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Dopamine turnover in the mediobasal hypothalamus in rat fetuses
Pergamon
Neuroscience Vol. 89, No. l, pp. 235-241. 1999 Copyright ~ 1998 |BRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved PII: S0306-4522(98)00278-4 0306-4522/99 $19.00+0.00
D O P A M I N E T U R N O V E R IN THE MEDIOBASAL H Y P O T H A L A M U S IN RAT FETUSES V. M E L N I K O V A , * M. O R O S C O , t A. CALAS,~ A. S A P R O N O V A , * R. G A I N E T D I N O V , § N. D E L H A Y E - B O U C H A U D , ¶ S. N I C O L A I D I S , t K. R A Y E V S K Y § and M. UGRUMOV*![** *Laboratory of Hormonal Regulations, Institute of Developmental Biology, Russian Academy of Sciences, Moscow 117808, Russia tCNRS UPR 9054, Neurobiologie des Regulations, Coll6ge de France, Paris, France ~:D6partement de Neurobiologie des Signaux Intercellulaires, Institut des Neurosciences, CNRS URA 1488, Universit6 P. et M. Curie, Paris, France §Laboratory of Neurochemical Pharmacolology, Institute of Pharmacology, Academy of Medical Sciences, Moscow, Russia ¶D6partement de D6veloppement et Vieillissement du Syst6me Nerveux, Universit6 P. et M. Curie. Paris, France llLaboratory of Neurohistology, Institute of Normal Physiology, Russian Academy of Medical Sciences, Moscow, Russia Abstrae~In this study, the dopamine turnover in the mediobasal hypothalamus, the key compartment of the neuroendocrine regulation of reproduction, was evaluated in fetal male and female rats. Highperformance liquid chromatography with electrochemical detection was used to measure 3,4dihydroxyphenylalanine, dopamine and 3,4-dihydroxyphenylacetic acid in the mediobasal hypothalamus of fetuses on the 21 st day of intrauterine development and in primary cell culture (cell extracts and culture medium) of the same brain region, explanted at the 17th fetal day and maintained for seven days. The same technique was applied to determine dopamine release from fetal neurons of the mediobasal hypothalamus in response to an excess of K ÷ in the perifusion system or in culture. L-3,4Dihydroxyphenylalanine, dopamine and 3,4-dihydroxyphenylacetic acid were detected both e x vivo and in culture. The ratios of the concentrations of L-3,4-dihydroxyphenylalanine/dopamine and 3,4dihydroxyphenylacetic acid/dopamine were significantly higher in vitro than e x vivo, showing a lower rate of dopamine production and a higher rate of its degradation in the experiments in vitro. Moreover, it has been demonstrated that an excess of K ÷, i.e. a membrane depolarization, resulted in a highly increased release of dopamine in the perifusion system and in culture. The dopaminergic activity in the developing mediobasal hypothalamus showed sexual dimorphism that was manifested in a greater concentration of 3,4-dihydroxyphenylalanine and dopamine, at least in cell extracts of cultures, as well as in a higher rate of dopamine release, both in the perifusion system and in culture in males compared to females. Thus, dopamine is synthesized and released in response to a membrane depolarization in the mediobasal hypothalamus of rats as early as the end of intrauterine development, suggesting its contribution to the inhibitory control of pituitary prolactin secretion. © 1998 IBRO. Published by Elsevier Science Ltd. K e y words: development, hypothalamus, dopamine, e x vivo, culture, rat.
The tuberoinfundibular dopaminergic system is the key compartment of the neuroendocrine regulation of prolactin secretion in adult mammals. 19 It consists of dopaminergic neurons of the arcuate nucleus, including their axonal projections to the median eminence. D o p a m i n e (DA) produced in this system is transferred via axons to the median eminence and **To whom correspondence should be addressed. DOPAC, 3,4-dihydroxyphenylacetic acid; E, embryonic day; EDTA, ethylenediaminetetra-acetate; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; HPLC-ED, highperformance liquid chromatography with electrochemical detection; L-DOPA, L-3,4-dihydroxyphenylalanine; MBH, mediobasal hypothalamus.
Abbreviations: DA, dopamine;
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discharged into the hypophysial portal circulation. Finally, it reaches pituitary lactotropes inhibiting the prolactin secretion via D 2 receptors.IS'24'32 There are controversial data in the literature about the development of the tuberoinfundibular dopaminergic system and the onset of the D A inhibitory control of prolactin secretion during ontogenesis. 14,2o,21 In this context, studies of the differentiation of D A producing neurons in the tuberoinfundibular system are of particular importance. So far, only tyrosine hydroxylase, the first enzyme of D A synthesis, has been detected with certainty in neurons of the mediobasal hypothalamus (MBH; the arcuate nucleus and median eminence) of fetal rats. 6'31 This raised the question whether D A is synthesized in this region
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( D Fig. 1. Dissection of the rat MBH on El7. (A) Whole brain. (B) Coronal thick slice of the brain. (C) Coronal thick slice of the brain with the dissected MBH. (D) Drawing of the coronal slice of the brain with the outlined MBH. until birth. Therefore, the present study aimed to evaluate the D A t u r n o v e r in differentiating n e u r o n s o f the M B H in rats at the end o f intrauterine development.
EXPERIMENTAL PROCEDURES
Animals and dissection of the mediobasal hypothalamus Wistar rats (Nursery "Stolbovaya", Russian Academy of Sciences, Moscow) pregnant females and fetuses on embryonic day (E) 17 and E21 (the day of conception being El) were used in this study. Pregnant rats were decapitated and fetuses were removed, followed by processing of male and female materials separately. Fetuses were decapitated and their brain was removed from the skull (Fig. 1A) and placed on its dorsal surface. Thereafter, a thick frontal slice was made through the whole forebrain at the level of the MBH under binocular loupe by making frontal cuts just caudal to the pituitary stalk and just rostral to the primary portal plexus (Fig. 1B). Finally, the MBH--the hypothalamic area around the bottom of the third ventricle--was dissected from the thick frontal slice (Fig. 1C, D) and the meninges were removed. Ex vivo stud), In the first series of experiments, the MBH of fetuses at E21 was dissected at 4°C, weighed, homogenized in 0.1 N HC104 (20% w/v) with the ultrasonic homogenizer and centrifuged for 20 min at 15,000 r.p.m. The supernatant of male and female materials was frozen in liquid nitrogen and kept at -80°C until L-3,4-dihydroxyphenylalanine (LDOPA), DA, noradrenaline and 3,4-dihydroxyphenylacetic acid (DOPAC) assay by high-performance liquid chroma-
tography with electrochemical detection (HPLC-ED). L-DOPA was measured only in males. In the second series of experiments, the MBH of fetuses was incubated in a buffer composed of (in mM): NaC1 136, KC13.0, CaC12 2.0, MgSO 4 1.2, NaH2PO 4 0.5, ascorbic acid 0.13, HEPES 20, D-glucose 10. Four MBHs were placed in each chamber, 400 ~tl in volume, similar to those described by Gallardo and Ramirez. 1° This was followed by perifusion with a rotary pump (Zaling, Poland) at a rate of 250 ~d/min. The chambers were placed in a water bath at 37°C. After passage through the chambers, effluent samples were discarded for the first 30 min, followed by collection of two 5-min fractions. Also, the initial buffer was replaced by that containing 60 mM KC1, and two additional 5-min fractions were collected. The osmolarity of the perifusion medium was corrected by proportional reduction of the NaC1 concentration. All effluent samples were frozen in liquid nitrogen and kept at -80°C until the DA assay. Cell culture Dissociated primary culture of the MBH neurons was mainly performed as described by Tixier-Vidal and FaivreBauman. 29 For this aim, the MBHs of male and female fetuses at El7 were pooled separately and dissociated mechanically in Fl2/Dulbeceo's modified Eagle's medium (Gibco) supplemented with 10% fetal calf serum. The cell suspension was centrifuged for 10 min at 800 r.p.m. The pellet was resuspended in serum-free medium (F12/ Dulbecco's modified Eagle's medium) supplemented with transferrin (100 ~tg/ml), putrescin (10 -4 M), insulin (5 p.g/ ml), glutamine (5 x 10 -3 M), sodium selenite (2 x 10 -5 M), progesterone (2 x 10 8 M), 17!3-estradiol (10-i2 M), corticosterone (10-7M), triiodothyronine (10-8M), HEPES (3.57 g/l), sodium bicarbonate (2.44 g/l), as well as arachidonic acid (1 p.g/ml) and doeosahexaenoic acid (0.5 ~tg/ml)
DA turnover in the mediobasal hypothalamus
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Table 1. Dopamine, 3,4-dihydroxyphenylacetic acid, L-3,4-dihydroxyphenylalanine and noradrenaline contents (pg/mg tissue) in the rat mediobasal hypothalamus at embryonic day 21 Sex Male Female
DA
DOPAC
L-DOPA
Noradrenaline
145:1:55 (8) 77 :t: 35 (9)
130-- 35 (7) 83 + 25 (8)
510d: 210 (10)
90+ 17 (12) 38 + 20* (t0)
Data are mean :1:S.E.M. *P<0.05. Numbers of measurements are given in parentheses. adsorbed on bovine serum albumin (fatty acid free, 37.5 pg/ ml), at a density of 1 x 106 cells in 35-mm Petri dishes (final volume 2 ml of medium), precoated with gelatine and poly-L-lysine. The cell survival in the primary cell suspension usually exceeded 85%, which was estimated using a hemocytometer following cell staining with Trypan Blue. All procedures described above were made in sterile conditions. Cultures were maintained at 37"C in a humidified atmosphere of 5% CO2. Culture medium was renewed on the fourth and sixth days, and from the fourth day cytosine arabinoside (10 8M) was included in the medium for inhibition of proliferation of glial cells. On the seventh day of culture, cell extracts and culture media were sampled for L-DOPA, DA and DOPAC assay. Moreover, in some dishes, the culture medium was replaced successively with 1 ml of buffer containing 3 mM KC1 (see ex vivo study) for 30 min, 1 ml of the same buffer (3 mM KC1) for 15 min, and l ml of buffer containing 60 mM KCI for 15 min. The second and third incubation media were collected and frozen in liquid nitrogen until HPLC-ED assay. High-perjormance liquid chromatography with electrochemical detection HPLC-ED was used to measure the concentrations of L-DOPA, DA, noradrenaline and DOPAC in the nerve tissue and cell extracts, as well as in the per±fusion and culture media. Catecholamines were extracted on acidic activated alumina and eluted in 0.6 N acetic acid. After centrifugation for 5 min at 5000 r.p.m., the supernatants were analysed by means of reverse-phase liquid chromatography with electrochemical detection (Albedo 100R, Europhor) at a potential of 700 inV. The chromatographic system consisted of a 20-gl sample loop leading to a 25-cm column (Kromasi, A.I.T.) with a 4.6-mm internal diameter and 5 pm (_'-18 packing. The mobile phase consisted of acetate buffer containing 134gM EDTA, 80pM octanesulfonic acid and 5% v/v methanol at pH 4.1. Statistics The results were statistically treated using the nonparametric Wilcoxon test. RESULTS
Ex vivo study DA, D O P A C , L-DOPA and noradrenaline were detected in the M B H at E21 (Table 1). Although the concentrations of D A and D O P A C tended to be higher in males than in females, no significant sex difference was observed, perhaps because of the large differences between individual values. Conversely, the concentration o f noradrenaline in males significantly exceeded that in females. The ratio of L-DOPA to D A in males was 3.5 +0.6, while that of D O P A C to D A was about 1:1 both in males (0.896±0.12) and females (1.07 +0.21), showing no significant sex difference in any case.
E21
[] 3 mM KCl ] E17(7 DIV)
350
--
300 L
*
[
350
(8)
300
60 mM KCI
(9
(9)
Male
Female
Male
Female
Fig. 2. DA release. (Left) From the dissected MBH of the rat on E21. (Right) On the seventh day of culture (7 DIV) of the rat MBH taken on El7. Mean+S.E.M. *P<0.05; n, number of measurements.
D A release from the dissected M B H increased significantly in the presence of excess K + (60 m M KC1) in the per±fusion system in both males and females at E21 (Fig. 2). Although there was no sexual dimorphism in the absolute values of spontaneous release and K+-stimulated release of DA, the difference between the former and the latter in males significantly exceeded that in females (187=t=29 in males vs 85 + 12 in females, P<0.05). Cell culture D A , D O P A C and L-DOPA were detectable in cell extracts of the M B H dissected on E l 7 and maintained for seven days in culture (Table 2). The concentrations of L-DOPA and D A in males were greater than those in females. Although the same tendency was found for D O P A C , no significant sex differences were observed (Table 2). The concentration of L-DOPA comfortably exceeded that of DA, with no sex difference ( L - D O P A / D A = 11.2+-0.71 in males vs 11.7+2.78 in females). The concentration of D O P A C was also greater than that of DA, showing a ratio 2.4+-0.15 in males and 2.84+ 0.44 in females, with no sex difference. In contrast to cell extracts, there were no sex differences in the concentrations of L-DOPA, D A and D O P A C in the culture medium (Table 2). The concentration of L-DOPA comfortably exceeded that of D A in the culture medium in both males and females, with no sex difference (L-DOPA/ DA--36.1 ±2.27 in males vs 40.3± 12.26 in females).
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Table 2. Dopamine, 3,4-dihydroxyphenylacetic acid and c-3,4-dihydroxyphenylalanine concentrations in cell extracts (pg/dish) and in the incubation medium (pg/ml) of culture of the rat mediobasal hypothalamus taken on embryonic day 17 and cultured for seven days in vitro DA
DOPAC
c-DOPA
Sex
Cell extract
Culture medium
Cell extract
Culture medium
Cell extract
Culture medium
Male Female
2504-30 (6) 1394-10"(6)
54+ 10 (9) 59±5 (7)
6004- 110 (7) 401 4-90 (8)
164±40 (8) 198±24 (7)
28004-169 (9) 16404-520*(10)
1950:t:480(10) 2380+480 (10)
Mean + S.E.M. *P<0.05; n, number of measurements.
This was also the case for the D O P A C / D A ratio (3.07+0.15 in males vs 3.3+0.18 in females). D A release was stimulated significantly in the presence of an excess of K + in culture of both male and female materials, but to a greater extent in the former than in the latter (210 • 26 in males vs 90 ± 23 in females, P<0.05; Fig. 2).
DISCUSSION Dopamine turnover in the mediobasal hypothalamus o f rat fetuses
Studies in the last decade have shown that the development of the hypothalamic dopaminergic system in rats is manifested in subsequent or chronologically overlapping processes: (i) genesis of the neurons I and their migration to their final locations; 28"31 (ii) expression of the enzymes of D A synthesis; ~3'3° (iii) onset of D A synthesis, specific uptake and K+-stimulated release; 3 (iv) establishment of efferent projections; (v) synaptic afferent innervation. 3° In immature-born animals, including rats, processes (i)-(iv) take place mainly during the second half of fetal life, while process (v) is generally a postnatal event. 3° Hypothalamic DA systems appeared to be quite heterogeneous in terms of morphology, function and developmental characteristics, justifying separate studies of the development of their major compartments. In this context, precise study of the differentiation of D A neurons of the tuberoinfundibular system is of particular importance, since they appear to differ in morphological and functional characteristics from D A neurons in other brain regions. For instance, these neurons are characterized by the relatively delayed expression of the enzymes of D A synthesis in ontogenesis, 31 as well as by the low level of DA catabolism and the absence of D A uptake and autoregulation mechanisms in adults. ~7 Moreover, the tuberoinfundibular system contains numerous peptidergic neurons coexpressing single enzymes of D A synthesis and, hence, are potentially capable of contributing to the D A metabolism in this particular hypothalamic region, s In the present study, the technique of precise dissection of the MBH in rat fetuses has been devel-
oped to define the local D A metabolism ex vivo and in culture using the HPLC-ED assay of DA, its immediate precursor L-DOPA and DOPAC, the product of DA degradation mainly by presynaptic monoamine oxidase B. 34 Only free, i.e. unconjugated c-DOPA, D A and DOPAC, were measured by this technique.~l It is generally considered that the concentrations of catecholamine metabolites and particularly the ratio of metabolite to amine can serve as indices of neuronal activity in the developing brain.~'26 According to our data, D A and DOPAC concentrations in the MBH of rat fetuses at E21 were essentially higher than those in both the whole brain and diencephalon of fetuses at the same age.5,~ In adults, the concentration of c-DOPA and its ratio to the D A concentration are almost zero for the MBH and other brain regions containing dopaminergic systems, because of the high activity of aromatic c-amino acid decarboxylase. 7'17 Surprisingly, the concentration of c-DOPA was significantly higher than that of D A in rat fetuses (L-DOPA/DA=3.5), suggesting a lower level of either synthesis of aromatic L-amino acid decarboxylase or its activity when compared to adults. In summary, our data point to a D A metabolism in the MBH of fetuses shortly before birth that is in agreement with the previous observation that the onset of hypothalamic D A inhibitory control of the pituitary prolactin secretion occurs from E20 to E22.16 In addition to D A synthesized in neurons of the arcuate nucleus, DA and other neurotransmitters may belong to the axons which arrive in the arcuate nucleus and the median eminence from outside of the MBH, e.g., from the zona incerta, substantia nigra, ventral tegmental region, etc., which is a characteristic of adults. 12"27 This hypothesis was indirectly supported by detection in the fetal MBH of noradrenaline (see Results) that apparently belonged to noradrenergic axons arriving from the brainstem. a2 Moreover, it cannot be excluded that at least a part of the measured L-DOPA and DOPAC originated in the maternal organism, since both appear to penetrate through the placental barrier. 4'23 In this study, particular attention was paid to spontaneous and K+-stimulated release of D A from the rat MBH on E21 in the perifusion system, since
DA turnover in the mediobasal hypothalamus this is one of the most important characteristics of the functional maturation of dopaminergic neurons. 3 Our data proved that DA release from the fetal MBH can be provoked by membrane depolarization, which is potentially an important point in the mechanism of the DA inhibitory control of pituitary prolactin secretion. However, in the perifusion model, we failed to differentiate DA release from the neurons of the arcuate nucleus and that from axons reaching the median eminence from outside of the MBH, which are not involved in the regulation of prolactin secretion. A subsequent study in culture has been undertaken to solve this problem.
Dopamine turnover & primary t&sue culture o f the fetal mediobasal hypothalamus
The primary culture of the MBH explanted at El7 and maintained for seven days was used to evaluate the DA metabolism and K+-stimulated release in proper neurons of the arcuate nucleus without any contribution of other potential sources of DA and metabolites, either in fetuses or in pregnant mothers. Although the differentiation of hypothalamic neurons in culture is principally similar to that in vivo, the former is still slightly retarded when compared with the latter.aS This means that the degree of neuron differentiation on the seventh day of culture of the MBH dissected at El7 should correspond approximately to that on E21-E22 in vivo. 3"~s According to our data, cell extracts and incubation media showed rather high levels of L-DOPA, DA and DOPAC. This could be considered as convincing evidence of DA synthesis by fetal neurons of the tuberoinfundibular system. It should be stressed that, in contrast to the ex vivo data, in culture the concentration of DOPAC considerably exceeded that of DA. These results suggest either a higher rate of DA catabolism or that a lower level of DA production occurs in vitro than ex vivo. The latter idea appears to be more probable, since there are two sources of DA in the MBH ex vivo--neurons of the arcuate nucleus and those distributed outside the MBH and projecting their axons to the median eminence--while there is only one source of DA in culture--proper neurons of the arcuate nucleus. Moreover, the ratio of the concentration of L-DOPA to that of DA in culture comfortably exceeded this index ex vivo, showing a lower level of either synthesis of aromatic L-amino acid decarboxylase or its activity. The greater ratio of DOPAC to DA in culture compared to that ex vivo might also indicate a higher activity of monoamine oxidase in the former than in the latter case. In contrast to DA metabolism, there was no principal difference in DA release in culture when compared with the perifusion system. In fact, an excess of K ÷ resulted in 1.8- to 3.6-fold increased release of DA, showing that the neurons of the
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arcuate nucleus are mature enough to discharge DA in response to membrane depolarization at the end of fetal life.
Sexual dimorphism in dopaminergic activity in the mediobasal hypothalamus
As follows from our study in culture, L-DOPA and DA concentrations in cell extracts of males significantly exceeded those in females. The same tendency, but without a significant difference, was the case for DOPAC in cell extracts, as well as for DA and DOPAC in the MBH ex vivo. This might be a result of either a greater number of DA-producing neurons or of the higher activity of enzymes of DA synthesis and degradation in males than in females. This should be elucidated in future studies. It remains uncertain whether sex differences in the DA concentration in fetal rats are determined genetically or mediated by sex steroids or other sex-related factors. The former idea seems to be preferable, because the MBH was explanted in culture on El7, i.e. before the apearance of sexual differences in the concentration of circulating testosterone. 2 Furthermore, the primary cultures of male and female materials were maintained in the same medium, which included some sex steroids at lower concentrations (e.g., 2 x 10 ~ M progesterone) when compared with those in the plasma of fetuses (e.g., 4.45 x 10 5 M progesterone). 33 Furthermore, it has been demonstrated that sex steroids do not influence tyrosine hydroxylase activity and, hence, DA synthesis in differentiating neurons of the mouse MBH either in vivo or in culture. 9 Clear sexual dimorphism is a characteristic of K+-stimulated release of DA in both the perifusion system and in culture. DA release occurred at a higher rate in males than in females, which may be explained either by the presence of a greater number of DA neurons or by the advanced differentiation of these neurons in males compared to females. The latter suggestion is supported indirectly by earlier observations of the gradually increasing K +stimulated release of neurotransmitters in the course of neuron differentiation.3 On the other hand, the same suggestion would contradict earlier studies showing that the origin and initial differentiation of the hypothalamic tyrosine hydroxylase-expressing neurons in female rats precede those in males both in vivo and in culture.l"25 Hence, sexual dimorphism in K+-stimulated release of DA is most probably genetically programmed and does not depend on sexual steroids. Indeed, there were no sexual differences in the concentrations of circulating sex steroids in fetuses before explanting the MBH to culture, 2'33 as well as in the culture media of male and female materials. It should be stressed that, in adults, there is a reverse in the rate of DA release from the neurons of the tuberoinfundibular system, being much higher
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in females t h a n in males u n d e r the influence of estrogen. 24
p r o d u c i n g system shows sexual d i m o r p h i s m t h a t is m o s t p r o b a b l y genetically determined.
CONCLUSION D A is synthesized a n d m a y be released in response to m e m b r a n e d e p o l a r i z a t i o n in the M B H of rats as early as the end of intrauterine development, suggesting its c o n t r i b u t i o n to the i n h i b i t o r y control of the pituitary prolactin secretion. The developing D A -
Acknowledgements--This work was supported by grants from the French Minist6re de l'Enseignement Sup6rieur et de la Recherche (1994 1997), Minist6re de l'Education Nationale (1993-96), INSERM (93 EO 02, 1994-95), NATO (OUTR.CRG 970131), INTAS-RFBR (95-1246) and the Russian Foundation for Basic Research (930420105; 1995-98), PICS (98-04-22018).
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Simon H., Moal M. and Calas A. (1979) Efferents and afferents of the ventral tegmental A10 region studied after local injection of [3H]leucine and horseradish peroxidase. Brain Res. 178, 17~,0. Specht L. A., Pickel V. M., Joh T. H. and Reis D. J. (1978) Immunocytochemical localization oftyrosine hydroxylase in processes within the ventricular zone of prenatal rat brain. Brain Res. 156, 315-321. Tixier-Vidal A. and Faivre-Bauman A. (1990) Use of hypothalamic cell culture to study role of diffusible factors in phenotypic expression of central nervous system neurons. In Methods in Neurosciences (ed. Conn P. M.), Vol. 2, pp. 355 369. Academic, New York. Ugrumov M. V. (1994) Hypothalamic catecholaminergic systems in ontogenesis: development and functional significance. In Phylogeny and Development of Catecholamine Systems in the CNS ~?f Vertebrates (eds Smeets W. J. A. J. and Reiner A.), pp. 435452. Cambridge University Press, Cambridge. Ugrumov M. V., Taxi J., Tixier-Vidal A., Thibault J. and Mitskevich M. S. (1989) Ontogenesis of tyrosine hydroxylase immunopositive structures in the rat hypothalamus. An atlas of neuronal cell bodies. Neuroscience 29, 135 156. Weiner R. l., Findell P. R. and Kordon C. (1988) Role of classic and peptide neuromediators in the neuroendocrine regulation of LH and prolactin. In The Physiology of Reproduction (eds Knobil E., Neill J. et al.), pp. 1235 1281. Raven, New York. Weisz J. and Ward 1. L. (1980) Plasma testosterone and progesterone titres of pregnant rats, their male and female fetuses, and neonatal offspring. Endocrinology 106, 306-316. Yu P. H. (1986) Monoamine oxidase. In Neuromethods. Neurotransmitter Enzymes (eds Boulton A. A., Baker G. B. and Yu P. H.), Vol. 5, pp. 235-272. Humana, Clifton.
(Accepted 1 May 1998)
Neuroscience Vol. 89, No. l, pp. 235-241. 1999 Copyright ~ 1998 |BRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved PII: S0306-4522(98)00278-4 0306-4522/99 $19.00+0.00
D O P A M I N E T U R N O V E R IN THE MEDIOBASAL H Y P O T H A L A M U S IN RAT FETUSES V. M E L N I K O V A , * M. O R O S C O , t A. CALAS,~ A. S A P R O N O V A , * R. G A I N E T D I N O V , § N. D E L H A Y E - B O U C H A U D , ¶ S. N I C O L A I D I S , t K. R A Y E V S K Y § and M. UGRUMOV*![** *Laboratory of Hormonal Regulations, Institute of Developmental Biology, Russian Academy of Sciences, Moscow 117808, Russia tCNRS UPR 9054, Neurobiologie des Regulations, Coll6ge de France, Paris, France ~:D6partement de Neurobiologie des Signaux Intercellulaires, Institut des Neurosciences, CNRS URA 1488, Universit6 P. et M. Curie, Paris, France §Laboratory of Neurochemical Pharmacolology, Institute of Pharmacology, Academy of Medical Sciences, Moscow, Russia ¶D6partement de D6veloppement et Vieillissement du Syst6me Nerveux, Universit6 P. et M. Curie. Paris, France llLaboratory of Neurohistology, Institute of Normal Physiology, Russian Academy of Medical Sciences, Moscow, Russia Abstrae~In this study, the dopamine turnover in the mediobasal hypothalamus, the key compartment of the neuroendocrine regulation of reproduction, was evaluated in fetal male and female rats. Highperformance liquid chromatography with electrochemical detection was used to measure 3,4dihydroxyphenylalanine, dopamine and 3,4-dihydroxyphenylacetic acid in the mediobasal hypothalamus of fetuses on the 21 st day of intrauterine development and in primary cell culture (cell extracts and culture medium) of the same brain region, explanted at the 17th fetal day and maintained for seven days. The same technique was applied to determine dopamine release from fetal neurons of the mediobasal hypothalamus in response to an excess of K ÷ in the perifusion system or in culture. L-3,4Dihydroxyphenylalanine, dopamine and 3,4-dihydroxyphenylacetic acid were detected both e x vivo and in culture. The ratios of the concentrations of L-3,4-dihydroxyphenylalanine/dopamine and 3,4dihydroxyphenylacetic acid/dopamine were significantly higher in vitro than e x vivo, showing a lower rate of dopamine production and a higher rate of its degradation in the experiments in vitro. Moreover, it has been demonstrated that an excess of K ÷, i.e. a membrane depolarization, resulted in a highly increased release of dopamine in the perifusion system and in culture. The dopaminergic activity in the developing mediobasal hypothalamus showed sexual dimorphism that was manifested in a greater concentration of 3,4-dihydroxyphenylalanine and dopamine, at least in cell extracts of cultures, as well as in a higher rate of dopamine release, both in the perifusion system and in culture in males compared to females. Thus, dopamine is synthesized and released in response to a membrane depolarization in the mediobasal hypothalamus of rats as early as the end of intrauterine development, suggesting its contribution to the inhibitory control of pituitary prolactin secretion. © 1998 IBRO. Published by Elsevier Science Ltd. K e y words: development, hypothalamus, dopamine, e x vivo, culture, rat.
The tuberoinfundibular dopaminergic system is the key compartment of the neuroendocrine regulation of prolactin secretion in adult mammals. 19 It consists of dopaminergic neurons of the arcuate nucleus, including their axonal projections to the median eminence. D o p a m i n e (DA) produced in this system is transferred via axons to the median eminence and **To whom correspondence should be addressed. DOPAC, 3,4-dihydroxyphenylacetic acid; E, embryonic day; EDTA, ethylenediaminetetra-acetate; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; HPLC-ED, highperformance liquid chromatography with electrochemical detection; L-DOPA, L-3,4-dihydroxyphenylalanine; MBH, mediobasal hypothalamus.
Abbreviations: DA, dopamine;
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discharged into the hypophysial portal circulation. Finally, it reaches pituitary lactotropes inhibiting the prolactin secretion via D 2 receptors.IS'24'32 There are controversial data in the literature about the development of the tuberoinfundibular dopaminergic system and the onset of the D A inhibitory control of prolactin secretion during ontogenesis. 14,2o,21 In this context, studies of the differentiation of D A producing neurons in the tuberoinfundibular system are of particular importance. So far, only tyrosine hydroxylase, the first enzyme of D A synthesis, has been detected with certainty in neurons of the mediobasal hypothalamus (MBH; the arcuate nucleus and median eminence) of fetal rats. 6'31 This raised the question whether D A is synthesized in this region
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( D Fig. 1. Dissection of the rat MBH on El7. (A) Whole brain. (B) Coronal thick slice of the brain. (C) Coronal thick slice of the brain with the dissected MBH. (D) Drawing of the coronal slice of the brain with the outlined MBH. until birth. Therefore, the present study aimed to evaluate the D A t u r n o v e r in differentiating n e u r o n s o f the M B H in rats at the end o f intrauterine development.
EXPERIMENTAL PROCEDURES
Animals and dissection of the mediobasal hypothalamus Wistar rats (Nursery "Stolbovaya", Russian Academy of Sciences, Moscow) pregnant females and fetuses on embryonic day (E) 17 and E21 (the day of conception being El) were used in this study. Pregnant rats were decapitated and fetuses were removed, followed by processing of male and female materials separately. Fetuses were decapitated and their brain was removed from the skull (Fig. 1A) and placed on its dorsal surface. Thereafter, a thick frontal slice was made through the whole forebrain at the level of the MBH under binocular loupe by making frontal cuts just caudal to the pituitary stalk and just rostral to the primary portal plexus (Fig. 1B). Finally, the MBH--the hypothalamic area around the bottom of the third ventricle--was dissected from the thick frontal slice (Fig. 1C, D) and the meninges were removed. Ex vivo stud), In the first series of experiments, the MBH of fetuses at E21 was dissected at 4°C, weighed, homogenized in 0.1 N HC104 (20% w/v) with the ultrasonic homogenizer and centrifuged for 20 min at 15,000 r.p.m. The supernatant of male and female materials was frozen in liquid nitrogen and kept at -80°C until L-3,4-dihydroxyphenylalanine (LDOPA), DA, noradrenaline and 3,4-dihydroxyphenylacetic acid (DOPAC) assay by high-performance liquid chroma-
tography with electrochemical detection (HPLC-ED). L-DOPA was measured only in males. In the second series of experiments, the MBH of fetuses was incubated in a buffer composed of (in mM): NaC1 136, KC13.0, CaC12 2.0, MgSO 4 1.2, NaH2PO 4 0.5, ascorbic acid 0.13, HEPES 20, D-glucose 10. Four MBHs were placed in each chamber, 400 ~tl in volume, similar to those described by Gallardo and Ramirez. 1° This was followed by perifusion with a rotary pump (Zaling, Poland) at a rate of 250 ~d/min. The chambers were placed in a water bath at 37°C. After passage through the chambers, effluent samples were discarded for the first 30 min, followed by collection of two 5-min fractions. Also, the initial buffer was replaced by that containing 60 mM KC1, and two additional 5-min fractions were collected. The osmolarity of the perifusion medium was corrected by proportional reduction of the NaC1 concentration. All effluent samples were frozen in liquid nitrogen and kept at -80°C until the DA assay. Cell culture Dissociated primary culture of the MBH neurons was mainly performed as described by Tixier-Vidal and FaivreBauman. 29 For this aim, the MBHs of male and female fetuses at El7 were pooled separately and dissociated mechanically in Fl2/Dulbeceo's modified Eagle's medium (Gibco) supplemented with 10% fetal calf serum. The cell suspension was centrifuged for 10 min at 800 r.p.m. The pellet was resuspended in serum-free medium (F12/ Dulbecco's modified Eagle's medium) supplemented with transferrin (100 ~tg/ml), putrescin (10 -4 M), insulin (5 p.g/ ml), glutamine (5 x 10 -3 M), sodium selenite (2 x 10 -5 M), progesterone (2 x 10 8 M), 17!3-estradiol (10-i2 M), corticosterone (10-7M), triiodothyronine (10-8M), HEPES (3.57 g/l), sodium bicarbonate (2.44 g/l), as well as arachidonic acid (1 p.g/ml) and doeosahexaenoic acid (0.5 ~tg/ml)
DA turnover in the mediobasal hypothalamus
237
Table 1. Dopamine, 3,4-dihydroxyphenylacetic acid, L-3,4-dihydroxyphenylalanine and noradrenaline contents (pg/mg tissue) in the rat mediobasal hypothalamus at embryonic day 21 Sex Male Female
DA
DOPAC
L-DOPA
Noradrenaline
145:1:55 (8) 77 :t: 35 (9)
130-- 35 (7) 83 + 25 (8)
510d: 210 (10)
90+ 17 (12) 38 + 20* (t0)
Data are mean :1:S.E.M. *P<0.05. Numbers of measurements are given in parentheses. adsorbed on bovine serum albumin (fatty acid free, 37.5 pg/ ml), at a density of 1 x 106 cells in 35-mm Petri dishes (final volume 2 ml of medium), precoated with gelatine and poly-L-lysine. The cell survival in the primary cell suspension usually exceeded 85%, which was estimated using a hemocytometer following cell staining with Trypan Blue. All procedures described above were made in sterile conditions. Cultures were maintained at 37"C in a humidified atmosphere of 5% CO2. Culture medium was renewed on the fourth and sixth days, and from the fourth day cytosine arabinoside (10 8M) was included in the medium for inhibition of proliferation of glial cells. On the seventh day of culture, cell extracts and culture media were sampled for L-DOPA, DA and DOPAC assay. Moreover, in some dishes, the culture medium was replaced successively with 1 ml of buffer containing 3 mM KC1 (see ex vivo study) for 30 min, 1 ml of the same buffer (3 mM KC1) for 15 min, and l ml of buffer containing 60 mM KCI for 15 min. The second and third incubation media were collected and frozen in liquid nitrogen until HPLC-ED assay. High-perjormance liquid chromatography with electrochemical detection HPLC-ED was used to measure the concentrations of L-DOPA, DA, noradrenaline and DOPAC in the nerve tissue and cell extracts, as well as in the per±fusion and culture media. Catecholamines were extracted on acidic activated alumina and eluted in 0.6 N acetic acid. After centrifugation for 5 min at 5000 r.p.m., the supernatants were analysed by means of reverse-phase liquid chromatography with electrochemical detection (Albedo 100R, Europhor) at a potential of 700 inV. The chromatographic system consisted of a 20-gl sample loop leading to a 25-cm column (Kromasi, A.I.T.) with a 4.6-mm internal diameter and 5 pm (_'-18 packing. The mobile phase consisted of acetate buffer containing 134gM EDTA, 80pM octanesulfonic acid and 5% v/v methanol at pH 4.1. Statistics The results were statistically treated using the nonparametric Wilcoxon test. RESULTS
Ex vivo study DA, D O P A C , L-DOPA and noradrenaline were detected in the M B H at E21 (Table 1). Although the concentrations of D A and D O P A C tended to be higher in males than in females, no significant sex difference was observed, perhaps because of the large differences between individual values. Conversely, the concentration o f noradrenaline in males significantly exceeded that in females. The ratio of L-DOPA to D A in males was 3.5 +0.6, while that of D O P A C to D A was about 1:1 both in males (0.896±0.12) and females (1.07 +0.21), showing no significant sex difference in any case.
E21
[] 3 mM KCl ] E17(7 DIV)
350
--
300 L
*
[
350
(8)
300
60 mM KCI
(9
(9)
Male
Female
Male
Female
Fig. 2. DA release. (Left) From the dissected MBH of the rat on E21. (Right) On the seventh day of culture (7 DIV) of the rat MBH taken on El7. Mean+S.E.M. *P<0.05; n, number of measurements.
D A release from the dissected M B H increased significantly in the presence of excess K + (60 m M KC1) in the per±fusion system in both males and females at E21 (Fig. 2). Although there was no sexual dimorphism in the absolute values of spontaneous release and K+-stimulated release of DA, the difference between the former and the latter in males significantly exceeded that in females (187=t=29 in males vs 85 + 12 in females, P<0.05). Cell culture D A , D O P A C and L-DOPA were detectable in cell extracts of the M B H dissected on E l 7 and maintained for seven days in culture (Table 2). The concentrations of L-DOPA and D A in males were greater than those in females. Although the same tendency was found for D O P A C , no significant sex differences were observed (Table 2). The concentration of L-DOPA comfortably exceeded that of DA, with no sex difference ( L - D O P A / D A = 11.2+-0.71 in males vs 11.7+2.78 in females). The concentration of D O P A C was also greater than that of DA, showing a ratio 2.4+-0.15 in males and 2.84+ 0.44 in females, with no sex difference. In contrast to cell extracts, there were no sex differences in the concentrations of L-DOPA, D A and D O P A C in the culture medium (Table 2). The concentration of L-DOPA comfortably exceeded that of D A in the culture medium in both males and females, with no sex difference (L-DOPA/ DA--36.1 ±2.27 in males vs 40.3± 12.26 in females).
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Table 2. Dopamine, 3,4-dihydroxyphenylacetic acid and c-3,4-dihydroxyphenylalanine concentrations in cell extracts (pg/dish) and in the incubation medium (pg/ml) of culture of the rat mediobasal hypothalamus taken on embryonic day 17 and cultured for seven days in vitro DA
DOPAC
c-DOPA
Sex
Cell extract
Culture medium
Cell extract
Culture medium
Cell extract
Culture medium
Male Female
2504-30 (6) 1394-10"(6)
54+ 10 (9) 59±5 (7)
6004- 110 (7) 401 4-90 (8)
164±40 (8) 198±24 (7)
28004-169 (9) 16404-520*(10)
1950:t:480(10) 2380+480 (10)
Mean + S.E.M. *P<0.05; n, number of measurements.
This was also the case for the D O P A C / D A ratio (3.07+0.15 in males vs 3.3+0.18 in females). D A release was stimulated significantly in the presence of an excess of K + in culture of both male and female materials, but to a greater extent in the former than in the latter (210 • 26 in males vs 90 ± 23 in females, P<0.05; Fig. 2).
DISCUSSION Dopamine turnover in the mediobasal hypothalamus o f rat fetuses
Studies in the last decade have shown that the development of the hypothalamic dopaminergic system in rats is manifested in subsequent or chronologically overlapping processes: (i) genesis of the neurons I and their migration to their final locations; 28"31 (ii) expression of the enzymes of D A synthesis; ~3'3° (iii) onset of D A synthesis, specific uptake and K+-stimulated release; 3 (iv) establishment of efferent projections; (v) synaptic afferent innervation. 3° In immature-born animals, including rats, processes (i)-(iv) take place mainly during the second half of fetal life, while process (v) is generally a postnatal event. 3° Hypothalamic DA systems appeared to be quite heterogeneous in terms of morphology, function and developmental characteristics, justifying separate studies of the development of their major compartments. In this context, precise study of the differentiation of D A neurons of the tuberoinfundibular system is of particular importance, since they appear to differ in morphological and functional characteristics from D A neurons in other brain regions. For instance, these neurons are characterized by the relatively delayed expression of the enzymes of D A synthesis in ontogenesis, 31 as well as by the low level of DA catabolism and the absence of D A uptake and autoregulation mechanisms in adults. ~7 Moreover, the tuberoinfundibular system contains numerous peptidergic neurons coexpressing single enzymes of D A synthesis and, hence, are potentially capable of contributing to the D A metabolism in this particular hypothalamic region, s In the present study, the technique of precise dissection of the MBH in rat fetuses has been devel-
oped to define the local D A metabolism ex vivo and in culture using the HPLC-ED assay of DA, its immediate precursor L-DOPA and DOPAC, the product of DA degradation mainly by presynaptic monoamine oxidase B. 34 Only free, i.e. unconjugated c-DOPA, D A and DOPAC, were measured by this technique.~l It is generally considered that the concentrations of catecholamine metabolites and particularly the ratio of metabolite to amine can serve as indices of neuronal activity in the developing brain.~'26 According to our data, D A and DOPAC concentrations in the MBH of rat fetuses at E21 were essentially higher than those in both the whole brain and diencephalon of fetuses at the same age.5,~ In adults, the concentration of c-DOPA and its ratio to the D A concentration are almost zero for the MBH and other brain regions containing dopaminergic systems, because of the high activity of aromatic c-amino acid decarboxylase. 7'17 Surprisingly, the concentration of c-DOPA was significantly higher than that of D A in rat fetuses (L-DOPA/DA=3.5), suggesting a lower level of either synthesis of aromatic L-amino acid decarboxylase or its activity when compared to adults. In summary, our data point to a D A metabolism in the MBH of fetuses shortly before birth that is in agreement with the previous observation that the onset of hypothalamic D A inhibitory control of the pituitary prolactin secretion occurs from E20 to E22.16 In addition to D A synthesized in neurons of the arcuate nucleus, DA and other neurotransmitters may belong to the axons which arrive in the arcuate nucleus and the median eminence from outside of the MBH, e.g., from the zona incerta, substantia nigra, ventral tegmental region, etc., which is a characteristic of adults. 12"27 This hypothesis was indirectly supported by detection in the fetal MBH of noradrenaline (see Results) that apparently belonged to noradrenergic axons arriving from the brainstem. a2 Moreover, it cannot be excluded that at least a part of the measured L-DOPA and DOPAC originated in the maternal organism, since both appear to penetrate through the placental barrier. 4'23 In this study, particular attention was paid to spontaneous and K+-stimulated release of D A from the rat MBH on E21 in the perifusion system, since
DA turnover in the mediobasal hypothalamus this is one of the most important characteristics of the functional maturation of dopaminergic neurons. 3 Our data proved that DA release from the fetal MBH can be provoked by membrane depolarization, which is potentially an important point in the mechanism of the DA inhibitory control of pituitary prolactin secretion. However, in the perifusion model, we failed to differentiate DA release from the neurons of the arcuate nucleus and that from axons reaching the median eminence from outside of the MBH, which are not involved in the regulation of prolactin secretion. A subsequent study in culture has been undertaken to solve this problem.
Dopamine turnover & primary t&sue culture o f the fetal mediobasal hypothalamus
The primary culture of the MBH explanted at El7 and maintained for seven days was used to evaluate the DA metabolism and K+-stimulated release in proper neurons of the arcuate nucleus without any contribution of other potential sources of DA and metabolites, either in fetuses or in pregnant mothers. Although the differentiation of hypothalamic neurons in culture is principally similar to that in vivo, the former is still slightly retarded when compared with the latter.aS This means that the degree of neuron differentiation on the seventh day of culture of the MBH dissected at El7 should correspond approximately to that on E21-E22 in vivo. 3"~s According to our data, cell extracts and incubation media showed rather high levels of L-DOPA, DA and DOPAC. This could be considered as convincing evidence of DA synthesis by fetal neurons of the tuberoinfundibular system. It should be stressed that, in contrast to the ex vivo data, in culture the concentration of DOPAC considerably exceeded that of DA. These results suggest either a higher rate of DA catabolism or that a lower level of DA production occurs in vitro than ex vivo. The latter idea appears to be more probable, since there are two sources of DA in the MBH ex vivo--neurons of the arcuate nucleus and those distributed outside the MBH and projecting their axons to the median eminence--while there is only one source of DA in culture--proper neurons of the arcuate nucleus. Moreover, the ratio of the concentration of L-DOPA to that of DA in culture comfortably exceeded this index ex vivo, showing a lower level of either synthesis of aromatic L-amino acid decarboxylase or its activity. The greater ratio of DOPAC to DA in culture compared to that ex vivo might also indicate a higher activity of monoamine oxidase in the former than in the latter case. In contrast to DA metabolism, there was no principal difference in DA release in culture when compared with the perifusion system. In fact, an excess of K ÷ resulted in 1.8- to 3.6-fold increased release of DA, showing that the neurons of the
239
arcuate nucleus are mature enough to discharge DA in response to membrane depolarization at the end of fetal life.
Sexual dimorphism in dopaminergic activity in the mediobasal hypothalamus
As follows from our study in culture, L-DOPA and DA concentrations in cell extracts of males significantly exceeded those in females. The same tendency, but without a significant difference, was the case for DOPAC in cell extracts, as well as for DA and DOPAC in the MBH ex vivo. This might be a result of either a greater number of DA-producing neurons or of the higher activity of enzymes of DA synthesis and degradation in males than in females. This should be elucidated in future studies. It remains uncertain whether sex differences in the DA concentration in fetal rats are determined genetically or mediated by sex steroids or other sex-related factors. The former idea seems to be preferable, because the MBH was explanted in culture on El7, i.e. before the apearance of sexual differences in the concentration of circulating testosterone. 2 Furthermore, the primary cultures of male and female materials were maintained in the same medium, which included some sex steroids at lower concentrations (e.g., 2 x 10 ~ M progesterone) when compared with those in the plasma of fetuses (e.g., 4.45 x 10 5 M progesterone). 33 Furthermore, it has been demonstrated that sex steroids do not influence tyrosine hydroxylase activity and, hence, DA synthesis in differentiating neurons of the mouse MBH either in vivo or in culture. 9 Clear sexual dimorphism is a characteristic of K+-stimulated release of DA in both the perifusion system and in culture. DA release occurred at a higher rate in males than in females, which may be explained either by the presence of a greater number of DA neurons or by the advanced differentiation of these neurons in males compared to females. The latter suggestion is supported indirectly by earlier observations of the gradually increasing K +stimulated release of neurotransmitters in the course of neuron differentiation.3 On the other hand, the same suggestion would contradict earlier studies showing that the origin and initial differentiation of the hypothalamic tyrosine hydroxylase-expressing neurons in female rats precede those in males both in vivo and in culture.l"25 Hence, sexual dimorphism in K+-stimulated release of DA is most probably genetically programmed and does not depend on sexual steroids. Indeed, there were no sexual differences in the concentrations of circulating sex steroids in fetuses before explanting the MBH to culture, 2'33 as well as in the culture media of male and female materials. It should be stressed that, in adults, there is a reverse in the rate of DA release from the neurons of the tuberoinfundibular system, being much higher
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V. Melnikova et al.
in females t h a n in males u n d e r the influence of estrogen. 24
p r o d u c i n g system shows sexual d i m o r p h i s m t h a t is m o s t p r o b a b l y genetically determined.
CONCLUSION D A is synthesized a n d m a y be released in response to m e m b r a n e d e p o l a r i z a t i o n in the M B H of rats as early as the end of intrauterine development, suggesting its c o n t r i b u t i o n to the i n h i b i t o r y control of the pituitary prolactin secretion. The developing D A -
Acknowledgements--This work was supported by grants from the French Minist6re de l'Enseignement Sup6rieur et de la Recherche (1994 1997), Minist6re de l'Education Nationale (1993-96), INSERM (93 EO 02, 1994-95), NATO (OUTR.CRG 970131), INTAS-RFBR (95-1246) and the Russian Foundation for Basic Research (930420105; 1995-98), PICS (98-04-22018).
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(Accepted 1 May 1998)