The effects of peripheral deafferentation on spontaneously bursting neurons in the somatosensory cortex of waking cats

Brain Research 750 Ž1997. 109–121

Research report

The effects of peripheral deafferentation on spontaneously bursting neurons in the somatosensory cortex of waking cats Harry H. Webster b , Iran Salimi a , Alexandre A. Myasnikov a , Robert W. Dykes a

a, )

Departement de Physiologie, Centre de Recherche en Sciences Neurologiques, UniÕersite´ de Montreal, ´ ´ C.P. 6128, Succ. Centre-Õille, Montreal, Que. H3C 3J7, Canada b Centre de Recherche, Hopital du Sacre-Coeur de Montreal, de Psychiatrie, UniÕersite´ de ˆ ´ ´ and Centre de Recherche Fernand-Seguin, Departement ´ Montreal, ´ Montreal, Que., Canada Accepted 29 October 1996

Abstract Single neurons Ž n s 356. were studied in the forelimb representation of awake, quietly resting cats. Thirty-five spontaneously bursting neurons in a sample of 206 cells recorded before forelimb deafferentation were compared to 39 spontaneously bursting neurons in a sample of 127 neurons studied 1–3 weeks after deafferentation. The probability of encountering bursting neurons increased significantly following deafferentation from 17% to 31% of the sample Ž P - 0.005.. The same 5 classes of bursting cells were observed after deafferentation but there were significant changes in the duration of interspike intervals in some classes, in the probability of observing certain classes, and in the proportion of spikes found in bursts. The probability of encountering class III cells, a class thought to consist primarily of non-inactivating pyramidal burst neurons, nearly doubled and the average interspike interval length within the burst increased from 1.9 to 3.0 ms. The burst structure in the other classes did not change but they were found less frequently. These other classes may include inhibitory interneurons which receive less excitatory drive after deafferentation and therefore provide less inhibition to class III cells. The differential behavior of the different classes of bursting cells may be one reason why the overall level of spontaneous activity does not change after deafferentation and it suggests that there are homeostatic mechanisms in primary somatosensory cortex that maintain a certain level of neural activity. Keywords: Cortical reorganization; Neuronal plasticity; Extracellular recording; Single unit activity; Activity pattern

1. Introduction Ongoing neural activity forms a backdrop against which experimenters study neural activity correlated with variables they can manipulate. It is difficult to determine the proportion of ongoing activity that is either of spontaneous Ži.e., arising from uncontrolled variables extrinsic to the cell being studied. or of intrinsic origin Ži.e., arising solely from properties of that cell’s membrane.. Nevertheless, at least some of this activity seems to arise from intrinsic properties of the cell. Early studies of neurons in undercut cat cortex showed that some neuronal activity continued when local circuits were disconnected from other regions of the central nervous system w9x. Later, ongoing neuronal activity was observed in intact cortex where communication among cells had been disrupted pharmacologically by )

Corresponding author. Fax: q1 Ž514. 343-2111.

iontophoretic application of a calcium chelating agent w33,34x. Some neurons in hippocampal slices fall silent, while others generate bursts of action potentials in the presence of blockers of synaptic transmission w14,27x. Intrinsically generated bursts of action potentials form distinctive temporal patterns apparently because of the distinctive membrane properties of particular classes of neurons Žsee refs. in w39x.. In slices of rat and mouse cortex, sustained intracellular depolarizing currents produce characteristic patterns of action potentials in at least three morphologically distinct classes of cells w1,2,11,14,38x. Regular-spiking cells do not generate bursts and are characterized by a train of action potentials which adapts within 50–100 ms to a slower rate. Fast-spiking cells with a very narrow action potential are capable of discharging long bursts of spikes at very high rates whereas intrinsically bursting cells discharge only 2–4 spikes in a burst before undergoing a long hyperpolarization. The first

0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 3 3 8 - 8

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of these discharge patterns has been correlated with a subset of pyramidal cells, the second with gamma-aminobutyric acid-containing ŽGABAergic., non-spiny stellate cells and the last with layer V pyramidal cells w2,49x. Baranyi et al. w6,7x studied these distinctive discharge patterns in the motor cortex of the waking cat. By combining data on intrinsic membrane characteristics with data from antidromic and orthodromic activation of the peduncular pyramidal tract and the ventrolateral thalamus, they found the three classes described above and divided the intrinsically bursting neurons into inactivating and non-inactivating burst neurons. We have used deafferentation by transection of peripheral nerves as another way to alter afferent drive in primary somatosensory cortex. We examined bursting activity of single neurons in the forelimb representation of primary somatosensory cortex of awake cats before and after deafferentation to test two hypotheses: first, we hypothesized that the bursting patterns observed in ongoing neuronal activity in some somatosensory cortical neurons would resemble the distinctive discharge patterns observed in cortical slice preparations. Some evidence supporting this view appeared as we developed the methodology to analyze bursts w39x. Second, we hypothesized that these patterns could be used to identify several classes of somatosensory cortical neurons as had been done in motor cortex w6,7x. This was useful in deafferented somatosensory cortex where we were unable to manipulate afferent drive on the studied cells.

2. Materials and methods Animal training, surgery, placement of the recording chamber and the details of the methods used for data analysis are described in the preceding report w39x. In brief, eight adult cats previously trained to sit quietly in a comfortable position for 2–4 h were anesthetized and a craniotomy was made to expose the somatosensory cortex over one hemisphere. A recording chamber w35x with a removable seal was affixed with dental cement over the craniotomy, and stimulating electrodes were implanted chronically in the thalamus and pyramidal tract. 2.1. Recordings When the cat was again able to sit quietly and comfortably for 2–4 h in a sphinx position in the recording apparatus, recordings were begun with tungsten-in-glass electrodes Žimpedance: 1–4 M V at 1 kHz.. The electrode was positioned in stereotaxic coordinates and inserted directly through the dura. Standard electrophysiological recording equipment was used to amplify the single unit activity. It was filtered to retain frequencies between 300 and 3000 Hz. Usually, a single microelectrode penetration was made each day and the coordinates of that penetration were recorded. Subsequent penetrations were adjusted in

relation to the information gained from the previous recordings. In each penetration the cortical surface was designated as the micrometer reading where the first spontaneous or evoked activity could be detected. Before peripheral nerve transections, penetrations were made 1.0 mm apart in the forearm somatosensory cortex at positions recorded on a grid in stereotaxic coordinates. Following nerve transections, penetrations were placed between those points made prior to the deafferentation. In this way we could be reasonably sure we were recording from the somatosensory cortex that had previously represented the forelimb. Data collection involved recording of: Ži. the depth of the neuron; Žii. the presence or absence of a receptive field and its location on schematics of the body part; Žiii. the intensity of the stimulus required to elicit a response; Živ. the nature of the adequate stimulus Žskin, deep or tap.; Žv. the presence or absence of spontaneous activity and its rate; and Žvi. a digital record of the neural activity. As well, the duration of the first component of the action potential or of its whole waveform Ž5–10 waveforms. was digitized with a digital storage oscilloscope ŽTektronix Model 2201. and transferred to a personal computer through an RS232C port. Although the filter settings mentioned above could modify the waveform of the action potential, we were able to compare the durations from different cells relative to one another, and to monitor the consistency of their shapes during the experiment. The activity of 356 neurons was recorded in 50 penetrations Ž30 from normal and 20 from deafferented cats.. Although the somatosensory cortex is only about 1.5–2.5 mm thick, the curvature of the postcruciate gyrus in the forelimb representation allowed trajectories of up to about 4.5 mm before the electrode left the cortex. On average, we encountered 7 cells per penetration. To facilitate our analysis we established a terminology for the structured nature of the spike train and created several graphical displays as analysis tools. Interspike intervals ŽISIs. were of two types: Ž1. intervals between bursts, between a spike and a burst, or between two spikes which were not part of a burst were called non-burst intervals ŽNBIs., and Ž2. intervals within a burst ŽWBIs. ŽFig. 1.. One graphical display was the burst-order ŽBO. histogram Že.g. Fig. 5E and Fig. 6E., a display of the number of times bursts of different lengths occurred. The ‘‘order’’ of a burst was determined by the number of intervals in a burst. Thus a first-order burst consisted of one interval between two spikes; a second-order burst consisted of two intervals and three spikes, and so on. In this context a single spike could be considered a degenerate burst of order zero. Of the 356 neurons isolated, 333 had sufficient spontaneous activity Ž1000 spikes at a rate greater than 1rs. to be analyzed in this manner. We also measured the length of each interval within a burst. Once obtained, the mean and standard deviation of each interval was plotted according to the cardinal order of

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3. Results

Fig. 1. Definitions used to describe bursting patterns within an ensemble of action potentials. Subjectively bursts are easy to distinguish when the between-burst interval is several times longer than the average length of the non-burst interval.

The receptive fields of the neurons studied before deafferentation were located on the contralateral forearm and paw, but most cells studied in the first 3 weeks after deafferentation lacked any obvious afferent drive. However, by placing the penetrations studied after deafferentation between those studied prior to deafferentation we were assured that most cells came from regions near those studied before deafferentation, and thus within the region originally serving the forearm. We divided the sample having high spontaneous activity into bursting Ž n s 74. and non-bursting Ž n s 112. groups and plotted the mean discharge rates for each. Fig. 2A shows the distribution of mean discharge rates for

that interval within the bursts analyzed for an individual neuron Že.g. Fig. 5D and Fig. 6D.. This display provided a measure of the average length of successive intervals within a burst, and a visual indication of the duration and variability of the intervals at each position within a burst. The trend in the graph showed the relationships among successive intervals. Finally, information about changes in burst structure among successive bursts was obtained by another display called a burst-versus-time display Že.g. Fig. 7.. In this graph the vertical axis represented the temporal position of spikes within the burst with a high temporal resolution Žmilliseconds. and the horizontal axis represented time at a lower temporal resolution Žseconds., thereby indicating the time of occurrence of each burst. This device allowed the user to inspect the structure of a series of sequentially encountered bursts within a spike train. Assignment to a particular class of bursting cells was made following the criteria established by Martinson et al. w39x. Class I neurons had long bursts of nearly equal short intervals, class II cells had similarly short intervals but bursts seldom exceeded a duration of 4 intervals. Class III cells were characterized by a short first interval that was followed by successively longer intervals. Class IV bursts began with a long interval and subsequent intervals became progressively shorter. Class V cells displayed bursts that were predominantly one interval long and so could not be classified further. 2.2. Histology At the end of the last recording session microlesions were made with a y10 m A current applied for a duration of 15 s at stereotaxic electrode placements. Each animal was perfused with saline followed by phosphate-buffered formalin Ž10% at 48C.. Serial sections stained for Nissl substance and glial fibrillary acidic protein w8,26x were used to reconstruct electrode trajectories and estimate recording positions.

Fig. 2. Distributions of the mean frequencies of the ŽA. non-bursting cells and ŽB. bursting cells in normal and deafferented cortex. The means of these distributions, as tested by a Student’s t-test as well as the actual distributions, as tested by the Chi-square test were not significantly different. Further examination described in the text revealed certain differences among the groups.

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Table 1 Average modal interspike interval length of each class of cells Class

I II III IV V

normal deafferented normal deafferented normal deafferented normal deafferented normal deafferented

n

x"S.D.

Coef. of variation

6 4 11 8 6 13 9 8 3 6

2.37"0.65 2.00"0.33 2.58"0.91 3.15"0.77 1.93"0.55 3.00"1.60 2.84"1.10 2.63"0.82 2.00"0.01 2.98"0.82

0.27 0.17 0.35 0.24 0.29 0.53 0.39 0.31 0.05 0.28

% change in modal ISI with deafferentation y16 q22 q55 y 7 q49

The lengths before and after deafferentation differed significantly for classes III and V.

non-bursting cells in normal and deafferented cortex Ž9.24 " 6.80 and 10.99 " 6.77 Žx " S.D... with a skewed distribution showing a few cells with relatively high rates. Fig. 2B shows the distributions of mean discharge rates Ž9.52 " 5.39 and 12.47 " 10.70 Žx " S.D... for the bursting neurons in normal and deafferented cortex. Although these means were not significantly different, because there was a particularly large range of average discharge rates in bursting neurons, we investigated the source of those differences by further analysis. Thirty-five bursting neurons were found before and 39 were found after deaffereaatation. These represented 17.0% and 30.7% of the two samples respectively. The proportion found after deafferentation was significantly larger Ž x 2 : P - 0.004.. Table 1 shows the number of cells found in each class. The relative proportions before and after deafferentation are illustrated in Fig. 3A. Classes I, II and IV were less commonly encountered while the proportion of bursting cells in classes III and V was increased. The organization of the discharge patterns before and after deafferentation was examined by calculating the average ISI for each group. Fig. 4A shows that the mean ISI and the modal ISI of non-bursting cells were reduced after deafferentation. Only the reduction in the mode was significant Ž t-test: P - 0.05; x 2 : P - 0.03.. Fig. 4B shows the same analysis for bursting cells before and after the dis-

Fig. 3. A: proportions of bursting neurons of each class found in normal and deafferented cortex. A greater proportion of cells of classes III and V were found after deafferentation. The digit inside each column indicates the number of cells within that class. The increase in the proportion for class III was significant at P - 0.006 while class V had a marginal significance value of P s 0.074, two-tailed Žone-tailed P - 0.05. by Chi-square test. B: lengths of ISI for each class before and after deafferentation. The increase in the lengths of the WBI is attributed primarily to classes III and V bursting neurons. The interval lengths of the other groups were statistically unchanged. C: the burst index was a measure of the proportion of intervals organized in bursts. Class I had the largest index because these cells discharged with unusually long trains of spikes, and those trains were especially long after deafferentation.

charges had been divided into WBIs and NBIs. The average ISI Žfor NBIs and WBIs together. increased following deafferentation, but when the mode of the WBIs and the mode of the NBIs were analyzed separately, it became apparent that this was due to an increase in the average value of the modes of the WBIs; the average value of the NBIs decreased slightly. These effects were only

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other three groups was negligible ŽFig. 3B and Table 1.. These observations suggest that there were differences in the way each class of bursting neurons responded to the deafferentation. A burst index ŽBI. was constructed by expressing the number of bursts as a fraction of all events where an event was either a burst or a single spike. In normal cortex about 40% of all events were bursts for each class of cells except class V where the proportion was closer to 20%. This index changed in class I and class V cells, showing in these classes that the number of bursts increased following deafferentation. 3.1. Class I: long bursts of short interÕals

Fig. 4. Interspike interval ŽISI. lengths in non-bursting ŽA. and in bursting ŽB. neurons. The significant decrease in modal ISI length after deafferentation suggests that there was an increase in the level of spontaneous activity in the population of non-bursting neurons. In the case of the bursting neurons, the duration of NBIs decreased and the lengths of WBIs increased, but these changes were not statistically significant.

marginally significant for the WBIs Ž P - 0.05; one-tailed t-test.. However, when the cells were divided into classes and the same comparisons were made to test the hypothesis that the change in WBIs produced by deafferentation might be more evident for one class than for another, an analysis of variance of the mean WBI length for each group showed significant differences among the groups, and a protected t-test Ž P - 0.05. narrowed this effect to classes III and V; the average modes of the WBIs for these classes were respectively 55% and 49% greater than the interval lengths found in normal cortex. Once these groups were removed from the average, the difference in the modal intervals before and after deafferentation for the

The most easily recognized and the most stereotyped discharge pattern consisted of long bursts of short intervals. This pattern was found in bursts of orders ranging from one to more than 10, in approximately equal proportions. The other identifying characteristic of this cell type was the uniformity of the WBIs for all cardinal positions within the burst. Fig. 5 Žleft panel. provides an analysis of one example of this class. The spike train has many distinct bursts separated by NBIs that are more than one order of magnitude longer than the WBIs ŽFig. 5A, left.. The records seldom contain individual action potentials; nearly all spikes appear in bursts. This is apparent also from the ISI histogram presented in Fig. 5B, left; the peak of short intervals shown in this figure was truncated because it was many times larger than the peak of long intervals. During the analysis of 5291 action potentials from this cell, 1741 Ž32.9%. were found as single events; the majority Ž67.1%. were members of a burst. The 491 bursts had a modal value of 9 spikesrburst. The average length of the WBIs was about 1.5 ms whereas the mode of the NBIs peak was around 60 ms, a value 40 times longer than the mean of the intervals within the burst. An autocorrelogram of this bursting pattern ŽFig. 5C, left. emphasizes the tightly structured nature of the discharge; once a spike occurs at time zero, there is a high probability of another spike occurring between 1.4 and 1.6 ms later and again at several multiples of the first interval. Autocorrelograms such as these are seen only when the WBIs are nearly equal; the autocorrelogram is very sensitive to the regularity of the pattern in the burst because each interval within the burst contributes equally along the time axis. The regularity of the burst structure is also seen clearly in the WBI length graph for bursts having nine intervals ŽFig. 5D, left.. Each interval is of approximately the same length Žabout 1.5 ms. with an S.D. of less than 0.5 ms. The BO histogram ŽFig. 5E, left. shows that bursts of all orders were equally probable; only 21% of the bursts had 1–3 intervals and many had 10 or more intervals ŽFig. 7.. This ensemble of characteristics was found for 10

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neurons or 13.5% of our sample of bursting cells, however, only four of these were found in deafferented cortex. The WBIs in deafferented cortex were as short as WBIs in normal cortex, but the proportion of action potentials found in the bursts was higher after deafferentation; the BI went from 0.39 to 0.60 ŽFig. 3C.. 3.2. Class II: short bursts of short interÕals Fig. 5 Žright panel. summarizes the characteristics of the second distinctive discharge pattern. That pattern shared many characteristics of class I except that the number of intervals per burst seldom exceeded 4; there were no very long bursts. The ISI histogram usually had a large peak of very short intervals ŽFig. 5B, right. and the autocorrelogram displayed clear peaks ŽFig. 5C, right.. The NBIs of this cell tended to be quite long, most exceeding 100 ms. The mean WBI lengths were nearly equal ŽFig. 5D, right., averaging about 2.5 ms each. Fig. 7B shows the rhythmic spike train generated by this pattern; nearly all bursts consisted of 3–4 spikes. Nineteen cells, or 25.7% of bursting cells, and 5.7% of the total sample were assigned to this category, with their occurrence being almost equally likely before and after deafferentation Ž11 and 8 respectively.. 3.3. Class III: bursts with interÕals of increasing lengths The third distinctive bursting pattern consisted of groups of short, high-frequency discharges in which the last interval was always noticeably longer than the preceding intervals in the burst. The bursts shown in Fig. 6A Žleft. illustrate this characteristic clearly. The ISI histogram of this cell type is bimodal ŽFig. 6B, left. but the variable lengths of intervals within the burst make it more difficult to distinguish between intervals inside the burst and those outside; the first peak of the ISI often mixes with the second peak because some of the intervals within the burst can be up to 100% longer than the first interval. This cell generated 2411 spikes in the analyzed record. Of these, 995 Ž41.3%. were found in bursts. The graph of WBIs ŽFig. 6D, left. shows that actually the burst is highly structured; in this example the first interval in the burst is only about 1.3 ms in duration; the second is about as long, but the third and fourth intervals become progressively longer; the last interval averaged 2.13 ms, 1.6 times longer

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than the first. This pattern was observed in 19 cells Ž25.7% of the bursting cells and 5.7% of all.. Twice as many Ž n s 13. were found after deafferentation as before. The proportion of spikes found in bursts did not change, but this class showed a 55% increase in modal WBI after deafferentation ŽTable 1., however, the pattern of gradually increasing WBIs was still characteristic of each cell studied after deafferentation. The successive WBIs were each more variable in length that their predecessor; the bursts of each order terminated with an interval having a larger standard deviation than did the early intervals in the same burst ŽFig. 8.. 3.4. Class IV: bursts with interÕals of decreasing length The fourth group of cells discharging in bursts was characterized by the first interval being longer than the following intervals. The average length of the first interval was around 4.5 ms with a large standard deviation. Both the interval length and the variability of that length decreased progressively until the last interval in the burst was only about 2.2 ms long. Sometimes the last interval was slightly longer than the next-to-the-last interval, but on average the last was still the shortest. This pattern of intervals produced an ISI histogram with a broad peak that was strongly skewed to the right. In the case illustrated in Fig. 6 Žright panel., the spike train ŽFig. 6A, right. is less structured than the spike trains of the previous classes; this appearance may be due to the greater variability of the interspike intervals within the burst. These characteristics, however, are not reflected in the ISI histogram ŽFig. 6B, right. where the broad peak of short intervals is clearly separated from the peak representing the NBIs. In the autocorrelogram, a significant amount of the signal is represented by short intervals ŽFig. 6C, right., but the changing interval length within the burst prevented more than two peaks from being visible in the autocorrelogram ŽFig. 6C, right.. Nevertheless the presence of peaks in the autocorrelogram was what showed this pattern to be different from a stochastic pattern, which would have generated a completely flat autocorrelogram. The number of intervals within a burst ŽBO histogram, Fig. 6E. was also quite variable. The bursts tended to be short, often 2–3 intervals, but could range up to 6. In an interspike interval ensemble of 555 spikes, 232 Ž41.8%. were single events and 323 Ž58.2%. were in bursts.

Fig. 5. Left panel: an example of a neuron from class I. A: the spike interval ensembles show the characteristic long trains of short intervals. B: the ISI histogram shows the predominance of very short intervals. C: the autocorrelogram of the same cell shows the elevated probability of firing at multiples of 1.7 ms. D: the WBI display shows that each interval within the burst is of nearly the same duration independent of cardinal order. This example was for bursts of 9 intervals. E: the BO histogram shows that this cell generated many, very long bursts, those longer than 10 intervals are counted in the same bin with those of 10 intervals. Bursts of lengths less than 10 intervals are almost equally probable. Right panel: an example of a neuron in class II. A: the spike interval ensemble shows short bursts with intervals of nearly equal lengths. B: the ISI histogram shows a peak denoting that a substantial number of very short intervals was present. C: the autocorrelogram shows the distribution of interval lengths at multiples of 2.5 ms. D: the WBI length display for bursts of the fourth order. There was very little variability for these intervals within the burst and they were all of nearly the same length. E: the BO histogram shows that the majority of bursts were 1–3 intervals long.

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Inspection of the individual class IV bursts suggests they were often generated with one spike missing from its expected position within the burst. This activity pattern

may be a partial explanation of the large variability in interval length seen in the WBI histogram and in the variability of the number of spikes per burst. In these cells,

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Fig. 8. The WBI length displays for bursts of order 1 through 6 for the neuron of class III illustrated in Fig. 6A. In each case where there were two or more intervals in a burst, the next interval was longer than the preceding one.

Fig. 7. Burst versus time displays for two neurons showing the intervals within each burst and the highly structured nature of the burst. A: 11 bursts occurring during 1 s from the class I neuron are illustrated in Fig. 5A. B: a series of 33 bursts of mainly 3 intervals occurring during 200 ms from the class II neuron are illustrated in Fig. 5B. In both figures the first interval in each burst occurs on the ordinate at time zero.

the proportion of events that were bursts was the lowest of any class except class V, being only 34% of all spikes. We found 9 cells of this class in normal cortex and 8 in deafferented cortex. None of the measured parameters changed with deafferentation. 3.5. Class V: cells haÕing bursts of one or two interÕals Because the assignment of a cell to a particular class depends upon the relationships among successive intervals within the burst, cells that discharge only bursts containing

one interval cannot be assigned to any of the first 4 classes. This occurred 16 times in our sample of 74 cells Ž22%.. We assigned these cells of ambiguous nature to a fifth class. We could not do several steps in burst analysis such as the calculation of the position of the shortest and longest intervals and the generation of the BO histogram. The only relevant measures that we were able to obtain were a mean WBI of 2.66 " 0.81 Žx " S.D.. and a BI of 0.26 " 0.11. The BI was significantly different Ž P - 0.05. from BIs of all other classes except for class IV Ž P ) 0.10. and it increased after deafferentation ŽFig. 3C.. The lack of more information prevented further analysis. 3.6. Antidromic stimulation Stimulating electrodes were placed in the ventroposterior thalamus and in the pyramidal tract in four animals Žcf. w45x for methods.. Of the cells tested with these electrodes we found no evidence of antidromic activation of class I cells even though six bursting cells were excited synaptically from the thalamus at a relatively long latency Ž3.68 " 2.4 ms.. In contrast, some class III and class V cells were activated antidromically from the thalamus at short latencies and several class IV cells were activated antidromically from the pyramidal tract as were several class II cells.

Fig. 6. Left panel: an example of a neuron in class III. A: the spike interval ensemble shows bursts of differing lengths but when they consist of 3 or 4 intervals the last interval is always the longest. B: this cell had a high spontaneous activity with a modal interval length near 30 ms but as well there are a large number of shorter intervals between 1 and 7.5 ms. C: the autocorrelogram shows the high probability of firing about 1.3 ms after the occurrence of the first spike and the enhanced probability of firing that continues up to about 7 ms after the previous spike. D: the WBI length display explains the autocorrelogram by showing that the first intervals of the burst are very short Žabout 1.3 ms. but that each successive interval is longer. E: the BO histogram shows that bursts of one interval were the predominant pattern in this cell with a second mode in the BO histogram around bursts having 3 WBIs. Right panel: an example of a neuron from class IV. A: the spike interval ensemble shows the long initial interval of the burst. B: the ISI histogram shows many short intervals. C: the autocorrelogram confirms the preference to discharge in intervals of less than 3 ms. D: the WBI length diagram shows that the burst begins with a long interval that is highly variable and ends with a much shorter interval that is very precisely calibrated at 2.6 ms. E: the BO histogram shows that most bursts consist of one interval with a large number having 2–4 intervals.

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3.7. Effects of deafferentation Following deafferentation, the proportion of bursting cells increased two-fold, but the mean rate of their ongoing spontaneous activity was not affected. There was no change in the proportion of classes I, II and IV but there was a higher proportion of classes III and V. The organization of bursts within each class seemed to be relatively unaffected by the deafferentation; there was no obvious change in the structure and only in classes III and V was there an important increase in the interval length Ž55% and 49% respectively.. 3.8. Bursting cells according to layers In seven of the cats, bursting cells were assigned to a specific cortical depth. The proportions found in the upper third of the cortex were the most affected by deafferentation. In putative layer III, 17 cells were recorded prior to transection, but only two cells were found there following transection. Conversely, in the lower third of the cortex, putatively in layer VI, only four cells were found prior to transection but 10 were recorded after transection. These changes were significant at P - 0.05 with two-tailed ttests. Bursting cells in the upper third of the cortex were mostly class II and class IV neurons whereas cells in the lowest third were divided about equally among the different classes. 4. Discussion Using several analytical tools described previously w3,5,39,43x, we examined five distinct classes of bursting neurons in the somatosensory cortex of awake cats before and after deafferentation. The same five classes were present after deafferentation but they were encountered more frequently Ž17% vs. 31%.. Most of this increase was due to encountering more cells in classes III and V after deafferentation. Nerve transection did not change the organization of the bursts very much. After deafferentation the mean number of intervals within a burst was unchanged, except for classes I and V which showed a small increase. Only in classes III and V did the WBI length change, increasing by 55% and 49% respectively. We suspect that these changes are one effect of reduced inhibition in somatosensory cortex following deafferentation. Since we will argue below that classes I and II are predominantly inhibitory interneurons and that classes III and IV are probably pyramidal cells, the changes in relative proportions suggest a reduction in the activity of inhibitory interneurons and increased activity from normally quiescent pyramidal cells. A similar reciprocity between the activity of pyramidal cells and interneurons was noted during sleep–waking cycles some time ago w49x. The hypothesis that deafferentation reduces inhibition in somatosensory cortex is consistent with reports from several

different laboratories. Peripheral deafferentation causes receptive field enlargement w10,18,19,44x, transient increases in spontaneous activity w45x, and increased bursting w17x. 4.1. Morphological correlates of burst pattern classes Some morphologically distinct classes of cells may be recognized by their electrophysiological properties w6,7,49x. For example, the discharges of the cerebellar Purkinje cell are physiognomic and even specify the origin of the excitatory influence w58x. The very narrow action potential of certain cortical neurons distinguishes one class of GABAergic interneurons w1,31,32,38x although one class of pyramidal cells also has an action potential that is very narrow w7,20x. McCormick et al. w38x, Mason and Larkman w40x, Larkman and Mason w37x, Kawaguchi w31,32x and Baranyi et al. w6,7x described characteristic bursting patterns of several classes of cortical neurons that seem to be generated from their intrinsic membrane properties. Because cortical neurons in tissue slices maintained in vitro show similar bursting behaviors to those in vivo, even when deprived of many connections w12,14,38x, and because intracellular injection of depolarizing current will repeatedly provoke the same, nearly stereotypic discharge patterns w31,32,40x, these bursting patterns seem to reflect mechanisms intrinsic to the cell. The elegant and technically very demanding intracellular experiments in whole animals of Baranyi et al. w6,7x, which showed that bursting discharge patterns produced in intact cat motor cortex are correlated with their membrane properties as well as with their morphology, support this hypothesis. We believe that we have identified some of the same discharge patterns that have been described in vivo w6,7x and in vitro w12,14,31,32,38,40x in waking cat somatosensory cortex. For example, class I cells having long bursts of short intervals were observed to have had very brief action potentials and were never activated antidromically from the thalamus or pyramidal tract in the few tests that we performed. We believe they are likely to be the fastspiking or thin-spiking neurons seen in vivo by others w6,7,36,42,47,48,52,51,53–57x and which are thought to be GABAergic interneurons w38,54,56x. Baranyi et al. w7x and Kawaguchi w31x showed that the spike width of the fast-spiking cells in their preparations overlapped with the spike widths of a second class of cells, but by using more than one parameter the groups could generally be divided. Any individual variable such as spike width, membrane resistivity or frequency adaptation when used alone, may be insufficient to uniquely identify a cell class because more than one cell class may share any one electrophysiological variable, but uniqueness may be conferred by some combination of these variables. We suggest, on the basis of the data presented here and in the preceding paper w39x, that one can look beyond the shape of the individual action potential or beyond a particular membrane property to the pattern of several spikes associated with a group to obtain additional identifying

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information. For example, a set of characteristics that could be used to identify GABAergic interneurons may be that Ži. they are thin spikes that cannot be activated antidromically from the thalamus or pyramdial tract, and Žii. they often discharge long trains of spikes at high frequency with little or no frequency adaptation. To test our hypothesis it would be necessary to fill and recover a number of class I bursting neurons. Nevertheless, even if our class I is limited to interneurons, it is likely to include more than one morphological cell type; Kawaguchi w32x recorded from, and filled, fast-spiking neurons that had no frequency adaptation Žsimilar to our class I cells.. The majority were shown to be parvalbumin-containing, non-spiny, multipolar neurons Žbasket cells w24x., which are now thought to be GABAergic interneurons. However, 3 of 29 filled, thin-spike cells were shown to be chandelier cells. The behavior of class II neurons was similar to the behavior of class I neurons in our sample, except that their bursts were significantly shorter and their WBIs were significantly longer. We suggest that many cells in this class may also be GABAergic interneurons, but that they were less intensely depolarized in our preparation than were class I cells. However, it should be remembered that high frequency bursts of up to four spikes are also seen in some pyramidal cells w7x. The 16 class V cells, which had only two and rarely three short spikes per burst, preventing the cells from being analyzed in detail, may also have been GABAergic interneurons. The very short WBI length and the low variability of the lengths of the single interval generated in class V cells suggest that these cells are similar to classes I and II but that they have an even lower excitatory drive. Consistent with this interpretation is the observation that in normal animals these cells discharged bursts less frequently than did any other class, but that after deafferentation, their BI increased. Our class III seems to be equivalent to the non-inactivating burst neurons of Baranyi et al. w6,7x. According to Connors and Gutnick w14x the intrinsically bursting cells are relatively rare by comparison with all pyramidal cells. However, since our analysis was focussed only on bursting cells, it is difficult to relate our relatively high proportion Ž19 of 74. to an actual proportion in the population of all neurons. Kawaguchi w31x described a cell type similar to the non-inactivating burst neurons having a low membrane resistance. He remarked on the significant frequency adaptation of these cells, but also noted that one class of non-pyramidal neurons tended to fire in bursts and showed significant frequency adaptation as well. Thus, to confirm that our class III cells are non-inactivating pyramidal burst neurons will require antidromic activation from an efferent pathway Ža positive sign we obtained only 7 times. or the filling and recovering of well-characterized examples. Their very characteristic burst beginning with a short interval Žsometimes less than 2 ms. and evolving into a last interval, three or four spikes later, that was twice as long,

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readily distinguished them from other classes. These cells often fired bursts Ž40% of events. but seldom had bursts longer than four intervals. Since the gradually lengthening sequence of intervals seen in class III was a pattern that might be explained by the temporal evolution of an underlying excitatory postsynaptic event, it is useful to mention the evidence that the burst of class III cells was intrinsic to the cell: first, the total duration of the burst was usually less than 12 ms whereas most EPSPs in cortical neurons last 20–50 ms. Second, the discharge pattern was highly stereotypic. For a given cell, each burst lasted about the same time, had about the same number of spikes and always started with a very short interval. A burst generated by a synaptic event should be much less stereotyped, since the synaptic events would be subject to temporal summation and the random fluctuations of ongoing cortical neuronal activity. The organization of class IV bursts was much less stereotyped than the others, but its pattern, beginning with a long interval and ending with a very short interval Ž; 2 ms. is difficult to explain by any known pattern of cortical postsynaptic membrane fluctuations. The progressively decreasing interspike intervals could be attributable to the rising phase of an excitatory postsynaptic potential, but the consistently abrupt termination of the burst after the shortest interval cannot be explained by any of the usual patterns of postsynaptic events. Rather, we believe that this pattern corresponds to a class of neurons that Baranyi et al. w7x termed inactivating burst neurons. Supporting this hypothesis is the fact that, of the cells tested, three were activated antidromically from the pyramidal tract. One weakness of a system that classifies cells using discharge patterns within bursts is the inability to classify the large population of cells Ž78% of this sample, Table 1. that do not burst and the unknown number of other cells that may not even have spontaneous activity. The dominant role of inhibition in the sensory cortical network w21,46x and the many cells that are silent for unknown reasons w18,19x mean that this classification scheme must remain incomplete and can only be an inclusive scheme when there is a burst pattern in the ongoing activity. There will always be the large class of cells that do not burst, which according to Baranyi et al. w6x may be either fast or slow PT cells or fast-spiking interneurons. Baranyi et al. w6,7x deliberately tried, but failed to produce bursting behavior in these cells, suggesting that all fast PT cells or all slow PT cells and all fast-spiking cells are not identical. It is reassuring to know that there is one or more classes of cells, distinguishable from the categories studied here, which are physiologically unable to generate the patterns observed in bursting neurons. Whether external excitatory activity is capable of generating patterns of action potentials that may be confounded with bursts arising from intrinsic membrane properties is not entirely clear. Oscillations attributable to properties of the system rather than properties of individual cells seem

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to be distinguishable in most cases by their time course. We inferred that some of the structure observed in the recorded spike trains was due to influences extrinsic to the neuron being studied because it followed slower temporal patterns, suggesting that it was driven by fluctuations in cortical excitability. Oscillatory behavior is characteristic of cortical neuronal circuits and has been recognized since the first electrophysiological studies. Its origins and properties have been the subject of research for decades w15x and this work continues today w3,4,13,22,25,41,49,50 x. However, the temporally structured oscillatory discharges, which are an intrinsic property of many complex circuits and are related to the nature of the interactions of the constituent elements, occur with a much lower base frequency and have interspike interval distributions with longer median intervals and broader variances than those described here. Distinguishing patterns generated by cortical circuitry from patterns attributable to the intrinsic properties of a neuron must be a primary and necessary step of the analysis of those circuits. 4.2. Cellular mechanisms The circuitry in which a cell is found may have a direct effect upon the frequency with which intrinsic bursts are generated even if the structure of the burst, once set in motion, is determined by membrane properties Že.g. w48,49x.. For example, the bursts in some pyramidal cells are controlled by low-voltage, transient calcium channels. These channels are normally inactive and the cell normally fires trains of single spikes. When the cell is hyperpolarized for more than 150 ms, the deactivated calcium channels become capable of being opened again and, as the membrane rebounds from the hyperpolarization such as after a long IPSP, the next depolarization is accompanied by a calcium current leading to a burst of sodium potentials that ride on top of the calcium potentials w16x. These sodium spikes occur at gradually increasing intervals as the calcium channels become inactivated again w23,28–30x. This mechanism probably explains the bursts that occur in the class III neurons. In those cells bursts occurred only 41% of the time and these could easily reflect only those discharges that followed a large IPSP.

Acknowledgements We are deeply appreciative of the efforts contributed by others. Mrs. Julia Martinson developed the software for analysis of the burst structure. Mrs. Lise Imbeault provided the kind and patient effort required to type numerous versions of this manuscript. Mr. Giovanni Filosi and Mr. Claude Gauthier are responsible for the excellent art work. Mrs. Feliciana Faraco-Cantin and Mrs. Jeanne Lavoie provided most of the histological ŽNissl. support for these studies. Mr. Richard Bouchoux designed and constructed

the microdrive and recording chamber. We followed Dr. R. Nelson’s suggestions while creating the graphical displays of bursts. This research work was supported by operating grants from Medical Research Council and The Scottish Rite Charitable Foundation of Canada awarded to R.W. Dykes. H.H. Webster was also supported by a postdoctoral fellowship from Fonds de la Recherche en Sante´ du Quebec. ´ References w1x Agmon, A. and Connors, B.W., Repetitive burst-firing in the deep layers of mouse somatosensory cortex, Neurosci. Lett., 99 Ž1989. 137–141. w2x Agmon, A. and Connors, B.W., Correlation between intrinsic firing patterns and thalamocortical synaptic responses of neurons in mouse barrel cortex, J. Neurosci., 12 Ž1992. 319–329. w3x Armstrong-James, M. and Fox, K., Effects of ionophoresed noradrenaline on the spontaneous activity of neurones in rat primary somatosensory cortex, J. Physiol. (Lond.), 335 Ž1983. 427–447. w4x Armstrong-James, M. and Fox, K., Evidence for a specific role for cortical NMDA receptors in slow-wave sleep, Brain Res., 451 Ž1988. 189–196. w5x Bair, W., Koch, C., Newsome, W. and Britten, K., Power spectrum analysis of bursting cells in area MT in the behaving monkey, J. Neurosci., 14 Ž1994. 2870–2892. w6x Baranyi, A., Szente, M.B. and Woody, C.D., Electrophysiological characterization of different types of neurons recorded in vivo in the motor cortex of the cat. I. Patterns of firing activity and synaptic responses, J. Neurophysiol., 69 Ž1993. 1850–1864. w7x Baranyi, A., Szente, M.B. and Woody, C.D., Electrophysiological characterization of different types of neurons recorded in vivo in the motor cortex of the cat. II. Membrane parameters, action potentials, current-induced voltage responses and electrotonic structures, J. Neurophysiol., 69 Ž1993. 1865–1879. w8x Benevento, L.A. and McCleary, L.B., An immunocytochemical method for marking microelectrode tracks following single-unit recordings in long surviving, awake monkeys, J. Neurosci. Meth., 41 Ž1992. 199–204. w9x Burns, B.D., The Uncertain NerÕous System, Arnold, London, 1968, 194 pp. w10x Calford, M.B. and Tweedale, R., Immediate and chronic changes in responses of somatosensory cortex in adult flying-fox after digit amputation, Nature, 362 Ž1988. 446–448. w11x Chagnac-Amitai, Y., Luhmann, H.J. and Prince, D.A., Burst generating and regular spiking layer 5 pyramidal neurons of rat neocortex have different morphological features, J. Comp. Neurol., 296 Ž1990. 598–613. w12x Connors, B.W., Gutnick, M.J. and Prince, D.A., Electrophysiological properties of cortical neurons in vitro, J. Neurophysiol., 48 Ž1982. 1302–1320. w13x Connors, B.W. and Kriegstein, A.R., Cellular physiology of turtle visual cortex: distinctive properties of pyramidal and stellate cells, J. Neurosci., 6 Ž1986. 164–177. w14x Connors, B.W. and Gutnick, M.J., Intrinsic firing patterns of diverse neocortical neurons, Trends Neurosci., 13 Ž1990. 99–104. w15x Creutzfeldt, O.D. and Kouchim, J., Neuronal basis of EEG waves. In: O.D. Creutzfeldt ŽEd.., Handbook of Electroencephalography and Clinical Neurophysiology, Vol. 2, Part C, Elsevier, Amsterdam, 1974, pp. 2C-5–2C-55. w16x Deisz, R.A., A tetrodotoxin-insensitive sodium current initiates burst firing of neocortical neurons, Neuroscience, 70 Ž1995. 341–351. w17x Dykes, R.W., Avendano, ˜ C. and Leclerc, S.S., Evolution of cortical responsiveness subsequent to multiple forelimb nerve transections:

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