Journal of Chemical Neuroanatomy 6162 (2014) 107119

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Journal of Chemical Neuroanatomy

journal homepage: www.elsevier.com/locate/jchemneu

Neuropeptide Y immunoreactivity in the cat claustrum:

A light- and electron-microscopic investigation
D.V. Hinova-Palova a, B. Landzhov a,*, E. Dzhambazova b, M. Minkov c,
L. Edelstein d, L. Malinova a, A. Paloff a, W. Ovtscharoff a
a

Department of Anatomy, Histology and Embryology, Medical University of Soa, 1431 Soa, Bulgaria
Department of Chemistry, Biochemistry, Physiology and Pathophysiology, Soa University St. Kliment Ohridski, 1407 Soa, Bulgaria
Department of Anatomy, Histology and Embryology, Medical University of Varna, 9002 Varna, Bulgaria
d
Medimark Corporation, P.O. Box 2316, Del Mar, CA 92014, USA
b
c

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 19 May 2014
Received in revised form 15 August 2014
Accepted 16 August 2014
Available online 23 August 2014

The claustrum is a telencephalic nucleus located ventrolateral to the basal ganglia in the mammalian
brain. It has an extensive reciprocal connectivity with most if not all of the cerebral cortex, in particular,
primary sensory areas. However, despite renewed and growing interest amongst investigators, there
remains a paucity of data concerning its peptidergic prole. The aim of the present study was to examine
the presence, morphology, distribution and ultrastructure of neuropeptide Y-immunoreactive (NPY-ir)
neurons and bers in the claustrum of the cat. Ten adult healthy cats from both sexes were used. All
animals received human and ethical treatment in accordance with the Principles of Laboratory Animal
Care. Subjects were irreversibly anesthetized and transcardially perfused with xative solution
containing glutaraldehyde and paraformaldehyde. Brains were promptly removed, postxed and
sectioned. Slices were incubated with polyclonal anti-NPY antibodies according to the standard avidin
biotinperoxidase complex method adopted by our Department of Anatomy, Histology and Embryology.
NPY-ir neurons and bers were found to be diffusely distributed throughout the claustrum, with no
obvious topographic or functional patterning other than larger numbers in its central/broadest part
(stereotaxic planes A12A16). Neurons were generally classied by diameter into three sizes: small
(under 17 mm), medium (1725 mm) and large (over 25 mm). Staining density is varied with some
neurons appearing darker than others. At the electron-microscopic level NPY immunoproduct was
observed within neurons, dendrites and terminal boutons, each differing relative to their ultrastructural
attributes. Two types of NPY-ir synaptic boutons were found. Lastly, it is of interest to note that genderspecic differences were not observed.
2014 Elsevier B.V. All rights reserved.

Keywords:
Cat
Claustrum
Neuropeptide Y
Light microscopy
Electron microscopy
Ultrastructure

1. Introduction
The claustrum is a telencephalic nucleus in the mammalian
brain that has long been known as a site of extensive reciprocal
heterosensory convergence (Olson and Graybiel, 1980; Ashwell
et al., 2004; Hinova-Palova et al., 2007, 2008). Typically, it is
subdivided into the dorsal claustrum (or insular claustrum) and
ventral claustrum (or endopiriform nucleus) (Guirado et al.,
2003; Edelstein and Denaro, 2004a; Ashwell et al., 2004). The

* Corresponding author at: Medical University of Soa, Faculty of Medicine,

Department of Anatomy, Histology and Embryology, 2 Zdrave Str., 1431 Soa,
Bulgaria. Tel.: +359 29172608; fax: +359 29172601.
E-mail address: landzhov_medac@abv.bg (B. Landzhov).
http://dx.doi.org/10.1016/j.jchemneu.2014.08.007
0891-0618/ 2014 Elsevier B.V. All rights reserved.

dorsal claustrum is located deep to the insular cortex and is

extensively connected with most if not all neocortex (Druga,
1966a,b; Otellin and Makarov, 1972; Kunzle, 1975, 1978; Norita,
1977; Riche and Lanoir, 1978; Olson and Graybiel, 1980; Carey
et al., 1980; Hinova-Palova et al., 1980a,b; Hinova-Palova, 1981;
Hinova-Palova and Paloff, 1982, 1984; Carey and Neal, 1985;
Edelstein, 1986; Neal et al., 1986; Sloniewski et al., 1986; TanneGariepy et al., 2002; Ashwell et al., 2004). The ventral claustrum is
situated beneath the piriform cortex; its interconnections with
prepiriform and entorhinal cortex are well documented (Druga,
1966a,b, 1971; Sherk, 1986; Witter et al., 1988; Dinopoulos et al.,
1992).
From a phylogenetic perspective, the dimensions of the
claustrum vary greatly. In lower mammals such as the mouse
and rat it is a small and discrete nucleus ventrolateral to the

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caudoputamen. In the cat it is a relatively large structure that

rivals, or even exceeds, the size of the putamen. In primates it is a
thin gray slab varying in thickness up to several millimeters,
bounded laterally by the extreme capsule and medially by the
external capsule (De Vries, 1910; Landau, 1923; Berlucchi, 1927;
Loo, 1931; Brockhaus, 1940; Macchi, 1984; Rae, 1954; Stelmasiak,
1955; Berke, 1960; Pilleri, 1961, 1962; Filimonoff, 1966; Druga,
1974, 1975; Zilles and Zilles, 1980; Paxinos and Watson, 1989;
Kowianski et al., 2004).
We have long been interested in the claustrums cytoarchitecture, ultrastructure, immunocytochemistry and connectivity
(Hinova-Palova et al., 1979, 1980a,b, 1988, 1997, 2001, 2007, 2008,
2012, 2013a,b,c; Hinova-Palova, 1981, 1986; Hinova-Palova and
Usunoff, 1981; Hinova-Palova and Christova, 1988; Hinova-Palova
and Braak, 1993; Edelstein et al., 2011a,b, 2012a,b). The
claustrums syncytium of GABAergic interneurons and glutamatergic projection neurons, as well as its heterosensory reciprocal
connectivity, leads one to ponder its functional interplay with the
rest of the brain.
Neuropeptide Y is a novel 36-amino acid peptide and one of the
key regulators of food intake, circadian rhythm and reproduction in
many mammalian species, including humans. Originally isolated
from the porcine hypothalamus (Tatemoto, 1982; Tatemoto et al.,
1982), NPY is widely distributed throughout the central and
autonomic nervous system of various vertebrates, such as
mammals (Adrian et al., 1983; Allen et al., 1983; Whale and
Albus, 1984; Emson and De Quidt, 1984; Smith et al., 1985;
Chronwall et al., 1985; Sasek and Elde, 1985; de Quidt and Emson,
1986; Dumont et al., 1992; Krivukuca et al., 2010), amphians
(Danger et al., 1985; Perroteau et al., 1988), teleosts (Pontet et al.,
1989; Danger et al., 1991), elasmobranchs (Vallarino et al., 1988;
Chiba and Honma, 1992), cyclostomes (Chiba et al., 1993) and the
white sturgeon (Chiba and Honma, 1994). With respect to
phylogenetic comparisons specic to the neurochemical architecture of the claustrum, a major focus of our lab, there are but a
handful of studies on NPY immunoreactivity, namely, in the
squirrel monkey (Smith et al., 1985), the cat (Edelstein et al.,
2011b) and humans (Edelstein et al., 2010).
The aim of the present study is to make evident the features and
characteristics of NPY-immunoreactive neurons and bers in the
cat claustrum at the light- and electron-microscopic level, as well
as to provide a description of their distribution.
2. Material and methods
2.1. Perfusion protocol
Ten adult healthy cats from both sexes with average weight 2.3 kg (from 1.9 to
3.0 kg) were used. Our perfusion protocol was previously described in detail
(Papantchev, 2008). In brief, all animals were deeply and irreversibly anesthetized
with an intraperitoneal injection of Urethan (40 mg/kg) and transcardially perfused
with 500 ml of heparinized saline, followed by 3000 ml of phosphate buffered
saline (PBS; pH 7.4) containing 2.5% glutaraldehyde and 4% paraformaldehyde.
After 2 h the brains were removed and postxed in the same perfusate for
additional 2 h. The portion of each cerebral hemisphere containing the claustrum
was removed, dissected and cut into a total of fourteen blocks. Each block was then
sectioned in the coronal plane at 4080 mm on a Vibratome (Technical Products
International, St. Louis, MO, USA). The claustrum was examined in accordance with
the stereotaxic atlas of Reinoso-Suarez (1961).
2.2. Immunohistochemistry
Our protocol for the immunohistochemical visualization of NP immunoreactivity
was previously described in detail (Paloff et al., 2004). In brief, all coronal sections as
described above were treated with sodium borohydride for 45 min followed by
three consecutive 2-min rinses in 0.01 M PBS. This was immediately followed by
30 min in a solution of 1% bovine serum albumin (BSA) and overnight incubation in
a 1:1000 solution of a polyclonal anti-NPY-antibody (Sigma, St. Louis, MO, USA).
Afterwards, sections were given three consecutive 2-min rinses in 0.01 M PBS,
incubated for 20 min in 1% BSA in 0.01 M PBS and followed by a 2-h incubation in
1:500 biotinylated anti-mouse IgG (Vector, Burlingame, CA, USA). This was then
followed by three consecutive 2-min rinses in 0.01 M PBS, with sections incubated

for 1-h in a solution of avidinbiotinperoxidase complex (Vector, Burlingame, CA,

USA). All incubations were carried out on a shaker table at room temperature. For
controls, fteen sections were incubated as described but with the omission of the
primary or secondary antibody. All controls were negative.
After this 1-h incubation a new series of rinses were performed according to a
protocol used in our Department (Paloff et al., 2004; Hinova-Palova et al., 2007)
rst in 0.01 M PBS and then in Tris buffer, pH 7.6 which preceded the visualization
of peroxidase activity using H2O2 and 3,30 -diaminobenzidine as substrates. The
protocol continued with three 2-min rinses in Tris buffer followed by another three
2-min rinses in phosphate buffer. Afterwards, some of the sections was processed
for electron microscopy. Sections were postxed for 1-h with 1% OsO4 in phosphate
buffer, dehydrated in graded series of ethanol and at-embedded in Durcupan
(Fluka, Buchs, Switzerland) between plastic sheets (Paloff et al., 2004; HinovaPalova et al., 2007). Sections were trimmed-out under a dissecting microscope and
cemented to epoxy blanks. Thin sections were cut with an ultramicrotome (LKB,
Stockholm-Bromma, Sweden), counterstained with uranyl acetate and lead citrate
according to a standard protocol used in our Department (Paloff et al., 2004;
Hinova-Palova et al., 2007) and examined with an Hitachi H-500 electron
microscope (Hitachi, Tokyo, Japan). A light microscope (Olympus, Tokyo, Japan)
was used for the low-power examination of NPY-ir neurons.
One-hundred neuronal perikarya were identied and measured via electron
micrographs. The NPY-ir neurons (or neuronal groups) were rst identied by light
microscope and then serial sections were performed. All consecutive measurements were performed on sections where a prominent nucleolus was present. The
ratio of the mean nuclear diameter to the mean diameter of the perikaryon was
calculated as the nucleocytoplasmic ratio (Paloff et al., 2004; Hinova-Palova et al.,
2007) using an ultrastructural size calculator (Ted Pella, Tustin, CA, USA). In
addition, a total of 200 NPY-ir terminal boutons were analyzed in order to evaluate
the synaptic morphology and characteristics of the vesicular populations.
2.3. Morphometric analysis
Our morphometric analysis protocol was previously detailed (Hinova-Palova
et al., 2007, 2008). Briey, stereotaxic planes from A10 to A19 (Reinoso-Suarez,
1961) were examined using an image analyzer (CUE-2, Olympus America, Center
Valley, PA, USA) and a 40 objective. A total of 42 slides (3 randomly selected slides
from each of the 14 tissue blocks examined) per stereotaxic plane were studied. The
number of NPY-ir neurons seen in each section were counted and stored in the
database. The number of NPY-ir neurons was calculated for each plane as an average
of the number of counted neurons from all analyzed sections per plane. The number
of NPY-ir neurons counted was presented as a percentage of all NPY-ir neurons seen
in the entire claustrum. Afterwards, standard planar morphometry and linear
analysis (i.e. line length and width) were performed. The maximum diameter of 500
neurons was measured, and the cells were divided into groups. A mean of the
maximum and minimum diameter of all neurons in each group was then calculated.
For all measurements, only those neurons with clearly visible nuclei were used.
Explanatory marks were added to all images using Adobe Photoshop CS3.

3. Results
3.1. Light microscopy
Neurons and bers immunopositive for NPY-ir were found
throughout the claustrum, from rostral to caudal planes (Figs. 1
and 2). In general, the NPY-ir neurons were typically stained with
immunoproduct visible in the cell cytoplasm and processes, while
the nucleus remained free of label (Fig. 3). Labeling intensity,
however, varied. Some neurons were lightly stained (Fig. 4), while
in others the staining was so dense that they appeared as if they
were silver impregnated (Fig. 5). Immunopositive neurons were
also found proximal to and within the external and extreme
capsules (Fig. 6).
Although NPY-ir neurons were seen throughout the claustrum,
their distribution displayed a lack of uniformity. Based on our
morphometric analysis, approximately 70% of labeled cells were
located in stereotaxic planes A12A16. Of these cells, the majority
appeared clustered within the claustrums central triangle region
(planes A13A15; Fig. 7), with but a handful found proximal to the
external and extreme capsules. Most commonly, the neurons were
situated parallel to the ber tracts, though some were positioned in
a perpendicular fashion. Moving caudally, the number of NPY-ir
neurons gradually diminished (Fig. 8), and at the level of
stereotaxic planes A10A11 only 15% of all counted NPY-ir
neurons were identied. Approximately 10% of all NPY-ir neurons

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Fig. 1. Coronal section through claustrum in different planes from rostral to caudal:
(A) 19 mm in front of the interaural line (arrows delineate claustrum). Nissl staining
20; (B) 15 mm in front of the interaural line (arrows delineate claustrum). Nissl
staining 20; (C) 11 mm in front of the interaural line (arrows delineate claustrum).
Nissl staining 20.

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Fig. 2. Schematic drawings of 5 coronal sections through the cat claustrum showing
the distribution of NPY-positive neurons (black dots) from rostral to caudal planes.
Cn caudate nucleus, Ic internal capsule, Put putamen, Sp septum pellucidum,
Pal pallidum (globus pallidus), Amg amygdala, Th thalamus, Ac anterior
commissure, Hy hypothalamus, Ent entopeduncular nucleus, NII second
cranial nerve (optic).

were identied in the more rostral stereotaxic planes A17A18.

Finally, the rostral pole of the claustrum contained no more than
5% of NPY-ir neurons.
The NPY-ir neurons differed in size and were generally
subdivided into large (over 25 mm), medium (1725 mm), small
(under 17 mm) and dwarf cells (under 8 mm). They were further
categorized into spiny and aspiny types relative to their dendritic
architecture. The various shapes of NPY-ir neurons seen was not
unlike what has previously been noted in similar studies (perhaps
owing to the likelihood that most if not all claustral neurons coexpress proteins and peptides of one type or another): polygonal
(multipolar), triangular, fusiform (bipolar), globular, elliptical, oval
and round.
3.1.1. Large spiny neurons
Large spiny NPY-ir neurons were classied as being greater than
25 mm in diameter, with polygonal (multipolar), triangular,
fusiform, or globular perikarya (Figs. 9 and 10). They were seen
giving rise to as many as ve thick dendritic processes, each with
multiple and multidirectional branches. These branches were
typically quite long and could sometimes be followed up to
700 mm from the cell body. The secondary and tertiary dendrites
were covered with spines. Axons with highly visible hillocks and
initial segments were frequently seen emanating from these cells.
The intensity of immunostaining varied widely from highly
dense (i.e. impregnation-like) to weakly stained. Typically, the
nuclei were non-immunoreactive.

Fig. 3. NPY-immunoproduct is clearly visible in the cell cytoplasm and processes,

with the nucleus stain-free. Scale bar = 50 mm.

3.1.2. Medium spiny neurons

Medium spiny NPY-ir neurons measured from 18 to 24 mm in
diameter with multipolar, fusiform, or oval perikarya. They usually
had long bifurcated dendrites, each covered with well-developed
spines (Fig. 11).

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Fig. 4. Large NPY-positive lightly stained neuron. Scale bar = 50 mm.

Fig. 5. Large spiny NPY-positive darkly stained neuron with long dendrites. Scale
bar = 50 mm.

Fig. 6. Large fusiform NPY-positive neuron close to external capsule. Scale

bar = 50 mm.

Fig. 7. Low magnication of triangular part of claustrum showing the distribution of

NPY-positive neurons. Scale bar = 1 mm.

Fig. 8. Low magnication of the caudal part of claustrum showing a lesser number
of NPY-positive neurons. Scale bar = 1 mm.

Fig. 9. Large spiny NPY-positive neuron with fusiform cell body. Scale bar = 50 mm.

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Fig. 10. Large spiny pear-shaped NPY-positive neuron. Scale bar = 50 mm.

111

Fig. 12. Small spiny NPY-positive neurons with long spiny dendrites. Scale
bar = 50 mm.

Fig. 11. Two medium spiny NPY-positive neurons with long bifurcated dendrites
rich with spines. Scale bar = 50 mm.

3.1.3. Small spiny neurons

Small spiny NPY-ir neurons measured from 15 to 17 mm in
diameter and were typically oval or fusiform in shape with two to
four long and thin dendrites covered with spines (Fig. 12).

Fig. 13. Large aspiny NPY-positive neuron with long prominent bulbous dendrites
(arrows show the varicosities) Scale bar = 50 mm.

3.1.4. Large aspiny neurons

Large aspiny NPY-ir neurons were similar to the large spiny
neurons. These cells also averaged over 25 mm in diameter. Their
perikarya were multipolar, triangular, fusiform, or oval in shape
(Fig. 13), each with as many as ve dendritic processes which
unlike the large spiny neurons were rarely seen to branch.
3.1.5. Medium aspiny neurons
Medium aspiny NPY-ir neurons ranged from 18 to 23 mm in
diameter. Typically, they had three or four dendrites (Fig. 14), with
the primary dendrites being smooth and normally non-branching,
becoming progressively thinner with irregularly spaced varicosities along their lengths.
3.1.6. Small aspiny neurons
Small aspiny NPY-ir neurons were typically 1417 mm in
diameter (Fig. 15) with predominantly oval or slightly elliptical
perikarya and typically two or three thin, varicose, non-branching
dendrites.

Fig. 14. Medium aspiny multipolar NPY-positive neuron with varicose dendrites
(arrows). Scale bar = 50 mm.

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Fig. 15. Small aspiny elliptical NPY-positive neuron with short varicose dendrites.
Scale bar = 50 mm.

3.1.7. Neurogliaform or Dwarf cells

While rarely seen in our sections, we made note of particularly
small cells, averaging under 8 mm, which we termed neurogliaform or dwarf. They had round or oval perikarya with numerous
short and intensely branching processes, most with varicosities of
differing shape and size (Fig. 16).
3.2. Electron microscopy
The ultrastructural analysis of NPY-ir neurons in our sections
clearly demonstrated immunoproduct within perikarya, dendrites,
dendritic spines, axons (both myelinated and unmyelinated), and
terminal synaptic boutons. The ultrastructural features of these
neurons differed relative to their assigned type (i.e. large spiny,
medium spiny, etc.).
3.2.1. Large NPY-ir neurons
Approximately 50% of all NPY-ir neurons were included in this
group (Fig. 17). These neurons were usually heavy stained and had
diameters over 27 mm. Ultrastructurally, they were seen to contain
an abundance of cytoplasm and organelles: mitochondria, Nissl
bodies, a well-developed Golgi apparatus, many free ribosomes, a
large centrally located nucleus (lled with diffusely distributed

Fig. 17. Part of large NPY-positive neuron. Immunoproduct is seen diffusely

distributed throughout the cytoplasm (C), while the nucleus (N) is free of label.
Scale bar = 3 mm.

euchromatin), and a prominent nucleolus. Many labeled and

unlabeled synaptic boutons terminated on the cell surface.
3.2.2. Medium NPY-ir neurons
Approximately 40% of all NPY-ir neurons were classied as
medium in size with a diameter from 18 to 22 mm (Fig. 18).
Typically, these neurons were darkly stained with a relatively large
cell nucleus, a large amount of euchromatin, many mitochondria, a
well-developed Golgi apparatus, and a small amount of Nissl
bodies. A greater number of synaptic boutons (both labeled and
unlabeled) were noted to have terminated on neuronal surfaces
compared with small NPY-ir neurons.
3.2.3. Small NPY-ir neurons
Approximately 10% of NPY-ir neurons were classied as small
in size, usually under 16 mm (Fig. 19). Most of these neurons were

Fig. 16. Two dwarf cells with irregular cell bodies and intensely branching short
varicose dendrites (arrows). Scale bar = 50 mm.

Fig. 18. Medium fusiform NPY-positive neuron. Immunoproduct is distributed

throughout the cytoplasm and nucleus. Scale bar = 5 mm.

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113

Fig. 19. Two small NPY-positive neurons. One of them contains heavy a deposition
of immunoproduct (D), the other, less so (L). Scale bar = 5 mm.
Fig. 20. Large NPY-positive dendrite (D) on which terminate two unlabeled boutons
(T). Scale bar = 1 mm.

darkly stained and evinced several dening characteristics

including a relatively large cell nucleus, a large volume of
heterochromatin, a thin rim of cytoplasm surrounding the nucleus,
a paucity of organelles (with few small mitochondria and free
ribosomes), scattered cisterns of granular endoplasmatic reticulum, and a well-developed Golgi apparatus. Also, a comparatively
smaller number of synaptic boutons (labeled and unlabeled) were
seen to have terminated on the neuronal surface. More often than
not, we had to view quite a few sections in order to spot one with
these cells. These heavily stained neurons usually had a deep
nuclear invagination.
3.2.4. Lightly and darkly stained NPY-ir neurons
Lightly and darkly stained NPY-ir neurons were analyzed
individually. In general, their ultrastructural appearance was
dissimilar. Lightly stained neurons were usually medium or large
in size, while darkly stained neurons were small and rarely
medium in size. The darkly stained small and medium neurons
often had a deep invagination of the nucleolar envelope. There was
also a difference in electron density, which was signicantly
greater in the subgroup of darkly stained NPY-ir neurons.
3.2.5. NPY-ir dendrites
NPY-immunoreactive dendrites differed in size from 0.5 mm
(small) up to 3 mm in diameter (large). Immunoproduct was
associated with the axial neurolaments in the dendrites (Fig. 20),
and was seen in both spiny and aspiny types. The majority of both
labeled and unlabeled synaptic boutons were found to terminate
on NPY-ir dendrites (Figs. 2123).
3.2.6. NPY-ir synaptic boutons
With regard to synaptic terminals, three main categories were
noted: axo-somatic, axo-dendritic and axo-spinous. They varied in
shape, size and vesicular morphology (Figs. 21, 22 and 24). In the
context of synaptic terminals, two distinct types of boutons were
identied relative to vesicular diameter: large round or LR
(Figs. 21, 22 and 24) and small round or SR (Fig. 26).
Approximately 75% of NPY-ir boutons were of the LR type
(Figs. 21, 22 and 24) with an irregular appearance and a diameter
from 1.5 to 3.5 mm (Figs. 21, 22 and 24). Their vesicles were round
with an average diameter of 40 nm (Figs. 21 and 24) and
mitochondria were always present (Figs. 21 and 24). Most often,
LR boutons terminated on medium and large dendrites (Fig. 23),
although unlabeled boutons were also found (Fig. 23). On occasion

Fig. 21. Large NPY-positive dendrite (D) on which terminates a large NPY-positive
synaptic terminal (T). Scale bar = 0.5 mm.

an LR bouton was seen to terminate on more than one dendrite

whiles some formed axo-somatic synapses (Fig. 25). Synaptic
contacts by LR boutons were of the asymmetric type.
Approximately 25% of all NPY-ir boutons were of the SR type
with a diameter from 0.5 to 1.2 mm (Fig. 26). The synaptic vesicles
were somewhat smaller in size than the LR type, with an average
diameter of 30 nm (Fig. 26). On rare occasion we observed heavily
labeled en passant boutons (Fig. 27).
3.2.7. NPY-ir neuropil
Immunoproduct was prevalent in dendrites, myelinated axons,
and terminal boutons throughout the claustrum.

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Fig. 24. Labeled NPY-positive large round synaptic bouton (T) terminates on two
unlabeled medium dendrites (D). Scale bar = 1 mm.

Fig. 22. NPY-positive terminal bouton (T) establishes synaptic contact with a large
labeled NPY-positive dendrite (D). Scale bar = 1 mm.

Fig. 23. NPY-positive dendrite (D) on which terminate two unlabeled boutons (T).
Scale bar = 2 mm.

Fig. 25. Labeled NPY-positive bouton (T) contacting an NPY-positive neuron. Scale
bar = 1 mm.

4. Discussion
To the best of our knowledge, the present study is the rst to
provide detailed insight into the topographical distribution and
morphological characteristics of NPY-ir neurons in the cat
claustrum at the light- and electron-microscopic level. Overall,
our ndings are consistent with those reported for the squirrel
monkey, Saimir sciureus (Smith et al., 1985), cat (Edelstein et al.,
2011b) and human (Edelstein et al., 2010) in which NPY-ir neurons
were recognized in the claustrum of those species.
In this investigation, we report the existence of at least seven
readily distinguishable types of NPY-ir neurons in the cat

claustrum based on shape, namely: polygonal (multipolar),

triangular, fusiform (bipolar), globular, elliptical, oval and round.
We further categorized each of these cells into four main subtypes
based on diameter: large, medium, small and dwarf, and then
into spiny and aspiny relative to their dendritic architecture. The
observed population of NPY-ir neurons was heterogeneous in
composition which, though seen in higher numbers within the
triangular dorsal region (A13A15) in comparison with more
rostral and caudal planes, was reected throughout the claustrum.
When ruminating on the function of the claustrum, we must
surely take into consideration its anatomy and connectivity, as

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Fig. 26. NPY-positive dendrites (D) and terminal boutons of differing size and shape
(arrows). Scale bar = 1 mm.

Fig. 27. Heavy labeled en passant NPY-positive bouton (arrows) terminates on the
periphery of an NPY-positive neuron. Scale bar = 2 mm.

well as the ultrastructure of its neurons. Our electron-microscopic

analysis of the cat claustrum revealed that large NPY-ir neurons
contained an abundance of cytoplasm rich in a variety of
organelles. In addition, their nuclei displayed a preponderance
of euchromatin, with mitochondria-laden terminal boutons.
Characteristics such as these are reective of cells with signicant
metabolic activity. Furthermore, the long and multibranched

115

dendrites of these large neurons especially those of the spiny

variety offer support the contention that these neurons receive a
signicant volume of synaptic input. Both the light- and electronmicroscopic characteristics of these large cells are indicative of
projection neurons rather than interneurons. This was conrmed
by our previous research (Hinova-Palova, 1986).
Medium NPY-ir neurons typically had a lesser volume of
cytoplasm with a relative paucity of granular endoplasmic
reticulum. In point of fact, several subtypes of medium neurons
have been found in the cat claustrum (Hinova-Palova, 1986).
However, for the purposes of this study, we were unable to conrm
that the medium NPY-ir neurons we found corresponded to a
specic subtype. Nonetheless, it is not unreasonable to assume
that some of these cells are projection neurons owing to their lightmicroscopic and ultrastructural morphology.
Small NPY-ir neurons corresponded to the subtypes previously
described by Hinova-Palova (1986). These neurons displayed a
relatively large nucleus, a thin rim of cytoplasm, a paucity of
organelles and very few axo-somatic synapses morphological
criteria that are commonly attributed to local circuit neurons, also
referred to as interneurons (Paloff, 1985; Paloff et al., 1989).
Furthermore, most of the small NPY-ir neurons that we found were
seemingly lled or impregnated with immunoproduct. On the
basis of their light-microscopic and ultrastructural characteristics,
we propose that these densely labeled small NPY-ir neurons are
part of a subpopulation of local circuit inhibitory interneurons.
We also observed NPY-ir dwarf cells in the present study.
Ramon and Cajal (1911) was the rst to recognize these cells in the
striatum by using the Golgi impregnation method. In previous
investigations (Hinova-Palova, 1986; Hinova-Palova et al., 1987)
we demonstrated the existence of dwarf cells in the cat claustrum
via light- and electron-microscopy. Mounting evidence leads us to
believe that these cells are also interneurons.
To elaborate, the small and medium aspiny NPY-ir neurons seen
in the present investigation were predominantly oval-to-fusiform
in shape. In contrast, the large and medium spiny NPY-ir neurons
were often multipolar-to-pyramidal in shape. Based on the extant
literarture on this topic, it is not unreasonable to assume that the
aspiny neurons represent a subpopulation of local circuit
inhibitory interneurons (along with the dwarf cells), while the
spiny neurons represent projection neurons. It was previously
reported that the large spiny neurons in the cat claustrum receive
direct input from cortex and were also shown to project back to the
same areas of cortex, establishing excitatory synapses (HinovaPalova, 1986; Hinova-Palova et al., 1988; Reynhout and Baizer,
1999). Using the Golgi impregnation method in the monkey
claustrum, Brand (1981) reported that the axons of large neurons
were efferent in nature, projected out of the claustrum. Moreover,
in some species, it was reported that the overwhelming majority of
claustral neurons are glutamatergic projection neurons, with only
612% of cells engaged in local network activity (Wojcik et al.,
2004).
The existence of projection neurons in the cat claustrum was
also supported by our present study concerning the features of
large, medium and small spiny NPY-ir neurons. In some sections,
the entire initial segment of an NPY-ir myelinated axon was
present. However, it must be noted that some of these axons were
found not to originate in the claustrum, and thus were afferent in
nature. Neverthless, the existence of afferent and efferent
projection bers in claustrum has been suggested by Wojcik
et al. (2004).
Golgi impregnation studies of the mammalian claustrum have
lead to the description of two major functional classes of neurons
projection neurons with polymorphic perikarya and spiny
dendrites, and inhibitory interneurons characterized by roundto-oval perikarya and aspiny dendrites (Brand, 1981; Braak and

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Braak, 1982; Spahn and Braak, 1985; Hinova-Palova, 1986; Druga

et al., 1993; Wojcik et al., 2004). Our current ndings also reveal
two major classes of NPY-ir neurons in the cat claustrum: spiny
and aspiny. In a previous study on the ultrastructure of the NPYlabeled cat claustrum (Edelstein et al., 2011b), we reported on the
existence of immunoproduct within dendritic spines of differing
shape and size. Thus, our light- and electron-microscopic
investigations strongly suggest that NPY-immunoreactivity is
present in all neuronal types within the claustrum, and that
NPY-ir neurons are not reective of a specic subpopulation. The
fact that NPY-immunoreactivity was observed in neurons of
varying shapes and sizes suggests that these cells play differing
roles in the context of claustral function (Hinova-Palova et al.,
2007, 2008). Present results were comparable to our previous
study of the synaptic organization of the cat claustrum (HinovaPalova, 1986).
It has been established that neurons of differing shapes and
sizes can have different functions (Papantchev et al., 2006). One of
the aims of the present study was to identify whether NPY
immunoproduct was present in possibly functionally distinct
subpopulations of claustral neurons. It is generally accepted that
the number and types of synapses are principal factors with
respect to inuencing neuronal function (Paloff, 1985; Paloff et al.,
1989). This explains why otherwise morphologically identical
neurons function and respond differently, as they are dependent
upon on the characteristics and spike codes of their synaptic
inputs. Therefore, one could speculate that the morphologically
similar NPY-ir neurons we have found dispersed throughout the
cat claustrum may not be as functionally similar as one might
think.
As we have shown in this study, the observed population of
NPY-ir neurons was heterogeneous in nature, consisting of small,
medium and large neurons of various shapes. In support of the
above-mentioned conclusions, we must emphasize that Golgi
impregnation studies of the claustrum in various species of
mammal have lead to the description of two major functional
classes of neurons: projection neurons with polymorphic perikarya, spiny dendrites, prominent mitochondria and numerous
axodendritic synapses, and inhibitory interneurons characterized
by round or oval perikarya and aspiny dendrites (Braak and Braak,
1982; Hinova-Palova, 1986; Wojcik et al., 2004). Another aim of
this study was to establish whether NPY-ir neurons in the
claustrum have a well-dened topographical distribution. In point
of fact, such a distribution was not found. On the contrary, despite
the fact that the central part of the claustrum (stereotaxic planes
A12A16) was seen to contain the majority of NPY-ir neurons,
these cells were diffusely distributed throughout the claustrum.
Indeed, though NPY-ir neuronal clustering or grouping was
apparent within the central stereotaxic planes (which generally
has the highest concentration of neurons), this was not seen within
the ascribed functional zones of claustrum.
Immunohistochemical and in situ hybridization studies have
shown that NPY is one of most abundant and widely distributed
peptides in the central and autonomic nervous system of
mammals, particularly in rats and humans. In addition to the
claustrum, NPY is highly expressed in the cerebral cortex,
hypothalamus, amygdala, hippocampus, nucleus of the solitary
tract, locus coeruleus, nucleus accumbens, periaqueductual gray,
nucleus raphe pallidus, striatum and the bed nucleus of the stria
terminalis (Allen et al., 1983; Chronwall et al., 1985; Sasek and
Elde, 1985; Smith et al., 1985; de Quidt and Emson, 1986; Dumont
et al., 1992; Todd and Spike, 1993; Krivukuca et al., 2010; Edelstein
et al., 2010, 2011a,b). Neuropeptide Y in involved in the control of a
broadly diverse array of physiological functions including feeding
behavior, water consumption, learning and memory, locomotion,
body temperature regulation, sexual behavior, emotional behavior,

neuronal excitability, cardiovascular homeostasis, hormone secretion and circadian rhythms (Baraban et al., 1997; Colmers and
Bleakman, 1994; Hokfelt et al., 1998; Inui, 1999; Munglani et al.,
1996; Vezzani et al., 1999; Wahlested and Reis, 1993). In addition,
it has been suggested that NPY plays a role in psychiatric disorders
including depression, eating disorders, anxiety and epilepsy
(Baraban et al., 1997; Colmers and Bleakman, 1994; Grundemar
et al., 1992; Hokfelt et al., 1998; Munglani et al., 1996; Wahlested
and Reis, 1993). What more, hypothalamic injections of NPY
increase food intake (Stanley and Leibowitz, 1985); in the
hippocampus, NPY has been suggested as an endogenous antiepileptic (Colmers et al., 1991); in the cortex NPY is co-expressed in
many inhibitory GABAergic neurons that modulate pyramidal cell
function in the other regions of the brain (Kubota et al., 2011); NPY
cells play a role in cardiovascular function and emotion (Colmers
and Wahlested, 1993).
From a clinical perspective, given that the claustrum and NPY
have been implicated in the development, symptoms and/or
sequelae of serious maladies such as autism, schizophrenia,
epilepsy, Alzheimers disease, Parkinsons disease, and Huntingtons disease (Wegiel et al., 2014; Cascella and Sawa, 2014; Venneri
and Shanks, 2014; Kalaitzakis, 2014; Corcoran, 2004; Halliday
et al., 1990; Koide et al., 1995; Croom and Taylor, 2001; Flint and
Martin, 1986; Ramos et al., 2006), it behooves both basic and
clinical investigators to learn as much as possible about the
interplay between the two.
5. Conclusion
In conclusion, this is the rst detailed investigation of the light
and electron-microscopic features of NPY-ir neurons and bers in
the cat claustrum. It is hoped that our results and wide-ranging
discussion will provide deeper insight into the functional
neuroanatomy of the claustrum, further elucidating its involvement with a host of critical physiological parameters and biologic
processes, especially those impacted by the aforementioned
neurological disorders. Lastly, much work remains to be done in
terms of studying the claustrum from an ontogenetic and
phylogenetic perspective, with respect to homologues in nonmammalian species as well as its contribution in the context of
higher cortical functions and prevalent hypotheses (Smythies
et al., 2012, 2014a,b). In this regard, it must be mentioned that one
of the greatest minds in modern biology, the late Nobel laureate
and co-discoverer of the structure of DNA, Francis Crick, spent the
last hours of his remarkable life dictating corrections to a draft of
what was to become his nal paper, entitled What is the function
of the claustrum? (Crick and Koch, 2005; Edelstein and Denaro,
2004b). Francis Crick and his co-author Christof Koch have laid the
foundation for future investigative efforts into the claustrums
putative role in that most complex and elusive of neurophysiologic
attributes, consciousness.
Ethical statement
All animals in this study were afforded human care in
compliance with the Principles of Laboratory Animal Care
formulated by the National Society for Medical Research and the
Guide for the Care and Use of Laboratory Animals prepared by
the National Institutes of Health (NIH publication No. 86-23,
revised 1996).
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