Excitatory and inhibitory effects of serotonin on spinal axons

15. C. Terhorst, R. Robb, C. Jones, J. L. StrominP. J. Durda, C. Shapiro, P. D. Gottlieb, J. Imwith 1251-labeled T lymphocytes (data not 9. munol. ger, Proc. Natl. Acad. Sci. U.S.A. 74, 4002 120, 53 (1978). shown). 10. J. A. Ledbetter and L. A. Herzenberg, Immu(1977). 16. We thank J. Distaso and K. LeClair for techninol. Rev. 47, 65 (1979). Comparison of the molecular size de- 11. P. J. Durda and P. D. Gottlieb, J. Exp. Med. cal assistance. The antiserum to ,82m was from Dr. L. Nadler (Sidney Farber Cancer Institute). termined for T4 62,000-dalton with that 144, 476 (1976). Supported by NIH grant AI 15066 and by a A. Boyum, Scand. J. Clin. Lab. Invest. Suppl. of the Lyt 1.1 antigens from murine 12. 21, 97 (1968). travel fellowship (to A;vanA.) from-the Netherlands Foundation for the Advancement of thymocytes shows a striking similarity. 13. U. K. Laemmli, Nature (London) 227, 680 Pure Research (Z.W.O.). Alloantiserum to Lyt 1.1 precipitated a 14. (1970). W. M. Bonner and R. A. Laskey, Eur. J. Biochem. 83 21 March 1980 46, (1974). 67,000-dalton glycoprotein and an 87,000-dalton glycoprotein that could be labeled with tritiated sodium borohydride and galactose oxidase but not with the 1251-labeled lactoperoxidase (9). A monoclonal xenoantiserum [termed 53- Excitatory and Inhibitory Effects of Serotonin on 7.3 (10)] detected a 70,000-dalton glyco- Sensorimotor Reactivity Measured with Acoustic Startle protein on 125_-labeled C57B1/6 thymoAbstract. Serotonin infused into the lateral ventricle in rats produced a dose-decytes (10). Murine Lyt 2 and Lyt 3 antigens were pendent depression of the acoustic startle reflex. When infused onto the spinal cord, found on cell surface glycoproteins from serotonin produced a dose-dependent increase in startle. Thus the same neurotransthymocytes labeled with 1251_labeled lac- mitter can modulate the same behavior in opposite ways, depending on which part of toperoxidase (10, 11). Immune precipi- the central nervous system is involved. tations carried out with alloantiserums to Serotonin is often considered to be an total of 10 ,ul of fluid was administered to Lyt 2 and Lyt 3 detected a 35,000-dalton protein for each marker on C57B 1/6 important behavioral inhibitor (1). How- each animal at the rate of 4 ,ud/min (12). thymocytes (11). However, in contrast, a ever, there are data that are difficult to The rats were returned immediately to monoclonal rat antibody (53-6.7) precipi- reconcile with this conclusion (2). The the test chamber and given noise bursts tated a complex of two subunits of ap- acoustic startle response, a simple reflex every 20 seconds for 20 minutes. Next, proximately 30,000 and 35,000 daltons behavior, is modified by changes in sero- the rats with cannulated ventricles were that was resolved under reducing condi- tonin levels. Small amounts of serotonin infused with 10 A.l of 0.5 percent Fast tions (10). Under nonreducing condi- or serotonin agonists, infused directly in- Green FCF dye. Fifteen minutes later tions, a 65,000-dalton glycoprotein was to the hippocampus (3) or the ventricles they were perfused, and their brains found. These molecular sizes are similar (4), depress startle (5), suggesting that were examined to ensure adequate into those determined for T5 (30,000 to serotonin inhibits this behavior. How- fusion of the dye. Animals in which one ever, markedly increasing the levels of or both ventricles were incompletely in32,000 daltons). Our results plus the previous function- serotonin in the brain and spinal cord (6, fused (8 percent) or whose catheters al data indicate that the glycoproteins 7) or administering drugs that mimic showed signs of being clogged (17 perrecognized by the monoclonal antise- serotonin, heightens startle (8), suggest- cent) were not included in the data. rums to T4 and T5, respectively, are ing that serotonin is excitatory. Figure 1 shows that serotonin caused a Excitatory or inhibitory effects of neu- rapid decrease in startle amplitude when the human homologs of the Lyt 1 and Lyt 2,3 antigens. Isolation and further rochemicals on behavior may depend on infused into the lateral ventricle and a characterization of these cell surface the location and nature of the receptors rapid increase when infused onto the spimarkers will be important in determining involved. We now report that serotonin nal cord. As shown in Fig. 2, both of the precise role of T4 and T5 in the func- depresses acoustic startle when infused these effects were directly related to the tions of the cells that express them. In- into the forebrain (intraventricularly), amount of serotonin infused. An overall formation obtained in such studies would but heightens this response when infused analysis of variance with doses common to both placements (saline and 12.5, 50, also aid in our understanding of the dif- onto the spinal cord (intrathecally). Male albino rats (300 to 400 g) were and 200 ,ug of serotonin) revealed a sigferentiative pathways of human T lymanesthetized with chloral hydrate and nificant dose x placement interaction, phocytes. Cox TERHORST, ANDRE VAN AGTHOVEN implanted with cannulas into the lateral F (3, 12) = 7.14, P < .01. Subsequent ELLIS REINHERZ, STUART SCHLOSSMAN ventricles or with catheters into the spi- analyses indicated a dose-related depresnal cord (9). Twenty-four hours later, an sion of startle when serotonin was inSidney Farber Cancer Institute, injection of the monoamine oxidase in- fused into the lateral ventricle, linear F Harvard Medical School, hibitor pargyline (25 mg/kg) was given (1, 12) = 20.04, P < .001, and a dose-reBoston, Massachusetts 02115 (10). One hour later, the rats were placed lated excitation of startle when serotonin in a startle apparatus and subjected to was infused onto the spinal cord, linearF References and Notes noise bursts every 20 seconds for 15 min- (1, 12) = 17.35, P < .001 (13). 1. E. Reinherz and S. Schlossman, Cell, in press. 2. E. L. Reinherz, P. C. Kung, G. Goldstein, S. F. utes (11). The animals were then infused Infusion of serotonin into the two Schlossman, Proc. Natl. Acad. Sci. U.S.A. 76, with various doses of serotonin dis- areas also had opposite effects on other 4061 (1979). 3. , J. Immunol., in press. solved in saline (pH 7.4). The animals types of motor activity. Doses (- 3.12 4. R. L. Evans, H. Lazarus, A. Penta, S. F. Schlossman,J. Immunol. 120, 1423 (1978); E. L. with cannulated lateral ventricles re- ,ug) that depressed startle when given inReinherz and S. F. Schlossman, ibid. 122, 1335 ceived saline or 0.78, 3.12, 12.5, 50, or traventricularly produced catalepsy (14). (1979). 5. H. Cantor and E. A. Boyse, Cold Spring Harbor 200 ,ug of serotonin. The spinal animals In contrast, -doses that increased startle Symp. Quant. Biol. 51, 23 (1977). received saline or 12.5, 25, 50, 100, or when given intrathecally produced trem6. C. G. Gahmberg and L. C. Anderson, Ann. N. Y. Acad. Sci. 312, 240 (1978). 200 ,ug of serotonin. (Doses are based on or of the hind quarters, indicative of a lo7. P. Cresswell, T. Springer, J. L. Strominger, M. the salt weight of serotonin creatinine calized serotonin "syndrome" (9, 15). J. Turner, H. M. Grey, R. T. Kubo, Proc. Natl. Acad. Sci. U.S.A. 71, 2123 (1974). Recent single-unit recording studies sulfate.) There were three rats in each of 8. J. Michaelson, L. Flaherty, E. Vitetta, M. D. the experimental and control groups. A indicate that there are different types of Poulik, J. Exp. Med. 145, 1066 (1977). 521 0036-8075/80/0725-0521$00.50/0 Copyright © 1980 AAAS SCIENCE, VOL. 209, 25 JULY 1980 serotonin receptors in the rat central nervous system (16, 17). One type is found in certain forebrain structures. Here serotonin depresses neuronal firing when applied microiontophoretically. Another type is found in the brainstem and spinal cord. Here serotonin facilitates the excitatory inputs of other neurotransmitters. Classical- serotonin antagonists attenuate the facilitatory effects of serotonin in the brainstem and spinal cord but fail to alter the inhibitory effects in the forebrain. These data predict that a serotonin antagonist would attenuate the excitatory effects of intrathecally administered serotonin on startle but would not block the inhibitory effects of intraventricularly administered serotonin. To test this hypothesis, ten rats were fitted with intrathecal catheters and ten with intraventricular cannulas. On the next day, all the rats were injected with pargyline (25 mg/kg) and 1 hour later were given noise bursts every 20 seconds for 15 minutes. Half of each group was then injected intraperitoneally with sa- FA 60 T 50 Intraventricular D E E line, and half with the serotonin antagonist cinanserin (10 mg/kg). The rats were immediately given another 15-minute series of noise bursts. Finally, the animals were infused with 100 Ag of serotonin intrathecally or 12.5 ,ug intraventricularly and then subjected to noise bursts for the next 20 minutes. Consistent with predictions based on single-unit studies, cinanserin significantly attenuated the excitatory effect of intrathecally given serotonin (55 versus 257 percent, P < .05) but did not block the depressant effect of intraventricularly given serotonin (-88 versus -72 percent), even though a lower amount of serotonin was administered intraventricularly. Our data indicate that a simple reflex behavior can be modulated in opposite directions by the same neurotransmitter, depending on where the neurotransmitter acts in the central nervous system. The inhibitory effects are probably mediated by structures that surround the lateral ventricles or cerebral aqueduct, since radioactive serotonin infused into serotonin Intrathecal serwrotonin .9 40 c 1i1 0 E ov 30 I Fig. 1. Mean amplitude of startle over 2-minute periods before and after infusion (time 0) of 200 Ag of serotonin into the lateral ventricle (A),or onto the spinal cord (B). ,4 A ISo 0 eN 20 D 9 X 1010 -10 0 10 20 -10 10 Time after infusion (minutes) 10 20 40 30 O 20 E 10 Intrathecal serotonin Fig. 2. Mean change in startle, computed as the mean amplitude of startle during the 20minute period after infusion minus the mean amplitude of startle during the 15-minute period before infusion, for each dose of serotonin infused (A) onto the spinal cord or (B) into the lateral ventricle (± standard error). 5 E 0) ._ Intraventricular serotonin L.-I-- -30 0 12.5 25 50 - / 100 Serotonin (mg) 522 -J 200 the lateral ventricles seems to reach sites in these areas only and is not distributed throughout the cerebrospinal fluid space, probably because it is actively taken up from cerebrospinal fluid by serotonin terminals before it can diffuse very far (18). The excitatory effects are probably mediated by a facilitation of the lower motor neuron's response to the excitatory volley initiated by the startle stimulusserotonin has been shown to facilitate lower motor neuron responsivity to afferent stimulation (17). The finding that forebrain and spinal serotonin affect startle in opposite ways has a number of implications. First, it can no longer be assumed that a neurochemical modulates a behavior in a simple, unidirectional fashion. There may be a critical balance between the inhibitory and excitatory effects of serotonin and other neurotransmitters on behavior. Procedures that change absolute levels of serotonin throughout the central nervous system may not actually change this balance, and hence fail to alter behavior markedly (7). Second, these data indicate that the spinal cord is a possible target organ for mediating the effects of drugs on behavior. Generally, it is assumed that when a drug alters behavior it does so by affecting the brain, but our data suggest that some of the effects seen after systemic administration of a drug may be mediated partially or even entirely in the spinal cord. This finding is particularly important in view of the fact that the spinal cord is the final common pathway for most behaviors measured in psychopharmacological studies (avoidance conditioning, bar pressing, locomotor activity, and stereotyped behavior). Third, serotonin may exercise reciprocal control over behaviors other than the startle reflex. For example, it has repeatedly been implicated in the control of mood (19). However, reported correlations between levels of serotonin metabolites in cerebrospinal fluid and psychiatric symptomatology may be more reflective of spinal serotonin metabolites associated with motor activity than with mood, since much of the fluid obtained in these studies was of spinal or lower brainstem origin (20). Finally, the wide shifts in mood seen in bipolar depressive illness may reflect an imbalance within the serotonin system from one extreme to the other. It would be of considerable interest, therefore, to determine how drugs, like lithium or tricyclic antidepressants, which are relatively successful clinically and which alter serotonin transmission, afSCIENCE, VOL. 209 fect the balance between inhibitory and excitatory effects of serotonin on behavior. The present model might be useful for testing such interactions. MICHAEL DAVIS DAVID I. ASTRACHAN ELIZABETH KASS Department ofPsychiatry, Yale University School ofMedicine and Connecticut Mental Health Center, New Haven 06508 References and Notes 1. J. B. Meyerson and M. Eliasson, Handbook of Psychopharmacology, vol. 8, Drugs, Neurotransmitters, and Behavior (Plenum, New York, 1977); L. S. Seiden and L. A. Dykstra, Psychopharmacology: A Biochemical and Behavioral Approach (Van Nostrand Reinhold, New York, 1977); M. H. Sheard and M. Davis, Eur. J. Pharmacol. 40, 295 (1976). 2. S. Barasi and M. H. T. Roberts, J. Physiol. (London) 236, 11(1976); J. Maj, W. Palider, L. Baran, J. Neural Transm. 38, 131 (1976); N. R. Myslinski and E. G. Anderson, J. Pharmacol. Exp. Ther. 204, 19 (1978); B. L. Jacobs, Life Sci. 19, 77 (1976); D. G. Grahame-Smith, Br. J. Pharmacol. 43, 856 (1971). 3. M. A. Geyer, Psychopharmacol. Commun. 1, 675 (1975). 4. M. A. Geyer, A. J. D. Warbritton, D. B. Menkes, J. A. Zook, A. J. Mandel, Pharmacol. Biochem. Behav. 3, 293 (1975). 5. M. Davis and M. H. Sheard, Eur. J. Pharmacol. 35, 261 (1976); Pharmacol. Biochem. Behav. 2, 827 (1974); M. Davis and J. K. Walters, ibid. 6, 427 (1977). 6. L. D. Fechter, Pharmacol. Biochem. Behav. 2, 161 (1974). 7. M. Davis, Neurosci. Biobehav. Rev., in press. 8. , D. I. Astrachan, P. M. Gendelman, D. S. Gendelman, Psychopharmacology, in press. 9. Cannulas were made from 23-gauge hypodermic needles turned down in a metal lathe to a hub 8 mm long and 4 mm wide, and were threaded inside to accept inner or infusion cannulas. Inner and infusion cannulas were made from 30-gauge hypodermic needles turned down and threaded to fit into the outer cannulas so that their tips protruded 1.5 mm beyond the outer tips. The cannulas were implanted 1.4 mm lateral to the lambda and 4 mm below the top of the skull in rats anesthetized with chloral hydrate, and were secured with skull screws and dental cement. Intrathecal catheters were made from PE 10 polyethylene tubing, as described by T. L. Yaksh and P. R. Wilson [J. Pharmacol. Exp. Ther. 208, 446 (1979)]. 10. In intact animals, excitatory motor effects caused by serotonin or its precursors occur only in the presence of a monoamine oxidase inhibitor. Therefore the rats were first treated with a moderate dose of pargyline, which preliminary studies indicated does not alter baseline startle levels. In order to equate conditions for both lateral ventricle and spinal placements, pargyline was given to all animals. After completing the dose-response curves, we found, however, that higher amounts of serotonin (200 to 400 ,ug) heightened the startle response when given intrathecally without pargyline pretreatment. Since Geyer et al. (4) already showed that serotonin given alone intraventricularly depresses startle, pretreatment with a monoamine oxidase inhibitor is not required to demonstrate forebrain versus spinal effects of serotonin on startle. 11. The apparatus used to measure startle is described by G. T. Weiss and M. Davis [Pharmacol. Biochem. Behav. 4, 713 (1976)]. Briefly, five separate stabiimeters were used to record the amplitude of the startle response. Each stabilimeter consisted of an 8 by 15 by 15 cm Plexiglas and wire mesh cage suspended between compression springs within a steel frame. Cage movement caused displacement of an accelerometer; the resultant voltage was proportional to the velocity of displacement. Startle amplitude was defined as the maximum accelerometer voltage during the first 200 msec after the stimulus and was measured with a sample-and-hold circuit. The stabilimeters were housed in a dimly lit, ventilated, sound-attenuated chamber 1.1 m SCIENCE, VOL. 209, 25 JULY 1980 12. 13. 14. 15. from a high-frequency speaker. The startle stimulus was a 90-msec, 115-dB burst of white noise with a rise-decay time of 5 msec. Background white noise, provided by a white noise generator, was 46 dB. Sound level measurements were made in the cages with a General Radio model 1551-C sound level meter (A scale). M. Davis and R. D'Aquila, Pharmacol. Biochem. Behav. 4, 469 (1976). B. J. Weiner, Statistical Principles in Experimental Design (McGraw-Hill, New York, 1962), pp. 70-77. Catalepsy was measured as described by P. Worms and K. G. Lloyd [Pharmacological Methods in Toxicology (Raven, London, 1979)]. B. L. Jacobs (2). However, the increase in startle cannot be explained by an artifact caused by the serotonin syndrome and mistaken for startle since measuring cage movement in the absence of a startle stimulus revealed that the amount of drug-induced movement recorded in this way was far below that recorded in the presence of the noise bursts. 16. G. K. Aghajanian and H. J. Haigler, Proceedings, Fifth International Congress of Pharmacology (Basel Press, Switzerland, 1972); R. B. McCall and G. K. Aghajanian, Brain Res. 169, 11(1979). 17. R. S. Neuman and S. R. White, Brain Res., in press. 18. K. Fuxe, T. Hokfelt, M. Ritzen, U. Ungerstedt, Histochemie 16, 186 (1968). 19. T. N. Chase and D. L. Murphy, Annu. Rev., Pharmacol. 13, 181 (1973); D. L. Murphy, I. Campbell, J. L. Costa, Psychopharmacology: A Generation of Progress (Raven, New York, 1978). 20. E. Garelis, S. N. Young, S. Lal, T. L. Sourkes, Brain Res. 79, 1 (1974). 21. This research was supported by NSF grant BMS-78-04170, NIMH grants MH-25642 and MH-18949, research scientist development award MH-00004 to M.D., and by the state of Connecticut. 8 February 1980 Female Mate Choice in a Neotropical Frog Abstract. Female Physalaemus pustulosus choose their mates and are more likely to choose larger males. There is a significant negative correlation between the size of the male and the fundamental frequency of one of the components of its advertisement call. Playback experiments demonstrate that females are capable of choosing larger males by distinguishing among differences in spectral components of the advertisement call. Darwin (I) proposed that sexual selec- manent identification. Numbered pieces tion has two principal components: com- of surveyors' flagging were stitched to petition among males (intrasexual selec- the middorsal surface, allowing undistion) and choice by females (intersexual turbed identification during behavioral selection). The role of male-male com- observations. petition has been demonstrated (2); howBreeding in P. pustulosus occurs ever, the importance of choice by fe- throughout the year, but is concentrated males, especially choice based on male during the wet season (April to Decemtraits, has been disputed since Darwin ber). As with most anurans that have a first proposed the theory (3). Studies prolonged breeding season, the sex ratio have shown that mate choice by females at the breeding site was skewed toward is based at least in part on resources con- males (9). Males advertised from calling trolled by males (4), and on male position sites, and the females approached and on a lek (5), or rank in a social hierarchy initiated amplexus. Usually, a female (6). Attempts to show that females in was present in the pool only on the night natural populations prefer certain male she mated. The females seemed to morphological or behavioral character- choose their mates freely, although on istics have been frustrated by difficulties several occasions noncalling males interin separating the effect of these charac- cepted females that were en route to callteristics from that of the other factors in- ing males. The males often fought each fluencing mate choice (7). Only laborato- other, but in over 500 hours of observary studies of Drosophila have adequately tions during 1978 and 1000 hours in 1979, demonstrated female choice based on I never saw an unmated male displace a male traits (8). I report that female male in amplexus. Males constructed choice of larger males influences male foam nests during amplexus by beating mating success in a neotropical frog, and the jelly matrix of the egg mass with their that this choice is based on one male hind legs as they fertilized the eggs (10). phenotypic characteristic, the funda- Nest building usually occurred 1 to 4 mental frequency of an advertisement hours after the beginning of amplexus. call component, which is correlated with The males were not territorial and did size and probably age. not defend resources. There was no relaThe breeding behavior of Phys- tion between a male's calling site and the alaemus pustulosus (Leptodactylidae) site used for oviposition or his ability to was monitored in a small cement pool on attract mates. Barro Colorado Island, Panama, for 12 In 1978, I marked 185 males and obweeks from June to August 1978. Indi- served 103 matings. As Fig. 1 shows, the viduals were captured, measured (snout larger males were more likely to acquire to vent), and given a toe clip for per- mates. However, only some of the males 0036-8075/80/0725-0523$00.50/0 Copyright © 1980 AAAS 523