EVIDENCE THAT TRANQUILIZING ACTION OF RESERPINE IS ASSOCIATED WITH CHANGE IN BRAIN SEROTONIN AND NOT IN BRAIN NOREPINEPHRINE

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1 835 EVIDENCE THAT TRANQUILIZING ACTION OF RESERPINE IS ASSOCIATED WITH CHANGE IN BRAIN SEROTONIN AND NOT IN BRAIN NOREPINEPHRINE EFFECTS OF RESERPINE, RAUNESCI NE, BENZOQUI NOLIZI NES, DIMETHYL- AMINOBENZOYL METHYLRESERPATE, AND STRESS B. B. BRODIE, K. F. FINGER,2 F. B. ORLANS,3 G. P. QUINN AND F. SULSER4 Laboratory of Chemical Pharmacology, National Hear! Institute, National Institutes of Health, Bethesda, Maryland Received for publication November 28, 1959 Numerous studies show that reserpine releases bound serotonin and norepinephrine throughout the body, and thereby lowers the content of these amines in brain and peripheral tissues. The question arises whether the central actions of reserpine are associated with the change in brain serotonin or with the change in brain norepinephrine, for the drug decreases the levels of these amines in rabbit brain at the same rate, to the same extent, and for the same period of time (Brodie et a!., 1957). Proof of which amine determines the central actions of reserpine might be provided by investigation of a drug that releases only one of the brain amines. K#{228}rkiand Paasonen (1959) concluded that depletion of brain norepinephrine is responsible for the central actions of the Rauwolfia alkaloids. Their experiments indicate that reserpine and raunescine decrease the level of norepinephrine more than the level of serotonin, and that the sedative action of raunescine is linked with the reduction in norepinephrine content. Pletscher et at. (1959) drew a similar conclusion from studies of the benzoquinolizine derivatives, Ro (compound I) and Ro (compound II). Our results show that reserpine, raunescine, and compounds I and II do not selectively release norepinephrine or serotonin in brain; hence it is not possible to associate the sedation produced Some of the findiigs have been presented in a preliminary report in Fed. Proc. 18: 388, 1959, and at the Fall Meeting of the American Society for Pharmacology and Experimental Therapeutics, Coral Gables, Florida, September Guest worker, Pfizer Laboratories. Ciba Fellowship. Fellowship of the Swiss Academy of Medical Sciences. 25 by these drugs with either one of the amines. We have demonstrated that a reserpine analogue, dimethylaminobenzoyl methylreserpate (Su 5171) can selectively release brain norepinephrine without producing sedation. In stressed animals, moreover, reserpine depletes most of the brain norepinephrine and only negligible amounts of the brain serotonin, though it does not elicit sedation. The formulas of the benzoquinolizine derivatives and of Su 5171 appear on page 251. METHODS AND MATERIALS. Materials. Solutions of Su 5171 or raunescine were prepared by dissolving 4 mg of the free bases in a few drops of glacial acetic acid and adding.2 ml of propylene glycol,.2 ml of ethanol, and water to a volume of 4 ml. Further dilutions were made with water. A reserpine solution was prepared by dissolving the lyophilized phosphate in water. The hydrochlorides of the benzoquinolizines (compounds I and II) were dissolved in saline. New Zealand White rabbits, male, were given drugs intravenously, and mice (NIH general purpose, male) and rats (Sprague-Dawley, male) intraperitoneally. Chemical methods. At various times after drug administration, rabbits were killed by an intravenous injection of air, mice and rats by decapitation. Brains were rapidly removed and immediately analyzed for amine content or stored in a freezer. Untreated animals served as controls. In previously published methods for the fluorimetric estimation of tissue serotonin (Bogdanski et al., 1956) and norepinephrine (Shore and Olin, 1958), a separate extraction procedure was described for each amine. We used the modified method of Mead and Finger (this laboratory, unpublished), who have shown that the butanol

2 196 CENTRAL ACTION OF RESERPINE 251 CH3 O)N CII, N-CH3 CH3 OOC_OCO_O Su 5171 (Ciba) Dimethylaminobenzoyl methylreserpate Oj O I N / \ CII, I / --CH HO CII, CH2 O CHZOL)/N HO K --CH CH (H, H-OCH3 Compound I Ro (Hoffmann-La Roche) H3 Compound II Ro (Hoffmann-La Roche) 1 8 -I z I TIME -HOURS FIG. 1. Serotonin and norepinephrine content of rat brain at various times after administration of reserpine (1 mg/kg, i.p.) and raunescine (2.5 mg/kg, i.p.). Each value is the mean of at least 3 experiments. For each experiment the brains of 3 rats were pooled for analysis. Normal values ± SE.; brain serotonin.48 g/g ±.17; brain norepinephrine.49 g/g ±.16.

3 252 BRODIE ET AL. Vol. 129 TABLE 1 Serotonin and norepinephrine content of rat brain 4 hours after i.p. injection of reserpine (1 mg/kg) and raunescine (2.5 mg/kg) The brains of 3 animals were pooled for each estimation. No. of Expts. Serotonin pg/g ± S.E. Norepinephrine pg/g ± SE. Control ± ±.16 Reserpine ±.8.16 ±.15 Raunescine 6.19 ±.8.18 ±.16 extraction procedure of Shore and Olin also extracts serotonin. In the modified procedure the amount of each amine is measured in separate aliquots of the final acid extract as described in the methods for norepinephrine and serotonin cited above. RESULTS. Effect of reserpine and raunescine on norepinephrine and serotonin levels in rat brain. Rats received 1. mg of reserpine per kg intraperitoneally. The drug reduced the levels of brain norepinephrine and serotonin at the same rates and to the same extents (fig. 1), and producecl deep sedation in 3 to 4 hours. In a separate experiment reserpine was given to a larger number of rats and the lowering of brain amines measured after 4 hours, at which time K#{227}rki and Paasonen (1959) indicated that reserpine selectively released norepinephrine. In this group of animals the brain levels of both amines were found to be about 35% of normal (table 1). Similar results were obtained with 2.5 mg of drug per kg. The intraperitoneal injection of 2.5 mg of raunescine per kg also lowered the levels of brain norepinephrine and serotonin at the same rate and to the same extent (fig. 1). In 4 hours, when the animals showed deep sedation, the brain level of norepinephrine was 37% of normal and that of serotonin was 42% of normal (table 1). Effect of the benzoquinolizines (compounds I and II) on norepinephrine and serotonin levels. Mice were given 5 mg of the drugs per kg intraperitoneally. Compound I, the more potent tranquilizer, depleted serotonin to about 3% and norepinephrine to about 4% of normal within 2 minutes; the serotonin level then rose rapidly and returned to 7% of normal in 2 hours (fig. 2). MICE z 4 8 $2 4 8 $2 TIME- MINUTES FIG. 2. Serotonin and norepinephrine content of whole brains of mice at various times after administration of compounds I and II. The animals received 5 mg/kg of drugs i.p. In each experiment the brains of 6 mice were pooled for analysis. Normal values ± S.E.; brain serotonin.71 ig/g ±.4; brain norepinephrine.55 g/g ±.4. Each normal value is the mean of 12 experiments. Other values in figure are the mean of 8 experiments. There was a significant difference betweeni serotonin levels in 2 minutes (P <.1) and norepinephrine levels in 2 minutes (P <.1). There was no difference for either amine at 6 minutes.

4 196 CENTRAL ACTION OF RESERPINE 253 NOREPINEPHRINE SEROTONIN C a: 2-4 COMPOUND I SE A--A COMPOUND ± SE 6 $2 $8 6 2 $8 TIME-MINUTES FIG 3. Serotonin and norepinephrine content of rabbit brain stem at various times after administration of the benzoquinolizines, compounds I and II. Animals received 5 mg/kg of drugs iv. Normal values ± S.E.; brain serotonin.76 /hg/g ±.5; brain norepinephrine.6 tg/g ±.3. Each normal value is the mean of 9 animals. Other values in figure are the mean of 3 animals. The drug induced a definite sedation which appeared shortly after administration and lasted for about 4 minutes. Compound II, the less potent drug, released in 2 minutes a smaller proportion of the brain amines; serotonin was lowered to about 5% of normal and the norepinephrine to about 55% of normal (fig. 2). This drug elicited relatively little sedation. Rabbits received 5 mg of the drugs per kg intravenously. Compound I produced a profound sedation, which appeared soon after the injection and was still pronounced after 3 hours. The levels of the brain amines were lowered to about the same extent, 2 to 25% of normal (fig. 3). In contrast, compound II, which elicited no noticeable sedation, reduced the content of brain serotonin to 75% of normal and norepinephrine to 6% of normal (fig. 3). Thus, comparison of brain amine levels after administration of compounds I and II does not indicate which of the amines, norepinephrine or serotonin, is connected more closely with the tranquilizing action. Effat of Su 5171 on serotonin and norepinep/rine levels in rabbit brain stein. After administration of a small dose of Su 5171,.25 mg/kg, the serotonin content of rabbit brain stem declined only slightly over a period of 4 hours (fig. C I a: z o /a TI ME- HOURS FIG. 4. Serotonin and norepinephrine content of rabbit brain stem at various times after administration of.25 mg of Su 5171 per kg, i.v. Normal values of brain amines are the same as in figure 3. Other values in figure are the mean of 4 to 7 animals. 4). In contrast, the level of norepinephrine declined over the same period to about 5% of normal. The animals were not sedated, though they showed slight miosis. After administration of a large dose of Su 5171,

5 254 BRODIE ET AL. Vol. 19 o a 4 S S TIME- HOURS FIG. 5. Serotonin and norepinephrine content of rabbit brain stem at various times after administration of 2 mg of Su 5171 per kg, i.v. Normal values of brain amines are the same as in figure 3. Other values in figure are the mean of 3 or 4 animals. 2 mg/kg, the levels of both amines were at first lowered to the same extent, 3% of normal within 1 hour (fig. 5). The norepinephrine content remained at this level over a period of 6 hours. In contrast, the level of serotonin increased progressively after 2 hours and in 6 hours was 65% of normal. The same dose of drug produced a deep sedation, loss of righting reflex, and adynamia. The effects were evident in 3 minutes, maximal for 2 hours, and gradually decreased over a period of 6 hours. At this time the animals were no longer obviously sedated but showed a slight miosis and a small decrease in locomotor activity. Effect of reserpine on brain levelsof serotonin and norepinephrine in stressed rats and rabbits. Rats were placed in a cold room at a temperature of 4#{176}C for a period of 4 hours. They were then injected, intraperitoneally, with 1 mg of reserpine per kg and kept in the cold room. No sedation was elicited over the next 4 hours. In contrast, rats placed in a cold room and given chlorpromazine (5 mg/kg, intraperitoneally) showed marked sedation. Four hours after reserpine administration, the cold-treated animals were killed and the brains analyzed for serotonin and norepinephrine. The results in figure 6 show that the release of serotonin was small compared to that in control animals not subjected to a cold temperature. On the other hand, norepinephrine release was about the same in the control animals and in animals subjected to cold. Rabbits placed in the cold for a period of 3 hours and then given 1 mg of reserpine intravenously, also did not become sedated. A small degree of miosis was evident, but there was no los.s of righting reflex, no enhanced salivation, and no active closure of the eyelids. As seen in figure 6, I a: z TIME- HOURS FIG. 6. Comparison of the effect of reserpine on brain norepinephrine and serotonin content of rats and rabbits kept at room temperature and of animals subjected to a temperature of 4#{176}C. For each experiment the brains of 3 rats were pooled for analysis. Circles represent serotonin and triangles norepinephrine. Normal values for rat brain are same as in table 1 and for rabbit brain stem are the same as in figure 3. Other values in figure are the mean of 9 experiments for rats and 5 experiments for rabbits.

6 196 CENTRAL ACTION OF RESERPINE 255 the level of brain norepinephrine was markedly lowered, but the level of brain serotomn was little affected. DIscussIoN. When it was first shown that reserpine releases stored serotonin, the conception arose that the central actions of the alkaloid are mediated through free indole (Brodie and Shore, 1957). The discovery that reserpine also releases norepinephrine has provoked other opinions including the pessimistic view that the results with reserpine are too ambiguous to interpret (Erspamer, 1959), and the positive view that the depletion of brain norepinephrine determines the central actions of reserpine (Pletscher et a!., 1959; K#{228}rkiand Paasonen, 1959). Supporting the hypothesis of norepinephrine depletion, Carlsson et a!. (1957) demonstrated that dopamine (from dopa administration) counteracts the tranquilizing action of reserpine, and he suggested that dopamine substitutes for the loss of norepinephrine (Carlsson, 1959). Though plausible, this idea overlooks the possibility that dopamine exerts a physiological antagonism. In further support of the norepinephrine-depletion view are reports that the tranquilizing action of reserpine-like compounds is more closely related to the decline in brain norepinephrine than in serotonin. K#{228}rkiand Paasonen (1959) used bioassay procedures, the rat stomach method for serotonin and cat blood pressure rise for norepinephrine, and reported control values less than one-half those reported by workers using fluorimetric assays. They concluded that sedation elicited in rats by reserpine and raunescine is associated with a selective loss of brain norepinephrine. Our results obtained with spectrophotofluorimetric procedures show that the two alkaloids release brain serotonin and norepinephrime to the same degree. Pletscher et al. (1959) also concluded that the sedative action of reserpine is related to the loss of brain norepinephrine. They found that after 1 hour Ro and Ro (compounds I and II) deplete brain serotonin to the same extent in mice, though compound I is the more potent tranquilizer. But our studies show that after 2 minutes compound I releases both amines to a greater extent than does compound II, but these differences disappear in 1 hour because of the unusually brief action of compound I in mice. In the rabbit, also, compound I releases both amines to a greater extent than does compound II, but since compound I has long duration of action the differences persist for a number of hours. Strong evidence for the hypothesis that the central actions of reserpine are associated with the change in brain serotonin is provided by the experiments with Su After small doses of Su 5171,.25 mg/kg, about 5% of brain norepinephrine in the rabbit is released, and relatively little of brain serotonin. The animals are not depressed despite the lowering of brain norepinephrine by 5%. With 2 mg/kg of drug, both brain amines are almost completely depleted and the animals are deeply sedated. The effects on the behavior of the animal are short-lasting and largely disappear within 6 hours. During this time, the serotonin level rises and returns to about 65% of normal; contrastingly, the norepinephrine level remains the same throughout this period, perhaps because of the slow biosynthesis of norepinephrine compared to that of serotonin. Thus, after 6 hours, the animals are largely recovered despite the almost complete depletion of brain norepinephrine. A similar picture is seen 24 hours after administration of tetrabenazine, followed by reserpine. Though the brain norepinephrine is still markedly reduced, the animals show no signs of sedation (Quinn et a!., 1959). The experiments with stressed rats and rabbits offer the strongest evidence that reserpine, itself, does not owe its sedative action to the loss of brain norepinephrine. These experiments show that rats and rabbits subjected to cold and then given reserpine are not sedated. Correspondingly, the level of brain norepinephrine but not brain serotonin is markedly decreased. Our preliminary results show that stress produced by the intradermal injection of formaldehyde and the intraperitoneal injection of pentylenetetrazol also prevents the action of reserpine, and the depletion of brain serotonin, but not of norepinephrine. Garattini and Valzelli (1958) have also shown that rats subjected to cold as well as heat and then given reserpine are not sedated, and brain serotonin is not lowered. These workers did not measure brain norepinephrine. Studies on the mechanism whereby stress blocks the action of reserpine are in progress and will be published shortly. The results described in this paper suggest,

7 256 BRODIE ET AL. Vol. 129 but do not prove, a causal relation between the release of serotonin and the central actions of reserpine. But a major obstacle to this view is removed by the evidence that the loss of brain norepinephrine is probably not the cause of reserpine-induced sedation. A dilemma concerning the role of norepinephrine in brain now confronts us since the decline in brain catecholamines caused by Su 5171 or by reserpine in stressed animals does not visibly affect the rabbit s behavior. Moreover, recent reports indicate that norepinephrine loss induced by reserpine does not lower and may even increase sympathetic discharge from the central sympathetic system (Iggo and Vogt, 1959). In considering this problem, it may be presumed that only the free amines exert biological activity. On release of stored brain amines, the total amine levels but not necessarily the free amine levels are lowered. Reserpine interferes with the mechanisms that concentrate the amines in body tissues; the drug not only causes the release of brain amines but makes the tissues incapable of taking up amines that are still produced (Shore et al., 1957). After depletion of the amine stores, the biological activity of the free amines will depend on the levels attained at the receptor sites. These levels will depend on rates of amine formation, inactivation, and removal by circulating fluids. Because the amines do not readily cross the bloodbrain barrier into the bloodstream, their levels at receptor sites may be sufficient to produce a persistent response. In a previous paper, we discussed the possibility that free serotonin exerts the predominant action because of its rapid synthesis even after reserpine treatment, although free norepinephrine may also exert some effect (Brodie et al., 1959). To clarify the role of free serotonin and norepinephrine in reserpine action, it is necessary to know the biological activity of the free amines in brain after the drug s administration. This problem is now under investigation. SUMMARY A reserpine analogue, dimethylaminobenzoyl methylreserpate (Su 5171), can release half the brain norepinephrine in rabbits without releasing significant amounts of brain serotonin and without eliciting sedation. Large doses release about 7% of both amines and induce marked sedation. Additional strong evidence that the sedation produced by reserpine itself is not due to the loss of brain norepinephrine is provided by experiments in which rats and rabbits subjected to stress are not noticeably affected by reserpine. The norepinephrine in the brains of these animals is depleted but serotonin is lowered only slightly. These results suggest that the tranquilizing actions of Rauwolfia alkaloids are related to the change in the level of brain serotonin rather than of norepinephrine. Our studies fail to confirm published observations that reserpine and raunescine in rats release more norepinephrine than serotonin in brain, and that raunescine produces sedation at doses that release considerable brain norepinephrine, but no serotonin. In addition, we are unable to confirm the published observation that Ro , a potent benzoquinolizine tranquilizing agent, releases brain serotonin in mice to the same extent as its weakly active congener, Ro The implications of these results to the role of serotonin in the action of reserpine are discussed. ACKNOWLEDGMENT. We thank Ciba Pharmaceutical Products, Inc., for the generous samples of Su 5171 and lyophilized reserpine phosphate, and Hoffmann-La Roche Inc. for the samples of Ro and Ro In particular we wish to acknowledge the helpfulness of Dr. A. Plummer who drew our attention to Su REFERENCES BOGDANSKI, D. F., PLETSCHER, A., BRODIE, B. B. AND UDENFRIEND, S.: THIS JOURNAL 117: 82, BRODIE, B. B. AND SHORE, P. A.: Ann. N. Y. Acad. Sci. 66: 731, BRODIE, B. B., OLIN, J. S., KUNTZMAN, R. AND SHORE, P. A.: Science 125: 1293, BRODIE, B. B., SPECTOR, S. AND SHORE, P. A.: Pharmacol. Rev. 11: 548, CARLSSON, A., LINDQvI5T, M. AND MAGNU55ON, T.: Nature 18: 12, CARLSSON, A.: Pharmacol. Rev. 11:49, ERSPAMER, V.: Proc. 21st mt. Congr. Physiol. Sciences, Symposia and Special Lectures, p. 216, GARATTINI, S. AND \ALZELLI, L.: Science 128: 1278, IGGO, A. AND VOGT, M.: J. Physiol. 174: 14, KARKI, N. T. AND PAASONEN, M. K.: J. Neurochem. 3: 352, PLETSCHER, A., BESENDORF, H. AND GEY, K. F.: Science 129: 844, QUINN, G. P., SHORE, P. A. AND BRODIE, B. B.: THIS JOURNAL 127: 13, SHORE, P. A., PLETSCHER, A., TOMI E. G., CARLSSON, A., KUNTZMAN, R. AND BRODIE, B. B.: Ann. N. Y. Acad. Sci. 66: 69, SHORE, P. A. AND OLIN, J. S.: THIS JOURNAL 122: 295, 1958.