Ovarian regulation of kisspeptin neurones in the arcuate nucleus of the rhesus monkey (macaca mulatta).

Tonic gonadotrophin secretion throughout the menstrual cycle is regulated by the negative-feedback actions of ovarian oestradiol (E2) and progesterone. Although kisspeptin neurones in the arcuate nucleus (ARC) of the hypothalamus appear to play a major role in mediating these feedback actions of the steroids in nonprimate species, this issue has been less well studied in the monkey. In the present study, we used immunohistochemistry and in situ hybridisation to examine kisspeptin and KISS1 expression, respectively, in the mediobasal hypothalamus (MBH) of adult ovariectomised (OVX) rhesus monkeys. We also examined kisspeptin expression in the MBH of ovarian intact females, and the effect of E2, progesterone and E2 + progesterone replacement on KISS1 expression in OVX animals. Kisspeptin or KISS1 expressing neurones and pronounced kisspeptin fibres were readily identified throughout the ARC of ovariectomised monkeys but, on the other hand, in intact animals, kisspeptin cell bodies were small in size and number and only fine fibres were observed. Replacement of OVX monkeys with physiological levels of E2, either alone or with luteal phase levels of progesterone, abolished KISS1 expression in the ARC. Interestingly, progesterone replacement alone for 14 days also resulted in a significant down-regulation of KISS1 expression. These findings support the view that, in primates, as in rodents and sheep, kisspeptin signalling in ARC neurones appears to play an important role in mediating the negative-feedback action of E2 on gonadotrophin secretion, and also indicate the need to study further their regulation by progesterone.

STX, a novel nonsteroidal estrogenic compound, induces rapid action in primate GnRH neuronal calcium dynamics and peptide release.

Previously, we reported that 1 nM 17ß-estradiol (E(2)) induces a rapid action, which is, in part, mediated through the G protein-coupled receptor GPR30 in primate GnRH neurons. Because it has been reported that the diphenylacrylamide compound, STX, causes estrogenic action in the mouse and guinea pig hypothalamus, the present study examined effects of STX in primate GnRH neurons and whether there is an action independent of GPR30. Results are summarized as follows. STX (10 nM) exposure increased 1) the oscillation frequency of intracellular calcium concentration ([Ca(2+)](i)), 2) the percentage of cells stimulated, and 3) the synchronization frequency of [Ca(2+)](i) oscillations. STX (10-100 nM) also stimulated GnRH release. The effects of STX on both [Ca(2+)](i) oscillations and GnRH release were similar to those caused by E(2) (1 nM), although with less magnitude. STX (10 nM)-induced changes in [Ca(2+)](i) oscillations were not altered by GPR30 small interfering RNA transfection, indicating that STX-sensitive receptors differ from GPR30. Finally, a higher dose of E(2) (10 nM) induced a larger change in [Ca(2+)](i) oscillations than that with a smaller dose of E(2) (1 nM), and the effects of 10 nM E(2) were reduced but not completely blocked by GPR30 small interfering RNA transfection, indicating that the effects of 10 nM E(2) in primate GnRH neurons are mediated by multiple membrane receptors, including GPR30 and STX-sensitive receptors. Collectively, the rapid action of E(2) mediated through GPR30 differs from that mediated through STX-sensitive receptors. The molecular structure of the STX-sensitive receptor remains to be identified.

Rapid action of estradiol in primate GnRH neurons: the role of estrogen receptor alpha and estrogen receptor beta.

Estrogens play a pivotal role in the control of female reproductive function. Recent studies using primate GnRH neurons derived from embryonic nasal placode indicate that 17ß-estradiol (E(2)) causes a rapid stimulatory action. E(2) (1nM) stimulates firing activity and intracellular calcium ([Ca(2+)](i)) oscillations of primate GnRH neurons within a few min. E(2) also stimulates GnRH release within 10min. However, the classical estrogen receptors, ERα and ERß, do not appear to play a role in E(2)-induced [Ca(2+)](i) oscillations or GnRH release, as the estrogen receptor antagonist, ICI 182,780, failed to block these responses. Rather, this rapid E(2) action is, at least in part, mediated by a G-protein coupled receptor GPR30. In the present study we further investigate the role of ERα and ERß in the rapid action of E(2) by knocking down cellular ERα and ERß by transfection of GnRH neurons with specific siRNA for rhesus monkey ERα and ERß. Results indicate that cellular knockdown of ERα and ERß failed to block the E(2)-induced changes in [Ca(2+)](i) oscillations. It is concluded that neither ERα nor ERß is required for the rapid action of E(2) in primate GnRH neurons.

Rapid direct action of estradiol in GnRH neurons: findings and implications.

Estradiol plays a pivotal role in the control of gonadotropin-releasing hormone (GnRH) neuronal function and female reproduction. While positive and negative feedback actions of estradiol that enhance and suppress release of GnRH and LH are primarily mediated through estrogen receptor alpha located in interneurons, a series of recent studies in our laboratory indicate that rapid excitatory actions of estradiol also directly modify GnRH neuronal activity. We observed this phenomenon in cultured primate GnRH neurons, but similar rapid direct actions of estradiol are also described in cultured GnRH neurons and green fluorescent protein-labeled GnRH neurons of mice. Importantly, rapid direct action of estradiol in GnRH neurons is mediated through membrane or membrane associated receptors, such as GPR30, STX-sensitive receptors, and ERß. In this review, possible implications of this rapid estradiol action in GnRH neurons are discussed.

Recent discoveries on the control of gonadotrophin-releasing hormone neurones in nonhuman primates.

Since Ernst Knobil proposed the concept of the gonadotrophin-releasing hormone (GnRH) pulse-generator in the monkey hypothalamus three decades ago, we have made significant progress in this research area with cellular and molecular approaches. First, an increase in pulsatile GnRH release triggers the onset of puberty. However, the question of what triggers the pubertal increase in GnRH is still unclear. GnRH neurones are already mature before puberty but GnRH release is suppressed by a tonic GABA inhibition. Our recent work indicates that blocking endogenous GABA inhibition with the GABA(A) receptor blocker, bicuculline, dramatically increases kisspeptin release, which plays an important role in the pubertal increase in GnRH release. Thus, an interplay between the GABA, kisspeptin, and GnRH neuronal systems appears to trigger puberty. Second, cultured GnRH neurones derived from the olfactory placode of monkey embryos exhibit synchronised intracellular calcium, [Ca(2+)](i), oscillations and release GnRH in pulses at approximately 60-min intervals after 14 days in vitro (div). During the first 14 div, GnRH neurones undergo maturational changes from no [Ca(2+)](i) oscillations and little GnRH release to the fully functional state. Recent work also shows GnRH mRNA expression increases during in vitro maturation. This mRNA increase coincides with significant demethylation of a CpG island in the GnRH 5'-promoter region. This suggests that epigenetic differentiation occurs during GnRH neuronal maturation. Third, oestradiol causes rapid, direct, excitatory action in GnRH neurones and this action of oestradiol appears to be mediated through a membrane receptor, such as G-protein coupled receptor 30.

Rapid action of oestrogen in luteinising hormone-releasing hormone neurones: the role of GPR30.

Previously, we have shown that 17beta-oestradiol (E(2)) induces an increase in firing activity and modifies the pattern of intracellular calcium ([Ca(2+)](i)) oscillations with a latency < 1 min in primate luteinising hormone-releasing hormone (LHRH) neurones. A recent study also indicates that E(2), the nuclear membrane impermeable oestrogen, oestrogen-dendrimer conjugate, and the plasma membrane impermeable oestrogen, E(2)-BSA conjugate, all similarly stimulated LHRH release within 10 min of exposure in primate LHRH neurones, indicating that the rapid action of E(2) is caused by membrane signalling. The results from a series of studies further suggest that the rapid action of E(2) in primate LHRH neurones appears to be mediated by GPR30. Although the oestrogen receptor antagonist, ICI 182, 780, neither blocked the E(2)-induced LHRH release nor the E(2)-induced changes in [Ca(2+)](i) oscillations, E(2) application to cells treated with pertussis toxin failed to result in these changes in primate LHRH neurones. Moreover, knockdown of GPR30 in primate LHRH neurones by transfection with human small interference RNA for GPR30 completely abrogated the E(2)-induced changes in [Ca(2+)](i) oscillations, whereas transfection with control siRNA did not. Finally, the GPR30 agonist, G1, resulted in changes in [Ca(2+)](i) oscillations similar to those observed with E(2). In this review, we discuss the possible role of G-protein coupled receptors in the rapid action of oestrogen in neuronal cells.

Estrogen receptor-alpha immunoreactive neurons in the brainstem and spinal cord of the female rhesus monkey: species-specific characteristics.

The distribution pattern of estrogen receptors in the rodent CNS has been reported extensively, but mapping of estrogen receptors in primates is incomplete. In this study we describe the distribution of estrogen receptor alpha immunoreactive (ER-alpha IR) neurons in the brainstem and spinal cord of the rhesus monkey. In the midbrain, ER-alpha IR neurons were located in the periaqueductal gray, especially the caudal ventrolateral part, the adjacent tegmentum, peripeduncular nucleus, and pretectal nucleus. A few ER-alpha IR neurons were found in the lateral parabrachial nucleus, lateral pontine tegmentum, and pontine gray medial to the locus coeruleus. At caudal medullary levels, ER-alpha IR neurons were present in the commissural nucleus of the solitary complex and the caudal spinal trigeminal nucleus. The remaining regions of the brainstem were devoid of ER-alpha IR neurons. Spinal ER-alpha IR neurons were found in laminae I-V, and area X, and were most numerous in lower lumbar and sacral segments. The lateral collateral pathway and dorsal commissural nuclei of the sacral cord and the thoracic intermediolateral cell column also contained ER-alpha IR neurons. Estrogen treatment did not result in any differences in the distribution pattern of ER-alpha IR neurons. The results indicate that ER-alpha IR neurons in the primate brainstem and spinal cord are concentrated mainly in regions involved in sensory and autonomic processing. Compared with rodent species, the regional distribution of ER-alpha IR neurons is less widespread, and ER-alpha IR neurons in regions such as the spinal dorsal horn and caudal spinal trigeminal nucleus appear to be less abundant. These distinctions suggest a modest role of ER-alpha in estrogen-mediated actions on primate brainstem and spinal systems. These differences may contribute to variations in behavioral effects of estrogen between primate and rodent species.

Gonadotrophin-releasing hormone (GnRH) release in marmosets I: in vivo measurement in ovary-intact and ovariectomised females.

In vivo hypothalamic gonadotrophin-releasing hormone (GnRH) release was characterised for the first time in a New World primate. A nonterminal and repeatable push-pull perfusion (PPP) technique reliably measured GnRH in conscious common marmoset monkeys. Nineteen adult females (n = 8 ovary-intact in the mid-follicular phase; n = 11 ovariectomised) were fitted with long-term cranial pedestals, and a push-pull cannula was temporarily placed in unique locations within the pituitary stalk-median eminence (S-ME) 2 days prior to each PPP session. Marmosets underwent 1-3 PPPs (32 PPPs in total) lasting up to 12 h. Plasma cortisol levels were not elevated in these habituated marmosets during PPP, and PPP did not disrupt ovulatory cyclicity or subsequent fertility in ovary-intact females. GnRH displayed an organised pattern of release, with pulses occurring every 50.0 +/- 2.6 min and lasting 25.4 +/- 1.3 min. GnRH pulse frequency was consistent within individual marmosets across multiple PPPs. GnRH mean concentration, baseline concentration and pulse amplitude varied predictably with anatomical location of the cannula tip within the S-ME. GnRH release increased characteristically in response to a norepinephrine infusion and decreased abruptly during the evening transition to lights off. Ovary-intact (mid-follicular phase) and ovariectomised marmosets did not differ significantly on any parameter of GnRH release. Overall, these results indicate that PPP can be used to reliably assess in vivo GnRH release in marmosets and will be a useful tool for future studies of reproductive neuroendocrinology in this small primate.

Search for neural substrates mediating inhibitory effects of oestrogen on pulsatile luteinising hormone-releasing hormone release in vivo in ovariectomized female rhesus monkeys (Macaca mulatta).

Neural substrates mediating the negative feedback effects of oestrogen on luteinising hormone-releasing hormone (LHRH) release were studied using the in vivo push-pull perfusion method in female rhesus monkeys. Twelve long-term ovariectomized female monkeys were implanted with Silastic capsules containing 17beta-oestradiol 2 weeks before the experiments and, on the day of the experiment, oestradiol benzoate (EB, 50 microg/kg) or oil was subcutaneously injected. Push-pull perfusate samples from the stalk-median eminence were collected in 10-min fractions from 4 h before to 18-20 h after EB or oil injection. LHRH and neuropeptide Y (NPY) levels in the same perfusates were measured by radioimmunoassay, and glutamate and GABA in the same perfusates were assessed by high-performance liquid chromatography (HPLC). The results indicate that EB significantly suppressed LHRH release (P < 0.005) starting within 2 h after EB, and continued for 18 h or until the experiment was terminated. Pulse analysis suggested that oestrogen suppressed the pulse amplitude, but not pulse frequency, of LHRH release. By contrast, EB did not alter any parameters (mean release, pulse amplitude or frequency) of pulsatile NPY release throughout the experiment. HPLC analysis further suggested that neither glutamate nor GABA levels in the stalk-median eminence were changed with oestrogen-induced LHRH suppression. Oil treatment did not alter LHRH, NPY, GABA and glutamate levels. It is concluded that oestrogen induces suppression of pulsatile LHRH release within 2 h, but the inhibitory effect of oestrogen on LHRH release does not appear to be mediated by NPY, GABAergic, or glutamatergic neurones.

Projections from estrogen receptor-alpha immunoreactive neurons in the periaqueductal gray to the lateral medulla oblongata in the rhesus monkey.

The periaqueductal gray (PAG) contains numerous estrogen receptor-alpha immunoreactive (ER-alpha IR) neurons that are distributed in a species-specific way. These neurons might modulate different types of behavior that are mediated by the PAG such as active and passive coping responses, analgesia, and reproductive behavior. In primates, it is not known whether ER-alpha IR PAG neurons represent local interneurons and/or neurons that project to brainstem areas that control these behaviors. In this double labeling study, we asked whether ER-alpha IR neurons in the PAG of the rhesus monkey project to the nucleus retroambiguus (NRA), an area in the ventrolateral caudal medulla oblongata that is involved in expiration, vocalization, and reproductive behavior. Tracer was injected into the caudal lateral medulla oblongata to retrogradely label PAG neurons, and ER-alpha was visualized immunohistochemically. Although ER-alpha IR neurons and NRA-projection neurons were present at similar levels of the PAG, their distributions hardly overlapped. ER-alpha IR PAG neurons that project to the lateral caudal medulla represented less than 2% of ER-alpha IR PAG neurons. These double-labeled neurons were mainly located in the ipsilateral caudal PAG. The cluster of neurons in the medial part of the lateral PAG that projects specifically to the NRA-region did not contain double-labeled cells. The results indicate that only a few ER-alpha IR PAG neurons project to the NRA-region. This might be related to the modest effects of estrogen on mating-related behavior in primates compared most other mammalian species. Remaining ER-alpha IR PAG neurons might act locally on other PAG neurons, or they might represent neurons that project to other areas. Furthermore, the finding that the distributions of ER-alpha IR neurons and neurons that project to premotor neurons in the NRA-region scarcely overlap illustrates that the PAG in primates is very highly organized into anatomically distinct regions compared with other species.

Effect of dietary sulfur amino acids on the taurine content of rat tissues.

The effect of dietary sulfur amino acids on the taurine content of rat blood and tissues was investigated. Three types of diet were prepared for this study: a low-taurine diet (LTD), normal taurine diet (NTD; LTD + 0.5% Met), and high-taurine diet (HTD; LTD + 0.5% Met + 3% taurine). These diets had no differing effect on the growth of the rats. The concentration of taurine in the blood from the HTD- and NTD-fed rats was respectively 1200% and 200% more than that from LTD-. In such rat tissues as the liver, the taurine content was significantly affected by dietary sulfur amino acids, resulting in a higher content with HTD and lower content with LTD. However, little or no effect on taurine content was apparent in the heart or eye. The activity for taurine uptake by the small intestine was not affected by dietary sulfur amino acids. The expression level of taurine transporter mRNA was altered only in the kidney under these dietary conditions: a higher expression level with LTD and lower expression level with HTD.

Synchronization of Ca(2+) oscillations among primate LHRH neurons and nonneuronal cells in vitro.

Periodic release of luteinizing hormone-releasing hormone (LHRH) from the hypothalamus is essential for normal reproductive function. Pulsatile LHRH release appears to result from the synchronous activity of LHRH neurons. However, how the activity of these neurons is synchronized to release LHRH peptide in a pulsatile manner is unclear. Because there is little evidence of physical coupling among LHRH neurons in the hypothalamus, we hypothesized that the activity of LHRH neurons might be coordinated by indirect intercellular communication via intermediary (nonneural) cells rather than direct interneural coupling. In this study, we used an in vitro preparation of LHRH neurons derived from the olfactory placode of monkey embryos to assess whether nonneuronal cells, play a role in coordinating LHRH neuronal activity. We found that cultured LHRH neurons and nonneuronal cells both exhibit spontaneous oscillations in the concentration of intracellular Ca(2+) ([Ca(2+)](i)) at similar frequencies. Moreover, [Ca(2+)](i) oscillations in both types of cell were periodically synchronized. Synchronized [Ca(2+)](i) oscillations spread as intercellular Ca(2+) waves across fields of cells that included LHRH neurons and nonneuronal cells, although waves spread at a higher velocity among LHRH neurons. These results suggest that LHRH neurons and nonneuronal cells are functionally integrated and that nonneuronal cells could be involved in synchronizing the activity of the LHRH neurosecretory network.

Monosynaptic projections from the nucleus retroambiguus region to laryngeal motoneurons in the rhesus monkey.

Vocalization and straining-related activities require the activation of laryngeal muscles. The control of laryngeal muscles during these activities is thought to be mediated by a pathway from the periaqueductal gray via premotor neurons in the nucleus retroambiguus to laryngeal motoneurons in the nucleus ambiguus. However, direct contacts between the nucleus retroambiguus and laryngeal motoneurons have never been demonstrated anatomically. Moreover, data in primates about the nucleus retroambiguus-nucleus ambiguus pathway are lacking. Therefore, the present study examines the projection from the nucleus retroambiguus region to laryngeal motoneurons in the rhesus monkey at the light and electron microscopic levels. Injections with wheat germ agglutinin-horseradish peroxidase were made into the nucleus retroambiguus in five rhesus monkeys to anterogradely label fibers in the nucleus ambiguus. In two of these animals, the cricothyroid muscle was injected with cholera toxin subunit b to identify the motoneurons that supply it. The results show that the nucleus retroambiguus region most densely projects to the compact formation of the nucleus ambiguus, whereas cricothyroid motoneurons, which surround the compact formation, receive a moderate projection. The projections are bilateral, with a contralateral predominance. Ultrastructurally, anterogradely labeled terminal profiles from the nucleus retroambiguus contact cholera toxin subunit b-labeled dendrites of cricothyroid motoneurons. The terminal profiles contain primarily spherical vesicles and form asymmetrical contacts with cricothyroid motoneurons. This study demonstrates that the nucleus retroambiguus region projects to the nucleus ambiguus in the primate. Some of these projections include monosynaptic connections to laryngeal motoneurons. This pathway is important for the control of the vocal folds during vocalization and straining-related activities.

Presence of luteinizing hormone-releasing hormone fragments in the rhesus monkey forebrain.

Previously, we have shown that two types of luteinizing hormone-releasing hormone (LHRH) -like neurons, "early" and "late" cells, were discernible in the forebrain of rhesus monkey fetuses by using antiserum GF-6, which cross-reacts with several forms of LHRH. The "late" cells that arose from the olfactory placode of monkey fetuses at embryonic days (E) 32-E36, are bona fide LHRH neurons. The "early" cells were found in the forebrain at E32-E34 and settled in the extrahypothalamic area. The molecular form of LHRH in "early" cells differs from "late" cells, because "early" cells were not immunopositive with any specific antisera against known forms of LHRH. In this study, we investigated the molecular form of LHRH in the "early" cells in the nasal regions and brains of 13 monkey fetuses at E35 to E78. In situ hybridization studies suggested that both "early" and "late" LHRH cells expressed mammalian LHRH mRNA. Furthermore, "early" cells predominantly contain LHRH1-5-like peptide and its cleavage enzyme, metalloendopeptidase E.C.3.4.24.15 (EP24.15), which cleaves LHRH at the Tyr5-Gly6 position. This conclusion was based on immunocytochemical labeling with various antisera, including those against LHRH1-5, LHRH4-10, or EP24.15, and on preabsorption tests. Therefore, in primates, a group of neurons containing mammalian LHRH mRNA arises at an early embryonic stage before the migration of bona fide LHRH neurons, and is ultimately distributed in the extrahypothalamic region. These extrahypothalamic neurons contain LHRH fragments, rather than fully mature mammalian LHRH. The origin and function of these neurons remain to be determined.

Neural mechanisms underlying the pubertal increase in LHRH release in the rhesus monkey.

Puberty is triggered by an increase in pulsatile release of luteinizing hormone-releasing hormone (LHRH) from the hypothalamus. Although the LHRH neurosecretory system is mature well before the onset of puberty, a central inhibitory mechanism restrains LHRH release in juvenile primates. Recent studies suggest that this central inhibition is primarily because of GABAergic neurotransmission. A reduction of GABAergic restraint appears to be essential for the initiation of puberty, but the mechanism that underlies the disinhibition process remains to be elucidated. Future research into the regulation of central inhibition should provide more effective treatments for the prevention of disease associated with abnormal pubertal development.

A novel mammalian receptor for the evolutionarily conserved type II GnRH.

Mammalian gonadotropin-releasing hormone (GnRH I: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) stimulates pituitary gonadotropin secretion, which in turn stimulates the gonads. Whereas a hypothalamic form of GnRH of variable structure (designated type I) had been shown to regulate reproduction through a cognate type I receptor, it has recently become evident that most vertebrates have one or two other forms of GnRH. One of these, designated type II GnRH (GnRH II: pGlu-His-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2), is conserved from fish to man and is widely distributed in the brain, suggesting important neuromodulatory functions such as regulating K+ channels and stimulating sexual arousal. We now report the cloning of a type II GnRH receptor from marmoset cDNA. The receptor has only 41% identity with the type I receptor and, unlike the type I receptor, has a carboxyl-terminal tail. The receptor is highly selective for GnRH II. As with the type I receptor, it couples to G(alpha)q/11 and also activates extracellular signal-regulated kinase (ERK1/2) but differs in activating p38 mitogen activated protein (MAP) kinase. The type II receptor is more widely distributed than the type I receptor and is expressed throughout the brain, including areas associated with sexual arousal, and in diverse non-neural and reproductive tissues, suggesting a variety of functions. Surprisingly, the type II receptor is expressed in the majority of gonadotropes. The presence of two GnRH receptors in gonadotropes, together with the differences in their signaling, suggests different roles in gonadotrope functioning.

Luteinizing hormone-releasing hormone (LHRH) neurons: mechanism of pulsatile LHRH release.

Many types of neurons and glia exhibit oscillatory changes in membrane potentials and cytoplasmic Ca2+ concentrations. In neurons and neuroendocrine cells an elevation of intracellular Ca2+ concentration is associated with neurosecretion. Since both oscillatory membrane potentials and intracellular Ca2+ oscillations have been described in primary LHRH neurons and in GT1 cells, it is evident that an endogenous pulse-generator/oscillator is present in the LHRH neuron in vitro. The hourly rhythms of LHRH neurosecretion appear to be the synchronization of a population of LHRH neurons. How a network of LHRH neurons synchronizes their activity, i.e., whether by the result of synaptic mechanisms or electrical coupling through gap junctions or through a diffusible substance(s), remains to be clarified. Even though LHRH neurons themselves possess an endogenous pulse-generating mechanism, they may be controlled by other neuronal and nonneuronal elements in vivo. NE, NPY, glutamate, and GABA are neurotransmitters possibly controlling pulsatile LHRH release, and NO, cAMP, and ATP may be diffusible substances involved in pulsatile LHRH release without synaptic input. Although synaptic inputs to the perikarya of LHRH neurons could control the activity of LHRH neurons, a line of evidence suggests that direct neuronal and nonneuronal inputs, especially those from astrocytes to LHRH neuroterminals, appear to be more important for pusatile LHRH release.

Neural circuits regulating pulsatile luteinizing hormone release in the female guinea-pig: opioid, adrenergic and serotonergic interactions.

We studied three neurotransmitters involved in the regulation of pulsatile luteinizing hormone (LH) release: opioid peptides, serotonin and norepinephrine, using the ovariectomized guinea-pig. This is an attractive animal model due to the regularity of its LH pulses, enabling any disruptions to be clearly ascertained. In all experiments, a specific agonist or antagonist was administered, either alone or serially to enable detection of interactions, and effects on mean LH concentrations, pulse amplitude and interpulse interval were determined by PULSAR analysis. In the ovariectomized guinea-pig, catecholamines are stimulatory (acting through the alpha1 and alpha2 but not beta receptors, unlike other species), opioids inhibitory and serotonin permissively stimulatory to pulsatile LH release. Stimulatory effects of the opiate antagonist were not blocked by pretreatment with an alpha1- or alpha2-adrenergic antagonist. Similarly, pretreatment with the opiate antagonist did not prevent the suppression of LH release by alpha1 and alpha2 antagonists. This suggests that, in the guinea-pig, effects of opiates and catecholamines on LH release are exerted by independent pathways to luteinizing hormone releasing hormone (LHRH) neurones. For the opiate-serotonin interactions, pretreatment with the serotonergic antagonist did not block the stimulatory effect of the opiate antagonist on LH release. However, pretreatment with the opiate agonist could not be overcome by the serotonergic agonist. This suggests that the effects of the serotonin system on LHRH release may be indirectly mediated by opioid neurones. Taken together, these studies demonstrate that the three neurotransmitter systems studied are critically involved in normal pulsatile LH release in the female guinea-pig, and demonstrate novel functional relationships between the opioid and the adrenergic and serotonergic systems.

Neurobiological mechanisms of the onset of puberty in primates.

An increase in pulsatile release of LHRH is essential for the onset of puberty. However, the mechanism controlling the pubertal increase in LHRH release is still unclear. In primates the LHRH neurosecretory system is already active during the neonatal period but subsequently enters a dormant state in the juvenile/prepubertal period. Neither gonadal steroid hormones nor the absence of facilitatory neuronal inputs to LHRH neurons is responsible for the low levels of LHRH release before the onset of puberty in primates. Recent studies suggest that during the prepubertal period an inhibitory neuronal system suppresses LHRH release and that during the subsequent maturation of the hypothalamus this prepubertal inhibition is removed, allowing the adult pattern of pulsatile LHRH release. In fact, y-aminobutyric acid (GABA) appears to be an inhibitory neurotransmitter responsible for restricting LHRH release before the onset of puberty in female rhesus monkeys. In addition, it appears that the reduction in tonic GABA inhibition allows an increase in the release of glutamate as well as other neurotransmitters, which contributes to the increase in pubertal LHRH release. In this review, developmental changes in several neurotransmitter systems controlling pulsatile LHRH release are extensively reviewed.

Monosynaptic projections from the nucleus retroambiguus to motoneurons supplying the abdominal wall, axial, hindlimb, and pelvic floor muscles in the female rhesus monkey.

The nucleus retroambiguus (NRA) consists of premotor neurons in the caudal medulla. It is involved in expiration, vomiting, vocalization, and probably reproductive behavior by means of projections to distinct motoneuronal cell groups. Because no information is available about the NRA and its efferent pathways in primates, the present study examines NRA projections to the lumbosacral spinal cord in female rhesus monkeys. To identify the NRA, wheat germ agglutinin-horseradish peroxidase (WGA-HRP) was injected into the lumbosacral cord in three monkeys. To study the distribution of NRA axons in the lumbosacral cord, WGA-HRP injections were made into the NRA in seven monkeys. To identify motoneuronal cell groups receiving input from the NRA, the same seven monkeys also received cholera toxin subunit b (CTb) injections into different hindlimb, axial, and pelvic floor muscles. The results show that NRA neurons projecting to the lumbosacral cord are mainly located between 1 to 4 mm caudal to the obex. They send numerous axons to external oblique and pelvic floor motoneurons, whereas projections to iliopsoas and axial motoneurons are less numerous. The projections are bilateral, but show a clear contralateral predominance in the iliopsoas, axial, and pelvic floor motoneuronal cell groups. At the ultrastructural level, NRA-terminal profiles make asymmetrical contacts with labeled and unlabeled dendrites in these motoneuronal cell groups and contain large amounts of spherical and a few dense core vesicles. It is concluded that the NRA is well developed in the monkey and that there exists a direct pathway from the NRA to lumbosacral motoneurons in this species. The finding that the NRA projects to a somewhat different set of motoneuronal cell groups compared with other species fits the concept that it is not only involved in expiration-related activities but also in species specific receptive and submissive behavior.