murofushi et al., 1995).
<60 Hz (Goodman and Art, 1996b). In fact, since It is the otolithic organs that are most likely
low frequency tuning requires smaller currents, BK responding to auditory stimuli. For instance, click-
current ¯uctuations would dominate cell response sensitive neurons were found in the posterior part of
[in a low-frequency cell, resting currents would only the superior branch or in the inferior branch of the
require the opening of 10 BK channels (Art and vestibular nerve, positions consistent with otolithic
Goodman, 1996)]. a€erents in the cat (Gacek, 1969) and guinea (Didier
Dynamic range is de®ned as ``the range of stimu- and Cazals, 1989). Furthermore, in the study of
lus intensities over which a given sensory receptor is Murofushi et al. (1995) all neurons responding to
able to respond'' (Torre et al., 1995). For the vestib- click also responded to static tilt. In this study, no
ular system, this range has to accommodate the click-response neurons responded to yaw rotation
small and slow head movements necessary to center suggesting that the horizontal SCC, at least, was not
in the visual ®eld a stationary nearby object and the involved. Contrariwise, Young et al. (1977) did ®nd
fast and large movements necessary to follow a that SCC a€erents respond to low frequency tone
moving object while traveling at high speed or to bursts. Lastly, the reader should be reminded that
avoid losing balance after a jump. In all vestibular the saccule sits close to the oval window and thus
organs, some high-gain cells are organized to could be subjected to intense auditory stimuli.
respond to very small accelerations, and saturate in Finally, vibratory frequencies in air <20 Hz are
the presence of large accelerations, while other low- referred to as infrasound. These low frequencies
gain cells only respond to larger inputs. Moreover, seem to be detected by the basilar papilla in the
the e€erent system appears to further extend vestib- pigeon (Schermuly and Klinke, 1990) and the otolith
ular dynamic range (see Section 5.2.1). organs in ®sh (Karlsen, 1992).
196 P. S. Guth et al.

Auditory and lateral line organs will only be within the hair cell (Hudspeth, 1989). Transduction
brought into the discussion when comparison occurs when the external mechanical forces cause
between them and vestibular organs illuminates the the hair cells' stereocilia to move. In their plane of
latter. Otherwise the central concern of this article is maximal sensitivity (Flock, 1965; Lowenstein and
the vestibular hair cell. Within the vestibular hair Wersall, 1959; Shotwell et al., 1981; Wersall et al.,
cell, this article will concern itself primarily with 1965) stereocilia are arranged in a `stair step' fashion
post-transductional signal processing. That is, an in- from the shortest to the tallest (Wersall and Bagger-
depth discussion of the structure and mechanics of Sjoback, 1974). When the appropriate mechanical
accessory structures such as the cupula and the oto- stimulus is applied to move the stereocilia, an ionic
lithic membrane and their in¯uences on the signal current, which ¯ows tonically from the endolymph
reaching the hair cell are outside the scope of this space into the apical end of the hair cell, is modu-
article, as are the well-reviewed phenomena associ- lated (Crawford et al., 1989; Flock, 1965). When
ated with transduction. However, for the nonspecia- movement is towards the tallest of the stereocilia,
list reader, a brief discussion of these topics seems this ionic current is increased. When movement is
important. towards the shortest of the stereocilia, current is
decreased (Flock, 1965; Lowenstein and Wersall,
1959; Shotwell et al., 1981; Wersall et al., 1965).
1.1. Accessory Structures
When the stereociliary movement is at an angle of
In the inner ear vestibular organs, environmental 908 to the plane of maximum sensitivity there is no
stimuli are mechanically coupled to the hair cells in change in transduction channel current (Shotwell et
one of two ways. al., 1981).
Vestibular hair cells are tightly packed within the
1. In the SCCs the kinocilia of the hair cells are
neuroepithelium and form tight junctions with their
embedded in channels within a gelatinous mass,
supporting cells (Hillman, 1976). These tight junc-
the cupula, which ®lls the space between the roof
tions, located in a subapical `belt' as in most epithe-
of the ampullated swelling of the canal and the
lia, prevent mixing of endolymph and perilymph,
apical surfaces of the hair cells themselves
which bathe the hair cell's apical and basolateral
(Hudspeth, 1989; Wersall, 1956). When the head
sides, respectively (Sterkers et al., 1988b). These two
is subjected to angular accelerations, inertial
¯uids have very di€erent ionic compositions: peri-
forces within the canals push the cupula in the
lymph is similar to extracellular ¯uids (i.e. high in
direction opposite to the acceleration (Wilson
sodium and low in K), while endolymph is similar
and Melvill-Jones, 1979). This in turn causes a
to intracellular ¯uids, that is, high in K and low in
bending or shearing movement of the hair bun-
sodium (Sterkers et al., 1988b; Maggio, 1966).
dles and either an opening or closing (depending
Chloride concentrations are almost equal in peri-
upon the direction of the angular acceleration-re-
lymph and endolymph (Sterkers et al., 1988b;
lated forces) of nonspeci®c cation channels
Maggio, 1966). Calcium is ca 1.2 mM in perilymph
(Corey and Hudspeth, 1979a; Hudspeth and
and 0.5 mM in endolymph (Ferrary et al., 1988). In
Corey, 1977) within or associated with the stereo-
fact, the concentration of free ionized calcium in en-
ciliary bundle.
dolymph is even lower (ca 20±30 mM), due to the
2. In the utricle and saccule (the macular organs)
presence of a strong bu€ering system (Ferrary et al.,
the hair cell cilia are also in contact with a gelati-
1988; Sterkers et al., 1988a). The regulation of endo-
nous membrane (Hudspeth, 1989; Hillman, 1976)
lymphatic Ca essential to the functioning of vestibu-
called the otolithic membrane. Sitting on this
lar organs, since a change in concentration would
membrane opposite to the side in contact with
a€ect adaptation of mechanoactivated currents
the hair cells are calcium carbonate crystals
(Pickles and Corey, 1992; Hudspeth and Gillespie,
known as otoconia or otoliths (ear stones)
1994; Ricci and Fettiplace, 1997), and a drastic drop
(Hudspeth, 1989; Hillman, 1976). Linear accel-
in concentration would even be able to impair the
erations applied to the head cause a shifting
mechanotransduction apparatus irreversibly (Assad
movement of the otoconia and gelatinous mem-
et al., 1991).
brane which translates into movement of the hair
Although there appears to be some disagreement
cell kinocilia and stereocilia (Wilson and Melvill-
as to the exact location of the transduction channel
Jones, 1979). Just as it does in the SCCs, this
[(Ohmori, 1985; Jaramillo and Hudspeth, 1991;
movement in the otolithic organs causes either an
Denk et al., 1995), see Hackney and Furness, 1995
opening or closing of the nonspeci®c cation chan-
for a review], there is general agreement that these
nels associated with the stereociliary bundle
channels are relatively nonspeci®c with regards to
(Corey and Hudspeth, 1979a; Hudspeth and
the cations which are allowed to pass through them
Corey, 1977). As a consequence, the hair cells de-
(Corey and Hudspeth, 1979a; Garcia-Anoveros and
polarize (or hyperpolarize) and this triggers (or
Corey, 1997).
inhibits) transmitter release (Hudspeth, 1989).
Since the predominant cation in the endolymph is
K, the opening of the nonspeci®c transduction chan-
1.2. Transduction nels on the hair cell's apical pole by mechanical
stimulation generates an inward K current which de-
In the inner ear mechanosensory systems, trans- polarizes the cell. The amplitude of the voltage re-
duction is the conversion of the mechanical energy sponse (receptor potential) depends in a nonlinear
contained in the external stimuli (sound pressure manner upon the de¯ection amplitude and can
waves and/or head motions) into electrical changes reach up to 45 mV for maximal de¯ections of the
The Vestibular Hair Cells 197

stereociliary bundle (Crawford and Fettiplace, and Melvill-Jones, 1979). But higher CNS centers
1980). There are controversies regarding the size of require not only acceleration data but also infor-
receptor currents [compare for example (Corey and mation concerning head velocity and head position.
Hudspeth, 1979b) and (Kros et al., 1992)]. These integrations of acceleration (velocity and pos-
Di€erences in mechanoactivated current amplitude ition) could take place in the vestibular nuclei or
between acoustical and vestibular hair cells have even more centrally but the information to the var-
been reported (Geleoc et al., 1997), and may be cor- ious CNS centers is more consistent and obtained
related with the number of stereocilia in the bundle, more eciently by having some of the vestibular
which ranges from <30 (see below) to 300 (Garcia- sensory cells directly reporting acceleration infor-
Anoveros and Corey, 1997). However, part of the mation, while others are processing their inputs and
di€erences in reported MET channel currents is reporting velocity or positional information.
likely due to the experimental setup. For example, Between the transductional processes at their hair
sodium, which has been used as the extracellular bundle and the release of transmitter(s) at their
cation in most cases, has been found to partially a€erent synapses, hair cells are busy modifying the
block mechanoactivated currents (Ricci and signal according to their individual biochemical and
Fettiplace, 1997). In addition, enzymatic treatment biophysical repertoires. These di€erences among
used for hair cell dissociation may reduce MET cur- vestibular hair cells have been increasingly appreci-
rents by digesting a component of the mechanosen- ated over the past decade. To bring these di€erences
sitive apparatus (Rusch and Eatock, 1996b). and their importance to the attention of interested
The receptor potential is shaped by both trans- readers is the main purpose of this review.
duction current (Baird, 1994a,b) and by currents
through voltage-dependent ion channels in the baso- 2.1. Hair Cell Morphology
lateral walls of the hair cells (Hudspeth and Lewis,
1988; Baird, 1994a,b; Correia et al., 1996). That is, Hair cells are rather variable in size (ranging 10±
the instantaneous depolarization due to MET chan- 50 mm in length and 5±10 mm in diameter) and
nel opening is proportional to the amplitude of shape. Vestibular hair cells have been classi®ed
MET currents (Corey and Hudspeth, 1983); this po- according to the shape of their apical hair bundles
tential change, in turn, a€ects voltage-sensitive ion or of their bodies, or by a€erent innervation pat-
channels (Hudspeth, 1986, 1989). The open prob- terns. Most likely, these classi®cations re¯ect di€er-
ability of these channels further modi®es the recep- ent properties of hair cells, since the hair bundle is
tor potential. Eventually, the modi®ed receptor involved in mechanotransduction while the hair cell
potential controls and is in¯uenced by voltage-sensi- body is involved in transmitter release. In this sec-
tive Ca channels which in turn control transmitter tion, we will outline the major morphological classi-
release. The resulting output is a partially processed ®cations of hair cell types. Innervation patterns in
(e.g. low-®ltered or band-passed) vestibular re- the vestibular system are quite complex, and will be
sponse. Subsequent processing due to a€erent nerve treated as a separate topic, since they probably add
properties is likely to occur. Therefore, caution must
be used when nerve ®ring is employed to monitor
hair cell activity.

2. MORPHO-PHYSIOLOGICAL DIFFERENCES
AMONG HAIR CELLS
When examined super®cially it may appear that
hair cells are merely passive transducers whereby
mechanical stimulation at the apical end of the cells
directly causes neurotransmitter to be released at the
basal end of the cells resulting in a direct increase or
decrease in impulse frequency in the a€erent neur-
ons. In actuality, the process is much more compli-
cated. In addition to a variety of anatomical
di€erences, hair cells from di€erent inner ear organs
and from di€erent species have markedly di€erent Fig. 1. Hair cell body shapes. Top, typical type I (left) and
physiological, biochemical and pharmacological type II (right) are outlined, with the a€erent terminals
properties (see Sections 3, 5 and 6). Even hair cells sketched in solid black. Dots represent synaptic bodies.
within the same organ have di€ering properties (see Bottom, hair cell shapes observed in anamniote vestibular
the following). It is, therefore, highly probable that organs. From left to right: cigar (ci), club (cb) and pear
these various properties control the manner in (pe) shapes observed in frog SCC (Guth et al., 1994b),
which individual hair cells respond to their input spherical cell (sf) observed in frog saccule (Holt and
Eatock, 1995); the cylindrical cells observed by Holt and
signals, process them and produce a modi®ed output
Eatock (1995) were similar to cigar cells (ci), although pea-
which is compatible with, and ready for, higher nut-shaped variants have been observed (ci'). Further var-
neural processing. iants, as small cylindrical cells (sc) or `tailed' (td) cells have
Most researchers agree that the appropriate mech- been observed in the sacculus (P. Perin, personal communi-
anical stimuli to the vestibular hair cells arise from cation); the latter may correspond to `gourd' cells observed
angular and linear accelerations of the head (Wilson in the ®sh sacculus (Sugihara and Furukawa, 1989).
198 P. S. Guth et al.

further complexity to the signal coming from hair length and structure (Hillman, 1976; Hackney and
cells. Furness, 1995). In the turtle SCC, for example,
The ®rst classi®cation of vestibular hair cells in there is a marked di€erence in stereociliary number
amniotes grouped them into Types I and II (Fig. 1) between Types I and II cells: the former bear 60 or
(Wersall, 1956) (anamniotes do not show Type I more stereocilia, while the latter display <50 (in the
hair cells, but see below). Type I hair cells are SCC periphery, Type II cells only have 15±35 stereo-
shaped like bottles or ¯asks with rounded bases and cilia (Peterson et al., 1996). Since the names used in
short necks, and are innervated by an a€erent nerve hair bundle classi®cation partially overlap those
calyx engul®ng the entire cell. Type II hair cells used in classifying cell shape, in this review we will
have instead fairly regular cylindrical shapes, are call hair bundle categories `classes' and cell shape
generally taller than Type I cells, and are innervated categories `types', to avoid confusion. The ®rst par-
by several ®ne a€erent nerve branches, mostly ameter used in ciliary tufts classi®cation is the ratio
synapsing with the basal part of the cell (Goldberg between kinocilium length and length of the tallest
and Fernandez, 1984). Type I and II hair cells also stereocilia [which is ca 1 in class I tufts, but much
show functional di€erences (see Sections 3 and 5). larger in class II (Hillman, 1976)].
However, in spite of the presence of both types in Table 1 summarizes hair bundle classes found in
all amniote vestibular organs, the di€erent vestibular the various vestibular organs in the bullfrog (Lewis
organs respond to mechanical stimuli in di€erent and Li, 1975; Hillman, 1974). Classes A and B hair
frequency ranges. In the 1970s, hair cells in di€erent cells display very short hair bundles (1±2 mm).
vestibular organs were found to display di€erences Classes C and F hair cells display tall (10 mm) hair
in their hair bundles (Hillman, 1974, 1976; Lewis bundles. The distinctive feature of classes D and E
and Li, 1975). It is, therefore, tempting to link the is a bulbed kinocilium; class D hair cells have
frequency (and adaptation) properties of the cells to shorter stereocilia than class E (4±5 mm vs 10±
their bundle properties, since the hair bundle is 15 mm). Class A-like and C-like cells, which are
where mechanotransduction takes place. The SCCs found in the SCCs, show an extremely long kinoci-
are mainly limited to the transduction of low-fre- lium (>60 mm).
quency stimuli [the response to mechanical stimuli These hair cell classes are di€erentially distributed
follows the stimulus timecourse only for frequencies across the vestibular organs of the bullfrog. For
below 10 Hz (Highstein et al., 1996)]. The saccule is instance, class A, which is thought to represent an
specialized to transduce small amplitude, high fre- immature hair cell (Lewis and Li, 1975), is found
quency linear accelerations such as those due to only in the peripheral region of the high-frequen-
rapid movements and substrate-borne vibrations cies-sensing organs (saccule, basilar papilla, amphi-
(Koyama et al., 1982; Lewis et al., 1992). The utri- bian papilla). Classes B and C are only found in the
cle, in addition to high frequency linear accelera- utricle and lagena, and could be gravistatic receptors
tions, also senses low frequency signals such as are (Lewis and Li, 1975). Class D is found in the high-
caused by static tilt (Baird, 1994a,b; Baird and frequencies-sensing organs (it is the predominant
Lewis, 1986). Part of these di€erences arise from type in the saccule). Classes E and F are found in
gross anatomical features of the organs [i.e. the the utricle, and display slowly adapting responses.
cupula-endolymph system has an higher inertia than Classes A-like and C-like are only found in the
the otolith-otolithic membrane system (Precht, 1978; SCCs, which respond to very low frequencies. In
Wilson and Melvill-Jones, 1979)]. However, hair fact, a very long kinocilium would act as a mechan-
cells also show morphological di€erences that corre- ical low-pass ®lter on MET currents.
late well with organ response.
As regards hair cell morphology, the following 2.1.2. Hair Cell Body
rule-of-thumb may be inferred: hair bundle mor-
phology is related to the frequency response of the Hair cell body features such as shape seem to cor-
cell, while cell body properties are related to the relate more to the position of the hair cell within an
posttransductional shaping of receptor potentials. organ than to the particular organ of origin itself.
The next section will deal with those topics. This probably depends on the di€erent packing den-
sities in di€erent epithelial zones as well as di€erent
a€erent and e€erent innervation patterns, and is
2.1.1. Hair Bundle very likely to re¯ect functional characteristics as
Hair cell bundles di€er in stereociliary number, well. As already mentioned, in amniotes both Type I
length, packing and diameter and in kinociliary and II hair cells are found in all vestibular organs

Table 1. Distribution of hair bundle types in the bullfrog labyrinth [from Lewis and Li (1975)]
Organ Dominant Other
bundle class bundle classes Frequency range Accessory structure
Utricle B C, E, F Low (gravistatic) Otolithic membrane
Lagena B C, E Low (gravistatic) Otolithic membrane
Sacculus D A Moderate (vibrational) Otolithic membrane
Amphibian papilla D, E A Moderate to high Tectorial membrane
(vibrational, auditory)
Basilar papilla D, F A High (auditory) Tectorial membrane
Semicircular canals Similar to C Similar to A Low (angular acceleration) Cupula
The Vestibular Hair Cells 199

(Goldberg and Fernandez, 1984). Moreover, even in further supporting the idea of Type I-likeness.
a quite primitive sensory epithelium (the undi€eren- Honrubia et al. (1989) reporting on claw-like
tiated vestibular macula of the lamprey) two di€er- synapses of Type II hair cells in the bullfrog
ent hair cell types can be found (Hoshino, 1975). suggested that these might be the ancestors of calyx
Two types of hair cells are found also in the gold- synapses.
®sh saccule: the rostral macula contains short ovoid Weisleder et al. (1995) provided a wonderfully
hair cells while the caudal macula contains long clear demonstration of the relationship between
cylindrical or gourd-shaped cells (Sugihara and Types I and II hair cells. The investigators damaged
Furukawa, 1989). Eighty-six percent of the short, vestibular hair cells in the chick with streptomycin.
oscillatory-type hair cells had a hyperpolarization- They then followed the generation of new hair cells
activated K±Na current (Ih) (Sugihara and beginning with the di€erentiation of supporting
Furukawa, 1996). This current was not observed in cells, as others have reported. First seen are new
long spike-type hair cells, while it was seen in all Type II hair cells. But then, the new Type II hair
hair cells of the frog saccule. A di€erent inwardly cells undergo further di€erentiation to Type I hair
rectifying current (Ik1) occurred only in cylindrical cells, a€erent calyx and all. These results provide
hair cells with a small width-to-length ratio (Holt evidence, in the words of the authors, ``that Type I
and Eatock, 1995) in the frog saccular hair cells. hair cells . . . are a more di€erentiated stage of Type
Remarkably, Ik1 also occurs only in long, spike-type II hair cells''.
hair cells of the gold®sh saccule (Sugihara and Using a classi®cation scheme similar to the one
Furukawa, 1989). At least with regard to these two outlined in Table 1, bullfrog utricle hair cell distri-
currents and these two species, there is a correlation bution, a€erent innervation patterns, biophysics and
between hair cell shape and conductances as ®rst functional properties have been studied (Baird and
reported by Sugihara and Furukawa (1989). Schu€, 1994). In this study, the utricular macula
Similarly, Steinacker and associates found oscillat- was divided into three zones, adding a juxtastriolar
ing and spiking hair cells (Steinacker and Perez, belt of cells between striolar and extrastriolar popu-
1992) and long and short cells (Steinacker and lations. Juxta- and extrastriolar regions were domi-
Romero, 1991) in toad®sh saccule, although they nated by class B, while classes C, E and F were
reported no regional localization of these hair cells.
mostly con®ned to striolar regions. A correlation
However, the ®sh saccule shows a division into
was found between bundle type and hair cell body
rostral and caudal parts that is not found in other
shape: in fact, class E hair cell body was always
vertebrate classes (Platt and Popper, 1981), and
cylindrical, while the majority of type F hair cell
therefore it is not known whether these di€erent
bodies were spherical; classes B and C hair cells had
hair cell populations have any counterparts in non-
a body shape that varied depending on macular pos-
®sh vertebrates. On the other hand (Chang et al.,
ition: extrastriolar class B cells had a cylindrical
1992), two hair cell types have been observed in the
utricle of the cichlid ®sh Astronotus, with di€erent shape, while striolar class B cells were similar to the
structure and distribution. The hair cells were `club' cells described in the leopard frog SCC (Guth
divided into striolar and extra-striolar: extra-striolar et al., 1994b). These cells have been suggested to
hair cells resembled Type II hair cells while striolar represent a transition stage between supporting and
hair cells had structures such as perinuclear cisterns, hair cells (Baird et al., 1997). Class C cells were
large perinuclear mitochondria and relatively large cylindrical in the outer striolar rows, but spherical
numbers of synaptic bodies. It is noteworthy that in inner striolar rows. Possibly, the spherical cells
the saccule of Astronotus likewise has two hair cell described in the bullfrog utriculus (Baird and
types, striolar and extra-striolar (Popper et al., Schu€, 1994) and in the leopard frog sacculus (Holt
1993) and that the distribution of these cells is remi- and Eatock, 1995) correspond to `pear' cells
niscent of Types I and II hair cells in the saccule of observed in the frog SCC (Guth et al., 1994b) and
the turtle (Jorgensen, 1974). And, while the striolar saccule (P. S. Guth et al., personal communication).
hair cells do not have chalice-type a€erent inner- In frog SCC, three types of hair cells [club, cigar
vation they do receive innervation by the largest of and pear (Guth et al., 1994b)] have been found to
the a€erent neurons. These considerations led be di€erentially localized in the crista: club cells pre-
Chang et al. (1992) to call the striolar cells Type I- dominate in the periphery while cigar cells predomi-
like. These authors discussed the evolutionary sig- nate in the central part (Masetto et al., 1994); pear
ni®cance of the Type I-like hair cell and their ®nding cells seem to be located in an intermediate zone
that anamniotes (i.e. all vertebrates not reptiles, (Gioglio et al., 1995; Prigioni et al., 1996), possibly
birds or mammals) have more than one type of ves- homologous of the juxtastriolar region of the oto-
tibular hair cell. lithic organs. Previous results suggested a central
Pursuing these interesting ®ndings, Lanford and localization of pear-shaped cells (Guth et al., 1994b)
Popper (1995) reported on basal outpouchings in in the frog SCC. These discrepancies may result
vestibular hair cells of the gold®sh that were sur- from methodological di€erences: in fact, Guth et al.
rounded by a€erent endings. This is reminiscent of (1994b) did not di€erentiate between a central and
the Type I hair cell-a€erent arrangement not gener- an intermediate region on the transverse axis, and
ally seen in anamniotes. Further, these authors saw the serial longitudinal sections used by them encom-
e€erent±a€erent synapses in the gold®sh, as did passed the whole transverse width of the crista,
Sans and Highstein (1984) in the toad®sh. E€erent- while Prigioni and Gioglio focused on the most cen-
a€erent synapses are also a feature of the Type I tral longitudinal slices (S. Masetto, personal com-
arrangement not generally seen in anamniotes munication). Therefore, data from Guth et al.
Fig. 2. Voltage-dependent ionic currents observed in vestibular hair cells. Currents observed in saccular
hair cells are shown on the left, currents from SCC hair cells are shown on the right. Top, saccular
macula (left). S, striola; ES, extrastriola; SCC crista (right). C, Central zone; P, peripheral zones. Ionic
current names are described in the main text. The protocols used are listed as (holding potential; step po-
tentials; duration). Saccular currents: IK1 (ÿ66 mV; ÿ66/ ÿ 116 mV; 300 msec); Ih (ÿ66 mV; ÿ96/
ÿ 146 mV; 500 msec); IBK (ÿ70 mV; ÿ50/ ÿ 30 mV; 15 msec. Inset: ÿ60; ÿ60/60; 30 msec); IA (ÿ105 mV;
ÿ56/3 mV; 100 msec); Ica (ÿ80 mV, ÿ60/0 mV; 160 msec); IK, V (ÿ60; ÿ60/30 mV; 25 msec); IK, L
(ÿ90 mV; ÿ120/0 mV; 160 msec). SCC currents: IK1 (ÿ60 mV; ÿ60/ ÿ 160 mV; 150 msec); Ih (ÿ60 mV;
ÿ60/ ÿ 120 mV; 250 msec); IBK (ÿ80 mV; ÿ80/40 mV; 150 msec. Inset, ÿ80 mV; ÿ20 mV; 150 msec); IA
(ÿ80 mV; ÿ80/40 mV; 125 msec. Inset, ÿ60; ÿ130/120 prestep subtraction; 200 msec); Ica (ÿ60 mV, ÿ60/
0 mV; 160 msec); IK, V (ÿ80; ÿ80/40 mV; 25 msec). Inset, ÿ100 mV; ÿ40 mV; 15 sec); IK,L (ÿ90 mV;
ÿ120/0 mV; 160 msec). (a) Modi®ed from Holt and Eatock (1995); (b) modi®ed from Hudspeth and
Lewis (1988); (c) P. Perin (unpublished data); (d) modi®ed from Steinacker and Perez (1992); (e) modi-
®ed from Ricci et al. (1996); (f) modi®ed from Masetto et al. (1994); (g) S. Masetto (unpublished data);
(h) modi®ed from Prigioni et al. (1996); (i) modi®ed from Norris et al. (1992); (l) modi®ed from Russo
et al. (1996). Scale bar indicates 500 pA for ICa, Ih and SCC IK1, 1 nA for other currents.
The Vestibular Hair Cells 201

(1994b) are probably an average between central single-channel conductance (Steinacker and Perez,
and intermediate regions. 1992; Art et al., 1995), and are blocked by TEA
Hair cell morphological types are summarized in (Hudspeth and Lewis, 1988; Goodman and Art,
Fig. 1. The physiological features of these cell types 1996b; Masetto et al., 1995b) and charybdotoxin
will be discussed in the following sections. (Steinacker and Perez, 1992; Masetto et al., 1995b),
A somewhat di€erent view of the question of hair but not by 4-aminopyridine (4-AP) (Goodman and
cell shape-conductance correlation was presented in Art, 1996b; Hudspeth and Lewis, 1988) or apamin
Prigioni et al. (1996). These authors examined the (Masetto et al., 1995b). BK channels begin activat-
conductances of pear-shaped hair cells of the frog ing at ÿ40 mV (Masetto et al., 1994) or above
SCC. Pear-shaped hair cells of the peripheral (Griguer et al., 1993; Lang and Correia, 1989) in
regions of the crista displayed mostly delayed recti- SCC hair cells, and above ÿ60 (Hudspeth and
®er and Ca-dependent K currents with a small con- Lewis, 1988) or ÿ50 mV (Steinacker and Perez,
tribution from the inactivating K current IA. In 1992; Roberts et al., 1990; Holt and Eatock, 1995)
contrast, pear-shaped hair cells of the zone near the in saccular hair cells. In turtle cochlea (Wu et al.,
center of the crista displayed an overwhelming Ca- 1995; Art and Fettiplace, 1987) and toad®sh saccu-
dependent K current and neither of the other two K lus (Steinacker and Perez, 1992), BK activation kin-
currents. These data seem to suggest that the more etics is greatly variable. BK activation kinetics
acceptable correlation is between conductances and re¯ect cytoplasmatic Ca concentrations (see Section
position rather than hair cell shape. At the end, it 6.3), since BK opening requires two Ca-dependent
may be more complicated than this view because steps (Hudspeth and Lewis, 1988). However, the
there is also a general correlation between hair cell presence of several splicing variants with di€erent
shape and position (and function) in the vestibular kinetic properties (Wu and Fettiplace, 1996) or
epithelia. di€erent channel modulation (Art et al., 1995) has
been suggested. In fact, in saccular hair cells, BK
channels appear less sensitive to Ca than in turtle
3. HAIR CELL CONDUCTANCES cochlear hair cells (Art et al., 1995). Moreover,
other di€erences in BK expression have been found.
Hair cell ion channels are operated by mechanical Pear shaped hair cells from the intermediate region
stimulation, voltage, and ligands, such as Ca. The of SCC crista express a noninactivating BK current
application of voltage and current clamp techniques similar to the one observed in acoustic and otolithic
to vestibular hair cells has revealed many di€erences organs, which accounts for 95% of total outward
in terms of their ion channels expression and func- currents in these cells (Prigioni et al., 1996). In the
tional roles. This supports the view that a major remaining frog SCC hair cells, a partially inactivat-
determinant of the a€erent nerve ®ber spiking pat- ing BK only accounts for 25±50% of total currents
tern is the hair cell processing of the input stimuli. (Masetto et al., 1994). This may re¯ect a lack of im-
The major ion channels expressed by vestibular hair portance of BK channels in low frequency re-
cells (Fig. 2) are potassium and calcium channels; sponses, as has been observed for low-frequency-
however, nonspeci®c channels (Holt and Eatock, tuned cochlear hair cells (Art et al., 1993).
1995; Sugihara and Furukawa, 1989) have also been Interestingly, inactivating BK currents have been
described. On the other hand, sodium channels are observed in most SCC hair cells (Masetto et al.,
usually not present in mature vestibular hair cells, 1994, 1995b) and in a subset of saccular hair cells
although they may be present in acoustic hair cells (Steinacker and Romero, 1991; Steinacker and
(Sugihara and Furukawa, 1989; Fuchs and Evans, Perez, 1992), suggesting a di€erent molecular com-
1988; Evans and Fuchs, 1987; Witt et al., 1994). position for these channels, in spite of their pharma-
cological similarity to other hair cell BK channels.
3.1. Potassium Channels An additional Ca-dependent K channel has been
observed in hair cells from the saccule and reptilian
Most vestibular hair cells display inactivating and cochlea (Art et al., 1995; Steinacker and Rojas,
noninactivating outward K channels. Inward K 1988). This low conductance channel is blocked by
channels are found in hair cells from the macular apamin and TEA (Yoshida et al., 1994; Tucker and
organs but rarely from the SCCs. Type II vestibular Fettiplace, 1996), is permeant to Cs as well as K
hair cells show a BK-like (Hille, 1992) Ca-dependent (Tucker and Fettiplace, 1996; Erostegui et al.,
K channel (Hudspeth, 1986). In saccular hair cells, 1994a) and displays a higher Ca-sensitivity than BK
similarly to what is found in lower vertebrate acous- (Art et al., 1995). Therefore, it has been classi®ed as
tic hair cells (Fuchs et al., 1988; Art and Fettiplace, SK (Hille, 1992). SK channels appear to be involved
1987), this current is very prominent (Hudspeth and in hair cell responses to cholinergic stimulation (see
Lewis, 1988; Roberts et al., 1990; Steinacker and Sections 5.2 and 5.7).
Perez, 1992; Holt and Eatock, 1995; Sugihara, The pool of Ca-independent K channels expressed
1994), while in SCC (Lang and Correia, 1989; by hair cells is quite variable: several types of I(A),
Griguer et al., 1993; Masetto et al., 1994) and most delayed recti®ers and inward recti®ers have been
likely in utricle (Baird, 1994a) it is only a minor found in vestibular hair cells, and appear to be dif-
component of total outward current. These channels ferentially expressed in di€erent hair cell types.
are de®ned as BK (Rudy, 1988; Hille, 1992) since IA currents are de®ned as voltage-dependent, inac-
they are voltage- and Ca-dependent (Hudspeth and tivating K currents that are blocked by 4-AP (Rudy,
Lewis, 1988; Art et al., 1995; Masetto et al., 1994; 1988). Inactivation properties, however, may be
Sugihara, 1994), display a large, voltage-dependent quite variable (Norris et al., 1992). In frog saccular
202 P. S. Guth et al.

hair cells, IA has been found to display half-inacti- (Sugihara and Furukawa, 1989, 1996; Holt and
vation voltages V1/2 ranging from ÿ80 to ÿ93 mV Eatock, 1995; Rusch and Eatock, 1996a,b). IK1 acti-
(Hudspeth and Lewis, 1988); however, this ®nding vates rapidly between ÿ80 and ÿ90 mV; Ih activates
may be an artifact (Goodman and Art, 1996a,b) due slowly below ÿ50 mV, but displays a considerable
to G-protein activation by F-based intracellular sol- open probability (ca 20%) even for more depolar-
ution (this raises the interesting possibility that hair ized potentials (Holt and Eatock, 1995). In the gold-
cell IA is modulated in vivo). On the other hand, ®sh saccule, most short, oscillatory hair cells, but
reported V1/2 for SCC hair cells IA currents were not long, spiking hair cells, display an Ih (Sugihara
ÿ53 mV (Steinacker, 1996), ca ÿ55 mV (Housley et and Furukawa, 1989, 1996); in the frog saccule, all
al., 1989), ÿ53.6 mV (Russo et al., 1995) and hair cells have been shown to express it (Holt and
ÿ49.6 mV (Griguer et al., 1993), and therefore these Eatock, 1995). Recently, this current has been also
currents are partially active at rest. Inactivation is found in hair cells from the central region of pigeon
time-dependent and voltage-independent; inacti- SCC cristae (Masetto and Correia, 1997). The IK1-
vation removal is instead voltage-dependent (Russo like inward recti®er is expressed by tall hair cells
et al., 1995; Norris et al., 1992; Masetto et al., from the frog and gold®sh saccule (Holt and
1994). Activation of IA seems more consistent: all Eatock, 1995; Sugihara and Furukawa, 1989), and
observed threshold potentials are between ÿ50 and by central hair cells in frog SCC (Masetto et al.,
ÿ60 mV (Hudspeth and Lewis, 1988; Masetto et al., 1994). This current shows Na-dependent inacti-
1994; Lang and Correia, 1989; Murrow, 1994). In vation during sustained voltage steps and is blocked
the frog crista, IA expression appears to be region- by external Cs, TEA and Ba (Masetto et al., 1994;
ally limited to peripheral cells (Masetto et al., 1994), Holt and Eatock, 1995; Sugihara and Furukawa,
and in amniote vestibular organs it is limited to 1996). At least in auditory hair cells, both mild and
Type II cells (Correia et al., 1996). Multiple com- strong recti®er subunits have been found to be
ponents have been observed: in frog SCC (Housley expressed (Glowatzki et al., 1995); these channels
et al., 1989; Norris et al., 1992) two IA components carry current in the outward direction for potentials
seem to be expressed by hair cells in di€erent rela- near EK (Holt and Eatock, 1995; Goodman and
tive proportions. Inactivation time constants were Art, 1996a), thus a€ecting hair cell resting potential
4.44 msec for fast channels and 36.4 msec [similar to and resting input impedance.
what found in SCC peripheral cells by (Masetto et Type I hair cells from all vestibular organs
al., 1994)] for slow channels; steady state inacti- (Griguer et al., 1993; Rennie et al., 1996; Ricci et
vation for the fast channels at rest was smaller than al., 1996; Rennie and Correia, 1994; Rusch and
for the slow channels. Separation of this current is Eatock, 1996a,b) express a unique 4-AP sensitive
however not always easy since hair cells express voltage-dependent K current, called IKI, which
other 4-AP sensitive channels [IK, L (Rusch and accounts for most of total current in these cells
Eatock, 1996a) and a delayed recti®er-type current (Ricci et al., 1996). This current is activated for very
(Goodman and Art, 1996b; Sugihara and negative potentials [ca ÿ100 mV (Rennie and
Furukawa, 1995)]. Correia, 1994; Rusch and Eatock, 1996a,b)] and
The slow-activating delayed recti®ers appear to be does not inactivate substantially: therefore, at the
expressed preferentially by cells that respond to resting potential this conductance acts as a steady
lower frequencies (Murrow, 1994; Art et al., 1993). K-selective leak. Interestingly, IK, L has been shown
Consistently, they are large in hair cells from the to be modulated by nitric oxide (NO) (Chen and
SCCs, especially in the central region (Masetto et Eatock, 1994).
al., 1994; Russo et al., 1996; Rennie and Ashmore,
1991). However, in SCC these currents are blocked
by TEA and charybdotoxin (Masetto et al., 1994, 3.2. Calcium Channels
1995b), while in acoustic organs the main com-
ponent is blocked by 4-AP (Goodman and Art, Hair cells continuously face the opposing necessi-
1996b; Sugihara and Furukawa, 1995). Multiple ties of having a sustained, fast-changing calcium
components are probably expressed: in frog SCC in¯ux and of avoiding the cytotoxic e€ects of large
hair cells from the central crista, the presence of two calcium concentrations in the cytoplasm. Several
delayed recti®er components has been suggested details of this delicate balancing process are now
based on slow inactivation properties (Russo et al., emerging, and suggest that Ca regulation is likely to
1996). Hair cell delayed recti®ers activate ca be speci®cally adapted to each hair cell's particular
ÿ60 mV, and a steady-state inactivation between 40 needs. In fact, calcium is necessary to hair cells for a
and 50% is obtained above ÿ50 mV (Masetto et al., wide variety of processes, that di€er more or less
1994; Russo et al., 1996; Sugihara and Furukawa, markedly among hair cells, from mechanosensitivity
1995; Goodman and Art, 1996b). and mechanotransduction current adaptation
Two types of inward rectifying currents have been [reviewed in Gillespie (1995)], to synaptic release at
found in vestibular hair cells: a K-selective, Cs- and a€erent active zones (Annoni et al., 1984), to e€er-
TEA sensitive inward recti®er similar to the cardiac ent stimulation [reviewed in Ohmori (1996) and
IK1 (Art and Goodman, 1996; Sugihara and Fuchs (1996)], and to highly specialized tasks such
Furukawa, 1989, 1996; Holt and Eatock, 1995; as the electrical tuning of lower vertebrate acoustic
Goodman and Art, 1996a,b; Ohmori, 1984; Masetto and seismic organs (Art and Fettiplace, 1987;
et al., 1994; Murrow, 1994) and a slow, mixed K/Na Hudspeth and Lewis, 1988) or cell motility in mam-
conductance which is blocked by external Ba and re- malian Type I hair cells and cochlear outer hair cells
sembles the Ih originally found in cardiac cells (OHCs). A recent short review on the role of Ca in
The Vestibular Hair Cells 203

hair cells may be found in Lenzi and Roberts 1800±2000 channels per cell and a noise-estimated
(1994). single-channel current of 0.8 pA at ÿ50 mV have
In both Type I and Type II cell, several voltage- been reported (Roberts et al., 1990); in SCC, there is
independent ways for Ca in¯ux in hair cell cyto- to date no estimate of single-channel current, and
plasm have been observed (most likely with di€erent the only data on channel density [1800 per cell
relative importance and functions): transduction (Prigioni et al., 1992)] assume a single-channel cur-
channels (Jorgensen and Kroese, 1994), neuronal- rent of 0.09 pA at ÿ12 mV, derived from other cell
type nicotinic receptors (Elgoyhen et al., 1994), types, which may not apply to SCC hair cells.
ionotropic glutamate receptors (Prigioni et al., 1990; In saccular hair cells, (Roberts et al., 1990; Issa
Dememes et al., 1995), ATP receptors (Rennie and and Hudspeth, 1994), VOCCs are clustered, in
Ashmore, 1993), and inositol 1,4,5 trisphosphate patches containing ca 90 Ca channels and 40 K(Ca)
(IP3)-sensitive intracellular stores activated by G- channels, at the 300 nm-wide presynaptic active
protein coupled receptors (Shigemoto and Ohmori, zones. By loose-patching active zones, Roberts et al.
1990; Kakehata et al., 1993). (1990) recorded from single active sites Ca currents
All Type II hair cells studied display voltage- with similar kinetics to whole-cell, suggesting that
dependent calcium channels, consistently with their all clusters in a cell display similar characteristics.
role as presynaptic elements. In sacculus, voltage- Each cluster regulates Ca concentration in an inde-
operated Ca channels (VOCCs) are clustered at pendent domain of hair cell cytoplasm (Roberts,
a€erent presynaptic active zones, together with K(Ca) 1993; Roberts et al., 1991); Ca appears to di€use
channels (Roberts et al., 1990; Issa and Hudspeth, only for an extremely short space in these domains
1994). This organization permits a large and fast Ca
(Roberts, 1993, 1994). As regards other vestibular
concentration rise at these active zones, with much
organs, there are no direct data about channel distri-
lesser changes in the surrounding cytoplasm. In ad-
bution.
dition, mobile Ca-binding proteins present at high
concentrations in saccular hair cells cytoplasm Vestibular hair cells VOCCs have been described
ensure the rapid removal of Ca from active zones as L-type based on the following observations.
(see Section 6.3). 1. Dihydropyridine (DHP) sensitivity: in sacculus,
On the other hand, data on voltage-dependent Ca (+)-202791 increased Ca currents and slowed
entry in Type I hair cells are scarce: in SCC Type I deactivation (Roberts et al., 1990); in SCC, Bay-
cells, Ca in¯ux has been shown to be activated by K 8644 5 mM increased Ca currents by 1222 19%
depolarizations (Rennie and Ashmore, 1991; Rennie at ÿ20 mV (Prigioni et al., 1992) and nifedipine
and Correia, 1994) or at the end of depolarizing partially blocked Ca currents (Rennie et al.,
steps (Sans et al., 1994; Griguer et al., 1995); no 1994). Similarly, in cochlear hair cells, Bay-K
complete study, however, has speci®cally addressed 8644 increased currents by 504267% at ÿ30 mV
Type I hair cell VOCCs, and experimental di€er- (Fuchs et al., 1990; Zidanic and Fuchs, 1995),
ences may explain the contrasting results. In fact, and DHP blockers at a concentration of 10 mM
Type I hair cells show lower densities of presynaptic suppressed from 43% (nifedipine) to 72% (nimo-
active zones than Type II (Schessel and Highstein, dipine) (Zidanic and Fuchs, 1995). DHP action
1981): therefore, the presynaptic voltage-dependent was voltage-dependent.
Ca currents could be quite small and dicult to 2. Currents increase when Ba is substituted for Ca
detect. The presence of subpopulations of Type I as charge carrier (Art and Fettiplace, 1987;
cells di€ering in VOCC expression is possible, since Fuchs et al., 1990; Lewis and Hudspeth, 1983;
these cells show di€erent Ca resting concentration
Perin et al., 1997a; Prigioni et al., 1992; Roberts
and high K-induced Ca increases (Chabbert et al.,
et al., 1990).
1994).
3. Stronger block by Cd than by Ni: the former
Di€erences in Ca channel expression among hair
shows a Kd of 10 mM for cochlear hair cells
cells are probably not as marked as for K channels
(see above). Fuchs et al. (1990) found a pharmaco- (Fuchs et al., 1990) and 5 mM for canal hair cells
logically and kinetically homogeneous VOCC popu- (Prigioni et al., 1992), while the latter has shown
lation in the di€erent regions of chick cochlea, while higher Kd in both cases [in Prigioni et al. (1992)
the same cells displayed clear di€erences in the ex- Ni 5 mM blocked 16.5 27.5% and Ni 40 mM sup-
pression of K channels; VOCCs also appeared to be pressed all Ca current, while in Fuchs et al.
similar in both tall and short hair cells (Zidanic and (1990) Ni 50 mM only blocked 39 28% and Ni
Fuchs, 1995). Ca current amplitudes, on the other 1 mM was needed to suppress all current. In both
side, varied tonotopically among cochlear hair cells: studies, at higher concentrations Ni block was no
apical cells, which respond to lower frequencies, longer completely reversible].
showed an average current of 46225 pA, vs the 4. Current rundown in the whole cell (Hudspeth
1252 70 pA of basal cells, which sense higher fre- and Lewis, 1988; Lang and Correia, 1989;
quencies. Ca current amplitudes vary widely also Ohmori, 1984; Parsons et al., 1994; Prigioni et
among the di€erent vestibular organs, ranging from al., 1992; Roberts et al., 1990) but not in the per-
the 50±150 pA observed in SCCs (Lang and forated patch con®guration of patch clamp
Correia, 1989; Rennie and Ashmore, 1991; Rennie (Parsons et al., 1994; Perin et al., 1997a),
and Correia, 1994; Prigioni et al., 1992) to currents suggesting the requirement of some di€usible in-
tenfold as large seen in frog saccular hair cells tracellular component. Rundown was not coun-
(Hudspeth and Lewis, 1988; Roberts et al., 1990; teracted by ATP, GTP, Mg or EGTA addition
Parsons et al., 1994). In the latter, a density of ca (Hudspeth and Lewis, 1988; Fuchs et al., 1990).
204 P. S. Guth et al.

5. No e€ect of holding potential changes between a1E (together with N-type a1B) is found in spiral
ÿ110 and ÿ70 mV (Prigioni et al., 1992) or ganglion neurons (Davis et al., 1997). It is possible
between ÿ80 and ÿ40 mV (Zidanic and Fuchs, that a1D is the main pore-forming subunit expressed
1995) on current amplitudes. in all hair cells, and di€erence in kinetics (and very
6. High single-channel conductance: a single chan- likely in regulation) result from di€erences in b sub-
nel current of 0.8 pA at ÿ50 mV in 4 mM Ca was unit expression [b subunits have been found to a€ect
extrapolated from noise estimates (Roberts et al., VOCC kinetics (Neely et al., 1993) and to be
1990) in saccular hair cells, and a channel con- involved in G-protein-mediated VOCC modulation
ductance of 26 pS in saccular hair cell (Su et al., (Campbell et al., 1995)].
1995) [but 14 pS in turtle cochlear hair cells (Art A fast transient component, with a maximal peak
et al., 1995)] was directly measured in hair cell amplitude ranging from 10 to 600 pA has been
membrane patches [interestingly, in saccular hair described (Rennie and Ashmore, 1991) in guinea-pig
cells another channel, with a 20 pS conductance, SCC Type II hair cells, but not in analogous cells
was also observed (Su et al., 1995)]. from other species (Prigioni et al., 1992; Rennie and
7. Steep voltage dependence of activation: reported Correia, 1994). This transient could be an artifact
e-fold current increases in the activation range of due to high series resistance. In most studies, saccu-
potentials are 6.7 mV (Art and Fettiplace, 1987) lar Ca currents at positive potentials show a clear
and 7.8 mV (Zidanic and Fuchs, 1995) for decay, which may result from an incomplete block
cochlear hair cells, and 4 mV (Roberts et al., of SK (Ohmori, 1984; Roberts et al., 1990; Tucker
1990) for saccular hair cells. and Fettiplace, 1996) or from the activation of Ca-
extrusion mechanisms (see Section 6.3). In the near-
However, hair cell VOCCs display some features resting potential window, however, Ca current does
which di€erentiate them from L channels. First of not inactivate since it is able to support a noticeable
all, the activation potential range is more negative resting discharge: in fact, Ca current activation is ca
than most other known L-type channels [see 2% at resting potential (Roberts et al., 1990).
(Zidanic and Fuchs, 1995) for discussion]. Based on kinetic data, Zidanic and Fuchs (1995)
Activation of vestibular hair cell VOCCs has been suggest the presence of a single homogeneous
shown to begin at potentials as low as ÿ60/ ÿ 50 mV VOCC population partially blocked by DHPs.
(Hudspeth and Lewis, 1988; Rusch et al., 1991; However, kinetic data may not be enough to reject
Lewis and Hudspeth, 1983; Fuchs et al., 1990; the presence of pharmacologically di€erent isoforms
Rennie and Ashmore, 1991; Lang and Correia, of kinetically indistinguishable channels (Penington
1989; Prigioni et al., 1992; Perin et al., 1997a), and and Fox, 1995).
maximal current is observed between ÿ20 mV (Art The modulation of cochlear OHC VOCCs by
and Fettiplace, 1987; Rusch et al., 1991; Prigioni et ATP has been recently reported (Chen et al., 1995).
al., 1992; Perin et al., 1997a) and ÿ10 mV Interestingly, ATP e€ects on Ca channels appear
(Hudspeth and Lewis, 1988; Lang and Correia, very variable in di€erent OHCs populations, ran-
1989; Fuchs et al., 1990) [an older study (Ohmori, ging from ca 50% increase to a 30% decrease, with
1984) gave a peak value of 0 mV]. Reversal poten- some cells being una€ected; the VOCC populations
tials, in several studies (Ohmori, 1984; Lang and a€ected appeared kinetically and pharmacologically
Correia, 1989; Prigioni et al., 1992), were lower than homogeneous (predominantly L-type). Therefore,
expected, due to a contaminating outward current VOCCs di€erential expression may re¯ect the need
(Ohmori, 1984; Prigioni et al., 1992) that could be of a speci®c modulation rather than of particular
SK (Tucker and Fettiplace, 1995). kinetic features. In the vestibular hair cells, ATP
In sacculus and cochlea, Ca currents are much (Rennie and Ashmore, 1993) and acetylcholine
faster than reported for other known VOCCs: (ACh) (Yoshida et al., 1994) have been shown to
measured activation times range between 100 and modulate Ca cytoplasmatic concentration. Although
800 msec (Ohmori, 1984; Hudspeth and Lewis, 1988; receptor-induced Ca increases are most likely par-
Roberts et al., 1990; Zidanic and Fuchs, 1995). On tially due to Ca-permeable ionotropic receptors (see
the other hand, SCC currents appear to be slower Neurotransmitters), preliminary data (Perin et al.,
[time to peak ranged from 5.2 20.3 msec at ÿ40 mV 1997b) suggest that ACh is also able to modulate
to 3.12 0.3 msec at ÿ20 mV (Prigioni et al., 1992)]. VOCCs in frog saccular hair cells. Moreover, in tur-
It is possible that this kinetic di€erence re¯ects a tle cochlear hair cells (Ricci and Fettiplace, 1997)
true dichotomy in VOCC expression, with fast- cyclic AMP (cAMP) is able to decrease the ampli-
transducers expressing one type and slow-transdu- tude of voltage-dependent Ca currents. The growing
cers another. Pharmacological and molecular data body of evidence seems to suggest that, as in neur-
are, however, confusing. Chick cochlear VOCCs ons, VOCC modulation plays a signi®cant role in
appear to be insensitive to o-conotoxin GVIA hair cell signal processing.
(Zidanic and Fuchs, 1995), while SCC VOCCs are
reversibly blocked by it (0.5 mM blocked 9225% Ca
current), as has been reported (Dolphin, 1995) for
3.3. Functional Roles
the cloned L-type class D a1-subunit. On the other
hand, the mouse cochlea has been found to express As we have just seen, channel subunits are not
mRNA for three di€erent a1 subunits (a1C, a1D, equally distributed among vestibular hair cells.
a1E) and three b subunits (b1, b3 and b4) (Green et These di€erences may be related to frequency sensi-
al., 1996), and preliminary results suggest that hair tivity, gain and dynamic properties, and may show
cells mainly express a1D, while the putative R-type species speci®city (Correia et al., 1996). The
The Vestibular Hair Cells 205

suggested physiological roles of the di€erent ion phology shifts EK, and therefore Vz, to more
channels will be overviewed here. positive values, thus allowing the activation of both
Ica acts as a link between receptor potentials and IK, L and ICa (Goldberg, 1996).
transmitter release, as happens at neuronal synapses. The role of IKI is mainly to set the Type II hair
The fast kinetics and low voltage of activation of cell resting potential: cells displaying this current
this current allow a very precise phase locking of have a more hyperpolarized Vz (Masetto et al., 1994;
hair cell a€erent transmission even for high frequen- Holt and Eatock, 1995). However, IK1 may also con-
cies. Moreover, in high-frequency-responding hair tribute to avoidance of large hyperpolarizations,
cells, ICa also sustains electrical tuning. In fact, there thus clamping the cell potential around Vz even
is a well-established correlation between Ca current upon strong negative stimulation. The mutually
amplitude and resonant frequency, since larger Ca exclusive expression of this current and IA in SCC
currents induce a faster increase in Ca concentration hair cells would agree with this secondary role, since
at the active zone, speeding up BK kinetics (Art and IA inactivation removal requires hyperpolarizations
Fettiplace, 1987; Hudspeth and Lewis, 1988). Ca (Masetto et al., 1994). In low-frequency-tuned acous-
in¯ux also has other important roles such as mediat- tic cells, an inward recti®er has been shown to
ing responses to ligands and triggering responses of enhance electrical tuning (Goodman and Art,
other ion channels (see Sections 3.2 and 6.3). 1996a); however, this role does not seem to be cov-
IBK is the second component of the resonant cir- ered in vestibular hair cells, where IK1 expression
cuit in high-frequency tuned hair (Hudspeth, 1986). correlates with lack of resonant behavior (Sugihara
In nonresonant frog SCC hair cells, IBK has been and Furukawa, 1989; Holt and Eatock, 1995).
suggested to play an important role in slow response Ih has been found in saccular (Holt and Eatock,
adaptation (Masetto et al., 1995a,b). Since IBK 1995), utricular (Rusch and Eatock, 1996b) and SCC
channels have relatively high conductance they can (Masetto and Correia, 1997) hair cells. The suggested
also be important in protecting the cells from role of this slow current, at least in the saccule, is
intense, long overstimulation. that of a depolarizing `leak', which would bring hair
ISK mediates the inhibitory cholinergic response cell resting potentials in the range where ICa and IBK
seen in acoustic and saccular hair cells. The extreme produce resonance. In fact, in the saccule only cells
Ca-sensitivity of this voltage-independent channel expressing Ih resonate at rest (Holt and Eatock,
[the Ca concentration necessary for half-opening is 1995; Sugihara and Furukawa, 1989), and the orig-
2.4 mM in Art et al. (1995)] enables this channel to inal model of resonance required the presence of a
respond to the opening of Ca-permeable nicotinic `leak current' with a reversal potential of ÿ30 mV
receptors (see Section 5.7.1), but also to a maximal (Hudspeth and Lewis, 1988). Ih would also prevent
activation of VOCCs (Tucker and Fettiplace, 1996). large and prolonged hyperpolarizations; this role
It is not clear, however, if physiological VOCC acti- may be important for SCC hair cells, but not for
vation is enough to activate this current. otolithic organs, where prolonged hyperpolarization
The main role of IKv is probably to avoid the are unlikely to occur. Ih has been found in both
buildup of K inside hair cells due to the steady Type I and II hair cells; its role in Type I cells is not
inward MET current responsible for resting dis- clear, although it may be related to resting potential
charge. The high-frequency responding hair cells setting (Rusch and Eatock, 1996b).
probably do not need such a system since MET IA is quickly activated in response to an excitatory
channel adaptation drastically reduces K in¯ow at stimulus, and quickly repolarizes cell membrane,
rest (Hudspeth and Gillespie, 1994). The role of the thus shutting out transmitter release. For longer
delayed recti®ers seem therefore to be that of an stimuli, however, IA inactivation would make its
homeostatic outward K leak activated by depolariz- contribution negligible. Therefore, IA decreases hair
ation; consistently, these channels never inactivate cell responses to rapid depolarizations, reducing
completely (Russo et al., 1996). In hair cells tuned synaptic noise and causing a€erent neuron ®ring to
to low frequencies from acoustic organs, delayed- become more regular. In fact, in frog SCC cristae,
recti®er currents, together with inward recti®ers, peripheral hair cells, which display a strong IA com-
have been shown to sustain membrane resonance ponent, contact units with a low coecient of vari-
(Goodman and Art, 1996b). ation (CV) in their ®ring patterns, while central hair
IKL shapes the responses of Type I hair cells. The cells, which lack IA, contact high CV units (Masetto
low activation range of this current makes Type I et al., 1994; Honrubia et al., 1989).
voltage responses linear in respect to receptor cur- Ionic currents are responsible for shaping hair cell
rents, in contrast to Type II cell rectifying response neurotransmitter release, both at rest and under
(Rusch and Eatock, 1996a,b): since this current stimulation. However, since a single a€erent unit in-
dominates the electric properties of Type I hair cells, nervates multiple hair cells, the response of a unit
the latter homogeneously display a hyperpolarized will be more complex than the responses of single
Vz (ca ÿ70 mV) and a very low input resistance. If hair cells. The integration of single cell responses in
these properties are exhibited by Type I cells in vivo, a€erent discharge patterns is the subject of the next
however, it is not clear how a very small receptor sections.
potential (due to the low input resistance) could de-
polarize the hair cell membrane potential from its
quite negative resting value up to a range positive 4. VESTIBULAR HAIR CELL MOTILITY
enough to activate VOCCs and evoke transmitter
release. One possible explanation is that K accumu- Vestibular hair cells exhibit motility and shape
lation due to the peculiar chaliceal synapse mor- changes. It has long been known that the kinocilia
206 P. S. Guth et al.

can carry out active ¯agella-like movements to the active stereociliary bundle movement reported
(Bowen, 1931). Also long known is that the kinocilia by Assad and Corey (1992). These latter authors
have the complete structure of motile cilia (Flock saw only minimal movements in vestibular hair cell
and Duvall, 1965; Hamilton, 1969). In 1977, Flock bodies or cuticular plates.
and Cheung (1977) demonstrated the presence of Griguer et al. (1993) suggest that ``stereociliary
actin ®laments in vestibular hair cells. This ®nding movement could act as a homeostatic mechanism
was elaborated and re®ned in a later paper (Flock, adjusting the mechanotransduction to the stimulus''.
1980) in which was reported the existence of three If homeostasis were its reason for being then one
sets of actin ®laments: one in the stereocilia; one in might reasonably expect stereociliary movement in
the cuticular plate; and one at the circumference of all vestibular cells. This is clearly not the case
the cell apex. Later, this laboratory (Sobin and (Griguer et al., 1993). In a more recent paper (Sans
Flock, 1983) and others (Drenckhahn et al., 1985; et al., 1994), this group made the very interesting
Sans et al., 1989; Valat et al., 1991) reported on the suggestion that the transductional and adaptation
presence of ®mbrin, myosin and tropomyosin as processes are regulated at the apex of Type I hair
well as actin in the vestibular hair cells of mammals. cells by a release of glutamate and substance P (SP)
Type I vestibular hair cells isolated from the gui- from the calyx by way of reciprocal synapses (see
nea pig exhibited changes of the `neck' region in re- Section 5.7).
sponse to perfusion with K solution of abnormally Spontaneous and electrically-induced beating
high concentration (Didier et al., 1990b; Valat et al., movements of the kinocilia and stereocilia were
1991) or current application (Griguer et al., 1993; observed (Rusch and Thurm, 1989) in the SCC
Zenner et al., 1992). ampulla of the eel, Anguilla anguilla. This beating
Studying Type I vestibular hair cells exclusively, was spontaneous and seen only with progressive de-
Griguer et al. (1993) and Sans et al. (1994) reported terioration of the preparation, and could also be
on a voltage-dependent sequence of motile events. induced by pressing on the tip of the kinocilium.
Depolarization of ca 10 mV produced neck shorten- When a voltage was imposed across the sensory epi-
ing while depolarizations up to 20 mV were required thelium a pointer-like rigid de¯ection of the hair
to elicit cuticular plate and stereociliary tilting. The bundles was induced. The direction of the de¯ec-
stereociliary tilting or pivoting was away from the tions depended on the sign of the imposed voltage.
kinocilium in response to depolarizations. The three When the kinocilia were disconnected from the
areas of movement (i.e. stereocilia, a cuticular plate stereocilia, the latter ones were still able to move,
and neck region) may be correlated with the localiz- rod-like, in response to imposed voltages. In frog
ation of the three sets of actin ®laments reported by saccular hair cells, hair bundle de¯ections with simi-
Flock (1980). lar voltage sensitivities and directions of response as
Employing isolated frog saccular hair cells (i.e. those of the eel ampulla have been reported (Assad
Type II only) Assad et al. (1989) similarly reported et al., 1989).
a pointer-like pivoting of stereocilia away from the In 1990, Didier et al. (1990a) demonstrated high
kinocilium when the cell was depolarized and K (80±125 mM) induced Type I hair cell (but not
toward the kinocilium during hyperpolarization. Type II) movement. In contrast, Zenner et al. (1990)
These investigations showed no evidence of move- reported shortening and elongation of both Types I
ments of the cuticular plate or the cell body. They and II mammalian vestibular hair cells in response
suggested that the stereociliary pivoting was driven to 80 mM K+ exposure. In a later publication this
by forces generated by the so-called gating springs same group (Zenner and Zimmermann, 1991)
which are assumed to have the same geometry as applied various stimuli such as electrical ®elds, K
the tip links connecting rows of stereocilia. In fact, concentration changes, kainate and temperature
in a later publication (Assad and Corey, 1992) these changes to isolated mammalian hair cells. Type I
authors demonstrated that perfusion with reduced hair cells responded to electrical ®elds with shape
Ca solution or with streptomycin, both of which are changes more reliably than did Type II. Similarly
known to reduce the transduction current, inhibited only Type I cells responded to temperature changes
active stereociliary bundle movement. The move- and kainate exposure. Both types responded to
ment of the stereocilia could occur independent of changes in K+ concentration. Vestibular hair cells,
any attachment to the kinocilium. That is, the in these investigators' hands, showed frequency
stereocilia themselves are fully capable of pivoting maxima of up to 85 Hz in response to electrical
movements about their bases (Assad and Corey, stimulation. A more re®ned version of this work
1992). Remember, that although the kinocilia are dealing exclusively with electrically-evoked motile
true cilia, the stereocilia are not and therefore their responses appeared in 1992 (Zenner et al., 1992).
movements are probably not caused by ciliary The shortening of the neck region and the tilting
motor mechanisms. of the cuticular plates of vestibular hair cells could
In comparison to movements in cochlear OHCs be seen as pulling on their associated accessory
which can follow frequencies up to tens of kHz, ves- structures, either the cupula or the otolithic mem-
tibular hair bundle movements do not exceed fre- branes. As discussed by Valat et al. (1991), hair cells
quencies of ca 100 Hz. at the top of the crista have short, free-standing
Other movements of hair cells such as voltage- stereocilia while those at the edges and bottom have
dependent elongation of cell bodies observed in tall stereo- and kinocilia inserted into the cupula
OHCs from mammalian cochlea (Ashmore, 1987; (Mbiene and Sans, 1986). In the otolithic organs,
Brownell et al., 1985; Dallos et al., 1991) or active the hair bundles of Type I hair cells of the periph-
tilting of the cuticular plate of OHCs are unrelated eral and parastriolar zones are inserted into the oto-
The Vestibular Hair Cells 207

lithic membrane lacunae while those in the striolar been found [Schessel et al. (1991) vs Yamashita and
region are freestanding (Lim, 1979). With these Ohmori (1990)]: nerve ®ber properties are therefore
arrangements, the hair cells at the periphery of the likely to contribute to a€erent signal processing,
vestibular organs could actively pull the cupula or possibly with di€erent mechanisms in high- and
otolithic membrane and thus in¯uence the signal to low-CV units.
the more centrally situated hair cells, analogously to Boyle et al. (1991) recorded from horizontal SCC
the roles of cochlear inner and OHCs. a€erent neurons in the toad®sh (Opsanus tau). This
animal, like the frog, has only Type II hair cells.
However, neurons with irregular or regular inter-
spike intervals which had characteristics similar to
5. NEUROTRANSMISSION those seen in mammals, birds and lizards were
observed. Although the morphological correlates
5.1. A€erent Innervation
were not exactly the same as those described above
Vestibular a€erent nerve ®bers, whose somata are for animals with both Type I and Type II hair cells,
located in Scarpa's ganglion, connect endorgan hair a€erents having low sensitivities and slow dynamics
cells with brainstem nuclei and cerebellum. These innervate the most peripheral areas of the crista
®bers convey hair cell responses, in both static and while a€erents with higher sensitivities and faster re-
dynamic conditions, to central systems involved in sponses innervate the more central areas of the
posture and movement control. Central processes, crista in the ray (Flock and Goldstein, 1978) frog
however, will not be covered here [see deWaele et al. (Honrubia et al., 1989) and toad®sh (Boyle et al.,
(1995) for a recent review], and most of this section 1991). The latter group also stimulated the e€erent
will deal with the relationships between peripheral nerves running from the brainstem back out to the
arrangement of primary vestibular a€erent ®bers hair cells, and noted that e€erent stimulation
(mainly distribution and shape of their synaptic end- increased a ®ber's resting ®ring rate, reduced its sen-
ings) and their roles in the conversion process taking sitivity, and, if a ®ber only responded to a portion
place in vestibular organs. of the sinusoidal stimulus cycle, e€erent excitation
From early morphological studies (Lorente de extended the period of this response. Boyle et al.
No, 1926) it was evident that vestibular a€erents (1991) pointed out that the large variability of re-
comprise a heterogeneous population of ®bers with sponse in vestibular neurons may be related to these
diameters ranging between 2 and 13 mm (Wersall, di€erent aspects of hair cell/neuron function:
1956). Further studies (Boyle et al., 1991; Goldberg 1. cupula motion de¯ecting apical stereocilia may
and Fernandez, 1984) suggested that this heterogen- be regionally variable;
eity may re¯ect the presence of separate pathways, 2. receptor potentials might not be uniform across
possibly conveying di€erent information to the hair cells;
CNS. This hypothesis is well-supported (Adrian, 3. hair cell basolateral surfaces contain ion channels
1943; Blanks et al., 1975; Estes et al., 1975). In fact, that might act as frequency-dependent ampli®ers
in all vertebrates: of receptor potentials;
1. Thick axons innervate the central region of cris- 4. ongoing neurotransmitter release from hair cells
tae ampullares and the striolae (i.e. their corre- might not be uniform across cells; and
sponding parts in otolith organs), thin ®bers the 5. a€erents might di€erentially innervate hair cells
peripheral regions and medium-sized ®bers the with respect to number and type or they might
intermediate regions of the various endorgans respond di€erentially to a given amount of trans-
(Baird et al., 1988; Baird and Schu€, 1994; mitter release.
Brichta and Peterson, 1994; Honrubia et al.,
1989; Myers and Lewis, 1990) 5.1.1. The Role of the Synaptic Body
2. Thick ®bers have irregular resting activity and
Synaptic bodies are large protein complexes with
give rise to phasic-tonic discharges in response to
no limiting membranes, that have a granule-like
mechanical stimuli; thin ®bers have regular rest-
appearance, and are found associated with presyn-
ing activity and give rise to tonic responses; med-
aptic active zones at graded synapses. Unlike the
ium-sized ®bers have, in general, intermediate
inert melanin or glycogen granules, however, synap-
properties. (Anastasio et al., 1985; Baird et al.,
tic bodies most likely play an active dynamic role,
1988; Baird and Schu€, 1994; Dickman and
since they are surrounded by a halo of synaptic ves-
Correia, 1989; Fernandez and Goldberg, 1971;
icles tethered to them (Gleisner et al., 1973;
Landolt and Correia, 1980).
Sobkowicz et al., 1982, No. 403).
3. In the eighth nerve, thicker a€erent axons are
They occur in the photoreceptors of retina
found in the center and thinner axons in the per-
(Cohen, 1963; Dowling, 1968, 1974; Fine, 1963;
iphery (Honrubia et al., 1989 Honrubia et al.,
Gray and Pease, 1971; Matsusaka, 1967; Missotten,
1984; Kuruvilla et al., 1985).
1960; Sjostrand, 1958) and in the acousticolateral
In the lizard Calotes versicolor, SCC neurons with systems (Flock, 1965; Gleisner et al., 1973; Hama,
the highest CV in their interspike intervals terminate 1965; Roberts and Ryan, 1971; Tanaka and Smith,
in a nerve calyx surrounding one or more Type I 1978) of birds, mammals, ®shes and amphibians.
hair cell (Schessel et al., 1991). Low-CV ®bers dis- They have also been found in some invertebrates
play a signi®cant post-spike hyperpolarization, (Osborne, 1966; Wood et al., 1977). In mammals,
(Schessel et al., 1991; Yamashita and Ohmori, synaptic bodies are primarily associated with sen-
1990), while in high-CV ®bers contrasting data have sory receptors. However, they are also in nonsen-
208 P. S. Guth et al.

sory cells such as the bipolar cells of the retina determined by the arrival of the a€erent dendrite.
(Dowling and Boycott, 1965) and pinealocytes That is, the synaptic body `settles' and becomes
(Lues, 1971; Wolfe, 1965) (N.B. there is a view that attached to the membrane at the synaptic site upon
the pineal gland does serve a light-sensing role, per- the arrival of the a€erent dendrite.
haps vestigial). In this connection, Vollrath (1973) Ross (1995) demonstrated plasticity of synapses
reported a large increase in synaptic body numbers and synaptic bodies in the utricle in response to
in hamster pinealocytes after exposure of the ani- space ¯ight. During space ¯ight there was an
mals to continuous light for 2 months. Satoh and increase in synapses of both Type I and Type II hair
Vollrath (1988) even tried administering melatonin, cells. The synaptic bodies became more sphere-like
the pineal hormone, to mice but found no e€ect on in both types of hair cells indicating to the author
synaptic body formation. As a further illustration of recent synaptogenesis.
synaptic body dynamic involvement in function, According to Guth et al. (1993) there are also
pineal gland synaptic bodies grow by ca 20% from dense granules in frog vestibular hair cells that are
day to night (Jastrow et al., 1997). similar to synaptic bodies in size, shape and electron
Not much is known about synaptic body function densities. However, these dense granules were found
but they do seem to locate at the site of a€erent throughout the hair cells, were not in the same
nerve contact and are generally surrounded by a focusing plane as the synaptic bodies and synaptic
halo of synaptic vesicles. For these reasons their vesicles and did not respond to drug treatment as
function is thought to be associated with synaptic the synaptic bodies did. This study was undertaken
transmission. In fact, in the retina of the frog, as a veri®cation and extension of the original provo-
mature electrophysiological activity and synaptic cative ®ndings (Osborne and Thornhill, 1972;
vesicles are observed only after the synaptic bodies Monaghan, 1975) that the synaptic bodies in amphi-
have formed (Nillson and Crescitelli, 1970). bian hair cells would decrease in electron density in
Furthermore, as in retina, developmental matu- response to monoamine-depleting drugs such as
ration of the hair cells involves the appearance of reserpine or guanethidine. This ®nding strongly
well-de®ned synaptic bodies and vesicles (Favre et suggested that the synaptic body was a possible sto-
al., 1986; Sobkowicz et al., 1975, 1982, 1986). In rage site of such monoamines. However, mono-
older animals, synaptic bodies also remain as per- amines have never been observed in hair cells, while
sistent ultrastructural markers of functional a€erent adrenergic stimulation has been found to a€ect the
synapses (Park et al., 1987; Woods and Park, 1987). endolymph-secreting cells in acoustic and vestibular
In hair cells, the synaptic bodies could be essential organs (Liu and Wangemann, 1997). Using a water-
for transmission because they are always associated soluble monoamine depleter, tetrabenazine, Guth et
with the synaptic apparatus (Siegel and Brownell, al. (1993) found not only a quanti®able decrease in
1986). electron density of the synaptic bodies after drug
In contrast to conventional synapses, which treatment but that this densitometric change was ac-
release neurotransmitter transiently, ribbon synapses companied by a marked decrease in ®ring rates of
release transmitter continuously and at high rates a€erent neurons of the semi-circular canal of the
(Devries and Baylor, 1993). The presence of a frog. The depleter used by Osborne and Thornhill
synaptic body has been correlated to an unusual (1972), guanethidine, also decreased the ®ring rates
syntaxin (Morgans et al., 1996), and to the absence of these a€erents (Guth et al., 1993). Neither mela-
of synapsins (Scarfone et al., 1988, 1991), which are nocytes nor nonsynaptic dense granules were
involved in the priming step (Rosahl et al., 1995), decreased in their optical densities by these drug
occurring between docking and exocytosis. treatments. Some evidence has been put forward for
Moreover, synaptic body-synapses display L-type a role of histamine in frog SCC neurotransmission
VOCCs, instead of the N- or P-type as usually (Housley et al., 1988). Histamine is also depletable
observed in neurons (Dunlap et al., 1995), and mul- by the drugs mentioned above (Douglas, 1965). But,
tiple active zones (Sakaba et al., 1997). Accordingly, attempts to restore the electron densities of synaptic
the Ca-dependence of these synapses di€ers from bodies or a€erent neuronal ®ring rates by the ad-
the neuronal ones: in fact, at the former synapses, ministration of histamine or its precursor histidine
Ca already stimulates transmitter release at 1 mM, after depleting drug treatment have so far failed.
while 100 mM are required in neurons (Lagnado et Parsons et al. (1994) recorded a continued
al., 1996). Therefore, it appears that resting neuro- increase in saccular hair cell membrane area, as
transmitter release at active zones does not interfere measured by capacitance changes, during main-
with BK activation, which requires Ca concen- tained depolarization. This increase was presumably
trations of ca 40 mM (Hudspeth and Lewis, 1988). due to fusion of synaptic vesicle membrane with
On the other hand, a Ca-sensor with lower anity hair cell membrane during exocytosis. They calcu-
could be involved in the depolarization-evoked re- lated that there would have to have been ten times
sponse (Heidelberger et al., 1994). Therefore, this more synaptic vesicles than the number actually pre-
change in hair cell synaptic body properties could sent to account for the capacitance changes. This
re¯ect alterations in whole organ homeostatic feed- ®nding suggested a rapid rate of vesicle membrane
back loops. replenishment. There is no direct evidence connect-
Because of their variations in shape synaptic ing the synaptic bodies with the re-supply of synap-
bodies are also called `ribbons', `bars', `round tic vesicles. However, cells with synaptic bodies,
bodies', `plates', `rings' and `spheroids' or `spher- such as hair cells, lack synapsins (Scarfone et al.,
ules'. During hair cell development (Sobkowicz et 1988, 1991; Favre and Sans, 1977; Mandell et al.,
al., 1982), the ®nal location of the synaptic body is 1990) and therefore the release machinery in these
The Vestibular Hair Cells 209

cells may be di€erent from cells without synaptic Regarding their functional connections (Favre
bodies that do have synapsins. One is led to the and Sans, 1977), it has been demonstrated that e€er-
possibility that the synaptic body is involved in ent neurons can respond to many sensory stimuli
transmitter release and perhaps vesicle replenish- thus suggesting that sensory information originating
ment and has special properties permitting the main- in vestibular end-organs combine in the medulla
tenance of high rates of transmitter release (Parsons with information coming from other sensory sys-
et al., 1994). Several authors had suggested that the tems (mainly the visual and the proprioceptive sys-
synaptic bodies are sites for storage of neurotrans- tem) (Highstein, 1991). The function of the e€erent
mitters or their precursors (Monaghan, 1975; system therefore appears to be more general than
Osborne and Thornhill, 1972; Osborne, 1977; just controlling vestibular a€erent activity.
Thornhill, 1972; Wagner, 1973). In lateral line of It is a common feature of e€erent vestibular sys-
salamander tadpoles, sensory stimulation caused the tems that a few e€erent neurons can control the ac-
synaptic bodies to move to the base of the hair cell tivity of many peripheral sensory units. This occurs
(Flock, 1974). Experiments with the insoluble mar- because the e€erent ®bers, after leaving the CNS,
ker horseradish peroxidase in extracellular ¯uid ramify in the eighth nerve with collaterals arising
have demonstrated that a large proportion of synap- from single parent axons that innervate several, if
tic vesicles associated with synaptic bodies become not all, of the end organs (Iurato et al., 1972; Llinas
labeled with the horseradish peroxidase (Siegel and and Precht, 1969; Prigioni et al., 1983; Valli et al.,
Brownell, 1986). This suggests that the vesicles are 1986).
open to the extracellular ¯uid at some point, most The e€erent terminals form direct synaptic con-
likely during exocytosis. Exocytosis-inhibiting elec- tacts on Type II hair cells but also form postsyn-
trolyte composition (i.e. high Mg, low Ca extracellu- aptic connections with a€erent calyx nerve terminals
lar ¯uid) blocks this labeling. of Type I hair cells. Amongst the anamniotes, the
If the synaptic body is some sort of transmitter e€erents synapse with the a€erents in ®sh (e.g. toad-
storage granule to which synaptic vesicles shuttle for ®sh) (Highstein, 1992) but not in frogs (Hillman,
repletion of their transmitter stores then it may be 1976).
possible to tag the synaptic vesicles with a normally Immunohistochemical, electrophysiological and
impermeant marker and determine if that tag ends pharmacological studies have provided evidence
up in the synaptic body. Despite the lack of its limit- that, although other substances have been proposed
ing membrane, the synaptic vesicles are somehow (see below), ACh is the major transmitter released at
tethered to the synaptic body. the majority of e€erent synapses (Ishiyama et al.,
An important new development in synaptic body 1994a; Iurato et al., 1971a,b; Kong et al., 1994;
research has been the isolation and puri®cation of Lopez and Meza, 1988, 1990; Sugai et al., 1992).
synaptic bodies from bovine photoreceptors However, there is considerable evidence indicating
(Schmitz et al., 1996). This should accelerate that some vestibular e€erents are not cholinergic
research into the nature, and ultimately, the func- (Dememes et al., 1983; Goldberg and Fernandez,
tion of these enigmatic structures. 1980; Perachio and Kevetter, 1989).

5.2.1. Physiological E€ects of the Vestibular


5.2. E€erent Innervation E€erents
It is well-known that auditory and vestibular The vestibular e€erent system has been suggested
receptors receive an e€erent innervation originating to extend the a€erent dynamic range (Boyle and
in the brain. The location of the cell bodies of e€er- Highstein, 1990), and to switch vestibular responses
ent ®bers, normally clustered in the brainstem near between `positional' and `volitional' modes
the vestibular nuclei, has been extensively investi- (Highstein, 1992), analogously to the g-motoneuron
gated, by means of electrophysiological and mor- system innervating neuromuscular spindle organs.
phological methods, in virtually all vertebrate In the mammalian cochlea, e€erents exert a protec-
classes. The e€erent nuclei generally lie in the tive role as well (Pujol, 1994); however, this is not a
medulla at the same level as the vestibular nuclei role imputed to the vestibular e€erents, probably
(Gacek and Lyon, 1974; Goldberg and Fernandez, because vestibular hyperstimulation is less likely to
1980) and are segmentally organized (Highstein and occur than auditory hyperstimulation.
Baker, 1985, 1986; Dickman and Correia, 1993; The stimulation of e€erent ®bers has been shown
Goldberg and Fernandez, 1980; Highstein, 1991; to hyperpolarize hair cells (Art et al., 1984, 1985;
Hillman, 1976; Klinke and Galley, 1974; Llinas and Ashmore and Russell, 1982; Flock and Russell,
Precht, 1969; Strutz et al., 1980; Warr et al., 1986). 1976; Sugai et al., 1992). However, intracellular
Hillman (1976) and Llinas and Precht (1969) recordings from single a€erent axons provided
suggested that, at least in the frog, Purkinje cells of results with evidence for inhibitory (Art et al., 1982,
the auricular lobe of the cerebellum might also send 1984, 1985; Art and Fettiplace, 1987; Ashmore and
e€erent axons to labyrinthine receptors. Russell, 1982; Flock and Russell, 1973, 1976;
Perachio and Kevetter (1989) described two Furukawa, 1981) facilitatory (Goldberg and
groups of vestibular e€erents in the gerbil. The lar- Fernandez, 1980; Highstein and Baker, 1985) and
ger of the two groups was found lateral to the genu mixed e€ects (Bernard et al., 1985; Prigioni et al.,
of the facial nerve and ventromedial to the vestibu- 1983; Rossi and Martini, 1991; Rossi et al., 1980;
lar nuclei. The smaller group of cells was ventral to Valli et al., 1986) on the a€erent discharge.
the facial genu. Moreover, while the inhibitory e€ects were always
210 P. S. Guth et al.

cancelled by curare or atropine, the facilitatory tained. In addition, high frequency gain increases
e€ects are not blocked by classical cholinergic block- are present.
ers (Bernard et al., 1985; Prigioni et al., 1983; Rossi Lagenar a€erents are also di€erentially a€ected
and Martini, 1991; Valli et al., 1986). Di€erences in by e€erent action. The most vibration-sensitive
the observed relative importance of facilitatory and a€erents are the largest and have an irregular dis-
inhibitory e€ects may arise from technical di€er- charge, and it is these that are most profoundly
ences: for example, in a work on the squirrel a€ected by e€erent stimulation (Locke and
monkey where e€erent e€ects were found to be Highstein, 1989, 1990).
mainly facilitatory (Goldberg and Fernandez, 1980) According to Highstein (1992), ``the e€erent con-
contralateral e€erents, which have been found to be trol of sensory processes is most likely in place to
inhibitory in the frog (Myers et al., 1997) only enhance the perception of certain sensations in bio-
accounted for 2% (15/604) of all units studied. Fast logically relevant contexts in complex, behaving
e€erent facilitation has been observed to a€ect organisms''. For instance, the di€erential e€ect of
mainly a€erents with response patterns consistent the e€erents on a€erents subserving di€erent func-
with a central or striolar location in the sensory tions, as described above, may also serve to enhance
epithelia of the SCC and utricle (Goldberg and contrast between these a€erents.
Fernandez, 1980). On the other hand, contralateral The predominant transmitter released by the e€er-
e€erents in the gerbil vestibular organs were found ents is ACh [although other transmitter/modulators
to innervate only the peripheral cristae or maculae, (T/Ms) are also released (see below)]. Several propo-
while ipsilateral mainly innervated central and strio- sals have been made for clarifying ACh action both
lar regions (Purcell and Perachio, 1995a,b, 1996a,b), on isolated hair cells and on primary a€erent vestib-
and a9 nicotinic receptors, which are thought to be ular neurons. The main idea emerging from these in-
involved in e€erent hyperpolarization of hair cells vestigations is that ACh can induce two distinct
(see below), are predominantly observed in the per- modi®cations of vestibular hair cell membrane po-
ipheral regions of SCC crista [see Fig. 3 in (Hiel et tential:
al., 1996)]. In the pigeon, contralateral e€erents
1. an increase in a€erent discharge by acting on a
have been shown to exert either strong inhibition or
muscarinic receptor. This e€ect, antagonized by
weak facilitation of SCC responses. Facilitation was
atropine, seems to be correlated with a hair cell
mostly observed on low-CV a€erents, and inhibition
depolarization (Guth et al., 1986; Housley et al.,
was observed on high-CV units (Dickman and
1990; Norris et al., 1988);
Correia, 1993). Di€erent e€erent pools seem there-
2. decrease in a€erent discharge, by acting on either
fore to contact hair cells and/or a€erent terminals
a nicotinic-like or a muscarinic-like receptor.
with di€erent location and properties; however,
This e€ect, blocked by curare, by strychnine and,
more work is needed to clarify the functional role of
partially, by atropine (Guth et al., 1994a; Norris
this multiple innervation.
et al., 1988; Rossi et al., 1980; Sugai et al., 1992)
E€erent neurons are tonically active and may be
seems to be correlated with a hair cell hyperpol-
stimulated by vestibular input as well as by stimuli
arization.
from other sensory modalities (tactile, acoustic or
visual); as regards a€erents±e€erents feedback, mul- According to some authors (Adams et al., 1987;
tiple pathways, both mono- and polysynaptic, have Sewell and Starr, 1991) e€erent facilitation might be
been found (Highstein, 1991). E€erent activation sustained not only by ACh but by a peptide as well,
increases the resting discharge of SCC a€erents, es- that is, by the calcitonin gene-related peptide
pecially in the low-resting discharge, high-gain and (CGRP). CGRP administration increased the sen-
acceleration-sensitive units, but decreases their gain sory discharge in lateral line organs and CGRP
(Boyle et al., 1991; Goldberg and Fernandez, 1980); immunoreactivity was found both in lateral line
low gain a€erents are on the other hand minimally e€erent ®bers (Adams et al., 1987) and in mamma-
a€ected (Highstein, 1992). lian vestibular and auditory system (Roberts et al.,
The connections between vestibular a€erents and 1994; Takeda et al., 1986; Tanaka et al., 1989a,b;
e€erents are considered to be monosynaptic Vetter et al., 1991). Therefore, the hypothesis may
(Highstein and Baker, 1985, 1986). The e€erents are be put forward that this substance acts as an excit-
tonically active and may be stimulated to even atory e€erent neurotransmitter in labyrinthine
greater activity not only by vestibular input but by organs. This might explain the existence of noncho-
stimuli from other sensory modalities (i.e. touch, linergic excitatory e€ects produced by e€erent
sound, sight). When activated the e€erents increase stimulation (Flock and Russell, 1973; Highstein,
the resting discharges of SCC a€erents, but may 1991; Rossi et al., 1980). Roberts et al. (1994), by
decrease their responses to appropriate physiological means of double-labeling experiments, demonstrated
stimuli. That is, low-gain a€erents are minimally fa- the presence of both choline acetyltransferase
cilitated, while high-gain and acceleration-sensitive (ChAT) and CGRP in many e€erent neurons that
a€erents are strongly facilitated. SCC a€erents with innervate the lateral line and the ear of the eel. Such
low levels of spontaneous activity are especially re- a coexistence of ACh and CGRP might be a com-
sponsive to e€erent activation (Highstein, 1991). mon feature of e€erent neurons since it was
In contrast, the sensitivities of high-gain and observed in both vestibular and cochlear e€erent
acceleration-sensitive a€erents are reduced by e€er- neurons of the rat (Tanaka et al., 1989a,b; Vetter et
ent stimulation. However, this reduction in sensi- al., 1991). Co-localization of ACh and CGRP raises
tivity is proportional across the stimulation range so the possibility that e€erent neurons can release ACh
that the shapes of the input±output curves are main- and CGRP independently, as occurs in Aplysia
The Vestibular Hair Cells 211

motoneurons (Whim and Lloyd, 1990), and there- Usami et al. (1991a) produced immunocytochemical
fore that the same group of e€erent neurons can evidence for the presence of SP, and Dechesne et al.
produce, according to the neurotransmitter released, (1997) for rab 3A, a dynamic element of the synap-
both inhibitory and facilitatory e€ects. Remember tic vesicle cycle, in the apical portion of the calyx.
also that ACh itself and alone is capable of produ- These workers postulate a feedback loop by which
cing both facilitatory and suppressive e€ects on the Type I hair cell activates the calyx which, in turn
a€erent vestibular discharge (Guth et al., 1994a) releases glutamate and SP to in¯uence the hair cell.
depending on which ACh receptor is activated.

5.3. Reciprocal Synapses 5.4. Synaptic Transmission

A reciprocal synapse, as described by Shepherd 5.4.1. Generalities


(1990) is one in which there is a synapse between Hallowell Davis' well-known aphorism that the
process (a) and process (b) and a return synapse inner ear is ``a room full of treasures'' was meant to
from (b) to (a). These were ®rst described in the describe the cochlea. It applies equally well to the
olfactory bulb where they amounted to 90% of all vestibular periphery and it is nowhere truer than in
dendrodendritic connections, and were later the consideration of vestibular neurotransmission.
reported in retina, lateral geniculate, ventrolateral For purposes of examining synaptic transmission,
nucleus of the thalamus, substantia gelatinosa of the the vestibular periphery may be viewed as a three-
spinal cord and carotid body [all cited in Sobkowicz component system:
et al. (1993)]. These synaptic specializations seem to
be characteristic of sensory pathways. 1. hair cell;
On the basis of limited physiological evidence, 2. a€erent;
reciprocal synapses may function as local `switches' 3. e€erent neurons.
in which synaptic transmission from (a) to (b) may This deceptively simple system is served by upwards
be excitatory whereas transmission from (b) to (a) of ten T/Ms: a€erent T/Ms both transsynaptic and
may be inhibitory reducing the ability of (a) to auto receptor-mediated; the e€erent T/Ms; and the
further excite (b). T/Ms of undetermined origin (such as histamine and
Amongst inner ear organs, the majority of work others). So, although the cellular elements are only
has been done in the organ of Corti (Nadol, 1988; three, the long list of T/Ms bespeaks a complexity
Sobkowicz et al., 1993). Pujol and Lenoir (1986) of function hitherto unappreciated. To add to this
have o€ered the following opinion: ``These synapses complexity, some of the T/Ms may be served by
may only represent trophic junctions: a€erent end- more than one receptor (see, for instance, Sections
ings occupying places on the OHC synaptic mem- 5.7.1 and 5.7.2). Why this seemingly extraordinary
brane where e€erents never arrived or where complexity and how do these T/Ms interact, com-
e€erents have pathologically disappeared''. pete or cooperate in the functioning of the vestibular
Reciprocal synapses, between hair cells and e€er- organs? This question deserves continued exper-
ent ®bers have been reported in the guinea pig imental scrutiny.
cochlea (Thorn et al., 1972) and the basilar papilla Transmitters and their functions seem to be con-
of the chick (Tanaka and Smith, 1978). Engstrom et served throughout vertebrate evolution. The under-
al. (1972) also reported on the occasional, apparent pinnings of this assertion come from the writings of
reciprocal synapse between hair cells and e€erents in Florey (1967, 1972) and Michelson (1974). The for-
the macula of the saccule and utricle of the squirrel mer wrote, ``A primitive transmitter, from which
monkey. another, perhaps more e€ective transmitter has
The reciprocal synapses between vestibular hair developed, never existed: the cells of our brain pro-
cells and a€erent ®bers were reported by Dunn duce the same transmitters as the nerve cells of
(1980) to occur in the crista ampullaris of the bull- lower worms''. The latter wrote, ``Choline esters
frog where they contribute 6±8% of synapses with other than ACh are often found throughout the ani-
the anterior ampullar nerve. mal kingdom but never act as transmitters''. Both
In the mammalian utricular and saccular maculae, authors agree that, although the transmitters them-
Ross and co-workers (Ross, 1997; Ross et al., 1990) selves are unchanged throughout the animal king-
report that ca 20% of synaptic connections between dom, their functions may vary from phylum to
Type II hair cells and a€erents are of the reciprocal phylum but within any one phylum (e.g. vertebrate)
type with presynaptic vesiculated endings of intra- their functions are the same. Only one minor excep-
macular origin. tion to this assertion is known to the authors: in
The relationship between Type I vestibular hair anurans the cardioaccelerator transmitter di€ers
cells and their calyceal a€erent endings constitutes a from the rest of the phylum by one methyl group. It
reciprocal synapse, albeit one of unusual mor- is epinephrine and not norepinephrine [reviewed in
phology. Sans and co-workers (Sans et al., 1994; Taxi (1976)].
Scarfone et al., 1988) reported on the existence of In a recent review article, Juusola et al. (1996)
synaptic features in the upper part of the calyx contrasted cells such as photoreceptors and hair
including synaptic vesicles, synapsin and synapto- cells whose information is carried by graded poten-
physin. Further, this group has shown the presence tial changes with cells such as motoneurons whose
of glutamate-like immunoreactivity in the calyx information is carried by action potentials. Hair
(Dememes et al., 1990) and glutamate receptors on cells and other graded-potential cells release their
Type I vestibular hair cells (Devau et al., 1993). transmitter tonically and modulate this release up or
212 P. S. Guth et al.

down in a graded fashion. This arrangement allows were acting on the a€erents themselves both
for early processing of information and synapses spontaneous and mechanically-evoked trans-
can provide signi®cant signal optimization before mission might be equally a€ected. Second, during
encoding into action potentials for distance trans- the passage of hyperpolarizing current, the a€er-
mission (Juusola et al., 1996). A few of the charac- ents may be caused to ®re by the application of a
teristics of graded potential neurons distinguishing solution containing 10 mM K (Ricci et al.,
them from spiking neuron are: many active zones; 1991b).
ribbon or synaptic body synapses; many postsyn- 7. When endolymphatic K+ is replaced by Na+
aptic connections; mainly L-type Ca channels and and tetraphenylboron, a K+ chelator, is added to
lack of synapsin I. the endolymph, (while perilymph is kept
At graded-potential synapses transmitter release unchanged), the a€erents become silent (Valli et
from vesicles can be sustained at much higher rates al., 1986).
than at spiking synapses. For instance, vesicle 8. D-Tubocurarine in endolymph (not perilymph)
release rates derived from membrane capacitance can completely reduce spontaneous and evoked
measurements (Parsons et al., 1994) are of the order a€erent neural activity (Valli et al., 1974) presum-
of 10 000 vesicles secÿ1. Release rates of this order ably by interfering with transduction.
must be maintained by tightly regulated and loca- 9. Electrical stimulation of the e€erents which end
lized changes in Ca concentrations such as are seen primarily, if not totally, on hair cells of the lat-
in hair cells (see Section 6.3). eral line (Russell and Roberts, 1972) or of the
frog SCC (Valli et al., 1986) can cause total sup-
5.4.2. The Activity of the A€erent Neurons Depends pression of spontaneous a€erent ®ring.
Completely on Transmitter Release from Hair Cells Therefore, a€erent neuronal ®ring seems to
depend completely on activation by neurotrans-
In the absence of stimulation of the mechanosen- mitters released from the hair cells.
sory epithelium the a€erent ®bers innervating hair
cells of vestibular organs and, most probably, the
other acousticolateralis organs exhibit a resting ®r-
ing rate (perhaps imprecisely called spontaneous ac- 5.4.3. Evoked and Resting Activities of the A€erents
tivity) which is believed to be entirely dependent on are Di€erentially Modulated
a `basal' release of the neurotransmitter from the As pointed out above, a resting current across the
hair cells. The evidence supporting this assertion is transductional elements is believed to account
as follows: (through the release of neurotransmitter by hair
1. Manipulations designed to inhibit neurotransmit- cells) for the generation of the resting a€erent ac-
ter release from hair cells such as replacement of tivity. Modulation of this current by the mechanical
normal perilymph with a solution containing stimulation of the sensory epithelium is responsible
high Mg and low Ca [SCC (Valli and Zucca, for the modulation of the ®ring rate that is called
1976) cochlea- (Siegel and Relkin, 1987)] or evoked activity.
cobalt application [lateral line (Sewell, 1990)] A series of observations (Guth et al., 1991;
cause the a€erent ®bers innervating these hair Drescher and Drescher, 1987a; Gleich et al., 1990;
cells to become silent. However, the role of Starr and Sewell, 1990), has led to the conclusion
charge screening caused by these divalent cations that there are di€erences between resting and
in the silence of the a€erents must be considered. mechanically-evoked a€erent activity in respect to
2. Application of excitatory amino acid antagonists mechanisms responsible for the release of the hair
[e.g. kynurenate) (Annoni et al., 1984; Soto and
Vega, 1988) or spider venom (Cousillas et al., Table 2. Evoked neurotransmitter release is a€ected more
1988)] can cause complete cessation of ®ring of than resting
the a€erents. (A) Glutamate antagonists reduced evoked more than
3. In the absence of hair cells, apparently normal spontaneous ®ring
cochlear a€erents exhibit no spontaneous activity 1. Lateral line (Bledsoe et al., 1988).
(Durham et al., 1989). 2. Cochlea (Cousillas et al., 1988).
4. A€erent ®bers became silent when deprived of 3. Saccule (Starr and Sewell, 1991).
hair cell input by acute (Bernard, 1983) or
chronic (Norris et al., 1994) aminoglycoside (B) In the cochlea, perfusions of glutamate caused a
treatment. reduction in tone-evoked activity without a change in
spontaneous rate (Gleich et al., 1990).
5. The frequency of a€erent ®ring in Type I neurons
goes to zero when the hair cells are transduction- (C) In the SCC electrical d.c. currents across the
ally hyperpolarized (Precht, 1976). neuroepithelium, in which the endolymph was negative
6. Passing D.C. currents across the neuroepithelium relative to the perilymph, decreased evoked more than
of the SCC in which endolymph is negative with resting a€erent activity. When of sucient amplitude the
regard to the perilymph can cause the a€erents to current can completely inhibit all a€erent activity
become silent when the current amplitude is su- (Ricci et al., 1991b).
ciently high (Ricci et al., 1991b). For several
reasons these currents are thought to act only on (D) In the lateral line, evoked activity has been shown
to be more a€ected than resting with decreasing calcium
the hair cells. First, passage of hyperpolarizing
and increasing magnesium concentrations (Drescher and
currents a€ects spontaneous and mechanically- Drescher, 1987a).
evoked a€erent ®ring di€erently. If the current
The Vestibular Hair Cells 213

Table 3. Resting neurotransmitter release is a€ected more An even more dramatic dissociation of resting
than evoked and evoked a€erent activity was recently reported
by Zucca et al. (1995). The application of hypertonic
(A) In the SCC an increase of cAMP produced an
increase in resting, but not evoked ®ring rates (Ricci et al., solutions to the whole labyrinth while recording
1991a). multiunit activity of the ampullar nerve produced an
increase in resting activity and a decrease in
(B) In the SCC ACh application produced an increase mechanically-evoked activity.
in resting but not evoked ®ring rates (Guth et al., 1991).

(C) In the SCC application of a calcium ionophore 5.4.4. Calcium Sources in the Resting and Evoked
caused an increase in resting but not evoked activity Modes of Transmitter Release
(Aubert et al., 1993).
The manipulations of Ca concentrations and
(D) In the SCC 4-AP caused a decrease in resting which ¯uxes listed in Tables 2 and 3 suggest that, although
was much greater than the decrease in evoked activity Ca is involved in both resting and evoked release of
(Ricci et al., 1991b). transmitter, its involvement in the two modes of
release may be di€erent. One explanation for the
(E) In the lateral line, salicylate decreased resting more results seen with calcium channel blockers and with
than evoked activity (Puel et al., 1989). low Ca-high Mg (Guth et al., 1991) (i.e. that resting
fails before evoked activity) could be that resting ac-
(F) tivity is more dependent on the entry of extracellular
1. In the SCC (Guth et al., 1991), substituting low
Ca, high Mg solutions for perilymph caused a marked
calcium through ion channels whereas the evoked
reduction in resting activity before the subsequent mode can cause the mobilization of intracellular
reduction in evoked activity occurred. stores. It is important to emphasize that the two
2. Likewise, diltiazem or streptomycin (thought to act sources of calcium, intra- and extracellular, are both
also on Ca channels) both caused resting activity to fail parts of a calcium circuit (i.e. Ca ¯ows into the cell
before evoked activity (A. through ion channels, is bound to intracellular orga-
Ricci, personal communication). nelles from which it may be mobilized and it may be
3. In the lateral line, resting activity is a€ected more transported back across the cell membrane to the
than evoked for 2±4 mM calcium (Drescher and Drescher, extracellular ¯uid). The intracellular pools, being
1987a).
much smaller than the extracellular source, are ulti-
(G) In the SCC, d.c. currents across the mately dependent on the extracellular source (i.e. if
neuroepithelium in which the endolymph is positive to the extracellular calcium is removed, intracellular cal-
perilymph increased resting activity much more than cium will in time be reduced or depleted). (See also
evoked (Ricci et al., 1991b). Section 6.3.)
The evidence implicating one or another of these
(H) Cromakalim, an opener of K+ channels regulated pools as proximate sources of Ca for spontaneous
by internal ATP, increased resting activity while the or evoked transmitter release in the intact organ is
amplitude of mechanically-evoked a€erent activity of the at the moment indirect.
SCC remained (Zucca et al., 1992a).
Another important factor in the di€erential
modulation of transmitter release in evoked and
cell neurotransmitter. Many of these studies employ resting modes may be the existence of a hair cell
a€erent ®ring as a dependable, if distant, means of autoreceptor (see Section 5.7.2.6). Brie¯y stated, it
monitoring release of transmitter from hair cells. may be that a positive-feedback autoreceptor is
These observations demonstrated that evoked and recruited in the evoked mode to cause even greater
spontaneous neurotransmitter release can be di€er- release of hair cell transmitter in that mode.
In any case, it seems clear that mechanisms re-
entially a€ected by a variety of chemical and physi-
sponsible for the a€erent neurotransmitter release
cal manipulations.
during resting and evoked activities are di€erentially
The observations given in Tables 2 and 3 illustrate modulated and should be the focus of new research
several general di€erences between spontaneous and activity.
evoked neurotransmitter release.
Note that evoked and spontaneous activities may
each be independently modulated to cause either an 5.5. Adaptation
increase or a decrease in the release of the hair cell
neurotransmitter. Note also that either evoked or Di€erences in adaptation observed in the various
spontaneous release of transmitter may be the more vestibular organs may re¯ect the involvement of two
readily a€ected. Taken together the observations of di€erent adaptation systems: while saccular hair
cells rely on a mechanical adaptation that can be
Tables 2 and 3 strongly suggest that the resting and
extremely fast [reviewed in Hudspeth and Gillespie
evoked modes of neurotransmitter release are modu-
(1994)]. SCCs appear to use more complex processes
lated by di€erent mechanisms and that these mech- (metabolic adaptation, or autoreceptors), whose
anisms operate presynaptically in the hair cells. That mechanism is at present only partially understood
is to say, the modulation probably does not occur in (see below). Utricular cells appear to use both pro-
the a€erent because any change in the a€erent den- cesses even on the same time scale [e.g. in type F
drite would likely a€ect both resting and evoked ac- and E cells (Baird, 1994a,b), ruling out speed as a
tivity similarly and not di€erentially. physiological basis for a double adaptation mechan-
214 P. S. Guth et al.

ism. The physiological basis, however, remains at organs. Moreover, perilymphatic K accumulation
present elusive. might exert cytotoxic e€ects.
Semicircular canals whole-nerve a€erent discharge Regarding the second point (i.e. the maintenance
adapts in several tens of seconds, both to continuous of the correct K gradient between the endolymph
(Rusch and Thurm, 1989; Zucca et al., 1993b) and and the perilymph), this can be achieved only if an
repetitive stimulation (P. Valli, unpublished obser- amount of K equivalent to that lost during receptor
vations). At present, the mechanism involved in this current ¯ows is regained by the endolymph. In SCC
relatively slow process is not fully understood; how- and utricle, dark cells, which are involved in endo-
ever, it appears to be a step downstream from MET. lymph secretion, seem to be responsible for these
Adaptation of the a€erent nerve depolarizing re- homeostatic mechanisms. In fact, dark cells strongly
sponse and ®ring rate, that could be elicited by both express Na/K ATPase on their basolateral side
sinusoidal and step mechanical stimulation, was (Burnham and Stirling, 1984a; Ichimiya et al.,
abolished by blocking Na/K-ATPase (Taglietti et 1994), and a slow K conductance (carried by the
al., 1977; Zucca et al., 1993b). Since canal hair cells weakly voltage-dependent IsK channels) on their
lack this pump, which is instead present on a€erent apical side (Vetter et al., 1996). K homeostatic
nerve terminals, the e€ect may well be postsynaptic. mechanisms may play a role in slow, ouabain-sensi-
On the other hand, ampullar microphonic current tive vestibular adaptation (Taglietti et al., 1977),
recorded from the isolated frog semicircular canal and have been suggested to shape the response of
showed adaptation per se (Masetto et al., 1995a,b), Type I hair cells (Goldberg, 1996).
suggesting the involvement of presynaptic factors as Dark cells, which are responsible for endolymph
well. Microphonic current adaptation was depen- secretion, are likely to be involved in both the
dent on perilymphatic Ca (Masetto et al., 1995a) above-mentioned K+ homeostatic processes. By
and impaired by Cd and charybdotoxin (Masetto et using K-sensitive microelectrodes, Valli et al. (1988)
al., 1995b), suggesting the involvement of an IK(Ca) showed that any variations in perilymphatic K con-
activated by Ca entering through VOCCs; the cellu- tent induced a prompt change in the rate of K trans-
lar localization of the channels involved is, however, port from the perilymph into the endolymph. This
still unknown. It is, therefore, either possible that indicates that vestibular organs are endowed by K
two distinct mechanisms are working at pre- and homeostatic mechanisms able to bu€er very e-
postsynaptic sites or that the data available re¯ect ciently the concentration of this ion in both the
partial aspects of a more complicated process. ¯uids bathing vestibular hair cells. This K homeo-
static mechanism may also play a role in the adap-
5.6. Potassium Fluxes in Vestibular Organs tation of the entire vestibular periphery but
especially of the a€erent ®bers to high frequency
As described elsewhere in this review the receptor mechanical or electrical stimulation (see also Section
current ¯owing across vertebrate hair cells and mod- 5.5).
ulating transmitter release at their synaptic pole is Saccular regulation of extracellular ¯uids may be
mainly carried by K. These ions enter sensory cells di€erent from what observed in other vestibular
through the nonspeci®c transduction channels organs. In facts, the saccule lacks dark cells (Sellick
located on their hair-bearing membrane, and exit and Johnstone, 1972; Sellick et al., 1972), and saccu-
through voltage- and Ca-dependent K channels lar hair cells appear more resistant to chronic altera-
located on their basolateral membrane. A direct tions of endolymphatic composition than hair cells
consequence of this K ¯ow is that a considerable from other vestibular organs (Vetter et al., 1996).
amount of this ion continuously passes from the en- Na±K±ATPase activity in hair cells is not signi®ca-
dolymph into the perilymph. As an example, in frog tively di€erent among the vestibular organs
SCCs, the amount of K needed to carry the receptor (Burnham and Stirling, 1984a,b; Kawasaki et al.,
current has been calculated to be ca 1992, No. 2850); on the other hand, Ca-extrusion
3  1011 ions secÿ1 in resting conditions, an amount mechanisms between saccular hair cells (which
that may vary from zero up to 15  1011 ions secÿ1 express large Ca currents) and SCC hair cells (which
during sinusoidal mechanical stimulation of the sen- express smaller currents) are probably di€erent. The
sory organ (Valli et al., 1990). homeostatic mechanisms involved in the delicate
Because of such an intense K ¯ow at least two balance of endolymphatic composition are currently
problems must be solved concomitantly by vestibu- under intense study.
lar organs: the ®rst is to prevent large K ¯uctu-
ations, especially in the narrow clefts surrounding
the hair cells basolateral membranes; the second is 5.7. Neurotransmitters and Neuromodulators
to maintain an appropriate K gradient at both ends 5.7.1. Acetylcholine
of hair cells needed for the transduction process.
Regarding the ®rst point, normal vestibular pro- 5.7.1.1. Generalities
cesses can take place in labyrinthine organs only Pride of place belongs to ACh. Not only was it
when the perilymphatic K content is normal. the ®rst transmitter ever established (e.g. in heart,
Vestibular receptors are, in fact, extremely sensitive autonomic ganglia and the neuromuscular junction),
to K changes in the perilymph (Valli et al., 1988, but it was the ®rst ever established in the inner ear.
1990) and variations as low as 20.25 mM are su- There were early clues (Gisselson, 1952) that choli-
cient to produce clear changes in the amplitude and nomimetic agents a€ected the latency of cochlear
the frequency of EPSPs and, as a consequence, in potentials, and (Churchill et al., 1956) that the e€er-
the spike discharges arising from the various end ent cochlear bundle contained acetylcholinesterase
The Vestibular Hair Cells 215

(AChE). These clues suggested that ACh was a mechanism is that there are two separate ACh
likely transmitter candidate in the cochlea. Building receptors serving these responses. In fact, appli-
on those clues, this laboratory established: cation of ACh to vestibular organs does produce
1. the synthesis of ACh by cochlear e€erents (Jasser both responses and therefore ACh, by itself, can
and Guth, 1973); mimic e€erent nerve stimulation (Guth et al., 1986;
2. the release of ACh upon e€erent stimulation Bernard et al., 1985). As it turns out, in the frog, the
(Norris and Guth, 1974); SCC responds to ACh administration primarily with
3. the e€ect of applied ACh on cochlear activity facilitation of a€erent ®ring (Guth et al., 1986)
(Amaro et al., 1966); while the saccule responds primarily with inhibition
4. the in¯uence of known cholinergic agents on (Guth et al., 1994a). The facilitation is most potently
cochlear activity (Guth and Amaro, 1969). antagonized by atropine and the suppression by
strychnine or D-tubocurarine.
Many other laboratories contributed to the estab-
lishment of ACh as the predominant transmitter of
the crossed olivocochlear bundle [reviewed in Guth 5.7.1.3. The hair cell ACh receptor
et al. (1976)] especially in regard to the early Cholinergic receptors belong to two di€erent
research, and later by Eybalin (1993)]. classes: muscarinic and nicotinic. Nicotinic receptors
In the vestibular periphery evidence in support of are ligand-gated ion channels composed of ®ve sub-
the cholinergic nature of the vestibular e€erents is units. The endplate nicotinic receptor is composed
sparse yet sucient to support that notion. ACh is of (a1)2, b1, g and E subunits in the adult muscle;
accepted as the predominant vestibular e€erent neuronal nicotinic receptors have a di€erent compo-
transmitter as much by evidence as by analogy to sition. There are two classes of neuronal nicotinic
the cochlea. Cholinergic e€erent labyrinthine ®bers receptors: heteromeric receptors have the structure
have been found by measurement of AChE and cho- (an)2(bm)3, where n = 2±6 and m = 2±4; homomeric
line acetyltransferase (ChAT) activities (Godfrey et receptors have the structure (an)5, where n = 7±9.
al., 1984; Lopez and Meza, 1990). Much earlier, his- Pharmacologically, these receptors may be distin-
tochemical studies demonstrated the association of guished because homomeric receptors are reversibly
the less reliable indicator AChE with the vestibular blocked by strychnine and a-BTX while heteromeric
e€erent neurons and endings (Cohen, 1987; Gacek receptors are insensitive to both. Endplate receptors
et al., 1965; Hilding and Wersall, 1962; Iurato et al., are also sensitive to a-BTX, but the block is irrevers-
1971a,b). ible (Karlin and Akabas, 1995). Homomeric neur-
ChAT-like immunoreactivity was traced to the onal receptors are highly permeable to Ca, and are
vesiculated e€erent endings in vestibular endorgans thought to be mainly presynaptic in the CNS
(Gonzalez et al., 1993; Kong et al., 1994). All ®ve (Wonnacott, 1997). The homomeric a9 receptor
vestibular endorgans had such immunoreactivity strikingly resembles the nicotinic receptors found in
with the SCCs and utricle having similar densities of acousticovestibular organs: this receptor, in fact,
staining and the saccule having less. In the SCC, was not very sensitive to either nicotine or muscar-
greatest density of staining was in the periphery ine, while both atropine and D-tubocurarine blocked
close to the semilunar plane and there was less den- it. Strychnine was the most potent antagonist, and
sity in the central area. This pattern is identical with the overall pharmacology displayed a mixed nic-
that of the innervation pattern of the vestibular otinic-muscarinic pro®le (Elgoyhen et al., 1994).
e€erent (Hunter-Duvar and Hinojosa, 1984; Muscarinic receptors are seven-transmembrane
Spoendlin, 1970; Wersall, 1956). receptors coupled to trimeric G-proteins. Five
Employing ¯uorescence detection with a-bungaro- muscarinic receptor subtypes (M1 to M5) have been
toxin (a-BTX), a nicotinic receptor ligand, Anniko found, and are grouped in an `even' series (M2/M4)
and Arnold (1991) also found generally less intense coupled to Gi/o and cAMP decrease and an `odd'
staining in the saccular macula than in the utricular series (M1/M3/M5) coupled to Gq/11 and to phos-
macula. Wackym et al. (1995) in the rat as did pholipase C (PLC)-IP3 stimulation. Other e€ects,
Anniko and Arnold (1991) in human, both found a- such as the activation of PLA2 or PLD, have
BTX binding in the vestibular periphery. a-BTX is a recently been ascribed to muscarinic receptor acti-
reversible ligand at inner ear and brain nicotinic vation. A good review on muscarinic receptors may
ACh receptors (Fex and Adams, 1978), but it is irre- be found in Felder (1995).
versible at the neuromuscular junction. Hair cells isolated from: mammalian cochlea (i.e.
OHCs) (Housley and Ashmore, 1991; Erostegui et
5.7.1.2. The pharmacology of ACh al., 1994a); frog SCC (Housley et al., 1990); chick
Stimulation of neurons e€erent to the vestibular cochlea (Ohmori, 1990) frog saccule (Yoshida et al.,
periphery produces both facilitation (Goldberg, 1994) all respond to the application of ACh
1979; Hartmann and Klinke, 1980; Rossi et al., bespeaking the presence of an ACh receptor on
1980) and inhibition (Hartmann and Klinke, 1980; these cells.
Rossi et al., 1980) of vestibular a€erent ®ring rates. From the perspective of the vestibular hair cells,
In some species, facilitation occurs almost exclu- there seem to be two easily distinguishable ACh
sively (Goldberg, 1979) and in others inhibition receptors. One of these is atropine-preferring,
appears to be the predominant response although it muscarinic-like (i.e. metabotropic) and usually med-
is possible to see facilitation as well (Rossi and iates depolarization and facilitation of a€erent ®ring
Martini, 1991; Rossi et al., 1980). If ACh is respon- (Guth et al., 1986; Norris et al., 1988). [There may
sible for both e€ects, then one possible (even likely) also be a muscarinically-mediated hyperpolarization
216 P. S. Guth et al.

(Ohmori, 1990; Steinacker and Rojas, 1988; OHC it did not (Housley and Ashmore, 1991).
Yoshida et al., 1994).] The other is strychnine-pre- The possibility that saccular cholinergic modu-
ferring, nicotinic-like, ionotropic and mediates lation is di€erent from that of the cochlea is how-
hyperpolarization and suppression of a€erent ®ring ever open and under active study. Sugai et al.
(Guth et al., 1994a). These are both unusual ACh (1992) also reported a cholinergically-induced
receptors with unorthodox pharmacology: neither hyperpolarization in frog saccular hair cells.
one of the classical ligands, muscarine or nicotine, 6. Steinacker and Rojas (1988), recording from iso-
activates either receptor very well; atropine antagon- lated toad®sh saccular hair cells, found that ap-
izes both (albeit at slightly di€erent concentrations) plication of ACh or oxotremorine, a muscarinic
and; strychnine, usually thought of as a glycine an- agonist, caused an increase in open time and
tagonist, is a potent antagonist at the nicotinic-like opening rate of a K+ channel. These e€ects
receptor (Guth et al., 1994a). There is good pre- occurred when the cell-attached mode of record-
cedent for both nicotinic and muscarinic receptors ing was used and the drugs were applied in the
to exist on a single cell type; for example, neurons bath suggesting the involvement of an intracellu-
of the chick ciliary ganglion have a muscarinic lar mediator. That and the ecacy of oxotremor-
receptor, and two di€erent nicotinic receptors ine suggested muscarinic mediation.
(Rathouz et al., 1995). 7. In unpublished work, this laboratory has results
demonstrating that an irreversible ligand selective
for muscarinic receptors [propylbenzilyl choline
5.7.1.4. Evidence for two ACh receptors in VHC mustard (Burgen et al., 1974)] antagonizes the
1. Recording from single a€erent units in the frog ACh-induced facilitation of a€erent ®ring in the
SCC Bernard et al. (1985) found that muscarinic SCC while not a€ecting the a€erent suppression
agonists caused an increase in frequency of ®ring induced by ACh in the saccule.
while nicotinic agonists produced both increases Regarding muscarinic receptor expression, in the
and decreases. rat cochlea M3 receptors were found to be expressed
2. Using multiunit recording of a€erent activity it by inner hair cells (IHCs), lower-turn OHCs and
was found that ACh application caused an a€er- a€erent Type I neurons (Sa®eddine et al., 1996). Rat
ent facilitation in the frog SCC (Guth et al., spiral ganglion and organ of Corti expressed M1/
1986; Norris et al., 1988) and a suppression in M3/M5 receptors (Upadhyay et al., 1997). The IP3
the frog saccule (Guth et al., 1994a). involvement in saccular cholinergic action would
(a) The ACh-induced suppression was antag- suggest the presence of the same receptor in the sac-
onized most potently by strychnine and culus as well. However, preliminary data reports the
showed marked long-lasting desensitization presence of mRNA for the M2 receptor (G. B.
(Guth et al., 1994a). Athas, personal communication) and a muscarinic
(b) The ACh-induced facilitation was not modulation of VOCCs in frog saccular hair cells
antagonized by strychnine but was antagonized (Perin et al., 1997b). All muscarinic receptors may
by atropine and did not show similar desensiti- inhibit or facilitate VOCCs depending on the path-
zation (Guth et al., 1994a). ways activated (Pemberton and Jones, 1997), and
therefore the VOCC data do not help in clarifying
3. Using whole-cell patch clamp recording of iso- the nature of receptor involved. The muscarinic
lated frog SCC hair cells, Housley et al. (1990), facilitatory e€ect could be due to the reduction in a
demonstrated that ACh produced either a hyper- (possibly Ca-dependent) M-like K current (Housley
or a depolarization (in di€erent cells). The de- et al., 1990).
polarization was antagonized by atropine. Thus, there seem to be at least two kinds of ACh
4. Anniko and Arnold (1991) found speci®c binding receptors on hair cells. Muscarine-like receptors
of a-BTX in human vestibular hair cells and cause both depolarization/a€erent facilitation and
Wackym found it on the basal aspect of Type II hyperpolarization/a€erent suppression. Nicotinic-
hair cells (Wackym et al., 1995), suggesting the like receptors cause hyperpolarization of hair cells
presence of a nicotinic-like ACh receptor with its and a suppression of a€erent ®ring. Individual hair
well-known a-BTX binding site. cells may have none, one or both. It is worth noting
5. Yoshida et al. (1994) using whole-cell patch- that ACh application (Bobbin et al., 1985) and e€er-
clamp recording adduced evidence for a G-pro- ent nerve stimulation (Russell and Roberts, 1972)
tein-coupled muscarinically-mediated hyperpolar- both caused biphasic e€ects on a€erent ®ring of the
ization by ACh on isolated frog saccular hair lateral line.
cells. In this paper, the authors suggested a mus-
carinically-mediated hyperpolarization. These ex-
periments do not, however, conclusively prove 5.7.1.5. The nicotinic-like receptor(s)
the muscarinic nature of K-channel activation in The nicotinic-like receptor found in the vestibular
saccular hair cells: in fact, in OHCs, a slow, system exhibits interesting pharmacological charac-
store-mediated cholinergic e€ect appears to be in- teristics. For instance, despite its name, nicotine is a
directly triggered by a Ca-permeable nicotinic weak agonist (Erostegui et al., 1994; Fuchs and
receptor activation (Sridhar et al., 1995, 1997). In Murrow, 1992b; Guth et al., 1994a; Kakehata et al.,
cochlear hair cells, the involvement of G-proteins 1993; Sugai et al., 1992; Yoshida et al., 1994);
has been observed by using a nonhydrolysable strychnine is often the most potent antagonist and
GTP-analog. GTPgS activated the cholinergic atropine is a reasonably potent antagonist (Guth et
channel (Shigemoto and Ohmori, 1991) while in al., 1994a; Sugai et al., 1992; Yoshida et al., 1994).
The Vestibular Hair Cells 217

This receptor shows long-lasting desensitization 2±3 mRNAs in Scarpa's ganglion neurons. The a6
(Bernard et al., 1985; Guth et al., 1994a). and b 2±3 riboprobes labeled all neurons while the
Most neuronal (or hair cell) nicotinic receptor other subunit mRNAs were selectively expressed.
channels are cation-selective, but unlike muscle nic- No expression of a3 or b4 was found. On the other
otinic receptors may have a notable permeability to hand, all hair cells (i.e. SCC, saccule and utricle) of
Ca (Sargent, 1993). Calcium entry through nicotinic the rat expressed the a9 subunit whether or not they
receptor channels may be sucient to activate cal- received direct cholinergic innervation. In the vestib-
cium-dependent K (Fuchs and Murrow, 1992a; ular periphery, a4 and b2 nicotinic subunits seem to
Housley and Ashmore, 1991; Housley et al., 1990; be coexpressed in a€erent neurons but not by hair
Yoshida et al., 1994; Fuchs and Murrow, 1992b) cells (Wackym et al., 1995; Ohno et al., 1993a). All
and calcium-dependent -Clÿ conductances (Sargent, a€erents seem to express a6, b2 and b3 mRNA,
1993) and even stimulate calcium-activated transmit- while only a subpopulation of a€erents expresses a4,
ter release. Therefore, it has been argued, depending a5 and a7. Therefore, e€erent±a€erent synapses will
on its colocalization with Ca-dependent K channels most likely display di€erent properties from e€erent
or not, the nicotinic ACh receptor could mediate synapses on hair cells. On the other hand, all vestib-
either the inhibition or facilitation of a€erents. ular hair cells of the rat expressed a9 mRNA regard-
However, a€erent facilitation induced by ACh is not less of their cholinergic innervation (Hiel et al.,
antagonized by nicotinic antagonists (Guth et al., 1996), and SCC hair cells also express mRNA for a3
1994a; Rossi et al., 1980). (Athas et al., 1997b). A note of caution was inserted
Some of the questions surrounding this unusual into the molecular biological literature concerning
nicotinic-like receptor are being answered. For ACh and ATP receptors in hair cell organs
example, the antagonism of ACh at this receptor by (Housley and Ryan, 1997). Particularly interesting is
strychnine, although nontraditional, is best the ®nding that, employing RT-PCR work in rat
explained by the notions of receptor superfamilies cochlea (Housley et al., 1994), the primers for the a5
and multiplicity of receptor subunits. The nicotinic subunit ampli®ed the cDNA of the b4 subunit, lead-
ACh receptor belongs to the same receptor super- ing to a false positive for the latter. Therefore, mol-
family as the glycine and g-amino butyric acid ecular data must be interpreted with caution, and
(GABA)-A receptors. The multiplicity of subunits functional, pharmacological and immunohistochem-
with di€erent pharmacological properties explains ical data are needed in concert with the molecular
the variety of nicotinic receptors and how it is that biological data to satisfactorily de®ne the presence
nicotine, the ligand for which this receptor was of a receptor.
named, may be such a weak ligand for the hair cell
receptor. Grenningloh et al. (1990) reported that the
strychnine binding subunit of the glycine receptor 5.7.1.6. The muscarinic-like receptor(s)
shows signi®cant structural homology with a subu- In SCC hair cells, a muscarinic-like receptor med-
nit of the nicotinic ACh receptor. Elgoyhen et al. iates depolarization and an increase in a€erent ®r-
(1994) reported on the pharmacology of a homomer ing. On the other hand, in OHC and saccular hair
of the a9 nicotinic subunit expressed in Xenopus cells receptor mediates hyperpolarization and, ulti-
oocytes. This homomer had pharmacological prop- mately, a decrease in a€erent ®ring. These di€er-
erties remarkably similar to those of the hair cell ences may be due to the presence of di€erent
nicotinic-like receptor. For instance, both nicotine muscarinic subtypes, di€erent G-proteins, di€erent
and cytisine were very weak agonists while the re- second messengers or the activation of di€erent ion
sponse to ACh was blocked by strychnine, D-tubo- channels. This receptor may cause the increase in
curarine and atropine, in that order. Furthermore, IP3 intracellular concentration (Caul®eld, 1993) in
the a9 subunit gene was detected in cochlear frog saccular hair cells (Yoshida et al., 1994), a
(Elgoyhen et al., 1994) and vestibular (Hiel et al., release of intracellularly-stored Ca and a subsequent
1996) hair cells in situ and in isolated OHC (Athas activation of a Ca-dependent K+ conductance lead-
et al., 1997a) and SCC hair cells (Athas et al., ing to hyperpolarization. Another possibility is that
1997b). the muscarinic-like receptor may inhibit the L-type
Other nicotinic receptor subunits have been Ca channel by activating PKC as was demonstrated
found. For instance, Wackym et al. (1995) and in NIH 3T3 cells (Pemberton and Jones, 1997).
Ohno et al. (1993a) by means of in situ hybridization More dicult to explain is the muscarinically-
histochemistry, mapped the distributions of the a2, induced depolarization, enhancement of hair cell
3, 4 and b2 subunits of the nicotinic ACh receptor in transmitter release and increase in a€erent ®ring
the vestibular periphery. They found that neither the rates seen in VHC especially of the SCC (Guth et
Type I nor Type II hair cells expressed these subunit al., 1986; Housley et al., 1990; Norris et al., 1988).
genes. However, the a4 and b2 subunit genes were The evidence is incomplete but it appears that Ca is
co-expressed by a€erent neurons in Scarpa's required for the depolarization and that the e€ect is
ganglion. These data suggested to the authors that due to a reduction in a current carried by K through
the nicotinic ACh receptors on the a€erent neuron what is probably a Ca-dependent K channel
chalices might contain these subunits, but that the (Housley et al., 1990). The mechanism of muscarini-
receptors on the Type II hair cells were di€erent. cally-mediated excitation in brain is discussed and
The results of Wackym et al. (1995) and Ohno et al. reviewed by Krnjevic (1993). The consensus is that
(1993a) were con®rmed and extended by Hiel et al. excitation occurs by suppression of an ongoing, out-
(1996) also using in situ hybridization. These last- ward K current. By analogy with mammalian brain
mentioned workers found expression of a 4±7 and b excitatory muscarinic receptors, and with the e€ect
218 P. S. Guth et al.

seen on cochlear a€erent neurons (Yamaguchi and receptors (Ambache, 1955). In another instance it
Ohmori, 1993) the K currents involved might be the was suggested that the ACh receptor in echinoderms
M current, the after-hyperpolarizing current or a was of low speci®city because it responded both to
voltage-dependent leak current (Dodd et al., 1981; nicotinic and muscarinic agents. On closer inspec-
Halliwell and Adams, 1982; Lancaster and Adams, tion, especially by the use of selective alkylating
1986; Madison et al., 1987; Hille, 1994). This de- agents, it was found that there were, in fact, two
polarizing metabotropic response to ACh is rarely ACh receptors, one resembling the nicotinic and the
seen in vestibular hair cells other than those of the other the muscarinic receptor (Michelson, 1974).
SCC. The exact mechanism of this reduction is The muscarinic receptors are single protein mol-
unknown, but it may be mediated by a G-protein ecules with seven transmembrane domains. The nic-
subunit directly, second-messenger or protein kinase otinic receptors are aggregates of ®ve separate
modulation (Hille, 1994). protein subunits with little or no structural hom-
Activation of the hair cell M3 muscarinic receptor ology to the muscarinic receptor. It is unlikely that
could cause inhibition of a€erent ®ring by a G-pro- they could be amalgamated so as to produce a single
tein-mediated inhibition of VOCCs (Hille, 1994; entity with the properties of both. And, there is a
Dolphin, 1995; Felder, 1995) resulting in a reduction simpler explanation. The di€erent conclusions
of hair cell transmitter release (See Section 3.2). arrived at by di€erent investigators [e.g. (Erostegui
Alternatively M3 receptor activation may lead to et al., 1994; Kakehata et al., 1993)] as to the nature
PLC stimulation, IP3 accumulation, increase in in- of ACh receptors would seem to reside very much in
tracellular Ca concentration, activation of the Ca- methodology.
dependent K channels, hyperpolarization and re- The metabotropic (i.e. muscarinic-like) ACh
duction in hair cell transmitter release (Felder, 1995; receptor relies on G-protein and second messenger
Clapham, 1995). Another possibility revolves activation and the consequent biochemical cascade.
around the ®nding of the message for the M2 Some of the components in this cascade could very
muscarinic receptor subtype in saccular hair cells well be unattached and mobile. Therefore, when
(G. B. Athas, personal communication). This recep- patch-clamp recordings are made in the whole cell
tor is known to reduce cAMP content by working mode, these mobile components could easily be
through the G-protein Gi (Felder, 1995). Since, diluted and/or di€used away from sites of action
cAMP seems to be involved in the increase in a€er- [e.g. the mobile Ca bu€er suggested by Roberts
ent ®ring seen in SCC, it follows that a reduction in (1993)]. In such a condition it might be dicult to
cAMP may be involved with the cholinergic re- obtain evidence of a metabotropic response to choli-
duction in a€erent ®ring. nomimetics.
Some investigators have suggested that the hair On the other hand, rather than using the whole
cells have but a single ACh receptor and that it is cell technique, Kakehata et al. (1993) employed the
mixed nicotinic-muscarinic, nonfastidious or even perforated-patch recording technique on isolated
promiscuous with regard to ligand binding sites OHC which would likely leave the metabotropic
(Housley and Ashmore, 1991; Sugai et al., 1992). It cascade undisturbed. Furthermore, these authors
is the novelty of the pharmacology of the hair cell chose to employ agonists and antagonists known to
ACh receptors that has led researchers to this con- favor muscarinic receptors to the exclusion of those
clusion. To quote Elgoyhen et al. (1994), ``Upon ex- favoring nicotinic receptors.
pression in Xenopus oocytes, all functional nAChR The response of saccular hair cells and saccular
a subunits cloned to date form either heteromeric or a€erents to e€erent stimulation and ACh appli-
homomeric receptor-channel complexes that are cation is almost uniformly hyperpolarization and a
activated by nicotine''. As mentioned above, it is the decrease in a€erent ®ring rates (Guth et al., 1994a;
pharmacology of the a9-homomeric receptor that Rossi et al., 1980; Sugai et al., 1992; Yoshida et al.,
matches most closely that of the hair cell nicotinic- 1994). (The saccular hair cells may be much more
like receptor. And, strikingly, nicotine did not elicit like cochlear hair cells in this regard and this may ®t
a response in a9 injected oocytes. Neither did they the saccule's vibratory-sensing function, particularly
respond to muscarine or the muscarinic agonists in `lower' vertebrates.) Basolateral Ca channels and
bethanechol or pilocarpine. They did respond to the Ca-dependent K+ channels are essentially co-loca-
nicotinic agonist dimethylphenyl piperazinium and lized in saccular hair cells (Roberts et al., 1990;
the muscarinic agonist oxotremorine, but the re- Roberts, 1993). So that whether the intracellular Ca
sponses were only ca 5% of the maximal current re- concentration was increased via Ca entry through
sponses seen with ACh application (Elgoyhen et al., ACh nicotinic receptor channels, Ca channels or by
1994). activating the muscarinic cascade, the end result
Pharmacology can be very useful when it provides could be an activation of the Ca-dependent K+
direction for studies using other techniques such as channels which would then cause a hyperpolariz-
immunohistochemistry and molecular biology. By ation.
itself it rarely provides the ®nal incisive data. After So, there are at least two ACh receptor types on
all, the ACh receptors, both muscarinic and nic- inner ear hair cells. Nicotinic-like ionotropic recep-
otinic, have sites that are suciently similar that tor(s) mediate hyperpolarization and are found on
both accommodate ACh. It is not so dicult to ima- both cochlear and vestibular hair cells. Muscarinic
gine that chemical structures having some selectivity metabotropic receptor(s) mediate hyperpolarization
for one of the receptors might also be accommo- in some cochlear (e.g. OHC) and in saccular hair
dated in the other. Early pharmacologists knew that cells. In SCC hair cells the metabotropic ACh recep-
atropine could act on nicotinic as well as muscarinic tor mediates depolarization. The metabotropic ACh
The Vestibular Hair Cells 219

receptors mediating the two di€erent e€ects may be If Clÿ is not the current carrier but substitutions
identical and the e€ect determined by coupling to for it reduce responses to e€erent stimulation or
di€erent G-proteins. ACh application, then what is its role? Mayer and
The distribution of these receptors among hair Westbrook (1983) and Takahashi (1990) have
cells is far from uniform. For instance, OHC from suggested that external Clÿ was essential for, or
the lower turns of the cochlea respond more reliably even activated, the inward K+ recti®er current seen
to ACh than those from the upper turns (Nenov in mouse spinal sensory ganglion cells. With this
and Bobbin, private communication). The distri- precedent in mind, it is conceivable that Clÿ may
bution and nature of these receptors are arguably exert a permissive or even an activating e€ect on a
the most important determinants of e€erent func- K+ conductance. There have recently been
tion in the inner ear. described Clÿ channels of very low conductances
(i.e. of the order of 1±2 pS) [reviewed in Valverde et
al. (1995)]. It is conceivable that these low conduc-
tance channels carry such small Clÿ currents as to
5.7.1.7. The putative role of chloride be generally undetected but yet, if localized, su-
Reports of the e€ects of e€erent stimulation or cient to in¯uence the major current-carrying K+
ACh application on hair cells are periodically punc- channels.
tuated with references to a role for chloride (Clÿ) in Mayer and Westbrook (1983) considered the
these e€ects. The current state of our understanding alternative possibility that the large anions, which
of the role for chloride channels in general is dis- were substituted for Clÿ, could themselves a€ect the
cussed by Valverde et al. (1995). conductance by some unspeci®ed processÐsome
In the inner ear, Desmedt and Robertson (1975) pharmacological e€ect. They rejected the idea that
believing that the e€erent cochlear bundle exerted these anions could physically block the channel
its inhibitory e€ect on cochlear function by increas- because their e€ects were not voltage-dependent.
ing the hair cell conductance for small anions, Recently, Nakagawa et al. (1994) examined the con-
replaced Clÿ with the larger-diameter anions gluco- ductance called IK, n (Housley and Ashmore, 1991),
nate and sulfate. They found that these replacements and found that conductance to be inhibited by
of Clÿ in perilymphatic perfusions caused the e€er- decreases in chloride concentration. Thus, we may
ent-induced inhibitions of neural responses to be be a step closer to understanding the modulatory
`reduced or eliminated reversibly'. role of Clÿ in hair cells.
Next, Rossi and Sacchi (1982) replaced Clÿ with At the time of this writing, the exact role of chlor-
isethionate in perfusions of the frog SCC and found ide is very uncertain. If, however, a means of inter-
that the inhibition of a€erent ®ring induced by e€er- fering with a Clÿ movement other than substitution
ent stimulation was reduced. by large anions, were to be used, and it likewise pro-
In the turtle half-head preparation, Art et al. duced an antagonism of ACh, then even more
(1984) examined the hyperpolarization of cochlear weight would have to be given to the idea of Clÿ
hair cells caused by e€erent stimulation or ACh ap- involvement. Chloride channel blocking drugs might
plication. They considered, as current carriers of the help but chloride channel blockers are notoriously
hyperpolarization, ions whose equilibrium potentials `dirty' drugs in that they do many things beside inhi-
were negative to resting membrane potentials, bit Clÿ-conductances. And, they are of many di€er-
namely Clÿ or K+. They distinguished between the ent chemical classes. However, if several Clÿ-
two by measuring reversal potentials of the induced channel blockers of di€erent structure and di€erent
current when K+ and Clÿ concentrations were pharmacological spectrum all antagonized the sup-
altered. The reversal potential was shifted by pressive e€ect of ACh or e€erent stimulation, then
changes in K+ concentration but not by large the hypothesis of Clÿ involvement would be more
changes in Clÿ concentrations. However, ``low Clÿ strongly supported. So far, it has been shown that
substitutions . . . halved the maximum size of the Clÿ-channel blockers such as ¯ufenamic acid, picro-
synaptic potentials: thus, although K+ is the major toxin and NPPB [5-nitro-2(3-phenylpropyl (amino)-
current carried, the involvement of Cl ÿ . . . cannot benzoic acid] do antagonize the e€ect of ACh on the
be entirely eliminated''. saccule (Guth et al., 1996).
Sugai et al. (1992) recorded from frog saccular Two other reports of Clÿ-dependent ACh-induced
hair cells using microelectrodes. Upon e€erent inhibitions bear mentioning. These are: Kehoe
stimulation they recorded a hyperpolarization of (1972) working in Aplysia reported on the presence
two phases a small, early and a larger, late one. of three ACh receptors. One of these, an inhibitory
Referring to an earlier paper [cited in Sugai et al. receptor, was Clÿ-mediated and antagonized by
(1992)], the authors wrote that the reversal poten- strychnine and a-BTX much as the vestibular inhibi-
tials for the two phases were ca ÿ65 and ÿ80 mV, tory ACh receptor, and Finkel (1983) working in
respectively, these reversal potentials could suggest Helix reported on the presence of a Clÿ-mediated in-
roles for both Clÿ (ÿ65 mV) and K+ (ÿ80 mV). hibition by ACh.
The response of frog saccular a€erents to ACh
application is generally a suppression of ®ring (Guth 5.7.2. The Hair Cell A€erent Transmitter
et al., 1994a). When 80% of the Clÿ is replaced by
large anions such as isethionate, methane sulfonate 5.7.2.1. Generalities
or methylsulfate, Guth et al. (1996) found that the There is general consensus that the hair cell trans-
response to ACh in the saccule is blunted or elimi- mitter is probably glutamate-like. Whether it is pre-
nated, as the previously mentioned authors. cisely glutamate (Glu) is still not certain. The other
220 P. S. Guth et al.

candidates, naturally-occurring excitatory amino released and its release to be Ca-dependent. Bobbin
acids, are: L-aspartate; L-homocysteate; L-cysteate; et al. (1990) performed the appropriate control of
quinolinate; N-acetyl aspartyl glutamate, and attempting to denude the cochlea of hair cells using
cysteine sul®nate. Aspartate may be removed from the ototoxic combination kanamycin-ethacrynic acid
the list of possible a€erent transmitters by the use of prior to the application of high K. These investi-
`Fonnum's Razor' (Fykse and Fonnum, 1996). That gators found that there remained a signi®cant non-
is, aspartate is not taken up by synaptic vesicles hair cell release of Glu after hair cell destruction
while Glu is. There are diculties that arise in deter- although it was (as expected) lower than in intact
mining whether Glu has a transmitter role that do cochlea.
not arise with other transmitters. For instance, Glu The two most elegant demonstrations of Glu
is incorporated into proteins and peptides, is release from cochlear hair cells are by Kataoka and
involved in fatty acid synthesis, contributes to the Ohmori (1996) and Anson and Ashmore (1994).
regulation of ammonia levels and control of osmotic These workers patch-clamped cochlear hair cells and
and anionic balance, serves as a precursor for cerebellar granule cells. The granule cells are extre-
GABA and for various Krebs cycle intermediates mely sensitive to Glu. They apposed these two cells
and is a constituent of important cofactors such as and recorded from the granule cell while depolar-
glutathione and folic acid. So, it is not surprising izing the hair cell. The greater the hair cell depolar-
that L-Glu is the most plentiful amino acid in the ization, the greater the current amplitude in the
CNS by three to four times the next most abundant. receptor cell. Kataoka and Ohmori (1996) were able
Its concentration in nervous tissue is of the order of to block the current response in the granule cell
10 mM (Mokrasch, 1973; Tower, 1960). Hence, all with the NMDA antagonist, 2-amino-5-phosphono-
nervous tissue has some Glu and its concentrations valerate. These studies are not only elegant but they
across the brain vary by little more than two-fold. are more convincing than the other release studies.
A second diculty with Glu's candidacy is that it It should be noted that the activity detected by the
excites virtually all neurons with which it comes in cerebellar granule cell and its antagonism by 2-
contact and that both D- and L- isomers are active amino-5-phosphonovalerate only indicate that the
(Curtis and Johnston, 1974). The activity of most responsible agent is glutamate-like and not necess-
other transmitters is generally restricted to speci®c arily Glu per se. We are once again indebted to
cells and not virtually universal. A further diculty, Eybalin (1993) for his wholly professional, balanced
related to its universal excitatory action is its elec- review of the evidence of Glu's candidacy for the
trogenic transport. At this writing there are three cochlear hair cell transmitter. To paraphrase his
high anity Glu transporters and all are electro- summary statement, although Glu has probably sat-
genic in the sense that all co-transport two Na+ is®ed several criteria, there remains some uncer-
ions with each Glu molecule and thus cause a de- tainty as to its complete acceptance. He says, ``from
polarization (Kanai et al., 1993). Therefore, a Glu- a formal point of view, establishing the molecule
induced depolarization could be receptor- or trans- glutamate as a neurotransmitter could well be some-
port-mediated, or both. Another related matter, dis- thing like an impossible task''.
cussed thoroughly by Eybalin, (1993), is the
presence of nonsynaptic receptors and/or binding 5.7.2.2. The presence of Glu
sites for excitatory amino acids. In inner ear organs
The presence of Glu-like immunoreactivity in ves-
these sites or the mRNA encoding Glu receptors
tibular organs has been demonstrated by several lab-
have been found in frog saccule (Dememes et al.,
oratories. Dememes et al. (1990) found strong
1983) and rat and guinea pig cochlea (Sa®eddine
immunostaining in all vestibular ganglion neurons
and Eybalin, 1992a,b). There is also electrophysio-
and their processes as well as both types of hair cells
logical/pharmacological evidence for nonsynaptic
(Types I and II). Supporting cells were devoid of
Glu receptors in cochlear spiral ganglion neurons
immunoreactivity. Panzanelli et al. (1994) found
(Nakagawa et al., 1991).
both Glu-like and carnosine-like immunoreactivities
The enzymes of synthesis for Glu, via di€erent
co-localized in vestibular hair cells of the frog.
pathways, are glutaminase, aspartate aminotransfer-
Harper et al. (1995) reported immunocytochemical
ase and ornithine transaminase. Antibodies to gluta-
localization of both Glu and aspartate in vestibular
minase and aspartate amino-transferase appear to
hair cells, nerve ®bers and ganglion cells. Usami and
label some terminal ®elds and a greater number of
Ottersen (1995) noted that Glu immunoreactivity
cell bodies but do not serve as a general marker for
was preferentially distributed in hair cells whereas
Glu or Asp terminal ®elds (Donoghue et al., 1985;
glutamine reactivity was distributed in supporting
Wenthold and Altschuler, 1983). In fact, Godfrey et
cells. This di€erential distribution suggested to the
al. (1986) measured aspartate aminotransferase ac-
authors a Glu-glutamine cycle such as has been
tivity in cochlear structures and found lower activity
suggested at glutamatergic synapses in brain.
in the organ of Corti than in stria vascularis. In the
organ of Corti, the sample containing mostly sup-
porting cells had about the same activity as the 5.7.2.3. The pharmacology of Glu
samples containing either the OHCs or IHCs. The ®rst reports of Glu's excitatory e€ect in ves-
Finally, there is the criterion of transmitter tibular organs were by Annoni et al. (1984) and
release. The cochlear Glu release literature is Dechesne et al. (1984). The former applied Glu and
thoroughly reviewed in Eybalin (1993). All investi- other excitatory amino acids to the isolated frog
gators who sought it, especially after exposure to labyrinth while recording single unit a€erent ac-
very high concentrations of K, found Glu to be tivity. Glu and its agonists consistently increased
The Vestibular Hair Cells 221

a€erent activity. When hair cell transmitter release cell transmitter release failed, the response to added
was inhibited by bathing the preparation in high Mg quisqualate, kainate or NMDA changed character
and low Ca, Glu produced a depolarization of the such that there was a loss of the facilitation of a€er-
a€erents. A variety of Glu antagonists decreased ent ®ring but not of the slow a€erent depolarization.
amplitudes of postsynaptic potentials without a€ect- Thus, these Glu agonists seem to act both on the
ing their frequencies indicating a postsynaptic block- hair cell and the dendrites as suggested by Valli et
ade. Dechesne et al. (1984) either applied Glu to the al. (1985). Quisqualate and kainate were more
membranous labyrinth or perfused it into the lateral potent than NMDA suggesting that the Glu recep-
SCC of the cat and observed a depolarizing action tors of the frog SCC were mainly of the nonNMDA
on a€erents and an increase in both spontaneous type (but see below).
and stimulus-evoked activity in vestibular a€erents. Finally, Starr and Sewell (1991) studied the gluta-
Valli et al. (1985) substantiated the earlier reports matergic pharmacology of the vestibular periphery
of Glu's postsynaptic depolarizing e€ect and its in the saccule of the gold®sh (Carassius auratus).
facilitatory e€ect on a€erent ®ring rates. Beyond First, they demonstrated that cobalt application
this, these workers were able to adduce evidence for does indeed reduce hair cell transmitter release, as
both pre- and post-synaptic Glu receptors and demonstrated by others, by showing that it reduces
demonstrate a tolerance of the response to Glu. the rate of occurrence of MEPSPs without reducing
This tolerance to repeated applications of Glu was their amplitude. Next, they found, exactly as others
thought to be due to a depolarization block of the had, that the Glu agonists L-Glu, kainate and quis-
a€erent dendrites. These authors also used the qualate produced an increase in a€erent ®ring rates
weakly selective antagonist D-a-amino adipate and and a postsynaptic depolarization. Lastly, they pro-
demonstrated a reduction in EPSPs amplitude and duced evidence that strongly supports the idea that
frequency in a€erent dendrites. A possible demon- resting and evoked hair cell transmitter release can
stration of the presynaptic e€ect of Glu is found in be di€erentially modulated (see Section 5.4.3). That
Valli et al. (1985), where the standing transductional is, the Glu antagonists g-D-glutamyl glycine and 5-
current was blocked by injecting the K-chelator tet- amino-phosphonovalerate both reduced the ampli-
raphenylboron into the endolymph. When the rest- tudes of sound-evoked EPSPs much more than that
ing transductional current was thus inhibited, the of spontaneous MEPSPs. These workers concluded,
resting discharge of the a€erents disappeared, and and presented as their major ®nding, that the Glu
application of Glu produced only a slow postsyn- antagonists operated both pre- and postsynaptically.
aptic depolarization and did not induce a€erent ®r- The postsynaptic action accounts for the reduction
ing. The presynaptic e€ect of Glu may be on the in EPSP amplitude. The authors argued that a pre-
autoreceptor suggested by Devau et al. (1993) and synaptic action, possibly on a Glu autoreceptor,
others and be an example of a positive feedback could a€ect transmitter release especially when there
autoreceptor enhancing transmitter release. is synchronized release of transmitter quanta. Thus,
Soto and Vega (1988) exposed the vestibular the postulated autoreceptor could be activated
organs of the axolotl to Glu and its analogs and during large depolarizations and where there is a
concurred with other reports that it increases a€er- high concentration of hair cell transmitter in the
ent ®ring rates. They also bathed their preparation synaptic cleft as during mechanical or sound stimu-
in high Mg±low Ca to inhibit hair cell transmitter lation (but not during resting release). The acti-
release and found that Glu's e€ect can be postsyn- vation of the autoreceptor would then recruit even
aptic. In addition these authors showed that Glu an- more hair cell transmitter release thus making den-
tagonists (in order of decreasing potency: dritic spike generation more likely.
kynurenate; L-Glu diethyl ester; D,L-2-amino-4- It is not possible to decide for certain on the
phosphono-butyrate; D-a-amino-adipate; and D,L-2- nature of the Glu receptors involved based on the
amino-5-phosphonovalerate) reduced both resting literature cited above. There are, to add further con-
and evoked activity of the a€erents. Later, the same fusion, two publications with apparently con¯icting
group (Perez et al., 1991) found, interestingly, that conclusions. Prigioni et al. (1994) inhibited hair cell
the ototoxic aminoglycoside antibiotic, streptomy- transmitter release with high Mg concentrations and
cin, antagonized the excitatory e€ects of the selec- then examined the depolarization of the a€erents
tive Glu agonists kainate and quisqualate. The produced by Glu. To determine the nature of the
aminoglycoside antibiotics, therefore, in addition to Glu receptor involved, they used CNQX (6-cyano-7-
their many other actions on hair cells are proposed nitroquinoxaline-2,3-dione) a nonNMDA receptor
to be nonselective Glu antagonists. However, the antagonist and manipulations of ions, especially Na.
actions of the aminoglycoside antibiotics may not be Both the CNQX and reductions in Na concen-
related to Glu at all since Norris et al. (1994) have trations dose-dependently blocked the Glu-induced
shown that these agents alone inhibited both resting depolarization. They concluded that the receptors
and evoked activities in the SCC. It is, therefore, involved were therefore nonNMDA. A problem can
not clear what the mechanisms and sites of action of be found with the design and that is that high Mg
these drugs are. concentrations will themselves inhibit NMDA recep-
Further data in support of a dual site of action tors thus biasing the results. The authors mention
for Glu (i.e. on the hair cell itself and on the a€erent this possibility.
dendrite) were adduced by Prigioni et al. (1990). Contrariwise, Zucca et al. (1993a,b) found that
These workers inhibited hair cell transmitter release NMDA antagonists (amino phosphonovalerate,
by adding increasing concentrations of Mg or Co to kynurenate, ketamine and Mg) reduced resting ®ring
the medium bathing the isolated frog SCC. As hair rates of a€erent ®bers of the SCC. NMDA agonists
222 P. S. Guth et al.

were not used. It may be possible to reconcile these onists to Type I hair cells isolated from guinea pig
two ®ndings by suggesting that they were each crista ampullaris and monitored intracellular Ca
monitoring two di€erent sites of action and two concentration changes. In addition to Glu appli-
di€erent receptors. One possibility is that the pre- cation, these authors also used Glu agonists with
synaptic receptor may be of the NMDA and/or certain selectivity for the various Glu receptors such
metabotropic type and the postsynaptic receptor of as NMDA, AMPA (a-amino-3-hydroxy-5-methyl
the AMPA type. Evidence in support of the pre- isoxazole-4-propionic acid) quisqualate and ACPD
sence of the NMDA receptor was found by Soto et (1-amino-1,3-cyclopentanedicarboxylic acid). All
al. (1994). Recording resting and mechanically- agonists produced an increase in Ca concentration
evoked a€erent neural activity in the vestibular suggesting the presence of all three Glu receptors
nerve of the axolotl, these workers found a dose- (i.e. NMDA, AMPA/kainate and the metabotropic
dependent facilitation of the resting discharge by Glu receptor). AMPA and NMDA applications pro-
NMDA. Antagonists acting at three di€erent sites duced somewhat greater responses at the base of the
on the NMDA receptor (i.e. D-2-amino-5-phospho- hair cell than at its apex suggesting a di€erential dis-
novalerate, 7-Cl-kynurenate and Mg) were used. Mg tribution of receptors and the possibility of Glu
antagonized the e€ect of applied NMDA. The other autoreceptors at the hair cell base. Selective antag-
two reduced the resting ®ring rates. Glycine, which onists [amino phosphonovalerate and MK-801 for
has a permissive e€ect on the NMDA receptor, NMDA receptors; 6,7 dinitroquinoxaline-2,3 dione
enhanced the response to NMDA. The authors say (DNQX) for AMPA receptors and D,L-2-amino-4-
that the NMDA receptors participate in the resting phosphonobutyric acid (APB) and D,L-2-amino-3-
discharge but that they are not critical for brief re- phosphonopropionic acid (APP) for the metabotro-
sponses to mechanical stimuli. pic receptors] all produced at least partial inhibition
These results comport with the notion that the of the elevated Ca response to Glu and its agonists.
a€erent discharge in the resting mode and evoked In summary, the pharmacological studies gener-
mode may be di€erentially modulated. Recently, ally provide support for the candidacy of a Glu-like
Cochran and Correia (1995) recorded from a€erents hair cell transmitter. The precise nature of the a€er-
of the turtle SCC maintained in vitro. Glu, aspartate ent transmitter(s) and its receptors await the concur-
and NMDA were all found to excite the a€erents. rence of biochemical, molecular biological,
Kynurenic acid reduced the resting ®ring rates and immunolocalization and pharmacological research.
antagonized Glu and aspartate.
It should be noted that many of the experimenters
cited with regard to Glu's candidacy were propo-
nents of it. Instead of adhering to Claude Bernard's 5.7.2.4. The release of Glu
admonition to remove one's prejudices when don- The laboratory of the Dreschers has most assidu-
ning one's lab coat many of them designed exper- ously addressed the question of transmitter release
iments to support their views in favor of Glu especially aspartate and Glu from vestibular organs.
whereas the more circumspect approach would have For this purpose they have developed and used a
been to perform experiments designed to disprove preparation of trout saccular macula. One advan-
the hypothesis. Some experiments did appear to dis- tage of this preparation is that a tissue sheet consist-
prove the hypothesis. For example, Guth et al. ing almost completely of hair cells (Type II from the
(1988) demonstrated that resting ®ring rates of the ®sh) may be isolated. Initially they (Potter et al.,
ampullar nerve of the frog SCC continued unabated 1986) incubated this preparation with radio carbon-
after the preparation was: labeled glutamine and then stimulated release by ex-
posure to depolarizing concentrations of K+.
1. made tolerant to Glu applications;
Chromatographic fractions corresponding to Glu
2. made unresponsive to Glu by ¯ooding it with ex-
and aspartate showed highly signi®cant increases in
ogenous Glu decarboxylase; and
radioactivity during high-K+ treatment.
3. made unresponsive to Glu by using diltiazem
In their next publication (Drescher and Drescher,
treatment.
1991) this preparation was extracted and the extract
Likewise, Gleich et al. (1990) found that Glu appli- examined for transmitter candidates. They found
cation to the point of desensitization did not a€ect high concentrations of Glu and b-alanine in the hair
the spontaneous rate of cochlear a€erents. cell layer. The saccular nerve had a di€erent spec-
Devau et al. (1993) made the very interesting sug- trum of primary amines from the hair cell layer. The
gestion that, by way of a reciprocal synapse arrange- nerve had high levels of histidine-containing com-
ment, the a€erent calyx may modulate hair cell pounds including peptides. They were especially
sensory transduction. This suggestion was based on: struck by the presence of N-acetylhistidine (see
the presence of Glu-like immunoreactivity; synaptic Section 5.7.10) in the hair cell layer. Lastly, they
vesicle-like structures in the calyx of Type I hair (Drescher and Drescher, 1992) again resorted to K-
cells (Scarfone et al., 1988, 1991) and studies that induced release from this preparation but only
have indicated that Glu is the transmitter of the ves- increased the K concentration to 14 mM (as com-
tibular nerve (Cochran et al., 1987; Dememes et al., pared to most other K+ release research that
1984; Raymond et al., 1984; Drescher and Drescher, employed very high concentrations). They found a
1987a,b). That is, that the calyx once activated by Ca-dependent, notable release of Glu from the hair
the hair cell neurotransmitter, then feeds back Glu cell layer. In fact, of the amines only Glu was
on to the Type I hair cell. Subsequently, this group released in a statistically signi®cant manner by this
(Devau et al., 1993) applied Glu agonists and antag- relatively mild treatment.
The Vestibular Hair Cells 223

Also employing judicious concentrations of K+ in vestibular ganglia almost all cells were immuno-
their release experiments were Zucca et al. (1992b). reactive for the NMDAR1 and the AMPA-type Glu
The investigators employed 25 frog SCC, placed ®ve R2/3 subunits. This localization suggests partici-
per bag in net bags. The canals were then exposed pation of both receptors in a€erent signaling. This
to 5 mM K+ solutions. [These investigators had pre- study also reported labeling of non-neuronal cells in
viously shown that exposure to this concentration of the inner ear.
K causes a dramatic increase in a€erent ®ring by This same laboratory (Fujita et al., 1994) using in
acting to depolarize the hair cells (Valli et al., situ hybridization found strong labeling for
1988)]. Any Glu which was released was detected by NMDAR1 receptor mRNA in rat and guinea pig
means of a bioluminescence-enzymatic method on- vestibular ganglia. These ®ndings con®rm the likely
line. These researchers demonstrated that there was participation of NMDA receptors in a€erent signal-
only a detectable out¯ow of Glu when the SCCs ing.
were intact. If the ampullae were removed, leaving The vestibular periphery of rat and guinea pigs
only the ducts or if high-Mg low-Ca was employed was examined for the presence of AMPA-type
to inhibit hair cell transmitter release there was sup- receptors using antibodies against peptides corre-
pression of Glu release. So, a Ca-dependent release sponding to the C-terminal portions of the receptor
of Glu in response to physiologically relevant con- (Dememes et al., 1995). Speci®cally, the subunits
centrations of K was demonstrated. It needs to be examined were Glu R1, Glu R2/3 and Glu R4. Glu
said that the only substance examined for and R1 immunoreactivity was found on the basolateral
detectable by this design was Glu. aspects of the Type I hair cells and on the calyceal
a€erents. Glu R2/3 was also found in the hair cells
5.7.2.5. Molecular biological evidence of Glu as hair and Glu R4 was undetectable. In the vestibular
cell transmitter ganglia the neurons were intensely stained with the
Glutamate receptors are an heterogeneous and antibodies to Glu R2/3 and Glu R4. The ®broblasts
growing family comprising several ionotropic and and Schwann cells were similarly stained. These
metabotropic receptors (Hollmann and Heinemann, authors suggested that the AMPA receptors (Glu
1994; Pin and Duvoisin, 1995). Many receptor subu- R2/3) may serve as autoreceptors controlling trans-
nits have been cloned, and since the terminology mitter release and/or act `postsynaptically' to the
used for them may be quite cumbersome, Table 4 calyx from which Glu may be released. The Glu R1
correlates the cloned subunit names with the func- detected in the a€erent ®bers supports Glu's candi-
tional groups of Glu receptors found in vivo. dacy as the hair cell transmitter. Early on Dechesne
Using receptor subunit-speci®c antibodies, Usami et al. identi®ed and localized a kainate-binding pro-
et al. (1995) examined the cellular localization of tein in the frog inner ear (Dechesne et al., 1991).
NMDA and AMPA receptor subunits in the rat, Niedzielski and Wenthold (1995) looked for the
guinea pig and monkey inner ear. In the spiral and expression of AMPA, kainate and NMDA receptor
subunits in vestibular ganglia of the rat. They stu-
Table 4. Cloned glutamate receptor subunits and their died 14 Glu receptor subunits: Glu R1±4 (including
physiological properties the ¯ip and ¯op variants); Glu R5±7; KA 1 and 2;
NR1; and NR2A±D. Reverse transcription of RNA
Subunit Receptor type followed by DNA ampli®cation using the polymer-
GluR1 (GluR-A) Ionotropic (AMPA) ase chain reaction was used. Immunocytochemistry
GluR2 (GluR-B) Ionotropic (AMPA) and in situ hybridization with subunit-speci®c oligo-
GluR3 (GluR-C) Ionotropic (AMPA) nucleotides were subsequently used for cellular
GluR4 (GluR-D) Ionotropic (AMPA) localization of receptor expression. The predomi-
GluR5 Ionotropic (low anity KA) nant receptors expressed by vestibular ganglion cells
GluR6 Ionotropic (low anity KA) were Glu R2, Glu R3, Glu R4, Glu R5 and NR1.
KA1 Ionotropic (low anity KA) Expressed at moderate levels were Glu R6, NR2B
KA2 Ionotropic (low anity KA)
KBP KA binding protein and NR2D. At still lower levels were KA1, KA2,
NMDAR1 Ionotropic (NMDA) NR2A and NR2C. The workers failed to detect Glu
NMDAR2A Ionotropic (NMDA) R1 and Glu R7. These ®ndings with regard to Glu
NMDAR2B Ionotropic (NMDA) R1 are at odds with the positive ®ndings of
NMDAR2C Ionotropic (NMDA) Dememes et al. (1995).
NMDAR2D Ionotropic (NMDA) Rabejac et al. (1997) examined mouse vestibular
mGluR1 Metabotropic Q IP3 (Group I) ganglion cells for AMPA-type receptors by immuno-
mGluR2 Metabotropic q cAMP (Group II)
cytochemistry and pharmacologically. They found
mGluR3 Metabotropic q cAMP (Group II)
mGluR4 Metabotropic q cAMP (Group III) clear evidence of Glu R2,3 and Glu R4 in neuron
mGluR5 Metabotropic Q IP3 (Group I) cell bodies and neurites. A weak signal for Glu R1
mGluR6 Metabotropic q cAMP (Group III) was seen in some cells. AMPA and Glu produced an
mGluR7 Metabotropic q cAMP (Group III) increase in intracellular Ca. The AMPA response
mGluR8 Metabotropic q cAMP (Group III) was blocked by DNQX.
Q, Strong increase; q, strong decrease; q, weak decrease.
There seems to be general agreement amongst all
Metabotropic receptors have been shown to exert other workers (Dememes et al., 1995; Niedzielski and
e€ects in addition to what has been reported in this table Wenthold, 1995; Usami et al., 1995) that the Glu
(Pin and Duvoisin, 1995) [adapted from (Hollmann and R2,3 and NMDAR1 subunits are expressed in ves-
Heinemann, 1994) and (Pin and Duvoisin, 1995)]. tibular ganglion neurons supporting the notion that
224 P. S. Guth et al.

the a€erents innervating vestibular hair cells are glu- involved seems to be metabotropic in that it is acti-
tamate-receptive. vated by ACPD (Vazquez et al., 1995) and that
The pharmacological, immunohistochemical and both PLC and protein kinase C (PKC) may be
molecular biological evidence thus tends to support involved. That is, PLC-liberated diacylglycerol may
Glu's candidacy as a hair cell a€erent transmitter. activate PKC which then inhibits a K channel
(Barrie et al., 1991; Vazquez et al., 1995). Glaum et
al. (1990) have reported that glutamate is capable of
5.7.2.6. Hair cell autoreceptors increasing intracellular free Ca concentrations by
Valli et al. (1985) found that glutamate and one a€ecting both Ca mobilization and in¯ux. Both of
of its antagonists, a-amino-adipate, exerted presyn- these processes may be involved in glutamate's
aptic as well as postsynaptic e€ects on the release of actions on the hair cell. The observation that gluta-
neurotransmitter from the hair cells of the SCC. mate and glutamate antagonists may act on a pre-
Prigioni et al. (1990) using excitatory amino acid synaptic receptor is not a new one (Colton and
agonists not only con®rmed the earlier ®nding (Valli Freeman, 1975; Cotman et al., 1986; Thie€ry and
et al., 1985) of both pre- and postsynaptic e€ects of Bruner, 1978; Usherwood and Machili, 1966).
excitatory amino acids but conjectured that these Nevertheless, such a positive feedback is unusual for
amino acids may function presynaptically in a posi- transmitters, the majority of them exerting a nega-
tive feedback manner. Starr and Sewell (1990) tive feedback control over their own release (Vizi,
suggested that glutamate antagonists act presynapti- 1979).
cally on the saccular hair cells to reduce the amount
of neurotransmitter released by sound stimulation.
These data, indicating the existence of a glutamate 5.7.2.7. The Sewell hair cell transmitter search
receptor on the hair cell, may be interpreted to Otto Loewi was able to detect an activity in ¯uid
suggest that there is normally a glutamate-mediated perfusing the frog heart, appearing after vagal
recruitment of the a€erent neurotransmitter during stimulation, which when that ¯uid was itself applied,
the evoked release but not during the resting release slowed the heart. This activity was present in the
mode. Guth et al. (1988) have produced evidence perfusion ¯uid during vagal stimulation but not
that suggests that glutamate may not to be the neu- before or after. Thus, Loewi (Loewi, 1921a,b) was
rotransmitter released at rest. These authors showed able to determine the presence of a humoral factor
that of two manipulations of the SCC leading to a without preconceptions as to its nature, structure,
loss in response to applied glutamate neither caused etc. The identical strategy was used by Sewell et al.
a change in the resting multiple unit a€erent ®ring (1978) in an attempt to determine the nature of the
rate. These two manipulations were: applications of hair cell transmitter at a time when there were few
glutamate in quick succession to cause glutamate clues to its identity. They detected a substance or
desensitization and bath application of the enzyme substances capable of increasing the ®ring rates of
glutamate decarboxylase to catabolize extracellular primary auditory ®bers in the perilymph of frogs or
glutamate. Likewise, Gleich et al. (1990) found that guinea pigs subjected to sound stimulation. An
glutamate application to the point of desensitization increase in ®ring rate occurred in single units of the
did not a€ect the spontaneous rate in cochlear a€er- frog auditory nerve after perilymph obtained from
ents. In contrast, Rebillard and Bryant (1989) found frogs or guinea pigs during sound stimulation was
that the sound-evoked cochlear action potential is infused into the frog perilymphatic sac. Perilymph
sensitive to perfusion of the Glu-degrading enzyme, obtained from animals maintained in quiet failed to
Glu dehydrogenase. Hence the notion of the di€er- cause an increase in ®ring rate. It is worth noting
ential modulation of hair cell transmitter release in that samples with or without the activity (called
the spontaneous and evoked modes (see Section auditory nerve activating substance, ANAS) had
5.4.3) is supported by these apparently contradictory similar contents of Glu, suggesting that the activity
studies. Further, it seems as though Glu may act on was not due to the presence of Glu.
its autoreceptor in a positive feedback manner in In a further attempt to isolate and purify the hair
the evoked mode (but not the resting mode) in cell transmitter and ®nally determine its structure,
cochlea as well as the vestibular organs. Sewell and Mroz (1990) turned to the ®sh as a
Thus, when the sensory epithelium is mechanically source since they have larger numbers of hair cells
stimulated, glutamate release by the hair cells may than frogs. They also began using a simpler assay
act on a hair cell autoreceptor that enhances neuro- for the transmitter candidates, namely the Xenopus
transmitter release and thereby greatly increases laevis lateral line, and found, after fractionation of
a€erent ®ring. Such a glutamate-mediated positive the extracts, at least two unidenti®ed excitatory sub-
feedback is unnecessary and even undesirable in the stances, a high molecular weight (HMW) and a low
resting condition. A derivative question is: what molecular weight (LMW) substance (Sewell and
would limit the e€ect of this putative receptor? Mroz, 1987). The HMW is present in both brain
Might receptor-desensitization perform the limiting and inner ear extracts but the LMW is found in
function? inner ear and present but not concentrated in brain
Such a presynaptic Glu receptor, which when acti- or muscle. Using a ®ve-step chromatographic pro-
vated augments transmitter release, has been cedure, Sewell and Mroz (1990) were able to report
reported in other structures and seems to be a com- that the LMW excitatory substance may be a zwit-
mon feature of glutamatergic synapses (Herrero et terionic compound with titratable anionic and cat-
al., 1992; Hu and Storm, 1991; Jones and Roberts, ionic groups. The structure of this compound is
1990; Vazquez et al., 1995). The Glu receptor currently being determined by mass spectrometry.
The Vestibular Hair Cells 225

(Note: a recent ®nding is that the neuroactive sub- e€erent ®bers might be responsible for noncholiner-
stance is in brain, but its activity is obscured by the gic (de®ned pharmacologically) a€erent excitatory
presence of a suppressive substance. The two can be e€ects produced by e€erent stimulation in lateral
separated by cation exchange chromatography.) (W. line (Flock and Russell, 1973) and in vestibular
F. Sewell, personal communication). organs of gold®sh (Hartmann and Klinke, 1980)
and frog (Rossi et al., 1980). Examining both e€er-
ent stimulation and CGRP application more closely,
5.7.3. Calcitonin Gene-related Peptide
Sewell and Starr (1991) reported some new and clar-
Calcitonin gene-related peptide is a 37-amino acid ifying ®ndings. Stimulation of e€erent ®bers to the
peptide produced by alternative splicing of the calci- X. laevis lateral line produced three distinguishable
tonin gene transcript. It has been reported to have e€ects on a€erent ®ring rates:
e€ects on heart (Saito et al., 1986) and on smooth
muscle (Brain et al., 1985). CGRP and its binding 1. a rapidly-occurring suppression followed by;
sites are distributed in many areas in the CNS 2. a rapidly-occurring facilitation and in about half
including those involved with sensory function the preparations there was seen;
(Sko®tsch and Jacobowitz, 1985; Tschupp et al., 3. a slowly-developing facilitation that persisted.
1985). For instance, CGRP-like immunoreactivity The rapidly-occurring suppressions and facilitations
has been demonstrated in dorsal root ganglia neur- could be blocked by curare, atropine and strychnine,
ons and spinal dorsal horn (Gibson et al., 1984). suggesting cholinergic mediation. The slowly-devel-
Release of CGRP from rat primary sensory neurons oping excitation was not blocked by these agents
has been demonstrated (Saria et al., 1986). It was suggesting noncholinergic mediation. These authors
suggested that CGRP may have as its role the o€ered the possibility that the slowly-developing fa-
enhancement of release of excitatory amino acids cilitation was produced by CGRP released from
from primary sensory endings (Kongrga and e€erent nerves, and that it was CGRP release that
Randic, 1990) (a role that it may also have in acous- explained noncholinergic facilitation in ®sh and frog
ticolateralis organs). vestibular organs. It is also noteworthy that the
CGRP co-exists with ACh in the motor nerve e€ects of CGRP showed a tolerance or desensitiza-
endings of the rodent neuromuscular junction tion, ®ring rates returning to baseline in the contin-
(Fontaine et al., 1986; New and Mudge, 1986) ued presence of CGRP.
where it is suggested to have: a regulatory role in In the lateral line paper of Adams et al. (1987)
the development of cholinergic synapses (Betz and there is mention of unpublished ®ndings that there
Changeux, 1979; Blosser and Appel, 1980; Stallcup are CGRP-containing e€erent ®bers in the vestibular
and Patrick, 1980); a role in increasing ACh recep-
end organs of gerbil and rat. Tanaka et al. (1989a,b)
tor biosynthesis (Fontaine et al., 1986; Laufer and
and Ohno et al. (1993a,b) found a dense plexus of
Changeux, 1987; New and Mudge, 1986) and a
CGRP-immunoreactive ®bers beneath the sensory
regulatory role in the phosphorylation and desensiti-
epithelium in SCC, utricle and saccule of the rat.
zation of the ACh nicotinic receptor (Mulle et al.,
Most of the CGRP-immunoreactive ®bers formed
1988).
direct contacts primarily with the a€erent calyces of
Among the acousticolateralis organs, most of the
neuroanatomical studies on CGRP have examined Type I hair cells.
its presence in the cochlea. As reviewed in Eybalin In contrast, Wackym et al. (1991) using both in
(1993), CGRP-like immunostaining was found in situ hybridization and CGRP-immunoreactivity
inner spiral and tunnel bundles and at the basal pole found contacts between CGRP neurons and: a€er-
of the OHCs. CGRP-like immunostaining was also ent neurons; Type I and; Type II hair cells. In a
observed in two groups of e€erent ®bers: those mak- later paper (Ishiyama et al., 1994b) this group con-
ing synapses with the radial a€erents; and those ®rmed the previous ®ndings and found vesiculated
synapsing with OHCs. CGRP has been reported to endings which were not CGRP-immunoreactive.
co-exist with ChAT, the enzyme that synthesizes Usami et al. (1991a,b) found CGRP- and SP immu-
ACh, in at least part of the mammalian cochlear noreactivity in nervous elements beneath hair cells
e€erent olivocochlear system (Wackym et al., 1991). of rat vestibular organs. All three possible combi-
In anamniotes, the octavolateralis e€erent system nations of SP-positive/CGRP-positive, SP-positive/
consists of a single nucleus, the octavolateralis e€er- CGRP-negative and SP-negative/CGRP-positive
ent nucleus (Meredith and Roberts, 1986). The out- were seen.
put neurons of this nucleus are known to be Finally, ACh, ATP, GABA, CGRP and Met- and
cholinergic (Brantley and Bass, 1988; Danielson et Leu enkephalins were applied to isolated vestibular
al., 1983). Roberts and Ryan (1971) examined these hair cells and compared for their abilities to increase
e€erent neurons in the eel, Anguilla anguilla, for intracellular free Ca. Of these, ACh, ATP and
both ChAT and CGRP immunoreactivity and con- GABA were e€ective while neither CGRP nor the
cluded that ACh and CGRP probably co-exist in enkephalins produced a signi®cant increase in intra-
many of these e€erent neurons innervating lateral cellular Ca (Yamashita et al., 1993).
line and inner ear organs. In summary, it seems likely that there are e€erent
CGRP was applied to the Xenopus lateral line by ®bers in vestibular end organs that contain both
Adams et al. (1987) and by Sewell and Starr (1991). ACh and CGRP. These ®bers make contact with
Both papers reported an increase in a€erent ®ring Types I and II hair cells and with a€erent endings.
produced by nano- to micromolar concentrations of CGRP may exert neurotransmitter function in caus-
CGRP. They suggested that CGRP released from ing a late a€erent facilitatory response (Sewell and
226 P. S. Guth et al.

Starr, 1991). It may also exert neuromodulatory GABA transaminase (Lopez et al., 1992); the bind-
e€ects on ACh receptors. ing of GABA to chick vestibular membranes and its
antagonism by bicuculline (Meza et al., 1981a,
5.7.4. Enkephalins and other Opioids 1985). Glutamic acid decarboxylase (GAD) the key
enzyme of GABA synthesis is present in vestibular
As with other T/Ms there is considerably more in-
homogenates from frog, chick, rat and guinea pig
formation about them in cochlea than in vestibular
(Lopez and Meza, 1988; Meza et al., 1981b, 1984),
organs. The cochlear literature on this subject is
although the frog had only 1/3 to 1/5 the activity of
very well reviewed by Eybalin (1993).
the other species. This group also localized GAD ac-
The enkephalins, methionine- and leucine-enkeph-
tivity to vestibular hair cells in two ways: by demon-
alin, are pentapeptides derived from the proenke-
strating that GAD activity remains after eighth
phalin precursor. To brie¯y summarize, in cochlea
nerve ablation (Lopez and Meza, 1988); and by
these peptides have been found in e€erent endings
demonstrating that hair cell destruction by strepto-
by immunohistochemical methods. Also, messenger
mycin results in GAD depletion (Meza et al., 1989).
RNA of the preproenkephalin precursor has been
GABA-like immunoreactivity was found in the cyto-
found in neurons of the lateral superior olive in
plasm of both Type I and Type II hair cells and
agreement with immunocytochemical data. There is
calyceal endings in the guinea pig crista ampullaris
also evidence of a population of medial cochlear
(Lopez et al., 1990). Didier et al. (1990c) found
e€erents expressing mRNA of preproenkephalin.
GABA-like immunoreactivity in the calyceal endings
(Eybalin, 1993) also discusses the evidence concern-
surrounding Type I hair cells in the guinea pig. The
ing relevant opioid receptors (m, d and k) and pro-
staining was most intense in the SCC and less so in
posed associated second messengers in the cochlea
the macular organs. No mention is made of any
(cAMP and IP3).
staining of Type II hair cells in this report.
The dynorphins and their precursor, pro-dynor-
More recently, Matsubara et al. (1995) examined
phin, were also found in cochlea localized to the
GABA-like immunoreactivity in the vestibular per-
e€erent ®bers. Both dynorphin A- and B-like and a-
iphery of several di€erent species. In chick and
neo-endorphin-like immunoreactivities were found
pigeon GABA-like immunoreactivity was found
in the cochlear ®bers usually associated with the lat-
within hair cells and in e€erents. In squirrel monkey
eral e€erent system. Eybalin (1993) also reviews evi-
GABA reaction products were seen only in e€erent
dence of a reduction in cochlear compound action
endings. Strikingly, in contrast to other studies, no
potential amplitude mediated through k opioid
GABA-like immunoreactivity was seen in guinea pig
receptors.
vestibular organs. Also, no di€erentiation was made
The presence of leu-enkephalin in vestibular e€er-
between hair cells of Types I or II. Examining all
ent neurons was noted by Carpenter et al. (1987). In
three immunoreactivities (GAD-like, GABA-like
vestibular organs, Soto et al. (1988) applied the
and GABA-transaminase-like) in one organ, the gui-
opioid antagonist naloxone (10ÿ6±10ÿ4 M) and
nea pig utricle, Meza et al. (1992) found: GAD-like
found that the resting discharge of vestibular a€er-
immunoreactivities exclusively in the cytoplasm of
ents in the axolotl was increased. In a later report,
Types I and II hair cells; GABA-like immunoreac-
the same group (Vega et al., 1991) con®rmed the
tivity in Types I and II hair cell cytoplasm as well as
earlier ®nding with naloxone, this time speci®cally
some nerve ®bers; and GABA-transaminase-like
in SCC a€erents of the axolotl. They extended the
immunoreactivity in Type I hair cell nerve chalices
®ndings, using three synthetic enkephalin agonistic
and ®bers and an occasional hair cell cytoplasm.
analogs [D-Ala-leu-enkephalin (DALE), D-Ala-Met-
The release of GABA from trout saccular macula
enkephalin (DAME) and D-Ala-Gly-phe-ol-enkeph-
but not saccular nerve preparations was reported by
alin (DAGO)]. DAGO produced a long-lasting
Drescher and Drescher (1987b). This release was
increase in a€erent ®ring, DAME increased at low
evoked by exposure to 53.5 mM K+ and was depen-
and suppressed at high concentrations while DALE
dent on the presence of Ca. The fact that GABA
produced the opposite biphasic e€ects. Thus, in
was not released from saccular nerve suggested that
both cochlea and vestibular organs the opioid pep-
it must have come from the hair cells and support-
tides exert important, generally suppressive, e€ects
ing cells of the saccular macula and not the nerve
on a€erent activity.
endings.
The following paragraphs are an attempt at a syn-
5.7.5. g-Amino Butyric Acid thesis of a sometimes confusing subject. The clearest
The idea that GABA might be involved in hair ®ndings seem to be in respect to GAD. GAD is
cell-a€erent neurotransmission seems to be sup- found most often in the cytoplasm of vestibular hair
ported more by the biochemical/anatomical studies cells especially Type I. It follows then that GABA,
and less by the functional/pharmacological studies. the product of GAD activity, should likewise be
For example, the laboratory of Graciela Meza has found in hair cells (of both types but especially Type
been most thorough in its study of GABA in the I). GABA is also seen in nerve ®bers and even ves-
vestibular periphery and most tenacious in its advo- tibular ganglion cells (Whitlon and Sobkowicz,
cacy of a role for GABA. This group has demon- 1989). The localization of GAD and GABA more in
strated: the synthesis of GABA in chick vestibular Type I hair cells than in Type II is, in a sense, sup-
cristae (Meza et al., 1981b); a possible terminating ported by the observation that the frog, having only
mechanism for GABA in the form of an energy- Type II hair cells had much less GAD activity in
and Na+-requiring uptake process (Meza et al., vestibular homogenate than did chick, rat or guinea
1981a); the presence of the degradative enzyme, pig (Lopez and Meza, 1990). This shading or tilting
The Vestibular Hair Cells 227

of the data will become important in explaining the was not blocked by picrotoxin, a GABA-A antagon-
e€ect of applied GABA in some species and its lack ist, suggesting mediation by a GABA-B receptor.
of e€ect in others in the next section. No results in Type II hair cells were reported.
GABA-like immunoreactivity is occasionally One possible explanation of these apparently dis-
found in a€erent endings and one may surmise that parate results might be that only Type I vestibular
a transport mechanism for GABA may exist in hair cell a€erents have GABA receptors. This would
these endings. If so then the presence of GABA- explain the lack of e€ect of GABA in frog (Annoni
transaminase in these endings (and not in the hair et al., 1984; Guth and Norris, 1984) and axolotl
cells) is put in context as the ®nal degradative step (Vega et al., 1987). This might also account for the
for GABA. very low GAD activity in the frog (Lopez and
It is clear that GABA is not the universal hair Meza, 1990). It might also explain the positive
cell-a€erent excitatory transmitter. The only phar- results of Felix and Ehrenberger (1982) and Lapeyre
macological research supporting GABA in this pos- et al. (1993) in mammals. Very recent immunohisto-
tulated role is that of Felix and Ehrenberger (1982) chemical evidence using a monoclonal antibody
who reported that GABA application excited a€er- speci®c for the b2±b3 subunits of the GABA-A
ent ®bers in the saccule of the cat. They also receptor protein would seem to support this idea.
reported that the GABA antagonists bicuculline and Foster et al. (1995) found no immunostaining in
picrotoxin not only blocked the e€ect of applied hair cells or supporting cells of the hamster, rat and
GABA but also decreased spontaneous activity in mouse crista ampullaris. However, they did ®nd
the saccular nerve. This same group (Ehrenberger et immunostaining in calyces surrounding Type I hair
al., 1982) tested picrotoxin in human trials and cells and in cell bodies in the vestibular ganglion but
reported a suppression of spontaneous nystagmus, not in Type II hair cells or, apparently, their a€erent
caloric excitability and labyrinthine vertigo. GABA endings. It is now that a warning about the treach-
does not a€ect sound-evoked cochlear potentials in ery of pharmacology seems in order. Drugs once
mammals except at high concentrations that are thought to be selective for the GABA-A receptor
likely to be nonspeci®c (Bobbin and Guth, 1970; such as picrotoxin and bicuculline can also antagon-
Bobbin and Thompson, 1978; Klinke and Oertel, ize ACh at its nicotinic-like receptor (see Section
1977). 5.7.1.5). And, of course, the selectivity of any drug
In contrast, the lateral line a€erents do respond is dependent upon the concentration used.
to the application of GABA (Bledsoe et al., 1983;
Bobbin et al., 1985). The Bobbin group argued
against GABA's candidacy as an a€erent transmit- 5.7.6. Substance P
ter because the GABA antagonist bicuculline, while All the studies reporting the presence of SP, a
blocking GABA suppressive e€ect, failed to in¯u- member of the tachykinin group of T/Ms in the ves-
ence mechanical- or glutamate-evoked a€erent ac- tibular periphery have employed immunohistochem-
tivity. They suggest that GABA may be involved in ical techniques. As reported by Ylikoski et al.
e€erent modulation of lateral line a€erent activity. (1984a) between 30 and 40% of the cells of the ves-
Mroz and Sewell (1989) report an initial brief excit- tibular ganglia showed SP-like immunoreactivity.
atory phase of action followed by a suppressive The neural elements of the vestibular sensory epithe-
phase. They were able to detect the brief excitatory lia, especially the calyces of Type I sensory cells of
phase because they averaged the discharge each sec- the macular organs, showed strong immunoreactiv-
ond as compared to Bledsoe et al. (1983) who aver- ity. In a second paper on the subject (Ylikoski et al.,
aged every 30 sec. 1984b) this group observed SP-like immunoreactiv-
In the SCC of the frog, GABA application has no ity within and below the sensory epithelia of both
e€ect (Annoni et al., 1984; Guth and Norris, 1984). utricular and saccular maculae and ampullary cris-
Furthermore, neither bicuculline (Annoni et al., tae of the rabbit. Once again particularly strong
1984) nor picrotoxin (Annoni et al., 1984; Guth and staining was seen in the macular organs and in the
Norris, 1984) had any e€ect on resting or evoked calyceal endings.
discharge. Similarly, Vega et al. (1987) recording Usami et al. (1991a) found SP-like immunoreac-
from a€erent ®bers of the lagena and anterior SCC tivity in the vestibular ganglia and end organs of the
in the axolotl reported that GABA and its agonist, guinea pig. SP-like immunoreactivity was found in
muscimol, produced a very weak excitatory e€ect thick and thin nerve ®bers as well as in the ganglia.
and then only in some ®bers and at relatively high The thick ®bers were found around or beneath sen-
concentrations (10ÿ4±10ÿ2 M). Bicuculline at high sory hair cells. These SP-positive ®bers were located
concentrations (10ÿ4±10ÿ3 M) had no e€ect on spon- on the slopes of the central region in the crista and
taneous activity while picrotoxin partially blocked the periphery of the macular organs. Electron mi-
spontaneous activity in 33% of the a€erent ®bers croscopy con®rmed that the immunostaining was
(10ÿ4±10ÿ3 M). Finally, Lapeyre et al. (1993) pub- con®ned to primary a€erent neurons. The thin SP-
lished their ®ndings that GABA (100 mM) in¯uenced positive ®bers were found in the stroma of vestibular
K currents in Type I hair cells isolated from guinea end organs, nerve trunk, vestibular ganglia and
pig SCC. GABA application elicited an increase in along blood vessels of vestibular ganglia. The thin
both the outward current produced by depolariz- and thick SP-positive ®bers in man have quite di€er-
ation and the inward current produced by hyperpol- ent functions. Next, this group (Usami et al., 1991b)
arization in some cells. The outward-going currents examined the coexistence of SP-like and CGRP-like
were probably Ca-dependent K currents. This e€ect immunoreactivities. All three possible combinations
was mimicked by baclofen, a GABA-B agonist, and of reactivities were found. Fibers having both im-
228 P. S. Guth et al.

munoreactivities were located within as well as (Ashmore and Ohmori, 1990; Dulon et al., 1991;
beneath the sensory epithelia. Housley et al., 1992; Ikeda et al., 1991; Nakagawa
Jin (1992) reported the presence of SP-like immu- et al., 1990; Rennie and Ashmore, 1993; Shigemoto
noreactivity in vestibular ganglion and ``peripheral and Ohmori, 1990; Yamashita et al., 1993). The
region of vestibular end organs''. consensus is, ®rstly, that there exist on inner ear
So, all studies agree that SP-like immunoreactivity hair cells receptors which respond to ATP. There
is located in vestibular ganglia and most suggest its are the intriguing reports that these ATP receptors
presence in a€erent ®bers. A problem arises with may be concentrated on the endolymphatic or apical
regard to the study of SP±CGRP coexistence in that ends of cochlear hair cells (Ashmore and Ohmori,
CGRP-like immunoreactivity is quite clearly associ- 1990; Housley et al., 1992). However, Aubert et al.
ated with e€erent ®bers (see above) while SP-posi- (1994) argue that ATP applied to the perilymphatic
tive ®bers are most likely a€erent. This could be compartment in the isolated SCC is unlikely to
explained by the presence of a local circuit (Ross, reach the well-isolated apical or endolymphatic ends
1997) in which a€erent terminals also bear e€erent of hair cells in an unmetabolized state in the short
recurrent synapses; however, more data are necess- time between application and response. There may
ary to clarify this point. well be ATP receptors on both apical and lateral
aspects of the hair cells.
It is interesting to note that in some hair cell types
5.7.7. ATP [e.g. in frog sacculus (Roberts et al., 1990)] transduc-
The ®rst evidence that ATP might be involved in tion channels are the only reported apical membrane
sensory transmission was provided by the Holtons ionic pathways, whereas in other cells [e.g. guinea-
(Holton and Holton, 1954; Holton, 1959). They pig cochlear OHCs (Housley et al., 1992)] an apical
demonstrated that ATP was released from sensory ATP-gated conductance with high Ca-permeability,
dendrites innervating blood vessels and was respon- most likely representing a P2x purinergic receptor
sible for antidromic vasodilation. In the later paper, (Nakagawa et al., 1990) has been found. In guinea
the Holtons cited the work of Blaschko et al. (1956) pig vestibular hair cells, ATP-dependent inward cur-
who found ATP associated with catecholamines in rents and Ca-in¯ux have been observed, but not
chroman particles of the adrenal medulla. The localized in a membrane compartment (Rennie and
Holtons said that it was likely that ATP might also Ashmore, 1993).
be liberated into the tissue spaces from sympathetic When activated, these ATP receptors on hair cells
nerves. Indeed, ATP has been found co-localized, cause an increase in intracellular Ca concentration
even co-packaged with several transmitters including and in this regard there might be an interaction with
ACh (Dowdall et al., 1974) catecholamines and 5- ACh (Ashmore and Ohmori, 1990; Ikeda et al.,
hydroxytryptamine (Zimmermann, 1994). 1991; Shigemoto and Ohmori, 1990). Dolezal et al.
At this writing at least ®ve subtypes of P2 purino- (1997) studying the increases in intracellular Ca
ceptors have been identi®ed (Abbrachio and caused by carbachol and ATP concluded that both
Burnstock, 1994; Fredholm et al., 1994; Burnstock agents released from the same intracellular store in
and Kennedy, 1985; Gordon, 1986; O'Connor et al., Chinese hamster ovary cell lines. The response to
1991): P2x; P2y; P2u; P2t; and P2z. There are as yet no ATP was strongly suppressed by previous carbachol
very selective antagonists and the initial di€eren- application and vice versa. None of the papers cited
tiation of these receptor subtypes depended on rela- suggested that the receptor upon which ATP acted
tive potencies of agonists acting upon them. In was anything but P2y. The identity of the ATP
recent times, the P2y and P2u purinoceptors have receptor as P2y was most clearly demonstrated phar-
been cloned and found to belong to the G-protein- macologically by Aubert et al. (1994). This receptor
coupled receptor superfamily (Lustig et al., 1993; belongs to the receptor superfamily that is G-protein
Webb et al., 1993). The gene for the P2x receptor coupled. Probably causally related to the ATP-
subtype has been identi®ed and this receptor has an induced increase in intracellular Ca is the demon-
intrinsic ion channel (Brake et al., 1994; Surprenant stration of an increase in inositolphosphates in the
et al., 1995; Valera et al., 1994). isolated organ of Corti after ATP application. So,
Bobbin and Thompson (1978) were the ®rst to the sequence might be: ATP, acting on a G-protein
suggest that ATP might a€ect neurotransmission in linked P2y receptor increases IP3 concentration in
the inner ear. More recently, this same group the hair cell (Niedzielski and Schacht, 1992); IP3 act-
(Kujawa et al., 1994a,b) demonstrated that the e€ect ing on its own receptor releases intracellular stores
of ATP on cochlear function might be mediated by of Ca which may then be responsible for increases
the P2y purinoceptor. In other systems, ATP has in hair cell transmitter release and thus an increase
been shown to increase ®ring rates of a€erent ®bers in a€erent ®ring.
in lateral line (Mroz and Sewell, 1989) and frog A remaining question is: from whence comes the
SCC (Aubert et al., 1994). Still in the frog SCC, ATP: e€erents or hair cells; supporting cells; a€erent
Aubert et al. (1995) adduced evidence that ATP was dendrites; some or all of these? The e€erents might
present and had a physiological role in vestibular be a source because of the by-now-well-known co-
neurotransmission. Aubert et al. (1994) produced packaging and co-release of ACh and ATP
convincing pharmacological evidence that ATP (Dowdall et al., 1974; Silinsky and Hubbard, 1973).
facilitatory e€ect on a€erent vestibular ®ring was However, the isolated SCC is unlikely to be under
also mediated by the P2y receptor. the in¯uence of tonic activity of the e€erents. The
Numerous studies have reported an e€ect of ATP e€erents are decentralized when the organ is iso-
on isolated cochlear and vestibular hair cells lated. Furthermore, if there were tonic e€erent ac-
The Vestibular Hair Cells 229

tivity in the isolated SCC then ACh antagonists that this e€ect is counteracted by addition of the
such as atropine, curare or strychnine (Guth et al., adenosine deaminase inhibitor deoxy-coformycin;
1986; Norris et al., 1988) would have e€ects on that addition of the adenosine receptor antagonist,
a€erent ®ring in the isolated SCC and they do not. theophylline, (in sub-phosphodiesterase-inhibiting
But, ATP antagonists and nucleotide pyrophospha- concentrations) increases a€erent ®ring and; that
tase do. Therefore, the ATP is unlikely to be coming inhibiting the re-uptake of adenosine prolongs its
from the e€erents. It would be interesting to deter- e€ect. Furthermore, these authors showed clearly
mine the e€ects of ATP antagonists and the cata- that the source of the adenosine in the SCC is not
bolic enzyme nucleotide pyrophosphatase on SCC the e€erent ®bers. Thus, if adenosine is present as a
physiology as Aubert et al. (1995) did but in a de- result of ATP catabolism, the ATP could not have
e€erented SCC. In summary, the most likely source come from the e€erents.
of ATP is the hair cell itself and that the liberated Pertinent to this discussion was the ®nding of A.
ATP acts on hair cell autoreceptors. However, at Aubert (personal communication) of indirect evi-
the neuromuscular junction the appearance of ATP dence that ATP is converted to physiologically-
in the extracellular ¯uid is antagonized by curare, active concentrations of adenosine in the frog SCC.
suggesting that its source is postsynaptic, from the Application of adenosine antagonists along with
muscle itself (Meunier et al., 1975). It is even poss- ATP produced a potentiation of ATP e€ect on a€er-
ible that the source of ATP in vestibular organs are ent ®ring such that the facilitatory response to ATP
the nonsensory cells. was enhanced when its a€erent-suppressive catabolic
There is also an intriguing interaction between product adenosine was antagonized. This suggests
ATP and ACh described in both cultured PC-12 that in vivo there may be an interesting purinergi-
(pheochromocytoma) cells (Surprenant et al., 1995) cally-mediated homeostatic arrangement whereby
and OHCs (Ashmore and Ohmori, 1990). In the lat- released ATP causes an e€erent facilitation which its
ter work, ACh and ATP were administered simul- product, adenosine, then suppresses or even turns
taneously and produced a less-than-additive o€. Zimmermann (1994) discusses similar inter-
response suggesting some interaction. In OHCs, actions of ATP and adenosine.
both produce an increase in intracellular Ca. It has
been suggested that both ligands may a€ect the
same ion channel (i.e. Ca channel) and/or the same
second messenger, but through two di€erent recep- 5.7.9. Histamine
tors. Both ligands are known to increase a€erent ®r- Interest in the possibility that histamine might be
ing rates in vestibular organs, ACh through a a T/M in the vestibular periphery was stimulated by
muscarinic receptor (M3,) which increases IP3, and the ®nding of Turner and Jackson (1983) and
ATP through a P2y receptor which also increases Jackson and Turner (1987) that an H-1 antagonist,
IP3. Thus, the less-than-additive interaction may be astemizole, thought not to enter the CNS (Richards
by convergence at the second messenger level after et al., 1984), suppressed vertigo, especially that of
activation of two di€erent receptors. peripheral origin. Earlier studies on the vestibular
There are also provocative reports of e€ects of site of action of the H-1 antihistamines had focused
ATP on the ACh nicotinic receptor to be con- on the vestibular nuclei (Schwartz et al., 1991;
sidered. ATP not only increases the opening fre- Zimmermann, 1994; Wang and Dutia, 1995) but
quency of the receptor channels but may directly astemizole represented a new, noncentrally-acting
activate this receptor as well [reviewed by class of H-1 antihistamines.
Zimmermann (1994)]. A recent review covering ATP Norris and Jackson (1987) went on to demon-
and ACh roles in the acousticovestibular system strate that astemizole applied to the isolated SCC of
may be found in Housley and Ryan (1997). the frog reduced ®ring rates in a€erent nerves. Then
Housley et al. (1988) produced evidence that hista-
mine is remarkably potent (threshold concentration
5.7.8. Adenosine ca 100 nM) in eliciting a small but robustly reprodu-
Adenosine has been widely implicated in neuro- cible increase in ®ring rates of a€erent neurons of
modulation in the CNS (Snyder, 1985). Its mechan- the isolated SCC. This facilitatory e€ect was antago-
ism of action is thought to be a receptor-mediated nized by H-1 antagonists and by atropine. The facil-
inhibition of transmitter release. Whether adenosine itatory e€ect was mimicked by histidine-containing
is released from cells per se or occurs as a degrada- compounds such as L-histidine, carnosine and thyro-
tive product of released ATP is as yet unclear. There tropin-releasing hormone. The H-1 antagonists, pyr-
is evidence for both [adenosine formed from ATP: ilamine and diphenhydramine, produced
(Fredholm and Hedqvist, 1980; McAfee and Henon, suppression of ®ring of the ampullar a€erents in
1986)] [adenosine released per se: (McAfee and concentrations well below those necessary to cause
Henon, 1986; Pull and McIlwain, 1972)]. local anesthesia (i.e. blockage of Na channels). An
In the only paper examining the possible neuro- inhibitor of histamine synthesis, a-¯uoro-methylhis-
modulatory role of adenosine in vestibular organs, tidine (Schwartz et al., 1991) reduced a€erent ®ring
Bryant et al. (1987) adduced evidence completely in the SCC in a dose-dependent manner (Housley et
supportive of such a role. They showed that adeno- al., 1988) suggesting that continual histamine syn-
sine is released during excitation; that it exerts an in- thesis was required for maintenance of a€erent dis-
hibitory e€ect on a€erent ®ring; that addition of the charge. This synthesis inhibitor is generally regarded
catabolizing enzyme, adenosine deaminase, to the as irreversible but in the currently-discussed research
isolated SCC of the frog increases a€erent ®ring and its e€ect was, inexplicably, reversible.
230 P. S. Guth et al.

The H-1 antagonists are known to exert anticholi- 5.7.10. N-Acetylhistidine


nergic activity (Garrison, 1990). The H-1 antagon- N-Acetylhistidine is present in both cold-blooded
ists blocked the a€erent facilitatory e€ect of vertebrates (Erspamer et al., 1965) and mammals
carbachol, a cholinomimetic, and atropine blocked (O'Dowd et al., 1988). It has been shown to be
the same e€ect of histamine. The most parsimonious released from anuran photoreceptors along with glu-
explanation of these results may be that histamine, tamate, aspartate, putrescine and cadaverine (Miller
ACh, and their agonists and their antagonists are all and Schwartz, 1983).
acting at the muscarinic-like ACh receptor (see Drescher and Drescher (1991) were able to extract
Section 5.7.1). In fact, there is a fair correlation N-acetylhistidine from the hair cell layer of the trout
between the antimuscarinic activity of H-1 antihista- saccule. The nerve fraction contained the histidyl
mines and their ecacy in motion sickness. dipeptides carnosine and homocarnosine. As men-
Unfortunately, astemizole is reported to be ine€ec- tioned in Drescher and Drescher (1991) P. S. Guth
tive at muscarinic receptors (Garrison, 1990). It may (unpublished data) had observed that N-acetylhisti-
therefore be necessary to implicate both antimus- dine (10 mM) caused a facilitation of a€erent ®ring
in the ampullar nerve of the frog SCC.
carinic and antihistaminic activities to explain the
It seems worthwhile to point out that an anti-
vestibular suppressant activities of the H-1 antagon-
serum which reacts with the histidyl dipeptides car-
ists. nosine, homocarnosine and anserine produced
In addition, if histamine is truly capable of facili- strong immunolabelling of hair cells of the frog
tating a€erent activity then there may be at hand an SCC (Panzanelli et al., 1994). Carnosine is also loca-
explanation for the vertigo that accompanies middle lized to other sensory structures such as primary
ear infections. That is, the histamine released by olfactory receptors (Bi€o et al., 1990) and retinal
basophils or mast cells recruited in the in¯ammatory neurons (Sassoe-Pognetto et al., 1995).
process could activate vestibular receptors and cause
a mismatch in neural activity between the two ears.
Kellog et al. (1965) and Thach and Graybiel 6. SECOND MESSENGERS AND EFFECTORS
(1968) have shown that removal of a€erent neural OF HAIR CELLS
input from the peripheral vestibular organs will Neurotransmitter e€ects are mediated by two cat-
block the initiation of motion sickness. Since there egories of receptors: ionotropic receptors are ligand-
is now evidence that antihistamines will reduce ves- gated channels that open (or close) in response to
tibular a€erent activity (Housley et al., 1988) a transmitter binding; a€ecting membrane potential
mechanism for the amelioration of motion sickness and input resistance; and in several cases increasing
and vertigo by actions at the detector organ cytoplasmic Ca concentrations; metabotropic recep-
(Anthony, 1991) itself by compounds classi®ed as tors are, on the other hand, linked to the activation
antihistaminics is a distinct possibility. of cytoplasmic `second messengers', that display a
Guth et al. (1985) have suggested that there may huge variety of e€ects. Activated metabotropic
be di€erences between the T/Ms of the various receptors bind to trimeric G proteins, activating
acousticolateralis synapses. The H-1 antagonists them. This in turn triggers a cascade of events that
may have di€erent e€ects in the various labyrinthine depends on the G-protein involved: G proteins of
Gs class activate, and Gi/o inhibit adenylate cyclases;
organs. As evidence for this, in the clinical literature,
Gq/11 activates PLC, while G12/13 a€ects ion pump-
these drugs have not been reported to exert e€ects ing (Neer, 1995). Further complexities are present,
on hearing other than a rarely reported episode of since both a and bg subunits of G-proteins are
tinnitus (Garrison, 1990). This contrasts with the active, and in addition to the mentioned e€ects,
e€ectiveness of astemizole in ameliorating chronic each G-protein may a€ect other processes (a typical
vertigo in patients su€ering from peripheral vestibu- example is the direct modulation of ion channels)
lar disorders (Jackson and Turner, 1987; Turner and (Neer, 1995; Hille, 1994). Hair cells have been found
Jackson, 1983). Similarly, histamine does not cause to express G0a (Valat et al., 1995) and Gi2a
facilitation of a€erent ®ring in the lateral line (Tachibana et al., 1994) but not Gs, that was instead
(Bledsoe et al., 1989; Mroz and Sewell, 1989), and expressed by cochlear supporting cells (Mizuta et
Bobbin and Thompson (1978) reported that intra- al., 1995). This would suggest an important role for
cochlear infusion of histamine in the guinea pig at cAMP in these cells; however, the observed role of
10 mM produced a slight decrease in the compound IP3 in cholinergic transmission would suggest the
action potential, results which contrast with histami- additional presence of a subunits of the Gq/11
ne's e€ects on the SCC neuroepithelium reported by family.
Housley et al. (1988). `Second messengers' are those components usually
thought of as being activated or inhibited by G-pro-
The paper of Bledsoe et al. (1989) is interesting in tein coupled receptors. Second messengers include
that, although histamine itself had no e€ect on lat- cyclic AMP, cyclic GMP, inositol phosphates, Ca,
eral line a€erent discharge rates [con®rmed by Mroz diacylglycerol. E€ectors are the last components in
and Sewell (1989)], the antihistaminic pyrilamine the cascade initiated by G-protein-coupled recep-
was e€ective in causing a€erent suppression as in tors. These are generally protein kinases although
the SCC. Furthermore, Mroz and Sewell (1989) G-protein subunits themselves have been shown to
found histidine and carnosine to cause facilitation of a€ect ion channels (Hille, 1994; Wickman and
a€erent discharge in the X. laevis lateral line. Clapham, 1995). [It is now clear that there must also
The Vestibular Hair Cells 231

be protein phosphatases in this series of reactions manipulations were considered due to increases in
(Hunter, 1995).] cAMP concentration. Of the parameters monitored,
Some ionotropic receptors can also activate sec- only an increase in the resting ®ring rate of a€erent
ond messengers (Dani and Mayer, 1995). For ®bers and a decrease in the transepithelial potential
instance, ligand-operated ion channels that admit occurred in response to all three manipulations.
Ca into the cell (e.g. some nicotinic receptors and These authors concluded that there may be two sites
some Glu receptors) could conceivably initiate Ca- of action of cAMP in the SCC: one in the hair cells
induced intracellular Ca release and/or activate Ca involved in transmitter release and; one in the non-
calmodulin kinase and/or activate or inhibit adeny- hair cell epithelial cells to reduce resistance (as in
lyl cyclases (ACs) (Mons and Cooper, 1995). the cochlea). Examining the distribution of AC by
the cytochemical detection of enzyme activity [by a
6.1. Cyclic Nucleotides modi®cation of the method of Mees (1984)]
Drescher et al. (1995a) found reaction product as-
There are currently several known isoforms of sociated with the stereocilia, the cuticular plate and
ACs, which are variously stimulated or inhibited by: the e€erent endings in the frog saccule. They, there-
a- and bg subunits of G-proteins; Ca and PKC fore, presume a role for cAMP in transduction and
(Mons and Cooper, 1995). in second messenger modulation of ACh synthesis.
Drescher et al. (1995a,b) demonstrated the ex- Koch and Zenner (1988) discuss the roles of cAMP
pression of Ca/calmodulin-sensitive isoforms of AC and G-protein in guinea pig inner ear. A cAMP-
in the inner ear. The highest basal activity of AC in gated channel has been detected in guinea pig inner
the inner ear was found in the cochlear lateral wall and OHCs and has been suggested to play a role in
and especially in the stria vascularis. Stria vascularis transduction (Kolesnikov et al., 1991).
had activity two to three times that of the spiral The presence of guanylyl cyclase (GC) and cGMP
ligament (Ahlstrom et al., 1975). In the saccule in the inner ear has been reported in the cochlea
(Paloheimo and Thalmann, 1977) utricle and SCC (Guth and Stockwell, 1977) but not in the vestibular
(Bagger-Sjoback et al., 1980) activity was similar to organs. A role for cGMP has been suggested (Chen
that measured in the cochlear lateral wall. and Eatock, 1994) by the ®nding that NO is able to
Histochemical studies localized the enzyme activity modulate IK, L currents (see Section 3.1) in Type I
to the infoldings of marginal cells (Mees, 1984; Zajic vestibular hair cells. Soluble GC is considered the
et al., 1983); and utricular dark cells (Zajic et al., main site of action of NO (Wang and Robinson,
1983). Sterkers et al. (1988a,b) discuss AC and 1997); therefore, the reported e€ect of NO strongly
cAMP in some detail and suggest that they may infers the presence of an active GC-cGMP system at
play a role in endolymph secretion. least in Type I cells.
Oudar et al. (1990) examined the frog SCC for
AC staining. These authors found such staining on 6.2. The Phospholipase C Cascade
the apical and basolateral membranes of dark cells
and the apical membranes of sensory cells. As inter- Many T/Ms in¯uence PLC through G-protein
esting as the localization is their observation that coupled receptors. This enzyme catalyzes the hy-
the AC localized to the apical membrane of the drolysis of phosphatidyl inositol 4,5 bisphosphate
dark cells required the presence of vasotocin for into IP3 and diacylglycerol (DAG). Both IP3 and
staining to be present. In the sensory cells and baso- DAG act as second messengers. IP3 acts on its own
lateral membranes of the dark cells the presence of receptor to increase the intracellular Ca concen-
vasotocin was not necessary. Once again, the tration by releasing Ca from intracellular stores
emphasis was on the role of cAMP in secretory ac- such as the endoplasmic reticulum. DAG activates
tivities but its role as second messenger was also Ca-dependent PKC. The presence of IP3 receptors
considered. on secretory vesicles has recently come to light
Doi et al. (1990, 1992) produced evidence that the (Blondel et al., 1995). These receptors, when acti-
AC/A-kinase cascade may be physiologically im- vated, could lead to the release of Ca from the ves-
portant in modulation of the endocochlear poten- icles thus activating or enhancing the `docking' of
tial. After considering several mechanisms vesicles with the secretory membrane. This activated
explaining this evidence these authors opted for the or enhanced `docking' could occur through the me-
one that suggested that an increase in cAMP diation of Ca-sensitive proteins such as synaptotag-
(through the AC activator forskolin) caused a min (Blondel et al., 1995).
decrease in the electrical resistance of the scala The IP3 second messenger system has been found
media. This is apparently accomplished by an in cochlea where it is responsive to purinergic P2y
increase in chloride permeability of the endolymph- and muscarinic M3 receptor activation (Bartolami
perilymph barrier. These ®ndings and mechanisms et al., 1990; Guiramand et al., 1990; Niedzielski and
have pertinence to vestibular systems in that Ricci et Schacht, 1991; Niedzielski and Schacht, 1992;
al. (1991a) made similar conclusions in regard to Schacht and Zenner, 1987).
cAMP and the SCC. In this work cAMP levels in There is strong evidence of a purinergic P2y recep-
the SCC were increased by a€ecting AC (with the tor in vestibular epithelia (Aubert et al., 1994, 1995)
activator forskolin), inhibiting phosphodiesterase but the subtypes of muscarinic receptor in vestibular
[with the inhibitor 3-isobutyl-l-methyl xanthine epithelia have not been delineated. Ogawa and
(IBMX)] or by using a membrane-permeant ana- Schacht (1993) applied purinergic P2 agonists and
logue, dibutyryl cAMP. Any changes in SCC func- cholinomimetics to isolated rat vestibular tissue and
tion that occurred in response to all three assayed it for IP3 synthesis. These authors found
232 P. S. Guth et al.

that the P2 agonist ATP-gS and the muscarinic ago- sence of multiple Ca-binding proteins is not clear;
nist carbachol both caused increases in IP3 levels. however, bu€er properties (KD, kinetics of binding)
The e€ect of carbachol was blocked by atropine. in¯uence Ca dynamic modulation (Wu et al., 1996),
Adenosine and the nicotinic agonist, dimethyl phe- and therefore the di€erent components may be dif-
nyl piperazinium, failed to cause IP3 increases. ferentially distributed across the cell or among cells
displaying di€erent physiological roles. In fact, calre-
6.3. Regulation of Cytoplasmic Calcium tinin expression in the saccule seems to be restricted
to club-like hair cells (Edmonds and Roberts, 1997;
One of the most amazing features of hair cells is Baird et al., 1997), and hair cell bodies and hair bun-
the presence in a small and compact cell of separate dles display di€erent calcium binding protein ex-
intracellular compartments whose functions are pression [e.g. S-100 is restricted to the cell bodies,
likely to be di€erentially regulated. Ca concen- while calretinin, in most cells, is only found in hair
tration, in particular, appears very strictly regulated bundles (Baird et al., 1997)]. A comparison between
(Lenzi and Roberts, 1994). As has now been exten- perforated patch and whole-cell recording of hair cell
sively pointed out, using both experimental and currents has shown that the Ca bu€ering power of
theoretical approaches, the dynamic regulation of in- the cell body is equivalent to 1 mM BAPTA
tracellular Ca in saccular hair cells depends essen- (Roberts, 1994), while the bu€ering capacity of the
tially on the presence of a large concentration of hair bundle is ca ten-fold less (Ricci and Fettiplace,
mobile endogenous bu€ers, which would provide ca 1997), con®rming the view that apical and basolat-
5 mM Ca-binding sites (Roberts, 1993, 1994). Dyna- eral Ca domains of hair cells are independent. Once
mic regulation of Ca is especially important at the bound to bu€er proteins and removed from active
a€erent presynaptic active zone, that in seismic hair zones Ca has to be extruded from the cell. This
cells is capable of following stimuli up to frequencies requires a massive Ca pumping across hair cell
of ca 1 kHz (i.e. Ca increase and reuptake cycle can- plasma membrane. Assuming that hair cell Ca-
not take longer than 1 msec). In the fast synapses of ATPase rate was similar to that of sarcoplasmic reti-
retinal bipolar neurons (that show specializations culum (200 ions secÿ1). Roberts et al. (1991) argued
similar to hair cell a€erent synapses), exocytosis that hair cells would need 300 000 maximally-acti-
timecourse is limited only by calcium current kinetics vated Ca±ATPases just to maintain resting Ca con-
(Mennerick and Matthews, 1996). It is reasonable to centrations. In fact, Ca±ATPases present in large
suggest that the same holds true at hair cell a€erent concentrations both in the plasma membrane and in-
synapses. A computer simulation of calcium and buf- tracellular stores appear to be the primary route for
fer di€usion (Roberts, 1994) showed that for low Ca Ca elimination from hair cell cytoplasm (Tucker and
in¯ux, as due to the opening of a single channel Fettiplace, 1995), while the role of a Na/Ca exchan-
around resting potential [I(Ca)=0.8 pA for a single ger appears to be very minor. Saccular hair cell Na/
active zone] a mobile bu€er would speed up but not K±ATPase can extrude up to 4 fmol(Na) minÿ1;
signi®cantly increase steady Ca reduction in respect (Mroz et al., 1993), a Na/Ca antiporter with a coup-
to a ®xed bu€er, whereas for strong ¯uxes, as occur ling ratio of 1.5 could therefore barely balance rest-
following a depolarizing stimulus, the mobility of the ing currents, which bring in the cell ca
bu€er is necessary not only to hasten Ca variations 5 fmol(Ca) minÿ1 (Roberts et al., 1991). It is there-
but also to lower steady Ca levels, especially in the fore possible that the Na/Ca exchanger is the `house-
regions outside the active microdomain. In close keeper' system that extrudes Ca at rest, Ca ATPases
proximity to Ca channel clusters, however, even would be necessary to eciently deal with stimulated
mobile bu€ers would not be able to bind all entering Ca in¯ux, that may be up to ten times as great as
Ca, thus leading to the formation of extremely steep resting in¯ux (Roberts et al., 1991; Roberts, 1994).
Ca gradients around presynaptic active zones. In The ER compartment involved in Ca pumping is not
fact, membrane depolarizations have been observed known; however, in a simulation of hair cell cyto-
to induce the formation of calcium `hot spots' of plasm, the positioning of ATPases was not crucial in
<1 mm diameter in turtle cochlea (Tucker and dynamic Ca regulation (Wu et al., 1996). The pre-
Fettiplace, 1995) and bullfrog sacculus (Issa and sence of a widespread intracellular reticular mem-
Hudspeth, 1994) hair cell cytoplasm. Fixed bu€er brane system in hair cells is well known; there are
seems to play a minor role at the a€erent synapse ultrastructural evidences that, at e€erent synapses,
(Issa and Hudspeth, 1994). The mobile bu€ering sys- these membranes take the form of ¯attened submem-
tem seems to involve several proteins, which are dif- brane sacs covering the whole postsynaptic area,
ferentially expressed in hair cells (Edmonds and which are called sub-surface cisterns (Tanaka and
Roberts, 1997; Baird et al., 1997) The ®rst bu€er to Smith, 1978). It is not yet known, however, whether
be identi®ed was calbindin D-28k, a protein that these sac-like cisterns represent IP3-sensitive Ca
bears four binding sites for Ca and is present in a stores or whether they only form a di€usional barrier
concentration of 1.5 mM in saccular hair cells cyto- for Ca entering the synaptic site through cholinergic
plasmatic fractions (Oberholtzer et al., 1988). In ad- receptors (Martin and Fuchs, 1992). Several pieces of
dition, hair cells from saccule and utricle have been evidence indicate the activation of an IP3-dependent
observed to express calmodulin, calretinin, parvalbu- pathway by e€erent stimulation, ACh (Yoshida et
min and S-100 (Edmonds and Roberts, 1997; Baird al., 1994; Kakehata et al., 1993) or ATP perfusion
et al., 1997; Dechesne et al., 1988a,b; Dememes et (Ashmore and Ohmori, 1990); in the guinea-pig
al., 1991, 1993; Gillespie and Hudspeth, 1991; Saidel cochlea, class P2y ATP receptors are coupled to PLC
et al., 1995; Sans et al., 1986, 1987; Shepherd et al., and IP3 pathways (Niedzielski and Schacht, 1992)
1989; Walker et al., 1993). The reason for the pre- (the cellular location of these receptors is still uncer-
The Vestibular Hair Cells 233

tain), and a calcium-induced calcium release from in- Caul®eld, M. D. (1993) Muscarinic receptorsÐ
tracellular stores has been suggested to explain characterization, coupling and function.
cholinergic slow e€ects in cochlear OHCs (Sridhar et Pharmac. Ther. 58, 319±379.
al., 1995, 1997). Diener, H. C. and Dichgons, J. (1988) On the
role of vestibular,visual and somatosensory infor-
mation for dynamic postural control in humans.
In: Vestibulospinal Control of Posture and
7. SUMMARY Locomotion, pp. 253±262. Ed. M. Pompeiano and
This article reviews the role of a long-neglected J. H. J. Allum. Elsevier: New York.
post-transductional processing by the hair cell. The Eybalin, M. (1993) Neurotransmitters and
neglect of this hair cell function has existed because neuromodulators of the mammalian cochlea.
of the lack of appropriate techniques for studying Physiol. Rev. 73, 309±373.
them. In the past decade immunohistochemical, Ferrary, E., Tran, P., Huy, B. A., Roinel, N.,
molecular biological, intracellular recording and Bernard, C. and Amiel, C. (1988) Calcium and
pharmacological techniques have become commonly the inner ear ¯uids. Acta Otolaryngol. (Stockh.)
available so that the hair cell has become accessible 460(Suppl.), 13±17.
and has begun to reveal its secrets. Garcia-Anoveros, J. and Corey, D. P. (1997)
Thanks to the laboratories of Hudspeth, Ohmori, The molecules of mechanosensation. A. Rev.
Flock and others transduction has been made com- Neurosci. 20, 567±594.
prehensible. Many laboratories have contributed to Gillespie, P. G. (1995) Molecular machinery of
our developing understanding of the biophysics of auditory and vestibular transduction. Curr. Opin.
the hair cell. With regard to the transmitters and Neurobiol. 5, 449±455.
modulators, it may be said that the era of identi®- Goldberg, J. M. (1979) Vestibular receptors in
cation is near its end and the next task is the under- mammals: a€erent discharge characteristics and
standing of the receptors serving them and the e€erent control. Prog. Brain Res. 50, 355±367.
reactions occurring subsequent to receptor acti- Goldberg, J. M. and Fernandez, C. (1984) The
vation. vestibular system. In: Handbook of Physiology:
The hair cells are upstream of the a€erents and as The Nervous System: Sensory Systems Part II,
such command them. The a€erents are silent in the pp. 977±1022.
absence of hair cell transmitter release. The hair cells Guth, P. S. and Norris, C. H. (1996) The hair
are not, as might have once been thought, merely cell acetylcholine receptors. A synthesis. Hear.
the passive, homogeneous agencies of transduction Res. 98, 1±8.
but are active and di€erent from one another in Guth, P. S., Norris, C. H. and Bobbin, R. P.
many ways. This was a watershed realization. By (1976) The pharmacology of transmission in the
virtue of their: morphological and physiological peripheral auditory system. Pharmac. Rev. 28,
di€erences; varying complements of biophysical and 95±125.
biochemical activities; varying complements of the Guth, P. S., Norris, C. H. and Sewell, W. F.
transmitters and modulators and their receptors (1985) Primary a€erent transmission in acoustico-
and; by the possibility that they behave di€erently in lateralis organs. In: Auditory Biochemistry, pp.
regard to resting and stimulated modes and adapt 42±49. Ed. D. G. Drescher. C. C. Thomas,
di€erently, the hair cells process the transductional Spring®eld, IL.
stimuli in innumerably di€erent ways. There are the Hackney, C. M. and Furness, D. N. (1995)
di€erences enumerated above but, of course, there is Mechanotransduction in vertebrate hair cells:
also some overlap and some redundancy among structure and function of the stereociliary bundle.
them. So, out of the variations in processing of the Am. J. Physiol. 268 (Cell Physiol. 37, C1±C13).
transductional stimuli comes a probabilistic swarm Highstein, S. M. (1991) The central nervous
of signals activating the a€erents. This leads to the
system e€erent control of the organs of balance
mosaic of a€erent neuronal activity necessary for
and equilibrium. Neurosci. Res. 12, 13±30.
the brain to perform its familiar miracles.
Hillman, D. E. (1976) Morphology of periph-
This article presents the current state of our infor-
eral and central vestibular system. In: Frog
mation regarding all the activities of vestibular per-
Neurobiology: A Handbook, pp. 452±480. Ed. R.
iphery between hair cell signal transduction and
Llinas and W. Precht. Springer-Verlag, Berlin.
CNS operations.
Hollmann, M. and Heinemann, S. (1994)
Cloned glutamate receptors. A. Rev. Neurosci. 17,
31±108.
8. PERTINENT REVIEW ARTICLES Hudspeth, A. J. (1983) Mechanoelectrical
transduction by hair cells in the acousticolateralis
For those readers wishing more information on sensory system. A. Rev. Neurosci. 6, 187±215.
some subjects touched on in this article, we present Hudspeth, A. J. (1989) How the ear's works
the following list of pertinent review articles: work. Nature 34, 397±404.
Bledsoe, S. C., Bobbin, R. P. and Puel, J. L. Hunter-Duvar, I. M. and Hinojosa, R. (1984)
(1988) Neurotransmission in the inner ear. In: Vestibule: sensory epithelia. In: Ultrastructural
Physiology of the Ear, pp. 385±405. Eds. A. E. Atlas of the Inner Ear, pp. 211±244. Ed. I.
John and J. Santos-Sacchi. Raven Press: New Friedmann and J. Ballantyne. Butterworth,
York. London.
234 P. S. Guth et al.

Iurato, S., Luciano, L., Pannese, E. and Reale, Vizi, E. S. (1979) Presynaptic modulation of
E. (1972) E€erent vestibular ®bres in mammalia: neurochemical transmission. Prog. Neurobiol. 12,
morphological and histochemical aspects. In: 181±290.
Basic Aspects of Central Vestibular Mechanisms, Volknandt, W. (1995) The synaptic vesicle and
Progress in Brain Research, Vol. 37, pp. 429±443. its targets. Neuroscience 64, 277±300.
Ed. A. Brodal and O. Pompeiano. Elsevier,
Amsterdam.
AcknowledgementsÐThe authors are grateful and deeply
Klinke, R. (1986) Neurotransmission in the
indebted to their colleagues who have provided detailed
inner ear. Hear. Res. 22, 235±243. and insightful comments on this review article. Deserving
Lenzi, D. and Roberts, W. M. (1994) Calcium special mention are Dr S. Masetto for helpful criticism and
signalling in hair cells: multiple roles in a compact for kindly allowing us to use his unpublished data, Drs G.
cell. Curr. Opin. Neurobiol. 4, 496±502. B. Athas and M. M. Garcia for help and criticism on the
Maggio, E. (1966) The humoral system of the molecular biology data, and Drs W. F. Sewell and D.
labyrinth. Acta Otolaryngol. (Stockh.) Webster. Chris Holt assistance is greatly appreciated.
Supported by NIH Grant No. R01DC00303.|
218(Suppl.), 1±35.
Norris, C. H. (1988) Drugs a€ecting the inner
ear. A review of their clinical ecacy, mechan-
isms of action, toxicity, and place in therapy. REFERENCES
Drugs 36, 754±772.
Abbrachio, M. P. and Burnstock, G. (1994) Purinoceptors: are
Ohmori, H. (1992) Ion channels for the there families of P2x and P2y purinoceptors. Pharmac.
mechano-electrical transduction and e€erent Therapeut. 64, 445±475.
synapse of the hair cell. Adv. Biophys. 28, 1±30. Adams, J. C., Mroz, E. A. and Sewell, W. F. (1987) A possible
Pappas, D. G. (1984) Barany's history of ves- neurotransmitter role for CGRP in a hair-cell sensory organ.
Brain Res. 419, 347±351.
tibular physiology-translation and commentary.
Adrian, E. D. (1943) Discharges from vestibular receptors in the
Ann. Otol. Rhinol. Laryngol. 110(93-Suppl.), 1± cat. J. Physiol. (Lond.) 101, 369±407.
16. Ahlstrom, P., Thalmann, I., Thalmann, R. and Ise, I. (1975) Cyclic
Pickles, J. O. and Corey, D. P. (1992) AMP and adenylate cyclase in the inner ear. Laryngoscope 85,
Mechanoelectric transduction by hair cells. TINS 1241±1258.
Amaro, J., Guth, P. S. and Wanderlinder, L. (1966) Inhibition of
15, 254±259. auditory nerve action potentials by acetylcholine and physostig-
Pin, J. P. and Duvoisin, R. (1995) Review: neu- mine. Br. J. Pharmac. 28, 207±211.
rotransmitter receptors I. The metabotropic glu- Ambache, N. (1955) The use and limitations of atropine for phar-
tamate receptors: structure and functions. macological studies on autonomic e€ectors. Pharmac. Rev. 7,
Neuropharmacology 34, 1±27. 467±494.
Anastasio, T. J., Correia, M. J. and Perachio, A. A. (1985)
Raymond, J., Dememes, D. and Nieullon, A. Spontaneous and driven responses of semicircular canal primary
(1988) Neurotransmitters in vestibular pathways. a€erents in the unanesthetized pigeon. J. Neurophysiol. 54, 335±
Prog. Brain Res. 76, 29±43. 347.
Rosahl, T. W., Spillane, D., Missler, M., Herz, Anniko, M. and Arnold, W. (1991) Acetylcholine receptor localiz-
ation in human adult cochlear and vestibular hair cells. Acta
J., Selig, D. K. Wol€, J. R., Hammer, R. E.,
Otolaryngol. (Stockh.) 111, 491±499.
Malenka, R. C. and Sudhof, T. C. (1995) Annoni, J. M., Cochran, S. L. and Precht, W. (1984)
Essential functions of synapsins I and II in synap- Pharmacology of the vestibular hair cell-a€erent ®ber synapse in
tic vesicle regulation. Nature 375, 488±493. the frog. J. Neurosci. 4, 2106±2116.
Schwartz, J. C., Arrang, J. M., Garbarg, M., Anson, L. C. and Ashmore, J. F. (1994) Evidence for release of an
excitatory amino acid from inner hair cells isolated from the gui-
Pollard H. and Ruat, M. (1991) Histaminergic nea pig cochlea. 1st International Symposium Inner Ear
transmission in the mammalian brain. Physiol. Neuropharmacology Vol. 1, p. 3.
Rev. 71, 1±51. Anthony, P. F. (1991) Partitioning of the labyrinth: application in
Smith, P. F. and Darlington, C. L. (1994) benign paroxysmal positional vertigo. Am. J. Otol. 12, 388±393.
Pharmacology of the vestibular system. Clin. Art, J., Crawford, A., Fettiplace, R. and Fuchs, P. (1982) E€erent
regulation of hair cells in the turtle cochlea. Proc. R. Soc. Lond.
Neurol. 3, 467±484. B Biol. Sci. 216, 377±384.
Snyder, S. H. (1985) Adenosine as neuromodu- Art, J., Crawford, A., Fettiplace, R. and Fuchs, P. (1985) E€erent
lator. A. Rev. Neurosci. 8, 103±124. modulation of hair cell tuning in the cochlea of the turtle.
Sterkers, O., Bernard, C., Ferrary, E., Sziklai, J. Physiol. (Lond.) 360, 397±421.
Art, J. and Fettiplace, R. (1987) Variation of membrane properties
I., Tran, P., Huy, B. A. and Amiel, C. (1988)
in hair cells isolated from the turtle cochlea. J. Physiol. (Lond.)
Possible role of Ca ions in the vestibular system. 385, 207±242.
Acta Otolaryngol. (Stockh.) 460(Suppl.), 28±32. Art, J., Fettiplace, R. and Wu, Y. (1993) The e€ects of low calcium
Sterkers, O., Ferrary, E. and Amiel, C. (1988) on the voltage-dependent conductances involved in tuning of
Production of inner ear ¯uids. Physiol. Rev. 68, turtle hair cells. J. Physiol. (Lond.) 470, 109±126.
Art, J. and Goodman, M. (1996) Ionic conductances and hair cell
1083±1128. tuning in the turtle cochlea. Ann. N.Y. Acad. Sci. 781, 103±122.
Sudhof, T. C. (1995) The synaptic vesicle cycle: Art, J., Wu, Y. and Fettiplace, R. (1995) The calcium-activated
a cascade of protein±protein interactions. Nature potassium channels of turtle hair cells. J. Gen. Physiol. 105, 49±
375, 645±653. 72.
Torre, V., Ashmore, J. F., Lamb, T. D. and Art, J. J., Fettiplace, R. and Fuchs, P. A. (1984) Synaptic hyperpol-
arization and inhibition of turtle cochlear hair cells. J. Physiol.
Menini, A. (1995) Transduction and adaptation (Lond.) 356, 525±550.
in sensory receptor cells. J. Neurosci. 15, 7757± Ashcroft, D. W. and Hallpike, C. S. (1934) On the function of the
7768. saccule. J. Laryngol. 49, 450±460.
The Vestibular Hair Cells 235

Ashmore, J. F. (1983) Frequency tuning in a frog vestibular organ. Betz, H. and Changeux, J. P. (1979) Regulation of muscle acetyl-
Nature 304, 536±538. choline receptor synthesis in vitro by cyclic nucleotide deriva-
Ashmore, J. F. (1987) A fast motile response in guinea-pig outer tives. Nature 278, 749±752.
hair cells: the cellular basis of the cochlear ampli®er. J. Physiol. Bi€o, S., Grillo, M. and Margolis, F. L. (1990) Cellular localization
(Lond.) 388, 323±347. of carnosine-like and anserine-like immunoreactivities in rodent
Ashmore, J. F. and Ohmori, H. (1990) Control of intracellular cal- and avian central nervous system. Neuroscience 35, 637±651.
cium by ATP in isolated outer hair cells of the guinea-pig Blanks, R. H. I., Estes, M. S. and Markham, C. H. (1975)
cochlea. J. Physiol. (Lond.) 428, 109±131. Physiologic characteristics of vestibular ®rst-order neurons in
Ashmore, J. F. and Russell, I. J. (1982) E€ect of e€erent stimu- the cat. II. Response to constant angular acceleration.
lation on hair cells of the frog sacculus. J. Physiol. (Lond.) 329, J. Neurophysiol. 38, 1250±1268.
25p. Blaschko, H., Born, G. V. R., D'Iorio, A. and Eade, N. R. (1956)
Assad, J. and Corey, D. (1992) An active motor model for adap- Observations on the distribution of catechol amines and adeno-
tation by vertebrate hair cells. J. Neurosci. 12, 3291±3309. sine triphosphate in the bovine adrenal medulla. J. Physiol.
Assad, J., Shepherd, G. and Corey, D. (1991) Tip-link integrity (Lond.) 133, 548±557.
and mechanical transduction in vertebrate hair cells. Neuron 7, Bledsoe, S. C., Bobbin, R. P. and Puel, J. L. (1988)
985±994. Neurotransmission in the inner ear. In: Physiology of the Ear,
Assad, J. A., Hacohen, N. and Corey, D. P. (1989) Voltage depen- pp. 385±405. Eds A. E. John and J. Santos-Sacchi. Raven Press:
dence of adaptation and active bundle movement in bullfrog sac- New York.
cular hair cells. Proc. natn. Acad. Sci. U.S.A. 86, 2918±2922. Bledsoe, S. C. Jr, Chihal, D. M., Bobbin, R. P. and Morgan, D.
Athas, G. B., Chen, C., Bobbin, R. P. and Garcia, M. M. (1997a) N. (1983) Comparative actions of glutamate and related sub-
Nicotinic acetylcholine receptor subunits in outer hair cells of stances on the lateral line of xenopus laevis. Comp. Biochem.
the guinea pig cochlea demonstrated by expression pro®ling. Physiol. [C] 75, 199±206.
Abstracts of the 20th ARO Midwinter research Meeting, 66. Bledsoe, S. C. Jr, Sinard, R. J. and Allen, S. J. (1989) Analysis of
Athas, G. B., Norris, C. H., Guth, P. S., and Garcia, M. M. histamine as a hair-cell transmitter in the lateral line of Xenopus
(1997b) Expression pro®ling of vestibular hair cells from the laevis. Hear. Res. 38, 81±93.
frog. Abstracts of the 20th ARO Midwinter research Meeting, Blondel, O., Bell, G. I. and Seino, S. (1995) Inositol 1,4,5,-trispho-
148. sphate receptors, secretory granules and secretion in endocrine
Aubert, A., Bernard, C. and Vaudry, H. (1993) E€ects of modi®- and neuroendocrine cells. TINS 18, 157±161.
cations of extracellular and intracellular calcium concentrations Blosser, J. C. and Appel, S. H. (1980) Regulation of acetylcholine
on the bioelectrical activity of the isolated frog semicircular receptor by cyclic AMP. J. Biol. Chem. 255, 1235±1238.
canal. Brain Res. 607, 301±306. Bobbin, R. P., Bledsoe, S. C., Winbery, S. L. and Jenison, G. L.
Aubert, A., Norris, C. H. and Guth, P. S. (1994) In¯uence of ATP (1985) Actions of putative neurotransmitters and other relevant
and ATP agonists on the physiology of the isolated semicircular compunds on Xenopus laevis lateral line. In: Auditory
canal of the frog (Rana pipiens). Neuroscience 62, 963±974. Biochemistry, pp. 102±122. Ed. D. G. Drescher. C. C. Thomas:
Aubert, A., Norris, C. H. and Guth, P. S. (1995) Indirect evidence Spring®eld, IL.
for the presence and physiological role of endogenous extracellu- Bobbin, R. P. and Guth, P. S. (1970) Evidence that gamma-anino-
lar ATP in the semicircular canal. Neuroscience 64, 1153±1160. butyric acid is not the inhibitory transmitter at the crossed olivo-
Bagger-Sjoback, D., Filipek, C. S. and Schacht, J. (1980) cochlear nerve-hair cell junction. Neuropharmacology 9, 567±
Characteristics and drug responses of cochlear and vestibular 574.
adenylate cyclase. Arch. Otorhinolaryngol. 228, 217±222. Bobbin, R. P. and Thompson, M. H. (1978) E€ects of putative
Baird, R. (1992) Morphological and electrophysiological properties transmitters on a€erent cochlear transmission. Ann. Otol.
of hair cells in the bullfrog utriculus. Ann. N.Y. Acad. Sci. 656, Rhinol. Laryngol. 87, 185±190.
12±26. Bobbin, R. P., Ceasar, G. and Fallon, M. (1990) Potassium
Baird, R. (1994a) Comparative transduction mechanisms of hair induced release of GABA and other substances from the guinea
cells in the bullfrog utriculus I. Responses to intracellular cur- pig cochlea. Hear. Res. 46, 83±93.
rent. J. Neurophysiol. 71, 666±684. Bowen, R. E. (1931) Movement of the so-called hairs in the ampul-
Baird, R. (1994b) Comparative transduction mechanisms of hair lar organs of ®sh ears. Proc. natn. Acad. Sci. U.S.A. 17, 192±
cells in the bullfrog utriculus. II. Sensitivity and response 194.
dynamics to hair bundle displacement. J. Neurophysiol. 71, 685± Boyle, R., Carey, J. P. and Highstein, S. M. (1991) Morphological
705. correlates of response dynamics and e€erent stimulation in hori-
Baird, R., Desmadryl, G., Fernandez, C. and Goldberg, J. (1988) zontal semicircular canal a€erents of the toad®sh, Opsanus tau.
The vestibular nerve of the chinchilla. II. Relation between a€er- J. Neurophysiol. 66, 1504±1521.
ent response properties and peripheral innervation patterns in Boyle, R. and Highstein, S. M. (1990) E€erent vestibular system in
the semicircular canals. J. Neurophysiol. 60, 182±203. the toad®sh: action upon horizontal semicircular canal a€erents.
Baird, R. and Lewis, E. (1986) Correspondences between a€erent J. Neurosci. 10, 1570±1582.
innervation patterns and response dynamics in the bullfrog utri- Brain, S. D., Williams, T. J., Tippins, J. R., Morris, H. R. and
cle and lagena. Brain Res. 369, 48±64. MacIntyre, I. (1985) Calcitonin-gene-related peptide is a potent
Baird, R. and Schu€, N. (1994) Peripheral innervation patterns of vasodilator. Nature 298, 240±244.
vestibular nerve a€erents in the bullfrog utriculus. J. Comp. Brake, A. J., Wagenbach, M. J. and Julius, D. (1994) New struc-
Neurol. 342, 279±298. tural motif for ligand-gated ion channels de®ned by an ionotro-
Baird, R., Steyger, P. and Schu€, N. (1997) Intracellular distri- pic ATP receptor. Nature 371, 519±523.
butions and putative functions of calcium-binding proteins in Brantley, R. K. and Bass, A. H. (1988) Cholinergic neurons in the
the bullfrog vestibular otolith organs. Hear. Res. 103, 85±100. brain of a teleost ®sh (Porichthys notatus) located with a mono-
Barrie, A. P., Nicholls, D. G., Sanchez-Prieto, J. and Sihra, T. clonal antibody to choline acetylcholinesterase. J. Comp. Neurol.
S. (1991) An ion channel locus for the protein kinase C poten- 275, 87±105.
tiation pf transmitter glutamate release from guinea pig cerebro- Brichta, A. M. and Peterson, I. H. (1994) Functional architecture
cortical synaptosomes. J. Neurochem. 57, 1398±1404. of vestibular primary a€erents from the posterior semicircular
Bartolami, S., Guiramand, J., Lenoir, M., Pujol, R. and Recasens, canal of a turtle, Pseudemys (Trachemys) scripta elegans.
M. (1990) Carbachol-induced inositol phosphate formation J. Comp. Neurol. 344, 481±507.
during rat cochlea development. Hear. Res. 47, 229±234. Brownell, W. E., Bader, C. R., Bertrand, D. and de Ribaupierre,
Bernard, C. (1983) Action of streptomycin and calcium on the api- Y. (1985) Evoked mechanical responses of isolated cochlear
cal membrane of hair cells of the frog isolated semicircular outer hair cells. Science 227, 194±196.
canal. Acta Otolaryngol. (Stockh.) 96, 21±30. Bryant, G., Barron, S., Norris, C. H. and Guth, P. S. (1987)
Bernard, C., Cochran, S. L. and Precht, W. (1985) Presynaptic Adenosine is a modulator of hair cell a€erent neurotransmission.
actions of cholinergic agents upon the hair cell-a€erent ®ber Hear. Res. 30, 231±238.
synapse in the vestibular labyrinth of the frog. Brain Res. 338, Bullock, T. H. and Horridge, G. A. (1966) Structure and Function
225±236. in the Nervous System of Invertebrates. Freeman: San Francisco.
236 P. S. Guth et al.

Burgen, A. S. V., Hiley, C. R. and Young, J. M. (1974) The bind- Crawford, A., Evans, M. and Fettiplace, R. (1989) Activation and
ing of (H3)-propylcholine mustard by longitudinal muscle strips adaptation of transducer currents in turtle hair cells. J. Physiol.
from guinea-pig small intestine. Br. J. Pharmac. 50, 145±151. (Lond.) 419, 405±434.
Burnham, J. A. and Stirling, C. E. (1984a) Quantitative localization Crawford, A. and Fettiplace, R. (1980) The frequency selectivity of
of Na±K pump site in frog inner ear dark cells. Hear. Res. 13, auditory nerve ®bres and hair cells in the cochlea of the turtle.
261±268. J. Physiol. (Lond.) 306, 79±125.
Burnham, J. A. and Stirling, C. E. (1984b) Quantitative localiz- Curtis, D. R. and Johnston, G. A. R. (1974) Amino acid transmit-
ation of Na±K pump site in the frog sacculus. J. Neurocytol. 13, ters in the mammalian nervous system. Ergebnisse Physiol. 69,
617±638. 97±188.
Burnstock, G. and Kennedy, C. (1985) Is there a basis for dis- Dallos, P., Evans, M. G. and Hallworth, R. (1991) Nature of the
tinguishing two types of P2-purinoceptor? Gen. Pharmac. 16, motor element in electrokinetic shape changes of cochlear outer
433±440. hair cells. Nature 350, 155±157.
Campbell, V., Berrow, N., Fitzgerald, E., Brickley, K. and Dani, J. and Mayer, M. (1995) Structure and function of glutamate
Dolphin, A. (1995) Inhibition of the interaction of G protein and nicotinic acetylcholine receptors. Curr. Opin. Neurobiol. 5,
G(o) with calcium channels by the calcium channel beta-subunit 310±317.
in rat neurones. J. Physiol. (Lond.) 485 (Pt 2), 365±372. Danielson, P. D., Zottoli, S. J., Corrodi, J. G., Rhodes, K. J. and
Carpenter, M. B., Chang, L., Pereira, A. B., Hersh, L. B., Bruce, Mufson, E. J. (1983) Localization of choline acetyltransferase to
G. and Wu, J. Y. (1987) Vestibular and cochlear e€erent neur- somata of posterior lateral line e€erents in the gold®sh. Brain
ons in the monkey identi®ed by immunocytochemical methods. Res. 448, 158±161.
Brain Res. 408, 275±280. Davis, K. G., Sheikchali, S. A., Drescher, M. S., Khan, K. M. and
Caul®eld, M. D. (1993) Muscarinic receptors-characterization,cou- Drescher, D. G. (1997) Alpha-1 isoform transcripts of voltage-
pling and function. Pharmac. Therapeut. 58, 319±379. gated calcium channels in subdissected fractions of the mamma-
Cazals, Y., Aran, J.-M. and Erre, J.-P. (1982) Frequency sensitivity lian cochlea. Abstr. Assoc. Res. Otolaryng. 20, 231.
and selectivity of acoustically evoked potentials after complete Dechesne, C., Raymond, J. and Sans, A. (1984) Action of gluta-
cochlear hair cell destruction. Brain Res. 231, 197±203. mate in the cat labyrinth. Ann. Otol. Rhinol. Laryngol. 93, 163±
Cazals, Y., Aran, J.-M., Erre, J.-P. and Guilhaume, A. (1980) 165.
Acoustic responses after total destruction of the cochlear recep- Dechesne, C. J., Hampson, D. R., Goping, G., Wheaton, K. D. and
tor: brainstem and auditory cortex. Science 210, 83±86. Wenthold, R. J. (1991) Identi®cation and localization of a kai-
Chabbert, C., Geleoc, G., Lehouelleur, J. and Sans, A. (1994) nate binding protein in the frog inner ear by electron microscopy
Intracellular calcium variations evoked by mechanical stimu- immunocytochemistry. Brain Res. 545, 223±233.
lation of mammalian isolated vestibular Type I hair cells. Dechesne, C. J., Kau€, C., Stettler, O. and Tavitian, B. (1997)
P¯ugers Arch. 427, 162±168. Rab3A immunolocalization in the mammalian vestibular end-
organs during development and comparison with synaptophysin
Chang, J. S., Popper, A. N. and Saidel, W. M. (1992)
expression. Dev. Brain Res. 99, 103±111.
Heterogeneity of sensory hair cells in a ®sh ear. J. Comp.
Dechesne, C. J., Thomasset, M., Brehier, A. and Sans, A. (1988a)
Neurol. 324, 621±640.
Calbindin (CaBP 28 kDa) localization in the peripheral vestibu-
Chen, C., Nenov, A., Norris, C. H. and Bobbin, R. P. (1995) ATP
lar system of various vertebrates. Hear. Res. 33, 273±278.
modulation of L-type calcium channel currents in guinea pig
Dechesne, C. J., Lavigne-Rebillard, M., Brehier, A., Thomasset,
outer hair cells. Hear. Res. 86, 25±33.
M. and Sans, A. (1988b) Appearance and distribution of neur-
Chen, W. and Eatock, R. A. (1994) Nitric oxide inhibits a low-vol-
on-speci®c enolase and calbindin (CaBP 28 kDa) in the develop-
tage-activated potassium conductance in mammalian Type I hair
ing human inner ear. Brain Res. 469, 221±230.
cells. Biophys. J. 66, A430.
Dememes, D., Eybalin, M. and Renard, N. (1993) Cellular distri-
Churchill, J. A., Schuknecht, H. F. and Doran, R. (1956)
bution of parvalbumin immunoreactivity in the peripheral ves-
Acetylcholinesterase activity in the cochlea. Laryngoscope 66, 1±
tibular system of three rodents. Cell Tissue Res. 274, 487±492.
15.
Dememes, D., Lleixa, A. and Dechesne, C. J. (1995) Cellular and
Clapham, D. (1995) Calcium signaling. Cell 80, 259±268.
subcellular localization of AMPA-selective glutamate receptors
Cochran, S. L. and Correia, M. J. (1995) Functional support of in the mammalian peripheral vestibular system. Brain Res. 671,
glutamate as a vestibular hair cell transmitter in an amniote. 83±94.
Brain Res. 670, 321±325. Dememes, D., Moniot, B., Lomri, N., Thomasset, M. and Sans,
Cochran, S. L., Kasik, P. and Precht, W. (1987) Pharmacological A. (1991) Detection of calbindin-D 28k mRNA in rat vestibular
aspects of excitatory synaptic transmission to second-order ves- ganglion neurons by in situ hybridization. Brain Res. molec.
tibular neurons in the frog. Synapse 1, 102±123. Brain Res. 9, 153±156.
Cohen, A. I. (1963) The ®ne structure of the visual receptors of the Dememes, D., Raymond, J. and Sans, A. (1983) Selective retro-
pigeon. Exp. Eye Res. 2, 88±97. grade labelling of vestibular e€erent neurons with [3H]choline.
Cohen, G. M. (1987) Acetylcholinesterase activity in the embryonic Neuroscience 8, 285±290.
chick's ear. Hear. Res. 28, 57±63. Dememes, D., Raymond, J. and Sans, A. (1984) Selective retro-
Colton, C. K. and Freeman, A. R. (1975) Dual response to lobster grade labeling of neurons of the cat vestibular ganglion with
muscle ®bers to glutamate. Comp. Biochem. Physiol. 51, 275± [3H]D-aspartate. Brain Res. 304, 188±191.
284. Dememes, D., Wenthold, R. J., Moniot, B. and Sans, A. (1990)
Corey, D. and Hudspeth, A. (1979a) Response latency of ver- Glutamate-like immunoreactivity in the peripheral vestibular
tebrate hair cells. Biophys. J. 26, 499±506. system of mammals. Hear. Res. 46, 261±269.
Corey, D. and Hudspeth, A. (1983) Kinetics of the receptor current Denk, W., Holt, J., Shepherd, G. and Corey, D. (1995) Calcium
in bullfrog saccular hair cells. J. Neurosci. 3, 962±976. imaging of single stereocilia in hair cells: localization of trans-
Corey, D. P. and Hudspeth, A. J. (1979b) Ionic basis of the recep- duction channels at both ends of tip links. Neuron 15, 1311±
tor potential in a vertebrate hair cell. Nature 218, 675±677. 1321.
Correia, M., Ricci, A. and Rennie, K. (1996) Filtering properties of Desmedt, J. E. and Robertson, D. (1975) Ionic mechanism of the
vestibular hair cells: an update. Ann. N.Y. Acad. Sci. 781, 138± e€erent olivo-cochlear inhibition studied by cochlear perfusion
149. in the cat. J. Physiol. (Lond.) 247, 407±428.
Cotman, C. W., Flatman, J., Ganong, A. H. and Perkins, M. (1986) Devau, G., Lehouelleur, J. and Sans, A. (1993) Glutamate recep-
E€ect of excitatory amino acids in synaptic transmission of the tors on type I vestibular hair cells of guinea-pig. Eur. J. Neurosci.
Scha€er collateral-commissural pathway of the rat hippo- 5, 1210±1217.
campus. J. Physiol. (Lond.) 378, 403±415. Devries, S. and Baylor, D. (1993) Synaptic circuitry of the retina
Cousillas, H., Cole, K. S. and Johnstone, B. M. (1988) E€ect of and olfactory bulb. Cell 72, 139±149.
spider venom on cochlear nerve activity consistent with glutami- Dickman, J. D. and Correia, M. J. (1989) Responses of pigeon
nergic transmission at hair cell-a€erent dendrite synapse. Hear. horizontal semicircular canal a€erent ®bers. II. High-frequency
Res. 36, 213±220. mechanical stimulation. J. Neurophysiol. 62, 1102±1112.
The Vestibular Hair Cells 237

Dickman, J. D. and Correia, M. J. (1993) Bilateral communication from hair cells by elevated extracellular potassium. J. Neurochem.
between vestibular labyrinths in pigeons. Neuroscience 57, 1097± 59, 93±98.
1108. Drescher, M. J., Kern, R. C., Hat®eld, J. S. and Drescher, D.
Didier, A. and Cazals, Y. (1989) Acoustic responses recorded from G. (1995b) Cytochemical localization of adenylyl cyclase activity
the saccular bundle on the eighth nerve of the guinea pig. Hear. within the sensory epithelium of the trout saccule. Neurosci.
Res. 37, 123±128. Lett. 196, 145±148.
Didier, A., Decory, L. and Cazals, Y. (1990) Evidence for potass- Dulon, D., Mollard, P. and Aran, J.-M. (1991) Extracellular ATP
ium-induced motility in Type I vestibular hair cells in the guinea elevates cytosolic Ca++ in cochlear inner hair cells. Neuroreport
pig. Hear. Res. 46, 171±176. 2, 69±72.
Didier, A., Dupont, J. and Cazals, Y. (1990c) GABA immunoreac- Dunlap, K., Luebke, J. and Turner, T. (1995) Exocytotic Ca chan-
tivity of calyceal nerve endings in the vestibular system of the nels in mammalian central neurons. TINS 18, 89±98.
guinea pig. Cell Tissue Res. 260, 415±419. Dunn, R. A. (1980) Reciprocal synapses between hair cells and ®rst
Diener, H. C. and Dichgons, J. (1988) On the role of vestibular,vi- order dendrites in the crista ampullaris of the bullfrog. J. comp.
sual and somatosensory information for dynamic postural con- Neurol. 193, 255±264.
trol in humans. In: Vestibulospinal Control of Posture and Durham, D., Rubel, E. W. and Steel, K. P. (1989) Cochlear abla-
Locomotion, pp. 253±262. Eds. M. Pompeiano and J. H. tion in deafness mutant mice: 2-deoxyglucose analysis suggests
J. Allum. Elsevier: New York. no spontaneous activity of cochlear origin. Hear. Res. 43, 39±46.
Dodd, J., Dingledine, R. and Kelly, J. S. (1981) The excitatory Eatock, R., Corey, D. and Hudspeth, A. (1987) Adaptation of
action of acetylcholine on hippocampal neurons of the guinea mechanoelectrical transduction in hair cells of the bullfrog's sac-
pig and rat maintained in vitro. Brain Res. 207, 109±127. culus. J. Neurosci. 7, 2821±2836.
Doi, K., Kitano, I. and Mori, N. (1992) Adenylate cyclase modu- Edmonds, B. and Roberts, W. M. (1997) Calretinin is present in a
lation of endocochlear potential during suppression of strial subset of hair cells of the frog sacculus. Biophys. J. 72, W-Pos
Na+±K+ ATPase. Hear. Res. 58, 221±226. 285.
Doi, K., Mori, N. and Matsunaga, T. (1990) E€ects of forskolin Ehrenberger, K., Benkoe, E. and Felix, D. (1982) Suppressive
and 1,9 dideoxy-forskolin on cochlear potentials. Hear. Res. 45, action of picrotoxin, a GABA antagonist, on labyrinthine spon-
157±164. taneous nystagmus and vertigo in man. Acta Otolaryngol.
Dolezal, V., Lisa, V. and Tucek, S. (1997) Di€erential e€ects of the (Stockh.) 93, 269±273.
M1-M5 muscarinic acetylcholine receptor subtypes on intracellu- Elgoyhen, A. B., Johnson, D. S., Boulter, J., Vetter, D. E. and
lar calcium and on the incorporation of choline into membrane Heinemann, S. (1994) Alpha 9: an acetylcholine receptor with
lipids in genetically modi®ed chinese hamster ovary cell lines. novel pharmacological properties expressed in rat cochlear hair
Brain Res. Bull. 42, 71±78. cells. Cell 79, 705±715.
Engstrom, H., Bergstrom, B. and Ades, H. W. (1972) Macula utri-
Dolphin, A. C. (1995) Voltage-dependent calcium channels and
culi and macula sacculi in the squirrel monkey. Acta
their modulation by neurotransmitters and G-proteins. Exp.
Otolaryngol. (Stockh.) 301, 75±126.
Physiol. 80, 1±36.
Erostegui, C., Nenov, A., Norris, C. H. and Bobbin, R. P. (1994a)
Donoghue, J. P., Wenthold, R. J. and Altschuler, R. A. (1985)
Acetylcholine activates a K+ conductance permeable to Cs+ in
Localization of glutaminase-like and aspartate aminotransferase-
guinea pig outer hair cells. Hear. Res. 81, 119±129.
like immunoreactivity in neurons of the cerebral neocortex.
Erostegui, C., Norris, C. H. and Bobbin, R. P. (1994) In vitro phar-
J. Neurosci. 5, 2597±2608.
macologic characterization of a cholinergic receptor on outer
Douglas, W. W. (1965) Histamine and Antihistamines. In: The
hair cells. Hear. Res. 74, 135±147.
Pharmacological Basis of Therapeutics, pp. 615±643. Eds. L.
Erspamer, V., Roseghini, M. and Anastasi, A. (1965) Occurrence
Goodman and A. Gilman. Macmillan: New York.
and distribution of N-acetylhistidine in brain and extracerebral
Dowdall, M. J., Boyne, A. F. and Whittaker, V. P. (1974)
tissues of poikilothermal vertebrates. J. Neurochem. 12, 123±130.
Adenosine triphosphate: a constituent of cholinergic synaptic
Estes, M. S., Blanks, R. H. I. and Markham, C. H. (1975)
vesicles. Biochem. J. 140, 1±13.
Physiologic characteristics of vestibular ®rst-order canal neurons
Dowling, J. E. (1968) Synaptic organization of the frog retina: an in the cat. I. Response place determination and resting discharge
electron microscopic analysis comparing the retinas of frogs and characteristics. J. Neurophysiol. 38, 1232±1249.
primates. Proc. R. Soc. Lond. 170, 205±228. Evans, M. G. and Fuchs, P. A. (1987) Tetrodotoxin-sensitive, vol-
Dowling, J. E. (1974) Synaptic arrangements in the vertebrate ret- tage-dependent sodium currents in hair cells from the alligator
ina: the photo-receptor synapse. Soc. Gen. Physiol. Series 28, cochlea. Biophys. J. 52, 649±652.
87±103. Eybalin, M. (1993) Neurotransmitters and neuromodulators of the
Dowling, J. E. and Boycott, B. B. (1965) Neural connection of the mammalian cochlea. [Review]. Physiol. Rev. 73, 309±373.
retina: ®ne structure of the inner plexiform layer. Cold Spring Favre, D., Dememes, D. and Sans, A. (1986) Microtubule organiz-
Harbor Symposium on Quantitative Biology, Ed. L. Frisch, Vol. ation and synaptogenesis in the vestibular sensory cells. Brain
30, pp. 393±402. Res. 390, 137±142.
Drenckhahn, D., Schafer, T. and Prinz, M. (1985) Actin, myosin Favre, D. and Sans, A. (1977) Synaptogenesis of the e€erent vestib-
and associate proteins in thevertebrate auditory and vestibular ular nerve endings of the cat: ultrastructural study. Arch.
organs: immunocytochemical and biochemical studies. In: Otorhinolaryngol. 215, 183±186.
Auditory Biochemistry, pp. 317±335. Ed. D. G. Drescher. C. C. Felder, C. (1995) Muscarinic acetylcholine receptors: signal trans-
Thomas: Spring®eld, IL. duction through multiple e€ectors. FASEB Jl 9, 619±625.
Drescher, D. G. and Drescher, M. J. (1987a) Calcium and mag- Felix, D. and Ehrenberger, K. (1982) The action of putative neuro-
nesium dependence of spontaneous and evoked a€erent neural transmitter substances in the cat labyrinth. Acta Otolaryngol.
activity in the lateral-line organ of Xenopus laevis. Comp. (Stockh.) 93, 101±105.
Biochem. Physiol. A 87, 305±310. Fernandez, C. and Goldberg, J. M. (1971) Physiology of peripheral
Drescher, M. J., Drescher, A. J., Khan, K. M. and Drescher, D. G. neurons innervating semicircular canals of the squirrel monkey.
(1995a) Nucleotide sequences of cDNA for adenylyl cyclase iso- II. Response to sinusoidal stimulation and dynamics of periph-
forms expressed in mouse cochlea. Proc. Assoc. Res. eral vestibular system. J. Neurophysiol. 34, 661±675.
Otolaryngol. 18, Abstr. 141. Ferrary, E., Tran Ba Huy, P., Roinel, N., Bernard, C. and Amiel,
Drescher, M. J. and Drescher, D. G. (1987b) Amino acids, includ- C. (1988) Calcium and the inner ear ¯uids. [Review]. Acta
ing neurotransmitter candidates, in a hair cell-enriched fraction Otolaryngol. (Stockh.) 460(Suppl.), 13±17.
from the lateral line of Xenopus laevis. Comp. Biochem. Physiol. Fettiplace, R. (1987) Electrical tuning of hair cells in the inner ear.
A 86, 553±558. TINS 10, 421±425.
Drescher, M. J. and Drescher, D. G. (1991) N-acetylhistidine, glu- Fex, J. and Adams, J. C. (1978) alpha-Bungarotoxin blocks reversi-
tamate, and beta-alanine are concentrated in a receptor cell layer bly cholinergic inhibition in the cochlea. Brain Res. 159, 440±
of the trout inner ear. J. Neurochem. 56, 658±664. 444.
Drescher, M. J. and Drescher, D. G. (1992) Glutamate, of the en- Fine, B. S. (1963) Synaptic lamellas in the human retina: an elec-
dogenous primary alpha-amino acids, is speci®cally released tron microscopic study. J. Neuropath. exp. Neurol. 22, 255±262.
238 P. S. Guth et al.

Finkel, A. S. (1983) A cholinergic chloride conductance in neurones Gacek, R. R., Nomura, Y. and Balogh, K. (1965)
of helix aspersa. J. Physiol. (Lond.) 344, 119±135. Acetylcholinesterase activity in the e€erent ®bers of the stato-
Flock, A. (1965) Electron microscopic and electrophysiological stu- acoustic nerve. Acta Otolaryngol. (Stockh.) 59, 541±553.
dies on the lateral line canal organ. Acta Otolaryngol. (Stockh.) Garcia-Anoveros, J. and Corey, D. (1997) The molecules of
333(Suppl.), 1±28. mechanosensation. A. Rev. Neurosci. 20, 567±594.
Flock, A. (1974) Information transfer at the synapse between hair Garrison, J. C. (1990) Histamine, bradykinin, 5-hydroxytryptamine
cell and sensory nerve ®bers. Rheinischer-Westfaelischer Akad. and their antagonists. In: The Pharmacological Basis of
Wissenschaften 53, 347±375. Therapeutics, pp. 575±617. Eds. A. G. Gilman, J. W. Rall, A. S.
Flock, A. (1980) Contractile proteins in hair cells. Hear. Res. 2, Nies and P. Taylor. Macmillan: New York.
411±412. Geleoc, G., Lennan, G., Richardson, G. and Kros, C. (1997) A
Flock, A. and Cheung, H. (1977) Actin ®laments in sensory hairs quantitative comparison of mechanoelectrical transduction in
of the inner ear receptor cells. J. Cell Biol. 75, 339±343. vestibular and auditory hair cells of neonatal mice. Proc. R. Soc.
Flock, A. and Duvall, A. J. (1965) The ultrastructure of the kinoci- Lond. B Biol. Sci. 264, 611±621.
lium of the sensory cells in the inner ear and lateral line organs. Gibson, S. J., Polak, J. M., Bloom, S. R., Sugate, I. M., Mulderry,
J. Cell Biol. 25, 1±7. P. M., Ghatei, M. A., McGregor, G. P., Morrison, J. F. B.,
Flock, A. and Goldstein, M. H. J. (1978) Cupular movement and Kelly, J. S., Evans, R. M. and Rosenfeld, M. G. (1984)
nerve impulse response in the isolated semicircular canal. Brain Calcitonin gene-related peptide immunoreactivity in the spinal
Res. 157, 11±19. cord of man and eight other species. J. Neurosci. 4, 3101±3111.
Flock, A. and Russell, I. J. (1973) The post-synaptic action of e€er- Gillespie, P. G. (1995) Molecular machinery of auditory and vestib-
ent ®bres in the lateral line organ of the burbot Lota lota. ular transduction. Curr. Opin. Neurobiol. 5, 449±455.
J. Physiol. (Lond.) 235, 591±605. Gillespie, P. and Hudspeth, A. (1991) High-purity isolation of bull-
Flock, A. and Russell, I. J. (1976) Inhibition of e€erent nerve frog hair bundles and subcellular and topological localization of
®bres: action on hair cells and e€erent synaptic transmission in constituent proteins. J. Cell Biol. 112, 625±640.
the lateral line organ of the burbot Lota lota. J. Physiol. (Lond.) Gioglio, L., Congiu, T., Quacci, D. and Prigioni, I. (1995)
257, 45±62. Morphological features of di€erent regions in frog crista ampul-
Florey, E. (1967) Neurotransmitters and modulators in the animal laris (Rana esculenta). Arch. Histol. Cytol. 58, 1±16.
kingdom. Fed. Proc. 26, 1164±1178. Gisselson, L. (1952) The e€ect of acetylcholinesterase inhibiting
Florey, E. (1972) Comparative physiology: transmitter substances. substances on the muscles of the middle ear and on the latency
J. evolut. Biochem. Physiol. 7, 3. of cochlear potentials. Acta Otolaryngol. (Stockh.) 42, 208±218.
Fontaine, B., Klarsfeld, A., Hokfelt, J. and Changeux, J.-P. (1986) Glaum, S. R., Holzworth, J. A. and Miller, R. J. (1990) Glutamate
Calcitonin-gene related peptide, a peptide present in spinal cord receptors activate Ca++-mobilization and Ca++ in¯ux into
motoneurons, increases the number of acetylcholine receptors in astrocytes. Proc. natn. Acad. Sci. U.S.A. 87, 3454±3458.
primary cultures of chick embryo myotubes. Neurosci. Lett. 71, Gleich, O., Johnstone, B. M. and Robertson, D. (1990) E€ects of
59±65. L-glutamate on auditory a€erent activity in view of its proposed
Foster, J. D., Drescher, M. J. and Drescher, D. G. (1995) excitatory transmitter role in the mammalian cochlea. Hear. Res.
Immunohistochemical localization of GABA-A receptors in the 45, 295±311.
mammalian crista ampullaris. Hear. Res. 83, 203±208. Gleisner, L., Flock, A. and Wersall, J. (1973) The ultrastructure of
Fredholm, B. B., Abbrachio, M. P., Burnstock, G., Daly, J. W., the a€erent synapse on hair cells in the frog labyrinth. Acta
Harden, T. K., Jacobson, K. A., Le€, P. and Williams, M. (1994) Otolaryngol. (Stockh.) 76, 199±207.
Nomenclature and classi®cation of purinoceptors. Pharmac. Rev. Glowatzki, E., Fakler, G., Brandle, U., Rexhausen, U., Zenner, H.,
46, 143±156. Ruppersberg, J. and Fakler, B. (1995) Subunit-dependent assem-
Fredholm, B. B. and Hedqvist, P. (1980) Modulation of neuro- bly of inward-recti®er K+ channels. Proc. R. Soc. Lond. B Biol.
transmission by purine nucleotides and nucleosides. Biochem. Sci. 261, 251±261.
Pharmac. 29, 1635±1643. Godfrey, D. A., Park, J. L. and Ross, C. D. (1984) Choline acetyl-
Fuchs, P. (1996) Synaptic transmission at vertebrate hair cells. transferase and acetylcholinesterase in centrifugal labyrinthine
Curr. Opin. Neurobiol. 6, 514±519. bundles of rats. Hear. Res. 14, 93±106.
Fuchs, P. A. and Evans, M. G. (1988) Voltage oscillations and Godfrey, D. A., Wiet, G. J. and Ross, C. D. (1986) Quantitative
ionic conductances in hair cells isolated from the alligator histochemistry of the cochlea. In: Neurobiology of Hearing: the
cochlea. J. comp. Physiol. [A] 164, 151±163. Cochlea, pp. 149±157. Eds. R. A. Altschuler, D. W. Ho€man
Fuchs, P. A., Evans, M. G. and Murrow, B. W. (1990) Calcium and R. P. Bobbin. Raven Press: New York.
currents in hair cells isolated from the cochlea of the chick. Goldberg, J. (1996) Transmission between the type I hair cell and
J. Physiol. (Lond.) 429, 553±568. its calyx ending. Ann. N.Y. Acad. Sci. 781, 474±488.
Fuchs, P. A. and Murrow, B. W. (1992a) Cholinergic inhibition of Goldberg, J. M. (1979) Vestibular receptors in mammals: a€erent
short (outer) hair cells of the chick's cochlea. J. Neurosci. 12, discharge characteristics and e€erent control. Prog. Brain Res.
800±809. 50, 355±367.
Fuchs, P. A. and Murrow, B. W. (1992b) A novel cholinergic Goldberg, J. M. and Fernandez, C. (1980) E€erent vestibular sys-
receptor mediates inhibition of chick cochlear hair cells. Proc. R. tem in the squirrel monkey: anatomical location and in¯uence
Soc. Lond. B Biol. Sci. 248, 35±40. on a€erent activity. J. Neurophysiol. 43, 986±1025.
Fuchs, P. A., Nagai, T. and Evans, M. G. (1988) Electrical tuning Goldberg, J. M. and Fernandez, C. (1984) The Vestibular System.
in hair cells isolated from the chick cochlea. J. Neurosci. 8, In: Handbook of Physiology: The Nervous System: Sensory
2460±2467. Systems Part III, Ed. I. Darian-Smith, pp. 977±1022, Bethesda,
Fujita, S., Usami, S., Shinkawa, H., Sato, K., Kiyama, H. and MD.
Tohyama, M. (1994) Expression of NMDA receptors subunit Gonzalez, A., Roberts, B. L. and Meredith, G. E. (1993) Choline
mRNA in the vestibular ganglion of the rat and guinea pig. acetyltransferase immunoreactive neurons innervating labyr-
Neuroreport 5, 862±864. inthine and lateral line sense organs in amphibians. J. comp.
Furukawa, T. (1981) E€ects of e€erent stimulation on the saccule Neurol. 332, 258±268.
of the gold®sh. J. Physiol. (Lond.) 315, 203±215. Goodman, M. and Art, J. (1996a) Positive feedback by a potass-
Fykse, E. M. and Fonnum, F. (1996) Amino acid neurotrans- ium-selective inward recti®er enhances tuning in vertebrate hair
mission: dynamics of vesicular uptake. Neurochem. Res. 21, cells. Biophys. J. 71, 430±442.
1053±1060. Goodman, M. and Art, J. (1996b) Variations in the ensemble of
Gacek, R. R. (1969) The course and central termination of ®rst potassium currents underlying resonance in turtle hair cells.
order neurons supplying vestibular end organs in the cat. Acta J. Physiol. (Lond.) 497(Part 2), 395±412.
Otolaryngol. (Stockh.) 254(Suppl.), 1±66. Gordon, J. L. (1986) Extracellular ATP: e€ects, sources and fate.
Gacek, R. R. and Lyon, M. (1974) Localization of vestibular e€er- Biochem. J. 233, 309±319.
ent neurons in the kitten with horseradish peroxidase. Acta Gray, E. G. and Pease, H. L. (1971) On understanding the organiz-
Otolaryngol. (Stockh.) 77, 92±1013. ation of the retinal receptor synapses. Brain Res. 35, 1±15.
The Vestibular Hair Cells 239

Green, G., Khan, K., Beisel, D., Drescher, M., Hat®eld, J. and Hartmann, R. and Klinke, R. (1980) E€erent activity in the gold-
Drescher, D. (1996) Calcium channel subunits in the mouse ®sh vestibular nerve and its in¯uence on a€erent activity.
cochlea. J. Neurochem. 67, 37±45. P¯ugers Arch. 388, 123±128.
Grenningloh, G., Schmieden, V., Scho®eld, P., Seeburg, P., Heidelberger, R., Heinemann, C., Neher, E. and Matthews,
Siddique, T., Mohandas, T., Becker, C. M. and Betz, H. (1990) G. (1994) Calcium dependence of the rate of exocytosis in a
Alpha subunit variants of the human glycine receptor: primary synaptic terminal. Nature 371, 513±515.
structures, functional expression and chromosomal localization Herrero, I., Miras-Portugal, M. T. and Sanchez-Prieto, J. (1992)
of the corresponding gene. EMBO Jl 9, 771±776. Positive feedback of glutamate exocytosis by metabotropic pre-
Griguer, C., Chabbert, C., Sans, A. and Lehouelleur, J. (1995) synaptic receptor stimulation. Nature 360, 163±165.
Transient increase in cytosolic free calcium evoked by repolari- Hiel, H., Elgoyhen, A. B., Drescher, D. G. and Morley, B. J. (1996)
zation in Type I vestibular hair cells of rats. Cell Calcium 17, Expression of nicotinic acetylcholine receptor mRNA in the
327±334. adult rat peripheral vestibular system. Brain Res. 738, 347±352.
Griguer, C., Kros, C. J., Sans, A. and Lehouelleur, J. (1993) Highstein, S., Rabbitt, R. and Boyle, R. (1996) Determinants of
Potassium currents in type II vestibular hair cells isolated from semicircular canal a€erent response dynamics in the toad®sh,
the guinea-pig's crista ampullaris [published erratum appears in Opsanus tau. J. Neurophysiol. 75, 575±596.
P¯ugers Archives 1994 May; 427(1±2): 193]. P¯ugers Arch. 425, Highstein, S. M. (1991) The central nervous system e€erent control
344±352. of the organs of balance and equilibrium. [Review]. Neurosci.
Res. 12, 13±30.
Guiramand, J., Mayat, E., Bartolami, S., Lenoir, M., Rumigny,
Highstein, S. M. (1992) The e€erent control of the organs of bal-
J. F., Pujol, R. and Recasens, M. (1990) A M3 muscarinic recep-
ance and equilibrium in the toad®sh, Opsanus tau. Ann. N.Y.
tor coupled to inositol phosphate formation in the rat cochlea?
Acad. Sci. 656, 108±123.
Biochem. Pharmac. 39, 1913±1919.
Highstein, S. M. and Baker, R. (1985) Action of the e€erent vestib-
Guth, P. S. and Amaro, J. (1969) A possible cholinergic link in
ular system on primary a€erents in the toad®sh, Opsanus tau.
olivo-cochlear inhibition. Int. J. Neuropharm. 8, 49±57.
J. Neurophysiol. 54, 370±384.
Guth, S. L. and Norris, C. H. (1984) Pharmacology of the isolated Highstein, S. M. and Baker, R. (1986) Organization of the e€erent
semicircular canal: e€ect of GABA and picrotoxin. Exp. brain vestibular nuclei and nerves of the toad®sh, Opsanus tau.
Res. 56, 72±78. J. comp. Neurol. 243, 309±325.
Guth, P. S., Aubert, A., Ricci, A. J. and Norris, C. H. (1991) Hilding, D. and Wersall, J. (1962) Cholinesterase and its relation to
Di€erential modulation of spontaneous and evoked neurotrans- the nerve endings in the inner ear. Acta Otolaryngol. (Stockh.)
mitter release from hair cells: some novel hypotheses. Hear. Res. 55, 205±217.
56, 69±78. Hille, B. (1992) Ionic Channels of Excitable Membranes. Sinauer:
Guth, P. S., Dunn, A., Kronomer, K. and Norris, C. H. (1994a) Sunderland, MA.
The cholinergic pharmacology of the frog saccule. Hear. Res. 75, Hille, B. (1994) Modulation of ion-channel function by G-protein-
225±232. coupled receptors. TINS 17, 531±536.
Guth, P. S., Fermin, C. D., Pantoja, M., Edwards, R. and Norris, Hillman, D. (1974) Cupular structure and its receptor relationship.
C. H. (1994b) Hair cells of di€erent shapes and their placement Brain Behav. Evol. 10, 52±68.
along the frog crista ampullaris. Hear. Res. 73, 109±115. Hillman, D. E. (1976) Vestibular and lateral line system 14.
Guth, P. S., Norris, C. H. and Barron, S. E. (1988) Three tests of Morphology of peripheral and central vestibular systems. In:
the hypothesis that glutamate is the sensory hair cell transmitter Frog Neurobiology, pp. 452±480. Eds. R. Llinas and W. Precht.
in the frog semicircular canal. Hear. Res. 33, 223±228. Springer-Verlag: Berlin.
Guth, P. S., Norris, C. H. and Bobbin, R. P. (1976) The pharma- Hillman, D. E. and Lewis, E. R. (1971) Morphological basis for a
cology of transmission in the peripheral auditory system. mechanical linkage in otolithic receptor transduction in the frog.
[Review]. Pharmacol. Rev. 28, 95±125. Science 174, 416±419.
Guth, P. S., Norris, C. H., Guth, S. L., Quine, D. B. and Williams, Hollmann, M. and Heinemann, S. (1994) Cloned glutamate recep-
W. H. (1986) Cholinomimetics mimic e€erent e€ects on semicir- tors. A. Rev. Neurosci. 17, 31±108.
cular canal a€erent activity in the frog. Acta Otolaryngol. Holt, J. R. and Eatock, R. A. (1995) Inwardly rectifying currents
(Stockh.) 102, 194±203. of saccular hair cells from the leopard frog. J. Neurophysiol. 73,
Guth, P. S., Norris, C. H. and Sewell, W. F. (1985) Primary 1484±1502.
A€erent Transmission in Acousticolateralis Organs, pp. 42±49. Holton, F. A. and Holton, P. (1954) The capillary dilator sub-
Ed. D. G. Drescher. C. C. Thomas: Spring®eld, IL. stances in dry powders of spinal roots; a possible role of adeno-
Guth, P. S., Pantoja, M. and Norris, C. H. (1996) Evidence that sine triphosphate in chemical transmission from nerve endings.
chloride may be involved in the suppressive e€ect of acetyl- J. Physiol. (Lond.) 126, 124±140.
choline in frog vestibular organs. Proc. Assoc. Res. Otolaryngol. Holton, P. (1959) The liberation of adenosine triphosphate on anti-
19, 398. dromic stimulation of sensory nerves. J. Physiol. (Lond.) 145,
494±504.
Guth, P. S., Pantoja, M., Norris, C. H. and Fermin, C. D. (1993)
Honrubia, V., Ho€man, L. F., Sitko, S. and Schwartz, I. R. (1989)
The correlated blanching of synaptic bodies and reduction in
Anatomic and physiological correlates in bullfrog vestibular
a€erent ®ring rates caused by transmitter-depleting agents in the
nerve. J. Neurophysiol. 61, 688±701.
frog semicircular canal. Hear. Res. 66, 143±149.
Honrubia, V., Sitko, S., Lee, R., Kuruvilla, A. and Schwartz, I.
Guth, P. S. and Stockwell, M. (1977) Guanylate cyclase and cyclic
R. (1984) Anatomical characteristics of the anterior vestibular
guanosine monophosphate in the guinea pig cochlea.
nerve of the bullfrog. Laryngoscope 94, 464±474.
J. Neurochem. 28, 263±270.
Hoshino, T. (1975) An electron microscopic study of the otolithic
Hackney, C. and Furness, D. (1995) Mechanotransduction in ver- maculae of the lamprey (Entosphenus japonicus). Acta
tebrate hair cells: structure and function of the stereociliary bun- Otolaryngol. (Stockh.) 80, 43±53.
dle. Am. J. Physiol. 268, C1±13. Housley, G. D. and Ashmore, J. F. (1991) Direct measurement of
Halliwell, J. V. and Adams, P. R. (1982) Voltage-clamp analysis of the action of acetylcholine on isolated outer hair cells of the gui-
muscarinic excitation in hippocampal neurons. Brain Res. 250, nea pig cochlea. Proc. R. Soc. Lond. B Biol. Sci. 244, 161±167.
71±92. Housley, G. D., Batcher, S., Kraft, M. and Ryan, A. F. (1994)
Hama, K. (1965) Some observations on the ®ne structure of the lat- Nicotinic acetylcholine receptor subunits expressed in rat cochlea
eral line organ of the Japanese sea eel, Lyncozymba nustromi. detected by the polymerase chain reaction. Hear. Res. 75, 47±53.
J. Cell Biol. 24, 193±210. Housley, G. D., Greenwood, D. and Ashmore, J. F. (1992)
Hamilton, D. W. (1969) The cilium on mammalian vestibular hair Localization of cholinergic and purinergic receptors on outer
cells. Anatom. Record 164, 253±258. hair cells isolated from the guinea-pig cochlea. Proc. R. Soc.
Harper, A., Blythe, W. R., Grossman, G., Petrusz, P., Prazma, Lond. B Biol. Sci. 249, 265±273.
J. and Pillsbury, H. C. (1995) Immunocytochemical localization Housley, G. D., Norris, C. H. and Guth, P. S. (1988) Histamine
of aspartate and glutamate in the peripheral vestibular system. and related substances in¯uence neurotransmission in the semi-
Hear. Res. 86, 171±182. circular canal. Hear. Res. 35, 87±97.
240 P. S. Guth et al.

Housley, G. D., Norris, C. H. and Guth, P. S. (1989) Progress in Brain Research, Vol. 37, pp. 429±443. Eds. A. Brodal
Electrophysiological properties and morphology of hair cells iso- and O. Pompeiano. Elsevier: Amsterdam.
lated from the semicircular canal of the frog. Hear. Res. 38, Jackson, R. T. and Turner, J. S. (1987) Astemizole: its use in the
259±276. treatment of patients with chronic vertigo. Arch. Otolaryngol.
Housley, G. D., Norris, C. H. and Guth, P. S. (1990) Head Neck Surg. 113, 536±542.
Cholinergically-induced changes in outward currents in hair cells Jaramillo, F. and Hudspeth, A. (1991) Localization of the hair
isolated from the semicircular canal of the frog. Hear. Res. 43, cell's transduction channels at the hair bundle's top by ionto-
121±133. phoretic application of a channel blocker. Neuron 7, 409±420.
Housley, G. D. and Ryan, A. F. (1997) Cholinergic and purinergic Jasser, S. and Guth, P. S. (1973) The synthesis of acetylcholine by
neurohumoral signalling in the inner ear: a molecular physiologi- the olivo-cochlear bundle. J. Neurochem. 20, 45±53.
cal analysis. Audiol. Neurootol. 2, 92±110. Jastrow, H., Von Mach, M. A. and Vollrath, L. (1997) The shape
Howard, J. and Hudspeth, A. (1987) Mechanical relaxation of the of synaptic ribbons in the rat pineal gland. Cell Tissue Res. 287,
hair bundle mediates adaptation in mechanoelectrical transduc- 255±261.
tion by the bullfrog's saccular hair cell. Proc. natn. Acad. Sci. Jin, H. (1992) Immunohistochemical studies on the guinea pig's
U.S.A. 84, 3064±3068. vestibular ganglion cellÐwith reference to the distribution of
Howard, J., Roberts, W. and Hudspeth, A. (1988) substance P and neuro®lament. Nippon Jibiinkoka Gakkai Kaiho
Mechanoelectrical transduction by hair cells. A. Rev. Biophys. 95, 391±399in Japanese.
Biophys. Chem. 17, 99±124. Jones, P. G. and Roberts, P. J. (1990) Ibotenate stimulates gluta-
Hu, G. Y. and Storm, J. F. (1991) Excitatory amino acids acting mate release from guinea pig cerebrocortical synaptosomes: inhi-
on metabotropic glutamate receptors broaden the action poten- bition by L-AP-4. Neurosci. Lett. 111, 228±232.
tial in hippocampal neurons. Brain Res. 568, 339±344. Jorgensen, F. and Kroese, A. B. A. (1994) Ionic selectivity of the
Hudspeth, A. (1983) Mechanoelectrical transduction by hair cells mechano-electrical transduction channels in the hair cells of the
in the acousticolateralis sensory system. A. Rev. Neurosci. 6, frog sacculus. Acta Physiol. Scand. 151, 7±16.
187±215. Jorgensen, J. M. (1974) The sensory epithelia of the inner ear of
Hudspeth, A. (1986) The ionic channels of a vertebrate hair cell. two turtles, Testudo graeca L. and Pseudemys scripta (Schoep€).
Hear. Res. 22, 21±27. Acta Zool. 55, 289±298.
Hudspeth, A. (1989) How the ear's works work. Nature 341, 397± Juusola, M., French, A. S., Uusitalo, R. O. and Weckstrom,
404. M. (1996) Information processing by graded-potential trans-
Hudspeth, A. and Corey, D. (1977) Sensitivity, polarity, and con- mission through tonically active synapses. TINS 19, 292±297.
ductance change in the response of vertebrate hair cells to con- Kakehata, S., Nakagawa, T., Takasaka, T. and Akaike, N. (1993)
trolled mechanical stimuli. Proc. natn. Acad. Sci. U.S.A. 74, Cellular mechanism of acetylcholine-induced response in disso-
2407±2411. ciated outer hair cells of guinea-pig cochlea. J. Physiol. (Lond.)
Hudspeth, A. and Gillespie, P. (1994) Pulling springs to tune trans- 463, 227±244.
duction: adaptation by hair cells. Neuron 12, 1±9. Kanai, Y., Smith, C. P. and Hediger, M. A. (1993) The elusive
Hudspeth, A. J. and Jacobs, R. (1979) Stereocilia mediate transduc- transporters with high anity for glutamate. TINS 16, 365±370.
tion in vertebrate hair cells. Proc. natn. Acad. Sci. U.S.A. 76, Karlin, A. and Akabas, M. H. (1995) Toward a structural basis for
1506±1509. the function of nicotinic acetylcholine receptors and their cou-
Hudspeth, A. J. and Lewis, R. S. (1988) Kinetic analysis of voltage- sins. Neuron 15, 1231±1244.
and ion-dependent conductances in saccular hair cells of the Karlsen, H. E. (1992) Infrasound sensitivity in the plaice
bullfrog Rana catesbeiana. J. Physiol. (Lond.) 400, 237±274. (Pleuronectes platessa). J. exp. Biol. 171, 173±187.
Hunter, T. (1995) Protein kinases and phosphatases: the yin and Kataoka, Y. and Ohmori, H. (1996) Of known neurotransmitters,
yang of protein phosphorylation and signaling. Cell 80, 225±236. glutamate is the most likely to be released from chick cochlear
Hunter-Duvar, I. M. and Hinojosa, R. (1984) Vestibule: Sensory hair cells. J. Neurophysiol. 76, 1870±1879.
Epithelia. In: Ultrastructural Atlas of the Inner Ear, pp. 211±244. Kawasaki, K., Yamamoto, A., Omori, K., Iwano, T., Kumazawa,
Eds. I. Friedmann and J. Ballantyne. Butterworth and Co.: T. and Tashiro, Y. (1992) Quantitative immunoelectron micro-
London. scopic localization of Na, K±ATPase alpha-subunit in the epi-
Ichimiya, I., Adams, J. C. and Kimura, R. S. (1994) thelial cells of rat vestibular apparatus. Hear. Res. 60, 64±72.
Immunolocalization of Na + , K(+)±ATPase, Ca(++)± Kehoe, J. (1972) Three acetylcholine receptors in Aplysia neurons.
ATPase, calcium-binding proteins, and carbonic anhydrase in J. Physiol. (Lond.) 225, 115±146.
the guinea pig inner ear. Acta Otolaryngol. (Stockh.) 114, 167± Kellog, R. S., Kennedy, R. S. and Graybiel, A. (1965) Motion sick-
176. ness symptomatology of labyrinthine defective and normal sub-
Ikeda, K., Saito, Y., Nishiyama, A. and Takasaka, T. (1991) E€ect jects during zero-gravity maneuvers. Aerospace Med. 36, 313±
of neuroregulators on the intracellular calcium level in the outer 318.
hair cell isolated from the guinea pig. Oto-Rhino-Laryngology Klinke, R. and Galley, N. (1974) E€erent innervation of vestibular
53, 78±81. and auditory receptors. Physiol. Rev. 54, 316±357.
Ishiyama, A., Lopez, I. and Wackym, P. A. (1994a) Choline acetyl- Klinke, R. and Oertel, W. (1977) Evidence that GABA is not the
transferase immunoreactivity in the human vestibular end-organ. a€erent transmitter in the cochlea. Exp. brain Res. 28, 311±314.
Cell Biol. Int. 18, 979±984. Koch, T. and Zenner, H. P. (1988) Adenylate cyclase and G-pro-
Ishiyama, A., Lopez, I. and Wackym, P. A. (1994b) Subcellular in- teins as a signal transfer system in the guinea pig inner ear.
nervation patterns of the calcitonin gene-related peptidergic Arch. Otorhinolaryngol. 245, 82±87.
e€erent terminals in the chinchilla vestibular periphery. Kolesnikov, S. S., Rebrik, T. I., Zhainazarov, A. B., Tavartkiladze,
Otolaryngol. Head Neck Surg. 111, 385±395. G. A. and Kalamkarov, G. R. (1991) A cyclic-AMP-gated con-
Issa, N. and Hudspeth, A. (1994) Clustering of Ca2+ channels and ductance in cochlear hair cells. FEBS Lett. 290, 167±170.
Ca(2+)-activated K+ channels at ¯uorescently labeled presyn- Kong, W.-J., Egg, G., Hussl, B., Spoendlin, H. and Schrott-
aptic active zones of hair cells. Proc. natn. Acad. Sci. U.S.A. 91, Fischer, A. (1994) Localization of Chat-like immunoreactivity in
7578±7582. the vestibular endorgans of the rat. Hear. Res. 75, 191±200.
Iurato, S., Luciano, L., Pannese, E. and Reale, E. (1971a) Kongrga, I. and Randic, M. (1990) Tachykinins and calcitonin-
Acetylcholinesterase activity in the vestibular sensory areas. Acta gene related peptide enhance release of endogenous glutamate
Otolaryngol. (Stockh.) 71, 147±152. and aspartate from the rat spinal dorsal horn slice. J. Neurosci.
Iurato, S., Luciano, L., Pannese, E. and Reale, E. (1971b) 10, 2026±2038.
Histochemical localization of acetylcholinesterase (AChE) ac- Koyama, H., Lewis, E. R., Leverenz, E. L. and Baird, R. A. (1982)
tivity in the inner ear. Acta Otolaryngol. (Stockh.) 279(Suppl.), Acute seismic sensitivity in the bullfrog ear. Brain Res. 250, 168±
1±50. 172.
Iurato, S., Luciano, L., Pannese, E. and Reale, E. (1972) E€erent Krnjevic, K. (1993) Central cholinergic mechanisms and function.
vestibular ®bres in mammalia: morphological and histochemical In: Cholinergic Function and Dysfunction, pp. 285±292. Ed. A. C.
aspects. In: Basic Aspects of Central Vestibular Mechanisms, Cuello. Elsevier: Amsterdam.
The Vestibular Hair Cells 241

Kros, C., Rusch, A. and Richardson, G. (1992) Mechano-electrical Lopez, I. and Meza, G. (1990) Comparative studies on glutamate
transducer currents in hair cells of the cultured neonatal mouse decarboxylase and choline acetyltransferase activities in the ver-
cochlea. Proc. R. Soc. Lond. B Biol. Sci. 249, 185±193. tebrate vestibule. Comp. Biochem. Physiol. [B] 95, 375±379.
Kujawa, S. G., Erostegui, C., Fallon, M., Crist, J. and Bobbin, R. Lopez, I., Wu, J. Y. and Meza, G. (1992) Immunocytochemical evi-
P. (1994a) E€ects of adenosine 5'-triphosphate and related ago- dence for an a€erent GABAergic neurotransmission in the gui-
nists on cochlear function. Hear. Res. 76, 87±100. nea pig vestibular system. Brain Res. 589, 341±348.
Kujawa, S. G., Fallon, M. and Bobbin, R. P. (1994b) ATP antag- Lorente de No, R. (1926) Etudes sur l'anatomie et la physiologis
onists cibacron blue, basilen blue and suramin alter sound- du labyrinth de l'oreille et du VIIIe nerf. Deuxieme partie.
evoked responses of the cochlea and auditory nerve. Hear. Res. Quelqes donnees au sujet de l'anatomie de organes sensoriels du
78, 181±188. labyrinthe. Trabajares Lab. Invest. Biol. Universidad Madrid 24,
Kuruvilla, A., Sitko, S., Schwartz, I. R. and Honrubia, V. (1985) 53±153.
Central projections of primary vestibular ®bers in the bullfrog. I. Lowenstein, O. and Wersall, J. (1959) A functional interpretation
The vestibular nuclei. Laryngoscope 95, 692±707. of the electron microscopic structure of the sensory hairs in the
Lagnado, L., Gomis, A. and Job, C. (1996) Continuous vesicle elasmobranch Raja clavata in terms of directional sensitivity.
cycling in the synaptic terminal of retinal bipolar cells. Neuron Nature 184, 1807.
17, 957±967. Lues, G. (1971) Die Feinstruktur der zirbeldense normaler, trachti-
Lancaster, B. and Adams, P. R. (1986) Calcium-dependent current ger und experimenteller beein¯uster Meerschweinchen.
generating the after-hyperpolarization of hippocampal neurons. Zeitschrift Zellforschung Mikroskopische Anat. 114, 38±60.
J. Neurophysiol. 55, 1268±1282. Lustig, K. D., Shiau, A. K., Brake, A. J. and Julius, D. (1993)
Landolt, J. P. and Correia, M. J. (1980) Neurodynamic response Expression cloning of an ATP receptor from mouse neuroblas-
analysis of anterior semicircular canal a€erents in the pigeon. toma cells. Proc. natn. Acad. Sci. U.S.A. 90, 5113±5117.
J. Neurophysiol. 43, 1746±1770. Madison, D. V., Lancaster, B. and Nicoll, R. A. (1987) Voltage
Lanford, P. J. and Popper, A. N. (1995) Calyx-like a€erents in the clamp analysis of cholinergic action in the hippocampus.
crista ampullaris of the gold®sh, Carassius auratus. Proc. Assoc. J. Neurosci. 7, 733±741.
Res. Otolaryngol. 18, 100. Maggio, E. (1966) The humoral system of the labyrinth. Acta
Lang, D. G. and Correia, M. J. (1989) Studies of solitary semicir- Otolaryngol. (Stockh.) 218(Suppl.), 1±35.
cular canal hair cells in the adult pigeon. II. Voltage-dependent Mandell, J. W., Townes-Anderson, E., Czernik, A. J., Cameron,
ionic conductances. J. Neurophysiol. 62, 935±945. R., Greengard, P. and De Camilli, P. (1990) Synapsins in the
Lapeyre, P. N., Kolston, P. J. and Ashmore, J. F. (1993) GABAB- vertebrate retina: absence from ribbon synapses and hetero-
mediated modulation of ionic conductances in type I hair cells geneous distribution among conventional synapses. Neuron 5,
isolated from guinea-pig semicircular canals. Brain Res.. 609, 19±33.
269±276. Martin, A. R. and Fuchs, P. A. (1992) The dependence of calcium-
Laufer, R. and Changeux, J.-P. (1987) Calcitonin-gene related pep- activated potassium currents on membrane potential. Proc. R.
tide elevates cyclic AMP levels in chick skeletal muscle: possible Soc. Lond. B Biol. Sci. 250, 71±76.
neurotrophic role for a coexisting neuronal messenger. EMBO Jl Masetto, S. and Correia, M. (1997) Electrophysiological properties
6, 901±906. of vestibular sensory and supporting cells in the labirynth slice:
Lenzi, D. and Roberts, W. (1994) Calcium signalling in hair cells: before and during regeneration. J. Neurophysiol. 78, 1913±1927.
multiple roles in a compact cell. Curr. Opin. Neurobiol. 4, 496± Masetto, S., Perin, P., Botta, L., Zucca, G. and Valli, P. (1995a)
502. Mechanisms for sensory adaptation in frog vestibular organs.
Lewis, E. R., Hecht, E. I. and Narins, P. M. (1992) Diversity of Neuroreport 7, 230±232.
form in the amphibian papilla of puerto rican frogs. J. comp. Masetto, S., Perin, P., Milesi, V., Botta, L., Zucca, G. and Valli,
Physiol. A 171, 421±435. P. (1995b) E€ects of ion channel blockers on frog semicircular
Lewis, E. R. and Li, C. W. (1975) Hair cell types and distributions canal. A comparison between whole organ and single cell study.
in the otolithic and auditory organs of the bullfrog. Brain Res. Primary Sensory Neuron 1, 129±142.
83, 35±50. Masetto, S., Russo, G. and Prigioni, I. (1994) Di€erential ex-
Lewis, R. S. and Hudspeth, A. J. (1983) Voltage- and ion-depen- pression of potassium currents by hair cells in thin slices of frog
dent conductances in solitary vertebrate hair cells. Nature 304, crista ampullaris. J. Neurophysiol. 72, 443±455.
538±541. Matsubara, A., Usami, S.-I., Fujita, S. and Shinkawa, H. (1995)
Lim, D. J. (1979) Fine morphology of the otoconial membrane and Expression of substance P,CGRP, and GABA in the vestibular
its relationship to the sensory epithelium. Scanning Electron periphery, with special reference to species di€erences. Acta
Microscopy 3, 929±938. Otolaryngol. (Stockh.) 519(Suppl.), 248±252.
Liu, J. and Wangemann, P. (1997) K+ secretion in vestibular dark Matsusaka, T. (1967) Lamellar bodies in the synaptic cytoplasm of
cells and strial marginal cells from the gerbil is stimulated via the accessory cone from chick retina as revealed by electron mi-
beta-1 adrenergic but not vasopressin receptors. Abstracts of the croscopy. J. Utrastructural Res. 18, 55±70.
20th ARO Midwinter Research Meeting, 688, Ed. G. Popelka, St. Mayer, M. L. and Westbrook, G. L. (1983) A voltage-clamp analy-
Petersburg. sis of inward (anomalous) recti®cation in mouse spinal sensory
Llinas, R. and Precht, W. (1969) The inhibitory vestibular e€erent ganglion neurones. J. Physiol. (Lond.) 340, 19±45.
system and its relation to the cerebellum in the frog. Exp. brain Mbiene, J. P. and Sans, A. (1986) Di€erentiation and maturation
Res. 9, 16±29. of the sensory hair bundles in the fetal and postnatal vestibular
Locke, R. E. and Highstein, S. M. (1989) E€erent modulation of receptors of the mouse: a scanning electron microscopy study.
synaptic noise and spike frequency in lagenar a€erents of the J. comp. Neurol. 254, 271±278.
toad®sh. Proc. Assoc. Res. Otolaryngol., Abstr. 99. McAfee, D. A. and Henon, B. K. (1986) Adenosine and ATP in
Locke, R. E. and Highstein, S. M. (1990) E€erent modulation of neurotransmitter actions. In: The Vertebrate Nervous System,
synaptic noise in lagenar a€erents of the toad®sh. Neurosci. pp. 481±502. Eds. M. A. Rogowski and J. L. Barker. Plenum
Abstr. 16, 735. Press: New York.
Loewi, O. (1921a) Ueber humorale uebertragbarkeit der McCue, M. P. and Guinan, J. J. J. (1995) Spontaneous activity and
Herznervenwirkung (I mitteilung.). P¯ugers Arch. gesamte frequency selectivity of acoustically responsive vestibular a€er-
Physiol. 193, 239±242. ents in the cat. J. Neurophysiol. 74, 1563±1572.
Loewi, O. (1921b) Ueber humorale uebertragbarkeit der Mees, K. (1984) Cytochemical localization of adenylate cyclase in
Herznervenwirkung (II mitteilung). P¯ugers Arch. gesamte the lateral wall of the inner ear. Arch. Otorhinolaryngol. 240, 55±
Physiol. 193, 201±213. 61.
Lopez, I., Juiz, J. M., Altschuler, R. A. and Meza, G. (1990) Mennerick, S. and Matthews, G. (1996) Ultrafast exocytosis eli-
Distribution of GABA-like immunoreactivity in guinea pig ves- cited by calcium current in synaptic terminals of retinal bipolar
tibular cristae ampullaris. Brain Res. 530, 170±175. neurons. Neuron 17, 1241±1249.
Lopez, I. and Meza, G. (1988) Neurochemical evidence for a€erent Meredith, G. E. and Roberts, B. L. (1986) Central organization of
GABAergic and e€erent cholinergic neurotransmission in the the e€erent supply to the labyrinth and lateral line receptors of
frog vestibule. Neuroscience 25, 13±18. the dog®sh. Neuroscience 17, 225±233.
242 P. S. Guth et al.

Meunier, F.-M., Israel, M. and Lesbats, B. (1975) Release of ATP Nakagawa, T., Komune, S., Uemura, T. and Akaike, N. (1991)
from stimulated nerve electroplaque junctions. Nature 257, 407± Excitatory amino acid response in isolated spiral ganglion cells
409. of guinea pig cochlea. J. Neurophysiol. 65, 715±723.
Meza, G., Gonzalez-Viveros, M. T. and Ruiz, M. (1985) Speci®c Neely, A., Wei, X., Olcese, R., Birnbaumer, L. and Stefani,
[3H]gamma-aminobutyric acid binding to vestibular membranes E. (1993) Potentiation by the beta subunit of the ratio of the
of the chick inner ear. Brain Res 337, 179±183. ionic current to the charge movement in the cardiac calcium
Meza, G., Hernandez, C. and Ruiz, M. (1981a) 3H-GABA uptake channel. Science 262, 575±578.
in isolated vestibullary cristae of chick inner ear. Soc. Neurosci. Neer, E. J. (1995) Heterotrimeric G proteins: organizers of trans-
Abstr. 7, 147. membrane signals. Cell 80, 249±257.
Meza, G., Lopez, I., Paredes, M. A., Penaloza, Y. and Poblano, New, H. V. and Mudge, A. W. (1986) Calcitonin gene-related pep-
A. (1989) Cellular target of streptomycin in the internal ear. tide regulates muscle acetylcholine receptor synthesis. Nature
Acta Otolaryngol. (Stockh.) 107, 406±411. 323, 809±811.
Meza, G., Lopez, I. and Ruiz, M. (1984) Possible cholinergic Niedzielski, A. S. and Schacht, J. (1991) Phospholipid metabolism
neurotransmission in the cristae ampullares of the chick inner in the cochlea: di€erences between base and apex. Hear. Res. 57,
ear. Neurosci. Lett. 49, 93±97. 107±112.
Meza, G., Ruiz, M. and Cuadros, P. (1981b) GABA synthesis on Niedzielski, A. S. and Schacht, J. (1992) P2 purinoceptors stimulate
isolated ampullar cristae of chick inner ear. Trans. Am. Soc. inositol phosphate release in the organ of Corti. Neuroreport 3,
Neurochem. 12, 256. 273±275.
Meza, G., Solano-Flores, L. P. and Poblano, A. (1992) Recovery Niedzielski, A. S. and Wenthold, R. J. (1995) Expression of
of vestibular function in young guinea pigs after streptomycin AMPA, kainate and NMDA receptor subunits in cochlear and
treatment. Glutamate decarboxylase activity and nystagmus re- vestibular ganglia. J. Neurosci. 15, 2338±2353.
sponse assessment. Int. J. Dev. Neurosci. 10, 407±411. Nillson, S. E. G. and Crescitelli, F. (1970) A correlation of ultra-
Michelson, M. J. (1974) Some aspects of evolutionary pharma- structure and function in the developing retina of the frog tad-
cology. Biochem. Pharmac. 23, 2211±2224. pole. J. ultrastruct. Res. 30, 87±102.
Miller, A. M. and Schwartz, E. A. (1983) Evidence for the identi®- Norris, C., Ricci, A., Housley, G. and Guth, P. (1992) The inacti-
cation of synaptic transmitters released by photoreceptors of the vating potassium currents of hair cells isolated from the crista
toad retina. J. Physiol. (Lond.) 334, 325±349. ampullaris of the frog. J. Neurophysiol. 68, 1642±1653.
Missotten, L. (1960) Etude des synapses de la retine humaine au Norris, C. H., Aubert, A. and Shea, J. J. (1994) Comparison of
microscope electronique. Ophthalmologica 140, 200±214. cochleotoxicity and vestibulotoxicity of streptomycin and genta-
Mizuta, K., Iwasa, K., Simonds, W. and Tachibana, M. (1995) micin. In: eniere's Disease: Perspectives in the 90's. Proceedings
Ultrastructural localization of G-protein Gs in the organ of of the third International Symposium on Meniere's Disease, pp.
Corti. Neurosci. Lett. 201, 147±150. 485±490. Eds. R. Filipo and M. Barbara. Kugler Publications:
Mokrasch, L. C. (1973) Chemical composition of nervous tissue. Amsterdam.
In: Biology Data Book, pp. 1206±1230. Eds. P. L. Altman and Norris, C. H. and Guth, P. S. (1974) The release of acetylcholine
D. S. Dittmer. Federation of Societies for Experimental Biology: (ACH) by the crossed olivo-cochlear bundle (COCB). Acta
Bethesda, U.S.A. Otolaryngol. (Stockh.) 77, 318±326.
Monaghan, P. (1975) Ultrastructural and pharmacological studies Norris, C. H. and Jackson, R. T. (1987) Inhibition by astemizole of
of the a€erent synapse of lateral line sensory cells of the African nerve activity from the isolated posterior semicircular canal.
clawed toad, Xenopus laevis. Cell Tissue Res. 163, 239±247. Arch. Otolaryngol. 113, 981±983.
Mons, N. and Cooper, D. M. F. (1995) Adenylate cyclases: critical Norris, C. H., Housley, G. D., Williams, W. H., Guth, S. L. and
foci in neuronal signaling. TINS 18, 536±542. Guth, P. S. (1988) The acetylcholine receptors of the semicircu-
Morgans, C., Brandstatter, J., Kellerman, J., Betz, H. and Wassle, lar canal in the frog (Rana pipiens). Hear. Res. 32, 197±206.
H. (1996) A SNARE complex containing syntaxin 3 is present in Oberholtzer, J. C., Buettger, C., Summers, M. C. and Matschinsky,
ribbon synapses of the retina. J. Neurosci. 16, 6713±6721. F. M. (1988) The 28-kDa calbindin-D is a major calcium-bind-
Mroz, E. A., Nissim, K. R. and Lechene, C. (1993) Rapid resting ing protein in the basilar papilla of the chick. Proc. natn. Acad.
ion ¯uxes in gold®sh hair cells are balanced by (Na + K+)± Sci. U.S.A. 85, 3387±3390.
ATPase. Hear. Res. 70, 22±30. O'Connor, S. E., Dainty, I. A. and Le€, P. (1991) Further subclas-
Mroz, E. A. and Sewell, W. F. (1989) Pharmacological alterations si®cation of ATP receptors based on agonist studies. TIPS 12,
of the activity of a€erent ®bers innervating hair cells. Hear. Res. 137±141.
38, 141±162. O'Dowd, J. J., Robins, D. J. and Miller, D. J. (1988) Detection,
Mulle, C., Benoit, P., Pinset, C., Roa, M. and Changeux, J.- characterization and quanti®cation of carnosine and other histi-
P. (1988) Calcitonin gene-related peptide enhances the rate of dyl derivatives in cardiac and skeletal muscle. Biochim. biophys.
desensitization of the nicotinic acetylcholine receptor in cultured Acta 967, 241±249.
mouse muscle cells. Proc. natn. Acad. Sci. U.S.A. 85, 5728±5732. Ogawa, K. and Schacht, J. (1993) Receptor-mediated release of
Murofushi, T., Curthoys, I. S., Topple, A. N., Colebatch, J. G. and inositol phosphates in the cochlear and vestibular sensory epithe-
Halmagyi, G. M. (1995) Responses of guinea pig primary vestib- lia of the rat. Hear. Res. 69, 207±214.
ular neurons to clicks. Exp. brain Res. 103, 174±178. Ohmori, H. (1984) Studies of ionic currents in the isolated vestibu-
Murrow, B. (1994) Position-dependent expression of potassium lar hair cell of the chick. J. Physiol. (Lond.) 350, 561±581.
currents by chick cochlear hair cells [published erratum appears Ohmori, H. (1985) Mechano-electrical transduction currents in iso-
in J Physiol (Lond.) 1995 Feb 1; 482(Pt 3): 725]. J. Physiol. lated vestibular hair cells of the chick. J. Physiol. (Lond.) 359,
(Lond.) 480(Part 2), 247±259. 189±217.
Myers, S. F. and Lewis, E. R. (1990) Hair cell tufts and a€erent in- Ohmori, H. (1990) Mechano-electrical transduction and muscarinic
nervation of the bullfrog crista ampullaris. Brain Res. 534, 15± cholinergic responses in the chick hair cell. Neurosci. Res.
24. 12(Suppl.), S51±S62.
Myers, S. F., Salem, H. H. and Kaltenbach, J. A. (1997) E€erent Ohmori, H. (1996) A€erent and e€erent synaptic transmissions in
neurons and vestibular cross talk in the frog. J. Neurophysiol. hair cells. News Physiol. Sci. 11, 161±166.
77, 2061±2070. Ohno, K., Takeda, N., Kiyama, H., Kato, H., Fujita, S.,
Nadol, J. B. (1988) Comparative anatomy of the cochlea and audi- Matsunaga, T. and Tohyama, M. (1993a) Synaptic contact
tory nerve in mammals. Hear. Res. 37, 253±266. between vestibular a€erent nerve and cholinergic e€erent term-
Nakagawa, T., Akaike, N., Kimitsuki, T., Komune, S. and Arima, inal: its putative mediation by nicotinic receptors. Brain Res.
T. (1990) ATP-induced current in isolated outer hair cells of gui- molec. Brain Res. 18, 343±346.
nea pig cochlea. J. Neurophysiol. 63, 1068±1074. Ohno, K., Takeda, N., Tanaka-Tsuji, M. and Matsunaga,
Nakagawa, T., Kakehata, S., Yamamoto, T., Akaike, N., Komune, T. (1993b) Calcitonin gene-related peptide in the e€erent system
S. and Uemura, T. (1994) Ionic properties of I-k,n on outer hair of the inner ear. A review. Acta Otolaryngol. (Stockh.) 501, 16±
cells of guinea pig cochlea. Brain Res. 661, 293±297. 20.
The Vestibular Hair Cells 243

Osborne, M. P. (1966) The ®ne structure of synapses and tight Prigioni, I., Russo, G. and Marcotti, W. (1996) Potassium currents
junctions in the central nervous system of the blow¯y larva. of pear-shaped hair cells in relation to their location in frog
J. Insect Physiol. 12, 1503±1512. crista ampullaris. Neuroreport 7, 1841±1845.
Osborne, M. P. (1977) Role of vesicles with some observations on Prigioni, I., Russo, G. and Masetto, S. (1994) Non-NMDA recep-
vertebrate sensory cells. In: Synapses, pp. 40±63. Eds. G. A. tors mediate glutamate-induced depolarization in frog crista
Gottrell and P. N. R. Usherwood. Academic Press: New York. ampullaris. Neuroreport 5, 516±518.
Osborne, M. P. and Thornhill, R. A. (1972) The e€ects of mono- Prigioni, I., Russo, G., Valli, P. and Masetto, S. (1990) Pre- and
amine depleting drugs upon the synaptic bars in the inner ear of postsynaptic excitatory action of glutamate agonists on frog ves-
the bullfrog (Rana catesbeiana). Z. Zellforschung 127, 3247± tibular receptors. Hear. Res. 46, 253±259.
3255. Prigioni, I., Valli, P. and Casella, C. (1983) Peripheral organization
Oudar, O., Ferrary, E. and Feldmann, G. (1990) Adenylate cyclase of the vestibular e€erent system in the frog: an electrophysiologi-
and carbonic anhydrase in the semicircular canal epithelium of cal study. Brain Res. 269, 83±90.
the frog Rana esculenta. An ultrastructural cytochemical localiz- Puel, J. L., Bledsoe, S. C. Jr, Bobbin, R. P., Ceasar, G. and Fallon,
ation. Cell Tissue Res. 262, 579±585. M. (1989) Comparative actions of salicylate on the amphibian
Paloheimo, S. and Thalmann, R. (1977) In¯uence of ``loop'' diure- lateral line and guinea pig cochlea. Comp. Biochem. Physiol. [C]
tics upon Na + ,K + ±ATPase and adenylate cyclase of the 93, 73±80.
stria vascularis. Arch. Otorhinolaryngol. 217, 347±359. Pujol, R. (1994) Lateral and medial e€erents: a double neurochemi-
Panzanelli, P., Valli, P., Cantino, D. and Fasolo, A. (1994) cal mechanism to protect and regulate inner and outer hair cell
Glutamate and carnosine in the vestibular system of the frog. function in the cochlea. Br. J. Audiol. 28, 185±191.
Brain Res. 662, 293±296. Pujol, R. and Lenoir, M. (1986) The four types of synapses in the
Park, J. C., Hubel, S. B. and Woods, A. D. (1987) Morphometric organ of corti. In: Neurobiology of Hearing: the Cochlea, pp.
analysis and ®ne structure of the vestibular epithelium of aged 161±172. Eds. R. A. Altschuler, D. W. Ho€man and R. P.
C57BL/6 NNia mice. Hear. Res. 28, 87±96. Bobbin. Raven Press: New York.
Parsons, T., Lenzi, D., Almers, W. and Roberts, W. (1994) Pull, I. and McIlwain, H. (1972) Adenine derivatives as neurohu-
Calcium-triggered exocytosis and endocytosis in an isolated pre- moral agents in the brain. Biochem. J. 130, 975±981.
synaptic cell: capacitance measurements in saccular hair cells. Purcell, I. and Perachio, A. (1995a) Regional distribution of e€er-
Neuron 13, 875±883. ent innervation in the utricular maculae of the gerbil. Soc.
Pemberton, K. E. and Jones, S. V. (1997) Inhibition of the L-type Neurosci. Abstr. 21, 399.
calcium channel by the ®ve muscarinic receptors (m1±m5) Purcell, I. and Perachio, A. (1995b) Three-dimensional analysis of
expressed in 3T3 cells. P¯ugers Arch.-Eur. J. Physiol. 433, 505±
biocytin-labeled vestibular e€erent neurons in the semicircular
514.
canal of the gerbil. Abstracts of the 18th Midwinter Meeting
Penington, N. J. and Fox, A. P. (1995) Toxin-insensitive Ca cur-
ARO, 12. Ed. G. Popelka, St. Petersburgh.
rents in dorsal raphe neurons. J. Neurosci. 15, 5719±5726.
Purcell, I. and Perachio, A. (1996a) Regional distributions of e€er-
Perachio, A. A. and Kevetter, G. A. (1989) Identi®cation of vestib-
ent neurons in the semicircular canals in the gerbil. Ann. N.Y.
ular e€erent neurons in the gerbil: histochemical and retrograde
Acad. Sci. 781, 680±682.
labelling. Exp. brain Res. 78, 315±326.
Purcell, I. and Perachio, A. (1996b) Regional innervation patterns
Perez, M. E., Soto, E. and Vega, R. (1991) Streptomycin blocks the
of vestibular e€erent neurons in the saccular macula of the ger-
postsynaptic e€ects of excitatory amino acids on the vestibular
bil. Abstracts of the 19th Midwinter Meeting ARO, 174.
system primary a€erents. Brain Res. 563, 221±226.
Rabejac, D., Devau, G. and Raymond, J. (1997) AMPA receptors
Perin, P., Masetto, S., Zucca, G., Botta, L. and Valli, P. (1997a)
in cultured vestibular ganglion neurons: detection and acti-
Calcium currents kinetics in frog saccular hair cells. P¯ugers
vation. Eur. J. Neurosci. 9, 221±228.
Arch., in press.
Rathouz, M. M., Vijayaraghavan, S. and Berg, D. K. (1995)
Perin, P., Masetto, S., Zucca, G. and Valli, P. (1997b) Cholinergic
Acetylcholine di€erentially a€ects intracellular calcium via nic-
modulation of calcium channels in frog saccular hair cells. Soc.
otinic and muscarinic receptors on the same population of neur-
Neurosci. Abstr. 23, in press.
ons. J. biol. Chem. 270, 14366±14375.
Peterson, E., Cotton, J. and Grant, J. (1996) Structural variation in
ciliary bundles of the posterior semicircular canal. Quantitative Raymond, J., Nieoullon, A., Dememes, D. and Sans, A. (1984)
anatomy and computational analysis. Ann. N.Y. Acad. Sci. 781, Evidence for glutamate as a neurotransmitter in the cat vestibu-
85±102. lar nerve: radioautographic and biochemical studies. Exp. Brain
Pickles, J. and Corey, D. (1992) Mechanoelectrical transduction by Res. 56, 523±531.
hair cells. TINS 15, 254±259. Rebillard, G. and Bryant, G. M. (1989) E€ects of in vivo perfusion
Pin, J. and Duvoisin, R. (1995) The metabotropic glutamate recep- of glutamate dehydrogenase in the guinea pig cochlea on the
tors: structure and functions. Neuropharmacology 34, 1±26. VIIIth nerve compound action potential. Brain Res. 494, 379±
Platt, C. and Popper, A. (1981) Fine structure and function of the 382.
ear. In: Hearing and Sound Communication in Fishes, pp. 3±38. Rennie, K. J. and Ashmore, J. F. (1993) E€ects of extracellular
Ed. A. P. a. R. F. WN Tavolga. Springer-Verlag: New York. ATP on hair cells isolated from the guinea-pig semicircular
Popper, A., Platt, C. and Edds, P. (1990) Evolution of the ver- canals. Neurosci. Lett. 160, 185±189.
tebrate inner ear: an overview of ideas. In: The Evolutionary Rennie, K. J. and Correia, M. J. (1994) Potassium currents in
Biology of Hearing, pp. 49±57. Eds. D. Webster, R. Fay and A. mammalian and avian isolated type I semicircular canal hair
Popper. Springer-Verlag: New York. cells. J. Neurophysiol. 71, 317±329.
Popper, A. N., Saidel, W. M. and Chang, J. S. (1993) Two types of Rennie, K., Ricci, A. and Correia, M. (1996) Electrical ®ltering in
sensory hair cell in the saccule of a teleost ®sh. Hear. Res. 64, gerbil isolated type I semicircular canal hair cells.
211±216. J. Neurophysiol. 75, 2117±2123.
Potter, A. J., Drescher, M. J. and Drescher, D. G. (1986) Rennie, K. J. and Ashmore, J. F. (1991) Ionic currents in isolated
Potassium-stimulated e‚ux of radiolabeled products formed vestibular hair cells from the guinea-pig crista ampullaris. Hear.
from L-[14C(U)]-glutamine in vitro by the saccule of the rainbow Res. 51, 279±291.
trout (Salmo gairdnerii R.). Comp. biochem. Physiol. A 84, 265± Rennie, K. J., Ricci, A. J. and Correia, M. J. (1994) Calcium cur-
270. rents in isolated mammalian semicircular canal hair cells. 1st
Precht, W. (1976) Physiology of the peripheral and central vestibu- International Symposium Inner Ear Neuropharmacology, Vol. 1,
lar system. In: Frog Neurobiology, pp. 481±512. Eds. R. Llinas p. 12. Montpellier, France.
and W. Precht. Springer Verlag: Berlin. Ricci, A., Norris, C. and Guth, P. (1991a) Cyclic AMP modulates
Precht, W. (1978) Neuronal Operation in the Vestibular System. sensory-neural communication at the vestibular end organ. Brain
Springer Verlag: Berlin. Res. 565, 78±84.
Prigioni, I., Masetto, S., Russo, G. and Taglietti, V. (1992) Ricci, A., Rennie, K. and Correia, M. (1996) A delayed recti®er
Calcium currents in solitary hair cells isolated from frog crista conductance shapes the voltage response of type I hair cells.
ampullaris. J. Vestibular Res. 2, 31±39. Ann. N.Y. Acad. Sci. 781, 690±692.
244 P. S. Guth et al.

Ricci, A., Wolverton, S., Aubert, A., Norris, C. and Guth, in the rodent peripheral auditory system. Molec. Brain Res. 40,
P. (1991b) Responses of the isolated semicircular canal to electri- 127±135.
cal stimulation. Proc. Assoc. Res. Otolaryngol. 14, 95. Sa®eddine, S. and Eybalin, M. (1992a) Co-expression of NMDA
Ricci, A. J. and Fettiplace, R. (1997) The e€ects of calcium bu€er- and AMPA/kainate receptor mRNAs in cochlear neurones.
ing and cyclic AMP on mechanoelectrical transduction in turtle Neuroreport 3, 1145±1148.
auditory hair cells. J. Physiol. (Lond.) 501, 111±124. Sa®eddine, S. and Eybalin, M. (1992b) In situ hybridization of
Richards, D. M., Brogden, R. N., Heel, R. C., Speight, J. M. and mRNA's encoding NMDA and AMPA/kainate receptors in
Avery, G. S. (1984) Astemizole: a review of its pharmacody- spiral ganglion of guinea pigs and rats. Proc. Workshop Inner
namic properties and therapeutic ecacy. Drugs 28, 38±61. Ear Biol. 29, 113.
Roberts, B. L., Maslam, S., Los, I. and Van der Jagt, B. (1994) Saidel, W. M., Lanford, P. J., Yan, H. Y. and Popper, A. N. (1995)
Coexistence of calcitonin gene-related peptide and choline acetyl- Hair cell heterogeneity in the gold®sh saccule. Brain, Behav.
transferase in eel e€erent neurons. Hear. Res. 74, 231±237. Evolut. 46, 362±370.
Roberts, B. L. and Ryan, K. P. (1971) The ®ne structure of the lat- Saito, A., Kimura, S. and Goto, K. (1986) Calcitonin gene-related
eral sense organs of dog®sh. Proc. R. Soc. London 179, 157±169. peptide as potential neurotransmitter in guinea pig right atrium.
Roberts, W. (1993) Spatial calcium bu€ering in saccular hair cells. Am. J. Physiol. 250, H693±H698.
Nature 363, 74±76. Sakaba, T., Tachibana, M., Matsui, K. and Minami, N. (1997)
Roberts, W. M. (1994) Localization of calcium signals by a mobile Two components of transmitter release in retinal bipolar cells:
calcium bu€er in frog saccular hair cells. J. Neurosci. 14, 3246± exocytosis and mobilization of synaptic vesicles. Neurosci. Res.
3262. 27, 357±370.
Roberts, W. M., Jacobs, R. A. and Hudspeth, A. J. (1990) Sans, A., Brehier, A., Moniot, B. and Thomasset, M. (1987)
Colocalization of ion channels involved in frequency selectivity Immuno-electronmicroscopic localization of ``vitamin D-depen-
and synaptic transmission at presynaptic active zones of hair dent'' calcium-binding protein (CaBP-28k) in the vestibular hair
cells. J. Neurosci. 10, 3664±3684. cells of the cat. Brain Res. 435, 293±304.
Roberts, W. M., Jacobs, R. A. and Hudspeth, A. J. (1991) The Sans, A., Etchecopar, B., Brehier, A. and Thomasset, M. (1986)
hair cell as a presynaptic terminal. Ann. N.Y. Acad. Sci. 635, Immunocytochemical detection of vitamin D-dependent calcium-
221±233. binding protein (CaBP-28K) in vestibular sensory hair cells and
Rosahl, T. W., Spillane, D., Missler, M., Herz, J., Selig, D. K., vestibular ganglion neurones of the cat. Brain Res. 364, 190±194.
Wol€, J. R., Hammer, R. E., Malenka, R. C. and Sudhof, T. Sans, A., Griguer, C. and Lehouelleur, J. (1994) The vestibular
C. (1995) Essential functions of synapsins I and II in synaptic type I hair cells: a self-regulated system? Acta Otolaryngol.
vesicle regulation. Nature 375, 488±493. (Stockh.) 513(Suppl.), 11±14.
Ross, M. (1995) Mammalian vestibular macula synaptic plasticity: Sans, A. and Highstein, S. M. (1984) New ultrastructural features
results from SLS-2 (space-life sciencesÐ2) space ¯ight. Proc. in the vestibular labyrinth of the toad®sh, Opsanus tau. Brain
Assoc. Res. Otolaryngol. 18, 3. Res. 308, 191±195.
Ross, M. (1997) Morphological evidence for local microcircuits in Sans, A. C., Atger, C., Cavadore, C. and Cavadore, J. C. (1989)
rat vestibular maculae. J. comp. Neurol. 379, 333±346. Immunocytochemical localization of myosin, tropomyosin and
Ross, M. D., Cutler, L., Meyer, G., Lam, T. and Varciri, P. (1990) actin in vestibular hair cells of human fetuses and cats. Hear.
3-D components of a biological neural network visualized in Res. 40, 117±125.
computer-generated imagery. Acta Otolaryngol. (Stockh.) 109, Sargent, P. B. (1993) The diversity of neuronal nicotinic acetyl-
83±92. choline receptors. A. Rev. Neurosci. 16, 403±443.
Rossi, M. L. and Martini, M. (1991) E€erent control of posterior Saria, A., Gamse, R., Patermann, J., Fischer, J. A., Theordorsson-
canal a€erent receptor discharge in the frog labyrinth. Brain Nerheim, E. and Lundberg, J. M. (1986) Simultaneous release of
Res. 555, 123±134. several tachykinins and calcitonin gene-related peptide from rat
Rossi, M. L., Prigioni, I., Valli, P. and Casella, C. (1980) spinal cord. Neurosci. Lett. 63, 310±314.
Activation of the e€erent system in the isolated frog labyrinth: Sassoe-Pognetto, M., Kirsch, J., Grunert, U., Greferath, U.,
e€ects on the a€erent EPSPs and spike discharge recorded from Fritschy, J. M., Mohler, H., Betz, H. and Wassle, H. (1995)
single ®bres of the posterior nerve. Brain Res. 185, 125±137. Colocalization of gephyrin and GABA-A receptor subunits in
Rossi, M. L. and Sacchi, O. (1982) E€ectiveness of some anions in the rat retina. J. comp. Neurol. 357, 1±14.
sustaining the e€erent inhibition in the frog labyrinth. Brain Res. Satoh, Y. and Vollrath, L. (1988) Lack of synaptic ribbons in the
233, 181±185. pineal gland of BALB/c mice. J. pineal Res. 5, 13±17.
Rudy, B. (1988) Diversity and ubiquity of K channels. Scarfone, E., Dememes, D., Jahn, R., De Camilli, P. and Sans,
Neuroscience 25, 729±749. A. (1988) Secretory function of the vestibular nerve calyx
Rusch, A., Kros, C. J., Richardson, G. P. and Russell, I. J. (1991) suggested by presence of vesicles, synapsin I, and synaptophysin.
Potassium and calcium currents in outer hair cells in organotypic J. Neurosci. 8, 4640±4645.
cultures of the neonatal mouse cochlea. J. Physiol. (Lond.) 434, Scarfone, E., Dememes, D. and Sans, A. (1991) Synapsin I and
52. synaptophysin expression during ontogenesis of the mouse per-
Rusch, A. and Eatock, R. A. (1996a) A delayed recti®er conduc- ipheral vestibular system. J. Neurosci. 11, 1173±1181.
tance in Type I hair cells of the mouse utricle. J. Neurophysiol. Schacht, J. and Zenner, H. P. (1987) Evidence that phosphoinosi-
76, 995±1004. tides mediate motility in cochlear outer hair cells. Hear. Res. 31,
Rusch, A. and Eatock, R. A. (1996b) Voltage responses of mouse 155±159.
utricular hair cells to injected currents. Ann. N.Y. Acad. Sci. 781, Schermuly, L. and Klinke, R. (1990) Infrasound sensitive neurons
71±84. in the pigeon cochlear ganglion. J. comp. Physiol. A 166, 355±
Rusch, A. and Thurm, U. (1989) Cupula displacement, hair bundle 363.
de¯ection and physiological responses in the transparent semicir- Schessel, D. A., Ginzberg, R. and Highstein, S. M. (1991)
cular canal of young eel. P¯ugers Arch. gesamte Physiol. 413, Morphophysiology of synaptic transmission between type I hair
533±545. cells and vestibular primary a€erents. An intracellular study
Russell, I. J. and Roberts, B. L. (1972) Inhibition of spontaneous employing horseradish peroxidase in the lizard, Calotes versico-
lateral-line activity by e€erent nerve stimulation. J. exp. Biol. 57, lor. Brain Res. 544, 1±16.
77±82. Schessel, D. A. and Highstein, S. M. (1981) Is transmission
Russo, G., Marcotti, W. and Prigioni, I. (1996) Inactivation of between the vestibular type I hair cell and its primary a€erent
delayed recti®er K+ current in semicircular canal hair cells. chemical? Ann. N.Y. Acad. Sci. 374, 210±214.
Neuroreport 7, 2143±2146. Schmitz, F., Bechmann, M. and Drenckhahn, D. (1996)
Russo, G., Masetto, S. and Prigioni, I. (1995) Isolation of A-Type Puri®cation of synaptic ribbons, structural components of the
K+ current in hair cells of the frog crista ampullaris. photoreceptor active zone complex. J. Neurosci. 16, 7109±7116.
Neuroreport 6, 425±428. Schwartz, J. C., Arrang, J. M., Garbarg, M., Pollard, H. and Ruat,
Sa®eddine, S., Bartolami, S., Wenthold, R. J. and Eybalin, M. (1991) Histaminergic transmission in the mammalian brain.
M. (1996) Pre- and postsynaptic M3 muscarinic receptor mRNA Physiol. Rev. 71, 1±51.
The Vestibular Hair Cells 245

Sellick, P. and Johnstone, B. (1972) The electrophysiology of the Sridhar, T. S., Brown, M. C. and Sewell, W. F. (1997) Unique
saccule. P¯ugers Arch. 336, 28±34. postsynaptic signalling at the hair cell e€erent synapse permits
Sellick, P., Johnstone, J. and Johnstone, B. (1972) The electrophysi- calcium to evoke changes on two time scales. J. Neurosci. 17,
ology of the utricle. P¯ugers Arch. 336, 21±27. 428±437.
Sewell, W. F. (1990) Synaptic potentials in a€erent ®bers innervat- Sridhar, T. S., Liberman, M. C., Brown, M. C. and Sewell, W.
ing hair cells of the lateral line organ in Xenopus laevis. Hear. F. (1995) A novel cholinergic ``slow e€ect'' of e€erent stimu-
Res. 44, 71±81. lation on cochlear potentials in the guinea pig. J. Neurosci. 15,
Sewell, W. F. and Mroz, E. A. (1987) Neuroactive substances in 3667±3678.
inner ear extracts. J. Neurosci. 7, 2465±2475. Stallcup, W. B. and Patrick, J. (1980) Substance P enhances cholin-
Sewell, W. F. and Mroz, E. A. (1990) Puri®cation of a low-molecu- ergic receptor desensitization in a clonal nerve cell line. Proc.
lar-weight excitatory substance from the inner ears of gold®sh. natn. Acad. Sci. U.S.A. 77, 634±638.
Hear. Res. 50, 127±137. Starr, P. A. and Sewell, W. F. (1990) E€ects of amino acid antag-
Sewell, W. F., Norris, C. H., Tachibana, M. and Guth, P. S. (1978) onists on auditory nerve synaptic potentials. Soc. Neurosci.
Detection of an auditory nerve-activating substance. Science Abstr. 14, 331.
202, 910±912. Starr, P. A. and Sewell, W. F. (1991) Neurotransmitter release
Sewell, W. F. and Starr, P. A. (1991) E€ects of calcitonin gene-re- from hair cells and its blockade by glutamate-receptor antagon-
lated peptide and e€erent nerve stimulation on a€erent trans- ists. Hear. Res. 52, 23±41.
mission in the lateral line organ. J. Neurophysiol. 65, 1158±1169. Steinacker, A. (1996) Ionic current contribution to signal proces-
Shepherd, G. M. (1990) The Synaptic Organization of the Brain. sing by toad®sh semicircular canal hair cells. Ann. N.Y. Acad.
Oxford University Press: New York. Sci. 781, 150±163.
Shepherd, G., Barres, B. and Corey, D. (1989) ``Bundle blot'' puri- Steinacker, A. and Perez, L. (1992) Sensory coding in the saccule.
®cation and initial protein characterization of hair cell stereoci- Patch clamp study of ionic conductances in isolated cells. Ann.
lia. Proc. natn. Acad. Sci. U.S.A. A 86, 4973±4977. N.Y. Acad. Sci. 656, 27±48.
Shigemoto, T. and Ohmori, H. (1990) Muscarinic agonists and Steinacker, A. and Rojas, L. (1988) Acetylcholine modulated pot-
ATP increase the intracellular Ca2+ concentration in chick assium channel in the hair cell of the toad®sh saccule. Hear. Res.
cochlear hair cells. J. Physiol. (Lond.) 420, 127±148. 35, 265±269.
Shigemoto, T. and Ohmori, H. (1991) Muscarinic receptor hyper- Steinacker, A. and Romero, A. (1991) Characterization of voltage-
polarizes cochlear hair cells of chick by activating Ca(2+)-acti- gated and calcium-activated potassium currents in toad®sh sac-
vated K+ channels. J. Physiol. (Lond.) 442, 669±690. cular hair cells. Brain Res. 556, 22±32.
Shotwell, S. L., Jacobs, R. and Hudspeth, A. J. (1981) Directional Sterkers, O., Bernard, C., Ferrary, E., Sziklai, I., Tran Ba Huy,
sensitivity of individual vertebrate hair cells to controlled de¯ec- P. and Amiel, C. (1988a) Possible role of Ca ions in the vestibu-
lar system. [Review]. Acta Otolaryngol. (Stockh.) 460(Suppl.),
tion of their hair bundles. Ann. N.Y. Acad. Sci. 374, 1±10.
28±32.
Siegel, J. H. and Brownell, W. E. (1986) Synaptic and Golgi mem-
Sterkers, O., Ferrary, E. and Amiel, C. (1988b) Production of inner
brane recycling in cochlear hair cells. J. Neurocytol. 15, 311±328.
ear ¯uids. [Review]. Physiol. Rev. 68, 1083±1128.
Siegel, J. H. and Relkin, E. M. (1987) Antagonistic e€ects of peri-
Strutz, J., Spatz, W. B., Schmidt, C. L. and Sturmer, C. (1980)
lymphatic calcium and magnesium on the activity of single a€er-
Origin of centrifugal ®bres to the labyrinth in the frog (Rana
ent neurons. Hear. Res. 28, 131±147.
esculenta). A study with the ¯uorescent retrograde neuronal tra-
Silinsky, E. M. and Hubbard, J. I. (1973) Release of ATP from rat
cer Fast Blue. Brain Res. 215, 323±328.
motor nerve terminals. Nature 243, 327±328.
Su, Z. L., Jiang, S. C., Gu, R. and Yang, W. P. (1995) Two types
Sjostrand, F. S. (1958) Ultrastructure of the retinal rod synapses of
of calcium channels in bullfrog saccular hair cells. Hear. Res. 87,
the guinea pig eye as revealed by three dimensional reconstruc-
62±68.
tions from serial sections. J. ultrastruct. Res. 2, 122±170.
Sugai, T., Yano, J., Sugitani, M. and Ooyama, H. (1992) Actions
Sko®tsch, G. and Jacobowitz, D. M. (1985) Autoradiographic dis-
of cholinergic agonists and antagonists on the e€erent synapse in
tribution of I-125 calcitonin gene-related peptide binding sites in
the frog sacculus. Hear. Res. 61, 56±64.
the rat central nervous system. Peptides 4, 975±986.
Sugihara, I. (1994) Calcium-activated potassium channels in gold-
Snyder, S. H. (1985) Adenosine as neuromodulator. A. Rev. ®sh hair cells. J. Physiol. (Lond.). 476, 373±390.
Neurosci. 8, 103±124. Sugihara, I. and Furukawa, T. (1989) Morphological and func-
Sobin, A. and Flock, A. (1983) Immunohistochemical identi®cation tional aspects of two di€erent types of hair cells in the gold®sh
and localization of actin and ®mbrin in vestibular hair cells. sacculus. J. Neurophysiol. 62, 1330±1343.
Acta Otolaryngol. (Stockh.) 96, 407±412. Sugihara, I. and Furukawa, T. (1995) Potassium currents under-
Sobkowicz, H. M., Bereman, B. and Rose, J. E. (1975) lying the oscillatory response in hair cells of the gold®sh saccu-
Organotypic development of the organ of Corti in culture. lus. J. Physiol. (Lond.) 489, 443±453.
J. Neurocytol. 4, 543±572. Sugihara, I. and Furukawa, T. (1996) Inwardly rectifying currents
Sobkowicz, H. M., Loftus, J. M. and Slapnick, S. M. (1993) Tissue in hair cells and supporting cells in the gold®sh sacculus.
culture of the organ of Corti. Acta Otolaryngol. (Stockh.) J. Physiol. (Lond.) 495, 665±679.
502(Suppl.), 3±36. Surprenant, A., Buell, G. and North, R. A. (1995) P-2X receptors
Sobkowicz, H. M., Rose, J. E., Scott, G. L. and Levenick, C. bring new structure to ligand-gated ion channels. TINS 18, 224±
V. (1986) Distribution of synaptic ribbons in the developing 229.
organ of Corti. J. Neurocytol. 15, 693±714. Tachibana, M., Asano, T., Wilcox, E., Yokotani, N., Rivolta, M.
Sobkowicz, H. M., Rose, J. E., Scott, G. E. and Slapnick, S. N. and Fex, J. (1994) G protein Gi2 alpha in the cochlea: clon-
M. (1982) Ribbon synapses in the developing intact and cultured ing and selective occurrence in receptor cells. Brain Res. molec.
organ of Corti in the mouse. J. Neurosci. 2, 942±957. Brain Res. 21, 355±358.
Soto, E., Flores, A., Erostegui, C. and Vega, R. (1994) Evidence Taglietti, V., Rossi, M. L. and Casella, C. (1977) Adaptive distor-
for NMDA receptor in the a€erent synaptic transmission of the tions in the generator potential of semicircular canal sensory
vestibular system. Brain Res. 633, 289±296. a€erents. Brain Res. 123, 41±57.
Soto, E., Perez, M. E. and Vega, R. (1988) Naloxone induces an Takahashi, T. (1990) Inward recti®cation in neonatal rat spinal
excitation e€ect on the vestibular primary a€erents. Soc. motoneurones. J. Physiol. (Lond.) 423, 47±62.
Neurosci. Abstr. 14, 331. Takeda, N., Kitajiri, M., Girgis, S., Hillyard, C. J., MacIntyre, I.,
Soto, E. and Vega, R. (1988) Actions of excitatory amino acid acid Emson, P. C., Shiosaka, S., Matsunaga, T. and Tohyama,
agonists and antagonists on the primary a€erents of the vestibu- Y. (1986) The presence of a calcitonin gene-related peptide in
lar system of the axolotl (Ambystoma mexicanum). Brain Res. the olivocochlear bundle in the rat. Exp. brain Res. 61, 575±578.
462, 104±111. Tanaka, K. and Smith, C. A. (1978) Structure of the chicken's
Spoendlin, H. (1970) Vestibular labyrinth. In: Ultrastructure of the inner ear: SEM and TEM study. Am. J. Anat. 153, 251±272.
Peripheral Nervous Systemand Sense Organs: Atlas of Normal Tanaka, M., Takeda, N., Senba, E., Tohyama, M., Kubo, T. and
and Pathologic Anatomy, pp. 263±273. Ed. A. Bischo€. Georg Matsunaga, T. (1989a) Localization and origins of calcitonin
Thieme Verlag: Stuttgart. gene-related peptide containing ®bres in the vestibular end-
246 P. S. Guth et al.

organs of the rat. Acta Otolaryngol. (Stockh.) 468(Suppl.), 31± Valli, P., Botta, L., Zucca, G. and Casella, C. (1986) Functional or-
34. ganization of the peripheral e€erent vestibular system in the
Tanaka, M., Takeda, N., Senba, E., Tohyama, M., Kubo, T. and frog. Brain Res. 362, 92±97.
Matsunaga, T. (1989b) Localization, origin and ®ne structure of Valli, P., Taglietti, V. and Rossi, M. L. (1974) E€ects of D-tubocur-
calcitonin gene-related peptide-containing ®bers in the vestibular arine on the ampullar receptors of the frog. Acta Otolaryngol.
end-organs of the rat. Brain Res. 504, 31±35. (Stockh.) 78, 51±58.
Taxi, J. (1976) Morphology of the autonomic nervous system. In: Valli, P. and Zucca, G. (1976) The origin of slow potentials in
Frog Neurobiology, pp. 92±150. Eds. R. Llinas and W. Precht. semicircular canals of the frog. Acta Otolaryngol. (Stockh.) 81,
Springer-Verlag: Berlin. 395±405.
Thach, J. J. and Graybiel, A. (1968) Behavioral responses of Valli, P., Zucca, G. and Botta, L. (1990) Perilymphatic potassium
unrestrained normal and labyrinthectomized squirrel monkeys to changes and potassium homeostasis in isolated semicircular
repeated zero-gravity parabolic ¯ights. Aerospace Med. 39, 734± canals of the frog. J. Physiol. (Lond.) 430, 585±594.
738. Valli, P., Zucca, G., Botta, L. and Casella, C. (1988) Sensitivity
Thie€ry, M. and Bruner, J. (1978) Direct evidence for a presynaptic and adaptation of ampullar receptors to rapid perilymphatic
action of glutamate at a cray®sh neuromuscular junction. Brain potassium changes. J. comp. Physiol. A 162, 173±178.
Res. 156, 402±406. Valli, P., Zucca, G., Prigioni, I., Botta, L., Casella, C. and Guth, P.
Thorn, L., Schinko, I. and Wetzstein, R. (1972) Synaptic bar in the S. (1985) The e€ect of glutamate on the frog semicircular canal.
e€erent part of a synapse in the organ of Corti. Experientia 28, Brain Res. 330, 1±9.
835±842. Valverde, M. A., Hardy, S. P. and Sepulveda, F. V. (1995)
Thornhill, R. A. (1972) The e€ect of catecholamine precursors and Chloride channels: a state of ¯ux. FASEB Jl 9, 509±515.
related drugs on the morphology of synaptic bars in the vestibu- Vazquez, E., Herrero, I., Miras-Portugal, M. T. and Sanchez-
lar epithelia of the frog Rana temporaria. Comp. gen. Pharmac. Prieto, J. (1995) Developmental change from inhibition to facili-
3, 89±97. tation in the presynaptic control of glutamate exocytosis by
Torre, V., Ashmore, J. F., Lamb, T. D. and Menini, A. (1995) metabotropic glutamate receptors. Neuroscience 68, 117±124.
Transduction and adaptation in sensory receptor cells. Vega, R., Soto, E., Budelli, R. and Gonzalez-Estrada, M. T. (1987)
J. Neurosci. 15, 7757±7768. Is GABA an a€erent transmitter in the vestibular system? Hear.
Tower, D. B. (1960) The Neurochemistry of asparagine and gluta- Res. 29, 163±167.
mine. In: The Neurochemistry of Nucleotides and Amino Acids, Vega, R., Soto, E. and Perez, M. E. (1991) Opioid receptors neuro-
pp. 173±204. Eds. R. B. Brady and D. B. Tower. Wiley: New pharmacology in the vestibular a€erents. Soc. Neurosci. Abstr.
York. 17, 317.
Tschupp, F. A., Henke, H., Petermann, J., Tobler, P., Janzer, R., Vetter, D. E., Adams, J. C. and Mugnaini, E. (1991) Chemically
Hokfelt, T., Lundberg, J. M., Cuello, C. and Fischer, J. (1985) distinct rat olivocochlear neurons. Synapse 7, 21±43.
Calcitonin gene-related peptide and its binding sites in the Vetter, D., Mann, J., Wangemann, P., Liu, J., McLaughlin, K.,
human central nervous system and pituitary. Proc. natn. Acad. Lesage, F., Marcus, D., Lazdunski, M., Heinemann, S. and
Sci. U.S.A. 82, 248±252. Barhanin, J. (1996) Inner ear defects induced by null mutation
Tucker, T. and Fettiplace, R. (1995) Confocal imaging of calcium of the isk gene. Neuron 17, 1251±1264.
microdomains and calcium extrusion in turtle hair cells. Neuron Vizi, E. S. (1979) Presynaptic modulation of neurochemical trans-
15, 1323±1335. mission. Prog. Neurobiol. 12, 181±290.
Tucker, T. R. and Fettiplace, R. (1996) Monitoring calcium in tur- Vollrath, L. (1973) Synaptic ribbons of a mammalian pineal gland.
tle hair cells with a calcium-activated potassium channel. Circadian changes. Z. Zellforschung Mikroskopische Anat. 145,
J. Physiol. (Lond.) 494, 613±626. 171±183.
Turner, J. S. and Jackson, R. T. (1983) Astemizole: its use inpati- Wackym, P. A., Popper, P., Lopez, I. and Micevych, P. E. (1995)
ents with chronic vertigo and ENG signs: a pilot study. Expression of alpha 4 and beta 2 nicotinic acetylcholine receptor
Laryngoscope 93, 898±902. subunit mRNA and localization of alpha-bungarotoxin binding
Upadhyay, S., Sheikhali, S. A., and Drescher, D. G. (1997). proteins in the rat vestibular periphery. Cell biol. Int. 19, 291±
Localization of muscarinic receptor subtype messages in the rat 300.
cochlea. Abstracts of the 20th ARO Midwinter Research Meeting, Wackym, P. A., Popper, P., Ward, P. H. and Micevych, P.
921. Ed. G. Popelka, St. Petersburg. E. (1991) Cell and molecular anatomy of nicotinic acetylcholine
Usami, S., Hozawa, J., Tazawa, M., Jin, H., Matsubara, A. and receptor subunits and calcitonin gene-related peptide in the rat
Fujita, S. (1991a) Localization of substance P-like immunoreac- vestibular system. Otolaryngol. Head Neck Surg. 105, 493±510.
tivity in guinea pig vestibular endorgans and the vestibular deWaele, C., Muhlethaler, M. and Vidal, P. P. (1995)
ganglion. Brain Res. 555, 153±158. Neurochemistry of the central vestibular pathways. Brain Res.
Usami, S., Hozawa, J. and Ylikoski, J. (1991b) Coexistence of sub- 20, 24±46.
stance P and calcitonin gene-related peptide-like immunoreac- Wagner, H. J. (1973) Darkness-induced reduction in the number of
tivities in the rat vestibular endorgans. Acta Otolaryngol. synaptic ribbons in ®sh retina. Nature 246, 53±55.
(Stockh.) 481(Suppl.), 166±169. Walker, R. and Hudspeth, A. (1996) Calmodulin controls adap-
Usami, S. I., Matsubara, A., Fujita, S., Shinkawa, H. and Hayashi, tation of mechanoelectrical transduction by hair cells of the bull-
M. (1995) NMDA (NMDAR1) and AMPA-type (GluR2/3) frog's sacculus. Proc. Natl Acad. Sci. U.S.A. 93(5), 2203±7.
receptor subunits are expressed in the inner ear. Neuroreport 6, Wang, J.-J. and Dutia, M. B. (1995) E€ects of histamine and beta-
1161±1164. histine on rat medial vestibular nucleus neurones: possible mech-
Usami, S.-I. and Ottersen, O. P. (1995) Di€erential cellular distri- anism of action of antihistaminergic drugs in vertigo and motion
bution of glutamate and glutamine in the rat vestibular endor- sickness. Exp. brain Res. 105, 18±24.
gans: an immunocytochemical study. Brain Res. 676, 285±292. Wang, X. and Robinson, P. (1997) Cyclic GMP-dependent protein
Usherwood, P. N. R. and Machili, P. (1966) Chemical transmission kinase and cellular signaling in the nervous system.
at the insect excitatory neuromuscular synapse. Nature 210, 634± J. Neurochem. 68, 443±456.
636. Warr, W. B., Guinan, J. J. and White, J. S. (1986) Organization of
Valat, J., Griguer, C., Lehouelleur, J. and Sans, A. (1991) Motile the e€erent ®bers: the lateral and medial olivocochlear systems.
responses of isolated guinea pig vestibular hair cells. Neurosci. In: Neurobiology of Hearing: The Cochlea, pp. 333±348. Eds. R.
Lett. 127, 231±236. A. Altschuler, R. P. Bobbin and D. W. Ho€man. Raven Press:
Valat, J., Scarfone, E., Travo, C., Homburger, V. and Sans, New York.
A. (1995) Immunocytochemical localization ofthe GTP-binding Webb, T. E., Simon, J., Krishek, B. J., Bateson, A. N., Smart, T.
protein GO alpha in the vestibular epithelium and ganglion of G., King, B. F., Burnstock, G. and Barnard, E. A. (1993)
the guinea pig. J. Neurocytol. 24, 916±924. Cloning and functional expression of a brain G-protein-coupled
Valera, S., Hussy, N., Evans, R. J., Adami, N., North, R. A., ATP receptor. FEBS Lett. 324, 219±225.
Surprenant, A. and Buell, G. (1994) A new class of ligand-gated Weisleder, P., Tsue, T. T. and Rubel, E. W. (1995) Hair cell repla-
ion channel de®ned by P2x receptor for extracellular ATP. cement in avian vestibular epithelium: supporting cell to Type I
Nature 373, 112. hair cell. Hear. Res. 82, 125±133.
The Vestibular Hair Cells 247

Wenthold, R. J. and Altschuler, R. A. (1983) Yamashita, T. and Ohmori, H. (1990) Synaptic responses to mech-
Immunocytochemistry of aspartate aminotransferase and gluta- anical stimulation in calyceal and bouton type vestibular a€er-
minase. In: Glutamine,Glutamate and GABA in the Central ents studied in an isolated preparation of semicircular canal
Nervous System, pp. 33±50. Eds. L. Hertz, E. Kvamme, E. G. ampullae of chicken. Exp. brain Res. 80, 475±488.
McGeer and A. Schousboe. Alan R. Liss: New York. Yamashita, T., Ohnishi, S., Ohtani, M. and Kumazawa, T. (1993)
Wersall, J. (1956) Studies on the structure and innervation of the E€ects of e€erent neurotransmitters on intracellular Ca2+ con-
sensory epithelium of the cristae ampullares in the guinea pig. A centration in vestibular hair cells of the guinea pig. Acta
light and electron microscopic investigation. Acta Otolaryngol. Otolaryngol. (Stockh.) 500(Suppl.), 26±30.
(Stockh.) 126(Suppl.), 1±85. Ylikoski, J., Eranko, L. and Paivarinta, H. (1984a) Substance P-
Wersall, J. and Bagger-Sjoback, D. (1974) Morphology of the ves- like immunoreactivity in the rabbit inner ear. J. Laryngol. Otol.
tibular sense organ. In: Handbook of Sensory Physiology Vol. 98, 759±765.
VI/i Vestibular System Part 1ÐBasic Mechanisms, pp. 53±78. Ylikoski, J., Paivarinta, H., Eranko, L., Mrena, I. and Lehtosalo,
Ed. H. H. Kornhuber. Springer Verlag: Berlin. J. (1984b) Is substance P the neurotransmitter in the vestibular
Wersall, J., Flock, A. and Lundquist, P.-G. (1965) Structural basis end organs? Acta Otolaryngol. (Stockh.) 97, 523±528.
for directional sensitivity in cochlear and vestibular sensory Yoshida, N., Shigemoto, T., Sugai, T. and Ohmori, H. (1994) The
receptors. Cold Spring Harbor Symp. 30, 115±132. role of inositol trisphosphate on ACh-induced outward currents
Whim, M. D. and Lloyd, P. E. (1990) Neuropeptide cotransmitters in bullfrog saccular hair cells. Brain Res. 644, 90±100.
released from an identi®ed cholinergic motor neuron modulate Young, E. D., Fernandez, C. and Goldberg, J. M. (1977)
neuromuscular ecacy in Aplysia. J. Neurosci. 10, 3313±3322. Responses of squirrel monkey vestibular neurons to audio-fre-
Whitlon, D. S. and Sobkowicz, H. M. (1989) GABA-like immunor- quency sound and head vibration. Acta Otolaryngol. (Stockh.)
eactivity in the cochlea of the developing mouse. J. Neurocytol. 84, 352±360.
18, 505±518. Zajic, G., Anniko, M. and Schacht, J. (1983) Cellular localization
Wickman, K. and Clapham, D. E. (1995) Ion channel regulation of adenylate cyclase in the developing and mature inner ear of
by G proteins. Physiol. Rev. 75, 865±885. the mouse. Hear. Res. 10, 249±261.
Wilson, V. J. and Melvill-Jones, G. (1979) Mammalian Vestibular Zenner, H. P., Reuter, G., Hong, S., Zimmermann, U. and Gitter,
Physiology. Plenum Press: New York. A. H. (1992) Electrically evoked motile responses of mammalian
Witt, C., Hu, H., Brownell, W. and Bertrand, D. (1994) type I vestibular hair cells. J. Vestibular Res. 2, 181±191.
Physiologically silent sodium channels in mammalian outer hair Zenner, H. P. and Zimmermann, U. (1991) Motile responses of
cells. J. Neurophysiol. 72, 1037±1040. vestibular hair cells following caloric, electrical or chemical stim-
Wolfe, D. E. (1965) The epiphyseal cell: an electron-microscopic uli. Acta Otolaryngol. (Stockh.) 111, 291±297.
study of its intercellular relationships and intracellular mor- Zenner, H. P., Zimmermann, U. and Gitter, A. H. (1990) Cell po-
phology in the pineal body of the albino rat. Prog. brain Res. 10, tential and motility of isolated mammalian vestibular sensory
332±386. cells. Hear. Res. 50, 289±293.
Wonnacott, S. (1997) Presynaptic nicotinic ACh receptors. TINS Zidanic, M. and Fuchs, P. A. (1995) Kinetic analysis of barium
20, 92±98. currents in chick cochlear hair cells. Biophys. J. 68, 1323±1336.
Wood, M. R., Feunninger, K. H. and Cohen, M. J. (1977) Two Zimmermann, H. (1994) Signalling via ATP in the nervous system.
types of presynaptic con®gurations in the insect central synapses: TINS 17, 420±426.
an ultrastructural study. Brain Res. 130, 24±45. Zucca, G., Akoev, G. N., Maracci, A. and Valli, P. (1993a)
Woods, A. D. and Park, J. C. (1987) Persistence of synaptic bodies NMDA receptors in frog semicircular canals. Neuroreport 4,
in saccular hair cells of senescent mice. Acta Otolaryngol. 403±404.
(Stockh.) 104, 193±201. Zucca, G., Botta, L., Barbieri, A., Grana, E. and Valli, P. (1992a)
Wu, Y., Art, J., Goodman, M. and Fettiplace, R. (1995) A kinetic E€ects of cromakalim (BRL 34915) on resting and evoked ac-
description of the calcium-activated potassium channel and its tivity in frog semicircular canals. Acta Physiol. Scand. 145, 423±
application to electrical tuning of hair cells. Prog. Biophys. 428.
molec. Biol. 63, 131±158. Zucca, G., Botta, L., Milesi, V., Dagani, F. and Valli, P. (1992b)
Wu, Y. and Fettiplace, R. (1996) A developmental model for gen- Evidence for L-glutamate release in frog vestibular organs. Hear.
erating frequency maps in the reptilian and avian cochleas. Res. 63, 52±56.
Biophys. J. 70, 2557±2570. Zucca, G., Botta, L., Milesi, V. and Valli, P. (1993b) Sensory adap-
Wu, Y., Tucker, T. and Fettiplace, R. (1996) A theoretical study of tation in frog vestibular organs. Hear. Res. 68, 238±242.
calcium microdomains in turtle hair cells. Biophys. J. 71, 2256± Zucca, G., Maracci, A., Milesi, V., Trimaschi, M., Mira, E.,
2275. Manfrin, M., Quaglieri, S. and Valli, P. (1995) Osmolar changes
Yamaguchi, K. and Ohmori, H. (1993) Suppression of the slow and neural activity in frog vestibular organs. Acta Otolaryngol.
(Stockh.) 115, 34±39.
K+ current by cholinergic agonists in cultured chick cochlear
ganglion neurones. J. Physiol. (Lond.) 464, 213±228.

You might also like