A quantitative study of temporal and spatial response patterns in a thalamic cell population electrically stimulated

BRAIN RESEARCH

255

A Q U A N T I T A T I V E S T U D Y OF T E M P O R A L A N D SPATIAL RESPONSE P A T T E R N S 1N A T H A L A M I C CELL P O P U L A T I O N E L E C T R I C A L L Y STIMULATED

J. S C H L A G AND J. V 1 L L A B L A N C A *

Department of Anatomy and Brain Research Institute, University of California, Los Angeles, Calif. (U.S.A.)

(Accepted November 13th, 1967)

INTRODUCTION

Our knowledge of functional thalamo-cortical relations is based mainly on experiments where the thalamus is electrically stimulated. However, there is very little information on what is the input signal to the cortex under these conditions. It is implicitly assumed that the shocks set up a massive and highly synchronized volley of impulses, and electrical cortical responses are interpreted on the basis of this assumption. The present investigation was designed to gather information on what thalamic neurons do when electrically stimulated: What proportion of them are affected? How far from the stimulated site? Are they all excited? We have considered mainly temporal and spatial parameters in analyzing the data. Nucleus ventralis lateralis was selected as the thalamic site of stimulation because of the interest of this laboratory in the induction of activity in the pyramidal tract system. There have been very few studies related to the problem outlined. Thalamic internuclear interactions have been investigated by Purpura's group2,n,10,11 and Sakata et al.lL lntranuclear interactions were recently studied by Marco et al. 7, but the latter authors have almost exclusively concentrated their attention toward a search for inhibitory interneurons. From these previous works, we could expect to find inhibitory effects of the stimulation in addition to the initial powerful excitatory drive. Brief reports concerned with some particular aspects of our data have been presented elsewhere16, 2o. METHODS

These experiments were performed on 22 cats. They were operated on under * U.S. Public Health Servicelnternational Postdoctoral Fellow (IF05_TW 998) on leave from C~itedra de Fisiopatologia, Escuela de Medicina, Universidad de Chile, Santiago (Chile). Brain Research, 8 (1968) 255-270

256

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ether anesthesia; the sites of incision and pressure points of the stereotaxic head holder were injected with procaine and the animals were kept motionless by intravenous injections of Flaxedil during the recording period. The body temperature was maintained by the use of a heating pad. Artificial respiration was adjusted at just the level sufficient for almost complete myosis. All necessary precautions were taken to prevent any severe or prolonged pain. As soon as the brain was exposed and the dura removed, the cerebral cortex was covered with mineral oil at 38°C. A small hole ~vas drilled in the occipital bone in order to implant an electrode through the cerebellum into the brachium conjunctivum (BC). Concentric electrodes used for subcortical stimulation consisted of a 22 gauge needle and a 150/z stainless steel wire protruding by less than 0.5 mm. Square pulses of 0.5 msec duration and 0.12-0.20 mA were applied, the inside wire always being the cathode. Cortical stimuli (0.1-0.5 msec, 2.0-4.2 mA) were applied through two silver ball electrodes on the pial surface, or through a silver ball on the pial surface and a stainless steel 150 # wire 2-3 mm beneath. Glass micropipettes filled with 2 M potassium citrate (1-5/z at the tip, 2-10 M~2 resistance) were used for unit recording. The microelectrode was implanted obliquely at a 20 ° angle (downward and medially), and aimed in such a way that its tip passed very close (0.5-1.5 mm) to the tip of the stimulating electrode. The potentials derived were fed through a cathode-follower to a 502 Tektronix cathode-ray oscilloscope and an Enhancetron 1024. The reference input of the recording circuit was connected to the stimulating circuit by a bridge of resistors and capacitors which served to equalize the impedance at both inputs and minimize the shock artifacts. The positioning of concentric electrodes in the nucleus ventralis lateralis of tile thalamus (VL) was guided (1) by recording potentials evoked on the anterior sigmoid gyrus by ipsilateral VL stimulation and (2) by recording VL responses to contralateral BC stimulation. Unit responses to this stimulation also served to identify VL neurons2,12,19; those firing in 0.8 msec to 3 msec are referred to in the text as 'BC-responding' neurons. The microelectrode tracks were examined in 80 ,~t histological sections stained with thionine, where the tip of the concentric electrode was marked by electrolytic iron deposits and potassium ferrocyanide reaction. Distances between the tips of the microelectrodes and concentric electrodes were evaluated on the basis of microdriver readings, the microdrivers being positioned on a reference frame as they were on the stereotaxic instrument. Distance measurements were disregarded when found to be inconsistent with the histological data (1 animal). RESULTS

Description o f unit responses to VL stimulation Once the stimulating electrode had been positioned in VL, it was not moved for the whole duration of the experiment, and the stimulus intensity was kept constant at a value sufficient to induce stable responses on the anterior sigmoid gyrus. The microelectrode was advanced until its tip reached about 4-5 m m from the stimulated Brain Research, 8 ( 1 9 6 8 ) 2 5 5 - 2 7 0

THALAMIC RESPONSES TO LOCAL STIMULATION

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point. From there, all cells encountered along the track (i.e. not only VL neurons) were recorded. Units were identified by the appearance of spikes. Often, injury discharges were provoked and the cells died before any record could be taken. Every effort was made to not select cells on the basis of the quality of recordings (e.g. spikes of large amplitude particularly suitable for illustration). It was necessary, however, to obtain a resolution sufficient for identifying discharges from the same unit without altering its activity by mechanical pressure. All these constraints undoubtedly introduced a bias in our sampling: (l) by ignoring silent cells, (2) by eliminating the ones most vulnerable to the pressure of the micropipette (maybe systematically the smallest cells), and (3) by discarding records obscured by the presence of spikes of unequal amplitude. These limitations have to be recognized in evaluating the significance of counts. Responsiveness to VL stimulation will be described by distinguishing three classes of cells:

A i-_l_ I

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Fig. 1. Post-stimulus histograms of unit responses to VL stimulation. Time of stimulation indicated by arrow at 0 msec. Vertical calibrations in this and following post-stimulus histograms: 100~ corresponds to an average of exactly 1 spike per bin. A, Cell silenced for more than 5130msec (class II); B, Cell initially fired and secondarily silenced (class III); right histogram shows detail of early firing (vertical calibration: 25 %). C, Cell considered as non-responsive (class 1); spikes were ascertained not to be time-locked to stimulus. D, BC-responding unit belonging to class 1I; the early spike in the response did not appear when the BC stimulus was preceded by a VL stimulus at a 100 msec interval (VL-BC). Record samples of this cell at right. Distances of cells from stimulated site are given in mm.

Brain Research, 8 (1968) 255-270

258

J. S C H L A G

A N D .1. V I L I . A B L A N ( A

Class I: Non-responsive cells. Their rates of firing did not appear to be altered at any time after the stimulus and there was no evidence of discharges time-locked to the stimulus. The decision to include cells in this class was based on the subjective impression o f unresponsiveness gained from an inspection o f individual records and a comparison o f pre- and post-stimulus histograms (Fig. 1C). It might be possible that averaging on a greater n u m b e r o f records (more than 40) or using finer methods of analysis could have revealed slight though consistent signs o f responsiveness. In addition, where the spontaneous activity was very low, a depression o f this activity caused by the stimulation would not be detected. Therefore, the p r o p o r t i o n of nonresponsive cells has been probably overestimated. ClassII: Initially silencedeells. The stimulus was immediately followed by a period o f complete absence of firing which lasted for 120-200 msec and sometimes more Cup to 600 msec in 1 case, Fig. 1A). The latency o f the depression could not he evaluated. It would have been necessary to measure how long spontaneous (i.e. non time-locked) firing continued following stimulation, but spikes practically never occurred, suggesting that the depression started very early. The silent period always terminated by a late burst o f firing at higher than normal frequency, which had the characteristics o f a post-inhibitory rebound (Fig. 3C). Class III: Initially fired cells. The stimulus was followed by time-locked dis-

A

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Fig. 2. Actual records from VL cells. A, Cell responding to stimulation of VL, posterior sigmoid gyrus (Cx), and combinations of pairs of these stimuli. In the latter cases, stimuli were separated by 100 msec and only the response to the second stimulus is shown. Response patterns were the same in all these conditions but more numerous early discharges were produced by paired stimuli. VL-VL. VL-Cx, Cx-VL, and Cx-Cx indicate the succession of the stimuli. Calibration: 100 msec. B, Same cell responding to BC stimulation (2 different sweep speeds, calibration: 20 and 5 msec, upper right record formed by 4 superimposed traces showing stability of latency). Details of responses to VL and VL-VL stimulation; small spikes were present in the records at that time; the responses of the small and large units had different latencies and both latencies were increased by repeating the stimuli (calibration: 20 msec). C, Response of another cell to 10/sec repetitive VL stimulation. The cell was silent after the first pulse but each following pulse evoked a discharge. Positivity downward.

Brain Research, 8 (1968) 255-270

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charges. There could be any number of spikes from 1 to about 15, the latency varied from less than 1 msec to 18 msec (median value, 7 msec), and bursts could last up to 40 msec. Latency, number of spikes, and duration of bursts remained rather constant in each case. Regularly, the initial firing was followed by a period of relative or complete silence and a rebound (Figs. 1B and 2A). Both had the same characteristics as the cells in class II. Therefore, some time after the stimulation (e.g. around 80120 msec) practically all neurons affected by the stimulus (i.e. classes II and III cells) were silent. During the period of depression seen in cells of classes lI and III, no response to BC stimulation could be seen (Fig. I D). In a few instances where the spikes were large (of the order of 30 mV) indicating close contact between the micropipette and the cell, the depression of firing was accompanied by a graded negative deflection (Fig. 2C), resembling a hyperpolarization as recorded intracellularly. That this graded potential could have been an actual hyperpolarization is consistent with other data obtained in similar conditions v. The percentages of cells falling in the three classes is given in Table l, for the whole population (167 units)* and for the neurons responding to BC stimulation in less than 3 msec (39 units) and in less than 1.5 msec (23 units). The ratio between initially silenced cells (class ll) and initially fired cells (class IIl) remained stable throughout the series of experiments, whereas the relative proportion of non-responsive cells (class I) varied appreciably from animal to animal. This accounts for slight differences in the percentages given here and in a preliminary reporO 6.

TABLE

I

DISTRIBUTION OF THE CELLS ACCORDING TO THE MODALITY OF RESPONSE TO W E STIMULATION (~oo)

Whole population BC-responding in less than 3 msec BC-responding in less than 1.5 msec

Class I

Class H

Class lli

51 41 30

13 13 26

36 46 44

These varieties of responses to VL stimulation could also be readily obtained by stimulating the ipsilateral pericruciate cortex, as previously shown by Sakata et al.lL In particular, the duration of the period of silence induced was of the same order of magnitude (Fig. 2A).

* These proportions are based on actual counts. But the numbers of cells sampled at various distances from the stimulated site were not proportional to the expected numbers of cells at those distances if it is assumed that the population is homogenously distributed in space. Making this assumption and weighing our figures accordingly, the proportions calculated were 55 ~ for class I, 9 % for class II, and 36% for class III in the whole population. Brain Research, 8 (1968) 255-270

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Changes o f unit responses by repetition o f the VL stimulus

W h e n VL stimuli are r e p e a t e d at a rate a r o u n d 10/sec the responses evoked on the pericruciate cortex u n d e r g o characteristic changes described as 'cortical augm e n t a t i o n 's. T h e m a i n feature o f a u g m e n t a t i o n is the p r o d u c t i o n o f a s e c o n d large surface-positive c o m p o n e n t in the cortical response 17 c o n c o m i t a n t with profuse discharges o f p y r a m i d a l tract cells14,15. It seemed interesting to inquire whether the initiation o f such p r o f u s e discharges could be traced d o w n to the site o f stimulation. The responses o f 161 t h a l a m i c cells to V L stimuli r e p e a t e d at 100 msec intervals were studied. T a b l e II s u m m a r i z e s the results, by showing the n u m b e r o f times that particular types o f changes were e n c o u n t e r e d when responses o f cells to a second V L stimulus were c o m p a r e d to their own responses to a single VL stimulus. T h e total figures have been b r o k e n d o w n to c o n s i d e r s e p a r a t e l y neurons r e c o r d e d at distances o f 0 - 2 m m and 2 - 4 m m from the s t i m u l a t e d sites. Characteristics o f responses as a function o f distance will be the object o f the next section. Entries A a n d B refer to the cells which were fired by the second, but not by the TABLE II FREQUENCIESOF CHANGESIN RESPONSIVENESSOBSERVEDWHEN VL STIMULUSWAS REPEATED AFTER 100 msec Types of changes in response to 2nd IlL pulse (as compared to 1st VL pulse)

A. B. C.

Number of times encountered 0-2 mm

From initial silence to initial burst 2 From no response to initial burst 5 From initial firing to initial firing with : (a) Increase in latency 19 (b) Increase in number of spikes but not in latency 7 (c) Increase in probability of firing only l Subtotal (a -'-b t c) 27 D. From no response to initialsilence 2 E. Decrease in latency of initial firing 5 F. No change in type of response 10 G. From no response to no response 35 Totals 86

2 4 mm

Subtotal

9 3

Il 8

1I 2 / 14 0 0 13 36 75

41 2 5 23 71 161

first o f two V L pulses. T h e i r p r o p o r t i o n a m o u n t e d to m o r e t h a n 1 0 ~ o f the total sample. T h e latencies o f the bursts were between 5 a n d 18 msec ( m e d i a n value: ! 2 msec}. Such results o b s e r v e d with 100 msec stimulus intervals could also be o b t a i n e d with l o n g e r intervals. T h e o n l y c o n d i t i o n to be m e t was t h a t the second stimulus be a p p l i e d before the occurrence o f the late b u r s t i n d u c e d by the first stimulus. In s o m e instances where the latency o f the late b u r s t v a r i e d a p p r e c i a b l y , the second VL pulse was t i m e d in such a w a y t h a t the late b u r s t had a l r e a d y occurred in a b o u t h a l f o f the trials and h a d n o t yet occurred in the o t h e r half. T h e results are illustrated by the h i s t o g r a m s C o f Fig. 3: after a late burst, no early t i m e - l o c k e d spike was ever t r i g g e r e d by the Brain Research, 8 (1968) 255-270

THALAMIC RESPONSESTO LOCAL STIMULATION

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Fig. 3. Post-stimulus histograms illustrating some typical changes in responsiveness occurring when the VL stimuli were repeated. Arrows indicate first and second stimuli. A, Response to first stimulus consisted in one isolated spike, whereas same cell responded to second stimulus by a burst of longer latency (samples of individual records in excerpt). B, Silence produced by first stimulus was deepened and lengthened by second stimulus. Stimulus intervals in A and B: 100 msec. C, Silence produced by first stimulus was followed by late burst of variable latency. Left histogram concerns all traces where second stimulus occurred after the late burst, no early discharge appeared after the second stimulus. Right histogram (same cell) concerns all traces where no late activity preceded the second stimulus, an early discharge time-locked to the second stimulus was triggered in 72 % of the cases. Negativity upward in CRO records. second stimulus, but it occurred 72 ~ of the time if no late burst preceded the second stimulus. This demonstrated that the mode of responding of cells might be experimentally shifted from one class to another. Entry C in Table H concerns those cells fired by the second as well as the first stimulus (class IIl) and exhibiting one or several of the following changes listed as sub-modalities: (a) the latency of the initial burst was lengthened (Fig. 3A), (b) the n u m b e r of spikes in the burst increased, (c) the probability of firing a burst at each trial was higher. These three characteristics are listed together because they are exactly the types of alterations undergone by pyramidal unit responses concomitantly with the appearance of cortical augmenting potentialsl4,15. Effects on the (a) latency, (b) intensity, and (c) probability of firing were no alternatives, they could exist simultaneously. Actually, they often did: most cells counted in (a) also had more spikes in their burst, but for those counted in (b) and (c), the latencies of the responses to the first stimulus were already in the upper values of the group, and they did not increase further upon repetition of the stimuli. The proportion o f class III cells varied from 3 6 ~ in response to a first VL pulse to 47 ~ after a second pulse. This proportion could still be increased by repeating the stimuli more often: some o f the cells listed in Table II under F and G also responded in an augmenting-like manner but required more than two stimuli at 10/sec to do so. Considering globally the n u m b e r o f cells listed under A, B, and C, it appears that the overall effect o f applying the stimuli twice at 100 msec intervals was (1) to Brain Research, 8 (1968) 255-270

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Fig. 4. Distribution of classes of responses in intact preparations as a function of distance of cells from stimulated VL site. Abscissa: distance ranges in ram. Ordinate: relative proportions of each class in each range, computed on the number (N) of cells indicated above each column. At left, responses to 1st stimulus; at right, responses to second stimulus applied 100 msec later. Class 1, no response, unfilled areas; class II, initial silence, hatched areas; class III, initial firing, black area.

produce a more intense firing, (2) by more thalamic neurons, but (3) with a slightly longer delay (Fig. 6). In many cases, the results obtained by pairing VL stimuli were duplicated by pairing VL-pericruciate cortex or pericruciate cortex-VL stimuli as shown in Fig. 2A.

Responses of thalamic cells as a .['unction of their distance from the site o["stimulation The data presented above were analyzed taking into account the relative position of each cell with respect to the site of stimulation. The simplest parameter to handle was distance. Results of this analysis are summarized in the histograms of Fig. 4. The left histogram refers to the situation where a single VL pulse was administered. As could be expected, the highest proportion of initially fired cells (class tII) was found in the region closest to the site of stimulation (between 0.5 and ! mm). More peripherally, their proportion decreased but it was still considerable even between 3 and 4 mm (38 %). Initial bursts recorded distally (farther than 2 mm) did not seem to differ appreciably from those recorded closer (within 2 mm), except for the fact that latencies longer than 10 msec were found only peripherally (Fig. 6). Non-responsive cells were present everywhere, even at less than 1 mm from the stimulating electrode. Most of the initially silenced cells (class lI) were situated between 2,0 and 2.5 mm (Fig. 4); actually, 82 % of them lay at distances greater than 2.0 mm in contrast with only 36 % of the initially fired cells. The probability of finding such an unequal distribution by chance in a population where class II and III cells would be randomly distributed, estimated by Fisher's exact method 4. was P -- 0.0054. Of course, the accuracy of measurements of distance can be questioned, but the possibility of systematic errors would be unlikely. In any event, the validity of our results could also be tested by analyzing responses along the individual tracks where the largest numbers of neurons were recorded, for the order in which cells were successively encountered is

Brain Research, 8 (1968) 255-270

THALAMIC RESPONSES TO LOCAL STIMULATION

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Fig. 5. Distribution of classes of responses along 3 individual tracks in intact animals. A, B and C, The 3 tracks along which the highest numbers of unit recordings were made are represented vertically and the positions of cells encountered are marked by symbols specifying the types of responses. Figures refer to latencies of initial bursts. Large black dots indicate the position of the tip of the stimulating electrode in relation to each track. Data were obtained in three different experiments. Calibration: 1 ram.

a much m o r e reliable i n f o r m a t i o n than estimates o f distances. Such an analysis (Fig. 5) confirmed that most initially silenced cells (class II) were located between a central zone where responsive cells showed initial bursts (class III) and a p e r i p h e r a l zone where the p r o p o r t i o n o f non-responsive cells (class I) became d o m i n a n t . As the p o s i t i o n s o f stimulating electrodes were chosen on the basis o f o b t a i n i n g the largest responses to BC s t i m u l a t i o n and p r o d u c i n g the largest responses on the m o t o r cortex (see Methods), the placements tended to be near the center o f the V L nucleus. Therefore, it is logical to t h i n k that m o r e V L neurons w o u l d be e n c o u n t e r e d closer to than far a w a y from the stimulated site. In fact, this seemed to be the case: T a b l e | I I shows t h a t the percentages o f B C - r e s p o n d i n g units were larger at shorter distances. Cells r e s p o n d i n g to BC stimuli at 0.8-1.5 msec were p r e s u m a b l y m o n o synaptically activated and, therefore, can be identified as VL cells2,12,19. The nature o f the units r e s p o n d i n g with a latency between 1.5 a n d 3 msec is less certain but, TABLE III PERCENTAGES OF THE UNITS, SITUATED AT VARYING DISTANCES, WHICH RESPONDED TO n c

Distances (mm)

0.5 1.5 1.5-2.0 2.0-2.5 2.5-3.0 3.0-4.0

STIMULATION

BC-responding in less than 3 msec

1.5 msec

39 26 26 18 5

31 8 9 4 5 Brain Research, 8 (1968) 255-270

264

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0~2 2-4 4-6 6-t0 10-20

0-2 2-4 4-6 6-1010-20 msec

0-2 mm

2-4 mm

Fig. 6. Histograms of latencies of initial firing in intact preparations. Abscissa: latency in msec. Ordinate: absolute number (N) of cells in each block. VL, responses to first VL stimulus (-histograms in black). VL-VL, Responses to second VL stimulus (hatched histograms). Left, cells located at distances shorter than 2 mm. Right, cells located between 2 and 4 ram.

undoubtedly, some of them were also VL units (as indicated by the occasional recording of a cell responding in more than 2 msec, situated between cells responding in about 1 msec). The fact that BC-responding units were found closer to the stimulated site introduces a bias in the sampling which has to be taken into consideration in evaluating the percentage differences in response modality presented in Table t. It can explain that relatively fewer BC-responding units belonged to class I (unresponsive to VL stimuli). But an important proportion of them belonged to class I1 (initially silenced), indicating that this responsiveness modality is a possibility for some VL as well as for non-VL neurons. The right histogram of Fig. 4 shows the distribution of response classes following the second of two VL stimuli separated by 100 msec. When comparing the two histograms of Fig. 4, the main differences appear to be an increase in the proportion of initially fired cells at all distances and a decrease in the number of initially silenced cells (see also Table II). Increases in latency of initial bursts were observed irrespective of distance (Fig. 6). Unit responses in isolated thalamus

We have mentioned the fact that cortical stimuli could induce in thalamic units the same types of response as those evoked by local thalamic stimulation. This raises the possibility of a cortical mechanism being involved in both situations. Therefore, it seemed interesting to pursue these investigations in animals where the cerebral cortex would be totally eliminated. The neocortex, corona radiata, corpus callosum, hippocampus, and caudate nucleus were removed bilaterally by suction in 3 cats. The exposed diencephalon was covered with mineral oil. The placements of stimulating electrodes in these preparations were less accurate (only once were they in the center Brain Research, 8 (1968) 255-270

THALAMIC RESPONSESTO LOCAL STIMULATION

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of VL) probably because the thalamus had been displaced during the surgical procedure. Thalamic cells seemed to be less spontaneously active in decorticate than in intact preparations, though we have no figures to substantiate this impression. If such was the case, inhibitory effects could be expected to be underestimated. Yet, in 102 cells recorded, the proportion of silenced cells was found to be higher (class II: 20.5 ~,,) than in intact animals (13 %). The proportion of initially fired cells was lower (23.5 ~o versus 36 ~,~), and that of non-responsive cells was about the same (56 ~o versus 51%). The distribution of responsive cells (classes 11 and l lI) as a function of distance was similar to that of intact animals (left histogram of Fig. 7, to be compared with that of Fig. 4). However, there were relatively more silenced cells in the proximity of the stimulating electrodes where they could hardly be found in intact animals (0.5-1 mm from the stimulating electrode).

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Fig. 7. Distribution of classes of responses in decorticate preparations as a function of distance of cells from stimulated VL site. Same presentation as for Fig. 4.

Repetition of the stimuli at 100 msec intervals considerably increased the proportion of cells responding by a burst. The global results illustrated by the right histogram in Fig. 7 resemble very much those obtained in intact animals (Fig. 4). Since there were more units which stopped firing after a single pulse in decorticate animals, the difference between the responses to one and two pulses was still more pronounced when the thalamus was isolated. The mean latency of firing (class Ill) which was already longer in decorticate than in intact animals still tended to increase upon repetition of the stimuli. Unit responses studied in other subcortical structures

I n 1 cat, stimulating and recording electrodes were placed in the medial geniculate nucleus (GM) instead of the VL. Although we did not collect enough data for quantitative evaluation (37 cells), this experiment essentially demonstrated the same phenomena : typical examples of the three classes of cells were found, and changes produced Brain Research, 8 (1968) 255-270

266

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Fig. 8. Post-stimulus histograms of mesencephalic unit responses to local stimulation. In all three cases 2 stimuli applied at 100 msec intervals (arrows). A, Response was a slow progressive increase in firing rate; in black, response to first stimulus alone; line shows continuation of histogram when second stimulus was administered. B and C, Two different unit responses consisting in a primary increase and a secondary decrease in firing rate. C concerns one of the few cells which were totally silenced for a short time. by repetition of the stimuli were similar to those reported for cells within VL or in its vicinity. In sharp contrast, stimulation and recording in the mesencephatic tectum and tegmentum yielded quite different results. Among 76 mesencephatic units studied in 5 cats, not a single one stopped firing immediately after the stimulus. Most:responses to local stimulation consisted of a rather sluggish augmentation o f the rate of firing, eventually followed by a diminution (Fig. 8B and C). Except for a few cases of discharges triggered within 1-2 msec, the latency of firing was rather long (median value: 16 msec). Using the classification adopted for thalamic cells, the distribution of mesencephalic neurons studied within 4 m m from the stimulating electrode was: class I, 73~o; class II, 0 ~ ; class 1II, 27~o. The characteristics of responses (latency, intensity, pattern, etc.) were not modified by repetition of the stimuli in any systematic fashion (Fig. 8A). DISCUSSION

Electrical stimulation of the thalamus is a standard procedure for studying thalamo-cortical relations. In the past, results have often been interpreted with the implicit assumption that each electrical shock triggers a synchronous volley ofimpulses~ as is the case when the shock is applied to a peripheral nerve. But this is only the first in a sequence of events. More recent studies have indicated that the neurons around the stimulated site are affected in a complex mannerr,7,i°,11: interactions probably occur, unit activity passes through different cycles including phases of quiescence which have been previously overlooked. The importance of t h e temporal dimension in patterned activity begins to be recognized and, as this work suggests, the spatial dimension also has to be considered. If it is assumed that the thalamic n e u r o n s t h a t we see discharging, or at least part of them, send impulses toward cortical areas, t h e Brain Research, 8 (1968) 255-270

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activity or inactivity of these neurons at particular instants cannot be overlooked in interpreting the successive components of the cortical responses induced. There is a close resemblance between the present study and that of Marco et a]. 7. The data are in good agreement on the estimated proportions of cells of different types. In both studies, non-responsive cells constituted the most numerous group (slightly more than 50 ~o). Among cells which were fired by local VL stimulation, Marco et al. 7 made a distinction between two kinds of responses: 1 or 2 short latency spikes and long, high frequency trains of spikes after longer delays. Making this distinction is of interest since convincing arguments were presented to regard these cells as inhibitory Renshaw-type interneurons. But analysis of our own data did not reveal the existence of two sharply separate populations: (1) there was no obvious bimodal distribution of either latencies (Fig. 6) or number of spikes; (2) these two parameters were not clearly correlated (e.g. short latency bursts and long latency isolated spikes were not rare); (3) 1 or 2 spike responses could be readily converted to burst responses by simply repeating the stimuli at 100msec intervals. Some of our individual records correspond to Marco's description of VL Renshaw-type cells. However, we have no absolute criterion for deciding how to separate these records from the others. The rarity of very early discharges was rather surprising: in 167 cells recorded in our intact animals, only 10 fired in less than 1 msec and 20 in less than 3 msec. This suggests that the population directly excited by the stimulating current was probably very small. In contrast, the numerous instances of discharges produced after 3 msec indicate the intervention of an interpolated mechanism (e.g. synaptic). In two cases, there was one isolated spike at 1 msec and a burst after 10 msec as if two different mechanisms were involved in succession. There is little doubt that the primary and secondary inhibitions observed (classes II and Ill cells) were of a postsynaptic nature. IPSPs have been recorded in the lateral ventral thalamus under a variety of circumstances~ a,~,v,~0-1z. In many cases, the same type of inhibition was provoked by both cortical and local thalamic stimulation (Fig. 2A). But the persistence of inhibition in completely decorticate preparations indicates that the postulated inhibitory neurons were thalamic. Several possibilities can be considered concerning their location and organization. They can be (1) interneurons dispersed within the thalamic nucleus itselfl,a, 7, (2) neurons from the thalamic reticular nucleus projecting backwards to the thalamus ~3 and perhaps organized in such manner that each sector of the thalamic reticulate region is associated with a particular nucleusZ, or (3) neurons of the midline and intralaminar regions 2,1°,11. It is not possible for us to decide among these alternatives. Our finding that primary inhibition (class 11 cells) was preferentially seen at some distance from the stimulated site has been submitted to controls and statistical tests which have practically ruled out the possibility of experimental artifacts. This suggests that a surrounding inhibition of the type observed in the thalamus by afferent stimulation 1,s can also be produced by local stimulation. Indeed, the primary inhibition (class II cells) appeared to be a function of distance, irrespective of direction around the stimulated site, rather than a feature particular to a given anatomical region. Brain Research, 8 (1968) 255-270

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In decorticate preparations, primary inhibition was seen much closer Io the stimulated site (Fig. 7). The proportion of class 11 cells increased at the expense of class III cells since the total for these two classes was little modified, li is suggested that many cells lost their capacity to respond by firing after decortication, and thus, shifted from class 111 to class II. Actually, such a shift would be practically possible since it occurred under conditions of repetition of the stimuli (Table !1). The reason why total decortication would diminish the propensity for early discharges is open for speculation. Repetition of the VL stimuli at 100 msec intervals brought about drastic changes in the response pattern, which are summarized in Table 1I. The overall effect of repetitive stimulation, artificially reconstituted by pooling together all individual responses, or only those of BC-responding units (1.5 or 3 msec latency), consisted of a more massive volley of impulses triggered with some longer delay after conditioned pulses (Fig. 6). Assuming that these represented impulses ultimately reaching the cortex, their number, grouping, and timing would suffice to explain why an "augmenting cortical potential' would occur and why it would be of larger amplitude, duration, and latency than the primary evoked potentiaH 7. The explanation holds if cortical cells were to respond passively to this increased input, though there is evidence that their reaction is not solely passive~L It is more difficult to interpret the mechanism of some of the changes seen by repeating the stimuli. Why would a second VL pulse trigger a discharge from cells which were silenced by a first pulse and had not yet recovered when the conditioned stimulus occurred? And, also, why would the second pulse increase the latency of the discharge from cells which were fired very early by the first pulse? The postponement of their response (i.e. the suppression of its early part) indicates that the inhibition was still going on a short time following the conditioned stimulus. Clearly, the conditioned response with its characteristic longer latency and duration cannot be attributed to the same mechanism as the unconditioned early firing of the same cells. At first sight, it may be thought that the discharges were simply facilitated, but the hypothesis of a facilitatory process seems incompatible with the finding of an increased latency. What looks like a facilitation could well be a different mechanism: e.g. an active release from inhibition (or disinhibition) as suggested before 14. The postulated process probably is thalamic since the potentiation of the responses by repetition of the stimuli has been observed in totally decorticate preparations. Control experiments performed in several parts of the midbrain showed results clearly different from those observed at thalamic levels. No cells were immediately silenced and very few of them were deeply inhibited by local stimulation. Repetition of the stimuli at 100 msec intervals did not produce the changes typical of 'augmentation' and, indeed, no incremental cortical potentials have ever been reported to result from mesencephalic stimulation. In contrast, the effects of GM stimulation were similar to those of VL stimulation. This suggests that the neuronal properties studied in this report are probably broadly represented throughout the thalamus but not further caudally. Results of unit studies in any cell population to any kind of stimulation most Brain Research, 8 (1968) 255-270

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often turn out in the form: X~o were excited, y ~ were inhibited, z~o did not respond. Only the proportions vary and the only way to find the significance of such data is lo be able to correlate the response modalities with other parameters. One of the most natural, of course, is the particular functional role of neurons in the network (depending, for instance, on whether they are relay cells or interneurons). But our results concerning VL cells identified by their short latency of response to BC stimulation indicate that this is not the only relevant factor. It was found that cells of the presumably same type responded differently, even in an opposite manner, to the local stimulus. In such cases, the response modalities may depend on the relative position of the cells with respect to the stimulated site or to the part of the target hit by afferent impulses. The organization of cervical motoneuron pools provides a good example where proximity could be used as a basis to explain neuronal interaction is. The same proximity principle may apply at other levels of the nervous system, such as the thalamus and the cerebral cortex. We think that topographical analyses can throw some light on heretofore puzzling unit data. SUMMARY

in locally anesthetized cats, unit activity was recorded in the immediate vicinity of a stimulating electrode implanted in nucleus ventralis lateralis of the thalamus (VL). Three classes of cells were distinguished: (I) non-responsive, (ll) immediately silenced, and (Ill) initially fired by the VL stimulus. The latter were also silenced following the initial discharge. Quantitative estimates of their relative proportions are given as a function of distance of the cells from the stimulated site. Immediately silenced cells were found preferentially located at more than 2 mm from the stimulated site. These results concern VL-identified neurons as well as other cells. Upon repetition of the VL stimuli at 100 msec intervals, the responses were changed in such a way that, globally, the second pulse produced a more intense firing, by more neurons, but with a slightly longer latency (augmentation). Similar observations were made while stimulating the VL in decorticate preparations and the medial geniculate nucleus (GM) in intact animals. This is in sharp contrast with the response patterns of mesencephalic cells locally stimulated. None of them were immediately silenced, and repetition of the stimuli at 100 msec intervals produced no systematic changes. The quantitative data gathered in this study are used to reconstruct a picture of the events occurring in a thalamic cell population submitted to local electrical stimulation. ACKNOWLEDGEMENTS

We are much indebted to Mr. D. Dearmore for his technical assistance, and to Mrs. B. Bedard and C. Rucker for the histological preparations. This investigation was supported by Grants NB-04955 and NB 21-633 and, in part, by Grant N B-02501 from the National Institutes of Health. Brain Research, 8 (1968) 255 270

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