Serotonin synthesis from tryptophan by hypothalamic cells in serum-free medium culture

142

Developmental Brain Reset~rch, 5.4 I 1990) 142--i46 Elsevier

BRESD 60364

Ser~.onin Iwnthee~ ~

try~n by hypothalamic cells in serum-free medium culture

Denis Becquet, Anne-Marie Francois-Bellan, Franqoise Boudouresque, Maxime Faudon, Francis Hery, Viviane Guillaume and Micheline Hery Facult# de M#decine Nord, Laboratoire de Neuroendocrinologie Exp#rimentale, 1NSERM U 297, Bd P. Dramard, 13326 Marseille (France) (Accepted 27 February 1990)

Key words: Serotonin; Tryptophan hydroxylase; Rat hypothalamic neuron; Serum free culture

The hypothalamus of both adult and fetal rats contains a population of cells which can exhibit some features of serotoninergic (5-HT) neurons under certain circumstances. However, their neuronal serotoninergic nature is still controversial. In fact the presence of tryptophan hydroxylase activity has not yet been clearly established. This study attempted to verify whether [3H]5-HT can be synthesized from [3I-I]tryptophan ([3H]TRP) in hypothalamic cell cultures from 16-day-old fetuses. Data showed that [3H]5-HT was synthesized from [3H]TRP and the amounts of [3H]5-HT increased linearly as a function of time for 60 min. Pargyline markedly increased the quantities of [3H]5-HT and decreased those of [3H]5-hydroxyindole acetic acid. [3H]5-HT synthesis was inhibited by p-chlorophenylalanine, while a-methyl-p-tyrosine had no effect. The present biochemical study shows the presence of an intrinsic 5-HT neuronal system in the hypothalamus of the fetal rat.

The hypothalamus of the rat is richly innervated by serotoninergic fibers (5-HT) 6 originating from the centralis superior and dorsalis raphe nuclei 3°. Moreover, 5-HT neurons have been detected in the hypothalamus. Fuxe and Ungerstedt 13 first identified a group of serotoninergic neurons (B10) in the dorsomedial nucleus (DMN) using formaldehyde-induced fluorescence histochemistry following intraventricular infusion of 5-HT. Since then, these 5-HT neurons have been observed using radioautography following intraventricular infusion of [3H]5-HT1 and more recently with immunochemistry after a systemic pretreatment by the monoamine oxidase inhibitor (MAO inhibitor), pargyline and the amino acid precursor of the 5-HT synthesis: L-tryptophan 12. Furthermore, the existence of an intrinsic 5-HT neuronal system is supported by the finding that complete deafferentation of the hypothalamus does not entirely deplete its 5-HT content 26. Recent studies by Ugrumov et al. 32'33 have provided a description of the 5-HT system in the developing hypothalamus. These authors detected by radioautography, sparse radioactively labeled cells at the 16th and 18th fetal days after the injections of [3H]5-HT into the cerebral ventricles. Immunocytochemistry failed to recognize hypothalamic 5-HT neurons in fetuses. However, 5-HT-immunoreactive neurons in the hypothalamus of fetuses (E 18) appeared after subsequent injections of

pargyline and L-tryptophan in pregnant females 2 h before the perfusion. Although these neurons can exhibit some characteristics of serotoninergic neurons, their serotoninergic nature is still debatable. Since the immunostaining of this neuronal group was completely blocked by a preliminary injection of a specific 5-HT uptake inhibitor, fluoxetine, Ugrumov et al. 33 concluded that the 5-HT immunostaining of hypothalamic neurons could be accounted for by the specific uptake of 5-HT from environmental 5-HT nerve terminals, rather than by intraneuronal synthesis from L-tryptophan. Nevertheless, as proposed by Ueda et al.3k it is possible that the absence of positively stained cells in experiments without preliminary treatment with an MAO inhibitor + Ltryptophan was due to a higher rate of serotonin catabolism rather than synthesis or. as assumed by Frankfurt et al. 12, to the fact that the synthesis of 5-HT in these cells may be limited by the availability of substrate. Therefore, the purpose of this study was to verify whether these hypothalamic neurons could synthesize 5-HT from tryptophan. Taking advantage of the presence of such neurons in the hypothalamus of 16-day-old rat fetuses 32, a culture system of hypothalamic neurons from fetal rats (E 16) was used to estimate the synthesis of [3H]5-HT from [3H]tryptophan. Hypothalamic cells taken from 16-day-old fetal rats

Correspondence: M. Hery, Facult6 de M6decine Nord, Laboratoire de Neuroendocrinologie Exp6rimentale, INSERM U 297, Bd P. Dramard, 13326 Marseille, France. 0165-3806/90/$03.50 (~ 1990 Elsevier ~,-i. . . . p,,hl;~h .... D w . . . . . . . . . . . .

143 (Sprague-Dawley) were mechanically dissociated in F12D M E M (50/50) medium supplemented with 10% fetal calf serum, 1 mM glutamine, 0.6% glucose and penicillin (50 U/ml) plus streptomycin (50 /~g/ml). After being resuspended in a serum-free medium, they were seeded at a density of 1.3-1.6 × 106 cells in 1.5 ml of medium per 35 mm plastic tissue culture dish. Tissue culture dishes were pretreated as follows: they were coated with t~-polylysine (10 /~g/ml) for 12 h, rinsed with distilled water, incubated for 2 h with 20% fetal calf serum which was then withdrawn to dryness. The synthetic culture medium used was that described by Bottenstein and Sato 2 supplemented with 17-/3-estradiol (10 -12 M), progesterone (2 × 10 -8 M), triiodothyronine (10 -9 M) and arachidonic acid (1/~g/ml) plus docosahexaenoic acid (0,5 /~g/ml). Cultures were maintained at 37 °C in 7% CO2/ 93% air and 92-95% humidity. The medium was first renewed 4 days after seeding and then every 2 days. After 12 days of culture the medium was withdrawn and cells were washed 2 times with 1 ml artificial CSF (in mM: NaC1, 126.5; NaHCO3, 27.5; KCi, 2.4; KHePO 4, 0.5; CaCl2, 1.1; MgCI2, 0.85; NazSO4, 0.5; glucose, 5.9; adjusted to pH 7.3 with an O2-CO 2, 95:5 v/v mixture with or without pargyline (5 x 10 -5 M). Cells were preincubated for 15 rain in 1 ml CSF and kept at 37 °C in an incubator. Freshly purified [3H]tryptophan ([3H]TRP) (10/~Ci; 25-30 Ci/mmol; Amersham Center, U.K.) was then introduced into the incubating medium. Sixty minutes later, incubation was stopped by adding a cold ethanol-water (1.5 ml) solution (74:16 v/v) into each well. Plates were placed at -20 °C for 30 rain, then cells were disrupted with a Teflon scraper. Cells were sonicated, kept overnight in a freezer, then centrifuged at 12,000 x g for 20 min. From the supernatant, [3H]indoleamines were separated from [3H]tryptophan and [3H]metabolites by successive ion exchange chromatography on Dowex AG50 WX4 microcolumns and Amberlite CG50 columns as previously described ~7. [3H]Metabolites were extracted on Sephadex G-10 columns ~6. [3H]Tryptamine and [3H]IAA were extracted

in benzene at pH 10 from their respective eluates. The identity of [3H]indoleamines and [3H]metabolites was verified by co-chromatography of their acetylated derivatives 2z. Proteins were measured according to Lowry et al. 23 using bovine serum albumin as standard. After a 60-min incubation period with [3H]TRP, the amounts of [3H]indoleamines and [3H]metabolites were measured in Amberlite and Sephadex eluates, respectively. Co-chromatographic analysis revealed the presence of [3H]5-HT and [3H]tryptamine as well as their metabolites, [3H]5-hydroxyindole acetic acid ([3H]5H I A A ) and [3H]indole acetic acid ([3H]IAA). [3H]5H I A A levels were 3.3 times greater than those of [3H]5-HT in the control group (Table I). In contrast, [3H]5-HT amounts were 2.8 times higher than those of [3H]5-HIAA when pargyline (5 x 10-5 M) was added to the incubating medium. Nearly undetectable [3H]tryptamine amounts were measured in the control group, although those found for [3H]IAA were 36 times higher. The opposite occurred with pargyline. Serotonin synthesis ([3H]5-HT + [3H]5-HIAA) was not affected by the presence of pargyline (5 x 10-5 M). Time course studies showed that 5-HT synthesis measured in the presence of an M A O inhibitor was linear for 60 rain (Fig. 1). These data also demonstrate that pargyline does not induce end product inhibition on 5-HT synthesis. p-Chlorophenylalanine (PCPA) (10 -4 M) added in a single dose for 24 h or a daily dose for 48 h into the culture medium induced a significant decrease in 5-HT synthesis, 25 and 36%, respectively (Fig. 2). Values were not significantly different between 24 and 48 h PCPA treatment, a-Methyl-p-tyrosine (10 -4 M) added for 48 h into culture medum did not modify 5-HT synthesis (Fig. 2). These results provide the first experimental evidence for the fact that fetal hypothalamic neurons may synthesize serotonin from tryptophan. In this study we used a system of rat fetal hypothalamic neurons taken on the 16th day of gestation and grown in a serum-free medium which allowed a functional maturation of postmitotic

TABLE I [3H] Indole metabolism in hypothalamic cell cultures [3H]Indole synthesis from [3H]tryptophan in hypothalamic cell cultures. Cells from the hypothalamus of 16-day-oldrat fetuses were cultured in a chemically defined medium. After 12 days, cells were incubated at 37 °C in artificial CSF with [3H]tryptophan and with or without pargyline (5 x 10`5 M). Sixty minutes later cultures were treated to measure [3H]5-HT, [3H]5-HIAA, [3H]tryptamine and [3H]indoleacetic acid ([3H]IAA) synthetized in cells. Results are expressed in nCi/mg protein. They are means _+ S.E.M. of data obtained in 12 wells for each group. Data were statistically evaluated by Student's t-test. n Ci/mg protein

[3H]5-H T

[3H]5-H1AA

[3H] Tryptamine

[3H]IAA

Control Pargyline

12.33 +_0.31 37.54 + 1.52"

41.24 + 1.86 13.43 +_0.72*

0.69 _+0.09 26.24 + 2.35*

25.13 _+2.26 1.56 _+0.19"

*P < 0.05 when compared to control values.

144 300" 0 13.

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20

40 TIME (min)

60

Fig. 1. Time course of [3H]5-HT synthesis. Hypothalamic cells were incubated in the presence of pargylin (5 x 10-s M) with [3H]tryptophan (10 /zCi) for 20, 40 and 60 min. Results are expressed in nCi/mg protein. They are means + S.E.M. of data obtained in 12 wells for each group.

hypothalamic neurons a°. The 5-HT metabolism was studied 12 days after seeding. By this time neurons had reached a mature synaptic organization22. A biochemical isotopic method was used to measure 5-HT synthesis; it consisted in measuring [3H]5-HT synthesized from [3H]TRP having a high specific activity. The method was thus highly sensitive and quantities of [3H]5-HT as low as 2 pg could be measured. Since tryptophan hydroxylase is selectively located in 5-HT neurons, we were certain that the 5-HT formed, originated from serotoninergic neurons and not from cells containing non-specific L-amino acid decarboxylase (AADC), i.e. cateeholaminergic or histidinergic cells as has been observed with 5-hydroxytryptophan5. Cells containing AADC also have a capacity for tryptophan uptake and can convert it to tryptamine3. In our study [3H]tryptamine was found in small amounts compared to the [3H]5°HT in the control group. The presence of pargyline markedly increased the quantities of [3H]tryptamine. [3H]Tryptamine was not stored in cells, thus in the absence of an MAO inhibitor, [3H]tryptamine was almost totally oxidated by MAO in [3H]indole acetic acid. Moreover, as has been previously described, efflux of [3H]tryptamine was not regulated 17. Consequently, amounts of [3H]tryptamine were extracted from all samples, but not measured in all experiments. The main advantage of using fetal hypothalamic cells in culture instead of performing in vivo studies on intact fetuses is the certainty that the synthetized 5-HT comes from intrinsic serotoninergic neurons and not from nerve terminals originating from raphe nuclei. In fact, by the 14th to 15th fetal day, some 5-HT-immunoreactive fibers arriving from raphe nuclei leave the medial forebrain bundle entering first the dorsal hypothalamus, then by the 16th fetal day few of them reach the ventral hypothalamus 32. Different arguments favor the presence

50

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14)

-iCO

CONTROL

PCPA 24 H

PCPA 48 H

~(-MPT 48H

Fig. 2. Effects ofp-chlorophenylalanine and a-methyl-p-tyrosine on [3H]5-HT synthesis. Hypothalamic cell cultures were performed as described in Table I except that before incubation procedures PCPA (10 -4 M) was added in a single dose (for 24 h) or daily dose (for 48 h) to the culture medium. In a third group, a-methyl-p-tyrosine (10-4 M) was added in a daily dose for 48 h, Incubation procedures were carried out in the presence of pargyline (5 x 1(I_5 M). Results are expressed as per cent of control values. They are means _+ S,E.M. of data obtained in 12 wells for each group, *P < 0.05 when compared to control values.

of a serotoninergic intrinsic neuronal system within the hypothalamus. Total deafferentation of the hypothalamus does not completely deplete the 5-HT content of the hypothalamus26. Moreover, surgical isolation of the hypothalamus results in only a 50% decrease in tryptophan hydroxylase and in 5-HT content of the DMN4. These findings concur with those of Frankfurt and Azmitia 11. The authors reported that after a unilateral injection of 5,7-DHT into the rostral midbrain, the majority of 5-HT immunoreactive fibers in the ipsilateral hypothalamus had disappeared while the 5-HT immunoreactive cells in the DMN remained on both sides of the hypothalamus. More recently, Weissman et al. 34 have identified a small group of tryptophan hydroxylasepositive cells in the rostrolateral portion of the DMN. Finally, using immunocytochemistry without an MAO inhibitor of L-tryptophan pretreatments, Ni and Jonakait z5 have observed 5-HT-positive cells located in the anterior hypothalamus of 15-day-old fetuses. Our study provides further evidence for the serotoninergic nature of the 5-HT immunoreactive neurons in the hypothalamus. We have shown that fetal hypothalamic cells synthesize [3H]5-HT from [3H]TRP. The formation of [3H]5-HT increased linearly as a function of time. The time course of [3H]5-HT synthesis resembles that reported for striatal slices ~5 or for hypothalamus slices (H6ry, F., unpublished observations) incubated with [3H]tryptophan. As is typical following the application of

145 an M A O inhibitor, oxidation of 5-HT into 5 - H I A A was inhibited in fetal cells, resulting in an increase in 5-HT ( + 2 0 4 % ) and a decrease in 5 - H I A A levels ( - 6 7 % ) . The 3-fold increase in [3H]5-HT amounts m e a s u r e d in the presence of pargyline suggest that 5-HT turnover was similar to that o b s e r v e d in the adult rat brain 16. [3H]5-HT synthesis in fetal h y p o t h a l a m i c cells is inhibited by P C P A , an inhibitor of t r y p t o p h a n hydroxylase ~9 while application of a - m e t h y l - p a r a t y r o s i n e , an inhibitor of tyrosine hydroxylase 29 had no effect on [3H]5-HT synthesis. A c c o r d i n g to the findings of Weissm a n et al. 34, our results confirmed the presence of cells containing t r y p t o p h a n hydroxylase in the hypothalamus, i.e. the presence of 5-HT neurons. In contrast, D e Vitry et al. 8 have r e p o r t e d that the h y p o t h a l a m i c cell line F7 synthesizes [3H]5-HT from [3H]5-hydroxytryptophan but not from [3H]tryptophan. The differences b e t w e e n the species studied and p r o b a b l y also the fact that hypothalamic cell line F7 initially o b t a i n e d by viral transformation of m o u s e e m b r y o s exhibited potentialities of a p l u r i p o t e n t cell 7, could explain the discrepancies b e t w e e n our results and those of D e Vitry. 5-HT synthesis in fetal

h y p o t h a l a m i c cells r e p r e s e n t e d 13% and 50% of 5-HT synthesis found in fetal cell cultures of anterior (B4-B9) and posterior (B1-B3) r a p h e nuclei, respectively is. T h e ratio of 5-HT synthesis in fetal h y p o t h a l a m u s versus that in fetal anterior raphe nuclei m a y be c o m p a r e d to the ratio of e n d o g e n o u s 5-HT levels in the D M N versus those m e a s u r e d in the anterior raphe nuclei 27'2s.

1 Beaudet, A. and Descarries, L., Radioautographic characterization of serotonin accumulating nerve cell group in adult rat hypothalamus, Brain Res., 160 (1979) 231-243. 2 Bottenstein, J.E. and Sato, G.H., Growth of a rat neuroblastoma cell line in serum free supplemented medium, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 514-517. 3 Boulton, A.A., The tyramines: functionally significant biogenic amines or metabolic accidents?, Life Sci., 23 (1978) 659-672. 4 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 Res., 117 (1976) 287-295. 5 Denoyer, M., Kitahama, K., Sallanon, M., Touret, M. and Jouvet, M., 5-Hydroxytryptophan uptake and decarboxylating neurons in the cat hypothalamus, Neuroscience, 31 (1989) 203-211. 6 Descarries, L. and Beaudet, A., The serotonin innervation of adult rat hypothalamus. In J.D. Vincent and C. Kordon (Eds.), Cell Biology of Hypothalamic Neurosecretion, Vol. 280, CNRS, Paris, 1978, pp. 135-153. 7 De Vitry, E, Growth and differentiation of a primitive nervous cell line after in vivo transplantation into syngenic mice, Nature (Lond.), 267 (1977) 48-50. 8 De Vitry, F., Catelon, J., Dubois, M., Thibault, J., Barritault, D., Courty, J., Bourgoin, S. and Hamon, M., Partial expression of monoaminergic (serotoninergic) properties by the multipotent hypothalamic cell line F7: an example of learning at the cellular level, Neurochem. Int., 9 (1986) 43-53. 9 De Vitry, F., Catelon, J., Tbibault, J., Bourgoin, S. and Hamon, M., Serotonin as a growth factor for the differentiation of monoaminergic neurons. In A. Dahlstr6m et al. (Eds.), Proc. 6th Int. Catecholamine Syrup., Alan R. Liss, New York, 1988. pp. 585-590. 10 Faivre-Bauman, A., Puymirat, J., Loudes, C. and Tixier-Vidal, A., Differentiated mouse fetal hypothalamic cells in serum free medium. In D. Barnes, D, Sirbasku and G. Sato (Eds.),

on the serotonin immunoreactive cell bodies and fibers in the adult rat hypothalamus, Brain Res., 261 (1983) 91-99. Frankfurt, M., Lauder, J.M. and Azmitia, E.C., The irnmunocytochemical localization of serotonergic neurons in the rat hypothalamus, Neurosci. Lett., 24 (1981) 227-232. Fuxe, K. and Ungerstedt, U., Histochemical studies on the distribution of catecholamines and 5-hydroxytryptamine after intraventricular injections, Histochemie, 13 (1968) 16-28. Gromova, H.A., Chubakov, A.R., Chumasov, E.I. and Konovalov, H.V., Serotonin as a stimulator of hippocampal cell differentiation in tissue culture, Int. J. Dev. Neurosci., 1 (1983) 339-349. Hamon, M., Bourgoin, S. and Glowinski, J., Feed back regulation of 5-HT synthesis in rat striatal slices, J. Neurochem., 20 (1973) 1727-1745, H6ry, F., Rouer, E. and Glowinski, J., Daily variations of central 5-HT metabolism in the rat brain, Brain Res., 43 (1972) 445-465. H6ry, F., Simonnet, G., Bourgoin, S., Soubri6, P., Artaud, E, Hamon, M. and Glowinski, J., Effect of nerve activity on the in vivo release of 3H-serotonin continuously formed from 3Htryptophan in the caudate nucleus of the cat, Brain Res., 169 (1979) 317-334. H6ry, M., Becquet, D., Boudouresque, F., Faudon, M., Guillaume, V. and H6ry, F., Synth6se de s6rotonine h partir de tryptophane darts des cellules hypothalamiques en cultures, Annales d'Endocrinologie, 50 (1989) 32 N. Koe, K.B. and Weissman, A., p-Chlorophenylalanine: a specific depletor of brain serotonin, J. Pharmacol. Exp. Ther., 154 (1966) 499-516. Kordon, C., H6ry, M., Szafarczyk, A., Ixart, G. and Assertreacher, I., Serotonin and the regulation of pituitary hormone secretion and of neuroendocrine rhythms, J. Physiol. (Paris), 77 (1981) 497-500. Laverty, R. and Sharman, D.F., The estimation of small quantities of 3,4-dihydroxyphenylethylamine in tissues, Br. J. Pharmacol., 24 (1965) 538-548. Legendre, P., Tixier-Vidal, A., Brigant, J.L. and Vincent, J.D., Electrophysiology and ultrastructure of mouse hypothalamic

In the adult rat, the i m p o r t a n c e of 5-HT transmission for induced or cyclic activation of pituitary secretion has been widely d o c u m e n t e d (for review see K o r d o n et al.2°). H y p o t h a l a m i c 5-HT neurons, as well as r a p h e structures and 5-HT nerve terminals in the suprachiasmatic nucleus, can thus be assumed to r e p r e s e n t parts of the neuronal circuitry of a central clock synchronizing n e u r o e n d o c r i n e rhythms. In early ontogenesis, 5-HT can play a neurotrophic role 9. Some support for this concept can be found in different studies 8'14"24. This intrinsic h y p o t h a l a m i c 5-HT neuronal system could act i n d e p e n d e n t l y before serotoninergic nerve terminals which originate from the raphe nuclei reach the hypothalamus. The authors thank Mrs. Qudrat for the typing.

12 13 14

15 16 17

18

19 20

21

Methods for Serum Free Culture of Neuronal and Lymphoid Cells, Alan R. Liss, New York, 1984, pp. 37-56.

11 Frankfurt, M. and Azmitia, E., The effect of intracerebral injections of 5,7-dihydroxytryptamine and 6-hydroxydopamine

22

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86-90. 30 Steinbusch, H.W.M. and Nieuwenhuys, R.. The raphe nuclei of the rat brainstem: a cytoarchitectonic and immunohistochemical study, In P.C. Emson (Ed.), Chemical Neuroanatomy, Raven. New York, 1983, pp. 131-207. 31 Ueda, S., Nishida, K., Nojyo, Y., Takeuchi, Y. and Sano, Y., Serotonin-containing neurons in the rat and cat brain, especially in the hypothalamus following monoamine oxidase inhibitor pretreatment: an immunohistochemical study using anti-serotonin antiserum, Arch. Histol. Jpn., 47 (1984) 405-410. 32 Ugrumov, M.V., Taxi, J., Mitskevich, M.S. and Tramu, G., Development of the hypothalamic serotoninergic system during ontogenesis in rats. Immunocytochemical and radioautographic study, Dev. Brain Res., 30 (1986) 75-84, 33 Ugrumov, M.V., Taxi, J., Steinbusch, H.W.M., Tramu, G. and Mitskevich, M.S., On the distribution and morphofunctionat characteristics of 5-HT-immunoreactive cells in the hypothalamus of fetuses and neonatal rats, Dev. Brain Re~., 46 (1989) 233-241. 34 Weissmann, D., Belin, M.F., Aguera, M., Meunier, C., Maitre, M., Cash, C.D., Ehret, M., Mandel, P. and Pujol, J.F., Immunohistochemistry of tryptophan hydroxylase in the rat brain, Neuroscience, 23 (1987) 291-304.