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Observations on strychninised isolated cortex
O B S E R V A T I O N S O N S T R Y C H N I N I S E D ISOLATED C O R T E X M. K. WRIGHT,M.Sc., W . K. ANDREW,B.Sc. a n d I. JACOBSON,B.Sc. Department o~ Anatomy, Witwatersrand University and the Neurosurgical unit, $ohannesburg General Hosl>ital, Johannesburg, South A~rica (Received for publication: September 7~ 1953) (Resubmitted, August 10, 1954) INTRODUCTION
The experiments described herein were originally planned to test the hypothesis that strychnine applied to a region of grey matter containing synapses would not elicit spikes in the absence of spontaneous electrical activity. Cerebral cortex isolated but for its blood supply was chosen for the purpose. The vitality of the isolated cortex was tested by electrical stimulation and it was then found that strychnine spikes could only be obtained from isolated cortex if it were artificially stimulated after strychninisation. The frequency at which spikes could be thus evoked, as well as the duration of a spike series that could be maintained, were found to bear a marked resemblance to the frequency and duration of the "spike and wave" pattern in petit real. This resemblance was followed up in more detail and the results form the basis of this communication. Burns (1950, 1952) has described cortical potentials after electrical, stimulation of isolated cortex but it has been found in this research that the effect of strychnine alters many features of the potentials thus evoked. Chang (1951) has stimulated stryehninised cortex electrically but the cortex was not isolated. The findings of both the above authors can, however, be correlated with the results reported herein. MATERIAL AND METHODS
the posterior half of the suprasylvian gyrus involving variable proportions of areas 21, 7 and 19 (Garol 1942). In one experiment the isolated cortex lay in the ectosylvian gyrus medial to the posterior ectosylvian fissure while in two others it lay on the convexity of the occipital pole. A curved strip of thin celluloid, which slid harmlessly beneath the pial blood vessels, was used initially to make the cuts perpendicular to the braia surface. In later experiments a curved steel knife with a blunt dorsal edge was devised. The undercuts were made with a thin pliable steel strip, 8 mm. wide, with a sharp leading edge. The site of each isolation included prominent pial arteries and veins and no damage was done either to these vessels or to the larger vessels in the neighbouring fissures into which they drained. At the conclusion of an experiment the vessels overlying the isolated cortex were opened to test for continued blood flow. Finally the brain was fixed in 10 per cent formalin and a block of cortex was removed after the whole brain had hardened. In 7 cases 20~, sections at 200~ intervals were stained with carbol fuchsin to confirm the success of the isolation and the area of acute neuronal damage in the isolated area. In the remaining experiments, in which the special steel knife was used, freehand sections of the hardened cortex were examined. Warm liquid paraffin was applied to the exposed brain when the dura was first opened. The excess paraffin was only temporarily removed during the isolation procedure. Bipolar recordings were made from the experimental cortex before it was isolated from the adjacent grey matter, after it was isolated and finally after it was isolated and undercut. Silver wire electrodes between 2 and 3 mm. apart were used. Bipolar or unipolar records from an adjacent gyrus were
Eight mature cats, anaesthetised by intraperitoneal injection of 35 to 40 rags. of veterinary Nembutal per kilogram of body weight, were used in these experiments. Cortical isolations were carried out in both hemispheres in 3 cats and 2 separate isolations were done on one side in 2 animals. The isolated cortex measured approximately 15 ram. x 5 ram. and in 10 cases lay in [ 635 ]
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M.K. WRIGHT, W. K. ANDREW and I. JACOBSON
obtained simultaneously with all records from u n d e r c u t cortex. The indifferent electrode was placed between temporal muscle and bone. A 2 channel M a r c o n i biological amplifier was used. The pen recorder was linear to 75 c/sec. ; the time constant was kept at 0.1 sec. and the attenuation was adjusted for each electrode placement such that strychnine spikes caused minimal pen overshoot peaks in the records. When recordings from unstrychninised cortex were taken and whenever the absence of cortical potentials was suspected, the sensitivity was increased to 100 ~V. and 50 ~V. per cm. deflection respectively. P a p e r speeds of 1.5, 3.0 and 6.0 cm/sec, were used as indicated in the figures. Local strychninisation with filter paper squares or triangles of less than 2 mm. side were applied in the manner described by McCulloch (1944). The concentration of strychnine used was 1.25 per cent. A thin layer of liquid p a r a f f i n over the brain does not interfere with local permeation of strychnine into the cortex. Electrical stimuli were derived from a t h y r a t r o n stimulator designed by N. C. Johnstone. Single, dual (with variable interval) and repeated condenser discharges of short duration were obtainable. These pulses were delivered through a high impedance coupling transformer across the secondary winding of which was placed a 10 k. potentiometer with the sliding contact grounded so that stimulus artefacts could be reduced or rendered monophasic, thus ensuring that they were not confused with strychnine spikes. Stimuli between 1.5 and 12 V. were applied through bipolar silver electrodes, both of which were on the isolated cortex. OBSERVATIONS
A. Isolated and undercut cortex. This group consisted of 11 experiments on 7 cats. In 2 experiments there was a small bridge of white matter joining the isolated with the adjacent cortex of the same gyrus, but if these fibres were functional they were not sufficient to change the characteristics of completely isolated cortex. Recordings were
generally taken from several parts of each isolated region during the course of om ~ experiment.
Spontaneous activity. Spontaneous potentials never occurred after the undercutting procedure, which was always the final step in the isolation of a cortical strip. The absence of spontaneous activity continued for between 2 and 5 hours before an experiment was terminated. This was in marked contrast to the appearance of such activity in 2 f u r t h e r experiments in which the cortex had an equally severe handling but in which either the undercutting or the vertical isolation failed by a margin of only a few square millimetres in area. Thus surgical trauma or avascularity with prolonged neuronal depression was not a sufficient explanation for the inactivity of u m stimulated acutely isolated cortex. The absence of spontaneous activity depended neither on the area of the isolate nor on the depth of the u n d e r c u t f r o m the surface. There were at least 5 experiments in which all cortico-cortical connections within the isolated strip must have been intact such that even a small active zone within the isolate could have affected its neighbouring cortex by means of fibres passing beneath the grey matter. Of the possible cytoarchitectural areas included in most of the isolated strips, only area 19 has limited intra-areal connections as revealed by strychnine neuronography (Garol 1942), and since no isolate was limited to area 19 it appears that the integrity of local cortieo-cortical connections does not relieve the electrical silence of acutely isolated cortex.
Local strychninisation of isolated cortex. Typical strychnine spikes were always recorded until undercutting was completed, after which spikes occurred only on very rare occasions when undue movement of the brain probably caused mechanical stimulation by one of the electrodes. On one occasion intracortical injection of strychnine produced slight muscular twitchings in the limbs but failed to evoke spikes from the isolated cortex.
OBSERVATIONS ON ISOLATED CORTEX
637
Electrical stimulation of isolated cortex with single pulses.
not much larger than the threshold response was reached.
Electrical stimulation with single pulses of brief duration produced neither after discharge nor temporary return of spontaneous activity in isolated cortex. Nonetheless comparable electrical stimulation of isolated and strychninised cortex evokes typical strychnine spikes. The occurrence of strychnine spikes following single electrical pulses was always a threshold phenomenon in the sense that there was a narrow range of approximately 0.2 V, within which a stimulus either produced nothing but its own artefact or else produced an artefact which merged with the first phase of a tri- or tetraphasic strychnine spike of much higher amplitude and longer duration. The precise threshold value with bipolar silver stimulating electrodes between 2 and 3 mm. apart was constant for a series of stimuli in any particular experiment and electrode placement, but it varied between 3.0 and 6.0 V. in different animals or in the same animal with different electrode placements. Electrical pulses, to be effective, must be applied within the isolated region not more than about 1.0 cm. from the strychninised zone. At distances greater than between 3 and 5 ram. from the strychnine, the stimulus threshold increased with distance but no precise relationship between the two could be established. The area of cortex strychninised had no consistent effect on the threshold of electrical stimulation. Spikes could be evoked for about half an hour after a single application of strychnine, but thereafter the threshold showed marked variations and then rose rapidly such that the effectiveness of electrical stimulation was often lost in a period of seconds rather than minutes. On re-strychninisation of the same site spikes could be evoked again. For some time after strychnine was applied to a particular site the spikes evoked usually followed the all or none principle. At other times, an increase in the stimulus increased the amplitude but not the form of the subsequent spike until a maximum response
Repetitive stimulation of isolated cortex. During the course of each isolation experiment the cortex was stimulated on numerous occasions at frequencies of 1, 2, 3.5, 7 and 10/sec. In one case the frequency was increased to 25/sec. In no instance did afterdischarge or regeneration of spontaneous activity occur, although the repetitive volley was sometimes maintained for 10 sec. With stimuli of threshold intensity as tested by single pulses, strychnine spikes were always evoked at pulse frequencies of 1/sec. and usually also at 2/sec. (fig. 1A). In seven experiments 3.5/sec. stimuli produced spikes at only every second pulse (fig. 1B). In 4 experiments 3.5/sec. volleys evoked spikes which followed the stimulation rate (fig. 1C), but even in these experiments 3.5/sec. strychnine spikes were only found with some of the stimulation sequences. At 7/sec. the maximum spike to pulse ratio was 1:2 (fig. 1D). The common observation with 7/sec. and higher frequencies was that a spike was produced at the initial stimulus and at variable intervals thereafter, though in such cases the time interval between spikes was longer than 0.5 sec. An increase in stimulus intensity generally had no effect on the amplitude of spikes evoked with repetitive stimuli, and in only two cases was the spike repetition rate increased from 2/sec. to 3.5/sec. Even stimulation at three times threshold strength never succeeded in " d r i v i n g " strychnine spikes at more than 3.5/sec. in any experiment. At all frequencies the individual spikes in a repetitive series were practically identical in amplitude and wave shape, but the first spike in a volley evoked soon after .strychninisation was occasionally up to 25 per cent greater in amplitude than the subsequent spikes. When spikes were driven at frequencies of 2 and 3.5/sec. the volley could only be maintained for periods varying between 7 and 30 sec. The termination of a volley of spikes was always sudden and unaccompanied by
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M.K.
WRIGHT, W. K. A N D R E W and I. JACOBSON
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Fig. 1 Repetitive stimulation of isolated cortex. Spikes recorded from closely spaced bipolar electrodes on brain surface, with time constant at 0.1 Second. Such conditions tend to render the second component of the spike diphasie and also reduce the duration of the third component. A . - D. Strychnine spikes evoked with the stimulus frequencies marked on the right of each strip. (S marks stimulus artefact in each record). E. Beginning of a series of evoked spikes (left) and end of the same series ( r i g h t ) . F. 3.5 per second spikes at the beginning of a series (left) ; transition to 1.75 per second (middle) and established 1.75 per second ( r i g h t ) .
OBSERVATIONS ON ISOLATED CORTEX amplitude decrement in the ultimate spikes (fig. 1E). A volley at 3.5/sec. occasionally d r o p p e d to 1.75/sec. for a variable time before terminating but at times it reverted to a frequency of 3.5/sec. before the end. The most constant, but not invariable, sign of approaching termination of a volley was an increase in the latency of the spikes; usually the stimulus artefact merged completely with the first phase of the spike, but towards the end of a volley the stimulus artefact and spike were joined by an increasingly prominent notch which, however, never reached the isoelectric line (fig. 1E). At the same time the first phase of the strychnine spike tended to diminish. This increase in the latency of the spike was not dependent on the relative positions of the stimulating and recording electrodes, for it was observed when the two sets of electrodes were separated from each other by distance varying between 1 and 5 mm.
Once a volley of spikes had failed it never recurred if the volley of electrical pulses continued, but if the stimulus volley was broken the strychnine spikes reappeared with the resumption of the stimulation. Figure 1F shows the transition in frequency, from 3.5 to 1.75 spikes/sec., that occurred in a typical case. During the transition there was a marked amplitude decrease and irregularity in all phases of the strychnine spikes though the latter returned to normal as soon as the lower frequency was established. This indicated that fatigue was not the reason for the termination of a volley, for if it were so, the spikes should not have become fully re-established at half the stimulus frequency. The abrupt manner in which all strychnine spike volleys terminate is also unlike a fatigue phenomenon. The probable mechanism determining both the frequency and duration of a spike volley will be discussed later.
Dual stimulation of isolated cortex. Dual stimuli of two thirds threshold intensity and less than 18 msec. interval evoked strychnine spikes following the second stimulus in only 3 experiments. The spikes were always of lower amplitude than those fol-
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lowing single or repetitive threshold stimuli and they did not occur invariably with each pair of pulses. There is, therefore, little opportunity for summation in the mechanism by which spikes are evoked.
B. Intact and incompletely isolated cortex. The observations in this section were made during the first part of the 11 experiments described above. In addition there were 2 cases in which subsequent attempts at isolation failed. Partially isolated cortex. A varying degree of spontaneous activity always remained in cortex enclosed by vertical incisions which had cut through the grey matter and some, but not all, of the cortico-cortical fibres under the grey matter. Spontaneous strychnine spikes always occurred though their frequency was dependent on the level of spontaneous activity. If the latter was much diminished, spikes could be evoked by electrical stimulation in the same way as from completely isolated cortex. If spontaneous activity remained prominent the partially isolated cortex behaved like the intact cortex described below. Figures 2A and B illustrate a common observation on partially isolated cortex in
Fig. 2 Partially isolated cortex. A. Two types of spontaneous spikes from partially isolated cortex. B. Shows that stimuli evoked only one type of spike.
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M . K . WRIGHT, W. K. ANDREW aad I. JACOBSON
which spontaneous strychnine spike; occurred but in which spikes could be elicited by repetitive stimulation up to 3.5/sac. Figure 2A shows two different spike shapes occurring spontaneously from two different loci in the strychninised zone. Figure 2B shows that stimulation evoked only one of these spike shapes though if the other occurred spontaneously during a stimulation sequence it disrupted temporarily the response to the repetitive stimuli. Since the apparent shape of a spike depends on its position of origin, relative to the recording electrodes, it follows that the excitation from the stiumlating electrodes took a consistent route to the sensitised zone and evoked a spike in approxinmtely the same focus at each stinmlus. The introduction Of a spontaneous spike at a different focus either temporarily blocked the route taken by the impulses generated by the stimulus, or else blocked the nerve cells participating in the commonly evoked spike. Intact cortex.
Electrical stimulation of stryehninised intact cortex with single pulses evoked spikes in an inconstant and unpredictable manner.. Repetitive stimulation never succeeded in evoking a rhythmic series of spikes at any frequency. I t should be emphasised that these observations referred to cortex which was spontaneously active and in which strychnine spikes were occurring spontaneously at irregular intervals. I) ISCUSSION Cortical mechanisms. The previous observations suggest the following hypothetical' interpretation of the mechanism underlying the interaction between electrical stimuli and strychnine spikes : Each strychnine, spike in a series produced by. repetitive electrical stimulation was initiated in the same group of neurones because the stimuli entered the strychninised zone from a constant direction. I f either .the cells in this group of n e u r o n e s or else the neurones which conducted the excitation from the point of stimulation to the s t r y c h n i n i s e d zone, were reactivated i n a later phase of the strychnine spike then they would not respond
to a subsequent stimulus for a time depend, ing on the characteristics of their refractor3~ period and the parameters of the applied stimulus. F u r t h e r observations (in p r e s s have shown that during the third (final surface positive) phase of a spike in nembutalised cats there is a relatively widespread activation ()f cortical neurones which ie~ a~ l,.ast i n ' p a r t transynaptic aml which couM account for the reactivation of neurones pos-. tulated above. Thus once a spike was initialed it wouht tend to interfere with the effects of the, subsequent stimulus and in the ease of the present experiments there would be an element of competition between activity induced by a spike ~nd that induced by the successive stimulus in a rei)eti~iw~ volley. [f the apt)lied stimulus failed to compete with the activity during the spike, then each st)ik~, in an evoke(t series would remain (.oilstare in shape and amplitude and the i;~ dividmd spike duration would determine the fre(ttieney at which a volley could be mai~,talmud. This, in fact, was th(, rule with isolated cortex. Itowever, towards tile end of a volley the latency of the evoked spikes increased while their first phases diminished, indicating that the stimulus took a longer route (probably involving several synapses) to the sensitised zone and triggered fewer cells therein. By thus blocking the path of the stinmlus, the strychnine spikes determh~ed the length, as well as the frequem'y of a~ evoked volley. In this compaction (/hang (1951) found that in stry~d~ninised intact~ cortex electrical stiumli failed to produce his secondary response for up to I).5 see. after a previous stimulation. This he attributed to refractoriness of the cortical internuncials, which interpretation fits the explanation offered here for the limitation of tile frequency of evoked strychnine spikes. ~ If, on the other hand, the applied .stimulus progressi.vely succeeded in r e n d e r i , g ref r a c t o r y some of the ~eurones responsible f o r a subsequent spike potential then the amplitude and form of a series of spikes evoked by relatively high frequency stimulation would rapidly disintegrate. This was ohserved (fig. 1 F ) when spikes changed from 3.5/see. to 1.75/see.
OBSERVATIONS ON ISOLATED CORTEX
The inconstant manner in which strychnine spikes were elicited from intact cortex was probably due to interference bet~veen the electrical stimuli and spontaneous activity. :There was also some evidence for mutual interference between spontaneous activity a n d ~trychnine spikes. The normal path by which stimuli reach the sensitised zone is probably that suggested by Burns (1952) for the propagation of his superficial response. The superficially applied stimuli excite the apical dendrites, or else the horizontal neurones, in the molecular layer; nerve impulses then travel along the molecular layer, descending into the deeper layers via the main shaft dendrites. Normally a strychnine spike is evoked with minimum latency when an impulse descends from the molecular layer directly into the deep layers of a strychninised zone. Thus the increase with distance of the threshold stimulus required to evoke a spike is correlated with the observations of Burns (1950, 1952) that the superficial response decays with distance from the point of stimulation and that a more intense stimulus produces a larger ~uperficial response. In those experiments when the latency of the spike increased, independently of the distance between stimulating and recording electrodes, the nerve impulses from the point of stimulation probably entered the deeper cortical laminae outside the strychninised zone and reached the sensitised zone by deep internuncial pathways involving several synapses. This route is the same as that travelled by the deep response of Burns, and appears to be less efficient for eliciting repetitive strychnine spikes than the more direct route through the molecular layer. Both routes, however, are cut off when an incision is made through the grey matter.
Comparison with the electroencephalography of petit real. The features of petit mal-attacks mimicked in these experiments are the upper frequency limit of 3.5/sec. and the duration of individual bursts. If these similarities are not merely coincidental then the following comments might throw some l i g h t on the mechanism of petit mal seizures:
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(a) The maximum frequency of the discharge is set by the excitability cycle of cortical neurone chains. The frequency of petit real discharges may therefore not necessarily reflect the frequencies emitted from any trigger zone. (b) Local cortical stimulation will elicit the discharges. Subcortical stimuli are therefore not essential.
Strychnine spikes and spontaneous activity.
These experiments demonstrate that strychnine per se is not adequate to produce neuronal discharge in nembutalised cats. Thus synchronised discharges in strychninised grey matter apparently depend on the presence and nature of the spontaneous activity. SUMMARY
1. Electrical stimulation of isolated and strychninised cortex was carried out in cats. 2. No spontaneous activity or strychnine spikes occur in isolated cortex. 3. Electrical stimulation of isolated and strychninised cortex elicits typical strychnine spikes. The threshold at which spikes are elicited increases with the distance between the strychninised zone and the point of stimulation. 4. The maximum frequency at which strychnine spikes occur is 3.5/sec. irrespective of the frequency of repetitive stimulation. A series of spikes at 2 or 3.5/sec. can only be maintained for approximately 30 sec. 5. Electrical stimulation of intact, spontaneously active cortex elicits spikes in an inconstant manner and a series of spikes cannot be maintained with repetitive stimulation. 6. The mechanism determining the length of a train of evoked spikes is discussed on the basis of interference between the activity produced by the stimulation and that produced by the strychnine spike itself. 7. The manner in which the mechanisms underlying these observations might elucidate some of the features of petit real discharges are discussed.
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M.K.
W R I G H T ~ W. K. A N D R E W
The senior a u t h o r g r a t e f u l l y acknowledges a g r a n t f r o m the Council for Scientific a n d I n d u s t r i a l Research ( S o n t h A f r i c a ) t o w a r d s the eost of electronic r e c o r d i n g a p p a r a t u s . We should like to tht~nk P r o f e s s o r R. A. D a r t in whose d e p a r t m e n t thi~ work wqs 1)erformed. We should also like to t h a n k Dr. Grey W a l t e r f o r several s u g g e s t i o n s which have clarified the p r e s e n t a t i o n of this work a n d have opened up f u r t h e r c o n s i d e r a t i o n s for i n v e s t i g a t i o n .
a n d [. ,JACOBS(JN
[mvut,ite foci within [I,, strj'~i~ii!iised zo:ic, Zl,v, ever, ;~ny one spike is a]nwst i!~'~linly CO[ll~)O!tlde~] of ~,omponents which :~rf prop:~g:m,d ~ith:i, i small eorti(m[ region , n d which ~b~,r~for,~ :i!'~ !le[th~r spa/ialJy nor temporally rlm.~rmmt q'hi~ propagal:iot~ 1~ generally, but not .~d.ireiy~ identical in r~speet ~i direction and extent m any !:vt* succ,:'ssive :pike-" - r l g i n a t i n g at the s~,rne f.('us. REF~RENCE~
A ddcnd.um : Dr. Grey W a R e r ( p e r s o n a l c o m m u n i c a t i o n ) has pointed out t h a t " i f the observed shape of a spike is f o u n d to depend on its position relative to the recording electrodes, this is ineluctable diagnostic evidence t h a t the spike is c o m p o u n d a n d t h a t the c o m p o n e n t s are not c o n g r u e n t in space a n d / o r t i m e ' '. In the ease of s t r y c h n i n e spikes recorded with bipolar electrodes which arc in direct c o n t a c t with the cortex a n d yet which are relatively small in relation to the a r e a of the s t r y c h n i n i s e d zone, the observed variations in w a v e - f o r m t~re p a r t l y due to p h a s e c h a n g e s produced when successive spikes o r i g i n a t e in d i f f e r e n t
B [ m x s . B. D. Some properties of the c a t ' s isola!;ed cerebral cortex. J. Physiol., 1950, 3: 50-68. BUaNS, B. D. a n d G~c*~'s'rmN, B. The f u n c t i o n a n d s t r u c t u r e of some lteurones ill the c a t ' s cerebral cortex. J. Physiol., 1952, :Y:¢: 412-433 CmxxG, It. T. C h a n g e s in excitability of cerebral cortex following single electric shock applied to cortical surface. ,l. ,Vc~trophysiol., 1951, ~ 4 : 9 5 ]II. GAROL, H. W. The f u n c t i o n a l o r g a n i z a t i o n of t,he sensory cortex of the cat, !1. 3", :Ve'uropath, exp N:'uro'[, 1942, I : 320-329. McCuLLOCtt, W. S. The f u n c t i o n a l o r g a n i z a t i o n of the cerebrM cortex. Physiol. Rev., 1944, 24: 390407,
Reference: WRIOHT, M. K., ANDREW, W. K. and J ACOBS0N, I. cortex.
EEG Clin. ~TeurophysioL, 1954, 6: 635-642.
Observations
on strychninised isolated
The experiments described herein were originally planned to test the hypothesis that strychnine applied to a region of grey matter containing synapses would not elicit spikes in the absence of spontaneous electrical activity. Cerebral cortex isolated but for its blood supply was chosen for the purpose. The vitality of the isolated cortex was tested by electrical stimulation and it was then found that strychnine spikes could only be obtained from isolated cortex if it were artificially stimulated after strychninisation. The frequency at which spikes could be thus evoked, as well as the duration of a spike series that could be maintained, were found to bear a marked resemblance to the frequency and duration of the "spike and wave" pattern in petit real. This resemblance was followed up in more detail and the results form the basis of this communication. Burns (1950, 1952) has described cortical potentials after electrical, stimulation of isolated cortex but it has been found in this research that the effect of strychnine alters many features of the potentials thus evoked. Chang (1951) has stimulated stryehninised cortex electrically but the cortex was not isolated. The findings of both the above authors can, however, be correlated with the results reported herein. MATERIAL AND METHODS
the posterior half of the suprasylvian gyrus involving variable proportions of areas 21, 7 and 19 (Garol 1942). In one experiment the isolated cortex lay in the ectosylvian gyrus medial to the posterior ectosylvian fissure while in two others it lay on the convexity of the occipital pole. A curved strip of thin celluloid, which slid harmlessly beneath the pial blood vessels, was used initially to make the cuts perpendicular to the braia surface. In later experiments a curved steel knife with a blunt dorsal edge was devised. The undercuts were made with a thin pliable steel strip, 8 mm. wide, with a sharp leading edge. The site of each isolation included prominent pial arteries and veins and no damage was done either to these vessels or to the larger vessels in the neighbouring fissures into which they drained. At the conclusion of an experiment the vessels overlying the isolated cortex were opened to test for continued blood flow. Finally the brain was fixed in 10 per cent formalin and a block of cortex was removed after the whole brain had hardened. In 7 cases 20~, sections at 200~ intervals were stained with carbol fuchsin to confirm the success of the isolation and the area of acute neuronal damage in the isolated area. In the remaining experiments, in which the special steel knife was used, freehand sections of the hardened cortex were examined. Warm liquid paraffin was applied to the exposed brain when the dura was first opened. The excess paraffin was only temporarily removed during the isolation procedure. Bipolar recordings were made from the experimental cortex before it was isolated from the adjacent grey matter, after it was isolated and finally after it was isolated and undercut. Silver wire electrodes between 2 and 3 mm. apart were used. Bipolar or unipolar records from an adjacent gyrus were
Eight mature cats, anaesthetised by intraperitoneal injection of 35 to 40 rags. of veterinary Nembutal per kilogram of body weight, were used in these experiments. Cortical isolations were carried out in both hemispheres in 3 cats and 2 separate isolations were done on one side in 2 animals. The isolated cortex measured approximately 15 ram. x 5 ram. and in 10 cases lay in [ 635 ]
636
M.K. WRIGHT, W. K. ANDREW and I. JACOBSON
obtained simultaneously with all records from u n d e r c u t cortex. The indifferent electrode was placed between temporal muscle and bone. A 2 channel M a r c o n i biological amplifier was used. The pen recorder was linear to 75 c/sec. ; the time constant was kept at 0.1 sec. and the attenuation was adjusted for each electrode placement such that strychnine spikes caused minimal pen overshoot peaks in the records. When recordings from unstrychninised cortex were taken and whenever the absence of cortical potentials was suspected, the sensitivity was increased to 100 ~V. and 50 ~V. per cm. deflection respectively. P a p e r speeds of 1.5, 3.0 and 6.0 cm/sec, were used as indicated in the figures. Local strychninisation with filter paper squares or triangles of less than 2 mm. side were applied in the manner described by McCulloch (1944). The concentration of strychnine used was 1.25 per cent. A thin layer of liquid p a r a f f i n over the brain does not interfere with local permeation of strychnine into the cortex. Electrical stimuli were derived from a t h y r a t r o n stimulator designed by N. C. Johnstone. Single, dual (with variable interval) and repeated condenser discharges of short duration were obtainable. These pulses were delivered through a high impedance coupling transformer across the secondary winding of which was placed a 10 k. potentiometer with the sliding contact grounded so that stimulus artefacts could be reduced or rendered monophasic, thus ensuring that they were not confused with strychnine spikes. Stimuli between 1.5 and 12 V. were applied through bipolar silver electrodes, both of which were on the isolated cortex. OBSERVATIONS
A. Isolated and undercut cortex. This group consisted of 11 experiments on 7 cats. In 2 experiments there was a small bridge of white matter joining the isolated with the adjacent cortex of the same gyrus, but if these fibres were functional they were not sufficient to change the characteristics of completely isolated cortex. Recordings were
generally taken from several parts of each isolated region during the course of om ~ experiment.
Spontaneous activity. Spontaneous potentials never occurred after the undercutting procedure, which was always the final step in the isolation of a cortical strip. The absence of spontaneous activity continued for between 2 and 5 hours before an experiment was terminated. This was in marked contrast to the appearance of such activity in 2 f u r t h e r experiments in which the cortex had an equally severe handling but in which either the undercutting or the vertical isolation failed by a margin of only a few square millimetres in area. Thus surgical trauma or avascularity with prolonged neuronal depression was not a sufficient explanation for the inactivity of u m stimulated acutely isolated cortex. The absence of spontaneous activity depended neither on the area of the isolate nor on the depth of the u n d e r c u t f r o m the surface. There were at least 5 experiments in which all cortico-cortical connections within the isolated strip must have been intact such that even a small active zone within the isolate could have affected its neighbouring cortex by means of fibres passing beneath the grey matter. Of the possible cytoarchitectural areas included in most of the isolated strips, only area 19 has limited intra-areal connections as revealed by strychnine neuronography (Garol 1942), and since no isolate was limited to area 19 it appears that the integrity of local cortieo-cortical connections does not relieve the electrical silence of acutely isolated cortex.
Local strychninisation of isolated cortex. Typical strychnine spikes were always recorded until undercutting was completed, after which spikes occurred only on very rare occasions when undue movement of the brain probably caused mechanical stimulation by one of the electrodes. On one occasion intracortical injection of strychnine produced slight muscular twitchings in the limbs but failed to evoke spikes from the isolated cortex.
OBSERVATIONS ON ISOLATED CORTEX
637
Electrical stimulation of isolated cortex with single pulses.
not much larger than the threshold response was reached.
Electrical stimulation with single pulses of brief duration produced neither after discharge nor temporary return of spontaneous activity in isolated cortex. Nonetheless comparable electrical stimulation of isolated and strychninised cortex evokes typical strychnine spikes. The occurrence of strychnine spikes following single electrical pulses was always a threshold phenomenon in the sense that there was a narrow range of approximately 0.2 V, within which a stimulus either produced nothing but its own artefact or else produced an artefact which merged with the first phase of a tri- or tetraphasic strychnine spike of much higher amplitude and longer duration. The precise threshold value with bipolar silver stimulating electrodes between 2 and 3 mm. apart was constant for a series of stimuli in any particular experiment and electrode placement, but it varied between 3.0 and 6.0 V. in different animals or in the same animal with different electrode placements. Electrical pulses, to be effective, must be applied within the isolated region not more than about 1.0 cm. from the strychninised zone. At distances greater than between 3 and 5 ram. from the strychnine, the stimulus threshold increased with distance but no precise relationship between the two could be established. The area of cortex strychninised had no consistent effect on the threshold of electrical stimulation. Spikes could be evoked for about half an hour after a single application of strychnine, but thereafter the threshold showed marked variations and then rose rapidly such that the effectiveness of electrical stimulation was often lost in a period of seconds rather than minutes. On re-strychninisation of the same site spikes could be evoked again. For some time after strychnine was applied to a particular site the spikes evoked usually followed the all or none principle. At other times, an increase in the stimulus increased the amplitude but not the form of the subsequent spike until a maximum response
Repetitive stimulation of isolated cortex. During the course of each isolation experiment the cortex was stimulated on numerous occasions at frequencies of 1, 2, 3.5, 7 and 10/sec. In one case the frequency was increased to 25/sec. In no instance did afterdischarge or regeneration of spontaneous activity occur, although the repetitive volley was sometimes maintained for 10 sec. With stimuli of threshold intensity as tested by single pulses, strychnine spikes were always evoked at pulse frequencies of 1/sec. and usually also at 2/sec. (fig. 1A). In seven experiments 3.5/sec. stimuli produced spikes at only every second pulse (fig. 1B). In 4 experiments 3.5/sec. volleys evoked spikes which followed the stimulation rate (fig. 1C), but even in these experiments 3.5/sec. strychnine spikes were only found with some of the stimulation sequences. At 7/sec. the maximum spike to pulse ratio was 1:2 (fig. 1D). The common observation with 7/sec. and higher frequencies was that a spike was produced at the initial stimulus and at variable intervals thereafter, though in such cases the time interval between spikes was longer than 0.5 sec. An increase in stimulus intensity generally had no effect on the amplitude of spikes evoked with repetitive stimuli, and in only two cases was the spike repetition rate increased from 2/sec. to 3.5/sec. Even stimulation at three times threshold strength never succeeded in " d r i v i n g " strychnine spikes at more than 3.5/sec. in any experiment. At all frequencies the individual spikes in a repetitive series were practically identical in amplitude and wave shape, but the first spike in a volley evoked soon after .strychninisation was occasionally up to 25 per cent greater in amplitude than the subsequent spikes. When spikes were driven at frequencies of 2 and 3.5/sec. the volley could only be maintained for periods varying between 7 and 30 sec. The termination of a volley of spikes was always sudden and unaccompanied by
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Fig. 1 Repetitive stimulation of isolated cortex. Spikes recorded from closely spaced bipolar electrodes on brain surface, with time constant at 0.1 Second. Such conditions tend to render the second component of the spike diphasie and also reduce the duration of the third component. A . - D. Strychnine spikes evoked with the stimulus frequencies marked on the right of each strip. (S marks stimulus artefact in each record). E. Beginning of a series of evoked spikes (left) and end of the same series ( r i g h t ) . F. 3.5 per second spikes at the beginning of a series (left) ; transition to 1.75 per second (middle) and established 1.75 per second ( r i g h t ) .
OBSERVATIONS ON ISOLATED CORTEX amplitude decrement in the ultimate spikes (fig. 1E). A volley at 3.5/sec. occasionally d r o p p e d to 1.75/sec. for a variable time before terminating but at times it reverted to a frequency of 3.5/sec. before the end. The most constant, but not invariable, sign of approaching termination of a volley was an increase in the latency of the spikes; usually the stimulus artefact merged completely with the first phase of the spike, but towards the end of a volley the stimulus artefact and spike were joined by an increasingly prominent notch which, however, never reached the isoelectric line (fig. 1E). At the same time the first phase of the strychnine spike tended to diminish. This increase in the latency of the spike was not dependent on the relative positions of the stimulating and recording electrodes, for it was observed when the two sets of electrodes were separated from each other by distance varying between 1 and 5 mm.
Once a volley of spikes had failed it never recurred if the volley of electrical pulses continued, but if the stimulus volley was broken the strychnine spikes reappeared with the resumption of the stimulation. Figure 1F shows the transition in frequency, from 3.5 to 1.75 spikes/sec., that occurred in a typical case. During the transition there was a marked amplitude decrease and irregularity in all phases of the strychnine spikes though the latter returned to normal as soon as the lower frequency was established. This indicated that fatigue was not the reason for the termination of a volley, for if it were so, the spikes should not have become fully re-established at half the stimulus frequency. The abrupt manner in which all strychnine spike volleys terminate is also unlike a fatigue phenomenon. The probable mechanism determining both the frequency and duration of a spike volley will be discussed later.
Dual stimulation of isolated cortex. Dual stimuli of two thirds threshold intensity and less than 18 msec. interval evoked strychnine spikes following the second stimulus in only 3 experiments. The spikes were always of lower amplitude than those fol-
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lowing single or repetitive threshold stimuli and they did not occur invariably with each pair of pulses. There is, therefore, little opportunity for summation in the mechanism by which spikes are evoked.
B. Intact and incompletely isolated cortex. The observations in this section were made during the first part of the 11 experiments described above. In addition there were 2 cases in which subsequent attempts at isolation failed. Partially isolated cortex. A varying degree of spontaneous activity always remained in cortex enclosed by vertical incisions which had cut through the grey matter and some, but not all, of the cortico-cortical fibres under the grey matter. Spontaneous strychnine spikes always occurred though their frequency was dependent on the level of spontaneous activity. If the latter was much diminished, spikes could be evoked by electrical stimulation in the same way as from completely isolated cortex. If spontaneous activity remained prominent the partially isolated cortex behaved like the intact cortex described below. Figures 2A and B illustrate a common observation on partially isolated cortex in
Fig. 2 Partially isolated cortex. A. Two types of spontaneous spikes from partially isolated cortex. B. Shows that stimuli evoked only one type of spike.
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M . K . WRIGHT, W. K. ANDREW aad I. JACOBSON
which spontaneous strychnine spike; occurred but in which spikes could be elicited by repetitive stimulation up to 3.5/sac. Figure 2A shows two different spike shapes occurring spontaneously from two different loci in the strychninised zone. Figure 2B shows that stimulation evoked only one of these spike shapes though if the other occurred spontaneously during a stimulation sequence it disrupted temporarily the response to the repetitive stimuli. Since the apparent shape of a spike depends on its position of origin, relative to the recording electrodes, it follows that the excitation from the stiumlating electrodes took a consistent route to the sensitised zone and evoked a spike in approxinmtely the same focus at each stinmlus. The introduction Of a spontaneous spike at a different focus either temporarily blocked the route taken by the impulses generated by the stimulus, or else blocked the nerve cells participating in the commonly evoked spike. Intact cortex.
Electrical stimulation of stryehninised intact cortex with single pulses evoked spikes in an inconstant and unpredictable manner.. Repetitive stimulation never succeeded in evoking a rhythmic series of spikes at any frequency. I t should be emphasised that these observations referred to cortex which was spontaneously active and in which strychnine spikes were occurring spontaneously at irregular intervals. I) ISCUSSION Cortical mechanisms. The previous observations suggest the following hypothetical' interpretation of the mechanism underlying the interaction between electrical stimuli and strychnine spikes : Each strychnine, spike in a series produced by. repetitive electrical stimulation was initiated in the same group of neurones because the stimuli entered the strychninised zone from a constant direction. I f either .the cells in this group of n e u r o n e s or else the neurones which conducted the excitation from the point of stimulation to the s t r y c h n i n i s e d zone, were reactivated i n a later phase of the strychnine spike then they would not respond
to a subsequent stimulus for a time depend, ing on the characteristics of their refractor3~ period and the parameters of the applied stimulus. F u r t h e r observations (in p r e s s have shown that during the third (final surface positive) phase of a spike in nembutalised cats there is a relatively widespread activation ()f cortical neurones which ie~ a~ l,.ast i n ' p a r t transynaptic aml which couM account for the reactivation of neurones pos-. tulated above. Thus once a spike was initialed it wouht tend to interfere with the effects of the, subsequent stimulus and in the ease of the present experiments there would be an element of competition between activity induced by a spike ~nd that induced by the successive stimulus in a rei)eti~iw~ volley. [f the apt)lied stimulus failed to compete with the activity during the spike, then each st)ik~, in an evoke(t series would remain (.oilstare in shape and amplitude and the i;~ dividmd spike duration would determine the fre(ttieney at which a volley could be mai~,talmud. This, in fact, was th(, rule with isolated cortex. Itowever, towards tile end of a volley the latency of the evoked spikes increased while their first phases diminished, indicating that the stimulus took a longer route (probably involving several synapses) to the sensitised zone and triggered fewer cells therein. By thus blocking the path of the stinmlus, the strychnine spikes determh~ed the length, as well as the frequem'y of a~ evoked volley. In this compaction (/hang (1951) found that in stry~d~ninised intact~ cortex electrical stiumli failed to produce his secondary response for up to I).5 see. after a previous stimulation. This he attributed to refractoriness of the cortical internuncials, which interpretation fits the explanation offered here for the limitation of tile frequency of evoked strychnine spikes. ~ If, on the other hand, the applied .stimulus progressi.vely succeeded in r e n d e r i , g ref r a c t o r y some of the ~eurones responsible f o r a subsequent spike potential then the amplitude and form of a series of spikes evoked by relatively high frequency stimulation would rapidly disintegrate. This was ohserved (fig. 1 F ) when spikes changed from 3.5/see. to 1.75/see.
OBSERVATIONS ON ISOLATED CORTEX
The inconstant manner in which strychnine spikes were elicited from intact cortex was probably due to interference bet~veen the electrical stimuli and spontaneous activity. :There was also some evidence for mutual interference between spontaneous activity a n d ~trychnine spikes. The normal path by which stimuli reach the sensitised zone is probably that suggested by Burns (1952) for the propagation of his superficial response. The superficially applied stimuli excite the apical dendrites, or else the horizontal neurones, in the molecular layer; nerve impulses then travel along the molecular layer, descending into the deeper layers via the main shaft dendrites. Normally a strychnine spike is evoked with minimum latency when an impulse descends from the molecular layer directly into the deep layers of a strychninised zone. Thus the increase with distance of the threshold stimulus required to evoke a spike is correlated with the observations of Burns (1950, 1952) that the superficial response decays with distance from the point of stimulation and that a more intense stimulus produces a larger ~uperficial response. In those experiments when the latency of the spike increased, independently of the distance between stimulating and recording electrodes, the nerve impulses from the point of stimulation probably entered the deeper cortical laminae outside the strychninised zone and reached the sensitised zone by deep internuncial pathways involving several synapses. This route is the same as that travelled by the deep response of Burns, and appears to be less efficient for eliciting repetitive strychnine spikes than the more direct route through the molecular layer. Both routes, however, are cut off when an incision is made through the grey matter.
Comparison with the electroencephalography of petit real. The features of petit mal-attacks mimicked in these experiments are the upper frequency limit of 3.5/sec. and the duration of individual bursts. If these similarities are not merely coincidental then the following comments might throw some l i g h t on the mechanism of petit mal seizures:
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(a) The maximum frequency of the discharge is set by the excitability cycle of cortical neurone chains. The frequency of petit real discharges may therefore not necessarily reflect the frequencies emitted from any trigger zone. (b) Local cortical stimulation will elicit the discharges. Subcortical stimuli are therefore not essential.
Strychnine spikes and spontaneous activity.
These experiments demonstrate that strychnine per se is not adequate to produce neuronal discharge in nembutalised cats. Thus synchronised discharges in strychninised grey matter apparently depend on the presence and nature of the spontaneous activity. SUMMARY
1. Electrical stimulation of isolated and strychninised cortex was carried out in cats. 2. No spontaneous activity or strychnine spikes occur in isolated cortex. 3. Electrical stimulation of isolated and strychninised cortex elicits typical strychnine spikes. The threshold at which spikes are elicited increases with the distance between the strychninised zone and the point of stimulation. 4. The maximum frequency at which strychnine spikes occur is 3.5/sec. irrespective of the frequency of repetitive stimulation. A series of spikes at 2 or 3.5/sec. can only be maintained for approximately 30 sec. 5. Electrical stimulation of intact, spontaneously active cortex elicits spikes in an inconstant manner and a series of spikes cannot be maintained with repetitive stimulation. 6. The mechanism determining the length of a train of evoked spikes is discussed on the basis of interference between the activity produced by the stimulation and that produced by the strychnine spike itself. 7. The manner in which the mechanisms underlying these observations might elucidate some of the features of petit real discharges are discussed.
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W R I G H T ~ W. K. A N D R E W
The senior a u t h o r g r a t e f u l l y acknowledges a g r a n t f r o m the Council for Scientific a n d I n d u s t r i a l Research ( S o n t h A f r i c a ) t o w a r d s the eost of electronic r e c o r d i n g a p p a r a t u s . We should like to tht~nk P r o f e s s o r R. A. D a r t in whose d e p a r t m e n t thi~ work wqs 1)erformed. We should also like to t h a n k Dr. Grey W a l t e r f o r several s u g g e s t i o n s which have clarified the p r e s e n t a t i o n of this work a n d have opened up f u r t h e r c o n s i d e r a t i o n s for i n v e s t i g a t i o n .
a n d [. ,JACOBS(JN
[mvut,ite foci within [I,, strj'~i~ii!iised zo:ic, Zl,v, ever, ;~ny one spike is a]nwst i!~'~linly CO[ll~)O!tlde~] of ~,omponents which :~rf prop:~g:m,d ~ith:i, i small eorti(m[ region , n d which ~b~,r~for,~ :i!'~ !le[th~r spa/ialJy nor temporally rlm.~rmmt q'hi~ propagal:iot~ 1~ generally, but not .~d.ireiy~ identical in r~speet ~i direction and extent m any !:vt* succ,:'ssive :pike-" - r l g i n a t i n g at the s~,rne f.('us. REF~RENCE~
A ddcnd.um : Dr. Grey W a R e r ( p e r s o n a l c o m m u n i c a t i o n ) has pointed out t h a t " i f the observed shape of a spike is f o u n d to depend on its position relative to the recording electrodes, this is ineluctable diagnostic evidence t h a t the spike is c o m p o u n d a n d t h a t the c o m p o n e n t s are not c o n g r u e n t in space a n d / o r t i m e ' '. In the ease of s t r y c h n i n e spikes recorded with bipolar electrodes which arc in direct c o n t a c t with the cortex a n d yet which are relatively small in relation to the a r e a of the s t r y c h n i n i s e d zone, the observed variations in w a v e - f o r m t~re p a r t l y due to p h a s e c h a n g e s produced when successive spikes o r i g i n a t e in d i f f e r e n t
B [ m x s . B. D. Some properties of the c a t ' s isola!;ed cerebral cortex. J. Physiol., 1950, 3: 50-68. BUaNS, B. D. a n d G~c*~'s'rmN, B. The f u n c t i o n a n d s t r u c t u r e of some lteurones ill the c a t ' s cerebral cortex. J. Physiol., 1952, :Y:¢: 412-433 CmxxG, It. T. C h a n g e s in excitability of cerebral cortex following single electric shock applied to cortical surface. ,l. ,Vc~trophysiol., 1951, ~ 4 : 9 5 ]II. GAROL, H. W. The f u n c t i o n a l o r g a n i z a t i o n of t,he sensory cortex of the cat, !1. 3", :Ve'uropath, exp N:'uro'[, 1942, I : 320-329. McCuLLOCtt, W. S. The f u n c t i o n a l o r g a n i z a t i o n of the cerebrM cortex. Physiol. Rev., 1944, 24: 390407,
Reference: WRIOHT, M. K., ANDREW, W. K. and J ACOBS0N, I. cortex.
EEG Clin. ~TeurophysioL, 1954, 6: 635-642.
Observations
on strychninised isolated