electrode inserted into this horn. The discharge spreads to the cerebral

Transcription

1 J. Physiol. (1967), 191, pp With 8 text-ft gure8 Printed in Great Britain SITE OF ORIGIN OF THE ABNORMAL DISCHARGE IN THE ELECTROCORTICOGRAM PRODUCED BY TUBOCURARINE PERFUSED THROUGH THE ANTERIOR HORN OF A LATERAL VENTRICLE BY W. FELDBERG AND K. FLEISCHHAUER* From the National Institute for Medical Research, Mill Hill, London, N. W. 7 (Received 3 February 1967) SUMMARY 1. In cats anaesthetized with chloralose, perfusion of tubocurarine through the anterior horn of a lateral cerebral ventricle produces a rhythmic discharge of high voltage negative spikes recorded from an electrode inserted into this horn. The discharge spreads to the cerebral cortex where it gives rise to synchronous surface negative deflexions of low voltage. They have previously been described as 'slow waves'. 2. The discharge results from an excitatory action of tubocurarine on the anterior limbic area, which is a cortical structure in the medial wall of the anterior horn lying rostral to the septum pellucidum. INTRODUCTION Tubocurarine perfused through the cerebral ventricles of an anaesthetized cat produces an abnormal discharge in the electrocorticogram (e.co.g.) which consists of two separate components. One is a discharge of high voltage surface negative spikes interrupted by episodes of fast activity. This discharge results from an action on the hippocampus reached by the tubocurarine when perfusing the inferior horn (Feldberg & Fleischhauer, 1963). The other is a discharge of low voltage surface negative deflexions of somewhat longer duration which were termed 'slow waves'. They result from an action on structures lining the anterior horn because they only occurred when the tubocurarine was perfused through this horn. They were therefore referred to as 'anterior horn discharge'. The actual structures, however, on which the tubocurarine acts when eliciting this discharge were not determined (Carmichael, Feldberg & Fleischhauer, * Wellcome Research Grant. Permanent address: Anatomisches Institut, Hamburg 20, Martinistrasse 52, Germany.

2 488 W. FELDBERG AND K. FLEISCHHA UER 1964b). This was done in the present experiments. Two procedures were used. Records were taken of the electrical activity of the structures lining the anterior horn during its perfusion with tubocurarine, and microinjections of tubocurarine were made into these structures. METHODS The experiments were done on cats under chloralose anaesthesia (50 mg/kg i.v.) induced with ethylchloride and ether to allow cannulation of the left femoral vein. A cannula was inserted into the trachea, and with the cat lying on its belly the head was fixed to the ear bars and mouthpiece of a Dell-Moruzzi stereotaxic instrument. Perfusion of the cerebral ventricles. The method of regional perfusion of the cerebral ventricles with drugs described by Carmichael, Feldberg & Fleischhauer (1964a) renders it possible to perfuse tubocurarine through an anterior horn alone or through an anterior horn and the third ventricle. Since perfusion of tubocurarine through the third ventricle produces no abnormal discharges on the e.co.g. but only an accentuation of background activity (Feldberg & Fleischhauer, 1962; Carmichael et al. 1964b), the procedure used was as follows. Tubocurarine was perfused from left anterior horn to aqueduct and was prevented entrance into left inferior horn and into right lateral ventricle by perfusion of artificial c.s.f. through Collison cannulae implanted into the middle of both lateral ventricles. The artificial c.s.f. used for perfusion and for dissolving the tubocurarine was that described by Merlis (1940). The rate of infusion through each cannula was 0 05 or 0-1 ml./min. Micro-injections of tubocurarine. Tubocurarine was introduced into small discrete regions of the brain by means of micro-injection. The method was that described by Myers (1966). The micro-injection device consisted of an infusion pump discharging 1 Al. of fluid in 46 see, and of two steel cannulae, one inside the other: the outer sleeve was of 22 gauge (0 7 mm) tubing, and the inner, the injection cannula, was 28 gauge (0-35 mm). The sleeve, fixed to a micromanipulator of the stereotaxic instrument, was brought into position in the desired sagittal and frontal planes of the brain without touching the dura, the bone having been removed from this region. The inner tube, filled with a solution of tubocurarine (3-6 x 10-2M) and connected by fine polythene tubing to the infusion pump, was introduced into the sleeve so that its tip was level with that of the sleeve. They were then inserted vertically into the brain to a point 1 mm above the desired spot, and finally the inner tube was lowered the further millimetre so that its tip protruded from the sleeve. The site of the tip was ascertained microscopically at the end of all experiments. For this purpose the brain was fixed by perfusion of saline solution followed by either formalin-saline (1/4 v/v) or Bouin's fluid from the aorta after opening the thorax, clamping the heart and cutting the jugular veins Sections were cut on the freezing microtome or after embedding the brain in paraffin. In order to find out how far the tubocurarine, introduced by a micro-injection, spreads in the brain tissue, micro-injections of 1 pl. of the fluorescent dye 3,6-diamino-acridine trihydrochloride (4-5 x 10-2M) were made into the brain substance; 30 min later the thorax was opened, the heart clamped and, with the jugular veins opened, Bouin's fluid was perfused through the aorta to fix the brain. The spread of the dye was determined histologically by means of fluorescence microscopy according to a method described elsewhere (Fleischhauer, 1964). In grey matter the dye had travelled about 2 mm from the tip of the cannula, in white matter the spread was even less. Recording of electrical activity. Electrical activity from various sites was recorded simultaneously with monopolar electrodes connected to an Offner pen recorder. The right metal ear bar of the stereotaxic instrument was earthed and served as indifferent electrode. The time constant of the Offner pen recorder was set at 0 3 sec and the high frequency limit at 500 c/s.

3 TUBOCURARINE AND ANTERIOR LIMBIC AREA 489 For recording the activity from the occipital cortex an epidural electrode consisting of platinum wire held in a nylon screw was inserted through a burr hole over the middle suprasylvian gyrus. The steel cannula used for perfusion of the anterior horn was insulated except at its tip and served as recording electrode from the anterior horn. Similarly, the sleeve of the micro-injection cannula was insulated except for its tip and served as an electrode recording from the site of injection. In a few experiments the electrical activity of additional deep structures of the brain was recorded from stereotaxically inserted insulated steel needle electrodes with a free tip of about 0 5 mm. The location of the tip was later determined histologically. RESULTS A rhythmic spike discharge recorded from the anterior horn Since an electrode inserted into the anterior horn should act as surface lead from the ventricular wall, it was expected to record changes in electrical activity synchronous with the 'slow waves' recorded in the e.co.g. on prolonged perfusion of tubocurarine through the anterior horn. And it did so. The results were the same if, instead of inserting a needle electrode, the perfusion cannula itself, insulated except at its tip, was used as the recording electrode, a procedure adopted in most experiments. On perfusion of tubocurarine (7.2 x x 10-4M) through an anterior horn, a rhythmic discharge of high voltage surface negative spikes was recorded within 5-10 min in the lead from this horn. The spikes increased in voltage and frequency as perfusion with tubocurarine continued. Such a discharge was not recorded from the anterior horn cannula when tubocurarine was perfused through an inferior horn, or through the third ventricle. The spikes in the lead from the anterior horn were recorded earlier than the 'slow waves' in the e.co.g. These appeared sometimes nearly 1 hr later, but they then occurred synchronously with the spikes which in the meantime had greatly increased in voltage. Figure 1 illustrates an experiment in which both anterior horns were perfused and the perfusion cannulae used as recording electrodes from the left (LAH) and the right (RAH) anterior horn. First, both horns were perfused with artificial c.s.f. (record a). Then tubocurarine (7-2 x 10-4M) was perfused through the left anterior horn whilst perfusion through the right was continued with artificial c.s.f. In the lead from the left horn negative spikes appeared after 7 min. At first they occurred infrequently and irregularly, but within the next few minutes they developed into a rhythmic discharge. Record b was taken 15 min, and record c 40 min, after the beginning of the tubocurarine perfusion, when the voltage of the spikes had increased to about 2 mv. In the cortical lead 'slow waves' did not appear until about 10 min later. They were at first just discernible above background activity. They stand out more clearly if the background activity is reduced by an intravenous injection of a small amount of

4 490 W. FELDBERG AND K. FLEISCHHA UER pentobarbitone sodium. This affects the voltage of the 'slow waves' and of the spikes recorded in the lead from the anterior horn to a small degree only, though it sometimes reduces the frequency of these discharges. In the experiment Fig. 1, an intravenous injection of 25 mg pentobarbitone sodium was given a few minutes before record d was taken. It shows the 'slow waves' on the e.co.g. occurring synchronously with the spikes in the lead from the anterior horn. Record e was taken at a faster speed. On the lead from the right anterior horn which was perfused with artificial c.s.f. throughout the experiment, only small spikes were recorded. They occurred synchronously with those in the lead from the left anterior horn, but their main deflexion was positive. LAH0 ME U & i RAH nwmww mmwm mnmu UWUMWMW~ w a b c. d e Fig. 1. Electrical activity recorded monopolarly from an epidural electrode on the left occipital cortex (LO) and from the bare tips of the insulated perfusion cannulae in the left (LAH) and right (RAH) anterior horn during perfusion of the cerebral ventricles in a cat anaesthetized with chloralose, immobilized with gallamine and artificially ventilated. Record a during perfusion of both anterior horns with artificial c.s.f. Records b, c, d and e, 15, 20, 40, 60 and 70 min after onset of perfusion with tubocurarine (7-2 x 10M) from the left anterior horn whilst perfusion of the inferior horn and of the right ventricle was continued with artificial c.s.f. Between c and d intravenous injection of 25 mg pentobarbitone sodium. Calibration 1 mv; negativity upward; time marker in seconds. Since the spikes in the lead from the anterior horn, like the 'slow waves' in the e.co.g., appear only during perfusion of tubocurarine through the anterior horn, both discharges must result from an action on structures lining this horn, and the synchrony of the two discharges points to a common site. As the 'slow waves' appear later, after the spikes have fully developed, stronger excitation of this site would be required for the 'slow waves' to be recorded in the e.co.g.

5 TUBOCURARINE AND ANTERIOR LIMBIC AREA 491 In order to find out where on the wall of the anterior horn the tubocurarine exerts its excitatory action when producing these changes in the electrical activity of the brain, the following regions had to be taken into consideration. The olfactory bulb reached from the olfactory recess, the corpus callosum forming the roof of the anterior horn, the caudate nucleus forming its lateral wall, and the septum pellucidum and anterior limbic area forming the medial wall. Figure 2 is drawn from a photograph of SC gc fx cc Fig. 2. Diagram of a mid-sagittal section of cat's brain to show septum (marked by horizontal lines) and anterior limbic area (marked by vertical lines). bo, Bulbus olfactorius; cc, corpus callosum; ch, chiasma opticum; fx, fornix; gc, gyrus cinguli; gp, gyrus proreus; hy, hypophysis; mi, massa intermedia; 8C, sulcus cruciatus. the left half of a cat's brain seen from the mid line. The septum extends between the descending column of the fornix and the anterior end of the corpus callosum. In the cat it consists of several well developed nuclei in which the cells are not oriented in layers (Andy & Stephan, 1964). The anterior limbic area is situated more rostrally. It is a cortical structure, an extension of the gyrus cinguli, and its cells are arranged in layers. Olfactory bulb. The following three results obtained on perfusion of tubocurarine (7-2 x 1O-4M) through the left anterior horn exclude an action of tubocurarine on the olfactory bulb as the cause for the 'slow waves'. (1) They appeared when the tubocurarine had not entered the olfactory recess, as shown on perfusion with bromophenol blue instead of tubocurarine at the end of the experiment. (2) On recording the electrical activity from the surface of the olfactory bulb as well as of the frontal and occipital cortex, 'slow waves' appeared on all three leads, but they were less pronounced on the lead from the olfactory bulb and did not appear earlier on this lead than on the other two.

6 492 W. FELDBERG AND K. FLEISCHHA UER (3) Destruction of the olfactory bulb before the onset of perfusion did not prevent the appearance of the 'slow waves'. Corpus callosum. An action of tubocurarine on the corpus callosum as the cause of a spike discharge would imply that tubocurarine has an excitatory effect on myelinated and unmyelinated nerve fibres in the central nervous system. However, micro-injections of tubocurarine into A B m(i Fig. 3. Electrical activity recorded monopolarly from the bare tips of the insulated micro-injection cannulae filled with tubocurarine (1-8 x 10-2M) and inserted into the left gyrus splenialis (A) and into the right corona radiata (B) in a cat anaesthetized with chloralose. The diagrams on top are drawn from photographs of frontal sections cut in a plane corresponding to A20 (A) and A 16 (B) of the Atlas of Snider & Niemer (1961), and show the positions of the micro-injection cannulae, given by the black rods, ending in grey (A) and white (B) matter. The records below are taken 30 min after insertion of the micro-injection cannula without injecting the tubocurarine (A) and 10 min after injection of 1,u. (12-5,ug) of the tubocurarine solution (B). Calibration 1 mv; negativity upwards; time marker in seconds. different regions of the cerebral cortex produced a spike discharge only when the tip of the micro-injection cannula was in grey, and not when it was in white matter. This difference is illustrated in Fig. 3. The left record shows the spikes produced when the injection cannula filled with tubocurarine (3-6 x 10-2M) was lowered into the left cerebral

7 TUBOCURARINE AND ANTERIOR LIMBIC AREA 493 cortex, so that the tip was in grey matter, as illustrated in the diagram A. Within a few minutes a spike discharge began, which increased quickly in voltage and then continued until the end of the experiment. There was no need to make the micro-injection since apparently sufficient tubocurarine had diffused from the cannula tip into the adjacent tissue. The right record was obtained by leading off from a micro-injection cannula inserted more rostrally on the other side, with the tip in the white matter of the corona radiata, as illustrated in diagram B. The injection of 25,ug tubocurarine produced no spikes. In the experiments in which the cannula tip was aimed so that it would rest in the corpus callosum, spikes developed in some of the experiments but not in others after the injection of 25 jg of tubocurarine. In those in which spikes developed, tubocurarine had escaped into the subarachnoid space of the sulcus callosus. This was evident when 1 ptl. of Indian ink was subsequently injected through the cannula. Post mortem, the ink was found to be partly distributed in the subarachnoid space around the cannula. Since a similar discharge occurred with micro-injections of tubocurarine into the subarachnoid space above the corpus callosum, or directly into the grey matter of the gyrus cinguli the spikes were due to an action, not on the corpus callosum, but on the cingulate gyrus. Caudate nucleus. The spike discharge recorded from the anterior horn as well as the discharge of the 'slow waves' is not caused by an action of tubocurarine on the caudate nucleus. The evidence is twofold: (1) When the electrical activity was recorded from an electrode inserted into the caudate nucleus and tubocurarine was perfused through the anterior horn, either no abnormal spikes appeared in the lead from the caudate nucleus or they appeared much later than those recorded from the anterior horn and remained at low voltage. Figure 4 shows such an experiment. In the lead from the left anterior horn (LAH) a rhythmic discharge of spikes appeared after 5 min but even after 20 min when record b was taken no spikes were recorded from the electrode in the caudate nucleus (LC). Later, small spikes appeared in this lead as shown in records c and d taken about 1 hr after the onset of tubocurarine perfusion; at this time the 'slow waves' had just become discernible in the e.co.g. (LO). (2) When micro-injections of tubocurarine (25 or 50,g) were made into the caudate nucleus at a depth of 1-2 mm below the surface no abnormal spikes developed in the lead from the micro-injection cannula and the background activity remained unchanged. The micro-injection of tubocurarine also did not initiate a spike discharge in the lead from the anterior horn or the appearance of 'slow waves' in the e.co.g. Septum pellucidum. The spikes recorded from the anterior horn and the

8 494 W. FELDBERG AND K. FLEISCHHAUER 'slow waves' in the e.co.g. do not result from an action of tubocurarine on the septum, although the septum is activated by tubocurarine and this activation may contribute to the discharge which occurs when tubocurarine is perfused through the anterior horn. Again the evidence is twofold: (1) On perfusion of tubocurarine through the anterior horn, spikes LC F'gllrtqTl [T LAH h Wh ill] LS ~ 1 a b c d Fig. 4. Electrical activity recorded monopolarly from needle electrodes in left caudate nucleus (LC) and left septum (LS), from the bare tip of the insulated perfusion cannula in left anterior horn (LAH) and from an epidural electrode on the left occipital cortex (LO) during perfusion of the cerebral ventricles in a cat anaesthetized with chloralose. Record a during perfusion with artificial c.s.f. Records b, c and d, 20, 60 and 61 min after onset of perfusion with tubocurarine (3-6 x 10-4M) from left anterior horn whilst perfusion of the inferior horn and of the right ventricle was continued with artificial c.s.f. Calibration 1 mv; negativity upwards. Time marker in seconds. appeared in the septum, but they appeared later than those in the lead from the anterior horn. (2) Micro-injections of tubocurarine into the septum caused a rhythmic discharge of spikes in the lead from this region, but no spikes were recorded from the anterior horn, or if so they were of low voltage.

9 TUBOCURARINE AND ANTERIOR LIMBIC AREA 495 In the experiment Fig. 4, an electrode was inserted into the left septum, and tubocurarine was then perfused through the left anterior horn. Within 5 min spikes appeared in the lead from this horn (LAH), but even 15 min later, when record b was taken, no spikes had yet appeared in the septal lead (LS). However, another 40 min later they had appeared in record c; they occurred synchronously with, but were of lower voltage than those in the lead from the anterior horn. fls sl LS i*f. I S ~~im _,SS.Si] LO 4 S # _p_-t _ 1 LAH a b c Fig. 5. Electrical activity recorded monopolarly from the bare tip of an insulated micro-injection cannula in left septum (LS inf.), from a needle electrode in left septum (LS) inserted 2 mm rostral to the micro-injection cannula, from an epidural electrode on the left occipital cortex (LO) and from the bare tip of the insulated perfusion cannula in anterior horn (LAH) during perfusion of the cerebral ventricles with artificial c.s.f. in a cat anaesthetized with chloralose. Record a before, and record b 25 min after micro-injection of 1 pl. tubocurarine (3-6 x 10-'M) (25 pug) into left septum. Record c 50 min after record b and 25 mi after a second microinjection of 25 pg tubocurarine. Calibration 1 mv negativity upward. Time marker in seconds. In the experiment in Fig. 5, a micro-injection of 25 gug tubocurarine was made into the septum, and a needle electrode was inserted into the septum 2 mm rostral to the micro-injection cannula. Within a few minutes a rhythmic discharge of spikes appeared in the lead from the site of injection (LS inf.). The spikes increased rapidly in size during the first minutes and then remained unchanged for at least an hour. They are shown in record b taken 25 min after micro-injection was made. At this time no spikes

10 496 W. FELDBERG AND K. FLEISCHHA UER were recorded from the anterior horn (LAH). Nor had the discharge spread within the septum from the site of injection to the needle electrode inserted 2 mm more rostrally (LS). Record c was taken after a second micro-injection of 25,ug at the same site. This did not increase the spike discharge recorded from the micro-injection cannula, but now a synchronous discharge of spikes developed in the lead from the other septal electrode and from the anterior horn. Micro-injections of tubocurarine into the septum did not produce the 'slow waves' in the e.co.g. and the septal spikes never reached the same voltage as the spikes recorded from the anterior horn on its perfusion with tubocurarine. LO_'a V.10" llf 410 LAH4 W I U ] LAL a b c d e Fig. 6. Electrical activity recorded monopolarly from an epidural electrode on left occipital cortex (LO) from the bare tip of the insulated perfusion cannula in left anterior horn (LAH) and from the bare tip of the insulated micro-injection cannula in left anterior limbic area (LAL) during perfusion of the cerebral ventricles in a cat anaesthetized with chloralose. Record a during perfusion with artificial c.s.f., records b, c, d and e, 5, 9, 12 and 20 min after onset of perfusion with tubocurarine (7-2 x 1O-4M) from the left anterior horn, whilst perfusion of the inferior horn and the right ventricle was continued with artificial c.s.f. Calibration 1 mv; except for e of record LAL where it corresponds to 2 mv; negativity upward; time marker in seconds. Anterior limbic area. The spikes recorded from the anterior horn and the 'slow waves' in the e.co.g. result from an action of tubocurarine on the anterior limbic area. This is evident from the results obtained when the electrical activity from this area was recorded during perfusion of the anterior horn with tubocurarine and when micro-injections of tubocurarine were made into this area. Figure 6 illustrates an experiment in which tubocurarine (7-2 x 10-4M) was perfused through the left anterior horn, whilst the electrical activity

11 TUBOCURARINE AND ANTERIOR LIMBIC AREA 497 was recorded from the left anterior limbic area. Record b is taken 5 min after the beginning of the tubocurarine perfusion and 1 min after spikes had appeared in the lead from the anterior limbic area (LAL). No spikes had yet appeared in the lead from the anterior horn (LAH). Even 4 min later when record c was taken they are scarcely discernible, although the voltage of the spikes in the anterior limbic area had increased. They are present, however, in record d taken another 3 min later, and have increased in voltage in record e taken 20 min after the onset oftubocurarine perfusion. They are of lower voltage than the spikes recorded from the anterior limbic area which had increased so much that the gain from this lead was reduced before record e was taken. LAH a iw LAL a b c d Fig. 7. Electrical activity recorded monopolarly from the bare tip of the insulated perfusion cannula in left anterior horn (LAH) and from the bare tip of the microinjection cannula in the left anterior limbic area (LAL) during perfusion of the cerebral ventricles in a cat anaesthetized with chloraloae. Record a, b and c, 6, 15 and 70 min after a micro-injection of 2,ul. tubocurarine (3-6 x 10-2M) (50 2tg) into the anterior limbic area. Cerebral ventricles perfused with artificial c.s.f. Between c and d, 10 min before d, perfusion from the left anterior horn was begun with tubocurarine (7.2 x 10-4M), whilst perfusion of the inferior horn and of the right ventricle was continued with artificial c.s.f. Calibration 1 mv; the gain was reduced at the middle of record b for LAL; from then onwards calibration from this lead corresponds to 2 mv; negativity upward; time marker in seconds. In the experiment in Fig. 7 a micro-injection of 50,tg tubocurarine was made into the anterior limbic area. After 6 min isolated spikes were recorded from the site of the micro-injection. The first two are shown in record a. The spikes increased in frequency and voltage, and, in record b taken 9 min later, had become so large that the gain was reduced. In the lead from the anterior horn they appeared much later and remained of low voltage. They are illustrated in record c taken 70 min after the microinjection. When the anterior horn was then perfused with tubocurarine (7-2 x 10-4M) they increased in voltage, but the tubocurarine perfusion 32 Physiol. 191

12 498 W. FELDBERG AND K. FLEISCHHA UER did not increase further the large spikes recorded from the anterior limbic area (record d). In some experiments the micro-injection of tubocurarine into the anterior limbic area finally resulted in the appearance of 'slow waves' in the e.co.g. which occurred synchronously with the spikes in the lead from the site of the micro-injection and from the anterior horn. This final phase is illustrated in Fig. 8. LO rr RO LAHkhS] LALW] Fig. 8. Electrical activity recorded monopolarly from epidural electrodes in left (LO) and right (RO) occipital cortices, from the bare tip of the insulated perfusion cannula in the left anterior horn (LAH) and of the insulated micro-injection cannula in the left anterior limbic area (LAL) during perfusion of the cerebral ventricles with artificial c.s.f. in a cat anaesthetized with chloralose. Micro-injection of 2 /ttl. tubocurarine (3.6 x 10-2M) (50,ug) 70 min earlier. Calibration 1 mv; negativity upwards; time marker in seconds. DISCUSSION In previous experiments in which tubocurarine was perfused through the anterior horn of a lateral ventricle, a rhythmic discharge of small surface negative deflexions or 'slow waves' was recorded in the e.co.g. This discharge was termed 'anterior horn discharge' because it could only be due to a spread of activity originating from an action of tubocurarine on structures lining the anterior horn. In fact, when an electrode was inserted into this horn, as in the present experiments, a rhythmic discharge of high voltage negative spikes was recorded long before the surface negative defiexions appeared in the e.co.g., but when they finally appeared

13 TUBOCURARINE AND ANTERIOR LIMBIC AREA 499 they occurred synchronously with the spikes recorded in the lead from the anterior horn. These spikes and the 'slow waves' are manifestations of the same discharge, and the structure from which it is elicited by the tubocurarine is the anterior limbic area, a cortical structure in the medial wall of the anterior horn rostral to the septum pellucidum. This conclusion is based on two findings, first that during perfusion of the anterior horn with tubocurarine the abnormal spike discharge is recorded in the lead from the anterior limbic area before it appears in the lead from the anterior horn; and, second, that with micro-injections of tubocurarine into different parts of the ventricular wall large negative spikes appear in the lead from the anterior horn only when the microinjections are made into the anterior limbic area. However, the finding that after micro-injections into the septum negative spikes appear in the lead of this region points to the possibility that activation of the septum contributes to the abnormal discharge recorded from the anterior horn on its perfusion with tubocurarine. Each large negative spike produced by a micro-injection of tubocurarine either into the anterior limbic area or into the septum pellucidum must result from a synchronous discharge of a great number of neurones. The absence of such a discharge following a micro-injection into the caudate nucleus means that in this structure either synchronization does not occur, or the tubocurarine lacks an excitatory effect. With monopolar recording from the tip of the micro-injection cannula, the method used in the present experiments, no distinction can be made between these possibilities, but the finding that background activity remained unchanged favours the view that the tubocurarine lacks an excitatory effect. The caudate nucleus contains a high concentration of acetylcholine, choline-acetylase, and acetylcholinesterase (MacIntosh, 1941; Feldberg & Vogt, 1948; Zetler & Schlosser, 1955; Hebb & Silver, 1956; Krnjevic & Silver, 1965). If tubocurarine were to lack an excitatory action on this structure it would mean that there is no association between a high concentration of these three substances in a structure of the central nervous system and responsiveness to tubocurarine which, at the neuromuscular junction and at the synapses of sympathetic ganglia, acts as a competitive inhibitor for acetylcholine. All central effects of tubocurarine so far observed appear to be excitatory and are most likely due to a depolarizing action which this alkaloid lacks in the peripheral nervous system. Here, tubocurarine is inactive on nerve fibres, and the same is true for the central nervous system because microinjections of tubocurarine into the white matter of the corona radiata, or of the corpus callosum, did not produce spikes. 32-2

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