Experience-dependent Plasticity of Rat Barrel Cortex: Redistribution of Activity across Barrel-columns

Mikhail A. Lebedev1,3, Giovanni Mirabella1, Irina Erchova1 and Mathew E. Diamond1,2

1 Cognitive Neuroscience Sector, International School for Advanced Studies, Via Beirut 9, 34014 Trieste and , 2 Department of Biomedical Sciences and Technologies, University of Udine, Via Gervasutta 48, 33100 Udine, Italy


    Abstract
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 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The redistribution of neuronal activity across rat barrel cortex following an alteration in whisker usage has been investigated. In adult rats, two mystacial vibrissae – D2 and one neighbor, D1 or D3 – were left intact while all other vibrissae on that side of the snout were clipped. Neurons in contralateral barrel cortex were sampled with a microelectrode array 3.5 days later. Stimulation of clipped vibrissae produced a narrow spatial distribution of cortical activity, whereas stimulation of intact vibrissae produced a widened spatial distribution. Simultaneous recordings from multiple cortical barrel-columns suggest that changes in the effective connectivity between barrel-columns may partially account for this redistribution of sensory responses. Evidence is also presented for a second mechanism, a release from inhibition in sensory-deprived cortical areas. A model is therefore proposed where these two mechanisms operate together to regulate the cortical distribution of evoked activity.


    Introduction
 Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Changes in sensory experience bring about a transformation in the way primary somatosensory cortex processes information from the peripheral receptors (Kaas, 1991Go). Somatosensory cortical plasticity is believed to be an essential element in improved tactile discrimination following behavioral training (Jenkins et al., 1990Go), tactile learning underlying Braille reading (Pascual-Leone and Torres, 1993Go) and in maladaptive changes such as phantom limb pain (Flor et al., 1995Go). The present study concerns the mechanisms of the somatosensory cortical plasticity produced by an innocuous shift in the usage of peripheral sensors.

The rat vibrissal system is an advantageous experimental model for several reasons. First, each cortical barrel-column is a morphological unit topographically related to a single principal whisker on the contralateral snout (Woolsey and Van der Loos, 1970Go; Welker, 1971Go; Simons, 1978Go). This arrangement provides a standard reference frame for detecting experience-dependent changes in cortical functional organization. Second, the usage of peripheral sensors can be altered simply by clipping the vibrissae, a manipulation not unlike the natural shedding of vibrissae (Ibrahim and Wright, 1975Go). Third, during the recording session the remaining part of a clipped whisker can be stimulated, allowing the experimenter to monitor the cortical response to both used and disused receptors.

In this study, we have conducted multielectrode recordings from rat barrel cortex following a brief period of whisker clipping. Our goals were: (i) to evaluate experience-dependent changes in spatial distribution across barrel cortex of peripherally evoked activity; and (ii) to determine whether such changes are accompanied by alterations in the correlated firing of neurons from different barrel-columns, and by alterations in the excitatory–inhibitory equilibrium of cortex. The evidence points to an interaction between two mechanisms: modification of excitatory connections within and between barrel-columns, and changes in intracolumnar inhibition.


    Materials and Methods
 Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Subjects and Manipulation of Tactile Experience

Adult male Wistar rats weighing ~350 g were used. In 14 subjects, two D-row vibrissae on the right side were ‘paired’, i.e. they were left intact while the other mystacial vibrissae on the same side were clipped close to the skin (Fig. 1A,BGo). These whisker-paired subjects were compared to 22 control subjects with all whiskers left intact. During the 3.5 day period subsequent to whisker clipping, the whisker-paired subjects, like the control subjects, were housed with littermates in a standard cage. We observed that whisker-paired subjects continued to utilize the remaining whiskers to explore their surroundings.



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Figure 1.  Altered sensory experience and subsequent sampling of cortical neural activity. (A) Whisker-pairing procedure. On the right side, all whiskers except two were clipped. All whiskers on the left side of the snout were left intact. (B) The ‘paired’ whiskers were D2 and one neighbor, either D1 or D3. (C) Three and a half days following the clipping of the whiskers, neurons in cortical barrel-columns D1, D2 and D3 (and occasionally the septa and adjacent barrel-columns) were sampled with an array of six microelectrodes. In off-line analysis, cortical single-units were discriminated based on waveform shape. For electrode 6, typical waveforms discriminated from real neural data are shown for two single-units.

 
Animal Preparation and Histology

Anesthesia was induced by urethane (1.5 g/kg body wt, i.p.). The subject was placed in a Narashige stereotaxic apparatus. The left somatosensory cortex was exposed by a 4 mm diameter craniotomy. During the recording session body temperature was maintained at 37.5°C and anesthesia was held at a depth characterized by the absence of withdrawal after a pinch applied to the forelimb; corneal and eyelid reflexes were present (Erchova et al., 1998Go). Respiratory rate was 85–130 breaths/min. Dominant ECoG frequency was 0.5–8 Hz in this state, in agreement with previous research (Kubicki and Rieger, 1968Go). All protocols adhered to NIH and local standards.

At the end of the experiment, subjects were perfused with saline followed by 4% paraformaldehyde. After postfixation in 20% sucrose, a flattened slab of neocortex was frozen, cut in 40 µm tangential sections and processed to label nitric oxide synthetase activity (Valtschanoff et al., 1993Go) in order to visualize barrel-columns in layer IV. To determine the columnar location of sampled neurons, electrode penetration sites were identified relative to the histological sections.

Data Acquisition

An array of six tungsten microelectrodes with 300 ± 50 µm separation between adjacent electrode tips was advanced into the cortical barrel field. Neurons in barrel-column D2 and surrounding D-row barrel-columns were sampled (Fig. 1CGo). The whole array was advanced in 100 µm steps and in every rat efforts were made to collect recording sites throughout the depth of cortex. Neuronal activity was amplified and band-pass filtered in the range 300–7500 Hz. Action potentials were digitized at 25 KHz, 32 points per waveform (Datawave Discovery, Boulder, CO) and stored on a Pentium PC for subsequent analyses. Comparisons between cortical layers (Diamond et al., 1994Go) are not reported here because the depth of the individual electrodes of the array could not be determined with a precision >200–300 µm

Vibrissal Stimulation

The short 1–2 mm hairs located between vibrissae were cut to prevent their incidental stimulation. The paired whiskers, 30–35 mm long, and the clipped whiskers, which had regrown to a length of ~4–5 mm, were all trimmed to a 3 mm length. Thus, test stimuli were equal for all whiskers, whether previously clipped or paired. Individual whiskers were deflected by a metal hook glued to a piezoelectric ceramic bimorph wafer (Morgan Matroc, Bedford, OH) positioned just below the whisker shaft, 2 mm from the skin. The stimulus was an up–down step function of 80 µm amplitude and 100 ms duration, delivered once per second 50 times for each vibrissa. The stimulated vibrissae were C1–3, D1–3, E1–3, gamma and delta. In 19 experimental subjects whisker D4 was also stimulated. Beginning 1 min after conclusion of whisker stimulation, neural data were acquired for a period of 3 min in the absence of any stimulus. This ‘background activity’ was utilized to construct neuronal cross-correlograms (see below).

Data Analysis

Single-unit action potentials were discriminated using a template-matching algorithm implemented by us in MATLAB (Mathworks, Inc, Natwick, MA). The algorithm separated waveforms by the differences in their shape and amplitude (Fig. 1CGo). Typically, one or two single units per electrode were discriminated. Classified action potentials were time-stamped with 0.1 ms resolution.

To calculate evoked response magnitude, the number of spikes occurring during the 100 ms duration of the stimulus was adjusted for ‘background’ activity by subtracting the number of spikes in the 100 ms interval preceding the stimulus. Response magnitudes for this 0–100 ms interval were compared by the appropriate non-parametric statistics. For multi-group comparisons, a Kruskal–Wallis one-way analysis of variance (ANOVA) on ranks was used. If statistically significant differences were found ({alpha} = 0.05), Dunn's pairwise multiple comparison procedure was used to identify those groups that differed from the others.

The next step was to characterize the functional connectivity between neuron pairs using cross-correlations (Palm et al., 1988Go; Eggermont 1992Go). Data were evaluated for all ‘triplets’, i.e. for simultaneous recordings from three single-units in barrel-columns D1, D2 and D3. For each neuron pair in a triplet, a cross-correlation histogram (CCH) for the 3 min period of background activity was constructed with 5 ms bins extending 200 bins before and after the central bin. To allow comparison of CCHs across neuron pairs with different firing rates, CCHs were normalized such that their scaling corresponded to the correlation coefficient (Palm et al., 1988Go; Aertsen et al., 1989Go; Eggermont, 1992Go).

All CCHs contained a clear central peak. The magnitude of the peak was taken to be the average value of the bin comprising the CCH maximum and the two bins to the left and the two bins to right of the maximum. Utilizing the peak magnitudes, a cross-correlation ‘bias’ was calculated for each triplet, according to equation (1):

(1)
where MD1D2 and MD2D3 are the magnitudes of the CCH peaks constructed from the D1–D2 and D2–D3 neuron pairs respectively. The bias can range from –100% to +100%, where a positive value indicates a stronger connectivity between the D1–D2 neuron pair than between the D2–D3 neuron pair, and a negative values indicates a stronger connectivity between the D2–D3 neuron pair.


    Results
 Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The main finding is that the 3.5 day period of whisker pairing altered the vibrissal-evoked activity in both deprived and non-deprived barrel-columns, as well as the functional connectivity between deprived and non-deprived barrel-columns. These results will be presented through (i) examination of the distribution of activity across multiple cortical barrel-columns evoked by stimulation of single-whiskers, (ii) analysis of the correlation between the activity of neurons from different barrel-columns, and (iii) assessment of inputs to deprived barrel-columns from disused receptors.

Distribution of Activity Evoked across Multiple Cortical Barrel-columns

Because multielectrode arrays were employed, we were able to sample single-unit activity from several barrel-columns in each subject. Two representative examples are illustrated (Fig. 2Go) a normal rat (n. 39) and a whisker-paired rat (n. 40). The electrode array was inserted to sample neurons in cortical barrel-columns D1–D4 and in the intervening septa (Fig. 2AGo). Peristimulus time histograms (PSTHs) representing responses to stimulation of vibrissae D1–D4 were calculated for each sampled single unit. Then, for each electrode track, the PSTHs were averaged across units to gain a representative sample of the cortical column. These average PSTHs are plotted as grayscale-coded horizontal stripes (Fig. 2BGo). To show the distribution of activity, the average PSTHs from different electrodes are aligned one below the other. Beneath the average PSTHs, the total evoked response recorded at each electrode is shown as a bar plot (Fig. 2CGo). These analyses show that in the normal rat stimulation of one whisker evoked a strong response in its associated barrel-column and the septum surrounding that barrel-column, and a weaker response in neighboring barrel-columns. In the illustrated control subject, the response in barrel-column D1 to stimulation of whisker D1 (the principal whisker response) exceeded the principal whisker responses in barrel-columns D2–D4. This observation reflects a general tendency for the principal whisker responses to be stronger for caudal than for rostral barrel-columns, as noted later.



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Figure 2.  Distribution of sensory-evoked activity in a normal and a D2–D3 paired rat. (A) Histologically reconstructed locations of the six electrode penetrations. Each electrode is represented by a black circle. The electrode numbers (1–6) in (B) refer to these sites, in increasing order from the left to the right. (B) Grayscale-coded population-average PSTHs for each of the electrodes for stimulation of vibrissa D1, D2, D3 and D4. (C) Average responses corresponding to PSTHs in (B) represented as bar plots.

 
The distribution of activity is different in the representative case in which whiskers D2–D3 were paired (Fig. 2Go). Stimulation of a clipped whisker (D1 or D4) evoked a strong response in its associated barrel-column and the septum surrounding that barrel-column, and a weaker response in non-deprived barrel-columns (D2 and D3). In contrast, stimulation of a paired whisker (D2 or D3) evoked a strong response in its associated barrel-column, in the barrel-column of the neighboring paired whisker, in the nearby septa, as well as in ‘deprived’ barrel-columns (D1 and D4). Thus, stimulation of clipped whiskers produced a spatial distribution of neuronal activity similar to that produced by intact whiskers in the normal case, whereas stimulation of paired whiskers produced a wider spatial distribution than in normal circumstances.

The distributions of activity across barrel columns in the two cases illustrated above were similar to those found by averaging the data from all cases for the two experimental conditions (subsequent section). Therefore the selected data, though limited, do not appear to reflect any bias in data collection.

To create a more general characterization of the experience-dependent changes in the distribution of cortical activity, we summed the responses of the neurons across all experimental subjects of a given condition. Single units recorded in the same barrel-column (D1, D2 or D3) were grouped together, while units located in the intercolumnar septa were excluded from the analysis. In control subjects, 83 neurons were recorded in barrel-column D1, 137 neurons in barrel-column D2 and 90 neurons in barrel-column D3. In Figure 3AGo, the average PSTHs are shown for these normal subjects in the interval –20 to 100 ms relative to the onset of stimulation (0 ms) of vibrissae D1, D2 and D3. The average response magnitude is given at the end of each trace. Note the short response latency (~7 ms) and high firing rate after the stimulation of the principal whisker of each barrel-column. After stimulation of the principal whisker of the adjacent barrel-column, responses occurred at a longer latency (~10 ms) and were weaker. Stimulation of the whisker located two steps from the principal whisker of a barrel-column produced a very weak, long-latency response.



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Figure 3.  Distribution of sensory-evoked activity in barrel-columns D1, D2 and D3 of (A) normal rats, (B) D1–D2 paired rats and (C) D2–D3 paired rats. Population-average PSTHs are presented as grayscale-coded plots. From left to right, responses to stimulation of whiskers D1, D2 and D3 are shown. On the right of each grayscale-coded PSTH, the corresponding average response is presented.

 
Asymmetries in the cortical representations of evoked activity were noted for control subjects as a function of the rostral– caudal position of the whisker. Principal whisker responses were strongest for barrel-column D1, weakest for barrel-column D3 and intermediate for barrel-column D2, consistent with the example presented in Figure 2Go. However, the difference did not reach statistical significance (ANOVA on ranks, P > 0.05). This suggests, therefore, that principal whisker inputs may be stronger in caudal barrel-columns than in rostral barrel-columns; confirming this tendency will require recordings from a more widely distributed array of electrodes.

Average response analysis in whisker-paired subjects was conducted separately for rats with whiskers D1–D2 paired (Fig. 3BGo) and those with whiskers D2–D3 paired (Fig. 3CGo). For D1–D2 paired rats, 37, 77 and 45 neurons were recorded in barrel-columns D1, D2 and D3 respectively. Stimulation of vibrissa D1 produced above-normal responses in barrel-column D2 (the ‘paired’ neighbor) (P < 0.001) and barrel-column D3 (‘deprived’ barrel-column) (P < 0.001). Stimulation of vibrissa D2 produced an increased response in both barrel-column D1 (the paired neighbor) (P < 0.001) and in barrel-column D3 (P = 0.002). Stimulation of the clipped whisker, D3, evoked responses in the paired barrel-columns, D1 and D2, that did not differ significantly from the controls (P > 0.1).

In D2–D3 paired rats, 37, 103 and 112 neurons were sampled in barrel-columns D1, D2 and D3 respectively. The changes in the distribution of responses were analogous to those found in D1–D2 paired subjects. Stimulation of vibrissa D3 produced an increased average response in the ‘paired’ barrel-column D2 (P = 0.002) (Fig. 3CGo). There was also an increased response in the deprived barrel-column, D1, but this did not reach statistical significance (P = 0.07). Stimulation of vibrissa D2 produced an increased average response in both the ‘paired’ barrel-column D3 (P <0.001) and the ‘deprived’ barrel-column D1, but the increased response in the latter column did not reach statistical significance (P = 0.09). Responses in barrel-columns D2 and D3 during the stimulation of the clipped whisker, D1, were not significantly different from the control values (P > 0.1 for both cases).

These experience-dependent changes indicated an expansion of the representation of the two intact vibrissae into the neighboring barrel-columns. The representation of the clipped whiskers in the neighboring ‘paired’ barrel-columns was unaltered compared to their representation under normal conditions. This latter result is consistent with the results of previous studies in which responses in one ‘paired’ barrel-column, D2, were examined after 3 days of whisker pairing (Diamond et al., 1993Go; Armstrong-James et al., 1994Go). These studies showed that the responsiveness of barrel-column D2 neurons to the stimulation of the intact neighboring whisker was increased, while the responsiveness to the stimulation of the clipped, neighboring whisker was unchanged.

The short-latency (<10 ms after stimulus onset) component of whisker-evoked response can be used to evaluate whether plastic changes occurred at subcortical levels (Diamond et al., 1993Go; Armstrong-James et al., 1994Go). Cortical events evoked at short latency (0–10 ms after stimulus onset) are related mainly to the thalamocortical volley (Armstrong-James and Callahan, 1991Go). If the increased responses of cortical neurons to the paired, neighboring whisker were due to changes in the response properties of the thalamic neurons projecting to the cortical barrel-column, or to changes in the thalamocortical synapse, one would expect some shift in these short latency responses. For normal control subjects stimulation of adjacent D-row whiskers produced a very small 0–10 ms response (on average, 2 spikes/100 stimuli) accounting for 5% of the total (0–100 ms) response. In all experimental conditions, the short-latency response to paired neighboring D-row whiskers was unchanged. Therefore, this analysis did not provide evidence of subcortical or thalamocortical modifications. All changes in response level occurred in the longer latency bin of 10–100 ms.

Experience-dependent Changes in the Functional Connectivity between Barrel-columns

As a second approach to detecting the intracortical contribution to experience-dependent plasticity, we estimated the functional connectivity between neurons located in different barrel-columns by measuring their coincident firing during 3 min periods of spontaneous activity. Whenever a neuronal ‘triplet’ was sampled (i.e. simultaneous recording of neurons located in barrel-columns D1, D2 and D3), CCHs were computed between the D1–D2 neuron pair and the D2–D3 neuron pair. The ‘bias’ in functional connectivity within the triplet was then computed (see Materials and Methods). We reasoned that if the redistribution of sensory-evoked responses described in the previous section were due to the facilitated transmission of signals between the two ‘paired’ barrel-columns, then we might detect a heightened functional connectivity between these barrel-columns, as compared to that between ‘unpaired’ barrel-columns.

In control subjects, there was no overall difference in spontaneous activity cross-correlation between barrel-columns D1–D2 and D2–D3. Consistent with our expectation, changes in sensory experience caused shifts in the intercolumnar balance. Representative CCHs are shown for a neuronal triplet recorded in a rat with whiskers D1–D2 paired (Fig. 4Go). The magnitude of the CCH was higher for the D1–D2 neuronal pair, and the bias was 22.9%. For all triplets, the proportions with different connectivity biases in relation to sensory experience are given in Table 1Go. It is evident that the pairing of two whiskers caused an increased frequency of connectivity bias in favor of the two ‘paired’ barrel-columns ({chi}2 = 23.2 with 2 degrees of freedom; P < 0.001). The mean values of the bias across all triplets were –1.8% (SEM 2.6%) for control rats, 7.5% (SEM 1.8%) for D1–D2 paired rats and –5.5% (SEM 2.0%) for D2–D3 paired rats. The dependence of the mean value of bias on experimental condition was statistically significant (ANOVA on ranks; P < 0.001). The change in the connectivity bias was greater for D1–D2 paired rats than for D2–D3 paired rats.



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Figure 4.  Example of cross-correlation histograms (CCHs) in a triplet from a D1–D2 paired rat. Three neurons were simultaneously recorded: one in column D1, one in column D2 and one in column D3. The peak in the D1–D2 CCH (left) is larger than that in D2–D3 CCH (right). The value of bias in this case (see text) is greater than the average.

 

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Table 1 Cross-correlation bias after whisker pairing
 
Release of Surround-whisker Responses in Deprived Cortical Barrel-columns

It has been proposed that one mechanism underlying cortical reorganization after peripheral denervation may be a change in the excitation–inhibition balance (Welker et al., 1990Go; Calford and Tweedale, 1991Go; Garraghty et al., 1991Go). In particular, inhibitory function could be down-regulated in sensory-deprived cortical areas, permitting the strengthened expression of sensory inputs that were weak before the manipulation. The present experimental model can be used to evaluate changes in inhibition by measuring the responses in deprived barrel-columns to the stimulation of clipped whiskers. If activity-dependent modifications of excitatory connections were the only plasticity mechanism at play, then the inactive whiskers would be likely to evoke a weakened response in deprived barrel-columns. On the other hand, if inhibition were diminished, there could be a ‘release’ of responses to inputs to sensory-deprived barrel-columns even from clipped whiskers. To evaluate these alternatives, we measured inputs to barrel-columns D1 and D3 from whiskers C1–3 and E1–3 when all whiskers were left intact (normal condition), and when only whiskers D1–D2 or D2–D3 were left intact. The analysis was carried out in the following way. For each neuron in column D1 or D3, the responses to the six vibrissae of interest, C1–3 and E1–3, were ranked according to magnitude. In Figure 5A,BGo, mean ranked responses are shown for neurons in barrel-column D1 and D3 respectively, under the three conditions of sensory experience. No significant differences between the conditions are present in Figure 5AGo (inputs to barrel-column D1), whereas in Figure 5BGo (inputs to barrel-column D3) a statistically significant increase in responses to clipped whiskers emerged when whiskers D1–D2 were paired. The four strongest inputs to deprived barrel-column D3 were significantly greater than were the four strongest inputs to barrel-column D3 in control subjects (P < 0.005). The increase was more prominent for higher-ranked inputs, i.e. strong inputs were ‘released’ to a greater degree than were weak inputs. This is consistent with the fact that potent sensory inputs elicit stronger inhibition in a cortical barrel-column (Moore and Nelson, 1998Go). In addition, when whisker D3 was paired with whisker D2, there was a tendency for the inputs from the clipped vibrissae C1–3 and E1–3 to be slightly suppressed compared to normal. Although this effect was not statistically significant, it may indicate a tendency for disused sensory receptors to be ‘excluded’ from this cortical barrel-column when its principal sensory input was left intact.



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Figure 5.  Ranked responses of neurons in columns D1 (top) and D3 (bottom) to stimulating whiskers 1–3 in C and E rows. Points represent mean values for the responses ranked 1–6, for normal, D1–D2 paired and D2–D3 paired subjects; error bars are SEM. Asterisks denote significantly different values from the control condition (P < 0.005).

 

    Discussion
 Top
 Footnotes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We report that a brief and innocuous change in receptor usage is sufficient to cause a reorganization in the way sensory-evoked activity is distributed across cortex. After two vibrissae were left intact for 3.5 days — all others on that side of the snout clipped — the paired vibrissae became more strongly represented both in deprived and in non-deprived cortical barrel-columns. These experience-dependent changes suggest an expansion of the representation of the two intact vibrissae into the neighboring barrel-columns.

Asymmetries in Normal Barrel Cortex

In our data there was a tendency that suggested asymmetries in the cortical representation of the whiskers, although the size of the studied cortical region was not sufficient to provide a clear demonstration. The caudal whiskers projected more strongly to their corresponding cortical barrel-column than did the rostral whiskers. Earlier evidence, both from electrophysiological data (Simons and Carvell, 1989Go) and from metabolic mapping (Durham and Woolsey, 1985Go; McCasland and Woolsey, 1988Go), have suggested similar asymmetries. The gradients in cortical responsiveness may be related to differences in the way rats utilize their whiskers. Caudal whiskers have been suggested to have a greater role in object localization, and rostral whiskers in analysis of the features of objects (Brecht et al., 1997Go).

A noteworthy observation that emerged from this study is that, generally, pairing of whiskers D1–D2 produced more prominent plastic changes than did pairing of whiskers D2–D3. Although we do not have a satisfactory explanation for the differences between the two whisker-pairing conditions, we emphasize that neurons of D1–D2 paired subjects exhibited a greater degree of plasticity for all three analyses reported here: changes in neuronal responsiveness to D-row whiskers, changes in cross-correlation during spontaneous activity, and changes in responsiveness to Cand E-row whiskers in the deprived barrel-column. This probably indicates that these plasticity effects are correlated phenomena, components of a single complex process.

‘Filling-in’ of Sensory-deprived Cortical Zones

The original reports of cortical plasticity showed that a somato-sensory cortical zone that loses its main sensory input, due to transection of a peripheral nerve, begins to respond to adjacent, intact receptors (Merzenich et al., 1984Go). Later, it was found that an auditory cortical zone deprived by cochlear lesion begins to respond to adjacent, intact receptors (Robertson and Irvine, 1989Go) and a visual cortical zone deprived by retinal lesion begins to respond to adjacent, intact receptors (Chino et al., 1992Go; Gilbert et al., 1996Go). These phenomena of ‘filling-in’ imply that the sensory system begins to utilize the initially ‘silent’ cortical zone to process information from the remaining sensory receptors. In our study, the 2-fold increase in the strength of input from whiskers D2 and its intact neighbor to the deprived barrel-column is analogous to the filling-in of deafferented cortical regions by intact receptors.

However, unlike studies employing a peripheral lesion to deafferent a cortical zone, in the present study the cortical response to the disused receptors was preserved because the sensory pathways from the clipped whiskers were left intact. Since the cortical modification was caused not by damage to the peripheral nerve or receptors, but by an innocuous manipulation similar to the natural shedding of vibrissae, we suggest that during changes in the pattern of sensory receptor usage cortical territories with reduced afferent activity can be rapidly ‘invaded’ by inputs from more active receptors.

Role of Intracortical Synaptic Plasticity

Several lines of evidence suggest that the response plasticity reported here was generated, at least in part, by activity-dependent modifications of excitatory intracortical connections. First, after 24 h of whisker pairing the responses of neurons in layer IV (targets of the primary thalamic pathway) are known to be unaltered, whereas the responses of neurons located above and below layer IV (targets of intercolumnar connections) show significant changes (Diamond et al., 1994Go). This finding argues that intercolumnar connections are particularly sensitive to changes in afferent activity, and could mediate rapid cortical plasticity. Second, intercolumnar connections utilize glutamatergic synapses with postsynaptic NMDA receptors (Armstrong-James et al., 1993Go) and are thus good candidates for activity-dependent modification. The NMDA receptor-mediated postsynaptic potential is slow, allowing integration of multiple inputs over time (Bear et al., 1987Go); moreover, NMDA receptors are Ca2+ channels, and post-synaptic Ca2+ influx is necessary for changes in synaptic strength. Indeed, blockade of NMDA receptors in cortex eliminates whisker-pairing induced plasticity (Rema et al., 1998Go). Third, the absence of any modification in the responses present at short poststimulus latency suggests that thalamocortical signals ascending to barrel cortex are not a major underlying factor. As in previous studies (Diamond et al., 1993Go, 1994Go; Armstrong-James et al., 1994Go), all the increases in response to the two paired whiskers in the present study occurred in the long-latency component of activation (>10 ms after stimulus onset). Under the anesthetic conditions used here, long-latency spikes evoked by a neighboring whisker are relayed almost entirely through the barrel-column of the neighboring whisker (Armstrong-James and Callahan, 1991Go; Armstrong-James et al., 1991Go). Fourth, in parallel with the current report, developmental expansion of the representation of a single intact whisker has been shown to have a major intracortical component (Fox, 1994Go). And finally, the CCHs showed higher effective connectivity between neurons in barrel-column D2 and its paired neighbor than between neurons in barrel-column D2 and its deprived neighbor. This supplements a previous finding of increased functional interaction between ‘paired’ barrel-columns during the response to whisker deflection (Lebedev et al., 1997Go). While the earlier finding of correlated activity between paired barrel-columns during whisker stimulation could reflect a more synchronized level of common ascending input to the two barrel-columns, this explanation is not likely to account for the increased cross-correlation observed during spontaneous activity. To propose that changes in intercolumnar spontaneous activity correlation are due to subcortical inputs, one would be forced to argue that the thalamic barreloids projecting to two barrel-columns are spontaneously active in a more synchronous manner, in spite of the fact that neurons in barreloids have no direct interconnection (Jones, 1985Go). It is simpler to suggest that repetitive co-stimulation of two whiskers, due to the peripheral manipulation, caused the corresponding pair of cortical barrel-columns to be repetitively coactive and to become more strongly connected, presumably through Hebbian mechanisms (Hebb, 1949Go). The degree of strengthening of intracortical connections apparently was sufficient to influence even the propagation of spontaneous activity through cortex.

The evidence summarized above is not intended to argue that subcortical stations cannot contribute to sensory cortical plasticity. Indeed, using the same whisker-pairing paradigm we have found evidence in favor of subcortical plasticity, but only after extended periods of sensory experience (Armstrong-James et al., 1994Go). Rapid response modifications have been detected at subcortical levels after manipulations that damage the peripheral sensory system or unbalance it by silencing one class of fiber (Faggin et al., 1997Go). In contrast, during innocuous shifts in sensory experience, or during sensory learning (Wang et al., 1995Go), the earliest response modifications seem to originate in cortex; cortex itself is reorganized before its ascending inputs.

Changes in the Excitation–Inhibition Equilibrium

If activity-dependent modification of corticortical synapses were the only plasticity mechanism at play, then the inactive whiskers would evoke a weakened response in deprived barrel-columns. In D1–D2 paired subjects, the opposite was true: the clipped vibrissae participated in the ‘invasion’ of the deprived barrel-column, D3. An additional mechanism is required, one that permits a change in cortical responsiveness without requiring a shift in synaptic strength. We suggest that inhibitory circuits are down-regulated in the deprived barrel-columns. Glutamic acid decarboxylase (GAD) expression, reflecting the synthesis of the inhibitory neurotransmitter GABA, is known to be regulated in an activity-dependent manner. In primates, expression of GAD decreases in deafferented cortical regions after nerve cut (Hendry and Jones, 1986Go; Hendry et al., 1990Go). In the barrel cortex, expression of GAD diminishes in sensory-deprived barrels within 3 days after vibrissa removal (Welker et al., 1989bGo), while it gradually increases over the course of weeks in the supragranular layers of those barrel-columns with principal vibrissa intact (Akhtar and Land, 1991Go). GAD expression also increases in the supragranular layers of barrel-columns whose principal whisker is hyperstimulated by continuous vibration (Welker et al., 1989aGo ). However, given that glutamatergic transmission may also be regulated by sensory activity (Carder and Hendry, 1994Go), histochemical changes in inhibitory and excitatory neurotransmitters are difficult to interpret until coupled with physiological studies. In primates, after digit amputation, receptive fields in denervated cortex expand to encompass intact skin regions more rapidly than might be expected if caused by synaptic modification or morphological changes (Calford and Tweedale, 1991Go).

In summary, the disinhibition proposal can account for the increased expression of disused receptors in deprived barrel-columns, a finding that eludes explanation by activity-dependent regulation of NMDA-mediated synapses.

A Unified View of Synaptic Modification and the Excitation–Inhibition Equilibrium

Based on the considerations given above, we argue that disinhibition and synaptic modification act together to regulate the distribution of evoked cortical activity. The Bienenstock, Cooper and Munroe (BCM) theory (Bienenstock et al., 1982Go) provides a flexible conceptual framework for bringing together synaptic modification and changes in the excitation–inhibition balance. The BCM theory asserts that each cortical neuron possesses a dynamic threshold for modification of the synapses it receives; the threshold increases when the neuron is active and decreases when the neuron is inactive. We expect that loss of the principal whisker would greatly reduce the activity level in the deprived barrel-columns and, correspondingly, diminishes the threshold for synaptic potentiation. Synapses bringing information from the active to the deprived barrel-columns would be quickly up-regulated under these circumstances; and disinhibition in the deprived barrel-columns could facilitate synaptic up-regulation by permitting convergent activity to evoke a higher level of postsynaptic activity (Kirkwood and Bear, 1994Go). In active barrel-columns (D2 and its paired neighbor) the synaptic modification threshold would remain sufficiently high to prevent potentiation of input from clipped whiskers (Benuskova et al., 1994Go). The end result is that transmission of information from barrel-columns with spared sensory input into barrel-columns deprived of sensory input would be facilitated, while transmission in the opposite direction would be constrained.


    Notes
 
We are grateful to O. Lebedeva for technical contributions. Supported by NIH grant NS32647, Telethon Foundation grant 984 and the Italian MURST.

Address correspondence to Mathew Diamond, Cognitive Neuroscience Sector, Neuroscience Programme, International School for Advanced Studies, SISSA, Via Beirut 9, 34014 Trieste, Italy. Email: diamond{at}sissa.it.


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3 Current address: Laboratory of Systems Neuroscience, National Institutes of Health Animal Center, PO Box 608, Poolesville, MD 20837-0608, USA Back


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