Hypothalamic Nerve Terminals Research Articles

Age and Long-Term Hormone Treatment Effects on the Ultrastructural Morphology of the Median Eminence of Female Rhesus Macaques

The median eminence (ME) of the hypothalamus comprises the hypothalamic nerve terminals, glia (especially tanycytes) and the portal capillary vasculature that transports hypothalamic neurohormones to the anterior pituitary gland. The ultrastructure of the ME is dynamically regulated by hormones and undergoes organizational changes during development and reproductive cycles in adult females, but relatively little is known about the ME during aging, especially in nonhuman primates. Therefore, we used a novel transmission scanning electron microscopy technique to examine the cytoarchitecture of the ME of young and aged female rhesus macaques in a preclinical monkey model of menopausal hormone treatments. Rhesus macaques were ovariectomized and treated for 2 years with vehicle, estradiol (E₂), or estradiol + progesterone (E₂ + P₄). While the overall cytoarchitecture of the ME underwent relatively few changes with age and hormones, changes to some features of neural and glial components near the portal capillaries were observed. Specifically, large neuroterminal size was greater in aged compared to young adult animals, an effect that was mitigated or reversed by E₂ alone but not by E₂ + P₄ treatment. Overall glial size and the density and tissue fraction of the largest subset of glia were greater in aged monkeys, and in some cases reversed by E₂ treatment. Mitochondrial size was decreased by E₂, but not E₂ + P₄, only in aged macaques. These results contrast substantially with work in rodents, suggesting that the ME of aging macaques is less vulnerable to age-related disorganization, and that the effects of E₂ on monkeys' ME are age specific.

Type 1 ryanodine receptor knock-in mutation causing central core disease of skeletal muscle also displays a neuronal phenotype

The type 1 ryanodine receptor (RyR1) is expressed widely in the brain, with high levels in the cerebellum, hippocampus, and hypothalamus. We have shown that L-type Ca(2+) channels in terminals of hypothalamic magnocellular neurons are coupled to RyRs, as they are in skeletal muscle, allowing voltage-induced Ca(2+) release (VICaR) from internal Ca(2+) stores without Ca(2+) influx. Here we demonstrate that RyR1 plays a role in VICaR in nerve terminals. Furthermore, in heterozygotes from the Ryr1(I4895T/WT) (IT/+) mouse line, carrying a knock-in mutation corresponding to one that causes a severe form of human central core disease, VICaR is absent, demonstrating that type 1 RyR mediates VICaR and that these mice have a neuronal phenotype. The absence of VICaR was shown in two ways: first, depolarization in the absence of Ca(2+) influx elicited Ca(2+)syntillas (scintilla, spark, in a nerve terminal, a SYNaptic structure) in WT, but not in mutant terminals; second, in the presence of extracellular Ca(2+), IT/+ terminals showed a twofold decrease in global Ca(2+) transients, with no change in plasmalemmal Ca(2+) current. From these studies we draw two conclusions: (i) RyR1 plays a role in VICaR in hypothalamic nerve terminals; and (ii) a neuronal alteration accompanies the myopathy in IT/+ mice, and, possibly in humans carrying the corresponding RyR1 mutation.

Syntillas Release Ca 2+ at a Site Different from the Microdomain Where Exocytosis Occurs in Mouse Chromaffin Cells

Spontaneous, short-lived, focal cytosolic Ca 2+ transients were found for the first time and characterized in freshly dissociated chromaffin cells from mouse. Produced by release of Ca 2+ from intracellular stores and mediated by type 2 and perhaps type 3 ryanodine receptors (RyRs), these transients are quantitatively similar in magnitude and duration to Ca 2+ syntillas in terminals of hypothalamic neurons, suggesting that Ca 2+ syntillas are found in a variety of excitable, exocytotic cells. However, unlike hypothalamic nerve terminals, chromaffin cells do not display syntilla activation by depolarization of the plasma membrane, nor do they have type 1 RyRs. It is widely thought that focal Ca 2+ transients cause “spontaneous” exocytosis, although there is no direct evidence for this view. Hence, we monitored catecholamine release amperometrically while simultaneously imaging Ca 2+ syntillas, the first such simultaneous measurements. Syntillas failed to produce exocytotic events; and, conversely, spontaneous exocytotic events were not preceded by syntillas. Therefore, we suggest that a spontaneous syntilla, at least in chromaffin cells, releases Ca 2+ into a cytosolic microdomain distinct from the microdomains containing docked, primed vesicles. Ryanodine (100 μM) reduced the frequency of Ca 2+ syntillas by an order of magnitude but did not alter the frequency of spontaneous amperometric events, suggesting that syntillas are not involved in steps preparatory to spontaneous exocytosis. Surprisingly, ryanodine also increased the total charge of individual amperometric events by 27%, indicating that intracellular Ca 2+ stores can regulate quantal size.

Role of premature leptin surge in obesity resulting from intrauterine undernutrition

Intrauterine undernutrition is closely associated with obesity related to detrimental metabolic sequelae in adulthood. We report a mouse model in which offspring with fetal undernutrition (UN offspring), when fed a high-fat diet (HFD), develop pronounced weight gain and adiposity. In the neonatal period, UN offspring exhibited a premature onset of neonatal leptin surge compared to offspring with intrauterine normal nutrition (NN offspring). Unexpectedly, premature leptin surge generated in NN offspring by exogenous leptin administration led to accelerated weight gain with an HFD. Both UN offspring and neonatally leptin-treated NN offspring exhibited an impaired response to acute peripheral leptin administration on a regular chow diet (RCD) with impaired leptin transport to the brain as well as an increased density of hypothalamic nerve terminals. The present study suggests that the premature leptin surge alters energy regulation by the hypothalamus and contributes to "developmental origins of health and disease."

Oxytocin is a cardiovascular hormone.

Oxytocin (OT), a nonapeptide, was the first hormone to have its biological activities established and chemical structure determined. It was believed that OT is released from hypothalamic nerve terminals of the posterior hypophysis into the circulation where it stimulates uterine contractions during parturition, and milk ejection during lactation. However, equivalent concentrations of OT were found in the male hypophysis, and similar stimuli of OT release were determined for both sexes, suggesting other physiological functions. Indeed, recent studies indicate that OT is involved in cognition, tolerance, adaptation and complex sexual and maternal behaviour, as well as in the regulation of cardiovascular functions. It has long been known that OT induces natriuresis and causes a fall in mean arterial pressure, both after acute and chronic treatment, but the mechanism was not clear. The discovery of the natriuretic family shed new light on this matter. Atrial natriuretic peptide (ANP), a potent natriuretic and vasorelaxant hormone, originally isolated from rat atria, has been found at other sites, including the brain. Blood volume expansion causes ANP release that is believed to be important in the induction of natriuresis and diuresis, which in turn act to reduce the increase in blood volume. Neurohypophysectomy totally abolishes the ANP response to volume expansion. This indicates that one of the major hypophyseal peptides is responsible for ANP release. The role of ANP in OT-induced natriuresis was evaluated, and we hypothesized that the cardio-renal effects of OT are mediated by the release of ANP from the heart. To support this hypothesis, we have demonstrated the presence and synthesis of OT receptors in all heart compartments and the vasculature. The functionality of these receptors has been established by the ability of OT to induce ANP release from perfused heart or atrial slices. Furthermore, we have shown that the heart and large vessels like the aorta and vena cava are sites of OT synthesis. Therefore, locally produced OT may have important regulatory functions within the heart and vascular beds. Such functions may include slowing down of the heart or the regulation of local vascular tone.

Histamine regulates food intake through modulating noradrenaline release in the para-ventricular nucleus

Histamine is one of the neurotransmitters suppressing appetite, but the interactions of histaminergic neurons with other neurons in satiety centers have not been clarified. Noradrenaline in the para-ventricular nucleus (PVN) has been shown to stimulate feeding. This study was designed to determine whether histamine modulates noradrenaline release via histamine H1-receptors in the PVN. Freely-fed rats were i.c.v. injected with an histamine H1-receptor antagonist, triprolidine (82 μg), and/or an alpha 2-adrenoceptor antagonist, rauwolscine (0, 20 and 40 μg), and food intake ( n=8 each) over 2 h was measured. Food intake was significantly ( p<0.01) increased in rats injected with triprolidine alone. The triprolidine-elicited increase in food intake was suppressed by rauwolscine at a dose of 40 μg. The noradrenaline content in perfusates collected by a microdialysis probe aimed at the PVN was significantly ( p<0.05) increased by the presence of triprolidine in the perfusates. The noradrenaline concentrations in perfusates collected from the PVN were elevated after tyramine (a noradrenaline uptake inhibitor) administration, but not when both tyramine and histamine were given. In conclusion, these results suggest that histamine inhibits noradrenaline release from hypothalamic nerve terminals in the PVN, and thus suppresses feeding behavior.

Neurochemical activity of noradrenergic neurons and pituitary-adrenal response after naloxone-induced withdrawal: the role of calcium channels.

The influence of the L-type calcium channel antagonist nimodipine on both the activity of noradrenergic neurons projecting to the hypothalamus and the pituitary-adrenal response during morphine tolerance and withdrawal was analysed. Tissue concentration of hypothalamic noradrenaline (NA) and its metabolite 3-methoxy-4-hydroxy-phenylethylen-glycol (MHPG) were determined by high-pressure liquid chromatography. Plasma corticosterone concentration (a marker of pituitary-adrenal activity) was measured by radioimmunoassay. Rats rendered tolerant to morphine decreased hypothalamic MHPG concentration, and reduced hypothalamic NA turnover. Chronic infusion of nimodipine concurrently with morphine prevented the decrease in NA turnover during tolerance. After naloxone administration to tolerant rats we found a striking parallelism between an increased activity of the hypothalamic-pituitary-adrenal axis and an enhanced activity of noradrenergic neurons projecting to the hypothalamus. However, hypothalamic NA turnover and MHPG concentration, both elevated during withdrawal, returned to control levels in rats infused chronically with nimodipine, concomitantly with a reduction of the secretion of corticosterone. Taken together, these data indicate that increased noradrenergic neuronal activity in the hypothalamic nerve terminals is associated with the neuroendocrine morphine withdrawal syndrome and suggest that an up-regulated calcium system might contribute to the activation of these neurons.

Interaction between glutamate and GABA on 3H-noradrenaline release from rat hypothalamus

Glutamate has been shown to stimulate noradrenaline (NA) release from hypothalamic nerve terminals. In the present study, we evaluated the possible interaction between the excitatory amino acid glutamate and gamma-aminobutyric acid (GABA), an inhibitory transmitter, on noradrenaline (NA) release from mediobasal hypothalamus (MBH) of adult male rats. Hypothalamic slices loaded in vitro with 3H-NA were superfused and exposed to glutamate, N-methyl- d-aspartic acid (NMDA), or kainate (KA). We found that 3H-NA release evoked by the excitatory amino acids glutamate and NMDA was dramatically decreased by GABA. The facilitatory effects of NMDA and KA were prevented concentration-dependently by the GABA B receptor antagonist 2-hydroxy saclofen which restored the NMDA effect. In addition, beclofen blocked K +-induced 3H-NA release. Activation of GABA A receptors by muscimol and THIP was ineffective. In conclusion, glutamate and GABA, through GABA B receptors, may interact to modulate NA release from the rat mediobasal hypothalamus.

A second gene for gonadotropin-releasing hormone: cDNAand expression pattern in the brain.

In vertebrates, the gonadotropin-releasing hormone (GnRH) decapeptide is secreted from hypothalamic nerve terminals to regulate reproduction via control of synthesis and release of pituitary gonadotropins. Only one GnRH peptide has been found in mammals, with one exception, although numerous other vertebrate species express more than one of the eight known decapeptide forms as shown by immunocytochemical labeling of distinct cell groups in the brain. However, neither the functional nor the evolutionary relationships among these GnRH forms are clear, because only one preprohormone gene sequence from any species has been reported. The most ubiquitous alternative form of GnRH is [His5,Trp7,Tyr8]GnRH (also referred to as chicken-II), which differs from the mammalian sequence at amino acids 5, 7, and 8. This peptide has been shown to have the most potent releasing-hormone activity, although immunocytochemical staining has suggested it is synthesized only in the mesencephalon. Here we report the cloning and expression pattern of the gene for the precursor of this form from the teleost fish Haplochromis burtoni. This is the second GnRH-encoding gene to be characterized in this species. The newly discovered preprohormone gene differs from that previously reported in two ways. First, whereas the original gene predicts only a single associated peptide, this one predicts two associated peptides, both of which appear to be unique. Second, the gene for [His5,Trp7,Tyr8]GnRH is expressed in only one cell group in the mesencephalon. In contrast, the previously reported gene is expressed only in the terminal nerve. The striking differences between the preprohormone structure and localization suggest that the genes coding for the two known GnRH forms in H. burtoni did not arise from a recent duplication event. Interestingly, neither of the two genes found to date in this species is expressed in cells which project from the hypothalamus to the pituitary, suggesting that yet a third gene coding for GnRH may exist.

GABA-mediated synaptic transmission in neuroendocrine cells: a patch-clamp study in a pituitary slice preparation.

Patch-clamp recording techniques were applied to thin slices of the rat pituitary gland in order to study synaptic transmission between hypothalamic nerve terminals and neuroendocrine cells of the intermediate lobe. Inhibitory postsynaptic currents (IPSCs) could be evoked by electrical stimulation of afferent neuronal fibres in the surrounding tissue of the slice. The IPSCs could be evoked in an all-or-nothing mode depending on the stimulus intensity, suggesting that single afferent fibres were stimulated. They had a chloride-dependent reversal potential and were blocked by bicuculline (Kd = 0.1 microM), indicating that they were mediated by gamma-aminobutyric acid A (GABAA) receptors. In symmetrical chloride solutions the current/voltage relation of the IPSC peak amplitudes was linear. The IPSCs were characterized by a fast (1-2 ms) rise time and a biexponential decay, with time constants of 21 +/- 4 ms and 58 +/- 14 ms at a holding potential of -60 mV (n = 6 cells). Both decay time constants increased with depolarization in an exponential manner. Spontaneously occurring IPSCs had a time course that was similar to that of evoked IPSCs. These miniature IPSCs, recorded in 1 microM tetrodotoxin, displayed an amplitude distribution that was well fitted by single Gaussian functions, with a mean value of its maxima of 18.1 +/- 2.3 pA (n = 4 cells). Amplitude histograms of evoked IPSCs were characterized by multiple peaks with a modal amplitude of about 18 pA (n = 6 cells). These findings indicate the quantal nature of GABAergic synaptic transmission in this system, with a quantal conductance step of about 280 pS. Single-channel currents underlying the IPSCs were studied by bath application of GABA to outside-out patches excised from intermediate lobe cells. Such GABA-induced currents revealed two conductance levels of 14 pS and 26 pS. In conclusion, GABAergic synaptic transmission in neuroendocrine cells of the pituitary has properties that are quite similar to those observed in neurones of the central nervous system.

Epoxygenase Products of Arachidonic Acid Are Endogenous Constituents of the Hypothalamus Involved in D2Receptor-Mediated, Dopamine-Induced Release of Somatostatin

The epoxyeicosatrienoic acids (EETs) were discovered as products of a cyclooxygenase/lipoxygenase-independent, cytochrome P-450 catalyzed metabolism of arachidonic acid (AA) termed the "epoxygenase" pathway. The rat hypothalamus is able to synthesize EETs from exogenous AA, and 5,6-EET has been found to release the neuropeptide somatostatin (SRIF) from hypothalamic nerve terminals of the median eminence (ME). In the present study, hypothalami from male rats were examined for the presence of endogenous EETs, using chemical, chromatographic, and mass spectral analysis procedures. The samples were initially separated in a C18 Sepralyte column, fractionated on TLC plates, and purified by reverse phase HPLC. Thereafter, they were esterified (pentafluorobenzyl esters) and subjected to negative ion chemical ionization/gas chromatography (GC)/mass spectral (MS) analysis. The GC retention time and the MS fragmentation patterns revealed the presence of a mixture of 8,9-, 11,12- and 14,15-EETs; instability of 5,6-EET during the isolation protocol precluded its identification. Total hypothalamic EET concentration was estimated to be 120 ng/g wet tissue. The 8,9-regiosomer released SRIF from ME nerve terminals with an ED50 of 5 x 10(-12) M; Dopamine (DA) and the D2 receptor agonist PPHT, but not the D1 receptor agonist SKF-38393, induced SRIF release from the ME. This effect was blocked by clotrimazole and ketoconazole, two inhibitors of microsomal cytochrome P-450 function and AA epoxygenase in particular. In contrast, the inhibitors failed to affect the increase in SRIF release induced by 8,9-EET. These results indicate that: 1) in addition to cyclooxygenase and lipoxygenase products, epoxygenase metabolites of AA are endogenous compounds of the hypothalamus, and 2) EETs may mediate the increase in SRIF release from hypothalamic neurons induced by the interaction of DA with D2 receptors.

Tryptophan Availability Modulates Serotonin Release from Rat Hypothalamic Slices

Application of a novel in vitro experimental system has allowed us to describe the relationship between tryptophan availability and serotonin release from rat hypothalamic slices. Superfusing hypothalamic slices with a physiologic medium containing l-tryptophan (1, 2, 5, or 10 microM) caused dose-dependent elevations in tissue tryptophan levels; the magnitude of the elevations produced by supplementing the medium with less than 5 microM tryptophan was within the physiologic range for rat brain tryptophan levels. Slice serotonin levels rose biphasically as the tryptophan concentration in the medium was increased. Superfusing the slices with medium supplemented with a low tryptophan concentration (1 or 2 microM) caused proportionally greater incremental changes in serotonin levels than the increases caused by further elevating the tryptophan concentration (5 or 10 microM). The spontaneous release of serotonin from the slices exhibited a dose-dependent relationship with the tryptophan concentration of the superfusion medium. Electrically evoked serotonin release, which was calcium-dependent and tetrodotoxin-sensitive, also increased in proportion to the medium tryptophan concentration. These data suggest that the rate at which serotonin is released from hypothalamic nerve terminals is coupled to brain tryptophan levels. Accelerations in hypothalamic serotonin synthesis, caused by elevating brain tryptophan levels, result in proportionate increases in the rates of serotonin release during rest and with membrane depolarization.

The effects of simultaneous or separate infusions of some pro-opiomelanocortin-derived peptides (β-endorphin, melanocyte stimulating hormone, and corticotrophin-like intermediate polypeptide) and their acetylated derivatives upon sexual and ingestive behaviour of male rats

Intraneuronal post-translational cleavage of pro-opiomelanocortin yields a variety of peptides including β-endorphin, melanocyte stimulating hormone and corticotrophin-like intermediate polypeptide, some of which are subsequently N-acetylated. Such peptides may be co-released from neuronal terminals, and so these experiments explored the effects of co-administration of some of them on sexual behaviour in the male rat, which is known to be sensitive to hypothalamic infusions of β-endorphin. Peptides were infused into the pre-optic-anterior hypothalamic area bilaterally in doses up to 320 pmol, and males allowed access to a sexually receptive female and/or a sweet solution (0.1% Acesulfame-K) for 15min, so that both sexual and ingestive behaviour could be studied. β-Endorphin(1–31) by itself inhibited sexual interaction, confirming our previous data. Acesulfame-K ingestion was inhibited in control-infused rats in the presence of a female, but this inhibition was released when sexual behaviour was itself diminished by β-endorphin(1–31). Both the acetylated and nonacetylated forms of melanocyte stimulating hormone (α-melanocyte stimulating hormone and des-acetyl melanocyte stimulating hormone) stimulate sexual behaviour; latencies both to ejaculation and to resumption of copulatory behaviour after an ejaculation (post-ejaculatory interval) were reduced. However, infusions of either corticotrophin-like intermediate peptide or N-acetylated β-endorphin(1–31) had no effect on either sexual or ingestive behaviour. Infusion of either acetylated melanocyte stimulating hormone or des-acetyl melanocyte stimulating hormone mixed with β-endorphin(1–31) prevented the inhibitory effect of the latter on sexual behaviour. Dose-response studies showed that the behavioural effect of such mixtures depended upon the molar ratios of the two peptides, rather than their absolute concentrations. The higher the ratio in favour of α-melanocyte stimulating hormone or des-acetyl melanocyte stimulating hormone, the greater the display of sexual behaviour. Infusing either corticotrophin-like intermediate polypeptides or N-acetyl β-endorphin(1–31) with β-endorphin(1–31) did not prevent the inhibition of sexual activity expected with β-endorphin(1–31) alone. These results are discussed in terms of the functional consequences of co-release of proopiomelanocortin peptides from hypothalamic nerve terminals.

Oxytocin: Neurobiologic considerations and their implications for affective illness

Demitrack, Mark A. and Philip W. Gold: Oxytocin: Neurobiologie Considerations and Their Implications for Affective Illness. Prog. Neuro-Psychopharmacol. and Biol. Psychiat. 1988, 12 (Suppl): S23–S51. 1. 1. Oxytocin is a nonapeptide hormone first isolated in the central nervous system in the supraoptic and paraventricular nuclei of the hypothalamus. Recent studies show that oxytocin is also found in brain regions outside of the hypothalamus and sites of synthesis have been reported in peripheral steroidogenic organs as well. 2. 2. Oxytocin is released from hypothalamic nerve terminals in the posterior pituitary for distribution in the systemic circulation, where it acts to stimulate uterine contraction during parturition and milk let-down during lactation. 3. 3. Although a definitive role for oxytocin in CNS function has not been elucidated, studies in experimental animals suggest involvement in cognition, tolerance and adaptation, and in complex maternal behaviors. Similarly, although oxytocin's function in steroid-producing tissues is not fully established, recent in vitro data has shown that oxytocin can Inhibit androgen and estrogen production, and thus may act in a local regulatory fashion. 4. 4. This selective review will highlight some current ideas regarding the functional significance of oxytocin in the central nervous system and review available data concerning the regulation of oxytocin secretion in humans and its potential role in neuropsychiatrie illness.

Coexistence of carriers for dopamine and GABA uptake on a same nerve terminal in the rat brain.

The ability of gamma-aminobutyric acid (GABA) to affect the release of [3H]-dopamine in rat brain synaptosomes prepared from corpus striatum, frontal cortex and hypothalamus and prelabelled with the radioactive catecholamine in the presence of desipramine was examined. GABA (10-300 microM) increased in a concentration-dependent way the basal release of [3H]-dopamine from striatum and cortical synaptosomes; however, its effect was much less pronounced in hypothalamic nerve terminals. 2,4-Diaminobutyric acid (DABA) mimicked GABA although less potently. Neutral amino acids such as leucine, valine or alpha-aminoisobutyric acid (100-300 microM) did not affect or increased minimally the release of [3H]-dopamine. The GABA-induced [3H]-dopamine release was not prevented by the GABAA-receptor antagonists, bicuculline or picrotoxin. The GABAA-receptor agonist, muscimol (10-300 microM), displayed only a very weak, not significant, enhancing effect on [3H]-dopamine release. The GABAB-receptor agonist (-)-baclofen (100 or 300 microM) had no effect. Three novel and selective inhibitors of GABA uptake, N-(4,4-diphenyl-3-butenyl)-nipecotic acid (SK&F 89976A), N-(4,4-diphenyl-3-butenyl)-guvacine (SK&F 100330A) and N-(4,4-diphenyl-3-butenyl)-homo-beta-proline (SK&F 100561) potently counteracted the enhancing effect of GABA on [3H]-dopamine release. Nipecotic acid also reduced the effect of GABA. It is concluded that carriers for the uptake of dopamine and GABA may coexist on the same nerve terminal in the rat brain.

Localization of a peptide sequence contained in the precursor to gonadotropin releasing hormone (GnRH)

The precursor protein that contains the sequence for the neurohormone gonadotropin releasing hormone (GnRH) also contains an additional fragment (amino acids 14–26, designated pHGnRH 14–26) that can release luteinizing hormone (LH) and follicle stimulating hormone (FSH) in vitro. An immunocytochemical study was carried out to determine if this sequence could be found in its processed form in hypothalamic nerve terminals. In rats, ewes and rhesus monkeys pHGnRH 14–26 was demonstrated in both neuronal cell bodies and in axon terminals. In mice, immunoreactivity was present in terminals only. No reaction was found in the hamster hypothalamus. Double label immunocytochemical studies for GnRH and pHGnRH 14–26 showed that both sequences could be found in the rat septal-preoptic-hypothalamic continuum in some but not all neuronal cell bodies. Control experiments strongly suggest that the 14–26 immunoreactivity represents the fragment after it has been cleaved from the precursor protein and that this peptide could be available for release into the hypophysial portal system.

Gonadal steroids and neurosecretion: Facilitatory influence on LHRH and neuropeptide Y

Luteinizing hormone releasing hormone (LHRH) is regarded as the primary hypothalamic signal that controls reproduction in the rat. Neuropeptide Y, a recently isolated hypothalamic peptide, appears to regulate LHRH secretion. Our studies show that gonadal steroids act in multiple ways to enhance the neurosecretory functions of each of these neuronal systems and, in addition, they promote excitation by NPY of LHRH release from the hypothalamic nerve terminals.

Diacylglycerol and phorbol esters enhance LHRH and prostaglandin E 2 secretion from median eminence nerve terminals in vitro

The present experiments were designed to evaluate the role of protein kinase C activation on the secretion of the neural peptide, LHRH, from hypothalamic nerve terminals in vitro. Two specific protein kinase C activators, diacylglycerol (1,2-didecanoylglycerol, DiC 10) and a phorbol ester (12,13-dibutyrate, PDBu) were used as probes. In addition to LHRH, secretion of prostaglandin E 2 (PGE 2) was also measured, since previous studies from our laboratory indicate that this arachidonic acid metabolite is intimately involved in the LHRH secretory process. PDBu at a dose of 200 nM significantly enhanced LHRH secretion from median eminence nerve terminals; in addition, a more modest but significant stimulation of PGE 2 release was also observed. DiC 10 (100 μM), on the other hand, enhanced PGE 2 release but had no clear effect on LHRH secretion. Release of LHRH, however, was clearly stimulated when the lipoxygenase inhibitor nordihydroguaiaretic acid was added to the medium, suggesting that some arachidonic acid metabolites are inhibitory to LHRH secretion. The results indicate that protein kinase C activation leads to an enhanced secretion of LHRH. In addition, they suggest that 1,2-diacylglycerol may also activate the formation of arachidonoyl residues inhibitory to LHRH release.

Calmodulin involvement on the Ca++-dependent release of LHRH and SRIF in vitro.

Mediobasal hypothalamic (MBH) slices of male adult rats were superfused at 37 degrees C with oxygenated Hepes-buffer Locke medium. Bacitracin (2 X 10(-5) M) was added to prevent enzymatic degradation of LHRH and SRIF. 6 min pulse of K+ (56 mM), veratridine (15 microM) or the ionophore A 23187 (10(-5) M), markedly stimulated the release of both neuropeptides. Trifluoperazine, a calmodulin inhibitor, decreased the K+-evoked LHRH and SRIF release in a dose-dependent manner; it was also effective in inhibiting the veratridine-induced neuropeptides release. Phenytoin, a calmodulin-dependent kinase inhibitor, also decreased in a dose-dependent manner the K+-induced LHRH and SRIF release; the basal release of both neuropeptides remained unaffected by either treatment. The ionophore-stimulated release of both neuropeptides was significantly inhibited as well. These data demonstrate that a Ca++-calmodulin kinase system may be involved in the mechanism of depolarization-induced LHRH and SRIF release from hypothalamic nerve terminals.

Insulin binding sites localized to nerve terminals in rat median eminence and arcuate nucleus.

Specific binding sites for blood-borne insulin were determined to be selectively localized on axons and axon terminals in the external median eminence and the hypothalamic arcuate nucleus by means of quantitative fine structural radioautography. This localization suggests that discrete populations of hypothalamic nerve terminals are potential targets for the direct effects of insulin and that insulin may act through synaptic mechanisms to influence hypothalamic circuits regulating energy balance and hypophyseal function.