1Department of Pharmacology and Therapeutics and 2The Rotary Hearing Centre, Department of Surgery, Faculty of Medicine, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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ABSTRACT |
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Tennigkeit, Frank,
Dietrich W. F. Schwarz, and
Ernest Puil.
Effects of Metabotropic Glutamate Receptor Activation in
Auditory Thalamus.
J. Neurophysiol. 82: 718-729, 1999.
Metabotropic glutamate receptors (mGluRs) are
expressed predominantly in dendritic regions of neurons of auditory
thalamus. We studied the effects of mGluR activation in neurons of the
ventral partition of medial geniculate body (MGBv) using whole cell
current- and voltage-clamp recordings in brain slices. Bath application of the mGluR-agonist, 1S,3R-1-aminocyclopentan-1,3-dicarboxylic acid or
1S,3R-ACPD (5-100 µM), depolarized MGBv neurons
(n = 67), changing evoked response patterns from
bursts to tonic firing as well as frequency responses from resonance
(~1 Hz) to low-pass filter characteristics. The depolarization was
resistant to Na+-channel blockade with tetrodotoxin (TTX;
300 nM) and Ca2+-channel blockade with Cd2+
(0.1 mM). The application of 1S,3R-ACPD did not change input conductance and produced an inward current
(IACPD) with an average amplitude of
84.2 ± 5.3 pA (at 70 mV, n = 22). The
application of the mGluR antagonist,
(RS)-
-methyl-4-carboxyphenylglycine (0.5 mM), reversibly blocked the
depolarization or IACPD. During intracellular application of guanosine
5'-O-(3-thiotriphosphate) from the recording electrode,
bath application of 1S,3R-ACPD irreversibly activated a large amplitude
IACPD. During intracellular application of
guanosine 5'-O-(2-thiodiphosphate), application of
1S,3R-ACPD evoked only a small IACPD. These
results implicate G proteins in mediation of the 1S,3R-ACPD response. A
reduction of external [Na+] from 150 to 26 mM decreased
IACPD to 32.8 ± 10.3% of control. Internal applications of a Ca2+ chelator,
1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; 10 mM), suppressed IACPD,
implying a contribution of a Ca2+ signal or
Na+/Ca2+ exchange. However, partial replacement
of Na+ with Li+ (50 mM) did not significantly
change IACPD. Therefore it seemed less
likely that a Na+/Ca2+ exchange current was a
major participant in the response. A reduction of extracellular
[K+] from 5.25 to 2.5 mM or external Ba2+
(0.5 mM) or Cs+ (2 mM) did not significantly change
IACPD between
40 and
85 mV. Below
85
mV, 1S,3R-ACPD application reversibly attenuated an inward
rectification, displayed by 11 of 20 neurons. Blockade of an inwardly
rectifying K+ current with Ba2+ (1 mM) or
Cs+ (2-3 mM) occluded the attenuation. In the range
positive to
40 mV, 1S,3R-ACPD application activated an outward
current which Cs+ blocked; this unmasked a voltage
dependence of the inward IACPD with a
maximum amplitude at ~
30 mV. The IACPD
properties are consistent with mGluR expression as a TTX-resistant,
persistent Na+ current in the dendritic periphery. We
suggest that mGluR activation changes the behavior of MGBv neurons by
three mechanisms: activation of a Na+-dependent inward
current; activation of an outward current in a depolarized range; and
inhibition of the inward rectifier, IKIR. These mechanisms differ from previously reported mGluR effects in the thalamus.
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INTRODUCTION |
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Neurons in the ventral part of the medial geniculate body
(MGBv) transform auditory input signals from the inferior colliculus en
route to the auditory cortex. These neurons also receive a strong
tonotopically organized input from the primary auditory cortex
(Andersen et al. 1980; Morel and Imig
1987
; Winer and Larue 1987
, see Jones
1985
for a review). Both afferent sensory and corticothalamic
inputs are glutamatergic in the medial and dorsal lateral geniculate
bodies (Deschênes and Hu 1990
; Hu et al.
1994
; McCormick and von Krosigk 1992
). However,
only corticothalamic inputs activate metabotropic glutamate receptors
(mGluRs), resulting in long-lasting excitatory responses (Eaton
and Salt 1996
; He 1997
; McCormick and von
Krosigk 1992
). In various thalamic nuclei, postsynaptic type 1 mGluRs mediate these effects (Eaton and Salt 1996
;
Godwin et al. 1996a
,b
; Martin et al.
1992
; Salt and Eaton 1996
).
The activation of mGluRs can couple to a variety of effector mechanisms
(see review by Pin and Duvoisin 1995). Synaptic
stimulation or application of 1S,3R-1-aminocyclopentan-1,3-dicarboxylic
acid (1S,3R-ACPD) can evoke a postsynaptic depolarizing current
associated with decreased membrane conductance to
K+ (e.g., Charpak and Gähwiler
1991
; Charpak et al. 1990
;
Guérineau et al. 1994
; McCormick and von
Krosigk 1992
). Other observed mechanisms include the activation
of a Na+/Ca2+ exchanger
(Keele et al. 1997
; Linden et al. 1994
;
Staub et al. 1992
) and
Ca2+-dependent or -independent cation currents
(Crépel et al. 1994
; Guérineau et al.
1995
; Mercuri et al. 1993
; Zheng et al.
1995
). In some neurons, the responses subsequent to activation
of mGluRs have multiple components (Crépel et al.
1994
; Guérineau et al. 1995
; Keele
et al. 1997
). In addition to these postsynaptic mechanisms, mGluRs have important roles in presynaptic modulation of transmission, synaptic plasticity, and neuronal death (see reviews by
Nicoletti et al. 1996
; Pin and Duvoisin
1995
).
Despite serious implications for signal transduction in the auditory
pathway, the effects of mGluR activation have not received examination
in MGBv neurons. This report is the first description of
mGluR-activated currents, frequency preferences, and 1S,3R-ACPD effects
on firing patterns in MGBv neurons. Preliminary results have appeared
in abstract form (Tennigkeit et al. 1996).
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METHODS |
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The preparation of thalamic slices and recording conditions were
similar to those described previously (Tennigkeit et al. 1996). The experiments followed a protocol approved by the
Committee on Animal Care of the University of British Columbia.
Sprague-Dawley rats (16-21 days old) were decapitated during deep
anesthesia with halothane. The brain was removed rapidly from the
cranium and submerged in cold (~4°C) artificial cerebrospinal fluid
(ACSF). The ACSF contained (in mM) 124 NaCl, 26 NaHCO3, 10 glucose, 4 KCl, 2 CaCl2, 2 MgCl2, and 1.25 KH2PO4. The ACSF was
saturated continuously with 95% O2-5%
CO2, maintaining a pH of 7.3. Using a Vibroslicer
(Campden Instruments, London, UK), we obtained coronal, 300-µm-thick
slices that contained the medial geniculate body. After 3 h
incubation of the slices at room temperature (22-25°C), we started
the recording session.
Whole cell patch-clamp electrodes were pulled (Narishige, Model PP83)
from borosilicate glass (WP-Instruments, Sarasota, FL). The electrode
solution (pH 7.3) contained (in mM) 140 K-gluconate, 10 HEPES, 5 KCl, 4 NaCl, 3 Na2ATP, 0.3 Na3GTP,
10 ethylene
glycol-bis-(-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), and 1 CaCl2 (~10 nM free
Ca2+, calculated using Max Chelator software). In
experiments with GTP analogues, 0.3 mM guanosine
5'-O-(3-thiotriphosphate) (GTP
S) or 0.3 mM
5'-O-(2-thiodiphosphate) (GDP
S) replaced GTP in the electrode solution. For solutions of low extracellular
Na+ concentration (26 mM), we replaced NaCl with
equimolar N-methyl-D-glucamine-Cl (NMDG). In the
case of Li+ applications, we replaced 50 mM NaCl
with equimolar LiCl. HEPES, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic
acid (BAPTA), EGTA, NMDG, S-glutamate, tetrodotoxin (TTX),
ATP (Na2ATP), GTP (GTP), GDP
S, GTP
S, and
inorganic salts were purchased from Sigma (St. Louis, MO). In selected
experiments, BAPTA (10 mM) replaced EGTA in the electrode solution.
Whole cell patch-clamp recordings were made with an Axoclamp 2A
amplifier (Axon Instruments, Foster City, CA) in current-clamp or
discontinuous single-electrode voltage-clamp mode (current-voltage switching frequency 4-5 kHz, 30% duty cycle, gain 3-5 nA/mV). Data
acquisition, storage, and analysis were controlled using pClamp 5 software (Axon Instruments) running on a PC. The experiments were
recorded continuously on a chart recorder (Gould), digitized (PCM
501ES, Sony) and stored on videotape (super Beta, Sony). The
voltage-current (V-I) relationships of 1S,3R-ACPD-evoked
currents were determined with slow voltage-ramp protocols; neurons were held for 400 ms at 50 or +10 mV, followed by a ramp at 60 ms/mV to
110 mV. This procedure yielded similar results when compared with the
steady-state current responses measured at the end of 500-ms
voltage-step protocols (n = 3, data not shown). Input
conductance was calculated from the voltage responses to injections of
hyperpolarizing current pulses or the slope of V-I
relationships (see RESULTS). The voltage values were
corrected for a measured junction potential of
11 mV.
The frequency responses of MGBv neurons were studied by injecting
swept-sinewave (ZAP function) current inputs and Fourier transformation
of the recorded voltage and current to the frequency domain, as
described previously (Puil et al. 1986, 1994
;
Tennigkeit et al. 1997
). The impedance (Z)
amplitude profile (ZAP) is the ratio of this output to input. To assure
approximate linearity, we adjusted the ZAP current amplitude to produce
a response of approximately ±5 mV (peak to peak) and blocked action
potentials and high-threshold Ca2+ spikes with
TTX (300 nM) and Cd2+ (100 µM). The frequency
response curves (Fig. 11B), representing the magnitude of
the complex-valued impedance as a function of frequency in a range of
0.1-20 Hz, were obtained from single ZAP records and smoothed with a
five-point moving average (cf. Tennigkeit et al. 1997
).
The ACSF was perfused at a flow rate of ~2 ml/min (bath volume 0.3 ml). Ion channel blockers and antagonists were applied in the ACSF for
a minimum of 10 min to allow a steady-state assessment of their
effects. The following glutamate receptor agonists and antagonists were
purchased from Precision Biochemicals (Vancouver, BC): 1S,3R-ACPD,
(RS)--methyl-4-carboxyphenylglycine (MCPG), D-2-amino-5-phosphonovalerate (D-APV), and
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In voltage-clamp
experiments, TTX (300 nM) and Cd2+ (0.1 mM) were
applied routinely to block action potentials and synaptic activity. In
10 experiments, 50 µM D-APV and 10 µM CNQX were applied
throughout the experiment to block inotropic glutamate receptors.
Quantitative data are presented as means ± SE. Differences were evaluated using Student's paired t-test and considered significant for P < 0.05.
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RESULTS |
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Effects of 1S,3R-ACPD in MGBv neurons
The application of the selective mGluR agonist, 1S,3R-ACPD,
depolarized all MGBv neurons (Fig. 1;
n = 67). At the concentrations used (5-100 µM), the
peak depolarization was in the range of 2-12 mV. On application of 50 µM to 15 neurons at ~60 mV, the depolarization reached threshold
in 12 neurons, eliciting tonic firing of action potentials (Figs.
1A and 5). In 2 of the 12 neurons, the applications led to
action potential bursting with plateau potentials (not shown) (cf.
Klink and Alonso 1997
; Tennigkeit et al
1997b
; Zheng et al. 1995
).
Co-application of TTX (300 µM) with 1S,3R-ACPD completely abolished
the action potentials and plateau potentials while only slightly
reducing the peak amplitude of the evoked depolarization. The
1S,3R-ACPD response was well maintained despite repeated applications (Fig. 1B). Applications of S-glutamate (50 µM
to 1 mM), in the presence of the ionotropic glutamate-receptor
blockers, CNQX (10 µM) and APV (50 µM), also elicited
depolarizations in two neurons (not shown). Recovery from the
depolarization elicited by an application (30-60 s) occasionally was
rapid but usually required 5-10 min (Fig. 1, A and
B).
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The amplitude of the 1S,3R-ACPD depolarization increased in a
concentration-dependent manner. We investigated this dependence in 35 neurons in the presence of TTX (300 nM) to block voltage-dependent Na+-currents and Cd2+ (100 µM) to block high-threshold Ca2+-currents (cf.
Tennigkeit et al. 1996). During their coapplication, a
brief application of 1S,3R-ACPD (30 s in Fig. 1B) produced a depolarization that slowly increased in amplitude and decayed even more
slowly. During an extended 1S,3R-ACPD application (7.5 min in Fig.
1B), we observed a small decline in the response (10-20%) over tens of seconds, possibly due to receptor desensitization. Full
recovery occurred within 5-20 min (Fig. 1B) despite such long applications (4-8 min; n = 6). Brief 1S,3R-ACPD
applications (50 µM for 30-60 s) were sufficient to elicit maximal
depolarizing responses. We measured these peak amplitudes for estimates
of the concentration-dependence, usually using only one concentration per neuron (n = 32). The concentration-response
relationship in Fig. 1C appears to saturate close to 50 µM. The half-amplitude for a maximal effect occurred on applications
of concentrations between 5 and 10 µM, close to the
EC50 value for mGluR2
(Pin and Duvoisin 1995
; Tanabe et al.
1992
). For subsequent investigations of the mechanism of the
depolarization, we chose 50 µM as the usual concentration for application.
Depending on the resting potential
(VR), the application of 1S,3R-ACPD
induced a depolarization as well as a transformation of the firing
pattern in response to injected current pulses. In neurons at a
negative VR (e.g., 75 mV in Fig.
2A), a small depolarizing
current pulse elicited low-threshold Ca2+ spike
(LTS) burst firing before an 1S,3R-ACPD application (Fig. 2A). During an 1S,3R-ACPD application, the depolarization
increased in amplitude and the pulse-evoked firing pattern changed at
first to firing of a single action potential with a delayed onset and then to tonic repetitive firing with a fast onset (Fig. 2A;
n = 17). The same changes in the response pattern
occurred during manual depolarization with DC when the neuron did not
reach threshold during an application of 1S,3R-ACPD or in its absence
(cf. Tennigkeit et al. 1996
). However, when DC was used
to hyperpolarize the neuron back to the original
VR, the response was reduced to a
single action potential on top of a LTS, possibly due to a maintained depolarization and T-current inactivation in distal dendrites. We also
observed similar reductions in the burst responses that occurred at the
offset of hyperpolarizing current pulses in 10 of 17 neurons. We
observed full recovery of these bursts in 7 of the 10 neurons within 15 min after terminating the 1S,3R-ACPD application.
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Effects of 1S,3R-ACPD on input conductance and impedance
The applications of 1S,3R-ACPD did not significantly change input conductance as measured from the voltage responses to constant hyperpolarizing current pulses. The failure of 1S,3R-ACPD to change input conductance was confirmed on applying DC compensation to return the membrane potential back to the original VR (Fig. 1A). 1S,3R-ACPD also did not significantly change the slope of the I-V relationship near rest. A small increase in input conductance (5.4 ± 2.8%; n = 12) occurred occasionally when the 1S,3R-ACPD depolarization evoked firing of action potentials, but we also observed similar increases without 1S,3R-ACPD application. When TTX application prevented the firing of action potentials, this increase in conductance was reduced to insignificant levels (0.6 ± 3.5%, n = 7; cf. Fig. 7A).
Because such conductance estimates rather poorly predict dynamic signal
generation, we measured the impedance amplitude profiles (ZAP)
(Puil et al. 1986) of MGBv neurons. At a
VR of
70 mV, the neuron of Fig. 2
exhibited a voltage-dependent resonance near 1 Hz due to interaction of
active currents, including the T-type Ca2+
current, with the passive properties (Tennigkeit et al. 1994
, 1997
). After DC depolarization, the apparent impedance
collapsed, resulting in low-pass filter characteristics (Fig.
2B, control). This occurred presumably as a result of
excessive T-current inactivation, which prevented voltage oscillations
at the resonance frequency (cf. Puil et al. 1994
). The
application of 1S,3R-ACPD dramatically reduced the impedances of MGBv
neurons in a functionally important frequency range near 1 Hz, but this
was mostly attributable to the depolarization. The frequency-response
curves of Fig. 2B (middle) show that a DC
hyperpolarization back to the initial
VR reversed the shift from nonresonant
to resonant behavior of the neuron. Hence the depolarization due to
activation of mGluRs likely shifted the frequency responses of MGBv
neurons from band-pass to low-pass filter properties.
MGluR-antagonism
The mGluR antagonist, MCPG (selective for the receptor
subtypes, mGluR1 and
mGluR2), reversibly blocked the depolarization induced by 1S,3R-ACPD. In the neuron of Fig.
3, for example, application of MCPG (0.5 mM of the racemic form) reduced the depolarization amplitude to 36.8%
of the control. In a different neuron and in the presence of TTX and
Cd2+, the same concentration reduced the
1S,3R-ACPD depolarization to 44.4% of the control value. Application
of MCPG alone did not evoke changes in membrane potential or input
resistance. Using voltage clamp, we observed that 1S,3R-ACPD
application evoked an inward current
(IACPD) which an additional
application (0.5 mM) of MCPG reduced to 43.8% of control (Fig.
3B). A similar blockade of 1S,3R-ACPD-evoked responses has
been observed in other thalamic neurons (cf. Salt and Eaton
1996). The partial blockade in MGBv neurons is consistent with
the known potencies of MCPG at mGluR1a (EC50 = 300 µM) and mGluR2 (EC50 = 500 µM) receptors.
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Mediation by G proteins
We considered the possibility that G proteins mediated the
1S,3R-ACPD response (Pin and Duvoisin 1995). On
replacement of GTP in the recording electrode with the nonhydrolyzable
GTP analogues, GTP
S or GDP
S (either at 0.3 mM), we proceeded to
record IACPD. When GTP
S was being
applied internally, the application of 1S,3R-ACPD irreversibly
activated IACPD (Fig.
4A; n = 3).
During such observations of IACPD
(recorded for
20 min), a second 1S,3R-ACPD application had no effect
(Fig. 4A). The magnitude of
IACPD was 148.3 ± 23.9 pA or
76.2 ± 28.4% (n = 3) greater than the averaged
IACPD amplitude without GTP
S
(84.2 ± 5.4 pA, n = 22; cf. Fig. 9D).
In contrast, internally applied GDP
S reduced
IACPD. This reduction was more pronounced during longer periods of recording and hence, longer applications of GDP
S (n = 3). The maximal amplitude
recorded 10 min after breaking through the cell membrane, for example, was 65.7 ± 3 pA, i.e., 22.0 ± 3.5% less than the average
control IACPD (Fig. 4B).
When measured 20-30 min after breakthrough, the maximal amplitude of
IACPD was 32 ± 1.7 pA,
corresponding to a reduction of 62.0 ± 2.1% (n = 3). The above results implicate an involvement of G proteins in the
activation of IACPD and a possible
role for GTP hydrolysis in its deactivation.
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Na+ dependence of IACPD and depolarization
Although 1S,3R-ACPD application did not reduce input conductance,
we occasionally found that it slightly increased input conductance. Hence we examined the possibility that an activation of a highly voltage-dependent inward current, rather than blockade of outward K+ currents, mediated the depolarization (cf.
McCormick and von Krosigk 1992). For a test of
Na+ involvement, we reduced the extracellular
Na+ concentration from 150 to 26 mM ("low
Na+") in two neurons. These conditions
resulted in several reversible changes in both neurons:
hyperpolarization (2 and 5 mV), reduced rate of rise and amplitude as
well as increased duration of action potentials, decreased amplitude of
the LTS, and reduced inward rectification at hyperpolarized potentials.
After DC compensation of the hyperpolarization, the low
Na+ conditions reversibly reduced the
1S,3R-ACPD-evoked depolarization to 10.7 and 14.1% of the control
responses (Fig. 5). We attributed the low
Na+ effects to reductions of the fast and
persistent Na+- and hyperpolarization-activated
cation (IH) currents.
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To investigate the possibility that a reduction in the persistent
Na+ current could have contributed to the
blockade of the 1S,3R-ACPD-evoked depolarization, we studied the
effects of low Na+ conditions and TTX application
on the ACPD depolarization. Alone the combined attenuation of
Na+ currents evoked a hyperpolarization of
2.8 ± 1.1 mV (n = 5) and, in voltage clamp, a
steady-state outward current of 48 ± 21.8 pA (n = 5). These conditions also reversibly reduced the control 1S,3R-ACPD-evoked depolarization by 70.4 ± 9.4%
(n = 4, not shown) or, with the additional application
of Cd2+, by 67.2 ± 10.3% (Fig.
6; n = 5, Vm = 70 mV). Using a voltage-ramp command (see METHODS), during combined TTX and
Cd2+ application, we recorded an inward
IACPD with a V-I
relationship that was nearly parallel to the voltage-axis between
40
and
85 mV (cf. Figs. 6-11, controls), reflecting an unchanged input
conductance (n = 29). Throughout this voltage range,
the lower external [Na+] greatly reduced
IACPD (Fig. 6B). These
results implicate a Na+-dependent current in the
1S,3R-ACPD-evoked depolarization.
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Is a Na+/Ca2+ exchanger involved in IACPD?
The activation of electrogenic transport or exchange mechanisms
could account for the Na+-dependent inward
current produced by 1S,3R-ACPD application. In amygdala neurons, for
example, mGluRs mediate an activation of a
Na+/Ca2+ exchange current
(Keele et al. 1997). Because Li+
ions cannot replace Na+ in this exchange
mechanism but penetrate Na+ and nonselective
cation channels, we studied the effects of partial replacement of
Na+ in the ACSF on the
IACPD (cf. Crépel et al.
1994
). Potentially, equimolar replacement of
Na+ with 50 mM Li+ could
reduce a Na+/Ca2+ exchange
current by ~65% (cf. Keele et al. 1997
). In MGBv
neurons, Li+ replacement by itself caused a
reversible depolarization of 4.8 ± 1.2 mV (n = 4)
and a steady-state inward current (Fig.
7B). This effect may be partly
attributable to inhibition of
Na+/K+-ATPase
(Padjen and Smith 1983
). Under Li+
replacement conditions, however, the
IACPD in the neuron of Fig. 7,
measured at
70 mV, was maintained at 93% of the control peak amplitude. In four neurons, the 1S,3R-ACPD-evoked depolarization remained unchanged in amplitude (8.3 ± 0.6 mV or 99.8 ± 12.1% of control). Li+ replacement or 1S,3R-ACPD
application also reduced inward rectification below
90 mV in a
reversible manner (Fig. 7, controls). The results imply that
Li+ can replace Na+ as a
charge carrier for IACPD and were not
consistent with a major involvement of a
Na+/Ca2+ exchanger in the
1S,3R-ACPD depolarization from rest (cf. Crépel et al.
1994
; Keele et al. 1997
) in MGBv neurons.
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Are IACPD and the depolarization influenced by Ca2+?
Replacement of Ca2+ with
Mg2+ in the ACSF did not significantly change the
1S,3R-ACPD depolarization. In the presence of TTX and Cd2+, the depolarization was 94.3 ± 4.6%
of control (n = 3, Fig.
8A). Previous studies
(Keele et al. 1997) have shown that replacement of
Ca2+ with 10 mM Mg2+
together with application of EGTA (1 mM) in the ACSF blocked an
1S,3R-ACPD-evoked inward current that had a V-I relationship that is similar to that described here. However, these conditions reversibly increased IACPD by 18.7%
(Vm =
70 mV, Fig. 8, B
and C). Hence, voltage-dependent Ca2+
currents did not seem to provide a major or specific contribution to
IACPD.
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The intracellular application of BAPTA (10 mM) by diffusion from the recording electrode caused a time-dependent reduction of the 1S,3R-ACPD depolarization and IACPD. On coapplication of TTX and Cd2+ to two neurons, the mean 1S,3R-ACPD depolarization was reduced by 49.4 and 48.1% at 12 or 30 min of BAPTA application, respectively. Under voltage-clamp conditions and continuous 1S,3R-ACPD application, the IACPD was present for 10 min before declining to an undetectable amplitude after 30 min. This decline in amplitude may have resulted from processes far from the electrode tip (see also preceding text). One possibility is that a rise in internal [Ca2+] in the dendrites may activate or potentiate the IACPD and depolarization.
Does a K+ current blockade contribute to IACPD?
A combined activation of inward and blockade of outward currents
could have resulted in the parallel V-I relationships (cf. Crépel et al. 1994; Shen and North
1992
). As a test of this possibility for
IACPD, we studied the effects of
changes in extracellular [K+] and the
K+-channel blockers, Ba2+
and Cs+. A lowering of extracellular
[K+] from the normal, 5.25, to 2.5 mM did not
cause significant or specific alterations in
IACPD (Fig. 9,
A-D; n = 3, Vm =
70 mV). Figure 9D
shows the changes in IACPD amplitude
under control, K+-channel blockade, and reduced
extracellular [K+]- and
[Na+]-conditions. In 9 of 20 neurons,
IACPD was nearly voltage-independent below
40 mV (Figs. 9A and
10A).
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|
Application of Ba2+ (0.1-1 mM) did not
significantly change IACPD (97.7 ± 4.2% of control; n = 3, Vm = 70 mV, Fig. 10B).
The current induced by Ba2+, however, outwardly
rectified at negative potentials and was roughly linear down to its
reversal near the calculated EK (
85 mV). This implied that Ba2+ blocked an inward
rectifier and a leak K+ current. The blockade of
inward rectifiers by Cs+ (2.5-3 mM) or
coapplication of Ba2+ and
Cs+ (Fig. 10C) gave similar
results
no significant effects on
IACPD (107.7 ± 8.4% of control;
n = 3, Vm =
70 mV).
In summary, the application of Ba2+,
Cs+, and reduced extracellular
[K+] produced only small changes
in IACPD and its V-I
relationship. We concluded that a blockade of K+
currents does not cause the generation of the
IACPD (between ~
40 and
85 mV).
MGluRs mediate a blockade of hyperpolarization-activated rectification
The V-I relationship of the 1S,3R-ACPD-induced current
had a striking voltage dependence in the hyperpolarized range beyond 85 mV (Fig. 11A, bottom),
leading to a reversal at
101.2 ± 1.7 mV within the investigated
voltage range (
130 to
10 mV) in 6 of 11 neurons. Application of
1S,3R-ACPD reduced the inward rectification, negative to
EK (Fig. 11A, top). The
application of extracellular Ba2+ (0.1 mM,
n = 1; not shown) or Cs+ (2.5 mM;
Fig. 11B, n = 3) completely blocked the
rectification and occluded the voltage dependence. This implicated a
blockade of an inwardly rectifying K+ current,
IKIR, during mGluR activation. Partial
replacement of Na+ in the ACSF also reversibly
eliminated the outward rectification in the hyperpolarized range (Fig.
6, n = 6). A mGluR-dependent blockade of a
hyperpolarization-activated cation current
(IH) was difficult to ascertain
because IKIR dominated the
hyperpolarization activated inward rectification, masking
IH in MGBv neurons (Tennigkeit et al. 1996
). However, the above results are consistent with a mGluR-mediated blockade of hyperpolarization-activated currents, including IKIR.
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Coactivation of a voltage-gated outward current with the IACPD
Using blockade of Na+ and
Ca2+ spikes by TTX and Cd2+
and large voltage ramps, we explored the effects of mGluR activation in
the depolarized voltage range. At potentials positive to 40 mV, the whole cell current induced by 1S,3R-ACPD application abruptly changed
to an almost linear outward slope reversing at
23.3 ± 3.8 mV
(Fig. 11A; n = 7). Extracellular application
of Cs+ (2.5 mM, n = 2) blocked
this outward contribution, unmasking a voltage dependence of the inward
IACPD in the depolarized range, with a
maximum near
30 mV (Fig. 10B). A parsimonious
interpretation of these results is that, in the depolarized range,
1S,3R-ACPD activated voltage-dependent, outward
K+ and inward cation currents, the latter with
close similarities to the TTX resistant, persistent
Na+ current (Cummins and Waxman
1997
), in MGBv neurons.
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DISCUSSION |
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The application of 1S,3R-ACPD to MGBv neurons had three distinct effects. First, we observed an inward current (IACPD) that was sensitive to blockade with a specific antagonist of mGluR activation. The consequences of the depolarization were shifts in subthreshold filtering from resonance to low-pass characteristics and signal generation from burst to tonic patterns. The other effects were a blockade of an inwardly rectifying current in the hyperpolarized range and an activation of a K+ current at suprathreshold potentials.
Ionic mechanism(s) of IACPD
To all appearances, IACPD was
independent of voltage throughout a wide range of membrane potentials
(40 to
85 mV). This led us to consider that, like other
mGluR-activated cationic currents (cf. Keele et al.
1997
; Mercuri et al. 1993
),
IACPD may participate in the
regulation of VR in MGBv neurons.
However, it seemed unlikely that a blockade of voltage-independent
K+ channels (Crépel et al.
1994
; McCormick and von Krosigk 1992
; Womble and Moises 1994
) produced
IACPD because 1S,3R-ACPD application did not reduce input conductance, and we could observe
IACPD during pharmacological blockade
of K+ channels.
The first clue that the ionic mechanism involved
Na+ came from our observations of the effects of
TTX blockade. Whereas IACPD survived
the blockade, the 1S,3R-ACPD depolarization decreased in amplitude, as
expected from a blockade of persistent Na+
current in MGBv neurons (Tennigkeit et al. 1997).
Indeed, we found that IACPD depended
on the extracellular [Na+] despite the blockade
of voltage-gated Na+ channels. This bears
resemblance to the mGluR-activated current in amygdaloid neurons
(Keele et al. 1997
) and Purkinje cells (Staub et
al. 1992
), produced by
Na+/Ca2+ exchange. For the
mGluR activation in MGBv neurons, however, such an exchange mechanism
seemed less likely because we found that Li+ can
replace Na+ as a charge carrier for
IACPD. Although we cannot entirely
exclude an involvement of
Na+/Ca2+ exchange, the more
likely possibilities are that 1S,3R-ACPD application caused a small
increase in a voltage-dependent Na+ conductance
(insensitive to blockade with 300 nM TTX) or acted on distal dendritic
receptors to produce the depolarizing current.
We investigated a possible contribution of a mGluR-activated,
Ca2+-sensitive nonselective cation current,
ICAN, as in hippocampal (Crépel et al. 1994) and locus coeruleus neurons
(Shen and North 1992
). There was some similarity because
intracellular application of the Ca2+ chelator,
BAPTA, also suppressed IACPD in MGBv
neurons. However, a marked conductance increase, voltage dependence,
and an insensitivity to MCPG blockade distinguished this current from
the IACPD in MGBv neurons.
We observed that IACPD became
dependent on voltage in the range positive to 40 mV. Here, a
Cs+ blockade of a K+
current, which coactivated with IACPD,
unmasked a voltage dependence that resulted in a peak amplitude near
30 mV. This was reminiscent of the TTX-insensitive, persistent
Na+ current in C-type dorsal root ganglion
neurons (Cummins and Waxman 1997
).
In summary, IACPD in MGBv neurons likely results from an increase in Na+ conductance that may depend on internal Ca2+ for activation. A persistent Na+ current enhances this depolarizing action, which, itself, may be highly voltage dependent or mostly of dendritic origin. At voltages above threshold, a K+ current can limit the extent of the 1S,3R-ACPD depolarization. This represents a novel sequence of mechanisms for mGluR-dependent excitation of thalamic neurons.
Receptors mediating IACPD
As suggested in the preceding text, the characteristics of
IACPD in MGBv neurons are consistent
with a dendritic location of greatest mGluR density (Jones and
Powell 1969; Vidanskzky et al. 1996
). An
abundance of mGluR1 protein occurs in rat MGBv (Fotuhi et al.
1993
; Martin et al. 1992
; Salt and Eaton
1996
). In other thalamic nuclei, there also are descriptions of
the mGluR1 mediation of postsynaptic responses (Eaton and Salt
1996
; Godwin et al. 1996a
,b
). The observed
partial blockade of IACPD with MCPG
(0.5 mM) would exclude a mGluR5 mediation of
ICAN, as in CA1 pyramidal neurons
(Congar et al. 1997
) but is compatible with an
involvement of mGluR1a (EC50 = 300 µM) or
mGluR2 (EC50 = 500 µM). The depolarization of
MGBv neurons caused by 1S,3R-ACPD application saturated at 50 µM.
This concentration is a rather low for an involvement of mGluR1
(EC50 = 105-170 µM) and entirely compatible
with a role of mGluR2 (EC50 = 5 µM). Thus while
different receptors may mediate the 1S,3R-ACPD effects on
hyperpolarization-activated rectification and the
depolarization-activated K+ current, the
candidates for IACPD mediation are
type 2 or 1 mGluRs.
Intracellular signal transduction
Using the GTP analogues, GTPS and GDP
S, we demonstrated an
involvement of G proteins in mGluR-mediated signal transduction for the
IACPD. While expecting G-protein
activation for "metabotropic" receptors, there is a report that
1S,3R-ACPD activation of a cationic current may not involve G proteins
but instead novel or membrane-delimited intracellular messenger systems
(Guérineau et al. 1995
). In MGBv neurons,
intracellular application of GTP
S irreversibly activated IACPD, locking G proteins in an
activated position (cf. Ross 1989
). Similar application
of GDP
S reduced IACPD with a delay,
attributable to diffusion into the distal dendrites, the well-known
location of mGluRs and corticothalamic synapses (Godwin et al.
1996a
; Jones and Powell 1969
; Vidnyanszky
et al. 1996
). Normally, the depolarization and
IACPD outlasted the brief 1S,3R-ACPD
application by several minutes; this is consistent with G-protein
activation of intracellular messenger systems (cf. Pin and
Duvoisin 1995
).
An intracellular Ca2+ signal appears to mediate
the link between the receptor-activation and
IACPD, although the source of the signal remains obscure. Internal application of BAPTA reduced IACPD after a delay that could have
resulted from slow diffusion of the chelator and/or slow
Ca2+-modulated processes. The signal does not
apparently depend on Ca2+ influx into the neuron
because IACPD, activated during
Ca2+-channel blockade with
Cd2+, remained unchanged in low
[Ca2+] and high [Mg2+]
media. An intracellular Ca2+ release from
inositol triphosphate (IP3)-sensitive stores is compatible
with a role of mGluR1s, which are colocalized with IP3 receptors in rat
MGB (Fotuhi et al. 1993). For example, a rise in
intracellular [Ca2+], mediated by mGluR1
activation in thalamus, has been implicated in seizure generation and
neurotoxicity (McDonald et al. 1993
; Tizzano et
al. 1995
; see review by Nicoletti et al.
1996
).
Modulation of the inward rectifier
About 50% of MGBv neurons displayed a prominent inward
rectification below 85 mV, which was reduced by application of
1S,3R-ACPD. A Cs+ or Ba2+
blockade of the rectifying current,
IKIR (Tennigkeit et al.
1996
; cf. Womble and Moises 1993
) occluded this
1S,3R-ACPD action. A similar occlusion occurred after partial
replacement of external Na+ with
Li+, possibly relating to an interdependence of
Na+ and K+ in the
IKIR channel function (Hille
1992
). A reduction of IKIR may
result from a dilution of soluble intracellular mediators during whole
cell recording. It is not surprising, therefore, that we did not
observe the inward rectification and its modification by 1S,3R-ACPD
application in all neurons.
A mGluR-dependent K+ current at depolarized potentials
On application of 1S,3R-ACPD, an outward current that was
sensitive to Cs+ blockade was coactivated with
the IACPD at potentials positive to
40 mV. This implies an endowment of MGBv neurons with a
mGluR-activated, voltage-dependent K+ current,
possibly activated by Ca2+ (cf. Budde et
al. 1992
). A balance between the outward current and the inward
IACPD may have contributed to the
apparent voltage independence (cf. preceding text) of the whole cell
current activated by 1S,3R-ACPD.
Functional significance
The depolarization due to IACPD
dramatically changed the functional behavior of MGBv neurons. Their
response mode shifted from resonant or oscillatory bursting to low-pass
filter behavior and tonic firing as a direct result of the
depolarization. This resembles mGluR-dependent behavior in the dorsal
lateral geniculate nucleus (Godwin et al. 1996b;
McCormick and von Krosigk 1992
) and the effects of
muscarinic agonists or raised extracellular [K+] in MGBv neurons (Hu 1995
;
Mooney et al. 1995
). The transformation in firing
pattern characterizes the transitions from sleep or drowsiness to
states of alertness which correlate to the release of several
neuromodulators, including acetylcholine and glutamate (McCormick 1992
; Salt and Eaton 1996
;
Steriade and Llinás 1988
). Modulators may interact
at the cellular level, sharing G-protein-controlled messenger pathways
(cf. Ross 1995) or converge with mGluR activation of the
same effector systems, as in hippocampal neurons
(Guérineau et al. 1994
, 1995
). Such mGluR and
muscarinic interactions may regulate the leak K+
current in the dorsal lateral geniculate nucleus (McCormick
1992
). Alternatively, the depolarization due to modulatory
mechanisms may invoke the tonic firing mode in different functional
contexts. For example, muscarinic K+-conductance
blockade may promote a general state of alertness, whereas the
corticifugal mGluR-dependent modulation may focus attention during
recognition of a visual signal in background (Sherman and Koch
1986
; Sillito et al. 1994
; Singer
1977
). In the auditory thalamus, an excitatory input of long
duration from the cortex enhances frequency tuning (He
1997
; Villa et al. 1991
; Zhang et al.
1997
), presumably by mGluR activation.
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ACKNOWLEDGMENTS |
---|
We thank L. Corey for excellent technical assistance.
The authors acknowledge the financial support of the Medical Research Council of Canada, the Rotary Hearing Foundation (Vancouver), and the Lion's MD19 Hearing Foundation.
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FOOTNOTES |
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Address for reprint requests: E. Puil, Dept. of Pharmacology and Therapeutics, The University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
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 17 November 1998; accepted in final form 26 March 1999.
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REFERENCES |
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