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Effects of neurotransmitters on in vitro release of LHRH from fragments of mediobasal hypothalamus were investigated. Incubations were carried out for 10 min in a Locke-HEPES medium in the presence of dithioerythritol (10−5M) and bacitracin (2 x 10−5M) to prevent catecholamine oxidation and peptide degradation, respectively. Addition of 3 x 10−9M to 10−7M concentrations of dopamine (DA) to the incubation medium released doserelated amounts of LHRH from hypothalamic fragments, including the medial and lateral palisadic zones of the median eminence, which contain most of the tubero-infundibular DA terminals. LHRH release did not occur when norepinephrine (10−7M) was added instead of DA or was it obtained when DA was incubated with the caudal portion of the mediobasal hypothalamus or the organum vasculosum of the lamina terminalis, which also contain LHRH but are poorly innervated by DA neurons. The effect of DA on palisadic structures was blocked by the addition of pimozide. Moreover, it seemed to be steroi...

Two estradiol antagonists, Parke-Davis CI 628 and CI 680, were studied for their inhibitory potency against labeled estradiol uptake within the hypothalamus, cerebral cortex, pituitary and uterus of the ovariectomized rat. Both drugs, tested from 15 min to 24 h prior to estradiol, induced a fast and long-lasting decrease of the hormone uptake. Three degrees of inhibition were observed depending on the tissue studied: (a) the most important inhibition concerned uterus and pituitary, where the estradiol uptake was prevented by up to 90% by the largest doses of antagonists used (6.3 and 6.1 mg, respectively, for CI 268 and CI 680); (b) in the hypothalamus, the estradiol uptake was counteracted to a lower degree; the inhibition being 39% for anterior hypothalamus and 22% for medial posterior hypothalamus; (c) cerebral cortex uptake was completely unaffected by pretreatments with the antagonists. After subcellular fractionation it was observed that the nuclear uptake of estradiol was completely abolished in both hypophyseal and hypothalamic tissues if the hormonal injection was preceded by the administration of 6.3 mg of CI 628.

A large number of articles have recently been published on the semantic aspects of the terms neurotransmitter, neurohormone and neuromodulator. It seems indeed that these terms have often been misused. Anatomical data and electrophysiological techniques, on the one hand, and the recent development of accurate in vitro methods for study of the hypothalamo-hypophysial axis, on the other hand, are making a major contribution to a better definition of these terms. These methods have facilitated the study of the various modulatory actions of monoamines, neuropeptides and also steroid hormones on neuroendocrine processes. Here William Rotsztejn describes more recent developments.

The effects of the anti-estrogen Ci-628 have been tested on both plasma LH and hypothalamic LH-RH content in ovariectomized or ovariectomized, estradiol (E2)-implanted rats, in order to correlate those parameters with the [3H]E2 retention in the hypothalamus and the pituitary. Incresing doses of CI-628 induced a dose-dependent inhibition of [3H]E2 retention in both cytosolic and nuclear fractions of the pituitary. In contrast, hypothalamic retention of [3H]E2 is only inhibited significantly with higher doses of CI-628 (2.4 and 24 mg/kg). In ovariectomized rats, only a high dose of CI-628 (24 mg/kg) is able to decrease elevated LH levels observed following castration. In the presence of E2, CI-628 has both estrogenic and anti-estrogenic properties on LH secretion. CI-628 acts at the pituitary level to decrease the tissue sensitivity to LH-RH, but has no effect on mediobasal hypothalamic (MBH) LH-RH content.

The luteinizing hormone-releasing hormone (LHRH) content of the two halves of the mediobasal hypothalamus (MBH) has been studied two weeks after unilateral or bilateral ovariectomy (OVX) in the rat. In control animals, the LHRH content of the right-side MBH was higher than that of the left side. Right-side OVX induced a significant increase in LHRH content on the right-side MBH while left-side OVX was followed by elevated LHRH content on the left-side MBH. In contrast, bilateral OVX induced a significant decrease on the right-side MBH. The observation that unilateral OVX induced increased LHRH content in the ipsilateral half of the MBH to OVX, supports the view that a neural pathway might connect the ovary and the MBH.

A suitable preparation to study the effect of dopamine (DA) on in. vitro LHRH secretion is presented. Enzymatic degradation of LHRH in the incubation medium is completely inhibited by bacitracin (2 × 10−5 M). A high potassium concentration (56mM) induces an increase in LHRH release from entire pieces of mediobasal hypothalamus (MBH), but not from a synaptosomal pellet; this release is inhibited when calcium is omitted. The depolarization-induced LHRH release in vitro is independent of the initial amount of the peptide present in the MBH. In contrast, DA (10−6M) is not effective in vitro on MBH from ovariectomized rats but induces a fast release of LHRH from MBH of normal male and estradiol-pretreated ovariectomized rats. The preparation presented here appears to be of great interest in investigating amine-steroid-LHRH interactions at the cellular level.

VIP stimulated prolactin secretion from incubated rat hemipituitaries. Under the same conditions, the secretion of GH, LH, FSH was not affected. The stimulation of prolactin was dose-dependent, with an apparent affinity of VIP of 10.9 ± 3.1 nM and a maximal stimulation of 57.7 ± 4.2%. Secretin, a structurally related peptide, was also active at higher concentrations whereas another partial analogue, glucagon, was ineffective. The effect of VIP was not blocked by α-flupentixol, a potent dopaminergic antagonist, at concentrations which antagonized the dopamine inhibition of prolactin secretion. Stimulation by VIP and TRH was additive. Neither Metenkephalin nor naloxone interfered with the response to VIP. It thus seems that specific VIP receptors are present on pituitary prolactin cells. VIP, present in the mediobasal hypothalamus and detected in the hypothalamo-hypophyseal portal blood therefore is a good candidate as a physiological PRF.

The effect of vasoactive intestinal peptide (VIP) was studied on the release of somatostatin (SRIF) from slices of several regions of the rat brain in vitro. VIP induced a dose-dependent inhibition of SRIF release from mediobasal hypothalamic slices but did not interfere with SRIF release from preoptic area, amygdala or cortex. VIP Inhibition had an apparent affinity: Kd = 6.8 ± 3.9 × 10−11 M. Secretin has a similar effect but at 600-fold higher concentrations (Kd secretin = 4.2 ± 0.6 × 10−8 M). Glucagon was ineffective in concentrations ranging from 10−10 M to 10−7 M. The data are consistent with a role of VIP in the hypothalamic control of growth hormone secretion.

Publisher Summary This chapter discusses brain amines and hypothalamic control of pituitary gonadotropic secretion. Brain neurotransmitters are mainly involved in gonadotropic modulation under conditions of hormone fluctuations. This is the case during circadian, cyclic, or pulsatile stimulation by the hypothalamus of hormone release. Pulsatile secretion is of particular importance whenever high levels of luteinizing hormone (LH) are secreted—for instance, after castration in rats during the mid-cycle LH peak observed in primates. Noradrenergic inputs to the preoptic area of the hypothalamus facilitate this mode of secretion, mainly via α-adrenergic receptors, whereas dopamine neurons antagonize it. This is an effect that likely accounts for the inhibitory effect of the amine on LH plasma levels observed after castration. Mesencephalic serotonin neurons, projecting to the suprachiasmatic nucleus of the hypothalamus, play a major role in controlling circadian hormone secretion; this account for the effect of drugs affecting 5-HT metabolism or receptors on the strictly timed ovulation processes of rodents. Dopamine and serotonin fibers, innervating the median eminence, can also affect directly LHRH release from neurosecretory nerve endings, respectively, by facilitating or inhibiting it.

The effect of castration and adrenalectomy was studied on vasoactive intestinal peptide (VIP) concentrations in various brain and peripheral structures of male rats. Castration has no effect on any of the structures studied, whereas 4 weeks adrenalectomy (ADX) decreases VIP concentrations in the hippocampus and increases them in the adenohypophysis, two structures known to contain large amounts of glucocorticoid receptors. Corticosterone administration restores VIP levels to control values only in the dorsal hippocampus. In contrast, dexamethasone counteracts both the decrease and the increase of VIP concentrations obtained after ADX in the hippocampus and in the adenohypophysis respectively. The present results suggest that corticosteroids can be involved in the regulation of VIP actions both in the brain and in the adenohypophysis.

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) at concentrations of 10(-7) and 10(-5) M, or GABA at 10(-7) to 5 x 10(-5) M affected LHRH release from mediobasal hypothalamic (MBH) fragments or the organum vasculosum of the laminae terminalis (OVLT). In contrast, serotonin (10(-7) and 10(-5) 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-endings.

We have studied the regional and subcellular distribution of vasoactive intestinal peptide (VIP) in the brain of adult male rat, using a specific radioimmunoassay. Selective deafferentation of the mediobasal hypothalamus (MBH) was also performed in order to investigate the origin of hypothalamic VIP. The highest concentrations of VIP were found in the neocortex, namely the occipital region. The brain stem, the posterior hypothalamus, and the pineal gland contained low amounts of the peptide. VIP was not detectable in the cerebellum and the neurohypophysis. After fractionation, most of the VIP was recovered from the crude mitochondrial fraction of the hypothalamus as well as the parietal cortex. However, a non-negligible portion of the activity was also found in the supernatant suggesting that the peptide is mainly located in nerve endings but also present in neuronal cell bodies and/or axons. Two weeks after complete deafferentation of the MBH, VIP concentrations of the caudal MBH (including the infundibular sulcus, the stalk, part of the ventromedial nucleus and premamillary structures) were decreased by 40%. In contrast, no change in VIP levels were observed in the rostral MBH, organum vasculosum of the lamina terminalis (OVLT), and cortex. This suggests that hypothalamic nerve endings containing the peptide derive from neuronal cell bodies located both outside and within the MBH.

Somatostatin, in concentrations ranging from 10−10 M to 10−7 M, induces a dose-dependent inhibition of LHRH release from mediobasal hypothalamic slices incubated in vitro. In contrast, VIP, secretin, glucagon, substance P, neurotensin and arginine-vasotocin do not affect spontaneous release of LHRH.

Incubated pituitary halves from ovariectomized, estrogen-implanted female rats were shown to be much more sensitive to LHRH than pituitaries from castrated, nontreated animals. LHRH in a concentration of 1,885 pg/ml increased the release of LH and FSH from 7.3 +/- 0.9 and 0.91 +/- 0.13 ng/h/hemipituitary respectively to 21.4 +/- 1.9 and 1.97 +/- 0.18 ng/h in animals implanted with the steroid. In contrast, 5,000 pg/ml of LHRH increased LH secretion from 3.4 +/- 0.3 to 8.4 +/- 0.4 ng/h in ovariectomized, nontreated animals. In pituitaries from both steroid and nontreated animals a highly significant dose response for LH and FSH secretion to the actual concentration of LHRH measured in each incubation tube by radioimmunoassay was observed. When expressed as percent of the corresponding control release, maximal stimulation of LH and FSH was comparable. Pituitaries from implanted animals provided a very sensitive bioassay for LHRH, in which amounts of the peptide lower than 100 pg/ml were detected. The apparent responsiveness to LHRH of pituitaries from estradiol-treated rats was found to be over 20 times greater than that of pituitaries from nontreated castrates.

Vasoactive intestinal peptide (VIP) stimulates both adenosine 3',5'-cyclic monophosphate (cAMP) accumulation and prolactin release in normal rat pituitary cells in culture. cAMP accumulation is significant (P less than 0.01) at VIP concentrations as low as 1 nM and reaches a maximum with 0.1 microM. Addition of dexamethasone as early as 15 min before VIP inhibits VIP stimulation of both cAMP production and PRL secretion. The rapid inhibition is dose-dependent: it appears at doses as low as 0.01 pM and is complete at 1 pM dexamethasone. Increasing concentrations of dexamethasone induce a noncompetitive type of inhibition, as shown by the decrease in Vmax with no change in the apparent Km for VIP. Cycloheximide (1 mM) counteracts the inhibitory effect of dexamethasone on VIP-induced cAMP production, which suggests the involvement of a rapid protein synthesis mechanism. Ru-26988, a specific glucocorticoid devoid of any mineralocorticoid activity and which does not bind to intracellular transcortin-like component, also produces an inhibition of VIP-induced cAMP accumulation. Corticosterone also inhibits VIP-induced cAMP production but at concentrations higher than those of dexamethasone. In contrast, aldosterone, progesterone, estradiol, and testosterone have no effect. These results demonstrate that, in normal rat pituitary cells in culture, glucocorticoids at physiological concentrations rapidly inhibit the cAMP production and prolactin release induced by VIP by acting through specific glucocorticoid receptors.

In order to investigate whether the capacity of hypothalamic and anterior pituitary tissue to concentrate and retain estradiol is affected by serotonin (5-HT), 3H-estradiol (3HE2) retention in these structures was measured after 5-HT synthesis inhibition by either parachlorophenylalanine (PCPA) or 6-fluoro-tryptophane (6 FTrp), or after destruction of midbrain raphe nuclei containing 5-HT cell bodies, as well as after administration of the 5-HT precursor 5-hydroxytryptophane (5-HTP). No modification in 3HE2 retention was observed after tryptophane hydroxylase inhibitors of raphe lesions; administration of the precursor only increased the steroid retention at very high, nonphysiological dose levels. It is concluded that the interaction of 5-HT with gonadotropic release cannot be accounted for by a direct effect on specific estrogenic receptors, but occurs at a different level of gonadotropic release regulating structures or directly on LH-RH neurons.

Pharmacological and neurosurgical approaches were used to determine whether central serotoninergic systems are essential to the diurnal fluctuation of plasma corticosterone (B) and the enhanced secret

THE morphinomimetic pentapeptides enkephalins1 have been identified in the central nervous system (CNS)2–5. In the rat, Met-enkephalin seems to be the predominant opiate-like pentapeptide6; it has been found in different structures of the brain, including the anterior and the mediobasal hypothalamus (MBH)2–4. Enkephalins, like morphine, have been shown to enhance the secretion of prolactin7–9 and growth hormone9,10, and to inhibit the release of thyroid stimulating hormone11, and luteinising hormone (LH) and ovulation12,13. These effects can be inhibited by naloxone, an opiate antagonist; most authors have postulated that they involve a direct action at the hypothalamic level8–14. In this report, we rule out a direct action of Met-enkephalin on LH releasing hormone (LHRH) release and postulate instead that Met-enkephalin modulates a dopamine-stimulated secretion.