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.

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.

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.

Intracellular Ca(2+) oscillations in luteinizing hormone-releasing hormone neurons derived from the embryonic olfactory placode of the rhesus monkey.

To understand the mechanism of pulsatile luteinizing hormone-releasing hormone (LHRH) release, we examined whether cultured LHRH neurons exhibit spontaneous intracellular Ca(2+) ([Ca(2+)](i)) signaling. The olfactory placode and the ventral migratory pathway of LHRH neurons from rhesus monkey embryos at embryonic ages 35-37 were dissected out and cultured on glass coverslips. Two to five weeks later, cultured cells were labeled with fura-2 and examined for [Ca(2+)](i) signaling by recording changes in [Ca(2+)](i) every 10 sec for 30-175 min. Cells were fixed and immunostained for LHRH and neuron-specific enolase. In 20 cultures, 572 LHRH-positive cells exhibited [Ca(2+)](i) oscillations at an interpulse interval (IPI) of 8.2 +/- 0.7 min and a duration of 88.8 +/- 2.9 sec. LHRH-negative neurons in culture exhibited only occasional [Ca(2+)](i) oscillations. In 17 of 20 cultures with LHRH-positive cells, [Ca(2+)](i) oscillations occurred synchronously in 50-100% of the individual cells, whereas [Ca(2+)](i) oscillations in cells in the remaining three cultures did not synchronize. Strikingly, in 12 of 17 cultures the synchronization of [Ca(2+)](i) oscillations repeatedly occurred in complete unison at 52.8 +/- 3.0 min intervals, which is similar to the period observed for LHRH release, whereas in 5 of 17 cultures the less tight synchronization of [Ca(2+)](i) oscillations repeatedly occurred at 23.4 +/- 4.6 min intervals. IPI of [Ca(2+)](i) oscillations in cells with tight synchronization and less tight synchronization did not differ from IPI in cells without synchronization. The results indicate that LHRH neurons derived from the monkey olfactory placode possess an endogenous mechanism for synchronization of [Ca(2+)](i) oscillations. Whether synchronization of [Ca(2+)](i) oscillations relates to neurosecretion remains to be investigated.

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.

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.

Developmental changes in the positive feedback effect of estrogen on luteinizing hormone release in ovariectomized female rhesus monkeys.

To examine developmental changes in the LH response to estrogen, eight neonatally ovariectomized monkeys received repeated injections (sc) of 50 micrograms/kg estradiol benzoate (EB) at approximately 4-month intervals starting at age 8-12 months and ending at 49-52 months. Serum samples were obtained 24 before and 0, 6, 12, 24, 36, 48, 60, 72, 94, 108, and 120 h after each EB injection. Serum LH and estradiol levels were measured by RIA. The baseline LH level before EB injection during the prepubertal period (greater than 20 months of age) was 14.4 +/- 2.2 ng/ml, and it increased progressively to 115.3 +/- 13.5 ng/ml at 41-44 months, the age shortly before the first ovulation in our intact colony animals, then declined slightly. EB first induced a typical LH response, which consisted of a negative phase (suppression) followed by a positive phase (surge), at the average age of 29.3 +/- 1.9 months (n = 8). This is similar to the age of menarche in our colony animals. The baseline LH level before EB injection at the time of the first typical response (with negative and positive phases) was 36.7 +/- 6.7 ng/ml, a level 2.5 times higher than that of the prepubertal age. The magnitude of LH suppression by EB was significantly correlated with the baseline level of LH; the higher the baseline LH before EB injection, the greater the degree of LH decrease (r = 0.968; P less than 0.001). Similarly, the amplitude of the LH peak from the trough of the negative phase was significantly correlated with the baseline LH; the higher the LH level before EB injection, the higher the LH increase (r = 0.863; P less than 0.001). The latency to the LH peak was shortest when baseline LH was highest; the peak latency (34.4 +/- 1.6 h) of the LH surge at 41-44 months of age was significantly shorter than the latency (46.5 +/- 2.7 h) of the first LH response occurring at 29.3 +/- 1.9 months of age (P less than 0.001). Finally, the pattern of circulating levels of estradiol after EB injection did not differ across the developmental stages examined. These results are interpreted to mean that an increase in LH release, presumably LHRH release, starts at the onset of puberty and continues until the age of first ovulation, and that the levels of LHRH release during the pubertal period may determine the effectiveness of estrogen on the LH surge.(ABSTRACT TRUNCATED AT 400 WORDS)

Negative feedback effects of estrogen on luteinizing hormone-releasing hormone release occur in pubertal, but not prepubertal, ovariectomized female rhesus monkeys.

In a previous study we found that ovariectomy resulted in an increase in both LHRH release and LH release in pubertal monkeys but not in prepubertal monkeys. To determine whether this castration-induced LHRH increase is due to the removal of estrogen, in the present study, the effects of estradiol benzoate (EB, 30 micrograms/kg body wt) on in vivo LHRH release were examined using a push-pull perfusion method in prepubertal (age 15-19 months, n = 5), early pubertal (24-29 months, n = 5), and midpubertal (36-48 months, n = 5) female rhesus monkeys that were ovariectomized 3 to 5 months earlier. LHRH in 10-min perfusate fractions from the stalk-median eminence was measured from -6 to +24 h after EB injection. Circulating LH levels were also monitored over the same period at various intervals. EB decreased LH levels in early pubertal and midpubertal monkeys, whereas it did not cause any significant effects on LH release in the prepubertal monkey. EB also resulted in suppression of LHRH release in both early and midpubertal monkeys; mean LHRH release before EB in the early and midpubertal groups was 6.6 +/- 0.6 and 7.0 +/- 0.6 pg/ml.10 min, respectively. EB decreased mean LHRH release beginning 3 h after EB with the nadir occurring at 18-21 h after EB (1.0 +/- 0.2 pg/ml.10 min) in early pubertal monkeys and 21-24 h after EB (1.2 +/- 0.1 pg/ml.10 min) in midpubertal monkeys. Decrease of mean LHRH release was due to a decrease in LHRH pulse amplitude and basal release but not pulse frequency. Oil injection alone (control) failed to suppress LHRH and LH release. In contrast to the results in pubertal monkeys, mean LHRH release in prepubertal monkeys was not altered by EB (before EB, 1.1 +/- 0.2 pg/ml.10 min; 18-21 h after EB, 1.5 +/- 0.3 pg/ml.10 min). These results suggest that the LHRH neurosecretory system in pubertal monkeys is responsive to the negative feedback effects of estrogen. However, the fact that estradiol failed to suppress LHRH release in prepubertal monkeys indicates that the LHRH neurosecretory system and/or its regulatory systems are not sensitive to estradiol before the onset of puberty. These findings are consistent with the hypothesis that the increase in pulsatile LHRH release at the onset of puberty is not dependent on changes in ovarian steroid feedback mechanisms.

In vivo release of luteinizing hormone releasing hormone increases with puberty in the female rhesus monkey.

To determine the regulatory mechanism of the LHRH release associated with puberty, episodic release of LHRH from the stalk-median eminence was measured using a push-pull perfusion technique in conscious prepubertal and peripubertal female monkeys. After insertion of a push-pull cannula into the stalk-median eminence, a modified Krebs-Ringer phosphate buffer solution was infused through the push-cannula, and perfusates were collected through the pull-cannula at 200 microliters/10 min. LHRH in perfusates was determined by RIA. Two 6-h sampling sessions, in the morning (0600-1200 h; lights on 0600 h) and in the evening (1800-2400 h; lights off 1800 h) were performed in each animal. LHRH release patterns were analyzed in prepubertal (15.7 +/- 0.7 months of age; mean +/- SEM, n = 6) early pubertal (premenarcheal; 26.1 +/- 1.0 months, n = 7), and midpubertal (40.0 +/- 1.4 months, n = 6) monkeys. Results were as follows: 1) LHRH release was pulsatile in all age groups. While LHRH release in five of six prepubertal animals consisted of small (amplitude less than 2.5 pg/ml) pulses, in all peripubertal animals LHRH release was a mixture of small and large (amplitude greater than 2.5 pg/ml) pulses. 2) There was a significant developmental increase in mean LHRH release (P less than 0.02), and this was particularly apparent in the evening. Mean LHRH release in the early and midpubertal groups was higher than that in the prepubertal group (P less than 0.05 for morning and P less than 0.01 for evening). The mean release in the evening of the midpubertal group further increased over that of the early pubertal group (P less than 0.05). 3) Similarly, LHRH pulse amplitude increased developmentally (P less than 0.01). Pulse amplitudes in early and midpubertal groups were higher than those in the prepubertal group (P less than 0.05 for morning and P less than 0.02 for evening). Again the amplitude in the evening further increased from the early pubertal to the midpubertal period (P less than 0.05). 4) There was also a developmental increase in basal LHRH release (P less than 0.01). The evening values in the early pubertal and midpubertal groups were higher than those in the prepubertal group (P less than 0.05). 5) The interpulse interval decreased developmentally (P less than 0.001). Interpulse intervals in early and midpubertal groups were shorter than those in the prepubertal group (P less than 0.01 for morning and P less than 0.025 for evening).(ABSTRACT TRUNCATED AT 400 WORDS)

A role for norepinephrine in the control of puberty in the female rhesus monkey, Macaca mulatta.

The onset of puberty in female rhesus monkeys is characterized by increases in pulsatile LHRH release. In this study we have tested the hypothesis that changes in input to the LHRH neurosecretory system from noradrenergic neurons contribute to this pubertal increase in LHRH release. In the first experiment, the ability of the LHRH neurosecretory system of prepubertal (12-20 months of age, no signs of puberty evident), early pubertal (24-30 months, premenarchial), and midpubertal (30-45 months, postmenarchial but prior to first ovulation) monkeys to respond to alpha 1-adrenergic stimulation was tested. LHRH release in the stalk-median eminence of conscious monkeys was measured using an in vivo push-pull perfusion method. During push-pull perfusion, perfusates were collected continuously in 10-min fractions, and the alpha 1-adrenergic stimulant methoxamine (MTX, 10(-8), 10(-5) M) or vehicle was infused through the push cannula for 10 min at 90 min intervals. LHRH levels in perfusates were estimated by RIA. Monkeys in all three age groups responded to MTX with significant increases in LHRH release, with the response of the prepubertal group being significantly greater than that of the older age groups. The results indicate that alpha 1-adrenergic receptors are present and functional prior to puberty. In the second experiment, norepinephrine (NE) release in perfusates collected from monkeys in the three age groups was measured by HPLC with electrochemical detection. NE release increased significantly from the pre- and early pubertal to the midpubertal stage. The enhanced sensitivity of prepubertal monkeys to MTX may be due to the absence of high levels of endogenous NE, which results in a situation similar to denervation hypersensitivity. During the early pubertal stage, increases in input from noradrenergic neurons to the LHRH neurosecretory system may occur, thereby resulting in increases in LHRH release, since early pubertal monkeys are highly sensitive to alpha-adrenergic input. Therefore, we propose that the increase in NE release during puberty contributes to the developmental increase in LHRH release.

Infusion of neuropeptide Y into the stalk-median eminence stimulates in vivo release of luteinizing hormone-release hormone in gonadectomized rhesus monkeys.

Studies in the rat and rabbit indicate that facilitatory effects of neuropeptide Y (NPY) as well as norepinephrine (NE) on LH and LHRH release are dependent on the presence of the ovarian steroid estrogen. However, we have previously found the NE and an alpha-1-adrenergic agonist are both stimulatory to pulsatile LHRH release in ovariectomized rhesus monkeys. In the present experiment the effects of NPY on LHRH release were examined in conscious monkeys using a push-pull perfusion method. Twelve gonadectomized monkeys (8 females and 4 males) were used. Perfusate samples from the stalk-median eminence (S-ME) were obtained through a push-pull cannula at 10-min intervals for 12 h, and the amount of LHRH in samples were determined with RIA. NPY dissolved in a modified Krebs-Ringer phosphate buffer solution at concentrations of 10(-8), 10(-7), 10(-6), and 10(-5) M was directly infused into the S-ME through the push cannula for 10 min at 90-min intervals. Vehicle was infused as a control. Since sex differences in LHRH response to NPY were not present, data from males and females were combined for analysis. NPY infusion into the S-ME stimulated LHRH release in a dose-dependent manner (P less than 0.001). The peak LHRH responses (mean +/- SEM) to NPY at different concentrations were: 10(-8) M = 2.1 +/- 0.4 pg/ml; 10(-7) M = 2.6 +/- 0.5 pg/ml; 10(-6) M = 6.5 +/- 1.1 pg/ml; 10(-5) M = 15.1 +/- 2.9 pg/ml, whereas to vehicle 0.37 +/- 0.17 pg/ml. All NPY doses tested were significantly effective as compared to vehicle (P less than 0.01). The LHRH response to 10(-6) M was greater (P less than 0.01) than that of 10(-8) M or 10(-7) M, and the response to 10(-5) M was greater (P less than 0.01) than that of all lower doses. The results indicate that NPY infusion into the S-ME elicits the release of LHRH in vivo in a dose-dependent manner in the monkey. The data further suggest that LHRH neurons and/or neuroterminals in the monkey are responsive to NPY stimulation in the absence of gonadal steroids. It is concluded that in addition to NE, NPY is an important regulator of pulsatile LHRH release in the nonhuman primate.

The role of neuropeptide Y in the progesterone-induced luteinizing hormone-releasing hormone surge in vivo in ovariectomized female rhesus monkeys.

Progesterone induces a LHRH surge in estrogen-primed ovariectomized rhesus monkeys, with a concomitant increase in the pulse frequency of neuropeptide Y (NPY) release. However, the role for NPY in the positive feedback action of progesterone on LHRH release in primates is unknown. The present study examines the effect of an antisense oligodeoxynucleotide for NPY messenger RNA (AS NPY) on the progesterone-induced LHRH surge in vivo using push-pull perfusion. The AS NPY was directly infused into the stalk-median eminence (S-ME), whereas perfusates were collected for assessment of LHRH release. For a control, a scrambled oligodeoxynucleotide was infused. The results indicate that 1) the scrambled oligodeoxynucleotide did not interfere with the progesterone-induced LHRH surge, 2) whereas AS NPY blocked the progesterone-induced increase in LHRH release, and 3) no LHRH surges were induced by oil as a control for progesterone, but the AS NPY also reduced LHRH release in oil controls. These data suggest that 1) AS NPY infusion into the S-ME results in reduction in LHRH release; and 2) NPY release in the S-ME is important for the positive feedback effects of progesterone on LHRH release in estrogen-primed ovariectomized monkeys.

Changes in pulsatile release of neuropeptide-Y and luteinizing hormone (LH)-releasing hormone during the progesterone-induced LH surge in rhesus monkeys.

In previous studies we have shown that the pulsatility of LH-releasing hormone (LHRH) release in gonadectomized monkeys is modulated by input from neuropeptide-Y (NPY) neurons: 1) the endogenous release of NPY in the stalk-median eminence (S-ME) was pulsatile; 2) NPY pulses were temporally correlated with LHRH pulses, with NPY pulses preceding LHRH pulses by approximately 5 min; and 3) infusion of NPY into the S-ME stimulated LHRH release, whereas 4) infusion of antiserum to NPY suppressed endogenous LHRH pulses. It is not known, however, whether ovarian steroid hormones alter the pulsatility of NPY and LHRH release or whether the temporal correlation of NPY and LHRH pulses is maintained during the LH surge. In the present study we examined the changes in pulsatile release of NPY and LHRH in ovariectomized monkeys treated with estradiol benzoate (EB) followed by progesterone or oil. Using push-pull perfusion, perfusate samples from S-ME were collected at 10-min intervals for 15 h. NPY and LHRH concentrations in the perfusates were measured by RIA. Circulating LH levels were also monitored by periodic blood sampling and RIA. Injection of progesterone (sc) after EB induced an LH surge with a peak latency of 7.3 +/- 1.3 h (mean +/- SE) in seven of seven monkeys, whereas oil injection after EB elicited an LH surge in none of seven monkeys. The progesterone-induced LH surge was associated with an increase in LHRH release; the mean, pulse amplitude, and pulse frequency increased significantly (for all, P < 0.05) 4-8 h after progesterone. NPY pulse frequency also increased significantly (P < 0.05) 4-8 h after progesterone treatment, whereas mean release and pulse amplitude did not change in response to progesterone. Oil treatment after EB administration did not alter any parameter of LHRH and NPY pulses. Interestingly, the NPY and LHRH pulses were highly correlated (P < 0.001) in monkeys treated with either EB-progesterone or EB-oil, and NPY pulses preceded LHRH pulses by 4.8 +/- 0.7 and 5.1 +/- 0.6 min, respectively. In summary, 1) an episode of increased LHRH release occurs before and during the progesterone-induced LH surge; 2) acceleration of LHRH pulse frequency and the increase in LHRH pulse amplitude after progesterone are accompanied by acceleration of NPY pulse frequency; and 3) ovarian steroids do not affect the temporal correlation between NPY and LHRH pulses.(ABSTRACT TRUNCATED AT 400 WORDS)

Posterior hypothalamic lesions advance the time of the pubertal changes in luteinizing hormone release in ovariectomized female rhesus monkeys.

To examine further the relationship between developmental changes in LH release and the onset of puberty, effects of posterior hypothalamic lesions were tested in ovariectomized (OVX), sexually immature female monkeys. In OVX females (n = 3) with sham hypothalamic lesions basal LH levels were suppressed during the prepubertal period until 25 months of age, when LH levels started to increase. The increase in basal LH continued; a 100% elevation from prepubertal levels was attained at 26.0 +/- 0 months of age, and a 200% elevation was attained at 31.0 +/- 3.2 months of age. A consistent appearance of LH circadian fluctuation (nocturnal LH increase) with a large amplitude accompanied the initial LH increase. Lesions of the posterior hypothalamus (PH) in OVX animals (n = 6) at 17-18 months of age, which we previously reported to be effective in advancing the onset of puberty by several months in ovarian intact monkeys, resulted in an early 100% increase in basal LH levels and the circadian LH fluctuation (19.5 +/- 1.0 months of age). Basal LH levels in these animals further increased, reaching a 200% elevation of prelesion levels at 24.2 +/- 0.7 months of age. All of these LH changes with PH lesions occurred significantly (P less than 0.01) earlier than those in sham-lesioned animals. Lesion of the PH in OVX animals (n = 4) at 13-14 months of age resulted in an increase in LH and the circadian LH fluctuation within 1 month postoperatively. However, 100% and 200% LH elevations did not occur until 20.8 +/- 1.0 and 24.8 +/- 1.4 months of age, respectively. These ages were similar to those of animals receiving lesions at 17-18 months of age, but much younger than those of sham controls (P less than 0.01). PH lesions in animals at 13-14 months of age also advanced the time of the first positive feedback effects of estrogen. In animals (n = 4) with PH lesions, estradiol benzoate induced a first LH response at 21.5 +/- 1.6 months of age, when basal LH was 276 +/- 83% increased from prelesion levels. This age was significantly (P less than 0.05) younger than that (29.3 +/- 1.9 months; n = 6) of the first LH surge induced by estrogen in control animals when basal LH levels attained 248 +/- 18% of prepubertal levels.(ABSTRACT TRUNCATED AT 400 WORDS)

An increase in single unit activity of the medial basal hypothalamus occurs during the progesterone-induced luteinizing hormone surge in the female rhesus monkey.

Accumulated evidence from our laboratory indicates that a positive feedback effect of progesterone (P) occurs at the hypothalamic level. The present study in female rhesus monkeys examined the effects of P on single unit activity of neurons in the hypothalamus and on LH release. Single unit activity was recorded by inserting a flexible stainless steel electrode into the hypothalamus of the monkey, which was restrained in a chair under light ketamine sedation. The firing rate of the single unit activity of the ventral hypothalamus (1.5 +/- 0.2 spikes/sec; n = 57) in ovariectomized estrogen-primed monkeys was slow and was slower in the ventral hypothalamus than in the dorsal hypothalamus (6.2 +/- 0.8 spikes/sec; n = 80). P injection resulted in a dramatic increase in unit activity of the ventral hypothalamus (4.7 +/- 0.6 spikes/sec; n = 111), but not of the dorsal hypothalamus (5.3 +/- 0.7 spikes/sec; n = 72), and induced a concomitant release of LH. Both increases in circulating LH and unit activity of the ventral hypothalamus were significantly correlated over time (P less than 0.02). In contrast, oil injections induced neither change. Therefore, in the rhesus monkey, P seems to activate neural substrates in the ventral hypothalamus to promote the release of LHRH and, subsequently, LH.

Persistent estrus and blockade of progesterone-induced LH release follows lesions which do not damage the suprachiasmatic nucleus.

Very small electrolytic lesions were made over the anterior or posterior portion of the optic chiasm in mature female rats showing normal estrous cycles. Lesions over the posterior portion of chiasm destroyed the suprachiasmatic nucleus of the hypothalamus (SCN) while the anterior lesions destroyed a small neural structure, here designated as the medial preoptic nucleus (MPN). Both lesions were effective in inducing persistent vaginal estrus, but when animals were ovariectomized and treated with exogenous and progesterone it was found that lesions including the MPN alone, but not the SCN alone, eliminated the positive feedback effects of this steroid regimen on LH release.

Effects of electrical stimulation of the medial basal hypothalamus on the in vivo release of luteinizing hormone-releasing hormone in the prepubertal and peripubertal female monkey.

In this study the hypothesis that the LHRH neurosecretory system of the prepubertal female monkey has the capacity to function in a manner comparable to that of monkeys in more mature stages of development was tested. Using push-pull perfusion in the stalk-median eminence, effects of electrical stimulation of the medial basal hypothalamus on in vivo LHRH release were determined in conscious prepubertal, early pubertal, and midpubertal monkeys. After a 180-min period of baseline sample collection, electrical stimulation was applied six times at 90-min intervals via a monopolar electrode, the tip of which was 1-2 mm rostro-dorsal to the perfusion site. Control experiments were performed in the same manner, but without electrical stimulation. During control perfusions, the mean LHRH level remained stable. Mean (+/- SEM) LHRH release for the entire perfusion period in control experiments was 0.5 +/- 0.2, 2.4 +/- 0.4, and 2.2 +/- 0.7 pg/ml.10 min for the prepubertal (n = 6), early pubertal (n = 4), and midpubertal (n = 6) groups, respectively. Mean LHRH release in the prepubertal group was significantly lower than that in either of the older groups (P less than 0.05). In contrast, in all three age groups, repeated electrical stimulation of the medial basal hypothalamus resulted in 1) a short latency increase in LHRH release occurring within 20 min after each stimulation, and/or 2) a gradual increase in mean LHRH release over several hours. In the prepubertal group (n = 4), mean LHRH levels were 0.8 +/- 0.5 pg/ml.10 min during the 90 min before the first electrical stimulation and increased to 6.1 +/- 2.9 pg/ml.10 min during the 90 min after the sixth stimulation. This degree of responsiveness was similar to that of the older age groups. Mean LHRH levels before stimulation were 1.3 +/- 0.6 and 2.4 +/- 1.1 pg/ml.10 min in the early pubertal (n = 5) and midpubertal (n = 5) groups, respectively, and increased to 7.8 +/- 3.5 and 6.0 +/- 1.8 pg/ml.10 min, respectively, after the sixth stimulation. These increases in LHRH concentration with electrical stimulation were significant for all three age groups (P less than 0.03-0.001), while there were no significant differences between age groups. The temporal patterns of these responses suggest that electrical stimulation elicits LHRH release with a similar magnitude in all three age groups by 1) depolarizing LHRH neurons directly, and/or 2) stimulating multineuronal systems that synapse with LHRH neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Transplantation of the fetal olfactory placode restores reproductive cycles in female rhesus monkeys (Mucaca mulatta) bearing lesions in the medial basal hypothalamus.

The purpose of this study was to determine whether loss of the reproductive cycle after lesions of the medial basal hypothalamus can be reversed by transplantation of the embryonic olfactory placode (OP) into female rhesus monkeys. Seven adult female rhesus monkeys with regular menstrual cycles received bilateral radiofrequency lesions in the arcuate nucleus and the median eminence. After confirmation of anovulation in these monkeys, four monkeys were stereotaxically implanted with the OP obtained from monkey fetuses on embryonic days 35-36. The remaining three monkeys were similarly implanted with embryonic cerebellum (CB) as a control. Fetuses were delivered by cesarean section, and the OP and CB were immediately dissected out using a stereomicroscope. Fetal tissue was then cut into small pieces (< 1 mm3), mixed with artificial cerebrospinal fluid containing small pieces of Gelfoam, and stereotaxically injected into the infundibular recess of the third ventricle. The recovery of ovulatory cycles in recipient monkeys was observed for at least 6 months; sex skin color changes and menstrual records were obtained daily, and serum samples for LH, estrogen, and progesterone were obtained twice a week. Three of four OP-transplanted monkeys resumed their ovulatory cycles within 2 months, whereas the fourth monkey, an elderly female, failed to recover her cycle. In contrast, none of the three CB-transplanted monkeys resumed ovulatory cycles. Histological examination indicated that 1) lesion scars were present in the median eminence-stalk region as well as the medial basal portion of the arcuate nucleus of all seven brains; and 2) cartilage was present in the third ventricles of the OP-implanted brains. Moreover, immunocytochemical staining revealed that in all OP monkeys, small, round, and immature LHRH-positive cells with fine short processes were found in the third ventricle and/or median eminence-stalk region, whereas no similar LHRH cells were found in CB-transplanted monkeys. It is concluded, therefore, that implantation of LHRH neurons derived from the fetal OP can result in resumption of the ovulatory cycle in female monkeys whose own LHRH pulse-generating mechanisms were impaired. Moreover, the results suggest that LHRH neurons derived from embryonic OP possess the physiological functions necessary for the stimulation of gonadotropin secretion.

Pulsatile release of luteinizing hormone-releasing hormone (LHRH) in cultured LHRH neurons derived from the embryonic olfactory placode of the rhesus monkey.

To study the mechanism of LH-releasing hormone (LHRH) pulse generation, the olfactory pit/placode and the migratory pathway of LHRH neurons from monkey embryos at embryonic age 35-37 were dissected out, under the microscope, and cultured on plastic coverslips coated with collagen in a defined medium for 2-5 weeks. First, we examined whether cultured neurons release the decapeptide into media. It was found that LHRH cells release LHRH in a pulsatile manner at approximately 50-min intervals. Further, LHRH release was stimulated by depolarization with high K+ and the Na+ channel opener, veratridine. However, whereas the Na+ channel blocker, tetrodotoxin suppressed the effects of veratridine, tetrodotoxin did not alter the effects of high K+. Subsequently, the role of extracellular and intracellular Ca2+ in LHRH release was examined. The results are summarized as follows: 1) exposing the cells to a low Ca2+ (20 nM) buffer solution suppressed LHRH release, whereas exposure to a normal Ca2+ solution (1.25 mM) maintained pulsatile LHRH release; 2) LHRH release from cultured LHRH cells was stimulated by the voltage-sensitive L-type Ca2+ channel agonist, Bay K 8644 (10 microM), whereas it was suppressed by the L-type Ca2+ channel blocker, nifedipine (1 microM), but not by the N-type channel blocker, omega-conotoxin GVIA (1 microM); 3) the intracellular Ca2+ stimulant, ryanodine (1 microM), stimulated LHRH release, whereas the intracellular Ca2+ transporting adenosine triphosphatase antagonist, thapsigargin (1 and 10 microM), did not yield consistent results; and 4) carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (1 microM), a mitochondrial Ca2+ mobilizer, stimulated LHRH release, whereas ruthenium red, a mitochondrial Ca2+ uptake inhibitor, did not induce consistent results. These results indicate that: 1) the presence of extracellular Ca2+ is essential for LHRH neurosecretion; 2) Ca2+ enters the cell via L-type channels but not N-type channels; and 3) mobilization of intracellular Ca2+ from inositol 1,4,5-triphosphate-sensitive stores, as well as mitochondrial stores, seem to contribute to LHRH release in these cells.

A role of gamma-amino butyric acid (GABA) and glutamate in control of puberty in female rhesus monkeys: effect of an antisense oligodeoxynucleotide for GAD67 messenger ribonucleic acid and MK801 on luteinizing hormone-releasing hormone release.

Previously we have shown that gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter restricting the pubertal increase in LHRH release in juvenile monkeys, and that interfering with GABA synthesis with an antisense oligodeoxynucleotide (AS) for glutamic acid decarboxylase (GAD67) mRNA results in an increase in LHRH release in prepubertal monkeys. GAD67 is a catalytic enzyme that synthesizes GABA from glutamate. To further clarify the role of GABA in puberty, we examined whether the inhibition of LHRH release by GABA continues after the onset of puberty and whether input from glutamatergic neurons plays any role in the onset of puberty when GABA inhibition declines, using a push-pull perfusion method. In Study I, the effects of the AS GAD67 mRNA on LHRH release in pubertal monkeys (34.3 +/- 1.5 months of age, n = 8) were examined, and the results were compared with those in prepubertal monkeys (18.5 +/- 0.4 months, n = 12). Direct infusion of AS GAD67 (1 microM) into the stalk-median eminence (S-ME) for 5 h stimulated LHRH release in both prepubertal and pubertal monkeys. However, the increase in LHRH release in pubertal monkeys was significantly (P < 0.01) smaller than that in prepubertal monkeys. Infusion of a scrambled oligo as a control was without effect in either group. In Study II, to examine the possibility that an increase in glutamate tone after the reduction of an inhibitory GABA tone contributes to the AS GAD67-induced LHRH increase, the effects of the NMDA receptor blocker MK801 (5 microM) on LHRH release were tested in monkeys treated with AS GAD67. MK801 infusion into the S-ME during the treatment of AS GAD67 (1 microM) suppressed the AS GAD67-induced LHRH release in both age groups. MK801 alone did not cause any significant effect in either group. The data are interpreted to mean that GABA continues to suppress LHRH release after the onset of puberty, although the degree of suppression is weakened considerably after the onset of puberty, and that the increased LHRH release after AS GAD67 treatment may be partly due to an increase in glutamate tone mediated by NMDA receptors, as well as due to the decrease in GABA release following the decrease in GAD synthesis. Taken together, the present results suggest that GAD may play an important role in the onset and progress of puberty in nonhuman primates.