Effects of pulsatile infusion of the GABA(A) receptor blocker bicuculline on the onset of puberty in female rhesus monkeys.

In order to test the hypothesis that GABA is an inhibitory neurotransmitter restricting the release of LHRH before puberty, we examined the effects of pulsatile infusion of the GABA(A) receptor blocker, bicuculline, on the timing of puberty. Eleven female monkeys at 14-15 months of age were implanted with a stainless steel cannula into the base of the third ventricle above the median eminence. Five monkeys received bicuculline infusion every 2 h at a dose of 1 microM with a gradual increase to 100 microM in 10 microl using a portable infusion pump. The remaining 6 monkeys received similar infusions of saline. An additional 11 colony monkeys without cannula implantation were used for controls. Results indicate that bicuculline infusion advances the timing of puberty. The age of menarche (17.8+/-0.5 months) in the bicuculline infusion animals was significantly earlier than that in the saline controls (28.2+/-2.3, P < 0.001) as well as in colony controls (30.6+/-0.9, P < 0.001). The age of first ovulation (30.5+/-3.3 months) in bicuculline-treated animals was much younger (P < 0.001) than that in both controls (44.8+/-1.8 and 44.7+/-1.2, respectively). Bicuculline also accelerated the growth curve. These results suggest that the reduction of tonic GABA inhibition of LHRH neurons advances the onset of puberty.

Role of glutamic acid decarboxylase in the prepubertal inhibition of the luteinizing hormone releasing hormone release in female rhesus monkeys.

To investigate further the role of GABA in the onset of puberty, this study examines whether glutamic acid decarboxylase (GAD), the catalytic enzyme for GABA synthesis, is involved in the suppression of luteinizing hormone releasing hormone (LHRH) before puberty in rhesus monkeys. First, both GAD67 and GAD65 mRNAs were detectable by reverse transcription-PCR analysis in the preoptic area, medio-basal hypothalamus, posterior hypothalamic area, and hippocampus of the monkey brain. Second, effects of antisense oligodeoxynucleotides (D-oligos) for GAD67 and GAD65 mRNAs on LHRH release were examined in conscious female rhesus monkeys at the prepubertal stage using a push-pull perfusion method. The GAD67 or GAD65 antisense D-oligos or scrambled D-oligos were infused directly into the stalk-median eminence. Both the GAD67 and the GAD65 antisense D-oligos induced a large and prompt increase in LHRH release, whereas the scrambled D-oligos did not induce any significant effect. The results suggest that the removal of GABA inhibition by interfering with GAD synthesis is effective in increasing LHRH release in prepubertal monkeys. Third, the specificity of the antisense D-oligos on GAD levels was examined by incubating basal hypothalami with D-oligos in vitro and subsequent Western blot analysis. The antisense D-oligos consistently decreased the proteins GAD67 and GAD65 compared with respective control D-oligos. We conclude that the decrease of tonic GABAergic inhibition and maturational changes in GAD synthesis may be critical factors for the onset of puberty in nonhuman primates.

A primary cell culture system of luteinizing hormone releasing hormone neurons derived from embryonic olfactory placode in the rhesus monkey.

The purpose of this study is to establish a primary LHRH cell culture system using embryonic olfactory placode and to examine whether LHRH cells derived from olfactory placode and the migratory pathway of LHRH neurons mature in vitro. Six monkey fetuses at the ages of E34-E36 were delivered surgically and the area including the olfactory placode (PL) and the areas that encompass the migratory pathway (MP) were dissected out. The tissues were cut into small pieces and plated on collagen- or poly-L-lysine-coated glass coverslips in medium M199. Cultures were maintained for up to 33 days and immunostained for LHRH, GnRH-associated peptide, neurofilament protein, neuron-specific enolase, and glial fibrillary acidic protein. LHRH positive cells were also positive for neurofilament proteins neuron-specific enolase, and GnRH-associated peptides, but negative for glial fibrillary acidic protein. In the first week of culture, LHRH cells remained within the explants of PL, were rounded (average dimensions: 13.0 x 11.3 microns) and stained lightly. By the second week a number of LHRH cells (15.7 x 13.6 microns) with neurites started to migrate out from PL explants, whereas some still remained in the PL. By the third week a large number of LHRH cells (19.3 x 9.4 microns) had migrated out from the PL. They were fusiform in shape with clear nuclei and extended long varicose neurites up to 500 microns in length. A few "pioneer" LHRH cells appeared to lead the migration of 100-400 LHRH cells forming 1-3 major migratory paths. In contrast, LHRH cells from MP explants migrated out sooner than those from PL explants. LHRH cells from the ventral part of the MP, which is close to the PL, migrated out by 1-2 weeks and formed several migratory paths, whereas LHRH cells from the dorsal part of the MP, which is farther from the PL, were scattered widely around explants and their neurites were extended tortuously. Cultured LHRH cells released LHRH into the media and responded to challenge with high K+. The results indicate that 1) primary LHRH neurons can be obtained from the embryonic PL and their migratory pathway, 2) these neurons migrate and mature in culture and 3) they are accessible for cellular and molecular studies.