J. Physiol. (1965), 181, pp. 317-323 317 With 3 text-figures Printed in Great Britain EFFECT OF YOSCINE ON TE OUTPUT OF ACETYLCOLINE INTO PERFUSED CEREBRAL VENTRICLES OF CATS BY R. L. POLAK* From the National Institute for Medical Research, Mill ill, London, N. W. 7 (Received 12 February 1965) Mitchell (1963) and Szerb (1964) have found that there is an increased release of acetylcholine from the cerebral cortex in sheep, cats and rabbits when atropine is administered either systemically or into the fluid within a Perspex ring applied to the cortex. In the present experiments it is shown that atropine and hyoscine (both non-convulsant drugs), produce a similar increase in the release of the acetylcholine from subcortical structures into the perfused cerebral ventricles. Furthermore, the interaction of hyoscine with leptazol is described. METODS The experimental procedure was essentially the same as described in the preceding paper (Beleslin, Polak & Sproull, 1965), with the exception that some of the cats were anaesthetized with intraperitoneal pentobarbitone sodium (36 mg/kg, with or without supplements of 4-6 mg/kg). As before, gallamine triethiodide (12 mg) i.v. was used for immobilization in the leptazol experiments. RESULTS Intravenous hyoscine and atropine. On prolonged perfusion of the cerebral ventricles with an anticholinesterase the acetylcholine output is known to rise gradually (Bhattacharya & Feldberg, 1958). This occurred also in the present experiments. owever, when hyoscine hydrobromide was injected during such perfusions, there was a steep rise in acetylcholine output of the samples collected during the subsequent 15-30 min (Table 1). The high acetylcholine output was not maintained as seen from the results of those experiments in which the output was followed up over a sufficiently long period of time. A similarly increased release of acetylcholine was obtained with intravenous atropine sulphate (Expt. 6). * NATO Science Fellow. Permanent address: Medisch-Biologisch Laboratorium RVO- TNO, Lange Kleiweg 139, Rijswijk (Z..), olland.
318 B. L. POLAK Experiments 2 and 4 of Table 1 were done under chloralose, the remaining four under pentobarbitone sodium anaesthesia with similar results. TABLE 1. Effect of intravenous injections of hyoscine or atropine on acetylcholine output from cerebral ventricles perfused with neostigmine 1/50,000 of six cats anaesthetized with intraperitoneal pentobarbitone sodium (expts. 1, 3 5 and 6) or intravenous chloralose (expts. 2 and 4). The figures with an asterisk refer to 15 mnin, those without an asterisk, to 30 min samples ACh output in ng/min of successive samples Expt. 1 Expt. 2 Expt. 3 Expt. 4 Expt. 5 Expt. 6 < 1*5 0*5 0*7 1-7 2-8 2-7 1.5 0*9 0*9 2-3 3-8 3-3 [yo8cine [yo8cine 0.9 3'1 44 4-2 0-14 mg/kg] 0-2 mg/kg] 1.0 [yo8cine 5.9 4-9 12-0* 2.7* [yo8cine 1 mg/kg] 7-0 4-8 8-3* 3.7* 1 mg/kg] 21-0* 7 0 4*0 5.5* 3.8* 6.3* 21.0* [yo8cine [Atropine 7-0* 17-0 1 mg/kg] 1 mg/kg] 7.0* 14*0 26*0 14.0* 7.0* 23*0 17.0* 5.9* 20*0 16-0 21*0 In expts. 1-5 of Table 1, the amounts of hyoscine injected varied between 0 14 and 1 mg/kg. The level of acetylcholine output reached did not appear to be dependent on the dosage, but there was a definite correlation with the level of acetylcholine output before the injection. In the two experiments in which this output was as high as 7 and 3 1 ng/min, it rose to 26 and 21 ng/min; in the two in which it was as low as 1 and 0*9 ng/min, it rose to only 7 and 3-8 ng/min; and in the one in which it was 1-5 ng/min it rose to 12 ng/min. The effects of repeated intravenous injections of hyoscine in increasing doses are shown in Fig. 1. Whether a first injection had resulted in a great or small rise in acetylcholine output, the increase produced by subsequent injections was comparatively small even when the dose of hyoscine was 5 or 10 mg/kg. Again there was no correlation of the increase in acetylcholine output with the dose of hyoscine, but this time there was also no correlation with the level of acetylcholine output prevailing before the injections. Perfuaion with hyo8cine. yoscine also increased the output of acetylcholine from the perfused cerebral ventricles when added in a concentration of 1/200,000 to the neostigmine solution delivered through the anterior horn cannula. With these perfusions, mydriasis, the typical peripheral effect of hyoscine on the pupils, was not observed. The results of two experiments are illustrated in Fig. 2. The acetylcholine output rose greatly in the first two 15 min samples collected after
YOSCINE AND ACh OUTPUT 319 the beginning of the hyoscine perfusion, but there was either no or only a small, further rise in acetylcholine output in the subsequent samples, although perfusion with hyoscine was continued. These experiments A 10 B 0 ± ± 1 5 20 5 10 5 0 2 3 4 hr L 0-2 1 5 10 80 Fig. 1. Acetyicholine output inng/min of successive 30 and 15 min samples collected from the cerebral ventricles perfused with neostiginine 1/50,000 in three anethetized cats. Expts. A, B ad C, sae as expts. 3, 1 and 2, of Table 1. At the arrows marked, or L, inltravenous injections of hyoscine or leptazol. The figures below the letters refer to the amounts in mg/kg. further illustrate that an intravenous injection of 1 mg/kg hyoscine given during the hyoscine perfusion after the acetylcholine output had reached its maxrimum, produced either no, or only a slight, further increase in acetylcholine output.
320 R. L. POLAK Interaction of leptazol with hyo8cine. An intravenous injection of leptazol still produced a large increase in acetylcholine output when hyoscine itself had lost its ability to do so, that is, after repeated intravenous injections or during prolonged perfusion through the cerebral ventricles. This is illustrated in expt. C of Fig. 1, and in expt. B of Fig. 2, both under chloralose anaesthesia. A 10 S 1 2 3 hr 5x10-* 40 1 35 25 20 1 5 10 5 0 1 2 3 4 hr t 1 90 Fig. 2. Acetylcholine output in ng/min of successive 30 or 15 min samples collected from the cerebral ventricles perfused with neostigmine 1/50,000 in two cats anaesthetized with chloralose. The horizontal bars indicate the perfusion with hyoscine 1/200,000 added to the neostigmine solutions delivered through the anterior horn cannula. At the arrows marked or L, intravenous injections of hyoscine or leptazol. The figures below the letters refer to the amounts in mg/kg. In expt. C of Fig. 1, an intravenous injection of leptazol was given after four intravenous injections of hyoscine in increasing doses. With the fourth injection of 10 mg/kg the acetylcholine output rose from 6-8 to
YOSCINE AND ACh OUTPUT 321 8*3 ng/min only, but with the subsequent injection of 80 mg/kg leptazol it rose to 30 ng/min. In expt. B of Fig. 2, hyoscine 1/200,000 was perfused through the anterior horn cannula. After 1 hr of this perfusion, when the output of acetylcholine had risen to 12 ng/min, an intravenous injection of 1 mg/kg hyoscine was given. It had no effect on the output of acetylcholine. owever, the output rose to 43 ng/min after a subsequent intravenous injection of 90 mg/kg leptazol. A B 20 r 10l S VE * I 1 2 hr 1 2 Sx10-6 1 x 4 1x0 I-.s hr L L L L 100 100 200 400 Fig. 3. Acetylcholine output in ng/min of successive 30 or 15 min samples collected from the cerebral ventricles perfused with neostigmine 1/50,000 in two cats anaesthetized with pentobarbitone sodium. At the arrow marked L, intravenous injections of leptazol. The figures below the letters refer to the amounts in mg/kg; the horizontal bars indicate the perfusion with hyoscine 1/200,000 (expt. A) and 1/100,000 (expt. B), added to the neostigmine solution delivered through the anterior horn cannula. When leptazol was given first, hyoscine still retained its ability to raise the acetylcholine output, even if the output had already been raised to a high level by the leptazol. The results of two such experiments are given in Fig. 3. The perfusion of the cerebral ventricles with hyoscine was begun after two intravenous injections of leptazol. The doses injected were large because the experiments were done under pentobarbitone anaesthesia and the effect of leptazol on the acetylcholine output was found to be smaller when this anaesthetic was used instead of chloralose. In expt. A, two injections of 100 mg/kg leptazol had increased the acetylcholine output from 16 to 8-3 ng/min and subsequent perfusion with 21 Physiol. 181
322 B. L. POLAK hyoscine 1/200,000 caused a further rise to 20 ng/min. In expt. B, two injections, one of 200 and one of 400 mg/kg leptazol had raised the output from 2*1 ng/min to the high level of 25 ng/min, yet the subsequent perfusion with hyoscine 1/100,000 produced a further rise to 32 ng/min. DISCUSSION yoscine and atropine are not convulsant drugs; hyoscine is in fact regarded as a central depressant. Therefore it is unlikely that they raise acetylcholine output from the caudate nucleus in the same way as leptazol and strychnine, which increase the activity in this nucleus by their convulsant action. It would seem that the increased output of acetylcholine produced by hyoscine and atropine results from a direct action of these drugs on the caudate nucleus, since the effect was also obtained when hyoscine was perfused through the cerebral ventricles, and the likeliest explanation is that proposed by Szerb (1964) for the increased output of acetylcholine from the cerebral cortex after atropine and by Giarman & Pepeu (1962, 1964) for the decrease in acetylcholine content of rat brain produced by hyoscine, atropine and atropine-like substances. To explain the present results it may be assumed that hyoscine and atropine compete with acetylcholine for certain receptor sites in the C.N.s. and prevent uptake of liberated acetylcholine, i.e. that they act in the same way as adrenergic blocking agents do when these elevate the output of the adrenergic transmitter on stimulation of adrenergic nerves, e.g. from the spleen (Brown & Gillespie, 1957; Brown, Davies & Gillespie, 1958; Brown, Davies & Ferry, 1961). This hypothesis would explain several features of the hyoscine-action, for instance, the finding that there was little further increase in the acetylcholine output after repeated injections of hyoscine or after its prolonged perfusion through the cerebral ventricles, an observation similar to that made with atropine by Szerb (1964). Should all the sites of uptake be blocked, additional hyoscine would have no further effect. The correlation between acetylcholine levels in the perfusates before and after the first injections of hyoscine would also be explained, thus: if hyoscine were to act only by blocking acetylcholine uptake, the output of acetylcholine after the hyoscine should be proportional to the level of activity in the cholinergic neurones at the time of injection, as reflected by the magnitude of the acetylcholine output then prevailing. Further, hyoscine should remain effective after leptazol, and leptazol should still continue to increase activity and thus raise the acetylcholine output when hyoscine has become ineffective. This was, in fact, found to be so.
YOSCINE AND ACh OUTPUT 323 SUMMARY 1. In anaesthetized cats an increased output of acetylcholine into the effluent from perfused cerebral ventricles was produced by hyoscine or atropine i.v. and also by intraventricular hyoscine, suggesting a direct action on the caudate nucleus. 2. Once the acetylcholine output had been raised by intravenous or intraventricular hyoscine further administration of hyoscine, even in large doses i.v., had relatively little effect. Yet leptazol i.v. produced a further large increase in acetylcholine output. 3. Since hyoscine and atropine antagonize the actions of acetylcholine in the c.n.s., their ability to increase the acetylcholine output is explained by competition with acetylcholine for certain sites of uptake in the C.N.S. The various features of this hyoscine effect and its interaction with leptazol are discussed along these lines. I wish to express my sincere thanks to Dr P. B. Medawar for the facilities to work in the National Institute for Medical Research, and to Professor W. Feldberg for his continued interest in this work. REFERENCES BELESLIN, D., POLAK, R. L. & SPROULL, D.. (1965). The effect of leptazol and strychnine on the acetylcholine release from the cat brain. J. Physiol. 181, 308-316. BATTACARYA, B. K. & FELDBERG, W. (1958). Perfusion of cerebral ventricles: assay of pharmacologically active substances in the effluent from cisterna and aqueduct. Brit. J. Pharmacot. 13, 163-174. BROWN, G. L. & GILESPIE, J. S. (1957). The output of sympathetic transmitter from the spleen of the cat. J. Phygiol. 138, 81-102. BROWN, G. L., DAvIs, B. N. & GILESPIE, J. S. (1958). The release of chemical transmitter from the sympathetic nerves of the intestine of the cat. J. Physiol. 143, 41-54. BROWN, G. L., DAVIES, B. N. & FERRY, C. B. (1961). The effect of neuronal rest on the output of sympathetic transmitter from the spleen. J. Phy8iol. 159, 365-380. GIARMAN, N. J. & PEPEU, G. (1962). Drug-induced changes in brain acetylcholine. Brit. J. Pharmacol. 19, 226-234. GIARmAN, N. J. & PEPEU, G. (1964). The influence of centrally acting cholinolytic drugs on brain acetylcholine levels. Brit. J. Pharmacol. 23, 123-130. MITCELL, J. F. (1963). The spontaneous and evoked release of acetylcholine from the cerebral cortex. J. Phy8iol. 165, 98-116. SZERB, J. C. (1964). The effect of tertiary and quaternary atropine on cortical acetylcholine output and on the electroencephalogram in cats. Canad. J. Physiol. Pharmacol. 42, 303-314. 21-2