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Effect of neutransmitters on in vitro release of luteinizing-hormone-releasing hormone from the mediobasal hypothalamus of male rats
Neuro~ience Letterm, 10. ~1978) 169--163 © Ekevie2'/North-Hoiland Scientific Publisl'.erc Ltd.
159
EFFECT OF NEUROTRANSMITTEIt~ ON IN VITRO RELEASE OF LUTEINIZING-HORMONE-RELEASI,'qG HORMONE FROM THE MEDIOBASAL HYPOTHALAMUS OF MALE RATS*
J.L. CHARLI, W.H. ROTSZTF.JN**,E. PATTOU and C. KORDON Unitts de Neuroendocrinolog.e, Centre Paul Broca de I'IN~ERM, 2ter rue d 'Al~sia, 75014
t~
(Fnmce)
(P~ceived Jmle 13th, 1978) (Revised version received August 12th, 1978) (Aecepte,l August 29tb, 1978)
SUMMAF:Y
Effects of neurotransmitters on in vitro release of lutein-hormone-releasing hormone (LHRH) from fragments of hypothalamic tissue were investigated. Neither acetylcholine (ACh) or histamine (HA) vt concentrations of 10 .7 and 10 -s M, o~' GABA at 10 -7 to 5 • 10 -5 M affected LHRH release from mediobasal hypothalamic (MBH) fragments or the organum vasculosum of the laminae terminalis (OVLT). In contrast, serotonin ( ] 0 -7 and 10 -s M) significantly inhibited the release of LHRH from the MBH but not from the OVLT. This response to serotonin (5-HT) is no longer observed in presence of methiothepin, a 5-HT receptor antagonist. The present results confirm in vivo data suggesting that the inhibitory effect of serotonin on the release of LHRH from median eminence involves a direct action on neurosecretory nerve~ndings.
Several putatwe neurotransmi~ers have been reported to interefere with the release of pituitary gonadotropins [12]. Most observations concerning these effects have been obtainc-d in vivo by pharmacological experiments. The level at which the interaction occurs cannot be de~rmined by such techniques. The recent development of in vitro methods [ 1,20] allows direct measurement of LHRH release and. makes it possible to investigate whether neurotransmitters act directly at the hypothalamic level. In the present work, we have tested the hypothesis that acetylcholine (ACh), 7-aminobutyric acid (GABA), histamine (HA) and serotonin (5.HT), all of wifich are present in high concentrations in * This work was supported by contracts No. 74/7/0804 from the D~l~gation G~n~rale ~ la Recherche Scientifique et Technique and No. 1891 from the Centre National de la Recherche Sckmtifique. ** Address corresponfience to: Dr. W.H. Rotsgtejn.
160
•
the hypothalamus [3,4,22,25], act directly on incubated hypothslamic fragments rich in LHRH nerve-endings. We had previously demonstrated that the release of the neurohormone from this p~paration can be induced by membrane depolarization in a c~eium-dependent m a n n e r [ 20] ; moreover, dol~ amine, but not norepinephrine were found to stimulate the release of LHRH from the palisadic zone of the median 'eminence [ 21] hltact male Sprague--Dawley rats (200--250 g body wt.) were used. The animals were killed by decapitation in the morning and tissue fragments containing either the whole mediobasal hypothalamus (MBH), the palisadic zone of the median eminence (rostral MBH), the infundibular sulcus (caudal MBH) or the organum vasculosum of the laminae terminalis (OVLT) were rapidly dissected as previously described [ 21] and incubated individually in 100/~l Locke Medium (in m M • NaCI 154, KCI 5.6, CaCl2 2.2, MgCI2 1, NaHCO3 6. glucose 10) buffered to pH 7.2 with 2 mM HEPES (N-2-dihydroxyet~ylpiperazine-N.2~thane sulfonic acid, Calbiochem) at 37°C in a shaking water bath in an atmosphere of 95% 02, 5% CO2. Bacitracin (2 • 10 -s M, Sigma) was added to prevent peptidasic degradation of LHRH [17], drugs and transmitters were diluted in the medium at the concentrations indicated in tables. At the end of incubation, tissue and medium were immediately extracted with 0.1 N HCI. LHRH radioimmunoassay was carried out with an anti-LH~tH antiserum raised by Kerdelhu~. et al. [ 9 ] as previously described [ 21], and factorial analysis to unequal subclass numbers was used in the statistical asse~ment of possible interacting effects [6 ]. As shown in Table I, neither ACh in the presence of eserine (10 -4 M) to .block acetylcholinesterase, nor histamine tested at concentrations of 10 -7 and 10 -5 M affected LHRH release from any of the structures studied. GABA (10 -7 to 5 • 10 -5 M) was equally ineffective on the postarior and the anterior portion of the MBH. In contrast, 5-HT (10 -~ mid 10 -s M) significantly (P < 0.01) inhibited the release of immunoreactive LHRH from the mediobasal hypothalamus, but not from the OVLT (Table II). This response to 5-HT is no longer observed in the presence of methiothepin (5 • 10 -6 M), a 5-HT receptor antagonist, although the interaction between both drugs is not statistically significant. The existence of an inhibitory effect o f fi-HT on the release of pituitary gonadotropins has already been described by several authors. Systemic injection of the amine was shown to block ovulation [ 18]. Intraventricular infusion of 5-HT [ 8,~z~4] or of drugs inducing an increase in endogenous stores of the transmitter [10,16] also result in LH blockade. T h e e effects cannot be explained by a direct action on pituitary gonadotro~in cells, since LH secretion is n~t affected by addition of ~HT to incuba~.~d pil~dtaries [23]. In addition, micropharma-.ological experiments, which have sbo~m that local increases in 5-HT levels were effective only when they occur within the arcuate-median eminence r e , on [11], are suggestive of an hypothal~.m~ic site of action. In spite of the fact that basal release of LHRH is low making it difficult to demonstrate inhibitory effects, the present data seer~ to confirm, an inhibitory
161
TABLE I E F F E C ~ O F V A R I O U S N E U R O T R A N S M I T T E R S ON T H E RELEASE OF L H R H FROM INCUBATED MBH F R A G M E N T S AND OVLT Values are r e p o rted as mean ± S.E.M. Numbers of rats are given in parentheses. LHRH release (pg/eq) MBH Control ACh 10-" M ACh 10 -s M
33.6 ± 2.6 (9) 29.6 ~ 3.5 (7) 32.6 ± 4.6 (8)
Control HA 1 0 - ' M HA 10-' M
19.1 ± 3.8 (7) 22.8 ± 6.5 (7) 28.3 ± 7.5 (V)
Rostral MBH
Caudal MBI!
O V LT 26.8 ± 3.9' (8) 23.4 ± 2.~. (8) 22.0 ± 3.4 (8)
Control GABA 10-" M GABA 1 0 - ' M
25.2 ± 2.2 (6) 23.2 ~ 5.0 (7) 26.0 ± 5.7 (5)
20.2 ± 3.2 (6) 28.0 ± 5.0 (7) 24.0 ± 1.7 (7)
Contro! GABA 10-' M GABA 5 . 1 0 s t~i
29.7 ± 3.0 (8) 36.7 ± 7.2 (8) 32.3 ± 5.0 (8)
25.5 ± 4.2 (8) 25.5 ± 4.0 (8) 28.2 ± 3.2 (8)
18.0 ~ 2.0 (8) 18.5 ± 3.7 (6) 15.3 ± 2.0 (8)
role for t h e ami~,e p r o b a b l y involving direct a c t i o n o n L H R H s e c r e t o r y nerveendings. This eff'~t s~ems to be hormone-sl~ecific, since 5-HT does not affect
the release of T R H under similar conditions [7]. Specific receptors of the amine also appear to mediate the response, since the transmitter does not TABLE II E F F E C T OF 5-HT A N D / O R ITS RECEPTORS ANTAGONIST METHIOTHEPIN ON IN VITRO L H R H R E L E A S E F R O M k ~ H AND OVLT Values are e x p r e ~ e d as mean ± S.E.M. Number of rats are given in parentheses. LHRH release (pgleq) MBH
OVLT
Control 5-HT 10 -~ M 5-HT 1 0 - ' M
28.0 ± 1.2 (16) 21.1 ± 1.0 (15)" 21.9 ± 1.1 (13)"
27.5 ~ 2.0 (7) 23.1 ± 1.7 (8) 23.2 ± 2.0 (8)
Control 5-HT 10 "~ M Me t h i o t h e p in 5.10" M 5-HT + M e t h i o t h e p i n
36.6 ± 5.5 (14) 19.2 ± 1.8 (16) a 27.9 ± 5.2 (13)
18.0 • 1.0 (6) 20 c, _.- 2.0 (6)
28.3 ± 6.1 (14)
!~.., ± 0.8 (6)
a p < 0.01 vs. control.
1~.. ~ : 2.o (5)
162
modify LHRH release in the presence of methiothepin. However, this conclusion must be considered with caution, since the interaction betwee.~ the drugs is not statistically significant. The concentzations of ~I~'I effectiv,~ in d~reasing LHRH l~lease under our experimental conditions however, are consistent with the affinity of the amine for its receptor detemdned through binding experimen'~ in other CNS structures [ 2]. GABA, HA and ACh do not seem to trigger directly the release of LHP,H from the median eminence nerve-endings containing ~ e neuropeptide. GABA was tested on the palisadic zone of the median em~.nence as well as on more posterior structures ~f the MBH, b e c a ~ t b ~ e structures have been shown to contain fairly high concentrations of both GABA [25] and LHRH [21]. The ineffectiveness of the transmitter under these conditions is in good agreement with physiological data on LH ~ecretion [19]. In contrast, both HA [14] mid ACh [13,15] have been shown to enhance LH secretion. Our results ~uggest that they act at a higher level in the CNS than the median eminence. In the case of ACh, this conclusion disagrees with that of Fiorindo and M~xtini [ 5]. The difference in experimental conditions may be responsible, in that the latter authors used much longer incubation periods and measured LHI~H release indirectly through its effect on LH release from pituitary tissue coincubated with hypothalamic fragments. It thus seems that, among neurotransmitters with gonadotropic control, only 5~HT and dopamine [21] exhibit a clear~ut effect on the basal release of LHRH from neurosecretory neurons. In both cases, the interaction is probably mediated by receptors to the amine located on ?.HRH pt'odueing cells in the median eminence. ACKNOWLEDGEMENT
We are indebted to Miss Marie-Christine Simon for her sldlled secrv~riai assistance. REFERENCES 1 Bennett, G.W., Edwardson, J.A., Holland, D., Jeffeoate, S.L. and White, N., Release of immunoreactive LHRH and TRH from hypothalamic synaptosomes, Nature (Lond.), 257 (1975) 323-325. 2 Bennett, J.P. Jr. and Snyder, S.H., Serotonin atld lysergic acid diethylamide binding in rat brain membranes. Relationship to post~ynaptie serotonin receptors, Mol. Pharmacol.,
12 (1976) 373--389. 3 Br.'~wnstein, M.J., Seavedra, J.M., Palkovits, M. and Axelrod, J., Histamine content of hypothaiamie nuclei of the rat, Brain Res., 77 (1974) 151--156. 4 Brownstein, M.J., Kobayashi, R., Palkovits, M. and Seavedrs, J.M., Choline acetyltrtmsferase levels in diencephalic nuclei of the rat, J. Neurochem., 24 (1975) 35--38. 5 Fiorindo, R.P. and Martini, L., Evidence for a cholinergie component in the neuroendocrine control of iuteinizing hormone (LH) secretion Neuroendocrlnology, 18 (1975) 322--332. 6 Harvey, W.R., I~ast~quare~ analysis of data with unequal subelus numbers, U.S.A. Agrie,~l~ural Research Service AR8 ?0-8 (1966).
163 7 Joseph-Bravo, P., C "arli, J.L., Palacios, J.M. and Kordon, C., Effects of nev'otr~nsmitters on the in vitro release, of immunoreac~ive thyrotropin releasing hormone from rat mediobasal hypothalamus, Endocrinology, in press. 8 Kamberi, I.A., Mical, R.S. and Porter, J.C., Effect of anterior pituitary perfusion and intraventricuiar injection of catecholamines and indolamines on LH release, Endocrinology, 87 (1970) 1--12. 9 Kerdelhu~, B., Catin, S., Kordon, C. and Jutisz, M., Delayed effects of in vitro LHRH immunoneutralization on gonadotropin and prolactin secretion in female rat, Endocrinology, 98 (1976) 1539--1549. 10 Kordon, C., Javoy, F., Vassent, G. and Glowinski, J., Blockade of superovulat'o, in the immature rat by increased brain serotonin, Eur. J. Pharmacol., 4 ( 1968) 169--174. 11 Kordon, C., Effect of selective experimental changes in regional hypothalamic monoamine levels on superovulation in the immature rat, Neuroe-docrinology, 4 (1969) 129--138. 12 Kordon, C. and Glowinski, J., Role of hypothalamic monoamergic neurones in the gonadotrophin release-regulating mechanisms, Neuropharmacology, 11 (1972) 153-162. 13 Libertun, C. and McCann, S.M., Blockade of the relea~ of gonadotropias and prolactin by subcu~aeous or intraventricular injection of atropine in male and female rats, Endocrinology, 92 (1973) 1714--1724. 14 Lil~rtun, C. and M¢Cann, S.M., The possible role of histamine in the control of prolactin anc'~ go~adotropin release, Neuroendocrinology, 20 (1976) 110--1 ~.0. 15 Lichtenst~iger, W., C/clic variations of catecholamine content in hypothalarr ic nerve cell~ during the estrcus cycle of the rat, with a concomitant study of the sub ~tantia nigra, J. Pharmacol...~xp. Ther., 165 (1969) 204--215. 16 Lippmann, W., Relationship between hypothalamic norepinephrine and serolonin and gonadotropic secreti,m in the hamster, Nature (Lond.), 218 (1968) 173--174. 17 MeKelvy, J.F., Leblanc, P., Loudes, C., Perrie, S., Grimm-Jorgensen, Y. and l~ordon, C., The use o~" bacitracir, as an inhibitor of the degradation of thyrotropin releas ~.ng factor and luteir.izing hormone releasing factor, Biochem. Biophys. Res. Commun., 73 (1976) 507--515. 18 O'Steen, W.K., Sero, onin suppression of luteinization in gonadotrophin-treated immature rats, Endoc~mlogy, 74 (1964) 885--888 19 Pass, K.A. and Ondo. J.G., The effects of ~r-aminobutyric acid on prolactin and gonadotropin secretion in the unanesthetized rat, Endocrinology, 100 (1977) 1437--1442. 20 Rotsztejn, W.H., C~a:li, J.L., Pattou, E., Epelbaum, J. and Kordon, C., In vitro release of luteinizir.g hormolte-releasing hormone (LHRH) from rat mediobasai hypothalamus: effects of potassium, calcium and dopamine, Endocrinology, 99 (1976) 1653--1666. 21 Rotsztejn, W.H., ChaAi, J.L., Pattou, E. and Kordon, C., Stimulation by doptmine of luteinizing hormone-releasing hormone (LHRH) release fron~ the mediobasal hypothalamus in male rats, Endocrinology, 101 (1977) 1475--1483. 22 Saavedra, J.M., Palkovits, M., Brownstein, M. and Axelrod, J., Serotonin distribution in the nuclei of the rat hypothalamus and preoptic region, Brain Res., 77 (1974) 157--165 23 Scemama, A., Effet des monoamines sur la s~crfition gonadotrope hypophysaire, In J. Benoit and C. Kordon (Eds.), l~.euroendocrinologie, C.N.R.S., Paris (1970), pp. 109-119. 24 Schneider, H.P.G. and McCann, S.M., Mono- and indolamines and control of L,H secretio:i. Endocrinology, 86 (1970) 1127--1133. 25 Tappaz, M.L., Brownstein, M.J. and Kopin, I.J., Glutamate decarboxylase (GAD) and GABA in discrete nuclei of hypothalamus and substantia nigra, Brain Res., 125 (1977) 109--121.
159
EFFECT OF NEUROTRANSMITTEIt~ ON IN VITRO RELEASE OF LUTEINIZING-HORMONE-RELEASI,'qG HORMONE FROM THE MEDIOBASAL HYPOTHALAMUS OF MALE RATS*
J.L. CHARLI, W.H. ROTSZTF.JN**,E. PATTOU and C. KORDON Unitts de Neuroendocrinolog.e, Centre Paul Broca de I'IN~ERM, 2ter rue d 'Al~sia, 75014
t~
(Fnmce)
(P~ceived Jmle 13th, 1978) (Revised version received August 12th, 1978) (Aecepte,l August 29tb, 1978)
SUMMAF:Y
Effects of neurotransmitters on in vitro release of lutein-hormone-releasing hormone (LHRH) from fragments of hypothalamic tissue were investigated. Neither acetylcholine (ACh) or histamine (HA) vt concentrations of 10 .7 and 10 -s M, o~' GABA at 10 -7 to 5 • 10 -5 M affected LHRH release from mediobasal hypothalamic (MBH) fragments or the organum vasculosum of the laminae terminalis (OVLT). In contrast, serotonin ( ] 0 -7 and 10 -s M) significantly inhibited the release of LHRH from the MBH but not from the OVLT. This response to serotonin (5-HT) is no longer observed in presence of methiothepin, a 5-HT receptor antagonist. The present results confirm in vivo data suggesting that the inhibitory effect of serotonin on the release of LHRH from median eminence involves a direct action on neurosecretory nerve~ndings.
Several putatwe neurotransmi~ers have been reported to interefere with the release of pituitary gonadotropins [12]. Most observations concerning these effects have been obtainc-d in vivo by pharmacological experiments. The level at which the interaction occurs cannot be de~rmined by such techniques. The recent development of in vitro methods [ 1,20] allows direct measurement of LHRH release and. makes it possible to investigate whether neurotransmitters act directly at the hypothalamic level. In the present work, we have tested the hypothesis that acetylcholine (ACh), 7-aminobutyric acid (GABA), histamine (HA) and serotonin (5.HT), all of wifich are present in high concentrations in * This work was supported by contracts No. 74/7/0804 from the D~l~gation G~n~rale ~ la Recherche Scientifique et Technique and No. 1891 from the Centre National de la Recherche Sckmtifique. ** Address corresponfience to: Dr. W.H. Rotsgtejn.
160
•
the hypothalamus [3,4,22,25], act directly on incubated hypothslamic fragments rich in LHRH nerve-endings. We had previously demonstrated that the release of the neurohormone from this p~paration can be induced by membrane depolarization in a c~eium-dependent m a n n e r [ 20] ; moreover, dol~ amine, but not norepinephrine were found to stimulate the release of LHRH from the palisadic zone of the median 'eminence [ 21] hltact male Sprague--Dawley rats (200--250 g body wt.) were used. The animals were killed by decapitation in the morning and tissue fragments containing either the whole mediobasal hypothalamus (MBH), the palisadic zone of the median eminence (rostral MBH), the infundibular sulcus (caudal MBH) or the organum vasculosum of the laminae terminalis (OVLT) were rapidly dissected as previously described [ 21] and incubated individually in 100/~l Locke Medium (in m M • NaCI 154, KCI 5.6, CaCl2 2.2, MgCI2 1, NaHCO3 6. glucose 10) buffered to pH 7.2 with 2 mM HEPES (N-2-dihydroxyet~ylpiperazine-N.2~thane sulfonic acid, Calbiochem) at 37°C in a shaking water bath in an atmosphere of 95% 02, 5% CO2. Bacitracin (2 • 10 -s M, Sigma) was added to prevent peptidasic degradation of LHRH [17], drugs and transmitters were diluted in the medium at the concentrations indicated in tables. At the end of incubation, tissue and medium were immediately extracted with 0.1 N HCI. LHRH radioimmunoassay was carried out with an anti-LH~tH antiserum raised by Kerdelhu~. et al. [ 9 ] as previously described [ 21], and factorial analysis to unequal subclass numbers was used in the statistical asse~ment of possible interacting effects [6 ]. As shown in Table I, neither ACh in the presence of eserine (10 -4 M) to .block acetylcholinesterase, nor histamine tested at concentrations of 10 -7 and 10 -5 M affected LHRH release from any of the structures studied. GABA (10 -7 to 5 • 10 -5 M) was equally ineffective on the postarior and the anterior portion of the MBH. In contrast, 5-HT (10 -~ mid 10 -s M) significantly (P < 0.01) inhibited the release of immunoreactive LHRH from the mediobasal hypothalamus, but not from the OVLT (Table II). This response to 5-HT is no longer observed in the presence of methiothepin (5 • 10 -6 M), a 5-HT receptor antagonist, although the interaction between both drugs is not statistically significant. The existence of an inhibitory effect o f fi-HT on the release of pituitary gonadotropins has already been described by several authors. Systemic injection of the amine was shown to block ovulation [ 18]. Intraventricular infusion of 5-HT [ 8,~z~4] or of drugs inducing an increase in endogenous stores of the transmitter [10,16] also result in LH blockade. T h e e effects cannot be explained by a direct action on pituitary gonadotro~in cells, since LH secretion is n~t affected by addition of ~HT to incuba~.~d pil~dtaries [23]. In addition, micropharma-.ological experiments, which have sbo~m that local increases in 5-HT levels were effective only when they occur within the arcuate-median eminence r e , on [11], are suggestive of an hypothal~.m~ic site of action. In spite of the fact that basal release of LHRH is low making it difficult to demonstrate inhibitory effects, the present data seer~ to confirm, an inhibitory
161
TABLE I E F F E C ~ O F V A R I O U S N E U R O T R A N S M I T T E R S ON T H E RELEASE OF L H R H FROM INCUBATED MBH F R A G M E N T S AND OVLT Values are r e p o rted as mean ± S.E.M. Numbers of rats are given in parentheses. LHRH release (pg/eq) MBH Control ACh 10-" M ACh 10 -s M
33.6 ± 2.6 (9) 29.6 ~ 3.5 (7) 32.6 ± 4.6 (8)
Control HA 1 0 - ' M HA 10-' M
19.1 ± 3.8 (7) 22.8 ± 6.5 (7) 28.3 ± 7.5 (V)
Rostral MBH
Caudal MBI!
O V LT 26.8 ± 3.9' (8) 23.4 ± 2.~. (8) 22.0 ± 3.4 (8)
Control GABA 10-" M GABA 1 0 - ' M
25.2 ± 2.2 (6) 23.2 ~ 5.0 (7) 26.0 ± 5.7 (5)
20.2 ± 3.2 (6) 28.0 ± 5.0 (7) 24.0 ± 1.7 (7)
Contro! GABA 10-' M GABA 5 . 1 0 s t~i
29.7 ± 3.0 (8) 36.7 ± 7.2 (8) 32.3 ± 5.0 (8)
25.5 ± 4.2 (8) 25.5 ± 4.0 (8) 28.2 ± 3.2 (8)
18.0 ~ 2.0 (8) 18.5 ± 3.7 (6) 15.3 ± 2.0 (8)
role for t h e ami~,e p r o b a b l y involving direct a c t i o n o n L H R H s e c r e t o r y nerveendings. This eff'~t s~ems to be hormone-sl~ecific, since 5-HT does not affect
the release of T R H under similar conditions [7]. Specific receptors of the amine also appear to mediate the response, since the transmitter does not TABLE II E F F E C T OF 5-HT A N D / O R ITS RECEPTORS ANTAGONIST METHIOTHEPIN ON IN VITRO L H R H R E L E A S E F R O M k ~ H AND OVLT Values are e x p r e ~ e d as mean ± S.E.M. Number of rats are given in parentheses. LHRH release (pgleq) MBH
OVLT
Control 5-HT 10 -~ M 5-HT 1 0 - ' M
28.0 ± 1.2 (16) 21.1 ± 1.0 (15)" 21.9 ± 1.1 (13)"
27.5 ~ 2.0 (7) 23.1 ± 1.7 (8) 23.2 ± 2.0 (8)
Control 5-HT 10 "~ M Me t h i o t h e p in 5.10" M 5-HT + M e t h i o t h e p i n
36.6 ± 5.5 (14) 19.2 ± 1.8 (16) a 27.9 ± 5.2 (13)
18.0 • 1.0 (6) 20 c, _.- 2.0 (6)
28.3 ± 6.1 (14)
!~.., ± 0.8 (6)
a p < 0.01 vs. control.
1~.. ~ : 2.o (5)
162
modify LHRH release in the presence of methiothepin. However, this conclusion must be considered with caution, since the interaction betwee.~ the drugs is not statistically significant. The concentzations of ~I~'I effectiv,~ in d~reasing LHRH l~lease under our experimental conditions however, are consistent with the affinity of the amine for its receptor detemdned through binding experimen'~ in other CNS structures [ 2]. GABA, HA and ACh do not seem to trigger directly the release of LHP,H from the median eminence nerve-endings containing ~ e neuropeptide. GABA was tested on the palisadic zone of the median em~.nence as well as on more posterior structures ~f the MBH, b e c a ~ t b ~ e structures have been shown to contain fairly high concentrations of both GABA [25] and LHRH [21]. The ineffectiveness of the transmitter under these conditions is in good agreement with physiological data on LH ~ecretion [19]. In contrast, both HA [14] mid ACh [13,15] have been shown to enhance LH secretion. Our results ~uggest that they act at a higher level in the CNS than the median eminence. In the case of ACh, this conclusion disagrees with that of Fiorindo and M~xtini [ 5]. The difference in experimental conditions may be responsible, in that the latter authors used much longer incubation periods and measured LHI~H release indirectly through its effect on LH release from pituitary tissue coincubated with hypothalamic fragments. It thus seems that, among neurotransmitters with gonadotropic control, only 5~HT and dopamine [21] exhibit a clear~ut effect on the basal release of LHRH from neurosecretory neurons. In both cases, the interaction is probably mediated by receptors to the amine located on ?.HRH pt'odueing cells in the median eminence. ACKNOWLEDGEMENT
We are indebted to Miss Marie-Christine Simon for her sldlled secrv~riai assistance. REFERENCES 1 Bennett, G.W., Edwardson, J.A., Holland, D., Jeffeoate, S.L. and White, N., Release of immunoreactive LHRH and TRH from hypothalamic synaptosomes, Nature (Lond.), 257 (1975) 323-325. 2 Bennett, J.P. Jr. and Snyder, S.H., Serotonin atld lysergic acid diethylamide binding in rat brain membranes. Relationship to post~ynaptie serotonin receptors, Mol. Pharmacol.,
12 (1976) 373--389. 3 Br.'~wnstein, M.J., Seavedra, J.M., Palkovits, M. and Axelrod, J., Histamine content of hypothaiamie nuclei of the rat, Brain Res., 77 (1974) 151--156. 4 Brownstein, M.J., Kobayashi, R., Palkovits, M. and Seavedrs, J.M., Choline acetyltrtmsferase levels in diencephalic nuclei of the rat, J. Neurochem., 24 (1975) 35--38. 5 Fiorindo, R.P. and Martini, L., Evidence for a cholinergie component in the neuroendocrine control of iuteinizing hormone (LH) secretion Neuroendocrlnology, 18 (1975) 322--332. 6 Harvey, W.R., I~ast~quare~ analysis of data with unequal subelus numbers, U.S.A. Agrie,~l~ural Research Service AR8 ?0-8 (1966).
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