Download Heterogeneous Integration of Bilateral Whisker Signals by Neurons

J Neurophysiol 93: 2966 –2973, 2005.
First published November 24, 2004; doi:10.1152/jn.00556.2004.
Innovative Methodology
Heterogeneous Integration of Bilateral Whisker Signals by Neurons in
Primary Somatosensory Cortex of Awake Rats
Michael C. Wiest,1,4 Nick Bentley,5 and Miguel A. L. Nicolelis1,2,3,4
1
Departments of Neurobiology, 2Biomedical Engineering, and 3Psychological and Brain Sciences, and 4Duke University Center for
Neuroengineering, Duke Medical Center, Durham, North Carolina; and 5Department of Neurobiology and Anatomy, Wake Forest
University School of Medicine, Winston-Salem, North Carolina
Submitted 27 May 2004; accepted in final form 17 November 2004
INTRODUCTION
One approach to understand the coding of whisker stimuli in
the rat primary somatosensory cortex (S1) focuses on a oneto-one relationship between anatomically discrete cortical
“barrels” in layer IV and a single principal whisker on the
contralateral whisker pad (Armstrong-James 1995; Brumberg
et al. 1996; Petersen et al. 2002). Because of the relative
scarcity of connections between layer IV excitatory cells in
different barrels, and because these cells tend to respond to
principal whisker stimulation with the shortest latency, the
layer IV barrel field can be interpreted as an array of dedicated
detectors for deflections of each contralateral whisker. The fact
that whiskers other than the principal whisker do modulate
responses in a barrel can be explained in a number of ways.
Brumberg et al. (1996) proposed that inhibitory circuitry within
barrels functions to “increase the ‘principal whiskerness’ of the
Address for reprint requests and other correspondence: M. C. Wiest, Department of Neurobiology, Box 3209, Room 339, Bryan Research Building,
Duke University Medical Center, 101 Research Drive, Durham, NC 27710
(E-mail: [email protected]).
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cortical column” by suppressing weaker responses so that
“only . . . the initial perturbation of the principal whiskers will
overcome the . . . cortical inhibition” (Brumberg et al. 1996).
For Armstrong-James (1995), a “transient insularity” between
barrels includes the whole cortical column centered over a
barrel. That is, for some time after each stimulus the barrel
columns can encode the activity of single whiskers. Similarly,
Petersen et al. (2002) recently noted that the earliest evoked
spike after an isolated punctate single-whisker stimulus contains most of the information about which the single whisker
was stimulated. From this they concluded that later activity,
reflecting interactions between different barrels, was largely
redundant for neural coding of stimulus identity.
In the supra- and infragranular layers intercolumnar connections are more common, and receptive fields in those layers are
correspondingly larger (Armstrong-James et al. 1992; Ghazanfar et al. 2000; Schubert et al. 2001). Moreover, these multiwhisker receptive fields are dynamic over poststimulus time
(Ghazanfar and Nicolelis 1999, 2001a). It is more difficult to
interpret the responses of cells in these layers primarily in
terms of the stimulation of individual whiskers. A complementary view emphasizes the integrative function of S1. Thus
although Simons (1985) considered that “each barrel is the
morphological correlate in layer IV of a functional cortical
column that extends throughout the thickness of cortex,” he
also concluded “that an important function of the SI cortex is
to integrate information from different, individual vibrissae on
the contralateral face.” He suggested intercolumnar integration
functioned to make SI neurons sensitive to spatiotemporal
patterns of whisker activity characterizing the movement of
particular objects across the contralateral whisker pad (Simons
1985), as have others (Ghazanfar and Nicolelis 1997; Shimegi
et al. 2000). Even for coding single-whisker stimuli, Ghazanfar
et al. (2000) showed that the relative contribution of temporal
interactions between neurons increases with the number of
whiskers to be discriminated. Inhibitory intercolumnar interactions have also been suggested to maintain a dynamic range
and prevent response saturation (Mirabella et al. 2001).
It is important to note that the different approaches to
inter-columnar interactions sketched above need not be exclusive, but could reflect different processing strategies appropriate to different behavioral contexts. This idea has arisen in the
whisker sensory-motor literature, in terms of different S1
neuronal response properties in the quiet waking state as
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Wiest, Michael C., Nick Bentley, and Miguel A. L. Nicolelis.
Heterogeneous integration of bilateral whisker signals by neurons in
primary somatosensory cortex of awake rats. J Neurophysiol 93:
2966 –2973, 2005. First published November 24, 2004; doi:10.1152/
jn.00556.2004. Bilateral single-unit recordings in primary somatosensory cortex (S1) of anesthetized rats have revealed substantial cross
talk between cortical hemispheres, suggesting the possibility that
behaviorally relevant bilateral integration could occur in S1. To
determine the extent of bilateral neural responses in awake animals,
we recorded S1 multi- and single-unit activity in head-immobilized
rats while stimulating groups of 4 whiskers from the same column on
both sides of the head. Results from these experiments confirm the
widespread presence of single units responding to tactile stimuli on
either side of the face in S1 of awake animals. Quantification of
bilateral integration by multiunits revealed both facilitative and suppressive integration of bilateral inputs. Varying the interval between
left and right whisker stimuli between 0 and 120 ms showed the
temporal integration of bilateral stimuli to be dominated on average
by suppression at intervals around 30 ms, in agreement with comparable recordings in anesthetized animals. Contrary to the anesthetized
data, in the awake animals we observed a high level of heterogeneity
of bilateral responses and a strong interaction between synchronous
bilateral stimuli. The results challenge the traditional conception of
highly segregated hemispheric processing channels in the rat S1
cortex, and support the hypothesis that callosal cross-projections
between the two hemispheres mediate rats’ known ability to integrate
bilateral whisker signals.
Innovative Methodology
BILATERAL INTEGRATION IN AWAKE S1
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unambiguous representation of the contralateral whisker pad in
each S1 hemisphere implied by the traditional models mentioned above. However, if bilateral receptive fields (Manns et
al. 2004; Shuler et al. 2001) observed in anesthetized preparations remain functional in the behaving animal, the conception
of the S1 barrel cortex as an encoder for exclusively contralateral whisker activity will need to be refined or replaced.
Previous behavioral experiments in our laboratory have
demonstrated that rats can integrate whisker signals from both
sides of the face in a tactile discrimination task (Krupa et al.
2001b; Shuler et al. 2002). In fact, the rat integrated bilateral
whisker signals even in an aperture width discrimination task
that does not force rats to use a bilateral strategy (Krupa and
Nicolelis 2004a; Oliveira et al. 2003), suggesting that bilateral
integration is a natural function of the rat whisker system. S1
is the first place where bilateral whisker sensory afferents
converge (Erzurumlu and Kilacky 1980; Smith 1973; Waite
1969). Accordingly, Shuler et al. (2002) found that intact S1
cortex in both hemispheres was required for successful performance of a bilateral tactile discrimination task.
If S1 is an actual site of integration, rather than merely a
conduit for sensory information that is integrated at higher
processing stages, then the influence of ipsilateral as well as
contralateral stimuli should be measurable in S1. It remains
unknown whether the bilateral connections between the S1
hemispheres are fully functional on millisecond timescales in
awake, behaving animals. Our experiments addressed this
question using bilateral multielectrode recordings in S1 of
head-immobilized awake rats during controlled bilateral stimulation of columns of whiskers. Following Shuler et al. (2001)
we looked for evidence of bilateral integration in layer V,
which contains the main output neurons of S1.
METHODS
Head-immobilized awake electrophysiology experiments
A sample of 5 female Long–Evans rats was used in the present
experiments. Details of surgery and recording procedures may be
found elsewhere (Nicolelis 1997; Nicolelis and Chapin 1994). Briefly,
surgery was performed under pentobarbital anesthesia to implant rats
with pairs of 16 electrode arrays (2 ⫻ 8) bilaterally in S1. The arrays
were positioned stereotactically and by means of tactile stimulation
during surgery to record in cortical layer V (⫺3 mm caudal from
bregma, 5.5 mm mediolateral, and about 1.3 mm depth from brain
surface). During the same surgery a brass head post (1/4-in. width;
Small Parts, Miami Lakes, FL) was fixed to the dental-cement head
cap for head-immobilized awake recordings. Rats were given ⱖ7 days
after surgery to recover.
Control of whisker stimulus delivery was achieved by combining
head-immobilization with a computer-controlled multiple individualwhisker stimulator (Krupa et al. 2001a) adapted for use on an awake
animal. For all experiments in this study the stimulator noose was
attached to the bottom 4 large whiskers in a single arc (B–E) (Fig. 1A).
Animals were partially acclimated before surgery to restraint in a
Plexiglas restraint tube with mild food deprivation and calorie-dense
liquid reward during sessions in the restraint tube, and wearing light
drawstring “jackets” to minimize traction against the Plexiglas tube
and prevent the subject from using her forepaws to remove the
stimulator arms.
The whisker stimulator was used to apply controlled 5-ms rostral
deflections of the bottom 4 whiskers in a single column on each side
of the subject’s face. To lasso 4 whiskers, the noose was first opened
wide, then fine forceps were used to pull through the whiskers one at
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compared with the whisking state (Castro-Alamancos 2004;
Fanselow and Nicolelis 1999; Moore 2004; Moore et al. 1999;
Nicolelis and Fanselow 2002). These authors proposed that
greater response magnitudes during the quiet state could perform a gross “detection” function, whereas relatively less spike
adaptation during the whisking state would allow for finer
“discrimination” of spatiotemporal patterns of stimulation.
Based on the finding that the horizontal extent of cortex
activated by stimulation of a vibrissa is relatively smaller
during whisking than during an isolated passive contact, some
authors (Castro-Alamancos 2004; Moore 2004; Moore et al.
1999) have further speculated that a more spatially “focused”
sensory representation during whisking could improve tactile
discrimination.
All the coding studies mentioned above have in common
that the whisker cortex is interpreted as a topographic map of
whisker deflections on the contralateral whisker pad, in line
with the cortical homunculus as a map of somatosensory
activity in general. A topographic organization does not prohibit lateral interactions, but implies that they should be relatively local. In particular, SI cortex is understood to encode
stimulations of the contralateral whisker pad. Such a view is
apparently supported by behavioral experiments demonstrating
that certain unilateral tactile detection tasks do not require
ipsilateral S1 (Hutson and Masterton 1986).
This picture is overly restrictive. It has long been known that
callosal cross-projections integrate the two S1 hemispheres
(Cauller et al. 1998; Koralek et al. 1990; Olavarria et al. 1984;
White and DeAmicis 1977). Interhemispheric connections are
concentrated in the homologous portions of the barrel field,
innervating primarily but not exclusively the supra- and infragranular layers (Hayama and Ogawa 1997). Those callosal
connections that do innervate layer 4 are concentrated in the
septal regions outside the barrel centers (Hayama and Ogawa
1997; Olavarria et al. 1984), as are unilateral interbarrel connections in layer IV (Kim and Ebner 1999). In the present
study we focus on the bilateral integration of whisker sensory
signals in S1 through these interhemispheric connections,
which offers an advantageous window on the larger issue of the
dynamic integration of distributed sensory representations because the peripheral signals to be integrated are clearly segregated in space and may be independently manipulated up to the
level of the S1 cortex.
Pidoux and Verley (1979) recorded S1 local field potential
(LFP) responses to ipsilateral whisker stimulation in anesthetized rats, the first electrophysiological evidence of cross talk
between the S1 hemispheres. Shuler et al. (2001) demonstrated
that whisker stimulation on either side of the face of an
anesthetized rat could evoke responses in the same layer V S1
neuron. This result has been recently confirmed by intracellular
recordings (Manns et al. 2004). Other studies have shown that
the responses of individual neurons in S1 are suppressed in the
absence of activity in the contralateral hemisphere (Shin et al.
1997). Rema and Ebner (2003) found similar response suppression as well as changes in whisker-pairing plasticity in all
layers arising from contralateral lesions.
One potentially relevant function of the commissural interhemispheric connections is to transfer learned associations
from one hemisphere to the other (Calford and Tweedle 1990;
Ebner and Myers 1962). Such transfer of plasticity on relatively long timescales might be imagined to preserve the
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M. C. WIEST, N. BENTLEY, AND M. A. L. NICOLELIS
冘
n
Dn ⫽
Posti ⫺
i⫽1
FIG. 1. A: close-up of a whisker-stimulator noose around 4 whiskers from
an arc. B: a Nissl-stained slice from one animal showing an electrode track
down to layer V (indicated by the arrow).
a time before tightening the noose. Two basic protocols were run. In
both experiments, stimuli were presented in random order at random
intertrial intervals of between 2 and 4 s. In expt. 1 the stimulus set
consisted of a left-column deflection, a right-column deflection, and
simultaneous bilateral deflections. In expt. 2 the stimulus set consisted
of a left- then right-column deflection at interstimulus intervals (ISIs)
of 0, 30, 60, 90, and 120 ms. TEMPO software (Reflective Computing, St. Louis, MO) controlled the stimulus administration and sent
time-stamped stimulus events with millisecond accuracy to the file of
neural recordings described below. The Duke University Institutional
Animal Use Committee approved all surgical and behavioral methods.
Single-unit (SU), multiunit (MU), and local field potential (LFP)
activity were recorded through a many-neuron acquisition processor
(MAP; Plexon, Dallas, TX) using electrode arrays built in-house by G.
Lehew. Units were sorted first on-line during each recording session,
to set each channel’s threshold for recording a spike (digitized at 40
kHz) and graphically defining unit waveforms on each channel with
spikes significantly (about ⱖ2 SDs) above background noise
(Nicolelis et al. 2003). These on-line–sorted units, which may include
spikes from multiple neurons, are termed MUs. Some units were
further sorted off-line according to their clustering in the principalcomponent space representing the spike waveforms. Those sorted
units that showed a distinct cluster in principal-component space from
the cluster of noise waveforms, and displayed fewer than 0.1% of ISIs
within a refractory period of 1 ms, are termed SUs.
Histology of coronal slices of the experimental animals’ brains
confirmed the localization of the electrode arrays in each animal to the
infragranular layers of S1. A Nissl-stained slice from one animal,
containing the track of an electrode, is shown in Fig. 1B.
Data analysis
Significant firing rate responses of SUs were identified from poststimulus time histograms (PSTHs) for each session using the standard
J Neurophysiol • VOL
冘
n
␭i
i⫽1
Here ␭i is the expected number of spikes per bin calculated from the
baseline, and Posti is the observed spike count in poststimulus bin i.
To assess the significance of deviations from the expected cumulative sum, an empirical distribution of the cumulative-summed spike
count at each time bin before the stimulus was constructed from 1,000
bootstrapped samples (with replacement) of the prestimulus spike
histogram. The prestimulus cumulative sums were counted starting
from 200 ms before stimulus onset. This empirical distribution of
prestimulus firing was then used to find the poststimulus bin (if any)
at which the cumulative-summed poststimulus spike count exceeded
or was ⬍99% of the cumulative spike counts from the baseline
distribution (Martinez and Martinez 2002). This bin was recorded as
the onset of an excitatory or inhibitory response.
The response offset was identified as the first zero crossing of the
derivative of the cumulative deviation from the baseline spike count,
meaning that the cumulative sum was no longer deviating from its
expected growth with time. This procedure identified the time at
which the PSTH returned to a baseline firing rate. The magnitude of
the response was quantified as the number of excess spikes between
onset and offset, compared with the baseline expected number, divided by the number of trials. This procedure gives the average
number of spikes per trial, over and above the baseline number
expected, fired by an MU in response to a particular whisker column
stimulation.
RESULTS
Bilateral integration by single units in S1
We isolated 361 layer V SUs recorded over 38 sessions in 5
animals. Of the 361 SUs, 198 (55%) showed significant responses to stimulation, on at least one side of the face, and 96
(27%) responded to both ipsilateral and contralateral stimulation. That is, 49% of those responding to contralateral stimulation also responded to ipsilateral stimulation. Several examples of SU responses to ipsilateral and contralateral stimulation
are shown in Fig. 2. These results strongly suggest that the
firing rates of individual neurons in S1 of awake rats are driven
by both ipsilateral and contralateral stimuli.
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statistical function built-in to the Nex data analysis software package
(Nex Technologies, Littleton, MA; Abeles 1982), which assumes
Poisson spike count statistics to derive confidence intervals on the
spike counts in each PSTH bin. To identify significant SU responses
we used a bin width of 10 ms and set the confidence level to 99%.
To achieve greater precision in response latencies and magnitudes
we constructed the MU PSTHs using 1-ms bins. However, despite the
greater numbers of spikes generally in the MU PSTHs, bin counts
could fluctuate above and below the 99% confidence limits, adding
uncertainty to a latency calculation. To circumvent this problem but
avoid smoothing the histograms, which would introduce complexities
into the determination of confidence intervals, we applied a method
based on the statistical distribution of cumulative-summed spike
counts (e.g., Ghazanfar et al. 2001b; Ushiba et al. 2002). This
approach takes into account the statistics of all the spiking activity up
to a given moment, as opposed to considering each bin independently.
Additionally, the method requires no assumption of a particular
parametric form of the spiking statistics.
To find the onsets of significant deviations from prestimulus activity, we calculated the deviation Dn of the poststimulus cumulative
summed spike count from the expected cumulative sum, based on the
average prestimulus firing rate ␭, at each poststimulus bin n
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layer IV inhibitory interneurons in rabbits, believed to relay
feedforward inhibition to excitatory cortical cells. Interestingly, in freely moving rats engaged in an active whiskerdependent discrimination task, 54% of infragranular S1 neurons responded to multiwhisker deflections with purely inhibitory or inhibitory followed by excitatory responses, indicating
a greater role for inhibitory coding in the discriminating animals compared with the passively stimulated animals (Krupa et
al. 2004b).
Multiunit responses in S1 to ipsi- and contralateral stimuli
FIG. 2. Single units (SUs) respond to contralateral and ipsilateral whisker
stimuli. Each row shows poststimulus time histograms (PSTHs) of one layer V
single unit in response to contralateral (left column) and ipsilateral (right
column) whisker column stimuli delivered at t ⫽ 0. Spike counts are based on
10-ms bins. Dashed lines show the 99% confidence limit for the spike counts,
based on the prestimulus baseline firing rate. Bottom row: example of an SU
whose firing was suppressed by whisker stimulation, a relatively rare type of
response.
Most SU responses were initially excitatory, occasionally
followed by a period of significantly reduced firing (e.g., the
SU in row 2 of Fig. 2). However, “direct” inhibitory responses
were observed occasionally, such as the SU on the bottom row
of Fig. 2. The firing of 11 SUs was suppressed by stimulation
on either side of the face, whereas 19 other SUs reduced their
firing in response to one unilateral stimulus but not the other.
Such inhibitory responses thus accounted for 30 (15%) of the
significant SU responses. Sachdev et al. (2000) reported direct
inhibitory responses in S1 of awake rats in response to stimulation of single whiskers. In that study most neurons exhibiting
direct inhibition were found to be located over the septal
regions outside the central barrel region of the cortex, in layers
II, III, IV, and V. They also noted that repetitive stimulation
faster than 6 Hz tended to turn the inhibitory responses into
excitation. Swadlow (2003) described the electrophysiology of
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FIG. 3. Multiunits (MUs) respond to contralateral and ipsilateral whisker
stimuli. Each row shows PSTHs of one layer V multiunit in response to
contralateral (left column) and ipsilateral (right column) whisker column
stimuli delivered at t ⫽ 0, in 1-ms bins. Dashed lines show the 99% confidence
limit for the spike counts, based on the prestimulus baseline firing rate.
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In the same experiments we recorded 1,103 multiunit channels (MUs) that showed significant modulation in response to
bilateral stimulation of 4 whiskers in a single column on each
side of the rat’s face. Overall, 984 MUs significantly modulated their firing rates in response to contralateral stimulation,
and 585 responded to ipsilateral stimulation. Of those that
responded to contralateral stimulation, 50% (491 MUs) responded to ipsilateral stimulation as well, in agreement with
the results observed for SUs (49%). Figure 3 shows several
examples of MU responses to ipsilateral and contralateral
whisker stimulation.
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M. C. WIEST, N. BENTLEY, AND M. A. L. NICOLELIS
FIG. 4. MU response onset latencies and magnitudes. A: histograms of
response onset latencies (in milliseconds) for bilateral (solid black line),
contralateral (gray line), and ipsilateral (dashed black line) whisker column
stimulation. Response onsets to ipsilateral stimulation tended to be longer than
those to contralateral or bilateral stimulation, consistent with a longer synaptic
path including interhemispheric connections in the corpus callosum. The range
of latencies shown in the figure includes 94, 95, and 88% of responses to
bilateral, contralateral, and ipsilateral stimulation, respectively. This range was
chosen for clarity in displaying the main peaks of the distributions; but all
responses were included in the calculation of average latencies. Data are
combined from 5 rats. B: histograms of response magnitudes (in spikes above
baseline per trial) for bilateral (solid black line), contralateral (gray line), and
ipsilateral (dashed black line) whisker column stimulation. Bilateral stimulation tended to evoke a significantly smaller response compared with unilateral
stimulation, although bilateral stimulation was more reliable in generating a
significant response. The range of magnitudes shown includes 100, 76, and
75% of responses to bilateral, contralateral, and ipsilateral stimulation, respectively; thus the distribution peaks are clearly visible. Nevertheless, all responses were included in calculations of average magnitudes and integration
factors (Fig. 6). Data are pooled from 5 rats.
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FIG. 5. Sessions with the stimulator attached on only one side of the face
continue to show ipsilateral responses. PSTHs are shown for 4 MUs recorded
at different electrodes, showing significant responses to ipsilateral whiskercolumn stimulation. Dashed line marks the 99% confidence limit. These
responses from unilateral sessions rule out the possibility that artifactual
ipsilateral responses arose from a motor response that led to self-stimulation of
contralateral whiskers against the stimulator arm.
However, they found that the whiskers’ movement signal
proceeded to S1 by “direct sensory activation,” implying that a
signal arising from a motor reaction would take at least the
time of the motor reaction plus the time of the sensory relay to
reach S1. The timing of ipsilateral responses is inconsistent
with what we know about the latency of a startle movement,
the fastest possible motor response the rats could make. Using
the same type of head restraint in a separate series of experiments (Wiest and Nicolelis 2003), we were able to record
occasional startle responses to whisker stimulation. These
manifested themselves as a distinct early peak (about 30 ms) in
the latency distribution of licking responses to stimulation.
Because a fast motor reaction can only occur ⱖ30 ms after
stimulation, a false ipsilateral response attributed to a motor
reaction would be observed only at still longer latencies. The
average latency of ipsilateral responses was 24 ms, and most of
the significant ipsilateral responses were observed at latencies
⬍30 ms. Therefore those ipsilateral responses we observed
could not have been a result of a motor reaction. Finally, the
ipsilateral responses we observed are similar to those recorded
in anesthetized animals, where whisker movements did not
occur.
Figure 4B shows the distribution of response magnitudes for
the 3 stimulation conditions, quantified in terms of excess
spikes per trial. The average response magnitudes were 0.82 ⫾
0.02, 0.64 ⫾ 0.04, and 0.83 ⫾ 0.03 spikes above background
per trial, in response to contralateral, ipsilateral, and bilateral
stimulation, respectively (SDs: 0.76, 0.84, and 0.84 spikes per
trial).
Direct inhibitory responses were rare, accounting for only 32
of 984 (3%) responses to contralateral stimulation, 30 of 585
(5%) responses to ipsilateral stimulation, and 35 of 1,103 (3%)
responses to simultaneous bilateral stimulation. The lower
incidence of inhibitory MU responses (3–5%) as compared
with SU responses (15%) suggests that excitatory responding
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Figure 4A shows the distribution of response latencies corresponding to contralateral, ipsilateral, and synchronous bilateral stimulation. The average response latencies for contralateral, ipsilateral, and bilateral stimuli were 14.67 ⫾ 0.02,
23.78 ⫾ 0.04, and 15.07 ⫾ 0.02 ms (SDs: 19, 24, and 17 ms,
respectively). For comparison, response latencies in S1 SUs in
pentobarbital-anesthetized rats, for contralateral and ipsilateral
whisker stimuli similar to those used in the present study, were
11 ⫾ 3.4 and 23 ⫾ 4.7 ms, respectively (Shuler et al. 2001),
consistent with the present study.
One potential confound in interpreting the bilaterally evoked
responses we observed is the possibility that a startle reaction
arising from an ipsilateral stimulus might lead to a movement
of contralateral whiskers against the (stationary) stimulator
noose, leading to a response resulting from contralateral selfstimulation. Such a response could be misinterpreted as an
evoked ipsilateral response. This scenario can be ruled out as
follows: First, sessions with the stimulator attached on only
one side of the face continue to show ipsilateral responses.
Four examples are shown in Fig. 5. This rules out the possibility that false ipsilateral responses resulted from contralateral
self-stimulation against the stimulator noose. That leaves the
possibility that a contralateral movement “in air” could generate a false ipsilateral response in S1.
This is possible in principle because Fee et al. (1997) found
that whisking movements modulate spiking activity in S1.
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Temporal integration of asynchronous bilateral stimuli in S1
FIG.
6. Heterogeneous integration of synchronous bilateral whisker stimuli.
A: histogram of the ratio of response magnitudes to bilateral vs. contralateral
whisker column stimulation, for all MUs recorded in 5 rats. Many MUs’
response ratios fall above or below 1, indicating that synchronous ipsilateral
stimulation can result in either inhibitory (⬍1) or excitatory (⬎1) modulation
of the contralateral response. B: histogram of the ratio of response magnitudes
to bilateral stimulation vs. the sum of contralateral and ipsilateral response
magnitudes, to assess whether bilateral stimuli are integrated sub- or supralinearly. A ratio ⬎1 indicates supralinear summation of the 2 whisker column
stimuli. Most MUs showed sublinear summation of bilateral stimuli, but
almost as many were supralinear.
from other neurons in the same MU sometimes masks inhibitory modulation within an MU.
The above results demonstrate that simultaneous ipsilateral
and contralateral whisker signals interact in each S1 hemisphere. To characterize the modulation of these bilateral interactions by the ISI between the left and right stimuli, we
recorded S1 responses at varying ISIs. The net excitatory
neuronal responses at each ISI are shown in Fig. 7. The
significant dip at 30 ms shows that response suppression
dominates at ISIs around 30 ms. This is another confirmation
that ipsilateral stimulation can modulate the spiking activity in
S1 on behaviorally relevant timescales.
DISCUSSION
Integration of synchronous bilateral stimuli in S1
The observation that single and multiunits respond to ipsilateral whisker stimulation clearly indicates that bilateral stimuli are integrated in rat S1. However, the average neuronal
response to synchronous bilateral stimulation (0.83 ⫾ 0.03
spikes per trial) is not significantly different from the response
elicited by contralateral stimulation alone (0.82 ⫾ 0.02 spikes
per trial).
To go beyond such average measures, Fig. 6A shows a
histogram of the ratios of bilateral response magnitude to
contralateral response magnitude for each MU for all rats. The
distribution of ratios is peaked near one, but many are less than
or greater than 1, indicating that ipsilateral stimulation can
result in either inhibitory or excitatory modulation of the
contralateral response. To assess whether the facilitative or
suppressive effects of ipsilateral stimulation on the contralateral response were statistically significant for the different
MUs, we estimated the statistical uncertainty in the response
magnitudes as the square root of the total excess spike count in
the response window (Poisson statistics). Then, at the 95%
confidence level, the bilateral response was significantly lower
than the contralateral response (i.e., suppressive modulation)
for 265 MUs (out of 345 MUs whose ratio was ⬍1), whereas
the bilateral response was larger compared with the contralateral response (i.e., facilitative modulation) for 392 MUs (out of
455 whose ratio was ⬎1). An additional 194 MUs (not deJ Neurophysiol • VOL
The main result of the present study is that neurons in S1 of
awake rats integrate bilateral inputs over ⬎100 ms. This result
suggests the potential importance of bilateral integration in S1
for whisker-guided behavior. Moreover, this finding further
challenges the traditional conception of the organization of rat
somatosensory cortex into independent representational “modules” in each hemisphere.
In awake, immobilized rats, neural responses in S1 to punctate whisker stimuli were similar in terms of response latencies
FIG. 7. Temporal integration of asynchronous bilateral whisker stimuli. Net
excitatory response magnitude, in spikes per trial above baseline, is plotted for
varying intervals between a left and a right whisker column stimulus. Response
suppression is dominant at intervals of about 30 ms. Data shown are pooled
from 5 animals.
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picted in the histogram of Fig. 6A) responded significantly to
bilateral but not to contralateral stimulation alone. For these
units also, the ipsilateral stimulation may also be considered to
have increased, or facilitated, the contralateral response.
Because ipsilateral stimulation can significantly modify the
response to contralateral stimulation, we also asked how the 2
responses added. Figure 6B plots the distribution of the ratios
of bilateral response magnitudes to the sum of response magnitudes for each side, to assess whether bilateral whisker
signals are summed sub- or supralinearly. The trend is toward
sublinear summation, but many MUs showed supralinear integration. Specifically, 552 MUs were significantly sublinear
(out of 663 with ratios ⬍1, 2 sigma confidence level), and 254
MUs were significantly supralinear (out of 313 with ratios ⬎1).
To these supralinear units may be added 127 more that responded significantly to bilateral but not to contralateral or
ipsilateral stimulation alone. Thus in awake S1, supralinear
summation of synchronous bilateral inputs is almost as common as sublinear summation.
Innovative Methodology
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M. C. WIEST, N. BENTLEY, AND M. A. L. NICOLELIS
to those recorded in anesthetized animals (Shuler et al. 2001).
In terms of bilateral integration, 49% of SUs responding to
contralateral whisker column stimulation also responded to
ipsilateral stimulation, compared with 62% in the anesthetized
study [M. Shuler, personal communication; the published report (Shuler et al. 2001) contains the percentage that responded
to either of 2 whisker columns (73%)]. In these terms, the
degree of bilateral integration is similar in the anesthetized and
waking S1 (but see following text).
Principles of spatiotemporal integration of multiple
unilateral stimuli
J Neurophysiol • VOL
Principles of spatiotemporal integration of bilateral stimuli
Shuler et al. (2001) found primarily inhibitory interactions
between pairs of bilateral whisker column stimuli in S1 layer V
of anesthetized rats, with greatest suppression occurring at ISIs
of 30 –90 ms. The responses in the anesthetized animals increased monotonically on average from ISIs of 60 to ⱕ180 ms.
In our study, the net excitatory responses increased from ISIs
of 30 ms to ISIs of 60 or 90 ms, but were relatively smaller for
ISIs of 120 ms (Fig. 7). Thus bilateral integration appears
somewhat more dynamic in the waking animals.
Furthermore, the anesthetized study found only a marginal
difference between the responses to synchronous bilateral stimuli
as compared with contralateral stimulation alone. This was expected because contralateral stimuli universally suppressed the
responses to ipsilateral stimuli. Because the latency for the contralateral response is shorter, the ipsilateral excitatory response is
suppressed before it occurs. We also found no significant difference between the average response to contralateral and bilateral
stimulation. However, in the awake rats many individual units
showed significant supralinear spatial summation (Fig. 5B) as well
as sublinear summation of synchronous bilateral inputs. This
difference suggests that the profile of excitatory and inhibitory
contributions to ongoing S1 responses to bilateral stimuli is
substantially altered in the anesthetized animal. The heterogeneity
of responses we observed in the awake animals could function to
enhance discrimination of patterns of whisker stimulation (Maass
et al. 2002).
We conclude that individual neurons in the cortical whisker
representation in the awake rat are influenced by nonlinear
combinations of inputs from multiple whiskers on both sides of
the face, over time spans up to and greater than a hundred
milliseconds. Moreover, the spatiotemporal integration of these
inputs can depend strongly on the animal’s behavioral state
(Castro-Alamancos 2004; Fanselow and Nicolelis 1999; Krupa
et al. 2004b; Moore 2004; Moore et al. 1999; Nicolelis and
Fanselow 2002). The fact that rats can perform a variety of
unilateral and bilateral whisker tasks points to the possibility
that the degree of tactile bilateral integration through the
corpus callosum can be modulated according to the animal’s
current behavioral context. This would amount to using different sensory codes for different behavioral contexts. If this is the
case, mechanisms for adaptive control of bilateral connectivity
revealed in the whisker system may provide insight into dynamical mechanisms for adjusting the functional connectivity
within and among other brain areas as well.
93 • MAY 2005 •
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Previous studies of spatiotemporal integration of multiwhisker inputs in the rat whisker sensory system have found
evidence of both inhibitory and excitatory interactions. Extraand intracellular studies of S1 responses to combinations of
unilateral stimuli found inhibitory interactions dominating between pairs of neighboring whiskers (Brumberg et al. 1996;
Higley and Contreras 2003; Simons 1985) at ISIs around
10 –20 ms. [Moore (2004) recently suggested that these relatively slow temporal interactions are supplemented by resonances at higher frequencies that could account for rats’ texture
discrimination ability.] Pairs of synchronous stimuli were
found to sum linearly (Mirabella et al. 2001; Simons and
Carvell 1989), whereas Mirabella et al. (2001) reported that
synchronous stimulation of 4 whiskers usually resulted in
sublinear summation. These studies focused on responses of
neurons in middle and supragranular layers of S1 in immobilized or anesthetized rats. Brumberg et al. (1996) suggested
that surround inhibition could function for contrast enhancement in layer IV and to “effectively increase the ‘principal
whiskerness’ of the cortical column,” in line with a model of
barrel columns as “single-whisker processing units.” Mirabella
et al. (2001) suggested that inhibitory interactions could serve
to avoid response saturation and maintain a dynamic response
range under naturalistic multiwhisker inputs. Recent recordings in freely moving rats engaged in an active whiskerdependent discrimination task have suggested (Krupa et al.
2004b) that inhibitory response modes in all layers are significantly more common in the discriminating animals as compared with passively stimulated animals.
In undrugged rats, similar temporal integration of multiple
unilateral inputs in S1 layer V was reported in Fanselow and
Nicolelis (1999). SU responses to the second of a pair of
unilateral electrical stimulations of the facial afferent infraorbital nerve in awake rats were suppressed most strongly at ISIs
around 25–50 ms. This suppression was most pronounced
during rats’ awake quiet behavioral state as compared with the
whisking and active exploring states.
However, not all unilateral interactions are inhibitory: synchronous multiple-whisker deflections have also been reported
to sum supralinearly. Shimegi et al. (2000) reported supralinear
summation of neighboring single-whisker stimuli in 37% of S1
neurons, primarily by cells in supragranular layers. They noted
that enhanced responses were often selective for specific combinations of stimulus features such as whisker position, angle
of deflection, and relative timing of multiwhisker stimuli,
supporting the idea that S1 cells code for spatiotemporal
patterns of whisker stimulation. Ghazanfar and Nicolelis
(1997) found that 81% of SUs in S1 layer V showed supralin-
ear responses to simultaneous stimulation of triplets of whiskers as compared with single-whisker stimulations. They reported a bias toward supralinear summation of within-column
triplets as compared with within-row triplets. They pointed out
that their findings were consistent with the reports of sublinear
summation of unilateral stimuli because sublinear spatial summation was reported in studies of responses in granular and
supragranular layers to pairs of neighboring whiskers, as opposed to the layer V responses to stimulation of triplets of
relatively distant whiskers in their study. Together these findings imply dynamic excitatory as well as inhibitory nonlinear
interactions among multiple whisker stimuli, which could
function to encode spatiotemporal patterns of unilateral whisker stimulations.
Innovative Methodology
BILATERAL INTEGRATION IN AWAKE S1
GRANTS
This work was supported by National Institute of Dental and Craniofacial
Research Grants DE-11451 and DE-13810.
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