Brain Research, 162 (1979) 45-54 © Elsevier/North-HollandBiomedicalPress
45
DIFFERENTIAL SEROTONERGIC INNERVATION OF INDIVIDUAL HYPOTHALAMIC NUCLEI AND OTHER FOREBRAIN REGIONS BY THE DORSAL AND MEDIAN MIDBRAIN RAPHE NUCLEI
L. D. VAN DE KAR and S. A. LORENS Departments of Pharmacology, University of Iowa, Iowa City, Iowa 52242 and (S.A.L.) Loyola University, Stritch School of Medic#1e, Maywood, Ill. 60153 (U.S.A.)
(Accepted May 18th, 1978)
SUMMARY Lesions in the midbrain median but not in the dorsal raphe nucleus significantly decreased the serotonin (5-HT) content of the hippocampus (61 ~o), medial preoptic area (49 ~), suprachiasmatic nucleus (70 ~) and anterior hypothalamic area (60 ~). Electrolytic lesions restricted to either the median or dorsal raphe nucleus produced significant reductions in the 5-HT concentration of the caudate-putamen, anterolateral hypothalamic area (45 ~o) and arcuate nucleus (48-58 ~). The fall in caudate-putamen 5-HT level was significantly greater after the dorsal (66 ~) than after the median (24 ~) raphe lesion. Neither lesion significantly affected the 5-HT content of the posterolateral hypothalamic area, the ventromedial hypothalamic nucleus or the dorsomedial hypothalamic nucleus. Thus, like their differential projections to the caudate-putamen and hippocampus, the mesencephalic dorsal and median raphe nuclei appear to innervate different preoptico-hypothalamic nuclei and areas. Whereas the median raphe nucleus seems to be the primary source of 5-HT fibers to the suprachiasmatic nucleus, anterior hypothalamic area and medial preoptic area, the 5-HT inputs to the anterolateral hypothalamic area and arcuate nucleus appear to derive from both the dorsal and median raphe nuclei.
INTRODUCTION Serotonin (5-hydroxytryptamine; 5-HT) has been implicated as playing a major role in a number of psychological and physiological processes, including sexual behavior and neuroendocrine regulation. Secretion of luteinizing hormonel~,19-~2,27, 31,40 and growth hormone z~ thus are controlled in part by 5-HT fibers. The hypothalamus and preoptic area are major regions involved in the regulation of both sexual behaviorr,9,10,16,~6 and neuroendocrine secretionT,14,ag. Since both the median
46 and dorsal midbrain raphe nuclei have been reported to innervate the hypothalamus ~, 4,13,1s,30, the present study was undertaken to determine whether different preopticohypothalamic areas and nuclei receive their 5-HT input from the mesencephalic dorsal or median raphe nucleus. METHODS
Animals Male Sprague-Dawley rats weighing 200-250 g at the time of surgery were obtained from Bio Labs (St. Paul, Minn.). The rats were housed in groups of 6 in a temperature (22 ~ 1 °C) controlled room with a 12:12 h light :dark cycle (lights on at 07.00). Food and water were available ad libitum.
Surgery, histology and biochemistry Lesions were produced electrotytically under ether anaesthesia in either the dorsal (n -- 25) or median (n -- 25) raphe nucleus as previously described z4. In brief, 2 mA DC was passed for 10-15 sec between an intracranial cathode and an anode attached to the wound margin. The cathode was a 30-gauge tungsten wire insulated with Epoxylite except for 0.5 mm of its tip. Operated control rats (n = 18) were treated in the same manner as the lesion rats, except that an electrode was not lowered into their brains. The midbrain was frozen (--16 °C) and serial coronal sections (40/~m) obtained. Every third section was saved and stained by the cresylecht violet method. Lesion cavitation and glial scarring was determined microscopically and reconstructed diagrammatically. Hypothalamic nuclei and preoptic areas from 3 rats were pooled and homogenized in 40 #1 0.1 N HCI. The hippocampus and caudate-putamen from individual animals were homogenized in 100 #1 0.1 N HCI. Ten #1 aliquots were removed for protein assay according to Lowry et al. 25. The microhomogenizers were then centrifuged and 10 #l aliquots of the supernatant analyzed in duplicate for 5-HT as detailed by Saavedra et al.~L
Procedure Surgery was performed 2 weeks after the rats had arrived in the laboratory. The rats were sacrificed by decapitation 17-20 days postoperatively between 13.00 and 16.00 h. The brains were removed rapidly and dissected over dry ice 24. The hippocampus and caudate-putamen were frozen in liquid nitrogen and stored at --85 °C until they were assayed for 5,HT. The brain stem was placed in formalin for at least 1 week prior to histological processing and analysis. The diencephalon was mounted on flat cork discs, frozen in liquid nitrogen, wrapped in Saran Wrap and aluminum foil, then stored at --85 °C. Preoptico-hypothalamic nuclei and areas from 3 different rats were pooled for the 5-HT assay. The lesions in these animals were matched histologically and, as far as
47 possible, by the decrease in 5-HT levels in the caudate-putamen (for the dorsal raphe lesions) and hippocampus (for the median raphe lesions) 18,15,1s,23,24. Individual diencephalic nuclei and areas were microdissected bilaterally from cryostat cut frontal sections (0.6-1.2 mm) as originally described by Eik-Nes and Brizzee 11 and according to Saavedra et al. 33 (see their Fig. 1). For the suprachiasmatic and arcuate nuclei hollow needles with a 304/~m inner diameter were used. For the anterior hypothalamic area, and the ventromedial and dorsomedial hypothalamic nuclei, a needle with a 500 #m inner diameter was used. For the medial preoptic area, and the anterior and posterior lateral hypothalamic areas, a needle with a 600/~m inner diameter was employed. RESULTS
Lesion analysis The midbrain dorsal and median raphe lesions are shown schematically in Figs. 1 and 2, respectively. The dorsal raphe lesions were well confined to the ventromedial central grey and destroyed at least 35 ~o of the dorsal raphe nucleus. Incidental damage to the oculomotor complex, trochlear nucleus and medial longitudinal fasciculus was observed in several animals. However, the superior cerebellar peduncles, tegmental nuclei of Gudden and linear nuclei were spared in all rats. The median raphe lesions ablated at least 40 ~ of the median raphe nucleus. In several animals (A-D groups, Fig. 2) the lesion extended rostrally dorsal to the caudal two-thirds of the interpeduncular nucleus. In 10 rats (E-G groups, Fig. 2) the lesions extended caudally into the periventricular B6 5-HT cell group and pontine raphe nucleus (B5). The linear nuclei were spared in all animals, but the superior cerebellar peduncle was damaged in 3 rats (B group, Fig. 2). All rats sustained uni- or bilateral damage to Gudden's tegmental nuclei and the medial longitudinal fasciculus.
Caudate-putamen and hippocampal 5-HT Reductions in the 5-HT content of the caudate-putamen and hippocampus were used as a second control for the efficacy of the lesions13,15,18,2a,24. Rats with dorsal raphe lesions showed a significant decrease in the 5-HT content of the caudateputamen (61 7oo),but no change, or even a slight increase, in the 5-HT content of the hippocampus (Table I). Median raphe lesions, in contrast, produced a large decrease (66 ~o) in the 5-HT concentration of the hippocampus, but only a small decrease (24 ~) in the caudate-putamen. As previously reported 24, the more rostral median raphe lesions (animals A-D, Fig. 2) produced greater falls in hippocampal 5-HT concentration. Probably because of the uniformity in the positions of the dorsal raphe lesions, no consistent variation in caudate-putamen 5-HT content was found 24.
Regional preoptico-hypothalamic 5-HT Most of the lesion-induced changes in 5-HT concentration were found in the anterior hypothalamus and preoptic area (Table II). Thus, the median raphe lesions resulted in significant 5-HT reductions in the suprachiasmatic nucleus (70 ~), medial
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Fig. 2. Lesion damage (blackened area) in animals receiving median raphe lesions. Details as in Fig. 1. Fig. 1. Diagrammatic reconstruction of damage (blackened area) to the dorsal raphe nucleus on serial coronal sections, each separated by approximately 0.6 mm. Numbers identify individual rats, while the letters indicate animals whose preoptico-hypothalamic nuclei and areas were pooled for 5HT analysis. Abbreviations: BC, brachium conjunctivum;BP, middle cerebellar peduncle; B6, 5-HT cell group; c, pars centralis of dtg; DBC, decussation of BC; dr, dorsal raphe nucleus; dtg, dorsal tegmental nucleus of Gudden; gp, basal pontine nuclei; III, oculomotor complex; il, intermediate linear nucleus; ipn, interpeduncular nucleus; IV, trochlear nucleus; lc, locus coeruleus; ML, medial lemniscus; MLF, medial longitudinal fasciculus; mp, mammillary peduncle; mr, median raphe nucleus; nr, red nucleus; rl, rostral linear nucleus; rp, pontine raphe nucleus; rtp, pontine reticular tegmental nucleus; TTS, tectospinal tract; vtg, ventral tegmental nucleus of Gudden.
50 p r e o p t i c a r e a (49 }o), a n t e r i o r h y p o t h a l a m i c a r e a (60~,,,), the r o s t r a l p o r t i o n o f the l a t e r a l h y p o t h a l a m i c a r e a (46 ~ ) a n d the a r c u a t e n u c l e u s (58 ~o). In c o n t r a s t , the dorsal r a p h e lesions p r o d u c e d a significant d e c r e a s e in 5 - H T c o n c e n t r a t i o n o n l y in the a n t e r o l a t e r a l h y p o t h a l a m u s (45 ~ ) a n d a r c u a t e n u c l e u s (48 ~ ) . A t t e m p t s to c o r r e l a t e the m a g n i t u d e o f d e c r e a s e s in r e g i o n a l p r e o p t i c o - h y p o t h a l a m i c 5 - H T c o n t e n t w i t h t h o s e in the c a u d a t e - p u t a m e n
and hippocampus
did n o t result in a n y significant
relationships.
TABLE I Differential effects of selective midbrain raphe lesions on regional forebra#l 5-hydroxytryptamine concentrations (ng/g wet weight) Number of rats in parentheses. Probabilities based on Student's t-test, one-tail ; Ps in parentheses are based on a two-tailed t-test and refer to differences between the lesion groups. Group
Area assayed Hippocampus
Caudate-putomen
Mean 4: S.E.M.
% Difference
P
Mean ~: S.E.M.
% Differeltcc
P
349 ± 30 (9) 411 ± 31 (10)
-~ 18
-n.s.
625 ± 64 (11) 214 ± 30(15)
-- 66
Median raphe lesion 135 ± 22(17)
--61
< 0.0005 ( < 0.0O05)
473 ± 54(16)
--24
-< 0.0005 ( < 0.0005) --, 0.05
Control Dorsalraphelesion
TABLE II Effects of discrete mesencephalic raphe lesions on the 5-hydroxytryptamine concentration (ng/rng protein) of preoptico-hypothalamic areas and nuclei Mean ± S.E.M. Number of determinations in parentheses. Control
Medial preoptic area Anterolateral hypothalamic area Suprachiasmatic nucleus Anterior hypothalamic area Arcuate nucleus Ventromedial nucleus Dorsomedial nucleus Posterolateral hypothalamic area
9.8 ± 1.0 (6) 18.5 31.4 12.6 22.7 5.9 11.3
Dorsal raphe lesion 8.1 ± 1.1 (6)
+ 3.4 (4) 8.5 ± 1.7 (7)* i 4.6 (6) 25.2 ~ 3.1 (5) ± 2.6 (5) 8.3 ± 2.1 (6) + 3.7 (5) 11.9 ± 2.0 (5)* :t= 1.5 (5) 4.2 ± 0.9 (6) ± 1.8 (6) 8.1 ± 1.7 (6)
11.3 ± 3.6 (6)
11.3 ± 3.0 (6)
% Median raphe Change lesion
°o Change
--17
4.8 ± 1.3 (7)*
--51
--54 --20 --34 --48 --29 --28
8.7 9.4 5.1 9.5 5.3 7.1
--53 --70 --60 --58 --10 --37
0
± ± ± ± ± ±
2.3 (7)* 3.2 (6)** 1.1 (6)* 2.4 (5)* 1.4 (7) 1.5 (7)
8.9 ± 2 . 6 ( 6 )
--21
Probabilities determined by Student's t-test, two-tailed: * P < 0.05, significant difference between lesion and control group; ** P < 0.01, significant difference between median raphe lesion group and the other two groups.
51 DISCUSSION It is now generally accepted that the dorsal and median raphe nuclei differentially innervate distinct forebrain regionsl,4,13,15,1s, 23,24. A dorsal raphe-striatal and median raphe-hippocampal 5-HT projection was confirmed in the present study. In addition, the results suggest that the median raphe nucleus is the principal origin of 5HT fibers to the suprachiasmatic nucleus, the anterior hypothalamic area and the medial preoptic area. No evidence was obtained for an exclusive origin in the dorsal raphe nucleus of a 5-HT projection to any of the preoptico-hypothalamic structures studied. At present we cannot exclude the possibility that some of the effects of median raphe lesions are due to interruption of 5-HT fibers arising in the pontine raphe (B5 and B6 cell groups) or nucleus raphe magnus (B3). Projections to the hypothalamus from these regions have been reported 4. The autoradiographic method employed, however, does not discriminate 5-HT from non-5-HT fibers. Electrolytic lesions which extensively damaged the pontine B5 and B6 cell groups without invading the B7 (dorsal raphe nucleus) or B8 (median raphe nucleus) cell groups, moreover, do not significantly affect the 5-HT concentration of the hypothalamus or of other forebrain regions, except the dorsal thalamus (35 ~o reduction) 23. In addition, Bloom et al. 3 have shown that stimulation of the median raphe nucleus produces a short-latency inhibition of neuronal firing in the suprachiasmatic nucleus, while Carter and Sawyer6 observed increased multiunit activity in the medial preoptic area after electrochemical stimulation of the median raphe nucleus. The lesions in the median and dorsal raphe nuclei were incomplete. Therefore, it is possible that the dorsal raphe nucleus contributes 5-HT fibers to the preopticohypothalamic nuclei and areas studied, especially since the dorsal raphe lesions produced small but insignificant reductions in the 5-HT concentrations of several regions assayed. On the other hand, Kellar et al. 17 found only a 10 ~ reduction in the hypothalamic uptake of [SH]5-HT and no significant change in hypothalamic tryptophan hydroxylase activity after dorsal raphe lesions. Both of these measures were affected (about 50 ~ reductions) after median raphe lesions. Geyer et al. la also found a greater reduction in hypothalamic tryptophan hydroxylase activity after median raphe lesions than after dorsal raphe lesions. Evidence has been advanced suggesting the presence of 5-HT-containing perikarya in the hypothalamus, particularly in the paraventricular region. Hypothalamic deafferentation produced by circular knife cuts reduced the 5-HT content and tryptophan hydroxylase activity of the median eminence, arcuate nucleus, ventromedial nucleus and dorsomedial hypothalamic nucleus 38-69 ~5. Prolonged intraventricular infusion of [aH]5-HT has resulted in labeling of paraventricular hypothalamic neurons2, 8. The failure of dorsal and median raphe lesions to affect the 5-HT content of the ventromedial and dorsomedial hypothalamic nuclei thus may be due to the presence of 5-HT cell bodies in these regions. The absence of a significant reduction in the 5-HT concentration of the posterolateral hypothalamic area was surprising. However, it may well be that our
52 punches were made dorsal to the ascending 5-HT fibers which appear to travel in the ventral part of the medial forebrain bundle1,4, 3s. The falls observed in the anterolateral hypothalamus, furthermore, may be due primarily to degenerated fibers of passage rather than degenerated terminals. The 5-HT input to the lateral hypothalamus thus may derive from fibers arising in the paraventricular hypothalamus 29, or in brain stem neurons other than those comprising the B7 and B8 cell groups. The results of the present study are in disagreement with those of Palkovits et al. a0, who reported that selective dorsal raphe lesions reduced by 63-64 ~ the 5-HT content of the arcuate nucleus, the suprachiasmatic nucleus and the medial forebrain bundle. However, these workers found that their dorsal raphe lesions also produced a decrease in hippocampal tryptophan hydroxylase activity (73 ~). This observation strongly suggests that their dorsal raphe lesions extended ventrally and interrupted a large number of 5-HT fibers originating in the median raphe nucleus, as both Geyer et al. 13 and Kellar et al. 18 found no change in hippocampal tryptophan hydroxylase activity after lesions restricted to the dorsal raphe nucleus, but large decreases in the hippocampal activity of this enzyme after selective median raphe nucleus lesions. That the 5-HT input to the hippocampus derives principally from the median raphe nucleus also is supported by autoradiographicl,4, 2s, horseradish peroxidase 34 and other lesionlS,2a, 24 studies. It has been demonstrated in the male rat that the medial preoptic area plays an important role in sexual behavior6,9,t°,16, 26, whereas the arcuate and ventromedial hypothalamic nuclei are involved in regulating the secretion of luteinizing hormone (LH) ~4,39. Serotonergic neurons also appear to be involved in the regulation of both sexual behavior36, 37 and L H secretion 12,16,17,21,27,31,4°. Since our data suggest a dominant 5-HT innervation of the medial preoptic area, anterior hypothalamic area and suprachiasmatic nucleus by the median raphe nucleus, whereas the arcuate nucleus seems to receive an almost equal innervation from both the dorsal and median raphe nuclei, it is possible that the 5-HT fibers originating in the dorsal raphe nucleus are involved in the regulation of L H secretion, whereas the fibers from the median raphe nucleus may be involved in the regulation of sexual behavior as well as LH secretion. ACKNOWLEDGEMENTS The authors express their gratitude to Isabel Bizet and Jon Levine for their technical assistance, and to Dr. L. S. Van Orden III for his guidance and provision of facilities. This research was supported in part by Grant G M 12675.
REFERENCES 1 Azmitia, E. C. and Segal, M., An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat, J. comp. Neurol., 179 (1978) 641-668. 2 Beaudet, A. and Descarries, L., A serotonin-containing nerve cell group in rat hypothalamus, Neurosci. Abstr., 1 (1976) 678.
53 3 Bloom, F. E., Hoffer, P. J., Siggins, G. R., Barker, J. R. and NicoU, R. A., Effects of serotonin on central neurons: microiontophoretic administration, Fed. Proc., 31 (1972) 97-106. 4 Bobillier, P., Seguin, S., Petitjean, F., Salvert, D., Touret, M. and Jouvet, M., The raphe nuclei of the cat brain stem : a topographical atlas of their efferent projections as revealed by autoradiography, Brain Research, 113 (1976) 449-486. 5 Brownstein, M. J., Palkovits, M., Tappaz, M. L., Saavedra, J. M. and Kizer, J. S., Effect of surgical isolation of the hypothalamus on its neurotransmitter content, Brain Research, 117 (1976) 287-295. 6 Caggiula, A. R., Antelman, S. M. and Zigmond, M. J., Disruption of copulation in male rats after hypothalamic lesions, Brain Research, 59 (1973) 273-287. 7 Carrer, H. F. and Sawyer, C. H., Changes in multiunit spike activity in the rat preoptic area induced by stimulating the midbrain, Exp. NeuroL, 52 (1973) 525-534. 8 Chan-Palay, V., Indoleamine neurons and their processes in the normal rat brain and in chronic diet-induced thiamine deficiency demonstrated by uptake of [aH]serotonin, J. comp. NeuroL, 176 (1977) 467-494. 9 Chen, J. J. and Bliss, D. K., Effects of sequential preoptic and mammillary lesions on male rat sexual behavior, J. comp. physioL Psychok, 87 (1974) 841-847. 10 Davidson, J. M., Activation of the male rat's sexual behavior by intracerebral implantation of androgen, Endrocrinology, 70 (1966) 783-794. ll Eik-Nes, K. B. and Brizzee, K. R., Concentration of tritium in brain tissue of dogs given [1,2-SH~]cortisol intravenously, Biochim. biophys. Acta (Amst.), 97 (1965) 320-333. 12 Gallo, R. V. and Moberg, G. P., Serotonin mediation of episodic luteinizing hormone release during electrical stimulation of the arcuate nucleus in ovariectomized rats, Endocrinology, 100 (1977) 945-954. 13 Geyer, M. A., Puerto, A., Dawsey, W. J., Knapp, S., Bullard, W. P. and Mandell, A. J., Histologic and enzymatic studies on the mesolimbic and mesostriatal serotonergic pathways, Brain Research, 106 (1976) 241-256. 14 Halasz, B. and Pupp, L., Hormone secretion of the anterior pituitary gland after physical interruption of all nervous pathways to the hypophysiotropic area, Endocrinology, 80 (1965) 608-622. 15 Jacobs, B. L., Wise, W. D. and Taylor, K. M., Differential behavioral and neurochemical effects following lesions of the dorsal or median raphe nuclei in rats, Brain Research, 79 (1974) 353-361. t6 Johnston, P. and Davidson, J. M., Intracerebral androgens and sexual behavior in male rats, Hormone Behav., 2 (1966) 345-357. 17 Kamberi, I. A., Mical, R. S. and Porter, J. C., Effects of anterior pituitary perfusion and intraventricular injection of catecholamines and indoleamines on LH release, Endocrinology, 87 (1970) 1-12. 18 Kellar, K. J., Brown, P. A., Madrid, J., Bernstein, M., Vernikos-Dannelis, J. and Mehler, W. R., Origins of serotonin innervation of forebrain structures, Exp. Neurol., 56 0977) 52-62. 19 Kordon, C., Effects of selective experimental changes in regional hypothalamic monoamine levels on supraovulation in the immature rat, Neuroendocrinology, 4 (1969) 129-138. 20 Labhsetwar, A. P., Effects of 5-HT on spontaneous ovulation. Theory for the dual hypothalamic control of ovulation, Acta endocr. (Kbh.), 68 (1971) 334-344. 22 Ladowsky, W. and Naronha, J. G. L., Inhibitory role of 5-HT in the control of ovulation, J. Endocr., 62 (1974) 677-678. 22 Leonardelli, J., Dubois, M. P. and Poulain, P., Effect of exogenous 5-HT on LH-RF secreting neurons in the guinea pig hypothalamus as revealed by immunofluorescence, Neuroendocrinology, 15 (1974) 69-71. 23 Lorens, S. A., Some behavioral effects of serotonin depletion depend on method: a comparison of 5,7-dihydroxytryptamine, p-chlorophenylalanine, p-chloroamphetamine, and electrolytic raphe lesions, Ann. N. Y. Aead. Sci., 305 (1978) 532-555. 24 Lorens, S. A. and Guldberg, H. C., Regional 5-hydroxytryptamine following selective midbrain raphe lesions in the rat, Brain Research, 78 (1974) 69-71. 25 Lowry, O. H., Rosebrough, N. Y., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. bioL Chem., 193 (1951) 265-275. 26 Malsbury, C. W., Facilitation of male rat copulatory behavior by electrical stimulation of the medial preoptic area, Physiol, Behav., 7 (1971) 797-805. 27 Mess, B.,Serotonin: aneurotransmitterinhibitingthethyrotrophicandgonadotrophic function of the hypothalamo-hypophyseal system. In Results in Neurochemistry, Neuroendocrinology, Neuro-
54
28 29 30
31 32 33 34 35 36
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physiology and Behavior, Neuropharmacology, Neuropathology, Qybernetics, Academia Kiado. Budapest, 1976, pp. 9--23. Moore, R. Y. and Halaris, A. E., Hippocampal innervation by serotonin neurons of the midbrain raphe in the rat, J. comp. Neurol., 164 (1975) 171-184. Millhouse, O. E., A Golgi study of the descending medial forebrain bundle, Brain Rese~,rch, 15 (1969) 341-363. Palkovits, M., Saavedra, J. M., Jacobowitz, D. M., Kizer, J. S., Zaborszky, L. and Brownstein, M. J., Serotonergic innervation of the forebrain: effect of lesions on serotonin and tryptophan hydroxylase levels, Brain Research, 130 (1977) 121-134. Porter, J. C., Mical, R. S. and Cramer, O. M., Effect of 5-HT and other indoles on the release of LH, FSH and prolactin, Gynecol. Invest., 2 (1971-72) 13-22. Saavedra, J. M., Brownstein, M. J. and Axelrod, J., A specific and sensitive enzymatic isotopic microassay for serotonin in tissues, J. Pharmaeol. exp. Ther., 186 (1973) 508-515. Saavedra, J. M., Palkovits, M., Brownsteirl, M. J. and Axelrod, J., Serotonin distribution in the nuclei of the rat hypothalamus and preoptic region, Brain Research, 77 (1974) 157-165. Segal, M. and Landis, S. C., Afferents to the hippocampus of the rat studied with the method of retrograde transport of horseradish peroxidase, Brain Research, 78 (1974) 1 -15. Smythe, G. A., Brandstater, J. F. and Lazarus, L., Serotonergic control of rat growth hormone secretion, Neuroendocrinology, 17 (1975) 245-257. S6dersten, P., Larsson, K., Ahlenius, S. and Engel, J., Sexual behavior in castrated male rats treated with monoamine synthesis inhibitors and testosterone, Pharmacol. Biochem. Behav., 5 (1976) 319 327. S6dersten, P., Larsson, K., Ahlenius, S. and Engel, J., Stimulation of mounting behavior but not lordosis behavior in ovariectomized female rats by p-chlorophenylalanine, Pharmacol. Biochem. Behav., 5 (1976) 329-333. Ungerstedt, U., 1. Stereotaxic mapping of the monoamine pathways in the rat brain, Acta physiol. scand., Suppl. 367 (1971) I 48. Voloschin, L., Joseph, S. A. and Knigge, K. M., Endocrine function in male rats following complete and partial isolations of the hypothalamo-pituitary unit, Neuroendocrinology, 3 (1968) 387-397. Wuttke, W., Bj6rklund, A., Baumgarten, H. G., Lachenmeyer, L., Fenske, M. and Klemm, H. P., De- and regeneration of brain serotonin neurons following 5,7-dihydroxytryptamine treatment: effects on serum LH, FSH and prolactin levels in male rats, Brain Research, 134 (1977) 317-331.
45
DIFFERENTIAL SEROTONERGIC INNERVATION OF INDIVIDUAL HYPOTHALAMIC NUCLEI AND OTHER FOREBRAIN REGIONS BY THE DORSAL AND MEDIAN MIDBRAIN RAPHE NUCLEI
L. D. VAN DE KAR and S. A. LORENS Departments of Pharmacology, University of Iowa, Iowa City, Iowa 52242 and (S.A.L.) Loyola University, Stritch School of Medic#1e, Maywood, Ill. 60153 (U.S.A.)
(Accepted May 18th, 1978)
SUMMARY Lesions in the midbrain median but not in the dorsal raphe nucleus significantly decreased the serotonin (5-HT) content of the hippocampus (61 ~o), medial preoptic area (49 ~), suprachiasmatic nucleus (70 ~) and anterior hypothalamic area (60 ~). Electrolytic lesions restricted to either the median or dorsal raphe nucleus produced significant reductions in the 5-HT concentration of the caudate-putamen, anterolateral hypothalamic area (45 ~o) and arcuate nucleus (48-58 ~). The fall in caudate-putamen 5-HT level was significantly greater after the dorsal (66 ~) than after the median (24 ~) raphe lesion. Neither lesion significantly affected the 5-HT content of the posterolateral hypothalamic area, the ventromedial hypothalamic nucleus or the dorsomedial hypothalamic nucleus. Thus, like their differential projections to the caudate-putamen and hippocampus, the mesencephalic dorsal and median raphe nuclei appear to innervate different preoptico-hypothalamic nuclei and areas. Whereas the median raphe nucleus seems to be the primary source of 5-HT fibers to the suprachiasmatic nucleus, anterior hypothalamic area and medial preoptic area, the 5-HT inputs to the anterolateral hypothalamic area and arcuate nucleus appear to derive from both the dorsal and median raphe nuclei.
INTRODUCTION Serotonin (5-hydroxytryptamine; 5-HT) has been implicated as playing a major role in a number of psychological and physiological processes, including sexual behavior and neuroendocrine regulation. Secretion of luteinizing hormonel~,19-~2,27, 31,40 and growth hormone z~ thus are controlled in part by 5-HT fibers. The hypothalamus and preoptic area are major regions involved in the regulation of both sexual behaviorr,9,10,16,~6 and neuroendocrine secretionT,14,ag. Since both the median
46 and dorsal midbrain raphe nuclei have been reported to innervate the hypothalamus ~, 4,13,1s,30, the present study was undertaken to determine whether different preopticohypothalamic areas and nuclei receive their 5-HT input from the mesencephalic dorsal or median raphe nucleus. METHODS
Animals Male Sprague-Dawley rats weighing 200-250 g at the time of surgery were obtained from Bio Labs (St. Paul, Minn.). The rats were housed in groups of 6 in a temperature (22 ~ 1 °C) controlled room with a 12:12 h light :dark cycle (lights on at 07.00). Food and water were available ad libitum.
Surgery, histology and biochemistry Lesions were produced electrotytically under ether anaesthesia in either the dorsal (n -- 25) or median (n -- 25) raphe nucleus as previously described z4. In brief, 2 mA DC was passed for 10-15 sec between an intracranial cathode and an anode attached to the wound margin. The cathode was a 30-gauge tungsten wire insulated with Epoxylite except for 0.5 mm of its tip. Operated control rats (n = 18) were treated in the same manner as the lesion rats, except that an electrode was not lowered into their brains. The midbrain was frozen (--16 °C) and serial coronal sections (40/~m) obtained. Every third section was saved and stained by the cresylecht violet method. Lesion cavitation and glial scarring was determined microscopically and reconstructed diagrammatically. Hypothalamic nuclei and preoptic areas from 3 rats were pooled and homogenized in 40 #1 0.1 N HCI. The hippocampus and caudate-putamen from individual animals were homogenized in 100 #1 0.1 N HCI. Ten #1 aliquots were removed for protein assay according to Lowry et al. 25. The microhomogenizers were then centrifuged and 10 #l aliquots of the supernatant analyzed in duplicate for 5-HT as detailed by Saavedra et al.~L
Procedure Surgery was performed 2 weeks after the rats had arrived in the laboratory. The rats were sacrificed by decapitation 17-20 days postoperatively between 13.00 and 16.00 h. The brains were removed rapidly and dissected over dry ice 24. The hippocampus and caudate-putamen were frozen in liquid nitrogen and stored at --85 °C until they were assayed for 5,HT. The brain stem was placed in formalin for at least 1 week prior to histological processing and analysis. The diencephalon was mounted on flat cork discs, frozen in liquid nitrogen, wrapped in Saran Wrap and aluminum foil, then stored at --85 °C. Preoptico-hypothalamic nuclei and areas from 3 different rats were pooled for the 5-HT assay. The lesions in these animals were matched histologically and, as far as
47 possible, by the decrease in 5-HT levels in the caudate-putamen (for the dorsal raphe lesions) and hippocampus (for the median raphe lesions) 18,15,1s,23,24. Individual diencephalic nuclei and areas were microdissected bilaterally from cryostat cut frontal sections (0.6-1.2 mm) as originally described by Eik-Nes and Brizzee 11 and according to Saavedra et al. 33 (see their Fig. 1). For the suprachiasmatic and arcuate nuclei hollow needles with a 304/~m inner diameter were used. For the anterior hypothalamic area, and the ventromedial and dorsomedial hypothalamic nuclei, a needle with a 500 #m inner diameter was used. For the medial preoptic area, and the anterior and posterior lateral hypothalamic areas, a needle with a 600/~m inner diameter was employed. RESULTS
Lesion analysis The midbrain dorsal and median raphe lesions are shown schematically in Figs. 1 and 2, respectively. The dorsal raphe lesions were well confined to the ventromedial central grey and destroyed at least 35 ~o of the dorsal raphe nucleus. Incidental damage to the oculomotor complex, trochlear nucleus and medial longitudinal fasciculus was observed in several animals. However, the superior cerebellar peduncles, tegmental nuclei of Gudden and linear nuclei were spared in all rats. The median raphe lesions ablated at least 40 ~ of the median raphe nucleus. In several animals (A-D groups, Fig. 2) the lesion extended rostrally dorsal to the caudal two-thirds of the interpeduncular nucleus. In 10 rats (E-G groups, Fig. 2) the lesions extended caudally into the periventricular B6 5-HT cell group and pontine raphe nucleus (B5). The linear nuclei were spared in all animals, but the superior cerebellar peduncle was damaged in 3 rats (B group, Fig. 2). All rats sustained uni- or bilateral damage to Gudden's tegmental nuclei and the medial longitudinal fasciculus.
Caudate-putamen and hippocampal 5-HT Reductions in the 5-HT content of the caudate-putamen and hippocampus were used as a second control for the efficacy of the lesions13,15,18,2a,24. Rats with dorsal raphe lesions showed a significant decrease in the 5-HT content of the caudateputamen (61 7oo),but no change, or even a slight increase, in the 5-HT content of the hippocampus (Table I). Median raphe lesions, in contrast, produced a large decrease (66 ~o) in the 5-HT concentration of the hippocampus, but only a small decrease (24 ~) in the caudate-putamen. As previously reported 24, the more rostral median raphe lesions (animals A-D, Fig. 2) produced greater falls in hippocampal 5-HT concentration. Probably because of the uniformity in the positions of the dorsal raphe lesions, no consistent variation in caudate-putamen 5-HT content was found 24.
Regional preoptico-hypothalamic 5-HT Most of the lesion-induced changes in 5-HT concentration were found in the anterior hypothalamus and preoptic area (Table II). Thus, the median raphe lesions resulted in significant 5-HT reductions in the suprachiasmatic nucleus (70 ~), medial
",4
¢,~ ~
r~ O
--*
--~ Co
~
~
~
I'-.~mm
~
O
i'D
X ~ 0,
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~
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"T
",4
X
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49
!~,
E 27
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Fig. 2. Lesion damage (blackened area) in animals receiving median raphe lesions. Details as in Fig. 1. Fig. 1. Diagrammatic reconstruction of damage (blackened area) to the dorsal raphe nucleus on serial coronal sections, each separated by approximately 0.6 mm. Numbers identify individual rats, while the letters indicate animals whose preoptico-hypothalamic nuclei and areas were pooled for 5HT analysis. Abbreviations: BC, brachium conjunctivum;BP, middle cerebellar peduncle; B6, 5-HT cell group; c, pars centralis of dtg; DBC, decussation of BC; dr, dorsal raphe nucleus; dtg, dorsal tegmental nucleus of Gudden; gp, basal pontine nuclei; III, oculomotor complex; il, intermediate linear nucleus; ipn, interpeduncular nucleus; IV, trochlear nucleus; lc, locus coeruleus; ML, medial lemniscus; MLF, medial longitudinal fasciculus; mp, mammillary peduncle; mr, median raphe nucleus; nr, red nucleus; rl, rostral linear nucleus; rp, pontine raphe nucleus; rtp, pontine reticular tegmental nucleus; TTS, tectospinal tract; vtg, ventral tegmental nucleus of Gudden.
50 p r e o p t i c a r e a (49 }o), a n t e r i o r h y p o t h a l a m i c a r e a (60~,,,), the r o s t r a l p o r t i o n o f the l a t e r a l h y p o t h a l a m i c a r e a (46 ~ ) a n d the a r c u a t e n u c l e u s (58 ~o). In c o n t r a s t , the dorsal r a p h e lesions p r o d u c e d a significant d e c r e a s e in 5 - H T c o n c e n t r a t i o n o n l y in the a n t e r o l a t e r a l h y p o t h a l a m u s (45 ~ ) a n d a r c u a t e n u c l e u s (48 ~ ) . A t t e m p t s to c o r r e l a t e the m a g n i t u d e o f d e c r e a s e s in r e g i o n a l p r e o p t i c o - h y p o t h a l a m i c 5 - H T c o n t e n t w i t h t h o s e in the c a u d a t e - p u t a m e n
and hippocampus
did n o t result in a n y significant
relationships.
TABLE I Differential effects of selective midbrain raphe lesions on regional forebra#l 5-hydroxytryptamine concentrations (ng/g wet weight) Number of rats in parentheses. Probabilities based on Student's t-test, one-tail ; Ps in parentheses are based on a two-tailed t-test and refer to differences between the lesion groups. Group
Area assayed Hippocampus
Caudate-putomen
Mean 4: S.E.M.
% Difference
P
Mean ~: S.E.M.
% Differeltcc
P
349 ± 30 (9) 411 ± 31 (10)
-~ 18
-n.s.
625 ± 64 (11) 214 ± 30(15)
-- 66
Median raphe lesion 135 ± 22(17)
--61
< 0.0005 ( < 0.0O05)
473 ± 54(16)
--24
-< 0.0005 ( < 0.0005) --, 0.05
Control Dorsalraphelesion
TABLE II Effects of discrete mesencephalic raphe lesions on the 5-hydroxytryptamine concentration (ng/rng protein) of preoptico-hypothalamic areas and nuclei Mean ± S.E.M. Number of determinations in parentheses. Control
Medial preoptic area Anterolateral hypothalamic area Suprachiasmatic nucleus Anterior hypothalamic area Arcuate nucleus Ventromedial nucleus Dorsomedial nucleus Posterolateral hypothalamic area
9.8 ± 1.0 (6) 18.5 31.4 12.6 22.7 5.9 11.3
Dorsal raphe lesion 8.1 ± 1.1 (6)
+ 3.4 (4) 8.5 ± 1.7 (7)* i 4.6 (6) 25.2 ~ 3.1 (5) ± 2.6 (5) 8.3 ± 2.1 (6) + 3.7 (5) 11.9 ± 2.0 (5)* :t= 1.5 (5) 4.2 ± 0.9 (6) ± 1.8 (6) 8.1 ± 1.7 (6)
11.3 ± 3.6 (6)
11.3 ± 3.0 (6)
% Median raphe Change lesion
°o Change
--17
4.8 ± 1.3 (7)*
--51
--54 --20 --34 --48 --29 --28
8.7 9.4 5.1 9.5 5.3 7.1
--53 --70 --60 --58 --10 --37
0
± ± ± ± ± ±
2.3 (7)* 3.2 (6)** 1.1 (6)* 2.4 (5)* 1.4 (7) 1.5 (7)
8.9 ± 2 . 6 ( 6 )
--21
Probabilities determined by Student's t-test, two-tailed: * P < 0.05, significant difference between lesion and control group; ** P < 0.01, significant difference between median raphe lesion group and the other two groups.
51 DISCUSSION It is now generally accepted that the dorsal and median raphe nuclei differentially innervate distinct forebrain regionsl,4,13,15,1s, 23,24. A dorsal raphe-striatal and median raphe-hippocampal 5-HT projection was confirmed in the present study. In addition, the results suggest that the median raphe nucleus is the principal origin of 5HT fibers to the suprachiasmatic nucleus, the anterior hypothalamic area and the medial preoptic area. No evidence was obtained for an exclusive origin in the dorsal raphe nucleus of a 5-HT projection to any of the preoptico-hypothalamic structures studied. At present we cannot exclude the possibility that some of the effects of median raphe lesions are due to interruption of 5-HT fibers arising in the pontine raphe (B5 and B6 cell groups) or nucleus raphe magnus (B3). Projections to the hypothalamus from these regions have been reported 4. The autoradiographic method employed, however, does not discriminate 5-HT from non-5-HT fibers. Electrolytic lesions which extensively damaged the pontine B5 and B6 cell groups without invading the B7 (dorsal raphe nucleus) or B8 (median raphe nucleus) cell groups, moreover, do not significantly affect the 5-HT concentration of the hypothalamus or of other forebrain regions, except the dorsal thalamus (35 ~o reduction) 23. In addition, Bloom et al. 3 have shown that stimulation of the median raphe nucleus produces a short-latency inhibition of neuronal firing in the suprachiasmatic nucleus, while Carter and Sawyer6 observed increased multiunit activity in the medial preoptic area after electrochemical stimulation of the median raphe nucleus. The lesions in the median and dorsal raphe nuclei were incomplete. Therefore, it is possible that the dorsal raphe nucleus contributes 5-HT fibers to the preopticohypothalamic nuclei and areas studied, especially since the dorsal raphe lesions produced small but insignificant reductions in the 5-HT concentrations of several regions assayed. On the other hand, Kellar et al. 17 found only a 10 ~ reduction in the hypothalamic uptake of [SH]5-HT and no significant change in hypothalamic tryptophan hydroxylase activity after dorsal raphe lesions. Both of these measures were affected (about 50 ~ reductions) after median raphe lesions. Geyer et al. la also found a greater reduction in hypothalamic tryptophan hydroxylase activity after median raphe lesions than after dorsal raphe lesions. Evidence has been advanced suggesting the presence of 5-HT-containing perikarya in the hypothalamus, particularly in the paraventricular region. Hypothalamic deafferentation produced by circular knife cuts reduced the 5-HT content and tryptophan hydroxylase activity of the median eminence, arcuate nucleus, ventromedial nucleus and dorsomedial hypothalamic nucleus 38-69 ~5. Prolonged intraventricular infusion of [aH]5-HT has resulted in labeling of paraventricular hypothalamic neurons2, 8. The failure of dorsal and median raphe lesions to affect the 5-HT content of the ventromedial and dorsomedial hypothalamic nuclei thus may be due to the presence of 5-HT cell bodies in these regions. The absence of a significant reduction in the 5-HT concentration of the posterolateral hypothalamic area was surprising. However, it may well be that our
52 punches were made dorsal to the ascending 5-HT fibers which appear to travel in the ventral part of the medial forebrain bundle1,4, 3s. The falls observed in the anterolateral hypothalamus, furthermore, may be due primarily to degenerated fibers of passage rather than degenerated terminals. The 5-HT input to the lateral hypothalamus thus may derive from fibers arising in the paraventricular hypothalamus 29, or in brain stem neurons other than those comprising the B7 and B8 cell groups. The results of the present study are in disagreement with those of Palkovits et al. a0, who reported that selective dorsal raphe lesions reduced by 63-64 ~ the 5-HT content of the arcuate nucleus, the suprachiasmatic nucleus and the medial forebrain bundle. However, these workers found that their dorsal raphe lesions also produced a decrease in hippocampal tryptophan hydroxylase activity (73 ~). This observation strongly suggests that their dorsal raphe lesions extended ventrally and interrupted a large number of 5-HT fibers originating in the median raphe nucleus, as both Geyer et al. 13 and Kellar et al. 18 found no change in hippocampal tryptophan hydroxylase activity after lesions restricted to the dorsal raphe nucleus, but large decreases in the hippocampal activity of this enzyme after selective median raphe nucleus lesions. That the 5-HT input to the hippocampus derives principally from the median raphe nucleus also is supported by autoradiographicl,4, 2s, horseradish peroxidase 34 and other lesionlS,2a, 24 studies. It has been demonstrated in the male rat that the medial preoptic area plays an important role in sexual behavior6,9,t°,16, 26, whereas the arcuate and ventromedial hypothalamic nuclei are involved in regulating the secretion of luteinizing hormone (LH) ~4,39. Serotonergic neurons also appear to be involved in the regulation of both sexual behavior36, 37 and L H secretion 12,16,17,21,27,31,4°. Since our data suggest a dominant 5-HT innervation of the medial preoptic area, anterior hypothalamic area and suprachiasmatic nucleus by the median raphe nucleus, whereas the arcuate nucleus seems to receive an almost equal innervation from both the dorsal and median raphe nuclei, it is possible that the 5-HT fibers originating in the dorsal raphe nucleus are involved in the regulation of L H secretion, whereas the fibers from the median raphe nucleus may be involved in the regulation of sexual behavior as well as LH secretion. ACKNOWLEDGEMENTS The authors express their gratitude to Isabel Bizet and Jon Levine for their technical assistance, and to Dr. L. S. Van Orden III for his guidance and provision of facilities. This research was supported in part by Grant G M 12675.
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