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Volume 17, Number 4,
Issue of February 15, 1997
pp. 1350-1362
Copyright ©1997 Society for Neuroscience
Ligand-Gated Ion Channel Subunit Partnerships: GABAA
Receptor  6 Subunit Gene Inactivation Inhibits  Subunit Expression
A. Jones1, a,
E. R. Korpi2, a,
R. M. McKernan3, a,
R. Pelz4, a,
Z. Nusser5,
R. Mäkelä2,
J. R. Mellor1,
S. Pollard6,
S. Bahn1,
F. A. Stephenson6,
A. D. Randall1,
W. Sieghart4,
P. Somogyi5,
A. J. H. Smith1, and
W. Wisden1
1 Medical Research Council Laboratory of Molecular
Biology, Medical Research Council Centre, Cambridge CB2 2QH, United
Kingdom, 2 Department of Pharmacology and Clinical
Pharmacology, University of Turku, Fin-20014 Turku, Finland, Department
of Alcohol Research, National Public Health Institute, Fin-00180
Helsinki, Finland, 3 Merck Sharp & Dohme Research
Laboratories, Neuroscience Research Centre, Harlow, CM20 2QR, United
Kingdom, 4 University Clinic for Psychiatry, Department of
Biochemical Psychiatry, 1090 Vienna, Austria, 5 Medical
Research Council Anatomical Neuropharmacology Unit, Oxford University
Department of Pharmacology, Oxford, OX1 3TH, United Kingdom, and
6 School of Pharmacy, University of London, Brunswick
Square, London, WC1N 1AX, United Kingdom
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Cerebellar granule cells express six GABAA receptor
subunits abundantly ( 1, 6,
 2, 3,  2, and  ) and
assemble various pentameric receptor subtypes with unknown subunit
compositions; however, the rules guiding receptor subunit assembly are
unclear. Here, removal of intact 6 protein from
cerebellar granule cells allowed perturbations in other subunit levels
to be studied. Exon 8 of the mouse 6 subunit gene was
disrupted by homologous recombination. In 6  /
granule cells, the subunit was selectively degraded as seen by
immunoprecipitation, immunocytochemistry, and immunoblot analysis with
subunit-specific antibodies. The subunit mRNA was present at
wild-type levels in the mutant granule cells, indicating a
post-translational loss of the subunit. These results provide genetic evidence for a specific association between the
6 and subunits. Because in 6 /
neurons the remaining 1, 2/3, and
2 subunits cannot rescue the subunit, certain
potential subunit combinations may not be found in wild-type cells.
Key words:
GABAA receptor;
6 subunit;
granule cell;
cerebellum;
homologous recombination;
gene targeting;
transgenic mice;
knockout mice;
ligand-gated ion channel;
subunit
sorting;
subunit assembly;
internal ribosome entry site;
dicistronic
mRNA;
muscimol;
SR95531;
Ro 15-4513;
flunitrazepam
INTRODUCTION
In vertebrate brains, GABAA receptors
are the principal mediators of inhibitory synaptic transmission. They
are agonist-gated anion channels formed of pentameric assemblies of
subunits arranged around an aqueous pore (Seeburg et al., 1990 ;
Sieghart, 1995; Stephenson, 1995; Tyndale et al., 1995; McKernan and
Whiting, 1996). The subunit genes (1-6,
1-3, 1-3, and ) are differentially
transcribed, and the polypeptides are assembled in many possible
combinations depending on cell type (Persohn et al., 1992; Wisden et
al., 1992; Fritschy and Möhler, 1995). There are several
important unresolved issues. Why is this receptor heterogeneity needed
for synaptic function? What is the subunit composition of native
subtypes of receptor? Are there rules guiding which subunits assemble
with each other?
The cerebellum is an excellent brain area for investigating these
questions. Its clearly defined circuitry allows an almost complete
account of which cerebellar cell types express which GABAA
receptor subunit genes (Wisden et al., 1996). For example, cerebellar
granule cells express six subunit genes abundantly (1,
6, 2, 3, 2,
and ), and so they probably have several distinct GABAA
receptor subtypes of unknown subunit stoichiometry. As for the
canonical muscle nicotinic acetylcholine receptor subunits (Verrall and
Hall, 1992; Green and Claudio, 1993; Kreienkamp et al., 1995), there is
likely to be selective discrimination between GABAA
subunits; however, assembling a neuronal receptor requires solving an
extensive combinatorial element. To form a native receptor, subunits
have to recognize and distinguish their neighbors. The assembly
pathways used by granule cells to sort the six principal subunits into
different receptor subtypes are not known. Granule cells receive a
single GABAergic input from Golgi interneurons onto their distal
dendrites. At this synapse, the GABAA receptor subtypes
might be colocalized and intermingled (Nusser et al., 1995, 1996), and
to date the 1, 6, 2/3, and
2 subunits have been demonstrated to be present in the
synaptic junction (Nusser et al., 1995, 1996; Somogyi et al.,
1996).
Despite the comparative simplicity of the system, the receptor subunit
composition of granule cell GABAA receptors is
controversial. Current views accommodate
12/32,
62/32,
162/32,
162/32, 12/32, and
62/3 combinations (Korpi and
Lüddens, 1993; Mertens et al., 1993; Caruncho and Costa, 1994;
Khan et al., 1994, 1996; Quirk et al., 1994; Caruncho et al., 1995;
Korpi et al., 1995; Pollard et al., 1995). The evidence for such
combinations is derived from antibody-based data and correlation of
pharmacological fingerprints of native binding sites, with binding
profiles of subunits expressed in cell lines. Here we have targeted the
6 subunit gene by homologous recombination techniques.
Removal of 6 protein from cerebellar granule cells
allowed perturbations in other subunit levels to be studied and
provided genetic evidence for a specific association between the
6 and subunits. Our results begin to reveal the
rules guiding receptor subunit assembly.
MATERIALS AND METHODS
Generation of mutant mice
The replacement vector for homologous recombination,
designed for positive-negative selection (Mansour et al., 1988), was generated from a 6 kb mouse 129 strain 6 subunit gene
fragment (Jones et al., 1996), comprising part of exon 4 through to the middle of intron 8. This fragment was subcloned into pBluescript (Stratagene, La Jolla, CA) (see Fig. 1A). Into this
plasmid, a BamHI cassette
(TAG3IRESlacZpAMC1neopA) (Nehls et al., 1996) was inserted
between the AflII and NcoI sites located in exon
8, after the site ends were modified by adding BamHI
adaptors. This cassette, designed to report target gene expression and
to provide a dominant marker to select for insertion into the gene,
consisted of stop codons (TAG) in all three frames, followed by an
internal ribosome entry site (IRES) linked to a lacZ reading frame and
SV40 polyadenylation sequences. It also contained a neomycin resistance
gene under independent transcriptional control (Nehls et al., 1996).
The IRES-lacZ cassette was inserted with the lacZ coding sequence in
the same transcriptional orientation as the 6 gene
coding sequences. For negative selection, a
XhoI-SalI fragment containing two MC1tk gene
head-to-tail repeats (Smith et al., 1995) was placed in the targeting
vector polylinker appending the longer homology arm (see Fig.
1A). The vector was linearized with SalI
and electroporated into 129 strain-derived embryonic stem (ES) cells
(ES line "CCB," kindly supplied by Drs. W. Colledge and M. Evans,
Wellcome/CRC, University of Cambridge).
Fig. 1.
GABAA receptor 6
subunit gene disruption by homologous recombination. A,
Wild-type 6 gene, targeting (replacement) vector, and
the disrupted 6 gene structures. Numbers
indicate exons. On the Replacement vector, the
broken line indicates pBluescript (pBS) sequences. On the wild-type allele, the
asterisk marks exon 8 where the IRES
lacZ/neo cassette was inserted. Only relevant restriction sites
are shown. A, AflII;
B, BamHI; N,
NcoI; S, SphI; X, XhoI. Arrows mark the
neo and tk gene promoter sites and
direction of transcription. The lacZ coding sequence
orientation is the same as the 6 gene, thus permitting
its translation from the IRES sequence (striped
box) to be initiated on the mRNA derived from the
6 promoter. Expected restriction fragment lengths
diagnostic for homologous recombination and the probes used to detect
these are marked by double-headed arrows and
horizontal bars, respectively. B,
Confirmation of 6 mutant allele germline transmission.
Biopsy tail DNA samples were digested with SphI,
electrophoresed, and Southern-blotted. The membrane was probed with
PROBE A (3 flanking). Wild-type (+/+) individuals give
a 15 kb band, the homozygous null (/) animals give a 9 kb band, and
heterozygotes (+/) give both bands.
[View Larger Version of this Image (60K GIF file)]
Transfected ES cells were grown on G418r primary embryonic
fibroblast feeder cells, in medium supplemented with leukemia
inhibitory factor (Life Technologies, Paisley, UK) and selected in G418
(Life Technologies) and FIAU (Bristol-Myers, Hounslow, UK) (Mansour et
al., 1988; Smith et al., 1995). Genomic DNA was isolated from individual colonies, digested with SphI, Southern-blotted,
and probed with an intron 8-derived SacI-XbaI
restriction fragment (see PROBE A in Fig.
1A). The addition of the neomycin gene creates an
additional SphI site in the 6 gene locus,
thus enabling the discrimination between wild-type (15 kb) and null (9 kb) alleles (see Fig. 1A,B). Confirmation of correct
targeting events was established with restriction fragment probes from
the lacZ and neo genes (marked on Fig. 1A) (data not
shown) and probe B (the complete sequence of probe B, which comprises
the promoter and 5 untranslated region, is deposited in the EMBL
database, accession number X97475[GenBank]; Jones et al., 1996) (see Fig.
1A). Additional diagnostic restriction digests of
targeted genomic DNA used BamHI (see Fig.
1A).
Male chimeras derived from targeted cells were mated with wild-type
C57BL/6. Mutation germ-line transmission was determined by Southern
blot analysis of agouti progeny tail DNA (see Fig. 1B). Heterozygotes were intercrossed to generate a
homozygous 6 / line.
-Galactosidase staining
Mice were transcardially perfused with 4% paraformaldehyde
(PFA) in PBS. Brains were removed, post-fixed for 1 hr in 4% PFA, and
then equilibrated at 4°C in PBS containing 30% sucrose. Sections (40 µM) were cut on a sliding microtome and incubated
free-floating in 5-bromo-4-chloro-3-indolyl--galactoside (X-Gal)
(Bonnerot and Nicolas, 1993). After X-Gal staining, some sections were
counterstained with neutral red (Sigma, Poole, UK), allowing
non-lacZ-expressing cells to be visualized. Alternatively, whole brains
(see Fig. 3C) were immersed in the X-Gal staining
reagent.
Fig. 3.
LacZ expression driven from the 6
gene locus in 6 / adult mouse brains illustrated in
horizontal (A), coronal (B), and whole-mount views (C); blue coloration
indicates lacZ activity. A and B show the
confined expression of the 6 gene to the cerebellar granule cell layer; C shows the expression in the dorsal
regions of the inferior colliculi; D shows higher-power
view of 6 gene expression in the molecular layer of the
cerebellum. The arrow indicates an example of the
numerous lacZ positive cells in the molecular layer. These are probably
nonmigrated granule cells. The arrowheads mark putative
parallel fiber staining; E, 6 gene expression in the dorsal cochlear nucleus granule cells.
BS, Brainstem; Cb, cerebellum;
CbGr, cerebellar granule cell layer; Ctx,
neocortex; Gr, cerebellar granule cells;
DCGr, dorsal cochlear nucleus granule cells;
H, hippocampus; IC, inferior colliculi;
Mol, cerebellar molecular layer. Scale bars:
A, 1.3 mm; B, C, 1 mm;
D, 150 µm; E, 300 µm.
[View Larger Version of this Image (116K GIF file)]
Staining of cultured granule cells. Cells on coverslips (see
Granule Cell Culture and Electrophysiological Analysis) were washed in
PBS and fixed in ice-cold 2% PFA/0.2% glutaraldehyde in PBS for 5 min. The coverslips were washed in PBS, incubated with X-Gal solution
at 37°C overnight, and counterstained with neutral red.
Antibodies
1-specific antibodies.
1(1-9), an N-terminal-specific antibody (Zezula and
Sieghart, 1991); 1-N, affinity-purified, raised against
rat N terminus residues 1-14 (S. Pollard and F. A. Stephenson,
unpublished data). 1(328-382)/1L was
prepared as described (Mossier et al., 1994). Rabbits were immunized
with an MBP-1(328-382)-7His fusion protein, and the
antibodies were purified with a GST-1(328-382)-7His
fusion protein. This antibody precipitates GABAA receptors
and is selective for the 1 subunit (R. Pelz and W. Sieghart, unpublished data).
6-specific antibodies. 6-N
(Batch R54XV), affinity-purified polyclonal, was raised to bovine
6 subunit N-terminal residues 1-16 (Thompson et al.,
1992); 6(429-434) batch P24, affinity-purified rabbit
polyclonal antibody, was raised to rat 6 subunit
residues 429-434 (Tögel et al., 1994); 6-C,
affinity-purified rabbit polyclonal, was directed against the
C-terminus sequence CSKDTMEVSSTVE (S. Pollard and F. A. Stephenson,
unpublished data).
-specific antibodies. (318-400), rabbit
polyclonal was raised against the rat cytoplasmic loop sequence between
TM3 and TM4 (Quirk et al., 1995); (1-44) (rabbit R7) polyclonal was
prepared by immunizing with an MBP-(1-44)-7His fusion protein and
purifying by affinity chromatography, as described (Mossier et al.,
1994; R. Pelz and W. Sieghart, unpublished data). This antibody is
specific for the subunit and does not precipitate 132
receptors (R. Pelz and W. Sieghart, unpublished data).
Immunocytochemistry
Five 6 / and five +/+ mice were
transcardially perfused with 4% PFA, 0.05% glutaraldehyde, and
~0.2% picric acid for 7-17 min. After perfusion the brains were
washed in 0.1 M phosphate buffer. Preembedding
immunocytochemistry was carried out on 70-µm-thick vibratome sections
(Somogyi et al., 1989). Floating sections were incubated in 20% normal
goat serum (NGS) diluted in Tris-buffered saline (TBS), pH 7.4, for 1 hr. The purified antibodies were diluted in TBS containing 1% NGS.
After they were washed, the sections were incubated for 2 hr in
biotinylated goat anti-rabbit IgG (diluted 1:50 in 1% NGS containing
TBS), followed by incubation in avidin-biotinylated horseradish
peroxidase complex (diluted 1:100; Vector Laboratories, Peterborough,
UK) for 90 min. Peroxidase enzyme reaction was with 3,3-diaminobenzidine tetrahydrochloride as chromogen and
H2O2 as oxidant. In some cases, Triton X-100
(0.1-0.3%) was added to the TBS throughout the experiment. The
antibody concentrations used for immunocytochemistry were (1-44)R7,
0.7-2.2 µg/ml; 6-N, 1.5-3.0 µg/ml.
For controls, selective labeling could not be detected when the
primary antibodies were either omitted or replaced by 5% normal rabbit
serum. No immunoreactivity was obtained when the antibodies were
preincubated with the appropriate peptides used for immunization (Nusser et al., 1996).
Ligand autoradiography
The procedures were slightly modified from Olsen et al. (1990)
and Wong et al. (1996). Cryostat sections (14 µm) from frozen nonfixed adult mouse brains were preincubated in 50 mM
Tris-HCl, pH 7.4, and 120 mM NaCl for 15 min at 0°C,
except for the GABA site assays when 0.31 M Tris-citrate
solution, pH 7.1, was used. Incubations with ligands used fresh buffers
of composition identical to those used for preincubation. For the
benzodiazepine (BZ) site, [3H]Ro 15-4513 (5 nM, Du Pont de Nemours, NEN Division, Dreieich, Germany)
was used with and without 100 µM diazepam (Orion, Espoo, Finland) for a 60 min incubation at 0°C, followed by three 30 sec
washes, a dip in distilled water, and rapid drying. The same conditions
and washes were used for the GABA site, with [3H]muscimol
(20 nM, Amersham, Buckinghamshire, UK) and
[3H]SR 95531 (20 nM, Du Pont), except that
the incubation time was 30 min. The sections were washed three times
for 15 sec in 10 mM Tris-HCl, pH 7.4, followed by dipping
in distilled water and air drying. Sections were exposed to
Hyperfilm-3H (Amersham) for 1-6 weeks. The images were
produced by scanning the films. The nonspecific binding components to
BZ and GABA sites were defined in the presence of 10 µM
Ro 15-1788 (Hoffmann-La Roche, Basel, Switzerland) and 100 µM GABA, respectively.
Radioligand binding
Radioligand binding on membranes prepared from individual mouse
cerebella was as described previously (Quirk et al., 1994). Membranes
prepared from each animal were used for saturation binding with
[3H]Ro 15-1788 (0.1-17.0 nM),
[3H]zolpidem (1-30 nM), and
[3H]Ro 15-4513 (0.8-60.0 nM) displaced with
Ro 15-4513 (10 µM) to define the total number of BZ
binding sites, or with flunitrazepam (1 µM) to define
binding to diazepam-sensitive sites only. Saturation binding with
[3H]muscimol (2-45 nM) used 1 mM
GABA to determine nonspecific levels. All assays used eight
concentrations of ligand, with total and nonspecific binding measured
in duplicate with 30-80 µg of protein/assay tube.
Bmax and Kd values were
determined by nonlinear least-squares fit of the saturation curves
using the data analysis software RS1 (Bolt, Beranek and Newman,
Cambridge MA).
Immunoprecipitation analysis
Immunoprecipitation of GABAA receptors solubilized
from individual mouse cerebella used antibody (318-400) bound to
protein A-Sepharose as described previously (Quirk et al., 1994, 1995). [3H]muscimol binding to the solubilized receptor was
measured after gel filtration through Sephadex G-25 to remove any
remaining endogenous GABA.
PAGE and immunoblotting
Membranes from individual +/+ and / cerebella were prepared,
and equal amounts of protein per slot were subjected to SDS-PAGE in
10% polyacrylamide gels and immunoblotted. For the
1(1-14), 6(1-16), and
6-C antibodies, the ECL Western blotting system (Amersham) was used for detection (Pollard et al., 1995). ECL blots
were quantitated by normalizing with an anti-neuron specific enolase
(NSE) antibody (Sigma) and then probing with 1(1-14) (S. Pollard and F. A. Stephenson, unpublished data). Multiple exposures
were taken for both anti-NSE and 1(1-14)
immunoreactivity and quantitated using a Molecular Dynamics Personal
Quantitator. For the (1-44)R7 and 1(328-382)
antibodies, membranes were incubated with digoxygenin-labeled
antibodies and were then treated with anti-digoxygenin-alkaline
phosphatase Fab fragments (Boehringer Mannheim, Mannheim, Germany).
Proteins were detected by fluorescence using the CSPD substrate
(Tropix). Blots were quantitated by densitometry of Kodak X-Omat S
films with the DocuGel 2000i gel documentation system using the
RFLPscan software (MWG-biotech).
Granule cell culture and electrophysiological analysis
Cell culture. Cerebellar granule cells, attached to
matrigel-coated coverslips, were cultured from postnatal day 5 (P5)
mice as described for rat cells (Randall and Tsien, 1995). Minimal essential medium was supplemented with glucose (5 mg/ml), transferrin (100 µg/ml), insulin (5 mg/ml), glutamine (0.3 mg/ml), and 10% fetal
calf serum. After 2 d, the cells were fed with media that was
supplemented further with 4 µM cytosine arabinoside, and
they were then fed every 5 d by a 50% replacement of the culture
media. Electrophysiological measurements were made after 14-17 d
in vitro.
Electrophysiology
Recordings were from single, visually identified, granule
cells using both outside-out patches and whole cells pulled away from
the underlying cell-attachment substrate. No differences were observed
between data from patches and whole cells, and results from both data
sets were therefore pooled. A piezoelectrically driven theta tube-based
application system delivered 120 msec pulses of GABA. Concentration
jumps from control to agonist and vice versa took place within ~1
msec. Five 120 msec 20 µM GABA pulses were applied at 0.1 Hz before and during the application of 1 µM
flunitrazepam (Sigma). Recovery from the actions of flunitrazepam were
studied with 20 additional GABA applications. Data were filtered at 2 kHz and sampled directly to computer at 10 kHz under control of the
pClamp software suite. Because of the presence of some application-to-application variability in the current peak amplitude generated by GABA, an arbitrary threshold was set, with a 15% increase
in the GABA response considered to be a potentiation above the baseline
variability.
In situ hybridization
In situ hybridization with 35S-labeled
oligonucleotide probes was as described (Wisden and Morris, 1994). The
oligonucleotide sequences used were 4:
5-TTCTGGACAGAAACCATCTTCGCCACATGCCATACTTTAAGCCTGT-3 (EMBL
accession number L08493[GenBank]) and :
5-AGCAGCTGAGAGGGAGAAAAGGACGATGGCGTTCCTCACATCCAT-3 (EMBL
accession number M60596[GenBank])
Behavioral observations
The animals (n = 23 for both +/+ and /
lines, from two generations) were observed in their normal activities.
Open field explorative activity was determined, under artificial
lighting, in a round area (diameter 83 cm) divided into 19 segments.
The mice were in the area for the first time. Their behavior was
recorded for 5 min with a video recorder, and the behavioral parameters (number of segment crossings with all four feet and number of rearings)
were scored blindly afterward. The number of fecal boli was counted
before the area was cleaned for the next animal. The ability of the
mice to learn to climb up onto a thin horizontal wire while initially
hanging from their forepaws was tested in three trials during 1 d.
Their ability to learn to stay on an accelerating rotating rod
(Rotamex, Columbus Instruments, Columbus, OH) for 180 sec was tested
during daily sessions. The initial session was 3 min on a nonmoving
rod. On subsequent sessions, the mice were placed on the stationary
rod, and the rotation speed was then set at 5 rpm and increased to 15 rpm over a 180 sec interval.
RESULTS
Creation of a mouse line with no GABAA receptor
6 subunit protein: mapping 6 expression
with a dicistronic RNA encoding lacZ
Homologous recombination in embryonic stem cells was used to
create a 129/Sv × C57BL/6 mouse line in which the 6
subunit gene was disrupted at exon 8 (Fig.
1A,B) (see Materials and Methods). The
mutation, located just after the TM2 (channel lining) region, consisted
of an insertion of stop codons in all three reading frames, an IRES
linked to a -galactosidase (lacZ) open reading frame and SV40
polyadenylation site, and finally a neomycin resistance gene expressed
from its own promoter (Mountford et al., 1994; Nehls et al., 1996)
(Fig. 1A). Translation of the 6
subunit mRNA from the mutant allele should terminate just after the TM2
region, resulting in a 300 amino acid protein designated
6M2. The stop codon-IRES insertion generates a
dicistronic mRNA in which -galactosidase protein translation is then
linked to an 6 gene expression, i.e., lacZ expression is
under 6 transcriptional regulatory element control.
Homozygous mutant mice had no overt defects and could breed normally
(see Behavioral Characterization of 6 / Mice). By the criteria of Nissl staining, the size, folding of folia, and histological appearance of the cerebellum in 6 /
animals were completely normal. Immunocytochemistry with the
6 N-terminal-specific antibody 6-N
(Thompson et al., 1992), however, demonstrated a total loss of
6-specific immunoreactivity from the cerebellar granule
cell layer of / mice (Fig. 2A,B).
Using both N- and C-terminal 6 subunit-specific
antibodies (Thompson et al., 1992; S. Pollard and F. A. Stephenson,
unpublished data), a complete loss of the 57 kDa immunoreactive
6 band was also seen on Western blots of cerebellar
protein extracts isolated from / animals (Fig. 2C).
Identical results were found with an additional C-terminal anti-peptide
antibody 6(429-434) (R. Pelz and W. Sieghart, data not
shown). Long exposure times of blots probed with the N-terminal antibody failed to show any 6-specific degradation
products in the / samples (data not shown), indicating that
6M2 is not a stable entity. In a 2
subunit gene knockout study, which similarly used an exon 8 disruption,
a truncated 2 form was also not detectable (Günther et al., 1995).
Fig. 2.
Immunodetection of the 6 subunit of
the GABAA receptor in 6 +/+ (A,
C) or 6 / (B, C) cerebella as
visualized with either light microscopic immunoperoxidase reactions
(A, B) or immunoblotting (C). A,
B, An intense immunoreactivity for the 6
subunits in the granule cell layer (gr)
disappeared in 6 / mice (B). The sections and one immunoblot were reacted with the same N-terminal domain-specific antibody. Scale bars: A, B, 500 µm.
C, The 57 kDa 6 protein is absent in
6 / cerebella, as shown with either 6-N, an N-terminal-specific antibody, or
6-C, a C-terminal-specific antibody to the
6 subunit.
[View Larger Version of this Image (52K GIF file)]
We mapped 6 gene expression in adult / animals using
-galactosidase staining (Fig. 3). An intense blue
coloration was seen in the cerebellar granule cell layer (Fig.
3A,B). The reaction product started to appear within the
first 5 min of incubating the sections at room temperature in X-Gal and
was fully developed within 30 min, thus demonstrating the extremely
high level of 6 locus expression. There were numerous
small blue cells in the cerebellar molecular layer. These are probably
ectopic granule cells (Fig. 3D) (cf. Thompson et al., 1992;
Gao and Fritschy, 1995; Gutiérrez et al., 1996). Strong, blue
coloration was also seen along the molecular layer outer edge, probably
corresponding to -galactosidase enzyme transported into granule cell
axons, the parallel fibers. As expected, there were many blue granule cells in the dorsal cochlear nuclei (Fig. 3E) (Varecka et
al., 1994). With little exception, the rest of the brain showed no detectable staining (Fig. 3A). Unexpectedly, however, many
cells in the inferior colliculi dorsal regions stained blue (Fig.
3C), and there were other minor cell populations in the
substantia nigra and thalamus (geniculate nuclei) with faint but
consistent blue staining (data not shown). These populations of stained
cells were not seen in wild-type animals, and thus were not
attributable to endogenous -galactosidase-like activity.
6 subunit expression has not been noted previously
in the inferior colliculi, substantia nigra, or thalamus by in
situ hybridization or immunoreactivity, although a rat
6 gene proximal promoter fragment consistently drives
lacZ expression in the inferior colliculi of transgenic mice (Jones et
al., 1996). The reasons for the lack of detection in previous studies
could include low 6 mRNA and protein levels.
Alternatively, 6 may be part of presynaptic receptors transported to distant axon terminals outside the inferior colliculi and so may escape detection in the inferior colliculi nucleus itself.
The long half-life of -galactosidase in mammalian tissue contributes
to the extreme sensitivity of the lacZ reporter method. Over time, low
levels of transcription from the 6-lacZ hybrid gene will
lead to accumulating amounts of -galactosidase. These results make
clear the usefulness of tracking gene expression using
dicistronic-based reporters (Mountford et al., 1994; Nehls et al.,
1996).
Pharmacological characterization of 6 /
cerebellar granule cells: BZ sensitivity
The 6 protein absence was established further
by pharmacological analysis. GABAA receptors containing the
6 subunit are insensitive to most types of BZs, such as
diazepam or flunitrazepam (Lüddens et al., 1990; Hadingham et
al., 1996). Ro 15-4513, however, is a BZ that binds to all subtypes of
GABAA receptor with x2 subunit combinations, including those containing the 6
subunit (Lüddens et al., 1990; Sieghart, 1995). Thus, a
diagnostic assay for 6 in cerebellar granule cells is
the high level of [3H]Ro 15-4513 binding on granule cell
membranes that is insensitive to full BZ agonists such as diazepam
(Sieghart et al., 1987; Malminiemi and Korpi, 1989; Lüddens et
al., 1990; Turner et al., 1991). Normally, more than half of the
[3H]Ro 15-4513 binding in the granule cell layer is
diazepam insensitive (DIS) but can be displaced by micromolar
concentrations of the BZ antagonist flumazenil (also known as Ro
15-1788). This profile is thought to be attributable to the abundant
expression of 62/32 receptors on granule cells (Lüddens et al., 1990; Korpi and
Lüddens, 1993; Korpi et al., 1993; for review, see Wisden et al.,
1996). In the 6 / animals, DIS binding over the
cerebellar granule cell layer is completely absent (Fig.
4A), whereas in wild-type brains
[3H]Ro 15-4513 still binds over the granule cell layer
even in the presence of 100 µM diazepam (Fig.
4A). From binding studies using isolated cerebellar
membranes, the contribution that the 6 subunit makes to
the number of total cerebellar Ro 15-4513 binding sites was estimated
to be ~40% (see Evaluation of 1 Subunit Levels; also
see Table 1).
Fig. 4.
Autoradiographic analysis of GABAA
receptor binding sites in wild-type (+/+) and 6 /
mice. A, Benzodiazepine sites labeled by 5 nM [3H]R0 15-4513 showing total and
diazepam-insensitive binding in the presence of 100 µM
diazepam. B, GABAA receptor sites labeled by
20 nM [3H]muscimol, showing total binding.
The nonspecific signal in the presence of 100 µM GABA was
at the film background level. C, GABAA receptor sites labeled by 20 nM [3H]SR 95531, showing total binding. The nonspecific binding signal in the presence
of 100 µM GABA was similar in wild-type and / brains
(data not shown). Similar distinct pharmacological profiles were
observed between the wild-type and 6 / brains in
each of seven pairs of adult mice studied. D, E,
In situ hybridization x-ray film autoradiographs of
adult mouse brains hybridized with 4 (D)
and -specific (E) 35S-labeled
oligonucleotide probes. Wild-type (+/+) brains are on the
left; 6 / brains are on the
right. No differences can be seen in subunit mRNA levels
between +/+ and / brains. Note also the very similar pattern of
4 and gene expression in the forebrain/thalamus regions, and the correlation with the distribution of
[3H]muscimol (B). Cbgr,
Cerebellar granule cells; CP, caudate-putamen; Ctx, cerebral cortex; Gr, cerebellar
granule cell layer; H, hippocampus; IC,
inferior colliculus; OB, olfactory bulb;
T, thalamus.
[View Larger Version of this Image (86K GIF file)]
The 6 subunit has a closely related homolog, the
4 subunit, which is expressed in certain forebrain areas
such as the thalamus (Wisden et al., 1991, 1992). The recombinant
4 subunit in an 4x2 configuration displays
a pharmacological profile identical to that of
6x2 receptors, and
4 mRNA is found at low levels in cerebellar granule
cells of adult rats (Wisden et al., 1991; Laurie et al., 1992). Thus we
looked to see whether there had been a compensatory change in
4 expression in the cerebellum of 6 /
mice (Fig. 4D); however, consistent with the absence of DIS binding in 6 / animals, there was no
upregulation of 4 mRNA in / cerebella (Fig.
4D).
The BZ sensitivity of GABAA receptors in
6 / cerebellar granule cells was investigated
directly using electrophysiology on cultured granule cells isolated
from P5 animals. After 14-17 d in vitro, we tested the
effects of BZ agonist flunitrazepam (1 µM) coapplication
on the current amplitude generated by 20 µM GABA. Results
of a typical culture are shown in Figure 5. Examination of wild-type cells revealed a heterogeneous response:
flunitrazepam-induced potentiation of the GABA response took place in
approximately half (20 of 36) of the cells tested (Fig. 5C,
top row). In those cells with flunitrazepam-potentiated GABA
responses, the average potentiation was 58 ± 7%. In contrast, in
age-matched cultures derived from 6 / cerebella, 16 of 18 cells tested had flunitrazepam-induced potentiations of their
GABA responses. The average potentiation was 62 ± 7% (Fig.
5C).
Fig. 5.
Electrophysiological characterization of
GABAA receptors in cerebellar granule cells from wild-type
and 6 / cells. Photomicrographs in A
and B show typical examples of lacZ-expressing
cerebellar granule cells, isolated from P5 6 / mouse
cerebella, and cultured for 3 weeks. A is a
low-magnification view, showing the mosaic of blue
(lacZ-positive) cells scattered throughout the culture. Both isolated and clustered blue cells can be seen.
Within any given cluster, not all the cells are blue and
therefore are not expressing the 6 gene. The cells have
been counterstained with neutral red. All electrophysiological
recordings were from isolated cells. Arrows in
B show examples of non-lacZ-expressing cells. Scale
bars: A, 200 µm; B, 30 µm.
C, Example responses to 120 msec applications of 20 µM GABA alone, and 20 µM GABA with 1 µM flunitrazepam (open arrows). The
top row shows an example of wild-type cells (+/+) with
GABAA receptors that responded to flunitrazepam
(left trace) or were insensitive to flunitrazepam
(right trace). The bottom row shows a
typical GABAA response from an 6 / cell and the associated flunitrazepam potentiation. From left to
right and top to bottom, the value
x on the scale bar corresponds to 200, 230, and 170 pA,
respectively. The traces were averages of three to five consecutive
records.
[View Larger Version of this Image (76K GIF file)]
To examine a possible reason for the heterogeneity of the
GABAA receptor response, the extent of gene expression from
the 6 locus in cultured 6 /granule
cells was analyzed with -galactosidase histochemistry. At 3 weeks in
culture, numerous cells strongly stained dark blue after incubation
with X-Gal (Fig. 5A,B); however, there were many adjacent
"granule-like" cells that either contained just a few blue
particles or were completely unstained (Fig. 5B). This
applied both to isolated cells and to cells in large clusters. There
was no obvious correlation between the location of cells (isolated or
in clusters) and lacZ expression. This mosaic of blue cells is evidence
that at least in culture, not all granule cells or granule-like cells
express the 6 gene (cf. Santi et al., 1994), and may
explain the heterogeneous nature of the BZ potentiation seen in our
cultures.
Selective subunit protein loss from cerebellar granule cells of
6 / mice
A key and controversial question for cerebellar granule cell
GABAA receptors has been which subunits co-assemble
in vivo (Wisden et al., 1996). To examine one aspect of
this, we immunoprecipitated deoxycholate-solubilized cerebellar
GABAA receptors from 6 / mice with a
-specific polyclonal antiserum, (318-400), raised against the
putative intracellular loop domain between TM3 and TM4 (Quirk et al.,
1994, 1995). In wild-type and 6 +/ cerebella, the
(318-400) antiserum precipitated the same number of muscimol binding sites (Fig. 6A). By this
assay, the protein was also present in both pure wild-type 129/Sv
and pure C57BL/6 cerebella (data not shown). In contrast,
immunoprecipitation of -containing receptors from 6
/ cerebella was greatly reduced (Fig. 6A). These
data were extended by Western blot analysis of membrane protein samples
isolated from individual +/+ and 6 / cerebella. With
use of a -subunit-specific antibody, (1-44)R7, raised against the N terminus (R. Pelz and W. Sieghart, unpublished data),
6 / samples showed a dramatic reduction in the
53 ± 1 kDa subunit band intensity to 25 ± 8% of +/+
levels (Fig. 6B). The residual protein in the
6 / tissue had the same molecular weight as that in wild-type
tissue (Fig. 6B).
Fig. 6.
Immunoprecipitation and immunoblot analysis of
GABAA receptor subunit levels in wild-type and
6 / cerebella. A, After cerebellar
GABAA receptors were solubilized in Triton
X-100/deoxycholate, the number of [3H]muscimol binding
sites immunoprecipitated by the (318-400) antiserum from +/+, +/,
and / cerebella was determined (n = 10-14).
B, Immunoblot analysis: the marked subunit reduction in 6 / cerebella detected with the (1-44)R7
antiserum. The identity of the low molecular weight doublet (33 and 31 kDa) seen in all samples is unknown. C, A 51 kDa
1 immunoreactive band is present in both / and +/+
cerebellar samples as detected with the 1(328-382)/1L
antibody.
[View Larger Version of this Image (35K GIF file)]
Immunocytochemistry with the (1-44)R7 antibody clearly supported
the Western blot and immunoprecipitation data (Fig. 7). Light microscopic immunocytochemistry with this antibody revealed a
very intense cerebellar granule cell layer staining in wild-type animals (Fig. 7A), similar to that reported earlier using a
different -specific antibody (Benke et al., 1991; Gao and Fritschy,
1995). The immunoreactivity originated mainly from staining of the
glomeruli, and granule cell bodies were only weakly outlined (Fig.
7C). The glomeruli appeared as dark rings of labeled granule
cell dendrites surrounding pale centers representing the unstained
mossy fiber terminals (Fig. 7C). In contrast to the
wild-type animals, in 6 / mice the granule cell
layer immunostaining for the subunit was virtually absent (Fig.
7B). In particular, no immunoreactivity could be detected in
the glomeruli (Fig. 7D), suggesting that 6
/ granule cell dendrites contain either no subunit protein or
an undetectably low level. Electron microscopic examination of the
immunoreactivity for the subunit in the granule cell layer further
confirmed the lack of subunit immunoreaction in 6
/ granule cells (not shown). Therefore, the residual subunit immunoreactivity seen on Western blots may represent a level of protein
undetectable by immunocytochemistry under our conditions, or it could
come from cell types other than granule cells, because whole cerebella
were used to prepare the protein extracts.
Fig. 7.
Immunodetection of the subunit of the
GABAA receptor in 6 +/+ (A,
C) or 6 / (B, D)
cerebella, using a polyclonal antibody R7 and immunoperoxidase
reaction. The granule cell layer showed intense immunoreactivity in
6 +/+ animals but almost no staining was observed in the
6 / mouse. C, At higher
magnification, it is evident that the subunit is localized mainly
in the glomeruli, granule cell bodies (gc) being
only weakly outlined. The glomeruli appear as dark rings of granule
cell dendrites surrounding a pale center (arrows)
representing the unstained mossy fiber terminal. D, In
the 6 / mice, both the granule cell bodies
(gc) and the glomeruli (asterisks)
are immunonegative for the subunit. C and
D were photographed using DIC optics. Scale bars:
A, B, 500 µm; C, D, 10 µm.
[View Larger Version of this Image (107K GIF file)]
The subunit loss occurs post-translationally
One possibility to explain the loss of protein from
6 / granule cells is through a change in regulation
at the mRNA level; however, the subunit mRNA level in the
cerebellar granule cells was at normal levels when examined by in
situ hybridization (Fig. 4E). High levels of mRNA were seen in both wild-type and / granule cells. mRNA
expression was also examined in both pure 129/Sv and C57BL/6 strain
wild-type animals and found not to differ (not shown). This result
suggests that the loss of subunit from the 6 /
cerebellar granule cells occurs post-translationally.
[3H]Muscimol and [3H]SR95531, two
ligands that mark out 6-and -containing
GABAA receptors
[3H]Muscimol and [3H]SR95531 are
ligands that highlight restricted GABA molecule conformations
(Sieghart, 1995). In particular, [3H]muscimol is the
classic GABAA ligand and has been used extensively for
mapping GABAA receptors in the brain (Palacios et al.,
1980; Olsen et al., 1990). We used [3H]muscimol and
[3H]SR95531 to autoradiographically probe the remaining
GABAA receptors in the subunit-deficient/6 / cerebellar granule cell layer. A clear cut, but completely unanticipated, pharmacological feature was
revealed: the selective and extensive loss of high-affinity [3H]muscimol (Fig. 4B) and
[3H]SR95531 (Fig. 4C) binding from the granule
cells. Binding over the cerebellar molecular layer with these ligands
remained unchanged, as did the levels of binding in the forebrain,
e.g., normal levels of [3H]muscimol binding remain over
the thalamus of / animals (Fig. 4B). The decrease
in [3H]muscimol binding seen by autoradiography in
6 / individuals was further quantified by studying
[3H]muscimol binding to membranes from whole cerebella
(Table 1). The level of high-affinity [3H]muscimol sites
was reduced to ~25% of that found in control animals (Table 1). No
significant reduction in [3H]muscimol binding was seen in
+/ animals (data not shown). Saturation analysis revealed no change
in the observed Kd values for
[3H]muscimol in / animals (Kd
is ~6 nM; Table 1). The residual binding is likely to
come from sites within the molecular layer, the granule cell layer, and
the deep cerebellar nuclei, all of which contain an
12/32 component. Under
autoradiographic conditions, however, [3H]muscimol does
not highlight these 1-containing receptors in the cerebellum.
Rather, [3H]muscimol and [3H]SR95531 seem
to selectively highlight 6-containing receptors.
Evaluation of 1 subunit levels in 6
/ and -deficient cerebella
The 1 protein is expected to account for the
majority of the remaining subunits in the cerebellum of
6 / mice (Sieghart, 1995; McKernan and Whiting,
1996; Wisden et al., 1996). To examine whether there was any
concomitant change in the 1-receptor population in the
6 / cerebella, the portion of 1
subunits complexed with the 2/3 and 2
subunits was measured using three different ligands targeting the BZ
binding site. These assays are not likely to measure any
1 subunits that are complexed with the but not the
2 subunits, e.g., 12/3,
because the GABA responses of these complexes cannot be modulated by
BZs (Saxena and Macdonald, 1994).
Full saturation analysis was carried out on cerebellar membranes to
determine the Kd and Bmax
values for [3H]Ro 15-1788 binding, diazepam-sensitive
[3H]Ro 15-4513 binding, and [3H]zolpidem
binding (Table 1). All three ligands identified approximately the same
number of binding sites in the cerebellar membranes (~990-1160 fmol/mg protein). There was no statistically significant difference between the number of binding sites for any ligand between the 6 +/+ and -deficient/6 null groups,
although the trend was always toward a reduced number of sites in the
/ animals. Total [3H]Ro 15-4513 binding sites (both
diazepam-sensitive and -insensitive components, with the nonspecific
binding being defined in the presence of 10 µM Ro
15-4513), however, were decreased by 44% in the / cerebella (Table
1), with a minor change in the affinity Kd
constant. This figure is in line with the 30-40% contribution that
the 62/32 component has
been reported to make to the total Ro 15-4513 binding sites in the
cerebellum (Sieghart et al., 1987; Turner et al., 1991; Korpi et al.,
1993; Quirk et al., 1994).
These binding results suggest that the amount of total
1 protein complexed with 2/3 and
2 in the cerebellum is essentially unchanged between
-deficient/6 null and wild-type animals. Furthermore, immunocytochemistry with a polyclonal 1-specific
antibody (P16) showed no overt change in granule cell layer
immunostaining at the light microscopic level in 6 /
cerebella compared with wild-type tissue (data not shown); however,
this method may not pick out small changes in subunit levels. In fact,
immunoblotting with 1-specific antibodies did show a
downward trend in 1 protein levels between
-deficient/6 null and +/+ cerebellar samples (Fig.
6C). In / animals, a small reduction with high
variability was seen in the 1 51 kDa band intensity, as
determined by densitometric measurements. This was observed
independently with three different 1-specific
antibodies: 1(1-14) (S. Pollard and F. A. Stephenson, unpublished data), 1(1-9) (Zezula and Sieghart, 1991),
and 1(328-382) (R. Pelz and W. Sieghart, unpublished
data) (see Materials and Methods).
Behavioral characterization of 6
null/-deficient mice
The total loss of 6 from the cerebellum and
the associated severe reduction of subunit levels might be expected
to have consequences for nervous system function in the mutant mice.
The cerebellum integrates sensory input needed for maintaining balance and orientation, has a prominent role in the refinement of motor action, and may also participate in motor memory storage (Raymond et
al., 1996). We looked for evidence of cerebellar-associated motor
deficits in the / mice. In terms of simple observable behavior,
mutant mice seem indistinguishable from wild-type littermates. The
adult 6 null/-deficient mice are active and agile,
whether they be in the cage or roaming freely, and exhibit spontaneous activity, such as walking upside down on the ceiling of their cages. In
an open field test, mutant mice showed as much exploratory activity as
individuals with normal levels of 6 and proteins (Table 2). Mutant as well as wild-type mice learned the
horizontal wire task (data not shown; see Materials and Methods). Both
the wild-type and 6 null/-deficient mice reached the
rotating rod test learning criterion (Table 2). With these tests, we
found no evidence to suggest any form of ataxia associated with
cerebellar dysfunction. Additionally, a detailed behavioral analysis on
an independently generated 6 / mouse line, where the
same exon was disrupted by insertion of a neo gene (exon 8, NcoI site) in a 129xC57BL/6 background, showed no
abnormalities in motor behaviors (Gregg E. Homanics, Departmen |
