Nrurophormacoloy?.,

1975, 14, X69-881.

Pergamon

Press

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COMPARATIVE EFFECTS OF CONVULSANTS THE ANTIDROMIC CORTICAL RESPONSE TO PYRAMIDAL TRACT STIMULATION

ON

R. M. JOY Department of physiological Sciences, University of California, School of Veterinary Medicine, Davis. California 95616 (Accepted 10 April

1975)

Summary-The cortical antidromic response to pyramidal tract stimuli parallels inhibitory postsynaptic potential generation by recurrent collaterals. The modifications induced in this response by topical and systemic administration of strychnine, Metrazol@ and dieldrin were compared. Topical application of 1% strychnine or 10% Metrazol suppressed the response and evoked cortical spiking. With strychnine complete loss of the response occurred without development of sustained, repetitive epileptiform activity. The relationships observed between pyramidal stimulation and spikes suggest that Metrazol and strychnine spikes are not equivalent phenomena. Systemic administration of convulsive amounts of strychnine decreased the response while Metrazol and dieldrin did not alter it. All three compounds shortened the time required for recovery during paired pulse analysis. The data suggest that reduction of recurrent collateral inhibition at pyramidal cells is not a significant contributor to convulsive activities following administration of any of these agents.

Stimulation of the pyramidal tract or of pyramidal cell fibres in the peduncle gives rise to a characteristic response on the pericruciate cortex of the cat (JABBUR and TOWE, 1961; STEFANIS and JASPER, 1964; HUMPHREY, 1968a, b). This potential is the response to the antidromic activation of pyramidal cell fibres. Single stimuli evoke a double positive potential at the cortical surface (JABBUR and TOWE, 1961) which arises in part by the antidromic invasion of pyramidal tract cells. Short trains of 3-6 shocks produce a positive-negative potential, the positive wave arising with a latency of 0.5-l msec. HUMPHREY (1968a) has shown that this ‘part of the response results from a number of events: in part from the antidromic conduction of action potentials in pyramidal tract axons, in part from the subsequent invasion of the soma and proximal dendrites of pyramidal cells, and in part from early excitatory postsynaptic potentials (EPSPs) mediated by recurrent collaterals. The surface negative component of the response, which peaks at about 10-15 msec following stimulation and which persists for 60-80 msec, has been correlated with the simultaneous development of recurrent collateral inhibition within the pyramidal cell population. Laminar field analysis (HUMPHREY, 1968a) demonstrated that surface negativity is associated with a large sink in layer V of pericruciate cortex. This is consistent with the concept that the surface negative wave is the surface expression of inhibitory postsynaptic potentials (IPSPs) generated in pyramidal cells through recurrent collaterals. It is of particular interest that strychnine causes the simultaneous loss of the surface negative wave and the intracellular hyperpolarization of the IPSP (POLLEN and AJMONE-MARSAN, 1965; STEFANIS and JASPER, 1965). The surface negative wave has been used as an indirect measure of recurrent collateral inhibition to study the action of convulsant drugs. VAN DUIJN, SCHWARTZKROIN and PRINCE (1973) have shown that penicillin, like strychnine, reduces or abolishes the surface negative wave following topical application. Convulsants such as dieldrin have no effect upon the surface negative wave (JOY, 1974b) when administered systemically. It was thought important to expand studies of drug effects on the surface negative wave with particular emphasis upon the comparative actions of topical versus systemic application of convulsants. For this purpose we compared strychnine, a convulsant thought to block postsynaptic inhibition in the spinal cord and in the CNS (ECCLES, 869

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R. M. JOY

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and KOKETSU, 1954; ECCLES, 1964; CURTIS, 1962; POLLEN and AJMONE-MARSAN, 1965; STEFANISand JASPER,1965) and METRAZOL@,a convulsant without known action on inhibitory processes (ECCLES,SCHMIDTand WILLIS, 1963; LEWIN and ESPLIN, 1961). We also examined dieldrin, a chlorinated hydrocarbon with convulsive properties similar to those of Metrazol (JOY, 1973; 1974a) in order to determine whether these compounds also produce similar changes in this situation. METHODS Subjects

Thirty-five male cats, 2.5-3.5 kg, were used as subjects. Cats were fasted 12 hr prior to use. Surgical procedures

Cats were anaesthetized with ether, tracheotomized, placed in a stereotaxic frame and maintained with halothane. Cannulae were placed in a brachiocephalic vein for infusion of gallamine, a femoral vein for drug injection and a femoral artery for monitoring blood pressure. After reflecting the skin and muscles overlying the skull, holes were drilled through the bone to place subcortical electrodes. Various sections of bone were removed, as necessary, to expose the cortex, and wells of dental wax were built up around the exposed areas. Subsequently, the dura was removed, and the wells were filled with mineral oil at 37°C to prevent the cortex from drying. At the end of surgery, pressure points and wound edges were infiltrated with dibucaine, and the wound edges were covered with mineral oil and wrapped in saline-soaked gauze. The animal was then paralyzed and artificial respiration begun. A Beckman LB1 CO2 analyzer was used to insure an end-expiratory COZ between 2.8 and 3.2%. Temperature was maintained between 37.5 and 38°C. Vital signs, e.g. blood pressure, heart rate, CO* and EEG were assessed continually to insure a viable and stable preparation. At the end of the experiment the cats were sacrificed with pentobarbital and 25 pm frozen sections were stained with hematoxylin-eosin or Luxol fast blue. Stereotaxic placements and verifications were based on the atlas of JASPERand AJMONE-MARSAN(1954). Data collection

Cortical records were obtained with 0.5 mm chlorided silver ball electrodes. Records were monopolar, the stereotaxic frame serving as reference. Subcortical stimulation and recording were performed with bipolar stainless steel electrodes. Data was amplified and paralleled to a Tektronic 564 oscilloscope for viewing and to a HewlettPackard 3955C tape recorder. Stimuli were rectangular pulses of constant current and 0.1 msec duration derived from a WPI digital stimulator. Unless otherwise indicated, a train of 3 such stimuli at 500 Hz was used to stimulate the pyramidal tract axons. Experimental

procedures

Animals were given 2 hr following completion of surgery to recover from effects of anaesthesia. During this time electrodes were located in the desired areas under stereotaxic and physiological control. After 1.5 hr, stimulation began and was continued without cessation at a rate of 05 per set throughout the remainder of the experiment. This procedure was selected to reduce the effects of habituation or changes in responses due to turning stimuli on and off. At 2 hr, control recording began and this period was extended for as long as necessary to assure stable baselines. This usually occurred within 10-20 min. Once controls were obtained, the convulsant drugs were administered in incremental doses, and data were collected continually on tape until the experiment was terminated. Later the data were played back from the tape and analyzed on a Mnemetron Computer of Average Transients. Stimulus intensity-response amplitude curves were gener-

Convulsantsand pyramidal cells

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ated by averaging 20 responses at a given intensity under a given situation to yield a single datum. Amplitudes of surface negative waves were measured from baseline to peak negative values. Many such data were used to generate the figures. Where statistical measurements are provided, they were determined from four repetitions of the indicated measure in the same animal during control periods. It was not possible to generate equivalent volumes of data after drug administration. Paired pulses were used to determine the recovery of the response to a stimulus administered l&100 msec earlier. The average responses to 20 pairs of pulses were used to establish the extent of recovery at that time interval. Recovery is defined as: (amplitude of second response/amplitude of first response x 100). Repetitions of this process allowed statistical measures to be determined for each subject’s control responses. In all figures vertical lines through control values indicate &2 standard deviations. Administrution

of drugs

Strychnine sulphate was dissolved in saline for systemic or topical application. Doses indicated refer to the salt. Metrazol was also dissolved in saline. For systemic administration, solutions containing 1 mg/kg per min were infused until an effect occurred. This procedure was chosen to produce comparatively stable levels of Metrazol within each analysis period. Convulsions are produced by this technique after about 05 hr of infusion. Dieldrin was solubilized in ethanol and administered so that the total volume of ethanol injected never exceeded 0.1 ml/kg. Strychnine and Metrazol were purchased while dieldrin was a gift from the Shell Chemical Co. RESULTS

ldentijication

of response

The identification of the cortical response was performed in a manner similar to that described by STEFANIS and JASPER (1964). Single stimuli of approximately 1 mA were employed as the stimulating electrode was lowered towards the peduncle. As the electrode entered the medial lemniscus, a typical response appeared on the pericruciate cortex. When the electrode was lowered further, the lemniscal response decremented, then disappeared. Lowering the stimulating electrode another 0.5-1.5 mm induced the appearance of c(and /? waves (JABBUR and TOWE, 1961) on the surface of the pericruciate cortex. At this time the stimulus was changed to a train of 3 stimuli, and the location of maximal negativity was determined on the cortex. The stimulating electrode was lowered further until a threshold response could be produced by stimuli below 0.25 mA. Small adjustments of the cortical recording electrode were sometimes necessary during this procedure. Threshold responses were produced in different experiments with currents of @l-O.25 mA. Finally, stimuli were applied directly to the cortical surface and the presence of a peduncular response at the stimulating electrode verified that it was in the pyramidal outflow. Histological verification of electrode placement was obtained in about one-third of the experiments and demonstrated that the electrode was always in the pyramidal tract. Control

responses

Control studies were carried out in every experiment prior to drug administration. Small variations in amplitude and waveform were observed, but in general the response was stereotyped. The response to a train of 3 stimuli consisted of a positive-negative wave sequence (Fig. 1). The initial positive component originated during the stimulus train and peaked l-2 msec after the last stimulus artifact. A small inflection usually separated the positive and negative components. The surface negative component peaked at 15-20 msec and persisted for 6G30 msec. The peak of the surface negative wave most often possessed a smooth contour, but in a few cases the peak was broadened or notched. The latter waveform was observed in approximately 20% of the experiments.

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25

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msec Fig. 1. Control characteristics of responses evoked by antidromic pyramidal stimulation. Characteristic response of pericruciate cortex is a positive negative wave complex which may be smooth (A) or notched (B) at the peak of the surface negative wave. Calibrations for responses are 20msec and 100 PV. C: Recovery of surface negative wave during paired pulse presentation. Data are the mean +2 standard deviations from 6 different subjects. Recovery is essentially complete in 6G70 msec.

The two types of waveforms reacted equivalently to the administration of the various drugs. Paired pulse analysis indicated that the response to the second of two stimulus trains (test pulse) was depressed by a preceding pulse (conditioning pulse). This depression was maximal at 15520 msec and recovery of the test pulse response to within 20% of initial amplitude occurred over the next 4s-60 msec. The shape of the recovery curve approximated the shape of the surface negative wave. This suggests that the interaction was strictly occlusive in nature with no post-excitatory inhibition of the interneuronal circuit involved (HUMPHREY, 1968a) at least over 100 msec. In&fence of level of arousal Spontaneous changes in the level of consciousness were associated with changes in amplitude and duration of the surface negative wave. Experimental periods characterized by low voltage, desynchronized EEG activity were associated with larger, shorter duration surface negative waves than periods characterized by slow wave and/or spindle EEGs. This is demonstrated in Figure 2A, where surface negative wave amplitudes to various stimulus intensities during different EEG periods are compared. At all tested stimulus intensities, the surface negative wave was reduced when the EEG was of a slow wave-spindle nature. Surface negative wave variability was increased during the latter situation. To reduce the influence of this variable, data obtained during EEG periods dominated by spindles or slow waves were discarded. This represented 25-50x of the experimental control period. Injluence of anaesthetics Administration of pentobarbital resulted in a dose-dependent depression of the surface negative wave. In Figure 2B, response amplitudes are plotted after pentobarbital as a function of stimulus train length. Pentobarbital, 10 mg/kg, affected responses to short stimulus trains much more severely than responses to long stimulus trains. Increasing the amount of pentobarbital led to further depression of the response at all train lengths. Anaesthetic levels reduced surface negative amplitudes to 2550% of control. Amplitude reduction was associated with an increased duration of the surface negative wave to a maximum of about 100 msec. Paired pulse analysis revealed that recovery of the surface negative response was also depressed by 10 mg/kg pentobarbital (Figure 2C). Test pulse amplitudes at peak

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Fig. 2. Changes in surface negative waves induced by changes in consciousness and anaesthetics. A: Relationship between surface negative wave and EEG state. Solid line depicts surface negative wave amplitudes in arbitrary units as a function of stimulus intenisty during periods of low voltage, high frequency EEG. Dashed line shows surface negative wave amplitudes during slow wave or spindle EEG periods. Abscissa: wave amplitude; Ordinate: stimulus currents in threshold units. B: Influence of pentobarbital. Surface negative waves in arbitrary units are graphed as a function of the number of stimuli in each train. All stimuli were 3 times threshold. C: Recovery of surface negative response after pentobarbital.

depression were only 50% of control level. Recovery was prolonged from 60-100 msec. The correspondence observed during the control period between surface negative wave duration and recovery of test pulse responses was observed with pentobarbital as well. The administration of pentobarbital resulted in recovery functions similar to those reported by HUMPHREY (1968a) in anaesthetized preparations. EfSect of topical

strychnine

Topical application of strychnine caused an abrupt, reversible depression of the surface negative wave. In the experiment shown in Figure 3, a 2 mm diameter cotton pellet was saturated with 1% strychnine sulphate and then placed on the left pericruciate cortex directly adjacent to the recording electrode. Another cotton pellet saturated with 0.9% NaCl was similarly placed on the right side. Stimuli were delivered alternately to both peduncles so that the comparative responses could be evaluated simultaneously. On the cortex receiving strychnine, surface negative wave amplitudes fell progressively until the response had disappeared. This effect took 3-5 min. The cotton pellet was removed after 15 min and the cortex was washed twice with artificial cerebrospinal fluid. Recovery of the surface negative wave occurred during the next 30 min. A final amplitude of 60% of control levels was achieved. Responses on the right cortex were slightly diminished during the period in which the pellet was in contact with the cortex. Amplitudes quickly returned to 100% levels after removal of the saline pellet. The reduction of the surface negative wave by strychnine corresponded to the period of occurrence and to the intensity of cortical strychnine spikes. A reciprocal relationship was observed between spike frequency and surface negative amplitude. Spikes were frequently driven by the stimulus, but only after a latency of 8&120 msec. Figure 3 also indicates that prior to strychnine, the surface negative wave was followed by a pronounced late wave with a latency of g&100 msec. This wave has been correlated with corticofugal discharge (HUMPHREY, 1968a). Spikes arising after strychnine appeared at

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Fig. 3. Effects of topical strychnine. A: Effect of strychnine on surface negative wave amplitude. Solid line represents cortex receiving strychnine (applied during the time indicated by the solid bar over the time line) and dashed line represents control cortex. Bottom portion of graph indicates the frequency of strychnine spikes during the same time period. B: Spikes evoked by antidromic stimulation. Eight responses before strychnine (top). Eight responses after strychnine showing the confinement of spike discharge (bottom). Calibrations for responses are 20 msec and 200 mV. C: Frequency histogram of spikes originating at various times (msecs) after antidromic stimulation. The horizontal line denotes mean spike frequency per time period.

about the same time or somewhat later than the appearance of the late wave during the control. No short latency spikes were observed after strychnine. In anaesthetized cats strychnine spikes were not triggered by the antidromic stimulus (POLLEN and AJMONE-MARSAN,

1965).

On the control side, responses were evoked by the strychnine spikes that developed on the contralateral cortex. Interaction between these effects and the stimulation applied to the peduncle may account in part for the moderate reduction in control surface negative wave amplitude. Stimulation of the contralateral peduncle did not drive spikes on the strychninized cortex. Effect of systemic

strychnine

The gradual administration of strychnine in doses up to those initiating seizures (0.2-l mg/kg) was accompanied by a moderate depression in surface negative wave amplitudes (Figure 4). Figure 4A indicates that preconvulsive doses (0.2-0.4 mg/kg) caused a progressive depression in evoked amplitude. A seizure was precipitated shortly after administering 0.6 mg/kg, but 15 min later the surface negative wave had recovered to preseizure values. In 5 subjects administered strychnine, the mean surface negative wave amplitude just prior to a seizure was 83% + 7% (sr + S.E.) of control. This value contrasts markedly from the nearly complete abolition of the surface negative wave by topical strychnine in amounts insufficient to induce sustained convulsive activity.

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msec Fig. 4. Systemic strychnine and the surface negative wave. A: Changes affected by strychnine in surface negative wave amplitudes (abscissa) as a function of stimulus intensity (ordinate). Values at 0.6 mg/kg were obtained 15 min after a spontaneous seizure precipitated by the injection. B: Effect of strychnine on surface negative wave recovery. Recovery is more rapid after strychnine. Second response amplitudes at paired pulse intervals of 4G50msec were frequently greater than first response amplitudes, a situation rarely observed during control.

Paired pulses were used to determine whether strychnine altered recovery of the surface negative wave response. The experiment shown in Figure 4B is typical of the effects observed. Strychnine consistently decreased the duration of the recovery function. Test responses of more than loo’/, were frequently observed after strychnine. Such facilitation was observed during control recording in only 1 of 28 subjects. Changes in the shape of the surface negative response were consistent with the changes observed during recovery. The duration of the surface negative wave was shortened, and a period of heightened positivity in the cortical response from 40-50 msec accompanied the development of facilitation in the recovery function. Eflect

oftopical Metrazol

Various concentrations of Metrazol were examined for their effect on the surface negative wave (Figure 5). Cotton pellets saturated with Metrazol or saline were placed adjacent to the recording electrode and left in place 2-3 min. Solutions of Metrazol from 2-10% depressed the surface negative wave. A 10% solution reduced the response by 50%. Amplitude changes were paralleled by the development of spikes. Like strychnine, Metrazol spikes could be driven by antidromic stimulation. However, driving was never intense, and the latency for Metrazol spikes was much shorter (30-50 msec) than for strychnine spikes. The relationships between the surface negative waves and spikes were examined in more detail. Amplitude histograms derived from 300 control surface negative responses and 300 responses after 10% Metrazol application revealed that Metrazol decreased the mean amplitude while increasing the dispersion of amplitudes. Further analysis revealed that small amplitude surface negative responses were most often accompanied by spike driving. Intermediate amplitudes were accompanied by spontaneous, non-driven spikes while the largest amplitude surface negative waves were noted when there were no spontaneous spikes. Thus, the probability of spike driving was inversely related to the amplitude of the preceding surface negative wave. EfSect of systemic Metrazol When Metrazol was infused at a rate of 1 mg/kg per min, no significant changes in surface negative amplitudes were observed until a generalized seizure occurred (Figure 6A). Immediately following a seizure, amplitudes were depressed, but quickly returned to control amplitudes. In 5 subjects administered Metrazol, the mean surface negative wave amplitude just prior to a seizure was 98% f 4% (X k S.E.) of control.

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Fig. 5. Effects of topical Metrazol. A: Effects on surface negative wave amplitudes, Amplitudes were determined during successive applications of Metrazol and washing of the cortical surface with artificial cerebrospinal fluid. Surface negative waves, expressed as ‘A of control, were reduced by Metrazol. Bottom of graph indicates frequency of occurrence of spikes during the experiment. M2, 5, 10: Metrazol 2%, 5x, 10%; W: wash, S: saline. B: The distribution of surface negative wave amplitudes during control (top) and after 10% Metrazol application (bottom) are compared. Abscissae represent amplitudes in PV. Eight responses before (C) and after (D) Metrazol 10% are compared. Metrazol spikes were generated with a much shorter latency than after strychnine. Response calibrations: 20 msec, 400 pV. E: The latency (msec) of Metrazol spikes.

Recovery functions were, however, consistently altered by Metrazol. Subjects displayed a greater peak depression of the test pulse response after Metrazol. In the example shown in Figure 6B, three consecutive recovery cycles were obtained during the infusion period. Peak depression and subsequent recovery were progressively enhanced. Changes similar to those shown in Figure 6 were observed in 4 of 5 subjects. In the other subject no changes occurred during the infusion period. Effect of dieldrin On three occasions a saturated solution of dieldrin in mineral oil was applied to the cortex. No changes were observed in spontaneous EEG activity or in surface negative 0

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msec Fig. 6. Effects of systemic Metrazol. A: Effect on surface negative wave amplitude. B: Effect on recovery of surface negative wave to paired pulses. Numbers, 1, 2, 3 indicate series taken at different times during constant infusion of 1 mg/kg per min. Series 1 taken at 15 min; 2 at 25 min; and 3 at 35 min after infusion began.

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Fig. 7. Effect of systemic dieldrin. Effect of dieldrin on surface negative wave amplitudes (A) to peduncle stimulation and on pyramidal cell responses to lemniscal stimulation (B). Ordinates are stimulus intensity in threshold units and abscissae are wave amplitudes. C: Alteration of surface negative wave recovery by dieldrin. Recovery is shortened.

waves. It is not known whether this resulted from the inability of dieldrin to pass from the mineral oil through the aqueous layer lying over the cortex and eventually into the cortex or to a lack of direct effect of dieldrin in the cortex. The extreme insolubility of dieldrin in aqueous solution makes the first alternative most probable. However, it is interesting that the same quantity of dieldrin-saturated mineral oil, when injected intravenously, produced convulsions in 2-5 min. Systemic administration of dieldrin affected the surface negative wave similarly to Metrazol (Figure 7). Evoked amplitudes were not consistently altered at doses up to those precipitating seizures. Responses were abolished immediately after a generalized convulsion, but they recovered within 1@15 min to control values. In 6 subjects the mean surface negative wave amplitude just prior to the first seizure was 97% + 5% (s7+_ SE.) of control. The excitability of pyramidal tract neurones to sensory stimuli was greatly increased at this time. When the lateral lemniscus above the penduncles was stimulated, pyramidal tract responses (and pericruciate responses) were increased 2-3 fold (Figure 7B). Lemniscal stimulation consistently drove large positive-negative spikes in the pericruciate cortex before a seizure. Peduncular stimulation did not drive such spikes, although spontaneous spikes were occasionally seen. Paired pulse analysis revealed that dieldrin altered test pulse recovery similarly to Metrazol. Peak depression was increased while recovery was shortened. Some facilitation of the response was noted from 30-50 msec. This was not a consistent finding in all experiments, however. Changes in surface negative waveform correlating with change in recovery function were observed. The period of increased recovery was associated with an increased positivity in the declining phase of the surface negative wave. DISCUSSION

Interest in drug effects on the pyramidally evoked surface negative wave stem from its relationships to the recurrent collateral inhibition evoked by the antidromic activa\.I’.

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R. M. JOY

tion of pyramidal fibres. Before discussing the data, it is important to consider how exact this relationship is. Is the surface negative wave representative of and produced by cellular IPSPs induced by recurrent collaterals? The studies of PHILLIPS (1959) and, ,in particular, of STEFANIS and JASPER(1964) have demonstrated that antidromic stimulation of pyramidal fibres can produce antidromic cell discharges, EPSPs and IPSPs in pyramidal tract neurones. In particular, the IPSPs were of long duration, often 100 msec or more. POLLEN and AJMONE-MARSAN (1965) subsequently demonstrated that cellular IPSPs and the surface negative wave corresponded both in terms of latency and peak response time. They also found that topical application of 1% strychnine produced a decline of both the surface negative wave and cellular IPSPs. The detailed studies of HUMPHREY(1968a, b) on the antidromic cortical response provide further evidence for IPSP origin of the surface negative wave. He demonstrated that there is a parallel increase in the amplitudes of IPSPs and the surface negative wave as the number of stimuli in the stimulus train is increased. The recovery of the surface negative wave also paralleled the recovery of IPSPs during testing with paired pulse trains. Both of the above functions possess the same time course as the inhibition of orthodromic activation of pyramidal tract cells produced by conditioning antidromic volleys. Further, the laminar analysis carried out by HUMPHREY(1968a) on the surface negative wave indicated that it was associated with a large positive source in the deep layers of the pericruciate cortex. This is consistent with the generation of the surface negative wave by IPSPs in deep-lying somatic and proximal dendrites. The variability of the surface negative wave, its susceptibility to anaesthesia and asphyxsia, its long latency to peak, and its long duration are consistent with a postsynaptic response. The amplitude of the surface negative wave is not depressed by topically applied yaminobutyric acid. y-Aminobutyric acid does depress cortical surface negative events associated with excitatory potentials generated in the superficial layers of the cortex (HANCE, WINTERS, BACH-Y-RITAand KILLAM, 1963). This preponderance of evidence has led to a general acceptance of the concept that the surface negative wave is a surface expression of IPSPs generated in pyramidal tract cells in the underlying cortex. Is the amplitude of the surface negative wave proportional to the summed IPSP activity in the population of pyramidal tract cells underlying the electrode? Changes in the amplitude of the surface negative wave should be reflective of changes in (a) the number of pyramidal tract cells receiving IPSPs, (b) the amplitudes of the IPSPs in individual cells, and (c) the synchrony of the process. There is ample data in support of this concept, at least during the lightly anaesthetized state. POLLENand AJMONE-MARSAN(1965) noted parallel changes in IPSP and surface negative wave amplitudes with changes in stimulus train lengths as well as after topical strychnine. They indicated, however, that the rate of decline of the surface negative wave was more rapid than that of IPSPs following strychnine. They suggest that such differences were probably accountable on the basis of attenuation of the electrotonic spread of hyperpolarization along fine apical dendritic shafts or, alternatively, was the result of nonspecific attenuation of potentials produced by the drops of strychnine solution around the electrode. HUMPHREY(1968a, b) also noted parallel changes in surface negative wave and IPSP amplitudes as a function of stimulus train length. Further, parallel changes in amplitudes of the second of two successively evoked IPSPs and simultaneously recorded surface negative waves as a function of the interval between stimulus trains were observed. Is the recovery function of the surface negative wave indicative of the mean excitability state of the total pyramidal tract cell population? Sufficient evidence is available to propose that the recovery function of the surface negative wave, in normal circumstances, is indicative of pyramidal cell excitability during the interval following the conditioning stimulus. This is primarily an effect of the IPSP produced by the conditioning stimulus. HUMPHREY(1968b) has provided evidence that over the time range of 2G-100 msec, inhibitory interneurones are capable of free response and are not themselves refractory or subjected to inhibition. The smooth recovery phase of the IPSP and the similarly

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smooth recovery function of the surface negative wave suggest that no additional significant consequences of the antidromic stimulus develop within the pyramidal cell population over this time interval. The correlations between the time courses of the surface negative wave, and the recovery functions of the surface negative wave and the IPSP, and recurrent inhibition as determined by reflex testing procedures (HUMPHREY, 1968b) are consistent with the concept that interactions between two successively evoked IPSPs are of an occlusive nature. Recovery of the surface negative wave with time using paired stimuli is an indication of the return of the membrane potential in an “average” pyramidal cell from its hyperpolarized condition. These concepts cannot be strictly adhered to, of course, after convulsant drug administration, but they provide a basis for interpreting recovery functions. STEFANISand JASPER (1964) have dealt in detail with the possible contamination of the antidromic response with electrodes placed in the peduncles for stimulation. Major sources of potential orthodromic input onto the pyramidal cell population stem from current spread to the overlying medial lemniscus or as a secondary consequence of the simultaneous orthodromic pyramidal volley synapsing at various subcortical sites which project back to the pyramidal cells. In our experiments there were no indications of lemniscal contribution. In a few instances the initial placement of the stimulating electrode was inadequate, and pyramidal and lemniscal axons were simultaneously activated. This produced a complex potential on the pericruciate cortex which was easily distinguishable from the pure pyramidal response. Repositioning of the electrode resulted in the typical pyramidal response. Additionally, lemniscal input is greatly enhanced by dieldrin and Metrazol (JOY, 1974a; OKUMA, 1960). Recording electrodes over somatosensory cortex indicated no such enhancement to peduncle stimulation when these drugs were administered. Lemniscal stimulation also evokes dieldrin and Metrazol “spikes” in preconvulsive doses. No such events were produced by stimuli from the electrodes in these experiments. The possibility that orthodromic pyramidal activity secondarily activated subcortical systems which project back to pericruciate cannot be ruled out. However, the shape of the surface negative wave and its recovery function during control periods were smooth, providing no evidence of an important contribution from this source. The recovery of the surface negative wave to the second of a pair of stimulus trains was more rapid in these experiments than in those of HUMPHREY (1968b). However, our subjects were immobilized and locally anaesthetized whereas Humphrey used subjects which were under pentobarbital. The administration of pentobarbital increased the duration of recovery in our preparations as well as decreasing the atiplitude of the surface negative wave. A similar reduction was found when subjects were drowsy. These findings substantiate those of others (SUZUKI and TUKAHARA, 1963; TAKAHASHI, KUBOTAand UNO, 1967; STEFANISand JASPER,1964). Topical administration of strychnine and Metrazol Topical application of both these compounds reduced the amplitude of the surface negative wave. The concentration of 1% strychnine produced a total loss of the surface negative wave, whereas 2-10% solutions of Metrazol produced a gradual reduction to 50%. The effects of strychnine could be predicted from previous observations LANDAU, 1965; POLLENand AJMONE-MARSAN, 1965; PURPURAand GRUNDFEST,1956; VANDUIJN et al., 1973). Both strychnine and Metrazol induced the reduction of the surface negative wave in proportion to the intensity of “spikes” that developed after their application. The spikes could be driven by the peduncular stimulation, though the characteristics of the driving were different for both drugs. This contrasts with the inability of peduncular stimulation to drive strychnine spikes reported by POLLEN and AJMONE-MARSAN (1965). Their subjects were under general anaesthesia, however, whereas our subjects were not. The strychnine spikes driven by peduncular stimulation appeared with a latency of 8G-120 msec, a period corresponding more or less to the late-wave (HUMPHREY,1968a)

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R. M. JOY

normally accompanied by pyramidal cell facilitation. No spikes were evoked by the stimulus between &50 msec from the onset of the stimulus. The frequency of spikes during this time interval was at or below that predicted for a random process. The distribution of evoked latencies may or may not be related to an alteration of postsynaptic, recurrent collateral inhibition. The paucity of spikes between O-50 msec and the late peak in spike probability may represent an increase in spike probability resulting from the wearing off of an inhibitory event. The duration of an “average” IPSP (HUMPHREY, 1968b) and the time to peak spiking probability are approximately equal. The studies of POLLEN and Lux (1966) have demonstrated that the changes in membrane conductance induced in pyramidal cells by antidromic stimulation persist after strychnine even though the hyperpolarization has disappeared. It is possible, therefore, that inhibitory function may be in part preserved even though hyperpolarization and the surface negative wave is absent. Systemic

eflects of strychnine

Intravenous administration of strychnine resulted in only a l&l 5% decrease in surface negative wave amplitude at the time of the first spontaneous convulsion. This is consistent with the propensity of strychnine for areas other than cortex. The recovery functions obtained at the same time indicate that IPSP generation and interneurone activity are not blocked by strychnine. The more rapid recovery after strychnine may be representative of increased depolarizing pressures on pyramidal cells in the form of increased EPSP activity, from disinhibition, or from nonspecific changes in pyramidal cell membranes. In any event, these data do not suggest that alterations in recurrent collateral inhibition play an important role in the convulsive response of cortex to systemically administered strychnine. Facilitatory actions of strychnine (MANN and TOWE, 1974) on pyramidal cells or an enhancement of excitatory input to cortex are likely to be more important. Systemic

eflects of Metrazol

and dieldrin

Neither of these compounds affected the amplitude of the surface negative wave on systemic administration. Recovery functions indicated that interneurone activity related to the recurrent collateral process was not depressed by either drug. The failure of Metrazol to alter cortical recurrent collateral inhibition is consistent with its lack of effect on other types of inhibition (LEWIN and ESPLIN, 1969; ESPLIN and ZABLOCKAESPLIN, 1969). The mechanism of the convulsant properties of dieldrin in the mammalian CNS are not known, but the many similarities that exist between Metrazol and dieldrin (JOY, 1973; 1974a) suggest they may be similar. In these experiments, at least, the compounds were not distinguishable in action. The only effect that these drugs did produce was a shortening of the recovery function during paired stimulus presentation. As was indicated for strychnine, this change is consistent with an increased depolarization pressure representing increased EPSP activity, disinhibition, or nonspecific changes in pyramidal tract cell membranes. JOY (1974a) has presented evidence that extracortical actions of dieldrin are very important in determining cortically recorded effects. In fact cortical “spikes” to systemic dieldrin appear to be initiated at an extracortical origin (JOY, 1974~). Similar concepts have previously been established for Metrazol (HUNTER and INGVAR,1955 ; GASTAUT,1969). An important extracortical action of all three compounds is reinforced by the distinctly different effects that have been reported from topical versus systemic administration. Acknowledgements-The author wishes to thank PATRICIA J. ANDERSON for her technical, histological, artistic assistance. The contribution of dieldrin by Shell Chemical Co. is gratefully acknowledged.

and

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and pyramidal

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881

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