Download Functional Characterization of GABAA Receptors in Neonatal

J Neurophysiol
88: 1655–1663, 2002; 10.1152/jn.00822.2001.
Functional Characterization of GABAA Receptors
in Neonatal Hypothalamic Brain Slice
REN-QI HUANG AND GLENN H. DILLON
Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas 76107
Received 10 October 2001; accepted in final form 19 June 2002
INTRODUCTION
GABAA 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). GABAA 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
ⱖ20 subunits (␣1– 6, ␤1–3, ␥1–3, ␦, ␲, ⑀, ␳1–3, ␲, and ␪), the
Address for reprint requests: G. H. Dillon, Dept. of Pharmacology and
Neuroscience, University of North Texas Health Science Center at Forth
Worth, 3500 Camp Bowie Blvd., Fort Worth, TX 76107 (E-mail:
[email protected]).
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molecular architecture of native GABAA 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 GABAA 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). GABAA 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 GABAA receptor
subunits that are purported to exist in the hypothalamus.
METHODS
Hypothalamic brain slices
Sprague Dawley rats (Indianapolis, IN), postnatal day (P) 1–14
(either sex) were rapidly decapitated. Chemical anesthesia was not
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0022-3077/02 $5.00 Copyright © 2002 The American Physiological Society
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Huang, Ren-Qi, and Glenn H. Dillon. Functional characterization of
GABAA receptors in neonatal hypothalamic brain slice. J Neurophysiol 88: 1655–1663, 2002; 10.1152/jn.00822.2001. The hypothalamus influences a number of autonomic functions. The activity of
hypothalamic neurons is modulated in part by release of the inhibitory
neurotransmitter GABA onto these neurons. GABAA receptors are
formed from a number of distinct subunits, designated ␣, ␤, ␥, ␦, ⑀,
and ␪, many of which have multiple isoforms. Little data exist,
however, on the functional characteristics of the GABAA receptors
present on hypothalamic neurons. To gain insight into which GABAA
receptor subunits are functionally expressed in the hypothalamus, we
used an array of pharmacologic assessments. Whole cell recordings
were made from thin hypothalamic slices obtained from 1- to 14-dayold rats. GABAA receptor-mediated currents were detected in all
neurons tested and had an average EC50 of 20 ⫾ 1.6 ␮M. Hypothalamic GABAA receptors were modulated by diazepam (EC50 ⫽ 0.060
␮M), zolpidem (EC50 ⫽ 0.19 ␮M), loreclezole (EC50 ⫽ 4.4 ␮M),
methyl-6,7-dimethoxy-4-ethyl-␤-carboline (EC50 ⫽ 7.7 ␮M), and
5␣-pregnan-3␣-hydroxy-20-one (3␣-OH-DHP). Conversely, these receptors were inhibited by Zn2⫹ (IC50 ⫽ 70.5 ␮M), dehydroepiandrosterone sulfate (IC50 ⫽ 16.7 ␮M), and picrotoxin (IC50 ⫽ 2.6 ␮M).
The ␣4/6-selective antagonist furosemide (10 –1,000 ␮M) was ineffective in all hypothalamic neurons tested. The results of our pharmacological analysis suggest that hypothalamic neurons express functional GABAA receptor subtypes that incorporate ␣1 and/or ␣2
subunits, ␤2 and/or ␤3 subunits, and the ␥2 subunit. Our results
suggest receptors expressing ␣3–␣6, ␤1, ␥1, and ␦, if present, represent a minor component of functional hypothalamic GABAA
receptors.
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R.-Q. HUANG AND G. H. DILLON
Effects of antagonists were analyzed using the equation:
I/Imax⫽[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 Imax, 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 ␮M); 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
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 510101G 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/Imax⫽ [agonist]n/
(EC50n ⫹ [agonist]n), where I and Imax 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.
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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 GABAA receptor antagonist
bicuculline (Fig. 1, A and B). These characteristics demonstrate
that the evoked currents were due to activation of GABAA
receptors.
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 ␮M) is
illustrated in Fig. 1C. The maximal amplitude of GABA-gated
currents, typically achieved at a concentration of 300 ␮M, was
2,735 ⫾ 189 pA (n ⫽ 27). EC50 values ranged from 11.4 to 44
␮M (median ⫽ 20 ␮M, 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 ␮M) 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 ␮M) with 10
␮M 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 ␮M (Fig. 2, A and D). The EC50 and Hill
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used because of its well-known influence on GABAA 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 ␮m) 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).
PROPERTIES OF HYPOTHALAMIC GABAA RECEPTORS
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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 ␮M). In the presence of 10 ␮M GABA, 1 ␮M 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 ␮M inhibited GABA-gated
currents with an IC50 of 70.5 ⫾ 17 ␮M 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
GABAA receptors express ␣, ␤, and ␥ subunits.
Loreclezole and DMCM sensitivity
3␣-OH-DHP and furosemide sensitivity
FIG.
1. GABA-activated currents in hypothalamic neurons. A: currentvoltage 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 ␮M GABA recorded before, during, and after coapplication with 10 ␮M 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.
coefficient for the diazepam effect was 60 ⫾ 17 nM and 1.57 ⫾
0.56, respectively (n ⫽ 3–7).
The affinity of zolpidem is also influenced by the ␣ subunit
isoform present in GABAA receptors. Zolpidem has high affinity for GABAA receptors containing an ␣1 subunit and is
nearly insensitive to GABAA receptors containing ␣5 subunits
(Hevers and Lüddens 1998; Wingrove et al. 1994). Zolpidem
(0.01–10 ␮M) markedly potentiated the current induced by 10
␮M GABA in all cells (n ⫽ 14) recorded from the hypothalamus (Fig. 2B). The average EC50 was 0.19 ⫾ 0.07 ␮M and 3
␮M zolpidem maximally potentiated the GABA current to
210 ⫾ 18% of the control GABA response (Fig. 2D).
Inhibition of GABAA receptors by Zn2⫹ is influenced by the
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3␣-OH-DHP, an endogenous progesterone metabolite, has
been shown to differentially modulate GABAA 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 GABAactivated 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 GABAA receptor-Cl⫺
channels at concentrations ⬎1 ␮M (Ueno et al. 1997), we did
not evaluate concentrations ⬎1 ␮M in the present investigation. Nevertheless, it is apparent that hypothalamic GABAA
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 ␮M (n ⫽ 7) or 1 ␮M 3␣-OH-DHP
(n ⫽ 8), respectively (Fig. 4B).
Furosemide is a loop diuretic that acts as a selective, noncompetitive antagonist of ␣4- or ␣6-containing GABAA receptors, with IC50 values in the micromolar range (Knoflach et al.
1996; Korpi and Lüddens 1997). In 26 hypothalamic neurons
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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
␮M) with 10 ␮M GABA enhanced the GABA current in all the
cells tested (n ⫽ 18) in a concentration-dependent manner.
Loreclezole was insoluble at concentrations ⬎30 ␮M. However, the potentiating effect appeared nearly saturated at that
concentration, so an EC50 value could be estimated (mean
EC50 ⫽ 4.4 ⫾ 0.7 ␮M, range ⫽ 1.8 –10.0 ␮M). The maximal
enhancement of peak current was 194 ⫾ 12% of control
(range: 119 –272%) with 30 ␮M loreclezole. At higher concentrations (⬎10 ␮M), loreclezole produced an increase in the
current decay rate (Fig. 3A).
Positive modulation of GABAA receptors by the ␤-carboline
DMCM is also dependent on the ␤ subunit isoform. Figure 3B
shows that concentrations of DMCM at ⱖ10 ␮M caused a
modest potentiation of the GABA response. The maximal
effect (to 141 ⫾ 12.8% of control) was observed at 30 ␮M
DMCM. The average EC50 from 11 cells was 7.7 ⫾ 3.2 ␮M.
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tested, 300 ␮M furosemide, a concentration that inhibited the
response to GABA by 39 ⫾ 2% of control in rat recombinant
␣6␤2␥2 receptors (n ⫽ 7, data not shown), had no effect on
GABA-activated current. Increasing the furosemide concentration ⱕ1 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 GABAA 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), subunitspecific differences in affinity of picrotoxin for GABAA receptor blockade do exist (Bell-Horner et al. 2000). Examples of
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typical responses of a hypothalamic neuron to picrotoxininduced inhibition of the GABA response are illustrated in Fig.
5A. When coapplied with 10 ␮M GABA, picrotoxin (0.1–30
␮M) 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 ␮M, and the Hill coefficient was 1.1 ⫾ 0.1 (Fig. 5C).
DHEAS is a noncompetitive antagonist of GABAA receptors
in a number of preparations (Majewka et al. 1990). Figure 5B
illustrates the DHEAS-mediated inhibition of the response to
10 ␮M GABA in a hypothalamic neuron. DHEAS reversibly
inhibited the current amplitude and accelerated current decay
in a concentration-dependent fashion. Coapplication of 300
␮M DHEAS with 10 ␮M GABA reduced the GABA steadystate current to 7.7 ⫾ 3.5% of control. The IC50 and Hill
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2⫹
FIG. 2. Effects of diazepam, zolpidem, and Zn
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 ␮M). 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 ␮M GABA without drug.
Each data point represents the average of ⱖ3 cells. The data
were best fitted to a sigmoidal function. See Table 1 for
mean values.
PROPERTIES OF HYPOTHALAMIC GABAA RECEPTORS
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cultured cells and at the same time permits the rapid and
consistent exposure of neurons to various exogenous ligands.
Analysis of ␣ subunit isoform
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 ␮M 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.
coefficients were 16.7 ⫾ 2.3 ␮M 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 GABAA 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.
DISCUSSION
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 GABAA 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 GABAA 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
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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 ␮M 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.
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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 (BellHorner et al. 2000; Huang and Dillon 1998). The GABA EC50
for these latter receptors is 15–30 ␮M (Bell-Horner et al.
2000), which is comparable to the value we obtained in hypothalamic neurons (20 ␮M). The fact that the individual EC50
values were relatively evenly distributed around the mean does
not support the existence of subsets of GABAA 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.
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TABLE 1. Relative efficacy and affinity of drugs tested
for their modulatory effect on GABAA receptor current
in rat hypothalamic neurons
Drug
n
Maximal Response
(% of control)
Diazepam (0.03–1 ␮M)
Zolpidem (0.01–10 ␮M)
*ZnCl2 (1–1000 ␮M)
Loreclezole (0.3–30 ␮M)
DMCM (0.03–30 ␮M)
3␣-OH-DHP (0.3, 1 ␮M)
Furosemide (up to 1 mM)
*Picrotoxin (0.1–30 ␮M)
*DHEAS (1–300 ␮M)
3–7
9–14
5
18
11
8
26
5
6
206 ⫾ 12
210 ⫾ 18
7.6 ⫾ 2.7
194 ⫾ 12
141 ⫾ 12.8
423 ⫾ 48 at 1 ␮M
No effect
7.3 ⫾ 1.3
7.7 ⫾ 2.8
EC50/IC50, ␮M
0.060 ⫾ 0.017
0.19 ⫾ 0.07
70.5 ⫾ 17.3
4.5 ⫾ 0.86
7.7 ⫾ 3.2
ND
2.6 ⫾ 0.43
16.7 ⫾ 2.3
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
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.
(1997) for diazepam stimulation of recombinant ␣1␤2␥2 receptors. The efficacy of diazepam is significantly greater in
␣3-expressing compared with ␣1-expressing GABAA receptors. Ducic et al. (1993) reported a 350% potentiation by
diazepam of the GABA response in ␣3␤1␥2 receptors,
whereas ␣1␤1␥2 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 ␣3␤2␥2 GABAA receptors, which is approximately three times greater than that of rat ␣1␤2␥2 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
GABAA 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
J Neurophysiol • VOL
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).
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Maximal response is expressed as a percentage of the control response to
GABA (100%). Data are the means ⫾ SE for the indicated number (n) of cells
studied. Multiple values for n indicate varying numbers of cells tested at
different concentrations of those concentration-response profiles. The number
in brackets indicates the concentration range tested. EC50 or IC50 values were
calculated from the concentration-response relationships fitted in Figs. 1– 6 as
described in METHODS. ND, not determined. Values for antagonists (*) represent effect on steady-state GABA currents; values for agonists represent effect
on peak GABA current amplitude. DMCM, methyl-6,7-dimethoxy-4-ethyl-␤carboline; 3␣-OH-DHP, 5␣-pregnan-3␣-hydroxy-20-one; DHEAS, dehydroepiandrosterone sulfate.
PROPERTIES OF HYPOTHALAMIC GABAA RECEPTORS
Analysis of ␤ subunit isoform
The potentiating action of loreclezole depends on the presence of either ␤2- or ␤3-containing GABAA 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 GABAA 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 ␣1␤2␥2
receptors (unpublished observations). Thus in the present report the effect on GABAA 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 GABAA 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 (ⱖ10 ␮M) caused a modest potentiation of hypothalamic GABAA 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 abJ Neurophysiol • VOL
sence 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
GABAA 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
GABAA 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
GABAA receptors. For instance, the IC50 for Zn2⫹ is significantly greater in ␣␤␥ receptors (⬃50 ␮M) than in ␣␤ receptors
(⬃1 ␮M) (Gingrich and Burkat 1998; Smart et al. 1991). We
obtained in hypothalamic neurons a value consistent with the
former (70.5 ␮M). 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 ␣1␤1␥2 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 ␣2␤1␥1/3
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 GABAactivated current in response to 1 ␮M 3␣-OH-DHP. The maximal potentiation is similar to values reported for ␣1␤2␥2
receptors (Maitra and Reynolds 1998; Zhu et al. 1996) and is
several-fold less than observed with ␣1␤2 receptors (Maitra
and Reynolds 1998). Finally, the maximal efficacy we noted
for 3␣-OH-DHP also suggests the ␦ subunit does not coexpress 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 arcuateventromedial region of the hypothalamus via immunohistochemistry and in situ hybridization (Whiting et al. 1997).
GABAA receptors expressing ␣, ␤, and ⑀ subunits have high
sensitivity to GABA (average EC50 of 4 ␮M in ␣1␤1⑀ recep-
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to the diuretic furosemide. Furosemide blocks ␣4- and ␣6expressing receptors with ␮M 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 GABAA 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.
1661
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R.-Q. HUANG AND G. H. DILLON
This work was supported by a grant from the American Heart Association
and in part by National Institute of Environmental Health Services Grant
ES-07904.
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