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Endocrinology Vol. 140, No. 2 705-712
Copyright © 1999 by The Endocrine Society

ARTICLES

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 Release1

Etsuko Kasuya, Christopher L. Nyberg2, Kazutaka Mogi3 and Ei Terasawa

Wisconsin Regional Primate Research Center (E.K., C.L.N., K.M., E.T.) and Department of Pediatrics (E.T.), University of Wisconsin-Madison, Madison, Wisconsin 53715-1299

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: terasawa{at}primate.wisc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 µM) 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 µM) on LHRH release were tested in monkeys treated with AS GAD67. MK801 infusion into the S-ME during the treatment of AS GAD67 (1 µM) 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CONCEPT THAT an increase in pulsatile LHRH release is critical for the onset of puberty in nonhuman primates has been well established (1, 2, 3, 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 {gamma}-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, 12, 13). N-methyl-D-aspartate (NMDA), a stimulant for the glutamate receptor NMDA subtype, or glutamate, induces LHRH release in prepubertal and pubertal rat hypothalami in vitro (11, 14, 15), LH and LHRH release in vivo in prepubertal as well as in pubertal monkeys (16, 17, 18, 19, 20), and administration of NMDA induced precocious puberty in female rats and male monkeys (21, 22), whereas treatment with MK801, a specific antagonist of NMDA receptors, delayed the timing of puberty in female rats (23).

Because both GABA and glutamate are major neurotransmitters for inhibitory and excitatory signals in the hypothalamus, respectively, it is possible that an increase in glutamate tone is associated with the decrease in GABA tone at the onset of puberty. To understand the role of GABA and glutamate in puberty, therefore, we conducted two experiments. In Study I we examined whether GABA inhibition continues after the onset of puberty by testing the effects of an antisense oligodeoxynucleotide for GAD67 mRNA (AS GAD67) on LHRH release in both prepubertal and pubertal monkeys using a push-pull perfusion method in vivo. Because in the previous study (8) AS GAD67 induced substantially larger effects than AS GAD65, we used AS GAD67 in this study. 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 on LHRH release were tested in monkeys treated with AS GAD67.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All female rhesus monkeys (Macaca mulatta) in this study were born and raised at the Wisconsin Regional Primate Research Center (Madison, WI). Animals at two developmental stages were used: prepubertal stage (18.5 ± 0.4 months of age) and pubertal stage (34.3 ± 1.5 months of age). Monkeys were housed in pairs under controlled lighting conditions (12 h light, 12 h dark) with temperature maintained at 22 C. Monkeys were fed a standard diet of Purina Monkey Chow daily, supplemented with fresh fruit several times per week. Water was available ad libitum. The protocol for this study was reviewed and approved by the Animal Care and Use Committee, University of Wisconsin, and all experiments were conducted under the guidelines established by the NIH and USDA.

Push-pull perfusion
The push-pull perfusion method was very similar to that described elsewhere (24, 25). A cranial pedestal was implanted on the skull under isoflurane anesthesia. The third ventricle and bone structures were visualized with x-ray ventriculograms before pedestal implantation. The monkeys were allowed to recover for at least 4 weeks before the initiation of push-pull perfusion experiments. Monkeys were adapted to a primate chair, the experimental environment, and the investigators.

Three days before the push-pull perfusion, the animals were anesthetized with ketamine (10 mg/kg BW) and xylazine (1–2 mg), and were placed in a stereotaxic apparatus for cannula implantation. An outer cannula (20 gauge) with a stylet (27 gauge) was inserted into the S-ME using a microdrive unit (MO95-B, Narishige, Tokyo, Japan). x-ray ventriculograms at the time of pedestal implantation were used as reference points for the placement of the cannula tip. After cannula implantation, the monkey was placed in a primate chair. On the day of the perfusion experiment, the stylet was replaced with an inner cannula (29 gauge) and infusion was started. A modified Krebs-Ringer phosphate buffer solution (artificial CSF) was infused using a peristaltic pump (Minipulse 3, Gilson Electronic, Middleton, WI) into the S-ME through the push cannula at a rate of 23 µl/min, while perfusates were collected on ice through the pull cannula using an identically calibrated pump. Perfusate samples were collected at 10-min intervals, and each 150 µl of sample was aliquoted into a vial and stored at -70 C until assayed for LHRH.

Experimental design
Study I. Previously we showed that infusion of an antisense oligodeoxynucleotide for GAD67 mRNA (AS GAD67) into the S-ME of prepubertal monkeys resulted in a large increase in LHRH release. To determine whether AS GAD67 also increases LHRH release in pubertal monkeys (34.8 ± 1.9 months), AS GAD67 was infused into the S-ME using a push cannula, whereas perfusate samples were collected through a pull cannula. The results were then compared with those in prepubertal monkeys (18.7 ± 0.6 months). Since in a previous study (8) AS GAD67 induced substantially larger effects on LHRH release than AS GAD65, we used AS GAD67 in this study. The methods for AS GAD67 infusion were similar to those described previously (8). Based on DNA sequence of monkey GAD67 (8), AS GAD67 (5'-GAA GAT GGG GTC GAA GAC GC-3') and an oligodeoxynucleotide that contains the same bases in scrambled sequence (SC GAD67, 5'-TAG GAG CAG ACT GAG AGG CG-3') for control were synthesized at the Biotechnology Center, University of Wisconsin-Madison (Madison, WI). The oligodeoxynucleotides were desalted and resuspended in artificial CSF under sterile conditions. After 4 h of control perfusion, AS GAD67 or SC GAD67 (1 µM) was continuously infused into the S-ME for 5 h, which was followed by an additional 5 h of control perfusion. LHRH levels in the perfusate samples were measured by RIA.

Study II. There is a possibility that an increase in glutamate tone after the reduction of an inhibitory GABA tone contributes to the AS GAD67-induced LHRH increase. To test this possibility, the effects of the NMDA receptor blocker, MK801 (5 µM, Research Biochemicals International, Natick, MA), on LHRH release were examined in monkeys treated with AS GAD67. Five micromolars of MK801, a noncompetitive NMDA receptor antagonist, was infused simultaneously with AS GAD67 into the S-ME of prepubertal (18.9 ± 0.5 months) and pubertal (33.6 ± 2.4 months) monkeys. The infusion protocol was similar to that in Study I: after 4 h of control infusion, artificial CSF containing MK801 (5 µM) and AS GAD67 (1 µM) was infused for 5 h, followed by additional 5 h of control perfusion. As a control, MK801 in artificial CSF was similarly infused. LHRH levels in the perfusates were determined by RIA. We conducted studies I and II concurrently, so that the effects of MK801 plus AS GAD67 could be compared with those of AS GAD67 alone in each age group.

LHRH RIA
LHRH in perfusates (150 µl) was measured by RIA using antiserum R1245, kindly provided by Dr. T. Nett (Colorado State University, Fort Collins, CO), as described previously (24). Synthetic LHRH (Richelieu Biotechnologies Inc., Montréal, Québec, Canada) was used for the radiolabeled antigen and reference standard. The antigen-antibody complex was precipitated with a sheep antirabbit {gamma}-globulin. Sensitivity of the assay was 0.1 pg/tube, and intra and interassay coefficients of variation were 11.7% and 15.7%, respectively.

Data analysis
The effects of AS GAD67 on mean LHRH levels and the effects of MK801 on the AS GAD67-induced LHRH release were determined by 2-way ANOVA for repeated measures, followed by post hoc analysis with the Student-Newman-Keuls’ test. For statistical analysis the data during the first hour of the experiment was not included because high LHRH levels were sometimes observed during the initial period of infusion. Mean LHRH levels in each hour period before the oligodeoxynucleotide infusion were compared with those in each hour period during and after the infusion of the oligodeoxynucleotides. In Study I, hourly mean values of AS GAD67 were compared with corresponding hourly mean values of SC GAD67 within the same age group. Hourly mean values of AS GAD67 between the two age groups were also compared. Further, the magnitude of the LHRH response to AS GAD67 between prepubertal and pubertal groups was compared by calculating the difference between mean LHRH levels during the control period and those during and after AS GAD67 infusion. In Study II, hourly mean values of the AS GAD67 and MK801 treatment were compared with those of the MK801 treatment alone. In addition, hourly mean values of AS GAD67 treatment with MK801 from Study II were compared with corresponding data of AS GAD67 from Study I because the two studies were conducted concurrently. For graphic expression, normalized data are used: in each animal, the mean LHRH levels in the 3-h period before the oligodeoxynucleotide infusion was designated as 100%, and the mean LHRH levels in each 1-h period before and after the initiation of the oligodeoxynucleotide infusion were calculated accordingly. Statistical significance was attained at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of AS GAD67 on LHRH release in prepubertal and pubertal monkeys (Study I)
The direct infusion of AS GAD67 into the S-ME resulted in an increase in LHRH release in both prepubertal and pubertal monkeys (Figs. 1Go and 2Go). An increase in LHRH release started 2–3 h after the initiation of AS GAD67 infusion and lasted until after the termination of AS GAD67 infusion in prepubertal (Fig. 1AGo) and in pubertal monkeys (Fig. 1BGo). However, the AS GAD67-induced LHRH release in pubertal monkeys was consistently smaller than in prepubertal monkeys. SC GAD67 infusion did not cause any significant effects on LHRH release in either prepubertal or pubertal animals (Fig. 1Go, C and D).



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  		Figure 1. Effects of an antisense oligodeoxynucleotide for GAD67 mRNA (AS GAD67, 1 µM) on LHRH release in prepubertal (A) and pubertal (B) female rhesus monkeys. Representative cases from each group are shown. AS GAD67 was directly infused into the S-ME for the period indicated by shading, whereas perfusates were continuously collected. In contrast, the infusion of a scrambled oligodeoxynucleotide for GAD67 mRNA (SC GAD67, 1 µM) did not cause any significant effect on LHRH release in both prepubertal (C) and pubertal (D) monkeys. Note that the magnitude of the AS GAD67-induced LHRH increase in prepubertal monkeys is larger than in pubertal monkeys.

 


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  		Figure 2. Effects of AS GAD67 (1 µM) on LHRH release in prepubertal (A, n = 12) and pubertal (B, n = 8) monkeys and effects of SC GAD67 on LHRH release in prepubertal (C, n = 4) and pubertal (D, n = 8) monkeys. AS or SC GAD67 was infused for 5 h as indicated by shading. Hourly mean LHRH values in percent were calculated from the mean LHRH levels during the 3 h period before AS or SC GAD67 infusion. There was a significant treatment effect by time (P < 0.01 for both) for AS GAD67 vs. SC GAD67 in both age groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. mean LHRH levels before the AS GAD67 or SC GAD67 infusion (within treatment comparison); {dagger}, P < 0.05 and {dagger}{dagger}, P < 0.01 vs. corresponding hourly mean values of SC GAD67 between treatments in the same developmental stage. Note that the magnitude of the AS GAD67-induced LHRH increase in prepubertal monkey is significantly (P < 0.05) larger than in pubertal monkeys.

 
Comparisons of hourly means between AS GAD67 and SC GAD67 groups revealed that hourly means in the AS GAD67 treatment were significantly higher than those in the SC GAD67 treatment in both prepubertal (P < 0.01, Fig. 2Go, A and B) and pubertal monkeys (P < 0.01, Fig. 2Go, C and D). The post hoc analysis further indicated that, in prepubertal monkeys, an increase (P < 0.01 to 0.05) in mean LHRH release occurred during the second hour after the initiation of AS GAD67 infusion, and the increase lasted until 5 h after the termination of the antisense infusion when compared with the preinfusion levels (Fig. 2AGo). Similarly, in pubertal monkeys, an increase (P < 0.01 to 0.05) in mean LHRH release started during the third hour after the initiation of AS GAD67 infusion, and the increase lasted for the entire period of the experiment (Fig. 2BGo). Although the difference between the hourly mean LHRH over time in pubertal monkeys (Fig. 2BGo) vs. prepubertal monkeys (Fig. 2AGo) did not attain statistical significance (P = 0.08), the LHRH increase (7.6 ± 1.2 pg/ml) in response to AS GAD67, calculated by the difference in LHRH levels during/after AS GAD67 infusion and before AS GAD67 treatment, in the prepubertal group were significantly (P < 0.01) larger than that (3.2 ± 0.6 pg/ml) in the pubertal group.

Effects of MK801 on the AS GAD67-induced LHRH release in prepubertal and pubertal monkeys (Study II)
Simultaneous infusion of MK801 with AS GAD67 suppressed the AS GAD67-induced LHRH increase in both prepubertal (Fig. 3AGo) and pubertal (Fig. 3BGo) monkeys. Infusion of MK801 alone did not induce any significant changes in LHRH release in prepubertal monkeys (Fig. 3CGo). However, in pubertal monkeys MK801 infusion suppressed LHRH release in five of eight cases, as shown in one example in Fig. 3DGo, whereas MK801 failed to change LHRH release in the remaining three cases.



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  		Figure 3. Effects of MK801 (5 µM) on the AS GAD67-induced LHRH release in prepubertal (A) and pubertal (B) female rhesus monkeys. Representative cases from each group are shown. MK801 was infused simultaneously with AS GAD67 for 5 h, as indicated by shading. Note that MK801 suppressed the AS GAD67-induced LHRH release. In contrast, MK801 (5 µM) infusion alone did not result in any significant effects on LHRH release in prepubertal monkeys (C). In midpubertal monkeys MK801 alone suppressed LHRH release in some cases, as shown in (D), although in other cases MK801 did not cause any significant effects.

 
Suppression of the AS GAD67-induced LHRH increase by MK801 infusion was significant in both prepubertal (P < 0.01, Fig. 4AGo) and pubertal (P < 0.01, Fig. 4BGo) monkeys. Infusion of MK801 alone did not induce any significant changes in LHRH release in prepubertal monkeys (Fig. 4CGo) or in pubertal monkeys (Fig. 4DGo). The overall mean LHRH levels of the MK801 plus AS GAD67 treatment were not different from that of the MK801 treatment in prepubertal (Fig. 4Go, A and C) and pubertal monkeys (Fig. 4Go, B and D). However, LHRH release in prepubertal monkeys treated with MK801 plus AS GAD67 (Fig. 4AGo) was significantly (P < 0.01) lower than in prepubertal monkeys treated with AS GAD67 alone (Fig. 2AGo). Similarly, LHRH release in the pubertal monkeys treated with MK801 plus AS GAD67 (Fig. 4BGo) was lower (P < 0.01) than in pubertal monkeys treated with AS GAD67 alone (Fig. 2BGo).



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  		Figure 4. Effects of MK801 (5 µM) on the AS GAD67-induced LHRH release in prepubertal (A, n = 6) and pubertal (B, n = 8) female rhesus monkeys, and effects of MK801 (5 µM) alone on LHRH release in prepubertal (C, n = 4) and pubertal (D, n = 7) monkeys. MK801 with or without AS GAD67 was infused for 5 h as indicated by shading. Hourly mean LHRH values in percent were calculated from the mean LHRH levels during the 3 h period before MK801 infusion. Note that the AS GAD67-induced LHRH increase, shown in Fig. 2Go, was absent with MK801 treatment in both prepubertal and pubertal monkeys. MK801 alone did not cause significant changes in LHRH release. In both age groups there was a significant treatment effect by time when compared with AS GAD67 plus MK801 vs. GAD67 alone (P < 0.01 for both). a, P < 0.05; aa, P < 0.01 vs. mean LHRH release in AS GAD67.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The findings of the present study are summarized as follows. First, the direct infusion of AS GAD67 into the S-ME stimulated LHRH release in female rhesus monkeys at the pubertal stage, although the magnitude of the response was much smaller than that in monkeys at the prepubertal stage. Second, the infusion of MK801, a noncompetitive NMDA receptor antagonist, suppressed AS GAD67-induced LHRH release in both prepubertal and pubertal monkeys. These results are interpreted to mean that the inhibitory effect of GABA on LHRH release continues after the onset of puberty but is weakened considerably after the onset of puberty, and that the increased LHRH release with AS GAD67 may be partly due to an increase in glutamate tone, mediated by NMDA receptors, in addition to the decrease in GAD synthesis and subsequent decrease in GABA release.

In Study I, we observed that AS GAD67 stimulated LHRH release in pubertal as well as prepubertal monkeys. The effects of AS GAD67 on LHRH increase in prepubertal monkeys in this study started earlier and lasted longer than that observed in a previous study conducted in our laboratory (8). It is unclear why this difference occurred because the infusion protocol was essentially similar, except that the period of AS GAD67 infusion was 1 h shorter in this study than in the previous study. Nonetheless, the LHRH increase induced by AS GAD67 in this study is consistent with what we reported previously (8). In our previous study, we also found with Western blot analysis that AS GAD67 reduced GAD67 protein (8). Preliminary data further suggest that GABA levels decreased after AS GAD67 infusion (26). Therefore, infusion of AS GAD67 into the S-ME probably interfered with GAD67 synthesis, effectively reducing GABA release.

In pubertal monkeys treated with AS GAD67, a significant LHRH increase occurred 1 h later than in prepubertal monkeys, and the amount of the LHRH increase induced by AS GAD67 infusion in pubertal monkeys was smaller than that in prepubertal monkeys. These results suggest that there is an age-dependent difference in LHRH increase in response to AS GAD67. Although whether the presence of ovarian steroids in pubertal monkeys is due to the smaller LHRH response to AS GAD67 remains to be determined, the data can be interpreted to mean that GABA inhibition is considerably reduced in pubertal monkeys when compared with prepubertal monkeys. This view is supported by the fact that GABA levels in the S-ME of early and midpubertal monkeys were lower than those in prepubertal monkeys (7) and that the bicuculline-induced LHRH increase was much smaller in pubertal monkeys than in prepubertal monkeys (7). Therefore, inhibition of LHRH release by GABA in the S-ME "weakens" at the onset of puberty, but it remains even after the initiation of puberty.

In Study II we found that the AS GAD67-induced increase in LHRH release was suppressed by the NMDA receptor blocker, MK801. The results indicate that the AS GAD67-induced LHRH increase is not only due to the reduction of GABA tone in the S-ME, but also an increase in glutamate tone in the S-ME. In fact, preliminary data from our laboratory (26) suggest that glutamate increases following the decrease in GABA with treatment of AS GAD67. There are several explanations for the reduction of GABA tone resulting in the increase in glutamate tone. First, the developmental reduction of GABA release results in an increase in glutamate release by a presynaptic mechanism. This possibility is supported by an observation in monkeys that the perikarya of LHRH neurons are directly innervated by glutamate neurons, not GABA neurons, but glutamate neurons are innervated by GABA neurons (27). Second, the reduction of GABAergic input may allow an increase in the relative strength of excitatory glutamatergic input to LHRH neuroterminals, via a nonsynaptic mechanism, such as by a volume transmission (28). It has been shown that glutamate neuroterminals abut LHRH neuroterminals in the ME of rats (29), and nonsynaptic transmission could be mediated by nitric oxide (30, 31). Third, developmental reduction of GADs may lead to the accumulation of metabolic glutamate in GABA neurons because glutamate is a major precursor for GABA synthesis. Accumulated glutamate could be transported out of the cell resulting in excitation of the postsynaptic membrane. In this case, glutamate would not be released by Ca2+-dependent vesicular exocytosis, but would be released by a mechanism involving a Na+-dependent glutamate transporter. Such mechanisms have been reported in other types of neurons (32). Finally, a small elevation of estradiol, as a consequence of an increase in LHRH release due to GABA reduction, may increase glutamate release as well as receptor sensitivity to glutamate/NMDA, which in turn further increases glutamate tone.

It has been reported that GABA is inhibitory to LHRH and LH release in adult rats (33, 34, 35, 36, 37), whereas GABA is stimulatory in juvenile rats (38, 39). Moreover, Moguilevsky and his colleagues (40) reported that 1) GABA agonists (muscimol for GABAA and baclofen for GABAB) stimulated glutamate release as well as LHRH release in prepubertal male rats, but they inhibited both glutamate and LHRH release in adults, and 2) GABA antagonists (bicuculline for GABAA and phaclofen for GABAB) inhibited glutamate release as well as LHRH release in prepubertal male rats, whereas they stimulated both LHRH and glutamate release in adults. For the explanation of this paradoxical effect of GABA on LHRH release during sexual development, Bourguignon and his colleagues (41) postulate a reciprocal innervation between GABAergic and glutamatergic neurons. That is, during the juvenile period, inhibitory GABA neurons suppress glutamate neurons, which control LHRH neurons as well as GABA neurons, whereas in adults, reduction of GABA inhibition to glutamate neurons occurs, resulting in an increase in LHRH release.

The mechanism initiating puberty in rodents, represented by the studies in rats, appears to differ from that in primates. First, in monkeys, castration induces an elevation of gonadotropin release during the neonatal period and after the onset of puberty, but not during the juvenile period before the onset of puberty (42, 43), whereas in rats the castration-induced elevation of gonadotropin release occurs from the neonatal period throughout life (12). Second, NMDA-induced precocious puberty in monkeys does not lead to normal reproductive cycles as adults after cessation of the NMDA infusion (22), whereas in rats NMDA-induced precocious puberty continues with ovulatory cycles (21). Third, in primates there is a tonic GABA inhibition on LHRH release during the juvenile period, which is expressed by high GABA levels in the S-ME and an enhanced stimulatory effect of bicuculline on LHRH release (7). Moreover, as observed in this study, in primates the reduction of GABA inhibition appears to allow the activation of the excitatory glutamatergic system, which further contributes to increase LHRH release. In contrast, in rats comparable tonic central inhibition may not exist because bicuculline is inhibitory to LHRH release in the juvenile stage, and this bicuculline effect is reversed during the pubertal stage (40, 41). Thus, establishment of glutamatergic and other facilitatory neuronal systems may be more important for the onset of puberty in rats. Nonetheless, Bourguignon and colleagues (44) recently reported that treatment of an 11-month-old child who exhibited severe epileptic seizures and precocious puberty with the GABA agonists loreclezole and vigabatrin regressed all signs of precocious puberty, as well as seizure attacks, indicating that tonic GABA inhibition plays a key role in low levels of LHRH release before the onset of puberty in primates.

In summary, there is a tonic GABA inhibition of LHRH release before the onset of puberty in primates. At the onset of puberty, GABA inhibition declines, but is not completely removed. The reduction of GABA inhibition appears to be followed by a concurrent increase in glutamate tone. Subsequently, norepinephrine and neuropeptide Y neurons may also contribute to the progress of puberty (45, 46, 47). Because GAD is the enzyme controlling GABA synthesis, GAD may play an important role in the onset and progress of puberty.


   Acknowledgments
 
The authors would like to thank Laurelee Luchansky, Kim L. Keen, Fritz Wegner, and Dennis Mohr for their technical assistance, Harold Pape for animal care, Drs. Carol Emerson, Christine O’Rourke and Jan Ramer for their veterinary care, and Dr. David Fernandez for the comments on this manuscript.


   Footnotes
 
1 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. Back

2 Present address: State University of New York-Morrisville, Morrisville, New York 13408. Back

3 Present address: University of Tokyo, School of Veterinary Medicine, Tokyo, Japan. Back

Received June 10, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wildt L, Marshall G, Knobil E 1980 Experimental induction of puberty in the infantile female rhesus monkey. Science 207:1373–1375
  2. Terasawa E, Claypool L, Watanabe G, Gore A 1989 The timing of the onset of puberty in the female rhesus monkey. In: Delemarre-Van de Waal HA, Plant TM, van Rees GP, Shoemaker J (ed) Control of the Onset of Puberty III. Elsevier, Amsterdam, pp 123–136
  3. Plant TM 1994 Puberty in primates. In: Knobil E, Neill JD (ed) The Physiology of Reproduction. Raven Press, New York, pp 453–485
  4. Terasawa E 1995 Mechanisms controlling the onset of puberty in primates: the role of GABAergic neurons. In: Plant TM, Lee PA (eds) The Neurobiology of Puberty. Journal of Endocrinology Ltd., Bristol, pp 139–151
  5. Watanabe G, Terasawa E 1989 In vivo release of luteinizing hormone-releasing hormone increases with puberty in the female rhesus monkey. Endocrinology 125:92–99[Abstract]
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