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Proc. Nati. Acad. Sci.

USA
Vol. 91, pp. 10952-10956, November 1994
Neurobiology

Changes in y-aminobutyrate type A receptor subunit mRNAs,


translation product expression, and receptor function during
neuronal maturation in vitro
T. M. ZHENG, W. J. ZHU, G. PUIA, S. VICINI, D. R. GRAYSON*, E. COSTAt, AND H. J. CARUNCHO
Fidia-Georgetown Institute for the Neurosciences, Georgetown University School of Medicine, Washington, DC 20007

Contributed by E. Costa, August 2, 1994

The amounts of mRNAs encoding al, a'6, 132,


ABSTRACT
133, y2, and 8 subunits of y-aminobutyrate type A (GABAA)
receptors and the gold immunolabeling density of their translation products were monitored during the growth of neonatal
rat granule cells in primary culture. We investigated possible
correlations (i) between temporal changes in mRNA content
and expression density of their respective translation products
and (ii) between the quantitative changes of receptor subunit
expression, the GABA EC5. for Cl- channel activation, and
diazepam efficacy in modulating GABA action on the Clchannels. At 3 days in vitro, the amount of GABAA receptor
subunit mRNAs and the expression of their respective translation products were very low. During the next 2 weeks both
parameters for every subunit studied increased asynchronously; moreover, at 14 days in vitro the sum of V2 and 8
subunit expression was smaller than the expression of the al or
a6 or 182/133 subunits. This suggests that during in vitro
maturation each subunit may be regulated independently and
invites speculation as to possible changes in specific GABAA
receptor subtype abundance during development in vitro. The
maximal current intensity elicited by GABA failed to increase
from 5 to 14 days in vitro, though the amount of mRNA
encoding various subunits and the expression density of their
respective translation products increased. Thus, qualitative
changes in the GABAA receptor subtypes expressed and/or
abnormalities in the subunit assembly very likely account for
the uniformity of the maximal current intensity elicited by
GABA during in vitro development. Also, during maturation of
neuronal cultures from 5 to 20 days in vitro the extent of the
positive modulation of GABA action by diazepam decreased
dramatically. This finding might be related to an increase in the
abundance of GABAA receptors including the a6 subunit
and/or to the expression, during granule cell maturation in
vitro, of GABAA receptors devoid of y2 subunits.

Cerebellar granule cells receive a GABAergic input from


Golgi type II cells (1) and express numerous y-aminobutyrate
type A (GABAA) receptors in both their dendrites and their
soma (2-5). There are at least 16 genes encoding structurally
different GABAA receptor subunits, which are assembled
into pentameric complexes forming a variety of structurally
different GABAA receptor subtypes with different pharmacological properties (6, 7). Transient-expression studies with
cDNAs encoding various subunit combinations have demonstrated that the GABA potency in various structural forms
of recombinant GABAA receptors and their sensitivity to
benzodiazepines acting as positive allosteric modulators of
GABA action relate to the specific subunit combinations
(8-12).
To study whether changes in GABAA receptor subunit
mRNA and protein expression occur synchronously during

neuronal maturation in vitro (13) and whether eventual maturational variations in subunit expression could be associated
with changes in GABAA receptor function, we used a competitive reverse transcription-polymerase chain reaction
(RT-PCR) technique (14-16) to quantify specific GABAA
subunit mRNAs, the label-fracture immunogold technique to
monitor the expression of GABAA receptor subunits in the
plasma membrane of cerebellar granule neurons (4, 5), and
whole-cell recordings to assess GABA action and its modulation by diazepam (8).

MATERIALS AND METHODS


Cell Culture. Primary cultures of cerebellar granule cells
were prepared from 8-day-old Sprague-Dawley rat pups (13).
Cells were dispersed with trypsin and plated at 2 x 10W cells
per cm2 on 100- or 35-mm Nunc dishes coated with poly(Dlysine). Cultures were maintained in a humidified 6% CO2
atmosphere at 370C in basal Eagle's medium containing 10%6
heat-inactivated fetal bovine serum, 2 mM glutamine, gentamicin (50 tkg/ml), and 25 mM KCl. Cytosine arabinoside (10

,IM) was added to each cell culture 20 hr after plating, to


inhibit nonneuronal cell proliferation. As reported (13),
>90%o of the cells in these cultures are glutamatergic granule
neurons. Cells maintained in culture for >2 weeks were
supplemented with 5 mM glucose.
Quantitation of Receptor Subunit mRNAs by Competitive
RT-PCR. Cultured cells were harvested in 4 M guanidinium
isothiocyanate at 3, 7, and 14 days in vitro (DIV). Total RA
was isolated by gradient ultracentrifugation over a CsCl
cushion (17), and selected receptor subunit mRNAs were
quantified by a RT-PCR assay with specific competitive
internal standard cRNAs (14-16). The internal standard for
each GABAA receptor subunit analyzed contained a targeted
restriction site (Bgl II) positioned midway between the amplification primers, and the sense-strand cRNA was synthesized by in vitro transcription (14-16). The RNA/cRNA
mixtures were reverse transcribed and the cDNA aliquots
containing 100% of the reverse-transcribed material were
amplified for 28-30 cycles with Hot Tub DNA polymerase
(Amersham) in a thermal cycler (Perkin-Elmner). The amplification mixture (100 #l) contained cDNA, 10 mM MgCl2, 25
mM Tris HCl (pH 9.5), 50 mM KCl, 0.5 pM receptor subunitspecific primers, 200 ,uM dNTPs with trace amounts of
[a-32P]dCTP, and 2.5 units of Hot Tub polymerase. Amplification products from both the mRNA and cRNA templates
were distinguished in a postamplification step by Bgl II
digestion and agarose gel electrophoresis. The amount of
Abbreviations: DIV, day(s) in vitro; GABA, y-aminobutyrate; RT,

reverse transcription.

*Present address: Neuroscience Research Center, Allegheny-Singer


Research Institute, 320 East North Avenue, Pittsburgh, PA 15212.
tTo whom reprint requests should be addressed at: Nathan S. Kline
Institute for Psychiatric Research, Center for Neuropharmacology,
140 Old Orangeburg Road, Orangeburg, NY 10962.

The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. 1734 solely to indicate this fact.
10952

Neurobiology: Zheng et al.

Proc. Natl. Acad. Sci. USA 91 (1994)

was also very low at 3 DIV (97 4 amol/,g); it increased to


243 39 amol/,g by 7 DIV but failed to change significantly
by 14 DIV (276 21 amol/,ug). However, the amount of (33
mRNA (Fig. 1D) was 154 5 amol/,g by 3 DIV, increased
to 305 55 amol/,g by 7 DIV, and continued to increase, to
464 48 amol/,ug by 14 DIV. The 8 mRNA (Fig. 1E) was 42
1 amol/,g by 3 DIV, increased to 216 33 amol/,Lg by 7
DIV, and failed to increase significantly by 14 DIV (259 50
amol/,ug).
GABAA Receptor Density in the Plasma Membrane of
Cerebellar Granule Cells During Maturation in Vitro. The
density of colloidal gold particles labeling the GABAA receptor subunits under investigation was very low at 3 DIV
and then increased significantly by 14 DIV (Fig. 2). However,
the density of various receptor subunits increased asynchronously. For example, the number of al receptor subunits
increased about 2-fold by 7 DIV and >5-fold by 14 DIV. In
contrast, the a6 subunit failed to increase significantly by 7
DIV but increased >4-fold by 14 DIV. In the membranes of
cells at 7 DIV, the a6/al labeling ratio was 1.03, and it was
1.2 after 14 DIV. The density of labeled (2/,(3 subunits
increased 2-fold by 7 DIV and >4-fold by 14 DIV. The
labeling density of the (32/13 subunits approximated the sum
of the cr1 and a6 labeling densities. The labeling density of
either y2 or 8 subunits increased about 2-fold by 7 DIV and
>3-fold by 14 DIV. However, the sum of the density of y2 and
8 subunits at 14 DIV was much lower than that of either al
or a6 subunits (Fig. 2). The data in Fig. 3 offer an example of
the density changes of a6 and y2 subunits during 14 DIV and
also indicate the formation of small subunit clusters.
GABA-Gated Cl- Currents and Their Allosteric Modulation
by Diazepam During Granule-Cell Maturation in Vitro. The
fast (<6-ms onset) application of various GABA concentrations evoked inward currents, when symmetrical Cl- concentrations were present in the intracellular recording pipette
solution and in the extracellular bath solution (at a holding
potential -50 mV) (Fig. 4 A and B). From analysis of the
normalized dose-response relationships, GABA was shown
to be less potent in cerebellar granule neurons at S DIV than
at 10, 15, or 20 DIV. No significant differences in the Hill
coefficients of the GABA responses were recorded with
increasing time in culture (nH ranged from 1.1 to 1.5). In
primary cultures of cerebellar neurons at 5 DIV, the maximal
Cl- current elicited by GABA was not significantly different
from that observed at 10, 15, or 20 DIV (Fig. 4C Inset).
However, the extent of current desensitization during GABA
application, as measured by the ratio between peak and
steady state of the Cl- current elicited by GABA, was
significantly smaller at 5 DIV (3.2 0.4) than at the other
times tested (range, 6.2-6.5). The GABA-activated Cl- cur-

specific receptor subunit mRNA is reported as amol/pg of


total RNA.
Label-Fracture Immunocytochemistry. Cultures maintained for 3, 7, and 14 DIV were fixed and processed for
label-fracture immunocytochemistry (4, 5). For immunolabeling of GABAA receptor subunits, we used immunopurified
rabbit polyclonal antibodies against specific amino acid sequences of the al (3), a6 (18, 19), y2 (20), and 8 subunits (21).
The specificity ofthese antibodies was demonstrated with the
transient transfection of cDNA encoding the various subunits
in the human embryonic kidney cell line 293 (5). A mouse
monoclonal antibody was used to label the (32 and ,83 subunits
(22, 23). The antibody dilutions used were 1:20,000 (al),
1:10,000 (282/33), and 1:100 (a6, 'y2, 8). After overnight
incubation with the first antibody the cells were treated for 1
hr with the corresponding gold-conjugated second antibody.
Controls were prepared by omitting the primary antibody or
using nonimmune serum. Colloidal gold particles in the
plasma membrane were counted in squares of a grid superimposed on electron micrographs at x50,000 final magnification, so that each square corresponded to 1 gm2.
Electrophysiology. Primary cultures of cerebellar granule
neurons were voltage-clamped in the whole-cell configuration by the patch-clamp technique at room temperature (24).
The bath solution contained 145 mM NaCl, 5 mM KCl, 2 mM
CaCl2, and 5 mM Hepes/NaOH (pH 7.4). The recording
pipette contained 145 mM CsCl, 1 mM MgCl2, 11 mM EGTA,
and 10 mM Hepes/CsOH (pH 7.2). GABA or GABA plus
diazepam was diluted in bath solution and applied with a Y
tubing (25). Currents were monitored with a patch amplifier
(EPC-7; List Electronics) and digitized in an IBM-PC computer with the software AXOTAPE 2 (Axon Instrument). After
normalization, fitting of the dose-response relationship was
performed with the logistic equation % Imax = 100/Imax{1+(EC5o/[GABA]nH} where Imax is the maximal Cl- current
elicited by GABA, EC50 is the GABA concentration eliciting
the half-maximal response, and nH is the Hill coefficient.
ORIGIN (Microcal, Amherst, MA) was used for data analysis
and figure preparation. Statistical analysis of data was performed with ANOVA.

RESULTS
Expression of GABAA Receptor Subunit mRNAs During in
Vitro Maturation of Cerebellar Granule Cells. The al and a6
subunit mRNAs increased significantly from 90 31 and 97
54 amol/,ug of total RNA at 3 DIV, to 338 40 and 462 +
32 amol/,ug by 7 DIV, respectively (Fig. 1 A and B). By 14
DIV, these mRNAs had increased to 524 30 and 685 44
amol/,ug, respectively. The amount of (2 mRNA (Fig. 1C)
800- B

0-- <

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14

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14

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400-

200

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400-

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no

<

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800- E
600.
400.

*:
200

**

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3 7 14
Days in culture

2t

03

14

Days in culture
FIG. 1. Temporal expression of the
al (A), c6 (B), P2 (C), (33 (D), and 8 (E)
GABAA receptor subunit mRNAs in primary granule-cell cultures differentiating
in vitro. The results represent the mean
SEM absolute values for three or more
independent competitive RT-PCR experiments. Statistically significant differences from the values obtained at 3 DIV
are indicated: *, P < 0.05; **, P < 0.001
(Dunnett's t test).

Neurobiology: Zheng et al.

10954

Proc. Natl. Acad. Sci. USA 91 (1994)


3 DIV
7 DIV
* 14 DIV

100-

o
80-

60-

~0 401i
z

d201
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Ya6

[2/f33

FIG. 2. Temporal changes in the level of specific GABAA receptor subunit proteins (a1, a6, 82/P3, y2, and 8) in cerebellar granule
cells during maturation in vitro. Proteins were detected by immunogold labeling. The results represent the mean SEM of gold
particles (n = 10). Statistically significant differences from the values
obtained at 3 DIV are indicated: *, P < 0.05; **, P < 0.001 (Dunnett's
t test).

rents obtained from representative neurons at 7 and 14 DIV


were compared with the diazepam (10 ,uM)-elicited positive
allosteric modulation ofthis GABA response (Fig. 5 A and B).
We used a GABA concentration near the EC50 (0.3 and 0.5
AM GABA at 14 and 7 DIV, respectively) in order to compare
the extent of positive modulation elicited by diazepam at
different times in vitro. The diazepam dose-response relationship (Fig. SC) shows a dramatic downregulation of the
diazepam-elicited positive modulation of GABA-evoked Clcurrents in cells kept in culture for 14 days or longer.

DISCUSSION
In primary cultures of cerebellar granule cells maintained in
vitro for 2 weeks, there is a continuous increase in the content
of various GABAA receptor subunit mRNAs and their translation products. The increase in al, a6, 832//33, y2, and 8
GABAA receptor subunits in the plasma membrane (Fig. 2)
approximates the increase in the respective mRNAs occurring either in vivo (15, 16, 26-32) or in vitro (Fig. 1 and refs.
15, 33, and 34). However, the rate of these increases differs
for the various subunits. Further, the electrophysiological

data demonstrate that the affinity of GABA for the various


GABAA receptor subtypes expressed in granule cells is lower
at 5 DIV than at 10 DIV. In contrast, the diazepam-elicited
positive allosteric modulation of GABA-activated Cl- currents decreases dramatically after 7 DIV (Fig. 5); such a
decrease is consistent with another report (34).
In the present study, cells prepared from 8-day-old rat pups
showed a 3.7-fold increase in al mRNA content between 3
and 7 DIV, followed by a 1.6-fold increase between 7 and 14
DIV. A parallel analysis of this subunit expression in the
plasma membrane of granule neurons in vitro showed a
1.8-fold increase between 3 and 7 DIV and a 3-fold increase
between 7 and 14 DIV. Bolos et al. (34) reported that in
granule-cell cultures prepared from 8-day-old rat pups, the al
subunit mRNA increased 2- to 3-fold during 3 weeks in vitro.
However, a study (33) using cultured cerebellar granule cells
prepared from 10-day-old rat pups failed to show that the
mRNA encoding the al subunit increased during in vitro
maturation. Is there a critical period for preparing the granule-cell cultures in order to observe an increase in al mRNA
during in vitro maturation? A definitive answer to this question requires further study.
In the mature rat brain, the expression of a6 GABAA
receptor subunit appears to be restricted to cerebellar granule
cells (16, 18, 35). RT-PCR analysis of the changes in the a6
mRNA level during development in vivo showed a 100-fold
increase in a6 mRNA between postnatal days 7 and 21 (16).
The present data show a significant increase in a6 mRNA
during 2 weeks of maturation in vitro (Fig. 1). Moreover, the
expression density of the a6 subunit translation product also
increases during maturation in vitro (Figs. 2 and 3). However,
while the ratio of a6 mRNA levels is 1.7 between 7 and 14
DIV, that for the respective translation products expressed in
the plasma membrane is 3.6, suggesting either an increase in
the translation rate of the mRNA or a decrease in a6 protein
metabolism with time in culture. An immunocytochemical
electron microscopy study of the distribution in rat cerebellum of GABAA receptors including the a6 receptor subunit
showed that these receptors were restricted to postsynaptic
sites (granule-cell neurites), whereas the GABAA receptors
containing the a1 subunit were present in both synaptic and
extrasynaptic locations (neurites and soma) (36). Our previous study of expression of GABAA receptors in cultured
cerebellar granule cells (5) showed that at 14 DIV, a6-

FIG. 3. Electron micrographs oflabel-fractured replicas of cultured rat cerebellar granule cells labeled with GABAA receptor subunit-specific
antibodies followed by gold-conjugated second antibodies. (A and B) Immunogold labeling of the a6 subunit in the plasma membrane of granule
cells cultured for 3 and 14 DIV, respectively. (C and D) Immunogold labeling of the y2 subunit in the plasma membrane of granule cells cultured
for 3 and 14 DIV, respectively. Squares signal the presence of small receptor clusters. Please note that the density of the a6 subunit is much
greater than that of the y2 subunit at 14 DIV. (Bar = 200 nm.)

Proc. Natl. Acad. Sci. USA 91 (1994)

Neurobiology: Zheng et al.


A

GABA

GABA

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10955

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15 DIV, EC50 = 0.4 AM
20 DIV, EC5O 0.7 pM
=

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FIG. 4. Dose-response curves of GABA-elicited Cl- currents in cerebellar granule neurons. Representative whole-cell Cl- currents (at a
holding potential -50 mV) were elicited in cerebellar granule neurons at 5 DIV (A) and 15 DIV (B) with 20-sec applications of increasing
concentrations of GABA (0.03, 0.3, 3.0, 30, and 300 A&M) via a Y-tubing device. The dose-response curves normalized to the maximal current
for the cerebellar granule neurons are presented in C. The results are an average of 10 cells. (Inset) Mean SEM ofthe maximal current intensity
elicited by GABA in the neurons studied at various DIV.

densities of the 6 and y2 receptor subunits changed with time


in vitro, the total expression of the respective protein was
very low. Thus the sum of the immunogold labeling density
of y2 and 6 subunits was substantially lower than the sum of
al and a6 or (32 and (33 subunit densities.
The GABA potency in activating Cl- currents in GABAA
receptors increased significantly from 5 to 10 DIV but failed
to change from 10 to 20 DIV (Fig. 4), whereas the current
desensitization susceptibility increased from 5 to 10 DIV. The
maximal current elicited by GABA failed to change with time
in vitro (Fig. 4 Inset). Moreover, the positive modulation of
GABA-evoked Cl- influx by diazepam was markedly reduced from 7 to 14 DIV, and this modulatory activity
remained low during the subsequent week (Fig. 5). This
marked decrease of diazepam efficacy correlated with a
robust increase in a6 subunit expression and a much lower
expression of the 'y2 subunit as determined by freeze-fracture
immunogold labeling (Fig. 2). From these findings one might
infer that with time in vitro the number of GABAA receptors
including an a6 subunit increases. An increase in the number
of GABAA receptors including the a6 subunit is also consistent with the shift to the left of the GABA dose-response
curve occurring between 5 and 10 DIV and with the observed
maturation (time in vitro)-related decrease in the ability of
diazepam to positively modulate GABA-elicited currents. In
recombinant GABAA receptors, GABA potency to gate C1channels is greater in receptor assemblies including the a6
subunit (11); moreover, these receptors are less sensitive
than receptors including the al subunit to the positive

containing receptors were expressed both in neurites and in


soma.
The mRNAs encoding the (32 and (33 GABAA receptor
subunits increase significantly during the second week of
maturation in vivo (29, 31). Also, in granule-cell cultures
prepared at postnatal day 10, the 32 mRNA increases 4-fold
from 3 to 10 DIV (33). Figs. 1 and 2 demonstrate an increase
in the (32 mRNA of2.5-fold from 3 to 7 DIV, but the level fails
to increase further at 14 DIV. In contrast, the 833 mRNA
increased 2-fold during the initial time frame and an additional 1.5-fold from 7 to 14 DIV. During the same time frames,
we found 2- and 2.2-fold increases in 832/(33 subunit expression, respectively, suggesting a moderate translation rate of
these two mRNAs.
Previous work showed that the total amount of y2 mRNA
changed very slightly as granule cells matured in vitro,
whereas there was a pronounced difference in the maturational abundance of the two y2 mRNA splice variants. The
present study, using a polyclonal antibody that does not
differentiate between the two protein isoforms, showed that
'y2 receptor subunit increased 2.5-fold between 3 and 7 DIV,
and only a slight further increase occurred during the second
week in vitro (Fig. 2). Thus, the y2 subunit density at 14 DIV
was much lower than the density of either al or a6 subunits
(Fig. 2).
The mRNA for the 8 subunit increased 5-fold between 3
and 7 DIV but only 1.2-fold between 7 and 14 DIV. The 8
receptor subunit increased 2.3-fold from 3 DIV to 7 DIV and
increased an additional 1.5-fold by 14 DIV. Although the
A

GAMA0.5 A1

7DIV + Diazepam 10 PM
GA1 05pM

C
160

I 100 pA
10s

Diazepam 10
GABA 0.3 pM

1220

14 DIV
GABA0.3 pM

o 7 DIV
* 14DIV

A 21 DIV

FIG. 5. Whole-cell inward Cl-

_
r
/
1

PAM

440-

0- .
o.0l

.
0.1

Diazepan, JLM

cur-

rents elicited by GABA (0.5 pM) in

..

10

cerebellar granule cells at 7 DIV (A),


and by GABA (0.3 ,uM) at 14 DIV (B)
are potentiated to a different extent by
diazepam (10 IAM). (C) reports the dose
response of diazepam induced facilitation of GABA elicited currents. Each

point (mean SEM) is the response in


10 cells at various DIV.

10956

Neurobiology: Zheng et al.

allosteric modulation by diazepam (9-11). Therefore, one


might assume that the decrease in the GABA ECQo and in
diazepam's efficacy in modulating the action ot GABA in
cells kept 14 DIV may relate to the presence 6f a higher
proportion of GABAA receptors that contain the a6 subunit
instead ofthe al subunit. Though the inclusion of a6 subunits
may play some role in increasing GABA affinity (Fig. 4) and
decreasing diazepam efficacy (Fig. 5), this change per se may
not be the only factor that accounts for our results. Perhaps
the presence of GABAA receptors including a 8 subunit
instead of a y2 subunit may also contribute to the dramatic
decrease in diazepam efficacy with time in culture (37).
Further, since the sum of y2 and 8 receptor subunit expression at 14 DIV is lower than the expression of either al or a6
or the sum of (32 and (33 subunits, one can surmise that the
GABAA receptor subunit composition during maturation in
vitro might change to include a greater number of GABAA
receptors which are devoid of 'y2 subunits. Indeed receptors
including only a and ( subunits have been shown to produce
Cl- channels with a reduced conductance (38) which are
virtually insensitive to the positive modulation by diazepam.
This could explain the lack of increase in the maximal current
elicited by GABA and the resistance to the positive modulation by diazepam in cells kept >14 DIV. However, the lack
of increase of the GABA-elicited maximal current with time
in vitro might also be related to a maturation-related desensitization to the GABA action (Fig. 4); this desensitization
might cause an underestimation of the GABA-elicited currents.
In conclusion, while an in vitro maturation-dependent
increase in the subunit mRNAs of granule cells is associated
with an increase in the expression of their translation products, these two increases are asynchronous, suggesting a
different metabolic regulation of the structurally different
GABAA receptor subunits. The lack of an in vitro timerelated increase in the maximal current elicited by GABA
occurring in the face of an increase of al, a6, and (32/(3
receptor subunit expression allows us to speculate that during
in vitro maturation the granule cells lose the capacity to
express heterooligomeric GABAA receptors including 'y2
and/or 8 subunits.
We thank Drs. H. Mohler and P. Seeburg, who kindly provided the
subunit-specific antibodies, and Dr. T. Shirazaki for assistance with
the Y-tubing device. We thank Dr. T. Narahashi and Dr. R. Miledi
for their constructive suggestion. This work was supported in part by
National Institutes of Health Grant R01 NS30537 (D.R.G.) and by the
Fidia Research Foundation.;
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