Neuroscience Vol. 67, No. 3, pp.

583-593, 1995

Pergamon

0306-4522(95)00065-8

Elsevier ScienceLtd
Copyright (~'?1995IBRO
Printed in Great Britain. All rights reserved
0306-4522/95 $9.50+ 0.00

THE D E N S I T Y A N D D I S T R I B U T I O N OF SIX G A B A A
R E C E P T O R S U B U N I T S IN P R I M A R Y C U L T U R E S OF R A T
C E R E B E L L A R G R A N U L E CELLS
H. J. C A R U N C H O , * G. P U I A , * t H. MOHLER:~ and E. COSTA*
*Center for Neuropharmacology, N.S. Kline Institute for Psychiatric Research, Orangeburg, NY 10962,
U.S.A.
++Pharmakologisches lnstitut der Universit/it Zfirich, 8006 Ziirich, Switzerland
Abstract--In cultured cerebellar granule neurons (seven days/n vitro) the expression of GABAA receptor
subunits was quantified by using freeze-fracture immunocytochemical techniques with antibodies that
specifically recognize the a 1, a6, fl2-3, ?,2 and 6 subunits of the GABAA receptor. In some experiments
we have also used a less specific antibody that recognizes several c~ receptor subunits (a-total). The
specificity of these antibodies was verified in human embryonic kidney cell line no. 293 cells transfected
with complementary DNAs codifying for various GABAA receptor subunits. The most abundant labeling
in granule cells was generated by the antibody against the fl2-3 subunits (~44 colloidal gold
particles/pm2), while the specific antibodies against a l and a6 subunits show a labeling of about 16
colloidal gold particles/#m 2. The a-total antibody shows a labeling of ~ 37 gold particles//zm 2. Both the
),2 and ~ antibodies show a labeling of about 10 gold particles//~m2. In granule cells, the relative proportion
of the label density revealed with antibodies against a-total, f12 3, ~2 and 6 subunits is approximately
4:4:1 : 1. Assuming that one molecular form of the a subunit is assembled in a GABA A receptor, it can
be estimated that in granule cells about 50% of receptors include the a! subunit. A similar relative
abundance can be estimated for the a6 subunit. The proportion of GABAA receptors containing the ~,2
or ~ subunits can be estimated to be about 50% in each case.
Cerebellar granule cells express various abundances of GABA A receptor subunits which can be
estimated by freeze-fracture immunocytochemistry. Fifty to sixty percent of these subunits form small
receptor clusters, which appear to be associated with neuronal cytoskeleton proteins.

G A B A is the principal inhibitory neurotransmitter in

the m a m m a l i a n brain and acts via GABAA receptors
or via a metabotropic receptor coupled to a Gprotein (GABA~ receptor). 44 The GABAA receptor is
a hetero-oligomeric integral membrane protein, probably with a pentameric configuration. 34 So far, 16
genes encoding for GABAA receptor subunits have
been detected; these are classified into five families (~,
/L 7, 6, p) according to the subunit structural homology. 7'3 In addition, pharmacological and electrophysiological studies have demonstrated that the
subunit composition of the GABAA receptor is an
important determinant of G A B A ' s potency and the
maximal current intensity generated by G A B A gating; also the potency of allosteric modulators of
G A B A action that bind t o the GABAA receptor
ectodomain (i.e. benzodiazepines) is related to the
GABAA receptor s t r u c t u r e J 2'j6'29"38'42'48
G A B A A receptors are expressed in the plasma
membrane of granule cell somata and dendrites. 45

tPresent address: Department of Pharmacology, University

of Modena, Modena, Italy.
To whom correspondence should be addressed.
Abbreviations: BSA, bovine serum albumin; HEK-293, human embryonic kidney cell line no. 293; PBS, phosphatebuffered saline.

The dendrites receive GABAergic synapses from

Golgi type II neurons, while the somata are virtually
devoid of any synaptic contact. 35 The structure of the
G A B A A receptors present in the postsynaptic sites of
granule cell dendrites differs from that of receptors
located at extrasynaptic sites in somata. Specifically,
according to Baude et al., 2 the c~6 subunit is present
only in receptors located on postsynaptic membranes
(dendrites), while the ~ 1 subunit is present at both
synaptic (dendrites) and extrasynaptic receptors
(dendrites and soma).
Since cultured cerebellar granule cells show a high
degree of morphological homogeneity, they are a
suitable model to quantify the expression dynamics of
GABAA receptor subunits in the absence of those
multiple factors that modulate their expression in vivo
(such as cell migration necessary for the formation of
cerebellar circuits and neuronal networks). A polymerase chain reaction assay carried out with canonic
and mutated primers as internal standards has allowed the quantification of the content of m R N A s
encoding different GABAA receptor subunits that are
expressed in this neuronal culture. 5,6 These reports
have demonstrated that the m R N A s encoding for c~1,
~6, /~3 and 72 GABAA receptor subunits are
a b u n d a n t in these cells. Moreover, electrophysiological experiments performed by Bovolin et al. 6 have

583

H . J . Caruncho et al.

584

indicated t h a t different G A B A A receptor subtypes

m a y be expressed in granule cells. Finally, two recent
studies have s h o w n t h a t the s u b u n i t c o m b i n a t i o n s ct 1
fl2-3 72, ~6 fl2-3 y2, ct6 fl2-3 6 are a b u n d a n t in
cerebellar granule neurons. 8'39
In a n a t t e m p t to gain insight a b o u t the relative
a b u n d a n c e of the different G A B A k receptor subunits
expressed in granule cells, we used freeze-fracture
i m m u n o c y t o c h e m i c a l techniques t h a t permit the
labeling of the extracellular d o m a i n of integral
m e m b r a n e proteins a n d their visualization in freezefracture replicas. 37
EXPERIMENTAL PROCEDURES

Culture of cerebellar granule cells

Primary cultures of cerebellar granule ceils were prepared
from seven-day-old Sprague-Dawley (Zivic Miller, Allison
Park, PA) rat cerebella as described) Briefly, cells were
dispersed with trypsin (0.25 mg/ml, Sigmal Chem. Co., St.
Louis, MO) and plated at a density of 1 x 106 cells per plate
on 35 mm Nunc dishes coated with poly-L-lysine (10 #g/ml;
Sigma). The cultures were maintained in basal Eagle's
medium, 10% fetal bovine serum (Gibco BRL, Grand
Island, NY), 25raM KCI, 2raM glutamine (Sigma),
100 l~g/ml gentamicin (Gibco), for one week. Twenty-four
hours after plating, the incubation medium was replaced
and 1 # M cytosine arabinofuranoside was added to inhibit
replication of non-neuronal cells.
eDNA/transfection in human embryonic kidney 293 cell line
Human embryonic kidney 293 (HEK-293) cells (ATTC
CRL 1573) were grown in minimal essential medium
(Gibco) at 37C in a 6% CO2 incubator. Exponentially
growing cells were dispersed with trypsin and seeded at
2 x 105 cells per 35 mm dish in 2 ml of culture medium.
Transfection was performed with the calcium phosphate
precipitation technique. H Rat GABAA receptor subunit
cDNAs singly inserted into the expression vector pCIS2
were used to transfect the HEK-293 cells, t7 Cells were
incubated in the presence of supercoiled plasmids (3 pg of
each plasmid per 35mm dish) for 12 16h at 37C under
3% CO 2. The cells were then fixed and processed for
label-fracture.
Label-fracture
Cultures were fixed at 4C with a solution of 0.1%
glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). After 2-4 h the cells were rinsed in
buffer for several hours, labeled (see Immunocytochemical
procedures), embedded in glycerol, frozen in Freon 22
cooled by liquid nitrogen, and then fractured and replicated
in a Balzers 400D freeze-fracture unit (at -130C and
10-6 mbar). The replicas were cleaned directly in distilled
water (for at least 6 h), before being picked on to Formvarcoated grids.
Antibodies
Affinity purified polyclonal antisera raised in rabbits were
used as primary antibodies against unique amino acid
sequences corresponding to the N-terminal area of the ~t12,
7 23 and fi4 GABA A receptor subunits. We also used a mouse
monoclonal antiserum against a common epitope for the fl 2
and r 3 subunits} 8a9 For details of the characterization of
these affinity purified antibodies developed in the laboratory
of Dr Mohler, see Refs 3, 4 and 20. The ~6 antibody has
been prepared and fully characterized by the laboratory of
Dr Seeburg, this is also an affinity purified polyclonal
antiserum raised in rabbits against a unique sequence of the
~6 subunit. 2~,29Finally, we also used an antibody selective

for the family of ~t subunits prepared against a structural

peptide for the ct 1 subunit; its selectivity for the ct subunits
was documented in a previous paper. 9 Moreover, as is stated
in this report, the specificity of the primary antibodies was
tested in label-fracture experiments with HEK-293 cells
transiently transfected with cDNAs codifying for specific
GABAA receptor subunits.

Immunocytochemical procedures
Cell cultures were incubated for 30 min in RPMI medium
(Gibco) and then for another 30 rain in 1% bovine serum
albumin (BSA) in phosphate-buffered saline (PBS). Afterwards the samples were incubated with the primary antibody for 20 h at 4C. After testing the labeling background
obtained using different antibody concentrations, the final
dilutions employed in these experiments were: 1:20,000 (~ 1);
1 : 10,000 (fl~3); 1:1000 (or-total) and 1:100 (ct6, 72, 6). The
samples were washed several times in 1% BSA in PBS and
incubated for 1 h at room temperature with the corresponding secondary antibody [goat anti-rabbit immunoglobulin G
(ct 1, ct6, ct-total, y 2, 6), or goat anti-mouse irnmunoglobulin
G (f12 3)] conjugated with colloidal gold 10 nm in size. The
samples were washed several times in 1% BSA in PBS and
bi-distilled water and processed for label-fracture. Controls
were prepared by omitting the primary antibody, or replacing the primary antibody with non-immune serum.
Statistical analysis
The density of colloidal gold particles was counted in
squares of a grid that was superimposed on electron micrographs at x 50,000 final magnification, so that each square
corresponded to I pm 2. In each case the gold particles were
counted in 10 cells from at least three different experiments.
Quantitative data were obtained exclusively from unfractured membranes (see Discussion). The only experiments in
which the density of colloidal gold labeling was calculated
from data harvested from fractured membranes were those
in which we compared the labeling density of a given
receptor subunit expression in dendrites to that in soma. In
fact, it is difficult to detect colloidal gold labeling of receptor
subunits in the soma of unfractured cells, because such
labeling can be detected almost exclusively in the border
regions of unfractured cells. 9
The density of subunits labeling is reported as the
mean _+ S.E. of the number of colloidal gold particles
counted. We noted that in granule neurons GABA A receptors tended to be located in clusters. 8 To quantify the
percentage of subunits located in clusters, these were defined
by the presence of at least four gold particles in an area of
about 0.01 p m 2 which were clearly separated from contiguous gold particles. With the receptor density typical of
granule cells cultured from seven days in vitro, the probability of finding four gold particles within 0.01 #m 2 is lower
than 0.01% according to our morphometic measurements.
We have considered a density of four gold particles in
0.01 # m 2 as the threshold for cluster definition because by
studying recombinant receptors expressed in transfected
cells we failed to detect (with very few exceptions) gold
particle aggregations greater than three gold particles in
0.01 p m ~. Statistical comparison of data was performed
using the one-way ANOVA test/4
RESULTS
In label-fracture replicas from cultured n e u r o n s or
transiently transfected H E K - 2 9 3 cells we f o u n d b o t h
fractured cells (Fig. 1A) a n d u n f r a c t u r e d cells
(Fig. 1B) that remain a t t a c h e d to the replica because
a small section o f the cell was fractured o n the
intracellular face. In these p r e p a r a t i o n s the replicas
were washed only with water (in c o n t r a p o s i t i o n to

Topology of GABAA receptors in granule cells

conventional freeze-fracture, where the residual tissue
is digested with a solution of sodium hypochloride);
these unfractured cells are sandwiched between the
replica and the Formvar membrane when the samples
are picked up on to electron microscope grids. Under
the electron microscope they appear to have a gray to
black tonality, depending on the quantity of cytoskeleton underlying the membrane. In fractured
membranes the gold particles can be easily differentiated from intramembrane particles because these are
characterized by a white shadow due to the lower
amount of platinum deposition in these areas.
GABA A receptor topology in cerebellar granule cells
Label-fracture replicas of cerebellar granule cells
(seven days in vitro) stained with the or-total, ct 1, ct6,
fl2-3, ),2 and 5 primary antibodies are shown in
Figs 2 A - C and 3A-C, while Fig. 4 shows an electron
micrograph from a control preparation in which the
primary antibody was omitted; in this case the density

585

of colloidal gold particles is only 0.05 + 0.003//tm 2.

These images show that at least half of the labeling
(from 47 to 73% depending of the subunit labeled)
aggregates in clusters (see Table 1). Interestingly,
most of these receptor subunit clusters are located in
places where there is no evidence of postsynaptic
membrane specializations (see Figs 2A, 3A-C). The
total density of labeled subunits (gold particle counts
both inside and outside the clusters) reaches its
highest values in the immunostaining with either the
fl2-3 specific antibody ( ~ 4 4 gold particles/pm 2, see
Table 1) or the a-total antibody ( ~ 3 6 gold particles//~m:; Table 1). The images obtained by the
immunostaining with antibodies raised against
specific ~ subunits show that receptors containing
either ~l or ~t6 subunits are frequently expressed
( ~ 15-16 gold particles/#m2; see Table 1). The antibody against the 72 subunit labels ~nine gold particles//~m~ and the one against the 5 subunit
labels about I I gold particles//~m2 (see Table 1). The

Fig. 1. Low magnification electron micrographs of label-fracture replicas from rat cerebellar granule cells
in primary culture. (A) Granule cell fractured along the membrane external leaflet (extracellular face) of
the cell soma, from which protrudes four neurites. 18,000. Scale bar = 1 #m. Inset: high magnification
of the area surrounded by a square showing colloidal gold particles labeling the ~ 1 subunit of the GABAA
receptor, x 75,000. (B) Granule cell partially fractured along the membrane internal leaftlet (intracellular
face; lower part of the picture) and unfractured in other areas of the soma (central area of the picture).
x 10,000. Scale bar = 1 pm. Inset: high magnification of the area surrounded by a square showing the
labeling of the//2/3 subunits of the GABAA receptor, x 115,000.

Fig. 2. Electron micrographs of granule cell replicas stained with specific primary antibodies against the
a-total, ct 1 and ct6 GABA A receptor subunits. (A) Labeling with the ~t-total antibody. The mierograpb
shows the membrane (extracellular face) where the existence of colloidal gold particles clusters is evident
(stars). A diagonal shadow in the picture represents a neurite underlying the fractured membrane.
120,000. Scale bar = 200 nm. (B) Labeling with the ~t1 primary antibody. Extracellular face of a granule
cell soma with underlying unfractured processes (darker area in the left side of the micrograph). Small
clusters of colloidal gold particles are clearly evident (stars). x 85,000. Scale bar = 200 nm. (C) Labeling
with the ~6 primary antibody. Border area of the soma of an unfraetured granule cell (see, for example,
Fig. IB). As explained in more detail in the text, in unfractured cells the labeling is evident only at the
soma border. The central zone, where the nucleus is located, is too dark to differentiate any labeling (lower
area of the micrograph). Small clusters of gold particles are clearly seen (stars). x85,000. Scale
bar = 200 nm.
586

Topology of GABA A receptors in granule cells

Fig. 3. Electron micrographs of granule cell replicas stained with specific primary antibodies against fl2/3,
";2 and 6 GABA A receptor subunits. (A) Labeling with the fl2/3 primary antibody. Extracellular (e) and
intracellular (p) faces of granule cell dendrites. Clusters of gold particles are designated by stars. 85,000.
Scale bar = 200 nm. (B) Labeling with the ?,2 primary antibody. Intracellular faces of two granule cell
dendrites. A cluster of gold particles is denoted by a star. 85,000. Scale bar = 200 nm. (C) Labeling with
the ,5 primary antibody. Extracellular face of a granule cell dendrite where a cluster of gold particles is
evident (star). The micrograph also shows fractures of intracellular faces of two cells (probably glial cells)
where no labeling is evident (asterisks). x 85,000. Scale bar = 200 nm.

587

H. J. Ca~uncho et al.

588

Table 2. Quantification of GABAA receptor subunit

labeling in recombination GABAA receptors expressed in human embryonic kidney-293-tra.nsfected
cells
Antibodies
used

cDNAs
transfected

No. of gold
particles//a m 2

or-total
~t-total
~1
ctl
ct6
~6
82-3
82-3
~2
y2
6
&

ctl
ct6
ctl
~6
ct6
~I
~tl
ctl
~t6
ct6
~6
ct6

8.7 + 2.5
7.2 + 2.8
9.0___2.2*
0.034-0.005
12.1 + 2.8*
0.08__+0.002*
9.84-2.0*
0.094-0.008
3.8 ___0.05*
0.04 ___0.006
5.0 ___0.8*
0.06 +_.0.002*

f12
//2
f12
f12
82
82
82
81
82
82
82
82

~2
~,2
~2
~2
~2
~2
72
72
~2
t5
t5
72

*Data from Ducic et al. ~5

Fig. 4. Control replica in which the incubation with a

primary antibody was omitted. Note the almost complete
absence of colloidal gold particles. The black spots with a
white shadow represent intramembranous particles (with the
label-fracture technique the gold particles do not show a
white shadow), x 45,000. Scale bar = 200 nm.
difference between the labeling density of these two
subunits is not significant. The ratio between the label
with the or-total antibody (taken as an index of the
subunits of the class ~ subunits expressed in these
cells), 82-3, 72 and 6 subunits is approximately

4: 4:1 : 1. Interestingly, with the ~ 1 and ct6 specific

antibodies we found no significant differences in the
labeling density of GABAA receptor subunits in soma
or neurites. Such a comparison had to be made in
fractured membranes of soma and dendrites because
for technical reasons it is almost impossible to detect
subunit immunolabeling in the soma of unfractured
cells (see Discussion).
Comparing the number of colloidal gold particles
per cluster and the cluster density on dendritic granule cell membranes using the ~-total, ~ 1 or ~6 specific
antibodies, we found that when the labeling was
performed with the a-total or ct6 antibodies, the
subunit densities inside the clusters are significantly
higher (P < 0.05) than in experiments with the ~t 1
subunit-specific antibody (see Table 1). When the
data from the labeling with or-total and fl2-3 antibodies were compared, the number of gold particles
per cluster and the density of clusters were similar,
but the number of receptors outside clusters detected
with the 8 2 - 3 antibody was higher than that detected
with the a-total antibody.

Antibody specificity documented by label-fractured

replicas o f cells expressing recombinant GABA A receptors
To test the specificity of the antibodies prepared
against peptides specific for molecular forms of

Table 1. Quantification of the GABA A receptor subunit density in cultured rat

cerebellar granule ceils (seven days in vitro) using antibodies against specific receptor
subunits
Subunits
labeled
or-total
ctl
~t6
fl2-3
72
&

Gold/cluster*

No. of
clusters/#m21 "

6.3 + 0.50
4.5+__0.12
7.3 4- 0.82
5.8 +__0.16
5.7 ___0.27
4.4___0.13

3.7 + 0.15
1.64-0.25
1.7 4- 0.20
4.2 4- 0.43
1,1 4- 0.13
1,6+_0.22

Gold out
cluster/#m21"
12 4- 0.51
8.24- 1.0
4.5 + 0.83
19 4- 1. l
2.5 ___0.37
3.54-0.33

*Mean of the number of colloidal gold particles per cluster + S.E.M.

"~Mean of the number of colloidal gold particles/#m 2 + S.E.M.

Total
gold/#m21"
36 ___2.2
15+ 1.7
16.3 + 1.4
44 4- 2.8
9.4 ___0.95
11 4- 1.4

Topology of GABAA receptors in granule cells

GABAA receptor subunits, we immunostained HEK293 cells transfected with cDNAs codifying for various GABAA receptor subunits. Table 2 shows that
when the immunostaining is made with a specific
antibody for one of the subunits codified by one of
the cDNAs transfected, the labeling density is more
than two orders of magnitude higher than the labeling background found in cells stained with antibodies
that do not recognize any of the subunits expressed.
This is valid for the ~ l, c~6, fl2-3, 72 and ~ subunit
antibodies, while the ~t-total antibody recognizes
both the ~t1 and ct6 subunits (Table 2). In every case
around 25-50% of HEK-293 cells showed a specific

589

labeling, while the rest of the cells were not stained

(the intensity of labeling was similar to that found in
non-transfected cells). Electron micrographs illustrating examples of these experiments are shown in
Fig. 5A-D.
DISCUSSION
The present report is a study of the density
and distribution of G A B A A receptor subunits expressed in primary cultures of cells prepared from
cerebella of seven-day-old rats. The study was carried
out in label-fracture replicas stained with primary

Fig. 5. Electron micrographs of replicas of HEK-293 cells transfected with cDNAs codifying for ct6 f12
72 GABAA receptor subunits. Each of the micrographs show the immunostaining with a different
antibody: (A) ct1; (B) fl2/3; (C) ~2; (D) ~6. Note the absence of gold particles in the sample stained with
the ct1 antibody (A) that specifically recognizes a subunit that is not expressed. 50,000. Scale
bar = 200 nm.

590

H.J. Caruncho et al.

antibodies that recognize specific GABA A receptor

subunits. The counts, unless otherwise stated, were
performed on unfractured dendrite membranes. A
more detailed account of this technique and its
significance in terms of receptor topology has been
given in a previous report. 9
Assumptions and limitations

Although the antibodies used in this study

were characterized with great care in earlier reports
(see "Antibodies" subsection in Experimental Procedures), we have further characterized the specificity
of these antibodies and their suitability for the immunolabeling of GABAA receptor subunits in labelfracture replicas, by using a system of transformed
cells transfected with cDNAs codifying for various
subunits of the GABAA receptor. These experiments
have shown a lack of cross-reactivity among these
antibodies (with the exception of the or-total antibody, which was made against a peptide of the ~1
subunit but cross-reacts with the ~6 subunit). Thus,
these experiments have supported the use of immunogold labeling combined with freeze-fracturing
to quantify epitopes located in the extracellular domain of specific subunits of GABA-gated chloride
channels.
The immunogold labeling of GABAA receptor
subunits can be detected in both fractured and unfractured membranes (although in the latter case, one
can visualize the labeling only in dendrites and in the
borderline regions of the neuronal somata). An evaluation of the immunogold labeling in unfractured
membranes is important to avoid that part of the
labeling might be lost during membrane fracturing. 9
Hence, whenever possible, we have evaluated
GABAA receptor subunit density from counts made
in unfractured membranes. Of course, this was not
possible in the comparison of receptor subunit density in somata and neurites; hence, such a comparison
(based on immunogold counts in fractured membranes) could be affected by a different percentage
loss of receptors during membrane fracture. However, it is our experience that the percentage of
GABAA receptor subunit labeling lost during membrane fracturing is around 50%, independent of the
subunit labeled. 9 Moreover, when comparing the
density of subunits labeled with different antibodies,
one is making the tacit assumption that the antibody
affinity for their epitopes is similar. To attempt to
minimize the error that the possible differences in
primary antibody affinity may have on our evaluation
of subunit densities, we have used in every case
antibody concentrations that saturate the target binding sites and do not give a high background level.
The presence of gold particle clusters was not due
to an artefact due to the use of polyclonal secondary
antibodies, because receptor clusters were also detected using protein A-gold complexes (for images of
these experiments, see Ref. 9), which generally yield
a single gold particle per epitope. 27. However, for

comparative purposes, since the primary antibodies

used in the labelings were raised in different animals
(rabbit and mouse), we preferred to use goat goldconjugated secondary antibodies instead of protein
A-gold complexes, whose affinity for primary antibodies from different species varies greatly, a3
Finally, one must also consider that we are probably detecting some receptor subunits that are not
assembled into a receptor complex; therefore, we are
talking preferentially about the density of receptor
subunits and refer to receptor complexes only when
performing double-immunolabeling studies that can
give an indication of the co-localization of two
different subunits in the same receptor complex. 8
GABA~ receptor subunits expressed in granule cells

Earlier reports using in situ hybridization 24'25'36'5

and/or immunohistochemical techniques 24,2'4'45'53
have shown evidence supporting the presence of
different GABAA receptor subunits in cerebellar
granule cells. This evidence allows to infer that
structurally different GABAA receptors may be expressed in granule cell membranes. The present study
indicates that, in cerebellar granule cells, substantial
amounts of ~1, ~6, f12, f13, ~2 and 6 subunits are
expressed. Among the GABA A receptor subunits
abundantly expressed in cerebellar granule cells, the
ct l, f12, f13 and 7 2 subunits are commonly expressed
in neurons present in other brain a r e a s . 24'25'36,49 Only
the expression of the ~6 subunit appears to be
restricted to cerebeilar granule cells. 2,28,46The 6 subunit, which is abundant in granule cells, 4'4~is also well
expressed in the hippocampal dentate gyrus and
thalamus. 4 While we could not detect significant
differences between the immunolabeling density with
1 and ~6 antibodies in soma and dendrites (as stated
in the Experimental Procedures this study was carried
out only in fractured membranes), immunohistochemical studies of rat cerebellar slices have shown
that receptors containing the ct6 subunit are restricted
to postsynaptic sites located in granule cell dendrites, z
Such a discrepancy between the results of the present
study and those reported by Baude et al. 2 may be due
to differences in subunit expression between cultured
and naturally developed granule ceils. In support of
this possibility one must recall that, in vivo, the
expression of m R N A encoding for ~t6 begins only
about 10 days after birth, when the cerebeilar granule
cells migrate and contact with the Bergmann cells; 51
moreover, an involvement of granule cell dendrites
in specific synaptic circuits including Golgi type II
neurons also appears to be essential for the expression of GABA A receptors. 32 Recently, we have
used a double-immunolabeling approach to study the
preferential co-localization of two receptor subunits
in the same receptor complex. These experiments
were based on the assumption that, due to the
steric hindrance associated with the bulky antibody
complexes used in the immunolabeling procedure, the
probability of labeling a specific subunit in a receptor

Topology of GABAA receptors in granule cells

complex will decrease significantly if another subunit
present in the same receptor complex has been prelabeled. These results suggest that granule neurons
cultured for seven days include three major GABAA
receptor subtypes, c~l fl2-3 72, ~6 /32-3 72 and ~6
fl2-3 6 (see Ref. 8). Interestingly, a recent report by
Quirk et al. 39 has reached similar conclusions by using
a completely different approach (immunoprecipitation of GABA A receptors with subunit-specific antibodies). The above-mentioned study was performed
in rat cerebellar membranes and shows the existence
of GABA a receptors containing the subunit combinations a 1-72, ~6-72 and ~6-6, while indicating the
lack of receptors combining ~1-6 or ~ 1 ~ 6 . While
Quirk et al. 39 show for the whole cerebellum a
percentage of receptors containing the ~6 subunit of
about 58% and for the ~ 1 subunit about 33%, the
results of the present study suggest that for cerebellar
neurons cultured for one week in vitro around 50%
of GABA A receptors will contain the ~ 1 subunit and
an equivalent number (around 50%) will contain the
6 subunit. This discrepancy may be due not only to
differences in expression in vivo or in vitro, or that our
study is restricted to granule neurons while Quirk
et al.'s 39report refers to the whole cerebellum, but one
must also take into account the differences in the
maturational stage of the neurons. In this sense, two
recent reports 3~'52 have documented that, during
maturation in vitro of cerebellar granule neurons, the
percentage of GABA A receptors containing the a6
subunit with respect to those containing the a l
subunit increases with time in culture to a number
fairly close to the one reported by Quirk et al. 39 for
adult rats in vivo.
R e c e p t o r clusters

In granule cells, at least 50-60% of GABAA receptors are aggregated in clusters. Interestingly, in HEK293 cells transfec.ted with cDNAs encoding for
different subunits of the GABA A receptor, clustering
as defined in this report (see Experimental Procedures) is not clearly apparent? At this stage of
in vitro maturation the number of synaptic contacts

591

among different granule cells is scarce and most of the

clusters are located at extrasynaptic sites. In fact, preor postsynaptic membrane specializations easily detected in freeze-fracturing replicas of nervous tissue ~
were seldom found. While a clustering of GABA A
receptors at extrasynaptic sites has been documented
in other reports, 9'47most of the other members of the
ligand-gated ion channel family appear to form clusters almost exclusively at postsynaptic sites (for the
glycine receptor see Refs 22 and 23; for the acetylcholine receptor see Ref. 26; for the ~-amino-3hydroxy-5-methyl-4-isoxazolepropionatereceptor see
Ref. 13).
CONCLUSIONS

The present study documents quantitative differences in the expression of different GABAA receptor
subunits in primary cultures of rat cerebellar granule
cells. Structural differences of various GABAA receptor subtypes are important determinants of GABA
potency and of the maximal current intensity generated by GABA gating of chloride channels/6 Since
the positive and negative allosteric modulation of
GABA action by benzodiazepines depends on the
properties of GABA's action on chloride channels,
the pharmacological profile of various benzodiazepines also relates to the structure of GABAA
receptors. 38'43'48Thus, the understanding of the subunit assembly of various GABAA receptor subtypes
expressed in granule cells in culture may allow us to
begin to study whether there is a dynamic regulation
of receptor structure that changes with the topology
and physiology of neuronal circuits. Moreover, one
can verify whether subunit assembly can be affected
during tolerance to the therapeutic action of benzodiazepines.
Acknowledgements--The authors wish to thank Dr E. Slo-

bodyansky (Georgetown University, Washington, DC) and

Dr P. Seeburg (University of Heidelberg, Germany), who
kindly provided the c~-total and c~6 subunit antibodies,
respectively. This work was supported in part by ROI
MH49486-O1A3.

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