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Endocrinology Vol. 142, No. 10 4448-4453
Copyright © 2001 by The Endocrine Society

ARTICLES

Direct Regulation of Postnatal GnRH Neurons by the Progesterone Derivative Allopregnanolone in the Mouse

Joan A. Sim1, Michael J. Skynner2 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 mechanisms through which gonadal steroids exert critical feedback actions upon the activity of the GnRH neurons are not understood. We have examined here whether progesterone may modulate the electrical activity of the GnRH neurons following its rapid metabolism to the neuroactive steroid allopregnanolone within the brain. Using an acute brain slice preparation, whole-cell, patch-clamp recordings were made from GnRH neurons of juvenile (postnatal d 15–20) and adult (postnatal d 60–70) female mice in the presence of tetrodotoxin. Progesterone (1 µM) was not observed to have any actions (up to 5 min exposure) upon GnRH neurons. However, allopregnanolone (500 nM-1 µM) exerted rapid (<1 min) effects upon the baseline membrane potential of all GnRH neurons and also significantly (P < 0.01) enhanced their GABA responses by up to 4-fold. All GABA and allopregnanolone responses were abolished by the GABAA receptor antagonist bicuculline. No differences were detected in the allopregnanolone sensitivity of GnRH neurons recorded from juvenile and adult GnRH neurons. These results provide the first evidence for a direct action of the neurosteroid allopregnanolone on postnatal GnRH neurons and suggest a new mechanism through which fluctuating progesterone levels may influence the secretory activity of these important neurons in the female mouse.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GnRH neurons located in the basal forebrain represent the final output neurons of the neuronal network controlling fertility in mammals. Although it is now well established that feedback actions of gonadal steroid hormones play an important role in regulating the function of the GnRH neurons, the precise nature of this influence is not understood (1, 2, 3). Progesterone, one of the principal ovarian steroid hormones, is known to exert inhibitory as well as stimulatory effects upon gonadotropin secretion in several species, including humans (4, 5, 6), and these actions involve the modulation of pulsatile GnRH secretion (7, 8, 9).

The precise mechanisms through which progesterone influences the activity of the GnRH neurons are presently unknown. In many cases, progesterone requires the presence of E (1, 2, 3, 4, 5, 6) and is known to act through the PR (2, 9, 10, 11). However, evidence for the expression of PRs in GnRH neurons is not robust with investigators either being unable to find PR immunoreactivity in GnRH neurons (12, 13) or detecting its presence in only very small subpopulations of GnRH neurons (14). As such, indirect and nonclassical mechanisms of progesterone influence have been proposed (1, 2). For example, work in the rat has recently shown that progesterone’s enhancement of the E-induced GnRH surge occurs through the anteroventral periventricular nucleus where, interestingly, the PR appears to act as a ligand-independent transcription factor (1, 11). A further possibility is that progesterone may have nongenomic, membrane effects upon GnRH neurons or other cells in the GnRH network. In this regard, progesterone has been shown to exert a stimulatory influence upon GnRH secretion at the level of the GnRH nerve terminals in vitro (15, 16). Whether such effects of progesterone are exerted directly upon the GnRH neuron remain unknown.

A variety of studies have also indicated that neuroactive steroids, derived from progesterone within the brain (17, 18), may influence gonadotropin secretion although, again, the mechanism of action is unclear (19, 20, 21, 22). One of the most important of these neuroactive steroids is allopregnanolone (3{alpha},5{alpha} tetrahydroprogesterone or 5{alpha}-pregnan-3{alpha}-ol-20-one), a direct allosteric modulator of the GABAA receptor, which is believed to be of physiological significance in the regulation of brain function (17, 23, 24, 25, 26). As GnRH neurons express functional GABAA receptors (27, 28), it seems reasonable to speculate that this neurosteroid may represent a mechanism through which progesterone could regulate the activity of the GnRH neurons in a direct manner. At present, direct evidence supporting this hypothesis is lacking. Although, the immortalized GT1-1 cells are known to express allopregnanolone-sensitive GABAA receptors (29), the relevance of this finding to GnRH neurons in vivo is unknown. Furthermore, as the GABAA receptor subunit composition required for allopregnanolone sensitivity is not yet established (30, 31, 32) and also modulated substantially by the phosphorylation state of the receptor (33), it is not possible to predict whether the GABAA receptors expressed by native GnRH neurons in situ will be sensitive to allopregnanolone.

In this study, we have undertaken electrophysiological analyses in the postnatal female mouse to examine whether progesterone and allopregnanolone may influence directly the electrical properties of GnRH neurons. As described previously (28, 34), cells of defined location, characteristic morphology, orientation, and size were recorded and then subsequently confirmed (or not) to be GnRH neurons using postrecording single cell RT-PCR for GnRH mRNA. Because of the possible dependence of allopregnanolone action on GABAA receptor subunit composition (30, 31, 32), we have examined allopregnanolone’s influence upon both juvenile and adult GnRH neurons which express different GABAA receptor subunit mRNAs in the female mouse (28).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice (C57BL/6J x CBA/CA) were bred and housed at The Babraham Institute under conditions of 12 h of light (lights on at 0600 h) with constant access to food and water and treated in accordance with UK Home Office requirements under projects 80/972 and 80/1005. Vaginal smearing was used to assess the stage of estrous cycle of the adult mice. Coronal brain slices (150 µm thick) were prepared from juvenile (d 15–20) and adult (d 60–70) female mice as reported previously (28, 34). In brief, mice were anesthetized with isofluorane, decapitated, and their brains removed into an ice-cold artificial cerebrospinal fluid (ACSF) of the following composition (in mM): 118 NaCl, 3 KCl, 0.5 CaCl2, 6.0 MgCl2, 10 D-glucose, 10 HEPES, and 25 NaHCO3 (pH 7.4 when bubbled with 95% O2 and 5% CO2). Brain slices were cut on a vibratome and incubated at 30 C for 30 min in oxygenated, recording (r)ACSF (as for ACSF except CaCl2 2.5 mM and MgCl2, 1.2 mM) and thereafter for at least 1 h at room temperature (20–24 C) before recording. Slices were transferred to the recording chamber, continuously superfused with rACSF at a rate of 6 ml/min, and viewed with an upright Axioskop FS microscope (Carl Zeiss, Jena, Germany) with a x40 water-immersion objective (Achroplan 0.75 W, pH 2, Carl Zeiss) and Normaski differential interference contrast optics. As previously (28, 34), putative GnRH neurons on the top of the slice were identified according to their size, location within the MS and rPOA, orientation and predominant bipolar morphology. All recordings were made at room temperature (20–23 C) in the presence of tetrodotoxin (0.5 µM), which blocks action potentials, and thus enables the examination of only direct effects of test compounds upon the recorded neuron.

Patch pipettes with final resistance of 6–12 M{Omega} were pulled from thin-walled borosilicate glass capillary tubing (1.5 mm outer diameter, Clark Electromedical Ins., Reading, UK) on a Flaming/Brown puller (P-97; Sutter Instruments Co., Novato, CA). The internal pipette solution contained (in mM) 140 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 4 MgATP, 0.1 Na2GTP, 10 EGTA with pH adjusted to 7.3 with KOH and was passed through a 0.22 µm filter before use. The reference electrode was a glass bridge containing 4% agar-saline, of which one end was placed in the recording chamber and the other end in a 3 M KCl-containing side chamber connected to ground, via an Ag/AgCl pellet. Whole-cell recordings were performed as previously described (28, 34) using an Axoclamp-2B amplifier (Axon Instruments, Union City, CA) operating in bridge mode. Current and voltage were simultaneously generated and sampled on-line using a Digidata 1200 (Axon Instruments) interface connected to an IBM PC/AT clone. Signals were filtered (0.3–10 kHz, Bessel filter of Axoclamp-2B) before digitizing. Acquisition and subsequent analysis of the acquired data were carried out using the PClamp6 suite of software (Axon Instruments). In addition, applied current, and voltage signals were recorded simultaneously onto a chart recorder (Gould TA 240, Valley View, OH) and DAT recorder (DTR 1204, Biologic Science Ins., France). Changes to input resistance were monitored by passing 200 msec hyperpolarizing pulses (0.04–0.08 nA in amplitude) at 0.1 Hz. Following recordings of up to 1 h duration, the cytoplasmic content of the recorded neuron was harvested under visual control and single cell RT-PCR used to examine for the presence of GnRH transcripts as reported previously (28, 34). Only those cells expressing GnRH transcripts were classified as GnRH neurons.

All drugs were applied via the superfusing rACSF solution. Solutions were switched manually by means of a six-way tap, ensuring that the bath was completely exchanged with control solution between drug application (approximately 20 sec). For adult and juvenile GnRH neurons, dose response relationships using 1–100 µM GABA were first established and allopregnanolone then tested using the lowest effective GABA concentration. In the case of adult GnRH neurons, this was always 3 µM whereas, in juvenile GnRH neurons, the lowest effective dose ranged from 1–10 µM GABA. As the concentration of allopregnanolone at the synapse is presently unknown, we used 500 nM and 1 µM concentrations similar to that used by others and ourselves in investigating electrophysiological actions of allopregnanolone within hypothalamic slice preparations (33, 35). Statistical analysis was undertaken by paired t test using GABA-induced membrane depolarization values before and after exposure to allopregnanolone from each cell. Drugs used were GABA (Sigma, Poole, UK), bicuculline methobromide (Tocris, Bristol, UK), tetrodotoxin (Alexis Corp., San Diego, CA), progesterone (Sigma), and allopregnanolone (Sigma). Progesterone and allopregnanolone were dissolved in dimethylsulfoxide (DMSO) and diluted in rACSF so that the final concentration of DMSO was <1 µM. Control experiments showed that the DMSO vehicle alone had no effect upon GnRH neuron electrical properties.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Whole-cell, current-clamp recordings were made from 15 postnatal female GnRH neurons (7 diestrous adult and 8 juvenile), identified by postrecording single cell RT-PCR for GnRH transcripts. Using a high chloride ion solution in the recording pipette, the resting membrane potential of GnRH neurons was found to be between -63 and -72mV, with input resistances ranging from 1.2–1.6G{Omega}. These values are identical to previous electrophysiological recordings undertaken in our laboratory under the same recording conditions (28, 29). In the presence of tetrodotoxin, action potentials were blocked revealing persistent miniature events (Fig. 1Go), the majority of which represent quantal GABA release and tonic GABAA receptor activation (28).



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  		Figure 1. Whole-cell, patch-clamp recordings from two adult GnRH neurons which display insensitivity to 1 µM progesterone (A) but sensitivity to 1 µM allopregnanolone (B). A, The membrane potential of this GnRH neurons is not changed by the application of progesterone for 4 min and the cell remained equally sensitive to 10 µM GABA before and during the progesterone treatment. B, This GnRH neuron displays a dose-dependent change in membrane potential to 3, 10, and 30 µM GABA and then marked enhancement of the 3 µM GABA response in the presence of 1 µM allopregnanolone. Note, in B, membrane potential was always reset to control value with direct current injection (downward arrow and *) before the application of GABA in the presence of allopregnanolone.

 
Adult GnRH neurons
As found previously, all adult female GnRH neurons responded to 3–30 µM GABA concentrations in a dose-dependent manner (Figs. 1Go and 2CGo). Progesterone (1 µM) applied to the bathing solution for up to 5 min was not found to have any effect upon the membrane potential of GnRH neurons or alter their response to GABA (Fig. 1AGo; n = 4). In contrast, allopregnanolone (500 nM-1 µM) increased the baseline membrane potential of all 7 adult GnRH neurons within 1–2 min and also exerted a direct, concentration-dependent potentiation of GABA-evoked membrane depolarization in 6 of these cells (Figs. 1BGo and 2BGo). Allopregnanolone alone at 500 nM (n = 2) produced small 0–6mV depolarizations, while 1 µM concentrations (n = 5) produced a slow 7–27 mV (mean 15.8 ± 4.2mV) change in membrane potential (Figs. 1BGo and 2BGo). Because of the effects of allopregnanolone itself, the membrane potential of GnRH neurons was offset to control pre-allopregnanolone values with direct current (1 in Figs. 1Go and 2Go) just before applying the test dose of 3 µM GABA. Allopregnanolone at 1 µM resulted in significant 1.7- to 3.4-fold enhancements in the 3 µM GABA response of GnRH neurons with a mean 2.2 ± 0.4-fold change (n = 6, Fig. 2BGo; P < 0.01, paired t test). In two of these GnRH neurons, we also tested 500 nM allopregnanolone, and this induced a mean 1.5-fold enhancement of the 3 µM GABA response (Fig. 2BGoC). The effects of GABA and allopregnanolone on membrane potential were abolished completely by the addition of the GABAA receptor antagonist bicuculline (10 µM), with subsequent withdrawal of the direct current (** in Fig. 2Go) returning the membrane potential to pre-test levels in all 7 GnRH neurons (Fig. 2BGo).



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  		Figure 2. Effect of allopregnanolone on membrane potential and GABA-induced responses in juvenile (A) and adult (B and C) GnRH neurons. A, Responses evoked in a juvenile GnRH neuron to 45 sec application of GABA (3 µM) at resting membrane potential of -66 mV in the absence (control) and presence of 1 µM allopregnanolone (gray bar). B, Responses evoked in an adult GnRH neuron to 45 sec application of 3 µM GABA at resting membrane potential of -70 mV in the absence (control) and presence of 500 nM or 1 µM allopregnanolone (gray bar). C, Graph shows the mean (±SEM) change in membrane potential (mV) of 7 adult GnRH neurons to 3, 10, and 30 µM GABA (circles) and enhancement of the 3 µM GABA response by 500 nM (triangle) and 1 µM (square) allopregnanolone. Numbers of GnRH neurons tested with allopregnanolone are given in brackets. Note that in A and B, membrane potential was always reset to control value with direct current injection (downward arrow and *) before the application of GABA in the presence of allopregnanolone. In all cases, the effect of allopregnanolone was abolished by the application of bicuculline (10 µM), with the removal of dc injection (upward arrow and **), restoring membrane potential to control levels.

 
Juvenile GnRH neurons
As observed previously (28), all GnRH neurons in the juvenile female mouse responded to GABA in a dose-dependent manner but with enhanced individual variability, compared with adult GnRH neurons. The lowest effective GABA concentrations were 1 µM (n = 2), 3 µM (n = 2), and 10 µM (n = 4). Bath application of allopregnanolone (1 µM) alone evoked membrane depolarizations ranging from 0–28 mV (mean 13.8 ± 3.8 mV) in 8 neurons held around -70 mV with direct current injection (Fig. 2AGo). Allopregnanolone (1 µM) was found to significantly enhance the responses of GnRH neurons to 1 (n = 1), 3 (n = 3), and 10 µM (n = 4) GABA by 1.2-fold, 2.2 ± 0.4-fold, and 4.3 ± 1.0-fold, respectively (Fig. 2AGo; P < 0.01, paired t test). As with adult GnRH neurons, application of bicuculline (10 µM) completely abolished the effect of allopregnanolone on membrane potential in all 8 juvenile GnRH neurons (Fig. 2AGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We demonstrate here for the first time that the neuroactive steroid allopregnanolone exerts a rapid and direct effect upon native GnRH neurons through allosteric modulation of their GABAA receptors. In contrast, we have not found any evidence for a rapid influence of progesterone itself on the membrane properties of GnRH neurons. As allopregnanolone is rapidly, and exclusively, produced from progesterone within the brain (17, 18), these findings indicate a novel mechanism through which progesterone could modulate the secretory activity of the GnRH neuron in vivo (Fig. 3Go). Intriguingly, in vitro studies have suggested that the GnRH-expressing GT1-1 cell lines can themselves synthesize allopregnanolone from progesterone (29, 36). Alongside the demonstration that GnRH neurons in the guinea-pig respond directly to E (37), our present observations support further the important concept that gonadal steroids can regulate the behavior of the GnRH neurons in a direct, nongenomic manner.



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  		Figure 3. Schematic diagram indicating two possible mechanisms of progesterone influence upon the GnRH neurons. As GnRH neurons lack PRs, progesterone seems likely to exert indirect effects on GnRH neurons through a transsynaptic mechanism. However, progesterone may influence GnRH neurons directly in a rapid, nongenomic manner following its conversion to allopregnanolone in the brain.

 
Investigations in vitro have demonstrated that either the continuous or episodic administration of progesterone to hypothalamic tissue results in elevated basal GnRH secretion with a 30–60 min latency (15, 16, 38). This effect requires E pretreatment, is both calcium and membrane dependent, and, importantly, blocked by tetrodotoxin (15, 16, 38). Although it remains unclear how this is brought about, the 30–60 min delay suggests that it is not a rapid, nongenomic action in the normal sense (39), and the dependence upon action potential propagation indicates that it may not be exerted directly upon the GnRH nerve terminals in the median eminence. Our present results with progesterone in the mouse are similar as we have found that progesterone does not influence the excitability of GnRH cell bodies in any direct or rapid manner. This is in contrast to other cell types where direct and rapid progesterone actions have been observed (39). Thus, although not clarifying our understanding of how progesterone stimulates GnRH secretion in vitro, the present results provide important evidence discounting the possibility of rapid and direct actions of progesterone itself upon the GnRH cell body.

Allopregnanolone is well established as a potent neuroactive steroid, derived from progesterone, which modulates GABAA receptor open time to influence neuronal function throughout the brain including the hypothalamus (17, 18, 23, 24, 30, 31, 32, 33). Our electrophysiological studies now demonstrate that 100% of postnatal GnRH neurons are modulated directly by allopregnanolone in the mouse. An interesting electrophysiological feature of GnRH neurons in their normal environment is the variable, but nonetheless substantial, number of miniature postsynaptic events observed in the presence of tetrodotoxin. We have shown previously that these events occur almost entirely as a result of tonic, action potential-independent release of GABA (28, 34). Although such tonic GABA release may contribute significantly to the total GABAA receptor-mediated current within a cell (40), the physiological regulation and roles of this random "quantal" release from presynaptic GABAergic inputs remains unclear (41). Nevertheless, in the present experiments, it is seems very likely that the direct membrane effects of allopregnanolone observed on GnRH neurons result from the potentiation of GABAA receptors activated by this quantal GABA release.

GnRH neurons in the preoptic area of the adult female mouse express mRNAs for {alpha}1, {alpha}5, ß1, and {gamma}2 subunits of the GABAA receptor (28) and we have so far verified the presence of {gamma}2 (41) and {alpha}5 (K. Yuri and A. E. Herbison, unpublished observations) subunit protein in GnRH neurons. Despite intensive analysis, it remains that the subunits critical for the allosteric modulation of the GABAA receptor by neurosteroids have not yet been firmly established, and it is likely that the allopregnanolone sensitivity of this receptor depends upon both subunit composition and the cell type within which it is expressed (30, 31, 32, 33, 35). In this context, it would appear that GABAA receptors comprised of {alpha}1, {alpha}5, ß1, and/or {gamma}2 subunits are one combination which results in GABAA receptors sensitive to allopregnanolone when expressed in native GnRH neurons. As juvenile GnRH neurons (28) synthesize a much wider variety of GABAA receptor subunits ({alpha}1, {alpha}2, {alpha}3, {alpha}5, ß1, ß2, ß3, {gamma}2, and {gamma}3), we thought it would be interesting to also examine allopregnanolone effects in juvenile GnRH neurons. In essence, we have found little difference between the allopregnanolone responses of juvenile and adult GnRH neurons; in both cases, 1 µM allopregnanolone potentiated the 3 µM GABA response by 2.2-fold. From a functional perspective, this indicates that allopregnanolone-sensitive GABAA receptors are expressed by GnRH neurons throughout postnatal development.

The physiological significance of the current findings have not yet been established. It is clear that the stimulatory effects of progesterone on LH secretion are entirely dependent upon E pretreatment and the PR (1, 2, 10, 11) and, therefore, seem unlikely to involve allopregnanolone. Indeed, progesterone’s ability to enhance LH secretion has been shown to be independent of GABAA receptor activation in the prepubertal rat (22). Thus, it seems most likely that the increase in LH secretion observed in females following the administration of allopregnanolone (19, 20, 21, 22) is occurring through the activation of GABAA receptors not normally involved in the positive feedback actions of progesterone.

Allopregnanolone is also known to mimic the inhibitory influence of progesterone on LH secretion (9, 19) and may, therefore, have a physiological role in mediating part of progesterone’s negative feedback actions. It is interesting to note that basal LH levels in PR knockout mice are intermediate between those of intact mice and ovariectomized PR knockout animals (10), suggesting that other non-PR-dependent gonadal influences such as E or allopregnanolone are active in suppressing LH secretion. In direct support of a role for allopregnanolone in the suppression of LH secretion, the intracerebroventricular administration of allopregnanolone has been shown to suppress ovulation in adult female rats (43) and also inhibit GnRH release from hypothalamic tissue in vitro through a GABAA receptor-mediated mechanism in adult male rats (44). It is noteworthy, however, that recent work has failed to find any substantial effect of intracerebroventricular allopregnanolone on LH secretion in the ewe (9). Future studies will need to address the role of allopregnanolone in regulating LH secretion in the mouse in vivo.

In terms of understanding the direction of allopregnanolone’s influence upon the GnRH neuron, it is important to note that the depolarizing electrophysiological responses to GABA reported here do not enable us to determine whether GABA inhibits or excites GnRH neurons in vivo. In common with most electrophysiological studies examining the GABAA receptor, we have employed a high chloride ion concentration in our internal patch pipette solution, and this results in all GABAA responses being magnified as well as appearing depolarizing in direction. Ongoing studies in our laboratory are establishing the precise nature of GABA’s influence upon the firing of GnRH neurons under conditions in which the intracellular chloride ion concentration is not disturbed. However, there is some evidence to suggest that, like most other neurons, GnRH cells are excited by GABA in embryonic (45) and early postnatal development (46), but inhibited by GABA in the adult (46, 47). As the sensitivity of GnRH neurons to allopregnanolone does not appear to alter postnatally, this developmental switch in GABA action may underlie the stimulatory effects of allopregnanolone observed on GnRH release from GT1-1 cells (29) and LH secretion from prepubertal rats (21, 22), compared with its inhibitory actions at the level of the cell body in the adult (43, 44). Finally, in terms of the potential physiological impact of direct allopregnanolone actions upon adult GnRH neurons, it is worth noting that allopregnanolone concentrations are maximally elevated during the luteal phase of the cycle as well as in pregnancy (25, 26, 42), two periods when GnRH secretion is at its lowest.

In conclusion, we provide here the first evidence for a direct effect of neuroactive steroids upon the electrical excitability of postnatal GnRH neurons. We also provide the first examination of whether progesterone itself may exert direct membrane actions upon the GnRH neuron but find no evidence that this is the case. Thus progesterone may have at least two modes of action for regulating the activity of the GnRH neuron in vivo; one through the classic genomic, PR-dependent pathway (1, 2) and another following its rapid conversion to allopregnanolone and the consequent direct allosteric modulation of GABAA receptor function in GnRH neurons (Fig. 3Go).


   Acknowledgments
 
We thank Dr. John Bicknell for critical appraisal of the manuscript.


   Footnotes
 
This work was supported by the Biotechnology and Biological Sciences Research Council (UK).

1 Present address: Institute of Molecular Physiology, University of Sheffield, UK. Back

2 Present address: Pfizer Global Research and Development, Cambridge, UK. Back

Abbreviations: ACSF, Artificial cerebrospinal fluid; DMSO, dimethylsulfoxide.

Received May 23, 2001.

Accepted for publication June 28, 2001.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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