J. Phy8iol. (1967), 188, pp. 131-140 131 With 3 text-fitgurem Printed in Great Britain MONOAMINE OXIDASE INHIBITION: EFFECT ON 5-HYDROXYTRYPTAMINE OUTPUT FROM PERFUSED THIRD VENTRICLE AND BODY TEMPERATURE BY M. B. E. EL HAWARY,* W. FELDBERG AND V. J. LOTTIt From the National Institute for Medical Research, Mill Hill, London, N. W. 7 (Received 27 July 1966) SUMMARY 1. In cats anaesthetized with pentobarbitone sodium the third ventricle was perfused with artificial c.s.f., the effluent was collected in 30 min samples and assayed for 5-hydroxytryptamine (5-HT) on the rat stomachstrip preparation. Rectal temperature was monitored continuously. 2. On perfusion of artificial c.s.f. through the third ventricle, small amounts of 5-HT appeared in the effluent; the amounts decreased with successive samples. 3. When tranylcypromine (Parnate), an inhibitor of amine oxidase, was added to the perfusion fluid and perfusion was continued, the 5-HT output increased. This increase was associated with shivering and a rise in temperature which was not, however, maintained. 4. When tranylcypromine was injected intraperitoneally, during the fall in temperature produced by the pentobarbitone sodium anaesthesia, the 5-HT output also increased, shivering occurred and the fall in temperature was halted or reversed. The effect on temperature was maintained. 5. When the cat was killed and perfusion was continued, the 5-HT output, already elevated by the tranylcypromine before death, increased even further in the next few samples. INTRODUCTION The present experiments in anaesthetized cats are concerned with the effect of the monoamine oxidase inhibitor tranylcypromine on body temperature and on the output of 5-HT from the perfused third ventricle. * Riker Fellow. Permanent address: Faculty of Medicine, Kasr El Aini, Department of Pharmacology, University of Cairo, U.A.R. t Supported by a U.S.A. Public Health Service Fellowship no. 1-F 2-GM-22, 826-01, from the National Institute of General Medical Sciences. 9-2
132 M. B. E. EL HA WARY AND OTHERS The tranylcypromine was either added to the fluid perfusing the third ventricle or injected intraperitoneally whilst rectal temperature was recorded continuously. The release of 5-HT within the anterior hypothalamus of cats is thought to be the mechanism whereby body temperature is raised. This conclusion is based on the findings that 5-HT is present in this part of the brain (Amin, Crawford & Gaddum, 1954), and that injected into the cerebral ventricles, or directly into the anterior hypothalamus, it produces fever (Feldberg & Myers, 1964, 1965). It was further shown that 5-HT appears in the effluent from the perfused third ventricle (Feldberg & Myers, 1966). The amounts, however, were small, and it became apparent that in order to investigate the correlation between 5-HT release and body temperature some means were required whereby the 5-HT output could be substantially increased. Recently it was shown that the addition of 5-hydroxytryptophan (5-HTP), the precursor of 5-HT, to the fluid perfusing the third ventricle greatly increased the 5-HT content of the effluent, and that this increase was often accompanied by a rise in body temperature (El Hawary & Feldberg, 1966). In these experiments the increased 5-HT content in the effluent was not derived from the hypothalamus, but resulted from an action of the decarboxylating enzyme in the walls of the third ventricle on the 5-HTP in the perfusion fluid. In contrast, an increased output of 5-HT produced by the action of an amine oxidase inhibitor would signify an increase in endogenous 5-HT which had escaped enzymic destruction after its release, and if this increased output were associated with a rise in body temperature this would provide evidence of the physiological role of 5-HT as mediator in the hypothalamus for a rise in body temperature. The evidence would be similar to that obtained with the use of inhibitors of cholinesterase for the release of acetylcholine from cholinergic neurones. METHODS Cats of both sexes weighing 2*-83*2 kg were anaesthetized with intraperitoneal pentobarbitone sodium (30 mg/kg). The trachea was cannulated, a polythene tube was tied into the right femoral vein and the head was fixed in a stereotaxic instrument whilst the cat was lying on its belly, insulated from the table by a cotton-wool pad. Rectal temperature was measured by a thermistor probe inserted into the rectum and was monitored continuously by a Kent multichannel recorder as described by Feldberg & Myers (1964). The figures in this paper are plotted directly from the tracings obtained in this way. The third ventricle was perfused with artificial c.s.f. from a cannula inserted into the third ventricle with its opening ventral to the massa intermedia, and the outflow was collected from the cannulated aqueduct. The method of perfusion has been described and illustrated elsewhere (Feldberg & Myers, 1966). The rate of perfusion was 0 05 ml./min and the effluent was collected usually in half-hour samples and assayed for 5-HT on the fundus strip
5-HT AND TEMPERATURE AFTER MAO INHIBITION 133 of the rat's stomach suspended in a 5 ml. bath according to the method described by Vane (1957). Drugs used. The monoamine oxidase inhibitor tranylcypromine sulphate was kindly supplied to us by Dr P. Hey of Smith Kline and French. All values given refer to the salt. The 5-HT used was the creatinine sulphate, but all values are expre3sed in terms of the base. RESULTS Cessation of shivering in response to light pressure applied to the floor of the fourth ventricle To cannulate the aqueduct the cerebellum was slightly raised with a soft metal spatula 3-5 mm wide in order to guide the cannula into the aqueduct. In experiments where shivering was present, this procedure stopped it for varying periods of time, sometimes for several hours. Lifting the cerebellum in itself did not have this effect, but light pressure applied with the spatula to the region ofthe floor ofthe fourth ventricle near the aqueduct immediately stopped shivering. Sometimes merely touching this region was sufficient. The effect was not examined further, but care was taken to avoid it when cannulating the aqueduct. Effect of tranylcypromine o?t the fundus strip of the rat's stomach In relatively large doses, tranylcypromine caused contraction followed by relaxation. Several,ug of tranylcypromine were required to contract the fundus strip suspended in a 5 ml. bath, whereas it responded to less than 1 ng of 5-HT. This is illustrated in Fig. 1, in which the effects of 0*25, 0 5 and 1 ng of 5-HT are compared with those of 10, 20 and 40,tg of tranylcypromine. In this preparation the relaxations which followed the contractions produced by tranylcypromine began before, in others only after the tranylcypromine was washed out, but the muscle always relaxed more rapidly and more fully than when it had been contracted by 5-HT and the 5-HT was washed out; the muscle then remained relaxed for several minutes. There was usually no difference in the time course of the contractions elicited by tranylcypromine or by 5-HT, but in some preparations the muscle contracted more rapidly to tranylcypromine. The specific antagonist of 5-HT, 2-bromolysergic acid diethylamide (BOL), abolished the contractions, but not the relaxations produced by tranylcypromine, which became more pronounced. Figure 1 shows at B the effect of 40,zg tranylcypromine after the fundus strip had been treated with BOL. The small initial contraction may not be an effect of the tranylcypromine but may be due to stopping the overflow, since this alone (at St) produced a similar contraction. The concentration of tranylcypromine used for perfusion of the third ventricle was either 1/100,000 or 1/50,000, and effluent in a volume of not more than 0-5 ml. and usually less than 0-2 ml. was added to the 5 ml. bath
134 M. B. E. EL HA WARY AND OTHERS containing the fundus strip. The amounts of tranylcypromine present in these small volumes were either too small to contract the fundus strip or produced a weak contraction only. However, in order to exclude any possible interference of the tranylcypromine in the perfusion fluid with the assay, the standard solutions of 5-HT were made up in a solution of artificial c.s.f. containing tranylcypromine in the same concentration as present in the perfusion fluid. 5-H7 Tr 5-HT Tr 5-HT Tr Tr St 0-25 Q0 05 20 1 40 40 Fig. 1. Fundus strip of rat's stomach suspended in 5 ml. Krebs solution. At the white spots, responses to 0 25, 0 5 and 1 ng 5-HT and to 10, 20 and 40 /tg tranylcypromine (Tr) kept in the bath for 90 sec. (St), stoppage of overflow for 90 sec. Between A and B, 10,ug BOL added to the bath for 10 min. Effect of tranylcypromine on 5-HT output from perfused third ventricle and on rectal temperature Tranylcypromine added to the perfusion fluid. Perfusion of the third ventricle 'with artificial c.s.f. was begun between 2 and 3 hr after the intraperitoneal injection of the anaesthetizing dose of pentobarbitone sodium. At this time the temperature, which had fallen during the first hour after the injection, had returned to between 36-5 and 400 C. The effluent collected from the cannulated aqueduct in successive half-hour samples contained small amounts of 5-HT. In most experiments the 5-HT output was sufficient for assay on the fundus strip of the rat's stomach. In these experiments the output was found to decrease in the first few samples, and
5-HT AND TEMPERATURE AFTER MAO INHIBITION 135 then remained relatively constant without rising again. It did rise, however, when tranylcypromine was added to the perfusion fluid, as seen from the experiments of Table 1. TABLE 1. Effect on 5-HT output of tranylcypromine perfused in a concentration 1/100,000 (Expts. 3-6), and in Expt. 7, 1/50,000, through third ventricle of anaesthetized cats at a rate of 0-05 ml./min. The bold figures refer to samples collected during perfusion with tranylcypromine, the others during perfusion with artificial c.s.f. No. of 5-HT ng/ml. effluent in successive half-hour samples expt., A 1 4-0 2-0 1-0 0-8 0-8 0-6 0-5 0-6 0-6 0-6 0-6 0-S - 2 5-0 1-0 0-5 0-5 0-4 0-6 0-6 0-5 0-5 0-5 0-6 0-6 - 3 3-8 1-6 1-2 1-6 - 1-0 1-6 1-7 2-1 2-2 3-5 3-0 2-4 4 1-7 1-2 1-0 2-1 3-0 2-7 2-3 2-2 2-3 2-0 - - - 5 1-4 1-0 0-9 0-7 1-1 1-2 1-5 1-8 1-5 1-3 1-3 - 6 0-9 <0-3 <0-3 <0-3 1-3 2-0 2-0 2-0 3-5 3-0 4-0 - 7 1-7 0-8 0-7 0-5 1-0 1-6 1-8 1-4 1-6 1-0 - - The first two experiments show the 5-HT output during perfusion with artificial c.s.f. alone, but in the other five experiments perfusion with artificial c.s.f. was changed after 1-21 hr to perfusion with tranylcypromine 1/100,000 and in one experiment with 1/50,000. In each of these experiments the 5-HT content of the effluent rose in the first or second halfhour sample collected during the tranylcypromine perfusion, reached a maximum in the next few samples, and then remained at this level, or decreased somewhat, although perfusionwith tranylcypromine was continued. The perfusion with tranylcypromine resulted within 2-20 min in onset of shivering, or if shivering was present it became more vigorous. Shivering increased during the following minutes and often spread from the flanks, where it began, to all over the body; however, in the course of 60-90 min it subsided although perfusion with tranylcypromine was continued. The onset of shivering was followed by a rise in rectal temperature which again was not maintained. In fact, in a few experiments the initial rise was followed by a steady decline in temperature. Figure 2 illustrates an experiment in which the tranylcypromine perfusion was begun whilst temperature was rising. The result was a steeper rise which lasted for about 1 hr. Tranylcypromine injected intraperitoneally. Tranylcypromine also increased the 5-HT output when injected intraperitoneally. This is illustrated by the experiments of Table 2. In these, perfusion was begun earlier in the pentobarbitone sodium anaesthesia whilst temperature was still falling, and two half-hour samples were collected before the injection of either 2-5 mg/kg (Expts. 1, 2) or 10 mg/kg (Expts. 3-7). In Expt. 7 tranylcypromine was not only injected intraperitoneally but also perfused (in a concentration of 1/100,000) through the third ventricle. The effect was not greater than when either procedure alone was used. The intraperitoneal injection of tranylcypromine into the anaesthetized
136 1M. B. E. EL HAWARY AND OTHERS cat produced shivering, usually within 5-10 min, which persisted for j-2 hr, whilst the steep fall in temperature was halted, or even reversed, the rise being not more than 0.50 C. The effect on temperature was the same whether the dose of tranylcypromine injected was 2-5 or 10 mg/kg and whether the initial 5-HT output was low or high. This is illustrated by the two experiments of Fig. 3. C.s.f. Tr 1/50,000 42 415-0C41 /_ 2 405 E1 5 0 1 2 3 4 Hours Fig. 2. Record of rectal temperature in a cat anaesthetized by intraperitoneal pentobarbitone sodium during perfusion of the third ventricle at a rate of 0O05 ml./ min with artificial c.s.f., and with tranylcypromine (Tr) 1/50,000. The block diagram below the temperature curve represents 5-HT output in ng/ml. of effluent (Expt. 7 of Table 1). Relase of 5-HT after death Previously it was shown that on perfusion of the third ventricle with artificial c.s.f. the 5-HT content of the effluent increased for some time after the cat was killed (Feldberg & Myers, 1966). Such an increase after death was observed also when the third ventricle was perfused with 5-HTP, although its perfusion before death had already increased the 5-HT output 44-200 times (El Hawary & Feldberg, 1966). In the present experiments the 5-HT content of the effluent from the perfused third ventricle, after it had been increased by tranylcypromine, either added to the perfusion fluid or injected intraperitoneally, increased further after the cat was killed by an overdose of intravenous pentobarbitone sodium. For instance, in Expt. 6 of Table 1 it rose from 4 ng/ml. to
5-HT AND TEMPERATURE AFTER MAO INHIBITION 137 A 36 \2-5 3- m2g/kg 35 B C \ 10 37 * mg/kg 36 6 + E~~~~~~~~~~~~~~~- 44 1 2 3 4 5 Hours Fig. 3. Records of rectal temperature in two cats anaesthetized by intraperitoneal pentobarbitone sodiuim anaesthesia. Records begin shortly after starting perfusion of third ventricle with artificial c.s.f. at a rate of 0-05 ml./min. At the arrow in A, 2-5 mg/kg, at B, 10 mg/kg tranylcypromine injected intraperitoneally. The block diagram below the temperature curves gives 5-HT output in ng/ml. of effluent (A, Expt. 1, B, Expt. 6 of Table 2). TABLE 2. Effect of an intraperitoneal injection of tranylcypromine (2-5 mg/kg, Expts. 1, 2; 10 mg/kg, Expts. 3-7) on 5-HT output from the third ventricle perfused with artificial c.s.f. at a rate of 0-05 ml./min. In addition, in Expt. 7, tranylcypromine was perfused through the third ventricle in a concentration of 1/100,000, and the bold figures refer to the samples collected during this perfusion 5-HT ng/ml. effluent in successive half-hour samples Before i.p. tranylcy- No. of promine After i.p. tranylcypromine expt., A 1 0-8 0-3 0-3 0-3 0-8 1-2 1-5 1-4 1-7 1-4 2 1-6 1-3 2-0 2-7 - - - 6-0 - 3 0-3 < 0-3 0-5 2-5 4-8 5.9 5-0 - - 4 0-8 <0-3 <0-3 4-0 4-0 4.5 - - - 5 2-1 <0-5 1-2 3-3 5-0 - 6-3 - - - 6 2-5 2-0 1-7 3-1 6-0 6-0 3-8 3-7 2-9 3-0 7 0-3 0-3 0-7 2-0 3-0 3-5 4-0 - - -
138 M. B. E. EL HA WARY AND OTHERS 14 ng/ml. in the second 20 min sample collected after death; in Expt. 1 of Table 2, in which the 5-HT output had varied between 1-4 and 1*7 ng/ml. in the half-hour samples collected before death, it rose to 3*8 and to 7-5 and then fell to 5-5 ng/ml. in the first three 20 min samples collected after death. In Expt. 7 of Table 2, with tranylcypromine injected intraperitoneally and perfused through the third ventricle, the 5-HT output after death rose from 4 to 21 ng/ml. in the first, and then decreased to 20 and 14 ng/ml. in the second and third 20 min samples. DISCUSSION The mechanism by which shivering is stopped when short-lasting light pressure is applied to the floor of the fourth ventricle has not been elucidated. One possibility is that the pressure stimulates monoaminergic nerve fibres which terminate in the anterior hypothalamus, and act through the release of noradrenaline. Such a mechanism would account for the long duration of the effect-cessation of shivering for many hoursin response to the brief mechanical stimulus. The contractions of the fundus strip of the rat's stomach produced by tranylcypromine are most likely due to a direct effect on the smooth muscle (or on some nervous structure) in the wall of the stomach and not produced indirectly as a result of inhibition of monoamine oxidase activity. Otherwise, the contractions as well as the subbequent relaxations on washing-out the tranylcypromine would last longeir than the contractions after 5-HT, whereas the reverse was found. The coittractions appear to be a 5-HT-like effect since they were blocked by BOL. According to Tsai & Fleming (1965) tranylcypromine also has a direct stimulating effect on the isolated nictitating membrane of the cat. The main outcome of the present experiments is the finding that the amount of 5-HT in the effluent from the perfused third ventricle increased when the inhibitor of monoamine oxidase, tranylcypromine, was either added to the fluid perfusing the third ventricle or injected intraperitoneally, and that this increase in 5-HT output coincided with onset of shivering and a rise in temperature. The inhibition by tranylcypromine of monoamine oxidase activity in the hypothalamus explains the increased output of 5-HT since, when no longer destroyed after its release, it would diffuse in greater amounts into the fluid perfusing the third ventricle. Persistence of undestroyed 5-HT could also fully explain the shivering and the rise in temperature following the administration of tranylcypromine, as both effects are produced by 5-HT acting on the anterior hypothalamus. Some direct stimulating 5-HT-like effect on the hypothalamus, however, cannot be excluded because tranylcypromine has such an effect on peripheral structures. Yet
5-HT AND TEMPERATURE AFTER MAO INHIBITION 139 the finding of an increased 5-HT output favours the view that the shivering and rise in temperature are due wholly, or mainly, to released 5-HT, and that the experiments therefore provide 'physiological' in addition to the previous 'pharmacological' evidence for a rise in temperature with shivering produced by the release ofendogenous 5-HT in the hypothalamus. Although the 5-HT output remained high for some time during perfusion of tranylcypromine, temperature did not continue to rise and was sometimes followed by a steady fall with cessation of shivering. The mechanism for this fall in temperature is still unexplained. Several possibilities suggest themselves. The inhibition of monoamine oxidase activity may result in accumulation in the hypothalamus not only of 5-HT but also of catecholamines, which probably would not have been detected in the effluent because they are quickly destroyed in the slightly alkaline artificial c.s.f. Yet they would still have exerted their hypothermic effect on the anterior hypothalamus. On the other hand, the tranylcypromine may inhibit the enzymic destruction of 5-HT but not that of the catecholamines since, in cats and dogs, amine oxidase inhibitors are found to increase the level of 5-HT in the brain, but not that of noradrenaline (Vogt, 1959; Spector, Shore & Brodie, 1960; Pscheidt, Morpurgo & Himwich, 1962). Other possibilities would be that the fall in temperature results either from a direct action of tranylcypromine on the hypothalamus or from blockage of the 5-HT sensitive receptors due to persistence of 5-HT at the site of action. With regard to a direct action it is interesting to note that tranylcypromine has two actions on the fundus strip of the rat's stomach; like 5-HT, it produces a contraction, but it also acts in the opposite way causing relaxation, as fully revealed after BOL. In favour of blockage of the 5-HT sensitive receptor due to persistence of 5-HT as the cause of the fall in temperature, is a previous observation obtained with 5-HTP (El Hawary & Feldberg, 1966). Its perfusion through the third ventricle resulted in a greatly increased output of 5-HT which was maintained, whereas in several experiments the effect on temperature was a rise followed by a fall. The 5-HT detected in the effluent originates probably not only from the anterior hypothalamus but also from many other structures contained in the walls of the third ventricle. A similar ubiquitous origin can be assumed for the increase in 5-HT output produced by tranylcypromine. Its effect on the 5-HT output from the perfused lateral ventricle has not been examined, but it is likely that the output would also increase. The increase, after death, in the 5-HT content of the effluent from the third ventricle perfused with artificial c.s.f. (Feldberg & Myers, 1966) could be a sign either of increased release of 5-HT, or of its increased formation from the precursor 5-HTP, or of inhibition of the enzymic destruc-
140 M. B. E. EL HA WARY AND OTHERS tion of 5-HT after its release, or of a combination of all three factors. Inhibition of the enzymic destruction of 5-HT is excluded by the results of the present experiments since the rise in 5-HT output after death occurred when monoamine oxidase activity had been inhibited by tranylcypromine. At present it is not possible to decide between the other possibilities. It was previously shown (El Hawary & Feldberg, 1966; Feldberg & Myers, 1966) that an increase in 5-HT output after death also occurred when the third ventricle was perfused with 5-HTP or when this precursor was injected intraperitoneally, although the 5-HT output had increased to extremely high levels during life. The further increase after death could be the outcome of increased activity ofthe 5-HTP decarboxylating enzyme, since most of the 5-HT detected in the effluent is derived from the 5-HTP perfused through the ventricle or injected intraperitoneally. On the other hand, an increased release of 5-HT after death is not excluded because the administration of 5-HTP increases the 5-HT content of brain (Udenfriend, Bogdanski & Weissbach, 1956). More 5-HT would therefore be available for release after death, and the amounts might well be sufficient to greatly increase the already high 5-HT content of the effluent. Inhibition of the 5-HTP decarboxylase by specific inhibitors of this enzyme might decide between these alternatives. REFERENCES A&nN, A. N., CRAWFORD, T. B. B. & GADDUM, J. H. (1954). The distribution of substance P and 5-hydroxytryptamine in the central nervous system of the dog. J. Phy8iol. 126, 596-618. EL HAWARY, M. B. E. & FELDBERG, W. (1966). Effect of 5-hydroxytryptophan acting from the cerebral ventricles on 5-hydroxytryptamine output and body temperature. J. Physiol. 186, 401-415. FELDBERG, W. & MYERS, R. D. (1964). Effects on temperature of amines injected into the cerebral ventricles. A new concept of temperature regulation. J. Physiol. 173, 226-237. FELDBERG, W. & MYERs, R. D (1965). Changes in temperature produced by micro-injections of amines into the anterior hypothalamus of cats. J. Physiol. 177, 239-245. FELDBERG, W. & MYERw, R. D. (1966). Appearance of 5-hydroxytryptamine and an unidentified pharmacologically active lipid acid in effluent from perfused cerebral ventricles. J. Physiol. 184, 837-855. PSCIIDT, G. R., MORPURGO, C. & HIMWICH, H. E. (1962). Norepinephrine and 5-hydroxytryptamine in various species. Regional distribution in the brain, response to monoamine oxidase inhibitors, comparison of chemical and biological assaymethods for norepinephrine. Comparative Neurochemistry. Proc. 5th Int. Neurochem. Symp. 1964, ed. RICHTER, D., pp. 401-412. Oxford: Pergamon Press. SPECTOR, S., SHORE, P. A. & BRODIE, B. B. (1960). Biochemical and pharmacological effects of the monoamine oxidase inhibitors iproniazid, 1-phenyl-2-hydrazinopropane (JB 516) and I-phenyl-3-hydrazinobutane (JB 835). J. Pharmac. exp. Ther. 128, 15-21. TsA, T. H. &ZFLEMNG, W. W. (1965). Sympathomimetic action.of monoamine oxidase inhibitors in the isolated nictitating membrane of the cat. Biochem. Pharmac. 14, 369-371. UDENFRIEND, S., BOaDANSKI, D. F. & WEISSBACH, H. (1956). Increase in tissue serotonin by administration of its precursor 5-HTP. Fedn Proc. 15, 493. VANE, J. R. (1957). A sensitive method for the assay of 5-hydroxytryptamine. Br. J. Pharmac. Chemother. 12, 344-349. VoGT, M. (1959). Catecholamines in brain. Pharmacol. Rev. 11, 483-489.