1Department of Neurobiology and 2Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, California 94305-5122
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ABSTRACT |
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Browne, S. H.,
J. Kang,
G. Akk,
L. W. Chiang,
H. Schulman,
J.
R. Huguenard, and
D. A. Prince.
Kinetic and Pharmacological Properties of GABAA
Receptors in Single Thalamic Neurons and GABAA Subunit
Expression.
J. Neurophysiol. 86: 2312-2322, 2001.
Synaptic inhibition in the thalamus plays
critical roles in sensory processing and thalamocortical rhythm
generation. To determine kinetic, pharmacological, and structural
properties of thalamic -aminobutyric acid type A
(GABAA) receptors, we used patch-clamp techniques
and single-cell reverse transcriptase polymerase chain reaction
(RT-PCR) in neurons from two principal rat thalamic nuclei
the reticular nucleus (nRt) and the ventrobasal (VB) complex.
Single-channel recordings identified GABAA
channels with densities threefold higher in VB than nRt neurons, and
with mean open time fourfold longer for nRt than VB [14.6 ± 2.5 vs. 3.8 ± 0.7 (SE) ms, respectively]. GABAA receptors in nRt and VB cells were
pharmacologically distinct. Zn2+ (100 µM)
reduced GABAA channel activity in VB and nRt by
84 and 24%, respectively. Clonazepam (100 nM) increased inhibitory
postsynaptic current (IPSC) decay time constants in nRt (from 44.3 to
77.9 ms, P < 0.01) but not in VB. Single-cell RT-PCR
revealed subunit heterogeneity between nRt and VB cells. VB neurons
expressed
1-
3,
5,
1-3,
2-3, and
, while nRt cells
expressed
3,
5,
2-3, and
. Both cell types expressed more
subunits than needed for a single receptor type, suggesting the
possibility of GABAA receptor heterogeneity
within individual thalamic neurons.
subunits were not detected in
nRt cells, which is consistent with very low levels reported in
previous in situ hybridization studies but inconsistent with the
expected dependence of functional GABAA receptors
on
subunits. Different single-channel open times likely underlie distinct IPSC decay time constants in VB and nRt cells. While we can
make no conclusion regarding
subunits, our findings do support
subunits, possibly
1 versus
3, as structural determinants of
channel deactivation kinetics and clonazepam sensitivity. As the
2
and
subunits previously implicated in Zn2+
sensitivity are both expressed in each cell type, the observed differential Zn2+ actions at VB versus nRt
GABAA receptors may involve other subunit differences.
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INTRODUCTION |
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The thalamus is both the gateway
to the cerebral cortex and an information processing unit. Impulses
arriving from sensory, motor, and even "limbic system" pathways are
processed in specific thalamic relay nuclei and then transmitted to
appropriate cortical areas. The nucleus reticularis (nRt) surrounds the
thalamus like a shield. Fibers interconnecting thalamus and cortex
traverse nRt and establish synapses on reticular neurons (Jones
1985). Reciprocal connections between relay nuclei and nRt
provide an anatomic substrate for intrathalamic oscillations that occur
during slow-wave sleep and the pathophysiological state of absence
epilepsy (Huguenard 1999
; Steriade et al.
1993
). Recurrent inhibitory synapses between nRt neurons serve
to control the output of nRt onto thalamic relay cells and thus form an
important part of intrathalamic connectivity (Huguenard and
Prince 1994b
; von Krosigk et al. 1993
).
Gamma-aminobutyric acid type A (GABAA)
receptor-mediated currents in nRt and relay nuclei are pivotal in
controlling activities in this circuit. Hyperpolarizing inhibitory
postsynaptic currents (IPSCs), which occur following GABA release from
nRt terminals onto relay neurons, evoke rebound bursts by
deinactivating low-threshold T-type calcium channels. The bursts, in
turn, result in a glutamatergic reactivation of nRt neurons. Together
these events are important in development of recurrent oscillatory
thalamocortical rhythms (Huguenard 1999;
Huguenard and Prince 1994a
; von Krosigk et al. 1993
), while the inhibitory connections between nRt cells serve to regulate such rhythms (Huguenard 1999
;
Huguenard and Prince 1994b
; Ulrich and Huguenard
1997
; von Krosigk et al. 1993
). A detailed
understanding of the structure and functional properties of
GABAA receptors in nRt and neurons of relay
nuclei would provide vital information on generation and control of
thalamic oscillations and the processing of sensory information,
improve our understanding of currently used antiepileptic drugs, and
provide opportunities for rational drug design.
The GABAA receptor is a pentameric, ligand-gated
Cl channel. Seventeen mammalian subunits have
been identified. Thirteen subunits in four subfamilies are present in
rat:
1-
6,
1-
3,
1-
3, and
. Studies in
heterologous systems have shown that subunit composition defines
pharmacological and biophysical properties (MacDonald and Olsen
1994
). However, analysis of subunit composition and function of
native receptors has proved difficult.
In these experiments, we examined aspects of structural and functional characteristics of GABAA receptors in neurons of nRt and the ventrobasal nucleus (VB). The aim was to correlate kinetic and pharmacological properties of single channels and whole cell IPSCs in individual nRt and VB neurons with data obtained using RT-PCR to examine expression of GABAA subunits in the same neurons.
GABAA-receptor-mediated IPSCs recorded in
nRt cells have decay time constants (d)
approximately threefold longer than in VB neurons (Ulrich and
Huguenard 1995
; Zhang et al. 1997
). We tested
single-channel properties that might account for this, including
channel density, conductance, and open/closed times. We also
examined the effects of Zn2+ and the
benzodiazepine, clonazepam. Previous experiments on
GABAA receptor-modulated responses in VB
and nRt neurons have suggested that the therapeutic action of
clonazepam in absence epilepsy may be related to differential effects
of the drug in nRt versus thalamic relay nuclei (Gibbs et al.
1996
; Hosford et al. 1997
; Huguenard and
Prince 1994b
; Oh et al. 1995
). Clonazepam
decreases GABAergic output from the nRt nucleus by enhancing
intranuclear recurrent inhibition but has little direct effect on
GABAA responses in the VB nucleus
(Huguenard and Prince 1994b
). To further examine these
actions at a cellular level, we investigated clonazepam's effect on
monosynaptic IPSCs in nRt and VB neurons.
Knowledge of the subunit mRNAs of single nRt and VB neurons would
provide information on the molecular basis for kinetic and pharmacological differences. Using a multiplex-nested strategy, we
performed single-cell RT-PCR on multiple GABAA
receptor subunits simultaneously, following functional characterization
of each cell. We chose RT-PCR as it provides an extremely sensitive
method for detecting mRNA expression of GABAA
subunit isoforms present in individual cytosols. The nested design
(Chiang et al. 1994; Sucher and Deitcher
1995
) ensured a high level of specificity for each subunit
a
necessity, given the evolutionary development of the
GABAA receptor family with its extensive nucleic
acid homology. By sequencing the RT-PCR products, we confirmed the
specificity of the GABAA subunits detected.
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METHODS |
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Brain slices
Brain slices containing thalamus were prepared and maintained in
vitro using previously described techniques (Huguenard and Prince 1994a). Eight- to 13-day-old (P8-P13) Sprague-Dawley
rats of either sex (Simonsen Breeders) were anesthetized with
pentobarbital sodium (55 mg/kg ip) and decapitated. The brain was
rapidly removed, and 300 µm horizontal thalamic slices were cut with
a vibratome (TPI, St. Louis, MO) in a solution containing (in mM) 2.5 KCl, 1.25 NaH2PO4, 10 MgSO4, 0.5 CaCl2, 10 glucose, 26 NaHCO3, and 230 sucrose. Slices
containing nRt and VB were incubated in standard slice solution
containing (in mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 MgCl2, 2 CaCl2, 10 glucose,
and 26 NaHCO3 (pH = 7.4 when gassed with
95% O2-5% CO2) at room
temperature (23-25°C).
Patch-clamp recordings
Slices were incubated for 1-8 h and transferred to the
recording chamber (1.5 ml volume) where they were perfused with the standard slice solution at a rate of 2 ml/min at room temperature. The
chamber was mounted to the fixed stage of a microscope and viewed with
a ×63 objective (Zeiss Axioskop) and DIC optics and infrared
illumination during recordings. Visualization of slices with a
low-power objective (×2.5) allowed identification of nRt and VB.
Cell-attached and outside-out patch clamp configurations (Hamill
et al. 1981) were used to obtain single-channel activity from
visualized neurons in VB and nRt. Recording electrodes with resistances of 4-7 M
were pulled from KG-33 glass capillaries (1.0 mm ID, 1.5 mm OD, Garner Glass) using a PP-83 electrode puller (Narishige, Japan). Seal resistances less than 3 G
were rejected. The solution for filling cell-attached patch pipettes contained (in mM)
130 KCl, 5 TEA-Cl, 2 MgCl2, 10 HEPES, and 4 glucose and 2 µM GABA (pH adjusted to 7.2 with KOH). The
intracellular solution for filling outside-out pipettes contained (in
mM) 130 KCl, 5 EGTA, 2 MgCl2, 10 HEPES, and 4 dextrose (pH adjusted to 7.2 with KOH). Single-channel activities were
recorded with an Axopatch 200A amplifier (Axon Instruments, Burlingame,
CA) and filtered through an eight-pole Bessel low-pass filter with
1-kHz cutoff frequency. Signals were stored both on a videotape
recorder via a Neurocorder converter (Neurodata Instrument) and on
computer. In outside-out patch recordings, 10 µM GABA was pressure
applied (at a rate of 0.05 Hz with 20-ms, 20-kPa pulses) from a puff
pipette positioned about 10 µm from the outside-out patch. The
perfusion solution for Zn2+ experiments contained
126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 MgCl2, 2 CaCl2, 10 glucose,
and 10 HEPES (pH adjusted to 7.4 with NaOH). Dipotassium ATP (ATP),
HEPES, tetra-ethylammonium chloride monohydrate (TEA), ethylene
glycol-bis (b-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), bicuculline, and
-aminobutyric acid (GABA) were purchased from Sigma. Other chemicals
were purchased from Mallinckrodt Specialty Chemicals (Paris, KY).
Whole cell IPSC recordings and clonazepam application
Whole cell voltage-clamp recordings were obtained at a holding
potential (Vhold) of 60 mV using an
Axopatch 1A amplifier (Axon Instruments, Foster City, CA). Spontaneous
IPSCs (sIPSCs) were recorded in the presence of 10 µM
6,7-dinitroquinoxaline-2,3-dione (DNQX) and 5 µM of
R(
)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid
(D-CPP) to block ionotropic glutamate receptors. The
pipette solution contained (in mM) 135 CsCl, 10 EGTA, 2 MgCl2, 5 N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium chloride
(QX-314), and 10 HEPES, 290 mosm, pH 7.3. Signals were filtered
at 2 kHz (
3 dB, 8-pole Bessel filter) and digitized. CDR and SCAN
software, used to collect continuous records and detect spontaneous
events, were provided by Dr. J. Dempster. The sIPSCs were characterized
according to their amplitude, decay time constant and integrated area
(total charge). To evaluate potential presynaptic effects of
clonazepam we compared sIPSC frequencies in the absence and
presence of the drug. In all instances, whole cell activity was
recorded for several minutes to establish baseline values. The
perfusion media was then changed to one containing the indicated
concentration of the agent and after a 2- to 3-min delay to allow the
drug-containing media to fill the recording chamber and the drug to
diffuse into the slice, sIPSC characteristics were again evaluated. Two
minutes was judged to be sufficient for replacement of media in the
chamber since addition of bicuculline methiodide blocked all GABA
receptor-activated currents approximately 1.5 min after addition to
perfusion media (data not shown).
Single-channel data analysis
Single-channel currents were sampled every 50 µs with Fetchex and analyzed with Fetchan programs (Axon Instruments, v 6.0). A 50% threshold criterion was used to determine the durations of open and closed events. The collected open and closed intervals were binned with Pstat, and logarithmic distributions of open and closed durations were exponentially fitted using the Maximal Likelihood method. For I-V analysis, amplitudes were measured manually from long-duration openings. Because most outside-out patches from VB contained multiple channels, we calculated average channel open probability (Po) using equation: Po = Q/(NAt), where Q is the integrated charge, N is the channel number in a patch, A is the amplitude of single channel current, and t the duration of integration. To obtain Q, a 1,000-ms (t) period of current following application of GABA was integrated by Clampfit. N was calculated by dividing the maximal current by A.
RNA harvest and reverse transcription with random primers
NRt and VB neurons were initially identified by anatomical
position in the thalamic slice and morphological appearance under Nomarski optics. All cells from which cytosols were harvested were
characterized electrophysologically prior to PCR subunit analysis and
had characteristic nucleus-specific IPSCs (Huntsman and
Huguenard 2000)
i.e., sIPSCs in VB neurons occurred at a
higher frequency and had more rapid decay kinetics than those in nRt cells.
Positive pressure on the silanized patch pipette as it was lowered into
the preparation prevented contamination of the samples (Chiang
1998). After whole cell voltage-clamp recordings, negative pressure was applied to the pipette and the contents of the cell aspirated into a silanized micropipette; successful harvest was monitored via microscopy. The cytosol was expelled into an RNase-free silanized microcentrifuge tube containing 10 µl of reverse
transcription reagents: 0.5 mM each deoxynucleotide (dNTP; Boehringer
Mannheim, Indianapolis, IN), 5 µM random hexamer oligonucleotide (New
England Biolabs, Beverly, MA), 10 U/µl reverse transcriptase
(Superscript II, Gibco BRL, Grand Island, NY), 2 U/µl RNase inhibitor
(RNasin, Promega, Madison, WI), 10 mM dithiothreitol (DTT), and 50 mM
Tris-HCl (pH, 8.3). The mixture was incubated for 15 min at room
temperature followed by 1 h at 37°C. The reaction was terminated
by 5 µg yeast tRNA (Sigma, St. Louis, MO), 0.3 M sodium acetate (pH,
7.0) and 250 µl ethanol, and the mixture stored at
20°C. Prior to
PCR, the single cell cDNA template was recovered by centrifugation, washed with 70% aqueous ethanol, and resuspended in 3 µl RNase-free H2O, i.e., Millipore filtrated water (Water
Purification Systems, Bedford, MA) autoclaved for 45 min in baked glassware.
PCR amplification
Multiplex PCR with a nested design strategy was performed using
two approaches: in single cytosol experiments, an individual cell
precipitate was used as cDNA template for PCR and in the combined
cytosol experiment, 10 nRt single cytosol and 10 VB single cytosol cDNA
precipitates were combined according to cell type to produce two
templates for PCR. In the first round of PCR, outer primers for each
individual GABAA subunit tested were employed simultaneously (for primer sequences see Fig.
1B). In the single-cytosol experiments, primer sets for 1, -2, -3, and -5 and
2 subunits were used and in the combined single cytosol experiment, 12 primer sets
were used in the first round of PCR. The primer pair sets used for
1
subunit performed inadequately and thus have been excluded from this
analysis.
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The PCR mixture consisted of 50 mM Tris-HCl (pH 8.3), 500 µg/ml bovine serum albumin (BSA), 200 µM dNTP, 0.5 µM of each primer pair, 2 U Taq polymerase (Perkin Elmer Cetus, Foster City, CA) + Taq Start antibody(1:1) (Clontech, Palo Alto, CA), 3 mM MgCl2 and template to a total volume of 10 µl. The PCR conditions were 2 min at 94°, 30 cycles with an annealing temperature of 55°C and 2 min at 72°C (i.e., temp D = 94, A = 55, E = 72; time instantaneous, instantaneous, 0.15; slope, 2.0) performed on Rapid Thermocycler Model No. 1002 (Idaho Technology).
The resulting multiplex amplification product was then diluted into 80-100 µl milliQ H2O. One to 3 µl of the diluted first round PCR product were then used as template for a second round. In this portion of the experiment, parallel PCRs containing one pair of gene-specific nested primers per reaction were performed. The PCR mixture and PCR conditions were the same as in the first round. Positive and negative control samples were run simultaneously with the GABAA subunits through the first and second rounds of PCR. Positive controls included reactions with adult rat brain of cDNA (1 ng/µl) and single-cell template with primers for glyceraldehyde phosphate dehydrogenase (GAPDH). Negative control reactions included template of RNase free H2O and template from mock patch-pipette insertion into preparation without aspiration of a cytosol to monitor harvest contamination.
Gene expression was detected by agarose gel electrophoresis (Metaphor Agarose 3%, FMC, Rockland, ME), stained with ethidium bromide, of the entire product from the second round PCR run in parallel with known molecular weight markers (Lambda DNA restricted with BgII; pBR322 plasmid DNA restricted with HpaII). Expected sizes of the amplified GABAA subunit fragments were calculated from the published sequences (see Fig. 1A). To confirm the identity of the amplified fragments, each product was nucleotide sequenced.
Sequencing protocol
PCR product from each subunit nested primer pair set was gel purified and prepared for sequencing using the PE Applied Biosystems Terminator Ready Reaction Mix (Perkin Elmer Cetus): 8.0 µl terminator ready reaction mix, approximately 85 ng PCR product, 3.2 pmol of 5' nested primer with deionized water to volume of 20 µl. After a brief spin, the Ericomp TwinBlock System thermal cycler Model No. 1002 was used for the following reaction: 96°C for 20 s, repeat for 25 cycles; 96°C for 10 s, 50°C for 5 s, 60°C for 4 min, 20°C for 30 min. Extension product was purified by phenol-chloroform extraction and ethanol precipitation. Air-dried DNA pellet was sent to the Protein and Nucleic Acid (PAN) Facility of Stanford University Medical Center for sequencing.
Statistical analysis
All results are provided as means ± SE, and statistical significance assessed with a two-sided t-test, except in the case of the effects of clonazepam on sIPSC decay, in which an ANOVA with post hoc Tukey test was used.
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RESULTS |
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Single-channel properties of GABAA receptors in cell-attached patches from nRt and VB neurons
The single-channel properties of GABAA
receptors in nRt and VB neurons were examined using the cell-attached
patch configuration. GABA (2 µM) was added to the pipette solution.
GABAA channel activities were seen in 8 of 10 patches from nRt neurons and in 5 of 5 patches from VB neurons. Channel
density was higher for VB (2.8 ± 0.4/patch, n = 5 patches) versus nRt neurons (0.9 ± 0.2/patch, n = 10 patches). The observed channels were GABAA
receptors because no openings were observed in patches obtained with
pipettes filled with solution containing 20 µM bicuculline
(n = 5 patches) and extrapolated reversal potentials
(50 to
60 mV) were close to the calculated ECl (Fig.
2B). The channels from nRt
patches showed longer openings than those from VB patches (Fig.
2A). Both nRt and VB channels showed a major conductance
(Fig. 2A, o) and a subconductance (Fig. 2A, sub)
state. The major conductances were 16 ± 0.5 pS (n = 5 patches) and 17 ± 1.7 pS (n = 5 patches) for
nRt and VB channels, respectively (Fig. 2B). Neither the
single-channel conductance nor the extrapolated reversal potential
showed significant differences between nRt and VB channels (Fig.
2B). Kinetic analysis indicated that open time distributions
for nRt channels were longer than for VB channels (Fig. 2C).
Mean open time for nRt channels was 14.6 ± 2.5 ms
(n = 5 patches), which was significantly longer than
that for VB channels (3.8 ± 0.7 ms, n = 5 patches; P < 0.01).
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Single-channel properties of GABAA receptors in outside-out patches from nRt and VB neurons
Outside-out patches were directly excised from somata of nRt or VB
neurons in slices. When patches were held at 80 mV, very few channel
openings were observed in the absence of GABA (Fig. 3, Aa and Ba,
before
). When 10 µM GABA was applied to the tip of patch
pipettes, frequent channel openings were observed (Fig. 3,
Aa and Ba, after
). In agreement with the
results from cell-attached recordings (above), channel
numbers in outside-out patches from VB neurons (7.17 ± 1.80/patch, n = 6 patches) were
larger than those from nRt neurons (2.14 ± 0.14/patch,
n = 7 patches). As expected, nRt channels showed longer
openings (Fig. 3A, a and b) than VB channels
(Fig. 3B, a and b). All point histograms showed a
well-defined peak associated with the open state for the nRt channels
(Fig. 3Aa, graph at right) but not for VB
channels (Fig. 3Ba, graph at right), likely
because system filtering attenuated the shorter VB openings. Both nRt
and VB channels were sensitive to 20 µM bicuculline (Fig. 3,
Ac and Bc) and showed a major conductance and a
subconductance state (Fig. 3, Ab and Bb, o and
sub). The major conductance was 27.0 ± 0.4 pS (n = 6 patches) and 27.5 ± 1.2 pS (n = 2 patches)
for nRt and VB channels, respectively (Fig. 3C).
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Interestingly, it has previously been reported that miniature IPSC
(mIPSC) amplitude is comparable in nRt and VB cell recordings (Ulrich and Huguenard 1996). The threefold higher
GABAA channel density in somatic VB membranes,
obtained with both outside-out and cell-attached patches, suggests that
synaptic channel counts are unrelated to somatic channel density.
Sensitivity of nRt and VB channels to Zn2+
We further tested pharmacological properties of nRt and VB channels by perfusing outside-out patches with a solution containing 100 µM Zn2+. The activity of nRt channels was slightly inhibited (Fig. 4A, top), while VB channel openings were markedly attenuated by Zn2+ (Fig. 4A, bottom). To quantitatively analyze Zn2+ effects, we calculated channel open probability (Po). Because most patches from VB neurons contained multiple channels, a value of averaged Po was obtained by integration (see METHODS). In control recordings, nRt and VB channels showed similar average Po (0.60 ± 0.04, n = 8 patches and 0.55 ± 0.05, n = 5 patches, respectively) after exposure to GABA (Fig. 4B, left). When a solution containing 100 µM Zn2+ was perfused, Po for VB channels was reduced to 0.09 ± 0.01 (n = 5 patches) while Po for nRt channels decreased to 0.45 ± 0.03 (n = 8 patches). Reduction of VB channel activity by 100 µM Zn2+ (84%) was significantly larger than that for nRt channel activity (24%) (Fig. 4B, right, P < 0.01). This result indicates that VB GABAA receptor channels are more sensitive to Zn2+ than nRt channels.
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Spontaneous IPSCs in nRt and VB cells and the effect of clonazepam
In the neurons of the present experiments, the IPSC decay was
reasonably described by a single exponential decay. As previously reported (Zhang et al. 1997), sIPSC decay time constants
were slower for nRt (45.2 ± 12.1; n = 9) than for
VB neurons (17.3 ± 5.3 ms; n = 8). The 10-90%
rise times were 1.7 ± 0.5 ms for nRt and 1.8 ± 0.5 ms for
VB neurons. Typical sIPSCs are shown in Fig.
5. The frequency of sIPSCs was 1.2 ± 1.0 Hz in nRt neurons and 5.6 ± 5.0 Hz in VB cells. sIPSCs
were completely blocked by 20 µM bicuculline methiodide applied to
the bath (data not shown).
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We studied the effect of nM concentrations of clonazepam (CZP) on sIPSCs from 17 nRt and 15 VB neurons. Figure 6A shows the effect of 50 nM clonazepam on sIPSCs from nRt and VB. The traces are averaged spontaneous events (n > 20) recorded before and after the addition of CZP. At this concentration, CZP selectively prolonged the time course of sIPSCs in nRt.
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These effects in nRt were concentration dependent, as shown in Fig. 6,
B and C, for 10-100 nM CZP. Results are
summarized in Table 1. In the presence of
100 nM CZP, d in nRt increased 76% from 44.3 to 77.9 ms (P < 0.01); however, in VB the increase was
insignificant, from a baseline value of 15.6 to 21.4 ms (37%; P > 0.05). In the reticular nucleus, all cells
(n = 8) were influenced by clonazepam, and the
integrated area of sIPSCs increased by 79%, from 1.85 pC in control to
3.31 pC after CZP (P < 0.01), while in VB, the total
charge was not affected (0.92 and 0.99 pC in control and CZP,
respectively; P > 0.05; Fig. 6C).
Clonazepam had no effect on amplitude of sIPCSs at either site (data
not shown).
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In nRt cells, the effects of 100 nM CZP on d
and total charge were statistically significant (P < 0.05 and P < 0.01, respectively), whereas in VB, there
was a small, statistically insignificant increase in
d (Fig. 6B). The frequency of
sIPSCs was not altered significantly in either nucleus by clonazepam.
Control sIPSC amplitudes in nRt and VB neurons were not significantly
different, and at these concentrations, clonazepam had no significant
effect on amplitude.
Expression of GABAA subunit mRNA
To elucidate the molecular basis for the differences observed, we used multiplex nested single-cell RT-PCR. We found it was not possible to use degenerate primers and amplify each subunit equivalently in our multiplex system. This was due to the heterogeneity of nucleotides observed, even within families. For this reason, individual primer pair sets were used for each subunit (see Fig. 1A). Primer pairs were carefully and extensively tested; all first round primers were optimized to the same conditions allowing simultaneous amplification (data not shown). Nested primers were designed to cross the highly variable transmembrane region (see Fig. 1A). Despite this, it was necessary to amplify smaller fragments than initially expected to ensure specific subunit amplification.
We performed two experiments, the first on single cytosols looking at 5 GABAA subunits simultaneously and the second on
samples combining 10 cytosols of each cell type looking at 12 GABAA subunits. Initially, PCR amplification was
performed on single-cell cytosols. Cells without PCR product were
rejected, 11 single cells were analyzed: 7 VB cells and 4 nRt cells.
Both positive and negative controls were performed through both rounds
of PCR (see experimental procedures). Table
2 shows expression of 1-
3 and -5 and
2 in these cells. Expression of
subunits was markedly
different between VB and nRt cells. In general, VB cells expressed more
subunits. All seven VB cells expressed
1, and some expressed all
four
subunits. The only
subunits observed in nRt cells were
3 and
5 (1 each in 1 of 4 cells).
2 signal was detected in
both types of cells. Figure 7 provides an
example of
1 signals detected in 4/4 VB and 0/3 nRt cells and
2
detected in all cells.
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Cell-to-cell differences in signal detection may reflect heterogeneity in levels of single-cell gene expression. Signal detection is also affected by the sensitivity of the assay and the adequacy of the cytosol harvest. As regards sensitivity, we estimated, using serial dilution of template, that under optimal multiplex RT-PCR assay conditions for 6 and 12 primers, approximately 30 molecules of RNA could be detected (data not shown). In other words, to detect a signal, at least 30 molecules of mRNA had to be present in the cytosol harvested.
To avoid limits associated with unequal harvest of cytosols from
individual cells, we combined and analyzed 10 cytosols of each type. By
combining cytosols the likelihood of missing contributing portions of
cytosol was minimized. Further, the increased template concentration
allowed simultaneous use of 12 subunit primer pairs. Table
3 shows expression of
GABAA subunits in 10 pooled VB and 10 nRt cells.
Figures 8 and
9 show the corresponding
ethidium-bromide-stained metaphor agarose gels. There were marked
differences between VB and nRt cells in the expression of subunit
subtypes in the combined cytosol experiment, a finding similar to that
in single cells. The nRt cells analyzed appeared to express only
3
and -5 subtypes, whereas VB neurons expressed
1-
3 and -5. As
expected, alpha 6 was not seen in either cell group (McKernan
and Whiting 1996
). The
4 subunit was not found in either
cell type, although, it was present in all the positive controls.
2
(both splice variants) and
3 were observed in both nRt and VB cells
(Fig. 9B). No obvious signal for any
subunit was found
in nRt, while all three
subunits were found in VB cells. Signal for
the delta subunit was also found in both cell types.
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Summary of results
In this study, we provide evidence that kinetic and
pharmacological differences in GABAA receptors in
single thalamic neurons are associated with heterogeneity of
GABAA subunit expression. VB cells had shorter
single-channel open times than nRt cells, and VB single-channel
activity was significantly reduced by 100 µM
Zn2+. Whereas nRt cells showed a significant
increase in IPSC d when exposed to nanomolar
clonazepam, VB cells did not. VB cells studied expressed
1-
3 and
-5,
1-
3,
2 and
3, and
, while nRt cells expressed
3
and -5,
2 and -3, and
GABAA receptor subunits.
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DISCUSSION |
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Distinct single-channel properties of GABAA receptors (GABAARs) in nRt and VB cells underlie differences in IPSCs
Openings of nRt GABAA channels, recorded in
both outside-out and cell-attached patches, were about threefold longer
than for VB channels. Therefore the previously reported difference in
sIPSC duration between nRt and VB neurons (Zhang et al.
1997) can largely be explained by differences in single-channel
kinetics. Differences in rise times have not been observed, which
suggests that the on-rate for GABA is not different for the two
GABAA channel types. Further, differences in
single-channel amplitudes or subconductance states were not observed.
Channel density was much higher in VB cells, which is in agreement with
the differences in evoked IPSC amplitude in the two cell types
(Zhang et al. 1997
).
GABAAR subunit mRNA expression in VB vs. nRt cells
Our results indicate that the major difference in subunit
expression in neurons of VB versus nRt is in the and
subunits. VB cells expressed
1-
3 and -5 and
1-
3, while nRt cells
studied expressed
3 and -5. Neurons of both structures expressed
2 and -3 and
. Our findings are relevant for rat brain aged 8-12
days (i.e., P8-P12). The GABAA receptor in the
thalamus reaches a mature configuration by the end of the third
postnatal week (Bentivoglio et al. 1990
). Few prior
studies of postnatal GABAA subunit mRNA distribution in the rat brain have distinguished between nRt and VB
nuclei; rather, results have focused on expression in the entire thalamus or diencephalon (Laurie et al. 1992
;
Poulter et al. 1993
). Because of this, we have compared
our results to those in both immature and adult rats.
Our findings of 1-
3 and -5 subunits in VB cells are consistent
with literature for rats of this age (Fritschy et al.
1994
; Laurie et al. 1992
).
3 subunits are
also known to be expressed in nRt (Fritschy et al. 1994
;
Laurie et al. 1992
; Wisden et al. 1992
).
2 subunits (both splice variants) have been detected in both VB and
nRt in immature (Laurie et al. 1992
; Poulter et
al. 1993
) as well as in adult animals (Fritschy et al.
1994
; Wisden et al. 1992
). Similarly,
3 is
present in thalamus in immature animals (Laurie et al.
1992
; Poulter et al. 1993
) and in both nuclei in
adults (Wisden et al. 1992
). The
subunit, which we found in both cell types, has been detected in thalamic nuclei from p12
(Laurie et al. 1992
) but not consistently
(Poulter et al. 1993
). In adult animals,
is
reportedly present in nRt and VB (Fritschy and Mohler
1995
) or in ventroposterior medial nucleus only (Wisden
et al. 1992
).
Interestingly, we did not observe a signal for 4 in either cell
type, although strong signals were found in all positive controls. This
is in contrast to previous in situ hybridization and immunostaining
results (Fritschy and Mohler 1995
; Laurie et al.
1992
; Pirker et al. 2000
) and might be
due to the developmentally immature animals studied or could be a false
negative result.
All three isoforms were present in VB cells. We did not detect
isoforms in nRt, a finding consistent with previous immunohistochemical and in situ hybridization studies which failed to detect
2/
3 protein or mRNA in nRt (Fritschy and Mohler 1995
;
Fritschy et al. 1994
; Spreafico et al.
1993
). However, a recent study using specific antibodies
demonstrated
1- and
3-like immunoreactivity in neuropil and
neuronal processes of nRt (Pirker et al. 2000
). Similarly, an in situ hybridization study of
GABAA receptors in monkey thalamus demonstrated
low but detectable levels of
1 and
3 subunits in the reticular
nucleus (Huntsman et al. 1996
). Recently, Huntsman et al. (1999)
found that mice devoid of
3
have severely disrupted intra-nRt inhibition and display thalamic
hypersynchrony, which strongly implicates a role for
3 in nRt
function. The results of the latter three studies suggest that the very
low (or in this case, even below the threshold for detection) levels of
subunit mRNA (Wisden et al. 1992
; this study) are
sufficient for expression of functionally relevant levels
subunit
protein. Alternatively, nRt neurons may possess functional
GABAA receptors with no
subunits. Fritschy et al. (1992)
and Fritschy and Mohler
(1995)
suggested that a minor GABAA
receptor subtype, composed of
subunits, exists in rat brain. In
recombinant systems
,
and
subunits are generally considered
necessary for fully functional GABAA receptors
(Pritchett et al. 1989
). However, there are multiple examples of functional GABAA receptors composed
of
,
subunit combinations that exhibit GABA-gated chloride
channels modulated by benzodiazepine site ligands (Granja et al.
1997
; Im et al. 1993
; Knoflach et al.
1996
; Wong et al. 1992
, in HEK cells;
Angelotti et al. 1993
, in L929 fibroblasts). Such
receptors have a main-conductance level of 30pS (Verdoorn et al.
1990
), similar to those in neuronal GABAA
receptor channels (Angelotti and Macdonald 1993
), and
could potentially subserve GABAA receptor
function in nRt.
subunits appear pivotal in the differential kinetics of
GABAARs in VB and nRt cells
Consideration of the kinetic analysis of IPSCs, together with
subunit expression patterns, suggests that the significant differences in decay time constants in nRt and VB cells may correlate with differences in the or
subunits. Considerable work from
recombinant systems suggests the structural determinant of
GABAA receptor channel activation/deactivation
kinetics resides with the
subunit (Gingrich et al.
1995
; Lavoie et al. 1997
; Tia et al.
1996
; Verdoorn 1994
). Gingrich et al.
(1995)
indicated that substitution of
3 for
1 in a
recombinant receptor slowed deactivation threefold. Verdoorn
(1994)
also found that the decay time courses of currents mediated by receptors containing
3 were slower than those containing
1, or, both
1 and
3. Given such work in recombinant systems, it is likely that the slower
d in nRt versus
VB neurons relates to the presence of
1 in VB and not nRt cells, and
from our data and that of others (Fritschy et al. 1994
;
Laurie et al. 1992
), the incorporation of an
3
subunit in nRt neuronal GABAA receptors.
Pharmacological differences in GABAARs in nRt and VB cells are associated with heterogeneity in subunit expression
The reduction of single GABAA channel
activity in VB neurons by 100 µM Zn2+ was
significantly larger than that produced in nRt neurons (Fig. 4). This
is consistent with recent results obtained using whole cell recordings
of acutely isolated thalamic neurons (Gibbs et al.
2000). Our RT-PCR data indicate the presence or absence of subunits but do not describe the stoichiometry of the receptor. This
limitation must be kept in mind in interpreting these results. We
observed differential expression of
and
subunits in nRt and VB
cells. In heterologous systems, three subunits have been shown to have
significant roles in Zn2+ sensitivity:
subunits are associated with zinc insensitivity;
subunits are
associated with Zn2+ sensitivity, even in the
presence of
subunits (Saxena and MacDonald 1994
);
and the second transmembrane domain of
subunits has been identified
as a Zn2+ binding site on the murine
GABAA receptor complex (Wooltorton et al.
1997
). VB cells were Zn2+ sensitive and
yet expressed
subunits, a finding also reported in a single-cell
RT-PCR study of dentate gyrus granule cells (Berger et al.
1998
). In fact, our RT-PCR data from native cells showed
and
subunits in both nRt and VB neurons. If the
Zn2+ binding site is on the rat
subunits, as
in the mouse, our data would suggest that nRt cells failed to respond
to Zn2+ due to the low expression of
subunits.
We showed that clonazepam at nanomolar concentrations specifically
increases IPSC d in nRt and not VB, while the
sIPSC frequency was not affected. Clonazepam enhances recurrent
inhibitory strength within the reticular nucleus (Huguenard and
Prince 1994b
). This results in a decreased ability of
neighboring inhibitory neurons to fire synchronously and produce the
powerful inhibitory responses required for network synchronization
(Huguenard 1999
). Our results suggest that enhanced
recurrent intra-nRt inhibition produced by clonazepam is due to the
slowing of
d of IPSCs in individual nRt neurons.
Prior studies examined the effect of a broader range of clonazepam
concentrations on GABA responses in acutely isolated neurons. While
clonazepam affects the GABAA currents in both nRt
and VB cells, it consistently has a higher potency in nRt cells
(Gibbs et al. 1996; Oh et al. 1995
). Work
from recombinant systems has shown that clonazepam has greater efficacy
as a positive modulator of GABA-elicited currents in receptors
containing the
3 as opposed to the
1 subunit (Puia et al.
1991
; Verdoorn 1994
). The pattern of subunit
expression we observed in nRt and VB cells did show differences in
1
subunit expression (Tables 2 and 3), suggesting this difference may be
relevant to diazepam/clonazepam sensitivity in native systems.
We found evidence of more subunits expressed in the cytosol than are
necessary for the formation of a single receptor type, suggesting each
cell may have more than one type of GABAA
receptor. Other single-cell RT-PCR studies on
GABAA subunits in native cells have also found
supernumerary subunits (e.g., dentate gyrus) (Berger et al.
1998) and evidence for heterogeneity of
GABAA receptors in different portions of
individual neurons has been reported in the cerebellum (Brickley
et al. 1999
; Nusser et al. 1996a
). Immunogold
localization analysis (Nusser et al. 1996a
, 1998
) could provide useful data on possible GABAA receptor
heterogeneity within individual VB and nRt cells.
This study provides evidence via RT-PCR for molecular heterogeneity of GABAA receptors within the thalamus. Molecular heterogeneity has long been suspected as having important consequences for generation and control of thalamic oscillation and processing of sensory information. This has been difficult to directly demonstrate. However, when we combine our kinetic, pharmacological, and RT-PCR analyses in VB and nRt cells with observations from heterologous systems, interesting insights emerge.
Here we provide evidence that subunit heterogeneity contributes to
different IPSC durations in VB and nRt cells. Further, differential
expression of
1 versus
3 likely results in differential pharmacology such that inhibitory connections between nRt cells are
specifically enhanced by low concentrations of clonazepam. The net
effect is an enhanced intra-nRt recurrent inhibition that decreases
inhibitory output to VB, an action that can explain the therapeutic
effect of clonazepam in absence epilepsy (Huguenard and Prince
1994b
). Combined molecular and electrophysiological approaches
promise to provide valuable information about the operation of the
thalamic networks and selective modulation of circuit activities by
existing and new therapeutic agents.
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ACKNOWLEDGMENTS |
---|
We thank N. Sotelo-Kury for sequencing PCR products and excellent technical assistance and C. L. Cox and D. Ulrich for participating in preliminary experiments critical to this project.
This work was supported by National Institutes of Health Grants NS-06477, NS-34774, NS-07280, and GM-40600 and by the Pimley research and training funds.
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FOOTNOTES |
---|
Address for reprint requests: J. R. Huguenard (E-mail: john.huguenard{at}stanford.edu).
* S. H. Browne and J. Kang contributed equally to this work.
Received 8 November 2000; accepted in final form 31 July 2001.
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REFERENCES |
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