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Previously we have shown that release of gamma-aminobutyric acid (GABA) in the stalk-median eminence (S-ME) is high in prepubertal monkeys and that a decrease in GABA release triggers the onset of puberty. However, it is still unclear how disinhibition of the luteinizing hormone releasing hormone (LHRH) neuronal system from GABA input is followed (or accompanied) by an increase in stimulatory signals, such as glutamatergic input to LHRH neurons. To clarify the temporal relationship between the reduction of the GABAergic inhibitory signal and the enhancement of the glutamatergic stimulatory signal in the control of LHRH release at the onset of puberty, we conducted two experiments using a push-pull perfusion method. In the first experiment, we measured developmental changes in release of LHRH, GABA, and glutamate in the S-ME. LHRH levels were very low in prepubertal monkeys, increased to higher levels in early pubertal monkeys, with the highest LHRH levels occurring in mid-pubertal monkeys. As we previously observed, GABA levels were high in prepubertal monkeys and then decreased in early- and mid-pubertal monkeys. In contrast, glutamate levels were very low in prepubertal monkeys, increased dramatically in early pubertal monkeys, and then slightly decreased in mid-pubertal monkeys, although mid-pubertal levels remained much higher than prepubertal levels. In the second experiment, we measured GABA, glutamate and LHRH in the same samples obtained from prepubertal monkeys which were infused with an antisense oligodeoxynucleotide (AS) for glutamic acid decarboxylase (GAD) 67 mRNA into the S-ME. GAD67 is a catalytic enzyme for GABA synthesis from glutamate, and AS GAD67 mRNA interferes with GAD67 synthesis. Infusion of the AS GAD67 induced a decrease in GABA release, which subsequently resulted in an increase in LHRH release. Surprisingly, glutamate release also increased several hours after the decrease in GABA release, and the increased LHRH release continued. These data are interpreted to mean that a decrease in GABA synthesis by interference with GAD67 synthesis and the reduction of GABA release in the S-ME trigger an increase in LHRH release, but that a subsequent increase in glutamate release in the S-ME further contributes to the pubertal increase in LHRH release at the onset of puberty. The data further support our hypothesis that GAD plays an important role in the mechanism of the onset of puberty.

For several years, it has been well accepted that insulin-like growth factor 1 (IGF-1) plays a critical role in peripubertal growth. Recently, we have provided evidence to suggest that this peptide may also be involved in the sexual maturation process, via an action to stimulate hypothalamic luteinizing hormone releasing hormone release. Because ethanol (ETOH) delays puberty, an event that is associated with depressed growth rates and decreased growth hormone and luteinizing hormone (LH) secretion via actions at the hypothalamic level, we investigated whether this drug is capable of altering the expression of genes encoding IGF-1 in liver and brain, as well as the expression of the type 1 IGF receptor (IGF-1R) within the median eminence (ME). Also, we wanted to determine if any regional changes in the expression of these genes were associated with concomitant alterations in the serum levels of IGF-1 and LH. Rats were implanted with gastric cannulae on day 24 and began receiving specific control or ETOH diets on day 29. Rats were killed on day 34, determined to be in the late juvenile stage of development, and their tissues and blood were collected. Results indicate that the ETOH-fed rats showed a decrease (p < 0.01) in the expression of hepatic IGF-1 mRNA when compared with the controls, and this paralleled depressions in both serum IGF-1 (p < 0.01) and LH (p < 0.01). In contrast, no changes were detected in IGF-1 mRNA expression in the preoptic area and hypothalamus, as well as in IGF-1R mRNA expression within the ME. These results suggest that the well-known detrimental effects of ETOH on growth rates and the progression of the female pubertal process in the rat may be associated with the drug's ability to depress the hepatic synthesis of IGF-1 and the subsequent prepubertal circulating levels of the protein.

This research was designed to determine 1) whether changes occur in the levels of N-methyl-D-aspartic acid (NMDA) receptor (NMDA-R) messenger RNA (mRNA) in the reproductive hypothalamus of female rats as they approach puberty, 2) whether NMDA-R stimulation would promote differential LH responses during the specific stages of peripubertal development, and 3) whether ethanol (ETOH), which is known to affect the NMDA-R in other brain systems, can alter NMDA-R-activated LH secretion at puberty. In the first experiment, female rats were killed at 15, 20, 25, and 34-36 days of age to determine the levels of mRNA that code for the NMDA-R, specifically NMDA-R1, in the arcuate nucleus-median eminence (AN-ME) and preoptic area (POA) during pre- and peripubertal development by a ribonuclease protection assay. Results indicate that in juvenile animals, NMDA-R mRNA levels in the AN-ME increased at 25 days (P < 0.01). In the POA, the levels increased at 20 days (P < 0.05), but were unchanged at 25 days. During the peripubertal period, NMDA-R gene expression in the AN-ME did not change; however, gene expression in the POA increased (P < 0.05) during first proestrus, then declined during first estrus. In the second experiment, NMDA-R stimulation with N-methyl-D,L-aspartic acid (NMA; 2.5 mg/kg) produced differential stimulatory effects on LH release depending upon the stage of pubertal development. In this regard, significant post-NMA percent increases in LH released over pre-NMA (basal) levels occurred during anestrus (46%; P < 0.01) and first proestrus (95%; P < 0.01), with nonsignificant increases of 18% and 28% during first estrus and diestrus, respectively. Finally, a 3 g/kg dose of ETOH given intragastrically 90 min before the NMA challenge blocked (P < 0.05) NMA-induced LH release during first proestrus. In conclusion, these findings demonstrate regional differences in the timing of NMDA-R gene expression in the reproductive hypothalamus during pubertal development, show differential responses of LH to NMDA-R activation during the peripubertal period, and continue to demonstrate the vulnerability of the hypothalamic-pituitary axis to the detrimental effects of ETOH at this critical time of development.

In the present study, we have evaluated the effects of ethanol (ETOH) on luteinizing hormone releasing hormone (LHRH) secretion induced by N-methyl-DL-aspartic acid (NMA) in vitro and the ability of NMA to induce precocious puberty in vivo. For the in vitro experiments, the basal and NMA-stimulated release of LHRH from arcuate nucleus-median eminence (AN-ME) fragments obtained from immature female rats was measured by RIA following static incubation in medium consisting of Krebs-Ringer bicarbonate glucose buffer. Control vials contained medium only and the test vials contained medium plus ETOH doses of 30, 50, and 70 mM. These data demonstrate that ETOH did not alter basal LHRH release, but dose-dependently blocked (p < 0.01) the NMA-induced release of the peptide during anestrus, as well as first proestrus and estrus. For the in vivo experiment, 26-day-old females began receiving saline or saline-ETOH (3 g/kg) solution by gastric gavage daily at 12.30 p.m. Each day at 2.00 and 4.00 p.m., each of the animals received subcutaneous injections of a 40-mg/kg solution of NMA in saline. Other control animals received saline gastrically, as well as subcutaneously. The timing of puberty was assessed in all animals by monitoring vaginal opening (VO) and first diestrus (D1). The mean (+/- SEM) age at VO for animals in the non-ETOH group, which received NMA, was 31.7 +/- 0.40 days. Vaginal smears revealed that D1 occurred at a mean (+/- SEM) age of 33.0 +/- 0.42 days.(ABSTRACT TRUNCATED AT 250 WORDS)