Properties and Sex-Specific Differences of GABAA Receptors in Neurons Expressing gamma 1 Subunit mRNA in the Preoptic Area of the Rat

Sholeen T. Nett1, Juan Carlos Jorge-Rivera1, Margaret Myers1, Ann S. Clark3, and Leslie P. Henderson1, 2

1 Department of Physiology and 2 Department of Biochemistry, Dartmouth Medical School; and 3 Department of Psychology, Dartmouth College, Hanover, New Hampshire 03755 

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Nett, Sholeen T., Juan C. Jorge-Rivera, Margaret Myers, Ann S. Clark, and Leslie P. Henderson. Properties and sex-specific differences of GABAA receptors in neurons expressing gamma 1 subunit mRNA in the preoptic area of the rat. J. Neurophysiol. 81: 192-203, 1999. Gamma-aminobutyric acid type A (GABAA) receptors expressed within the medial preoptic area (mPOA) are known to play a critical role in regulating sexual and neuroendocrine functions. In the rat brain, high levels of expression of the gamma 1 subunit mRNA of the GABAA receptor are restricted to a limited number of regions that mediate sexual behaviors, including the mPOA. The biophysical and pharmacological profiles of native gamma 1-containing receptors in neurons are unknown. Here, we have characterized the properties of GABAA receptor-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) and currents elicited by fast perfusion of GABA to isolated mPOA neurons of juvenile male and female rats. No significant sex-specific differences were evident in the mean peak amplitude, distribution of event amplitudes, kinetics of current decay, or the frequency of sIPSCs. The profile of modulation of sIPSCs by diazepam, beta -CCM and zolpidem, allosteric modulators that act at the benzodiazepine (BZ) site of the GABAA receptor, support the assertion that mPOA neurons of both sexes express functional gamma 1-containing receptors. The ability of zolpidem to modulate both sIPSC amplitude and currents elicited by rapid perfusion of GABA to mPOA neurons differed significantly between the sexes. Zolpidem reversibly induced negative modulation of currents in mPOA neurons isolated from male rats, but had no effect in mPOA neurons from female rats. Concentration-response analysis of responses in neurons acutely isolated from male rats indicated an IC50 of 58 nM with maximal decreases of ~50% of control peak current amplitude. In situ hybridization analysis demonstrated that levels of the gamma 1 subunit mRNA are significantly higher in mPOA neurons from male than female rats. No significant sex-specific differences were detected in the levels of alpha 1, alpha 2, or alpha 5 mRNAs. These results suggest that native gamma 1-containing receptors are expressed in primary neurons of the mPOA and that sex-specific differences in the expression of this subunit may contribute to sexual dimorphism in GABAA receptor modulation by compounds acting at the BZ site.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Sexual dimorphism in neural structure has been described best in the medial preoptic area (mPOA) of the mammalian brain (for review Döhler 1991; Gorski et al. 1978; Sakuma 1995). This region is critical for the regulation of neuroendocrine function and for the production of sexual behaviors (for review, Madeira and Lieberman 1995; Theodosis and Poulain 1993). Within the mPOA, transmission mediated by gamma -aminobutyric acid type A (GABAA) receptors has been shown to regulate gonadotropin secretion (Adler and Crowley 1986; Moguilevsky et al. 1991) and to modulate the expression of both female sexual receptivity (for review; MCCarthy 1995) and male copulatory behavior (Fernández-Guasti et al. 1986; for review, Meisel and Sachs 1994). Although GABAergic transmission within the mPOA plays a key role in these hypothalamic functions, no study to date has assessed the physiological or pharmacological properties of GABAA receptors within this region.

The GABAA receptor is a heterooligomeric protein for which five families of mammalian subunit genes (alpha 1-6, beta 1-3, gamma 1-3, delta , and epsilon ) have been identified (Davies et al. 1997; for review, Macdonald and Olsen 1994; Whiting et al. 1997). It is believed that alpha 2, beta 3, and gamma 1 subunits comprise the predominant class of endogenous receptors in neurons of the mPOA in the adult rat brain (Herbison and Fénelon 1995). Although alpha 2 and beta 3 subunit mRNAs are expressed throughout the rat brain, high levels of gamma 1 subunit mRNA are limited to a small subset of interconnected forebrain regions that are known to be important in the regulation of sexual behaviors. Specifically, high levels of gamma 1 expression are detected in the mPOA, the medial amygdala, the lateral septal nuclei, and the bed nucleus of the stria terminalis (Araki et al. 1992, 1993; Herbison and Fénelon 1995; Wisden et al. 1992; Ymer et al. 1990). The highly restricted distribution in expression of this subunit mRNA has led to the suggestion that gamma 1-containing GABAA receptors may play a critical role in the regulation of hypothalamic function (Herbison and Fénelon 1995; Ymer et al. 1990).

Chloride flux through the GABAA receptor is modulated by a number of compounds acting at the benzodiazepine (BZ) recognition site (for review, Macdonald and Olsen 1994; Sieghart 1995). The effects of specific modulators depend on both alpha  and gamma  subunit composition (Macdonald and Olsen 1994; Sieghart 1995). gamma 1 subunits primarily coassemble with alpha 2 subunits in vivo (Wisden and Seeburg 1992) to create a receptor with a BZ type II pharmacological profile (for review, Sieghart 1995). Two classes of compounds that act at the BZ site, the imidazopyridines and the beta -carbolines, exhibit significantly different effects at recombinant receptors containing gamma 1 versus gamma 2 subunits. Specifically, the imidazopyridine, zolpidem, is a positive modulator of recombinant receptors containing alpha 2 and gamma 2 subunits, but is either ineffective or induces negative modulation (an inverse agonist) at receptors containing alpha 2 and gamma 1 subunits (Puia et al. 1991; Wafford et al. 1993). The effects of zolpidem on native receptors presumed to contain gamma 1 subunits have not been tested. The beta -carbolines act as inverse agonists at both native and recombinant receptors containing gamma 2 subunits (Rogers et al. 1994; Vicini et al. 1987), but potentiate or have no effect at either recombinant receptors containing alpha 2 and gamma 1 subunits (Puia et al. 1991; Wafford et al. 1993) or receptors in glial cells that are believed to contain a gamma 1 subunit (Bormann and Kettenmann 1988). In contrast to zolpidem and the beta -carbolines, the BZ, diazepam, potentiates both alpha 2beta xgamma 2- and alpha 2beta xgamma 1-containing heterologous receptors; however, the potentiation is less when the gamma 1 subunit is expressed (Puia et al. 1991).

Although numerous studies have suggested that gamma 1-containing neurons may play a unique role in the expression of behaviors mediated by the hypothalamus, no study to date has characterized either the biophysical or pharmacological properties of native gamma 1-containing receptors in neurons. In the present study, we have assessed the properties of GABAA receptor-mediated currents in mPOA neurons both in intact slices and in isolated cells exposed to ultrafast applications of GABA and GABAA receptor allosteric modulators. In addition, we have correlated the physiological and pharmacological profiles of currents elicited from female or male animals with levels of gamma 1, alpha 1, alpha 2, and alpha 5 subunit mRNAs.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Electrophysiological recording

For recording and analysis of spontaneous inhibitory postsynaptic currents (sIPSCs), animals, (postnatal day 10, PN10, to postnatal day 20, PN20) were decapitated, and the brains quickly dissected and placed in ice-cold saline. A thick coronal section was mounted with cyanoacrylic ester (Krazy glue) on the chuck of a Campden Vibroslice microtome (Stoelting, Wood Dale, IL), and 300 µm slices at the level of the anterior commissure were prepared. Initial recordings were made "blind," according to techniques described by Blanton et al. (1989), with the use of a model QL-2 recording chamber (Fine Science Tools, Foster City, CA) and a Nikon SMZ-2T microscope (MVI, Avon, MA). Later recordings were made under infrared optics using an Olympus BX50 microscope and a Dage VE1000 CCD camera system (Optical Analysis, Nashua, NH). No differences in data analyzed were noted between the two experimental set-ups. All recordings were made from slices superfused with 95% O2-5% CO2-saturated saline and from the central portion of the mPOA, a region containing the sexually dimorphic nucleus of the mPOA (SDN-POA) (Jacobson et al. 1980).

Ultrafast perfusion of 1 mM GABA for 3 ms to acutely isolated neurons dissected from the mPOA was made as previously described (Smith et al. 1996) with the minor modification that a LSS-3100 High Speed Positioning System (Burleigh Instruments, Fishers, NY) was used for solution switching. Assessment of open tip currents elicited by a switch from bath saline to bath saline diluted 50:1 with water (Lester and Jahr 1992) indicated that 10-90% of the peak on and off responses that reflect time of solution exchange were achieved in <500 µs with this system, and that applications were stable with repetitive exposures. For recordings made from both slices and isolated cells, electronic compensation was used to reduce the effective series resistance and the time constant of membrane charging during whole cell recordings and to provide measurements of access resistance and cell membrane capacitance. Series resistance compensation was 50-75%. All recordings were made in artificial CSF (aCSF) (in mM): 125 NaCl, 4 KCl, 26 NaHCO3, 2 CaCl2, 1 MgCl2, and 10 glucose. CNQX (10 µM) and CPP (20 µM) were added to block glutamatergic transmission, as described previously (Smith et al. 1996). Pipette saline consisted of (in mM) 153 CsCl, 1 MgCl2, 5 ethylene glycol-bis (beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 10 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) to which 2 mM MgATP was added each day. Unless otherwise indicated, standard chemicals were purchased from Sigma Chemical (St. Louis, MO). Recordings were made at 20-22°C; at a holding potential (VH) of -80 mV; and for slice recordings, preferentially from the superficial surface. The imidazopyridine, zolpidem hemi-tartrate (N,N-6-trimethyl-2-(4-methylphenyl)-imidazo[1,2-a]-pyridine-3 acetamide; a gift from Synthélabo Recherche, Bagneux Cedex, France) was dissolved in water (pH 4.8) and added to aCSF at final concentrations of 1 nM to 20 µM. beta -CCM (9H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester) and diazepam (7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one) were purchased from RBI (Natick, MA), dissolved in dimethyl sulfoxide (DMSO), and added to aCSF at a final concentration of 10 and 1 µM, respectively (0.01% DMSO). To determine a baseline for establishing drug effects, averaged peak current amplitudes of responses in the absence of any drug were measured at 3 min intervals for a period of 12 min. The standard deviation of response amplitudes over this time period was ±8% for all cells (n = 5). This measurement reflected inclusion of a few outlying individual data points. Exclusion of these few points provided an estimate of ±7% deviation of peak current amplitude over time in the absence of drug. BZ modulators were categorized as "without effect" if drug-induced changes were <7% of the initial value.

For slice recordings, BZ modulators were applied via the bath using gravity flow and a Hamilton multiport valve to switch between aCSF with and without BZ modulators. For all recordings, 3-5 min of predrug data were acquired. The bath was then changed to aCSF containing diazepam, beta -CCM or zolpidem, and 3 min later, data were again acquired for 3-5 min. The bath was then switched back to aCSF alone and 3 min were allowed to pass before postdrug data were collected. For application of GABA plus BZ modulators to acutely isolated cells, solution exchange of the double barrelled theta glass was accomplished by use of solenoid valves attached to a vacuum line (Zhu and Vicini 1997), thus neurons were pre-equilibrated with modulators and saline before being exposed to BZ modulators plus GABA. Concentration-response data for modulation of peak current amplitude by different concentrations of zolpidem was fitted according to Kapur and Macdonald (1996).

Data from slices and isolated cells were acquired and analyzed as described previously (Smith et al. 1996) with the use of a List EPC-7 amplifier (ALA Scientific Instruments, Westbury, NY) and either an Atari Mega 4 computer system or a PowerMac 8600. For analysis of sIPCSs, the acquisition program Acquire (Instrutech, Elmont, NY) was adapted to acquire randomly occurring, spontaneous synaptic currents. To avoid distortion due to cable properties, only events with 10-90% rise times <= 2 ms were included in data averages (Smith et al. 1996). For sIPSCs, >50 individual responses from each cell were averaged, and the software program, Review, was used to estimate rise times and to provide multiple iterative, exponential fits to current decays. Responses elicited with ultrafast perfusion were acquired with either the program Acquire or Pulse and analyzed by either Review or PulseFit (Instrutech). Data from both acutely isolated cells and slice recordings were digitized at 23.6 kHz and filtered at 4 kHz for analysis. Values are means ± SE. Statistical significance was determined using a two-tailed Student's t-test for comparison of means and a chi 2 test of association for comparison of percentages.

In situ hybridization

In situ hybridizations were carried out according to Wisden et al. (1992), with minor modifications, for animals of both sexes at PN14 (the mean age of animals used for physiological experiments). Oligonucleotides corresponding to subunit residues 341-354 of the gamma 1 cDNA (Ymer et al. 1990) and corresponding to subunit residues alpha 1: 342-356, alpha 2: 340-344, and alpha 5: 355-369 (Wisden et al. 1992) were synthesized by Operon Technologies (Alameda, CA) and 3' end-labeled using a 10:1 molar ratio of alpha -35S-dATP (Dupont/New England Nuclear, Boston, MA; 1,000-1,270 Ci/mmol) to oligonucleotide using terminal deoxynucleotide transferase (Promega, Madison, WI). Unincorporated, free nucleotides were removed using Centri-Sep columns (Princeton Separations, Adelphia, NJ). Labeled probe (specific activity 0.5-1 × 109 cpm/µg) was resuspended in a hybridization mixture consisting of 50% deionized formamide, 4× SSC, 1× Denhardt's solution, 100 µg/ml salmon sperm DNA, 150 µg/ml yeast tRNA, 50 mM dithiothreitol, and 10% dextran sulfate at a concentration of 1× 106 cpm/100 µl. Animals were decapitated, brains quickly removed, frozen on dry ice, and stored at -80°C for 1-4 wk. Sections (20 µm were cut using a Hacker-Bright model OTF cryostat, mounted on poly-L-lysine-coated slides, fixed with 4% paraformaldehyde (Fisher Scientific, Springfield, NJ) for 20 min, washed once with 3× and then twice in 1× phosphate-buffered saline (PBS), and dehydrated in serial ethanol (EtOH) (30, 60, 80, 95, and 100%). Before hybridization, slides were brought to room temperature, washed once with 0.1 M glycine in 1× PBS for 3 min, twice for 10 min each in 1× PBS, incubated for 10 min in 0.1 M triethanolamine/0.25% acetic anhydride, washed twice for 15 min each in 2× SSC, dehydrated in serial EtOH, and air dried. Hybridization was carried out in a moistened chamber at 42°C overnight. After hybridization, sections were washed for 5 min at room temperature in 1× SSC, followed by three 30 min washes at 55°C in 1× SSC in a shaking water bath, and a final 5 min wash in 1× SSC at room temperature. Sections were dehydrated in 30% EtOH/0.6 M NaCl, 60% EtOH/0.6 M NaCl, 80% EtOH, 95% EtOH, and 100% EtOH, air dried for ~1 h, and apposed to Biomax MR film (Eastman Kodak, New Haven, CT) for 2-3 wk. Included in each film cassette was a slide mounted with 14C microscales (Amersham, Arlington Heights, IL), which was used to quantify densitometric signals (Huntsman et al. 1994) and slides incubated with labeled probe and a 50-fold excess of unlabeled oligonucleotide to assess nonspecific hybridization. Films were developed using Kodak GBX developer and replenisher. Subsequent to exposure to film, slides were stained with cresyl violet. Quantitative analysis of the autoradiograms was performed using the MCID system (Imaging Research, St. Catherines, Ontario, CA). Densitometric measurements were taken from the central portion of the mPOA, a region containing the SDN-POA. The region analyzed by densitometry was comparable in extent to the region sampled for slice recording. The density values are expressed as means ± SE from 5-10 sections of the mPOA for each animal examined. Sex-specific comparisons of levels of signal corresponding to each subunit-specific probe were always made within a single experiment with probe labeled on the same day and all sections hybridized on the same day. Data were analyzed using a one-way analysis of variance (ANOVA) followed by posthoc comparisons using the Scheffé test.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Functional analysis of GABAA-mediated sIPSCs in neurons from the mPOA

Although putative gamma 1-containing receptors in astroglia have been characterized (Bormann and Kettenmann 1988), no study to date has defined the properties of native, neuronal gamma 1-containing receptors. Because steroid hormones can act both during early postnatal development (Smith et al. 1996) and during adulthood to regulate GABAA-mediated responses (Brussaard et al. 1997; Herbison and Fénelon 1995), experiments were performed on neurons from rats between postnatal day 10 (PN10) and PN20, ages corresponding to a developmental window subsequent to the critical period for most of the organizational actions of gonadal steroids, but preceding activational changes associated with the onset of puberty (for review, Döhler 1991; Rhees et al. 1990a,b). Properties of synaptic GABAA receptors were assessed from analysis of sIPSCs recorded in intact brain slices. Recordings were made from the central portion of the mPOA in the presence of pharmacological blockers of glutamatergic transmission (see METHODS). Under these conditions, sIPSCs were observed in >90% of mPOA neurons recorded from both sexes (Fig. 1A). Spontaneous IPSCs were completely and reversibly blocked by 10 µM bicuculline (n = 5; data not shown). Analysis of the frequency of sIPSCs indicated that values were similar for recordings from mPOA neurons of both male and female rats (1.44 ± 0.23 s-1 for females, n = 21 and 1.57 ± 0.23 s-1 for males, n = 20). Current decays were best fitted by the sum of two exponential components with time constants, tau 1 and tau 2, of 11.43 ± 0.37 ms and 44.08 ± 2.01 ms for females (n = 68) and 12.51 ± 0.51 ms and 46.06 ± 1.85 ms (n = 57) for males (Fig. 1, B and C). No significant differences were evident for recordings from females versus males in the values of tau 1 or tau 2 or the percentage of the peak current amplitude contributed by each kinetic component. Mean peak amplitudes were 112.9 ± 4.7 pA for females and 116.0 ± 6.7 pA for males (Fig. 1D). Further assessment of the distribution of current amplitudes indicated peaks corresponding to 43, 85, and 131 pA for males (Fig. 1E), and a similar distribution with peaks corresponding to 44, 76, and 134 pA for females (data not shown). Average peak current amplitudes of miniature IPSCs (mIPSCs) recorded in the presence of 1 µM tetrodotoxin were 48.0 ± 2.0 pA (n = 3), suggesting that the observed peak in the distribution of sIPSCs at 44 pA corresponds to single mIPSCs and subsequent peaks to multiples of these events. No statistical differences were evident in the distribution of current amplitudes between the sexes, and the coefficients of variation for the amplitude distributions were 0.60 for males and 0.57 for females.


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FIG. 1. gamma -Aminobutyric acid type A (GABAA) receptor-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) in neurons from the medial preoptic area (mPOA). A: representative superimposed sIPSCs recorded in the whole cell configuration from in an intact slice preparation. VH = -80 mV. Scale bar: 50 pA; 20 ms. B: representative averaged sIPSC from this neuron. Current decay was fitted by 2 exponential components with time constants, tau 1 and tau 2. Heavier line: exponential fit to the data. Lighter lines: individual components of the fit that correspond to tau 1 and tau 2. Numbers in parentheses: contribution of each kinetic component to the peak current. Scale bar: 50 pA; 20 ms. C: average time constants (tau 1 and tau 2) were calculated for each cell and averaged for recordings made from mPOA neurons from male and female rats. Error bars: SE. n: number of cells. D: average peak current amplitudes were calculated for each cell and averaged for recordings made from mPOA neurons from male and female rats. Error bars: SE. n: number of cells. E: distribution of individual event amplitudes calculated for 10 neurons from the mPOA of male rats. Events (>100) were analyzed for each cell. Peaks were evident at 43, 85, and 131 pA. Distribution of events for mPOA neurons from females was similar (data not shown). Bin width: 5 pA.

Modulation of sIPSCs by compounds acting at the BZ recognition site of the GABAA receptor

The presence of a gamma  subunit is required for modulation of GABAA receptor function by compounds acting at the BZ site. The imidazopyridine, zolpidem, and the benzodiazepine, diazepam, are considered classic agonists for BZI and BZII type GABAA receptors, respectively. Conversely, beta -carbolines are characterized as classical inverse agonists that negatively modulate GABAA receptor function (for review, Sieghart 1995). Previous studies have demonstrated that specific subtypes of recombinant receptors expressed in heterologous cells and native receptors in glial cells show a paradoxical pattern of modulation by zolpidem and the beta -carbolines that has been attributed to the presence of a gamma 1 versus a gamma 2 subunit (Bormann and Kettenmann 1988; Puia et al. 1991; Wafford et al. 1993). Specifically, while diazepam potentiates either gamma 1- or gamma 2-containing receptors, zolpidem acts as an inverse agonist (or is without effect), and the beta -carboline, beta -CCM, acts as a positive modulator (or is without effect) at receptors containing a gamma 1 subunit. To determine if the high levels of gamma 1 subunit mRNA in the mPOA result in the expression of neuronal receptors with functional properties consistent with those reported for recombinant receptors, we analyzed modulation of sIPSCs in mPOA neurons by the benzodiazepine, diazepam, the beta -carboline, beta -CCM, and the imidazopyridine, zolpidem. Drugs were applied to acutely isolated slices at 1-20 µM, concentrations previously shown to modulate the moderate affinity alpha 2beta xgamma (1 or 2) recombinant receptors (Puia et al. 1991), as well as receptors in glial cells (Bormann and Kettenmann 1988).

Analysis of modulation of sIPSCs in neurons from the mPOA indicated that BZ-like compounds elicited a paradoxical pattern of modulation of sIPSCs similar to that reported for GABA-elicited responses from recombinant alpha 2beta xgamma 1 receptors and receptors in glial cells. The overall effect of diazepam was to potentiate sIPSCs, while beta -CCM induced no significant effect and zolpidem induced a negative modulation of current amplitudes (Figs. 2 and 3). Specifically, diazepam elicited potentiation of sIPSCs in 77% of the mPOA neurons tested (n = 22), increasing either peak amplitude (68% of cells) and/or prolonging current decay (59% of cells). In contrast to the profile of pharmacological sensitivity typically seen with gamma 2-containing receptors, zolpidem induced positive modulation in only 22% of the neurons tested (n = 37), and beta -CCM induced negative modulation in only 20% of the cells examined (n = 37). All three pharmacological profiles are consistent with those reported for recombinant gamma 1-containing receptors or receptors in glial cells. Both zolpidem and beta -CCM were able to elicit classical responses predicted for gamma 2-containing receptors in other brain regions. Consistent with previous studies (Hollrigel and Soltesz 1997; Rovira and Ben-Ari 1993; Soltesz and Mody 1994; Vicini et al. 1987), zolpidem and diazepam acted as positive modulators of sIPSCs, and beta -CCM acted as an inverse agonist in neurons of the ventromedial nucleus of the hypothalamus (VMN) and the dentate gyrus (data not shown), two regions known to express high levels of gamma 2 subunit mRNA (Wisden et al. 1992).


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FIG. 2. Modulation of sIPSCs in neurons from the mPOA by compounds acting at the BZ modulatory site indicating pharmacological profiles consistent with expression of gamma 1-containing receptors. Representative, averaged sIPSCs recorded before modulator was added to the bath and >= 3 min after addition of (A) 1 µM diazepam, (B) 10 µM beta -CCM, and (C) 20 µM zolpidem. Response amplitudes were enhanced and/or current decays prolonged by diazepam, no change was observed in response to beta -CCM, and zolpidem either induced negative modulation of current amplitude or had no effect. Scale bar: 50 pA; 20 ms.


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FIG. 3. Sex-specific differences in modulation of GABAA-mediated sIPSCs by compounds acting at the BZ binding site. A: representative, superimposed, averaged responses in mPOA neurons in intact slices from female rats (top) illustrating potentiation of currents by diazepam (left), no change in response to beta -CCM (middle), and no change in response to zolpidem (right). Representative averaged responses in mPOA neurons in intact slices from male rats (bottom) illustrating potentiation of currents by diazepam (left), no change in response to beta -CCM (middle), and diminution of the response by zolpidem (right). Scale bar: 50 pA; 20 ms. B: scatter plot indicating potentiation of depression of peak current amplitudes observed in individual neurons of the mPOA from male or female animals exposed to diazepam, beta -CCM, or zolpidem. Each data point represents the percentage change in peak current amplitude for an individual neuron for females (square ) or males (black-square). C: cumulative data indicating the percentage enhancement or percentage diminution of control peak sIPSC amplitude elicited by diazepam (left), beta -CCM (middle), and zolpidem (right) for neurons from females and males (as indicated). Modulation of average peak current amplitudes was assessed by plotting the ratio of the average peak current estimated during exposure to modulator to the average peak current prior to its addition. No significant sex-specific differences were evident in the ability of diazepam or beta -CCM to modulate the responses, however zolpidem decreased the peak current amplitude in mPOA neurons of male rats to 77 ± 4% of the initial amplitude (P < 0.05), but had no effect on amplitudes of sIPCSs in mPOA neurons from female rats (104 ± 4%). *The average ratio (Ipeak zol/Ipeakcon) for responses from mPOA neurons from males was significantly lower than that for females (P < 0.001). Error bars: SE. n: number of cells.

Sex-specific differences in modulation of sIPCSs by zolpidem

Further analysis of the effects of modulators acting at the BZ site of the GABAA receptor revealed significant sex-specific differences in the efficacy of zolpidem to modulate sIPSC amplitudes in neurons of the mPOA (Fig. 3). Zolpidem significantly decreased the average peak current amplitude to 77.3 ± 4.2% (n = 19) of the control amplitude for neurons from male (P < 0.05), but had no significant effect on peak amplitudes in neurons from female (104.1 ± 3.9%; n = 18) rats. The average ratio of peak amplitude in the presence of the modulator to peak amplitude in control saline from mPOA neurons from males was significantly lower than that for females (P < 0.001) (Fig. 3B). Moreover, zolpidem was found to induce negative modulation in 85% of mPOA neurons tested from males (n = 19), but in only 11% of neurons recorded in the mPOA from females (n = 18; P < 0.005). No significant effects on the values of tau 1, tau 2, or the relative contribution of each component to the total peak current amplitude were evident on analysis of sIPSCs exposed to zolpidem. With regard to other modulators that act at the BZ binding site, no significant sex-specific differences were detected in the ability of beta -CCM to modulate sIPSC amplitude or kinetics in mPOA neurons (Fig. 3). Diazepam induced slightly greater modulation of peak current amplitude in mPOA neurons from females than from males; an effect that approached, but did not attain significance (Fig. 3). Diazepam was found to prolong current decays in 77% of neurons from females and in 60% of neurons from males. Diazepam did not induce changes in current decay kinetics in neurons that did not also show enhancement of amplitudes. For those neurons in which diazepam did increase the peak current amplitude, current decay, as estimated by a monexponential fit, was increased 1.3-fold in neurons from females and 1.2-fold in neurons from males. Neither increase in current decay was significant.

Zolpidem modulation of currents elicited by ultrafast perfusion of GABA to isolated mPOA neurons

Modulation of sIPSCs by compounds acting at the BZ site was assessed at concentrations shown previously to modulate currents for recombinant receptors (Puia et al. 1991) and receptors in glial cells (Bormann and Kettenmann 1988). It has been suggested, however, that drug diffusion may be limited in slice recordings and that the actual concentrations of modulators present at synapses is far less than those applied (Brussaard et al. 1997; Rovira and Ben-Ari 1993). To examine the ability of zolpidem to modulate GABAA receptor-mediated currents under conditions where drug concentrations delivered are equal to those reaching the cell, access is not restricted, and washout of drugs is not problematic, we also assessed the effects of zolpidem on currents elicited by ultrafast perfusion of 1 mM GABA onto neurons acutely isolated from the mPOA. Application of 1 mM GABA for 3 ms was chosen to mimic GABA concentrations present at the synaptic cleft (Jones and Westbrook 1995). Greater than 90% mPOA neurons from either sex for which GOmega seals were obtained were responsive to GABA (Fig. 4A). Current decays were best fitted by the sum of three exponential components (Fig. 4A) with average values of tau 1 = 8.39 ± 0.60 ms, tau 2 = 57.11 ± 2.49 ms, and tau 3 = 145.90 ± 16.71 ms for females (n = 32) and tau 1 = 8.55 ± 0.37 ms, tau 2 = 54.22 ± 1.77 ms, and tau 3 = 220.74 ± 48.37 ms for males (n = 103) (Fig. 4B).


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FIG. 4. Responses elicited by ultrafast perfusion of GABA to isolated mPOA neurons. A: representative individual current response elicited by exposure of an mPOA neuron to 1 mM GABA for 3 ms. Current decays were best fitted by the sum of 3 exponential components described by tau 1, tau 2, and tau 3. Numbers in parentheses: percentage of the peak amplitude attributed to each kinetic component. VH = -80 mV. Scale bar: 200 pA; 100 ms. B: time constants (tau 1, tau 2, and tau 3) for recordings made from mPOA neurons from male and female rats were calculated for individual cells and averaged. Error bars: SE. n: number of cells.

Previous studies have shown that BZs modulate currents elicited by millimolar concentrations of GABA (Lavoie and Twyman 1996; Mellor and Randall 1997). To assess the effects of zolpidem on currents elicited by ultrafast perfusion of GABA, solutions were switched from saline alone in one port of the theta glass and saline plus 1 mM GABA in the second port to saline plus zolpidem and saline plus 1 mM GABA plus zolpidem by changing the reservoirs of solution for each side of the theta tubing. Control experiments in which solution switching was made from one reservoir containing 1 mM GABA to a separate reservoir containing 1 mM GABA (but no zolpidem) showed no change in current amplitude (99.5 ± 9.3% of control, n = 11). When zolpidem was co-applied with GABA, negative modulation of current amplitudes was induced in 100% of mPOA neurons from male rats tested with concentrations of zolpidem ranging from 100 nM to 20 µM (n = 29) (Fig. 5, A and B). The maximum inhibition induced was ~50% of the peak control amplitude and was observed with 1, 10, and 20 µM zolpidem. The negative modulation in current amplitude induced by zolpidem was reversible (Fig. 5A), and the concentration response relationship indicated an IC50 = 58 nM zolpidem (Fig. 5B). No significant differences in the time constants (tau 1, tau 2, and tau 3) of current decay were induced by 10-20 µM zolpidem. However, the percentage of the peak current amplitude attributed to tau 1 was increased significantly (P < 0.01) from 25.14 ± 3.90% of the peak response in control solutions to 41.86 ± 3.37% during zolpidem application (n = 21). This effect was completely reversible on return to control saline plus GABA alone. In marked contrast to the maximal decrease elicited by co-application of µM concentrations of zolpidem and 1 mM GABA to mPOA neurons isolated from male rats, no modulation of currents was produced by 10 µM zolpidem in neurons isolated from females (100.0 ± 7.9% of peak control amplitude; n = 8) (Fig. 5, A and B). These data are consistent with the negative modulation of sIPSCs induced by zolpidem in intact slices from male rats and the lack of effect of this modulator on sIPSCs recorded from mPOA neurons in intact slices from females.


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FIG. 5. Dose-response relationship for zolpidem modulation of GABA-elicited responses in acutely isolated neurons of the mPOA. A: representative whole cell responses elicited by a 3 ms/1 mM application of GABA to mPOA neurons from a female (top) and male (bottom) rat (VH = -80 mV). Solutions were then exchanged to preequilibrate the cell by exposing it to aCSF + 10 µM zolpidem and subsequent responses elicited by a 3 ms application of 1 mM GABA + 10 µM zolpidem. In the presence of µM zolpidem, peak current amplitudes were decreased in mPOA neurons isolated from male rats. Returning solutions to aCSF and applying a 3 ms pulse of 1 mM GABA alone (wash), indicated that the decrease in peak amplitude elicited by zolpidem in males was reversible. In contrast, this concentration of zolpidem elicited no effect on GABA-induced currents in neurons from female rats. Scale bar: 200 pA; 100 ms. B: concentration-response relationship for mPOA neurons acutely isolated from male rats (bullet ) indicating the modulation of control responses by the indicated concentrations of zolpidem. Error bars: SE. n: number of cells. Maximal inhibition of ~50% was observed with 1, 10, and 20 µM zolpidem, and the decrease was reversible. IC50 = 58 nM. No effect of 10 µM zolpidem was observed in mPOA neurons from female rats (black-square).

In situ hybridization of subunit-specific mRNAS in the mPOA

Previous studies using both in situ hybridization and immunocytochemical analyses have demonstrated that gamma 1 subunit mRNA and protein are both highly expressed in and highly restricted to the mPOA and a limited number of other forebrain nuclei (Araki et al. 1992, 1993; Herbison and Fénelon 1995; Wisden et al. 1992; Ymer et al. 1990), while levels of gamma 2 and gamma 3 mRNA are low to negligible (Wisden et al. 1992). However, these previous studies did not compare levels of GABAA receptor subunit mRNAs in male versus female rats. To determine if the ability of zolpidem to modulate currents in the mPOA is correlated with sex-specific differences in the levels of alpha  or gamma  subunit mRNAs, in situ hybridization experiments were performed. As has been reported previously (Wisden et al. 1992), signals corresponding to either the gamma 2 or gamma 3 mRNA were weak to negligible in the mPOA (data not shown). Consistent with the pharmacological modulation of GABAergic currents by zolpidem, densitometric analysis indicated that high levels of gamma 1 mRNA were detected in the mPOA for both sexes with signal intensities strongest in the central region of the mPOA (Fig. 6). Although expression in the mPOA was clearly evident in both sexes, signal corresponding to the gamma 1 subunit-specific probe was significantly higher (P < 0.05) for male than female animals (Fig. 6). The efficacy and potency of zolpidem is dependent on alpha , as well as gamma , subunit composition (for review, Sieghart 1995). Neither alpha 4 nor alpha 6 subunit mRNA is expressed to an appreciable extent in the mPOA (Wisden et al. 1992); however, levels of alpha 1, alpha 2/3, and alpha 5 are predicted to correlate with high affinity BZI type receptors, moderate affinity BZII receptors, and very low affinity BZIII receptors, respectively (for review, Sieghart 1995). Densitometric analysis of the signal corresponding to 35S-labeled oligonucleotides specific for alpha 1, alpha 2, and alpha 5 indicated that all three transcripts are expressed within the mPOA of both male and female rats; however, no significant sex-specific differences in the levels of any of these alpha  subunit mRNAs were detected (Fig. 7). Previous studies have shown that neither the beta 1 nor the beta 2 mRNA is strongly expressed in the mPOA at any developmental age and that levels of beta 3 mRNA are comparable from birth to adulthood (Zhang et al. 1991). Moreover, because beta  subunits are not believed to play a major role in conferring sensitivity to BZ-like modulators (for review, Sieghart 1995), sex-specific differences in the levels of beta  transcripts were not examined.


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FIG. 6. Sex-specific differences in gamma 1 mRNA levels in the mPOA. Representative dark field autoradiograms of coronal sections from a male (top) and a female (bottom) rat brain hybridized with an 35S-labeled oligonucleotide specific for the gamma 1 subunit mRNA (left). High levels of gamma 1 mRNA are evident in the mPOA of both sexes; however, densitometric analysis of film autoradiograms (right) indicated that signal intensity for the gamma 1 subunit mRNA was significantly greater in the mPOA of male than female rats (*P < 0.05). Error bars: SE. Numbers above bars indicate animals.


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FIG. 7. Comparison of levels of alpha 1, alpha 2, and alpha 5 mRNAs in the mPOA of female vs. male rats. Coronal sections at the level of the mPOA were hybridized with 35S-labeled oligonucleotides specific for the alpha 1, alpha 2, or alpha 5 mRNA. Densitometric analysis of film autoradiograms indicated no sex-specific differences in the mPOA for any of the three alpha  transcripts examined. Four animals of each sex were examined for each alpha  subunit mRNA-specific probe. Error bars: SE.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Region-specific regulation of GABAA receptor subunit genes has provided for a vast degree of diversity in receptor function (for review, Macdonald and Olsen 1994; Sieghart 1995). A number of previous reports have described the unique distribution of the gamma 1 subunit mRNA within the rat brain and hypothesized that this distribution may contribute critical aspects to hypothalamic function (Herbison and Fénelon 1995; Ymer et al. 1990). The properties of native, neuronal gamma 1-containing receptors have not been described previously. Biophysical characterization of GABAA-mediated currents from neurons of the mPOA indicated that sIPSCs had unitary peak current amplitudes of ~40 pA and decayed biexponentially (Fig. 1). No significant differences were evident in the amplitudes, frequencies, or decay kinetics of sIPSCs from mPOA neurons of female versus male rats.

Analysis of macroscopic currents elicited by ultrafast perfusion of GABA to acutely isolated cells indicated that current decays were best fitted by three exponential functions. The first two time constants of current decay agree well with those assessed from analysis of sIPSCs (Figs. 1 and 4). The third, slower time constant, evident in some cells exposed to GABA with ultrafast perfusion, was only rarely observed in sIPSCs. Differences in kinetics of currents elicited by ultrafast perfusion versus IPSCs have been observed for other GABAA receptor-mediated responses in other brain regions (Mellor and Randall 1997). These differences may reflect activation of a population of receptors with distinct kinetic properties from those present in the subsynaptic membrane or may arise if some parts of the whole cell are exposed to GABA with a slower time course than would be expected for an excised patch (Rossi and Hamann 1998). The parameters describing current amplitudes and decay for both sIPSCs and fast perfusion responses fall within a range of values reported for GABAA-receptor mediated responses within many different brain regions with highly heterogeneous receptor subunit composition. Although detailed descriptions of the biophysical properties of recombinant gamma 1-containing receptors have not been made, the main conductance state and burst durations for GABAA receptors in neurons and in glial cells are similar, suggesting that expression of the gamma 1 versus the gamma 2 subunit does not lead to dramatic differences in single channel conductance or gating (Bormann and Kettenmann 1988; Macdonald et al. 1989; Vicini et al. 1987).

In contrast to the basic biophysical properties of GABAA receptor-mediated responses, neurons in the mPOA demonstrated pharmacological profiles markedly distinct from other brain regions, but consistent with the expression of heterologous receptors containing a gamma 1 subunit (Puia et al. 1991; Wafford et al. 1993). Specifically, zolpidem, which is considered a classical positive modulator of BZI and, with lower potency, of BZII receptors (for review, Sieghart 1995), acted as an inverse agonist, or was without effect, in the mPOA. In contrast, beta -CCM, a classical inverse agonist (for review, Sieghart 1995), did not diminish GABAA receptor-mediated responses in the mPOA. In addition to the paradoxical response of neurons in the mPOA to modulation by compounds acting at the BZ site, preliminary data indicates that steroid modulators also act as inverse agonists in the mPOA, while inducing positive modulation in regions with high levels of gamma 2 mRNA (Jorge-Rivera and Henderson 1998). Taken together, these data raise the possibility that preferential expression of the gamma 1 subunit may confer a unique profile of pharmacological properties to neurons in the mPOA that extends to a broad range of allosteric modulators of the GABAA receptor.

Positive allosteric modulators are believed to enhance the rate of GABA association to the first agonist binding site (for review, Sieghart 1995), and experiments using ultrafast perfusion of GABA and diazepam support this hypothesis (Lavoie and Tywman 1996). Under saturating concentrations of GABA, positive BZ modulators are predicted to prolong current decay, but to have little effect on peak current amplitude. Numerous previous studies indicate that positive BZ modulators do not enhance mIPSC amplitudes, suggesting that at many central synapses release results in saturating concentrations of GABA in the synaptic cleft (for discussion, Mellor and Randall 1997). In the present study, diazepam was observed to potentiate sIPSC amplitudes. Similar potentiation of GABAA receptor-mediated current amplitudes by positive modulators has been reported for other central synapses (Frerking et al. 1995; Mellor and Randall 1997; Nusser et al. 1997) and for currents elicited by ultrafast perfusion of 1 mM GABA to recombinant receptors in heterologous cells (Lavoie and Twyman 1996). Spontaneous IPSCs recorded in the mPOA appear to reflect responses arising from single and multiple quanta (Fig. 1), and enhancement of sIPSC amplitudes in the mPOA may reflect the integration of multiple mIPSCs whose durations are prolonged by the action of positive BZ modulators (Mody et al. 1994). Alternatively, it has been suggested that the ability of BZ compounds to potentiate current amplitudes arises because GABA concentrations at some central synapse are not saturating (Frerking et al. 1995; Nusser et al. 1997) or that BZ compounds may act to recruit receptors (Lavoie and Twyman 1996).

In contrast to diazepam, zolpidem induced no modulation of sIPSCs recorded in mPOA neurons from female rats and negative modulation of peak sIPSC amplitude and peak amplitude of currents elicited by ultrafast perfusion of 1 mM GABA recorded in mPOA neurons from male rats. Zolpidem-induced decreases in the amplitudes of either sIPSCs or responses elicited by ultrafast perfusion of GABA to mPOA neurons from male rats were not accompanied by significant changes in the values of individual time constants of current decay. However, co-application of 1 mM GABA with 10-20 µM zolpidem, concentrations that induced a maximal decrease of peak current amplitude of ~50%, did induce a significant increase in the contribution of tau 1 to the peak response. Similar decreases in peak current amplitude accompanied by speeding of deactivation have been reported for the partial inverse agonist Ro15-4513 (Mellor and Randall 1997).

In addition to the unique profile of BZ sensitivity of GABAA receptor-mediated currents in mPOA neurons versus other brain regions, our data also demonstrate that within a given region the efficacy of BZ modulators may vary between the sexes. Specifically, we have demonstrated that zolpidem acts as an inverse agonist of both sIPSCs and responses elicited by fast perfusion of GABA in neurons of the mPOA from male rats, but does not modulate either sIPSCs or responses elicited by fast perfusion in neurons from female rats. Results from in situ hybridization experiments indicate that levels of gamma 1 subunit mRNA in the mPOA are higher in male than female rats. It is clear that BZ sensitivity will be dependent on alpha  as well as gamma  subunit composition (for review, Sieghart 1995). In situ hybridization and immunocytochemical analyses indicate that alpha 2 and beta 2/3 subunits are the most abundantly expressed transcripts and subunits in the mPOA of juvenile female rats (Jung et al. 1998) and adult rats for which sex was not determined (Wisden et al. 1992). Results from in situ hybridization experiments presented here indicate that alpha 1, alpha 2, and alpha 5 mRNAs are all detectable within the mPOA of both sexes and that alpha 2 is the most prevalent transcript. Moreover, our results show that there are no significant differences in the expression of any of the three alpha  transcripts between male and female rats. That alpha 1, alpha 2, and alpha 5 transcripts in the mPOA of female and male rats are equivalent suggests that the difference in gamma 1 mRNA levels cannot be attributed simply to an overall lower level of GABAA receptor subunit gene expression or to simply fewer cells in the female versus in the male rat mPOA. Although the sex-specific difference in zolpidem sensitivity appears to be best correlated with specific differences in levels of gamma 1 subunit mRNA between the two sexes, it is clear that the relationship between mRNA levels of the nucleus as a whole and pharmacological profiles of individual cells is at best a broad correlation. Emulsion autoradiography of gamma 1 mRNA expression in the mPOA of adult rats indicates that within the SDN-POA, there is heterogeneity in both the numbers of neurons expressing detectable levels of this transcript and in the relative levels expressed (Clark et al. 1998). It is therefore likely that some of the heterogeneity in the ability of BZ-like compounds to modulate sIPSCs in the mPOA arises from cell to cell differences in the levels of receptors containing not just gamma 1 subunits, but heterogeneous complements of alpha  subunits as well. Analysis of mRNA levels by single cell polymerase chain reaction techniques in individual neurons assayed for zolpidem sensitivity will provide the best assessment as to the role of differential expression of the gamma 1 subunit mRNA in the observed sex-specific difference in the ability of this modulator to affect GABAA receptor-mediated responses.

There is a noted paucity of published data on the effects of BZs in the hypothalamus or other forebrain structures important for the production of sexual and reproductive behaviors (for review, Wilson 1996). The different profile of sensitivity to BZ modulation for neurons in the mPOA compared with other brain regions may have implications for regulation of hormonally-controlled functions. Specifically, endogenous BZ compounds have been reported to be synthesized by ependymal cells in the hypothalamus (Malagon et al. 1993; Tong et al. 1991), and are believed to exert beta -carboline-like effects at the GABAA receptor (Bormann et al. 1985), regulate aggressive behaviors (Kavaliers and Hirst 1986), and modulate GnRH expression in the hypothalamus (Li and Pelletier 1995). Our results would predict that, in contrast to other hypothalamic regions, GABAergic transmission in neurons in the mPOA would not be negatively modulated by these putative, endogenous beta -carboline-like compounds. In addition, gonadal steroid-dependent regulation of gamma 1 mRNA levels may produce differences in the sensitivity of mPOA neurons to BZ modulators within a given sex. In particular, levels of gamma 1 subunit mRNA have been shown to be increased in the mPOA of adult, gonadectomized female rats given exogenous 17beta -estradiol (Herbison and Fénelon 1995) and to be regulated across the estrous cycle in gonadally-intact female rats (Clark et al. 1998). Recent reports have demonstrated that fluctuations in gonadal steroids in female rats alter GABAA receptor subunit composition and result in significant changes in the sensitivity of neurons to both neurosteroid (Brussaard et al. 1998) and BZ (Smith et al. 1998) modulators. These steroid-induced changes in receptor subunit composition, in turn, are believed to contribute to the execution of appropriate reproductive behaviors (Brussaard et al. 1998) and to underlie the insensitivity to BZ anxiolytic drugs that is associated with PMS and postpartum syndrome (Smith et al. 1998).

Although the number of studies is limited, BZ compounds are known to act both in a sex-specific manner and to affect sexual behaviors. Sensitivity to diazepam has been shown to vary both in a sex-specific and hormone-dependent fashion in rats (Fernández-Guasti and Picazo 1990; Fernández-Guasti and Picazo 1997; Pesce et al. 1994) and in humans (Sundström et al. 1997). In addition, intracranial infusion of diazepam has been shown to facilitate sexual receptivity (MCCarthy et al. 1995). Finally BZs have been shown clinically to result in sexual dysfunction (Fava and Borofsky 1991; Ghadirian et al. 1992). These studies suggest that BZ modulation of GABAA receptor function may play an important role in sex-specific behaviors. Our results suggest that the high level of the gamma 1 subunit mRNA within the mPOA results in expression of GABAA receptors with a distinct pharmacological sensitivity. Moreover, the ability of gonadal steroids to regulate gamma 1 expression in the mPOA (Clark et al. 1998; Herbison and Fénelon 1995), and thus sensitivity to allosteric modulators, suggests that GABAA receptors within the hypothalamus may show a degree of plasticity in function that is dependent on hormonal state.

    ACKNOWLEDGEMENTS

  We thank S. Vicini for many helpful discussions and suggestions concerning fast perfusion techniques.

  This work was supported by the National Science Foundation (IBN-9412203 to A. S. Clark and L. P. Henderson, and DBI-9707828 to J. C. Jorge-Rivera), The American Heart Association (91001490 to L. P. Henderson), the National Institutes of Health (RO1-NS28668 to L. P. Henderson), and (F30 MH11391 to S. T. Nett).

    FOOTNOTES

  Address for reprint requests: L.P. Henderson, Dept. of Physiology, Dartmouth Medical School, Hanover, NH 03755.

  Received 29 May 1998; accepted in final form 24 September 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

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