Department of Neurology and Neurological Sciences, Stanford University Medical Center, Stanford, California 94305-5300
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ABSTRACT |
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Huntsman, Molly M. and
John R. Huguenard.
Nucleus-Specific Differences in
GABAA-Receptor-Mediated Inhibition Are Enhanced During
Thalamic Development.
J. Neurophysiol. 83: 350-358, 2000.
Inhibitory
postsynaptic currents (IPSCs) mediated by GABAA receptors
are much slower in neurons of the thalamic reticular nucleus (RTN)
versus those in the ventrobasal complex (VB) of young rats. Here we
confirm and extend those findings regarding GABAA response
heterogeneity especially in relation to development. Whole cell
patch-clamp recordings were used to investigate GABAA spontaneous and electrically evoked IPSCs (sIPSCs/eIPSCs) in RTN and VB
cells of different aged rats. Consistent with earlier findings, sIPSC
duration at P8-12 was considerably longer in RTN (weighted decay time
constant: D,W = 56.2 ± 4.9 ms; mean ± SE) than in VB (
D,W = 15.8 ± 1.0 ms) neurons.
Decay kinetics in RTN neurons did not differ at P21-30 (45.5 ± 4.7 ms) or P42-60 (51.6 ± 10.6 ms). In contrast, VB sIPSCs were
significantly faster at both P21-30 (
D,W = 10.8 ± 0.9 ms) and P42-60 (
D,W = 9.2 ± 0.4 ms) compared with P8-12 animals. IPSCs displayed differential
outward rectification and temperature dependence, providing further
support for nucleus-specific responses.
D,W increased
with membrane depolarization but with a net larger effect in VB. By
contrast,
D,W was always smaller at higher temperatures
but with relatively greater difference observed in RTN. Thus nuclear
differences in GABAA IPSCs are not only maintained, but
enhanced in the mature rodent under physiological conditions. These
findings support our hypothesis that unique GABAA receptors
mediate slowly decaying RTN IPSCs that are a critical and enduring
feature of the thalamic circuit. This promotes powerful intranuclear
inhibition and likely prevents epileptiform thalamocortical hypersynchrony.
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INTRODUCTION |
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The reticular nucleus (RTN) is composed of
GABAergic neurons and provides nearly all of the inhibitory
neurotransmission to relay cells in the rodent ventrobasal complex (VB)
(Benson et al. 1992; Houser et al. 1980
;
Ohara and Lieberman 1993
; Williams and Faull
1987
). RTN neurons send a major projection to relay nuclei and
receive extensive innervation from collaterals of corticothalamic and
thalamocortical fibers (Jones 1975
). However, they also
are connected internally to each other through sparse axon collaterals and dendrodendritic connections in rodents and cats (Cox et al. 1996
; Deschênes et al. 1985
;
Pinault et al. 1997
; Scheibel and Scheibel
1966
; Yen et al. 1985
). The intra-RTN
connections are critical for regulating inhibitory output and phasic
bursting activity in rodents and ferrets (Huguenard and Prince
1994
; Huntsman et al. 1999
; Sanchez-Vives
et al. 1997
; von Krosigk et al. 1993
). Thus
during thalamocortical oscillations, this intranuclear pathway may
prevent the pathological hypersynchrony of absence epilepsy, although
intracortical mechanisms are also likely to be important in the genesis
of such synchrony (e.g., Steriade and Contreras 1998
).
The synaptic properties of RTN neurons indicate that these sparse
intra-RTN connections, are compensated functionally with long-lasting
GABAA-receptor-mediated inhibitory postsynaptic
currents (IPSCs). These IPSCs decay three times slower than those in VB neurons (Zhang et al. 1997
) and are differentially
modulated by GABAA receptor ligands (Akk
et al. 1997
; Huguenard and Prince 1994
). These
properties are likely the result of nucleus-specific expression of
GABAA receptor subunits in the thalamus
(Fritschy and Möhler 1995
; Huntsman et al.
1996
; Wisden et al. 1992
).
GABAA receptor heterogeneity and function is
determined ultimately by the combination of subunits within a
pentameric chloride ion channel (Macdonald and Olsen
1994). Subunit mRNAs exhibit strict regional expression
throughout the CNS (Wisden et al. 1992
), and in the
thalamus, the distribution is specific to individual nuclei
(Huntsman et al. 1996
). In addition, thalamic
GABAA receptor subunits are regulated
developmentally (Gambarana et al. 1991
; Laurie et
al. 1992
; Poulter et al. 1992
). In most thalamic
nuclei, there is a developmental turnover of subunits somewhere between postnatal day 6 (P6) and P12 (Laurie et al. 1992
).
However, there are exceptions to this switch, especially in the RTN,
where expression of the early postnatal subunits remain in the adult
animal (Fritschy and Möhler 1995
; Huntsman
et al. 1996
; Laurie et al. 1992
; Wisden et al. 1992
).
Previous studies have shown that differences in kinetic properties of
IPSCs recorded in VB and RTN are likely due to distinct combinations of
native GABAA receptors (Zhang et al.
1997). However, these experiments were carried out in young
animals (P8-P12) and thus during the proposed developmental switch in
subunit composition. To determine if developmental changes in receptor
isoforms result in altered kinetic properties, spontaneous and evoked
IPSCs were studied in the maturing and adult rat. In the present study,
the biophysical properties of both spontaneous and evoked IPSCs were examined at three different ages: one early, at a stage when inhibitory synaptic activity first can be detected reliably (P8-12), which is
during the switch; another time point 11 days subsequent to this switch
(P21-30); and a final time point in the fully mature rat (P42-60). We
also examined the effects of temperature and voltage-dependent
modifications of IPSCs in VB and RTN neurons from adult rats to provide
further support for functional heterogeneity in
GABAA receptors.
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METHODS |
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Preparation of thalamic slices
All experiments were carried out in accordance with approved procedures (Protocol 4450/0) established by the Administrative Panel on Laboratory Animal Care at Stanford University. In these experiments, Sprague-Dawley rats of either sex were used from three different age groups: P8-12, P21-30, and P42-60. Animals were anesthetized with an intraperitoneal injection of 55 mg/kg pentobarbital sodium (Nembutal) until unresponsive. The anesthetized animals were decapitated, and brains were blocked, removed and placed in ice-cold, oxygen equilibrated (95% O2-5% CO2) choline chloride slicing solution for ~2-3 min. The slicing solution consisted of (in mM) 111 choline chloride (C5H11ClNO), 26 NaHCO3, 10 glucose, 2.5 KCl, 1.25 NaH2PO4*H2O, 10 MgSO4*7H2O, and 0.5 CaCl2*2H2O and was adjusted to 290 mOsm. Horizontal slices were cut at a 200-µm thickness with a Vibratome (TPI, St. Louis, MO), hemi-sected, and submerged in preheated (32°C), oxygen-equilibrated physiological saline [which contained (in mM): 126 NaCl, 26 NaHCO3, 10 glucose, 2.5 KCl, 1.25 NaH2PO4*H2O, 2 MgCl2*6H2O, and 2 CaCl2*2H2O] for 1 h, and sections were allowed to cool to room temperature.
Electrophysiology
Whole cell patch-clamp recordings were obtained similarly to our
previous study with noted exceptions (Zhang et al.
1997). Recordings were obtained from slices placed in a chamber
with a continuous perfusion of physiological saline (2 ml/min) at room temperature. In some cases, the temperature was increased to 36°C by
preheating the saline immediately before entering the recording chamber. A fine temperature probe (Cole Parmer, Vernon Hills, IL) was
placed within 200 µm of the recording electrode to assure accurate
measurements of the recorded cell. Recording pipettes were filled with
cesium chloride adjusted to 7.3 pH and 290 mOsm. The solution contained
(in mM) 135 CsCl, 5 lidocaine N-ethylbromide (QX-314), 2 MgCl2, 10 ethyleneglycol-bis (
-aminoethyl ether)-N, N,N',N'-tetraacetate acid (EGTA; Sigma, St. Louis, MO),
and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES; Sigma).
Thalamic neurons were voltage-clamped at a holding potential of 60 mV
for continuous records of spontaneous and evoked inhibitory events
using a List-Medical Patch Clamp amplifier (model L/M-EPC7, Darmstadt,
Germany). Neurons were visualized using a fixed staged, upright
microscope (Zeiss) equipped with a ×63 insulated objective, infrared
(IR) illumination, Nomarski optics and an IR-sensitive video camera
(COHU). Glass electrodes (KG-33 borosilicate glass, 1.0 mm ID, 1.5 mm
OD; Garner Glass Company, Claremont, CA) were pulled in four stages
with a Flaming/Brown micropipette puller (Model, P-87, Sutter
Instruments, San Francisco, CA). All micropipette electrodes had a
final resistance between 2.0 and 3.3 M
when filled with internal
pipette solution. A bipolar tungsten electrode (impedance 0.1-1 M
,
FHC, Brunswick, ME) was placed in RTN to activate neurons (0.5-2mA,
30-100 µs) and evoke inhibitory responses. Voltage dependence was
assessed by determining the rectification ratio. For this purpose,
amplitude,
D,W and charge were measured at both +60 and
60 mV for responses in individual neurons. Temperature dependence was
estimated for all the following biophysical parameters: rise time, peak
current amplitude,
D,W,
D1, and
D2 (1st- and 2nd-order exponential decay) using the
equation, Q10 = (X2/X1)10/(T2
T1),
where X = parameter and T = temperature (Otis and Mody 1992
).
GABAA-receptor-mediated IPSCs were isolated
pharmacologically by bath application of the ionotropic excitatory
amino acid receptor blockers: 6,7-dinitro-quinoxaline-2,3-dione (DNQX,
20 µm, RBI, Natrick, MA) and (±)-2-amino-5-phosphonopentanoic acid (AP-5, 100 µm, RBI) in physiological saline. All such glutamatergic excitatory responses were blocked within 2-3 min after the onset of
perfusion. GABAB receptors were blocked with
Cs+ and QX-314 in the internal pipette solution. In some
cases, the identity of the GABAA-dependent IPSCs was
confirmed by blockade with bath application of 10 µm bicuculline
methiodide. Access resistance of all recorded cells included in this
study were monitored constantly throughout each experiment and only
used for analysis if it was <14 M and stable (25% tolerance) for
the duration of the experiment.
Data analysis
IPSCs were filtered at 1 kHz and stored in digitized form on
videotape (model DR484, Neurodata). Continuous records of spontaneous events initially were collected with Axotape v2.0 (Axon Instruments, Foster City, CA), sorted with the customized software Detector v. 4.8 (J. R. Huguenard) and Metatape v14.0 (J. R. Huguenard) and
analyzed with Scan software (courtesy of J. R. Dempster). Background root mean square (RMS) noise was determined from event-free portions in the control record or from recordings in which sIPSCs had
been blocked with 10 µM bicuculline methiodide. A software trigger
was set to only accept sIPSCs that were more than three times the RMS
level for that particular recording. Evoked responses were collected
with Clampex version 5.5.3 (Axon Instruments) and traces were fit with
Clampfit version 6.0.1 (Axon Instruments) and Metatape. Data were
analyzed with Origin (MicroCal Software, Northhampton, MA) and Excel
(v7.0, Microsoft). Instat (v2.03 GraphPad, San Diego, CA) software was
used to measure significance of all biophysical parameters using
Student's t-tests, one-way ANOVA and Tukey-Kramer post
hoc multiple comparisons test when one-way ANOVA tests yielded
P < 0.05. The decay of averaged IPSCs
(N > 50 events/cell) was fit to the following
equation: I = A1expt/
D1 + A2e
t/
D2 and weighted
decay time constant (
D,W) was determined by the following formula:
D,W = (
D1A1 +
D2A2)/(A1 + A2).
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RESULTS |
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Neurons in the RTN and VB complex were identified by their
position and morphology in the ventral and dorsal thalamus. In horizontal sections, the RTN is positioned medial to the large fiber
bundle of the internal capsule and lateral to VB and separated from the
latter by the thin fiber bundle of the external medullary lamina
(Jones 1985). All recordings were made
50 µm within
these borders for each respective nuclear group to guarantee recordings were obtained from identified neurons. An additional physiological criterion for cell identification was the demonstration of the characteristically slower inactivation of the transient calcium current
in RTN versus VB neurons (Huguenard and Prince 1992
).
All GABAA-receptor-mediated IPSCs were recorded
from RTN and VB neurons with cesium-chloride-filled electrodes. IPSCs
reversed near the chloride equilibrium potential (~0.5 mV) and thus
were inward events at a 60-mV holding potential (Fig.
1).
GABAA-receptor-mediated IPSCs were isolated from
GABAB and ionotropic glutamate receptors (see
METHODS) and completely blocked by bath application of
bicuculline methiodide (not shown). The data in this report represent
voltage-clamp recordings from 128 neurons (58 RTN and 70 VB) in
thalamic slices from rats of the three different age groups.
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sIPSCs in RTN versus VB neurons in young rats within postnatal days 8-12
sIPSCs recorded in the RTN of P8-12 rats were slowly decaying
events with a mean weighted decay time constant
(D,W) of 56.2 ± 4.9 ms
(n = 10; Fig. 1A). Consistent with previous
results (Zhang et al. 1997
), sIPSCs in VB neurons
decayed about three times faster (
D,W = 15.8 ± 1.0 ms, n = 18, P < 0.0001, Student's t-test) than sIPSCs from RTN neurons at
this age (Fig. 1B). As expected, peak current amplitude was
highly variable in both types of thalamic neurons (Zhang et al.
1997
). Mean sIPSC amplitude and frequency were lower in RTN
neurons (
35.7 ± 4.0 pA, 3.0 ± 1.0 Hz, n = 10) than in VB neurons (
71.0 ± 8.9 pA, 6.7 ± 1.2 Hz,
n = 18; amplitude: P < 0.01;
frequency: P < 0.05; Student's t-test).
RTN sIPSC amplitudes in these immature neurons were somewhat smaller
than reported in our earlier study, likely reflecting improved
methodology for recording and detecting these events (see
DISCUSSION). However, consistent with our earlier report,
sIPSC rise times (calculated as the time required to rise from 10 to
90% of peak amplitude) were similar for RTN (1.2 ± 0.1 ms,
n = 10) and VB (1.1 ± 0.1 ms, n = 18, P = 0.9, Student's t-test) neurons. The
higher frequency and amplitude of events in VB neurons were correlated
with increased baseline noise levels. The different background noise
values in VB (5.9 ± 0.5 pA) versus RTN neurons (2.7 ± 0.2 pA, n = 10, P < 0.0001) may reflect
differences in ambient GABA levels (Tia et al. 1996
)
between these two nuclei.
Developmental changes of sIPSCs in older animals
To determine if similar decay kinetics exist in older animals,
sIPSCs were recorded from both RTN and VB cells in juvenile (P21-30)
and adult (P42-60) rats and compared with IPSCs from P8-12 rats
(Figs. 2 and
3). sIPSCs in RTN neurons were variable from cell to cell at all ages but no significant changes in decay rate
were found at either P21-30 (D,W of 45.5 ± 4.7 ms, n = 27) or P42-60 (51.6 ± 10.6 ms,
n = 10; P = 0.48, 1-way ANOVA; Fig. 2,
A and C). Mean peak IPSC amplitude in RTN neurons
showed a decreasing trend in older animals (P21-30:
31.2 ± 2.0 pA, n = 28; P42-60:
25.5 ± 2.6 pA,
n = 10); however, these values failed to reach
statistical significance (P = 0.1, 1-way ANOVA; Fig. 2,
A and C). Rise times in RTN neurons were
indistinguishable at all ages, showing similar values at P21-30
(1.12 ± 0.1 ms, n = 27) and at P42-60 (1.15 ± 0.1 ms, n = 10, P = 0.9, 1-way
ANOVA; Figs. 1C and 2C). The frequency of sIPSCs
in RTN neurons did not show significant changes at P21-30 (2.4 ± 0.3 Hz) or P42-60 (3.8 ± 1.3 Hz, P = 0.4, 1-way
ANOVA; Fig. 2, A and C). Baseline noise levels
were also similar at all three ages. Overall, sIPSCs in RTN appear to
have taken on a mature form as early as P8-12.
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We next examined the biophysical properties of sIPSCs in VB neurons in
the maturing rat (Fig. 2, B and D). By contrast
with RTN neurons, sIPSCs in VB neurons were significantly faster at both P21-30 (D,W 10.8 ± 0.9 ms,
n = 29) and P42-60 (9.2 ± 0.4 ms,
n = 11; P < 0.0001, 1-way ANOVA;
Tukey-Kramer multiple comparisons test for P8-12 vs. P21-30 and
P8-12 vs. P42-60: P < 0.001). Mean peak current
amplitude was also significantly smaller at P21-30 (
35.2 ± 2.4 pA, n = 29) and like the weighted decay values, these differences were maintained at P42-60 (
38.2 ± 3.4 pA,
n = 11; P < 0.0001, 1-way ANOVA;
Tukey-Kramer multiple comparisons test for 8-12 vs. 21-30:
P < 0.001, 8-12 vs. 42-60: P < 0.01; Fig. 2, B and D). sIPSC properties for
typical VB neurons at P8-12 and P21-30 are shown in Fig. 3. The
averaged sIPSC is seen to be smaller, faster and overall much less
effective (lower total charge) in the P21-30 neuron. Histograms
representing peak amplitude and duration measured at 50% of the total
decay (or half-width) for the two neurons illustrate different
distributions of events at these two developmental stages (Fig.
3B). Note that the amplitudes and half-widths were largely
overlapping in their distributions but that the peak of the
distribution was shifted toward faster and briefer events in the more
mature neuron. In general, these distributions could not be readily
fitted with simple Gaussian distributions, suggesting that each was
composed of at least two subpopulations. These differences in kinetics
may be the result of a different degree of electrotonic filtering in
young versus mature neurons. The latter have a more extensive dendritic
elaboration (Warren and Jones 1997
) that might result in
increased filtering of electrotonically distal events. Yet the trend
was the opposite
events in more mature neurons were briefer. Further,
the rise times measured in older animals were not significantly
different (P21-30: 1.1 ± 0.1 ms, n = 29;
P42-60: 1.2 ± 0.1 ms, n = 11, P = 0.9, 1-way ANOVA) from those measured at the youngest ages,
suggesting the electrotonic changes do not contribute to the observed
developmental differences in VB IPSC kinetics. sIPSC frequency in VB
neurons varied slightly at different developmental stages, but this
effect was not significant (P21-30: 9.1 ± 1.1 Hz,
n = 29; P42-60: 8.2 ± 1.6 Hz, n = 11, P = 0.4, 1-way ANOVA; Fig. 2D). We
also did not observe any changes in baseline noise levels between VB
neurons recorded at the youngest and oldest age groups.
Electrically evoked IPSCs in RTN and VB
To further compare IPSC kinetics at different stages of
development, the properties of electrically evoked IPSCs (eIPSCs) were
examined in both nuclei at all three age groups (Fig.
4). eIPSCs were evoked from stimulation
of fibers in the RTN using a bipolar tungsten electrode. Test pulses
were given at varying intensities (500 µA to 2 mA) until an inward
IPSC occurred. The intensity then was adjusted downward until a minimal
response was obtained, and the stimulus duration (90-160 µS) was
increased by 50% to obtain a 1.5-threshold response. eIPSC decay
kinetics could not be well fitted with double exponential decay
functions and therefore were quantified as the time necessary for both
50% (half-widths) and 90% of total decay. All estimates were from averages of 20 individual events from 15 individual RTN and VB neurons each held at -60 mV.
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The half-widths of eIPSCs in RTN neurons were slightly longer at P8-12
(50.9 ± 4.9 ms, n = 7) compared with both P21-30
(34.7 ± 3.7 ms, n = 17) and at P42-60 (30.6 ± 6.6 ms, n = 7, P < 0.05, 1-way
ANOVA); however, post hoc Tukey-Kramer comparison tests did not reveal
any pairwise differences among age groups (Fig. 4, A and
C). The times required for 90% decay of total peak
amplitude were similarly affected, (P8-12: 222.4 ± 19.9 ms,
n = 7; P21-30: 173.3 ± 21.3 ms,
n = 17; P42-60: 168.1 ± 35.1 ms,
n = 7), although these differences were not
statistically significant. The peak current amplitude of eIPSCs in RTN
neurons was comparable at all ages, at P8-12 (390.4 ± 96.6 pA,
n = 7), P21-30 (
362.8 ± 70.3 pA,
n = 17), or P42-60 (
274.4 ± 69.1 pA,
n = 7).
In contrast with the developmental change in sIPSCs, eIPSCs half-widths
in VB neurons did not show any significant changes from P8-12
(16.8 ± 1.4 ms, n = 11) to P21-30 (15.4 ± 2.3 ms, n = 8) and the P42-60 (16.5 ± 3.0 ms,
n = 7, P = 0.4, 1-way ANOVA) age groups
(Fig. 4, B and D), suggesting that different
mechanisms regulate decay of spontaneous and evoked responses (see
DISCUSSION). Similarly, the 90% decay time was unaffected
by development (P8-12: 58.1 ± 4.1, n = 11;
P21-30: 67.8 ± 7.5 ms, n = 8; P42-60: 80.2 ± 14.4 ms, n = 7, P = 0.2, 1-way
ANOVA; Fig. 4D). The peak current amplitude did not change
significantly during development: (P8-12: 868.3 ± 298 pA,
n = 11; P21-30:
601.9 ± 192 pA,
n = 8; P42-60:
655 ± 204 pA, n = 7, P = 0.7, 1-way ANOVA; Fig. 4D).
Voltage dependence of sIPSCs in RTN and VB in adult rats
We have shown that sIPSCs reach full maturity by the third
postnatal week in the rodent thalamus, therefore further analysis of
sIPSCs was obtained after P21. Voltage dependence of sIPSCs was
determined by examining peak current amplitude, weighted time constant
and overall charge with cells voltage clamped at +60 mV (Fig.
5) compared with -60 mV. In RTN neurons,
sIPSC amplitude was not voltage dependent, with similar peak absolute
values at -60 mV (26.6 ± 3.7 pA) and at +60 mV (22.8 ± 2.8 pA, P = 0.4, n = 6; Fig.
5A). In contrast, the
D,W of sIPSCs
recorded in the RTN at
60 mV (44.3 ± 6.0 ms) was much briefer
than sIPSCs recorded at +60 mV (94.2 ± 6.7 ms, P
<0.001, n = 6). Overall charge measured at
60 mV
(1.25 ± 0.29 nC) was much lower than the values estimated at +60
mV (2.16 ± 0.24 nC, P < 0.05, n = 6). By contrast, in VB neurons, peak current amplitude at
60 mV was
smaller (27.9 ± 4.5 pA) but not significantly (P = 0.1, n = 8) than at +60 mV (37.0 ± 3.4 pA; Fig.
5B). Similar to the RTN responses, the
D,W of sIPSCs in VB neurons decayed at about
half the rate at
60 mV (9.7 ± 0.5 ms) than at +60 mV (23.5 ± 1.8 ms, n = 8, P < 0.0001; Fig.
5B). The overall charge of sIPSCs in VB neurons was also greatly affected by voltage with much lower values at
60 mV
(0.26 ± 0.05 nC) compared with +60 mV (0.86 ± 0.1 nC,
P <0.0001, n = 8). Voltage dependence
likely involves subtype selective mechanisms (Burgard et al.
1996
) that might dictate further differences between these two
types of thalamic neurons. We compared the degree of voltage dependence
(see METHODS) in terms of amplitude,
D,W and charge at +60 mV/
60 mV and found
that VB neurons were slightly more sensitive to changes in membrane
potential than RTN neurons (Fig. 5C). Ratios for both RTN
and VB were analyzed in a single sample t-test measured
against a hypothetical value of 1.0. From these data, it was determined
that amplitude was not enhanced at positive holding potentials for
either RTN or VB; however,
D,W and charge were
found to be voltage dependent in both RTN and VB neurons of adult rats.
VB neurons yielded larger rectification ratios than RTN neurons for
amplitude (RTN: 0.92 vs. VB: 1.50, P > 0.05),
D,W (RTN: 2.22 vs. VB: 2.42, P > 0.05) and charge (RTN: 1.9 vs. VB: 3.8, P <0.05). These
data indicate outward rectification of sIPSCs in both nuclei; however,
the net responses in VB neurons were more sensitive to holding
potentials than RTN neurons.
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Temperature dependence of sIPSCs in RTN and VB of adult rats
We were interested in determining whether the nucleus-specific
IPSC kinetics in thalamus were maintained under conditions of
physiological temperature. Figure 6
illustrates temperature-dependent changes in sIPSC decay kinetics
recorded in 12 RTN and VB neurons in adult rat thalamic slices. An
increase in temperature from 26 to 36°C resulted in a significant
reduction in the decay of sIPSCs in both RTN
(D,W at 26°C: 42.1 ± 4.9 ms, and
36°C: 22.3 ± 2.4 ms, P < 0.01; Fig. 6,
A and D) and VB neurons
(
D,W at 26°C: 12.0 ± 1.1 ms and
36°C: 5.8 ± 0.51 ms, P < 0.001; Fig. 6,
B and D). The Q10 of the
decay rate measured by
D,W or the first
(
D1) or second (
D2)
time constants obtained from biexponential fits were 2.0-2.5 (Fig.
6C) and in agreement with temperature-dependent changes in
hippocampal neurons (Otis and Mody 1992
). Interestingly, temperature dependent changes in amplitude were observed specifically in RTN neurons and resulted in a significant increase in peak current
amplitude in all RTN neurons examined at higher temperatures (26°C:
29.14 ± 2.2 pA vs. 36°C:
54.43 ± 3.7 pA,
n = 5, P < 0.001; Fig. 6D).
The increased amplitude of sIPSCs in RTN neurons had a significant
effect on the overall charge such that even with an accelerated decay,
there was no significant difference between the charge measured at room
(1.2 ± 0.1 nC) or physiological (1.2 ± 0.2 nC,
n = 5, P = 0.9) temperatures (Fig.
6D). Conversely, the lack of increased peak current
amplitude in VB neurons resulted in a decrease in charge at 36°C
(0.2 ± 0.03 nC) compared with room temperature (0.3 ± 0.05 nC, n = 7, P < 0.05; Fig.
6D). Overall with the exception of rise times in RTN neurons
and amplitude of VB neurons, the remaining biophysical parameters
displayed similar temperature dependent alterations.
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DISCUSSION |
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In a previous report on
GABAA-receptor-mediated IPSCs in the thalamus of
young rats, quantitative analysis of IPSCs revealed decay time
constants were two to three times slower in RTN neurons than IPSCs
recorded in the VB complex (Zhang et al. 1997). In the
present study, consistent with the previous findings, the decay
kinetics of IPSCs in RTN within this same age range were also three
times slower than IPSCs in VB. Using additional measures of decay
kinetics, the present study detailed four principal findings. First,
decay kinetics of synaptic sIPSCs in RTN neurons were similar at all
age groups examined. Second, sIPSC duration and amplitude in VB neurons
varied with development. This indicates that the functional IPSC
heterogeneity between VB and RTN actually is increased in the fully
mature rat compared with that previously reported for immature animals
(Zhang et al. 1997
). Third, further characterization in
adult animals revealed that sIPSCs in VB neurons were more voltage
dependent than sIPSCs in RTN, providing further support for
GABAA receptor heterogeneity. Fourth, sIPSCs
recorded at physiological temperatures (36°C) were much briefer in
both RTN and VB respective to their normal control decay times at room
temperature. But the relative difference remains with RTN sIPSCs almost
four times slower than VB responses.
In the present study, the values obtained for the decay of
GABAA-receptor-mediated sIPSCs in rats within the
P8-12 age range were slightly faster in both RTN and VB neurons than
previously reported (Zhang et al. 1997). We attribute
this discrepancy to the way in which spontaneous events were detected.
In the present study, we used an objective criterion to collect all
events greater than the background noise level (3 times rms noise).
This increased sensitivity in sIPSC detection resulted in detecting
smaller events that would have otherwise been rejected incorrectly.
Differences in sIPSCs are dependent on differential distribution of thalamic GABAA receptors
The present results show a significant reduction in peak current
amplitude and decay kinetics of sIPSCs in developing VB neurons. Moreover, VB neurons had a greater degree of outwardly rectifying responses. The unique temperature-, voltage-, and age-dependent properties of GABAA-receptor-mediated responses
in VB neurons likely reflect region selective expression of
GABAA receptor subunits. The temporal changes in
synaptic inhibitory events follow a similar trend toward faster IPSCs
observed in hippocampal (Hollrigel and Soltesz 1997) and
cerebellar (Tia et al. 1996
) neurons. In all these
studies, the time course of the transformation to faster IPSCs
correlates with the timing of subunit turnover (Fritschy et al.
1994
; Gambarana et al. 1991
; Laurie et
al. 1992
). In addition, the developmental stabilization of
sIPSC decay kinetics in VB neurons in the present study also correlates
with maintained levels of subunit mRNAs in the adult rodent. Unaltered
GABAA-receptor-mediated IPSCs throughout
postnatal development in RTN may reflect a lack of developmental change
of subunits expressed in this nucleus (Huntsman and Jones
1995
; Huntsman et al. 1996
; Wisden et al. 1992
). Thus we find little evidence for a developmental switch in GABAA receptor function in RTN postnatally.
These data contrast the study by Gibbs et al. (1996)
,
who found an age-related increase in GABA-evoked chloride currents from
dissociated cultures of RTN neurons. The discrepancy between these two
findings may be due to differences in receptors and/or extracellular
space. The present study examined synaptic receptors, whereas
Gibbs et al. (1996)
studied the responses to bulk
application of exogenous GABA, which will presumably activate both
synaptic and extrasynaptic receptors. In addition, factors that
influence GABA levels in the extracellular space, and therefore
receptor activation, such as uptake or diffusion (see following text)
(Draguhn and Heineman 1996
; Roepstorff and
Lambert 1992
; Thompson and Gahwiler 1992
), will
be very different in the two preparations.
Heterogeneity and modulation of synaptic
GABAA-receptor-mediated sIPSCs are governed by the
properties created by differing combinations of postsynaptic receptors.
A number of studies have suggested that synaptic
GABAA-receptor-mediated currents (Hollrigel and
Soltesz 1997; Jones et al. 1998
; Otis and
Mody 1992
; Poisbeau et al. 1999
; Tia et
al. 1996
; Xiang et al. 1998
; Zhang et al. 1997
) likely result from expression of a specific combination of GABAA receptor subunits (Fritschy and
Möhler 1995
; Laurie et al. 1992
;
Wisden et al. 1992
). This is supported further by the
differential effects of specific GABAA ligands (e.g.,
zolpidem, clonazepam, loreclezole, flunitrazepam, and midazolam) on the decay of synaptic IPSCs (Akk et al. 1997
;
Hollrigel and Soltesz 1997
; Otis and Mody
1992
; Perrais and Ropert 1999
; Strecker
et al. 1999
). Other more subtle and dynamic modulation of
synaptic GABAA-receptor-mediated IPSCs also may involve
subunit-specific phosphorylation of serine and threonine residues
(Jones and Westbrook 1997
; Poisbeau et al.
1999
).
GABAA-receptor mediated-currents are likely influenced by
gating, deactivation (dependent on unbinding of agonist), and
desensitization (Angelotti and Macdonald 1993;
Burgard et al. 1996
; Gingrich et al.
1995
; Jones et al. 1998
; Puia et al.
1991
; Sigel et al. 1990
; Verdoorn et al.
1990
; Wafford et al. 1993
). Different receptor subtypes likely have different binding and unbinding properties. Thus
receptors that mediate synaptic responses with longer deactivation times are more efficient at binding GABA than others (Jones et al. 1998
). It appears that
3 subunits may be a
crucial component of receptors that exhibit long-lasting GABA-mediated
currents as opposed to receptors containing
1 and
2 subunits (Gingrich et al. 1995
;
Serafini et al. 1998
; Verdoorn 1994
). The
prolonged IPSCs in RTN neurons (Zhang et al. 1997
) may
reflect in part the predominance of
3 subunits in this
nucleus compared with limited expression in relay nuclei
(Fritschy and Möhler 1995
; Huntsman et al.
1996
; Wisden et al. 1992
). When examined at both
the mRNA and protein levels,
3 subunits are recognized
as a primary
subunit in the RTN of adult rodents (Fritschy
and Möhler 1995
; Wisden et al. 1992
) and
monkeys (Huntsman et al. 1996
), suggesting that
3-mediated long-lasting IPSCs may be a
species-independent phenomenon.
Although subunit turnover is a likely cause of the developmental
changes observed with sIPSCs in the VB complex, there are other factors
that should be considered, especially with regard to electrically
evoked events. In VB neurons, we did not observe a simultaneous
decrease in eIPSC decay as we did with sIPSCs in both the older ages.
Conversely, we did see a significant change in RTN eIPSC half-widths.
This difference between spontaneous and evoked responses may lie in the
differences behind the basic mechanisms of these two types of
inhibitory events. Compared with a spontaneous event, an evoked
response is likely to result in higher neurotransmitter concentration
due to the prolonged and highly synchronous release of GABA
(Mody et al. 1994). This higher concentration of GABA is
thought to result in a spillover of neurotransmitter into the
extracellular space, activate receptors there and thus influence eIPSC
decay times. Extrasynaptic GABAA receptors are likely made
up of different subunits than those located in the synapse
(Brickley et al. 1999
; Nusser et al.
1998
) and may have different binding, unbinding and/or
desensitization properties than synaptic receptors. Tiagabine, a
selective GABA uptake blocker, has been shown to dramatically enhance
the decay times of evoked but not spontaneous responses, presumably
through the activation of extrasynaptic receptors (Draguhn and
Heineman 1996
; Roepstorff and Lambert 1992
;
Thompson and Gahwiler 1992
).
Functional significance of long-lasting IPSCs and network activity
Our main purpose in this study was to test whether the slowly
decaying IPSCs in RTN neurons persist into maturity and under conditions of physiological temperature. The present study shows that
the long-lasting IPSCs are present in adult rats and that temperature-dependent decreases in decay times do not alter the functional capacity of RTN IPSCs. Why are we so interested in the
preservation of these characteristic IPSCs? One reason is because the
powerful IPSCs in RTN are fundamentally important for regulating the
output of inhibition from this nucleus. It has been shown that
inhibition between RTN neurons ultimately affects intrathalamic
circuits during the propagation of synchronized thalamocortical
oscillatory activity in vivo (Steriade et al. 1987) and
in vitro (Bal et al. 1995
; Huguenard and Prince
1994
; Huntsman et al. 1999
; Sanchez-Vives
et al. 1997
; Sohal et al. 1999
). Intra-RTN
connections appear to be instrumental in some conditions in propagating
oscillatory activity in this nucleus in cat (Bazhenov et al.
1999
; Destexhe et al. 1994
; Steriade et al. 1987
). By contrast, in ferret, mice and rats, it appears
the major role for these internal RTN connections is to dampen this activity. Prolonged IPSCs emanating from intra-RTN connections can
effectively shunt other RTN neurons through lateral inhibition (Sanchez-Vives et al. 1997
; Ulrich and Huguenard
1997
). When these prolonged IPSCs are hampered or blocked in
the RTN, there is a transformation of oscillatory activity from
asynchronous to hypersynchronous oscillations similar to those observed
in absence epilepsy (Bal et al. 1995
; Huguenard
and Prince 1994
; Huntsman et al. 1999
; von Krosigk et al. 1993
). Despite a lack of multiple
GABAA receptor isoforms in this nucleus
(Huntsman and Jones 1995
; Huntsman et al.
1996
), RTN neurons are primarily dependent on these receptors for postsynaptic inhibition (Sanchez-Vives et al. 1997
;
Ulrich and Huguenard 1996
) therefore modulation of
GABAA-receptor-mediated IPSC decay is a key
target for antiepileptic medications. The therapeutic utility of
benzodiazepines in absence epilepsy (Mattson 1995
)
suggests that in man, as in rat (Huguenard and Prince
1994
), enhanced intra-RTN inhibition could lead to
thalamacortical desynchronization.
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ACKNOWLEDGMENTS |
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This study was supported by National Institute of Neurological Disorders and Stroke Grants NS-06477, NS-07280, NS-34774, and NS-10768, and the Pimley Research Fund.
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FOOTNOTES |
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Address for reprint requests: J. R. Huguenard, Dept. of Neurology and Neurological Sciences, Rm M016, Stanford University Medical Center, Stanford, CA 94305-5122.
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 2 August 1999; accepted in final form 23 September 1999.
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REFERENCES |
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