J Neurophysiol 99: 1435–1450, 2008.
First published January 9, 2008; doi:10.1152/jn.01131.2007.
Encoding Network States by Striatal Cell Assemblies
Luis Carrillo-Reid,1 Fatuel Tecuapetla,1 Dagoberto Tapia,1 Arturo Hernández-Cruz,1 Elvira Galarraga,1
René Drucker-Colin,2 and José Bargas1
1
Departamentos de Biofı́sica and 2Neurociencias, Instituto de Fisiologı́a Celular, Universidad Nacional Autónoma de México,
Mexico City, Mexico
Submitted 12 October 2007; accepted in final form 8 January 2008
Carrillo-Reid L, Tecuapetla F, Tapia D, Hernandez-Cruz A,
Galarraga E, Drucker-Colin R, Bargas J. Encoding network states
by striatal cell assemblies. J Neurophysiol 99: 1435–1450, 2008. First
published January 9, 2008; doi:10.1152/jn.01131.2007. Correlated
activity in cortico-basal ganglia circuits plays a key role in the
encoding of movement, associative learning and procedural memory.
How correlated activity is assembled by striatal microcircuits is not
understood. Calcium imaging of striatal neuronal populations, with
single-cell resolution, reveals sporadic and asynchronous activity
under control conditions. However, N-methyl-D-aspartate (NMDA)
application induces bistability and correlated activity in striatal neu-
rons. Widespread neurons within the field of observation present burst
firing. Sets of neurons exhibit episodes of recurrent and synchronized
bursting. Dimensionality reduction of network dynamics reveals func-
tional states defined by cell assemblies that alternate their activity and
display spatiotemporal pattern generation. Recurrent synchronous
activity travels from one cell assembly to the other often returning to
the original assembly; suggesting a robust structure. An initial search
into the factors that sustain correlated activity of neuronal assemblies
showed a critical dependence on both intrinsic and synaptic mecha-
nisms: blockage of fast glutamatergic transmission annihilates all
correlated firing, whereas blockage of GABAergic transmission
locked the network into a single dominant state that eliminates
assembly diversity. Reduction of L-type Ca2⫹-current restrains syn-
chronization. Each cell assembly comprised different cells, but a small
set of neurons was shared by different assemblies. A great proportion
of the shared neurons was local interneurons with pacemaking prop-
erties. The network dynamics set into action by NMDA in the striatal
network may reveal important properties of striatal microcircuits
under normal and pathological conditions.
INTRODUCTION
A central pattern generator (CPG) produces specific activity
patterns in the absence of sensory inputs (Grillner et al. 2005b)
and can transform afferent inputs into detailed spatiotemporal
outputs (Grillner 2006; Yuste et al. 2005). In vitro experiments
with circuits containing CPGs demonstrate that tonic excitation
produced by the glutamate agonist N-methyl-D-aspartate
(NMDA) can activate the stereotyped electrical behavior that
neuronal networks exhibit in more intact preparations (such as
fictive locomotion). This is observed as recurrent bursting
activity in single neurons, while simultaneous unitary and
population recordings demonstrate synchronicity and alterna-
tion of cell assemblies activity during bursting (e.g., Gordon
and Whelan 2006; Grillner et al. 1981; Guertin and Hounsgaard
1998; Hsiao et al. 1998; Kiehn 2006; Takakusaki et al. 2004b).
Basal ganglia (BG) contain CPGs that activate innate be-
havioral routines, procedural memories, and learned motor
Address for reprint requests and other correspondence: J. Bargas, Instituto
de Fisiologı́a Celular UNAM, PO Box 70-253, Mexico City, DF 04510 Mexico
(E-mail: jbargas@ifc.unam.mx).
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programs (Barnes et al. 2005; Graybiel 1995; Grillner et al.
2005a,b; Takakusaki et al. 2004a). A major component of the
BG is the striatum, which receives a widespread input from the
cerebral cortex and thalamus. Striatal circuits process cortico-
thalamic inputs to produce specific outputs consisting of bursts
of action potentials during the execution of motor tasks, prob-
ably following voltage transitions to depolarized “up-states”
(Hikosaka et al. 2000; Kasanetz et al. 2006; Mahon et al. 2006;
Romo et al. 1992; Schultz et al. 1993; Wilson 1993). As in
other isolated nervous tissue preparations known to contain
CPGs (e.g., Guertin and Hounsgaard 1998), addition of NMDA
to neostriatal circuits in vitro (Vergara et al. 2003) and in vivo
(Herrling et al. 1983) induces recurrent bursting and pattern
generation in single neurons. Moreover, intrastriatal applica-
tion of NMDA in vivo generates turning behavior when ad-
ministered unilaterally in freely moving animals (Ossowska and
Wolfarth 1995); demonstrating that motor behaviors arise from
the striatal processing of enhanced excitatory drives. This evi-
dence suggests that the striatum posses the connectivity and
intrinsic mechanisms to orchestrate pattern generation (Grillner
2006).
To test this hypothesis, we used calcium imaging of neuro-
nal populations in a corticostriatal slice preparation to monitor,
with single-cell resolution, dozens of cells simultaneously and
then discern pattern generation produced at the microcircuit
level by the activity of cell assemblies. Simultaneous electro-
physiological recordings from striatal neurons demonstrated
that calcium transients result from neurons bursting on top of
suprathreshold up-states (Kerr and Plenz 2002).
METHODS
Slice preparation
Transverse corticostriatal slices (300 ␮m thickness) were obtained
from PD14-29 Wistar rats as previously described (Kawaguchi et al.
1989; Vergara et al. 2003). All procedures conformed to the guide-
lines of the UNAM’s Animals Scientific Procedures Committee.
Slices were obtained with ice-cold saline (4°C) containing in mM: 123
NaCl, 3.5 KCl, 1 MgCl2, 1 CaCl2, 26 NaHCO3, and 11 glucose (25°C;
saturated with 95% O2-5% CO2; pH ⫽ 7.4; 298 mosM/l). Slices were
then transferred to saline at room temperature (21–25°C) where they
remained for ⱖ1 h before recording. The cationic concentration of this
saline favors the appearance of up states in vitro (Vergara et al. 2003).
Although the present results were performed mainly in young animals
(PD14-21), basically the same network behavior was observed in older
animals (PD25-29) (J. Bargas, unpublished data and see RESULTS).
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1436 CARRILLO-REID ET AL.
Calcium imaging
Slices were incubated at room temperature in the dark for 20 –30
min in the presence of 10 –20 ␮M fluo 4-AM (Tef Labs, Austin, TX)
in 0.1% dimethylsulphoxide (35°C), equilibrated with 95% O2-
5% CO2. Slices were perfused with control saline (see preceding text)
in a perfusion chamber on the stage of an upright microscope
equipped with a ⫻10 water-immersion objective (Eclipse E600FN;
Nikon, Melville, NY). Excitation at 488 nm was performed with a
Lambda LS illuminator (Sutter instruments, Novato CA). Experi-
ments were performed at room temperature.
Images were acquired with a cooled digital camera (SenSys 1401E,
Roper Scientific, Tucson, AZ) at 250 –500 ms/frame. Imaging soft-
ware used was RS Image (Photometrics; Roper Scientific). The
imaged field was 800 ⫻ 600 ␮m in size. Short movies (100 –250 s, 50-
to 100-ms exposure, 2– 4 image/s) were taken at time intervals of 5–20
min during 1 h.
The number of fluo 4-loaded neurons in the field was determined at
the end of the experiment with a 5-s puff of 50 mM KCl. This
maneuver disclosed all fluo-4-labeled neurons (either active or silent
during the experiment). Cells active during the experiment were ana-
lyzed, and the ratio of active/silent cells was obtained. Spontaneous or
evoked calcium transients together with voltage responses were re-
corded electrophysiologically in some cells, both in control saline and
during the application of 5–12 ␮M NMDA (Sigma-Aldrich-RBI, St.
Louis, MO). In some experiments, cortical sensory motor areas were
stimulated with a concentric bipolar electrode (12 ␮m; FHC, Bow-
doinham, ME). Stimuli consisted of different trains of 5–10 stimuli at
20 Hz. Each stimulus was 100 –200 ␮s and 50 –120 ␮A. In some
experiments, we used the minimal stimulus intensity necessary to
evoke peaks of synchrony with amplitudes above chance (P ⬍ 0.05).
This allowed us to study changes of electrical evoked synchrony
under different pharmacological conditions.
Immunohistochemistry
Sections were processed to fix fluo-4 fluorescence— or cells active
during experiments—with N-(3-dimethylaminopropyl)-N⬘-ethylcarbodi-
imide hydrochloride (EDAC), and to perform conventional immunocy-
tochemistry in fluorescent cells to demonstrate either substance P (SP) or
enkephalin (ENK) on fluo-4-labeled cells using commercially available
antisera (Peninsula Labs, San Carlos, CA) conjugated to CY3 or CY5.
Slices were not processed for both antisera, but one was chosen in each
case. Thus in each trial, either SP or ENK positive and negative neurons
could be observed. Briefly, sections were rinsed in PBS and incubated for
18 –24 h at 4°C with primary rabbit antibody against ENK or SP (diluted
1:200). Sections were mounted in an anti-quenching media (Vectashield,
Vector Laboratories) and examined under a confocal microscope (MRC-
1024; Bio-Rad, Natford, UK) equipped with a krypton–argon mixed-gas
laser. Immunostained cells were studied in either single confocal images
or reconstructed sections made by projecting z-series of 10 – 40 consec-
utive confocal images 1 ␮m apart collected throughout the thickness of
the section. The background noise was reduced averaging three to six
images. Digitized images were transferred to a personal computer (Con-
focal Assistant, T. C. Brelje). More than 80% of fluo-4-loaded cells were
medium spiny neurons.
Drugs
Stock solutions were prepared before each experiment and added to
the perfusion solution in the final concentration indicated. NMDA,
APV, nicardipine, CNQX, biocytin, and bicuculline methiodide or
hydrochloride were obtained from Sigma (St. Louis, MO).
Electrophysiology
Calcium imaging and simultaneous electrophysiological recordings
were obtained from areas of the dorsal striatum previously shown as
receiving numerous cortical fibers (Vergara et al. 2003).
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An Axoclamp 2B amplifier (Axon Instruments, Foster City, CA) was
used to perform whole cell current- and voltage-clamp recordings. Sig-
nals were filtered at 1–3 kHz and digitized at 3–9 kHz with an AT-MIO-
16E4 board (National Instruments, Austin, TX) in a PC computer. Data
acquisition used a software designed in the LabView environment
(Lemus-Aguilar et al. 2006). Patch pipettes (3– 6 M⍀) were filled with (in
mM) 115 KH2PO4, 2 MgCl2, 10 HEPES, 0.5 EGTA, 0.2 Na2ATP, and
0.2 Na3GTP. In some experiments, biocytin 0.5%, and fluo-4 salt (20 –30
␮M) were added to the recording pipettes.
Image analysis
Image processing was carried out with Image J (v.1.36, National
Institutes of Health), Multicell 2.0 (kindly supplied by Robert Froemke),
and custom-made programs written in IDL (Cossart et al. 2003; Mao
et al. 2001; Schwartz et al. 1998) or MATLAB (The Math-Works,
Natick, MA).
All active neurons in a field were semi-automatically identified, and
their mean fluorescence was measured as a function of time. Single-
pixel noise was discarded using a 5-pixel ratio mean filter. Calcium-
dependent fluorescence signals were computed as (Fi – Fo)/Fo, where
Fi: fluorescence intensity at any frame and Fo: resting fluorescence,
i.e., average fluorescence of the first four frames of the movie.
Calcium signals elicited by action potentials were detected based on
a threshold value given by their first time derivative (2.5 times the SD
of the noise value). Thus we obtained a C ⫻ F binary matrix; were C
represents the number of active cells and F the number of frames for
each movie. Spike onsets were signaled by ones in the matrix represent-
ing transitions to the up states. Recordings were inspected manually to
remove artifacts and slow calcium transients which are likely to corre-
spond to glial cells (Ikegaya et al. 2005; Sasaki et al. 2007).
Statistical methods
To determine if calcium transients recorded from different cells
were correlated, the numbers of simultaneous activations per trial
(onset of signals occurring within 3 frame windows) were detected.
To determine the P value of simultaneous transients occurring by
chance, the distribution under the null hypothesis of independent
transients using Monte Carlo simulations with 1,000 replications were
computed (Mao et al. 2001).
The degree of correlation between active cells was calculated with
the Jaccard correlation coefficient. Nevertheless the magnitude of the
correlations is difficult to discern when many lines are superimposed.
Thus we constructed cross-correlation maps of Jaccard correlation
coefficients to show the magnitude of the correlations between all
cells pairs.
In addition, we identified the sets of cells activated simultaneously
over time. To identify peaks of synchronous activity (i.e., that in-
cluded more cells than those expected by chance), Monte Carlo
simulations were also used to estimate the significance of their firing
together. The threshold corresponded to a significance level of P ⬍
0.05. We then examined peaks that were significant at P ⬍ 0.05.
Peaks of synchronous and recurrent bursting activity that remained
significant during the experiment were selected for further analysis.
To analyze the dynamics of cell assemblies over time (network
dynamics), D ⫻ N matrices were constructed, where D represents the
number of active neurons in a set of experiments, and N denotes the
firing of cells during 250-ms to 1-s time bins (NMDA-induced up
states last between 0.5 and 5 s) (Vergara et al. 2003). Peaks of
recurrent synchronous activity were vectorized so that bursting over
time was associated with different neurons. Each vector element is
formed by the sum of the number of calcium spikes displayed by a
single neuron during the time bin, where peak derivatives denote the
time onset of electrophysiologically recorded up states (see preceding
text and RESULTS). Therefore the set of these vectors denote network
activity as a function of time (Brown et al. 2005; Sasaki et al. 2007).
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 STRIATAL NETWORK DYNAMICS 1437

To measure the similarity index between the network vectors, we RESULTS

computed the normalized dot product of all possible vector pairs,
which is equivalent to the cosine of the angle between the vectors
(Sasaki et al. 2006; Schreiber et al. 2003). Then we plotted the
similarity indexes as a pseudocolor matrix in which functional
states sustained by significantly correlated or synchronized burst-
ing cell assemblies appear as cluster-like structures (Sasaki et al.
2006).
To reduce the dimensionality of the network vectors, locally linear
embedding (LLE) was chosen. LLE is an unsupervised learning
algorithm that discloses nonlinear structures from multi-dimensional
data (Roweis and Saul 2000). Multidimensional scaling (MDS) (Sys-
tat, Richmond, CA) gave results quantitatively similar from those
obtained with LLE. Nevertheless LLE depicted more effectively the
trajectories of the functional states in the network. The trajectories of
our high-dimensional data were not well described by linear dimen-
sionality reduction methods (see Brown et al. 2005). To choose the
optimal number of states depicted from the LLE reduction, we used
hard and fuzzy clustering algorithms taking the Dunn’s index as a
validity function (Bezdek et al. 1997; Sasaki et al. 2007). To deter-
mine the number of neurons in each state, hierarchical cluster analysis
was computed using Euclidean distances and the nearest neighbor
single linkage method (Systat Software, San Jose, CA).
Optical imaging from populations of striatal neurons
To study striatal microcircuits in vitro, we used Ca2⫹ imag-
ing (Fig. 1) to measure electrical activity in many cells simul-
taneously with single-cell resolution. Activity was measured
indirectly, as changes in fluorescence. Fields from the dorsal
striatum were imaged in slices loaded with the Ca2⫹ indicator
fluo-4 AM in 174 experiments performed in 86 corticostriatal
slices. Figure 1A shows all dye-loaded neurons (see METHODS).
Contours of cells both active and inactive during the experi-
ment are depicted in Fig. 1B (filled circles and empty contours,
respectively). Only neurons active during an experiment were
analyzed. Under control conditions (with no drugs added), only
a few cells were active, and their firing was asynchronous
(filled circles in Fig. 1B). Records of Ca2⫹ transients from
three spontaneously active neurons are shown (Fig. 1C). These
experiments confirm that the striatal circuitry is mostly quies-
cent (n ⫽ 40 slices) under control conditions.
Simultaneous voltage recordings and Ca2⫹ imaging in me-
dium spiny neurons (Fig. 1, D and E) demonstrate the corre-

FIG. 1. Optical recording in striatal neurons. A: neurons in a striatal slice loaded with fluo-4 AM. Picture is the result of averaging 200 consecutive frames
and background subtraction (see METHODS). Scale bar: 100 ␮m. B: automatic contour detection of 376 cells from A. Dark circles indicate neurons exhibiting
spontaneous calcium transients under control conditions with no drugs added (14/376 or 3.7%). Under control conditions, most striatal cells loaded with fluo-4
remain silent. C: recurrent calcium transients recorded from 3 of the active cells shown in B (280 ms per frame). D: a fluo-4-loaded cell targeted for
electrophysiological recording (1). Fluo-4 salt was also administered through the recording pipette (bottom). Scale bar: 10 ␮m. Voltage responses (top) to current
steps (bottom) recorded from the neuron shown in D1 (2). Inward rectification and long latency to 1st spike are characteristics of medium spiny neurons.
Steady-state current-voltage relationship measured in current-clamp mode from traces shown in D2 (3). Most calcium transients and electrophysiological
recordings shown in the next figures are from this class of neurons. E: simultaneous recordings of voltage transitions (1) and calcium transients (2) from the cell
shown in D1 induced by the presence of N-methyl-D-aspartate (NMDA) in the bath. The duration of first derivatives of the calcium transients [dashed line
indicates 2.5 times the SD of the noise; 3; peaks in d(⌬F/F)/dt— gray stripes] match the duration of electrophysiological up states. Dots indicate events where
d(⌬F/F)/dt ⬎2.5 times SD; used to build raster plots (see following text). Histogram showing a bimodal distribution of membrane potential (4); taken from
electrophysiological recordings in 1. Current-voltage relationship measured in voltage-clamp configuration in the presence of NMDA (5). Note 3 crossing points
in the voltage axis and a negative slope conductance region.

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1438 CARRILLO-REID ET AL.
spondence of suprathreshold activity and somatic calcium
transients (n ⫽ 15 cells). As it has been observed in many other
central circuits (e.g., Gordon and Whelan 2006; Grillner et al.
1981; Guertin and Hounsgaard 1998; Hsiao et al. 1998; Kiehn
2006; Takakusaki et al. 2004b), we were able to generate
bistability in striatal neurons after bath application of NMDA
(Vergara et al. 2003), a transmitter known to induce motor
behavior when administered in the striatum (Fig. 1E) (Ossowska
and Wolfarth 1995). This treatment induces persistent bursting
behavior in neostriatal neurons (⬎1 h) without the need of
electrical stimulation (spontaneous). Most active cells during a
given experiment had the electrophysiological characteristics
of medium spiny neurons (Fig. 1D). Ca2⫹ transients (⌬F/F)
corresponding to up states, had time derivatives [d(⌬F/F)/dt]
that matched bursts duration (Cossart et al. 2003; Kerr and
Plenz 2002). Clearly membrane potential distribution is bi-
modal in bursting cells (Fig. 1E4). The current-voltage rela-
tionship (I-V plot) measured in voltage clamp at the end of 400-
to 500-ms commands shows a negative slope conductance
region (NSCR), indicating bistability, in NMDA-treated cells
(Fig. 1E5) (e.g., Hsiao et al. 1998; Izhikevich 2007; Vergara
et al. 2003). Ca2⫹ imaging and simultaneous electrophysiolog-
ical recordings also show that only bursts with two or more
action potentials produce detectable Ca2⫹ transients in striatal
neurons (n ⫽ 10 neurons; Fig. 2). These experiments con-
firmed that the striatal neurons can be activated in vitro by
NMDA bath application. Therefore the next step was to ask if
this activity was correlated and synchronous throughout the
network, in which case, it may correspond to a network
dynamics capable of producing pattern generation.
Cortical stimulation synchronizes widespread
striatal neurons
In corticostriatal slices, electrical stimulation of the cortex
evokes long-lasting depolarizations with overriding spikes in
medium spiny neurons (Bargas et al. 1991; Vergara et al.
2003). Trains of cortical stimuli (Fig. 3A) also result in pro-
20 mV
A
200 ms
-75 mV
B Arrows point to the same records shown below in a
5% ∆F/F
15 s
C threshold (dashed line) was set at 2.5 SD of the
10%/s
d(∆F/F)/dt
D
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longed synaptic depolarizations with action potentials (Fig.
3B). Figure 3B illustrates an experiment where whole cell
current-clamp recordings (Fig. 3B1) and calcium imaging (Fig.
3B2) were performed simultaneously from a medium spiny
neuron (stimulus frequency: 0.1 Hz; stimuli are signaled with
arrows at the bottom). A calcium transient accompanies each
orthodromic response to the cortical stimulus (Fig. 3B2, arrows
at the bottom). The time derivatives of the calcium responses
are shown in the third row (Fig. 3B3); note similar duration of
derivative positive peaks and voltage responses. Accordingly,
each peak from the differentiated calcium signal generates a
dot used to build raster plots as that illustrated in Fig. 3D. Each
row in the raster plot represents an active neuron (filled circles
in Fig. 3C). Filled circles in Fig. 3C indicate fluo-4-loaded
neurons within the field of observation that responded to
cortical stimulation in one representative experiment. Empty
circles indicate loaded neurons that did not respond to cortical
stimulation (see METHODS). Responsive cells are a minority of
the total number of neurons which are scattered throughout
the observational field among many unresponsive neurons
(Fig. 3C).
Cortical stimulation activated and synchronized ⬃10 –20%
of fluo-4-loaded cells. Only a few neurons were active before
and after the stimulus train, and these cells present a lack of
correlation (Fig. 3, D and E). At each trial, ⬎90% of the
responsive neurons followed the cortical stimulus (Fig. 3D,
bottom). Some neurons spontaneously active before the stim-
ulus did not follow the electrical stimulation (blue lines).
Targeted electrophysiological recordings of cells responsive to
cortical stimulation revealed that most active neurons were
medium spiny neurons (Fig. 3B).
Network dynamics set into action by NMDA
Ca2⫹ imaging allowed us to observe a population of striatal
cells activated by cortical stimulation in control conditions
(Fig. 3) and in the bath presence of NMDA (5–12 ␮M; Fig. 4).
Simultaneous voltage recordings of imaged neurons (Fig. 4A)
FIG. 2. Action potentials necessary to produce
detectable calcium transients. A: example of action
potentials evoked by brief intracellular depolarizing
current steps (not shown) in a medium spiny neuron.
slower time base. B: actions potentials were evoked
repetitively (as in Fig. 1A) in successive series (de-
limited by dashed boxes) of 1– 4 action potentials.
C: simultaneous recording of calcium transients ac-
companying the action potentials shown in B. Notice
that significant calcium transients follow voltage
responses with ⱖ2 action potentials. D: 1st deriva-
tive of calcium records shown in C. Detection
noise. Threshold is reached when stimulus evokes
ⱖ2 action potentials.
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 STRIATAL NETWORK DYNAMICS 1439

FIG. 3. Widespread distribution of striatal neurons synchronized by cortical stimulation. A: scheme of the experimental arrangement: field stimulation was
delivered to corticostriatal afferents (electrode in cortex) while neurons from a dorsal striatum area (square) were recorded with simultaneous calcium imaging
and whole cell, patch-clamp recording techniques. B: synaptic response from a medium spiny neuron to a train of synaptic potentials (10 field stimuli at 20 Hz;
arrows indicate train stimuli); note firing of action potentials on top of synaptic response. A sequence of synaptic responses such as that illustrated at left (1).
Calcium transients recorded from the spiny neuron (280 ms/frame) responding to cortical stimulation in 1 (2). Note that each synaptic response has a
corresponding calcium transient. First derivative obtained from calcium recordings (3). Dots indicate the onset of the responses used to build the raster plots
shown in D. C: mapping of all neurons present in the field of view (circles). Cells triggered by cortical stimulation: 33/252 (13%) are indicated with red filled
circles. Cells unresponsive to cortical stimulus but excitable, as shown after high K⫹ application, are indicated by empty circles in this and other figures. Scale
bar: 50 ␮m. D: population raster plot: each row represents an active neuron. Some cells present spontaneous activity before and after the stimulus. Note
synchronized responses from neurons several hundreds of microns apart during cortical stimulation (red). Histogram at the bottom represents the percentage of
responding cells triggered by each cortical stimulus. E: cross-correlation map including all active cells (P ⬍ 0.05; see METHODS). One spontaneously active cell
never responded to cortical stimulation (navy blue lines).

show that voltage transitions (Vergara et al. 2003) were ac-
companied by Ca2⫹ transients during NMDA activation (Kerr
and Plenz 2002, 2004). Cell activity in the circuit was followed
by recording their Ca2⫹ transients and plotting them as raster
plots (Fig. 4B). Similar NMDA-induced electrical activity has
been observed in neurons that are part of a CPG (e.g., Grillner
et al. 1981; Guertin and Hounsgaard 1998; Hsiao et al. 1998;
Takakusaki et al. 2004b). Interestingly, the raster plot (Fig. 4B,
top) shows that several neurons are involved in the activity.
Moreover, the time histogram (Fig. 4B, bottom; asterisks, red
lines) clearly shows periods of increased synchrony and cor-
related firing that occur spontaneously in different sets of
neurons (Cossart et al. 2003; Ikegaya et al. 2004). Statistically
significant (threshold P ⬍ 0.05) spontaneous peaks of syn-
chrony occurred in sets of neurons from n ⫽ 38/45 slices in
these conditions. On average, there were 2.5 ⫾ 0.2 peaks of
synchronous bursting per 201 ⫾ 10-s time epoch. Peaks of
recurrent synchronous activity showed no apparent periodicity,
and occurred with a mean interval of 46 ⫾ 10 s. Notably,
neurons firing synchronously during NMDA treatment could
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be hundreds of microns apart intermingled with silent cells
(Fig. 4C, filled circles). Spatial correlation maps (Fig. 4D)
show the pairs of neurons that exhibit statistically significant
(P ⬍ 0.05) correlated activity (line thickness is proportional to
the degree of correlation). On average, 77 ⫾ 4% of active cells
had correlated activity (n ⫽ 38 slices) at any given moment.
Cells active during synchrony peaks represent most of the
correlations between cells. Correlation plots of this activity
(Fig. 4E; P ⬍ 0.05) show that the degree of synchronization
among active neurons is heterogeneous, thus the network
activity is not due to chance correlation between any pair of
neurons but to the spatiotemporal dynamics of several cells.
Furthermore, application of NMDA to neostriatal circuits in
vitro (Vergara et al. 2003) and in vivo (Herrling et al. 1983)
induces bursting activity and generates turning behavior in
freely moving animals (Ossowska and Wolfarth 1995), sug-
gesting a general mechanism preserved in a broad range of
ages. Structured network dynamics with the same characteris-
tics have been shown in the cortex of young mice in vitro
(PD13-22) and adult cats in vivo, demonstrating that network
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1440 CARRILLO-REID ET AL.

A B
50

Active cell #
-75 mV 40

30

20
80 mV
10

% coactive cells
5 sec
10 * P<0.05
* **
20% 5
∆F/F

50 s
C D E
46 47 43 44 45

jaccard correlation
41 42
40 40
37 39 38 0.75
36
35

neuron i
34
33 32
29 31 30
28 30
27
2523
19 20 21 2226 24
20
18
1716 14 15 13
12
10 11
7 8 9 10 0
6
5 4
12 3 10 20 30 40
neuron i
FIG. 4. NMDA-induced network activity. A: simultaneous electrophysiological and calcium imaging recordings from a spiny neuron in the presence of 8 ␮M
NMDA. Cortical stimulus (arrows) produces plateau potentials with detectable calcium transients. Recurrent membrane potential transitions, with bursting and
corresponding calcium transients, are visible several minutes (up to ⬎1h) after interrupting cortical stimulation. B: raster plot of NMDA-induced activity (no
cortical stimulation). Each row represents an active cell. Activity can be followed in several neurons simultaneously with calcium imaging. Histogram at the
bottom shows the percentage of co-active neurons as a function of time. Peaks of spontaneous synchrony (red), indicated with asterisks, show that recurrent
bursting is shared by sets of neurons in the circuit, suggesting the emergence of a spatiotemporal pattern (250 ms/frame). C: mapping of neurons in the field of
observation that exhibited recurrent bursting (filled red and black circles), after NMDA administration (without cortical stimulation; 47/291 cells; 16%). Scale
bar: 100 ␮m. Red circles indicate cells active during peaks of synchronous activity. D: spatiotemporal correlation map of NMDA-induced activity. Lines connect
neurons the firing of which was significantly correlated (P ⬍ 0.05): n ⫽ 43/47 (91%). Red circles indicate active neurons involved in the synchrony peaks. Note
the widespread distribution of cells firing together. E: cross-correlation map of all possible pairs of neurons. Note the heterogeneous distribution of the correlation
coefficients. (For better visualization, cross-correlation of the same cells was removed from the map; black line).

activity, intrinsic to specific nuclei, is preserved in slices
(Ikegaya et al. 2004). But to address this issue in the striatal
microcircuit, we divided our data into age groups to discern
possible maturational variables. Figure 5 shows the age inde-
pendency of NMDA-induced network dynamics. In spite of the
previously described reduction in the number of loaded cells as
a function of age (PD14-29; Fig. 5, A–C) (Froemke et al. 2002;
Peterlin et al. 2000), the number of peaks of synchronous
bursting per time epoch was maintained across PD14-29 (Fig.
5D), confirming a robust mechanism, across this age range, for
the network behavior described here (Ikegaya et al. 2004).
These experiments demonstrated the following. 1) NMDA
treatment induces pattern generation in the neostriatum in vitro. 2)
Activity is distributed throughout the circuit, and it is shared by
a significantly higher percentage of neurons than those active
under control conditions. 3) Activity may be synchronous and
correlated firing is seen in sets of neurons: 4) these phenomena
continue for an extended period of time without electrical
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stimulation once the network becomes active. 5) The network
dynamics observed occur in a wide range of ages (PD14-29).
Accordingly, we hypothesized that the network dynamics set
into action by NMDA should reveal sets of related neurons
(cell assemblies) that alternate their activity to generate spa-
tiotemporal patterns of synchronization.
Visualizing functional states during network dynamics
We next investigated network dynamics in the striatum over
long periods of time (n ⫽ 15 slices). Brief movies (100- to
250-s epochs) were taken at different intervals for up to an hour
(e.g., Fig. 6C, 1–5, separated by lines). To analyze network
dynamics, we binned the raster plots of all the neurons in-
volved in the peaks of synchrony (Fig. 6). Time segments
represent vectors coding coactive burst activity of individual
neurons (see METHODS). Network dynamics consisted of a set of
N vectors (bins) in D dimensions (active neurons). Vectoriza-
99 • MARCH 2008 • www.jn.org
 STRIATAL NETWORK DYNAMICS 1441

A B

PD 14 PD 29

C D
400 5

4
Loaded cells

# Peaks/epoch
350

3
300

2
250
1
200

14 18 22 26 30 14 18 22 26 30
Postnatal Day Postnatal Day
FIG. 5. Age independency of network dynamics. Neurons in a striatal slice loaded with fluo-4 AM, PD 14 (A) and PD 29 (B). Pictures are the result of
averaging 720 consecutive frames (background is not subtracted). Scale bar: 100 ␮m. C: number of loaded cells as a function of postnatal day. Each point
represents one brain slice. The line represents an exponential fit. D: number of peaks per 201 ⫾ 10 s time epoch as a function of postnatal day. F, 1 brain slice.
—, best linear fit. Note that although the number of loaded cells decreases with age, the network dynamics remains constant.

tion allowed us to compare different states of network activity
rigorously over time (Brown et al. 2005; Sasaki et al. 2007).
The normalized inner product (see METHODS) of all possible
vector pairs provides a measure of the similarity among states
(Sasaki et al. 2006; Schreiber et al. 2003). To evaluate this
similarity along network activity, we plotted all vector pairs as
a matrix (Fig. 6A). Notably, abrupt transitions in the similarity
index showed the presence of cluster-like structures (Sasaki
et al. 2006).
To compare network responses induced either by cortical
stimulation or by NMDA bath application, we reduced the
dimensionality of the vectors using LLE (see METHODS), a
technique for nonlinear dimensionality reduction (Brown et al.
2005; Roweis and Saul 2000; Stopfer et al. 2003). The new
vectors were projected in two dimensions (Fig. 6B). Clusters of
points formed trajectories representing the responses over time
to specific stimuli. Trajectories illustrate different subgroups of
neurons coactive within each functional state (Brown et al.
2005; Stopfer et al. 2003). Thus changes in the functional state
of the network can easily be followed. In Fig. 6B, state 1
represents network response to cortical stimuli in control con-
ditions. State 2 depicts NMDA-induced network activity with-
out electrical stimulation. State 3 represents network response
to the same cortical stimuli (given in the state 1) in the bath
presence of NMDA. Figure 6C, top, shows the raster plot used
to reconstruct network states, whereas bottom shows histo-
grams with the percentage of co-active neurons along time.
Cortical stimulation in the presence of NMDA recruited many
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more striatal neurons (Fig. 6C3, blue dots and peaks) than in
control conditions (Fig. 6C1, green dots and peaks) and pro-
duced peaks of synchrony much larger than those produced by
NMDA only (Fig. 6C, 2, 4, and 5, red dots and peaks).
Spontaneous peaks of synchrony induced by bath NMDA were
⬃20% the size of those induced by cortical stimulation (cf. Fig.
6C, 1–5, bottom). Nonetheless the general behavior of the
active network after a cortical stimulus (Fig. 6C, 4 and 5)
remained essentially comparable to the activity before the
stimulus (Fig. 6C2 and see following text). In fact, time
histograms showed similar numbers of spontaneous peaks of
synchronous activity (Fig. 6C, bottom; P ⬍ 0.05) before and
after the cortical stimulus. Moreover, cortical stimulation (Fig.
6C3) elicited a trajectory looping back to the NMDA-induced
dynamics (Fig. 6B), showing recurrent activation of the same
assembly after a perturbation (Stopfer et al. 2003). Figure 6D
shows the spatial distribution of the neurons involved in the
different states. Hierarchical cluster analysis revealed the ex-
istence of different neuronal subsets (assemblies) that underlie
network states (Fig. 6F). Each state was basically sustained by
a different neuronal assembly. A state transition implies a
change of neuronal assembly (alternation). However, some
elements are shared by different states (core neurons). Thus
using a simple algorithm we could distinguish recursive peaks
of synchronization reflecting functional states that involve
different sets of neurons. We conclude that spatiotemporal
firing patterns codified in multidimensional vectors are suffi-
cient to reconstruct the dynamics of a given network.
99 • MARCH 2008 • www.jn.org
1442 CARRILLO-REID ET AL.

A B 2
60

similarity index
1 state 2
state 3
vector i (t)
40

LLE 2
0

-1
20

0 -2 state 1

-3
0 20 40 60 -2 -1 0 1 2 3
vector i (t) LLE 1
C
1 2 3 4 5
140
120
Active cell #

100
80
60
40
20
0
% coactive cells

10 P<0.05
* *
* * * * *
* *
5

1 min
% coactive cells

D E
state 1 state 2 state 3 20

10

0
1&2 1&3 2&3
# states
F

state 1
state 2
state 3
137
135
126
123
120
115
112
109
104
92
88
82
77
61
43
37
6
1
3
34
40
60
66
80
87
91
102
105
110
114
116
122
125
127
136
70
51
10
4
20
69
134
128
103
98
95
85
73
63
53
49
46
42
39
35
30
24
16
8
11
22
28
32
36
41
44
47
50
58
72
76
94
96
99
106
140
143
129
81
64
52
33
27
48
54
74
84
138
55
5
29
132

Active cell #
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 STRIATAL NETWORK DYNAMICS 1443

Pattern generation denoted by functional states sustained by specific states (Fig. 7F). Nevertheless there is a small core of
cell assemblies cells shared by all the network states (⬃8% of neurons in-
The preceding analyzed states included some with imposed volved in the synchrony peaks).
synchronization, i.e.: with electrical cortical stimulation. If We concluded that functional states, denoted by spontaneous
network dynamics induced by NMDA are mediated by cell peaks of synchrony (Barnes et al. 2005), are generated by the
assemblies, then one may expect the appearance of distinguish- coordinated participation of cell assemblies, as it is the case of
able functional states evoked by the excitatory tonic drive “unit CPGs” (Grillner 2006). The transitions from one state to
brought about by NMDA only (without electrical stimulation). the other display long-term network dynamics. The return of
To address this issue, we investigated the relationships be- the same cell assemblies after net traveling through different
tween the peaks of synchronous activity induced only by trajectories shows that the elements of these modules are
NMDA (similar results were obtained in n ⫽ 14/15 slices that robustly associated, allowing alternating participation. Also, a
presented NMDA-induced peaks of synchrony). core of neurons is shared by all the states.
To observe alternation between functional states, we inves-
tigated the NMDA-induced network dynamics in the striatum Mechanisms underlying network dynamics
over long periods of time (Fig. 7, n ⫽ 15 slices). Brief image for pattern generation
series (100 –250 s in duration) were obtained at different
intervals for ⱕ1 h (Figs. 7C, 1-,3 top, representative image For a preliminary investigation into the synaptic and intrin-
series obtained at different times during the experiment are sic requirements for multistable network dynamics, we chal-
shown separated by vertical lines). lenged network activity with both synaptic and intrinsic ion
A similarity correlation matrix (similarity index, Fig. 7A) channel antagonists. Fast synaptic inhibition within the striatal
between these vectors clearly disclosed abrupt transitions in- circuit (Czubayko and Plenz 2002; Koos et al. 2004; Tepper
dicating the presence of statistically significant cluster-like et al. 2004; Tunstall et al. 2002) was tested with the GABAA
structures (Sasaki et al. 2006). We projected these vectors into receptor antagonist, bicuculline, which was applied once the
two dimensions (Fig. 7B, colored circles) by further reducing network became active.
vectors dimensionality with LLE (Brown et al. 2005; Roweis Figure 8 shows that on exposure to 10 ␮M bicuculline, the
and Saul 2000; Stopfer et al. 2003). This projection allowed us number of state transitions is drastically reduced in the
to observe a variety of functional states in the network as NMDA-treated slices (n ⫽ 6 slices). The network becomes
clusters that follow a series of trajectories in sequence. We locked in a preferred state, which is recurrently revisited.
found a different set of neurons (cell assemblies) with corre- Similarity indexes representing network dynamics suggest the
lated synchronous firing for each functional state (Brown et al. absence of diverse cell assemblies (Fig. 8A). Only occasional
2005; Stopfer et al. 2003) (Fig. 7D; blue, red, green). Therefore transitions to another state occurred (Fig. 8B). Paradoxically,
the method allows us to follow the evolution in time of the the raster plot of network activity showed an increased fre-
functional states within the network. Experiments similar to quency of recurrent synchrony peaks: from 2.7 ⫾ 0.2 peaks in
that illustrated in Fig. 7B demonstrated the existence of robust the NMDA condition to 8.3 ⫾ 2 peaks in the presence of
nonrandom cell assemblies displaying co-active neurons along bicuculline (P ⬍ 0.01). There was also an increase in the
time with recurrent and alternating activity (Figs. 7, B and C) number of coactive cells supporting the preferred state (Fig.
(Sasaki et al. 2006). States were continuously revisited with no 8C). Most synchrony peaks, however, correspond to the same
state having a preference or a significantly higher probability of dominant state. Neurons involved in network sates are shown
recurrence (Fig. 7B; see percentages of trajectories out of each in Figs. 8, D and E. Hierarchical cluster analysis did not reveal
state including recurrences), demonstrating the existence of cluster-like structures in spite of synchronization (Fig. 8F),
various semi-stable network attractors. Time histograms with meaning that all active neurons were embedded into the same
several synchrony peaks (asterisks; Fig. 7C, bottom) show the state. Cortical stimulation in the presence of bicuculline re-
alternation of activity between different cell assemblies thus cruited even more striatal neurons than those obtained in
demonstrating multistable dynamics. control conditions or in the presence of NMDA alone (data not
Figure 7D shows the spatial distribution of neurons gener- shown). Thus fast GABAergic transmission is a necessary
ating the patterns and producing the different functional states. requirement for the network to exhibit multistable dynamics
Network states share some elements but most of the neurons and different functional states.
are dissimilar (Fig. 7E). Finally, hierarchical cluster analysis We then tested the role of fast AMPA glutamatergic trans-
confirmed the recruitment of different cell assemblies during mission in the orchestration of cell assemblies. Note that
FIG. 6. Visualizing network states. A: similarity indices of all vectors representing network dynamics as a time function (see METHODS). Note cluster-like
structures in the pseudocolored matrix (Sasaki et al. 2006). B: multidimensional reduction of vectors using locally linear embedding (LLE). Each point represents
a vector at a given time (see METHODS). Consecutive time points form different trajectories representing specific experimental conditions, each producing a
different network state. State 1: cortical stimulation under control conditions. State 2: NMDA-induced network activity. State 3: cortical stimulation in the
presence of NMDA in the bath. C: raster plot used to reconstruct the network states (370 ms/frame; top). Rows represent activity of individual cells. Vertical
lines delimit time series C1–C5 (186 s/image series; C1: t ⫽ 10 min, C2: t ⫽ 35 min, C3: t ⫽ 40 min, C4: t ⫽ 45 min, C5: t ⫽ 50 min). Histogram (bottom)
represents percentage of coactive cells over time in the same experiment. Peaks of synchrony without electrical stimulation (asterisks) were present both before
and after cortical stimuli (perturbation). Cortical stimulation (imposed synchrony or perturbation) apparently did not change the NMDA-induced network
dynamics (spontaneous). Note that patterns of active cells change over time. The heights of synchrony peaks during cortical stimulation appear truncated (1 and
3). D: spatial distribution of neurons involved in the different states. Scale bar 100 ␮m. E: percentage of coactive cells during different functional states. Note
the participation of very few cells in different states. F: hierarchical cluster analysis of cells participating in the network states (n ⫽ 92 cells). colored boxes
indicate the different states. Note that different cell assemblies sustain each recurrent state (imposed or spontaneous) although some cells are shared by different
states.

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1444 CARRILLO-REID ET AL.

A B 33% 67%
1
30 1

similarity index
state 1
vector i (t)

LLE 2
0 33%
20

state 2

-1 50%
state 3
10

0 67%
50%
-2
0 10 20 30 -2 -1 0 1
vector i (t) LLE 1
C 1 2 3
90
80
70
Active cell #

60
50
40
30
20
10
0
% coactive cells

10
P<0.05 * *
* * * * *
* *
5

1 min

D E
state 1 state 2 state 3
% coactive cells

20

10

0
1&2 1&3 2&3
# states
F
state 1
state 2
state 3
88
78
71
68
64
54
27
10
1
5
25
37
58
67
69
75
79
89
91
39
48
76
62
57
52
47
23
19
29
49
55
60
73
80
38
87
32
4
43
93
33
83
81
65
46
34
24
20
3
8
21
31
41
50
72
82
90

core ensemble
Active cell #
FIG. 7. Network states and striatal cell assemblies. A: similarity indices of all possible vector pairs of representative NMDA-induced activity—without cortical
stimulation. Note cluster-like structure of distribution indices (Sasaki et al. 2006). B: different states depicted by LLE. Note jumps from 1 state to the other, as
well as recurrent revisiting of the same state along time. Percentages over trajectories signal probability of leaving a given state. Probability of jumping from
1 state to the other is high. C: raster plot and time histogram of network dynamics. Recurrent peaks of synchronous bursting activity are marked with asterisks.
Vertical lines separate different series of images (186 s each; C1: t ⫽ 35 min, C2: t ⫽ 45 min, C3: t ⫽ 50 min). Colors denote different states. Note transitions
between states. D: spatial maps of cell assemblies sustaining the different states. Scale bar 100 ␮m. E: percentage of cells coactive in 2 states. Note overlap of
some cells active in different states. F: hierarchical clustering of cells involved in the peaks of synchrony revealed the participation of different cell assemblies
in different functional states. Note there is also a core of neurons active in all functional states.

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 STRIATAL NETWORK DYNAMICS 1445

A B
14
2.0 12%
1

similarity index
12
vector i (t)

10

LLE 2
1.0
state 1
8 state 2 88%
0.0
6
4 0 -1.0
100%
2
-2.0
-3.0 -2.0 -1.0 0.0 1.0
2 4 6 8 10 12 14
vector i (t) LLE 1
C NMDA + bicuculline
80
Active cell #

60

40

20
% coactive cells

20
*
P<0.05
10 *
** ** * *
* *

1 min

D E
% coactive cells

15
state 1 state 2
10

0
1&2
# states
F

state 1
state 2
53
12
3
1
8
13
80
71
68
65
61
57
55
48
45
40
37
31
29
27
22
19
15
11
9
14
16
20
26
28
30
36
38
43
47
51
56
60
64
66
69
75
81

Active cell #
FIG. 8. Fast GABAA inhibition is necessary for state transitions. A: similarity index matrix of the NMDA-induced dynamics in the bath presence of
bicuculline. Note the heterogeneous distribution of the indices showing the absence of cluster-like structures. B: in the bath presence 10 ␮M bicuculline, the LLE
algorithm reveals the recurrence of a preferred state with occasionally jumps to another state. C, top: the figure shows the raster plots of 2 different series of
images (220 s each): in 8 ␮M NMDA (35 cells; t ⫽ 25 min; left) and in 10 ␮M bicuculline (56 cells; t ⫽ 40 min; right), in the continuous presence of NMDA.
In this experiment, 8 cells were active in both series of images (440 ms/frame). Rows represent activity of individual cells. The vertical line delimits series of
images 1 and 2. Note overall synchronization of NMDA-induced activity in the presence of bicuculline. C: histogram representing percentage of coactive cells
over time in the same experiment (bottom). Peaks of synchrony (asterisks) increased in frequency and amplitude after bicuculline application (in the presence
of NMDA). D: neurons involved in the 2 states. Scale bar: 100 ␮m. E: percentage of coactive neurons in the 2 states. Note that all the cells of the state 2 also
participate in state 1. F: hierarchical cluster analysis of cells active in the synchrony peaks shows only 1 group of active cells.

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1446 CARRILLO-REID ET AL.

A B
1 NMDA + CNQX 1 NMDA + nicardipine
50 60
Active cell #

Active cell #
40

30 40

20
20
10

0
% coactive

% coactive
15 *
*
cells

cells
* P<0.05 10 * * P<0.05
10
5 5

1 min 1 min

2 NMDA + CNQX 2 NMDA + nicardipine

28 22 34
15 11 15 20 16
10

FIG. 9. Blockade of AMPA transmission or L-type calcium channels disrupts NMDA-induced network dynamics. A1: raster plot shows activity during the
bath presence of 8 ␮M NMDA (left, 36 cells; t ⫽ 20 min) and 10 ␮M 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX, right, 19 cells; t ⫽ 35 min, top). In this
experiment, 4 cells had activity in both conditions. The figure shows 2 series of images (180 s each; 250 ms/frame), 1 for each condition. Rows represent activity
of individual cells. The vertical line delimits series of images 1 and 2. Histogram represents percentage of coactive cells over time in the same experiment
(bottom). Peaks of synchrony (asterisks) were abolished after CNQX application (in the presence of NMDA) and overall activity was drastically reduced.
A2: spatial maps of cells involved in the peaks of synchronous activity. Scale bar: 100 ␮m. Gray circles represent cells involved in the synchrony peaks that
continued active in the presence of CNQX in the bath (n ⫽ 3 neurons). B1: the raster plot illustrates activity from 2 series of images (220 s each, top). Active
cells in the bath presence of 8 ␮M NMDA (left, 41 cells; t ⫽ 25 min) and after 5 ␮M nicardipine (right, 35 cells; t ⫽ 40 min). In this experiment, 10 cells were
active in both series of images (440 ms/frame). Rows represent activity of individual cells. The vertical line separates series of images 1 and 2. Histogram
represents percentage of coactive cells over time in the same experiment (bottom). Peaks of synchrony (asterisks) were abolished after nicardipine application
(in the continuous presence of NMDA). B2: spatial maps of active cells in the peaks of synchrony. Gray circles represent cells active in the presence of nicardipine
in the bath (n ⫽ 6 cells). Scale bar: 100 ␮m.

NMDA blockers cannot be used: either the withdrawal of
NMDA or the inhibition of NMDA receptors stops multistate
dynamics in slices from this quiescent circuit under control
conditions.
Active networks were challenged with the AMPA receptor
antagonist CNQX (10 ␮M) (Fig. 9A). As shown in the raster
plot of Fig. 9A1, CNQX drastically reduced the number of
active cells (cf., left and right) and disrupted the appearance of
synchrony peaks in all NMDA-treated slices (Fig. 9A, bottom;
n ⫽ 6 slices). Interestingly, a few neurons that were active
during synchrony peaks maintained spontaneous activity in the
absence of fast AMPA transmission (Fig. 9A, 1 and 2, gray
circles) (Vergara et al. 2003). Also in the presence of CNQX,
cortical stimulation was unable to recruit striatal neurons (data
not shown) and all correlated activity was suppressed (Fig. 9A).
Intrinsic conductances are known to participate in pattern
generation in single cells. In particular, in spiny neurons during
activity set into action by a tonic excitatory drive, the activity
of voltage-gated L-type calcium channels has been shown to be
essential to produce bistability and I-V plots with a negative
slope conductance region (see Fig. 1E5) (see also Vergara et al.
2003). L-type calcium channels promote the generation of
plateau potentials capable of sustaining repetitive firing that
outlasts input duration. We therefore tested the effects of
blocking L-type calcium channels with the antagonist nicardi-
pine (5 ␮M). As shown in the raster plot of Fig. 9B1, nicardi-
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pine only slightly reduces the number of active neurons (n ⫽
5 slices; cf., left and right panels; n ⫽ 41 vs. 35 neurons), it
nevertheless reduces the number of peaks with spontaneous
synchronous activity. Many cells defining previous functional
states continued spontaneously active in the presence of nicar-
dipine (Fig. 9B, 1 and 2, gray circles), but they were rarely
synchronized to produce functional states. We conclude that
plateau potentials resulting from the activity of voltage-gated
L-type calcium channels are required for network synchroni-
zation (Yuste et al. 2005) and pattern generation produced by
cell assemblies in NMDA-treated slices.
Shared core of neurons
In most slices presenting multistable network dynamics (n ⫽
20/25 slices), we found a core of neurons, which were shared
by all states. So we aimed at recording electrophysiologically
some of these neurons after their identification as a part of a
core.
These neurons were 8 ⫾ 2% of all active cells in the network
states (n ⫽ 20 slices). Some of these core neurons exhibited
periodic calcium transients during NMDA treatment or CNQX.
Bicuculline disrupted the periodic firing of these cells. Cell-
attached (Fig. 10A1) or whole cell current-clamp recordings
(Fig. 10, C1 and D1) were performed on some of these cells,
and we found that 40% of the core assembly (n ⫽ 4/10 cells)
showed firing properties characteristic of GABAergic inter-
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 STRIATAL NETWORK DYNAMICS 1447

FIG. 10. GABAergic interneurons are part of the core assembly. A: cell-attached (top) and calcium imaging recordings (bottom) from a neuron with regular
bursting activity in the presence of 8 ␮M NMDA that belongs to a core of shared neurons (1). Records show high-frequency regular bursts with their
correspondent calcium transients. Interspike interval histogram of intraburst activity. Note a peak value around 11 ms. B: fluorescence image of the cell recorded
in A. Scale bar 15 ␮m. C: voltage responses (top) to current steps (bottom) recorded from another neuron showing regular (pacemaker) bursting activity (1). The
same neuron filled with biocytin, showing that it corresponds to a cell with aspiny varicose dendrites. Scale bar 15 ␮m (2). D: cell in C exhibits regular bursting
during NMDA (8 ␮M). Note the periodicity of the up states (1). Histogram (from 1) shows the bimodal distribution over time of membrane potential (2).
D3: power spectrum from D1. Note activity with a period of ⬃2.18 s.
neurons (Plenz and Aertsen 1996; Tepper et al. 2004), such ity patterns. Although most striatal cells are silent under
as high-frequency bursts (Fig. 10A) and periodic pacemak- control conditions, either cortical stimulation or bath applica-
ing activity (Fig. 10D, 1–3). However, by fixing fluorescence tion of NMDA, induce synchronized burst firing among as-
of cells active during a given experiment (see METHODS), Fig. 11 semblies of striatal neurons. Cell assemblies defined functional
shows that most active cells in the different assemblies were states during a given experiment and alternate their activity as
immunoreactive to either substance P or enkephalines. though belonging to unit CPGs (Grillner 2006). Alternation of
activity and frequent recurrences of the same states suggest
DISCUSSION attractor network dynamics.
Ca2⫹ imaging of neuronal populations (Mao et al. 2001;
We demonstrate that an isolated striatal slice has the neces- Sasaki et al. 2006; Schwartz et al. 1998; Stopfer et al. 2003)
sary circuitry to transform a tonic excitatory drive into sequen- and simultaneous whole cell recordings show that calcium
tial correlated activity of several cell assemblies that exhibit transients correspond to burst firing in single cells, the
recurrent, alternating, and synchronized spatiotemporal activ- synchronization and recurrence of which generates the ac-
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1448 CARRILLO-REID ET AL.

Fluo4 ENK Merged

A B C

Fluo4 SP Merged
D E F

FIG. 11. Most active cells in cell assemblies are medium spiny projection neurons. A: confocal image of a field of view of the dorsal striatum showing cells
active during the experiment: loaded with fluo-4 AM. Fluorescence with EDAC, Scale bar 10 ␮m. B: confocal image of the same field showing that several of
these neurons were immunoreactive for ENK, a specific marker of spiny neurons. C: superimposition of A and B. D: confocal image of a field of view with some
cells loaded with fluo-4 AM in the dorsal striatum. Fluorescence with EDAC, scale bar 10 ␮m. E: confocal image of the same field showing that several of these
neurons were immunoreactive for SP, another specific marker of spiny neurons. F: superimposition of D and E.

tivity patterns. Pattern generation during network states
depend on synaptic and intrinsic properties as well as the
activity of a core of active neurons (Berke et al. 2004). In
summary, this work shows how pattern generation and
correlated activity is generated in the striatal microcircuitry
in vitro identifying the neuronal elements involved. The
connections and modulation of these elements deserve fur-
ther study because they may reveal basic general properties
of striatal microcircuitry and may further suggest in vivo
experiments.
Voltage transitions in striatal neurons
Striatal neurons, both in vivo and in vitro, exhibit spontane-
ous voltage transitions that sustain burst firing. Transitions
occur between a quiescent down state and a recurrent bursting
up state (Herrling et al. 1983; Kerr and Plenz 2002, 2004;
Mahon et al. 2006; Vergara et al. 2003; Wilson 1993). It is
largely unknown how these transitions reflect network activity
and how they propagate through the network. It is probable that
different classes of bursts exist, for example, during different
functional states (e.g., sleep vs. movement) (N. Vautrelle,
personal communication). Here we took advantage of the tonic
drive provided by NMDA to induce the transitions because in
the striatum (Ossowska 1995), as in other circuits (Gordon and
Whelan 2006; Grillner et al. 1981; Guertin and Hounsgaard
1998; Hsiao et al. 1998; Kiehn 2006; Takakusaki et al. 2004b),
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NMDA sets into action patterned activity that generates move-
ment. Accordingly, it was demonstrated that NMDA induces
correlated activity that evolves in time. In contrast to control
conditions in which the striatum has only a few active cells
with no correlated activity, both cortical stimulation and the
tonic excitatory drive provided by NMDA exposure (Vergara
et al. 2003) recruit neurons spread over a wide area; which, in
the case of NMDA, spontaneously express particular spatio-
temporal dynamics, indicating that the striatal microcircuit in
vitro preserves a set of “unit CPGs” (Grillner 2006), that is,
even if most connections present in more intact preparations
are severed, remaining sets of neurons, probably belonging to
larger CPGs or modules in vivo, preserve some of the proper-
ties of these larger modules. In support of this inference,
independent evidence in the cortex suggests that cell assem-
blies activity is preserved along different spatial scales (Plenz
and Thiagarajan 2007).
Mechanisms of network dynamics
The conditions that generate circuit dynamics reside in both
the synaptic and intrinsic properties of striatal neurons. NMDA
induces synchronous peaks of neuronal activity emerging from
the co-activation of robust and recurrent cell assemblies that
alternate in their patterns displaying a sequence of trajectories.
These results suggest the presence of robust mechanisms that
maintain and stabilize the cells participating in the assemblies.
99 • MARCH 2008 • www.jn.org
 STRIATAL NETWORK DYNAMICS
In particular, AMPA transmission appears to be necessary for
turning on network dynamics. In fact, the number of active
neurons drastically falls when AMPA transmission is blocked.
Because all glutamatergic synapses in the striatum originate
from cortical or thalamic afferents, cortico-thalamic afferents
are likely to be the source of tonic excitatory drive underlying
synchronous activity. NMDA presumably generates an in-
crease in the tonic excitatory drive conveyed by cortico-
thalamic afferents (Grillner 2006; Grillner et al. 1981), and
although some neurons maintain spontaneous activity in the
presence of CNQX— or after dissecting away the cerebral
cortex (Vergara et al. 2003)—the analysis shows that this
behavior is asynchronous so that the emergence of striatal cell
assemblies appears to require fast AMPA glutamatergic trans-
mission, presumably originating from cortical and thalamic
neurons (Kasanetz et al. 2006; Yuste et al. 2005). The most
parsimonious interpretation of these results is that cortico-
thalamic afferents are essential for the appearance of multistate
network dynamics. That is, network dynamics are commanded
by the cortex and/or the thalamus (Kasanetz et al. 2006; Magill
et al. 2001). However, the actual relationship between cortical
or thalamic activation and cell assembly activity in the striatum
needs further investigation. It is known that activation of
NMDA receptors is enough to induce bistability in striatal
neurons, amplifying the excitatory drive conveyed by cortical
or thalamic inputs (Grillner 2006; Grillner et al. 1981) so that
activity (e.g., from the cortex or thalamus) can be relied on.
And although cell assembly activity has been recorded in the
cortex using NMDA plus dopamine, it has not been recorded
after NMDA only (Tseng and O’Donnell 2005). Finally, stri-
atal assemblies receive the convergence of widespread corti-
cal areas, and slices cannot contain all these areas (Parthasar-
athy and Graybiel 1997). However, cortical cell assemblies
driving striatal cell assemblies are not excluded and may be
possible in several conditions (e.g., with added dopamine
receptor agonists). Therefore striatal assemblies may represent
the coordinated activity between cortical areas, that once
formed by development and learning, only need a cortical
trigger or a subset of the original stimuli for activation.
In addition, our data show that the striatum does not simply
follow cortical commands. The role of the striatal circuit
proper in the management of cortical drives is revealed by the
result of inhibiting GABAA receptors. Blockade of GABAA
inhibition not only increases the peaks of synchronous activity
and recruits more cells during synchrony peaks but also shifts
the network toward a preferred state that recruits most active
neurons. Without inhibition, the striatal circuit gets tied up in
a preferred state, from which alternation and selection among
different cell assemblies becomes unlikely. Therefore we con-
clude that inhibitory circuits in the striatum are responsible for
transforming cortical commands into a sequential activity of
striatal cell assemblies (Ossowska 1995; Wickens and Oorschot
2000). In addition, the blockade of L-type calcium channels
disrupts the peaks of synchrony. This result confirms that
intrinsic conductances, such as voltage-gated calcium chan-
nels, by generating plateau potentials may work as synchroni-
zation enablers of neuronal networks (Kiehn 2006; Yuste et al.
2005). Thus NMDA-induced network dynamics in the striatum
requires the participation of both synaptic and intrinsic mech-
anisms.
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1449
Finally, electrophysiological recordings of neurons exhibit-
ing Ca2⫹ transients showed that most of the active neurons in
cell assemblies were medium spiny projection neurons. This
was confirmed by immunocitochemistry (Fig. 11). Neverthe-
less, a subpopulation of neurons shared by all functional states
(see Parthasarathy and Graybiel 1997) exhibited periodic Ca2⫹
transients and the firing properties of pacemaking GABAergic
interneurons (e.g., Berke et al. 2004; Tepper et al. 2004). The
fact that striatal GABAergic interneurons comprise 2–5% of
striatal cells and yet make up 40% of the neurons active in
multiple assemblies suggests that striatal interneurons partici-
pate in the orchestration of multistate dynamics (Berke et al.
2004). Activity of pacemaker neurons is a frequent finding in
CPGs (Grillner 2006; Yuste et al. 2005).
Functional implications and perspectives
Recurrent and alternating bursting is characteristic of cell
assemblies included in CPGs in vivo and in vitro (Barnes et al.
2005; Grillner 2006; Ikegaya et al. 2004). Synchronized activ-
ity of these modules exhibit attractor dynamics (Cossart et al.
2003), providing a unified description for circuits encoding the
storage and retrieval of long-term and working memory. At-
tractors are seen as memory traces retrieved through excitatory
tonic driving or partial cues useful for executing motor pro-
grams. The persistence of cell assemblies along time is due to
recurrent connectivity (Barnes et al. 2005; Tsodyks 2005). The
alternation of their activity is under neuromodulatory control
(Yuste et al. 2005). Apparently, the properties of these micro-
circuits are at the interface between small networks and global
brain functions (Plenz and Thiagarajan 2007). Their distur-
bance provokes abnormal processes of synchrony, associated
with different disorders such as schizophrenia and Parkinson
Disease (Schnitzler and Gross 2005; Uhlhaas and Singer
2006). We show that an isolated circuit set into action, in vitro,
can exhibit synchronized states in specific cell assemblies
emerging and returning during relatively long periods of time.
ACKNOWLEDGMENTS
We thank Drs. Ranulfo Romo, Roman Vidaltamayo, and Nicolas Vautrelle
for critically reading the present manuscript. We thank R. Velázquez for
programming a part of the software for analysis, O. Jaidar and A. Hernández
for some experiments in older animals, and also A. Laville, C. V. Rivera, T.
Fiordelisio, and N. Jiménez, for technical support and advice.
GRANTS
This work was supported by grants from a Project Program grant IMPULSA 03
to J. Bargas, A. Hernandez-Cruz, E. Galarraga, and R. Drucker-Colin, by
Consejo Nacional de Ciencia y Tecnologı́a (México) Grants 42636 to E.
Galarraga and 49484 to J. Bargas, and by Dirección General de Asuntos del
Personal Académico, Universidad Nacional Autónoma de México Grants
IN201607 to J. Bargas and IN201507 to E. Galarraga.
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