1Department of Anatomy and Neurobiology and 2Developmental and Cell Biology, University of California, Irvine, California 92697-1280
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ABSTRACT |
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Dunning, D. D.,
C. L. Hoover,
I. Soltesz,
M. A. Smith, and
D. K. O'Dowd.
GABAA Receptor-Mediated Miniature Postsynaptic
Currents and -Subunit Expression in Developing Cortical Neurons.
J. Neurophysiol. 82: 3286-3297, 1999.
Previous
studies have described maturational changes in GABAergic inhibitory
synaptic transmission in the rodent somatosensory cortex during the
early postnatal period. To determine whether alterations in the
functional properties of synaptically localized GABAA
receptors (GABAARs) contribute to development of inhibitory transmission, we used the whole cell recording technique to examine GABAergic miniature postsynaptic currents (mPSCs) in developing cortical neurons. Neurons harvested from somatosensory cortices of
newborn mice showed a progressive, eightfold increase in GABAergic mPSC
frequency during the first 4 wk of development in dissociated cell
culture. A twofold decrease in the decay time of the GABAergic mPSCs,
between 1 and 4 wk, demonstrates a functional change in the properties
of GABAARs mediating synaptic transmission in cortical neurons during development in culture. A similar maturational profile
observed in GABAergic mPSC frequency and decay time in cortical neurons
developing in vivo (assessed in slices), suggests that these changes in
synaptically localized GABAARs contribute to development of
inhibition in the rodent neocortex. Pharmacological and reverse
transcription-polymerase chain reaction (RT-PCR) studies were conducted
to determine whether changes in subunit expression might contribute to
the observed developmental alterations in synaptic GABAARs.
Zolpidem (300 nM), a subunit-selective benzodiazepine agonist with high
affinity for
1-subunits, caused a reversible slowing of the mPSC
decay kinetics in cultured cortical neurons. Development was
characterized by an increase in the potency of zolpidem in modulating
the mPSC decay, suggesting a maturational increase in percentage of
functionally active GABAARs containing
1 subunits. The
relative expression of
1 versus
5 GABAAR subunit mRNA
in cortical tissue, both in vivo and in vitro, also increased during
this same period. Furthermore, single-cell RT-multiplex PCR
analysis revealed more rapidly decaying mPSCs in individual neurons in
which
1 versus
5 mRNA was amplified. Together these data suggest
that changes in
-subunit composition of GABAARs contribute to the maturation of GABAergic mPSCs mediating inhibition in
developing cortical neurons.
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INTRODUCTION |
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Inhibitory synaptic transmission in the mature
cerebral cortex, mediated by GABAARs, plays a
vital role in regulating normal cortical activity. Although GABAergic
neurons are present and GABAARs are expressed in
the neocortex of newborn rodents (Del Rio et al. 1992;
Laurie et al. 1992
), the inhibitory network is not fully
mature at birth. Anatomic studies indicate a paucity of inhibitory
synaptic contacts in animals <1 wk old (Blue and Parnavelas
1983
; Miller 1986
). In addition, the robust,
short-latency GABAAR-mediated postsynaptic
responses to stimulation of afferent fibers in the adult cortex are
seen only rarely before the second postnatal week (Agmon and
O'Dowd 1992
; Burgard and Hablitz 1993
; Luhmann and Prince 1991
). More recent studies have
identified symmetrical (presumed inhibitory) synapses at
postnatal day 4 (P4) (De
Felipe et al. 1997
), and demonstrated that thalamic stimulation can evoke labile, asynchronous GABAAR-mediated synaptic
currents in the mouse somatosensory cortex as early as
P0 (Agmon et al. 1996
). An immature
chloride gradient appears to be responsible for the elevated reversal
potential of the GABAergic synaptic currents observed in young cortical
neurons (Owens et al. 1996
). Age-related differences in
GABA-evoked currents in acutely isolated cortical neurons suggests that
alterations in the properties of the GABAARs may also
contribute to maturation of GABAergic transmission (Oh et al.
1995
). To address this question directly, however, it is
necessary to examine the functional properties of the subpopulation of
GABAARs that are specifically localized at synapses in
developing cortical neurons.
The functional properties of ionotropic GABAARs can be
influenced by a number of factors including receptor subunit
composition, desensitization rates, phosphorylation state, as well as
reuptake rates of the ligand (Angelotti and Macdonald
1993; Draguhn and Heinemann 1996
;
Galarreta and Hestrin 1997
; Haas and Macdonald 1999
; Jones and Westbrook 1995
,
1997
; Macdonald and Olsen 1994
; Serafini et al. 1998
; Verdoorn et al.
1990
). Although some or all of these properties may be altered
during development, a combination of biophysical and pharmacological
studies suggest that changes in
-subunit composition contribute to
early postnatal maturation of receptors mediating GABAergic currents in
cerebellar (Mathews et al. 1994
; Tia et al.
1996
; Vicini 1999
) and hippocampal neurons (Hollrigel and Soltesz 1997
; Kapur and Macdonald
1999
; Rovira and Ben-Ari 1993
). These findings,
in combination with previous reports of a large increase in
1 and a
decrease in
5 GABAAR subunit expression in the
developing rodent neocortex (Golshani et al. 1997
;
Laurie et al. 1992
; Paysan et al. 1994
),
suggest that changes in expression of these
-subunits may contribute to maturational changes in the functional properties of
GABAARs mediating inhibition in cortical neurons.
In this study whole cell recordings were used to examine the
biophysical features of the GABAergic miniature postsynaptic currents
(mPSCs), which reflect the functional properties of synaptically localized GABAARs, in cortical neurons developing in vivo
and in dissociated cell culture. Having observed a developmental change in the kinetic properties of the GABAergic mPSCs, we sought to identify
the molecular events that might underlie them. The sensitivity of the
GABAergic mPSCs to modulation by zolpidem, a benzodiazepine type I
(BZ1) agonist with high affinity for receptors containing 1- versus
5-subunits (Faure-Halley et al. 1993
;
Macdonald and Olsen 1994
; Pritchett and Seeburg
1990
), was used to probe for possible alterations in the
relative contribution of these subunits in the functionally active
synaptic GABAARs. Semiquantitative reverse
transcription-polymerase chain reaction (RT-PCR) analysis was
conducted to examine the developmental regulation of
-subunits (1, 2, 3, and 5) in RNA harvested from cortices or cultures at different
ages. Finally, by combining whole cell recordings and gene expression
in single neurons (Brooks-Kayal et al. 1998a
,b
; Eberwine et al. 1992
; Lambolez et al.
1992
; O'Dowd and Smith 1996
; Ruano et
al. 1997
), the correlation between
1 and
5 subunit expression and biophysical properties of the GABAergic mPSCs in individual cortical neurons was examined. Our data suggest that changes
in
-subunit composition of synaptically localized
GABAARs contribute to the maturation of inhibition in
developing cortical neurons.
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METHODS |
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Tissue culture
Primary neuronal cultures were prepared from mouse somatosensory
cortex as previously described (Li et al. 1997;
Massengill et al. 1997
). Briefly, P0 mice
(ICR, Harlan Sprague Dawley, San Diego, CA) were anesthetized by
hypothermia before decapitation. The brain was removed, and pieces of
the somatosensory cortex were dissected out and treated with papain (10 U/ml) for 30 min at 37°C. The tissue was mechanically dissociated, in
neurobasal medium with B27 supplements (NMB + B27; Life Technologies,
Gaithersburg, MD), using sterile glass micropipettes. The cells were
plated onto poly-D-lysine-coated glass coverslips (Bellco
Glass, Vineland, NJ) and maintained in a 5% CO2
incubator at 37°C overnight. The following day, coverslips were
transferred to dishes containing confluent nonneuronal feeder cultures
in NBM + B27 or fed with media conditioned by feeder cultures.
Coverslips were subsequently transferred to new feeder cultures or
supplemented with fresh conditioned medium every 3 to 5 days, and were
maintained in this manner for up to 4 wk.
Immunocytochemistry
Neuronal cultures were rinsed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde-PBS for 1 h on ice, washed in PBS, and permeabilized in PBS containing 0.02% saponin, 0.05% NaN3 for 30 min on ice. Incubation with the primary antibody at a dilution of 1:1,000 (polyclonal anti-GABA antibody, Sigma) was carried out in 2% BSA-PBS containing 0.05% NaN3 overnight at 4°C. Incubation with the secondary antibody at a dilution of 1:200 (Texas Red-conjugated anti-rabbit IgG, Vector Laboratories) was performed in 2% BSA-PBS containing 0.05% NaN3 for 2 h at room temperature. Cultures were washed three times with 2% BSA-PBS after each incubation. To determine the mean incidence of GABA-positive neurons, cultures were viewed with Hoffman optics to count the total number of neurons in randomly chosen fields of view. Fluorescent illumination of the same fields was used to count the number of labeled neurons. The mean percentage of GABA-positive neurons was determined from counts obtained from five fields on seven or more coverslips at each developmental stage.
Electrophysiology (cultured neurons)
Whole cell recordings were made from cultured somatosensory
cortical neurons [3-28 days in vitro (DIV)] using unpolished
pipettes with an open tip resistance of 1-3 M. The internal pipette
solution contained (in mM) 120 KCl, 20 NaCl, 2 MgCl2, 0.1 CaCl2, 1 EGTA, and 10 HEPES, pH 7.2. Cultured neurons were bathed in an external solution containing (in mM) 140 NaCl, 3 KCl, 4 MgCl2, 1 CaCl2, and 5 HEPES, pH 7.2. The following drugs were added to the external solution
and bath applied in various combinations as required: 1 µM
tetrodotoxin citrate (TTX), 5 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 50 µM D(
)-2-amino-5-phosphonopentanoic acid
(APV), and 2 µM (
)-bicuculline methochloride (BMC) and 3-3,000 nM
N,N,6-trimethyl-2-(4-methylphenyl)-imidazo [1,2-a]
pyridine-3-acetamide (Zolpidem) (all from Research Biochemicals). Data
were acquired using a patch-clamp amplifier (List EPC-7; Axopatch 1-D),
a D-A board (Labmaster or DigiData 1200A; Axon Instruments), and
pClamp6 (Axon Instruments) or SCAN (Courtesy of Dr. J. Dempster,
Strathclyde Electrophysiology Software, Strathclyde University,
Strathclyde, UK) software running on a Dell 386 or Pentium PC. The
signal was filtered at 2.5-5 kHz and digitized at 1-10 kHz.
DATA ANALYSIS.
mPSC frequency was determined in each neuron from 30, 1-s
current traces, filtered at 2.5 kHz, and digitized at 1 kHz using pCLAMP software. Individual events were counted when the amplitude was
>20 pA (4-fold greater than the average RMS noise level of 5 pA).
mPSC biophysical properties were determined from records filtered at 2.5-5 kHz and digitized at 5-10 kHz using SCAN. The mean
amplitude and 10-90% rise time were determined by averaging the
values obtained from 50 single events in each neuron. Decay kinetics
were evaluated by two different measures. The T50% value was defined
as the time required for the ensemble average mPSC from each neuron to
decay to 50% of the peak amplitude (Hajos and Mody
1997
). Second, the decay time constant for each neuron was
determined by fitting a single exponential to the falling phase of the
ensemble average mPSC.
Electrophysiology (acute slice preparation)
Neonatal mice age 8 and 20-23 days postnatal were used for
electrophysiological recordings in an acute slice preparation as previously described (Hollrigel and Soltesz 1997;
Hollrigel et al. 1998
). Briefly, mice were anesthetized
by halothane inhalation before killing by decapitation. The brain was
removed and placed in ice-cold oxygenated (95%
O2-5% CO2) artificial
cerebrospinal fluid (ACSF) containing (in mM) 126 NaCl, 2.5 KCl, 26 NaHCO3, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, and 10 D(+)-glucose. Coronal brain slices prepared with a
vibratome tissue sectioner (Lancer Series 1000), were equilibrated for
1 h in ACSF at room temperature before recording. Individual
slices were transferred to a submersion recording chamber perfused with
ACSF bubbled with 95%O2-5%
CO2 containing 10 µM APV, 5 µM CNQX, and 1 µM TTX. The internal pipette solution contained (in mM) 120 KCl, 20 NaCl, 2 MgCl2, 0.1 CaCl2, 1 EGTA, and 10 HEPES, pH 7.2. Blind whole cell recordings were obtained
as described previously (Blanton et al. 1989
) using an Axopatch-200A amplifier. All recordings, from neurons in layers V and
VI, were performed at room temperature.
RT-PCR
Mice at P7, P14, P21, and P28 were
anesthetized by halothane inhalation before decapitation. Brains were
removed and the somatosensory cortex dissected free from the
surrounding tissue. Total RNA was isolated from acutely dissociated
cortical tissue and cultured neurons at 7, 14, 21, and 28 DIV, by a
single step method (Chomczynski and Sacchi 1987). First
strand cDNA was synthesized by reverse transcription of 100 ng total
RNA as described (O'Dowd et al. 1995
). RT-PCR
amplification of
1,
2,
3,
5: PCR products were amplified in separate single round RT-PCR reactions, using primer pairs
specific for each of the four distinct GABAAR
-subunits (Table 1). PCR parameters
for amplification were as follows: 1 cycle at 94°C for 1 min, 20 cycles at 94°C for 30 s, 55°C for 1 min, and 72°C for 1.5 min, and a final elongation cycle at 72°C for 6 min. The identity of
each of the PCR products was confirmed by sequencing. Multiplex
RT-PCR amplification of
1 and
5: Co-amplification of the
products for
1 and
5 was accomplished by combining the individual
primer sets in a single round RT-PCR reaction using the same
amplification conditions stated above.
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In both the single and multiplex RT-PCR reactions, forward primers were radiolabeled by a T4 DNA kinase reaction (Promega) with 32P-ATP (NEN-DuPont), and ~5 × 105 cpm of 32P-labeled primer was added to the PCR. The radiolabeled PCR products were separated by electrophoresis on a nondenaturing 8% polyacrylamide gel. Quantitative analysis was performed using a phosphorimager (Molecular Dynamics, Sunnyvale, CA).
Single-cell multiplex RT-PCR
Analysis of GABAA receptor -subunit
mRNA expression was performed on RNA harvested from single cells as
described (Massengill et al. 1997
). Briefly, after whole
cell electrophysiology, mild suction was used to aspirate the contents
of the cell into the tip of the recording electrode that was then
expelled into a tube containing reverse transcription buffer.
First-strand cDNA synthesis was initiated by the addition of 100 units
of Moloney murine leukemia virus (MMLV) reverse transcriptase
(Life Technologies), and the reaction was allowed to proceed for 1 h at 37°C. After termination of the reaction, the resulting cDNAs for
the
1- and the
5-subunit of the GABAAR were
co-amplified in a multiplex PCR reaction, using two rounds of
amplification, with distinct sets of nested primers specific for
1
and
5 (Table 1). First round PCR parameters for co-amplification of
1 and
5 were as follows: 1 cycle at 94°C for 1 min, 40 cycles
at 94°C for 30 s, 55°C for 1 min, and 72°C for 1.5 min, and
a final elongation cycle at 72°C for 6 min. The first round products
were diluted at 1:1,000 and amplified in a second-round under the
following conditions: 1 cycle at 94°C for 1 min, 40 cycles at 94°C
for 30 s, 54°C for 1 min, and 72°C for 1.5 min, and a final
elongation cycle at 72°C for 6 min. PCR products were labeled by
inclusion of 32P-labeled primer, separated by gel
electrophoresis, and analyzed on a phosphorimager.
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RESULTS |
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To determine whether there are maturational changes in the
properties of GABAAR mediating synaptic
transmission in the neocortex, GABAergic mPSCs were examined in
cortical neurons during early postnatal development, both in vivo and
in vitro. A previous study from our lab demonstrated that
GABAAR-mediated currents could be recorded from
cortical neurons in brain slices as early as the day of birth
(Agmon et al. 1996). Here we demonstrate that GABAARs also mediate functional transmission
between cortical neurons, harvested from P0 mouse
somatosensory cortices, grown in dissociated cell culture. Fast, action
potential (AP)-independent postsynaptic potentials and currents (mPSPs
and mPSCs) were observed in the majority of cultured neurons examined
(Fig. 1A). These were mediated
by GABAARs based on the demonstration that the
mPSPs and mPSCs were reversibly blocked by BMC, a
GABAAR antagonist, but were not affected by the
glutamate receptor antagonists CNQX and APV (Fig. 1A). In
addition, the mPSCs exhibited a reversal potential near the calculated
chloride equilibrium potential (ECl =
2 mV) as expected for currents mediated by activation of
GABAARs (Fig. 1B).
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Frequency of GABAergic mPSCs in cortical neurons developing in vitro and in vivo
GABAergic mPSCs could be recorded from some neurons as early as the third day in culture (3 DIV). However, because of the low incidence of observing neurons with these currents at 3-4 DIV, quantitative analysis of GABAergic mPSCs in the first postnatal week was limited to cells between 5 and 7 DIV. In this age range, GABAergic mPSCs were recorded from ~75% of the neurons examined (Fig. 2A). From the second week on, GABAergic mPSCs were present in nearly all of the neurons examined (Fig. 2A). A progressive change in the average frequency of GABAergic mPSCs was observed, ranging from <1 Hz at 5-7 DIV up to 8 Hz at 4 wk (Fig. 2, B and C). A large increase in the frequency of GABAergic mPSCs also occurred in cortical neurons developing in vivo. Recordings from neurons in slices prepared at P8 revealed an average mPSC frequency of 0.15 ± 0.04 Hz (mean ± SE, n = 9) as compared with 2.7 ± 1.01 Hz (mean ± SE, n = 5) in neurons examined in slices made from animals at P20-23.
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To determine whether developmental changes in the number of GABAergic neurons might contribute to the increase observed in the mPSC frequency, staining with anti-GABA antibodies was used to assess the percentage of GABA-positive neurons at different times in culture (Fig. 3, A and B). The average percentage of GABA-positive neurons (15%) did not change significantly between 1 and 4 wk in vitro (Fig. 3C) demonstrating that the developmental increase in mPSC frequency is not a consequence of a change in the number of GABAergic neurons.
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Biophysical properties of GABAergic mPSCs in cortical neurons developing in vitro and in vivo
To address the question of whether there may be alterations in the functional properties of synaptic GABAARs, the biophysical properties of GABAergic mPSCs were assessed through analysis of 50-1,000 individual mPSCs recorded from single neurons between 1 and 4 wk in culture. Qualitative comparison of currents observed in old versus young neurons suggested that the currents decayed more rapidly in the older neurons (Fig. 4A). Two independent measures were employed to quantitatively evaluate this potential change in decay kinetics. First, a decay time constant was determined by fitting an exponential function to the falling phase of the ensemble average mPSC in each neuron. Although the falling phase of some individual mPSCs could be best fit by the sum of more than one exponential, the ensemble average in the vast majority of neurons at all ages examined were adequately fit by a single exponential (Fig. 4A). Second, the time for the mPSC to decay to half-amplitude (T50%) was determined from the ensemble average mPSC in each neuron. Examination of both the mean decay time constant and the T50% values as a function of age revealed a two- to threefold decrease during the first 4 wk in culture from >30 ms during the first 2 wk, to ~15 ms during the 4th week (Fig. 4, B and C). The mean mPSC amplitude and rise time were determined by averaging the values obtained from 50 or more single mPSCs in each neuron. In contrast to the change in decay kinetics, there was no significant change in either the mean amplitude or rise time of the mPSCs during the first 4 wk in culture (Fig. 4, D and E). The developmental increase in the rate of decay of the GABAergic mPSCs in the absence of alterations in amplitude supports the hypothesis that regulation of channel properties, rather than simply an age-dependent alteration in electronic filtering, underlies this change.
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Analysis of GABAergic mPSCs from neurons in acute slices prepared from animals at P8 and P20-23 revealed ensemble average mPSCs with more rapid decay kinetics in the older versus younger neurons (Fig. 4F). The mean mPSC decay time constant was significantly faster (3.5-fold) at P20-P23 when compared with P8 (Fig. 4G). Similar to the results in vitro, there was no significant change in the amplitude of the GABAergic mPSCs during this developmental period in vivo (Fig. 4H). These data suggest that changes in functional properties of synaptically localized GABAARs contribute to development of inhibition in the rodent neocortex.
Zolpidem sensitivity
The developmental increase in 1 and decrease in
5
GABAAR subunit expression in the rodent neocortex
(Golshani et al. 1997
; Laurie et al.
1992
; Paysan et al. 1994
) occurring over the
same time period as the changes we report in GABAergic mPSCs, suggests that developmental regulation of these subunits may contribute to the
maturational changes in the functional properties of
GABAARs in cortical neurons. To test this
hypothesis we examined the sensitivity of the GABAergic mPSCs to bath
application of zolpidem, a benzodiazepine agonist with a high affinity
for recombinant GABAARs containing
1-subunits
compared with receptors containing
5-subunits (Faure-Halley et al. 1993
; Macdonald and Olsen 1994
;
Pritchett and Seeburg 1990
). Exposure of a neuron at 28 DIV to 300 nM zolpidem caused a reversible increase in the amplitude of
the ensemble average mPSC (Fig.
5A). There was also a
reversible slowing of the decay time constant, from 8.4 to 13.0 ms, in
this same neuron (Fig. 5C). Evaluation of mPSCs recorded
from 12 neurons at 4 wk revealed a significant increase in amplitude
(23 ± 8%; P < 0.01, paired Student's
t-test) and decay time constant (34 ± 10%,
P < 0.01, paired Student's t-test)
following exposure to 300 nM zolpidem. A less pronounced, but still
significant, change was observed in the amplitude and decay time
constant of neurons at 7 DIV (Fig. 5, B and D).
Zolpidem (300 nM) enhanced the mPSC amplitude by 14 ± 6%
(P < 0.05, paired Student's t-test) and
decay time constant by 21 ± 5% (P < 0.01, paired Student's t-test) in the 16 cells examined at 5-7
DIV.
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To further explore differences in the sensitivity of neurons to
zolpidem, we plotted the percent change in decay kinetics and amplitude
as a function of zolpidem concentration in neurons at 5-7 versus
22-28 DIV (Fig. 5, E and F). The mPSCs recorded from neurons in both age groups show a dose-dependent increase in their
decay time constant. However, the shift to the left in the curve
generated from the older neurons demonstrates a developmental increase
in sensitivity to zolpidem with a significant difference observed at 30 nM zolpidem (P < 0.05, unpaired Student's
t-test; Fig. 5E). We were not able to determine
whether there was an age-related difference in the amplitude
enhancement due to the relatively large variability observed in the
neurons at both 1 and 4 wk (Fig. 5F). Taken together, these
data suggest that while zolpidem sensitive subunits (e.g., 1)
contribute to formation of the GABAARs mediating the mPSCs at all ages, their contribution increases during development.
Maturational changes in GABAAR -subunit expression
To determine whether an increase in expression of 1 and/or a
decrease in expression of
5-subunit mRNA is likely to contribute to
developmental regulation of GABAAR function,
semiquantitative RT-PCR analysis was used to examine expression of
these subunits in neurons developing both in vitro and in vivo. The
expression of the individual subunits was assessed in separate RT-PCR
reactions using 100 ng total RNA prepared from cultures at 7, 14, 21, and 28 DIV (Fig. 6, A, C, and
E). Twenty cycles of amplification using an end-labeled
forward primer generated PCR products within the linear range of
amplification when quantified on a phosphorimager. RNA harvested from
at least three different platings, for each of the developmental ages,
was used to quantitatively assess the relative changes in expression of
the subunits during the first 4 wk in culture. The most pronounced
change was a six- to eightfold increase in the expression of
1
between 7 and 28 DIV (P < 0.01, paired Student's
t-test; Fig. 6C). A decrease in
5 expression occurred over this same time period (P < 0.02, paired
Student's t-test; Fig. 6F). Using identical
amplification conditions and 100 ng total RNA, a similar developmental
change was observed in the expression of the
1- and
5-subunits in
RNA prepared from the somatosensory cortices of mice from
P7-P28 (Fig. 6, B, D, and F). RT-PCR
analysis of two additional subunits,
2 and
3, demonstrated that,
although these were expressed throughout the developmental period
examined, no significant change in expression was consistently observed
either in vivo or in vitro (data not shown).
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In these initial experiments, RNA was prepared from both somatosensory
cortical tissue and dissociated cell cultures that are composed of a
mixed population of cells including neurons and nonneuronal cells. To
determine whether the changes in 1- and
5-subunit expression were
representative of changes occurring in the neurons, we used a single
cell multiplex RT-PCR approach to assess the expression of both
subunits in single neurons at different ages in culture. After two
rounds of amplification using nested primers, PCR products representing
1 and/or
5 were amplified from the majority of neurons between 7 and 28 DIV (70/104), in which GABAergic mPSCs were recorded. Regardless
of developmental age, both
1 and
5 products were amplified from
most neurons (Fig. 7A).
However, consistent with our analysis of RNA from whole culture, the
frequency of encountering a single neuron in which only
5 was
detected decreased with age in culture, whereas the frequency of
encountering neurons in which only
1 was detected increased with age
in culture (Fig. 7B). These data suggest that changes in the
relative expression of
1- and
5-subunits contribute to changes in
the GABAergic synaptic currents in developing cortical neurons.
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-Subunit expression in single neurons is correlated with mPSC
decay kinetics
To examine the relationship between -subunit expression and the
rate of decay of mPSCs in individual neurons, cells were grouped into
three categories based on the PCR products amplified (i.e.,
5 only,
1 +
5, and
1 only). A representative GABAergic mPSC from
single neurons in each of the PCR categories is shown in Fig.
8A. Cell 1, in
which only
5 was amplified had a relatively slow decay time
constant. Cell 2 expressing both
1 and
5 had an
intermediate rate of decay, whereas cell 3, in which only
1 was amplified, had the fastest rate of decay. Analysis of all the
neurons in which at least one PCR product was amplified revealed that
the decay time constant in cells in which only
5 mRNA was detected
was significantly slower than in those cells in which only
1 mRNA
was detected (Fig. 8B). This was not simply an age-dependent phenomenon because a similar correlation was observed when analysis was
restricted to neurons in the third week in culture (Fig.
8C). In contrast, there was no correlation between
-subunit expression and mPSC amplitude (data not shown). These data
support the hypothesis that an increase in the ratio of
1:
5-subunit expression contributes to an increased rate of decay
in the GABAergic mPSCs.
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DISCUSSION |
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This study documents changes in the kinetic properties of
GABAergic mPSCs in mouse cortical neurons during the first
postnatal month, both in vivo and in vitro, indicating that
functional alterations in active, synaptically localized
GABAARs contribute to development of inhibition
in the rodent neocortex. Sensitivity of young cultured neurons to low
concentrations of zolpidem and the expression of GABAAR 1-subunits, in RNA harvested from
cortical tissue and single neurons in the first week, suggest that
1-subunits contribute to receptors mediating the mPSCs as early as
the first postnatal week. However, developmental increases in zolpidem
sensitivity, mPSC rate of decay, and the ratio of
GABAAR
1 to
5-subunit mRNA, suggest that an
increase in the proportion of synaptic GABAARs containing
1-subunits contributes to the maturation of GABAergic transmission in mouse cortical neurons.
Maturation in the functional properties of GABAARs mediating synaptic transmission in cortical neurons
Previous studies have provided evidence of developmental changes
in the functional properties of synaptically localized
GABAARs that are likely to contribute to
development of inhibition in the mammalian CNS. In cerebellar granule
cells, the fast exponential component of the decay phase of spontaneous
inhibitory postsynaptic currents (IPSCs) becomes more prominent, and
the IPSC amplitude decreases during the early postnatal period
(Tia et al. 1996). A dramatic increase in the mPSC
frequency and a twofold decrease in the decay time of the ensemble
average mIPSCs, in the absence of a change in mIPSC amplitude, has been
observed in dentate granule cells in rats between P0-P14
and adult (Hollrigel and Soltesz 1997
). In both of these
cases, a temporal correlation between eye opening/exploration of the
environment (P14) with the changes in properties of the
GABAergic currents has led to the suggestion that alterations in
sensory stimulation may trigger, directly or indirectly, the changes in
GABAAR function. Our results from cortical
neurons illustrate changes in mPSC frequency and kinetics that are
similar to those reported in dentate granule cells, suggesting that the
factors influencing development of these properties might be similar in
neocortex and hippocampus. However, the observation that the temporal
sequence and direction of the changes in GABAergic mPSCs in cortical
neurons developing in dissociated cell culture are similar to that seen
in vivo demonstrates that alterations in sensory input are not
necessary to initiate the change.
Although GABAergic mPSCs get faster during the first 4 wk of
development, the magnitude of the change we observed was greater in
cortical neurons maturing in vivo versus in vitro. A number of
variables may contribute to this difference. It is possible, for
example, that although the signals necessary for initiating the changes
in GABAAR function are intrinsic to the cortex,
or even cell autonomous, additional factors important in completion and/or maintenance of the more differentiated state may be absent in
the cultures. Recent studies have also demonstrated differences in the
decay kinetics of GABAergic sIPSCs between pyramidal and interneurons
in the rodent cortex, providing evidence of cell-specific expression of
functionally distinct GABAARs (Xiang et
al. 1998). In the present study, GABAergic mPSCs were recorded
from randomly selected neurons in the culture dish and within layers V
and VI in the slice preparations. Thus sampling from different
subpopulations could also contribute to the differences in mean mPSC
decay time between neurons examined in culture and slice.
1-Subunits contribute to functional GABAergic mPSCs during the
first postnatal week
Zolpidem has been a useful tool in gathering information regarding
the subunit composition of native receptors. Sensitivity to modulation
by low concentrations of zolpidem suggest that 1-subunit containing
receptors contribute to the GABA-evoked currents or IPSCs in a variety
of cells including dentate granule neurons (De Koninck and Mody
1994
; Soltesz and Mody 1994
), cultured
hippocampal neurons (Schonrock and Bormann 1993
),
cerebellar Purkinje cells (Itier et al. 1996
), and
cortical neurons (Gibbs et al. 1996
; Perrais and
Ropert 1999
). A reversible increase in the mPSC amplitude and a
slowing in the decay time, induced by bath exposure to zolpidem (300 nM) in the population of neurons examined between 5 and 7 DIV,
demonstrate that BZ1-sensitive receptors contribute to the GABAAR-mediated synaptic currents in the first
week. In addition, our single-cell RT-mPCR analysis revealed that both
1- and
5-subunits could be amplified from the majority of neurons
examined, even in the youngest age group. These findings suggest that
1-subunits contribute to the formation of functional receptors
mediating GABAergic mPSCs as early as the first postnatal week.
Evidence that changes in GABAAR -subunit expression
contribute to the development of GABAergic transmission in cortical
neurons
An increase in the expression of the 6
GABAAR subunit has been shown to play an
important role in developmental changes in inhibitory synaptic currents
in cerebellar granule neurons (Tia et al. 1996
;
Zhu et al. 1996
). Studies in hippocampal neurons indicate an increased potency of zolpidem in modulating
GABAAR-mediated currents during maturation,
consistent with the hypothesis that an increase in
1-subunit-containing receptors contributes to the developmental
changes observed in the GABAergic synaptic currents (Hollrigel
and Soltesz 1997
; Kapur and Macdonald 1999
;
Rovira and Ben-Ari 1993
). In this study, we present
three lines of evidence supporting the hypothesis that developmental
changes in
-subunit expression also contribute to alterations in
GABAergic transmission in cortical neurons. First, an increase in the
sensitivity of mPSCs to bath application of zolpidem during development
in vitro is consistent with an increase in the contribution of
1-subunits to GABAARs mediating synaptic
transmission. Although we did not investigate the pharmacological
properties of neurons developing in the animal, a recent study
demonstrates that zolpidem, at room temperature, enhances both the
amplitude (38%) and the duration (63%) of the mIPSC recorded from
layer V pyramidal neurons located in the visual cortex of rat
(Perrais and Ropert 1999
). These latter studies were
done in slices obtained from animals at P15-25, and the
magnitude of the modulation by zolpidem is consistent with that seen in
our older age group. Second, the striking increase in the level of
1
mRNA expression suggests that an increase in the number of
1-subunit-containing receptors contributes to the increase in mPSC
frequency that occurs during the same developmental period. Finally,
our single-cell RT-PCR analysis supports the hypothesis that a relative
increase in expression of
1:
5 is important in the functional
maturation of the decay kinetics of synaptic
GABAARs.
In addition to 1 and
5,
2-
4 are also expressed in the
developing rodent neocortex. A recent report demonstrating that the
majority of single pyramidal neurons in slices of the visual cortex
expressed more than two, and as many as five (
1-
5)
GABAAR subunits (Ruano et al.
1997
), raises the question of the role of other
-subunits in
maturational changes in the GABAergic mPSCs. Developing cultured
cortical neurons showed little change in expression of
2- and
3-subunits, making it unlikely that these subunits contribute to the
developmental changes in the kinetic and pharmacological properties of
the mPSCs we report. However, changes in
4-subunits, that were not
monitored in this study, may contribute to maturation of the GABAergic
mPSCs based on the report of an increase in
4 mRNA expression during
early postnatal development in the mouse somatosensory cortex
(Golshani et al. 1997
) .
Factors important in regulating the development of GABAergic synaptic transmission
Cortical function is critically dependent on the normal maturation
of the intrinsic membrane properties of its component neurons and the
pattern of excitatory and inhibitory connections formed between the
individual neurons. In the present study, comparison of the development
of cortical neurons in dissociated cell culture with those developing
in vivo resulted in identification of functional and molecular
properties of the GABAergic synaptic transmission system that can
develop in the absence of the normal pattern of afferent and efferent
connections. However, it is clear from a number of studies that
patterns of innervation (Paysan et al. 1997) and
receptor activation (Poulter et al. 1997
) can regulate GABAAR subunit expression in developing cortical
neurons. Thus continued exploration of the functional and molecular
properties of GABAARs mediating synaptic
transmission in developing cortical neurons, under a variety of
conditions, will be important in identifying environmental and genetic
factors that can influence GABAergic synaptic transmission in cortical neurons.
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ACKNOWLEDGMENTS |
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The authors thank A. Agmon for comments on the manuscript and B. Nicolas for expert technical assistance.
This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-27501 and NS-01854 to D. K. O'Dowd, NS-33213 to M. A. Smith, and by American Epilepsy Society Grant EFA-24106 to I. Soltesz.
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FOOTNOTES |
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Address for reprint requests: D. K. O'Dowd, Dept. of Anatomy and Neurobiology, University of California, Irvine, CA 92697-1280.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 June 1999; accepted in final form 20 August 1999.
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REFERENCES |
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