INTRODUCTION

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GABA_A receptors consist of a pseudosymmetrical, pentameric array of transmembrane subunits that form a receptor/Cl ion channel complex. GABA changes from an excitatory to an inhibitory neurotransmitter within 2 wk of birth, due to reversal of the Cl gradient (Obrietan and van den Pol 1995; Rivera et al. 1999). GABA_A receptor function is allosterically modulated by a variety of endogenous factors such as phosphorylation, pH, neurosteroids and Zn²⁺ (Hevers and Lüddens 1998; Huang and Dillon 1999; Moss and Smart 1996). In addition, a number of pharmacologic agents modulate the receptor, including benzodiazepines, barbiturates, general anesthetics, and convulsants (see Hevers and Lüddens 1998). Based on a repertoire of 20 subunits (1-6, 1-3, 1-3, , , , 1-3, , and ), the molecular architecture of native GABA_A receptors is extremely heterogeneous (Bonnert et al. 1999; Hevers and Lüddens 1998). Pharmacological studies of recombinant receptors have shown that individual subunits and their subtypes confer different sensitivities to GABA_A receptor modulators (Hevers and Lüddens 1998).

The hypothalamus plays an important role in regulation of a number of autonomic functions, including food intake, body temperature, cardiorespiratory activity, nociception/analgesia, circadian rhythms, and the endocrine system (for review, see Meister 1993). GABA suppresses the activity of hypothalamic neurons and has been suggested to be the dominant inhibitory neurotransmitter in the hypothalamus (Decavel and van den Pol 1990). GABA_A receptors as measured by [³H]muscimol binding are found throughout the hypothalamus (Xia and Haddad 1992). In situ hybridization studies of the hypothalamus have demonstrated that mRNA for 2, 3, 2, and  subunits is highly expressed, whereas mRNA for 1, 3, 5, 1, and 1 subunits is moderately expressed (Whiting et al. 1997; Wisden et al. 1992). In addition, message for 2 and 3 subunits is minimally expressed, whereas that for 4, 6, and  subunits is negligible or absent (Wisden et al. 1992). Immunocytochemical studies suggest the existence of 1, 2, 2, 3, and 2 subunits in hypothalamic magnocellular neurons (Fenelon and Herbison 1995) and 1, 2, 5, 2/3, and 2 in other hypothalamic regions (Davis et al. 2000; Fritschy and Mohler 1995; Pirker et al. 2000). Although the aforementioned techniques are clearly informative, it is accepted that conclusions derived using them may be limited. For instance, mRNA levels do not necessarily correlate with expression of functional receptors (Saha et al. 2001). Moreover, results from immunohistochemical studies may be impacted by tissue preparation and integrity, antibody selectivity, and the possible labeling of incompletely assembled and/or subcellular receptors. Thus the aim of the present study was to conduct a pharmacological analysis of a physiologically intact system to obtain functional evidence of expression of GABA_A receptor subunits that are purported to exist in the hypothalamus.

	METHODS

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Hypothalamic brain slices

Sprague Dawley rats (Indianapolis, IN), postnatal day (P) 1-14 (either sex) were rapidly decapitated. Chemical anesthesia was not used because of its well-known influence on GABA_A receptors (Franks and Lieb 1994). All procedures were conducted in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. All stages of brain dissection and tissue slicing were conducted in ice-cold (~4°C) artificial cerebrospinal fluid (ACSF) of the following composition (in mM): 124 NaCl, 5.0 KCl, 1.3 MgSO₄, 26 NaHCO₃, 1.24 KH₂PO₄, 2.4 CaCl₂, and 10 glucose; 300 mosM and pH ~7.4 after equilibration with a 95%-5% CO₂ gas mixture (carbogen). Thin hypothalamic slices (~200 µm) were cut with a vibratome (VSL, World Precision Instruments); slices were submerged in ACSF (22-25°C) aerated with the carbogen gas mixture. Slices were transferred to a recording chamber (~2 ml) and superfused continuously (7-10 ml/min, 22-25°C) with saline. To minimize synaptic influences on neurons under investigation, experiments were conducted in a synaptic blockade medium consisting of the following (in mM): 128 NaCl, 3.0 KCl, 11.4 MgCl₂, 10 HEPES, 2.0 CaCl₂, and 10 glucose, 300 mosM and pH 7.3. This superfusion medium has been shown to block both evoked and spontaneous chemical synaptic potentials in medullary neurons (Dean et al. 1997).

Individual hypothalamic neurons within the slice were visualized using an upright, fixed stage microscope (Nikon Optiphot-2UD) equipped with standard Hoffman modulation contrast (HMC) optics and a video camera system (Sony model XC-75 CCD video camera module, Tandy video monitor). Pipette tip location was acquired at low magnification (×4) via the CCD camera. The anatomical location of each recorded neuron was determined by comparison to plates in a stereotaxic atlas (Paxinos and Watson 1986).

Whole cell patch-clamp recording

Whole cell patch recordings were made at room temperature (22-25°C). Except during acquisition of current-voltage relationships, cells were voltage-clamped at 60 mV. Patch pipettes of borosilicate glass (M1B150F, World Precision Instruments) were pulled (Flaming/Brown, P-87/PC, Sutter Instrument, Novato, CA) to a tip resistance of 7-8 M. The pipette solution contained (in mM): 140 CsCl, 10 EGTA, 10 HEPES, and 4 Mg-ATP; pH 7.2. GABA was prepared in the extracellular solution and was applied (10 s in most cases) to cells via gravity flow using a Y-shaped tube positioned near the target cell. With this system, the 10-90% rise time of the junction potential at the open tip was 60-120 ms. GABA-induced Cl currents from the whole cell configuration were obtained using a patch-clamp amplifier (PC-501A, Warner Instruments, Hamden, CT) equipped with a 5101-01G headstage. GABA-induced Cl currents were low-pass filtered at 5 kHz, monitored on an oscilloscope and a chart recorder (Gould TA240), and stored on a computer (pClamp 6.0, Axon Instruments) for subsequent analysis. Sixty to 80% series resistance compensation was applied at the amplifier. To monitor the possibility that access resistance changed over time or during different experimental conditions, at the initiation of each recording, we measured and stored on our digital oscilloscope the current response to a 5-mV voltage pulse. This stored trace was continually referenced throughout the recording. If a change in access resistance was observed throughout the recording period, the patch was aborted and the data were not included in the analysis.

Data analysis

Concentration-response profiles were generated for GABA and a number of modulatory compounds. Agonist concentration-response profiles were fitted to the following equation: I/I_max= [agonist]ⁿ/(EC₅₀ⁿ + [agonist]ⁿ), where I and I_max represent the normalized GABA-induced current at a given concentration and the maximum current induced by a saturating concentration of agonist, respectively. EC₅₀ is the half-maximal effective agonist concentration, and n is the Hill coefficient. Ligand applications were separated by 3-min intervals to allow for recovery from desensitization when present.

Effects of antagonists were analyzed using the equation: I/I_max=[drug]ⁿ/([drug]ⁿ+ IC₅₀ⁿ), where I is current at a given concentration of drug, IC₅₀ = the concentration of drug yielding a current half of I_max, and n is the Hill coefficient. All data are presented as means ± SE. Student's t-test (paired or unpaired) was used to determine statistical significance (P < 0.05). Analysis of the effects of both agonists and antagonists was performed using the GABA EC₂₅ concentration (10 µM); this concentration generates a stable current, elicits minimal receptor desensitization and allows for several-fold drug-induced potentiation of the GABA-evoked response. The effects of antagonists were expressed as inhibition of the steady-state GABA currents. Effects of agonists were quantified as the magnitude of potentiation of peak GABA current amplitude.

Drugs

GABA, ()-bicuculline methiodide, picrotoxin, zolpidem, ZnCl₂, furosemide, dehydroepiandrosterone sulfate (DHEAS), diazepam, methyl-6,7-dimethoxy-4-ethyl--carboline (DMCM), and 5-pregnan-3-hydroxy-20-one (3-OH-DHP) were obtained from Sigma (St. Louis, MO). Loreclezole was a gift from Janssen Pharmaceutical (Natick, MA). Picrotoxin, 3-OH-DHP, lorecelezole, zolpidem, DMCM, DHEAS, and furosemide were initially prepared in a dimethyl sulfoxide (DMSO) stock solution. The final concentration of DMSO was <0.05% (vol/vol) when diluted to the desired concentration in saline.

	RESULTS

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In all hypothalamic neurons tested, application of GABA activated an inward current when the membrane potential was voltage clamped at 60 mV. The recorded currents were outwardly rectifying, reversed at the Cl equilibrium potential, and completely blocked by the GABA_A receptor antagonist bicuculline (Fig. 1, A and B). These characteristics demonstrate that the evoked currents were due to activation of GABA_A receptors.

View larger version (12K): [in this window] [in a new window]   Fig. 1. GABA-activated currents in hypothalamic neurons. A: current-voltage relationship for GABA in hypothalamic neurons. In nearly symmetrical [Cl], ([Cl]o/[Cl]i = 157.4 mM/140 mM), EGABA was near 0 mV (4.2 mV). Note the outward rectification of the current response. B: inhibitory response to bicuculline in hypothalamic neuron. Traces from a single cell show current activated by 20 µM GABA recorded before, during, and after coapplication with 10 µM bicuculline (BIC). The horizontal bars above the current traces indicate duration of drug application. C: GABA concentration-response relationship for hypothalamic neurons. Each point represents the mean of six neurons. The peak current amplitude was normalized to the maximal current. See text for EC50 and Hill coefficient values.

GABA sensitivity

The sensitivity to GABA provides some information about receptor subunit composition. The concentration-dependent response of hypothalamic neurons to GABA (1-3,000 µM) is illustrated in Fig. 1C. The maximal amplitude of GABA-gated currents, typically achieved at a concentration of 300 µM, was 2,735 ± 189 pA (n = 27). EC₅₀ values ranged from 11.4 to 44 µM (median = 20 µM, n = 11) and Hill coefficient values ranged from 1.2 to 2.25 (median = 1.6, n = 11). The mean GABA EC₅₀ (20 ± 1.6 µM) and Hill coefficients (1.7 ± 0.2) of the entire group were similar to the median values, suggesting a single population of cells.

Diazepam, zolpidem and Zn²⁺ sensitivity

The benzodiazepine-site ligand diazepam is known to be dependent on the presence of the  subunit and is influenced by the  subunit present in the receptor (Knoflach et al. 1996; Mckernan et al. 1995). We assessed diazepam's ability to potentiate hypothalamic GABA-activated current in the present investigation. Coapplication of diazepam (0.01-3 µM) with 10 µM GABA significantly enhanced GABA-gated current; threshold for the stimulatory effect was 30 nM, and maximal potentiation (to 206 ± 12% of control) was achieved at a concentration of 3 µM (Fig. 2, A and D). The EC₅₀ and Hill coefficient for the diazepam effect was 60 ± 17 nM and 1.57 ± 0.56, respectively (n = 3-7).

View larger version (20K): [in this window] [in a new window]   Fig. 2. Effects of diazepam, zolpidem, and Zn2+ on GABA-activated currents in hypothalamic neurons. A-C: the traces were recorded from a single cell. Varying concentrations of diazepam (A), zolpidem (B), or Zn2+ (C) were coapplied with GABA (10 µM). Drug application was 10 s in all cases. D: concentration-response relationships for the traces shown in A-C. All GABA responses were normalized to the peak current induced by 10 µM GABA without drug. Each data point represents the average of 3 cells. The data were best fitted to a sigmoidal function. See Table 1 for mean values.

The affinity of zolpidem is also influenced by the  subunit isoform present in GABA_A receptors. Zolpidem has high affinity for GABA_A receptors containing an 1 subunit and is nearly insensitive to GABA_A receptors containing 5 subunits (Hevers and Lüddens 1998; Wingrove et al. 1994). Zolpidem (0.01-10 µM) markedly potentiated the current induced by 10 µM GABA in all cells (n = 14) recorded from the hypothalamus (Fig. 2B). The average EC₅₀ was 0.19 ± 0.07 µM and 3 µM zolpidem maximally potentiated the GABA current to 210 ± 18% of the control GABA response (Fig. 2D).

Inhibition of GABA_A receptors by Zn²⁺ is influenced by the receptor subunit composition, in particular the presence of the  subunit (Gingrich and Burkat 1998; Smart et al. 1991). As shown in Fig. 2C, GABA-activated currents in hypothalamic neurons were sensitive to varying concentrations of Zn²⁺ (1-1,000 µM). In the presence of 10 µM GABA, 1 µM Zn²⁺ caused a slight but significant potentiation of steady-state currents in 5/5 cells tested (to 114 ± 4.9% of control, P < 0.05). Concentrations of Zn²⁺ beyond 1 µM inhibited GABA-gated currents with an IC₅₀ of 70.5 ± 17 µM and a Hill coefficient value of 0.9 ± 0.14 (n = 5, Fig. 2D). Currents were decreased to ~10% of control in the presence of 1 mM Zn²⁺. The responses to both diazepam and Zn²⁺ indicate hypothalamic GABA_A receptors express , , and  subunits.

Loreclezole and DMCM sensitivity

Loreclezole is often used to distinguish 2-3-containing receptors from 1-containing receptors because it is more potent in the former receptors than the latter (Wingrove et al. 1994). As Fig. 3A shows, coapplication of loreclezole (0.3-100 µM) with 10 µM GABA enhanced the GABA current in all the cells tested (n = 18) in a concentration-dependent manner. Loreclezole was insoluble at concentrations >30 µM. However, the potentiating effect appeared nearly saturated at that concentration, so an EC₅₀ value could be estimated (mean EC₅₀ = 4.4 ± 0.7 µM, range = 1.8-10.0 µM). The maximal enhancement of peak current was 194 ± 12% of control (range: 119-272%) with 30 µM loreclezole. At higher concentrations (>10 µM), loreclezole produced an increase in the current decay rate (Fig. 3A).

View larger version (20K): [in this window] [in a new window]   Fig. 3. Effect of loreclezole (LZ) and the -carboline methyl-6,7-dimethoxy-4-ethyl--carboline (DMCM) on GABA-activated currents in hypothalamic neurons. A and B: representative traces were recorded from a single cell. Concentrations of loreclezole (A) or DMCM (B) coapplied with 10 µM GABA are shown above the traces. The duration of all drug application is 10 s. C: concentration-response relationships for the traces shown in A and B. Each data point represents the average of 11-18 cells. The data were best fitted to a sigmoidal function. See Table 1 for mean values.

Positive modulation of GABA_A receptors by the -carboline DMCM is also dependent on the  subunit isoform. Figure 3B shows that concentrations of DMCM at 10 µM caused a modest potentiation of the GABA response. The maximal effect (to 141 ± 12.8% of control) was observed at 30 µM DMCM. The average EC₅₀ from 11 cells was 7.7 ± 3.2 µM.

3-OH-DHP and furosemide sensitivity

3-OH-DHP, an endogenous progesterone metabolite, has been shown to differentially modulate GABA_A receptor function in a subunit-selective manner (Brussaard et al. 1997; Maitra and Reynolds 1999; Wohlfarth et al. 2002; Zhu et al. 1996). As shown in Fig. 4A, 3-OH-DHP potentiated GABA-activated currents in a concentration-dependent manner. Because the effect of 3-OH-DHP could not be readily washed out, it was necessary to use a new slice for determination of effects of different concentrations of 3-OH-DHP. Thus full concentration-response profiles were not collected. In addition, because 3-OH-DHP can directly open GABA_A receptor-Cl channels at concentrations >1 µM (Ueno et al. 1997), we did not evaluate concentrations >1 µM in the present investigation. Nevertheless, it is apparent that hypothalamic GABA_A receptors are markedly sensitive to 3-OH-DHP, as GABA currents increased to 187 ± 20 and to 423 ± 48% of the control in response to 0.3 µM (n = 7) or 1 µM 3-OH-DHP (n = 8), respectively (Fig. 4B).

View larger version (13K): [in this window] [in a new window]   Fig. 4. Effect of 5-pregnan-3-hydroxy-20-one (3-OH-DHP) and furosemide on GABA-activated currents in the hypothalamus. A: the traces were recorded from 2 different neurons. Concentrations of 3-OH-DHP coapplied with 10 µM GABA are shown above the traces. B: histogram summarizing the mean effect of 3-OH-DHP on GABA response. Each data point contains 7-8 cells.

Furosemide is a loop diuretic that acts as a selective, noncompetitive antagonist of 4- or 6-containing GABA_A receptors, with IC₅₀ values in the micromolar range (Knoflach et al. 1996; Korpi and Lüddens 1997). In 26 hypothalamic neurons tested, 300 µM furosemide, a concentration that inhibited the response to GABA by 39 ± 2% of control in rat recombinant 622 receptors (n = 7, data not shown), had no effect on GABA-activated current. Increasing the furosemide concentration 1 mM was also without effect in all hypothalamic neurons tested (n = 4).

Picrotoxin and DHEAS sensitivity

The CNS convulsant picrotoxin exerts its effects via blockade of GABA_A receptors. Although picrotoxin appears to be an effective blocker in most preparations (Bell-Horner et al. 2000; Krishek et al. 1996; Newland and Cull-Candy 1992), subunit-specific differences in affinity of picrotoxin for GABA_A receptor blockade do exist (Bell-Horner et al. 2000). Examples of typical responses of a hypothalamic neuron to picrotoxin-induced inhibition of the GABA response are illustrated in Fig. 5A. When coapplied with 10 µM GABA, picrotoxin (0.1-30 µM) induced a modest inhibition of peak GABA current and subsequently markedly enhanced the rate of current decay. The IC₅₀ value for picrotoxin inhibition of steady-state current was 2.6 ± 0.4 µM, and the Hill coefficient was 1.1 ± 0.1 (Fig. 5C).

View larger version (19K): [in this window] [in a new window]   Fig. 5. Inhibition of GABA-activated currents by picrotoxin (PTX) and dehydroepiandrosterone sulfate (DHEAS). A and B: representative traces recorded from individual neurons. Both antagonists inhibited peak current and accelerated current decay in a concentration-dependent manner. The duration of all drug application was 10 s. C: mean concentration-response profiles for inhibition of GABA current by picrotoxin or DHEAS. Each point is a mean response (n = 5-6) normalized to the control GABA response. Mean effects of PTX and DHEAS are presented in Table 1. Values represent percentage inhibition of GABA steady-state currents.

DHEAS is a noncompetitive antagonist of GABA_A receptors in a number of preparations (Majewka et al. 1990). Figure 5B illustrates the DHEAS-mediated inhibition of the response to 10 µM GABA in a hypothalamic neuron. DHEAS reversibly inhibited the current amplitude and accelerated current decay in a concentration-dependent fashion. Coapplication of 300 µM DHEAS with 10 µM GABA reduced the GABA steady-state current to 7.7 ± 3.5% of control. The IC₅₀ and Hill coefficients were 16.7 ± 2.3 µM and 0.9 ± 0.1, respectively, for DHEAS inhibition of GABA-activated current in hypothalamic neurons (n = 6, Fig. 5C).

The efficacies and potencies of the different ligands modulating GABA_A receptors in hypothalamic neurons are summarized in Table 1. Locations of all hypothalamic neurons (n = 69) studied in this investigation are shown in Fig. 6. All neurons tested were in the periventricular hypothalamus, and were located in the posterior, dorsomedial, lateral, ventomedial, and arcuate hypothalamic nuclei.

View larger version (27K): [in this window] [in a new window]   Fig. 6. Schematic representation of majority of recording sites studied in the experiment. Slices in A and D represent the rostral and caudal extremes, respectively, encompassing the hypothalamic region that was investigated. ARC, arcuate hypothalamic nucleus; DM, dorsomedial hypothalamic nucleus; DMD, dorsomedial hypothalamic nucleus, diffuse; LH, lateral hypothalamic area; PH, posterior hypothalamic area; VMH, ventromedial hypothalamic nucleus. The drawing sections were adapted from a stereotaxic atlas for adult rats (Paxinos and Watson 1986).

	DISCUSSION

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The involvement of GABA in regulating activity of hypothalamic neurons is well documented (Decavel and van den Pol 1990; Shonis and Waldrop 1995). Whereas several studies have assessed the potential expression of GABA_A receptor subunits in the hypothalamus, a functional characterization of these receptors has not been conducted. Thus we have performed a pharmacologic and physiologic analysis of hypothalamic GABA_A receptors. We chose to use the thin brain slice preparation in this analysis. This system more closely resembles the physiologic condition of the intact brain than isolated cultured cells and at the same time permits the rapid and consistent exposure of neurons to various exogenous ligands.

Analysis of  subunit isoform

The response to GABA itself varies significantly between receptors, depending on which  subunit is present. The sensitivity to GABA of 6- and 5-containing receptors (Hevers and Lüddens 1998; Knoflach et al. 1993) is 5- to 20-fold greater than those expressing 1, 2, or 3 subunits (Bell-Horner et al. 2000; Huang and Dillon 1998). The GABA EC₅₀ for these latter receptors is 15-30 µM (Bell-Horner et al. 2000), which is comparable to the value we obtained in hypothalamic neurons (20 µM). The fact that the individual EC₅₀ values were relatively evenly distributed around the mean does not support the existence of subsets of GABA_A receptors with widely disparate GABA affinities.

Effects of diazepam are also influenced by  subunit isoform. The EC₅₀ of diazepam for stimulation of hypothalamic GABA-gated current was 60 nM in the present investigation. This is nearly identical (65 nM) to that reported by Amin et al. (1997) for diazepam stimulation of recombinant 122 receptors. The efficacy of diazepam is significantly greater in 3-expressing compared with 1-expressing GABA_A receptors. Ducic et al. (1993) reported a 350% potentiation by diazepam of the GABA response in 312 receptors, whereas 112 receptors were potentiated ~125%. In agreement with these previous studies, we also observed that the maximal response to diazepam was 311% of control in rat recombinant 322 GABA_A receptors, which is approximately three times greater than that of rat 122 receptors (data not shown). In the present study, the average maximal stimulation of an EC₂₅ GABA response by diazepam was just over 100% (range = 68-155%). Thus significant expression of 3-containing receptors appears minimal. In addition, none of the neurons tested appeared to express as their predominant GABA_A receptor those incorporating 4 or 6 subunits. This is based on responses to both diazepam and furosemide. All hypothalamic neurons responded to diazepam, and recombinant receptors expressing 4 or 6 subunits are insensitive to diazepam (Knoflach et al. 1996). Sensitivity to diazepam does not of course eliminate the possibility that receptors expressing 4 or 6 subunits may also be present in hypothalamic neurons. However, all hypothalamic neurons were also insensitive to the diuretic furosemide. Furosemide blocks 4- and 6-expressing receptors with µM affinity but is ineffective in other receptor configurations (Knoflach et al., 1996; Korpi and Lüddens 1997). Thus the results together suggest few hypothalamic neurons utilize receptors incorporating 4 or 6 subunits as the predominant  subunit isoform.

Zolpidem has essentially no effect in 5-containing receptors and displays high affinity for 1- and 2-containing receptors (Hadingham et al. 1993; Lüddens et al. 1995). All neurons tested in the present investigation were stimulated by zolpidem, suggesting a lack of neurons that express predominantly 5-containg receptors. Taken together, results with GABA, diazepam, and zolpidem are consistent with the existence of functional hypothalamic GABA_A receptors that express predominantly 1 and/or 2 isoforms of the  subunit. Receptors incorporating 3, 4, 5, and 6 subunits, if present, represent minority populations. Our findings expand significantly on experiments that have used visualization techniques to evaluate subunit expression in the hypothalamus. Wisden et al. (1992) found minimal and undetectable levels of 4 and 6 mRNA, respectively. mRNA for all other  subunits was present to varying degrees, with mRNA for the 2 isoform being most consistently and strongly evident. Immunohistochemical studies indicated the existence of both 1 and 2 subunits and a lack of expression of 4 or 6 subunits in the most parts of hypothalamus (Davis et al. 2000; Fritschy et al. 1994; Pirker et al. 2000). Davis et al. (2000) has shown that the density of immunoreactivity for 1 and 2 subunits varies with specific hypothalamic nuclei and that the relative density of these subunits in some hypothalamic nuclei switches by P20. It should be noted that because our recordings were obtained only in animals up to P14 in age, conclusions regarding functional subunit expression must be restricted to this age range.

Analysis of  subunit isoform

The potentiating action of loreclezole depends on the presence of either 2- or 3-containing GABA_A receptors and is absent in 1-containing receptors (Hevers and Lüddens 1998; Wingrove et al. 1994). In the present study, loreclezole potentiated GABA-activated currents in all hypothalamic neurons tested. Loreclezole at high concentrations can also directly gate the GABA_A receptor, and this effect is also influenced by the  subunit isoform (Sanna et al. 1996). We have confirmed this direct activation effect of loreclezole in recombinant 122 receptors (unpublished observations). Thus in the present report the effect on GABA_A receptors of high loreclezole likely comprise both modulatory and direct gating effects. However, the subunit-selective effects for both allosteric modulation and direct activation are the same (Sanna et al. 1996). Thus our conclusions are unchanged.

Positive modulation of GABA_A receptors by the -carboline DMCM has been suggested to be due to interaction at the same site (Asn290) responsible for loreclezole stimulation of receptors expressing 2/3 subunits (Stevenson et al. 1995). DMCM (10 µM) caused a modest potentiation of hypothalamic GABA_A receptors, suggesting the functional expression of receptors incorporating 2 and/or 3 subunits. It should be noted that we cannot rule out the possible coexistence of 1 with 2/3 subunits in the same neuron because the sensitivity to loreclezole or DMCM does not necessarily mean the absence of 1 subtype. In general, the 2 subunit is recognized as the most ubiquitous in the CNS. With regard to expression of mRNA, the 3 isoforms appears to be the most highly expressed in the hypothalamus (Wisden et al. 1992), whereas protein detection using immunocytochemical studies indicate the presence of 1-3 subunits (Pirker et al. 2000). Our data provide functional confirmation that 2/3 subunit-containing GABA_A receptors are widely expressed in the hypothalamic areas.

Analysis of   and  subunits

In vivo,  and  subunits generally combine with either a  or  subunit (Hevers and Lüddens 1998). However, binary receptors of only  and  subunits can readily form functional GABA_A receptors, and they are known to exist in specific brain regions (Brickley et al. 1999; Kawahara et al. 1993). None of our experiments suggested the existence of binary receptors in hypothalamic neurons. Moreover, our pharmacological analyses favor expression of  over  subunits in hypothalamic GABA_A receptors. For instance, the IC₅₀ for Zn²⁺ is significantly greater in receptors (~50 µM) than in receptors (~1 µM) (Gingrich and Burkat 1998; Smart et al. 1991). We obtained in hypothalamic neurons a value consistent with the former (70.5 µM). In addition, neurons in the present investigation were sensitive to diazepam and zolpidem, which requires the presence of a 2 subunit in combination with an  and a  subunit (Pritchett et al. 1989). The maximal efficacy of diazepam to enhance an EC₂₀ [GABA] in 112 receptors was 86-135% (similar to the maximal efficacy of 106% we recorded in hypothalamic neurons), whereas only approximately a 25-58% potentiation was observed in 211/3 receptors (Ducic et al. 1993; Wafford et al. 1993a).

Responses to the neurosteroid 3-OH-DHP also favor expression of the  subunit over expression of the  subunit. In the present studies, we observed 323% potentiation of GABA-activated current in response to 1 µM 3-OH-DHP. The maximal potentiation is similar to values reported for 122 receptors (Maitra and Reynolds 1998; Zhu et al. 1996) and is several-fold less than observed with 12 receptors (Maitra and Reynolds 1998). Finally, the maximal efficacy we noted for 3-OH-DHP also suggests the  subunit does not co-express with the , , and  subunits (thus forming an receptor), as the presence of  along with these other subunits diminishes the efficacy of 3-OH-DHP several-fold below that which we observed in the present investigation (Zhu et al. 1996; but see Mihalek et al. 1999). As with our investigation of which  subunits may be functional in the hypothalamus, our data confirm and expand the in situ hybridization studies of Wisden et al. (1992) and immunohistochemical studies of Peng et al. (2002). They found no evidence for the existence of the  subunit, while mRNA for all  subunits (although mainly 1 and 2) was present. Our functional studies revealed no evidence for  subunit expression and are most consistent with the suggestion that the  subunit expressed in the hypothalamus is likely the 2 isoform.

The  subunit has been reported to express in the arcuate-ventromedial region of the hypothalamus via immunohistochemistry and in situ hybridization (Whiting et al. 1997). GABA_A receptors expressing , , and  subunits have high sensitivity to GABA (average EC₅₀ of 4 µM in 11 receptors) and are insensitive to stimulation by benzodiazepines (Kasparov et al. 2001; Whiting et al. 1997). In the present report, no hypothalamic neurons had a GABA EC₅₀ <11 µM, and all were responsive to stimulation by diazepam. Thus functional expression of neurons with the configuration may be minimal.

In summary, results of pharmacological analysis of hypothalamic GABA_A receptors have provided insight about likely subunit configurations expressed in these neurons. The predominant  and  isoforms are likely 1 and/or 2 and 2 and/or 3, respectively. Our data suggest these subunits generally coassemble with the 2 subunit. We further suggest that 3, 4, 5, 6, 1, and  subunits express minimally in functional receptors. Given that the GABA_A receptor Cl channels participate in many hypothalamic functions, modulation of GABA_A receptor function by both endogenous and exogenous modulators may have a profound influence on synaptic transmission in this region. In addition, the knowledge we obtained here should prove to be useful in predicting and explaining effects on various hypothalamic functions as newer therapeutics with greater subunit specificity continue to be developed.