1Department of Neurobiology, Institute of Life Sciences, Hebrew University, Jerusalem 91904, Israel; and 2Institute of Pharmacology and Toxicology, University of Zurich, CH-8057 Zurich, Switzerland
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ABSTRACT |
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Devor, Anna,
Jean-Marc Fritschy, and
Yosef Yarom.
Spatial Distribution and Subunit Composition of GABAA
Receptors in the Inferior Olivary Nucleus.
J. Neurophysiol. 85: 1686-1696, 2001.
GABAergic inhibitory
feedback from the cerebellum onto the inferior olivary (IO) nucleus
plays an important role in olivo-cerebellar function. In this study we
characterized the physiology, subunit composition, and spatial
distribution of -aminobutyric acid-A (GABAA) receptors
in the IO nucleus. Using brain stem slices, we identified two types of
IO neuron response to local pressure application of GABA, depending on
the site of application: a slow desensitizing response at the soma and
a fast desensitizing response at the dendrites. The dendritic response
had a more negative reversal potential than did the somatic response,
which confirmed their spatial origin. Both responses showed voltage
dependence characterized by an abrupt decrease in conductance at
negative potentials. Interestingly, this change in conductance occurred
in the range of potentials wherein subthreshold membrane potential
oscillations usually occur in IO neurons. Immunostaining IO sections
with antibodies for GABAA receptor subunits
1,
2,
3,
5,
2/3, and
2 and against the postsynaptic anchoring
protein gephyrin complemented the electrophysiological observation by
showing a differential distribution of GABAA receptor subtypes in IO neurons. A receptor complex containing
2
2/3
2 subunits is clustered with gephyrin at presumptive synaptic sites, predominantly on distal dendrites. In addition, diffuse
3,
2/3, and
2 subunit staining on somata and in the neuropil presumably represents extrasynaptic receptors. Combining electrophysiology with
immunocytochemistry, we concluded that
2
2/3
2 synaptic receptors generated the fast desensitizing (dendritic) response at
synaptic sites whereas the slow desensitizing (somatic) response was
generated by extrasynaptic
3
2/3
2 receptors.
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INTRODUCTION |
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The inferior olivary (IO)
nucleus is highly innervated by GABAergic projections derived from
various sources, including deep cerebellar nuclei (De Zeeuw et
al. 1989), raphe nuclei (Walberg and Dietrichs
1982
), nucleus prepositus hypoglossi, dorsal column nuclei, and
reticular formations (Nelson and Mugnaini 1989
). Most of
the projections from the deep cerebellar nuclei, the largest GABAergic
input to the olive (De Zeeuw et al. 1989
), terminate in
glomerular structures. Each olivary glomerulus usually contains 5-8
spines, derived mostly from distal dendrites of different IO neurons,
coupled by gap junctions and interdigitated with both excitatory and
inhibitory synapses of extrinsic origin (De Zeeuw et al.
1990a
).
Coupling by gap junctions has a central role in the generation of
subthreshold oscillations, a striking feature of IO neurons (Bleasel and Pettigrew 1992; Lampl and Yarom
1997
; Llinas and Yarom 1986
; Manor et al.
1997
; Yarom 1991
). Subthreshold oscillatory activity of assemblies of olivary neurons was hypothesized to play a
key role in olivo-cerebellar physiology by controlling the synchrony
and rhythmicity of complex spikes in the cerebellar cortex (Lang
et al. 1999
). The coexistence of inhibitory terminals and gap
junctions in IO glomeruli suggests that GABAergic inputs may control
electrotonic coupling among IO neurons (Llinas 1974
) and
may thereby control subthreshold oscillatory activity. Consistent with
this possibility, physiological experiments in vivo have demonstrated
that activation of
-aminobutyric acid-A
(GABAA) receptors modulates the degree of
synchronization of complex spike activity in Purkinje cells
(Lang et al. 1996
; Llinas and Sasaki 1989
).
The variability in the subunit composition of
GABAA receptors is well documented. Nineteen
different mammalian GABAA receptor subunit genes
have been described: (1-6),
(1-4),
(1-3),
,
,
and
(1-3). A number of studies on recombinant receptors have shown that
the biophysical and pharmacological properties of
GABAA receptors depend on their subunit
composition (Mehta and Ticku 1999
; Whiting et al.
1999
). Moreover, synaptic and extrasynaptic receptors were
shown to differ in subunit composition (Brickley et al.
1999
; Nusser et al. 1998
).
In view of the importance of GABA transmission in olivo-cerebellar function, and of the variety of GABAA receptor subtypes, each with its own distinct kinetics and composition, it is essential to identify the type of receptors that mediate extrinsic inhibitory control of the IO nucleus. Both the location of the receptors and their functional characteristics, particularly their voltage dependence and kinetics, are likely to determine and limit the range of possible regulatory functions.
In the present study, we characterized the physiological properties of
GABAA receptors in the IO nucleus and analyzed
their spatial distribution and subunit composition. Postsynaptic
receptors were identified by their colocalization with the clustering
protein gephyrin (Essrich et al. 1998; Kneussel
et al. 1999
; Sassoe-Pognetto and Fritschy 2000
;
Sassoe-Pognetto et al. 2000
). Because gephyrin labels
GABAergic and glycinergic synapses, and because glycinergic synapses
were not found in the inferior olivary nucleus (De Zeeuw et al.
1994
), gephyrin was used in our study as a specific marker for
GABAergic postsynaptic sites. The strength of this study lies in the
comparison of electrophysiological with immunocytochemical findings,
which enabled us to substantiate our conclusions.
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Methods |
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Slice preparation
Slices 300-µm thick were prepared from the brain stems of Wistar-derived Sabra strain rats (18-25 days old). The animals were anesthetized intraperitonally with 60 mg/Kg pentobarbitone and perfused through the heart with 100 ml cold (0-1°C) physiological solution (Table 1, solution A). After decapitation, the brain stem was quickly removed and sliced (752 M vibroslice, Campden Instruments) in cold sucrose solution (Table 1, solution C). The slices were incubated in the sucrose solution at room temperature for 60 min. During this time, the sucrose solution was slowly replaced by solution A and the brain stem slices were then kept at room temperature until they were transferred into the recording chamber. Use of the sucrose solution was critical for increasing the viability of IO neurons.
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Recordings
The recording chamber was mounted on an upright microscope stage
(Zeiss Axioskop), was maintained at a constant temperature of 35°C by
a temperature control unit, and was continuously perfused with solution
B (Table 1). Whole-cell patch recordings were performed under visual
control using infrared differential interference contrast optics (DIC).
Recordings were made throughout the IO nucleus from neurons whose cell
bodies, which lie below the surface of the slice, were visually
identified. The pipettes were filled with the intracellular solution
(Table 1). In some experiments, chloride concentration in the pipette
was increased to 20 mM by replacing K-gluconate with KCl.
Bicuculline (Sigma) was added to the recording solution (solution A) to
reach a final concentration of 100 µM. In some experiments
bicuculline was applied by pressure pulses through a micropipette (tip
diameter ~2 µm) filled with 100 µM bicuculline in solution A
locally over the cell body. GABA was applied by pressure pulses through
a micropipette filled with 1 mM GABA in solution A, either in the
immediate vicinity of the cell body or in the dendritic area (15 µm
from the cell body). Because the majority of IO neurons have dendrites
that curl back toward the cell body, the most electrically distal
dendrites may not necessarily be the most spatially remote ones.
The patch pipettes were pulled on a Narishige pp-83 puller and had a DC
resistance of 10-15 M. The seal between the electrode tip and the
cell membrane was greater than 1 G
. Cell capacitance and series
resistance were not compensated. Recordings were made with an Axoclamp
2B (Axon Instruments) in continuous voltage clamp mode. Electrical
signals were stored on videocassette (Neurocorder DR-484) for off-line
analysis by the LabVIEW data acquisition and programming system
(National Instruments). All results are expressed as means ± SD.
Immunohistochemistry
Eighteen 21-30 day old Wistar rats were used for the
immunolabeling of GABAA receptor subunits 1,
2,
3,
5,
2 (Fritschy and Mohler 1995
), and
2/3 (bd17, Chemicon, Temecula, CA). An antibody against the synaptic
anchoring protein gephyrin (mAb7a, Connex, Martinsried, Germany) was
used as a marker for GABAergic synapses (Sassoe-Pognetto et al.
2000
). All of the animals were anesthetized intraperitonally
with pentobarbitone and were perfused with the physiological solution
(Table 1, solution A).
In seven animals in which GABAA receptor subunits
and gephyrin were codetected by double immunofluorescence staining, the following procedure was used: the brains were removed and frozen in dry
ice immediately after perfusion and were stored at 80°C for at
least three days. Cryostat sections (12 µm) were defrosted for
30 s at room temperature and were then fixed with 0.5%
paraformaldehyde in a 150 mM phosphate buffer (PBS, pH 7.4) during
30 s of microwave irradiation (Fritschy et al.
1998b
). The cryostat sections were rinsed with PBS and
were then incubated overnight at 4°C with primary antibodies for
GABAA receptor subunits and gephyrin in PBS
containing 4% normal goat serum. The following antibody dilutions were
used:
1, 1:40,000;
2, 1:4,000;
3, 1:6,000;
5, 1:3,000;
2/3, 1:20,000;
2, 1:2,000; gephyrin, 1:300. The sections were again rinsed with PBS and were incubated for 30 min in a mixture of the
corresponding secondary antibodies conjugated with Alexa 488 (Molecular
Probes, Eugene, OR), Cy3, or Cy5 (Jackson ImmunoResearch Laboratories,
West Grove, PA). After incubation, the sections were washed and
coverslipped with buffered glycerol.
In the remaining 11 animals, IO neurons were triple-labeled with neurobiotin, antibodies to GABAA receptor subunits, and antibodies to gephyrin. In this group, the brain stem was dissected after perfusion and was kept in cold (0-1°C) oxygenated sucrose solution (Table 1, solution C). The IO nucleus was pressure injected through a glass micropipette with 4.5% neurobiotin in sucrose solution. Within minutes, 300-µm brain slices were cut on a vibratome, fixed for 4 min with 4% paraformaldehyde in 150 mM phosphate buffer (pH 7.4), rinsed with PBS, and re-cut on a cryostat in 25-µm sections. The thin sections were then processed for immunofluorescence staining as described in the previous paragraph. Streptavidin conjugated with Cy2 (Vector Laboratories, Burlingame, CA) was used as a marker for neurobiotin.
In control experiments, preadsorption of primary antibodies against GABAA receptor subunits with 1-3 µg/ml of the corresponding peptide antigen resulted in a complete loss of specific staining (not shown).
Both morphological procedures gave identical staining of the
GABAA receptor subunits. Therefore, the results
of labeling produced by both techniques were pooled. Fluorescent images
were captured with a confocal laser-scanning microscope (Leica, TCS SP)
at a magnification of 60-75 nm per pixel by using simultaneous
acquisition of double- and triple-labeled images in separate channels.
Care was taken to sample images over the entire dynamic range of the photodetectors and to individually adjust the intensity of the excitation lines (488, 568, and 640 nm) to avoid cross-excitation of
the fluorochromes. Images were processed with Imaris image analysis
software (Bitplane, Zurich, Switzerland). Colocalization of
GABAA receptor subunits and gephyrin in
presumptive postsynaptic clusters was assessed in raw images as
described previously (Sassoe-Pognetto et al. 2000) by
using a "colocalization" algorithm that selects and marks
double-labeled pixels above a user-defined threshold. Clusters were
defined based on size (larger than 0.04 µm2,
which corresponds to three adjacent pixels at the magnification used)
and relative staining intensity (>30%). Colocalization between different GABAA receptor subunits was assessed
using the same approach but without the size criterion of labeled
structures, thus including diffuse staining of neuronal somata and the
neuropil. The results are derived from a total of 10 measurements in
three sections from three different animals and are expressed as
means ± SD.
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RESULTS |
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Two types of response of IO neurons to local GABA application
The response to local application of GABA was analyzed in 34 olivary neurons recorded under voltage clamp conditions. At resting potential (52.1 ± 2.6 mV), 20-ms pulses of GABA, applied near the cell body, elicited an outward current (Fig.
1A). The response appeared
after a delay of 6.1 ± 5.1 ms (n = 9, measured
from application onset) and reached an averaged peak amplitude of
89.3 ± 45.6 pA with a rise time of 38.1 ± 13.1 ms. These
parameters depended, to different degrees, on the distance between the
GABA-filled pipette and the cell body.
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An increase in the injection pressure (using 20 ms application) was
followed by an increase in the amplitude without an effect on the rise
time of the response (Fig. 1A), which suggests that larger
numbers of GABA receptors were activated. Increasing the duration of
the application increased both the amplitude and the duration of the
response (Fig. 1B). A lack of increase in the amplitude for
durations longer than 100 ms indicates either that 1 mM GABA saturated
the GABAA receptors present or that the dynamics of receptor activation and receptor desensitization reached an equilibrium steady state. Because prior reports have shown that 1 mM
GABA is not enough to saturate the receptors (Hutcheon et al.
2000; Jones and Westbrook 1995
;
Maconochie et al. 1994
), we conclude that an equilibrium
was reached in our experiments because of the slow application of GABA.
When GABA was applied for more than 1 s, the amplitude of the
response started to decrease before the end of the stimulus (Fig.
1B). An exponential fit to the decay showed a time constant of 0.98 ± 0.11 s (n = 3). This decay could
result either from a change in the driving force for
Cl ions or from desensitization of the
receptors. To rule out the former possibility, we analyzed the time
course of conductance change by introducing
15-mV pulses before and
during GABA application (Fig. 1C). The response to voltage
pulses alone was subtracted from the response to voltage pulses during
GABA application. The amplitude of the current induced by each pulse,
which tracks the change in conductance, was plotted as a function of
time. As shown in Fig. 1C, the time course of conductance
change followed the GABA-induced current almost exactly. Therefore, the
decrease in response in the presence of GABA must be explained by slow
desensitization of the receptors. However, the desensitization was
incomplete. As follows from Fig. 1B, when GABA was applied
for 3 s and longer, a residual nondesensitized current was clearly
observed. Consistent with this, when 1 mM GABA was added to the
recording chamber a permanent small increase in conductance was
measured (not shown).
When GABA was applied in the dendritic area, the response (to 20-ms pulses) had lower amplitude (19.0 ± 8.4 pA, n = 4), faster rise time (19.8 ± 1.7 ms), and shorter duration (Fig. 1D) than was observed during somatic application. An increase in stimulus duration failed to prolong the dendritic response, which decayed rapidly with a time constant of 0.11 ± 0.03 s. Instead, a prolonged stimulus activated a slow and delayed outward current that resembled the somatic response. A further increase in the duration of the stimulus increased the amplitude and duration of the second response component without affecting the first. Furthermore, analyzing the time course of conductance change (see the previous paragraph) during the dendritic response revealed that, in this case, it also followed the time course of GABA-induced current. The second component resembled the response to somatic application except for its much longer delay (150 ms in Fig. 1D). Both components were reversibly blocked by 100 µM bicuculline, which indicates that both were mediated by GABAA receptors (Fig. 1E, n = 3). Moreover, when bicuculline was applied locally over the cell body, only the second component was blocked (Fig. 1F).
To further characterize the response of IO neurons to somatic and dendritic GABA application, we used trains of 10 consecutive pulses (20 ms duration, 200 ms interpulse interval) (Fig. 2). This paradigm further distinguished between the two types of responses. Near the soma (Fig. 2A), responses summated to an almost steady-state level. An increase of injection pressure increased this steady state level, which was followed by a moderate decrease, as was observed with a single prolonged injection (Fig. 1). In contrast, at dendritic locations, only the first GABA pulse elicited a response, which was almost unaffected by an increase in pressure (Fig. 2B). The inability to follow a train of pulses indicates that desensitization at the dendritic location occurred much faster than at the somatic location.
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The spatial distribution of the two types of response was further
confirmed by measuring reversal potentials. To elicit both responses,
we used prolonged (up to 500 ms) injections in the dendritic area of IO
neurons and measured GABA-induced current at different membrane
potentials. As shown by the individual traces in Fig.
3A, the compound outward
current at 50 mV was almost a mirror image of the compound inward
current observed at
100 mV. Both components were clearly
distinguishable, which indicates the activation both of somatic and of
dendritic receptors. However, the biphasic appearance of the response
at
68 mV indicated that the late (somatic) component reversed at a
less negative potential.
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The plot in Fig. 3B shows the currents, measured at the
times denoted by the dashed lines, as a function of the holding
potential. Two points are of interest. First, as expected from cable
theory (Rall 1989), the reversal potential of the
somatic response was 13 mV more depolarized than that of the dendritic
response (
63 and
76 mV, respectively). The average reversal
potentials of the somatic and dendritic responses were
58.1 ± 4.2 mV and
67.9 ± 6.0 mV, respectively (n = 8).
In one cell, we further confirmed this difference by independently
measuring the reversal potentials of the somatic and dendritic
responses using short (20 ms) GABA pulses applied locally at the soma
or at a distance of 40 µm from the soma. The results fell well within
the range that was calculated for the compound response (
58 mV for
somatic application and
70 mV for dendritic application).
Second, both responses, somatic and dendritic, showed voltage
dependence with an abrupt decrease in conductance at a membrane potential of approximately 65 mV (n = 6). We fitted
the experimental data with two linear segments corresponding to two
conductance states and defined a break point as the intersection of
these two linear segments (Fig. 3B, arrows). The ratio
between the two conductance states varied in different cells (from 6.08 to 19.35, n = 6). This apparent voltage dependence may
be caused by an inadequate voltage clamp, by a nonsymmetric flow of
Cl
caused by a concentration difference between
inside and outside (Goldman rectification), or by voltage dependence of
the receptor channel. To rule out the first possibility, we linearized
the membrane by adding Cs (5 mM), harmaline (0.1 mg/ml),
tetraethylammonium (10 mM), and 4AP (0.1 mM) and by replacing
Ca2+ with Co2+ in the bath
solution (Yarom and Llinas 1987
). The linearity of the
membrane was examined by 300-ms, 5-mV voltage steps of both polarities
and the resultant currents were used to calculate the membrane
conductance at each level of membrane potential. Indeed, as shown in
Fig. 3D, the slope membrane conductance under these conditions was about 2 nS throughout the entire range of holding voltages. The GABA-induced current, on the other hand, still showed a
pronounced rectification. We ruled out the second possibility by
increasing the Cl
concentration in the
intracellular pipette solution from 4 to 20 mM. The results are shown
in Fig. 4. As expected, the reversal potentials of both responses shifted to lesser negative values (
52.3 ± 8.5 mV for the somatic components and
56.0 ± 7.6 mV for the dendritic components, n = 4). However,
the value of the break point was unaffected by the change in
Cl
concentration (Fig. 4B).
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We therefore concluded that GABA-induced current in olivary neurons is voltage dependent. This finding is especially interesting because the abrupt decrease in conductance occurred in the range of potentials wherein subthreshold oscillations usually occur in IO neurons.
Nonhomogeneous distribution of GABAA receptor subunits in IO neurons
To further support the conclusion that the slow- and fast-
desensitizing responses to GABA application are separated spatially, we
studied the subunit distribution of GABAA
receptors in different subcellular compartments of IO neurons.
Immunostaining of olivary sections was performed for the 1,
2,
3,
5,
2/3, and
2 GABAA receptor
subunits. The
1 and
5 subunits were not detected in IO neurons
although they were intensely stained in other brain stem neurons in the
same section. The
3 subunit staining was weak and diffuse and was
most apparent on IO somata. Finally, the
2,
2, and
2/3
subunits exhibited prominent puncta, irregularly distributed in the
neuropil of the IO, as well as some diffuse staining. In comparison,
gephyrin staining revealed only brightly stained puncta with a
distribution similar to that of the
2,
2/3, and
2 subunits.
Immunolabeling of the 2 subunit in neurobiotin-stained IO neurons
revealed that the strong punctate staining was concentrated around
distal dendrites; cell bodies and proximal dendrites were virtually
devoid of punctate
2 subunit staining (Fig.
5). In previous studies (Fritschy et al.
1998b
; Giustetto et al. 1998
; Sassoe-Pognetto et al. 2000
), punctate labeling of
GABAA receptor subunits was attributed to
GABAA receptors aggregated postsynaptically with
gephyrin. Therefore, to determine the synaptic specificity of the
2
subunit puncta, sections were triple-labeled with gephyrin (Fig.
6). Similar to the
2 subunit,
gephyrin-positive clusters were concentrated along dendrites and only a
few were apposed to neurobiotin-positive IO somata. Gephyrin and the
2 subunit were extensively colocalized (Fig. 6, A and
A', and Table 2) in both
double- and triple-labeled sections, which indicates the almost
exclusive localization of
2 subunits at synaptic sites.
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To further unravel the subunit composition of olivary GABA receptors,
sections were processed for codetection of the 2 and
2/3 subunits
along with neurobiotin. The results showed that the
2 subunit was
highly colocalized with
2/3 (Table 2 and Fig. 6B), which
indicates that a complex of
2
2/3 subunits clustered with gephyrin
at presumptive synaptic sites. Immunoreactivity of the
2/3 subunit
was more extensive than was the
2 subunit labeling and was, in
addition, diffusely distributed throughout the neuropil of the IO and
on neuronal somata.
Like the 2/3 subunit, staining for the
2 subunit exhibited both
intensely labeled clusters on dendrites of IO neurons and diffuse
staining of the neuropil and of individual somata (Fig. 7A). A majority of the
2
subunit-positive clusters was double-labeled with gephyrin (Fig. 7,
B-D), but the incidence of colocalization was
lower than that for the
2 subunit (Table 2). Moreover, clusters of
2 and
2/3 subunits were largely colocalized throughout the IO
nucleus (Table 2).
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Finally, double immunofluorescence staining of the IO for the 3
subunit (Fig. 8, left) and
gephyrin (Fig. 8, right) revealed weak and diffuse
3
subunit immunoreactivity on cell somata and proximal dendrites, with an
occasional cluster colocalized with gephyrin (Fig. 8, arrowheads).
Together these results indicate that a GABAA
receptor subtype comprising the
2
2/3
2 subunits was clustered
with gephyrin on distal dendrites of IO neurons. This receptor subtype
is likely to correspond to the fast desensitizing receptors that were
identified electrophysiologically. A second major subtype that contains
the subunit complex
3
2/3
2 is distributed extrasynaptically on
IO somata, presumably corresponding to the slow desensitizing receptor
population.
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DISCUSSION |
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In this work we characterized two types of responses of olivary
neurons to local application of GABA: a slow desensitizing response at
the soma and a fast desensitizing response at the dendrites. Both
responses were mediated by GABAA receptors and both were reversibly blocked by bicuculline. These two types of responses differed in their kinetics as well as in their spatial distribution. These observations were complemented by
immunocytochemistry, which showed that a complex containing
2
2/3
2 subunits was clustered with gephyrin at presumptive
synaptic sites, predominantly on distal dendrites, whereas a complex
containing
3
2/3
2 was distributed extrasynaptically,
predominantly on IO somata.
During prolonged stimulation at dendritic locations, first the dendritic and then the somatic responses were evoked in sequential order with a noticeable delay between them. This sequential activation strongly suggests that two spatially separated and kinetically different populations of GABA receptors coexist in olivary neurons. The spatial segregation of the two responses was further confirmed by the somatic application of bicuculline, which specifically and reversibly blocked the somatic response. We conclude that the activation of dendritic receptors gave rise to the first, fast desensitizing response component whereas somatic receptors generated the delayed, slow desensitizing response.
Both the fast and the slow desensitizing response components showed
clear voltage dependence. This conclusion was reached after we
eliminated the possibilities that nonlinear membrane or Goldman-like
rectification generated the apparent voltage dependence. The abrupt
decrease in conductance, which occurred close to the membrane resting
potential, reflected a low conductance state at hyperpolarized
potentials and a high conductance state at depolarized potentials. This
arrangement is expected to result in a much weaker inhibitory effect at
hyperpolarized than at depolarized membrane potentials. Although
voltage dependence of GABA channels has been described (Kerrison
and Freschi 1992; Weiss 1988
), an approximately 10-fold change in conductance over the 15-mV range of potentials described here has been demonstrated regarding glycine receptors only
(Faber and Korn 1987
). Moreover, the switch between the
two conductance states took place in the voltage range wherein
subthreshold oscillations usually occur in olivary neurons
(Lampl and Yarom 1997
). Therefore, one is tempted to
speculate that voltage dependence serves as an autoregulatory mechanism
that ensures a constant effect of transient inhibitory inputs. That is,
the increased excitability during the depolarizing phases of the
voltage oscillations is compensated for by an increase in the efficacy
of GABA inhibition caused by its voltage dependence.
The significant delay between the dendritic and somatic
responses, as well as their distinct kinetics, implies spatial
separation of the two types of receptors. This implication was
complemented by the morphological part of this study, which
demonstrated the existence of GABAA receptors
composed of 2
2/3
2 subunits located mainly on distal dendrites
as well as the existence of receptors composed of
3
2/3
2
subunits on the cell bodies. The
2
2/3
2 subunit composition is
likely to form a predominant receptor subtype because these subunits
appeared to be extensively colocalized with each other and with
gephyrin within individual clusters. There was considerable
variability in staining intensity among individual clusters (Fig.
6A'), which suggests that the number and/or density of
GABAA receptors per synaptic site is variable. In
contrast to this receptor subtype, the diffuse staining not colocalized
with gephyrin that was observed for the
3,
2/3, and
2 subunits
on IO somata and in the neuropil might correspond to one or several
subtypes of extrasynaptic receptors. Indeed, colocalization of the
diffuse
2 and
2/3 subunit staining was only partial (Table 2).
Unfortunately, the faint
3 subunit immunoreactivity precluded a detailed analysis of its codistribution with the
2/3 subunits.
If indeed 2
2/3
2 and
3
2/3
2 GABAA
receptor compositions generate the dendritic and somatic responses,
respectively, they should correspond to the fast and slow desensitizing
responses that were observed electrophysiologically. A number of
previous studies showed that the kinetics of native as well as
recombinant ligand-gated ion channels depend on subunit composition
(Brussaard et al. 1997
, 1999
; Gingrich et
al. 1995
; Hutcheon et al. 2000
; Liu and
Cull-Candy 2000
; Verdoorn 1994
). One of the most
consistent observations is that the presence of the
3 subunit slows
the activation, deactivation, and desensitization processes. Although the slow GABA application used in our study prevented us from fully
characterizing the kinetics of desensitization, our results regarding
the involvement of the
subunit in receptor kinetics are in
agreement with previous reports (Gingrich et al. 1995
; Hutcheon et al. 2000
; Verdoorn 1994
).
Additional or alternative determinants may contribute to receptor
kinetics, such as specific interactions with the proteins of the
postsynaptic apparatus (Rosenmood and Westbrook 1993) or differences in posttranslational regulation such as receptor
phosphorylation levels (Jones and Westbrook 1997
;
Moss et al. 1992
; Wan et al. 1997
).
Therefore, synaptic and extrasynaptic receptors are likely to have
distinct interactions with cellular cytoskeletons and to be
differentially exposed to cytosolic proteins. Receptor location may
also contribute to the difference in kinetics that was observed in our
study. Interestingly, CA1 hippocampal pyramidal neurons were
demonstrated to have fast synaptic receptors and slow extrasynaptic receptors (Banks and Pearce 2000
).
Despite the different subunit compositions of the dendritic and somatic
receptors, both somatic and dendritic responses showed rectification
around the resting potential. Subunit composition per se is unlikely to
contribute to this behavior because voltage-dependent rectification has
not been reported for recombinant 2 and
3 receptors.
The observation of clusters immunoreactive for the 2 and
2/3
subunits but lacking gephyrin was unexpected. Previous studies reported
consistent colocalization of
2 subunit immunoreactivity with
gephyrin (Essrich et al. 1998
; Sassoe-Pognetto et
al. 2000
). Moreover, gephyrin knockout mice were shown to lack
2 clustering (Kneussel et al. 1999
). Therefore, to
explain gephyrin-independent
2 and
2/3 clusters, one has to
assume either local aggregation of extrasynaptic receptors or the
presence of gephyrin isoforms not recognized by the antibody used in
our study (Bedford et al. 1999
). Either way, most
gephyrin-independent clusters of
2 and
2/3 subunits are not
associated with the
2 subunit, which suggests that other subunits,
such as
4 (Chang et al. 1995
), are expressed in the
IO, which adds to the heterogeneity of GABAA
receptors in this nucleus.
Assuming that antibody 7a labels gephyrin in a majority of postsynaptic
inhibitory synapses, the soma of olivary neurons must be largely devoid
of inhibition. This conclusion is consistent with the electron
microscopic studies of De Zeeuw et al. (1989) who
demonstrated that most of the inhibitory projections to the IO nucleus
terminate on olivary glomeruli on distal dendrites and that only few of
them terminate on cell bodies. Moreover, the majority of the glomerular
inhibitory synapses was derived from the deep nuclei of the cerebellum
whereas the somatic inhibitory synapses had a noncerebellar
origin. The lack of strong synaptic inhibition on the soma of
olivary neurons contradicts the classical view of neuronal integration
wherein the most effective target of inhibition is the cell soma.
However, it was shown that olivary neurons are endowed with axonal
spines that form gap junctions and receive GABAergic inhibition
(De Zeeuw et al. 1990b
).
Close apposition of GABAergic synapses to gap junctions suggests that
GABA released in the glomeruli may disrupt (uncouple) electrotonic
connections and therefore alter subthreshold activity in the olivary
network. The fast desensitization of the dendritic receptors found in
our study implies that if GABA induces functional uncoupling it would
be restricted to less than one cycle of oscillatory activity.
Alternatively, brief interruption of electrotonic coupling at certain
phases of the oscillatory cycle may be sufficient to induce a
long-lasting blockade of oscillatory activity. A similar phenomenon was
demonstrated in cardiac myocytes where a brief electric stimulus,
appropriately timed, may permanently stop the oscillations
(Chialvo and Jalife 1987; Guevara et al.
1981
).
The dendritic GABAergic responses of olivary neurons described in this study, as well as the heterogeneity of the subunit composition of GABAA receptors, adds a new level of complexity to the functional organization of the olivo-cerebellar circuit. Although the precise relationship between the inhibitory input and the subthreshold oscillatory activity of olivary neurons remains to be elucidated, the strategic location of inhibition on distal dendrites strongly suggests that it has a prominent role in the modulation of network interactions among IO neurons.
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ACKNOWLEDGMENTS |
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This study was supported by the US-Israel Binational Science Foundation and by the European Commission.
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FOOTNOTES |
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Address for reprint requests: Y. Yarom (E-mail: yarom{at}vms.huji.ac.il).
Received 27 September 2000; accepted in final form 18 December 2000.
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REFERENCES |
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