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Endocrinology Vol. 143, No. 4 1459-1466
Copyright © 2002 by The Endocrine Society

NEUROENDOCRINOLOGY

Effect of GABA on GnRH Neurons Switches from Depolarization to Hyperpolarization at Puberty in the Female Mouse

Seong-Kyu Han, Istvan M. Abraham and Allan E. Herbison

Laboratory of Neuroendocrinology, The Babraham Institute, Cambridge CB2 4AT, United Kingdom

Address all correspondence and requests for reprints to: Dr. Allan E. Herbison, Laboratory of Neuroendocrinology, The Babraham Institute, Cambridge CB2 4AT, United Kingdom. E-mail: . allan.herbison{at}bbsrc.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The amino acid {gamma}-aminobutyric acid (GABA) plays an important role in the regulation of the GnRH neurons. We examined whether GABA depolarizes or hyperpolarizes GnRH neurons over postnatal development using gramicidin, perforated-patch electrophysiology combined with GnRH-LacZ transgenic mice in whom GnRH neurons can be made to fluoresce. The basic membrane properties and GABA responsiveness of GnRH neurons were not altered by transgene expression or fluorescence. Ten of 12 immature GnRH neurons (10–17 d) were depolarized by GABA in a direct and dose-dependent manner that was blocked by a GABAA receptor antagonist. In peripubertal GnRH neurons (25–30 d), GABA exerted depolarizing (4/11) as well as hyperpolarizing (5/11) effects on GnRH neurons. In adult female mice, GABA was found to exert exclusively hyperpolarizing actions on GnRH neurons (9/10) that were direct and mediated by the GABAA receptor. GABA switched from depolarizing to hyperpolarizing actions around postnatal d 31, the time of vaginal opening. Unidentified preoptic area neurons exhibited predominantly hyperpolarizing responses to GABA at all three postnatal stages. These findings demonstrate that GnRH neurons display an unusually late postnatal switch in their response to GABA. They also provide the first direct evidence that GABA inhibits the electrical activity of postpubertal GnRH neurons.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH NEURONS REPRESENT the final output neurons of the neuronal network controlling fertility in all mammals. As such, an understanding of the neural control of the GnRH neurons is critical. The amino acid transmitter {gamma}-aminobutyric acid (GABA) is thought to be one of the most important neural regulators of GnRH neurons and has been proposed to play key roles in the developmental migration (1), pubertal activation (2), pulsatility, and gonadal steroid feedback regulation (3) of the GnRH neurons. During embryogenesis, GABA depolarizes GnRH neurons through the direct activation of GABAA receptors (4), and this is likely to aid in their correct temporal and spatial pattern of migration and biosynthetic activity (5, 6, 7). Following birth, GABAA receptor signaling changes in GnRH neurons during the late postnatal period (8), and studies in the monkey indicate that alterations in GABAergic input are involved in the pubertal activation of the GnRH neurons (9, 10). In the adult, investigations in several species have suggested that GABA acts through GABAA receptors located within the vicinity of the GnRH perikarya to modulate their pulsatile activity (11, 12, 13). Furthermore, a fall in local GABA release is implicated in the generation of the preovulatory GnRH/LH surge in the female (14, 15, 16, 17, 18), and steroid-dependent changes in GABAergic transmission are believed to help bring about negative feedback in both the male and female (19, 20, 21, 22).

Despite the wealth of data indicating a critical role for GABA in the neural control of the GnRH neurons, several key questions remain to be answered. Arguably, the most important of these is that of determining whether GABA excites or inhibits the electrical activity of postnatal GnRH neurons. Although recent studies in normal (10, 23) and transgenic (24) mice have shown that all GnRH neurons express GABAA receptors, the experimental conditions used in these studies did not allow the investigators to determine whether GABA depolarized or hyperpolarized these cells. It is well established that GABA acts through GABAA receptors to excite embryonic neurons but, under normal circumstances, then switches in the first to second postnatal weeks to exert its inhibitory actions (25). Although this depolarization-to-hyperpolarization pattern of GABAergic influence is believed to occur in the great majority of neurons within the brain, some neuronal phenotypes continue to exhibit depolarizing responses to GABA in the adult brain (26, 27, 28). In relation to the GnRH neurons, the GT1 immortalized cell line is depolarized by GABAA receptor activation (29, 30), but whether this reflects the embryonic nature of the GT1 cells or is, instead, a true reflection of adult GnRH neurons is unknown. Furthermore, despite the great majority of in vivo studies reporting inhibitory actions of GABA on LH secretion in adult mammals (3), it is intriguing that the very early investigations all showed stimulatory effects of GABA on LH release (31, 32).

In the current series of studies, we have investigated the effects of GABAA receptor activation upon the electrical activity of GnRH neurons over the course of postnatal development using gramicidin perforated-patch electrophysiology in the female mouse. This method represents the only patch-clamp technique through which the electrophysiological responses of a neuron can be assessed without altering the intracellular chloride environment; a critical point when assessing GABAA receptor responses (33, 34, 35). In the past, we had undertaken electrophysiological investigations on GnRH neurons in the nontransgenic mice through morphological identification and postrecording gene profiling (8, 23). However, the gramicidin approach does not easily permit us to extract the recorded cell’s cytoplasmic contents for postrecording GnRH genotyping. In this study, we have therefore used GnRH-LacZ (GNLZ) transgenic mice (7), in association with fluorescein di-ß-D-galactosidase compounds (36), to preidentify GnRH neurons through fluorescence in the acute brain slice procedure.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
GNLZ transgenic mice
The production and analysis of the 5.5-GNLZ-3.5 lines of mice have been reported in detail elsewhere (7). In brief, transgenic mice (C57/Bl6J x CBA/Ca) were produced by pronuclear injection of fertilized mouse eggs with a construct comprising all the introns and exons of GnRH-1, in addition to 5.5 kb of upstream 5' and 3.5 kb of downstream 3' sequence, into which a nuclear-localized LacZ cassette was engineered into exon 2 sequences. As assessed by the presence of ß-galactosidase (ßgal) immunoreactivity, approximately 80% of postnatal GnRH neurons in these mice express nuclear-located transgene (7). Homozygous and heterozygous 5.5-GNLZ-3.5 mice, which appear normal in all respects including their fertility, were bred and housed under conditions of 12 h of light (lights on at 0700 h) with constant access to food and water and treated in accordance with UK Home Office requirements under projects 80/1475. The day of vaginal opening occurs between postnatal d 28 and 31 in female mice under these housing conditions. Vaginal smears were taken from adult female mice to determine the stage of the estrous cycle.

Brain slice electrophysiology
Immature (postnatal d 10–17), peripubertal (postnatal d 25–30), and adult (postnatal d 36–55) female 5.5-GNLZ-3.5 and wild-type mice were killed by cervical dislocation, decapitated between 1000 and 1200 h, their brains rapidly removed, and placed in ice-cold, bicarbonate-buffered, artificial cerebrospinal fluid (ACSF) of the following composition (in mM): 118 NaCl, 3 KCl, 0.5 CaCl2, 6.0 MgCl2, 11 D-glucose, 10 HEPES, and 25 NaHCO3 (pH 7.4 when bubbled with 95% O2 and 5% CO2). Brains were blocked and glued with cyanoacrylate to the chilled stage of a vibratome and 150- to 200-µm-thick coronal slices containing the medial septum and preoptic area prepared. Slices from transgenic mice were then incubated in ACSF containing 10–40 µM 5-chloromethyl-fluorescein di-ß-D-galactopyranosidase (CMFDG, Molecular Probes, Inc., Eugene, OR) at room temperature in the dark. The slices were then incubated at 30 C for 30 min in oxygenated recording ACSF (rACSF) consisting of (in mM): 118 NaCl, 3 KCl, 2.5 CaCl2, 1.2 MgCl2, 11 D-glucose, 10 HEPES, and 25 NaHCO3, and thereafter kept at room temperature in rACSF for at least 1 h before recording. Slices were transferred to the recording chamber, held submerged, and continuously superfused with rACSF at a rate of 4–5 ml/min. The slices were viewed with an upright fluorescence Axioskop FS microscope (Carl Zeiss, Jena, Germany) with 10x or 40x immersion objectives (Achroplan 0.75W, Ph2, Carl Zeiss) and either fluorescence illumination using the fluorescein isothiocyanate filter block 15 (H546) (Carl Zeiss) or Normaski differential interference contrast (DIC) optics. Slices were initially examined under fluorescence with the low-power objective to determine the distribution of fluorescent cells. A single GnRH neuron was then brought into focus under the high-power objective using fluorescence for 5–10 sec before switching to DIC optics. Following patching of the cell under DIC optics, it was then briefly (5 sec) examined under fluorescence again to confirm its fluorescent identity. All recordings were made at room temperature (20–23 C).

Patch pipettes were pulled from thin-wall borosilicate glass capillary tubing (GC150TF, 1.5 mm outer diameter, Harvard Apparatus Ltd., Edenbridge, UK) on a Flaming/Brown puller (P-97; Sutter Instruments Co., Novato, CA). The patch pipette solution was passed through a disposable 0.22-µm filter before use and contained (in mM): 130 KCl, 5 NaCl, 0.4 CaCl2, 1 MgCl2, 10 HEPES, 1.1 EGTA, with pH adjusted to 7.3 with KOH. Gramicidin (Sigma, St. Louis, MO) was first dissolved in dimethylsulfoxide (Sigma) to a concentration of 2.5–5 mg/ml and then diluted in the pipette solution just before use to a final concentration of 2.5–5 µg/ml and sonicated for 10 min. Before backfilling the electrode with the gramicidin-containing solution, the tip of the electrode was always loaded with a small volume of gramicidin-free pipette solution. The gramicidin-perforated patch clamp recordings were performed using an Axoclamp-2B amplifier (probe gain, x0.1 MU with HS-2 probe; Axon Instruments, Foster City, CA) operating in bridge mode. The tip resistance of the electrodes was 6–8 M{Omega}. The junction potential between the patch pipettes and bath solution was nulled before giga-seal formation. Experiments began 15–20 min after giga-seal formation when the resting membrane potential of the cell reached a stable level below -45 mV (33). Spontaneous activities were sampled online using a Digidata 1200 interface (Axon Instruments) connected to an IBM PC/AT clone (Evesham, Cambridge, UK). Signals were filtered (3 kHz, Bessel filter of Axoclamp-2B) before digitizing at a rate of 2 kHz. Acquisition and subsequent analysis of the acquired data were performed using the Clampex7 suite of software (Axon Instruments). Traces were plotted using Origin 5 computer software (MicroCal Software, Northampton, MA).

Test compounds were dissolved in the rACSF solution and tested by adding to the perfusing rACSF at known concentrations. Previous studies in the mouse have shown that GABA responses are mediated entirely by the GABAA receptor (8, 22, 23, 24). Accordingly, we used GABA (10, 30, 100 µM) to test GnRH neuron responses and bicuculline methiodide (20–50 µM) to confirm the GABAA receptor dependency. Tetrodotoxin citrate (TTX, 0.5 µM) was used to demonstrate direct effects of GABA on recorded cells. All compounds were purchased from Tocris Cookson (Bristol, UK).

Immunocytochemistry
Slices treated with CMFDG as above were placed in 4% paraformaldehyde in phosphate-buffered solution (pH 7.6) for 20 h at 4 C. Following fixation, slices were rinsed several times in Tris-buffered saline (TBS) at room temperature and then incubated with a polyclonal rabbit antibody specific for GnRH (LR1, 1:5000; gift of Dr. R. Benoit, Montréal, Canada) for 48 h at 4 C. After washing in TBS, slices were incubated in Texas Red-labeled, antirabbit immunoglobulins (1:200, Vector Laboratories, Inc., Peterborough, UK) for 2 h at room temperature, washed, mounted onto slides, and coverslipped with Vectashield (Vector Laboratories, Inc.). All antibodies were dissolved in TBS containing 0.3% Triton X-100 and 0.3% BSA. Slices were viewed using a DMRB fluorescence microscope (Leica Corp., Nussloch, Germany) with Texas and I3 filter blocks (Leica Corp.) at 40x or 100x objective magnification to determine whether the cells were double labeled.

Statistical analysis
Experimental data were expressed as mean ± SEM and the number of neurones tested and analyzed was presented by n. The relationship between postnatal days and membrane potential changes was determined by nonlinear regression analysis using Genstat 3.2 statistical software (Lawes Agricultural Trust, Rothamsted, UK). One-way ANOVA was performed to test the spontaneous firing frequency and resting membrane potential differences among the postnatal age groups. One population t test was used to assess significant changes in GABA-induced membrane potential in immature, peripubertal, and adult groups. A level of P less than 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CMFDG fluorescence in GNLZ mice
The identity of fluorescent cells located within the medial septum (MS) and rostral preoptic area (rPOA) was examined by fixing CMFDG-treated brain slices and processing them for GnRH immunofluorescence (n = 5). The pattern of GnRH immunofluorescence was identical with that reported previously in the adult mouse brain (37). The CMFDG treatment resulted in the fluorescence of two populations of neurons: one that overlapped that of the GnRH neurons in the MS and rPOA and another in the lateral septum. Approximately 30% of all GnRH-immunofluorescent neurons exhibited CMFDG fluorescence regardless of their location within the MS and rPOA (Fig. 1Go). The CMFDG fluorescent cells detected in the lateral septum lacked GnRH immunofluorescence. The distribution of these lateral septal cells was the same as that reported for ßgal expression in 5.5-GNLZ-3.5 mice (7) and all other GnRH-LacZ transgenic mice (38) and represent cells that continue to express ßgal despite the cessation of GnRH biosynthesis in the adult (37). In relation to the CMFDG fluorescence, all cells located within the midline MS, and more than 90% of those around the organum vasculosum of the lamina terminalis (OVLT) in the rPOA, were also found to be immunofluorescent for GnRH (Fig. 1Go). Within the region of the OVLT, approximately one CMFDG-fluorescent cell that did not exhibit detectable GnRH immunofluorescence was identified in each brain slice. It is unclear whether these cells represent GnRH neurons with low levels of GnRH peptide or are cells expressing the transgene in an ectopic manner. The CMFDG fluorescence was equivalent in heterozygous and homozygous mice, but brain slices from nontransgenic mice did not exhibit any CMFDG fluorescence.



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  		Figure 1. Low- and high-power photomicrographs of fluorescent GnRH neurons in acute brain slices of the preoptic area prepared from GnRH-LacZ mice. The CMFDG fluorescence is green and the GnRH immunofluorescence is red. Note in A and B that the five fluorescent cells (arrowheads) lying at different focal planes in the slice are all immunofluorescent for GnRH, although one GnRH neuron (arrow) is clearly not. Scale bars represent 10 µm.

 
Electrophysiological recordings from GnRH neurons
To evaluate whether the CMFDG treatment and/or brief fluorescence exposure had any effects on GnRH neurons, we made recordings from four immature CMFDG-fluorescent GnRH neurons under whole-cell, patch-clamp conditions identical with those used previously to investigate nontransgenic immature GnRH neurons (8). Equivalent values for nontransgenic immature GnRH neurons (n = 10–21; 8) are given in parentheses. All cells were spontaneously active with mean spontaneous firing rates of 0.34–0.86 Hz (0.02–1.0 Hz) and a mean resting membrane potential of -63 ± 1.2 mV (-64 ± 2 mV). To assess GABAA receptor responses, we examined the effect of 30 µM GABA and found it to evoke a mean membrane depolarization of 37 ± 5 mV (27 ± 6 mV). These observations suggest that there are no major deleterious effects of the fluorescence procedure and that GABA responsivity is maintained.

Using the gramicidin-perforated patch technique, recordings were made from 12 immature, 11 peripubertal, and 10 adult GnRH neurons located within the rPOA. In nearly all cases, only a single recorded cell was obtained from each mouse and animal n numbers were 11, 10, and 9, respectively. The majority of recorded cells were adjacent to the OVLT, and the rest were located dorsally in the midline extending up into the MS. Under perforated patch, the mean resting membrane potential was -51.8 ± 2.2, -49.9 ± 1.4, and -49.0 ± 1.7 mV; action potential amplitude 14.7 ± 1.9, 13.4 ± 3.0, and 13.2 ± 1.3 mV; and mean firing frequency 3.9 ± 0.5, 3.1 ± 0.7, and 3.6 ± 0.7 Hz, respectively. No significant differences (ANOVA) were detected in any of these parameters across the postnatal age groups examined.

In immature mice (postnatal d 10–17), a 1-min exposure to 10, 30, and/or 100 µM GABA was found to evoke 4.8–18.1 mV membrane depolarizations in 10 of 12 GnRH neurons that were dose dependent, blocked completely by bicuculline, and repeatable (Fig. 2Go). In all cases, cell firing was reduced following the membrane depolarization although a short-lived, transient increase in firing was usually observed immediately following the onset of the depolarization (Fig. 2Go). The depolarizing effect of GABA on immature GnRH neurons was maintained in the presence of TTX (n = 2). In two cells, GABA was not found to exert any effect on membrane potential but reduced the action potential firing of one cell.



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  		Figure 2. GABA depolarizes immature GnRH neurons. Gramicidin, perforated-patch recording of an immature GnRH neuron showing a depolarizing response to 1-min exposure (black bar) to 30 µM GABA that is blocked completely by bicuculline (Bic) and repeatable after washout. Resting membrane potential = -53 mV.

 
In peripubertal female mice (postnatal d 25–30), a range of different effects of 10, 30, and 100 µM GABA were observed in GnRH neurons. Four of the 11 cells responded to GABA with membrane depolarization, five exhibited membrane hyperpolarizations, and two displayed no change in membrane potential. One of the depolarized GnRH neurons increased its firing rate, but the other depolarized cell and all hyperpolarized cells reduced their overall firing frequency during the GABA application.

In adult female mice (diestrus or estrus, postnatal d 36–55), testing with 10, 30, and/or 100 µM GABA resulted in 2.8–7.0 mV hyperpolarizations of the membrane potential (Fig. 3Go) in 9 of 10 cells with the remaining cell showing no response. The membrane hyperpolarization was always associated with a decrease in action potential firing and was dose dependent (Fig. 4AGo), blocked completely by bicuculline, and repeatable (Fig. 3Go). As for the membrane depolarizations in younger mice, the GABA-evoked hyperpolarizations persisted in the presence of TTX (n = 2). The stage of the estrous cycle did not effect the hyperpolarizing response to GABA.



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  		Figure 3. GABA hyperpolarizes adult GnRH neurons. Gramicidin, perforated-patch recording from an adult GnRH neuron showing a hyperpolarizing response to 1-min exposure (black bar) to 100 µM GABA that is blocked completely by bicuculline (Bic) and repeatable after washout. Resting membrane potential = -46 mV.

 


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  		Figure 4. Effect of GABA on GnRH neurons switches over postnatal development. A, Plots showing the mean membrane potential responses of immature (n = 12), peripubertal (n = 11), and adult (n = 10) GnRH neurons to 10, 30, and 100 µM GABA exposure. Whereas immature GnRH neurons display a dose-dependent membrane depolarization (open circles), peripubertal GnRH neurons show no overall response (open triangles) and adult GnRH neurons (filled squares) exhibit a dose-dependent hyperpolarizing action of GABA. B, Scatter plot showing the individual membrane potential changes recorded for all GnRH neurons in response to 100 µM GABA. The curve (y = -50 + 64/[1 + 0.0089 * x]) was derived from a hyperbolic regression model generated by the Genstat 3.2 statistical software package.

 
The plotting of mean data from the different age groups (Fig. 4AGo) revealed that GABA evoked a clear dose-dependent depolarization of membrane potential in immature GnRH neurons in parallel to the hyperpolarization of adult cells, but no overall directional effect of GABA on membrane potential was recorded from peripubertal GnRH neurons. Nonlinear regression analysis of the membrane response of each individual GnRH neuron indicated that the time point at which GABA responses were switching from depolarization to hyperpolarization within the recorded population of GnRH neurons was around postnatal d 30–31 (Fig. 4BGo).

Electrophysiological recordings from non-GnRH neurons
To assess the specificity of the late postnatal switch in GABA response displayed by GnRH neurons, we also made gramicidin perforated-patch recordings from unidentified non-GnRH neurons located within the preoptic area of both CMFDG-treated transgenic slices and wild-type mice at the same postnatal time points. Under perforated patch, the mean resting membrane potential of these cells was -49.2 ± 2.7, -49.4 ± 1.3, and -48.8 ± 2.1 mV, respectively. In immature mice, four of nine (44%) preoptic neurons were hyperpolarized by GABA (Fig. 5AGo), one was depolarized, and the remaining four neurons were unresponsive. No significant effect of GABA on membrane potential was detected when the results were averaged (Fig. 6Go). All hyperpolarized neurons responded with a decrease in firing (Fig. 5AGo). In peripubertal animals, 8 of 13 (62%) preoptic neurons were hyperpolarized by GABA, two were depolarized, and three were unaffected. As a group, this resulted in a significant (P < 0.05) hyperpolarizing response to GABA (Fig. 6Go). In adult animals, four of five (80%) preoptic neurons were hyperpolarized by GABA (Fig. 5BGo), and the remaining cell did not respond. As a group, a significant (P < 0.01) hyperpolarizing response was observed. Thus, in this heterogeneous group of preoptic neurons, both the percentage of neurons hyperpolarized by GABA (44–80%) and the amplitude of the hyperpolarizing effects of GABA (Fig. 6Go) were found to increase with age. As with the responses in GnRH neurons, the GABA-evoked hyperpolarizations were blocked completely by bicuculline and persisted in TTX (n = 3).



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  		Figure 5. Unidentified preoptic area neurons are hyperpolarized by GABA in immature and adult mice. A, Gramicidin, perforated-patch recording from a preoptic area neuron in a immature female mouse showing a hyperpolarizing response to 100 µM GABA (bar). Resting membrane potential = -47 mV. B, Gramicidin, perforated-patch recording from a preoptic area neuron in an adult female mouse displaying a hyperpolarizing response to 100 µM GABA (bar). Resting membrane potential = -54 mV.

 


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  		Figure 6. Comparison of GABA responses between GnRH and unidentified preoptic area neurons in the female mouse. Histograms show mean (+ SEM) membrane potential responses to 100 µM GABA by GnRH (open bars) and preoptic area (shaded) neurons recorded from brain slices prepared from immature, peripubertal, and adult female mice. *, P < 0.05; **, P < 0.01.

 
The differences in the membrane responses of GnRH and unidentified preoptic neurons to GABA are clearly evident in the graph depicting mean membrane changes to 100 µM GABA throughout postnatal development (Fig. 6Go). Whereas GABA significantly (P < 0.01) depolarized immature GnRH neurons, it had no significant effect on immature preoptic cells. In contrast, in peripubertal animals, GABA had no significant effects on GnRH neurons but hyperpolarized preoptic cells (P < 0.05). In adults, GABA hyperpolarized (P < 0.01) both GnRH and preoptic cells (Fig. 6Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report here the use of LacZ-based fluorescence to preidentify GnRH neurons in the acute brain slice procedure. This method (36), which has been used previously in neuronal preparations (39), provides an alternative to the use of green fluorescent protein (24, 40) to identify GnRH neurons in situ. A disadvantage of this method is that it permits us to visualize only approximately 30% of the GnRH neuronal population, compared with 65–90% when using green fluorescent protein tagging (24, 40). Because more than 80% of the GnRH neurons in GNLZ mice express immunoreactive ßgal, it seems most likely that the nonfluorescent GnRH neurons have insufficient enzymatically active ßgal to catalyze the CMFDG into its fluorescent derivatives. It is important to be aware that some cells targeted by GnRH promoter transgenics do not represent authentic GnRH neurons in the adult mouse brain (37, 38). However, these cells are easily distinguishable from GnRH neurons through their distinctive location in the lateral septum.

Using a protocol that involves minimal fluorescence illumination, we show here that the membrane potential and firing rate of fluorescent GnRH neurons, as well as the responsiveness of their GABAA receptors, are not altered when compared with unmodified GnRH neurons (8). This is important evidence indicating that the transgene and/or CMFDG fluorescence have no major deleterious effects on the cell. When comparing fluorescent GnRH neurons recorded under different patch-clamp conditions, we did, unexpectedly, find that their firing rate was substantially faster in perforated patch mode, compared with whole-cell recordings. This suggests that intracellular constituents, which are quickly replaced by the electrode solution in whole-cell patch-clamp but not in perforated patch recordings, are likely to be critical for GnRH neurons to fire at their normal frequency. The less negative resting membrane potentials found under perforated patch conditions is typical of this mode and likely to be caused by the inevitable higher series resistance encountered, compared with the whole-cell approach (33, 34).

We have found that GABA depolarizes GnRH neurons in immature female mice but then switches about the time of puberty to a hyperpolarizing action in the adult. Although this pattern holds true for the great majority of GnRH neurons we recorded, some cells were found that were unresponsive to GABA at all three developmental stages. We have encountered variability in GABA responses with immature GnRH neurons in earlier recordings (8) and this may reflect true heterogeneity within the GnRH population. Alternatively, the heterogeneity may result from experimental variability. For example, we have been unable to confirm GnRH immunoreactivity in approximately 10% of fluorescent cells located in the OVLT region in our transgenic mice, and it is possible that one or two of the 33 cells we recorded were not GnRH neurons.

In good agreement with previous results (8, 23, 24), the effects of GABA on mouse GnRH neurons were blocked completely by bicuculline. This indicates that the GABAA receptor mediates all fast GABA actions at the level of the GnRH perikarya in this species. This appears to be different in the guinea pig in whom mediobasal hypothalamic GnRH neurons express functional GABAB receptors (41). Although our results show that the GABAA receptor-mediated depolarizing-to-hyperpolarizing switch exists for GnRH neurons in much the same way that it does for most other neurons (25), a major difference is that it occurs relatively late in neuronal development. In regions such as the cortex and hippocampus, GABA becomes hyperpolarizing at different times in specific neuronal phenotypes but is predominantly inhibitory by the second postnatal week (25, 42, 43, 44). Within the hypothalamus, GABA loses its excitatory actions around postnatal d 10 in arcuate nucleus neurons (45), and oxytocin neurons switch from GABA-mediated excitation to inhibition in the second postnatal week (46). Indeed, we show here that unidentified preoptic neurons, residing in the same general region as the GnRH neurons, are mostly hyperpolarized by GABA even within the second postnatal week. Thus, the depolarization-hyperpolarization switch observed here in the fifth postnatal week with GnRH neurons is very unusual.

Chloride and bicarbonate are the principal permeant ions of the GABAA receptor. Whereas bicarbonate has been implicated in mediating depolarizing effects of the GABAA receptor under specific circumstances within the adult brain (26), it is chloride that is considered the principal ionophore responsible for the developmental shift from depolarizing to hyperpolarizing actions of the receptor (25, 44). When intracellular chloride ion concentrations within a cell result in a chloride equilibrium potential (ECl) above the resting membrane potential of that cell, opening of the GABAA receptor will result in an efflux of chloride ions and, consequently, membrane depolarization. In contrast, when chloride ion concentrations are relatively low, setting the ECl below the resting membrane potential of the cell, opening of the GABAA receptor will result in an inward flux of chloride and hyperpolarization of the cell. As such, the principal determinant of whether GABA depolarizes or hyperpolarizes a neuron is the relationship between its intracellular chloride ion concentration and membrane potential. Accordingly, the slow fall in intracellular chloride levels within neurons during early postnatal life is thought to underlie the developmental switch in GABA actions (25, 44). Because the resting membrane potential of GnRH neurons (-65 to -70 mV) does not change across postnatal development (23), our present data imply that ECl approaches the resting membrane potential of the majority of GnRH neurons around postnatal d 31 and thereafter falls below this level to result in GABAA receptor hyperpolarization in adulthood.

Of the molecules now established to play a role in determining chloride ion homeostasis in neurons, the chloride ion extruder KCC2 and importer NKCC1, alongside the voltage-dependent chloride ion extruder ClC2, are all thought to be developmentally regulated (28, 47, 48). However, it seems most likely that the induction of KCC2 expression during the first postnatal week is the principal determinant of the slow fall in intracellular chloride concentrations within neurons and the eventual hyperpolarizing effects of GABA (28). Importantly, Ganguly et al. (49) have recently demonstrated that it is the increasing GABA-mediated depolarization occurring with development that initiates KCC2 expression within a cell and the consequent switch to GABA hyperpolarization. The identification and expression profiling of transporter molecules determining chloride ion homeostasis in GnRH neurons has yet to be established. However, it is intriguing to speculate that the very delayed GABA depolarization-hyperpolarization switch observed here in GnRH neurons may result simply from a relatively delayed onset of excitatory drive to the GnRH neurons and resultant absence of KCC2 expression until puberty.

From a physiological perspective, our present findings are of importance to the understanding of the process of puberty and the neural control of GnRH secretion in the adult female. Although GABA-evoked depolarization was observed to ultimately suppress GnRH neuron firing in prepubertal mice, this inhibition is very likely to be an artifact and result from the sustained concentrations of exogenous GABA elevating the membrane potential into a range that inactivates the Na+ channel and also allows shunting inhibition (50, 51). Many immature GnRH neurons displayed an initial excitation to GABA before the spike inhibition, and this excitation is very likely to be the physiological effect of GABA in these cells. An increase in GnRH neuron firing is observed when allpregnanolone, an allosteric enhancer of GABAA receptor activity (22), is applied to gramicidin-patched immature GnRH neurons (Han, S.-K., and A. E. Herbison, unpublished data). Thus, GABAA receptor activation will excite immature GnRH neurons, whereas postpubertal GnRH neurons will be uniformly inhibited by GABA. As a consequence, it would be predicted that the direct activation of GABAA receptors on GnRH perikarya would stimulate LH release in prepubertal mice but inhibit LH in adults. Interestingly, a series of in vivo studies by Moguilevsky et al. (52) and Szwarcfarb et al. (53) have demonstrated precisely this phenomenon in the female rat; an elevation in whole-brain GABA levels increases LH release in immature rats but decreases LH secretion in adults. The transition phase for that effect was around postnatal d 25, the time from which there is likely to be a progressive increase in the frequency of GnRH pulses occurring within the median eminence (54).

Precisely how GABA may participate in the activation of the GnRH neurons at puberty in the mouse remains unclear. If the final postnatal maturation of neural inputs to GnRH neurons follows a similar course to that of other neuronal phenotypes (55), albeit at a much later postnatal time point, then GABA may have an important role. In that scenario, GABA would provide the principal stimulatory input to the prepubertal GnRH neurons right up into the fourth postnatal week and then switch to an inhibitory role as glutamate becomes the dominant excitatory transmitter. It is known that N-methyl-D-aspartate receptor expression in GnRH neurons is increased greatly between postnatal d 5 and 15 in the mouse (56). If correct, this would mean that the process of GnRH secretory activation in the mouse at puberty is fundamentally different from that occurring in the primate in which a release from an inhibitory GABA restraint is clearly involved in the female monkey (2, 9, 10).

In terms of understanding the role of GABA in regulating the electrical activity of mature GnRH neurons, this study provides evidence that GABAA receptor activation is indeed inhibitory. The suppressive influence of GABA on LH secretion reported in the past (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) is therefore likely to have resulted from direct activation of GABAA receptors located on GnRH cell bodies. In contrast, the stimulatory effects of GABA reported for GT1 cells (29, 30) probably reflect their embryonic nature. Proof of the inhibitory effect of GABA on adult female GnRH neurons is critical for the current hypothesis that gonadal steroid negative feedback involves an increased GABAergic influence on GnRH cell bodies (3, 19, 20, 21, 22) as well as the idea that GABAergic disinhibition of GnRH neurons is important for the generation of the GnRH/LH surge (14, 15, 16, 17, 18). The physiological significance and role of GABA acting directly or indirectly through GABAA and/or GABAB receptors at the level of the GnRH terminal in the regulation LH secretion remains unclear (57, 58).

In summary, using gramicidin, perforated-patch electrophysiology, we show here that GnRH neurons in the female mouse are depolarized in a direct manner by GABAA receptor activation up until the time of puberty whereupon GABA switches to exerting a hyperpolarizing action. The time of onset of this depolarization-hyperpolarization switch in the fifth postnatal week is very delayed, compared with other developing neurons, but in keeping with the other late changes in GABAA receptor signaling that occur in mouse GnRH neurons (8). We show that unidentified preoptic area neurons, residing in the same region as the GnRH neurons, are predominantly hyperpolarized by GABA from the second postnatal week onward. The late onset of the GABAA receptor-mediated depolarization-hyperpolarization is most likely because of delayed maturation of chloride ion homeostasis within the developing GnRH neuron. Although the contribution of these changes to the onset of puberty in the mouse may only be speculated on at present, the demonstration here that GABAA receptor activation does indeed inhibit the firing of adult female GnRH neurons is critical for our understanding of the neural regulation of these important neurons.


   Acknowledgments
 
Drs. John Bicknell and Martin Todman are thanked for critical appraisal of the manuscript.


   Footnotes
 
This work was supported by the Biotechnology and Biological Sciences Research Council (UK) and a Marie Curie Fellowship (IMA) of the European Community Human Potential Programme under contract number HPMF-CT-2000-00512.

Abbreviations: ACSF, Artificial cerebrospinal fluid; ßgal, ß-galactosidase; CMFDG, 5-chloromethyl-fluorescein di-ß-D-galactopyranosidase; DIC, differential interference contrast; ECl, chloride equilibrium potential; GABA, {gamma}-aminobutyric acid; GNLZ, GnRH-LacZ; MS, medial septum; OVLT, organum vasculosum of the lamina terminalis; rACSF, recording ACSF; rPOA, rostral preoptic area; TBS, Tris-buffered saline; TTX, tetrodotoxin citrate.

Received October 11, 2001.

Accepted for publication December 3, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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