Centre de Recherche en Sciences Neurologiques, Département de Physiologie, Université de Montréal, Montreal, Quebec H3C 3J7, Canada
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ABSTRACT |
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Gee, Christine E.,
Gavin Woodhall, and
Jean-Claude Lacaille.
Synaptically Activated Calcium Responses in Dendrites of
Hippocampal Oriens-Alveus Interneurons.
J. Neurophysiol. 85: 1603-1613, 2001.
Activation of metabotropic
glutamate receptors (mGluRs) by agonists increases intracellular
calcium levels ([Ca2+]i)
in interneurons of stratum oriens/alveus (OA) of the hippocampus. We
examined the mechanisms that contribute to dendritic
Ca2+ increases in these interneurons during
agonist activation of mGluRs and during synaptically evoked burst
discharges, using simultaneous whole cell recordings and confocal
Ca2+ imaging in rat hippocampal slices. First, we
found that the group I/II mGluR agonist
1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD; 100 µM)
increased dendritic
[Ca2+]i and depolarized
OA interneurons. Dendritic Ca2+ responses were
correlated with membrane depolarizations, but Ca2+ responses induced by ACPD were larger in
amplitude than those elicited by equivalent somatic depolarization.
Next, we used linescans to measure changes in dendritic
[Ca2+]i during
synaptically evoked burst discharges and somatically elicited
repetitive firing in disinhibited slices. Dendritic
Ca2+ signals and electrophysiological responses
were stable over repeated trials. Peak Ca2+
responses were linearly related to number and frequency of action potentials in burst discharges for both synaptic and somatic
stimulation, but the slope of the relationship was steeper for
responses evoked somatically. Synaptically evoked
[Ca2+]i rises and
excitatory postsynaptic potentials were abolished by antagonists of
ionotropic glutamate receptors. The group I/II mGluR antagonist
S--methyl-4-carboxyphenylglycine (500 µM) produced a significant
partial reduction of synaptically evoked dendritic Ca2+ responses. The mGluR antagonist did not
affect synaptically evoked burst discharges and did not reduce either
Ca2+ responses or burst discharges evoked
somatically. Therefore ionotropic glutamate receptors appear necessary
for synaptically evoked dendritic Ca2+ responses,
and group I/II mGluRs may contribute partially to these responses.
Dendritic [Ca2+]i rises
mediated by both ionotropic and metabotropic glutamate receptors may be
important for synaptic plasticity and the selective vulnerability to
excitotoxicity of OA interneurons.
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INTRODUCTION |
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In neurons, increasing
concentrations of intracellular calcium
([Ca2+]i) mediates
processes ranging from presynaptic transmitter release to modification
of protein phosphorylation and gene expression (for review see
Ghosh and Greenberg 1995). The excitatory
neurotransmitter glutamate increases neuronal
[Ca2+]i by a number of
mechanisms, including influx through calcium-permeable ionotropic
glutamate receptors (iGluRs), influx through voltage-dependent Ca2+ channels, and release from internal stores
via activation of metabotropic glutamate receptors (mGluRs)
(Berridge 1998
; Ghosh and Greenberg
1995
). Very high
[Ca2+]i may be induced by
excessive glutamatergic stimulation, as during seizure activity, and is
toxic to cells (Choi 1994
). In temporal lobe epilepsy,
principal cells of the hippocampus are lost as a result of such
glutamate-mediated excitotoxicity (Dingledine et al.
1990
; Meldrum 1995
). Gamma-aminobutyric
acid-containing interneurons of the hippocampus provide crucial
inhibition of projection cells and control their excitability (for
review see Freund and Buzsáki 1996
). In models of
epilepsy, some interneurons of the hippocampus are lesioned in addition
to projection cells (Sloviter 1987
). In the CA1 region,
interneurons in stratum oriens/alveus (OA) and stratum pyramidale are
particularly sensitive and are lesioned preferentially in the kainate
model of epilepsy (Best et al. 1993
; Morin et al.
1998
). The mechanism responsible for the specific loss of these
interneurons remains unknown.
In models of epilepsy and ischemia, activation of group I mGluRs
enhances neurotoxic mechanisms, whereas activation of the group II/III
mGluRs has a neuroprotective role (see Nicoletti et al.
1996). In the hippocampus, group II/III mGluRs are largely confined to axons and terminals, whereas the group I mGluRs are situated peri- and extrasynaptically on dendrites of hippocampal neurons (Luján et al. 1996
; Shigemoto et
al. 1997
). Microapplication of glutamate induces mGluR-mediated
somatic [Ca2+]i
oscillations in OA interneurons, but not in interneurons of stratum
radiatum/lacunosum-moleculare (Carmant et al. 1997
). In addition, application of the group I/II mGluR agonist
1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) evokes
oscillations of membrane potential and
[Ca2+]i in OA
interneurons through activation of voltage-dependent Ca2+ channels and Ca2+
release from intracellular stores but has little or no effect on
stratum radiatum/lacunosum-moleculare interneurons (Woodhall et
al. 1999
). The presence of these mGluR and
Ca2+-mediated mechanisms specifically in OA
interneurons raises the possibility that group I/II mGluR activation
may be involved in the selective vulnerability of OA interneurons to
excitotoxicity. In addition, mGluRs may contribute to synaptic
plasticity in OA interneurons as long-term potentiation of excitatory
synapses, dependent on mGluR activation, has been reported in these
neurons (Ouardouz and Lacaille 1995
; Perez et al.
2000
; but see Maccaferri and McBain 1996
).
However, if mGluR activation plays a role in excitotoxicity or synaptic
plasticity in OA interneurons, then mGluRs should be activated during
synaptic transmission and contribute to rises in
[Ca2+]i. To address this
question, we synaptically evoked epileptiform discharges in OA
interneurons from disinhibited rat hippocampal slices and recorded
whole cell current-clamp responses while simultaneously monitoring
changes in dendritic
[Ca2+]i with confocal
microscopy. Some of these data were published in abstract form
(Gee et al. 1998
).
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METHODS |
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Slice preparation
Transverse hippocampal slices were prepared from young (13-19
day) male Sprague-Dawley rats using similar procedures as
Woodhall et al. (1999). The rats were anesthetized with
halothane and decapitated, and the brains were rapidly dissected in
cold (5°C), oxygenated (95% O2-5%
CO2) solution containing (in mM) 120 choline-Cl,
2.5 KCl, 26 NaHCO3, 1.25 NaH2PO4, 8 MgSO4, 10 glucose, and 0.4 L-ascorbic acid, pH 7.35-7.4, ~305 mOsm. Blocks of brain containing the
hippocampus were affixed with cyanoacrylate to a vibratome stage and
cut into 300-µm-thick slices. A cut was made to remove the CA3 region
from the hippocampus. Slices were transferred to artificial
cerebrospinal fluid (ACSF; in mM: 124 NaCl, 2.5 KCl, 2 CaCl2, 26 NaHCO3, 1.25 NaH2PO4, 2 MgSO4, 10 glucose, 0.4 L-ascorbic
acid, and 4 myo-inositol, pH 7.35-7.4, ~305 mOsm) saturated with
95% O2-5% CO2 at room
temperature (22-24°C). Slices were allowed to recover for at least
1 h before use.
Electrophysiology
Slices were transferred to a recording chamber attached to the
stage of an upright laser scanning confocal microscope (Olympus BH5,
BioRad MRC-600, Mississauga, Ontario, Canada). The chamber was perfused
with oxygenated ACSF (17-19°C, 1-3 ml/min). Patch pipettes were
pulled from borosilicate glass (1 mm OD, A-M Systems, Everett, WA) and
filled with 145 mM K-methylsulfate, 1 mM MgCl2, 8 mM NaCl, 2 mM ATP, 0.4 mM GTP, 10 mM HEPES, 1 mM EGTA, 0.15% biocytin,
and 10-20 µM calcium green I or oregon green BAPTA-I (Molecular
Probes, Eugene, OR), titrated with KOH to pH 7.2-7.25, and adjusted to
275-285 mOsm (electrode resistance 4-8 M). In some experiments,
the electrodes were filled with the Ca2+
indicator and (in mM) 100 Cs-gluconate, 10 CsCl, 10 NaCl, 2 ATP, 0.4 GTP, 1 EGTA, 10 HEPES, 20 QX-314 bromide, 0.15% biocytin, titrated to
7.2-7.25 with acetic acid, and adjusted to 275-285 mOsm (4-8 M
).
Whole cell patch-clamp recordings were obtained, under visual control
using a long working-distance water-immersion objective (Olympus
WPlanFL ×40 UV, 0.7 numerical aperture, 3.1 mm working distance), from
CA1 OA interneurons, with horizontally oriented primary dendrites and
somata near the oriens/alveus border, as previously described
(Woodhall et al. 1999
). Changes in membrane voltage were
monitored using an Axoclamp-2B amplifier (Axon Instruments, Foster
City, CA) in bridge mode. Signals were digitized at 22 kHz and recorded
to videotape. In addition, signals were filtered at 1 kHz, digitized at
2 kHz (TL-1, Axon Instruments), stored on a PC, and analyzed using
pClamp software (Axon Instruments). The bridge balance was monitored
and adjusted using the bridge circuit. Cells with an initial resting
membrane potential more negative than
45 mV, overshooting action
potentials, and an input resistance >200 M
were accepted. To
control for the effects of membrane potential on input resistance and
to reduce spontaneous action potentials, current (approximately ±50
pA) was injected to set the membrane potential near
60 to
65 mV. At
this membrane potential, the input resistance of the OA interneurons
was 403 ± 27 M
(mean ± SE, n = 37).
A monopolar tungsten microelectrode was positioned in stratum radiatum
(or occasionally in the alveus) to synaptically activate OA
interneurons via their excitatory input from CA1 pyramidal cells (e.g.,
Blasco-Ibanez and Freund 1995; Lacaille et al.
1987
). OA interneurons were also activated by somatic
depolarizing current injection via the recording electrode.
Calcium imaging
After obtaining the whole cell configuration, at least 20 min
were allowed for intracellular diffusion of the fluorophore. The
fluorophore was excited using a 488-nm argon laser attenuated to 1% of
the maximum power. Emission was detected through a long-pass filter
(cutoff 515 nm) and recorded to a PC using the MPL software (BioRad).
The confocal aperture was opened fully. Linescans were taken from
dendrites approximately 100-150 µm from the soma at a rate of 12 ms
per line for a total scan time of 6.144 s. Alternatively, time lapse
images were collected at 0.2 Hz. The images were analyzed off-line
using Cfocal and Bfocal software (provided by M. Charlton, University
of Toronto, Toronto, Ontario, Canada). For linescans, the fluorescence
intensity (Fline) of a line was
averaged for a delimited region of interest representing a section of
the dendrite. Changes in fluorescence were calculated for each line
relative to the averaged baseline fluorescence prior to stimulation
(Frest) and expressed as
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Linescans were initiated by a digital trigger pulse. After a 1-s delay, synaptic stimulation was applied via the tungsten electrode, or somatic current injection was applied via the recording electrode. To compensate for small variations in the start time of the linescans, electrophysiological and Ca2+ responses were temporally aligned by eye. This correction and the relatively slow scan time used (12 ms per line) prevented a more precise temporal analysis of the Ca2+ and voltage responses.
Pharmacology
Experiments were carried out in the presence of 20 µM
bicuculline methiodide to block GABAA-mediated
inhibition. In some experiments, (±)-2-amino-5-phosphopentanoic acid
(APV, 200 µM) and 6-cyano-7-nitroquinoxaline-2,3,-dione (CNQX, 40 µM) were used to block ionotropic glutamate receptor (iGluR)
activation. During experiments with the mGluR agonist ACPD,
tetrodotoxin (TTX, 0.5 µM) was used to prevent action potentials and
indirect effects of the agonist. (S)--methyl-4-carboxyphenylglycine (S-MCPG) was used to antagonize mGluRs. ACPD and MCPG were purchased from Tocris-Cookson (Ballwin, MO). APV and CNQX were purchased from RBI
(Nantick, MA). K-methylsulphate and choline chloride were purchased
from ICN (Costa Mesa, CA). L-Ascorbic acid was purchased
from Fisher (Ottawa, Ontario, Canada). Other chemicals were purchased
from Sigma (Oakville, Ontario, Canada).
Histology
After recording, the slices containing biocytin-filled cells
were transferred to a freshly prepared solution of 4% paraformaldehyde in 0.1 M phosphate buffer and fixed overnight at 4°C. Slices were washed and stored in 0.1 M phosphate buffer for up to 2 wk then embedded in agarose and resectioned at 50-80 µm with a vibratome. The sections were then processed using the Vectastain ABC kit (Vector
Laboratories, Burlingame, CA) followed by nickel-intensification as
previously described (Woodhall et al. 1999). Sections
were mounted with D.P.X. mounting medium (Electron Microscopy Sciences, Ft. Washington, PA) and examined under a light microscope.
Statistics
Data are expressed as means ± SE. Data were tested for normality, and equal variance and appropriate parametric or nonparametric tests were applied using SigmaStat statistical software (SPSS). Significance level was set at P < 0.05.
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RESULTS |
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Intradendritic Ca2+ increases mediated by mGluR activation
Activating mGluRs by bath application of the group I/II agonist
(1S,3R)-ACPD increases intrasomatic Ca2+ and
causes depolarization of OA interneurons (McBain et al.
1994; Woodhall et al. 1999
). To assess whether
similar Ca2+ responses are produced in dendrites,
we applied ACPD (100 µM) in the presence of TTX (0.5 µM) to block
action potentials and possible indirect effects. Confocal images were
collected at 0.2 Hz from dendrites of OA interneurons filled with the
Ca2+ indicator oregon green BAPTA-I. In the
presence of ACPD, 11 of 13 cells depolarized (range, 4.0-31.3 mV; mean
depolarization, 13.7 ± 2.2 mV), and 2 cells showed no change in
membrane potential. In these experiments, the mean membrane potential
prior to ACPD application was
60.2 ± 0.6 mV. Dendritic
[Ca2+]i increased in the
cells that depolarized (mean peak Ca2+ response
57.7 ± 14.9%
F/F, n = 11) and did not change in the cells that did not depolarize. Figure
1 shows a typical response from one of
the cells that depolarized and showed an increase in dendritic
[Ca2+]i in the presence
of ACPD. We occasionally saw oscillatory responses to bath application
of ACPD (see Woodhall et al. 1999
). Cell input resistance was monitored, with hyperpolarizing current pulses, in three
cells during ACPD responses. It decreased in two cells (by 92 and
44%), and increased by 6% in the other.
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We examined the relationship between dendritic
Ca2+ responses and depolarizations induced by
ACPD. The peak amplitude of ACPD-induced dendritic
Ca2+ rises and depolarizations were significantly
correlated (r2 = 1.453, P = 0.023, n = 11, Fig.
2A). ACPD-induced
Ca2+ and depolarizing responses were also
correlated in time with an average latency to peak of 338 ± 30 and 349 ± 31 s, respectively, following the onset of ACPD
(r2 = 0.826, P < 0.001, n = 11, Fig. 2B). This latency to
peak was consistent with the time required for solution exchange in our perfusion system. We used somatic current injections to evoked dendritic Ca2+ responses of nonsynaptic origin.
The peak amplitude of these dendritic Ca2+
responses was significantly correlated with the somatic depolarizations (r2 = 0.193, P = 0.022, n = 27 observations from 11 dendrites, Fig. 2A). The slope of the linear regression for the
Ca2+-voltage relation was less for somatic
depolarizations than for ACPD responses (Fig. 2). The soma
depolarizations required to evoke dendritic Ca2+
responses similar to those induced by ACPD (54.3 ± 12.8%F/F, n = 11) were larger
(27.8 ± 2.8 mV, n = 11) than the ACPD-induced depolarizations (Fig. 2A). We also used somatic
depolarizations as control Ca2+ responses before
and after ACPD responses (Fig. 1). Ca2+ responses
(82.0 ± 25.9%
F/F) evoked by soma
depolarizations (29.0 ± 3.1 mV) were not significantly different
after the ACPD application (P = 0.22, n = 10, t-test).
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Biocytin-labeled cells that responded to ACPD had horizontally oriented cell bodies with dendrites confined to stratum oriens/alveus (9/10 cells), or that also crossed stratum pyramidale and entered strata radiatum/lacunosum-moleculare (1/10 cells). The axons of these cells were seen exclusively in stratum oriens/alveus (2/5 cells), in strata oriens/alveus, radiatum and lacunosum-moleculare (1/5 cells), or in radiatum and lacunosum-moleculare (2/5 cells). The biocytin-labeled ACPD-insensitive cells had horizontally oriented dendrites in stratum oriens/alveus (1/2 cells) or also dendrites that crossed stratum pyramidale and entered strata radiatum/lacunosum-moleculare (1/2 cells). The axons of the nonresponsive cells projected to stratum oriens/alveus (1/2 cells) or to strata pyramidale/radiatum (1/2 cells). Thus no clear relationship was observed between the morphology of OA interneurons and the electrophysiological and/or Ca2+ responses induced by ACPD.
Intradendritic Ca2+ responses evoked by synaptic activation
To examine synaptically evoked responses, we combined
current-clamp recordings of excitatory postsynaptic potentials (EPSPs) and burst discharges in OA interneurons held at membrane potentials near 60 to
65 mV, with imaging of dendritic
Ca2+ responses in linescan mode. OA interneurons
were activated polysynaptically, with a tungsten electrode placed in
stratum radiatum in the presence of 20 µM bicuculline. Large
epileptiform EPSPs triggering multiple action potentials were recorded
at the soma in response to single stimuli (20-130 µA for 500 µs)
in stratum radiatum (Fig. 3D). When the stimulating electrode was placed in the alveus, smaller but
qualitatively similar responses were recorded in OA interneurons (not
shown). Dendritic Ca2+ increased during the
synaptically evoked burst discharge (Fig. 3D). Dendritic
[Ca2+]i rose rapidly
during the responses and decayed slowly over seconds. All linescans
were filtered to remove fast transients before measuring response
parameters (see METHODS). The sample
Ca2+ responses shown in Fig. 3, D-F,
show both filtered and unfiltered traces. Whereas the filtering
reduced the peak amplitude of Ca2+ responses by
8.7 ± 7.1%
F/F (n = 7 cells randomly selected), it did not affect the time-to-peak or the
ability to resolve smaller peaks associated with individual action
potentials. The filtering was useful in reducing background
fluctuations. Ca2+ responses associated with
individual action potentials were more clearly resolved when the
interspike interval exceeded about 30 ms (e.g., Fig. 3, E
and F) but were not observed in all cells (e.g., Fig. 8).
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In 22 of 30 cells, Ca2+ levels were stable during the prestimulus baseline period (e.g., Fig. 3, E and F). In 17 of these 22 cells, Ca2+ responses returned to baseline levels before the end of the linescans (within 5.2 s from the time of stimulation). In the remaining 8 of 30 cells, fluorescence declined during the prestimulus period (e.g., Figs. 3D and 4), possibly due to bleaching of the dye. This decline was nonlinear and was not corrected. In five of these eight cells, Ca2+ responses returned to baseline levels during the linescans (e.g., Fig. 4).
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To distinguish between action potential and synaptically generated dendritic Ca2+ responses, changes in dendritic [Ca2+]i were monitored while a similar number of action potentials was generated by somatic current injection (Fig. 4, A1 and B1). During these experiments, exposure to the laser was kept to a minimum to avoid phototoxic damage to the cells. For each condition, two linescans, one during synaptic stimulation and one during somatic activation, were collected at the same dendritic location and usually at 1- to 2-min intervals, for a total of six to eight linescans per cell. Fluorescence levels usually returned to baseline during the 1- to 2-min intervals. Occasionally, changes in baseline fluorescence occurred between longer intervals. To control for the possibility of time-dependent changes in either synaptically or somatically evoked responses, we collected several linescans over longer intervals (Fig. 4). The mean peak Ca2+ response and the mean area under the Ca2+ response were not significantly different during 75 min of recording for both synaptic stimulation (peak Ca2+, F = 0.197, P = 0.823; area under Ca2+ response, F = 0.654, P = 0.534, n = 7 cells) and somatic activation (peak Ca2+, F = 0.284, P = 0.764; area under Ca2+ response, F = 0.30, P = 0.971, n = 3 cells; Fig. 4, 1-way ANOVAs). Similarly, the number of action potentials were stable over time whether evoked synaptically (H = 0.494, P = 0.781; n = 7 cells) or somatically (H = 0.935, P = 0.686; n = 3 cells; Fig. 4, Kruskal-Wallis).
We examined the relationships between peak Ca2+ response and burst characteristics for both synaptically and somatically evoked responses. Peak Ca2+ responses were plotted against the number of action potentials and mean spike frequency for the complete data set with both stimulation types (Fig. 5). Peak Ca2+ response was linearly related to the electrophysiological measure with either synaptic stimulation (number of action potentials: r2 = 0.153, P = 0.007; action potential frequency: r2 = 0.134, P = 0.014) or somatic stimulation (number of action potentials: r2 = 0.215, P = 0.009; action potential frequency: r2 = 0.234, P = 0.006). However, the slope of the relationships between peak Ca2+ response and the characteristics of the underlying bursts of action potentials was steeper for somatically than synaptically evoked responses (see Fig. 5, A and B), suggesting some differences between the two types of Ca2+ responses. Similar relationships were also found for the area under Ca2+ responses (not shown).
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EPSPs and dendritic Ca2+ responses require iGluR activation
We next examined the role of iGluRs in responses evoked by
synaptic and somatic stimulation. Bath application of the
N-methyl-D-aspartate (NMDA) receptor antagonist
APV (200 µM) and the
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate
receptor antagonist CNQX (40 µM) eliminated the electrophysiological
and dendritic Ca2+ responses evoked by synaptic
stimulation (peak Ca2+ response,
W =
45.0, P = 0.004; area under
Ca2+ response, W =
45.0,
P = 0.004; number of action potentials, W =
36.0, P = 0.008;
n = 9, Wilcoxon signed rank test; Fig.
6). These effects were reversible
following wash out of CNQX/APV. Peak Ca2+
responses recovered to 74.4 ± 7.4% of control, area under
Ca2+ responses to 119.5 ± 7.3% of control,
and number of spikes to 94.9 ± 17.4% of control
(n = 3). APV and CNQX did not affect either the
electrophysiological or dendritic Ca2+ responses
evoked by somatic activation (peak Ca2+,
t =
0.510, P = 0.626; area under
Ca2+ response, t =
0.275,
P = 0.826; number of action potentials, t = 0.0, P = 1.0, n = 9, Student's t-test; Fig. 6, C and
D). In the OA interneuron shown in Fig. 6, the stimulating
electrode placed in stratum radiatum also elicited an antidromic action potential. The dendritic Ca2+ response elicited
by this action potential was not blocked by the antagonists. This was
the only cell in which antidromic action potentials were elicited by
the synaptic stimulation. Similar Ca2+ responses
to individual spontaneous action potentials were seen in other cells
(e.g., Fig. 4). These results indicate that activation of iGluRs is
necessary for synaptically evoked burst discharges and dendritic
Ca2+ responses, but not for responses to somatic
activation.
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We attempted to visualize intradendritic Ca2+
responses to synaptic stimulation using an intracellular patch solution
containing cesium and QX-314 to block K+ and
voltage-gated Na+ channels. In these conditions,
mGluR-mediated activation of a Ca2+-dependent
nonselective cation current has been observed in CA1 pyramidal cells,
in the absence of iGluR activation (Congar et al. 1997).
With this protocol, dendritic Ca2+ responses were
detected during large amplitude and long duration EPSPs elicited by
single stimuli or 100-Hz/1-s trains. However, in the presence of APV
and CNQX, these dendritic Ca2+ signals, as well
as EPSPs, were completely abolished (Fig.
7, n = 11 cells). Thus in
our experimental conditions, no dendritic Ca2+
responses were detected in the presence of iGluR antagonists.
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mGluR activation contributes to synaptically evoked dendritic Ca2+ responses
To determine whether mGluRs contribute to synaptically evoked
responses, we examined the effects of the group I/II mGluR antagonist MCPG (500 µM; n = 14 cells). This antagonist required
long wash out periods (30-45 min). Therefore the time required to
1) load the cells with the calcium-sensitive dye,
2) obtain synaptically and somatically evoked responses, and
3) record responses following drug application and wash out
was longer than 70 min. Dendritic Ca2+ responses
to somatic activation and synaptic stimulation sometimes deteriorated
after the long wash out of MCPG from the slice. Therefore responses
were evoked in six cells, first in the presence of MCPG and then in
normal ACSF following a 30- to 45-min wash out. In the remaining cells
(n = 11), responses were obtained first in normal ACSF
and then in the presence of MCPG; in some of these cells
(n = 5/11), a wash out of MCPG was possible. In three
cells exposed to MCPG, the linescan intersected two distinct dendritic branches of the same cell. In two of these cells, MCPG reduced synaptically evoked Ca2+ responses to a greater
extent in one of the two branches (data not shown). Thus mGluR
contributions to synaptically evoked dendritic Ca2+ responses appeared spatially localized to
certain dendritic sites in certain cells, and consequently responses in
both dendrites of these three cells were analyzed as separate
observations. The mean peak amplitude and area under the response of
synaptically evoked Ca2+ responses were
significantly reduced in the presence of MCPG (Fig.
81; peak
Ca2+ response, t = 3.69, P = 0.002; area under Ca2+
response, t = 2.543, P = 0.022; paired
t-tests, n = 17). In the five cases with
MCPG wash out, the peak amplitude of synaptically evoked
Ca2+ responses was significantly reduced to
75.7 ± 8.4% of control in the presence of MCPG and recovered
partially to 82.8 ± 8.4% of control after wash out. By contrast,
MCPG did not significantly affect dendritic Ca2+
responses evoked by somatic activation in the same cells (Fig. 8D1; peak Ca2+ response,
t = 1.708, P = 0.119; area under
Ca2+ response, t = 0.820, P = 0.44, n = 10; paired
t-tests). EPSPs and burst discharges evoked by synaptic
stimulation were also not significantly changed in the presence of MCPG
(Fig. 8, A2 and 2-B4; number of action
potentials, t = 0.504, P = 0.69; action potential frequency, t = 1.06, P = 0.31; action potential amplitude, t = 1.48, P = 0.16; n = 14, paired
t-tests). Similarly, MCPG did not significantly affect
somatically evoked repetitive firing (Fig. 8, C2 and
D2-D4; number of action potentials, t = 1.562, P = 0.153; action potential frequency,
t =
1.20, P = 0.26; action potential
amplitude, t = 0.845, P = 0.42;
n = 10; paired t-tests). These results
suggest that mGluRs may contribute in part to dendritic Ca2+ responses but not to EPSPs and burst
discharges evoked by synaptic stimulation, and they do not contribute
to Ca2+ responses or repetitive firing evoked by
somatic activation.
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Biocytin-labeled cells that were responsive to MCPG had horizontally (4/5) or vertically (1/5) oriented cell bodies and horizontally oriented dendrites confined to stratum oriens/alveus. In one cell, the axon projected to stratum lacunosum-moleculare, whereas in the other cells it ramified in strata pyramidale/radiatum and lacunosum-moleculare. Biocytin-labeled cells that showed no effect of MCPG had dendrites confined to oriens/alveus (1/2) or also crossing into strata radiatum/lacunosum-moleculare (1/2). Their axon projected to stratum pyramidale (1/2) or strata pyramidale/radiatum (1/2).
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DISCUSSION |
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In the present study, we found that direct activation of mGluRs with the group I/II agonist ACPD increased dendritic Ca2+ levels and depolarized OA interneurons. We also observed that mGluRs may contribute in part to dendritic Ca2+ increases during synaptically evoked burst discharges in OA interneurons since the group I/II mGluR antagonist MCPG produced a significant partial reduction of synaptically evoked Ca2+ responses, without affecting electrophysiological burst discharges, nor dendritic Ca2+ responses elicited by somatic action potentials. Finally, synaptically evoked Ca2+ responses were abolished by blocking ionotropic glutamate receptors, suggesting that their co-activation may be necessary for mGluR-mediated Ca2+ responses. Thus ionotropic and metabotropic GluRs may jointly contribute to dendritic Ca2+ rises during synaptic activity in OA interneurons, which may be important for Ca2+-mediated synaptic plasticity or excitotoxicity.
Activation of mGluRs by ACPD increases dendritic [Ca2+]i
The Ca2+ response and depolarization of OA
interneurons produced by application of ACPD confirms previous findings
that ACPD increases
[Ca2+]i in somata and
proximal dendrites (Woodhall et al. 1999) and induces
Ca2+-dependent inward currents (McBain et
al. 1994
) in OA interneurons. Our results extend these findings
by demonstrating that
[Ca2+]i in more distal
dendrites also increases in response to ACPD. The effects of ACPD were
not mediated indirectly since TTX was used to block action
potential-driven synaptic transmission. The ACPD-induced increase in
dendritic Ca2+ levels and depolarization were
significantly correlated. However, larger current-evoked somatic
depolarizations were required to produce dendritic
Ca2+ responses similar to those induced by ACPD.
Since Ca2+ changes were measured in the
dendrites, this difference could be due to voltage attenuation of
somatic evoked depolarizations from the soma to the dendritic location.
Alternatively, ACPD-induced rises in dendritic
Ca2+ levels may involve other mechanisms in
addition to influx through voltage-dependent Ca2+
channels, likely through the previously reported mGluR-mediated coupling of voltage-dependent Ca2+ channels and
Ca2+ release from intracellular stores
(Woodhall et al. 1999
). The dendritic
Ca2+ responses and somatic depolarizations were
temporally correlated in our experiments. However, with the slow
acquisition rate of the confocal images we used (0.2 Hz), it was not
possible to determine more precisely the temporal relationship between
dendritic Ca2+ changes and membrane
depolarization (Bianchi et al. 1999
).
Postsynaptic group I mGluRs have been localized immunohistochemically
to peri- and extra-synaptic regions of dendrites on OA interneurons,
with the somatostatin/GABA containing interneurons in OA expressing
high levels of mGluR1 (Baude et al. 1993
;
Luján et al. 1996
). In contrast, group II mGluRs
are located presynaptically (Shigemoto et al. 1997
).
Thus the mGluR responses observed were likely due to direct activation
of dendritic postsynaptic group I mGluRs.
Mechanisms of synaptically evoked Ca2+ responses
We found that peak dendritic Ca2+ responses, evoked either synaptically or somatically, were linearly related to the number and frequency of action potentials in burst discharges, but that the slope of the relationship was steeper for somatic than synaptic responses. This linear relationship between peak Ca2+ response and action potential number or frequency may not be absolutely accurate since Ca2+ responses were obtained in different cells in these experiments. Still, since most synaptically and somatically evoked responses were recorded in the same cells, these results suggest that different mechanisms may underlie these two types of dendritic Ca2+ responses. This difference likely reflects the additional activation of glutamate receptor mechanisms during synaptic responses.
The absence of synaptically evoked Ca2+ responses
(or EPSPs) in OA interneurons when iGluRs were blocked suggests that
synaptically evoked mGluR-mediated Ca2+ responses
may require co-activation of iGluRs and/or voltage-dependent Ca2+ channels. QX-314 bromide salt may have
inhibited Ca2+ entry (Talbot and Sayer
1996) and therefore prevented the detection of mGluR-mediated
Ca2+ responses. However, in CA1 pyramidal cells
mGluR-mediated depolarization and Ca2+ responses
have been reported using such recording and stimulation protocols
(Congar et al. 1997
). Previous studies have reported a
similar requirement for Ca2+ entry via
iGluRs or voltage-dependent Ca2+ channels.
Cooperative actions of either iGluRs or voltage-dependent Ca2+ channels along with mGluRs are necessary for
oscillatory Ca2+ responses in OA interneurons
(Carmant et al. 1997
; Woodhall et al.
1999
). Analogously, mGluR-mediated enhancement of synaptically evoked Ca2+ responses can occur via potentiation
of spike-driven increases in
[Ca2+]i (Nakamura
et al. 1999
; Zheng et al. 1996
). Our observation of a partial contribution of mGluRs to synaptically evoked
Ca2+ responses with no enhancement of EPSPs or
burst discharges appears contradictory to our other observation that
ACPD elicited both a rise in dendritic
[Ca2+]i and a
depolarization. The depolarization evoked by ACPD is due, however, to
the activation of a Ca2+-dependent cationic
current (Crépel et al. 1994
; McBain et al. 1994
; Pozzo Miller et al. 1995
; Woodhall
et al. 1999
). The absence of depolarization may thus be due to
an insufficient elevation of
[Ca2+]i to activate this
current following synaptic stimulation.
An important consideration in the failure to detect pharmacologically
isolated synaptic mGluR responses is that the region of dendrite being
imaged may not have been in the vicinity of active synapses with
mGluRs. In control ACSF, synaptically evoked Ca2+
responses were due to activation of polysynaptic (Shaffer
collaterals/pyramidal cells) and monosynaptic inputs (pyramidal cells).
However, in the presence of APV and CNQX, only monosynaptic inputs were
activated, which reduced the likelihood that the region of interest
contained active synapses. Additionally, no action potentials
contributed to synaptically evoked Ca2+ influx in
the presence of APV and CNQX, resulting in a more spatially restricted Ca2+ response. Indeed, recent studies
in cerebellar Purkinje neurons have demonstrated the presence of highly
localized mGluR-mediated increases in dendritic
[Ca2+]i in response to
synaptic stimulation that are not accompanied by postsynaptic currents
(Finch and Augustine 1998; Takechi et al.
1998
). Likewise, MCPG reduces tetanus-induced dendritic
[Ca2+]i rises in CA1
pyramidal neurons with little or no effect on the postsynaptic current
(Frenguelli et al. 1993
). The dendrites of OA
interneurons lack the planar arrangement of Purkinje and pyramidal
neurons, making it difficult to image much of their dendritic trees. In
future experiments, it would be important to examine
Ca2+ responses at dendritic segments with clearly
identified monosynaptic excitatory inputs and activated at sub- and
supra-threshold levels. Our preliminary observation that MCPG could
differentially affect synaptically evoked dendritic
Ca2+ responses in different branches suggests
that mGluR-mediated Ca2+ responses could be
localized to certain dendritic segments in interneurons. In addition,
the mechanisms for mGluR-mediated intracellular Ca2+ release may not be present in all parts of
the dendritic tree (Berridge 1998
).
Conceivably, MCPG may have directly antagonized NMDA or AMPA receptors
(e.g., Contractor et al. 1998). However, our observation that MCPG did not affect burst discharges suggests that the effects on
Ca2+ responses were not due to nonspecific
antagonism of iGluRs. In disinhibited slices, antagonizing NMDA
receptors reduced the number of action potentials per burst, whereas
antagonism of AMPA receptors blocked burst discharges (data not shown)
(Williamson and Wheal 1992
). MCPG can also antagonize
group II mGluRs, which have been localized to presynaptic terminals in
the hippocampus (Shigemoto et al. 1997
). Preventing
activation of presynaptic group II mGluRs, which are negatively coupled
to cAMP, is expected to enhance glutamate release during synaptic
transmission (Conn and Pin 1997
). MCPG did not affect
synaptically evoked EPSPs and burst discharges, suggesting that there
was no increase in glutamatergic transmission due to antagonism of
presynaptic group II mGluRs at these synapses. Group III mGluRs are
also localized presynaptically to OA interneurons (Shigemoto et
al. 1996
, 1997
); however, MCPG does not
antagonize these receptors at the concentrations used (Conn and
Pin 1997
). Therefore the effects of MCPG we observed were
likely exerted via postsynaptic mGluRs.
Physiological implications of synaptically activated Ca2+ responses
Interneurons in OA and to a lesser extent in stratum pyramidale
are preferentially lost in experimental models of epilepsy, whereas
interneurons of strata radiatum/lacunosum-moleculare are not
(Best et al. 1993; Morin et al. 1998
).
While the exact mechanism of this cell loss is unknown, it likely
results from seizure-induced Ca2+-mediated
excitotoxicity (see Dingledine et al. 1990
;
Meldrum 1995
). Several lines of evidence suggest that
the selective vulnerability of OA interneurons may be due to a
Ca2+-mediated mGluR mechanism. First, high levels
of group I mGluRs are selectively expressed in OA interneurons
(Baude et al. 1993
; van Hooft et al.
2000
). Second, the group I/II mGluR agonist ACPD elicits large
rises in [Ca2+]i in OA
interneurons but not in interneurons of strata radiatum and
lacunosum-moleculare (Carmant et al. 1997
;
Woodhall et al. 1999
). Third, in the present study we
show that mGluR activation contributes in part to intracellular
Ca2+ rises during epileptiform synaptic burst
discharges. Although synaptically evoked mGluR-mediated intracellular
Ca2+ rises are less marked than mGluR
agonist-induced responses, they are likely to be more pronounced
during seizure activity. It is possible that other mechanisms like
Ca2+ influx through kainate receptors may
contribute to seizure-induced excitotoxicity in OA interneurons;
however, excitatory postsynaptic currents mediated by kainate are also
present in interneurons of strata radiatum and lacunosum-moleculare,
which are resistant to excitotoxicity (Cossart et al.
1998
; Frerking et al. 1998
).
Activation of mGluRs also plays a role in long-term potentiation of
excitatory synaptic transmission in OA interneurons (Cowan et
al. 1998; Ouardouz and Lacaille 1995
;
Perez et al. 2000
; but see Maccaferri and McBain
1996
). Interestingly, interneurons in strata radiatum and
lacunosum-moleculare lack this form of plasticity (Ouardouz and
Lacaille 1995
; Perez et al. 2000
). The
observation that group I/II mGluR activation produces large rises in
intracellular Ca2+ in OA interneurons but not in
interneurons of strata radiatum and lacunosum-moleculare
(Woodhall et al. 1999
) and that group I/II mGluRs
contribute in part to intracellular Ca2+ rises in
OA interneurons during synaptic burst discharges suggest that
mGluR-mediated Ca2+ transients may be involved in
the induction of long-term potentiation at OA interneuron synapses. In
other cell types, mGluRs are important for long-term potentiation
(e.g., Bortolotto et al. 1999
; Cohen et al.
1998
; Zheng et al. 1996
), particularly when
long-term potentiation is induced using weak tetanization protocols
that require Ca2+ release from internal stores
(Wilsch et al. 1998
).
Thus our results show that synaptic activation causes rises in dendritic [Ca2+]i in OA interneurons that require ionotropic glutamate receptor activation and are mediated in part by activation of group I/II mGluR receptors. These glutamate receptor-mediated rises in [Ca2+]i may have important functional implications for both normal and pathological hippocampal function.
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ACKNOWLEDGMENTS |
---|
We thank Dr. R. Robitaille for assistance with the confocal microscopy and for helpful discussions. Drs. S. Bertrand and N. Haddjeri provided critical comments on a previous version of the manuscript.
This research was supported by the Canadian Institutes of Health Research, the Fonds pour la Formation de Chercheurs et l'Aide à la Recherche (FCAR), and the Fonds de la Recherche en Santé du Québec. C. E. Gee was supported by postdoctoral fellowships from the Centre de Recherche en Sciences Neurologiques (J. P. Cordeau) and the Natural Sciences and Engineering Research Council of Canada. G. Woodhall received postdoctoral fellowships from the FCAR and Groupe de Recherche sur le Système Nerveux Central.
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FOOTNOTES |
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Address for reprint requests: J.-C. Lacaille, Département de Physiologie, Université de Montréal, c.p. 6128 succursale Centre-ville, Montreal, Quebec H3C 3J7, Canada (E-mail: lacailj{at}ere.umontreal.ca).
Received 13 June 2000; accepted in final form 21 December 2000.
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REFERENCES |
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