A Role of g-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 g-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 6 1.5 months of age, n 5 8) were examined, and the results were compared with those in prepubertal monkeys (18.5 6 0.4 months, n 5 12). Direct infusion of AS GAD67 (1 mM) 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 mM) on LHRH release were tested in monkeys treated with AS GAD67. MK801 infusion into the S-ME during the treatment of AS GAD67 (1 mM) 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. (Endocrinology 140: 705–712, 1999) T CONCEPT THAT an increase in pulsatile LHRH release is critical for the onset of puberty in nonhuman primates has been well established (1–4). LHRH release in prepubertal monkeys is low, and LHRH release increases at the onset of puberty (5, 6). However, the underlying mechanism triggering the pubertal increase in LHRH release is still unclear. Previously, we have proposed the hypothesis that g-amino butyric acid (GABA) is responsible for the low levels of LHRH release in prepubertal monkeys and the removal of this inhibition triggers the onset of puberty (4, 7). This hypothesis is based on the observations that 1) GABA release in the stalk-median eminence (S-ME) in prepubertal monkeys was much higher than that in pubertal monkeys (7); 2) the direct infusion of bicuculline, a GABAA receptor antagonist, into the S-ME induced a dramatic increase in LHRH release in prepubertal monkeys, whereas it increased LHRH release only slightly in pubertal monkeys (7); and 3) GABA infusion suppressed LHRH release in pubertal, but not in prepubertal monkeys (7). Subsequently, we have shown that infusion of antisense oligodeoxynucleotides for glutamic acid decarboxylase (GAD) mRNAs into the S-ME of prepubertal monkeys stimulated LHRH release (8). GAD is the catalytic enzyme for GABA synthesis from glutamate, and there are two forms of GAD (GAD67 and GAD65) with different molecular weights (67 kDa and 65 kDa, respectively) derived from two respective genes (9, 10). The antisense oligodeoxynucleotides presumably interfered with GAD synthesis, leading to a decrease in GABA synthesis and release, and resulting in the increase in LHRH release (8). Input from glutamatergic neurons has also been postulated as an important factor for the pubertal increase in LH release and in LHRH release (3, 11–13). N-methyl-d-aspartate (NMDA), a stimulant for the glutamate receptor NMDA subtype, or glutamate, induces LHRH release in prepubertal Received June 10, 1998. Address all correspondence and requests for reprints to: Ei Terasawa, Ph.D., Wisconsin Regional Primate Research Center, 1223 Capitol Court, Madison, Wisconsin 53715-1299. E-mail: [email protected]. * This study (publication number 38-012 from the Wisconsin Regional Primate Research Center) was supported by NIH Grants HD-11355, HD-15433, and RR-00167. † Present address: State University of New York-Morrisville, Morrisville, New York 13408. ‡ Present address: University of Tokyo, School of Veterinary Medicine, Tokyo, Japan. 0013-7227/99/$03.00/0 Vol. 140, No. 2 Endocrinology Printed in U.S.A. Copyright © 1999 by The Endocrine Society