1Program in Neuroscience and 2Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06030
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ABSTRACT |
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Signore, Armando P. and
Hermes H. Yeh.
Chronic Exposure to Ethanol Alters GABAA
Receptor-Mediated Responses of Layer II Pyramidal Cells in Adult Rat
Piriform Cortex.
J. Neurophysiol. 84: 247-254, 2000.
This study examined the effect of chronic exposure to ethanol
on -aminobutyric acid type-A (GABAA) receptor-mediated
responses of layer II pyramidal neurons of the piriform cortex. Slices
containing the piriform cortex were derived from pair-fed adult rats
maintained on ethanol-supplemented or control liquid diet for 30 days.
Responses of identified layer II pyramidal neurons to exogenously
applied GABA were monitored by whole-cell patch-clamp recording.
Chronic exposure to ethanol resulted in a rightward shift in the
EC50 of GABA and a decrease in the amplitude of maximal
GABA response. GABA-induced responses were modulated by acutely applied
ethanol (10-100 mM) in both chronic ethanol-treated and control
groups. No significant difference was found in the average change in
GABA response, suggesting that tolerance to acute ethanol exposure did
not develop. When the modulatory responses of individual cells were
classified and grouped as either being attenuating, potentiating, or
having no effect, the incidence of potentiation in the ethanol-treated group was significantly higher. Consistent with the absence of tolerance to acute ethanol, cross-tolerance to diazepam was not observed following 30 days of treatment with ethanol. These results are
discussed in light of regionally specific effects of chronic ethanol
treatment on GABAA receptor-mediated responses of layer II
piriform cortical neurons.
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INTRODUCTION |
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Several classes of
neurotransmitter receptors, among them the inhibitory ionotropic
-aminobutyric acid type-A (GABAA)
receptor, are implicated in contributing to the acute intoxicating
effects and long-term deleterious influences of ethanol on the CNS
(Deitrich et al. 1989
; Grant 1995
;
Grobin et al. 1998
; Littleton and Little 1994
; Nevo and Hamon 1995
; Samson and
Harris 1992
; Sytinsky et al. 1975
). Indeed,
there is considerable evidence that both acute and chronic exposure to
ethanol alters the function of GABAA receptors (Allan and Harris 1987
; Macdonald and Olsen
1994
; Sanna et al. 1993
). Nonetheless, in either
the acute or the chronic condition, the mechanisms underlying the
effects of ethanol on the GABAA receptor remain
to be elucidated.
Depending on whether the exposure is acute or chronic, ethanol appears
to modulate the functional properties of GABAA
receptors with variable outcomes. In the majority of cases, acute
exposure to ethanol results in enhanced GABAA
receptor function (Chandler et al. 1998; Nestoros
1980
; Nishio and Narahashi 1990
; Reynolds and Prasad 1991
; Soldo et al. 1998
;
Suzdek et al. 1986
). Chronic exposure to ethanol, on the
other hand, decreases GABAA receptor function,
consistent with the development of tolerance to the acute effects of
ethanol (Allan and Harris 1987
; Rastogi et al. 1986
; Ticku 1980
). In addition, an apparent
cross-tolerance develops consequent to chronic ethanol exposure since
there is a demonstrable concomitant loss of the allosteric potentiating
effects of certain GABAA receptor modulators,
such as benzodiazepines (Buck and Harris 1990
) or
barbiturates (Morrow et al. 1988
). Cross-tolerance to GABAA receptor modulators is seen in human
alcoholism as well as in animal models in which the effects of chronic
ethanol exposure have been tested (Miller 1995
).
Factors such as experimental preparation (Sapp
and Yeh 1998), regional selectivity (Givens and Breese
1990
; Matthews et al. 1998
; Soldo et al.
1994
), and developmental age (Lu and Yeh 1999
) are important considerations in investigating the effects of ethanol on
the CNS. For example, considerable research has been done using dissociated preparations, such as microsacs and brain homogenates (Allan and Harris 1987
; Buck and Harris
1990
; Morrow et al. 1988
; Rastogi et al.
1986
; Sanna et al. 1990
, 1993
). These
methodologies, while effective in delineating the essential effect of
ethanol on native GABAA receptors, do not
distinguish between different cell populations within a given brain
region or even among brain regions, thus compromising the ability to
discern cell type- or region-specific effects of ethanol. Dissociated
cells, tissue culture, and brain slices facilitate the analysis of
specific cell types (Tatebayashi et al. 1998
), but have
typically relied on tissue derived from neonatal or young postnatal
animals. Overall, it has been difficult to obtain an adult brain slice
preparation in which neurons remain viable for patch-clamp
electrophysiological analysis of GABAA receptor
function (Thibault et al. 1995
). In this light,
information on the effects of chronic ethanol on anatomically intact,
live adult CNS neurons is relatively scant (Freund et al.
1993
; Nestoros 1980
; Palmer and Hoffer
1990
) and has been limited to studies employing intra- and
extracellular recording approaches in vivo (Molleman and Little
1995
; Ripley et al. 1996
; Rogers and
Hunter 1991
; Whittington et al. 1991
).
To address issues related to the effects of chronic ethanol on the
adult brain, we developed an adult brain slice preparation and obtained
viable brain slices from rats that have been subjected to an
established chronic ethanol liquid-diet regimen (Liebar and
DeCarli 1982). In the present study, we focused on the piriform cortex, a region of the brain that subserves olfaction and is affected
by chronic use of ethanol in humans. Indeed, deficits in olfaction are
well-documented in alcoholics (Ditraglia et al. 1991
;
Gregson et al. 1981
; Kesslak et al. 1991
;
Shear et al. 1992
; Squires et al. 1985
).
In addition, plastic changes leading to degeneration of the piriform
cortex can be induced in rats by repeated exposure to ethanol
(Collins et al. 1996
, 1998
; Corso et al.
1998
; Crews et al. 1999
; Switzer et al.
1981
). In this study, we postulated that one of the plastic
changes might involve a change in the sensitivity of piriform cortical
neurons to GABA. The responses of pyramidal cells in layer II of the
rat piriform cortex to GABA were therefore examined by whole-cell
patch-clamp recording, and GABA concentration-response relationships
were compared between rats that were chronically treated with ethanol and pair-fed control animals. The sensitivity of GABA-mediated current
responses in piriform pyramidal neurons to modulation by acutely
applied ethanol and diazepam was also examined. We found that in adult
rats chronically exposed to ethanol, pyramidal cells in layer II of the
piriform cortex were less sensitive to GABA, but that sensitivity to
modulation by diazepam remained unaltered. Concomitantly, in the
chronic ethanol-treated group, there was an increase in the incidence
of pyramidal neurons with GABA-induced responses that were potentiated
by acute exposure to ethanol.
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METHODS |
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Chronic treatment with ethanol
Adult male Sprague-Dawley rats (200-280 g at start of study)
were pair-fed for 30 days with either a liquid diet (Research Diets,
Inc., New Brunswick, NJ) supplemented with ethanol (5% wt/vol;
"chronic ethanol," n = 12) or an isocaloric control
diet containing maltose-dextrin ("control," n = 14)
(Lieber and DeCarli 1982; Vavrousek-Jakuba et al.
1991
; Wiener et al. 1981
). The liquid diets were
prepared fresh each day and were available ad libitum along with water.
Following this regimen, rats in the chronic ethanol-fed group tended to
gain weight at a slower rate relative to the control group. To minimize
the weight differential at the end of the 30-day liquid-diet regimen
(377 ± 12 g in the control group versus 332 ± 6 g
in the chronic ethanol group), rats were weighed daily and the amount
of food given to each control rat was adjusted to match the intake of
the ethanol-fed rats for the entire 30-day treatment period.
Preparation of adult piriform cortical slices
Rats were killed and perfused transcardially with ice-cold, low-chloride artificial cerebral spinal fluid (low-chloride aCSF) containing (in mM): 140 sodium isethionate, 2.0 KCl, 4.0 MgCl2, 0.1 CaCl2, 25 NaHCO3, 25 glucose. Brains were quickly removed, bisected along the sagittal plane, immersed in ice-cold low-chloride aCSF, and 250-400 µM coronal slices containing the anterior piriform cortex were cut using a vibroslicer (Campden Instruments, Shelby, UK). Slices were then stored at room temperature in Dulbecco's modified Eagle's medium with high glucose (25 mM) and bicarbonate (25 mM) and used for electrophysiological recording within 6 h of cutting. All solutions used for cutting and incubating slices were bubbled continuously with 5% CO2/95% O2.
Electrophysiology
Adult piriform cortical slices were placed in a custom-made recording chamber, stabilized by an overlying platinum ring strung with a plastic mesh, and perfused with normal artificial cerebral spinal fluid (aCSF) containing (in mM): 124 NaCl, 5.0 KCl, 2.0 MgCl2, 2.0 CaCl2, 1.25 NaH2PO4, 25 NaHCO3, 10 glucose. The rate of perfusion was approximately 0.5 ml/min. Slices were viewed under Hoffman Modulation Contrast optics (Modulation Optics, Greenvale, NY) using a 40× extra-long working distance water immersion lens mounted on a fixed-stage upright microscope (Olympus, Woodbury, NY). A charge-coupled device (CCD) camera (Dage MTI, Michigan City, IN) was also used to view and collect images of the cells examined. Digitized images were collected and stored on a PC clone using image capture software (Flashpoint Integral Technologies, Indianapolis, IN).
Patch-clamp recording in the whole-cell configuration (Hamill et
al. 1981) was employed to assess GABAA
receptor-mediated activity. The recording electrodes were pulled from
fiber-filled borosilicate glass capillary tubes (Sutter Instruments,
Novato, CA) to an input resistance of 6-8 M
when filled with a
recording solution containing (in mM): 140 KCl, 1.0 MgCl2, 1.8 CaCl2, 3 Mg-ATP,
119.15 K-HEPES, pH 7.3 with KOH. The lidocaine derivative QX-222 (RBI,
Natick, MA) was included at a concentration of 5 mM to eliminate sodium
channel activity. Lucifer yellow (0.5%, Molecular Probes, Eugene, OR)
was included to facilitate visualization of the cells. During
whole-cell recording, the Lucifer yellow readily filled the soma and
processes of the cell under study; this was revealed by
epi-illumination using an Olympus U-MWIB filter (Fig.
1C).
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Seal formation and recordings were conducted using an EPC-7 amplifier
(Darmstadt, Germany). Cells were voltage clamped at 70 mV and the
recording was performed at room temperature. Liquid junction potentials
were nulled prior to the start of each recording. Membrane currents
were amplified and filtered through a four-pole Bessel filter. The
analog signals were monitored on-line using a chart recorder (Gould,
Valley View, OH). Digitized data were also acquired (DATAQ, JPM
Programming) and stored for off-line analysis.
Peak amplitude of GABA-induced current responses were determined using
Igor Pro (WaveMetrics, Inc., Lake Oswego, OR). From this data set, GABA
concentration-response relationships and estimations of
EC50 were made using Sigma Plot version 5.0 (SPSS, Chicago, IL). The effect of ethanol or diazepam on GABA-induced
responses were quantified by comparing the peak amplitude of GABA
responses averaged from a set of at least three identical and
consecutive trials each taken before (control), during, and after
exposure to ethanol or diazepam. The percentage change in the amplitude of GABA response from the control response was calculated as
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Focal applications of drugs
GABA, ethanol, and diazepam were dissolved in aCSF and loaded
into separate barrels of an eight-barrel glass pipette assembly. The
multibarrel assembly was pulled to a fine point, filled, and the tip
was broken under microscopic control such that the outer diameter of
each barrel was approximately 1.5 µM. Drug solutions were ejected by
regulated pressure (3 psi) and, as shown in Fig. 1B
(double arrowheads), were delivered within 10 µm of the cell under
study. The application of drugs was routinely directed to the basal
portion of the soma. One of the barrels of the multibarrel assembly was
routinely filled with aCSF, which was applied to clear drugs from the
vicinity of the cell and to control for mechanical artifacts due to
bulk flow.
For assessing the concentration-response relationship, the maximal amplitude of the GABA-induced response was obtained by a 2- or 3-s pressure application of GABA at varying concentrations (1-200 µM). In experiments involving tests of interaction between GABA and ethanol or diazepam, GABA was applied at equal intervals (10-30 s) at a concentration of 10 µM (approximately EC25 of the GABA response) for 100 ms to 1 s. Ethanol (10-100 mM) was prepared fresh immediately prior to each recording session. Diazepam was first dissolved in DMSO and stored frozen as stock at a concentration of 100 mM. On the day of the experiment, an aliquot of the stock was diluted and used at a concentration of 0.5 µM. Bicuculline methiodide was stored as 200 mM stock and diluted to 50 µM. Unless otherwise indicated, all drugs and chemicals were purchased from Sigma (St. Louis, MO).
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RESULTS |
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Identity of cells in piriform cortex
Figure 1A illustrates a coronal slice containing piriform cortex obtained from the adult rat brain. Pyramidal cells from layer II were identified in these slices. One such cell typically selected for patch-clamp recording is shown in Fig. 1B. This cell appeared phase bright and emitted a primary apical dendrite that was directed toward the lateral olfactory tract. Intracellular diffusion of Lucifer yellow included in the whole-cell recording solution allowed visualization of the neuronal cell body and the dendritic arbor under epi-illumination (Fig. 1C). Large layer II cells displaying an apical dendrite and not more than two basal dendrites were classified as pyramidal cells. Those that did not fit these criteria were rejected from the data pool.
GABA concentration-response relationship
Application of GABA elicited robust, reversible, and
concentration-dependent inward currents in pyramidal cells held at 70 mV. The penwriter record in the inset of Fig.
2A illustrates an experiment
to determine the GABA concentration-response relationship in a
pyramidal cell recorded in a control slice. In both control and chronic
ethanol-treated groups, pyramidal cells displayed a threshold for
response to GABA at approximately 3 µM, while GABA delivered at
60
µM resulted in a saturated maximal current response. The GABA-induced
current was blocked by 50 µM bicuculline methiodide, a competitive
antagonist of the GABAA receptor (Fig. 2B).
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To examine whether exposure to chronic ethanol shifted the sensitivity to GABA, concentration-response relationships were established for the control and ethanol-treated groups by plotting the peak amplitude of responses as a function of incremental concentrations of GABA. As shown in Fig. 2A, the EC50 of the control group (16 µM; n = 14) was significantly different (P < 0.01, analysis of covariance) from that of the ethanol-treated group (EC50 = 24 µM; n = 7). Thus based on these EC50 values, chronic treatment with ethanol resulted in a rightward shift in the apparent potency of GABA. In addition, the ethanol-fed group showed a reduced maximal current response to GABA (0.751 ± 0.027 nA) compared with the control group (1.027 ± 0.011 nA). This reduction was statistically significant (P < 0.05, unpaired Student's t-test) and indicated that chronic exposure to ethanol also altered the apparent efficacy of GABA.
Ethanol-GABA interaction
Acute exposure to ethanol has been reported to modulate neuronal
responses to GABA (Aguayo 1990; Nestoros
1980
; Peoples and Weight 1999
). Beyond
establishing a decreased sensitivity to GABA itself, a series of
experiments examined the interaction between acute ethanol exposure and
pyramidal cell responses to GABA and addressed the issue as to whether
this interaction was altered in chronic ethanol rats. GABA was applied
at a concentration of 10 µM, which approximated the
EC25 value (Fig. 2A). Ethanol (10, 25, 50, and 100 mM) or aCSF was continuously delivered between consecutive
pressure pulses of GABA. At all concentrations tested, ethanol exposure
resulted in a modulation of GABA-induced response in both control and
chronic ethanol groups. An example of 25 mM ethanol potentiating the
GABA response monitored in a pyramidal cell is given in the inset of
Fig. 3. Under both control and chronic ethanol conditions, the outcome of the modulation varied, as ethanol potentiated, attenuated, or had no effect on GABA responses in individually tested pyramidal cells. Figure 3 summarizes averaged ethanol-induced changes in GABA response irrespective of the direction of modulation. No clear-cut difference emerged at any of the ethanol concentrations tested [P > 0.2, two-way analysis of
variance (ANOVA)].
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The modulatory effect of acutely applied ethanol was then classified as
being either attenuating (GABA-induced current amplitude reduced by
>10% of control current), potentiating (GABA-induced current
increased by >10% of control current), or having no effect (change in
amplitude of GABA-induced current within 10% of control current). The
data are summarized in the form of a "scatterplot" (Fig.
4A), which plots the peak
amplitude of GABA-induced currents during the control period as a
function of that recorded during acute exposure to ethanol. Each point
represents data derived from an individual trial. The 45° line
predicts no effect of ethanol on the GABA-induced currents. The two
lines outlining the shaded "equivalence" zone represent either a
10% increase or decrease in the GABA-induced current response, which
was used in this study to determine whether GABA responses observed
during exposure to ethanol was potentiated (points lying above the
equivalence zone), attenuated (points lying below the equivalence
zone), or had no effect (points lying within the equivalence zone). As
seen in Fig. 4A, in the chronic ethanol group, 28 out of 52 cases lie above the equivalence zone, indicating potentiation of GABA
responses by ethanol. In the control group, only 9 out of 44 cases are
found above the equivalence zone. This is consistent with previous in vivo and in vitro studies in which GABA responses displaying
sensitivity to potentiation by ethanol were rarely encountered
(Carlen et al. 1982; Freund et al. 1993
;
Harris and Sinclair 1984
; Siggins et al.
1987
).
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Figure 4B represents the binned distribution of the pyramidal cells examined in which ethanol either potentiated, attenuated, or had no effect on GABA responses. An ethanol-induced attenuation of GABA responses was found in control slices (17% of modulatory responses encountered) but never in chronic ethanol slices. Concomitantly, there was a significantly higher percentage of ethanol-induced potentiation in the chronic ethanol group (overall, P < 0.001, chi-squared analysis). These observations, taken together, indicate that chronic ethanol treatment has the net effect of increasing sensitivity to ethanol-induced potentiation of GABA in piriform cortical pyramidal neurons.
Modulation by diazepam
At the cellular level, chronic exposure to ethanol has been
reported to produce cross-tolerance to the effects of a variety of
GABAA receptor modulators, including the
benzodiazepines (Mihic et al. 1992). The question arose
as to whether cross-tolerance to diazepam occurred in the adult rat
piriform cortex following chronic treatment with ethanol. In a series
of experiments, the identical protocol was used to assess the
interaction between acute ethanol and GABA, except that a maximally
effective concentration of diazepam (0.5 µM) was substituted for
ethanol. As expected of the allosteric modulator, potentiation of
GABA-induced currents was seen in all cells. Figure
5A illustrates an example of
such a potentiation. Within the population of cells sampled (Fig.
5B), the mean diazepam-induced potentiation of peak
GABA-mediated responses in the control (85% ± 15.0; n = 22) and ethanol-fed (182% ± 61.8; n = 20) groups
were not significantly different (P = 0.14, unpaired Student's t-test). Thus diazepam at the concentration
tested in this study did not reveal any cross-tolerance induced by
ethanol in piriform pyramidal neurons.
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DISCUSSION |
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This study addressed the issue of functional changes associated
with GABAA receptors in the adult rat piriform
cortex following chronic exposure to ethanol. A standard liquid-diet
regimen (Lieber and DeCarli 1982) was employed that is a
favored model for behavioral, neurochemical, and biochemical studies of
alcoholism. The major finding is that the sensitivity of layer II
piriform cortical pyramidal cells to GABA is attenuated but that this
does not appear to be associated with the development of tolerance to
the acute effects of ethanol nor to cross-tolerance insofar as
sensitivity to diazepam remained unchanged.
A potentially confounding factor that may affect the interpretation of
the data indicating a decrease in sensitivity of piriform cortical
pyramidal cells to GABA is weight loss, as chronic treatment with
ethanol in the form of a liquid diet, has been reported to have a
slight anorexic effect (Lieber and DeCarli 1982, 1989a
). The measures taken in this study to pair-feed a group of control rats
with an isocaloric liquid diet were designed with this effect of
ethanol in mind. Earlier studies have directly addressed this issue by
altering the ratio of ethanol to either fat content, minerals, or
vitamins in the diet and have reported that the effects of chronic
ethanol were independent of the liquid-diet regimen (Lieber and
DeCarli 1989b
; Lieber et al. 1965
). Nonetheless,
it is acknowledged that this study could have included additional control groups in which normal levels of food intake and body weight
are maintained.
The sensitivity of piriform cortical pyramidal neurons to GABA was
significantly reduced after 30 days of exposure to ethanol. Studies
employing synaptoneurosomes derived from brain tissue have reported
that GABA- or muscimol-stimulated chloride flux following chronic
ethanol is decreased, increased, or remains unchanged (Allan and
Harris 1987; Buck and Harris 1990
; Frye
et al. 1991
, 1996
; Morrow et al. 1988
, 1990
;
Sanna et al. 1993
; Tremwel et al. 1994
).
Such preparations routinely involve the use of large amounts of
cerebral cortical or cerebellar tissue. In contrast, the present study
is restricted to examining identified pyramidal cells in the piriform
cortex. The individually identified neurons in the slice preparation
are more likely to retain their physical attributes and are thus
studied in a milieu that approximates their environment in situ. This
consideration strengthens the conclusion that sensitivity to GABA is
attenuated in the piriform cortex following chronic exposure to ethanol.
A priori, the change in sensitivity to GABA observed in this study
could be accounted for by either a reduction in
GABAA receptor number, a change in subunit
expression, or both. Analysis of concentration-response relationships
reveals decreases in both apparent efficacy and potency following
chronic ethanol treatment. The observed decrease in efficacy can be
accounted for by a reduction in the number of
GABAA receptors, leading to the diminished
maximal response of piriform pyramidal neurons to GABA. Prevailing
evidence, however, does not uniformly support a chronic ethanol-induced
decrease in GABAA receptor binding or number
(Rastogi et al. 1986; Thyagarajan and Ticku
1985
). The observed decrease in potency, on the other hand,
implies the possible expression of GABAA receptor
isoforms that, when activated, shift the concentration-response curve
rightward. This would be consistent with the notion that chronic
ethanol treatment may lead to up- or down-regulation of
GABAA receptor subunits. Indeed, in a variety of
preparations, chronic exposure to ethanol has been shown to alter the
expression of certain GABAA receptor subunit
mRNAs and, in some cases, their encoded proteins (Devaud et al.
1997
; Kang et al. 1998
; Mahmoudi et al.
1997
; Matthews et al. 1998
; Mhatre and
Ticku 1992
; Montpied et al. 1991
).
While changes in receptor number and expression of subunits are not mutually exclusive, and the results of the present study do not rule out either possibility, the finding of a shift in apparent potency in chronic ethanol-treated rats is highly suggestive that a change in subunit expression underlies at least in part the diminished sensitivity to GABA. Interestingly, Purkinje cells examined in cerebellar slices derived from the same chronic ethanol-treated animals used in this study exhibited attenuated sensitivity to GABA that is associated with a change in apparent efficacy but not potency (DW Sapp and HH Yeh, manuscript in preparation). Ongoing studies are addressing the outstanding issue of profiling the GABAA receptor subunits expressed in layer II pyramidal neurons of the piriform cortex as well as the cerebellar cortex and how they may be affected by the regimen of chronic ethanol treatment used in this study.
An unexpected finding was the absence of tolerance to modulation by
acute exposure to ethanol. The hallmark of tolerance in animals
chronically exposed to ethanol is the development of an attenuated or
abolished response to acutely applied ethanol, as has been shown to
occur for GABAA receptor-mediated responses in
the CNS (Allan and Harris 1987; Mihic et al.
1992
; Morrow et al. 1988
, 1990
). Instead, our
data indicate that, on chronic exposure to ethanol,
GABAA receptors expressed in pyramidal neurons of the piriform cortex display increased sensitization to acute ethanol. Considering that tolerance to the effects of acute ethanol could not be
demonstrated in this study, it might have been predicted that
cross-tolerance would not develop either. Rodents chronically exposed
to ethanol exhibit diminished benzodiazepine-induced enhancement of
muscimol-stimulated chloride flux (Buck and Harris 1990
;
Sanna et al. 1993
), although no changes in
benzodiazepine binding have also been reported (Devaud and
Morrow 1994
; Karobath et al. 1980
; Mhatre
and Ticku 1989
; Rastogi et al. 1986
). In the
present study, the potentiating effect of diazepam on GABA-induced
responses in pyramidal cells was similar in the chronic ethanol-treated and control groups. It should be noted that a single concentration of
diazepam (0.5 µM) was tested which, in a previous study (Sapp and
Yeh, manuscript in preparation), was shown to be maximally effective in
potentiating GABA responses in cerebellar Purkinje cells. This
concentration of diazepam also effectively revealed cross-tolerance to
diazepam in cerebellar slices taken from the same ethanol-fed animals.
It may be that the detection of tolerance and cross-tolerance can be
enhanced by sampling more pyramidal neurons or by extending the chronic
ethanol treatment beyond 30 days. Given the data at hand, however, it
is proposed that layer II pyramidal neurons of the rat piriform cortex
may be more resistant to the development of tolerance to ethanol and
cross-tolerance to diazepam following chronic exposure to ethanol.
Nonetheless, it is acknowledged that a systematic analysis covering a
range of diazepam concentrations will need to be undertaken in future studies. Overall, in comparing layer II pyramidal cells to cerebellar Purkinje cells, an emerging picture is that the effect of chronic ethanol treatment on GABAA receptor function
involves multiple components that become manifested in a brain region-
and perhaps even cell type-specific manner.
Tolerance has been proposed to be a protective response on chronic
exposure to potentially harmful substances (Littleton
1983). The lack of tolerance to acutely applied ethanol and of
cross tolerance to diazepam after 30 days of chronic ethanol treatment suggest that layer II pyramidal neurons of the rat piriform cortex possess GABAA receptors that are resistant to
developing the degree of tolerance seen in other brain regions. The
functional implication of this resistance to tolerance remains to be
elucidated. However, it is tempting to speculate that this property of
piriform cortical pyramidal neurons may be a factor in contributing to
the susceptibility of the piriform cortex to seizures, such as those
that can occur on withdrawal from ethanol. In addition, it may play a
role in the development of experimentally induced neurodegeneration in animal models of alcoholism as well as of olfactory deficits in human alcoholism.
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ACKNOWLEDGMENTS |
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The authors thank Drs. Shao-Ming Lu and Douglas Sapp for critical reading of the manuscript.
This work was supported by National Institute on Alcohol Abuse and Alcoholism Grant P50 AA-03510.
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FOOTNOTES |
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Present address and address for reprint requests: H. H. Yeh, Center for Aging and Developmental Biology, AAB Institute of Biomedical Sciences, University of Rochester Medical Center, 601 Elmwood Ave., Box 645, Rochester, NY 14642 (E-mail: hermes_yeh{at}urmc.rochester.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 7 February 2000; accepted in final form 7 April 2000.
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REFERENCES |
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