Babraham, Cambridge. significance. It has been found that excitation of the cortex, either through

Transcription

1 98 J. Phy8iol. (1963), 165, pp With 6 text-figure8 Printed in Great Britain THE SPONTANEOUS AND EVOKED RELEASE OF ACETYL- CHOLINE FROM THE CEREBRAL CORTEX BY J. F. MITCHELL* From the Agricultural Research Council Institute of Animal Physiology, Babraham, Cambridge (Received for publication 30 April 1962) The possibility that acetylcholine (ACh) acts as a mediator of synaptic transmission in the cerebral cortex of mammals has been suggested both by experiment and by argument. It has, however, been difficult to obtain direct evidence in support of this view although many of the characteristics that might be expected of a central nervous transmitter have been clearly demonstrated for ACh in the cortex (Feldberg, 1945b, 1950, 1957; Crossland, 1960). They include the demonstration of enzymes for the synthesis and rapid destruction of ACh and the natural occurrence of this substance in the brain. ACh and drugs which are known to affect its action have been shown to influence the electrical activity of the cortex, and recently single cells in the cortex have been selectively activated by the iontophoretic application of ACh through micropipettes (Krnjevid & Phillis, 1961). However, at peripheral synapses the most important step in the identification of cholinergic transmission has been the demonstration of the release of ACh during stimulation of the appropriate nerve. Experiments of this kind, on the intact brain, offer certain difficulties and attempts by several groups of workers to demonstrate a central release of ACh during nervous stimulation have produced conflicting results (see Feldberg, 1945 b): but in 1950 Elliott, Swank & Henderson detected a release of ACh from the surface of the intact cortex and MacIntosh & Oborin (1953) showed that this release was related to the spontaneous electrical activity of the cortex. A technique similar to that of MacIntosh & Oborin has been used in the present experiments to study the effect of direct and indirect stimulation of the cortex on the local release of ACh, in an attempt to assess its significance. It has been found that excitation of the cortex, either through sensory nerves or by direct electrical stimulation, increases the ACh output * Present address: Department of Physiology, St Mary's Hospital Medical School, London, W. 2.

2 ACh RELEASE FROM THE CORTEX 99 in a manner which strongly suggests that it can only have been liberated from nervous structures within the cerebral cortex. The increases observed in the ACh output were dependent upon the pathway and frequency of the applied stimulation, the largest increases per stimulus being obtained by stimulation of peripheral nerves at low frequencies. Part of the work described in this paper has been communicated to the Physiological Society (Mitchell, 1960, 1961). METHODS Sheep were anaesthetized with ether or cyclopropane ( ml./min) and oxygen ( ml./min) from a Boyle's apparatus in closed circuit. Cats and rabbits were usually anaesthetized with allobarbitone (Dial, Ciba, ml./kg) or sodium pentobarbitone (Nembutal, Abbott Laboratories, 30 mg/kg) given intraperitoneally. In other experiments on cats anaesthesia was induced by ethyl chloride and maintained with ether, and in three experiments anaesthesia was maintained by chloralose (60-80 mg/kg) after induction with ethyl chloride. In all experiments the trachea was cannulated and a polythene cannula inserted in a forelimb vein. The head was clamped in a rigid frame and a large part of the cerebral cortex was exposed, usually on both sides of the mid line. The dura was opened and cylindrical Perspex cups, open at both ends and held in miniature universal clamps, were then lowered gently on to the surface of the exposed cortex and adjusted so that there was no obvious disturbance of the blood supply. The cups were each filled with 1.0 ml. of mammalian Ringer's solution (NaCl 9 0, KCI 0-42, CaCl2 0 24, NaHCO3 0-2, glucose 2-0 g/l.). Occasionally fluid leaked from the cups over the surface of the brain, but it was usually possible to seal these leaks by applying warm paraffin jelly to the junction between the brain and the cups. In experiments on sheep the opening of the cups covered 3 0 cm2 of cortex, but in experiments on cats and rabbits smaller cups covering 1-0 cm2 cortex proved more convenient. Two areas of the cortex were studied in all species, the primary somatosensory and the parietal cortex. In some experiments it was found that the volume of fluid in the cups steadily increased at a rate of about 0-02 ml./min. This increase was probably due to the entry of cerebrospinal fluid (c.s.f.) and could be overcome by introducing a needle in to the cisterna magna to connect it with an external reservoir so that the c.s.f. pressure was balanced against the fluid in the cups (Professor F. C. MacIntosh, personal communication). The solutions in the cups were usually stirred by a stream of oxygen bubbles delivered through fine polythene tubes, but in experiments on sheep, with the large diameter cups, vascular pulsation of the brain was sufficient to ensure efficient mixing of the solutions as judged by the behaviour of dye. The body temperature of sheep was maintained at C with an electric blanket. With cats and rabbits the temperature was maintained at 'C by the automatic heating device described by Krnjevi6 & Mitchell (1961 b). The temperature of the solutions in the cups was maintained at 'C by radiant heat if necessary and the temperature of the cortex monitored by a thermocouple, or more conveniently by a bead thermistor (Stantel type F. 2311/300) in a bridge circuit with DC amplification and oscilloscope or pen display. The collection, a8say and identification of ACh. Thirty minutes before beginning the collection of samples the Perspex cups were filled with Ringer's solution containing eserine sulphate 1F0 x 104 g/ml., and at the end of this period the solutions were removed and discarded. The chambers were refilled with 1-0 ml. of Ringer's solution containing fresh eserine, which was left in contact with the cortex for min and then removed by 7-2

3 100 J. F. MITCHELL suction into a graduated tube for assay on the eserinized frog rectus abdominis or leech muscle preparations in a small muscle bath (0 4 ml.). Before assay the cup samples were diluted with distilled water so as to make them isotonic with frog or leech Ringer's solution and they were compared with standard solutions of ACh chloride (Roche) containing eserine. All results are expressed in terms of ACh chloride. Since each sample from the cups had a volume of only 1-0 ml. it was not possible to prepare standard ACh solutions made up in inactivated cup solutions as a routine (Feldberg, 1945a), but in the course of each experiment a few single or pooled cup samples were treated with alkali (1/10th volume N-NaOH), allowed to stand at room temperature for 30 min and neutralized with N-HC1. When tested on the frog rectus or leech muscle preparations these samples were inactive. When known amounts of ACh were then added to the inactivated solutions their assay against standard ACh gave the expected result, suggesting that the cortex was not releasing interfering substances in significant amounts. A total of 46 cup samples from 12 experiments were cross-assayed on the cat (MacIntosh & Perry, 1950) or rat (Straughan, 1958) blood-pressure preparations and on every occasion the assays agreed well (± 10 %) in terms of ACh. On no occasion did the samples give a depression of the blood pressure after the assay preparation had been atropinized, and alkalitreated samples were all inactive. Pooled cup samples from two experiments were assayed on the rat duodenum preparation, and although the presence of eserine in the samples made the assays difficult agreement in terms of ACh was within %. The contraction of the frog rectus muscle, caused by activity in the cup samples, was increased after the muscle had been sensitized with eserine, and if eserine was omitted from the cup solutions no activity appeared in the samples (Fig. 1). Estimates of Na and K concentrations in the Ringer's solutions after they had been in contact with the brain were made during four experiments. Small changes ( ±2 %) were found in 10 % of the samples tested, but differences of this order were found not to affect the bioassay preparations. From this evidence it would seem likely that the active substance released into the cup fluid was ACh or some very similar choline ester. In three experiments on cats the depth at which ACh was released in the surface layers of the brain was studied in collaboration with Dr Szerb, using a push-pull cannula (Gaddum, 1961). The samples in these experiments were assayed for ACh on the dorsal muscle of a leech in a microbath (Szerb, 1961). Electrical stimulation and recording. When ACh release from the cortex was being studied in relation to either the depth of anaesthesia or the spontaneous electrical activity of the brain, or when it was important to maintain a constant level of anaesthesia, the electroencephalograph (e.e.g.) was recorded throughout the experiment. Steel needles or screws were inserted into the skull on either side of the mid line over the occipital cortex, and from these the e.e.g. was recorded and amplified for display on an oscilloscope and for recording by an Ediswan pen-writing unit. Part of the output from the amplifiers was rectified (full wave) and arranged to charge a 40,^F condenser which was automatically discharged every 30 sec. The voltage developed across the condenser was recorded by a second Ediswan pen and the height reached by the pen was taken to represent the summed voltage fluctuation about zero generated by the brain during each 30 sec period. These records (Fig. 3) provided a simple but convenient method for quickly assessing changes in the frequency and amplitude of the e.e.g. and made it possible to compare the level of electrical activity with ACh output. This type of integrated record could be misleading, because if anaesthesia became deep, with the appearance of typical high voltage slow waves, the integrated record would be similar to that produced by low-voltage waves of high frequency, and it was therefore essential to monitor the e.e.g. directly as well as in its integrated form. Direct electrical stimulation of nervous tissue was applied with bipolar silver electrodes with tips 1 5 mm apart and for stimulation of peripheral nerves steel needles were inserted aeeply into the limbs. Constant-voltage rectangular pulses were provided from a two-

4 ACh RELEASE FROM THE CORTEX 101 channel stimulator in which the intensity, frequency and pulse width were independently variable. Evoked potentials were recorded directly from the cortex with unipolar wick electrodes soaked in Ringer's solution, with a silver plate, inserted into the skin of the back, acting as the indifferent electrode. RESULTS ACh release from the unstimulated, anaesthetized cortex As is shown in the results summarized in Table 1, the variation in ACh output from any single sheep or cat over the total period of collection (75 min) was small, as was the difference between the outputs from the two sides of the cortex. Also there was no significant difference in ACh output from the somatosensory and parietal cortex in any one animal. Larger variations occurred from animal to animal and under different anaesthetics, TABLE 1. The spontaneous release of ACh from the primary somatosensory and the parietal cortex of cats and sheep during light anaesthesia. All animals were given atropine intraperitoneally (1-4 mg/kg). Cups were placed bilaterally on the cortex and solutions from these cups were assayed separately over 5 consecutive collection periods of 15 min each. Each result is the mean of 10 samples ACh release (ng/min/cm2 cortex I A + S.E.) Somatosensory Parietal Anaesthetic cortex cups cortex cups Sheep 1 Ether Sheep 2 Ether Sheep 3 Cyclopropane Sheep 4 Cyclopropane Sheep 5 Cyclopropane Sheep 6 Cyclopropane Cat 1 Dial Cat 2 Ether Cat 3 Nembutal *04 Cat 4 Chloralose < 0.3 < 0-3 Cat 5 Chloralose < 0.3 < 0.3 but with the exception of experiments under chloralose these variations were probably due to small differences in the depth of anaesthesia from animal to animal and to individual variations. In two experiments on cats in which chloralose was used no ACh output could be detected (< 0 3 ng/min/cm2 cortex), and in another experiment with ether the administration of chloralose (60 mg/kg) and the withdrawal of ether led to a fall in ACh output within 10 min. This effect, which appeared to be due to an action of chloralose, had previously been observed by MacIntosh & Oborin (1953). It was further found that the relative changes in ACh output produced by drugs or by stimulation occurred also in the presence of atropine. Figure 1, for instance, shows, in a typical experiment on a sheep, how the presence of atropine increased the amounts of ACh liberated in a given

5 102 J. F. MITCHELL time, but did not prevent the increase in ACh release produced by leptazol. No attempt has been made to investigate this action of atropine, but it has been utilized throughout the present experiments to facilitate the assay of ACh. 5 x4 0 NE C A B C D E < mi 6 30mi mn mn 13 Collection periods (15 min each) Fig. 1. The effect of eserine, leptazol and atropine on the ACh output from the left (-~)and right (- - -) parietal cortex of a sheep anaesthetized with cyclopropane. The collection periods were consecutive except for the 30 min intervals indicated. A, cup solutions eserinized (1 x 10-' g/ml.); B, D, E, Leptazol (150 mg/kg ][v.); C., Atropine (5 mg/kg i.v.). During two experiments in which anaesthesia was maintained at a constant depth for long periods (3-5 hr), the time course of the liberation of ACh from the cortex into the cup solutions was studied. Samples were left. in contact with the cortex for periods of 2-5, 5, 10, 15, 20 and 30 min. Figure 2 shows that during the first 15 min of collection the ACh liberated into the cups increased in an approximately linear manner. Over longer periods of collection, however, equilibrium between the ACh in the cup and the brain was approached. This suggests that the ACh leaves the cortex by simple diffusion, since the movement of ACh was proportional to time for %the first min and then, as the concentrations in the cups and the cortex approached each other, an equilibrium concentration was reached and maintained. After giving leptazol ( mg/kg i.v.),- which increased the release of ACh, it was found that an equilibrium was still reached in the same time but at a higher level, suggesting that the amount of ACh available for diffusion in

6 ACh RELEASE FROM THE CORTEX 103 the cortex had been raised. The results have usually been expressed as the average amount of ACh release (g)/min/cm2 of cortex during 15 min collection periods. Expressed in this way they are directly related to the equilibrium concentration that would eventually be reached in the cups. 8 _ 7 0 C4 0 v 3 c: ~ < Duration of collection (min) Fig. 2. The concentration of ACh reached in the cup solutions during collection periods of different durations. Sheep anaesthetized with cyclopropane. Release from left (0-0) and right (0-0) parietal cortex during light anaesthesia and from the right cortex after leptazol (120 mg/kg) (E-I). Each point represents the ACh concentration in one collection sample. If the transfer of ACh from the cortex to the cup solution was by simple diffusion a similar exchange might be expected to occur in the opposite direction. This was tested on four occasions when a known amount of ACh was added to the fluid in the cups while they were on the parietal cortex to give a concentration of 2-0 x 10-7g/ml., and samples were removed at intervals for assay. The ACh concentration in the cups fell steadily and it was found that it again reached an equilibrium concentration in min. The site of ACh release The following experiments suggest that the ACh recovered in the cup solution was released from the cortical tissue immediately beneath it. During 4 experiments on sheep, in which a consistent resting release of ACh was being obtained, the cortex underlying the cups (somatosensory area) was removed by suction and the cups replaced and sealed over the white matter. Sampling was resumed and continued for periods of up to 3 hr, and on no occasion were these samples found to contain detectable amounts

7 104 J. F. MITCHELL of ACh. During these periods simultaneous samples were taken from the intact cortex on the symmetrically opposite side of the brain and no changes in ACh output were found. Although these observations suggested that the ACh was liberated from the cortical layers of the brain they could be criticized because of the inevitable tissue damage caused by removing the cortex, but the cortical liberation of ACh that these experiments suggest is also supported by the experiments ofmacintosh & Oborin (1953), who found that ACh release was abolished from the isolated cortex with its blood supply intact. More evidence in favour of a cortical origin of the liberated ACh was obtained in three experiments on cats with push-pull cannulae. These cannulae, which perfuse only a small volume of tissue, were inserted in 1 mm stages through the surface layers of the brain, and at each stage the perfusate was collected for 10 min and assayed for ACh. The cannula tip was inserted to a total depth 5 mm and the procedure was repeated while withdrawing the cannula. When the tissue at the tip of the cannula had been previously perfused with an anticholinesterase (isopropylmethylphosphorofluoridate, 1.0 x 10-5 g/ml.) ACh (1P0-4-0 x 10-8 g/ml.) appeared in the perfusate as long as the cannula tip was within 3 mm of the surface. At greater depths, when the whole cannula was in the white matter, no ACh was detected (< 1.0 ng/ml.). The ACh collected in the eserinized cup solution did not originate from the blood flowing beneath the cup. Blood samples were withdrawn from the posterior part of the sagittal sinus and from fine needles inserted into veins on the surface of the cortex draining the collection areas. The blood samples collected varied from 5 0 to 0 5 ml. and were immediately assayed on the cat B.P. preparation, the precautions described by MacIntosh & Perry (1950) for preserving ACh in whole blood being observed. In all the samples of blood the ACh level was less than 5-0 ng/ml. In these experiments, in order to simulate the usual experimental conditions and to eliminate the complication of ACh release into the blood from tissue other than the cortex, eserine was not given to the whole animal and so, despite the presence of eserine in the collection cup, some destruction of ACh might occur in the blood in the brief interval between leaving the cup area and being collected for assay. The ACh was also not derived from the c.s.f., since it was found not to diffuse into this fluid under the conditions of the experiment. In three experiments on sheep in which leptazol (100 mg/kg i.v.) increased the release of ACh into the eserinized cup solutions on the somatosensory cortex, c.s.f. was withdrawn from the cisterna magna. The samples were assayed for ACh on the sensitized leech-muscle preparation and were all found to contain less than 3 0 ng/ml.

8 ACh RELEASE FROM THE CORTEX 105 Factors influencing the rate of ACh release Depth of anaesthesia. MacIntosh & Oborin (1953) found that the output of ACh from the surface of the cortex depended on the depth of anaesthesia and was approximately proportional to the spontaneous electrical activity of the cortex. This observation was confirmed in the present experiments when cyclopropane, ether, sodium pentobarbitone or allobarbitone were used for anaesthesia. Medium Light anaesthesia Cortical activity (arbitrary units) Iso,gv _ 2 4 6,/6w, e Mild convulsions 7 8 / I I I /. 1,13.AJ, i 60 sec Fig. 3. The release ofach from the parietal cortex of three sheep anaesthetized with cyclopropane and after atropine (3 mg/kg i.v.). Each point represents the mean of two samples, one from each side of the cortex, collected simultaneously. The abscissa represents the degree of excitation of the cortex expressed in arbitrary Units obtained from measurements of the integrated e.e.g. records. These records are Mustrated below, together with the e.e.g. recorded simultaneously.the figures above the integrated records refer to the arbitrary units assigned to them and correspond to those on the abscissa of the graph. Figure 3 shows a steady increase in the ACh output when, as judged by the e.e.g. and the integrated e.e.g., the depth of anaesthesia was reduced during three experiments on sheep. The output in this type of experiment reached ng/min/cm2 cortex in some animals after giving leptazol

9 106 J. F. MITCHELL (150 mg/kg) and on these occasions it was usual to observe large increases in the voltage of the e.e.g. and some signs of somatic convulsive activity. Direct stimulation of the cortex. Cups were placed bilaterally on the primary somatosensory cortex of sheep, cats and rabbits, and bipolar silver electrodes, insulated except at the tip, were placed in contact with the cortex inside the cups. The cortex was stimulated at fixed frequencies with 0-2 msec pulses during every alternate collection period. Three typical experiments on sheep anaesthetized with cyclopropane are illustrated in 200 Direct stimulation Transcallosal stimulation 150 c 'ZU co 50 C C Stimulus frequency (stimuli/sec) Fig. 4. The effect of direct, transcallosal and peripheral nerve stimulation on the amount of ACh released in 15 mini from the somatosensory cortex. Direct and transcallosal stimulation: each curve represents collections from single sheep and each point is the percentage increase in ACh concentration found in one sample collected during stimulation compared with the ACh concentration in samples collected before andafterstimulation. Periphera nerve stimulation: thecontralateral forepaw was stimulated during alternate collection periods in 9 cats. Each point represents the mean percentage increase in ACh concentration of 5-il samples collected during stimulation compared with the ACh concentration of unstimulated samples. The vertical lines show + S.E. Fig. 4, which shows that fromn the ipsilateral cortex there was a rise in ACh concentration during stimulation at pulses/sec and that the maximal output occurred at stimulus frequencies of about 30/sec when the output was increased by % over the resting level. From the contralateral cortes, which was stimulated simultaneously via the trans-

10 ACh RELEASE FROM THE CORTEX 107 callosal pathways, a small increase occurred which was maximal at 20 stimuli/sec but which was not observed at frequencies over 50/sec. In these experiments the anterior part of the corpus callosum was cut through in the mid line and the previous increase in release during stimulation was abolished, although the resting release remained unaffected. In Fig. 5 these results are expressed as the percentage increase in ACh release/ impulse plotted on log. paper. Experiments on 4 cats and 2 rabbits under s-or I-,.- x 04,P 0 EV 4) a- E C '4 u 4, U a 0._ U a C '4 4, 0*1 F- 1 * I051- I I I I U C t I I I ~~~~~~~a Stimulus frequency (stimuli/sec) Fig. 5. The calculated percentage increase in ACh concentration (log. scales) in the collecting cups in response to a single stimulus during direct (O-O), transcallosal (0-0) and contralateral peripheral nerve stimulation ({-C) at different frequencies. Experimental details same as in Fig. 4. allobarbitone anaesthesia gave similar results, the maximal increase during direct stimulation ( %) occurring at 30 stimuli/sec, whereas with transcallosal stimulation it occurred at 20 stimuli/sec and was % greater than the resting level. Stimulation of peripheral nerve. The effect of controlled afferent stimulation on the output of ACh from the somatosensory cortex was studied on

11 108 J. F. MITCHELL 9 cats anaesthetized with allobarbitone or ether. The sciatic nerve, or in the majority of experiments, the forepaw, was stimulated slowly (1 stimulus/5 sec) and the contralateral primary sensory cortex was explored with a wick electrode. A cup was placed over the point from which maximal evoked responses, having an initial positive deflexion, were recorded. A second cup was placed over the symmetrically opposite side of the cortex and a third on the contralateral parietal cortex. The voltage of the stimulus was twice that which just gave maximal evoked potentials when recorded from the contralateral somatosensory cortex and under these conditions no obvious evoked response could be recorded from the ipsilateral cortex. During alternate collection periods of 15 min each the sciatic nerve or forepaw was stimulated at a fixed frequency and the evoked potentials from the cortex underlying the cup were recorded. Stimulation caused an increase in ACh concentration in cups on the contralateral somatosensory cortex of cats. The result was similar during allobarbitone and ether anaesthesia. Figure 4 shows the increase in ACh concentration obtained At 100/sec no increase during stimulation at frequencies of /sec. was seen, but between 2 and 50 pulses/sec the increase amounted to % over the resting concentration and at 1 stimulus/sec the increase was about 90 %. At even lower frequencies ( stimuli/sec), however, the increase over the resting concentration was only about %. This reduction in the concentration does not mean a reduction in ACh released per stimulus at these low frequencies. If the amounts released were the same at 0 5 and 0-25 stimuli/sec then the increase in concentration would only have been 50 and 25 %. The fact that it was 65 and 50 % suggests that the amounts released per stimulus were greatest at 0.25/sec. This is in fact illustrated in Fig. 5, in which the results of Fig. 4 are expressed as the percentage increase in ACh release/impulse/cm2 cortex. The maximal release occurred at the lowest frequency tested and this is shown in Fig. 6 which gives the actual amount of ACh released/stimulus/cm2 cortex at different frequencies. Figure 6 shows that the characteristics of the ACh release from the somatosensory cortex in response to low-frequency afferent stimulation (< 10/sec) were similar to those found by Straughan (1960) and by Krnjevid & Mitchell (1961 a) at the neuromuscular junction of the rat diaphragm preparation. These results obtained on the neuromuscular junction are plotted in Fig. 6 together with those obtained from the somatosensory cortex. At both sites the ACh release was maximal at the lowest frequencies studied and declined rapidly as the frequency increased. The possibility that a higher release per impulse might occur from the somatosensory cortex at even lower frequencies was not tested, as the small changes in concentration involved could not be assayed reliably. Increases were not detected in samples taken simultaneously from the ipsilateral somatosensory or

12 ACh RELEASE FROM THE CORTEX109 parietal cortex. In one cat, in which chloralose was used as the anaesthetic, stimulation of the forepaw did not result in any detectable release of ACh from the contralateral somatosensory cortex. It is known that the amplitude of the primary surface waves of the cortical evoked potentials is affected by changes in the frequency of stimulation. In the present experiments the amplitude of the cortical potentials was recorded during the collection of ACh from the somatosensory cortex and the voltages of the primary positive and negative Stimulus frequency (stimuli/sec) Fig. 6. The amount of ACh liberated/stimulus/cm2 cortex from the somato-sensory cortex of 9 cats during stimulation of the contralateral forepaw (0-0). ACh release/impulse from the rat hemidiaphragm during stimulation ofthe phrenic nerve expressed as pg ACh/impulse/hemidiaphragm. Straughan, 1960, C9-C; Krnjevic & Mitchell, 1961a, 0-*. surface waves were measured separately from film records made at the start, the middle and the end of each period of stimulation. The results were averaged and expressed as a percentage of their maximum, which occurred at about 0*25 stim/sec. As the stimulus frequency rose from 0-25 to 5/sec the size of the potentials fell quickly to about 40 % of the maximum, and then much more slowly until they could not be detected at frequencies over 50/sec.

13 110 J. F. MITCHELL DISCUSSION MacIntosh & Oborin (1953) showed that undercutting the cortex abolished the spontaneous release of ACh and that cholinergic vasodilator nerve endings made no significant contribution to the liberated ACh. It therefore seemed likely that the ACh was cortical in origin, and the present experiments support this view, since after removing the cortex and collecting from the underlying white matter no ACh could be recovered. More direct evidence to suggest the release of ACh from within the cortex was obtained by using a push-pull cannula. The possibility could also be excluded that the ACh collected from the cortex was being liberated from some distant site in the brain, and was slowly transported by the c.s.f. to the cortex below the cups where it entered the collecting solutions by diffusion, because no ACh was found (< 3*0 ng/ml.), even during leptazol convulsions, in the uneserinized c.s.f. withdrawn from the cisterna magna. The only other way that ACh could reach the cup solutions, except directly from the cortex, would be from the blood, but no ACh was detected in the blood collected from the sagittal sinus or from the cortical veins during the collection of the ACh from the somatosensory cortex. The blood could, however, be removing ACh from the eserinized part of the cortex, so reducing the amounts collected, since it is known that the venous blood from the whole brain contains ACh provided its destruction is prevented by an anticholinesterase (Chang, Chia, Hsii & Lim, 1938; Chute, Feldberg & Smyth, 1940). In the present experiments the eserinization of the cortex under the cup would not be sufficient to allow such an accumulation of ACh in the blood leaving the brain. Nevertheless, an approximate calculation of the possible loss ofach from the cortex can be made on the assumption that the ACh content of the sagittal sinus blood lay on the threshold of the assay sensitivity, 5 ng/ml., and that 5% of the ACh was contributed by the cortex under the cup. If we assume a cortical blood flow of 0 7 ml./ min/g cortex (Landau, Freygang, Rowland, Sokoloff & Kety, 1955) and that the ACh collections were from a volume of cortex of 0-2 cm3, the total blood flow through this tissue in 1 min would be about 0 14 ml. and could remove ng ACh/min. The lowest outputs recorded in the present experiments were 01 ng/min/cm2 cortex under conditions of medium anaesthesia, but usually the outputs were times greater, which makes it unlikely that the values for the release of ACh are too low owing to escape into the blood stream. Of the anaesthetics used, only with chloralose was a release of ACh from the somatosensory cortex not detected. Chloralose, however, produced a depression of high-frequency cortical activity, as previously observed by Beecher & McDonough (1939) and by Adrian (1941). Since the ACh output

14 ACh RELEASE FROM THE CORTEX A11ill is directly related to spontaneous cortical activity the failure to detect ACh might be expected in the unstimulated cortex. Under chloralose, stimulation of afferent nerves also did not result in a detectable release of ACh. Unlike allobarbitone and ether, chloralose is known to abolish thalamic after-discharges following sensory stimulation (Adrian, 1941) which after-discharges have been shown to be particularly sensitive to the action of eserine and ACh applied topically to the cortex (Chatfield & Dempsey, 1942). If the nerve endings involved in the afterdischarges are cholinergic, as this evidence might suggest, then this would account for the absence of ACh release during peripheral nerve stimulation under chloralose anaesthesia. In this connexion it is interesting to note that Haase & van der Meulen (1961) found that small doses of chloralose abolished the inhibitory effect of antidromic ventral root stimulation on evoked spinal motoneurone activity. The chloralose was probably acting at the motor axon collateral synapses, which are known to be cholinergic, and the effect was not obtained with Nembutal or hydroxydione sodium. The amount of ACh liberated from the cortex in a given time has been shown to be roughly proportional to the spontaneous electrical activity of the cortex. This electrical activity is likely to provide an index of neuronal activity (Adrian, 1936) and therefore if the ACh is released by cortical nerve endings an association between these events might be expected. Directly relevant to this suggestion are the experiments of Tobias, Lipton & Lepinat (1946), Richter & Crossland (1949), Elliott et al. (1950) and Crossland (1953), who have shown that the amount of ACh present in rat brain increases during anaesthesia and decreases during convulsions. The increase in ACh content of the brain during anaesthesia cannot be attributed to stimulation of its synthesis or to inhibition of cholinesterase activity, and it has been suggested that it must be due to a decreased liberation of ACh from nervous tissue containing it (Crossland, 1953) with a consequent fall in the amount of ACh available for destruction. This suggestion is supported by the present experiments in which the ACh released in the brain was not destroyed because of the presence of an anticholinesterase and the ACh collected in the cup solutions fell during anaesthesia and increased during convulsions. Although ACh is being continually liberated from the cortex the amounts involved are extremely small when it is considered that the choline acetylase contained in 1 g of fresh cortical tissue from the cat is able to synthesize ug of ACh/min in vitro (Hebb & Silver, 1956). It is, however, unlikely that the enzyme system in vivo is synthesizing ACh to its full capacity; it is more likely that the situation resembles that found in the sympathetic ganglion, in which ACh release occurs at only yloth the rate

15 112 J. F. MITCHELL of synthesis (Birks & MacIntosh, 1961) and that the enzyme can synthesize ACh four times as fast in vitro in the presence of suitable substrates (Banister & Scrase, 1950). Release from the electrically stimulated cortex. Attempts to demonstrate the release of ACh from the brain during electrical stimulation have produced conflicting results (see reviews of Feldberg, 1945b; Schain, 1960). Direct stimulation of the brain, while a valuable technique in some types of investigations, is likely to provide misleading results when information is required from discrete systems. Not only is spread of current difficult to control but stimuli are applied unselectively to every structure near the electrodes, and the possibility of stimulating many combinations of inhibitory and excitatory pathways is always present. These difficulties may explain some of the previously conflicting results and to overcome them, in the present experiments, the somatosensory cortex was activated by transcallosal and peripheral nerve stimulation as well as by direct stimulation. The maximal calculated amounts of ACh released by a single stimulus were found to vary under these conditions. Peripheral nerve stimulation yielded the greatest release and the release on transcallosal stimulation of the cortex was the smallest. The amounts of ACh calculated to be released by a single stimulus over a wide range of stimulus frequencies were also different and may be largely attributable to the limitations imposed by frequency-sensitive synapses on the transmission of impulses to the cortex, and by the different number of cholinergic nerve endings activated. With direct stimulation, when the whole applied stimulus could reach every nearby cholinergic nerve ending without relying on transmission across synapses, the output would be expected to remain high over a wide range of frequency. However, for transcallosal and peripheral stimulation to be equally effective over this range of frequency the transmission of impulses across many synaptic relays would have to be complete, but there is abundant evidence that this is true only at low frequencies of stimulation. The transcallosal response, as judged by the surface evoked potential, rapidly deteriorates at stimulus frequencies above 100/sec (Clare, Landau & Bishop, 1961) but at frequencies below this the response is facilitated, (Grafstein, 1959; Clare et al. 1961) particularly the primary negative phase. At 20/sec facilitation is greatest and incremental spindles appear, while even at 2 stimuli/sec part of the response is facilitated. The finding that the ACh release fell simultaneously with the amplitude of the transcallosally evoked potential at 100 stimuli/sec, and was maximal at 20/sec when facilitation was greatest, suggested that a failure of nervous conduction in the transcallosal pathway rather than a property of cholinergic nerve endings determines the ACh output.

16 ACh RELEASE FROM THE CORTEX 113 Similarly, during peripheral nerve stimulation at high frequencies the fall in evoked ACh output per impulse is probably due to a failure of the afferent impulses to reach the cortex, because even at frequencies as low as 5/sec the peripheral volley is likely to be affected in the spinal cord, and at slightly higher frequencies (> 10/sec) the response would be further modified in the thalamus. It is known that when stimuli are separated by only 45 msec the evoked electrical response is abolished in the cortical radiation and in the cortex, although it remains almost maximal in the tegmentum (Marshall, 1941). The finding of a direct parallelism between the release of ACh from the primary sensory cortex and the amplitude of the evoked response during stimulation of the peripheral nerve at different frequencies, and the fact that these surface potentials are altered by ACh and prostigmine applied topically to the cortex or injected into the cerebral circulation (Chatfield & Dempsey, 1942; Beckett & Gellhorn, 1948; Bremer & Chatonnet, 1949; Marrazzi & Hart, 1950; Chatfield & Purpura, 1954) might suggest that a proportion of the ACh released from the cortex is derived from the structures responsible for the generation of these potentials. Yet it is more likely that the association between ACh release and the magnitude of the evoked potentials is a reflexion of a progressive reduction in afferent activity reaching the cortex as the frequency of peripheral stimulation increases. Since afferent volleys can be thought to reach the cortex without modification at frequencies below 5-10/sec, the characteristics at these frequencies of ACh release from cortical nerve endings can be compared during direct transcallosal and peripheral nerve stimulation. Under these conditions the maximal ACh release per stimulus was less during direct and transcallosal stimulation than during peripheral nerve stimulation. Therefore if every cholinergic nerve ending releases the same amount of ACh when activated by a single impulse it is likely that the first two types of stimulation involve fewer cholinergic endings than the third. This suggestion would also explain the failure of direct and transcallosal stimulation at frequencies below 5-10/sec to produce sufficient ACh to be detectable on bioassay. The possibility, however, of a release of ACh from the cortex by reverbatory circuits triggered by the stimulus cannot be excluded. It may be that the three types of stimulus activate directly a similar number of cholinergic nerve endings but a peripheral stimulus, and to a less extent the direct stimulus, excite reverbatory circuits, perhaps via the thalamus, thereby activating many additional cholinergic synapses. This view is supported by the action of chloralose or deep anaesthesia in reducing ACh release because both these procedures are known to reduce or abolish the thalamic after-discharges (Adrian, 1941), which are known to be sensitive to ACh and eserine (Chatfield & Dempsey, 1942). Physiol

17 114 J. F. MITCHELL The fact that the characteristics of ACh release from the somatosensory cortex and at the neuromuscular junction in response to low-frequency stimulation are rather similar may suggest that the basic mechanism subserving the release of ACh at peripheral and central cholinergic nerve endings is similar. The ACh released from the rat hemi-diaphragm, by stimulation of the phrenic nerve, is the result of activating approximately 10,000 cholinergic nerve endings (Krnjevi6 & Miledi, 1958; Krnjevic & Mitchell, 1961 a) and the maximal release of ACh, which occurred at 2 stimuli/sec, was 1-8 x g/stimulus/nerve ending. In the present experiments the maximal amount of ACh released has been calculated as being 93 pg/ stimulus/cm2 cortex. If the ACh entering a cup covering 1.0 cm2 cortex is assumed to be coming from a volume of cortical tissue of 0-2 cm3, and if the cortical nerve endings are each releasing the same amount of ACh per stimulus, as the nerve endings at the neuromuscular junction, then the number of cholinergic endings liberating ACh in response to a single peripheral afferent volley would be 255/mm3 or 51,000 below the cup. Although this estimate may be a little too low, since it assumes that all the liberated ACh is efficiently collected, the fact that the density of neurones in the cat cortex is about 25,000/mm3 suggests that only a small proportion of cortical synapses are cholinergic. They would appear to be involved in events triggered by the arrival of impulses from the primary afferent pathways, to a less extent by transcallosal and direct activation and also in association with the spontaneous electrical activity of the cortex. It may well be that the release under all these conditions results from secondary effects of cortical excitation such as the activation of corticothalamic or intracortical circuits which include cholinergic synapses within the cortex. The general characteristics of this release support the view, for which there already exists important but less direct evidence, that cholinergic transmission occurs in the cortex. SUMMARY 1. In the presence of a topically applied anticholinesterase ACh was found to be continuously released from the surface of the cerebral cortex of sheep, cats and rabbits anaesthetized with ether, allobarbitone, cyclopropane or Nembutal. 2. The rate of release was ng ACh/min/cm2 cortex and was roughly proportional to the electrical activity of the brain. 3. Atropine given intraperitoneally increased the output of ACh and chloralose reduced it to undetectable levels. 4. Direct electrical stimulation of the cortex or excitation by transcallosal or peripheral stimulation increased the rate of ACh release from

18 ACh RELEASE FROM THE CORTEX 115 the primary somatosensory cortex. This increase in the rate of ACh release depended on the frequency of stimulation. 5. A maximal release of ACh per stimulus was obtained by sensory nerve stimulation at 0X25 stimuli/sec and was 93 pg/stimulus/cm2 cortex. The maximal release per stimulus by transcallosal and direct stimulation was only % of this value and was only obtained at higher frequencies of stimulation, suggesting that fewer cholinergic nerve endings were activated by this type of stimulation. 6. It is suggested that the results provide further evidence for the existence in the cerebral cortex of cholinergic nerve endings which may lie in the primary afferent pathways and/or in other associated circuits which include cholinergic intracortical synapses. I am indebted to Miss C. Hebb for many helpful discussions and to Mr J. 0. Yates for his excellent technical assistance. REFERENCES ADRIAN, E. D. (1936). The spread of activity in the cerebral cortex. J. Physiol. 88, ADRIAN, E. D. (1941). Afferent discharges to the cerebral cortex from peripheral sense organs. J. Physiol. 100, BANISTER, J. & SCRASE, M. (1950). Acetylcholine synthesis in normal and denervated sympathetic ganglia of the cat. J. Physiol. 111, BECKETT, S. & GELLHORN, E. (1948). Role of acetylcholine in the activity of sensorimotor and suppressor areas of the cortex. Amer. J. Physiol. 153, BEECHER, H. K. & MCDONOUGH, F. K. (1939). Cortical action potentials during anaesthesia. J. Neurophysiol. 2, BIRKS, R. & MACINTOSH, F. C. (1961). Acetylcholine metabolism of a sympathetic ganglion. Canad. J. Biochem. Physiol. 39, BREMER, F. & CHATONNET, J. (1949). Ac6tylcholine et cortex cerebral. Arch. int. Physiol. 57, CHANG, H. C., CHIA, K. F., HsU, C. H. & LIM, R. K. S. (1938). Humoral transmission of nerve impulses at central synapses. II. Central vagus transmission after hypophysectomy in the dog. Chin. J. Physiol. 13, CHATFIELD, P. 0. & DEMPSEY, E. W. (1942). Some effects of prostigmine and acetylcholine on cortical potentials. Amer. J. Physiol. 135, CHATFIELD, P. 0. & PURPURA, D. P. (1954). Augmentation of evoked cortical potentials by topical application of prostigmine and acetylcholine after atropinization of the cortex. Electroenceph. clin. Neurophysiol. 6, CHUTE, A. L., FELDBERG, W. & SMYTH, D. H. (1940). Liberation of acetylcholine from the perfused cat's brain. Quart. J. exp. Physiol. 30, CLARE, M. H., LANDAU, W. M. & BISHOP, G. H. (1961). The cortical response to direct stimulation of the corpus callosum in the cat. Electroenceph. clitn. Neurophysiol. 13, CROSSLAND, J. (1953). The significance of brain acetylcholine. J. Ment. Sci. 99, CROSSLAND, J. (1960). Chemical transmission in the nervous system. J. Pharm., Lond., 12, ELLIOTT, K. A. C., SWANK, R. L. & HENDERSON, N. (1950). Effects of anaesthetics and convulsants on the acetylcholine content of brain. Amer. J. Physiol. 162, FELDBERG, W. (1945a). Synthesis of acetylcholine by tissue of the central nervous system. J. Physiol. 103, FELDBERG, W. (1945b). Present views on the mode of action of acetylcholine in the central nervous system. Physiol. Rev. 25, FELDBERG, W. (1950). The role of acetylcholine in the central nervous system. Brit. mcd. Bull. 6,

19 116 J. F. MITCHELL FELDBERG, W. (1957). Acetylcholine. In Metabolisn of the Nervous System. Ed. RICHTER, D. London: Pergamon Press. GADDUM, J. H. (1961). Push-pull cannulae. J. Physiol. 155, 1-2P. GRAFSTEIN, B. (1959). Organisation of callosal connections in suprasylvian gyrus of cat. J. Neurophysiol. 22, HAASE, J. & VAN DER MEULEN, J. P. (1961). Die spezifische Wirkung der Chloralose auf die recurrente Inhibition tonischer Montoneurone. Pflig. Arch. ges. Physiol. 274, HEBB, C. 0. & SILVER, A. (1956). Choline acetylase in the central nervous system of man and some other mammals. J. Physiol. 134, KRNJEVIC, K. & MILEDI, R. (1958). Motor units in the rat diaphragm. J. Physiol. 140, KRNJEVIC, K. & MITCHELL, J. F. (1961 a). The release of acetylcholine in the isolated rat diaphragm. J. Physiol. 155, KRNJEVIC, K. & MITCHELL, J. F. (1961 b). A simple and reliable device utilizing transistors for the maintenance of a constant body temperature. J. Physiol. 158, 6-8P. KRNJEVIC, K. & PHILLIS, J. W. (1961). Sensitivity of cortical neurones to acetylcholine. Experientia, 17, 469. LANDAU, W. M., FREYGANG, W. H., ROWLAND, L. P., SOKOLOFF, L. & KETY, S. S. (1955). The local circulation of the living brain; values in the unanaesthetized and anaesthetized cat. Trans. Amer. Neurol. Ass. 80, MACINTOSH, F. C. & OBORIN, P. E. (1953). Release of acetylcholine from intact cerebral cortex. Abstr. XIX int. physiol. Congr MACINTOSH, F. C. & PERRY, W. L. M. (1950). Biological estimation of acetylcholine. Methods med. Res. 3, MARRAZZI, A. S. & HART, E. R. (1950). Adrenergic and cholinergic influences on evoked potentials in pathways of simple synaptic pattern. Electroenceph. clin. Neurophysiol. 2, 116. MARSHALL, W. H. (1941). Observations on subcortical somatic sensory mechanisms of cats under nembutal anaesthesia. J. Neurophysiol. 4, MITCHELL, J. F. (1960). Release of acetylcholine from the cerebral cortex and the cerebellum. J. Physiol. 155, 22-23P. MITCHELL, J. F. (1961). Acetylcholine release from the cerebral cortex during stimulation. J. Physiol. 158, P. RICHTER, D. & CROSSLAND, J. (1949). Variation in acetylcholine content of the brain with physiological state. Amer. J. PhWsiol. 159, SCHAIN, R. J. (1960). Neurohumors and other pharmacologically active substances in cerebrospinal fluid: a review of the literature. Yale J. Biol. Med. 33, STRAUGHAN, D. W. (1958). Assay of acetylcholine on the rat blood pressure preparation. J. Pharm., Lond., STRAUGHAN, D. W. (1960). The release of acetylcholine from mammalian motor nerve endings. Brit. J. Pharmacol. 15, SZERB, J. C. (1961). The estimation of acetylcholine, using leech muscle in a microbath. J. Physiol. 158, 8-9P. TOBIAS, J. M., LIPTON, M. A. & LEPINAT, A. A. (1946). Effects of anaesthetics and coiivulsants on brain acetylcholine content. Proc. Soc. exp. Biol., N.Y., 61,