Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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Feigenspan, Andreas,
Stefano Gustincich, and
Elio Raviola.
Pharmacology of GABAA Receptors of Retinal
Dopaminergic Neurons.
J. Neurophysiol. 84: 1697-1707, 2000.
When the vertebrate retina is stimulated
by light, a class of amacrine or interplexiform cells release dopamine,
a modulator responsible for neural adaptation to light. In the intact
retina, dopamine release can be pharmacologically manipulated with
agonists and antagonists at GABAA receptors, and
dopaminergic (DA) cells receive input from GABAergic amacrines. Because
there are only 450 DA cells in each mouse retina and they cannot be
distinguished in the living state from other cells on the basis of
their morphology, we used transgenic technology to label DA cells with
human placental alkaline phosphatase, an enzyme that resides on the
outer surface of the cell membrane. We could therefore identify DA
cells in vitro after dissociation of the retina and investigate their
activity with whole cell voltage clamp. We describe here the
pharmacological properties of the GABAA receptors
of solitary DA cells. GABA application induces a large inward current
carried by chloride ions. The receptors are of the
GABAA type because the GABA-evoked current is
blocked by bicuculline. Their affinity for GABA is very high with an
EC50 value of 7.4 µM. Co-application of
benzodiazepine receptor ligands causes a strong increase in the peak
current induced by GABA (maximal enhancement: CL-218872 220%;
flunitrazepam 214%; zolpidem 348%) proving that DA cells express a
type I benzodiazepine-receptor (BZ1). GABA-evoked currents are
inhibited by Zn2+ with an
IC50 of 58.9 ± 8.9 µM. Furthermore, these
receptors are strongly potentiated by the modulator alphaxalone with an
EC50 of 340 ± 4 nM. The allosteric
modulator loreclezole increases GABA receptor currents by 43% (1 µM)
and by 107% (10 µM). Using outside-out patches, we measured in
single-channel recordings a main conductance (29 pS) and two
subconductance (20 and 9 pS) states. We have previously shown by
single-cell RT-PCR and immunocytochemistry that DA cells express seven
different GABAA receptor subunits (1,
3,
4,
1,
3,
1,
2S, and
2L) and by immunocytochemistry that all
subunits are expressed in the intact retina. We show here that
at least
1,
3 and
2 subunits are assembled into functional receptors.
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INTRODUCTION |
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In the vertebrate retina,
dopamine is synthesized by a class of neurons that can be either
amacrine or interplexiform cells. When the vertebrate retina is
stimulated by light, dopamine synthesis and release increase,
modulating many of the events that lead to neural adaptation to light.
These effects are mediated by metabotropic receptors that are expressed
by a large number of cell types throughout the entire retina (see
Djamgoz and Wagner 1992; Witkovsky and Dearry
1991
).
In contrast to the wealth of information available on the
pharmacological effects of dopamine, little is known about the
mechanisms that control its release in the dark and light. Several
experiments were carried out measuring the amount of dopamine
synthesized or released by the intact retina in different physiological
conditions or after addition of various pharmacological agents (see
Djamgoz and Wagner 1992). These studies have shown that
the release of dopamine can be manipulated with agonists and
antagonists at the GABAA receptors. GABA and its
agonist muscimol can in fact block the synthesis and release of
dopamine evoked by light (Kirsch and Wagner 1989
;
Morgan and Kamp 1980
, 1983
). Furthermore,
antagonists like bicuculline and picrotoxinin induce dopamine synthesis
and release in dark-adapted retinas (Critz and Marc
1992
; Ishita et al. 1988
; Kamp and Morgan
1981
; Kirsch and Wagner 1989
; Kolbinger and Weiler 1993
; Morgan and Kamp 1983
;
O'Connor et al. 1987
; Piccolino et al.
1987
).
These results suggest that GABAergic inhibition plays an important role in the regulation of dopamine release in both light and darkness. However, the identification of the target cell is left unsolved because dopaminergic amacrine cells (DA cells) receive multiple inputs and the pathway responsible for the control of dopamine release consists of multiple neurons.
DA cells are postsynaptic to various types of amacrine cells
(Kolb et al. 1990), and some of them are GABAergic
(Kolb et al. 1991
; Yazulla and Zucker
1988
) suggesting that GABA receptors reside on the DA cells
themselves. However, direct evidence can only be obtained by recording
from these cells.
Because there are only 450 DA cells in each mouse retina and they
cannot be distinguished from neighboring cells on the basis of their
morphology, we used transgenic technology to label the surface of DA
cells with human placental alkaline phosphatase (PLAP)
(Gustincich et al. 1997). We were then able to identify DA cells in vitro after dissociation of the retina.
We have previously reported that, in absence of synaptic inputs,
solitary DA cells fire action potential in a rhythmic fashion. Furthermore, extracellular application of GABA reversibly inhibits the
spontaneous discharge through bicuculline-sensitive receptors (Gustincich et al. 1997), thus proving that DA cells
express functional GABAA receptors.
GABAA receptors are pentameric structures that
consist of various combinations of at least 16 subunits (Barnard
et al. 1998; Bormann 2000
; Sieghart
1995
). In situ hybridization and immunocytochemical studies
demonstrated that each subunit has a unique distribution in the CNS
(Fritschy et al. 1992
; Laurie et al.
1992
; Wisden et al. 1992
); furthermore,
transfection experiments proved that the various combinations of
subunits found in specific regions of the brain have different
pharmacological properties (Rabow et al. 1995
).
We have recently shown by single-cell RT-PCR experiments that solitary
DA cells contain the messages for seven GABAA
subunits (1,
3,
4,
1,
3,
1,
2S, and
2L), and
immunocytochemistry with subunit-specific antibodies proved that all
subunits are expressed by DA cells in the intact retina, where they are
associated to form at least two types of synaptic and multiple
extrasynaptic receptors.
In this paper, we characterize the pharmacological properties of GABAA receptors of solitary DA cells, seeking evidence for the functional expression of their repertory of subunit transcripts.
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METHODS |
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Dissociation of the retina and identification of DA cells
Anesthetized, 1- to 3-mo-old mice homozygous for the PLAP
transgene (Gustincich et al. 1997) were used.
PLAP-expressing cells can be identified in the living state by labeling
of their membrane with a monoclonal antibody to PLAP (E6) (De
Waele et al. 1982
) conjugated to the fluorochrome Cy3 (E6-Cy3)
(Gustincich et al. 1997
). Dissociation of the retina by
enzymatic digestion and mechanical trituration was performed as
previously described (Gustincich et al. 1997
), with a
few small changes. After removal of cornea, lens, and vitreous body,
the eyecups including the retinas were transferred to 5 ml of digestion
buffer (20 U/ml papain, 200 U/ml DNAse I; both from Worthington
Biochemical, Freehold, NJ) in Earle's Balanced Salt Solution (EBSS;
Sigma, St. Louis, MO). After digestion (45 min, 37°C), the eyecups
were transferred to trituration buffer to stop papain activity (5 min,
37°C). This solution contained 1 mg/ml ovomucoid (Worthington), 1 mg/ml bovine serum albumin (Sigma), and 100 U/ml DNAse I (Worthington)
in EBSS. Following trituration with fire-polished Pasteur pipettes, the
cell suspension was centrifuged at 1,000 rpm (5 min), and the pellet
was resuspended in minimum essential medium (MEM; Sigma) containing
E6-Cy3 (1:100). The cell suspension was allowed to sediment on a
concavalin A (1 mg
ml
1)-coated coverslip at
the bottom of a recording chamber and kept at 37°C in 5%
CO2-95% O2.
Electrophysiology
Recording chambers with solitary retinal cells were mounted on
the stage of an inverted microscope (Diaphot 300, Nikon), and E6-Cy3-stained DA cells were identified by scanning the chamber in
epifluorescence (535 nm excitation filter; 610 nm barrier filter). Whole cell voltage-clamp and outside-out single-channel recordings were
performed with an Axopatch 200A patch-clamp amplifier (Axon Instruments, Foster City, CA) and viewed with an oscilloscope (BK
Precision, Chicago, IL) or directly on the screen of a Gateway 4DX2-66
computer. The sample frequency was 20 Hz and 10 kHz in whole cell and
single-channel experiments, respectively. Patch pipettes were
constructed from borosilicate glass (1.65 mm OD, 1.2 mm ID; A-M
Systems, Everett, WA) using a horizontal two-stage electrode puller
(BB-CH, Mecanex, Geneva); the electrode resistance ranged from 5 to 7 M. Electrodes were connected to the amplifier via an Ag/AgCl wire.
The electrode holder and the headstage were mounted on a piezoelectric,
remote-controlled device attached to a three-dimensional
micromanipulator (Burleigh Instruments, Fishers, NY). In voltage-clamp
experiments, the series resistance of the pipettes was in the range of
12-20 M
and could be compensated up to 95% following cancellation
of capacitive transients. Drugs were applied to single cells or excised
outside-out patches in the extracellular bath solution by gravity flow
through an array of microcapillary tubes. Drugs could be selected using
a Teflon Rotary Valve (Rheodyne, Cotati, CA). This application system
allowed for a complete solution exchange in the vicinity of the
recorded cell within 200-500 ms.
Recording solutions
The extracellular bath solution for recordings of both whole
cell and single-channel currents contained (in mM) 137 NaCl, 5.4 KCl,
1.8 CaCl2, 1 MgCl2, 5 HEPES, and 10 glucose, pH 7.4. The intracellular solution for
recordings of whole cell and single-channel currents contained (in mM)
120 CsCl, 20 TEACl, 1 CaCl2, 2 MgCl2, 11 EGTA, and 10 HEPES, pH 7.2. Flunitrazepam, zolpidem, alphaxalone, methyl
6,7-dimethoxy-4-ethyl--carboline-3-carboxylate (DMCM) (all
RBI, Natick, MA), CL-218872, and loreclezole were prepared as 10-mM
stock solutions in DMSO and stored at
20°C. The maximal final
concentration of DMSO was 0.03%. Bicuculline methiodide (RBI) and
picrotoxinin (Sigma) were freshly prepared and added to the
GABA-containing solution. A 10-mM stock solution of
ZnCl2 (Fluka, Buchs, Switzerland) and
pentobarbital (RBI) were prepared in extracellular solution and kept
frozen at
20°C. CL-218872 was a gift from Dr. J. Bormann, and
loreclezole was generously provided by Janssen Pharmaceutica (Beerse, Belgium).
Data analysis
Effects of modulatory drugs were expressed as
I/IC, the ratio of the
GABA-induced peak current in the presence of the drug (I)
relative to the control GABA response
(IC), or as percentage drug-induced
change of IC. Dose-response curves
were fitted with a logistic sigmoidal equation. GABA-activated currents
in outside-out patches were analyzed after low-pass filtering at 1 kHz
(3 dB, 4-pole Bessel filter). The analysis of single-channel
amplitudes was carried out with a semi-automatic procedure, where each
current step was measured on the computer screen using two cursors.
Current-voltage (I-V) relations were obtained by measuring
30 current amplitudes at different membrane potentials (range
70 to
70 mV). The single-channel conductance of the main state and
subconductance levels were derived from the slope of the I-V
curves by linear regression. Data are expressed as means ± SE;
n is number of DA cells.
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RESULTS |
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GABA concentration-response relationships
After enzymatic digestion and mechanical trituration of the
retina, DA cells were identified in the living state by labeling of
their membrane with E6-Cy3 (Gustincich et al. 1997).
Cells were voltage clamped at
70 mV with equal concentrations of
chloride on either side of the membrane. Under these experimental
conditions, we have previously shown that extracellular application of
GABA induced a large inward current carried by chloride ions and
blocked by bicuculline (Gustincich et al. 1997
).
In this paper we present whole cell patch-clamp recordings carried out
from 153 DA cells. A stable gigaseal was established in 82% of the
cells. GABA was applied at concentrations ranging from 0.3 to 1,000 µM. Desensitization of GABA receptor (GABAR) currents was
dose-dependent and became apparent at a GABA concentration of 10 µM
(Fig. 1A). The maximal current
measured at a saturating concentration of GABA (1,000 µM) was
variable and ranged in amplitude from 4.7 to
8.4 nA with a mean
value of
6.3 ± 0.7 nA (mean ± SE, n = 6).
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Dose-response curves for GABA were obtained from six individual
DA cells. Their responses to increasing concentrations of GABA were
uniform with an increase in relative current amplitude (Fig.
1B). EC50 values for individual cells
ranged from 5.9 to 10.1 µM with a median value of 7.4 µM. For each
concentration, the data from the cells were pooled and fitted to a
sigmoidal logistic function (Fig. 1C). The mean
EC50 value was 7.4 ± 0.7 µM with a Hill
coefficient of 1.6 ± 0.3 corresponding to two GABA binding sites
on each GABAR. The maximum value of the GABAR current derived by the
best fit to the logistic equation was 6,115 pA compared with a mean
peak current value of
6,283 pA measured in six cells. This indicated
that peak current amplitudes were not significantly affected by
desensitization or by redistribution of chloride ions even at
saturating GABA concentrations.
Inhibition by bicuculline and picrotoxinin
The competitive GABAA receptor antagonist bicuculline (concentration range 0.1-100 µM) was co-applied with 10 µM GABA. The block of GABAR currents by increasing concentrations of bicuculline is shown in Fig. 2A. Complete dose-response curves were obtained for six DA cells. The sensitivity of the GABAR current for various concentrations of bicuculline was homogeneous for all neurons examined. Therefore the responses of individual cells were pooled, and the data were fitted to a sigmoidal logistic function (Fig. 2B). Bicuculline (0.1 µM) inhibited GABAR currents by 5%, whereas the current was completely blocked by 100 µM (Fig. 2A). Low concentrations of bicuculline (0.1-1 µM) selectively reduced the peak amplitude of the GABA response, but did not have a significant effect on the steady-state value. The IC50 values for individual cells ranged from 0.8 to 2.2 µM with a median value of 1.4 µM. The mean IC50 value obtained from the fit was 1.4 ± 0.1 µM with a Hill coefficient of 1.0 ± 0.1. These results show that the GABA responses of solitary DA cells are exquisitely sensitive to the inhibitory action of bicuculline and are thus mediated by GABAA receptors.
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The open chloride channel blocker picrotoxinin also inhibited the GABA response of DA cells. At a concentration of 10 µM, picrotoxinin reduced the peak inward current evoked by GABA to 36% of control values (Table 1).
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Benzodiazepine binding site shows BZ1 receptor specificity
To study the effects of benzodiazepines on DA cells' GABAA receptors, we co-applied flunitrazepam (1 µM) and 3 µM GABA. Flunitrazepam potentiated the peak current induced by GABA in all cells tested (Table 1). In addition, GABAR currents were uniformly inhibited by the inverse benzodiazepine agonist DMCM (Table 1).
Next we determined the pharmacology of the benzodiazepine binding site by applying agonists with BZ1-receptor specificity.
When co-applied with 3 µM GABA, the imidazopyridine zolpidem (1 µM), a BZ1-selective drug, increased more than twofold the peak current evoked by GABA (Fig. 3A). Dose-response curves were obtained for seven DA cells; data were pooled and fitted with a sigmoidal logistic function (Fig. 3B). Zolpidem enhanced GABAR function of DA cells with an EC50 value of 126 ± 2 nM, a maximal enhancement of 348 ± 9% and a Hill coefficient of 0.9 ± 0.1.
|
CL-218872, another BZ1-selective drug with high affinity for the 1
subunit, had a very similar effect on the GABA response of DA cells
(Fig. 3C). When co-applied with 3 µM GABA, CL-218872 (1 µM) increased more than twofold the peak current induced by GABA
(Table 1), and its effects were completely reversible after wash out (2 min).
These data indicate that benzodiazepine binding sites of DA cell GABAA receptors conform to the traditional definition of the BZ1 type.
In addition, control GABA-induced currents of all DA cells were increased more than twofold by 50 µM pentobarbital (Table 1). At this concentration, application of pentobarbital alone did not induce an inward current.
Zn2+ inhibition of GABAR currents
The divalent metal cation Zn2+ has been
described as a noncompetitive blocker of GABAA
receptor channels (Celentano et al. 1991). The
inhibitory effects of Zn2+ were studied on GABAR
currents of DA cells by co-applying Zn2+
(concentration range, 1-1,000 µM) and 3 µM GABA (Fig.
4A).
Zn2+ inhibition was very similar in all cells
tested (n = 7) as reflected by the small standard
errors of the data points in the dose-response curve. Currents were
hardly affected by 1 µM Zn2+, and all were
inhibited by 100 µM. Since all DA cells were equally affected with
Zn2+, the individual data were pooled and fitted
to a sigmoidal logistic function (Fig. 4B). The
IC50 of Zn2+ inhibition of
GABAR currents was 58.9 ± 8.9 µM with a Hill coefficient of
0.8 ± 0.1, and the maximal inhibition of GABAR currents obtained with 1 mM Zn2+ was 94 ± 2%. Even higher
concentrations of Zn2+ did not completely block
the GABA response leaving a small but substantial current (Fig.
4A).
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The incomplete inhibition of GABAR currents could reflect the presence
of more than one subpopulation of GABAA
receptors, one Zn2+-sensitive and the other
Zn2+-insensitive. Likewise, we can assume a
single GABAR population displaying incomplete block by
Zn2+. Using recombinant expression systems, it
has been shown that the presence of a -subunit leads to lower
sensitivity to zinc ions (Fisher and MacDonald 1998
;
Horenstein and Akabas 1998
; Smart 1992
;
Smart et al. 1991
), and therefore
Zn2+-insensitive GABARs are likely to be
modulated by benzodiazepines. We then tested on seven DA cells the
possibility that the residual GABAR current after
Zn2+ block was mediated by a
flunitrazepam-sensitive GABAR subpopulation. First, inward currents
elicited by 3 µM GABA were potentiated by 1 µM flunitrazepam (Fig.
5A). After wash out of the
drug and complete recovery of the control response, GABA-induced
currents of the same neuron were inhibited by 100 µM
Zn2+ to 43 ± 4% of control value (Fig.
5B). When 1 µM flunitrazepam was applied in addition to
100 µM Zn2+, the residual current was 47 ± 6% of the control response augmented by flunitrazepam. While
flunitrazepam alone potentiated the GABA response of 214 ± 14%,
in the presence of 100 µM Zn2+ currents were
enhanced by flunitrazepam of 206 ± 10%. Thus
Zn2+-insensitive residual current was no more
sensitive to benzodiazepines than the
Zn2+-sensitive GABAR currents. The results of
this experiment are summarized in Fig. 5C.
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Steroid enhancement of GABAR currents
Varying concentrations of the neuroactive steroid alphaxalone (10-3,000 nM) were co-applied with 3 µM GABA. The lowest concentration tested (10 nM) augmented the GABA response by about 23%, and the GABAR currents were more than doubled with 0.3 µM alphaxalone (Fig. 6A). Dose-response curves were obtained from seven DA cells. Because the cells responded uniformly to increasing concentrations of alphaxalone, individual data were pooled and fitted with a sigmoidal logistic function (Fig. 6B). The EC50 value obtained from the best fit to the data points was 360 ± 4 nM with a Hill slope of 1.4 ± 0.2. The maximal potentiation obtained with 3 µM alphaxalone was 452 ± 37% which is close to the value obtained from the fit (473 ± 17%).
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Effect of loreclezole
Six DA cells were tested for enhancement of GABA (3 µM)-induced
currents by 1 and 10 µM loreclezole, a drug selective for the 2 or
3 subunits. Loreclezole uniformly potentiated the GABA response in
all of these cells in a concentration-dependent way, and the rate of
apparent desensitization was enhanced with higher concentrations (Fig.
7A). Loreclezole (1 µM)
increased GABAR currents by 43 ± 7%, whereas the GABA response
was enhanced by 10 µM loreclezole to 207 ± 14%. The effects of
different concentrations of loreclezole on GABAR currents are
summarized in Fig. 7C.
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Single-channel recordings
To determine the single-channel conductance, outside-out
patches were pulled from the cell bodies of solitary DA cells. Channel openings induced by 2 s GABA (1 µM) pulses were recorded at a holding potential of 70 mV. Because of the high-density of GABARs on
the surface of DA cells, it was not possible to obtain patches that
contained only a single GABAR channel. Therefore a detailed kinetic
analysis of single-channel properties could not be carried out.
Extracellular application of GABA induced bursts of channel openings to
multiple conductance levels (Fig.
8A). The main conductance and
a subconductance level were determined by recording GABA-induced single-channel openings at various holding potentials. The data points
were fitted with a linear regression line, and the resulting conductances of the current-voltage relationships were 29 and 20 pS,
respectively (Fig. 8B). Because of the small amplitude, the
current-voltage relation of the second subconductance state was
obtained by connecting the data points at
70 and 70 mV with a
straight line. The slope of this line was 9 pS, representing the
conductance of the second sublevel.
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The amplitude distribution of the main and two subconductance states
were determined in six patches at a holding potential of 70 mV with 1 µM extracellular GABA. The mean current values as determined by
Gaussian distributions were
2.06 ± 0.14 pA for the main level
and
1.55 ± 0.11 pA and
0.61 ± 0.08 pA, respectively, for the two sublevels (Fig. 8C).
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DISCUSSION |
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In this study a transgenic mouse line was used in which DA cells
were labeled with PLAP, an enzyme that resides on the outer surface of
the cell membrane (Gustincich et al. 1997). After
enzymatic digestion and mechanical trituration of the retina, DA cells
were identified in the living state by labeling of their membrane with a monoclonal antibody to PLAP conjugated to the fluorochrome Cy3 (E6-Cy3). We have previously shown by single-cell RT-PCR that E6-Cy3-positive neurons are dopaminergic because they contain TH mRNA.
Furthermore, we proved that the expression of PLAP on the cell surface
did not affect the properties of GABA-gated chloride channels
(Gustincich et al. 1997
). Many studies of GABAR
currents after enzymatic digestion of tissue have been reported,
showing no alterations of the responses compared with those recorded
from cultured neurons (Kapur and MacDonald 1996
).
The pharmacological properties of the GABAA receptors of DA cells were analyzed studying the effects of the application of various drugs that are known to have distinct actions in the presence or absence of specific subunits. Drugs were applied only when stable recordings were obtained, and responses to consecutive applications of 3 µM GABA remained constant. We never observed "run-down" of GABA-induced currents during the time course of the recordings (15-20 min). After application and wash out of the drug, GABA was repeatedly applied to ensure complete recovery of the control GABA response.
GABA sensitivity of DA cells
The EC50 value for acutely isolated DA cells
was 7.4 µM, which is slightly lower than that in acutely dissociated
pyramidal neurons (25.4 µM) (Celentano and Wong 1994),
in adult cortical neurons (40.3 µM), and in thalamic neurons (23 µM) (Oh et al. 1995
). It is one order of magnitude
lower than the value measured in unidentified cultured amacrine cells
of the rat retina (71 µM) (Feigenspan and Bormann
1994
). In cultures of rat hippocampus, rapidly and slowly
desensitizing responses to GABA have been observed with
EC50 values of 8.5 and 37.3 µM, respectively
(Schonrock and Bormann 1993
). High-affinity
(EC50 = 6-8 µM) GABA responses have been
reported for rat hippocampal neurons, rat nucleus tracti solitarii
neurons, and mouse retinal bipolar cells (Nakagawa et al.
1991
; Shirasaki et al. 1991
; Suzuki et
al. 1990
). The effect of subunit composition on the affinity of
GABAA receptors for GABA has been studied by
transient expression of
1
3
2L,
6
3
2L, and
6
3
isoforms in mouse
fibroblast cells (Saxena and MacDonald 1996
). The
isoform containing the
6 subunit revealed an increased affinity for
GABA (2.2 µM) when compared with the
1-containing receptor (16.4 µM). EC50 values in the range of 6-17 µM
have been reported with recombinant GABA receptors composed of
1
2
2,
5
2
2, and
5
1
2 (Ebert et al.
1994
; Sigel et al. 1990
, 1992
).
It has been recently shown that both
and
subunits interact to
influence EC50 values of
GABAA receptors (White et al.
1995
).
The individual EC50 values measured in this study were evenly distributed around the mean, within a range of 5-µM GABA concentration. The best fit to both the individual and the mean data points was obtained with a first-order sigmoidal function, suggesting a uniform sensitivity of the GABAA receptors of DA cells. It must be noted, however, that with only six different GABA concentrations tested, small differences in affinities of two or more hypothetical GABA receptor subpopulations could not be resolved.
Benzodiazepine pharmacology of DA cells
Central benzodiazepine receptors have been traditionally
classified into two different pharmacological subtypes. BZ1 sites show
high affinity for the triazolopyridazine CL-218872, zolpidem, and some
-carbolines, whereas BZ2 sites display low affinity for these
ligands and high affinity for flunitrazepam. It is now clear, however,
that benzodiazepine-binding sites of GABAA
receptors are much more heterogeneous in their affinity for the ligands listed above. For instance a subtype in cerebellar granule cells designated BZ3 is associated with the
6 subunit and is insensitive to diazepam (see MacDonald and Olsen 1994
).
We studied the effects of the BZ1-preferring agonists CL-218872 and zolpidem, as well as the benzodiazepine agonist flunitrazepam and the inverse agonist DMCM. The GABA response of all DA cells tested were modulated by these allosteric regulators.
The EC50 value for zolpidem was 126 nM and thus intermediate between BZ1 and BZ2 receptors. However, maximal enhancement of GABAA receptors currents was 348% compared with 214% by flunitrazepam and 220% by CL-218872.
The benzodiazepine sensitivity of DA cell GABAA
receptors can be explained by various combinations of subunits.
Expression in oocytes or mammalian cell lines suggested that
benzodiazepine sensitivity is conferred by the presence of the 2
subunit (Pritchett et al. 1989
). Since all DA cells
showed enhancement of the GABA response by flunitrazepam, a likely
explanation would be that all DA neurons express the
2 subunit.
Furthermore, all GABAA receptor currents were
positively modulated by CL-218872 and zolpidem, compounds indicative of
a BZ1 benzodiazepine binding site; these findings are therefore
consistent with the presence of the
1 subunit.
Recombinant GABAA receptors containing the 4
subunit (Wisden et al. 1991
) in combination with one
and the
2 subunits are insensitive to benzodiazepines such as
diazepam. Because DA cells, however, express multiple types of
GABAA receptors, the observed benzodiazepine
sensitivity does not rule out the presence of a functional receptor
containing
4.
Zn2+ sensitivity of the GABAA receptors
GABA receptor currents from all DA cells displayed low sensitivity
to Zn2+. Currents were maximally inhibited to 6%
of control values with an IC50 of 59 µM. The
effects of Zn2+ on GABA responses depend on the
isoforms of GABAA receptors that are expressed
(Celentano et al. 1991; Draguhn et al.
1990
; Smart and Constanti 1990
; Smart et
al. 1991
; Xie and Smart 1991
). Recombinant GABA
receptor isoforms expressed in human embryonic kidney cells composed of
1 or
1 subunits (Smart et al. 1991
) or both
(Draguhn et al. 1990
) were highly sensitive to
Zn2+ (IC50 < 2 µM). On
addition of the
2 subunit to these GABA receptor subtypes,
Zn2+ (100 µM) had no inhibitory effect on
GABA-induced currents. In addition, Zn2+
sensitivity was also modified by the
subunit, with
GABAA receptors that contained
6 being more
sensitive to Zn2+ than those that contained
1
(Saxena and MacDonald 1996
). Finally, expression of the
subunit together with
6 and
3 confers a 10-fold higher
sensitivity to Zn2+ when compared with the
6
3
2 GABA receptor isoform (Saxena and MacDonald
1996
). Therefore it has been proposed that when the
1 or
2 subunit is present in recombinant GABA receptor isoforms, the
sensitivity to Zn2+ inhibition is lost
(Draguhn et al. 1990
; Smart et al. 1991
).
As shown by Saxena and MacDonald (1996), the presence of
the
subunit causes a decrease in the susceptibility to
Zn2+ inhibition rather than a total loss. Thus
the moderate to low effect of Zn2+ on DA cell
GABA responses is compatible with the presence of
2 in all receptors
expressed. However, the incomplete inhibition of GABA-induced currents
by saturating concentrations of Zn2+ may result
from the presence of at least two subpopulations of GABA receptors, one
containing
2 (Zn2+ insensitive), the other
lacking
2 (Zn2+ sensitive). Based on the
studies of recombinant GABAA receptors, the
Zn2+-insensitive isoform containing
2 should
display high affinity for benzodiazepines like flunitrazepam. The
Zn2+-insensitive residual current, however, was
no more benzodiazepine sensitive than were the total GABA-induced
currents, resulting from the hypothetical activation of more than one
GABAA receptor population. These data suggested a
single population of moderately Zn2+-sensitive
GABA receptors rather than two or more subpopulations.
Modulation by neurosteroids and loreclezole
Although there is no absolute subunit specificity for steroid
modulation of GABAA receptor function, it has
been shown that subunit composition affects their action (Gee
and Lan 1991; Korpi and Luddens 1993
; Lan
et al. 1991
; Puia et al. 1990
,
1993
; Shingai et al. 1991
). The greater
potentiation of neurosteroids in the spinal cord has been attributed to
region-specific expression of
3 (Lambert et al. 1995
;
Lan et al. 1991
), whereas the
subunit inhibits
neurosteroid modulation of recombinant GABAA
receptors (Zhu et al. 1996
). Recombinant receptors
containing
1 are more sensitive to the modulatory activity of
neurosteroids than those containing
6, whereas the type of
subunit does not appear to play a significant role (Zhu et al.
1996
).
We have observed a large potentiation of GABA-induced chloride currents
(452%) when the neuromodulator alphaxalone was applied together with
GABA. The variability of the effect at higher steroid concentrations
might be due to a combination of allosteric modulation and direct
opening of GABA-gated chloride channels. The strong enhancement of DA
cell GABA responses by alphaxalone is in agreement with the expression
of a GABAA receptor that lacks the subunit.
A new allosteric modulatory site on the GABAA
receptor subunit has been described (Wafford et al.
1994
; Wingrove et al. 1994
). The action of the
broad-spectrum anticonvulsant loreclezole depends on the type of
subunit present in the receptor complex: receptors containing
2 or
3 have more than 300-fold higher affinity for loreclezole than
receptors containing
1. A single amino acid residue present in both
2 and
3, but not
1 has been shown to confer sensitivity to the
modulatory action of loreclezole (Wingrove et al. 1994
).
In addition, loreclezole potentiation does not require the presence of
either an
or a
subunit (Wafford et al. 1994
).
We have observed a dose-dependent potentiation of GABA responses by
loreclezole, indicating the presence of GABAA
receptors containing the 2 and/or
3 subunits. However, the
presence of
1 cannot be ruled out from these experiments, since a
loreclezole-insensitive receptor population would remain undetected.
The modulatory effects of loreclezole on GABA responses of DA cells
were less pronounced than previously reported for recombinant
receptors. Because receptors that contained
1 did not contribute to
the overall potentiation of GABA-evoked currents, these results could
indicate that DA cells did not express a unique population of GABA receptors.
Which GABAA receptor subunits are expressed by DA cells?
We have previously shown with single-cell RT-PCR experiments, that
DA cells contained the messages for seven GABAA
subunits: 1,
3,
4,
1,
3,
1,
2S, and
2L
(Gustincich et al. 1999
). By immunocytochemistry with
subunit-specific antibodies, we have confirmed that all subunits were
translated and expressed by DA cells in the intact retina.
Such a complexity in GABAA receptor composition
is not new. Three other types of neurons, hippocampal pyramidal cells
(Nusser et al. 1996) and granule cells of both
cerebellum and dentate gyrus (Laurie et al. 1992
;
Nusser et al. 1995
, 1998
), exhibit a
similar richness in subunits.
With the exception of 4, six of the subunits formed clusters on the
surface of DA cells that were interpreted as postsynaptic active zones
containing GABAA receptors. The postsynaptic
clusters distributed throughout the dendritic tree contained the
3
subunit associated with
3 and, less frequently,
1, whereas
clusters containing the
1 subunit were confined to large dendrites.
We were surprised that
subunits were not colocalized with the
3 subunit, but the antibodies to
1 and
2 stained fewer synapses.
We therefore speculated that a receptor consisting of 1,
1, and
2 subunits was localized at postsynaptic active zones on large
dendrites, whereas a second type of receptor containing
3 and
3
subunits was postsynaptic at contacts distributed throughout the
dendritic tree. Both types of synapses would be present on the vitreal
aspect of the perikaryon.
It is unclear which postsynaptic structures are present in solitary DA cells after enzymatic digestion and mechanical trituration of the retina. We do not know whether the recorded GABA-evoked currents derive exclusively from extrasynaptic receptors of the cell body or from postsynaptic receptors localized on the most vitreal aspect of the perikaryon and/or along the larger dendrites. Therefore the effects of drug application could reflect the participation of different receptor populations.
The physiological data show that 1,
3, and
2 subunits are
incorporated into functional receptors at the surface of solitary DA
cells. In fact, the BZ1 pharmacology indicates that these cells express
GABAA receptors containing
1 and
2
subunits. The combination
1
2
2 is the most abundant in the
brain (McKernan and Whiting 1996
) and is considered as
the equivalent to the BZ1 subtype, but the nature of the
subunit
does not appear to be essential for determining benzodiazepine
pharmacology (Benke et al. 1994
; Hadingham et al.
1993
). We can then speculate that we are recording currents
mediated by
1
1
2 or
1
3
2 receptors. These could be localized extrasynaptically and, for
1
1
2, on large dendrites. Furthermore, in the rat cerebral cortex, 19% of the
GABAA receptors have both
1 and
3 subunits
(Li and De Blas 1997
), raising the possibility
of a receptor consisting of
1
1
3
2.
Small populations of native receptors containing both 1 and
3
have been detected in the CNS, and evidence for the combination
1
3
2 has been obtained (Duggan et al. 1991
;
McKernan et al. 1991
; Mertens et al.
1993
). However, we failed to detect co-immunolocalization of
1 and
3, but we cannnot exclude the presence of this combination in the cell body. Because
3 has been colocalized with
3 on DA cells, we can speculate that these two subunits form a
3
3
2 receptor.
3
2/
3 constitutes approximately 17% of the total GABAA receptor repertory (McKernan and
Whiting 1996
). This combination confers BZ2 benzodiazepine
pharmacology, but unfortunately no specific agonists or antagonists at
this subtype are available. Interestingly,
3
2 is found in
cholinergic and monoaminergic neurons, and thus could be involved in
the control of the release of norepinephrine and dopamine in other
regions of the brain (Fritschy et al. 1992
; Gao
et al. 1993
). Alternatively,
3 and
3 could form a
functional receptor by themselves.
GABAA receptors containing the 4 subunit were
shown to be insensitive to benzodiazepines and to be colocalized with
the
subunit (Laurie et al. 1992
; Wisden et
al. 1992
), although
4 is also present in regions of the
brain where
is not expressed. Therefore
4 could be expressed
extrasynaptically in a distinct GABA receptor population of DA cells.
Small populations of receptors containing more than one type of subunit have been identified (Quirk et al. 1994
), but
most receptors appear to contain only one type (Mossier et al.
1994
; Quirk et al. 1994
). Immunoprecipitation
studies have shown that
2 and
3 can exist in the same receptor
complex, but
1 does not co-precipitate with another
subunit
(Quirk et al. 1994
). Since we have detected both
1
and
2 subunits, this could indicate the presence of at least two
receptor populations with different
subunits.
In conclusion, we have provided evidence that in DA cells at least the
1,
3, and
2 subunits are assembled into functional GABAA receptors. In absence of pharmacological
tools specific for the other subunits, we could not obtain definitive
evidence that also
3,
4,
1, and
1 are part of functional
receptors, although this is a very likely possibility. Thus the meaning
of the subunit diversity of the GABAA receptors
of DA cells remains to be clarified. On the basis of the physiology
alone, we could not predict the complexity of the
GABAA receptor subunit composition revealed in DA
cells by RT-PCR applied to individual cells. On the other hand, it is
well known that the results of immunocytochemistry are complicated by
the vagaries of the interaction between antibody epitopes and the
chemical fixatives required for microscopy. Our results therefore
emphasize the crucial importance of combining all available technical
approaches to the study of a complex receptor in a minuscule cell population.
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ACKNOWLEDGMENTS |
---|
We thank W. Sieghart for reading the manuscript.
This work was supported by National Eye Institute Grant EY-01344.
Present address of A. Feigenspan: Dept. of Neurobiology, University of Oldenburg, 26111 Oldenburg, Germany.
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FOOTNOTES |
---|
Address for reprint requests: E. Raviola, Dept. of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, MA 02115 (E-mail: elio_raviola{at}hms.harvard.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 31 March 2000; accepted in final form 8 June 2000.
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REFERENCES |
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