Neonatal Whisker Removal Reduces the Discrimination of Tactile Stimuli by Thalamic Ensembles in Adult Rats
Miguel A. L. Nicolelis1,
Rick C. S. Lin2, and
John K. Chapin2
1 Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710; and 2 Department of Neurobiology and Anatomy, Allegheny University School of Health Sciences, Philadelphia, Pennsylvania 19102
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ABSTRACT |
Nicolelis, Miguel A. L., Rick C. S. Lin, and John K. Chapin. Neonatal whisker removal reduces the discrimination of tactile stimuli by thalamic ensembles in adult rats. J. Neurophysiol. 78: 1691-1706, 1997. Simultaneous recordings of up to 48 single neurons per animal were used to characterize the long-term functional effects of sensory plastic modifications in the ventral posterior medial nucleus (VPM) of the thalamus following unilateral removal of facial whiskers in newborn rats. One year after this neonatal whisker deprivation, neurons in the contralateral VPM responded to cutaneous stimulation of the face at much longer minimal latencies (15.2 ± 8.2 ms, mean ± SD) than did normal cells (8.8 ± 5.3 ms) in the same subregion of the VPM. In 69% of these neurons, the initial sensory responses to stimulus offset were followed for up to 700 ms by reverberant trains of bursting discharge, alternating in 100-ms cycles with inhibition. Receptive fields in the deafferented VPM were also atypical in that they extended over the entire face, shoulder, forepaw, hindpaw, and even ipsilateral whiskers. Discriminant analysis (DA) was then used to statistically evaluate how this abnormal receptive field organization might affect the ability of thalamocortical neuronal populations to "discriminate" somatosensory stimulus location. To standardize this analysis, three stimulus targets ("groups") were chosen in all animals such that they triangulated the central region of the "receptive field" of the recorded multineuronal ensemble. In the normal animals these stimulus targets were whiskers or perioral hairs; in the deprived animals the targets typically included hairy skin of the body as well as face. The measured variables consisted of each neuron's spiking response to each stimulus differentiated into three poststimulus response epochs (0-15, 15-30, and 30-45 ms). DA quantified the statistical contribution of each of these variables to its overall discrimination between the three stimulus sites. In the normal animals, the stimulus locations were correctly classified in 88.2 ± 3.7% of trials on the basis of the spatiotemporal patterns of ensemble activity derived from up to 18 single neurons. In the deprived animals, the stimulus locations were much less consistently discriminated (reduced to 73.5 ± 12.6%; difference from controls significant at P < 0.01) despite the fact that much more widely spaced stimulus targets were used and even when up to 20 neurons were included in the ensemble. Overall, these results suggest that neonatal damage to peripheral sense organs may produce marked changes in the physiology of individual neurons in the somatosensory thalamus. Moreover, the present demonstration that these changes can profoundly alter sensory discrimination at the level of neural populations in the thalamus provides important evidence that the well-known perceptual effects of chronic peripheral deprivation may be partially attributable to plastic reorganization at subcortical levels.
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INTRODUCTION |
Peripheral sensory deafferentation is known to induce anatomic, biochemical, and functional changes at multiple levels of the somatosensory system (Florence and Kaas 1995
; Kaas 1991
; Rausell et al. 1992
). As a consequence of this reorganization process, the cortical and subcortical neurons that are deprived of their main sensory input become responsive to the stimulation of cutaneous territories surrounding the deafferented zone (Kaas 1991
; Kaas et al. 1983
). Despite the extensive literature devoted to the description of this phenomenon, little is known about the underlying consequences of long-term peripheral deafferentation for processing of tactile information by populations of somatosensory neurons. This issue is particularly relevant given recent suggestions that the occurrence and magnitude of plastic reorganization of the somatosensory cortex may be correlated with abnormal tactile sensations in amputees (Katz and Melzack 1990
; Lacroix et al. 1992
; Melzack 1990
), including referred sensations to the forelimb following a facial stimulus and excruciating chronic pain (Ramachandran 1993
; Ramachandran et al. 1992
).
Studies carried out in primates have revealed that long-term peripheral deafferentation can lead to an extensive area of cortical reorganization (Pons et al. 1991
). In these animals, cortical reorganization is paralleled by major histochemical changes in the ventral posterior complex (Rausell et al. 1992
), the main thalamic relay of the somatosensory system. Moreover, recent evidence supports the notion that sprouting or expansion of primary afferents at the level of the spinal cord and brain stem may play an important role in defining the extent of cortical reorganization (Florence and Kaas 1995
). These findings point to the relevance of investigating how functional alterations in subcortical structures, such as the somatosensory thalamus, may contribute to the emergence of altered tactile experience.
Previously we reported that a complete unilateral removal of facial whiskers in newborn rats induces extensive functional reorganization in the contralateral ventral posterior medial nucleus (VPM) of the thalamus (Nicolelis et al. 1991
). This plastic reorganization is characterized by the presence of neurons with large receptive fields (RFs) that span the face and other body parts such as the shoulder and forepaw. The existence of these face-body RFs raises the hypothesis that the process of plastic reorganization induced by a neonatal deafferentation could alter the ability of populations of VPM neurons to unambiguously represent the location of tactile stimuli in adult animals.
In the present study we tested this hypothesis by simultaneously recording the sensory responses of up to 20 thalamic neurons in adult rats
1 yr after rats had been subjected to a unilateral removal of facial whiskers. This experimental approach not only allowed us to characterize the long-standing effects of the neonatal deafferentation at the single-neuron level but also provided an unique opportunity to quantify the impact of plastic reorganization at the level of neuronal ensembles. This approach revealed that, after a long period of deafferentation, VPM neurons exhibited longer than normal sensory response latencies, long-lasting reverberatory firing following tactile stimulation of the deafferented face, and also large RFs covering the entire face and extensive regions of the body. Moreover, with the use of classical multivariate statistical techniques, such as discriminant analysis (DA), to characterize the encoding of tactile information by neural populations, we demonstrated that this long-standing peripheral deafferentation significantly reduced the probability of predicting the location of a tactile stimulus on the basis of the spatiotemporal patterns of thalamic ensemble activity. Overall, our results are consistent with the hypothesis that reorganization of the somatosensory thalamus may play an important role in cortical plasticity and thus in the generation of aberrant tactile sensations following peripheral deafferentation.
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METHODS |
Procedure for neonatal sensory deafferentation
Long-Evans (hooded) rats were used in all experiments. To produce a partial sensory deafferentation of the whisker pad, 1-day-old pups (n = 14, from 2 litters) were first anesthetized by hypothermia. After a few minutes on ice, the animals became paralyzed and remained deeply anesthetized while the large whisker follicles in the animal's right snout were cauterized. This procedure successfully removed all large facial vibrissae and severely limited whisker regrowth thereafter. After the completion of this surgery, the rats were warmed up and returned to their litters. During the first postnatal week, whenever new whiskers were observed, the surgical procedure described above was repeated. This criterion was used in all but one rat. In this animal, regrowth of two caudal whiskers occurred, but no further cauterization was carried out so that we could study the functional properties of VPM neurons following stimulation of these whiskers. Electrophysiological studies were carried out 12-24 mo after the neonatal sensory deafferentation.
Functional characterization of the plastic reorganization in the VPM thalamus
To quantify the long-term plastic modification of the functional organization of the VPM thalamus, simultaneous recordings of the extracellular activity of up to 20 thalamic neurons per animal were obtained in 12- to 24-mo-old rats (n = 5). For these neurophysiological experiments, animals were first anesthetized with pentobarbital sodium (50 mg/kg ip) and then transferred to a stereotaxic apparatus. Subsequently, arrays (16 wires per array, NBLABS, Dennison, TX) of Teflon-coated, stainless steel microwires (50 µm tip diameter) were chronically implanted in the VPM of the thalamus, contralateral to the lesioned face, as described elsewhere (Nicolelis and Chapin 1994
; Nicolelis et al. 1997
). In each animal, microwires were positioned in the region of the VPM in which neurons responded maximally to the stimulation of the bare skin in the lesioned (contralateral) whisker pad. The stereotaxic coordinates for these implants were determined in mapping experiments performed in two other animals. Five to 7 days after the microwire implant surgery, animals were again anesthetized with pentobarbital (50 mg/kg ip) and transferred to a recording chamber where all electrophysiological experiments were carried out. A detailed description of this experimental paradigm can be found elsewhere (Nicolelis and Chapin 1994
; Nicolelis et al. 1997
). Briefly, a head stage (NBLABS) was used to connect the 20-channel plastic connector cemented in the animal's head to a multichannel neuronal spike data acquisition processor (Spectrum Scientific, Dallas, TX). Time-voltage window discrimination combined with a spike-sorting algorithm were used to isolate single neurons on the basis of their extracellular action potentials (Nicolelis and Chapin 1994
). Once single neurons had been isolated and their spontaneous activity had been recorded, a computer-controlled vibromechanical probe was used to deliver mechanical stimulation to multiple sites on the animal's face, neck, shoulder, back, forepaw, and hindpaw. Stimuli consisted of step pulses (duration 100 ms) delivered at 1 Hz. Four hundred to 600 trials were collected for each cutaneous stimulation skin site. Control data were obtained by recording from the contralateral normal VPM in one of these animals and from normal adult rats (n = 12). In these animals, low-threshold tactile stimuli were delivered to the facial whiskers and fur. In one additional control animal, tactile stimuli were delivered to different regions of the face from which all facial whiskers and fur had been shaved. This allowed us to measure response latencies following stimulation of nonhairy skin of the face.
Histological reconstruction of the recording electrode tracks in the normal and whisker-deprived animals showed that all electrodes were arrayed within a radius of 0.5-1.0 mm of the center of the dorsal subregion of the VPM. As such, virtually all electrode recordings were obtained in the VPM mystacial whisker representation (in the normal animals) or in the equivalent region in the deprived animals.
Statistical analysis of single-neuron responses
At the single-neuron level, the effects of the neonatal sensory deafferentation were characterized by measuring neuronal response latencies, response magnitudes, and RF distributions following tactile stimulation of multiple skin sites (see above). Data analysis was carried out with the use of a neuronal ensemble data analysis software package (Stranger, Biographics, Winston-Salem NC) running on an IBM-PC compatible Pentium microcomputer with the use of Windows 3.11 (Microsoft, Redmond, WA) as the operating system. Sensory responses were expressed in poststimulus time histograms (PSTHs) and cumulative frequency histograms (CFHs). Response magnitudes (in spikes/s) for different poststimulus epochs were derived from PSTHs. Statistical significance of sensory responses was measured with the use of a new algorithm (A. Kirilov, personal communication) to calculate a one-way Kolmogorov-Smirnov statistic to test the probability that the distribution of the CFH, defining the overall sensory response of a given neuron, differed from its average firing rate. For each neuron, the time period used for the calculation of the CFHs extended from 50 ms prestimulus to 100 ms poststimulus (with the use of 1-ms bins). Each point of the CFH was obtained by calculating the sum of counts (spikes) in the preceding bins and then subtracting from this value the product of the average bin count (during the 150 ms) times the number of preceding bins. The significance level used for accepting a sensory response as valid was set at P < 0.01. An ellipse representing the confidence interval was superimposed on each CFH. CFHs were also used to measure sensory response latencies. Because these plots depict deviations from the neuron's average firing, response latencies could be measured by locating the inflection points that demarcate the initial change in firing rate that defines the onset of the neuronal response to a peripheral stimulus. Inflection points for long latency components were also observed in these plots. Pooled data from several animals were then used to define latency histograms for both control and deafferented animals.
Statistical analysis of neuronal ensemble responses
Discriminant analysis (CSS-Statistica; Tulsa, OK) (Dillon 1984
; Tabachnick and Fidell 1989
) was employed to statistically evaluate the differences between the responses of multineuronal ensembles to stimulation of various peripheral stimulation sites in the control (n = 3) and deprived animals (n = 4). The aim was to determine whether the sensory deprivation altered the statistical ability of the recorded thalamic neuronal ensembles to differentiate between different stimulus sites. DA was used because it not only provides a multivariate-analysis-of-variance-like treatment of the data set but also derives classification functions for trial-by-trial discrimination between the different experimental groups (i.e., stimulus sites). Here, the multivariate data set consisted of the spike counts measured during three different latency epochs (0-15, 15-30, and 30-45 ms) of the poststimulus responses of each neuron in a given animal (normal or deprived) to each stimulus applied to each cutaneous site. These results were then used to derive linear classification functions for optimally discriminating between the groups (stimulation sites). This provided an estimation of how well thalamic ensemble activity could predict the location of a tactile stimulus in either a sensory deafferented or a normal animal.
Because of the profoundly altered RF organization in the deprived VPM (see RESULTS) it was necessary to standardize the choice of the three stimulus sites used as groups for the DA. All of the electrode arrays, which spread to a radius of ~0.5-1 mm in the VPM whisker area, were found to yield populations of neurons whose RFs overlapped such that an "ensemble" RF could be identified. This typically consisted of a central area within which
50% of the sampled neurons could be strongly driven (>3 SD above background) by light stimulation. Here, this central area was "triangulated" by choosing three test stimulus sites around its edge at approximately even spacing. In the normal rats this typically included a caudal whisker, a dorsal whisker, and a rostral guard hair. In the deprived animals the three sites typically included a rostral guard hair, a location on the dorsal part of the deprived surface of face, and a location on the caudal face, neck, or forelimb. Thus many of the stimulus sites in the deprived animals were in undamaged cutaneous regions.
MATHEMATICAL PROCEDURES.
DA was used to derive a series of linear functions, the discriminant functions or roots, from a set of independent variables. The number of a priori defined groups (G) defines how many discriminant functions are calculated, because, at most, G
1 functions can be extracted. Each of these discriminant functions or roots is defined by a set of weights (w) associated with each of the independent variables (
)
|
(1)
|
where i = 1 ··· G
1, w is discriminant weights,
is independent variables, and n is number of variables.
In our neuronal ensemble analysis, discriminant functions were derived from a set of variables that measured the extracellular activity of up to 20 simultaneously recorded thalamic neurons. For each neuron, absolute spike counts were obtained for three sequential poststimulus epochs (see preceding text) in each of 600-900 trials of tactile stimulation. Each of these time epochs was defined as an independent variable, meaning that a total of three variables was used to describe the firing pattern of a single VPM neuron in the data matrix used for DA. The discriminant functions were derived with the use of a subset of the trials (i.e., "training" trials). Another subset of trials, named the "testing" trials, was used for evaluating how well the location of stimuli could be predicted by DA on a single-trial basis. All analyses carried out in this study used three experimental groups (G1, G2, and G3), which corresponded to three distinct skin locations stimulated in each animal (either 3 nonneighboring whiskers in controlsor 3 distinct body regions in deafferented rats). Sample sizes were always equal for the three groups.
A typical data matrix used for DA is depicted in Fig. 1. This matrix was defined by 60 independent variables (3 epochs per neuron for 20 VPM neurons), three groups (corresponding to 3 distinct stimulus sites), and 900 cases (300 trials per stimulus location). Once a data matrix was created, the DA software generated discriminant functions for classification of single trials into the three groups. Because there were three a priori groups (G1-G3) defined in our analysis, two discriminant functions (roots 1 and 2) were obtained for each data matrix (1 for each animal). Discriminant scores (Yi in Eq. 1) were then calculated for each trial by first multiplying the weight (w) associated with each variable by the value of the variable for the trial and then summing the obtained products. Each of these discriminant scores, therefore, represented the weighted neuronal ensemble response to a single tactile stimulus. Because two discriminant functions were generated for each data set, two discriminant scores were calculated for each trial (Y1j and Y2j, where j is the trial number). Just as discriminant scores were calculated for each trial, mean discriminant scores, called centroids, were obtained for each group. Centroids were calculated by multiplying the weights of each discriminant function by the mean value of the independent variables for each of the three groups and then adding the resulting products. The centroid of each group was then defined by two values (C1
and C2
, where
is the group number). Discriminant scores and centroids were used to evaluate how well the three groups could be separated by the discriminant functions. Wilks' Lambda and Mahalanobis distances (Dillon 1984
; Tabachnick and Fidell 1989
) were used as criteria to verify the statistical significance of the discrimination of trials. Once the Wilks' Lambda was obtained, an F value was calculated as described elsewhere (Tabachnick and Fidell 1989
). Whenever the F value obtained by applying DA to a data sample exceeded the critical F value for P < 0.01, we concluded that the three chosen stimulation sites could be distinguished on the basis of the activity of ensembles of VPM neurons. The contribution of each individual variable was quantified by calculating its F to remove (F-REMOVE) value. Each variable's F-REMOVE was then compared with a critical F value that was calculated by assuming P < 0.01. Variables with F-REMOVE higher than the critical F value contributed significantly for the discrimination of groups. For those variables, the absolute F-REMOVE value reflected the reduction in trial classification that would occur if that variable was removed from the ensemble.

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| FIG. 1.
Representation of data matrix structure used to perform discriminant analysis in both normal and deafferented rats. In these matrices, each column represents variable (V) that depicts the absolute number of spikes of a ventral posterior medial (VPM) neuron (NEURON 1 ··· NEURON n) in each of 3 poststimulus time (T) epochs (T1 = 0-15 ms; T2 = 15-30 ms; T3 = 30-45 ms). Conversely, each row of these matrices represents a single experimental trial defined by a mechanical stimulus (see METHODS) applied to a peripheral skin site. Discriminant analysis carried out here assumes that each matrix row represents case. Cases are clustered in 3 groups (G1-G3), each of which corresponds to 1 of the 3 distinct skin sites stimulated in each animal. Each group contained a maximum of 300 trials. In each group trials were further subdivided into "training" and "testing" trials. Training trials were used to derive discriminant functions, whereas testing trials were used to verify accuracy of these functions in determining stimulus location on a single-trial basis. With the use of this data structure, the population response of VPM ensemble to a single stimulus is depicted by a matrix row (e.g., trial 301 in dark blue). Moreover, the poststimulus time histogram (PSTH, light blue) of a neuron for a stimulus site can be obtained by averaging the 3 variables depicting the neuron's sensory response to a set of trials that define each group. Each element of the data matrix is defined by Vr,c where V represents variable, r is row position, and c is column position.
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In the second step of the DA, a set of 100-300 new trials (the testing trials) was used to evaluate how well one could predict the location of a tactile stimulus, on a trial-by-trial basis, in each animal. As mentioned above, these testing trials were not used to derive the discriminant functions. Classification functions were used to assign individual trials into each of the three groups. In general, DA generates one classification function per group. The simplest form of one of these functions is
|
(2)
|
where Ci is classification score, ci0 is a constant, cin is classification function coefficients,
is variables, i is group number, and n is variable number.
As in the case of discriminant functions, classification scores from each of these classification functions were calculated for each trial by multiplying the value of each variable in the trial by its classification function coefficient, summing the products, and then adding the value of the constant. Three classification scores (C1-C3) were generated for each individual trial, one score for each a priori defined group. Each trial was then classified as belonging to the group for which it had the highest classification score. This trial-by-trial prediction of stimulus location was then compared with the real location of each trial, and a percentage of correct predicted trials was obtained for all three groups in each animal. An average percentage of correct trials was then calculated for each normal and each sensory-deprived animal.
The effect of the ensemble size on classification of training and testing trials was also evaluated in both normal and sensory-deprived rats. To carry out this analysis, neurons in each of the simultaneously recorded VPM ensembles were rank-ordered according to the sum of the F values (F-REMOVE) of the three variables (T1, T2, and T3, see Fig. 1) used to describe each neuron's sensory response. As mentioned above, the higher the F-REMOVE value of a neuron, the larger the neuron's contribution to group discrimination. Thus the neuron with the highest F-REMOVE in each VPM ensemble was designated as the best predictor neuron of the ensemble. To study how the performance of each VPM ensemble (in terms of trial classification) varied as a function of the ensemble size, we then removed the best predictor neuron from the original data matrix and then carried out DA again to recalculate the percentage of correct trials for the reduced ensemble. The remaining neurons were rank-ordered again, because their F-REMOVE values changed with the removal of the best predictor neuron, and the whole procedure was repeated by removing, one by one, the neuron with the highest F-REMOVE value in the ensemble, until only two neurons (6 variables) were left in the data matrix. The results of this analysis were displayed in line plots that depicted the percentage of corrected classified trials as a function of ensemble size.
 |
RESULTS |
Twelve to 20 mo after the neonatal cauterization of whisker follicles, the deafferented snouts of these rats were typically covered with a central area of bare skin surrounded by common fur. This pattern was similar in all but one animal in which two caudal whisker follicles were spared.
Functional properties of deafferented VPM neurons
Overall, the sensory responses of a total of 76 deafferented VPM neurons (n = 5 rats) were characterized in this study. Initially, CFHs were used to determine the response latencies of each of these neurons to the stimulation of different regions of the face and body. In all animals, deafferented VPM neurons reliably responded to stimulation of the bare skin covering the whisker pad as well as of the fur surrounding the cauterized area. The minimal latencies (14.0 ± 7.5 ms, mean ± SD) of these responses were significantly longer than those observed in the VPM of normal animals (8.86 ± 5.33 ms, P < 10
6, Student's t-test). Analysis of the latency distribution for all 76 deafferented VPM neurons (Fig. 2) revealed that most of their sensory responses occurred during the 8- to 20-ms epoch (quartile range 9-18 ms), a period in which postexcitatory inhibition is commonly observed in normal VPM neurons. To rule out the possibility that these latency differences were purely attributable to inherent differences in the type of cutaneous receptors present in the whisker pad versus bare skin, a series of control experiments was carried out. These experiments revealed that stimulation of facial whiskers, fur, and bare facial skin in normal animals all produced initial excitatory responses in the same short latency range (5-8 ms, Fig. 3). These findings were consistent with the previous observation that stimulation of the hairy and glabrous skin of the rat forepaw produces similar minimal latencies in the primary somatosensory cortex (Chapin and Lin 1984
). We also observed that stimulation of the two whiskers that were spared in one deafferented animal (see METHODS) also produced longer response latencies than were observed when individual whiskers were stimulated in normal animals (Fig. 4).

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| FIG. 2.
Distribution of minimum sensory response latency calculated for a set of 76 VPM neurons isolated in 5 adult rats subjected to complete unilateral whisker removal at birth. Only sensory responses derived by stimulating facial regions were used to calculate this distribution. Response latency (14 ± 7.5 ms, mean ± SD) of these sensory-deprived neurons was found to be statistically higher than in normal animals.
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| FIG. 3.
PSTHs and cumulative frequency histograms (CFHs) depict sensory responses of 6 simultaneously recorded VPM neurons following stimulation of facial fur in a normal adult rat. Note that minimum response latencies, displayed in top right corner of each PSTH, are in 5- to 8-ms range.
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| FIG. 4.
PSTHs and CFHs depict sensory responses of 12 simultaneously recorded VPM neurons following stimulation of a single whisker in a neonatally deprived animal. For each neuron (N), identified by numbers 1-20, CFH was plotted atop corresponding PSTH. Perimeter of the ellipse in each CFH defined a confidence interval (P = 0.01) used to consider sensory response as statistically significant. First inflexion point in each CFH defined minimum sensory response latency of each neuron to stimulus. The value of this latency is displayed in top right corner of each PSTH.
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In addition to these latency increases, neurons located in the face representation of the VPM exhibited abnormal patterns of response to sensory stimuli. Whereas most normal VPM neurons responded to discrete cutaneous stimuli (duration 100 ms) with a characteristic series of phasic excitatory-inhibitory-excitatory responses (Fig. 5A), 69% (45 of 65) of the deafferented VPM neurons exhibited tonic, slowly adapting discharge during their responses to equivalent stimuli (Fig. 5B). Moreover, although the firing rate of normal VPM neurons returned to spontaneous levels soon after the end of the stimulus (at 100 ms), deafferented VPM neurons often displayed reverberant firing, with excitation-inhibition sequences recurring at intervals of ~100 ms for up to 700 ms after the stimulus offset. Nonetheless, normal and deafferented VPM neurons could not be distinguished solely on the basis of their spontaneous firing rate.

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| FIG. 5.
PSTHs represent sensory responses of 5 simultaneously recorded VPM neurons in a normal (A) and a neonatally deprived (B) adult rat. In the normal animal (A), discrete displacement (vertical ··· at 0 ms) of facial whisker (E1) induced a strong short-latency (4-8 ms) fast-adapting response in VPM neurons, followed by less intense long latency (15-25 ms) component. OFF responses ( ) can also be identified following stimulus offset (100 ms). In clear contrast, discrete stimulation of the face in a neonatally sensory-deprived adult rat (B) induced mainly slowly adapting firing of VPM neurons, which have longer than normal minimal response latencies. After stimulus offset (100 ms), deafferented VPM neurons exhibited reverberant firing every 100 ms for up to 700 ms. All PSTHs were built with the use of 1-ms bins.
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The neonatal sensory deprivation also altered the spatial organization of RFs in the VPM. Qualitative mapping of sensory responses, performed during the acute surgery for microwire implantation, revealed that VPM neurons exhibited much larger than normal RFs, a finding that was consistent with our previous mapping studies carried out a few weeks after the neonatal whisker cauterization (Nicolelis et al. 1991
). Most of these RFs covered the whole face, including the caudal facial regions that were not cauterized at birth. Moreover, many of these RFs extended further caudally to cover the neck, shoulder, and occasionally the forepaw. These observations were quantitatively confirmed through computer analysis of single-unit recordings obtained during subsequent chronic recordings through the same microwires. Such analyses were carried out on 67 of a total of 76 deafferented VPM neurons and involved construction of PSTHs of each neuron's response to stimulation of a range of body sites. As shown in Fig. 6, these results confirmed that, as a rule, the RFs of sensory-deprived VPM neurons were extremely large and spatially complex. Whereas all 67 neurons responded to the stimulation of the deprived face, their RFs extended to the caudal face (69%, 46 of 67), to other body regions such as the shoulder (33%, 22 of 67), the back, and the forepaw (51%, 34 of 67), and even to the ipsilateral normal whiskers (43%, 29 of 67). Typical examples of these face-body RFs are depicted in Fig. 6 from a set of 16 simultaneously recorded VPM neurons. Most of these neurons responded not only to the stimulation of the deprived face, but also to the forepaw, the hindpaw, and the ipsilateral whiskers. Figure 6 also shows that the longest response latencies in the deafferented VPM were observed following stimulation of the ipsilateral whiskers (23.7 ± 12.5 ms). As expected, neurons located in the VPM of normal adult rats or in the contralateral VPM of deafferented rats did not exhibit face-body RFs.

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| FIG. 6.
Large face-body receptive fields in the thalamus of neonatally deprived adult rats. PSTHs represent sensory responses of 16 simultaneously recorded VPM neurons in neonatally deprived adult rat following tactile stimulation of multiple skin sites (deprived face, forepaw, hindpaw, and ipsilateral whiskers). Notice that vast majority of neurons, identified by number in bottom right corner of each PSTH, respond to 2 extra skin sites in addition to deprived face, defining very large face-body receptive fields. For each PSTH, time is in ms and firing rate is in spikes/s (Hz).
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Neuronal population level analyses
DA was used to quantify the effects of the neonatal sensory deprivation on the ability of VPM ensembles (of 13-20 simultaneously recorded neurons) to "discriminate," on a trial-by-trial basis, the location of a cutaneous stimulus. By providing a rigorous statistical characterization of the predictive value of tactile sensory information contained within the recorded sample of VPM neurons, DA allowed us to assess the hypothetical ability of the population of thalamocortical somatosensory neurons to discriminate between sensory stimuli presented to different cutaneous locations.
This analysis revealed that, in normal adult rats, most VPM neurons provide significant contributions for the accurate discrimination of tactile stimuli (Nicolelis and Chapin 1994
; Nicolelis et al. 1993a
). As an example, Fig. 7 shows the significance levels, as statistically quantitated by F-REMOVE values, for 63 variables (derived from 21 VPM neurons) obtained in a normal adult rat. In this animal, 41 of the 63 variables (64%) contributed significantly (P < 0.01) to the classification of cases (trials) into three groups (stimulus locations). In Fig. 7, the critical F-REMOVE value (F = 2.85) for the P <0.01 significance level is represented by the horizontal line parallel to the X-axis. This significance threshold was used to justify the inclusion of a given variable in the model used to derive the discriminant functions.

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| FIG. 7.
Typical example of distribution of F to remove (F-REMOVE) values observed for each of 63 variables used to define 2 discriminant functions from a simultaneously recorded ensemble of VPM neurons in a normal rat. In this plot, variables are depicted in X-axis and magnitude of their F values is plotted in Y-axis. Horizontal line, plotted just above X-axis, represents minimal F value required for inclusion of given variable in definition of discriminant functions. This minimal value is also known as F-REMOVE. The higher the F value of variable, the higher its contribution for discrimination among groups. Variables with F values lower than F-REMOVE did not make statistically significant contribution for discrimination between groups. Notice that in this example, 41 of 64 variables (64%) contributed significantly for the classification of cases into 3 groups. Both short (T1 = 0-15 ms) and long latency epochs (T2 = 15-30 ms and T3 = 30-45 ms) of neuronal responses were found to be statistically significant in group discrimination.
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As might be expected, the shortest-latency poststimulus response epochs (0-15 ms; T1 in Fig. 1; 1st variable for each neuron in Fig. 7) usually had the highest F-REMOVE values (e.g., variables 4, 7, 19, 25, 40, 43, and 58). Interestingly, most of the longer latency components of these neurons' sensory responses (the 15- to 30- and 30- to 45-ms time epochs, T2 and T3 in Fig. 1) also provided significant contributions for the classification of cases. However, their F-REMOVE values were usually lower (e.g., variables 5, 8, 11, 14, 18, 20, 26, 27, 29, 38, 41, 45, 56, 59, and 60) than the values for the 0- to 15-ms time epoch. In fact, in two neurons in Fig. 7 (neuron 11, represented by variables 31-33, and neuron 15, represented by variables 43-45) the longer-latency responses were more significant. These observations are consistent with our previously published results suggesting that the representation of tactile information in the rat VPM is not only distributed in space but also in time (Nicolelis and Chapin 1994
; Nicolelis et al. 1993a
).
The same analysis carried out with data from the deprived animals revealed smaller percentages of significant F-REMOVE values, and those that were significant were mainly obtained from neurons with RFs centered distal to the whisker-deprived area. Those with RFs centered near or within the deprived area tended to have lower F-REMOVE values overall. Their highest F-REMOVE values were usually obtained for the longer latency epochs. Figures 8 and 9 illustrate two general patterns of F-REMOVE distributions observed in the whisker-deprived animals. The first (see Fig. 8) was characterized by the existence of a single, highly significant discriminant neuron (neuron 5, variables 13-15 in A, and neuron 1, variables 1-3 in B) in the ensemble whose F-REMOVE values (200 in A and 345 in B) far exceeded the F-REMOVE values for the remaining neurons. The percentages of statistically significant variables in these two cases (Fig. 8, A
and B
) were relatively low: 37% in A
and 41.7% in B
. As was typical of the highly discriminant neurons in these animals, this neuron had a large RF that extended into the deprived facial region but was centered outside this area.

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| FIG. 8.
Distribution of F values for variables used to define discriminant functions in 2 deprived animals. In these 2 examples, a single neuron (neuron 5, variables 13-15 in A, and neuron 1, variables 1-3 in B) made a much higher contribution for discrimination of groups than the rest of the ensemble. In addition, a much smaller number of neurons displayed significant F-REMOVE values. A and B : contribution of remaining variables without neurons 5 and 1, respectively. In A and B , critical F value (F-REMOVE = 2.85) is represented by horizontal line parallel to X-axis. In 1st animal (A and A ) 18 of 48 (37.5%) variables made significant contribution to discrimination of groups, whereas in 2nd animal (B and B ) 25 of 60 (41.7%) variables had statistically significant F values.
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| FIG. 9.
Two examples of F-REMOVE value distributions in deprived animals. Notice that in these 2 animals the number of variables with statistically significant contributions to discriminant functions (32 of 48, or 66.7%, in A; 32 of 51, or 62.7%, in B) is almost as high as in the distribution for the normal animal depicted in Fig. 7. Critical F: 2.85.
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Other whisker-deprived animals yielded higher percentages of significant F-REMOVE values but still underperformed the normal animals in their overall ability to discriminate stimulus location. For example, in the two ensembles depicted in Fig. 9, A and B, 32 of 48 (66%) and 32 of 51 (62.7%), respectively, were significant. Thus the highest F-REMOVE values obtained for these two ensembles (40 for neuron 2 in Fig. 9A and 72 for neuron 12 in Fig. 9B) were much closer to the ensemble average, in clear contrast to the cases illustrated in Fig. 7. Finally, the variables depicting longer latency components (15-30 and 30-45 ms, T2 and T3 in Fig. 1) often made highly significant contributions (i.e., their F-REMOVE was higher) for the discrimination of groups. For example, variables 5 (neuron 2) and 26 (neuron 9) provided the second and the third highest F-REMOVE values in the VPM ensemble represented in Fig. 9A; variable 50 (neuron 17) was the second most important discriminant variable in the population depicted in Fig. 9B.
Once the contributions of variables were defined for each VPM ensemble analyzed in this study, the next step was to evaluate how well each of these ensembles performed when DA was used to classify single trials into the three groups (i.e., 3 stimulus sites). Ensembles of VPM neurons in normal animals performed consistently better in this analysis than those in the sensory-deprived animals. In normal animals, multineuron ensemble activity correctly predicted the stimulus site in 90.6 ± 3.3% (mean ± SD) of training trials (range: 86.7-95%) and 88.2 ± 3.7% of testing trials (range: 82.7-92%). In sensory-deprived animals, correct prediction of stimulus location was reduced to 81.4 ± 8.8% for training trials (range: 70.6-90.6%) and 73.5 ± 12.6% for new trials (range: 41.8-88%). The difference in discrimination of testing trials between normal and deafferented animals was highly significant (P < 0.001, t-test). This reduction in prediction of stimulus location becomes even more meaningful when one considers that the distances between the discriminated stimulation sites were much greater in the deafferented animals. Whereas VPM ensembles in normal animals reliably discriminated between stimulation of whiskers spaced as close as 5-10 mm, those in deafferented animals were often unable to discriminate targets as far apart as the face and the forepaw.
Differences in single-trial discrimination between normal and deprived animals were better illustrated by graphs like those depicted in Fig. 10. In theses plots, the sensory response of each VPM ensemble for a single cutaneous stimulus (i.e., 1 trial) was depicted as a point whose X-Y position on the plane of the figure was defined by the canonical scores (Y1 and Y2) for roots 1 and 2 (see METHODS). For each ensemble, these roots defined the space that was optimal for differentiating between the neuronal ensemble responses obtained by stimulating the three cutaneous sites. For the normal VPM ensemble represented in Fig. 10A, plotting the Y1 and Y2 values for all training and testing trials revealed three clearly segregated clusters of trials. This demonstrated that the discriminant functions (or roots), formed by linear combinations of the variables depicting the neuronal ensemble activity, correctly classified a large percentage of the single trials depicting mechanical stimulation of one of the three possible sites: whiskers E1 (blue circles), E5 (red squares), or
(green triangle). On the other hand, Fig. 10B indicates that a much lower level of discrimination was achieved for roots derived from a sensory-deprived ensemble. In this case, plotting of the canonical scores Y1 and Y2 obtained for both training and testing trials revealed a high degree of overlap between trials describing stimulation of the face (blue circles), forepaw (red squares), and contralateral whiskers (green triangles).

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| FIG. 10.
Encoding of stimulus location by normal (A) and deafferented (B) VPM neuronal ensembles as measured by canonical correlation analysis. Scatter plots depict distribution of canonical scores, computed from 2 orthogonal canonical roots (root 1 in X-axis and root 2 in Y-axis), for a series of single trials obtained during stimulation of 3 different body locations. Each canonical score, depicted in these graphs by X- and Y-coordinates of colored symbol, represents the weighted neuronal ensemble response to a single stimulus trial. A: in normal animal, VPM ensemble activity defines well-separated clusters of trials, indicating that a very clear discrimination of stimulus location [whiskers E1 (blue circles), E5 (red squares), and (green triangles)] can be obtained with the use of either training trials (i.e., those used to derive canonical functions) or new trials. B: in neonatally deprived animal, single-trial discrimination of stimulus location [face (blue circles), forepaw (red squares), and ipsilateral whiskers (green triangles)] is clearly less accurate for both training and new trials. The difference in discrimination between the 2 animals is even more relevant when one considers that distance between stimulated whiskers was ~10 mm, whereas stimulated regions in deprived animal were much farther apart.
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An important factor in the use of DA to discriminate stimulus locations is the degree of spatial separation between these locations. In both normal and deprived animals, discrimination accuracy should increase with increasing distance between sites. The DA performed here, therefore, was strongly biased against finding significant differences between the normal and deprived animals. Our strategy (see METHODS) for standardizing the spacing of the stimulation sites was to choose them so as to triangulate the central areas of the composite RFs of the different recorded neuronal ensembles. In the normal ensembles, the composite RF centers tended to cover only the whisker field, and thus the three stimulus targets were always on whiskers around the edge of this field. In the deprived thalamic networks, the composite RFs and thus the three stimulus sites typically extended across the entire face and neck and often included the forepaw and/or the contralateral whisker field. In the normal thalamus such a wide separation of stimulus sites would have markedly increased the discrimination accuracy of thalamic ensembles, but in the deprived animals the accuracy of VPM networks was significantly reduced (see Fig. 11).

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| FIG. 11.
Discrimination of testing trials in 3 deprived animals. Notice that when closer targets are considered (A and B), such as nose, upper lip, and face (snout), discrimination of single trials is as deficient as that observed for far-apart skin regions (C), such as face (red squares) and back (green diamonds).
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Because the size of the neuronal ensemble was another variable that could have potentially influenced the percentage of trials correctly classified in both normal and sensory-deprived animals, we investigated how a systematic reduction in the size of normal (Fig. 12, A and C) and sensory-deprived VPM ensembles (Fig. 12, B and D) affected the outcome of the DA. As depicted in Fig. 12A, removal of the best discriminant neuron from a normal VPM ensemble (i.e., that with the highest F-REMOVE value) had little effect on the classification of trials given that three well-defined clusters could still be defined by plotting the pairs of canonical scores for a sample of testing trials (Fig. 12A). In fact, reducing the size of the ensemble from 17 to 12 neurons led to a 10% reduction in the number of trials correctly discriminated (Fig. 12C). Further reduction, however, produced a linear decrease in trial discrimination. Thus when the network was restricted to four neurons only 50% of the trials were correctly discriminated; with two neurons the discrimination of groups was close to random (33%). The effect of size on discrimination performance was similar for training (Fig. 12C,
) and testing trials (Fig. 12C,
) in normal animals. A similar reduction in size, however, considerably affected the discrimination ability of an ensemble of sensory-deprived VPM neurons (Fig. 12, B and D). On the basis of the F-REMOVE value distribution (Fig. 7A) of this ensemble, we removed neuron 5 from the population and then performed DA on the same set of training and testing trials. The scatter plot in Fig. 12B illustrates the considerable reduction (almost 20%) in the number of correctly discriminated trials that resulted from the removal of this single neuron. Moreover, Fig. 12D shows that further size reduction produced a linear decrease in trial discrimination that was more pronounced for testing trials (
) than for training trials (
). Thus only 40% of the trials were correctly classified when the network was reduced to seven neurons; random discrimination was reached when the network was reduced to two to four neurons. Figure 13 illustrates how reducing the size of VPM ensembles affected the classification of training trials in three normal (Fig. 13A) and four sensory-deprived (Fig. 13B) animals. In normal animals, sizable reductions in correct trial classification (<80%) were noticed only after VPM ensembles were reduced to <12 neurons. Subsequent size reduction of these normal ensembles led to a linear decay in the number of correctly classified trials which averaged 4.2% per neuron removed. On the other hand, all sensory-deprived VPM ensembles, including those with up to 20 neurons, displayed considerable reductions in trial classification when the highest predictor neurons were removed (Fig. 13B). Once these neurons were removed, subsequent size reduction resulted in a linear decay in trial classification that averaged 2.57% per neuron removed.

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| FIG. 12.
Removal of neurons from original neuronal ensemble reduced the percentage of trials correctly classified by discriminant analysis in both normal (A and C) and deprived animals (B and D). The reduction was more prominent in deprived animals (D) than in normal animals (C) when the neuron with highest F-REMOVE was removed from ensemble. Thus, although 3 clear clusters could still be observed in normal animals (A) after removal of the best discriminanting neuron, no clear discrimination between groups was evident in deprived animal (B) after similar manipulation. In addition, reduction of neuronal ensembles in deprived animals affected the classification of testing trials ( ) much more than training trials (D, ). In normal animals, on the other hand, training and testing trials were similarly affected by removing neurons from original ensemble. Neurons were removed, 1 at time, according to their F-REMOVE value.
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| FIG. 13.
Trial classification as a function of neuronal ensemble size for a sample of 3 normal (A) and 4 deprived animals (B). A: notice that in normal animals, minimal reduction in percentage of correct trials is observed when individual neurons are removed from ensembles containing 12-18 neurons. Subsequent reductions in ensemble size led to linear decrease in percentage of correct trials. B: in deprived animals, removal of individual neurons from ensembles containing 12-20 neurons may lead to dramatic reductions in ability to correctly classify the location of a tactile stimulus. Further reduction of these ensembles also led to linear decrease in number of correct trials.
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DISCUSSION |
The results of this study demonstrate that a neonatal sensory deafferentation induces a long-term plastic reorganization of the rat VPM that is characterized by 1) an increase in minimal response latencies to discrete stimulation of the deafferented skin; 2) prevalence of slowly adapting responses followed by reverberant discharges, in contrast to the rapidly adapting responses characteristic of normal VPM neurons; 3) existence of abnormally large face-body RFs; and 4) a reduction in the statistical ability to discriminate the locations of tactile stimuli with the use of measurements of poststimulus spiking activity in populations of sensory-deprived VPM neurons.
Overall, these results extend the findings from previous reports (Nicolelis et al. 1991
; Verley and Onnen 1981
), in which classical mapping techniques were used to describe the effects of neonatal whisker removal in the VPM, by demonstrating that the effects of the reorganization process in the VPM are not limited to the changes in receptive field size. Instead, they include a spectrum of functional modifications at the level of single neurons. Furthermore, the present study provides, for the first time, a quantitative description of the effects of a peripheral sensory deafferentation on the encoding of sensory information by populations of VPM neurons. By doing so, this study raises the interesting hypothesis that the altered representation of sensory information induced by the process of plastic reorganization during development or even during adulthood may account for a significant loss in perceptual performance. Finally, these results strengthen the hypothesis that substantial plastic reorganization can occur at subcortical levels of mammalian sensory systems, both at short (Nicolelis et al. 1993b
) and long (Garraghty and Kaas 1991
) time periods after deafferentation. Indeed, recent data obtained in primates subjected to therapeutic amputations suggest that sensory reorganization occurring at the brain stem level may have a profound impact on thalamic and cortical plasticity (Florence and Kaas 1995
).
Damage to whisker follicles or their sensory innervation is known to induce profound alterations in the organization of the cytochrome-oxidase-rich clusters of neurons in the VPM, also known as barreloids (Killackey and Shinder 1981
; Woolsey et al. 1979
). Most of the information concerning developmental reorganization in the VPM has been obtained in adult animals in which a neonatal sensory deafferentation was produced by partial or complete sections of the infraorbital (IO) nerve. Neonatal transection of the IO nerve disrupts the organization of thalamocortical terminals originating in the VPM (Jensen and Killackey 1987
) and tends to produce important degeneration in the nucleus (Chiaia et al. 1992
). A significant increase in dendritic length, area, and volume has also been observed in the VPM following IO nerve section (Chiaia et al. 1992
). Interestingly, neonatal whisker cauterization in mice has been reported to induce the opposite effect, that is, an increase in cell density in the VPM (Hamori et al. 1986
). The reasons for this discrepancy are not known.
Previous studies have demonstrated that sensory deafferentation of the rat face, produced by sectioning the IO nerve, leads to profound modifications in the peripheral innervation of the whisker pad (Renehan and Munger 1986a
,b
). These modifications include a considerable increase in the density of unmyelinated fibers and a reduction in the innervation of Merkel disks by myelinated fibers (Renehan and Munger 1986b
). Physiologically, this proliferation of unmyelinated fibers may account for the remarkable increase in the percentage of ganglion cells responding to noxious stimuli observed in rats deafferented at birth or during adulthood (Renehan et al. 1989
). It is also conceivable that the longer than normal latencies observed by us in the VPM of deafferented rats (see also Chiaia et al. 1992
) following the neonatal cauterization of whisker follicles resulted, at least in part, from an increase in the density of unmyelinated fibers paralleled by a loss of large myelinated fibers. It is interesting to note that marked reductions in conduction velocity in peripheral nerves are also present in patients suffering from median nerve damage (Hallin et al. 1981
). In addition, an increase in sensory response latency is observed in the somatosensory cortex of amputees (Drechsler and Schrappe 1981
), suggesting an impairment of the fast-conducting lemniscal pathway.
Whereas modifications in peripheral skin innervation may account for changes in thalamic response latency, it is likely that plastic reorganization within the VPM or in its afferent structures is responsible for the alterations in adaptation rate, firing pattern, and RF size observed in deafferented animals. Altogether, the predominance of slowly adapting responses, long-lasting reverberant firing, and large overlapping face-body RFs are all consistent with a reduction in the magnitude of
-aminobutyric acid (GABA)-mediated postexcitatory inhibition in the VPM. In fact, neonatal whisker removal in mice and rats has been reported to induce a reduction in GABA immunoreactivity in the primary somatosensory cortex (Akhtar and Land 1991
; Kossut et al. 1991
). Likewise, dorsal rhizotomy in primates was found to trigger a remarkable reduction in GABA receptors in the somatosensory thalamus (Rausell et al. 1992
). Unlike the primate ventral posterior complex, however, the rat VPM has no GABAergic interneurons (Harris 1986
). Thus all postexcitatory inhibition observed in this thalamic nucleus seems to be derived from GABAergic neurons located in the reticular nucleus (RT) of the thalamus (Lee et al. 1994a
). RT neurons receive collaterals from both thalamocortical projections originating in the VPM and from corticothalamic fibers derived from cortical neurons located in the infragranular layers of the somatosensory cortex (Chmielowska et al. 1989
). Recent studies have shown that in normal adult animals acute lesions of RT neurons produced a 3.2-fold increase in RF size of VPM neurons (Lee et al. 1994a
,b
). These changes in RF size began immediately and lasted for
1 mo after the RT lesion. Large VPM RFs, however, depended on the integrity of the subnucleus interpolaris of the trigeminal brain stem complex (SiV), which contains neurons that send projections to the VPM (Lee et al. 1994a
). Thus additional destruction of the SiV led to a dramatic reduction in RF size in the VPM (Lee et al. 1994a
). In addition, after the RT lesion virtually all VPM neurons exhibited sustained responses for the duration of the tactile stimulus (Lee et al. 1994a
).
The results of Lee et al. (1994a
,b
) suggest that some of the effects observed in the deafferented VPM may have resulted from a deficit in the GABA-mediated inhibition provided by RT neurons to the VPM. This could lead to an enhancement of the influence of paralemniscal projections, such as the ones derived from the SiV nucleus, to VPM neurons. SiV neurons have longer minimal response latencies and larger facial RFs than neurons in the principal nucleus of the trigeminal complex (PrV) (Nicolelis et al. 1995
), the main source of the trigeminal lemniscal pathway. Thus the enhancement of paralemniscal projections to the VPM could constitute one of the end products of the reorganization process triggered by the neonatal sensory deafferentation.
Further support for an important role played by the RT in the process of plastic reorganization of the thalamus was obtained recently by our observation that a neonatal sensory deafferentation induces a profound downregulation of parvalbumin (PV), a calcium binding protein, in the rat RT (Nicolelis, unpublished observations). Even though the physiological effects of such PV reduction remains unknown, one would predict that it may indicate a reduction in RT activity. It is important to emphasize that PV was also reduced in the VPM, a finding that confirms previous observations in the ventral posterior lateral nucleus of primates subjected to an extensive dorsal rhizotomy (Rausell et al. 1992
). It is not clear, however, whether this reduction of PV in the VPM was due to a reduction of RT terminals in the VPM, a reduction of terminals from the PrV that are known to contain PV (Bennett-Clarke et al. 1992
), or a reduction in PV intrinsic to VPM neurons.
An enhancement of the SiV-VPM pathway attributable to or combined with a reduction in the RT-mediated inhibition of VPM firing would account for most of the findings observed here, except for the presence of overlapping face-body RFs in the whisker-deprived VPM. To account for this observation one must assume that deprived VPM neurons receive aberrant inputs from the medial lemniscus or the spinal thalamic tract, which are normally destined for the ventral posterior lateral nucleus. Another possibility is that the face-body overlap occurred at the brain stem level and that brain stem afferents were responsible for the genesis of the large face-body RFs. Further experiments are required to elucidate this question.
It has always been difficult to estimate how changes in physiological recordings at the single-unit level might affect perceptual functions such as the localization of sensory stimuli. Nevertheless, the present use of many single-neuron recordings in combination with multivariate statistical techniques may shed light on this important issue by revealing the information that is available in the neuronal ensemble. Multivariate techniques, such as principal component analysis and DA, have recently been introduced in the study of neuronal ensemble data (Deadwyler et al. 1996
; Nicolelis et al. 1995
). In the present study, DA allowed us to quantify the impact of sensory plasticity on the function of populations of thalamic neurons. The results of our population analysis show that the information transmitted to the cortex from the deprived VPM represents a larger than normal cutaneous region, and that the spatiotemporal patterns of poststimulus activity in VPM neuron ensembles in deprived animals are much less specific than normal in determining the precise spatial location of a peripheral stimulus on a single-trial basis.
These results also show that information useful for defining the location of a stimulus tended to be fairly evenly distributed among the neurons of the ensembles recorded in normal animals (i.e., the neurons had relatively uniform F-REMOVE values), but that in the deprived ensembles this information tended to be segregated within a relatively small pool of neurons. Moreover, these tended to be neurons whose RF centers were outside the original deprived area, even though their surrounds did include this region. The locations of our electrode implants suggested that the recorded neurons would normally have been well within the whisker representation. One may conclude, therefore, that the neurons with near-normal discriminative ability (i.e., significant F-REMOVE values) were those that received sufficient sensory input from adjacent undamaged regions. Nonetheless, the combined contribution of the remaining neurons (with RF centered mainly within the deprived area) was often capable of compensating for the loss of a few high predictor neurons. The major difference between the high and low predictor neurons is that ensembles of the latter required more elements to make a correct discrimination.
Even though in this study we have not characterized the perceptual performance of neonatally deafferented animals, it is interesting to note that our results are in many respects similar to or consistent with physiological and psychophysical phenomena observed in human patients suffering from chronic pain following peripheral deafferentation. For example, the abnormal functional properties of VPM neurons in the deafferented rat are virtually identical to the aberrant thalamic firing patterns observed in the human patients (Gorecki et al. 1989
; Lenz et al. 1989
). In these patients, thalamic neurons exhibited a considerable increase of bursting activity, presumably mediated by calcium spikes (Lenz et al. 1989
), and very large RFs (Gorecki et al. 1989
). In addition, allodynia, a common complaint in amputees, could be elicited by microstimulation of the deafferented thalamus (Gorecki et al. 1989
).
Recent psychophysical evidence indicates that some patients with phantom limb sensation experience transitory difficulties in defining the precise location of cutaneous stimuli (Ramachandran 1993
). Immediately after a stimulus is delivered to a restricted region of the face, these patients report being touched in the face, but after a few seconds they insist that the stimulus was also felt in the ipsilateral phantom limb. It has been proposed (Ramachandran 1993
) that the most likely reason for this ambiguity in stimulus location is the extensive cortical reorganization, originally documented in deafferented primates (Merzenich et al. 1983a
,b
), that follows the long-term recovery from a limb amputation. Because this reorganization may involve, after long recovery periods, a considerable expansion of the cortical representation of the face (Pons et al. 1991
) at the expense of territories usually devoted to the representation of the upper limbs, it has been suggested that concomitant plastic modifications must also take place in the thalamus or even in the brain stem (Florence and Kaas 1995
; Pons et al. 1991
). Our results support this hypothesis by demonstrating that the profound plastic reorganization that occurs in the thalamus after a peripheral deafferentation can impair the ability of thalamic ensembles to predict stimulus location unambiguously. Neural ensemble analysis suggests that the basis of this deficit lies in alterations in the spatiotemporal encoding of tactile information in the VPM. Because these alterations are likely to reflect highly distributed modifications in the somatosensory cortex and in subcortical structures that project to the thalamus, further understanding of these effects will likely require simultaneous recordings at multiple processing levels (i.e., brain stem, thalamus, and cortex) of the trigeminal somatosensory system (Nicolelis et al. 1995
). Moreover, the presence of potential perceptual deficits will have to be investigated in the same pool of animals to test the hypothesis that the functional modifications induced by the process of plastic reorganization can account for an eventual impairment in performance in tactile discrimination tasks.
Even though difficulties in discriminating stimulus location seem to be more common in patients soon after the amputation, our data suggest that abnormal tactile perception might be expected to persist for years after a peripheral deafferentation. In fact, one of the predictions that emerged from this study is that rats may experience altered tactile sensations long after the sensory deafferentation. Thus we find it conceivable that long-lasting effects of the deafferentation-induced plastic reorganization of the somatosensory system could account for the chronic pain and other lingering changes in tactile perception reported in a large number of amputees (Katz and Melzack 1990
).
 |
ACKNOWLEDGEMENTS |
The authors thank Dr. Laura M. O. Oliveira, L. Andrews, H. Wiggins, and A. Kirillov for excellent technical assistance and A. Ghazanfar and K. T. Nguyen for comments on the manuscript.
This work was supported by National Institutes of Health Grants DE-11121-01 (to M.A.L. Nicolelis) and NS-26722 (to J. K. Chapin), the Whitehall Foundation and a Whitehead Scholar Award to M.A.L. Nicolelis, and Office of Naval Research Grant N00014-95-1-0246 to J. K. Chapin.
 |
FOOTNOTES |
Address for reprint requests: M.A.L. Nicolelis, Dept. of Neurobiology, Box 3209, Duke University Medical Center, Bryan Research Building, Durham, NC 27710.
Received 7 November 1996; accepted in final form 1 May 1997.
 |
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