Neuroscience 288 (2015) 156166

TRANSITION OF PATTERN GENERATION: THE PHENOMENON

OF POST-SCRATCHING LOCOMOTION
A. TREJO, J. A. TAPIA, B. DE LA TORRE VALDOVINOS,
N. HUIDOBRO, G. FLORES, J. FLORES-HERNANDEZ,
A. FLORES AND E. MANJARREZ *

networks, each exclusively producing a single motor

behavior, or if they overlap, sharing some neurons.
Most of the studies support the hypothesis that both
CPGs share common neurons.
Berkinblit et al. (1978) and Gelfand et al. (1988) suggested for the rst time that one CPG can be enough to
produce both scratching and locomotion. In 1999, Perreault et al. suggested that the similarity of eects from
extensor group I input on the rhythmicity during scratching
and locomotion supports the hypothesis that both rhythms
(scratching and locomotion in the cat) are generated by a
common network. Furthermore, there are studies in
literature indicating that Ia inhibitory interneurons are
rhythmically activated during scratching (Deliagina and
Orlovsky, 1980; Deliagina and Feldman, 1981) or during
locomotion (Feldman and Orlovsky, 1975; McCrea
et al., 1980; Pratt and Jordan, 1987), thus supporting
the hypothesis that both CPGs share the same Ia inhibitory interneurons during their rhythmical activation.
Recently, this hypothesis was directly demonstrated by
means of unitary recordings of Ia inhibitory interneurons
during both locomotion and scratching. Geertsen et al.
(2011) conrmed that at least some Ia inhibitory interneurons are rhythmically active during both the scratching
and the locomotion motor tasks in cats.
The question about the sharing of elements of
scratching and locomotion CPGs has been posed for a
variety of motor tasks, such as walking, ight,
swimming, escape behavior, and struggling, in dierent
animal species (for example see the study by Li et al.,
2007). For instance, in turtles, extracellular unitary recordings of spinal interneurons showed that most of the spinal
interneurons were rhythmically activated during scratching but also during forward swimming (Berkowitz, 2002).
Intracellular recordings combined with histological techniques showed that some of these swimming/scratching
interneurons had axon terminal arborizations in the spinal
ventral horn (Berkowitz, 2008; see also Berkowitz, 2005).
This direct histological-electrophysiological evidence suggests that some spinal interneurons belong to both the
scratching and the locomotor CPGs. Recently,
Berkowitz and Hao (2011), provided additional support
to this hypothesis in the turtle spinal cord. Other studies
support the idea of multifunctional interneurons and network reconguration during a variety of rhythmical motor
tasks (Meyrand et al., 1991; Weimann and Marder,
1994; Jing and Weiss, 2002; Popescu and Frost, 2002;
Briggman and Kristan, 2006; Frigon, 2009). In the larval
zebra sh, four dierent classes of commissural spinal

Instituto de Fisiologa, Benemerita Universidad Autonoma de

Puebla, 14 Sur 6301, Col. San Manuel, Puebla, Puebla CP
72570, Mexico

AbstractA fundamental problem in neurophysiology is the

understanding of neuronal mechanisms by which the central nervous system produces a sequence of voluntary or
involuntary motor acts from a diverse repertory of movements. These kinds of transitions between motor acts are
extremely complex; however, they could be analyzed in a
more simple form in decerebrate animals in the context of
spinal central pattern generation. Here, we present for the
rst time a physiological phenomenon of post-scratching
locomotion in which decerebrate cats exhibit a compulsory
locomotor activity after an episode of scratching. We found
exor, extensor and intermediate single interneurons rhythmically ring in the same phase during both scratching and
the subsequent post-scratching locomotion. Because no
changes in phase of these neurons from scratching to
post-scratching locomotion were found, we suggest that in
the lumbar spinal cord there are neurons associated with
both motor tasks. Moreover, because of its high reproducibility we suggest that the study of post-scratching ctive
locomotion, together with the unitary recording of neurons,
could become a useful tool to study neuronal mechanisms
underlying transitions from one rhythmic motor task to
another, and to study in more detail the central pattern generator circuitry in the spinal cord. 2015 IBRO. Published
by Elsevier Ltd. All rights reserved.

Key words: cat, involuntary movement, transition, interneurons, central pattern generator.

INTRODUCTION
An open question concerning both the locomotion central
pattern generator (CPG) and the scratching CPG in the
cat is whether or not they are separate spinal cord
*Corresponding author. Address: Instituto de Fisiolog a, Benemerita
Universidad Autonoma de Puebla, 14 sur 6301, Col. San Manuel,
A.P. 406, Puebla, Puebla CP 72570, Mexico. Tel: +52-22-22-295500x7326; fax: +52-22-22-33-4511.
E-mail address: eliasmanjarrez@gmail.com (E. Manjarrez).
Abbreviations:
CPG,
central
pattern
generator;
ENG,
electroneurograms; LGS, bilateral gastrocnemius plus soleus; MG,
medial gastrocnemius; PF, pattern formation; RG, rhythm generator;
sCDP, sinusoidal cord dorsum potentials; TA, tibialis anterior.
http://dx.doi.org/10.1016/j.neuroscience.2014.12.038
0306-4522/ 2015 IBRO. Published by Elsevier Ltd. All rights reserved.
156

A. Trejo et al. / Neuroscience 288 (2015) 156166

glycinergic inhibitory interneurons are active during swimming, escape, and struggling (Liao and Fetcho, 2008)
Likewise, by using calcium imaging, it was found that
two dierent sets of interneurons drive swimming and
escape tasks (Ritter et al., 2001).
We found that after the application of curare in the
precollicularpremammillary
decerebrate
cat,
the
spontaneous locomotion is abolished and the scratching
episode was followed by a locomotor-like motor pattern.
In this context, the aim of the present study is to
introduce
the
phenomenon
of
post-scratching
locomotion in the cat and show how it can be employed
as a tool to study neuronal mechanisms underlying the
organization of spinal CPGs. This has allowed us to
provide experimental evidence to support previous
studies showing that both scratching and locomotor
CPGs share common neurons.

EXPERIMENTAL PROCEDURES
Summary
We performed experiments in 16 precollicular
premammillary decerebrate cats. The scratching
episodes were elicited by stimulation of the ipsilateral
pinna. Electrical potentials of exor and extensor nerves
of the hindlimbs as well as from the spinal cord were
recorded during these episodes.
Preparation
We performed experiments in cats (weight range 24 kg),
in accordance with the European Communities Council
Directive of 24 November 1986 (86/609/EEC), the
guidelines contained in the National Institutes of Health
Guide for the Care and Use of Laboratory Animals (8523, revised in 1985) and the Norma Ocial Mexicana
NOM-062-ZOO-1999. Moreover, the Institute of
Physiology from the Benemerita Universidad Autonoma
de Puebla, Mexico, approved the present study. First,
the animals were gently handled and introduced in a
comfortable anesthesia-induction-box at a temperature
of 2229 C and 4070% relative humidity. The
anesthesia was induced with a mix of isourane (34%)
delivered in a mixture of 30% oxygen and 70% nitrous
oxide. The animals did not exhibit discomfort. Second,
when the animals were completely anesthetized we
employed an anesthesia face mask with the same
mixture of isourane (12%), 30% oxygen and 70%
nitrous oxide. Radiant heating and a heating pad were
employed to maintain the temperature of the animals at
about 37 C. Third, the administration of the anesthetic
mixture was maintained by a tracheostomy tube
inserted into the trachea. Fourth, blood pressure was
monitored from the right carotid artery using a
transducer. The radial vein was cannulated to
administer dextran and saline solution as necessary to
maintain blood pressure between 80 and 120 mmHg.
We administered atropine (0.05 mg/kg subcutaneous)
and dexamethasone (2 mg/kg intravenous). A buer
solution of glucose (5%) and bicarbonate (100 mM) was
continuously infused for blood pH maintenance. The

157

bladder was catheterized through the urethra. The level

of anesthesia was veried during the whole surgery by
monitoring arterial blood pressure, respiratory rate, heart
rate, color of mucous membranes, palpebral reex, and
by periodically testing the absence of muscle tone and
withdrawal reexes.
Surgical procedures. A laminectomy was performed to
expose C1C2 and L4S1 segments of the spinal cord
and the dura mater was removed. Bilateral
gastrocnemius plus soleus (LGS), medial gastrocnemius
(MG) and tibialis anterior (TA) nerves were dissected
and prepared for recording. Pools of mineral oil covered
the exposed cord (after placement of the electrodes)
and hindlimb nerves and the temperature was kept at
37 C. After surgery, the cat was xed on the
stereotaxic apparatus, and then decerebrated with a
transection at the precollicularpremammillary level and
removal of all rostral brain tissue. A stereomicroscope
(Leica) was used during the decerebration. During this
procedure great care was taken to avoid tearing the
venous sinuses. Bleeding was controlled with the
application of oxidized regenerated cellulose (Surgicel,
Johnson & Johnson) around the exposed tissues and
blood vessels. The cranial vault was lled with agar
(37 C). After decerebration and the agar application,
anesthesia was gradually discontinued. We did not
touch the animals for a period of about 7 min. We did
not observe any sign of pain or any sudden change in
blood pressure. Then the cats were paralyzed with
pancuronium bromide (Pavulon, Organon) and articially
ventilated. Finally, we applied a piece of cotton
impregnated with D-tubocurarine (0.1%) on the surface
of the C1C2 segments. At the end of the experiments
the animals were euthanized with an overdose of
pentobarbital of 175 mg/kg i.v., administered through the
radial vein. Arterial blood pressure was also monitored
during the euthanasia.
Electrophysiological recordings
We provoked scratching by mechanical stimulation of the
pinna, neck or head with duration of about 13 s; the
same duration in all the experiments. We recorded
electroneurograms (ENG) of the distal end of dissected
nerves. Spinal electrical activity (i.e., slow oscillations)
was monopolarly recorded with a multi-electrode array
of 30 AgAgCl electrodes (200 lm of diameter)
positioned on the surface of the lumbosacral L4S1
spinal cord against an indierent electrode placed on
the paravertebral muscles. We designed and
constructed this multielectrode array in our laboratory
and adapted it to the surface of the cat spinal cord
(Manjarrez et al., 2005). The distance between electrodes
was 5 mm along the rostrocaudal axis and 1 mm along
the mediolateral direction (see also Cuellar et al., 2009).
We used a Synamps (NeuroScan) electroencephalographic amplier (for details, see the methods section
from previous studies from our laboratory; Cuellar et al.,
2009; Perez et al., 2009). We obtained recordings in DC
mode of the cord dorsum potentials and ENG with a wider
DC-500-Hz band-pass lter. In the rst set of experiments

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A. Trejo et al. / Neuroscience 288 (2015) 156166

(four cats/16) we recorded the unitary activity of spinal

interneurons by means of glass micropipettes lled with
NaCl 1.2 M (715 MX). In a second set of experiments
(two cats/16) we employed a 5-channel single electrode
system (Mini matrix quartz/platinum-tungsten ber electrodes, Thomas Recording GmbH), for single-unit spinal
cord activity. Electrode impedance (12 MX) was
checked with a current metal electrode impedance tester
(5 nA/1-kHz sin wave). We employed the main amplier
system (MAF-05, Thomas RECORDING GmbH) with a
gain of 250 and 0.520-kHz bandpass lters. The spinal
interneurons were extracellularly recorded from the L6
segment.
Histology. Microelectrode placements were veried by
histology. At the end of each experiment the micropipette
used was cut and left in place. The animal was perfused
with 10% formalin. After complete xation and
dehydration, the lumbosacral spinal cord was placed in
a solution of methyl salicylate for clearing. The spinal
cord with the segment containing the microelectrode
was sectioned and photographed.
Data analysis. We used the program Scan 4.3
(Neuroscan) to measure the latency in seconds
(mean standard deviation, sd) from the end of the
scratching episode to the beginning of the postscratching episode. The pooled data were presented in
a raster plot. We used this same program to measure
the amplitude of the electrical sinusoidal spinal waves
and plotted this amplitude versus their recording
electrode. Furthermore, we calculated power spectra of
the sinusoidal spinal waves recorded during both ctive
scratching and post-scratch ctive locomotion using
Matlab software (MathWorks).
Histograms and statistical analysis. For comparative
statistics, parametric tests (e.g., Students t-test) were
applied when distributions passed tests of normality and
equal variance; otherwise, nonparametric tests (e.g.,
MannWhitney U test) were used.
We obtained histograms of the number of spikes of
each interneuron during the scratching cycle and the
step cycle, respectively. We used these histograms to

construct polar plots and analyze the rhythmical

discharge of these interneurons classied as exor,
extensor or intermediate, using circular statistics (Drew
and Doucet, 1991; Cuellar et al., 2014). The radial axes
were normalized. In order to establish statistical signicance of the vector related to the phase-preference bursting we applied the Rayleigh test.

RESULTS
In 14 of 16 cats, we recorded the electrical activity of the cat
spinal cord from L4 to S1 spinal segments during two motor
events in the same experiment: ctive scratching and postscratching ctive locomotion. Fig. 1 shows a typical
recording from our experiments. The scratching episode
was elicited by stimulation of the ipsilateral pinna. We
observed that after the end of the scratching episode, a
locomotion
episode
starts
(postscratch
ctive
locomotion). Fig. 1 illustrates sinusoidal cord dorsum
potentials (sCDP) during both motor tasks as well as the
patterns of exor and extensor electroneurographic activity
during scratching and locomotion. The lasting ring activity
in the exor (TA) trace during the post-scratching delay in
Fig. 1 was rare to observe among all recordings.
We observed that after the application of curare in
precollicularpremammillary decerebrate cats, the
incidence of spontaneous locomotion (those episodes
which we could not associate with scratching) was
about 2% of all locomotion episodes and was found in 4
of the cats: Experiment A (1/18) (number of nonassociated locomotion episodes/number of postscratching locomotion episodes); Experiment B (3/40);
Experiment C (3/40); Experiment D (1/10).
We also observed spontaneous scratching episodes
without locomotion (about 26.2% of all scratchings),
which mostly occurred at the beginning of the curare
application. After a period of about two or three minutes
we observed a high incidence of post-scratching
locomotion episodes.
In about 73.8% of the trials obtained from 14 cats, the
scratching episode was followed by a locomotor-like
motor pattern. Furthermore, the mean duration of the
scratching and post-scratching locomotion episodes was
8.6 2 s and 18.5 7 s, respectively. The latency from
the end of each scratching episode (magenta arrow in

Fig. 1. Scratching episode followed by a post-scratch locomotion episode. Top trace, sinusoidal cord dorsum potentials (sCDP) recorded from the
L6 spinal cord during both tasks. Middle trace, simultaneous recording of an extensor (Ext) nerve, MG. Bottom trace, simultaneous recording of a
exor nerve (Flex), TA. The rst arrow (green) indicates the beginning of the scratching episode, the second arrow (magenta) indicates its end, and
the third arrow (yellow) indicates the beginning of the post-scratch locomotion episode. Periods used to calculate power spectra are illustrated with
horizontal brackets. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

A. Trejo et al. / Neuroscience 288 (2015) 156166

Fig. 1) to the beginning of the post-scratch locomotion

episode (yellow arrow in Fig. 1), called post-scratching
latency, had a mean value of 6.2 3.5 s (mean sd)
for all the cats. We used the TA activity to perform the
measurements (blue trace, Fig. 1). To avoid bias this
mean latency was calculated as a mean of means for
the individual experiments. Furthermore, we measured
the latency from the beginning of the scratching episode
(see green arrow) to the beginning of the postscratching locomotion cycles (see yellow arrow) giving
us an additional measurement of the delay in which one
locomotor activity follows another. This mean latency
was 16.1 4.6 s.
Because in the present study we introduce for the rst
time the phenomenon of post-scratching locomotion, we
measured the relative amplitude and duty cycles of the
motor nerve activities. The duty cycles were calculated
as the ratio between the burst duration and the cycle
duration (Biewener and Gillis, 1999). We found that during
post-scratching locomotion the bursts of rectied and integrated ENG exhibit amplitudes of 21.8 5.3 lV for exion and 15.5 9.1 lV for extension, and duty cycles of
50 10% for exion and 50 3% for extension
(mean sd, 14 cats). The duty cycle represents the percentage of the step cycle in which the extensor or exor
nerves are active. These measurements could be useful
to compare this post-scratching locomotion with spontaneous or MLR locomotion previously described in classical studies.
Fig. 2 illustrates, in a pooled raster display, the end of
every burst ring activity of the exor TA nerve during
scratching, as well as during the subsequent onsets of
every burst during the post-scratching ctive locomotion.
These results are some typical examples (20 episodes)
obtained from 275 episodes in 14 animals. The triangles
indicate the beginning of the post-scratching locomotion
episodes.
The pooled raster display in Fig. 2 also illustrates that
the cycle duration of motoneuron rhythmic activity (i.e.,
the separation between consecutive vertical lines in
each row) was shorter during scratching (0.3 0.09 s)
than during post-scratching locomotion (1.7 0.6 s) for
all the animals. The cycle durations of these two motor
activities diered signicantly (MannWhitney U test,
p < 0.001).
Moreover, we calculated power spectra of sinusoidal
electrical waves recorded during scratching and postscratching locomotion for 14 cats (Fig. 3). The power
spectra analysis revealed that the range of common
frequencies of sinusoidal waves recorded during
scratching episodes (Fig. 3A) did not overlap with the
range of common frequencies of the spinal sinusoidal
waves recorded during post-scratching locomotion
(Fig. 3B). The mean frequency for the dominant peaks
of the spinal waves recorded during ctive scratching
was 3.5 0.9 Hz (mean sd, 14 animals), and those
recorded during locomotion episodes had a frequency of
0.9 0.4 Hz (the same 14 animals); these mean
frequencies diered signicantly (Students t-test,
p < 0.001).

159

Fig. 2. Examples of raster graphs for the exor rhythmical activity

during episodes of scratching and the subsequent post-scratching
ctive locomotion. Every vertical line represents the end of every
burst ring activity of the exor tibialis anterior (TA) nerve during
scratching, as well as the subsequent onsets of every burst during the
post-scratching ctive locomotion. All responses were aligned relative
to the end of the scratching episode. Triangles indicate the beginning
of the post-scratching locomotion. Two colors were employed to
easily visualize both rhythms. (For interpretation of the references to
color in this gure legend, the reader is referred to the web version of
this article.)

Furthermore, we compared the frequencies of sCDP

recorded during post-scratching locomotion in the
present experiments (14 cats) with those previously
recorded during spontaneous ctive locomotion without
application of topical curare (300 trials, 22 cats; Trejo
et al., 2009). The sCDP during spontaneous ctive locomotion exhibit an average frequency of 1.1 0.4 Hz.
We found that there was nostatistically signicant dierence in the mean frequency of the spinal waves recorded
during both types of locomotion (Students t-test,
p = 0.279).
In order to identify the rostrocaudal location of the
groups of neurons generating the sCDP occurring
during post-scratching locomotion, we analyzed their
amplitude distribution by plotting each amplitude versus
the position of their recording electrode. These
electrodes were positioned on the left (electrodes 18,
Fig. 4A, B) and right side (electrodes 2532, Fig. 4A, C)
of the spinal cord. On the top of the graphs we show
the spinal segment where each electrode was located.
We found that the amplitude of the spinal electrical
wave for post-scratching ctive locomotion is distributed
from the L4 to S1 spinal cord segments, and follows a
bell shape (white triangles, Fig. 4B, C) very similar to
the amplitude distribution for the sinusoidal electrical
waves associated with ctive scratching (black triangles,
Fig. 4B, C). Moreover, we compared statistically these
sCDPs amplitudes, recorded during scratching and
post-scratching locomotion, and only found statistical

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Fig. 3. Power spectra for the sinusoidal cord dorsum potentials

during (A) scratching and (B) post-scratching ctive locomotion and
for 14 cats as indicated in dierent colors. During scratching the
dominant peaks are between 2 and 6 Hz. In contrast, during postscratching locomotion the dominant peaks are below 2 Hz. Note that
the power spectra of the post-scratching locomotion do not include a
dominant peak between 2 and 4 Hz (i.e., in the range of scratching).

signicant dierence in the recording electrodes number 8

and 32, positioned respectively on the left and right S1
spinal segment (Students t-test, p < 0.05, Fig. 4).
We recorded the activity of 13 single interneurons with
glass micropipettes in four cats and 14 single
interneurons with the Thomas Recording mini matrix
system in two other cats (see Experimental
procedures) during scratching and post-scratching
ctive locomotion. In the experiments with glass
microelectrodes we recorded interneurons rhythmically
active during 19 episodes of scratching and postscratching ctive locomotion. Three of these six cats
were also employed for the analysis of interneurons

during sinusoidal traveling waves in the spinal cord during

scratching (Cuellar et al., 2009). We noted that these cats
exhibited both types of episodes, scratching alone, or
scratching followed by post-scratching locomotion. For the
analysis we selected only those episodes of scratching followed by post-scratching ctive locomotion (Fig. 5B, D),
as illustrated in Fig. 5A. The histological reconstructions
are illustrated in Fig. 5C. Fig. 5A illustrates the recording
of an interneuron that res rhythmically during the scratching cycles, as well as during the locomotor cycles. The raster display for 10 interneurons and 13 episodes from 4 cats
is illustrated in Fig. 5B. The phase in which every neuron
red, during scratching and during post-scratching locomotion is indicated on the right: exor indicates the exor
phase, Extensor is the extensor phase, and Interm is
the intermediate phase. Moreover, in the experiments with
the Thomas Recording mini matrix system we recorded 5
Flexor, 4 Extensor and 5 Intermediate interneurons rhythmically active during scratching and post-scratching ctive
locomotion (Fig. 5 D).
We did not observe a change in phase of these
recorded neurons; e.g., we did not observe that a
neuron ring during scratching extensor phase can
change to the exor phase during locomotion or vice
versa.
We constructed polar plots from all the recorded
interneurons. We employed the same methodology
described in a previous report (Cuellar et al., 2014). Circular statistics was employed in order to assign the specic
phase where a neuron red. We constructed histograms
of the number of spikes (gray lines, Fig. 6) and calculated
their average vector to nd the preferred phase of discharge. We found that all interneurons red in the same
phase during scratching and during post-scratching locomotion. Fig. 6 illustrates the polar plots calculated for the
experiments with microelectrodes. Fig. 6A shows the
polar plots of the discharge of 5/13 interneurons recorded
and classied as exor during scratching (left panel) and
post-scratching locomotion (right panel). The blue traces
in the plot represent the average of the electroneurographic exor activity, and the vectors show the preferred
phase of discharge (Rayleigh test, p < 0.05). Moreover,
we found that 4/13 interneurons red in the extensor
phase during both, scratching and post-scratching locomotion (Rayleigh test, p < 0.05), as indicated in the polar
plots in Fig. 6B. The extensor phase is marked by a red
trace. Finally, 4/13 interneurons showed an intermediate
phase preferred discharge during both motor tasks, as
illustrated in Fig. 6C, the intermediate phase is indicated
by magenta traces.
The change of motor pattern without circuit
reconguration is rare (except Li et al., 2014). The change
of phase in interneuron activity would imply a very signicant reconguration, which was not observed in our study.
Some modest conguration, however, may come from the
change of spike numbers when the network activity
switches from one to another, e.g. one neuron will re
the biggest number of spikes in locomotion but re the
smallest number in scratching (among all recorded interneurons). In this context, we analyzed the number of
spikes per cycle in both scratching and locomotion for

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Fig. 4. Lumbo-sacral amplitude distribution of sinusoidal cord dorsum potentials (sCDP) occurring during scratching and post-scratching
locomotion. (A) Diagram of the experimental arrangement, a 32-channel multielectrode was positioned on the dorsal surface of the L4S1 spinal
cord. (B) sCDP amplitude versus recording electrodes 18 and (C) sCDP amplitude versus recording electrodes 2532, positioned on the left and
right sides of the spinal cord, respectively. There is a statistically signicant dierence between the sCDPs amplitudes recorded during scratching
(black triangles) and post-scratching locomotion (white triangles) in electrodes 8 and 32 (Students t-test p < 0.05).

the interneuron recordings and we found signicant

changes in the number of spikes. The interneurons ring
during the exor phase exhibited change from 13 3
(scratching) to 79 22 (post-scratching locomotion)
spikes per cycle. The interneurons ring during the extensor phase exhibited change from 7 1 (scratching) to
26 9 (post-scratching locomotion) spikes per cycle.
The interneurons ring during the intermediate phase
exhibited change from 4 1 (scratching) to 12 3
(post-scratching locomotion) spikes per cycle. These
changes in the number of spikes for every phase were statistically signicant (p < 0.001, MannWhitney U test).
We also calculated the ratio between the number of spikes
per cycle during post-scratching locomotion and the number of spikes per cycle during scratching locomotion. We
obtained the ratios for the three groups of interneurons.
The ratios were scaled up and the values were as follows:
6 for exor interneurons, 3.7 for extensor interneurons and

3 for intermediate interneurons. This suggests that activities of the three groups of recorded interneurons are not
proportionally scaled up. Moreover, we computed the ring frequencies of these three groups of neurons during
scratching and post-scratching locomotion; these values
are consistent with the number of spikes per cycle previously analyzed for these neurons. These results suggest
that the change of the motor pattern from scratching to
post-scratching locomotion could involve a circuit reconguration as well; however, this possibility must be carefully taken into account (see Discussion).

DISCUSSION
Interneurons associated with scratching and postscratching locomotion
In the present study we show for the rst time the
occurrence of a phenomenon we call post-scratching

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Fig. 5. Interneurons ring during scratching and post-scratching locomotion. (A) Simultaneous recordings of the sinusoidal cord dorsum potential
(sCDP), a single exor interneuron (INT) ring during scratching and post-scratching ctive locomotion, and the corresponding MG and TA
electroneurograms. (B) Pooled raster graphs for the rhythmical cycle of 10 single interneurons recorded with the glass micropipette during
scratching and the subsequent post-scratching ctive locomotion. All responses were aligned relative to the end of the scratching episode. Triangles
indicate the beginning of the post-scratching locomotion. The right vertical boxes indicate the phase in which every neuron red: blue for exor, red
for extensor and magenta for Interm (intermediate). All neurons maintain the same phase during both rhythmical activities. (C) Histological location
of interneurons ring rhythmically during both motor tasks. The symbols represent the location of the recorded interneurons illustrated in B. (D) The
same as B, but for 14 other interneurons recorded with the mini matrix Thomas Recording system during scratching and the subsequent postscratching ctive locomotion. All neurons also maintain the same phase during both rhythmical activities. (For interpretation of the references to
color in this gure legend, the reader is referred to the web version of this article.)

ctive locomotion. Moreover, we found exor, extensor

and intermediate single interneurons rhythmically ring
in the same phase during both scratching and the
subsequent post-scratching locomotion. Because no
changes in phase of these neurons from scratching to
post-scratching locomotion were found we, suggest that
in the lumbar spinal cord there are shared neurons
associated with both motor tasks. Thus we can
speculate that these neurons could belong to both

scratching and locomotion CPGs. It is important to

mention that even when two dierent recording systems
were used the results were similar and comparable.
This highlights our results because they can be
reproducible.
Our results are consistent with the hypothesis that
the spinal rhythmic circuitry for pattern generation is
constituted by shared interneurons which are involved
in the generation of dierent rhythmic motor tasks

A. Trejo et al. / Neuroscience 288 (2015) 156166

163

Fig. 6. Polar plots of the analyzed interneurons. Vectors show the preferred phase of discharge (p < 0.05). The 0 represents the onset of the exor
activity. Data are shown counterclockwise. All neurons red in the same phase during scratching (left panel) and post-scratching locomotion (right
panel). (A) 5/13 interneurons red in the exor phase (indicated by the blue traces). (B) 4/13 interneurons were rhythmically active during the
extensor phase (red trace). (C) 4/13 interneurons were classied as intermediate during both rhythmic tasks (magenta traces indicate the
intermediate). (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

(Berkowitz, 2001a; Stein, 2005). In the studies reported

by Berkowitz (2001a) and Stein (2005), the interneurons
were active during dierent forms of scratching; furthermore, some interneurons were active in the same phase
during three dierent forms of scratching and during
swimming, while others were only active during scratching but not during swimming (Berkowitz and Stein, 1994;
Berkowitz, 2001a, 2002). The rhythmic activity of interneurons during three dierent forms of scratching was
concentrated mainly in a particular phase of the ipsilateral hip exor activity cycle, suggesting that some of
these neurons may contribute to the generation of the
hip rhythm for all three types of scratching, strengthening
the idea that vertebrate pattern-generating circuitry for
distinct behaviors may be overlapped (Berkowitz,
2001a). In agreement with these observations, a network
reconguration of neurons was also reported in other
species (Li et al., 2007; McLean et al., 2007;
Berkowitz, 2008; Liao and Fetcho, 2008). Because in
the present article the single interneurons were grouped
simply according to their activity phase we do not know if
the synaptic connections between them are excitatory or
inhibitory. Therefore, any conclusions on whether circuit

re-conguration takes place or not must be carefully

taken into account.
The combined use of multifunctional and specialized
neurons is now established as a principle of
organization across a range of vertebrates. The
mechanisms involved in selecting and generating
dierent motor patterns have been explored in several
studies made in the larval zebrash and the adult turtle.
Such studies (Berkowitz et al., 2010) have revealed that
distinct kinds of motor patterns are selected and generated by combinations of multifunctional and specialized
spinal interneurons. One possibility is that a combination
of multifunctional and specialized neurons could be
shared between motor circuits during scratching and
post-scratching locomotion.
However, intriguing evidence shows a dierent
regulation of the cycle period, phase durations, and
phase transitions during ctive locomotion compared to
scratching in the cat, thus providing evidence of
specialized rhythm-generating mechanisms in each
motor task (Frigon and Gossard, 2010; Gossard et al.,
2011). This suggestion should carefully be explored in
more detail in future studies.

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A. Trejo et al. / Neuroscience 288 (2015) 156166

D-Tubocurarine and the occurrence of postscratching locomotion

A possible explanation for the post-scratching ctive

locomotion phenomenon is that the D-tubocurarine
(applied at C1C2 to elicit scratching), in a preparation
where spontaneous ctive locomotion is mostly absent,
increases the excitability of neurons that are shared
between the two rhythms, scratching and locomotion. In
other words, the phenomenon we are reporting implies
that both CPGs share neurons which are overexcited
when D-tubocurarine is applied at C1C2, thus allowing
for locomotion to be elicited after a scratching episode.
It is well known that besides its peripheral eect on
muscular transmission, curare shows an excitatory
eect on the central nervous system (Bernhard and
Taverner, 1951) when injected intravenously (Cohnberg,
1946; Bernhard and Taverner, 1951; Bernhard et al.,
1951), or when applied topically (Feldberg et al., 1956),
and even after intraventricular injection (Feldberg et al.,
1956). Eccles (1946) applied curare on the isolated frog
spinal cord and found that large doses set up sustained
spontaneous convulsant activity. Intravenous injection of
curare was found to increase the amplitude of the monosynaptic extensor reex (Bernhard and Taverner, 1951).
In anesthetized and decerebrate cats, d-tubocurarine
applied topically on C1C2 spinal segments not only produces but facilitates the scratch reex (Feldberg and
Fleischhauer, 1960; Domer and Feldberg, 1960;
Berkinblit et al., 1978; Deliagina et al., 1983). This behavior suggests a selective activation of the long descending
propriospinal neurons in the lateral column of the spinal
cord, which on stimulation of the skin mediate the scratching activity (Feldberg and Fleischhauer, 1960).
The time-window of post-scratching locomotion
In our experiments, we found that after scratching, this
stereotypical post-scratching locomotion episode occurs
with a mean latency of 6.2 3.5 s (mean sd, 14
cats). This long latency between both episodes could
explain why in previous experiments this phenomenon
was not detected even though scratching and
locomotion have been recorded in the same
experiments (Degtyarenko et al., 1998; Perreault et al.,
1999; Stecina et al., 2005; Lafreniere-Roula and
McCrea, 2005; Frigon and Gossard, 2009, 2010). It
seems likely that in previous studies the time window for
observation was too small to observe the phenomenon.
In fact, after a reanalysis of four of our previous experiments (Cuellar et al., 2009; Perez et al., 2009) with a
larger time window of 20 s we found about 70% of postscratching ctive locomotion episodes.
Electrical sinusoidal waves associated to locomotion
In the present study we observed that the post-scratching
ctive locomotion was associated with sinusoidal
electrical waves recorded on the surface of the spinal
cord. The sinusoidal waves during scratching were
similar to those described in previous studies from our
laboratory (Cuellar et al., 2009; Perez et al., 2009). The

dominant frequency in the power spectrum of the electrical sinusoidal waves recorded during scratching episodes
did not overlap with the dominant frequencies of the spinal
waves recorded during post-scratching locomotion. Moreover, because the frequency of ctive spontaneous locomotion (Trejo et al., 2009) occurs in the same range as
the post-scratching locomotion we suggest that both
motor behaviors and spinal waves could share similar
neuronal circuits.
Speculations in terms of the two-layer CPG
architecture
Regarding the architecture of the CPG, McCrea and
Rybak (2008) proposed a two-level CPG architecture containing a half-center rhythm generator (RG) and a pattern
formation (PF) network. The latter is controlled by the RG
and projects to exor and extensor motoneuron populations. We could suggest that the scratching CPG and the
post-scratching CPG could share the PF circuits but not
the rhythmicity-generating systems in McCrea and
Rybaks hierarchy. This possibility is supported by our
nding that the scratching and the post-scratching CPG
always occurs at dierent frequencies, but the same exor
and extensor nerves (and possibly the same motoneuron
pools) can exhibit either scratching or post-scratching
locomotion. Future experiments conrming that the same
motoneurons can exhibit either scratching or post-scratching locomotion will be necessary to support the hypothesis
that both CPGs could share the same PF circuits.
Hart and Giszter (2010) (see also Hart and Giszter,
2004; Giszter and Hart, 2013) found that some sets of
interneurons in the intermediate nuclei of the spinal cord
may organize individual spinal primitives (i.e., pulsed premotor drives of xed duration that recruit a covarying set
of muscles, yielding particular force patterns). These
authors showed that these interneurons and their primitives are shared across motor behaviors. Our nding
about post-scratching locomotion is consistent with the
Hart and Giszters study. It is tempting to speculate that
both motor behaviors scratching and post-scratching
locomotion may be generated by the same set of interneurons and their primitives. Moreover, in the context of
McCrea and Rybaks CPG architecture, we could suggest
that Hart and Giszters neuronal circuits conform modular drives in the pattern-shaping systems, thus allowing
the transition from scratching to post-scratching
locomotion.
Descending pathways producing scratching and
post-scratching locomotion
We suggest that both rhythms, scratching and postscratching
locomotion,
originate
from
dierent
supraspinal structures. There are several observations
that support this idea. The stimulation of descending
pathways eliciting locomotion (e.g., mesencephalic
locomotor region, MLR) evokes a bilateral coordinated
rhythm in the four limbs; whereas, stimulation of
descending pathways for scratching can evoke
unilateral rhythm in only one hindlimb. It is plausible that
the same supraspinal structures that evoke ctive

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A. Trejo et al. / Neuroscience 288 (2015) 156166

locomotion can also evoke post-scratching locomotion.

Some of these structures have been very well described
in literature (for review, see Orlovsky et al., 1999). On
the other hand, the scratching rhythm can be elicited by
activation of one system of reticular neurons located in
the region of the obex (see Tapia et al., 2013), which
are on-o and o-on neurons that presumably send
descending information via the reticulospinal pathways
in order to activate the scratching CPG.
Transitions from one rhythmic motor task to another
Other forms of behaviors that involve transitions in motor
patterns have also been described in invertebrates. For
example, the craysh escape behavior is followed by
rhythmic swimming. Reichert et al. (1981) interpreted this
behavior by an extension that occurs as a chained reex
following a giant-neuron-mediated exion, with subsequent control of the extensors passing to a CPG circuit.
The sequenced behavioral grooming pattern in
rodents is generally preserved in decerebrate animals
(Berridge, 1989; Zor et al., 2011). Such experimental
preparations are necessary to dene neural mechanisms
underlying the smooth transition between behaviors normally expressed in a sequence. The present studies,
together with those on rodent grooming (Berridge, 1989;
Zor et al., 2011), indicate that the activity of shared neuronal mechanisms of the CPG circuits could contribute
importantly to normal transitions between behaviors.
Other switching-like transitions associated with the
CPGs have been found by means of stimulation of
the tectum. Grillners group found that depending on the
location and stimulus duration within the tectum in
lampreys, dierent types of eye, trunk and locomotion
movements can occur (Kazuya et al., 2007; Kozlov
et al., 2009). Grillner called this type of behavior tectal
control of locomotion, steering, and eye movements
(Kazuya et al., 2007). Because the stimuli were intentionally applied, the transitions between the dierent classes
of movements and steering of locomotion were not measured. In Grillners studies (Kazuya et al., 2007; Kozlov
et al., 2009), the experimenter changed the type of movement by the characteristics of the stimuli applied on the
tectum; however, in our case we observed the transition
from scratching to locomotion without any class of stimulus to elicit the locomotion.
It is tempting to speculate that the transition of pattern
generation reported in the present study could be
explained by the sequential action of brainstem
supraspinal commands which generate a variety of
dierent movements by selecting dierent combinations
of spinal modules (Roh et al., 2011). Other preparations
of decerebrate animals could be useful to explore the
shared interaction between spinal cord networks for locomotion and scratching. Recently, Berkowitzs group demonstrated that the turtle exhibits strong interactions
between spinal cord networks for locomotion and scratching (Hao et al., 2011). This nding suggests that turtles
could also exhibit transitions in pattern generation.
AcknowledgmentsThis work was supported by the following
grants: CONACYT under projects: F1-153583, 229866 and

Catedra Marcos Moshinsky (EM), Mexico. AT and JAT acknowledge PhD fellowship support from CONACYT Mexico. BDTV
acknowledge postdoctoral fellowship support from CONACYT
Mexico. AF and EM acknowledge support from Cuerpos
Academicos and PROMEP-PIFI-VIEP-BUAP Mexico. We thank
Pablo Linares for technical assistance. We thank John Reid for
proof reading the English manuscript.

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(Accepted 20 December 2014)

(Available online 31 December 2014)