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0022-3565/05/3123-1082–1089
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
U.S. Government work not protected by U.S. copyright
JPET 312:1082–1089, 2005
Vol. 312, No. 3
75663/1196385
Printed in U.S.A.
Differential GABAB Receptor Modulation of Ethanol Effects on
GABAA Synaptic Activity in Hippocampal CA1 Neurons
Peter H. Wu, Wolfgang Poelchen, and William R. Proctor
VA Eastern Colorado Health Care System, Medical Research Service, Denver, Colorado (P.H.W., W.R.P.); Department of
Psychiatry, University of Colorado Health Sciences Center, Denver, Colorado (P.H.W., W.R.P.); and the Institut fur
Neurophysiologie, Heinrich-Heine-Universitat, Dusseldorf, Germany (W.P.)
Received August 3, 2004; accepted December 17, 2004
GABA is a major inhibitory neurotransmitter in the brain
and generates both fast and slow inhibitory synaptic activity
in many types of neurons. There are two major types of
GABA receptors (GABAA and GABAB) that mediate GABA
neurotransmission. The GABAA receptor is a ligand-gated
chloride channel consisting of mainly pentameric subunit
proteins that form the chloride ion channel pore. In the brain,
the GABAA receptors consist of ␣, ␤, and ␥ subunits and have
selective cellular localization (Mohler et al., 2002). In hippocampal CA1 neurons, the ␣1 subunit of GABAA receptors is
distributed primarily on the dendritic region of the cell (Somogyi et al., 1989), whereas receptors with ␣2 subunits are
more dense in the somal region (Fritschy et al., 1998). The
This work was supported by a Department of Veterans Affairs-Merit Review award, National Institutes of Health Grant AA03527, and Colorado
Tobacco Research Program Grant 4I-007.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.104.075663.
that ethanol alone increases the probability of GABA release at
proximal but not distal regions. Changes by ethanol on the
probability of GABA release are only seen at distal locations
during GABAB blockade. Finally, when the modulation of presynaptic GABAB receptors is minimized by the local application
of 10 mM GABA directly onto somatic or dendritic GABAergic
synaptic regions, postsynaptic GABAB receptors seem to exert
significant negative (inhibiting) influence on the effects of ethanol on GABAA IPSCs in the distal subfields of CA1 pyramidal
neurons. Together, our data suggest that differences in both
presynaptic and postsynaptic GABAB receptor activity at these
GABAergic synapses may modulate the differential ethanol
sensitivity of proximal and distal GABAA IPSCs in hippocampal
CA1 pyramidal neurons.
particular type of subunits that compose a GABAA receptor
determines the channel kinetics, affinity for GABA, rate of
desensitization, and the modification of the channel activity
by other pharmacological agents, including benzodiazepines,
barbiturates, anesthetics, and most likely ethanol (Mohler et
al., 2002: Harris and Mihic, 2004).
Studies to demonstrate ethanol potentiation of stimulusevoked GABAA inhibitory postsynaptic currents (IPSCs) at
GABAergic synapses, however, have yielded conflicting results (Siggins et al., 1987; Aguayo, 1990; Allan et al., 1991;
Reynolds and Prasad, 1991; Proctor et al., 1992a,b; Soldo et
al., 1994; Lovinger, 1997; Weiner et al., 1997; Grobin et al.,
1998; Mihic, 1999). Despite these disparate findings, the bulk
of evidence indicates that ethanol can directly enhance the
activity of postsynaptic GABAA receptor-mediated chloride
channels (Faingold et al., 1998; Grobin et al., 1998; Harris
and Mihic, 2004). Recent studies have shown that ethanol
could enhance GABAA IPSCs via presynaptic interactions at
ABBREVIATIONS: EtOH, ethanol; IPSC, inhibitory postsynaptic current; aCSF, artificial cerebrospinal fluid; NMDA, N-methyl-D-aspartic acid;
AMPA, ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; APV, DL(⫺)-2-amino-5-phosphonovaleric acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt; BMI, bicuculline methiodide; CGP-52432, 3[[(3,4-dichlorophenyl)methyl]amino]propyl](diethoxymethyl) phosphinic
acid; PPI, paired-pulse index; ANOVA, analysis of variance.
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ABSTRACT
We tested the hypothesis that differential sensitivity to ethanol
of synaptic GABAA somatic and dendritic inhibitory postsynaptic currents (IPSCs) in hippocampal CA1 pyramidal neurons
could be due to differences in the extent of GABAB receptor
activity at GABAergic synapses in these two hippocampal subfields. Our present results show that dendritic (distally evoked)
GABA IPSCs contain a larger GABAB IPSC component of the
total GABA IPSC than the somatic (proximally evoked) subfield.
The inhibition of GABAB receptors by pretreatment of hippocampal slices with CGP-52432 [3[[(3,4-dichlorophenyl)methyl]amino]propyl](diethoxymethyl) phosphinic acid], a selective
GABAB receptor antagonist, changes the basal ethanol-insensitive, distally evoked GABAA IPSCs to become more sensitive
to ethanol. In addition, paired-pulse stimulation of the proximal
and distal subfields of hippocampal pyramidal neurons shows
GABAB Activity Modulates Ethanol Action on GABAA Currents
Materials and Methods
Slice Preparation. As described previously (Poelchen et al.,
2000), transverse hippocampal slices (400 ␮m) were prepared from 6to 8-week-old male Sprague-Dawley rats with a Sorvall tissue chopper (Sorvall, Newtown, CT). Slices were made in ice-cold artificial
cerebrospinal fluid (aCSF) and were stored in a submersion chamber
at 32°C in aCSF containing 126 mM NaCl, 3 mM KCl, 1.5 mM
MgCl2, 2.4 mM CaCl2, 1.2 mM NaH2PO4, 11 mM glucose, and 26 mM
NaHCO3 saturated with 95% O2/5% CO2.
Electrophysiological Recordings and Drug Application.
Hippocampal slices were transferred to a submersion recording
chamber (H and H Custom Machining, Denver, CO) and superfused
with gassed (95% O2/5% CO2) aCSF at a flow rate of 2 ml/min.
Whole-cell patch-clamp recordings were made from CA1 pyramidal
neurons at 32°C using an Axoclamp-2A amplifier (Axon Instruments
Inc., Union City, CA) operating in voltage-clamp mode. Cells were
not included in this study if their resting membrane potential was
depolarized more than ⫺65 mV (corrected for the liquid junction
potential), if their access resistance was greater than 30 M⍀, or if
this value changed by ⬎15% over the course of the recording. Cells
were voltage-clamped to approximately ⫺55 mV to increase the
chloride driving force to obtain GABAA IPSC responses of 50 to 100
pA (approximately 10 –30% of the maximal response). Recording
electrodes were constructed from fiber-containing borosilicate glass
(1.5 mm o.d., 0.86 mm i.d.; Sutter Instrument Company, Navato, CA)
and had resistances of 6 to 9 M⍀ when filled with the following
internal K⫹-gluconate solution: 125 mM potassium gluconate, 1.5
mM KCl, 10 mM HEPES, 0.1 mM CaCl2, 1.0 mM EGTA, 2.0 mM
Mg2⫹-ATP, and 0.2 mM GTP, pH adjusted to 7.25 with KOH, 290 ⫾
5 mOsM. This solution was kept on ice until immediately before use.
All drugs used to make up the aCSF and internal recording solution
were purchased from Sigma-Aldrich (St. Louis, MO). Ethanol and
the receptor antagonists listed below were added to the artificial
cerebrospinal fluid in known concentrations immediately before flowing into the slice chamber by direct infusion via calibrated syringe
pumps (Razel Scientific Instruments, Stamford, CT). Glutamatergic
N-methyl-D-aspartic acid (NMDA) and ␣-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) receptor antagonists DL(⫺)-2-amino-5phosphonovaleric acid (APV) and 6-cyano-7-nitroquinoxaline-2,3-dione
disodium salt (CNQX) and the GABAA receptor antagonist bicuculline
methiodide (BMI) were also purchased from Sigma-Aldrich. The
GABAB receptor antagonist CGP-52432 was purchased from Tocris
Cookson Inc. (Ellisville, MO). An 8.0 M ethanol solution (in deionized
water) was prepared immediately before each experiment from a 95%
stock solution (Aaper Alcohol and Chemical, Shelbyville, KT) and kept
in a glass storage bottle at 4°C.
Proximal and Distal GABAA and GABAB Receptor-Mediated IPSCs. Evoked GABAA IPSCs were pharmacologically isolated
by superfusion with the glutamatergic receptor antagonists APV (50
␮M) and CNQX (20 ␮M) to block NMDA and AMPA receptors,
respectively. Synaptic stimulation was delivered with the use of two
bipolar twisted tungsten wire electrodes (0.2-ms pulses of 3– 8 V),
one placed in the stratum lacunosum-moleculare layer (for distal
stimulation) and the other within 250 ␮m of the recorded cell soma
in the CA1 stratum pyramidale (for proximal stimulation), as illustrated in Fig. 1A. An interstimulation interval of 20 s was used, and
the stimulus intensities were adjusted to evoke proximal and distal
IPSCs of similar amplitude. When GABAB responses were isolated
by superfusion with the GABAA receptor antagonist BMI, the remaining GABAB component was completely blocked by CGP-52432.
For determinations of the extent of the GABAB fraction in proximal
and distal responses, the percentage of the total GABA IPSC remaining following BMI treatment was calculated. Paired-pulse depression in these CA1 pyramidal neurons was investigated by delivering
the stimuli to the electrodes located at both the proximal and distal
subfields of the neuron. The paired-pulse stimuli were determined at
an interpulse interval of 30 or 50 ms. Both intervals gave similar
results, so data from both conditions were combined for the data
analysis. The amplitudes of the evoked responses were measured,
and the paired-pulse index (PPI) was calculated as the ratio of the
two responses (second/first). Curve fitting was determined from responses to single pulses obtained throughout each experiment.
In some experiments, differential interference contrast microscopy
was used to measure the effect of ethanol from brief, local, direct
pressure application of GABA to the soma or dendrites of CA1
pyramidal neurons. Micropipettes containing 10 mM GABA were
placed 3 to 5 ␮m from the cell body or major dendritic process under
visual control. GABA was delivered through the micropipette (5–10
ms at 10 –20 psi) via a Picosprizer II (General Valve, Fairfield, NJ).
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GABAergic synapses (Weiner et al., 1997; Roberto et al.,
2003; Ariwodola and Weiner, 2004).
Activation of GABAA receptors in the septohippocampal
pathway has also been shown to potentiate behavioral effects
of several anesthetics (Ma et al., 2002) as well as ethanol
(Mihic et al., 1997). Electrophysiological studies have shown
that GABAA IPSCs evoked in the CA1 hippocampal stratum
pyramidale (proximal) subfield are enhanced to a greater
extent by ethanol than GABAA IPSCs evoked in the stratum
lacunosum-moleculare (distal) subfield (Weiner et al., 1997);
however, the mechanisms that mediate the differential effect
of ethanol on GABA currents evoked at these two pyramidal
cell subfields are not well understood. The fact that zolpidem
produced a similar enhancement of GABAA IPSCs at both
proximally evoked (proximal GABAA IPSCs) and distally
evoked (distal GABAA IPSCs) locations (Weiner et al., 1997)
would seem to preclude the possibility that differences in
GABAA receptor composition may underlie the differential
ethanol sensitivity of GABAA IPSCs evoked at these two
subfields in hippocampal CA1 pyramidal neurons. Other
studies (Wan et al., 1996; Roberto et al., 2003; Ariwodola and
Weiner, 2004) showed that a GABAB receptor antagonist can
augment the ethanol enhancement of GABAA IPSCs, implying the involvement of GABAB receptors in modulating ethanol effects of GABAA responses in the hippocampus, but the
mechanisms are not well understood.
GABAB receptors mediate the slow synaptic inhibitory response of GABA transmission (Solis and Nicoll, 1992) and are
found in both the presynaptic and postsynaptic elements of
GABAergic terminals. The GABAB receptor is a typical seven
transmembrane G protein-coupled receptor and is known to
exert its action through the interaction with Go/␣i and G␤␥
subunits that modulate presynaptic calcium channels (Dittman and Regehr, 1996; Kubota et al., 2003) to regulate
GABA release. The GABAB receptor has also been shown to
modify postsynaptic potassium conductance (Newberry and
Nicoll, 1984; Gahwiler and Brown, 1985; Osmanovic and
Shefner, 1988), thereby altering cell activity. Although ethanol had little direct effect on the GABAB receptor response
(Frye and Fincher, 1996; Roberto et al., 2003), blockade of the
GABAB receptor in CA1 neurons was shown to increase ethanol potentiation of the GABAA response (Wan et al., 1996).
In the current study, we test the hypothesis that differences in GABAB receptor function may underlie the differential ethanol sensitivity of GABAA IPSCs observed in proximal
and distal locations of CA1 pyramidal neurons. The results of
this study support this concept and implicate a significant
modulatory role of postsynaptic GABAB receptor activity in
the differential ethanol sensitivity between proximal and
distal subfields in hippocampal CA1 pyramidal neurons.
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Control responses were recorded, and then ethanol (80 mM) and/or
CGP-52432 was applied by bath superfusion, and the GABA-evoked
responses were measured before, during, and after washout of
ethanol.
Statistical Analysis. Drug effects were quantified as the percentage of change in amplitude under the curve of the GABAA IPSCs
following drug application relative to the mean control and washout
values. Statistical analyses were carried out with the use of SigmaStat (SPSS Inc., Chicago, IL). The minimal significance level was set
at p ⬍ 0.05.
Results
Effects of Ethanol on Proximally and Distally
Evoked GABAA IPSCs in the Hippocampus. GABA IPSCs were evoked by electrical stimulation (3– 8 V) by a bipolar stimulating electrode placed in the stratum pyramidale
(proximal) or stratum radiatum (distal) subfields of the hippocampus (Fig. 1A) and isolated by blocking AMPA and
NMDA receptors with 20 ␮M CNQX and 50 ␮M APV, respectively. This pharmacologically isolated GABA current usually contained two components: a fast GABAA response (peak
amplitude latency of 5–10 ms) and a late GABAB component
(peak amplitude latency of 150 –200 ms) that was completely
blocked by CGP-52432, a potent GABAB receptor antagonist.
Ethanol (at concentrations of 40 mM and above) produced
enhancements of proximally evoked GABAA IPSCs (proximal
GABAA IPSCs) but much smaller increases by distally
evoked GABAA IPSCs (distal GABAA IPSCs; Fig. 1, B and C),
exhibiting time and ethanol concentration dependence (Fig.
1, D and E, respectively). A partial dose-response relationship for ethanol indicates that both proximal and distal
GABAA IPSCs were enhanced by ethanol (40, 80, 100, and
120 mM) [F(4,74) ⫽ 11.646, p ⬍ 0.001, two-way ANOVA] and
that proximal GABAA IPSCs were potentiated to a significantly greater extent than distal GABAA IPSCs [F(1,74) ⫽
6.151, p ⬍ 0.015, two-way ANOVA]. These results are consistent with those reported by Weiner et al. (1997). Statistical
analysis of the present data shows that there is a significant
ethanol concentration versus hippocampal subfield interaction [F(4,74) ⫽ 3.447, p ⬍ 0.012, two-way ANOVA]. Thus,
ethanol exhibited differential potentiation of GABAA IPSCs
in the proximal and distal subfields of hippocampus over this
ethanol concentration range tested (40 –120 mM).
Effect of GABAB Receptor Activity on Ethanol Potentiation of GABAA IPSCs. GABAA IPSC traces typically
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Fig. 1. Differential effects of ethanol enhancement of GABAA responses evoked by proximal and distal stimulation. A, stimulating electrodes placed
in the stratum pyramidale and stratum radiatum alternately triggered so that proximal and distal GABAA IPSCs could be evoked in the same cell.
GABAA synaptic responses were electrically evoked either in stratum pyramidale (proximal sweeps, B) or stratum radiatum (distal sweeps, C). Bath
application of 80 mM EtOH significantly enhances the proximally evoked GABAA IPSC responses compared with control responses (Con), whereas
ethanol has little effect on the distally evoked IPSCs. Full recovery from ethanol (Wash) is usually seen within 10 to 20 min. As shown by the time
course of the peak amplitude (D), the effect of ethanol peaks in 3 to 5 min during ethanol perfusion and is reversible upon washout of ethanol from
the brain slice. E, proximally evoked GABAA IPSCs are significantly more sensitive to ethanol treatment than distally evoked GABAA responses from
ethanol concentrations of 40 to 120 mM. Regression determinations are shown for proximal (closed circles, solid line) and distal (open circles, broken
line) response pairs evoked from 36 CA1 pyramidal cells (n ⫽ 7, 13, 9, and 7 at ethanol concentrations of 40, 80, 100, and 120 mM, respectively). Each
cell was given only one concentration of ethanol to avoid the possibility of ethanol desensitization. r2 values for proximal (0.98, solid line) and distal
(0.90, broken line) were determined by nonlinear curve fitting. Calibration bars in B and C: 15 pA, 40 ms.
GABAB Activity Modulates Ethanol Action on GABAA Currents
activity on the ethanol enhancement of the GABAA responses, we repeated the ethanol experiments in the presence of CGP-52432, a selective GABAB receptor antagonist,
to block the GABAB IPSC component (Fig. 3, A1 and B1).
Pretreatment of hippocampal slices with CGP-52432 shows
that 80 mM ethanol now potentiates both the proximal and
distal GABAA IPSCs (Fig. 3, A2 and B2, respectively), and the
enhancement is reversed upon washout of ethanol; however,
the CGP-52432 block of the GABAB component was not completely reversed after 30 min of washout (Fig. 3, A3 and B3).
Composite results show that, in the presence of the GABAB
antagonist, 80 mM ethanol potentiates GABAA IPSCs from
the distally evoked location similarly to the ethanol enhancement via proximal stimulation (Fig. 3C) and that the proximal GABAA IPSCs were not further enhanced by the pretreatment of slices with CGP-52432.
Ethanol Enhancement of GABAA IPSCs: Presynaptic
and/or Postsynaptic? A paired-pulse stimulation paradigm
was used to examine a possible presynaptic role in the differential ethanol enhancement of proximal and distal
GABAA IPSC responses. An increase in the second of two
Fig. 2. GABAB activity modulates ethanol enhancement of distally evoked GABAA responses. Synaptic GABA receptor-mediated responses usually
consist of two components: a faster GABAA and slower GABAB component. Blockade of the GABAA responses by 10 ␮M BMI reveals a small GABAB
response from proximal stimulation (A1), whereas the distally evoked IPSC response from the same cell (B1) typically has a much larger GABAB
component. Neither proximally (A2) nor distally (B2) evoked GABAB responses are significantly modified by 80 mM ethanol (mean proximal GABAB
IPSCs: 103 ⫾ 9.8% of control, n ⫽ 7; mean distal GABAB IPSCs: 102 ⫾ 7.2, n ⫽ 7). C, bar graph showing the paired mean and S.E.M. values for the
proximal and distal GABAB component (16.8 ⫾ 5.24, n ⫽ 13; 49.1 ⫾ 8.61, n ⫽ 13, respectively) of the total GABA IPSC response in these cells. ⴱⴱ, p ⬍
0.01. The relationship of ethanol enhancement of the GABAA response and the proportion of the GABAB component is shown in D. These results are
from proximally and distally evoked GABAA responses measured in 13 cells treated with 80 mM ethanol. There is an inverse relationship between the
sensitivity to ethanol and the proportion of the GABAB component in the response, determined by blocking the GABAA component following ethanol
treatment and washout. Proximally evoked GABAA responses (closed circles) showed greater ethanol enhancement and had the least GABAB
influence, whereas ethanol had a smaller effect on the distal responses (open circles) usually comprising a greater GABAB component. A linear
least-squares fit is shown (solid line) that has an r2 value of 0.38, which is statistically significant (p ⬍ 0.001, n ⫽ 26). Calibration bars in A and B:
10 pA, 40 ms.
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show that, under most recording conditions, the proximal
GABA IPSC responses have a smaller late GABAB IPSC
component than those evoked distally (Fig. 2, A1 and B1).
Regardless of the proportion or size of the GABAB component, after pharmacological isolation with a GABAA antagonist, BMI (10 ␮M), ethanol (80 mM) treatment did not significantly modify these GABAB responses by either proximal
(103 ⫾ 9.8% of control, n ⫽ 7) or distal (102 ⫾ 7.2% of control,
n ⫽ 7) stimulation (Fig. 2, A2 and B2). Systematic subfield
stimulation studies in the same CA1 pyramidal neurons in
which GABA IPSCs were alternately evoked from proximal
and distal locations show that the mean distally evoked
GABA IPSC contains a significantly larger late GABAB IPSC
component than the mean proximally evoked GABA IPSC
(Fig. 2C) (Student’s t test, t ⫽ 3.209, p ⬍ 0.006). Furthermore,
a significant correlation is seen between the ethanol potentiation of GABAA IPSC and GABAB content; neurons having
a smaller GABAB component have greater responses to ethanol [F(1,24) ⫽ 14.878, p ⬍ 0.001, linear regression analysis]
(Fig. 2D).
To determine the effect of the possible GABAB receptor
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closely paired stimulations (30 –50 ms apart) is thought to
mainly be due to a presynaptic mechanism involving the
phenomena of a higher probability of neurotransmitter vesicular release. Single and double (paired-pulse) GABAA
IPSC responses were alternatively evoked in both proximal
and distal locations on CA1 hippocampal pyramidal neurons
(Fig. 4). The PPI was measured before and during ethanol (80
mM) treatment (Fig. 4, A1 and B1). The PPI was determined
as the ratio of the second peak amplitude response (P2, above
the decay phase of the first peak) divided by the peak amplitude of the first peak (P1) as shown in Fig. 4B1. Ethanol
treatment resulted in a significant increase in the proximally
stimulated PPI (Fig. 4A1) (control: 0.63 ⫾ 0.039, n ⫽ 11;
ethanol: 0.90 ⫾ 0.085, n ⫽ 10, p ⫽ 0.005), but no significant
effect was measured by distal stimulation (Fig. 4B1) (control:
0.64 ⫾ 0.028, n ⫽ 13; ethanol: 0.75 ⫾ 0.070, n ⫽ 10, p ⫽
0.186). However, in the presence of the GABAB antagonist
CGP-52432, ethanol at both proximally and distally evoked
sites show increased PPI (proximal, Fig. 4A2: 0.86 ⫾ 0.051,
n ⫽ 11, p ⫽ 0.013; distal, Fig. 4B2: 0.87 ⫾ 0.052, n ⫽ 12, p ⫽
0.003). The composite data from all cells tested are shown in
Fig. 4C. Interestingly, CGP-52432 treatment alone significantly increased the PPI at both proximal (0.85 ⫾ 0.073, n ⫽
11, p ⫽ 0.018) and distal (0.86 ⫾ 0.064, n ⫽ 13, p ⫽ 0.004)
stimulation sites.
Finally, local application of GABA directly onto the somatic
or dendritic subfields of CA1 neurons was used to test for the
effects of ethanol on postsynaptic GABA responses. Brief
pressure application (10 –20 psi, 5–15 ms) of GABA onto the
proximal subfield produces a GABA response that is enhanced by ethanol in the presence or absence of the GABAB
antagonist CGP-52432 (Fig. 5, A1 and A2). In contrast, al-
though the application of GABA onto the dendritic subfield of
CA1 neurons produces a robust GABA response, ethanol in
the absence of the GABAB receptor antagonist does not significantly enhance the GABA response (Fig. 5B1). However,
in the slices pretreated with CGP-52432, ethanol application
produces a significant increase in the GABA response (Fig.
5B2). A comparison of the mean values during ethanol and/or
CGP-52432 treatment (Fig. 5C) shows that, in the presence of
CGP-52432, ethanol enhances responses to local GABA application to a similar extent in both proximal and distal
subfields.
Discussion
Although it has been suggested that the differential sensitivity to ethanol in various brain preparations could be attributed to the composition of GABAA receptor subtypes
(Harris, 1999; Mihic, 1999; Blednov et al., 2003), recent studies have shown that ethanol increased spontaneous GABA
IPSC frequency in rat CA1 pyramidal neurons (Ariwodola
and Weiner, 2004) and rat amygdala neurons (Roberto et al.,
2003; Nie et al., 2004). These studies support the concept that
ethanol may also increase the GABAA IPSC response via
enhancement of presynaptic GABA release. Since pretreatment of hippocampal slices with a threshold concentration of
500 nM baclofen prevented ethanol (80 mM) from enhancing
the proximally evoked GABAA IPSCs and can even cause
IPSC inhibition by ethanol at a higher baclofen concentration
(1.25 ␮M), Ariwodola and Weiner (2004) concluded that presynaptic GABAB receptors may modulate the actions of ethanol in the proximal subfield of CA1 pyramidal neurons in
the hippocampus. On the other hand, our data show that
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Fig. 3. GABAB antagonist modifies the ethanol
sensitivity of GABAA IPSCs evoked by distal, but
not proximal, stimulation. A, proximal stimulation. After the GABAB response is blocked by
bath perfusion with 0.5 ␮M CGP-52432 (CGP;
A1; Control, control response), the remaining
GABAA response is enhanced by 80 mM EtOH
(A2); however, this enhancement is the same
with or without the presence of the GABAB
blocker. The CGP-52432 effect can be nearly reversed after a prolonged washout period (A3). B,
distal stimulation. The GABAB antagonist CGP52432 does not alter the GABAA response alone
(B1), but in the presence of CGP, 80 mM ethanol
now enhances the GABAA IPSC responses from
distally evoked stimulation (B2). Washout of
EtOH followed by CGP washout is shown in B3.
Calibration bars in A and B: 10 pA, 30 ms. C,
summary graph of the GABAB influence on the
ethanol modulation of GABAA receptor-mediated
responses. The composite data show that proximally evoked GABAA IPSCs are enhanced (30 –
50%) by 80 mM ethanol with (142 ⫾ 18.5, n ⫽ 25)
or without (135 ⫾ 15.2, n ⫽ 27) the presence of a
GABAB antagonist (0.5 ␮M CGP-52432). In contrast, significant modulation by 80 mM ethanol
in distally evoked GABAA IPSCs is only seen in
the presence of the GABAB antagonist (145 ⫾
17.2% of control, n ⫽ 27) compared with ethanol
alone (105 ⫾ 8.5% of control, n ⫽ 25). ⴱ, p ⬍ 0.05;
NS, not significant.
GABAB Activity Modulates Ethanol Action on GABAA Currents
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Fig. 5. GABAB antagonist modulates the
GABAA response to ethanol in distal, but not
proximal, responses to direct application of
GABA. The local application of 10 mM GABA
to the soma of CA1 pyramidal neurons (A1)
produces a GABAA response that is enhanced
by the bath perfusion of 80 mM ethanol and
blocked by BMI (not shown). Local application
of GABA to the distal dendrites (B1) also produces a GABAA response; however, this response is not enhanced by the bath perfusion
of ethanol. In the presence of a GABAB antagonist (CGP-52432), however, responses by distal GABA application are now enhanced by
ethanol (B2). The enhancement of ethanol on
proximal GABA responses is not changed by
the presence of CGP-52432 (A2). The mean
values for all the local GABA application conditions are shown in C. Direct application of
GABA enhances the GABAA response approximately 15 to 20% at distal sites only in the
presence of the GABAB antagonist. In addition, ethanol enhances the GABAA response by
direct proximal GABA application only about
15 to 20% compared with the 30 to 50% enhancement of the synaptic GABAA IPSC response. Calibration bars: A1 and A2, 20 pA, 30
ms; B1 and B2, 10 pA, 30 ms. The mean percentage of control values, S.E.M., and number
of cells tested for each condition in C are as
follows: proximal EtOH, 116 ⫾ 6.6, n ⫽ 27;
CGP ⫹ EtOH, 114 ⫾ 7.47, n ⫽ 6; distal EtOH,
104 ⫾ 3.7, n ⫽ 20; CGP ⫹ EtOH, 120 ⫾ 3.0,
n ⫽ 6. ⴱ, p ⬍ 0.05; NS, not significant.
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Fig. 4. Paired-pulse response ratios of GABAA
IPSCs are modulated by ethanol. Paired-pulse
stimulation (30–50-ms interval) in both the proximal (A1, Control) and distal (B1, Control) regions of
CA1 pyramidal neurons show paired-pulse depression (ratio, ⬍1.0) of GABAA IPSCs determined by
measuring the ratio of the amplitude of the second
pulse, P2, and the amplitude of the first pulse response, P1, as depicted in B1. The second pulse
amplitude values were obtained by subtracting the
influence of the decay phase of the first, unconditioned pulse (shown as the dashed sweep for the
mean single-pulse condition) from the absolute
peak amplitude of the second pulse. These GABA
IPSC responses were acquired using an alternating single- then double-pulse stimulation paradigm with a 20-s interval between to allow for full
recovery of the previous response. Ethanol (80
mM) enhances both peaks by proximal stimulation
(A1) but has little effect on either peak by distal
stimulation (B1). In other pyramidal cells, the effects of the GABAB antagonist CGP-52432 alone
and in the presence of 80 mM ethanol were tested
(proximal, A2; distal, B2). Both conditions increased the PPI by proximal and distal stimulation, giving support to presynaptic modulation by
these drugs. C, bar graph showing the summary
data for these PPI determinations measured from
8 to 13 cells in each condition. ⴱ, p ⬍ 0.05 and ⴱⴱ,
p ⬍ 0.01. Calibration bars in A and B: 30 pA,
50 ms.
1088
Wu et al.
Acknowledgments
We thank Drs. Kevin Staley and Ronald Freund for helpful suggestions.
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GABAB receptor activity (our present results) or that ethanol
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low presynaptic GABAB activity at proximal GABAergic synapses would imply that inhibition of GABAB receptors would
have little or no effect on the increase in probability of GABA
release measured by paired-pulse stimulation. However, our
experiments show that CGP-52432 enhances the pairedpulse ratio to a similar extent in both the proximally and
distally evoked GABAA IPSCs, suggesting similar endogenous presynaptic GABAB receptor activity in both subfields
of CA1 pyramidal neurons. Therefore, the differential sensitivity to ethanol in proximally and distally evoked GABAA
IPSCs cannot be explained readily by differences in presynaptic GABAB receptor activity alone. In addition, presynaptic
GABAB receptors have been shown to be inoperative under
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Evidence for a postsynaptic GABAB receptor-mediated
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channels and inhibition of N-type Ca2⫹ channels (Bertrand
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was thought to enhance neuronal inhibition at GABAergic
synapses (Pham et al., 1998). To circumvent the effect of
presynaptic GABAB receptor regulation of GABA release at
the GABAergic synapses, we pressure-applied 10 mM GABA
locally onto proximal or distal subfields of CA1 pyramidal
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absence or presence of a GABAB receptor antagonist, suggesting a minimal role for GABAB regulation of ethanol effects on these proximal GABAA IPSCs. In contrast, ethanol
alone did not enhance the distally evoked GABAA IPSCs, but
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GABAB receptor antagonist. Thus, our data suggest that the
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of postsynaptic GABAB receptor number and/or activity between these two CA1 subfields. Since ethanol alone failed to
increase the probability of GABA release in the distal
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1089
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Address correspondence to: Dr. William R. Proctor, Dept. of Psychiatry
(C-261), University of Colorado Health Sciences Center, 4200 E. 9th Avenue,
Denver, CO 80262. E-mail: [email protected]
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017