1Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43614-5804; and Departments of 2Neurology and 3Physiology, University of Michigan, Ann Arbor, Michigan 48104-1687
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ABSTRACT |
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Tietz, Elizabeth I.,
Jaideep Kapur, and
Robert L. Macdonald.
Functional GABAA receptor heterogeneity of acutely
dissociated hippocampal CA1 pyramidal cells. CA1 pyramidal
cells were voltage clamped, and GABA was applied to individual cells
with a modified U-tube, rapid drug application system. With
Vh = 50 mV, inward currents elicited by 10 µM GABA were inhibited by GABAA receptor (GABAR)
antagonists and were baclofen insensitive, suggesting that GABA actions
on isolated CA1 pyramidal cells were GABAR mediated. GABA
concentration-response curves averaged from all cells were fitted best
with a two-site equation, indicating the presence of at least two GABA
binding sites, a higher-affinity site (EC50-1 = 11.0 µM)
and a lower-affinity site (EC50-2 = 334.2 µM), on two or
more populations of cells. The effects of GABAR allosteric modulators
on peak concentration-dependent GABAR currents were complex and
included monophasic (loreclezole) or multiphasic (diazepam) enhancement, mixed enhancement/inhibition (DMCM, zolpidem) or multiphasic inhibition (zinc). Monophasic (70% of cells) or biphasic (30% of cells) enhancement of GABAR currents by diazepam suggested three different sites on GABARs (EC50-1 =1.8 nM;
EC50-2 = 75.8 nM; EC50-3 = 275.9 nM)
revealing GABAR heterogeneity. The imidazopyridine zolpidem enhanced
GABAR currents in 70% of cells with an EC50 = 222.5 nM,
suggesting a predominance of moderate affinity
2 (or
3-)
subtype-containing BZ Type IIA receptors. A small fraction of cells
(10%) had a high affinity for zolpidem, something that is suggestive
of
1 subtype-containing BZ Type I receptors. The remaining 30% of
cells were insensitive to or inhibited by zolpidem, suggesting the
presence of
5 subtype-containing BZ Type IIB receptors. Whether BZ
Type I and Type II receptors coexist could not be determined. The
-carboline methyl
6,7-dimethoxy-4-ethyl-
-carboline-3-carboxylate (DMCM) inhibited
GABAR currents in all cells at midnanomolar concentrations, but in
addition, potentiated GABAR currents in some cells at low nanomolar
concentrations, characterizing two groups of cells, the latter likely
due to functional assembly of
5
x
2GABARs. In all cells, GABAR
currents were moderately sensitive (EC50 = 9 µM) to
loreclezole, consistent with a relatively greater
3 subtype, than
1 subtype, subunit mRNA expression. Two populations of cells were
identified based on their sensitivities to zinc(IC50 = 28 and 182 µM), suggesting the presence of at least two GABAR isoforms
including
5
3
2 GABARs. Consistent with the heterogeneity of
expression of GABAR subunit mRNA and protein in the hippocampus and
based on their differential responses to GABA and to allosteric modulators, distinct populations of CA1 pyramidal cells likely express
multiple, functional GABAR isoforms.
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INTRODUCTION |
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-Aminobutyric acid (GABA) mediates fast
inhibitory synaptic transmission by opening the GABAA
receptor (GABAR) chloride ion channel, a hetero-oligomeric pentamer
(Nayeem et al. 1994
). GABAR currents are enhanced by
several clinically useful drugs, including barbiturates,
benzodiazepines, and imidazopyridines that act at different
allosteric regulatory sites on GABARs. Additional modulatory sites have
been described for negative modulators of GABARs such as the
-carbolines and zinc (Macdonald and Olsen 1994
;
Smart et al. 1991
). Heterogeneity of native GABARs was
suggested by classical pharmacological studies of allosteric modulators
that identified receptors with different drug sensitivities (BZ Type I
and II) (Burt and Kamatchi 1991
; Ehlert et al.
1983
). Heterogeneity of GABARs was supported by the
identification of different GABAR subunit families with multiple
subtypes [
(1-6),
(1-4),
(1-4),
(1), and
(1)]
displaying different pharmacological properties in recombinant
expression systems (Davies et al. 1997
; Macdonald and Angelotti 1993
; Macdonald and Olsen 1994
;
Vincini 1991
).
Studies in heterologous expression systems have shown that distinct
pharmacological properties are conferred by varying GABAR subunit
composition. For example, the subtype is a primary determinant of
benzodiazepine and imidazopyridine sensitivity [
1 (Type I);
2
and
3 (Type IIA);
5 (Type IIB) and
4 and
6 (Type III)] (Doble and Martin 1992
; Pritchett et al.
1989
; Wieland et al. 1992
). Loreclezole, a novel
antiepileptic drug, enhances currents from GABARs assembled with
2
and
3 subtypes but not with the
1 subtype (Wafford et al.
1994
). A
subunit is required to achieve the full range of
benzodiazepine effects (Pritchett et al. 1989
) and also
results in relative zinc insensitivity (Draguhn et al. 1990
; Smart et al. 1991
), depending on the
subtype present (Saxena and Macdonald 1996
). GABARs
containing
4 or
6 subunits are benzodiazepine insensitive and
have relatively high zinc sensitivity (Davies et al.
1997
; Saxena and Macdonald 1994
, 1996
). Thus
GABAR pharmacological properties are determined by
,
, and
subunit subtypes (Ducic et al. 1995
; Ebert et al.
1994
; Hadingham et al. 1993
).
GABARs play a prominent role in modulation of CNS excitability
(Stelzer 1992) and have a dense, heterogeneous
distribution in the CA1 region of the hippocampus (Olsen et al.
1990
). In situ hybridization studies of the CA1 region of the
hippocampus have demonstrated that
2,
5,
1,
3, and
2
subtype mRNAs are highly expressed,
1 and
4 subtype mRNAs are
moderately expressed,
3,
2, and
1 subtype mRNAs are minimally
expressed, and
6,
3, and
1 subtype mRNAs are negligibly
expressed or are absent (Wisden et al. 1992
). The
relative expression of GABAR subunit mRNAs may reflect their subunit
protein expression on CA1 pyramidal cells, and certain
immunohistochemically identified GABAR subunits exist only at subsets
of synapses on CA1 pyramidal cell somata and dendrites (Fritschy
and Möhler 1995
; Nusser et al. 1996
;
Somogyi et al. 1996
; Sperk et al. 1997
;
E. I. Tietz, S. Chen, and W. Sieghart, unpublished observations).
The diversity of subtype mRNA and protein expression and the
compartmentalization of subunit subtypes suggest that multiple GABAR
isoforms may be assembled on individual CA1 pyramidal cells to produce
functionally distinct GABARs. The heterogeneity of CA1 pyramidal cell
GABARs was deduced from early pharmacological studies in in vitro
hippocampus (Alger and Nicoll 1982
). Functional heterogeneity of GABARs was later reported in hippocampal CA1 cells in
culture (Schönrock and Bormann 1993
) and more
recently physiologically distinct GABAR currents, which may arise from different classes of GABAergic interneurons (Freund and
Buszáki 1996
; Lacaille et al. 1989
;
Miles et al. 1996
; Nusser et al. 1996
), were shown to be anatomically segregated on CA1 pyramidal cell somata
and dendrites (Banks et al. 1998
; Pearce
1993
).
The goal of the present study was to characterize the sensitivity of individual CA1 pyramidal cells to GABA and allosteric modulators and to compare the functional properties of GABARs to their proposed subunit composition. The concentration-dependent effects of various allosteric modulators that have subunit subtype-dependent actions (diazepam, zolpidem, loreclezole, DMCM, and zinc) on GABAR currents were studied in mature, acutely dissociated cells using the whole cell patch-clamp technique.
Portions of this work have appeared in Soc. Neurosci. Abstr. 21: 1346, 1995.
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METHODS |
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Isolation of CA1 pyramidal cells
CA1 pyramidal cells were dissociated acutely from 28- to
35-day-old Sprague-Dawley rats (Harlan, Indianapolis, IN) using
modifications of the original procedures of Kay and Wong
(1986) and others (Celentano and Wong 1994
;
Kapur and Macdonald 1996
; Oh et al.
1995
). Rats were euthanized with CO2 and
decapitated, and the brain was rapidly dissected free. The region
containing the hippocampus was blocked and placed for 1 min in ice-cold
(4°C), oxygenated (95% O2-5% CO2)
1,4-piperazinebis(ethanesulfonic acid (PIPES) buffer containing (in mM)
120 NaCl, 2.5 KCl, 1 MgCl2, 1.5 CaCl2, 25 D-glucose, and 20 PIPES, pH 7.0. Coronal slices (500 µm)
were cut on a vibroslice (Campden Instruments, Campden, U.K.) in
preoxygenated, PIPES buffer maintained at 4°C, then were incubated
for a minimum of 1 h at room temperature (24°C) in continuously
oxygenated PIPES buffer. One or two slices were incubated in Type XXIII
protease (3 mg/ml, Sigma Chemical, St. Louis, MO) in oxygenated PIPES
buffer for 30 min at 32°C. After an additional 15-60 min recovery in
PIPES buffer at room temperature, cells were isolated acutely by
trituration of two to three 1-mm fragments microdissected from the CA1
region of the right or left hippocampus. Cells were plated in
extracellular recording buffer (see next section) on
poly-L-lysine-coated (0.1 mg/ml in 0.1 M boric acid/0.1 M
Na tetraborate, pH 8.4) 35-mm culture dishes, were allowed to adhere to
the plate for
10 min, and were used for electrophysiological
recording within 1 h of dissociation.
Electrophysiological recording
Whole cell voltage-clamp recordings (Vh = 50 mV) were made from isolated pyramidal cells according to
Hamill et al. (1981)
. The bathing solution contained (in
mM) 142 NaCl, 1 CaCl2, 8.1 KCl, 6 MgCl2, 10 D-glucose, and 10 HEPES (Burgard et al.
1996
; Saxena and Macdonald 1996
). The pH was
adjusted to 7.4 with 5 M NaOH, and the osmolarity to 322-326 mOsm.
Patch pipettes were thin-wall, filamented borosilicate glass
capillaries (1.5 mm OD, World Precision Instruments, Sarasota, FL)
pulled to a tip resistance of 6-10 M
on a Flaming-Brown electrode
puller (P-87, Sutter Instruments, San Rafael, CA) using a two-stage
pull. To facilitate gigaohm seal formation, the patch pipettes were
front-filled (500 µm) with internal solution [which contained (in
mM) 155.3 KCl, 1 MgCl2, 10 HEPES, and 5 EGTA, pH 7.3]
adjusted to 285-287 mOsm. The shank was back-filled with the same
internal solution containing an ATP regeneration system (50 U/ml
creatinine phosphokinase, 22 mM phosphocreatine, and 4 mM MgATP,
297-299 mOsm).
Currents were recorded at room temperature using a List EPC-7 patch-clamp amplifier (List Electronics, Eberstadt, Germany) and low-pass filtered at 2 kHz with an eight-pole Bessel filter (Frequency Devices, Haverhill, MA). Whole cell currents were displayed on a Gould 2400S chart recorder and recorded for later analysis onto computer hard disk using a TL-1 AD/DA converter and Axotape 2.0 acquisition and analysis software (Axon Instruments, Foster City, CA). Peak current responses were measured off-line from the digitized current traces using Axotape 2.0 analysis software. Peak current (pA) was defined as the initial maximal negative deflection from the baseline value determined immediately before the onset of the drug response.
Drug solutions and drug application
Drug application was gravity driven through a glass micropipette
tip (30- to 50-µm tip diameter) positioned within 50-100 µm of an
individual pyramidal cell. All drug solutions were applied to
individual cells at 1-min intervals using this modified U-tube "multipuffer" drug-application system (Greenfield and
Macdonald 1996). A constant negative pressure prevented the
flow of buffer (or drug) from the tip of the drug-application
micropipette. The length of the drug-application bar (Figs. 2-8)
represented the total activation time (5-12 s) of a normally
"open" solenoid valve. The solenoid, when closed, interrupted the
negative pressure, allowing solution to flow. The response time of the
solenoid valve was 8-15 ms. An additional "lag time" was
introduced between solenoid closure and drug application, before the
onset of the GABA-mediated current response, due to the small amount of
buffer that flowed through the coupled micropipette before test drug
flow. The duration of solenoid activation was varied as a function of
the exact placement of the multipuffer relative to the recorded cell.
The response characteristics of the "multipuffer" were determined
previously by measuring the change in tip potential of an open
electrode to step changes of K+ (
= 23.0 ms; total
solution exchange time 101.4 ms) (Greenfield and Macdonald
1996
). Before each recording session, the area of drug
application was visualized with Fast Green and included the entire area
occupied by individual cells. Solutions were dissolved in extracellular
buffer to the desired concentrations from the following stock
solutions: 100 mM GABA in H20; 10 mM bicuculline methobromide in H2O; 20 mM picrotoxin in 100% EtOH; 10 mM
diazepam in DMSO; 0.4 mM zolpidem in H2O; 100 mM
loreclezole in DMSO; 10 mM DMCM in DMSO; and 100 mM ZnCl2
in H2O. Drugs dissolved in DMSO or ethanol were diluted
further in extracellular buffer to a final concentration of <0.05%.
The vehicles alone or when coapplied with 5 or 10 µM GABA had no
effect on CA1 pyramidal cell GABAR whole cell currents. Coapplied drug
solutions were maintained in the same drug reservoir. GABA, bicuculline
methobromide, picrotoxin, and ZnCl2 and all other chemicals
were purchased from Sigma Chemical. Diazepam was a gift from Hoffmann
La-Roche Incorporated (Nutley, NJ). Zolpidem was from Research
Biochemicals International (Natick, MA). Loreclezole was a gift from
Janssen Pharmaceutical (Belgium).
GABA and allosteric modulator effects on CA1 pyramidal cells
To evaluate GABA concentration-response relationships in CA1
pyramidal cells, the maximal currents induced at each of five to eight
GABA concentrations were compared in the 19 cells tested. To evaluate
allosteric modulator effects on CA1 pyramidal cell GABA-induced
currents, diazepam, zolpidem, loreclezole, DMCM, and zinc were
coapplied with 10 µM GABA. Because loreclezole only minimally
enhanced currents induced by 10 µM GABA, loreclezole also was
coapplied with 5 µM GABA. The concentration of GABA was chosen to be
on the linear portion of the GABA concentration-response curve, thus
allowing a reliable measure of both the potentiation and inhibition
produced by the various modulators. The effect of GABA-induced current
desensitization on the assessment of peak current also was minimized at
lower GABA concentrations (Celentano et al. 1991).
Data analysis
Peak GABAR current amplitudes were analyzed for each cell as
described above. To evaluate the CA1 pyramidal cell population responses, individual peak current amplitudes were normalized, i.e.,
defined as 100% for each cell. Individual and averaged GABA concentration-response data were fit to a one-site model by nonlinear regression with the equation
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(1) |
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(2) |
The concentration-response relationships for allosteric modulators were constructed by evaluating the degree of potentiation or inhibition achieved by coapplication of each allosteric modulator and were expressed as a fraction of the control GABAR whole cell current. The peak amplitudes of the GABAR currents in the presence of various concentrations of each allosteric modulator were taken as fractions of the average responses to GABA applied 1 min before and 1 min after coapplication of each concentration. The fractions of the average GABA-response were multiplied by 100 and expressed as percent control, i.e., the control GABA-responses were set equal to 100%.
For diazepam and zolpidem, which also binds to the benzodiazepine
binding site, a comparison of the peak current amplitudes obtained with
each allosteric-modulator concentration were compared with an equation
for a one-site model
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(3) |
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(4) |
No a priori assumptions were made with regard to the mechanism of
action of loreclezole and zinc at the GABAR, and their effects on GABAR
currents were compared with a four-parameter logistic equation
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(5) |
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RESULTS |
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Acutely dissociated CA1 pyramidal cell morphology
Because response heterogeneity may depends on the cell
preparation, it was important to specify the cells used in the study. In each dish 5-20 CA1 pyramidal cells were suitable for whole cell
recording based on their phase-bright appearance and distinct morphological characteristics (Fig. 1).
Cells selected had a diameter >20 µm and were pyramidal or
polygonal in shape. The cells had a single large principle dendrite
of 60-80 µm in length, although a majority of cells selected had
apical dendrites >120-150 µm long. Longer dendrites, i.e., 200 µm, occasionally were branched. Fusiform cells with an ovoid
appearance, multipolar cells with diffuse processes, and small granule
cells from the overlying dentate gyrus occasionally were present and
were rejected for study.
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CA1 pyramidal cell currents
Whole cell recordings were made in nearly symmetrical chloride ion
solutions ([Cl]o = 164 mM;
[Cl
]i = 155 mM;
ECl
= 1.4 mV). GABA evoked inward currents at
negative membrane holding potentials and outward currents at positive
holding potentials (Fig. 2A).
The GABAR current reversal potential (EGABA) was
1.6 ± 1.7 (SE) mV (n = 4 cells) equivalent to the
ECl
predicted by the Nernst equation. A plot
of the peak I-V relationship generated in an individual CA1
pyramidal cell for Vhs ranging from
90 to +50
mV is shown in Fig. 2B.
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Without ATP in the recording pipette, repetitive application of GABA
produced currents that declined in amplitude with each successive GABA
application (current rundown). To minimize current rundown, ATP and an
ATP regeneration system were included in the intrapipette recording
solution (Oh et al. 1995; Stelzer et al. 1988
). Stable recordings could be maintained for
1 h,
although a slow rundown or run-up occasionally was detected. Therefore to control for any small degree of rundown that may have occurred despite inclusion of an ATP regeneration system in the patch pipette, the effects of varying concentrations of allosteric modulators were
assessed as a fraction of the averaged baseline GABAR current before
and after test drug applications. Cells with current rundown of >80%
over the lifetime of the recording were not included in the analysis.
Although multiple test drugs occasionally were applied to the same
cells, it was generally not possible to obtain full concentration-response curves for more than a single drug to make reliable cross-drug comparisons of EC50s.
Unless otherwise noted, inward currents were elicited from pyramidal
cells by 10 µM GABA at Vh = 50 mV. The
competitive antagonist bicuculline methobromide (10 and 50 µM)
completely and reversibly inhibited the GABAR currents (data not
shown). After GABAR activation, the noncompetitive antagonist
picrotoxin (100 µM) also inhibited GABAR currents (data not shown).
The inhibition by picrotoxin reversed slowly (>5 min). The
GABAB agonist baclofen (0.1 and 1.0 µM;
Vh =
50 to +30 mV) was applied to pyramidal
cells 5 min after current activation by 10 µM GABA. Baclofen failed
to evoke currents in four of four cells (data not shown). Taken
together, these results suggested that GABA currents elicited from CA1
pyramidal cells were mediated by GABARs.
Heterogeneous GABA concentration response in CA1 pyramidal cells
Peak current amplitude increased with increasing GABA concentration (Fig. 3, A and B). Maximal current amplitude in single neurons ranged from 120 to 2,056 pA. For each cell, the one- or two-site best fit to the GABA concentration-response curves were statistically compared (P < 0.05). GABAR currents were monophasic in 8 of 19 cells and biphasic in 11 of 19 cells tested. There were no apparent morphological differences between CA1 pyramidal cells with monophasic or biphasic GABA concentration-response curves. Current traces for cells representative of each type are shown in Fig. 3, A and B. Examples of the best fit to GABA concentration-response curves derived from the current traces for the two cells shown in Fig. 3, A and B, displaying monophasic (EC50 = 8.2 µM; nH = 2.9) and biphasic (EC50-1 = 15.0 µM; EC50-2 = 370.4 µM; nH = 2.3) concentration-response curves, are shown in Fig. 3C. For individual cells fit best to a single site, estimates of EC50s ranged from 4.3 to 30.7 µM (median 11.3 µM). EC50s estimated from the best-fit curve to the averaged normalized data from the eight cells exhibiting a monophasic response was 10.3 µM [95% confidence interval (CI) = 6.5-14.4 µM] with a Hill slope (nH) of 1.6. Analysis of the GABA concentration-response curves for each cell that fit best to a two-site model indicated a similar range of EC50 estimates for the high-affinity site (EC50 = 6.1-15.0 µM). The EC50 for the low-affinity site in 9 of 11 cells ranged from 108 to 908 µM. EC50 estimates for the low-affinity site in 2 of 11 cells could not be accurately determined due to the fewer number of data points at very high GABA concentrations. GABA EC50s for higher- and lower-affinity sites, estimated from the best-fit line to the averaged, normalized data from the 11 cells with biphasic responses, were 12.3 µM (95% CI = 7.8-16.7 µM) and 315.6 µM (95% CI = 153.4-477.9 µM), representing 42 and 54% of sites, respectively. The averaged relative amplitude curve derived from normalized peak GABAR currents elicited in all cells (n = 19) is shown in Fig. 3D. The curve was fit best (P < 0.05) to high-affinity (EC50-1 = 11.0 µM; 95% CI = 3.6-25.9 µM) and low-affinity (EC50-2 = 346.2 µM; 95% CI = 121.6-570.8 µM; nH = 1.7) sites representing 38 and 62% of sites, respectively.
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Diazepam enhancement of CA1 pyramidal cell GABAR currents
The benzodiazepine agonist diazepam had variable effects on CA1 pyramidal cell GABAR currents recorded from acutely dissociated CA1 pyramidal cells. Diazepam (1-3,000 nM) was coapplied with 10 µM GABA and enhanced GABAR currents in a concentration-dependent manner (Fig. 4). GABAR current enhancement by 10 nM diazepam in individual cells varied widely. In six cells, 10 nM diazepam had little or no effect, i.e., diazepam (10 nM) enhanced the control GABAR current < 10%. However, in five cells, 10 nM diazepam enhanced GABAR currents to 110-135% of control. In one cell, 10 nM diazepam enhanced GABAR currents to 160% of control. Application of 10 nM diazepam failed in one cell. A similar heterogeneous response was obtained with 100 nM diazepam. Whereas GABAR currents in all of the 13 cells were enhanced (>110%) by 100 nM diazepam, GABAR currents in 8 cells were potentiated to 120-135% of control. In the remaining five cells, GABAR currents were enhanced to 145-200% of control. At higher diazepam concentrations, GABAR currents were potentiated to ~130-175% (300 nM); ~150-275% (1 µM); and ~155-375% (3 µM) of control.
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In 3 of the 13 cells, the concentration-response was biphasic (Fig. 4, A and C), and in the remaining 10 cells, it was monophasic (Fig. 4, B and C). EC50s for diazepam potentiation varied widely (2.1-1167.0 nM, median 68.5 nM) as did the maximal enhancement achieved (155-375%, median 175). There was no correlation (r = 0.01) between the estimated EC50 and the magnitude of potentiation.
Representative current traces from two cells in which diazepam was
coapplied with GABA are shown in Fig. 4, A and B. The concentration-response curves derived from these individual CA1
pyramidal cell responses are shown in Fig. 4C. The lines
represent the best fit (P < 0.05) of the data to one
or two sites using the equations described in METHODS (Fig.
4B). EC50s were derived from the equation for the best-fit lines. Diazepam had a biphasic effect (EC50-1 = 2.8 nM; EC50-2 = 335.3 nM) to enhance GABAR currents in some pyramidal cells as shown in Fig. 4A or a monophasic
effect (EC50 = 57.1 nM, Emax = 178.3 ± 3.3%) in other pyramidal cells as shown in Fig. 4B. The
range of EC50s estimated from fits of the data from
individual cells, shown in Fig. 4D, inset, suggested three EC50 groupings. The averaged data from all 13 cells
were fitted best (P < 0.05) with the equation for a
three-site fit (Fig. 4D). EC50s estimated from
the three-site model (nH = 1) were 1.8, 75.8, and 275.9 nM, representing 15, 20, and 65% of the effect of diazepam
to enhance 10 µM GABAR currents. Two-site fits of the diazepam data
with nH = 2 (Oh et al. 1995) or
with a variable Hill slope, gave similar EC50 estimates. In
the former case (nH = 2), a two-site fit
(EC50-1 = 5.6 nM and EC50-2 = 179.2 nM) was
statistically indistinguishable from a three-site fit
(EC50-1 = 3.3 nM, EC50-2 = 38.3 nM, and
EC50-3 = 222.3 nM).
Zolpidem effects on CA1 pyramidal cell GABAR currents
The imidazopyridine, zolpidem had variable effects on GABAR currents recorded from acutely dissociated CA1 pyramidal cells (Fig. 5). Zolpidem (1-3,000 nM) produced concentration-dependent enhancement of GABAR currents in seven of nine pyramidal cells (Fig. 5, C and D). Maximal GABAR current enhancement in these seven cells was either modest (141.9 ± 9.5%, n = 4) or large (308.1 ± 23.3%, n = 3) and could be distinguished statistically (P < 0.02, t-test with Welch's correction). The EC50 estimated from the best-fit line (Fig. 5D) to the averaged data for the seven cells (EC50 = 3.8-470.8 nM) in which zolpidem enhanced GABAR currents was 222.5 nM (95% CI: 23.0-468.1 nM). In three of the nine cells (33%), GABAR currents were inhibited slightly by low concentrations of zolpidem (10-100 nM, n = 2) (Fig. 5, B and D) or were unchanged by zolpidem (1-1,000 nM, n = 1). The insensitivity to zolpidem or the inhibition of GABAR currents by zolpidem were explored further in 19 additional cells by coapplying 1 µM zolpidem with 10 µM GABA. In 8 of the 19 additional cells (42% of cells), 1 µM zolpidem inhibited (65-95% of control, n = 6, 32% of cells) or did not significantly enhance (110% of control, n = 2, 11% of cells) GABAR currents. In the remaining cells (58%), zolpidem enhancement of GABAR currents ranged from 118.0 to -201.6% (mean 153.9 ± 6.9). The responses of all cells (n = 28) to 1 µM zolpidem are shown in Fig. 5D, inset.
|
DMCM effects on CA1 pyramidal cell GABAR currents
The -carboline DMCM had variable effects on GABAR currents
recorded from acutely dissociated CA1 pyramidal cells (Fig.
6). DMCM was applied with 10 µM GABA
onto eight cells. DMCM inhibited GABAR currents in half of the cells
(Fig. 6, B-D) and had a biphasic effect on the remainder of
the cells (Fig. 6, A, C, and D).
Traces from two representative cells are shown in Fig. 6, A
and B. The current traces in Fig. 6A show
concentration-dependent enhancement of GABAR currents at low
nanomolar DMCM concentrations and concentration-dependent inhibition of
GABAR currents at higher concentrations. In the second cell, isolated
from the same hippocampus, DMCM inhibited GABAR currents at
concentrations >100 nM (Fig. 5, B and C). A few
cells also were tested with 100 nM zolpidem. In the cell shown in Fig.
6A, the 10 µM GABA current enhanced by 3 nM DMCM was less sensitive to zolpidem than the cell in which GABA currents were only
inhibited by DMCM (Fig. 6B). The data from individual cells were fitted best to a sigmoidal curve, and the IC50s and
nHs were derived from the best-fit line. Cells
in which GABAR current was enhanced by DMCM (to >110% of control)
were grouped and were compared with cells in which DMCM coapplication
resulted in GABAR current inhibition. Comparisons of the mean log
IC50s indicated a significant difference (Student's
t-test, P = 0.04) between groups
(IC50 = 53.3 ± 31.6 nM vs. 268 ± 103.7 nM). The
pooled data from each group are shown in Fig. 6D. The mean
IC50s derived from the averaged data were 87.7 nM (95% CI:
27.3-281.4 nM) and 150.2 nM (95% CI 20.5 nM to 1.1 µM),
respectively.
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Loreclezole enhancement of CA1 pyramidal cell GABAR currents
Loreclezole had variable effects on GABAR currents recorded from
acutely dissociated CA1 pyramidal cells (Fig.
7). Potentiation of GABAR currents (10 µM GABA) by loreclezole (3 nM to 10 µM) was evaluated in four
cells. The maximal GABAR current enhancement produced by 10 µM
loreclezole was 110-120% of the control currents (data not shown).
Therefore, loreclezole (30 nM to 50 µM) was coapplied with 5 µM
GABA to an additional eight pyramidal cells. Further increases in the
loreclezole concentration applied were limited by its insolubility.
Loreclezole concentration-dependently potentiated GABAR currents in
these cells at concentrations >1 µM. A representative pyramidal cell
GABAR current that was potentiated to 180% of control with 50 µM
loreclezole is shown in Fig. 7, A and C. A
representative pyramidal cell GABAR current that was relatively
insensitive to loreclezole, i.e., potentiated to 130% of control with
50 µM loreclezole, is shown in Fig. 7, B and C. At higher concentrations (>10 µM), loreclezole produced an increase in the rate of apparent desensitization (Fig. 7, A and
B) (Donnelly and Macdonald 1996). Individual
loreclezole concentration-response curves were obtained for all cells
(Fig. 7C). The EC50s derived from individual
fits ranged from 2.9 to 30.6 µM. In seven of eight cells, GABAR
currents were potentiated to 155-215% of control, similar to the peak
potentiation (185%) derived from the best-fit curve to the pooled data
(Fig. 7D). The mean EC50 estimated from the
best-fit line of the averaged responses (n = 7) to
loreclezole was 8.6 µM (95% CI: 4.9-15.1 µM).
|
Zinc inhibition of CA1 pyramidal cell GABAR currents
Zinc had variable effects on GABAR currents recorded from acutely
dissociated CA1 pyramidal cells (Fig. 8).
Zinc (100 nM to 1 mM) was coapplied with GABA (10 µM) to eight
pyramidal cells and reduced GABAR currents in all cells in a
concentration-dependent fashion. On the basis of sensitivity to 100 µM zinc (Smart et al. 1991), data from individual
cells were pooled into two groups. In five of eight cells, zinc reduced
GABAR currents by ~50% (IC50 = 125-978 µM), whereas
in three of eight cells, zinc reduced GABAR currents to <10% of
control (IC50 = 18-54 µM, Fig. 8C).
Representative current traces from two cells exposed to zinc are shown
in Fig. 8, A and B. In cells with a lower
sensitivity to zinc, nearly complete inhibition of GABAR currents was
achieved only with 1 mM zinc. A comparison of the log IC50s
estimated from individual concentration-response curves for each cell
indicated a significant difference (Student's t-test,
P = 0.04) between the mean log IC50s of the
two groups of cells (IC50 = 32.5 ± 10.9 µM vs.
377 ± 156.9 µM). The sigmoidal fit of the
concentration-response curve for the pooled data are shown in Fig.
8D. The IC50s estimated from the averaged data
were 28.0 µM (95% CI: 20.8-37.9 µM, n = 3) and 182 µM (95% CI: 74.4-447 µM, n = 5).
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DISCUSSION |
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GABAR currents in acutely dissociated CA1 pyramidal cells
GABA concentration-dependently evoked inward currents on
morphologically identified CA1 pyramidal cells that reversed at the predicted ECl (Fig. 1). The voltage
dependency of the GABA-induced currents in dissociated CA1 cells, i.e.,
outward rectification of the I-V relationship, was
characteristic of hippocampal pyramidal cells (Ashwood et al.
1987
; Burgard et al. 1996
; Gray and
Johnston 1985
; Segal and Barker 1984
). The
insensitivity of pyramidal cells to baclofen indicated that
GABA-induced currents were not GABAB receptor mediated.
Similarly, Lenz et al. (1997)
reported an absence of
G-protein-mediated conductances using a high internal
[Cl
] to record whole cell currents from CA1 pyramidal
cells in hippocampal slices. Moreover, because inward currents in
dissociated cells were blocked by bicuculline and potentiated by
diazepam, it was unlikely that a component of GABA-activated currents
in dissociated pyramidal cells were GABAC receptor-mediated
(Bormann and Feigenspan 1995
).
Heterogeneity of GABAR currents
The actions of GABA on dissociated CA1 pyramidal cells suggested a
functional heterogeneity of GABARs within individual cells and among
different cells that was likely dependent on a variable subunit subtype
composition of different GABAR isoforms. Molecular heterogeneity of
GABARs was reflected in high and low affinities for GABA in receptor
binding studies (c.f. Sieghart 1995). In the present study, two
functionally different populations of acutely dissociated hippocampal
CA1 pyramidal cells were identified based on their monophasic and
biphasic GABA concentration responses, suggesting at least two affinity
states of GABARs in a majority of CA1 pyramidal cells. Both populations
of cells had an apparent high-affinity site for GABA similar to that
reported for trypsinized CA1 pyramidal cells (6.4-14.5 µM)
(Celentano and Wong 1994
; Shirasaki et al.
1991
). One population of dissociated cells had an apparent lower-affinity GABA site. A somewhat narrower range of GABA responses was reported for dorsal root ganglion cells isolated from adult rats
(26-107 µM) (White 1992
), consistent with the
diversity of GABAR subunit mRNAs in those cells (Persohn et al.
1991
). The range of GABA EC50s in dissociated CA1
cells was within the range of EC50s for recombinant GABARs
assembled in oocytes and embryonic kidney 293 cells from various
subunit combinations (1.3-42 µM, Levitan et al. 1988
;
9-985 µM, Sigel et al. 1990
; 17-103 µM,
Verdoorn et al. 1990
) and may reflect the 15-fold
difference in sensitivity of at least two
-subunit polypeptides
(Mr 55,000 and 58,000) to GABAA
agonists (Bureau and Olsen 1990
).
The range of EC50s associated with the GABAR high-affinity
state in dissociated cells also could represent more than one
high-affinity site. For example, two cultured hippocampal cell
populations, differentiated according to their GABA-induced
desensitization rates, both had high affinities for GABA (8.5 and 37.3 µM) (Schönrock and Borrmann 1993), similar to
the range of high-affinity EC50s detected in dissociated
cells. Conversely, discrepancies between GABAR potencies in cell
culture and young adult rat brain are not unexpected. Expression of
GABAR subunit mRNAs is regulated developmentally in the CA1 region
(Gambarana et al. 1991
; Möhler et al.
1995
), coincident with the late appearance of
-aminobutyric acid decarboxylase (GAD) immunoreactive cells (Potier et al.
1992
) and of functional GABAergic inhibition in the CA1 region
(Harris and Teyler 1983
; Michelson and Lothman
1992
).
Overall, the data suggested that the two GABAR affinity states detected
may reflect that at least two functionally distinct populations of
GABARs were localized to different regions of the pyramidal cell and/or
that different GABAR isoforms existed among distinct populations of
pyramidal cells. These data are consistent with earlier reports of
pharmacologically and electrophysiologically distinguishable GABAR
responses in CA1 pyramidal cell somata and dendrites (Alger and
Nicoll 1982). More recently, two anatomically and functionally
distinct GABAR-mediated synaptic responses, i.e., GABAA,
fast and GABAA, slow, were detected using both
intracellular and whole cell recordings in CA1 pyramidal cells in in
vitro hippocampal slices (Banks et al. 1998
;
Pearce 1993
). On the basis of immunocytochemical colocalization of the
2 and
1 subtypes, the former on the axon initial segment (Benke et al. 1994
; Chen et al.
1996
; Möhler et al. 1995
; Nusser et
al. 1996
; E. I. Tietz, S. Chen, and W. Sieghart,
unpublished observations),
2
1
2 receptors are likely prominent
CA1 pyramidal cell GABAR isoforms, perhaps mediating the
GABAA,fast response (Pearce 1993
). The
colocalization of
1 and
3 subtypes on pyramidal cell processes
(Chen et al. 1996
; Nusser et al. 1996
;
Sperk et al. 1997
; E. I. Tietz, S. Chen, and W. Sieghart, unpublished observation) suggests that GABARs composed of
1
3
2 subtypes could represent the second class of functionally and anatomically distinct GABARs, perhaps mediating the dendritic GABAA,slow response proposed to underlie the classical,
early inhibitory postsynaptic potential (Banks et al.
1998
; Pearce 1993
). The immunostaining pattern
of other subtype antibodies would allow a role for the
5, but not
3, subtype in mediating either of the proposed
GABAA,fast or GABAA,slow responses
(Fritschy and Möhler 1995
).
Cell-to-cell heterogeneity
In contrast to the biphasic GABA responses in CA1 pyramidal cells,
GABA concentration-response curves obtained in young adult dentate
granule cells, many isolated from the same hippocampi used in the
present study, were all monophasic (EC50 = 47 µM) (Kapur and Macdonald 1996). Because pyramidal cells and
granule cells display a different complement of GABAR subtype mRNAs
(Wisden et al. 1992
) and pattern of GABAR subtype
protein immunostaining (Fritschy and Möhler 1995
;
Nusser et al. 1996
; Somogyi et al. 1996
;
Sperk et al. 1997
; E. I. Tietz, S. Chen, and
W. Sieghart, unpublished observations), these contrasting findings
strengthen the likelihood that the unique pharmacological actions of
GABA on hippocampal principal cell types was dependent on GABAR subunit subtype composition.
The contrasting responses of hippocampal CA1 pyramidal cells, dentate
granule cells, and other neuron populations to GABA and allosteric
modulators supports the hypothesis that GABAR isoform heterogeneity is
one basis for the diversity of GABA inhibitory function within the
hippocampus and other neuronal circuits (Banks et al.
1998; Kapur and Macdonald 1996
;
Möhler et al. 1995
; Pearce 1993
).
Nevertheless, posttranslational modifications or other factors, e.g.,
phosphorylation state of the receptor, also might contribute to the
different GABAR-mediated current pharmacology among hippocampal
neuronal subtypes (Geynes et al. 1994
;
Leidenheimer et al. 1991
; Macdonald and Olsen
1994
; Smart 1997
; Stelzer 1992
). This possibility could not be distinguished in the present study. Moreover, heterogeneity of GABAR isoforms also probably is related to
differences in chloride channel properties, e.g., rectification and
desensitization, ascribed to hippocampal neuronal populations (Birnir et al. 1994
; Burgard et al. 1996
;
Gray and Johnston 1985
; Pearce 1993
;
Schönrock and Bormann 1993
).
Heterogeneity of allosteric regulation of GABAR currents
Added variation in the functional response of GABARs in pyramidal
cells was only evident in the presence of allosteric modulators, entirely consistent with classical binding studies that identified two
GABA binding sites and a diverse allosteric modulator pharmacology (c.f. Doble and Martin 1992; Sieghart
1995
). Allosteric modulators that interact with the
benzodiazepine binding site on GABARs, i.e., diazepam, zolpidem, and
DMCM, produced multiphasic modulation of GABAR currents consistent with
a mixed Type I/Type II benzodiazepine pharmacology (Doble and
Martin 1992
). The modulation of CA1 pyramidal cell GABAR
currents by allosteric modulators disclosed a large degree of GABAR
heterogeneity that was not always readily predicted from the GABAR
subtype mRNA and protein expressed in CA1 pyramidal cells
(Fritschy and Möhler 1995
; Nusser et al.
1996
; Somogyi et al. 1996
; Sperk et al.
1997
; Wisden et al. 1992
; E. I. Tietz, S. Chen, and W. Sieghart, unpublished observations).
The responses to diazepam were consistent with the existence of
multiple GABAR isoforms containing low- and mid- or low- and high-affinity benzodiazepine binding sites. Multiple nanomolar EC50s also were reported for clonazepam potentiation of
GABAR currents in thalamic and cortical neurons dissociated from early postnatal, juvenile and young adult rats (Oh et al.
1995). Alternately, potentiation of dentate granule cell GABAR
currents by diazepam was reported to be monophasic (EC50 = 158 nM) (Kapur and Macdonald 1996
). The distribution and
relative abundance of
subunit subtype mRNAs (
5 >
2
1 =
4 >>>
3) in CA1 pyramidal cells
(Williamson and Pritchett 1994
; Wisden et al.
1992
) and the pattern of
subtype immunostaining in the CA1
region (c.f. Fritschy and Möhler 1995
) suggest
that native GABARs composed of
1,
2, and/or
5 subtypes most
likely contributed to the heterogeneity of the diazepam responses in
CA1 pyramidal cells. The overall benzodiazepine sensitivity of CA1
cells also was expected to relate to their
2 subtype mRNA and
protein expression, although a contribution of
1 subtype- containing
native GABARs to the multiphasic effects of diazepam to enhance GABAR
currents could not be excluded (Knoflach et al. 1991
;
Somogyi et al. 1996
; Wisden et al. 1992
).
Pyramidal cell GABARs also might contain multiple
(McKernan
et al. 1991
; Verdoorn 1994
) or
subtypes
(Li and De Blas 1997
). Studies of the structural domains
of the benzodiazepine binding sites reinforce the primary importance of
variations in
and
GABAR subunit subtype composition in
determining benzodiazepine pharmacology (Smith and Olsen
1995
). Nonetheless, diazepam potentiation of GABAR currents in
oocytes was shown to vary 4- to 12-fold with
subtype substitution
(Sigel et al. 1990
), suggesting that the quaternary
structure of native GABARs also may affect benzodiazepine pharmacology.
The monophasic zolpidem concentration-response function was consistent
with the presence of primarily moderate affinity, BZ Type IIA
receptors, although a small population of higher-affinity BZ Type I
receptors might be represented by the lower percentage of cells with
the lowest EC50, consistent with the absence of 1
subtypes on the soma proximal dendrites of ~35% of pyramidal cells
(Nusser et al. 1996
). The remaining cells were
insensitive to or inhibited by 1 µM zolpidem, consistent with the
presence of BZ Type IIB (or BZ Type III) receptors with negligible
zolpidem affinity (Lüddens et al. 1994
;
McKernan et al. 1991
; Pritchett et al.
1989
). Zolpidem-insensitive CA1 pyramidal cells exhibited more
rectification and less desensitization at depolarized potentials than
zolpidem-sensitive cells, similar to properties described in
recombinant
5
1
2L and
5
3
2L GABARs expressed on mouse
L929 fibroblasts (Burgard et al. 1996
). Combined with
the results of binding studies using recombinant GABARs
(Lüddens et al. 1994
) and in situ hybridization
studies (Wisden et al. 1992
), these findings suggested
that an
5
3
2L GABAR isoform was also likely to be expressed on
CA1 pyramidal cells. The pattern of zolpidem sensitivity/insensitivity
may reflect the presence of
1,
2, and
5 subtype mRNAs and
protein (Fritschy and Möhler 1995
; Nusser et al. 1996
; Williamson and Pritchett 1994
;
Wisden et al. 1992
) consistent with the detection of
three binding sites (KD = 15 nM, 225 nM and 6 µM) for [3H]zolpidem in the hippocampus (Ruano
et al. 1992
) and a triphasic diazepam concentration response.
DMCM both enhanced and inhibited GABAR currents in dissociated
pyramidal cells. The positive modulatory effect of DMCM at low
concentrations in 50% of the cells tested suggested the presence of
either the 5 or
1 subtypes, shown in recombinant GABARs to confer
positive modulatory actions on DMCM (Puia et al. 1991
; von Blankenfeld et al. 1990
). The relatively low
expression of the
1 subtype, in comparison with the
5 subtype,
mRNA (Fritschy and Möhler 1995
; Wisden et
al. 1992
), suggested that potentiation of GABAR currents by
DMCM may more likely be due to the functional assembly of
5
x
2
GABARs, consistent with their reduced sensitivity to zolpidem. The
monophasic inhibitory response of hippocampal dentate granule cells to
DMCM (EC50 = 60 nM) (Kapur and Macdonald 1996
) also might be explained by the relatively lower levels of
5 subtype mRNA and protein expression compared with CA1 pyramidal cells (Fritschy and Möhler 1995
; Wisden et
al. 1992
). The relative contribution of
1 or
2
subtype-containing GABARs to the inhibitory effects of DMCM on GABAR
currents (Puia et al. 1991
; von Blankenfeld et
al. 1990
) could not be deduced from the data.
Loreclezole enhanced GABAR currents in the majority of cells with an
EC50 similar to that reported in dentate granule cells (9 µM), although a larger fraction (50%) of granule cells were loreclezole insensitive (Kapur and Macdonald 1996).
These findings were consistent with the relatively lower levels of
expression of
1 subtype mRNAs and protein in granule cells than in
CA1 pyramidal cell somata (Wisden et al. 1992
; E. I. Tietz, S. Chen and W. Sieghart, unpublished observations).
Two distinct populations of CA1 pyramidal cells were separated by the
extent of inhibition produced by zinc. The relative insensitivity of
some CA1 pyramidal cells to 100 µM zinc, a concentration previously
used to establish zinc insensitivity of 2 subtype-containing GABAR
isoforms (Smart et al. 1991
), was consistent with the
presence of the
2 subtype (Fritschy and Möhler
1995
; E. I. Tietz, S. Chen and W. Sieghart, unpublished
observations) and the sensitivity of dissociated cells to
benzodiazepine agonists. Nevertheless, this could not explain the
moderate zinc sensitivity of the remaining fraction of pyramidal cells
tested. Dentate granule cells also showed a uniformly moderate zinc
sensitivity (IC50 = 29 µM) (Kapur and Macdonald
1996
). Consistent with the ability of
subtypes to modulate
zinc sensitivity (Fisher and Macdonald 1997
;
Saxena and Macdonald 1996
), GABAR currents in mouse
fibroblasts containing
5
3
2L subtypes were moderately zinc
sensitive (IC50 = 22 µM) (Burgard et al.
1996
). This finding raises the possibility that zolpidem-insensitive GABAR isoforms in CA1 pyramidal cells may have
contributed to the appearance of two populations of cells with
different zinc sensitivities.
Comparison of heterogeneity of allosteric regulation and GABAR subtype expression
On the basis of allosteric modulator responses in dissociated
cells and responses obtained in recombinant receptors expressing known
subunit subtype combinations, certain functional GABAR subtype combinations were likely to be predominant on CA1 pyramidal cells. Although classical benzodiazepine agonists have relatively equivalent binding affinities among 1,
2, or
5 subtype-containing
recombinant receptors (cf. Doble and Martin 1992
), their
relative potencies at functional pentameric receptors assembled in
heterologous systems varied more widely (Sigel et al.
1990
), consistent with the range of action of diazepam in
dissociated CA1 pyramidal cells as indicated by the three diazepam
affinity states detected. The lack of
1 subtype immunoreactivity on
a fraction of pyramidal cell somata, the localization of the
2
subtype on the axon initial segment (Nusser et al.
1996
), coupled with the high levels of
5 mRNA and protein
expression on CA1 pyramidal cells (Fritschy and Möhler 1995
; Wisden et al. 1992
), would suggest a
predominance of Type IIA (
2 subtype-containing) and Type IIB (
5
subtype-containing) benzodiazepine receptors on CA1 pyramidal cell
GABARs (Doble and Martin 1992
). Although less prominent,
the existence of functional BZ Type I (
1 subtype) receptors cannot
be ruled out based on the presence of
1 subtype-immunopositive
synapses on a subpopulation of CA1 pyramidal cell somata and on their
processes (Nusser et al. 1996
; Zimprich et al.
1991
) and the increased sensitivity of a small fraction of
dissociated cells to zolpidem.
The predominance of BZ Type IIB (5 subtype-containing) receptors was
consistent with the responses to all of the allosteric modulators
tested. Given the insensitivity of some CA1 pyramidal cell GABAR
currents to zolpidem, the positive modulatory actions of DMCM in some
cells and their loreclezole sensitivity, an
5
3
2 GABAR isoform
was likely present on
30% of individual pyramidal cells. The
enhancement of GABAR currents by diazepam in all cells studied implies
that a large fraction of GABAR isoforms contained the
2 subtype
(Fritschy and Möhler 1995
, Sperk et al.
1997
; Wisden et al. 1992
; E. I. Tietz, S. Chen and W. Sieghart, unpublished). Nevertheless, the sensitivity of
some cells to zinc supports the possibility that a portion of native
pyramidal cell GABAR isoforms coexpress the
5 subtype as found with
5
3
2 recombinant receptors (Burgard et al.
1996
).
Taken together, the findings of the present study suggest that the
subtype-specific expression of GABARs can provide an additional method
for modulating the functional properties of individual hippocampal
pyramidal cells, consistent with the hypothesis that the diversity of
GABA actions in the CNS are related to the assembly of multiple GABAR
isoforms on individual neurons, among populations of neurons and within
specific functional neuronal pathways or brain regions (Burt and
Kamatchi 1991; Möhler et al. 1995
;
Nusser et al. 1996
) and reinforcing the likelihood that
GABAR heterogeneity plays an important role in regulating GABAR
inhibition within the hippocampal trisynaptic circuit.
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ACKNOWLEDGMENTS |
---|
We thank Dr. Andrew Beavis for assistance with the derivation of model equations and N. Esmaeil for technical assistance.
This work was supported by National Institutes of Health Grants K02-DA-00180 (E. I. Tietz), K08-NS-01748 (J. Kapur), and R01-NS-33300 (R. L. Macdonald).
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FOOTNOTES |
---|
Address for reprint requests: E. I. Tietz, Dept. of Pharmacology, Medical College of Ohio, Block Health Science Bldg., 3035 Arlington Ave., Toledo, OH 43614-5804.
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 1 December 1997; accepted in final form 17 December 1998.
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REFERENCES |
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