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The Journal of Neuroscience, November 15, 1999, 19(22):9698-9704
GABA Receptors Inhibited by Benzodiazepines Mediate Fast
Inhibitory Transmission in the Central Amygdala
Andrew J.
Delaney and
Pankaj
Sah
Division of Neuroscience, John Curtin School of Medical Research,
Australian National University, Canberra ACT 2601, Australia
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ABSTRACT |
The amygdala is intimately involved in emotional behavior, and its
role in the generation of anxiety and conditioned fear is well known.
Benzodiazepines, which are commonly used for the relief of anxiety, are
thought to act by enhancing the action of the inhibitory transmitter
GABA. We have examined the properties of GABA-mediated
inhibition in the amygdala. Whole-cell recordings were made from
neurons in the lateral division of the central amygdala. Application of
GABA evoked a current that reversed at the chloride equilibrium
potential. Application of the GABA antagonists bicuculline or SR95531
inhibited the GABA-evoked current in a manner consistent with two
binding sites. Stimulation of afferents to neurons in the central
amygdala evoked an IPSC that was mediated by the release of
GABA. The GABAA receptor antagonists bicuculline and
picrotoxin failed to completely block the IPSC. The
bicuculline-resistant IPSC was chloride-selective and was unaffected by
GABAB-receptor antagonists. Furthermore, this current was
insensitive to modulation by general anesthetics or barbiturates. In
contrast to their actions at GABAA receptors, diazepam and
flurazepam inhibited the bicuculline-resistant IPSC in a
concentration-dependent manner. These effects were fully antagonized by
the benzodiazepine site antagonist Ro15-1788. We conclude that a new
type of ionotropic GABA receptor mediates fast inhibitory transmission
in the central amygdala. This receptor may be a potential target for
the development of new therapeutic strategies for anxiety disorders.
Key words:
GABAC; fear; anxiety; diazepam; bicuculline; amygdala
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INTRODUCTION |
GABA is the major inhibitory
transmitter in the mammalian CNS (Nicoll et al., 1989 ). As with
many other types of receptor, two broad types of GABA receptor are
recognized: ionotropic ligand-gated channels and metabotropic
G-protein-coupled receptors. Ionotropic GABA receptors are further
subdivided into the bicuculline-sensitive GABAA
receptors (MacDonald and Olsen, 1994; Johnston, 1996a) and the
bicuculline-insensitive GABAC receptors (Qian and
Dowling, 1994; Bormann and Feigenspan, 1995).
GABAA receptors gate a chloride ionophore and
have modulatory binding sites for benzodiazepines, barbiturates, and
anesthetics, all of which potentiate the response to GABA (MacDonald
and Olsen, 1994; Johnston, 1996a). These receptors are potently
inhibited by the competitive antagonists bicuculline and SR95531 and
the plant alkaloid picrotoxin (Sieghart, 1995).
GABAA receptors are assembled from a large family
of which fifteen members have so far been identified: 6 , 4 , 3 ,
1 , and 1 (Barnard et al., 1998). Heterologous expression of
different subunits has shown that functional GABA receptors can form as homomers or as heteromultimers of different subunits. However, most
GABAA receptors in the CNS are thought to contain
both and subunits, with one or more of the , , or subunits (Barnard et al., 1998). The subunit combination of a
particular GABA receptor determines its pharmacological properties
(Costa, 1998; MacDonald and Olsen, 1994). For example, amplification of
GABA action by benzodiazepines is only seen in receptors that contain
one of 1, 2, 5 subunits and either a 2 or a 3 subunit.
Receptors that contain 4, 6, or 1 are unaffected by
benzodiazepines (MacDonald and Olsen, 1994; Costa, 1998).
GABAC receptors also gate a chloride channel, but
they are not blocked by bicuculline or SR95531, and are markedly less
sensitive to picrotoxin. They are also insensitive to modulation by
benzodiazepines and barbiturates (Qian and Dowling, 1993; Bormann and
Feigenspan, 1995; Johnston, 1996b). GABAC
receptors are assembled from  subunits (1, 2, 3), which
share some homology with GABAA receptor subunits, but do not appear to coassemble with them. GABAC
receptors have only clearly been demonstrated in the retina (Qian and
Dowling, 1994; Enz et al., 1995). Bicuculline-resistant responses to
GABA have been reported in several brain regions (Drew et al., 1984; Arakawa and Okada, 1988; Strata and Cherubini, 1994). However, the
importance of these receptors outside of the retina has yet to be demonstrated.
The amygdala is intimately involved in emotional behavior, and its role
in the generation of anxiety and conditioned fear is well known (Kluver
and Bucy, 1939; LeDoux, 1995). Benzodiazepines, which are commonly used
for the relief of anxiety, are thought to produce their therapeutic
effect by enhancing the action of GABA (Tallman and Gallager, 1985;
Costa and Guidotti, 1996). The action of benzodiazepines on GABA
receptors within the amygdala is likely to be responsible for the
antianxiety action of these agents because binding sites for
benzodiazepines are present in the amygdala at high density (Niehoff
and Kuhar, 1983; Richards and Möhler, 1984). In this study we
have examined the properties of ionotropic GABA receptors in the
central amygdala. We find that neurons in the central amygdala express
two types of ionotropic GABA receptor. One is the well known
GABAA type, which is blocked by bicuculline and
is typically modulated by benzodiazepines, barbiturates, and
anesthetics. The other type is relatively resistant to bicuculline and
picrotoxin, and like GABAC receptors is not modulated by barbiturates and anesthetics. However, unlike
GABAC receptors, these bicuculline-resistant GABA
receptors are inhibited by benzodiazepines. Both receptor types
contribute to fast inhibitory transmission in the central amygdala.
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MATERIALS AND METHODS |
All experiments were done on acute brain slices maintained
in vitro. All procedures were in accordance with the
Institutional Animal Care and Ethics Committee guidelines. Wistar rats
(17- to 20-d-old) were anesthetized with intraperitoneal pentobarbitone (50 mg/kg), and coronal brain slices (400 µM)
were prepared using standard methods. Slices were superfused at 200 ml/hr with oxygenated Ringer's solution containing (in
mM) NaCl 118, KCl 2.5, NaHCO3 25, glucose 10, NaH2PO4 1.2, MgCl2 1.3, and CaCl2 2.5, in a bath volume of 1 ml. Kynurenic acid (2 mM)
or CNQX (10 µM) and D-APV (30 µM) were included in the external solution
to block glutamatergic receptors. In other experiments (our unpublished
observations), we have confirmed that at these concentrations
these compounds completely block glutamatergic synaptic transmission.
Tetrodotoxin (0.5 µM) was added to the
Ringer's solution to block synaptic transmission during experiments
with iontophoretically applied GABA. Whole-cell recordings were made
from neurons in the lateral division of the central amygdala (CeL) or
from pyramidal neurons in the CA1 region of the hippocampus using the
"blind" approach. Borosilicate glass electrodes (3-5 M ) were
filled with a cesium-based internal solution to eliminate the effects
of GABAB receptors. The solution was either
high-chloride, containing (in mM) CsCl 130, MgCl2 1, EGTA 10, HEPES 10, Mg2ATP 2, and Na3GTP 0.2 (pH 7.3 with CsOH, 290 mOsm), or low-chloride, containing (in
mM) cesium gluconate 107.5, CsCl 17.5, NaCl 8, HEPES 10, BAPTA 10, Mg2ATP 2, and
Na3GTP 0.2 (pH 7.3 with CsOH, 290 mOsm). Membrane potentials recorded were corrected for a junction potential of +17 and
 10 mV for the high-chloride and low-chloride internals, respectively.
Normalized I-V relations were constructed by normalizing the current measured at a holding potential of 80 mV. IPSCs were evoked electrically using stainless steel bipolar stimulating electrodes (Frederick Haer) placed near the lateral border of the CeL
or near stratum pyramidale in the CA1 region of the hippocampus. Stimuli were 10-30 V in amplitude and 50 µsec in duration.
Iontophoresis pipettes (>5 M) were filled with either 300 mM GABA (pH 3) or 300 mM
glycine (pH 3) and placed adjacent to recorded neurons in CeL. Negative
retention current (50-100 nA) and positive ejection current (100-200
nA; 0.1-1 sec duration) were generated by a Dagan 6400 iontophoresis
unit. Signals were filtered (5 kHz) and amplified using an Axopatch 1D
amplifier (Axon Instruments, Foster City, CA), digitized at 10 kHz
(Instrutech, ITC 16), and recorded and analyzed using Axograph 4.0 software (Axon Instruments) on a Macintosh computer. Series resistance
(5-30 M) was monitored online throughout the experiment, and
experiments were rejected if resistance changed by >10%. No series
resistance compensation was used.
All values are expressed as mean ± SEM, and all
statistical comparisons were done using Student's t test.
Drugs used were CNQX (Tocris Cookson), bicuculline methiodide,
D-APV, propofol (Research Biochemicals, Natick,
MA), kynurenic acid, picrotoxin (Sigma, St. Louis, MO), tetrodotoxin
(Alamone Laboratories, Jerusalem, Israel), diazepam (a gift from Prof.
P. Gage), 1,2,5,6-tetrohydropyridine-4-yl)methylphosphinic acid
(TPMPA), flurazepam, Ro 15-1788 (gifts from Associate Prof. G. A. R. Johnston), and pentobarbitone (Bomac Laboratories).
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RESULTS |
Whole-cell recordings were made from neurons in the CeL.
Iontophoretic application of GABA evoked a current that reversed at the
chloride equilibrium potential (Fig.
1A,B). Bath application of the competitive GABAA receptor antagonist
bicuculline methiodide (BIC) blocked the GABA-activated current in a
manner that was best fit assuming two binding sites with
IC50 values of 0.12 and 23.1 µM (Fig. 1C). Similar results were
also obtained with another competitive GABAA
antagonist SR95531 (Hamann et al., 1988). On average, 10 µM BIC blocked the GABA-activated current by
65 ± 6%, and 100 µM by 92 ± 3%
(n = 11; Fig. 1E); 1 and 10 µM SR95531 blocked the iontophoretic current by
77 ± 6 and 93 ± 6% (n = 3), respectively.
These results suggest that two types of ionotropic GABA receptor are
present on CeL neurons. To rule out the possibility that the low
sensitivity of the GABA response might be caused by inadequate access
of the bath-applied antagonists to their site of action, we examined a
block of iontophoretically applied glycine by the selective antagonist
strychnine. Glycine activated a chloride-mediated current in all cells
tested (data not shown). Strychnine blocked this current at a single,
high-affinity site with an IC50 of 79 nM (Fig. 1D), close to the
reported IC50 for strychnine in isolated cells
and membrane patches (Shirasaki et al., 1991; Jonas et al., 1998). At
10 µM BIC, the contribution of the
high-affinity BIC sites to the GABA response will be negligible. We
therefore used this concentration of BIC to examine the properties of
the BIC-resistant GABA response.
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Figure 1.
GABA activates two types of ionotropic receptors
in the central amygdala. A, Responses to
iontophoretically applied GABA in a CeL neuron at membrane potential of
80, 60, 40, 20, 0, and +10 mV using low-chloride internal
solution. B, The current-voltage relationship for the
records shown in A (filled
circles) and responses similarly recorded from a different cell
using high-chloride internal solution (open squares).
The reversal potential in low-chloride internal was 50 mV, whereas in
high internal chloride, it was 2 mV, showing that the GABA activates
a chloride-selective current. C, Average responses to
iontophoretically applied GABA, recorded in increasing concentrations
of bicuculline. The graph below plots the percentage
inhibition of the GABA response for each dose of bicuculline
(n = 3-6 for each concentration point, except 0.2 µM, where n = 2). The solid
line is a fit to the equation a/(1 + (IC50/c)2) + b/(1 + (IC50/c)) with
IC50 values of 0.12 and 23.1 µM
(a + b was constrained to equal 1).
D, Inhibition curve for antagonism of the response to
iontophoretically applied strychnine (n = 2-6 for
each concentration point). The solid line is a fit to
the equation 1/(1 + (IC50/c)) with an
IC50 of 0.079 µM. E, Summary
of inhibition of the response to GABA at bicuculline concentrations of
10 and 100 µM (n = 11), SR95531 at 1 and 10 µM (n = 3), and strychnine (1 µM; n = 3).
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The relative insensitivity of the GABA response to BIC and SR95531
suggests that a GABAC like receptor might be
present. GABAC receptors can be blocked by high
concentrations of picrotoxin (Polenzani et al., 1991) and the selective
antagonist TPMPA (Ragozzino et al., 1996). In confirmation of this, we
found that the GABA response resistant to BIC was blocked by 88 ± 1% by 100 µM picrotoxin (n = 3) and by
73 ± 1% by 60 µM TPMPA
(n = 3; Fig. 2). These
results show that iontophoretically applied GABA activates two
pharmacologically distinct receptors. One has a high affinity for BIC
and SR95531, and represents activation of GABAA
receptors, the other is relatively resistant to BIC and SR95531 but is
antagonized by TPMPA.
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Figure 2.
GABAC-like receptors are present in
the central amygdala. A, Responses to iontophoretically
applied GABA recorded in control, 10 µM bicuculline, and
100 µM picrotoxin. B, The
trace on the left is the response to
iontophoretically applied GABA in the presence of 10 µM
BIC. This current is blocked by application of the selective
GABAC antagonist TPMPA (60 µM).
C, Summary data showing the average reduction of the
BIC-resistant response by picrotoxin and TPMPA.
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We next asked if these two types of GABA receptor were also activated
by synaptically released GABA. Stimulation of local afferents in the
presence of glutamatergic antagonists evoked an IPSC that reversed near
the chloride equilibrium potential (Fig.
3A,B),
showing that it is a chloride-selective current. Application of
bicuculline at a concentration that abolishes inhibitory transmission
at GABAA synapses (10 µM)
(Jonas et al., 1998) only reduced IPSC amplitude to 67 ± 3% of
the control response (n = 19; Fig. 3C,E).
Raising the concentration of BIC to 100 µM
further blocked the IPSC to 87 ± 3% (n = 4) of
control. Another GABAA antagonist picrotoxin was
also ineffective in blocking the IPSC; 25 µM
picrotoxin reduced IPSC amplitude to 63.6 ± 9.1% of control (n = 3), and 100 µM picrotoxin
reduced it to 96.7 ± 0.5% (n = 7; Fig.
3D). For comparison, GABAA
receptor-mediated IPSCs recorded in the CA1 region of the hippocampus
were inhibited by 98.3 ± 0.6% (n = 5) in 10 µM BIC and by 99.1 ± 0.3%
(n = 4) with 25 µM picrotoxin
(Fig. 3E). Thus in the central amygdala, a component of the
inhibitory synaptic current is resistant to block by bicuculline and
picrotoxin.
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Figure 3.
GABAergic inhibitory synaptic currents in the
central amygdala are not blocked by low doses of bicuculline or
picrotoxin. A, Synaptic currents in response to local
electrical stimulation in the presence of blockers of glutamatergic
receptors (see Materials and Methods) recorded at membrane potentials
of 80, 60, 40, 20, 0, and 10 mV using low-chloride internal
solution. B, Normalized current-voltage relationships
for synaptic currents recorded using low-chloride (closed
circles) (n = 4) and high-chloride internal
solutions (open squares) (n = 2).
The reversal potential in low-chloride internal was 52 mV, whereas in
high-internal chloride it was 0 mV. C, Average CeL IPSCs
recorded in control, in 10 µM bicuculline, and in 100 µM bicuculline. The IPSC was blocked to 67 ± 3% of
control by 10 µM and 87 ± 3% in 100 µM BIC. D, IPSCs recorded in control, in
25 µM picrotoxin (PTX), and 100 µM PTX. IPSC amplitude was blocked by 64 ± 0% in
25 µM PTX. E, Summary of effects of
antagonists bicuculline and picrotoxin on IPSC peak amplitude recorded
from CeL neurons (filled columns) or from CA1
pyramidal neurons (open columns) (n = 5, 3, 3, 7, 5, and 4, respectively).
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The BIC-resistant IPSC was a chloride-selective current as it reversed
near the chloride equilibrium potential (Fig.
4A,B). It was
unaffected by the glycine receptor antagonist strychnine (1 µM; n = 3) or the
GABAB antagonist CGP58854 (20 µM; n = 3). Application of the
GABA uptake inhibitor NO711 (n = 3) slowed its decay
(Fig. 4C), confirming that it is mediated by the release of
GABA. Thus, as with iontophoretic application of the GABA, a component
of the current activated by synaptically released GABA is resistant to
blockade by BIC and picrotoxin.
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Figure 4.
A, Synaptic currents recorded in
the presence of 10 µM bicuculline at membrane potentials
of 60, 40, 20, 0, and +10 mV using low-chloride internal
solution. B, Normalized current-voltage relationship
for IPSCs recorded in 10 µM bicuculline using
low-chloride (closed circles) (n = 4) and high-chloride internal solutions (open squares)
(n = 6). The reversal potential in low-chloride
internal was 44 mV, whereas in high-internal chloride it was 0 mV.
C, The GABA uptake blocker NO-711 slows the decay of
IPSCs. Each panel shows a train of five IPSCs at stimulated at 20 Hz.
Application of NO-711 slows the decay of the synaptic current; after
washout of NO-711, the decay returns back to the control
response.
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We next tested if the BIC-resistant IPSC was caused by activation of
GABAC receptors. TPMPA reversibly inhibited the
BIC-resistant IPSC (Fig. 5A,B)
in a dose-dependent manner with an IC50 of 18 µM (n = 5). Consistent with
previous results (Ragozzino et al., 1996) the
GABAA-receptor IPSC recorded in area CA1 was
unaffected by 60 µM TPMPA (Fig. 5B;
n = 7). Because GABAC receptors
are relatively insensitive to barbiturates, anesthetics, and
benzodiazepines we next tested the actions of these agents on the
BIC-resistant IPSC. In each case, we compared the action of these
agents on the BIC-resistant IPSC with their effects on the
GABAA-mediated IPSC recorded from pyramidal
neurons in the CA1 region of the hippocampus. Propofol, an intravenous
anesthetic agent that enhances GABAA receptor
responses (Manuel and Davies, 1998) increased the half-width of the
GABAA IPSC by 137 ± 22% (n = 3; p < 0.05) with little effect on the peak
amplitude. In contrast, propofol had no significant effect on either
the peak amplitude or the half width of the BIC-resistant IPSC in the
CeL (n = 3; Fig. 5C,D). The barbiturate
pentobarbitone also had a reduced effect on the BIC-resistant IPSC.
Pentobarbitone (25 µM) increased the half width
of GABAA-receptor mediated IPSCs by 166 ± 29% (n = 4; p < 0.001) with no change
in amplitude. In contrast, neither the amplitude nor the half-width of
the BIC-resistant IPSC were significantly affected by pentobarbitone.
Peak amplitude was reduced by 21.2 ± 7.4%
(p = 0.16), and half-width increased by 32 ± 16% (n = 3; p = 0.07; Fig.
5E,F).
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Figure 5.
GABAC receptors are insensitive to
general anesthetics and barbiturates. A, The IPSCs
recorded in the presence of 10 µM bicuculline are
reversibly blocked by TPMPA (60 µM). B,
Summary data for the effect of TPMPA on IPSCs recorded from neurons the
CeL and at GABAA synapses recorded in pyramidal neurons in
the CA1 region of the hippocampus. TPMPA (60 µM) reduced
the IPSC in the CeL by 65 ± 8% (n = 6), but
had no effect on IPSCs in area CA1 (3 ± 4%;
n = 7). C, D, Propofol (10 µM) slowed the decay of IPSCs in area CA1 (137 ± 22%; n = 3) with no effect on peak amplitude, but
had no effect on BIC-resistant IPSCs in the CeL. Traces recorded in the
presence of propofol are indicated by an asterisk.
E, F, Pentobarbitone (25 µM) prolonged the decay of IPSCs in area CA1 (half decay
166 ± 29% of control; n = 4) while having
relatively little effect on BIC IPSCs in the CeL (half decay 32 ± 29% of control; n = 4). Traces recorded in the
presence of pentobarbitone are indicated by an
asterisk.
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The 1,4-benzodiazepines act as positive modulators of some
GABAA receptors (MacDonald and Olsen, 1994;
Costa, 1998) by increasing the affinity of the receptor for GABA
(Lavoie and Twyman, 1996), whereas GABAC
receptors are insensitive to these agents. In the CeL, flurazepam (1 µM) reduced the amplitude of the BIC-resistant IPSC by
37 ± 5% (n = 9; Fig.
6A). Diazepam (1 µM), another 1,4 benzodiazepine, reduced the
amplitude of the BIC-resistant IPSC by 28 ± 7%, and 10 µM diazepam reduced it by 42 ± 5%
(n = 5; Fig. 6C). This effect was fully
antagonized by the benzodiazepine receptor antagonist Ro 15-1788
(Hunkeler et al., 1981) (Fig. 6C), showing that it was not a
nonspecific action of these benzodiazepines. There was no effect on the
kinetics of the IPSC with either diazepam or flurazepam (Fig.
6A,C). To confirm that the effects of the benzodiazepines on IPSC amplitude were caused by their postsynaptic actions on GABA receptors, we tested the action of diazepam on iotophoretically applied GABA. Diazepam (10 µM)
reduced the amplitude of the BIC-resistant GABA-evoked current by
27 ± 8% (n = 5; Fig. 6D),
showing that the effects of diazepam are postsynaptic.
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Figure 6.
Diazepam and flurazepam inhibit GABAC
receptors in the central amygdala. A, The effect of
flurazepam on the BIC-resistant IPSC. Flurazepam (1 µM)
reduced the peak amplitude (37 ± 5% of control;
n = 9) with no effect on the kinetics of the
current. B, The benzodiazepine site antagonist
Ro15-1788 (1 µM) has no effect on IPSC amplitude but
blocks the inhibitory effect of flurazepam. After washout of
Ro15-1788, a second application of flurazepam now inhibits the IPSC.
Individual records taken from the times indicated are shown above.
C, Summary data showing the effects of flurazepam and
diazepam on BIC-resistant IPSCs in control conditions
(n = 9) and Ro 15-1788 (n = 3). D, The effects of benzodiazepines are postsynaptic
because diazepam (10 µM) blocks the bicuculline-resistant
response to iontophoretically applied GABA by 27 ± 8%
(n = 5).
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We performed two control experiments to ensure that the benzodiazepines
were active at GABAA receptors in our hands.
First, we checked the action of these drugs on
GABAA synapses recorded from CA1 neurons in the
hippocampus. Diazepam (1 µM) increased the amplitude of
hippocampal GABAA receptor-mediated IPSCs by 34 ± 16% and its half decay by 17 ± 12%
(n = 5), and at 10 µM, the IPSC
amplitude and half width increased by 41 ± 16 and 28 ± 18%, respectively (data not shown). These effects are typical of the
actions of benzodiazepines at GABAA synapses
(Otis and Mody, 1992; Zhang et al., 1993). Second, we isolated the
GABAA-mediated IPSC in CeL neurons by performing
experiments in the presence of TPMPA. TPMPA (60 µM) blocked the control IPSC by 27 ± 2%
(n = 4). In the presence of TPMPA, bicuculline (10 µM) blocked the IPSC (96 ± 1% of
control; Fig. 7A), confirming
it was caused by activation of GABAA receptors.
Application of flurazepam in the presence of TPMPA had no effect on the
peak amplitude but increased the half width of the IPSC by 121 ± 5% (n = 3; Fig. 7B), showing that
GABAA receptors that contribute to the IPSC have
a typical pharmacology.
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Figure 7.
Pharmacology of GABAA receptors in the
CeL. GABAA receptors were isolated by blocking
GABAC receptors with TPMPA (60 µM).
A, The IPSC remaining in the presence of TPMPA is
effectively inhibited by 10 µM bicuculline.
B, The GABAA receptor-mediated IPSC is
positively modulated by benzodiazepines. Flurazepam had no effect on
peak amplitude but increased the half width by 121 ± 5%.
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DISCUSSION |
We have shown that in the CeL, both exogenously applied GABA and
synaptically released GABA activate two types of ionotropic GABA
receptor. One is a classical GABAA receptor,
inhibited by BIC and positively modulated by benzodiazepines. The other
is relatively insensitive to the classical GABAA
receptor antagonists bicuculline and picrotoxin. This BIC-insensitive
response is blocked by the GABAC antagonist
TPMPA. Furthermore, like GABAC receptors, the
BIC-insensitive component is not affected by the anesthetic propofol or
by the barbiturate pentobarbitone, agents that potentiate the response
to GABA at GABAA receptors. However, unlike
GABAC receptors, the BIC-resistant response is
inhibited by the benzodiazepines diazepam and flurazepam.
BIC is a competitive antagonist of GABA, and it is possible that the
"BIC-resistant" response simply represents the current caused by
activation of GABA receptors as BIC unbinds in the presence of GABA.
This is unlikely for several reasons. (1) The GABA response resistant
to BIC had a pharmacological profile different to that of
GABAA receptors. (2) If BIC was being competed
off, we would expect that the response in the presence of BIC would
have a slower rising phase than the control response. However, neither
the iontophoretic response to GABA nor the synaptic current remaining
in BIC had slower rising phases. (3) If BIC was unbinding, then we
would expect that the fractional block of the synaptic current by BIC would be significantly higher than during iontophoresis because GABA
will be present for a much shorter duration when it is released synaptically (Clements, 1996). However, the fractional block of the
GABA response during iontophoretic application of GABA was very similar
to that of the synaptic current (34 vs 33%). (4) The much higher
affinity antagonist SR95531 also revealed two types of GABA response.
Thus, we are confident that the BIC-resistant response represents
activation of a different type of GABA receptor.
GABAC receptors were first found in the retina
where they have been extensively characterized in a number of species
in bipolar and horizontal cells (Bormann and Feigenspan, 1995). These
receptors are thought to be assembled from subunits of which three
genes, 1, 2, and 3 have been found (Cutting et al., 1991;
Shimada et al., 1992). Homomeric 1 receptors are sufficient to form
ion channels with properties consistent with those of
GABAC receptors. However, 1 and 2 can also
assemble as heterooligomers with properties different from those of
homomeric channels (Enz and Cutting, 1999), raising the possibility of
some diversity in GABAC receptors. Although subunits have been detected in brain (Enz et al., 1995; Boue-Grabot et
al., 1998; Enz and Cutting, 1999), and BIC-resistant responses to GABA
have also been reported (Drew et al., 1984; Arakawa and Okada, 1988;
Strata and Cherubini, 1994), the presence of
GABAC receptors in central neurons has not been
clearly demonstrated (Johnston, 1996b). Our results suggest the
presence of a GABAC-like receptor in the central
amygdala. It is not known whether neurons in the CeL express subunits. However, it is unlikely that the BIC-insensitive responses we
have recorded are caused by receptors assembled from subunits alone
as such receptors, like GABAC receptors, are
unaffected by bicuculline and benzodiazepines (Bormann and Feigenspan,
1995), whereas the BIC-resistant response the CeL is blocked by high
concentrations of BIC and is negatively modulated by benzodiazepines.
GABAA receptors are assembled from 15 different
subunits. These subunits can form functional channels as homomers or as
heteromultimers of various subunit combinations. However, , , and
subunits are required to reproduce the full pharmacological profile
of native GABA receptors. Both and subunits determine
benzodiazepine sensitivity. The responses of receptors containing subunits are amplified by benzodiazepines, whereas receptors lacking
subunits are insensitive to benzodiazepines. Receptors
containing and 6 subunits are also benzodiazepine-insensitive
(Costa, 1998). In situ hybridization studies have shown that
1, 2, 3, 1, 2, 3, 1, 2, and 3 subunits are
expressed in the central amygdala (Wisden et al., 1992). Thus, subunits
that could produce GABA receptors positively modulated by
benzodiazepines are present, consistent with the presence of such
receptors on CeL neurons. Although inverse agonists of GABA receptors
are known, no GABA receptor examined so far has been found to be
negatively modulated by the 1,4 benzodiazepines (Costa, 1998). The
presence of the 1 subunit is known to produce atypical
benzodiazepine pharmacology turning the inverse agonist Ro15-4513 into
an agonist (Wafford et al., 1993). Furthermore, small changes in the
primary structure of receptor subunits can dramatically change the
pharmacological profile of that receptor (Wang et al., 1995; Valfa and
Schofield, 1998). Thus, one possibility is that the receptors we have
characterized here are assembled from variants of known
GABAA receptor subunits. Alternatively, it is not
inconceivable that an as yet undiscovered subunit might confer the
unusual benzodiazepine pharmacology in CeL neurons. The fact that the
effect of benzodiazepines was inhibited by Ro 15-1788 suggests that
the binding site for these agents on GABAC-like
receptors might be the same as in other, positively modulated
GABAA receptors.
The amygdala is a key structure in the processing of emotional
information (LeDoux, 1996) and has been implicated in the genesis of
fear responses. Dysfunction of the amygdala has been suggested to
underlie anxiety-type disorders (Davis, 1992; LeDoux, 1995). The
benzodiazepines, which are widely used in the treatment of such
disorders, are thought to act by enhancing the actions of GABA at
GABAA receptors (Tallman and Gallager, 1985;
Costa and Guidotti, 1996). The presence of a GABA receptor in the
amygdala that is inhibited by benzodiazepines suggests that the actions of these agents in the amygdala are more complex than previously thought. This receptor might be a possible new target in the
development of therapeutic agents for disorders involving the amygdala.
| |
FOOTNOTES |
Received June 1, 1999; revised July 30, 1999; accepted Sept. 1, 1999.
This work was supported by grants from the National Health and Medical
Research Council of Australia. P.S. is a Charles and Sylvia Viertel
Senior Medical Research Fellow. We thank Prof. Graham Johnston for
discussion throughout the course of this study and for providing us
with several compounds. We thank John Bekkers, Rowland Taylor, and Luli
Faber for comments on this manuscript.
Correspondence should be addressed to Pankaj Sah, Division of
Neuroscience, John Curtin School of Medical Research, GPO Box 334, Canberra ACT 2601, Australia. E-mail: pankaj.sah{at}anu.edu.au.
| |
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ABSTRACT
INTRODUCTION
