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Endogenous GABA Release Inhibits the Firing of Adult Gonadotropin-Releasing Hormone Neurons

Laboratory of Neuroendocrinology (S.-K.H., M.G.T., A.E.H.), The Babraham Institute, Cambridge CB2 4AT, United Kingdom; and Centre for Neuroendocrinology and Department of Physiology (S.-K.H., A.E.H.), University of Otago School of Medical Sciences, Dunedin, New Zealand

Address all correspondence and requests for reprints to: Allan E. Herbison, Department of Physiology, University of Otago School of Medical Sciences, P.O. Box 913, Dunedin, New Zealand. E-mail: allan.herbison{at}stonebow.otago.ac.nz.

Abstract

The effect of endogenous -aminobutyric acid (GABA)_A receptor-mediated signaling on the excitability of adult male and female GnRH neurons was examined using gramicidin perforated-patch electrophysiology in GnRH-LacZ and GnRH-GFP (green fluorescent protein) transgenic mouse models. In both lines of mice, approximately 80% of GnRH neurons (n = 42) responded to the selective GABA_A receptor antagonist bicuculline (20 µM) with a rapid and reversible membrane depolarization and/or increase in firing rate. Approximately 16% of GnRH neurons gave no response, and two neurons were inhibited by bicuculline. The same depolarizing responses (78%) were obtained from adult gonadectomized GnRH-GFP mice. The depolarizing response to bicuculline persisted in the presence of tetrodotoxin, demonstrating that even action potential-independent GABA release was acting to reduce GnRH neuron membrane potential. These observations show that endogenous GABA signaling through the GABA_A receptor exerts a powerful net inhibitory effect upon the excitability of mature GnRH neurons.

THE AMINO ACID neurotransmitters -aminobutyric acid (GABA) and glutamate are thought to provide the principal neuronal inputs to GnRH neurons (1), and an understanding of the nature of their influence upon these cells is critical. In vivo experiments undertaken in adult rodents, sheep, and primates all show that GABA acts through GABA_A receptors within the vicinity of the GnRH neuron soma to inhibit LH secretion (2, 3, 4, 5, 6, 7). Because GnRH neurons express functional GABA_A receptors (8, 9), the inhibitory effect of GABA is thought to result from the direct inhibition of GnRH neuron excitability. With the recent ability to undertake electrophysiological recordings from GnRH neurons in the mouse, two laboratories have recently provided conflicting accounts of direct GABA_A receptor-mediated GABA actions on adult GnRH neurons. Using gramicidin perforated-patch electrophysiology and a GnRH-LacZ transgenic model, we reported that GABA exerts depolarizing actions upon GnRH neurons up until the time of puberty when, thereafter, this switches to a hyperpolarizing response (10). DeFazio et al. (11), using the same patch-clamp approach with a GnRH-GFP (green fluorescent protein) transgenic mouse, reported that GABA exerted consistent depolarizing effects on GnRH neurons throughout postnatal development. Although the reasons for the discrepancy between the two studies are not known, the use of different transgenic mouse models may be important. Another is the concentration and application method of GABA; whereas we used prolonged (1 min) bath applications of 10–100 µM GABA, DeFazio et al. (11) used short puffs of 1 mM GABA.

Although the above two studies provided an appropriate start to the investigation of the role of GABA in GnRH neuron regulation, it is readily apparent that the sudden application of large amounts of exogenous GABA onto a cell lacks physiological relevance. Individual neurons receive a highly organized array of different GABAergic inputs, and each terminal releases GABA in both a precise, temporally regulated phasic manner, and a low level tonic fashion (12). Furthermore, within the individual neuron, the effect of discrete GABA_A receptor activation in any one dendritic or somal domain is very likely to depend upon the ongoing activity of surrounding glutamatergic synapses (13). Also, the chloride and bicarbonate ion flow through the GABA_A receptor is often delicately balanced and high concentrations of exogenous GABA can easily collapse ion gradients (14, 15). Thus, we reasoned that a better way to evaluate the physiological role of GABA actions upon adult GnRH neuron excitability would be to block endogenous GABA_A receptor signaling and observe the effects upon GnRH neuron excitability. To do this, we used the selective antagonist bicuculline at a concentration (20 µM) known to block all GABA_A receptor activity in GnRH neurons (9). The great advantage of antagonist over agonist studies is that the effects of on-going, endogenous GABA signaling upon GnRH neurons can be evaluated. To address the important possibility that different transgenic models may confound observations, we have examined bicuculline effects in both GnRH-LacZ and GnRH-GFP transgenic mice.

Materials and Methods

Animals
All transgenic mice [5.5-GNLZ-3.5 (Ref. 10) and GnRH-EGFP-mut5 (see Generation of GnRH-EGFP-mut5 transgenic mice)] were produced at the Babraham Institute under UK Home Office Project license 80/1475 and transferred for breeding to the University of Otago. All experimentation was approved by both The Babraham Institute and University of Otago Animal Welfare and Ethics Committees. Recordings were undertaken at both institutions. Both male and female mice were maintained under 12-h light, 12-h dark conditions (lights on 0700 h) with food and water available ad libitum. Because the effects of exogenous GABA upon GnRH neurons do not appear to depend upon the stage of the estrous cycle (10, 11), the female mice used in the study were not tested at any specific stage of the cycle. However, to be sure that gonadal steroids are not critical in either sex, a further group of mice were gonadectomized 2 wk before experimentation.

Generation of GnRH-EGFP-mut5 transgenic mice.
The V163A/S175G and I167T mutations characterized in mGFP5 constructs (16) were incorporated into a humanized enchanced GFP (EGFP) (CLONTECH, Palo Alto, CA) to create EGFP-mut5 (Gilthorpe, J., University College London, unpublished). The EGFP-mut5 sequence was initially subcloned between a 5' synthetic intron (ß-globin/IgG) and a 3' polyadenylation signal (late simian virus 40). To drive EGFP-mut5 within GnRH neurons, we used a previously reported GnRH genomic clone (17) that contained the entire GnRH transcription unit, 5.5 kb of 5' sequence, 3.5 kb of 3' sequence, a mutated GnRH start codon and a unique SmaI site within GnRH exonII, into which we subcloned the intron/EGFPmut5/polyadenylation sequence. Pronuclear injection was carried out using an approximately 12 kb SalI/Eco91I fragment. A single expressing founder mouse was identified and heterozygous offspring (CBA/Ca X C57BL6/J) were identified by PCR. Fluorescence immunocytochemistry using the LR1 polyclonal rabbit antisera was undertaken as described previously (17) to evaluate the relationship between GFP expression and the GnRH neurons.

Electrophysiology.
Gramicidin-perforated-patch clamp electrophysiology was undertaken as reported previously (10) with the exception that brains were blocked and glued with cyanoacrylate to the chilled stage of a vibratome (VT1000S, Leica, Nussloch, Germany) and 200-µm-thick coronal slices containing the medial septum and preoptic area prepared. The gramicidin perforated-patch approach, that maintains intact chloride ion homeostatsis, is the only experimental option available for examining the role of the GABA_A receptor in regulating neuronal membrane potential and firing rate (18). Slices were viewed with a fixed-stage upright microscope (BX51WI, Olympus, Tokyo, Japan) with x5 or x40 achromat objectives (MPL5X and LUMPLFL40XW/IR, Olympus) and either fluorescence illumination using the reflected light fluorescence illuminator (BX-RFA, Olympus), filter (U-MWIBA2, BA510–550, Olympus) or Nomarski differential interference contrast optics. Gramicidin (Sigma, St. Louis, MO) was first dissolved in dimethylsulfoxide (Sigma) to a concentration of 2.5 mg/ml and then diluted in the pipette solution [in mM; 130 KCl, 5 NaCl, 0.4 CaCl₂, 1 MgCl₂, 10 HEPES, 1.1 EGTA (pH 7.3)] just before use to a final concentration of 2.5 µg/ml and sonicated for 15 min. After cell attachment, access resistance was monitored and experiments begun when access resistance stabilized at 60–100 M. This typically required 5–20 min with bicuculline then being tested upon the cell within 20 min of pore formation. The gramicidin-perforated-patch clamp recordings were performed using a Multiclamp 700A (CV7B, Axon Instruments, Foster City, CA). The tip resistance of the electrodes was 4–7 M. Spontaneous activities were sampled online using a Digidata 1322A interface (Axon Instruments) connected to an IBM personal computer. Signals were filtered (10 kHz, Bessel filter of Multiclamp 700A) before digitizing at a rate of 1 kHz. Acquisition and subsequent analysis of the acquired data were performed using the Clampex9 suite of software (Axon Instruments). Bicuculline methiodide and tetrodotoxin citrate (TTX) were purchased from Tocris Cookson (Bristol, UK) and tested by adding to the perfusing ACSF (118 NaCl, 3 KCl, 2.5 CaCl₂, 1.2 MgCl₂, 11 D-glucose, 10 HEPES, 25 NaHCO₃, in mM) at known concentrations. Bicuculline (20 µM) was applied to the bathing solution for 2–5 min. Any cell that displayed a shift in membrane potential of over 2.5 mV or a change in firing rate more than 50% was considered to have responded.

Results

GnRH-EGFP-mut5 mice
Neurons expressing EGFP were found to have a pattern of expression in the brain very similar to that of LacZ-expressing cells in GnRH-LacZ transgenic mice (17). Immunofluorescence studies showed that 100% of bipolar EGFP-expressing neurons in the medial septum and rostral preoptic area expressed GnRH immunofluorescence and this accounted for approximately 60% of all GnRH neurons in the brain. Cells expressing EGFP in the lateral septum were not found to express GnRH immunoreactivity in adult mice but are easily distinguishable from the fluorescent GnRH neurons on the basis of location and morphology (19). No sex differences were observed. Under whole-cell, patch-clamp conditions, fluorescent GnRH neurons displayed properties similar to those of wild-type GnRH neurons (20) with mean firing rates of 0.03–0.52 Hz and resting membrane potentials of -68.1 ± 1.32 mV (n = 8).

Electrophysiology
Gramicidin perforated-patch recordings were obtained from 26 GnRH neurons originating from adult male (n = 15) and female (n = 11) GnRH-EGFP-mut5 mice. Typically, only a single recording was obtained from each mouse so that sample size represents both animal and cell number. Under perforated-patch conditions, the mean resting membrane potential of GnRH neurons in GnRH-EGFP-mut5 mice was -59.3 ± 0.9 mV. Twenty of the 26 GnRH neurons (77%), responded to 20 µM bicuculline with a rapid membrane depolarization and/or an increase in firing rate (Fig. 1A; Table 1). Five GnRH neurons showed no response (Fig. 1C) and a single cell (Fig. 1D) responded with a 2.7 mV hyperpolarization and reduced firing. In the presence of TTX (0.5 µM), which blocked all action potential-dependent transmission, bicuculline continued to elicit membrane depolarization (n = 3 of 3, Fig. 1B). The percentage of GnRH neurons excited by GABA_A receptor blockade was 73% in males and 82% in females (Table 1). In those neurons responding to bicuculline with depolarization, the magnitude of the membrane potential change was not different in males and females (Fig. 2A).

View larger version (43K): [in this window] [in a new window]   FIG. 1. Gramicidin perforated-patch voltage recordings showing the depolarizing (A, B, E–G), absent (C), and hyperpolarizing (D) responses of mature GnRH neurons to the GABAA receptor antagonist bicuculline (Bic). A, Female 67-d-old GnRH-EGFP-mut5 mouse, resting membrane potential (RMP) -60 mV. B, Male 61-d-old GnRH-EGFP-mut5 mouse in the presence of tetrodotoxin (TTX), RMP -62 mV. C, Female 58-d-old GnRH-EGFP-mut5 mouse, RMP -65 mV. D, Male 74-d-old GnRH-EGFP-mut5 mouse, RMP -61 mV. E, Male 60-d-old male GnRH-LacZ mouse, RMP -48 mV. F, Female 43-d-old GnRH-LacZ mouse, RMP -58 mV. G, Male 100-d-old gonadectomized GnRH-EGFP-mut5 mouse, RMP -57 mV; each asterisk represents a more than 20-mV oscillation in membrane potential. Note the remarkable induction of regular 15-sec oscillations by bicuculline.

View larger version (21K): [in this window] [in a new window]   FIG. 2. Histograms comparing sexes and genotype show the mean (+SEM) depolarizing membrane potential change to bicuculline in adult (A) GnRH-EGFP-mut5 males and females and (B) GnRH-LacZ, GnRH-EGFP-mut5 intact (male and female data from A combined) and gonadectomized (GDX) male and female GnRH-EGFP-mut5 mice. The N number is given at the base of each bar.

To be sure that gonadal steroid status was not altering GABA_A receptor functioning in GnRH neurons, we undertook a further series of recordings from nine GnRH neurons obtained from 2 wk-gonadectomized GnRH-EGFP-mut5 mice (four males and five females). Seven of these cells (78%) responded to 20 µM bicuculline with a rapid membrane depolarization and/or an increase in firing rate (Table 1). The degree of membrane depolarization evoked by bicuculline was not different to that observed in GnRH neurons obtained from intact mice (Fig. 2B). One GnRH neuron (female) showed no response, and the other (female) exhibited a rare hyperpolarization to bicuculline (Table 1). One of the GnRH neurons excited by bicuculline displayed a remarkable oscillatory behavior (Fig. 1G). Initially, this neuron displayed erratic, cyclical changes in membrane potential greater than 20 mV in magnitude with interspersed firing. After bicuculline, it demonstrated a dramatic switch to regular 15-sec membrane oscillations and, upon washout, returned to its baseline level of activity (Fig. 1G).

Discussion

We report here that the blockade of endogenous GABA_A receptor signaling results in increased excitability in most adult male and female GnRH neurons. In total, 33 of 42 (79%) GnRH neurons displayed increased electrical excitability after the removal of endogenous GABA_A receptor signaling, with 16% showing no response and two cells (5%) exhibiting decreased excitability. Importantly, we found no differences in the bicuculline responses of GnRH neurons recorded from GnRH-LacZ or GnRH-EGFP transgenic mouse models. The conditions under which GnRH neurons were examined in this study were very similar to those of DeFazio et al. (11) with the exception that we used lower gramicidin concentrations to form the perforated patches (2.5 vs. 10–50 µg/ml) and GnRH neurons in our GnRH-EGFP-mut5 exhibited lower resting membrane potentials of around -60 mV (compared with -50 mV). No effect of gonadectomy or sex differences were detected in the present study, suggesting that endogenous GABA signaling through the GABA_A receptor exerts a net inhibitory action upon the great majority of adult GnRH neurons over a range of GnRH neuron secretory states.

With the exception of the very first investigation by Ondo (21), every subsequent in vivo study undertaken in adult rats, sheep and monkeys has indicated that GABA acts through the GABA_A receptor to inhibit LH secretion (22, 23, 24). Studies examining the role of the GABA_B receptor suggest that major species differences exist, and that GABA_B receptors located in specific regions of the hypothalamus may exert different effects upon LH release (5, 25, 26). Although guinea pig GnRH neurons have been shown to express GABA_B receptors that rapidly inhibit GnRH neurons (27), studies to date in the mouse indicate that rapid GABA signaling at the GnRH neuron soma does not involve any appreciable GABA_B receptor component (8, 9). As before (10), our current findings demonstrate a clear inhibitory role for the GABA_A receptor by showing that endogenous GABA release acts to suppress the electrical excitability of GnRH neurons. Our experiments in the presence of TTX further show that endogenous GABA release is suppressing GnRH neuron firing even under conditions in which action potential-dependent GABA release is blocked. Previous studies have shown that many neuronal populations in the brain are subject to action potential-independent GABA release (28). Over 25% of GABA release in the vicinity of the GnRH cell bodies is thought to be of this nature (29), and we have demonstrated previously that GnRH neurons are subjected to a substantial and continuous barrage of action potential-independent GABA release (9, 20). Because the physiological role of this quantal GABA release is unknown (30), it is intriguing that it should be found here to play a role alongside regular action potential-dependent GABA release in suppressing GnRH neuron excitability.

The GABAergic neurons of the GnRH network appear as key elements of several central hypotheses concerning the neural regulation of fertility. One of the most prominent is their involvement in the estrogen-negative feedback regulation of GnRH neurons (24). In this regard, it is noteworthy that preliminary studies by Moenter and DeFazio (31) showed that estrogen treatment capable of generating LH-negative feedback, increased the frequency of spontaneous GABA_A receptor-mediated currents in GnRH neurons. Thus, with our present demonstration of the inhibitory role of endogenous GABA signaling in GnRH neurons, these results provide good evidence that estrogen increases GABA release upon GnRH neurons to suppress their activity at times of LH-negative feedback in the mouse. Such an hypothesis is in good agreement with many in vivo observations in other species (24).

Although we show that nearly 80% of GnRH neurons are inhibited by endogenous GABA release, approximately 16% of cells showed no response to bicuculline, and two neurons gave results demonstrating a net excitatory response to GABA_A receptor signaling. Thus, as has often been encountered in the study of the GnRH phenotype, a degree of cellular heterogeneity exists. The basis for the small degree of heterogeneity in GABA response is unknown and may result from differences in the expression of transporters regulating the intracellular chloride environment of GnRH neurons (32), or differences in the arrangements of GABAergic inputs on GnRH neurons. For example, it has recently been shown that the response of adult cortical pyramidal cells to GABA_A receptor activation depends critically upon the temporal and spatial relationship of individual GABAergic terminals to depolarizing inputs such as glutamatergic synapses (13). Thus, it remains possible that individual GABA_A receptors on a GnRH neuron may generate a local excitatory response depending on the state of the dendritic microdomain. However, as a whole, the net effect of all GABA_A receptor activation upon an individual GnRH neuron is to suppress its activity. It is also apparent that heterogeneity exists in the functional expression of several ion channels that will shape the electrical behavior of the GnRH neurons (20). This heterogeneity is well illustrated by the GnRH neuron shown in Fig. 1G that exhibits rapid, large amplitude membrane oscillations. Such cells are rare in our experience and the remarkable response to the withdrawal of GABA_A receptor activation is unlike that observed in any of the other 41 cells recorded. Determining the functional significance of oscillatory GnRH neurons such as this may provide substantial insight into our understanding of episodic pulsatility within this network.

In summary, using both GnRH-EGFP-mut5 and GnRH-LacZ transgenic mouse models and gramicidin-perforated-patch electrophysiology, we show here that endogenous GABA acts through the GABA_A receptor to exert a net inhibitory action upon nearly 80% of adult GnRH neurons. This inhibitory effect of endogenous GABA release upon adult GnRH neuron excitability supports the general concept that primary afferent GABAergic neurons represent a predominantly inhibitory component of the GnRH neuronal network.

Acknowledgments

We thank Dr. J. Gilthorpe (University College London, London, UK) for the EGFP-mut5 plasmid and Drs. R. Campbell, D. Grattan, and C. Jasoni and for valuable comments on a draft of the manuscript.

Footnotes

This work was funded by the Biotechnology and Biological Sciences Research Council (United Kingdom) and the Wellcome Trust.

Abbreviations: EGFP, enhanced GFP; GABA, -aminobutyric acid; GFP, green fluorescent protein; TTX, tetrodotoxin citrate.

Received October 6, 2003.

Accepted for publication November 4, 2003.

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