Divisions of 1Psychiatry and 2Neurology Research, Durham Veterans Administration Medical Center, and Departments of 3Psychiatry and 4Pharmacology, Duke University Medical Center, Durham, North Carolina 27705
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ABSTRACT |
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Calton, Jeffrey L., Maeng-Hee Kang, Wilkie A. Wilson, and Scott D. Moore. NMDA-Receptor-Dependent Synaptic Activation of Voltage-Dependent Calcium Channels in Basolateral Amygdala. J. Neurophysiol. 83: 685-692, 2000. Afferent stimulation of pyramidal cells in the basolateral amygdala produced mixed excitatory postsynaptic potentials (EPSPs) mediated by N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors during whole cell current-clamp recordings. In the presence of GABAA receptor blockade, the mixed EPSPs recruited a large "all-or-none" depolarizing event. This recruited event was voltage dependent and had a distinct activation threshold. An analogous phenomenon elicited by exogenous glutamate in the presence of tetrodotoxin (TTX) was blocked by Cd2+, suggesting that the event was a Ca2+ spike. Selective glutamatergic blockade revealed that these Ca2+ spikes were recruited readily by single afferent stimulus pulses that elicited NMDA EPSPs. In contrast, non-NMDA EPSPs induced by single stimuli failed to elicit the Ca2+ spike even at maximal stimulus intensities although these non-NMDA EPSPs depolarized the soma more effectively than mixed EPSPs. Elongation of non-NMDA EPSPs by cyclothiazide or brief trains of stimulation were also unable to elicit the Ca2+ spike. Blockade of K+ channels with intracellular Cs+ enabled single non-NMDA EPSPs to activate the Ca2+ spike. The finding that voltage-dependent calcium channels are activated preferentially by NMDA-receptor-mediated EPSPs provides a mechanism for NMDA-receptor-dependent plasticity independent of Ca2+ influx through the NMDA receptor.
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INTRODUCTION |
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Calcium influx into the postsynaptic membrane is
considered a critical event in most cellular models of neuroplasticity.
For N-methyl-D-aspartate (NMDA)-dependent
long-term potentiation (LTP), it is commonly thought that
Ca2+ influx through the channel associated with
the NMDA receptor is a requirement for induction of LTP either through
direct activation of Ca2+-sensitive substrates
and/or subsequent liberation of intracellular Ca2+ stores (Bliss and Collingridge
1993; Bliss and Lynch 1988
; Madison et
al. 1991
). Consistent with this, high-resolution imaging
studies showed that Ca2+ entry through NMDA
receptors produced a local rise in Ca2+ at the
dendritic spines of hippocampal pyramidal neurons (Müller and Connor 1991
; Petrozzino et al. 1995
).
However, other studies have localized voltage-dependent calcium
channels (VDCCs) in the shafts and spines of dendrites of CA1 pyramidal
neurons (Jaffe et al. 1994; Mills et al.
1994
; Segal 1995
), and
Ca2+ influx through these channels during
synaptic stimulation has been shown to be potentially greater than
through ligand-gated channels (Miyakawa et al. 1992
;
Regehr and Tank 1992
). Furthermore Ca2+ influx through VDCCs has been shown
necessary for certain forms of NMDA-independent LTP (Aniksztejn
and Ben-Ari 1991
; Grover and Teyler 1990
, 1995
;
Huber et al. 1995
). In light of these findings, it is
useful to address the relative abilities of NMDA- and
non-NMDA-receptor-mediated synaptic potentials to activate VDCCs. This
is especially relevant for those models addressing differences between
NMDA-dependent and -independent forms of synaptic plasticity.
The amygdala is a group of related nuclei in the basal forebrain
implicated in emotional learning such as fear and anxiety (e.g.,
Bechara et al. 1995; Fanselow and Kim
1994
; Kapp et al. 1992
). Both NMDA- and
non-NMDA-receptor-mediated components contribute to excitatory
neurotransmission in the amygdala (Gean and Chang 1992
;
Rainnie et al. 1991
), whereas
GABAA- and
GABAB-receptor-mediated components contribute to
inhibitory neurotransmission in this brain area (Maren
1996
). Similar to other regions of the brain, neurons in the
amygdala have been shown to exhibit NMDA-receptor-dependent and
-independent synaptic plasticity (Chapman et al. 1990
;
Gean et al. 1993
; Huang and Kandel 1998
;
Li et al. 1998
; Maren 1996
; Shindou et al. 1993
; Wang et al. 1997
).
Recent work using dissociated cells has shown that amygdaloid neurons
have a variety of high- and low-threshold
Ca2+ channels (Foehring and
Scroggs 1994
; Kaneda and Akaike 1989
; Yu
and Shinnick-Gallagher 1997
).
In the present study, we observed an all-or-none depolarizing event evoked by low levels of synaptic activation in disinhibited pyramidal cells of basolateral amygdala. After determining that this event is mediated by VDCC activation, we found that the Ca2+ spike can be induced by NMDA-receptor-mediated EPSPs but not by AMPAR-mediated EPSPs. In addition, this Ca2+ spike can be induced by AMPA EPSPs when K+ channels were blocked. These results provide for a mechanism of NMDA-receptor-dependent plasticity that is independent of Ca2+ influx through the NMDA-receptor complex.
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METHODS |
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Brain slice preparation
Brain slices containing the basolateral amygdaloid nucleus were
prepared from 14- to 18-day-old male Sprague-Dawley rats. Under
halothane anesthesia, the rats were decapitated and the brains were
removed and chilled in ice-cold oxygenated artificial cerebrospinal
fluid (ACSF) containing (in mM) 120 NaCl, 25 NaHCO3, 25 dextrose, 3.3 KCl, 1 MgCl2, and 2 CaCl2. After
hemisection, coronal brain slices (300-µm thick) were cut from each
hemisphere using a vibrating tissue slicer. Slices were transferred to
a holding chamber containing room-temperature ACSF bubbled with 95%
O2-5% CO2 and were
incubated for 1 h before being used in an experiment.
Drugs
The recording chamber was perfused with ACSF (2-3 ml/min) using a mechanical pump (Cole Palmer) and a mechanical valve was used for exchange of drug solutions. At this perfusion rate, fluid turnover in the recording chamber occurred in 20-30 s. All drugs were made as stock solutions and then diluted to final concentrations in ACSF. Final concentrations of drugs in ACSF were: dizocilpine (MK-801), 100 µM; D-2-amino-5-phosphonovaleric acid (APV), 50 µM; bicuculline methiodide (BMI), 20 µM; tetrodotoxin (TTX), 0.5 µM; and 6,7-dinitroquinoxaline (DNQX), 20 µM. All stock solutions were made using distilled water except for DNQX, which was made with dimethyl sulfoxide (DMSO; final DMSO concentration in ACSF was never >0.2%). To avoid junction errors from BMI application, bicuculline methochloride (BMC, 20 µM) was substituted for BMI when blockade of GABAA transmission was desired during ongoing recordings (i.e., to demonstrate "wash on" and "wash off" effects of bicuculline in Fig. 1A). BMC, BMI, APV, and DNQX were all obtained from Sigma (St. Louis). MK-801 was obtained from Research Biochemicals International (Natick, MA). TTX was obtained from Calbiochem (La Jolla, CA).
Electrophysiological recordings
For each experiment, slices were transferred to a submerged
recording chamber mounted on an upright microscope (Zeiss Axioscope) equipped with infrared illumination and differential interference contrast optics. Temperature in the recording chamber was held at
30 ± 1C throughout the recording period. For recording,
individual pyramidal cells of the basolateral amygdala were visualized
under high magnification. Whole cell patch-clamp electrodes were pulled from Corning 7740 glass (1.5 mm OD, WPI) using a Flaming-Brown micropipette puller (Sutter) and had input resistances of 2-4 M.
The standard electrode solution contained the following (in mM): 110 K-gluconate, 10 NaCl, 10 HEPES, 10 K4BAPTA, 4 QX314, and 2 MgATP. In some experiments, K-gluconate and
K4BAPTA were replaced by Cs-gluconate (100 mM)
and Cs4BAPTA (10 mM). Voltage and current
recordings were made using a Warner PC-501A patch-clamp amplifier.
Synaptic stimulation was triggered by electrical stimulation applied
through a monopolar tungsten electrode with a bath return placed in the
medial basolateral nucleus adjacent to the external capsule. Stimuli
were square wave current pulses (1-250 µA, 0.1 ms) delivered at
0.033 Hz. In some experiments, exogenous glutamate (500 µM) was
pressure applied (10-15 psi, 10-100 ms) through a small diameter
glass pipette placed within 100-200 µm of the recorded cell.
Although the disinhibited amygdala slice preparation often develops
epileptiform bursts (Gean and Chang 1991), we attempted to minimize burst activity by restricting the recording to 30 min after
superfusion of BMC or BMI. Slices showing spontaneous bursts (<10%)
were discarded. The absence of significant epileptiform bursting in our
preparation may have resulted also from the use of relatively thin
slices (300 µm).
Waveform analysis
Signals were filtered (2 kHz) and amplified before being
digitized (5 kHz) and saved to hard disk for off-line data analysis. To
enhance the detection of the recruited Ca2+ spike
during the rising phase of the evoked response, unaveraged raw
waveforms were expanded and differentiated using data acquisition and
analysis software (LabView, National Instruments).
Ca2+ spikes imbedded in synaptic potentials were
detected readily in differentiated records by the presence of a second
slope peak at the onset of the Ca2+ spike.
Differentiation of the raw waveform provided a sensitive measure for
subtle changes in the onset kinetics of the evoked response (e.g.,
Stasheff et al. 1993).
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RESULTS |
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Whole cell current- and voltage-clamp recordings were taken from
74 pyramidal cells of the basolateral amygdala. At initial patch
rupture, cells typically had a resting membrane potential of negative
60-70 mV [mean Vm = 62.3 ± 0.65 mV (mean ± SE); n = 24] and an
input resistance of 150-250 M
(mean
RN = 218.2 ± 10.75 M
). In
normal ACSF and with QX314 in the recording pipette to block sodium
spikes, low-intensity stimulation applied to the basolateral nucleus
evoked small EPSPs (average latency, 3.3 ms) previously demonstrated to
be mediated by both NMDA and non-NMDA glutamatergic receptors
(Calton et al. 1997
; Gean and Chang 1992
; Rainnie et al. 1991
). After perfusion of bicuculline to
block GABAA-mediated inhibition, stimulation at the same
intensity evoked a larger EPSP that recruited a slower onset,
large-amplitude depolarizing potential resembling a Ca2+
spike (Fig. 1A). This
putative Ca2+ spike occurred at an average latency of 19.69 ±2.46 ms, had a distinct stimulus threshold, and varied little in
amplitude with stimulus intensity (Fig. 1B). When raw
waveforms were differentiated (Fig. 1C), the slope of
the response during the rising phase clearly showed a second peak
corresponding to the regenerative event.
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Ionic mechanism of the recruited potential
Several experiments were conducted to verify that the recruited depolarizing potential is mediated by VDCCs. First, to demonstrate that this secondary depolarizing event occurs via a voltage-gated rather than a ligand-gated conductance, cells were alternately stimulated in current- and voltage-clamp recording modes (n = 4). Figure 2A shows traces from a typical experiment. As before, while recording under current-clamp conditions, EPSPs were evoked, and the EPSPs recruited the secondary depolarizing potential at higher stimulus intensities. In voltage-clamp mode, however, stimulation at similar intensities failed to elicit the secondary events, providing evidence that this secondary event is mediated by a voltage-gated conductance rather than by a ligand-gated conductance. Second, to determine that this secondary depolarizing potential is a Ca2+ spike, we examined the sensitivity of this potential to the Ca2+ channel blocker Cd2+ (Fig. 2B). To avoid the effect of the nonselective Ca2+ channel blocker on synaptic transmission, the synaptic glutamatergic response was replaced with one induced by exogenous glutamate (500 µM). Glutamate was applied through pressure pulses (10-15 psi, 10-100 ms) in the presence of TTX (0.5 µM). Brief application (10-30 ms) of glutamate evoked depolarizing potentials comparable with EPSPs that increased in amplitude with application duration. Glutamate at longer application times (20-100 ms) evoked the secondary depolarizing potential similar to one evoked by EPSPs. This secondary depolarizing potential was eliminated by bath application of the Ca2+ channel blocker Cd2+ (100 µM; n = 8), whereas the primary glutamate-induced depolarizing potentials were unaffected. This result showed that the secondary depolarizing potential is mediated by Ca2+ influx through VDCCs.
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Relative abilities of NMDA EPSPs and non-NMDA EPSPs to recruit Ca2+ spikes
Given that excitatory neurotransmission in pyramidal cells of the
basolateral amygdala is mediated by both NMDA and non-NMDA receptors
(Gean and Chang 1992; Rainnie et al.
1991
), we sought to determine if both forms of excitatory
transmission were equally effective at recruiting the voltage-dependent
Ca2+ spikes.
First, NMDA EPSPs were isolated by bath application of non-NMDA-receptor blocker, DNQX (Fig. 3A). Although the addition of DNQX resulted in a shift to the right in the input/output plot, isolated NMDA EPSPs nevertheless evoked the Ca2+ spike at higher stimulus intensities in all cells tested (n = 16, Fig. 3B). In contrast, non-NMDA EPSPs isolated by bath perfusion of a competitive NMDA-receptor antagonist APV (50 µM) failed to recruit the Ca2+ spike in 9 of 11 cells, even when stimulus intensity was increased by a factor of 10 (Fig. 4, A and C).
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We considered the possibility that the two cells showing Ca2+ spikes in the presence of APV may have done so because APV is a competitive NMDA-receptor blocker and thus subject to competitive displacement under conditions of increased glutamate concentrations in the synapse after high-intensity stimulation. Therefore we also studied the ability of non-NMDA EPSPs isolated by the noncompetitive NMDA antagonist MK-801 to generate the Ca2+ spike (Fig. 4B). After perfusion of MK-801 and 3 min of conditioning stimulation (0.1 Hz at subthreshold intensity) to activate the use-dependent block of NMDA receptors, the non-NMDA EPSPs failed to recruit Ca2+ spikes at maximal stimulus intensities in all cells tested (n = 7). The conditioning stimulation alone failed to eliminate the Ca2+ spikes in control cells (n = 2; data not shown). This result showed that Ca2+ spikes mediated by VDCCs can be evoked by isolated NMDA EPSPs but not by isolated non-NMDA EPSPs.
To further confirm that non-NMDA-receptor-mediated EPSPs cannot
generate the Ca2+ spikes, cells were stimulated
in current-clamp mode while manually held at different membrane
voltages (90 to
40 mV) by DC current injection in the presence of
DNQX, APV, or MK801. When synaptically stimulated at different holding
potentials, the amplitude of non-NMDA EPSPs is expected to increase as
the cell is increasingly hyperpolarized (e.g., Hestrin et al.
1990
). The same prediction can be made for NMDA EPSPs in the
absence of extracellular Mg2+ (Nowak et
al. 1984
). However, increasing hyperpolarization eventually should eliminate the recruitment of the Ca2+
spike, as the hyperpolarized resting potential prevents the EPSP from
depolarizing the membrane to the threshold of the VDCCs that mediate
the Ca2+ spike (e.g., Miyakawa et al.
1992
).
Figure 5 shows findings from a typical experiment examining NMDA EPSPs at different holding potentials in Mg2+-free ACSF. In all cells tested (n = 8), there was a clearly discernable membrane voltage at which the evoked amplitudes increased with further depolarization due to recruitment of the Ca2+ spike. In contrast, after isolating non-NMDA EPSPs by the perfusion of APV (n = 6, Fig. 4B) or MK801 (n = 7, data not shown), the amplitude of the response varied linearly with holding voltage in all cells tested with larger-amplitude responses occurring at more hyperpolarized holding potentials. This result provided further evidence that the Ca2+ spike can be elicited by isolated NMDA EPSPs but not by the isolated non-NMDA EPSPs. We found that maximal intensity non-NMDA-receptor-mediated EPSPs that failed to generate Ca2+ spikes were much larger in peak amplitude when measured at the cell body than mixed EPSPs that were threshold for evoking a spike (48.5 ±1.65 vs. 20.2 ±1.8 mV, respectively).
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Effect of elongation of non-NMDA EPSPs on Ca2+ spikes
Previous studies have reported that non-NMDA EPSPs, although
usually larger in peak amplitude, tend to be shorter in duration than
NMDA EPSPs due to rapid desensitization (Kiskin et al.
1986; Trussell and Fischback 1989
). In our
preparation, the mean half time to decay for non-NMDA EPSPs was 68.9 ms
compared with 96.2 ms for mixed EPSPs (although this more rapid decay
appeared to be accounted for in part by the greater peak depolarization
with increasing stimulus intensity). Therefore we sought to determine if an elongated non-NMDA EPSP could elicit a Ca2+
spike. First, we studied the effect of cyclothiazide, a drug that
elongates non-NMDA EPSPs by inhibiting desensitization of the AMPA
receptor (Yamada and Tang 1993
). After evoking a maximal non-NMDA EPSPs in the presence of APV or MK801, some cells were perfused with cyclothiazide. Cyclothiazide had no effect on peak amplitude of non-NMDA EPSPs but increased the mean duration (214 ± 21 ms) and half time to decay (101 ms), resulting in an average increase in non-NMDA EPSP area of 100.2 ±22.6% (Fig.
6). The prolonged non-NMDA EPSPs still
failed to evoke the Ca2+ spike in seven of the
eight cells tested (Fig. 6).
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Second, trains of stimulation were employed to elongate non-NMDA EPSPs. In the absence of NMDA-receptor blockade, Ca2+ spikes could be elicited reliably by either single stimulation pulses or by a stimulus train (4 pulses at a frequency of 100 Hz; Fig. 7). As expected, the stimulus intensity required to elicit the spike was less for the train stimuli, providing evidence that a stimulus train is more effective at depolarizing the cell than a single stimulus. After perfusion of APV (50 µM), stimulus trains elicited the Ca2+ spike in five of five neurons tested (Fig. 7). Again, this might be because APV is a competitive NMDA-receptor blocker, and thus subject to competitive displacement under conditions of high glutamate concentrations in the synapse. Therefore we also used train stimulation in the presence of the noncompetitive antagonist, MK801. In this case, although the duration of the postsynaptic depolarization was greatly prolonged relative to single stimuli, stimulus trains failed to elicited the Ca2+ spike in six of seven neurons tested (Fig. 7).
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Third, we examined the possibility that K+
channels may reduce the duration of non-NMDA EPSPs, preventing them
from evoking the Ca2+ spike. Such regulation of
EPSPs by K+ channels has been demonstrated in the
hippocampus (Hoffman et al. 1997). To block
K+ channels and otherwise eliminate the
voltage-dependent K+ conductance,
K+ in the internal electrode solution was
replaced by Cs+ (see METHODS). Under
these conditions, single non-NMDA EPSPs (n = 5) were
consistently able to activate the Ca2+ spike even
at relatively low stimulus intensity (Fig. 8). As seen in
Fig. 8, the blockade of K+ channels with Cs+
allowed the generation of Ca2+ spikes by non-NMDA EPSPs.
The elongated time course of the Ca2+ spikes reflects the
removal of voltage dampening normally provided by K+ efflux
through voltage-dependent K+ channels.
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DISCUSSION |
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We have demonstrated that in the presence of
GABAA-receptor blockade, synaptic activation will
evoke large depolarizing spikes mediated by VDCCs in pyramidal cells of
the basolateral amygdala. In addition, in this model system,
NMDA-receptor-mediated synaptic transmission is required for activating
these voltage-dependent Ca2+ spikes. We
considered the possibility that non-NMDA EPSPs failed to activate the
Ca2+ spike because of insufficient postsynaptic
depolarization. However, this seems not to be the case. Large-amplitude
non-NMDA EPSPs, which greatly exceeded NMDA EPSPs capable of eliciting
the Ca2+ spikes, failed to activate the VDCCs.
Considering that non-NMDA EPSPs, although usually larger in peak
amplitude, tend to be shorter in duration than NMDA EPSPs due to rapid
desensitization (Kiskin et al. 1986; Trussell and
Fischback 1989
), the duration of non-NMDA-receptor EPSPs were
increased by application of cyclothiazide or trains of stimulation.
These manipulations still failed to recruit the Ca2+ spike. This finding suggests that there are
other mechanisms for preferential activation of VDCCs by NMDA
receptors. These mechanisms may play a critical role in the
neuroplasticity underlying learning and memory in the amygdala
(McKernan and Shinnick-Gallagher 1997
). However, because
we obtained brain slices from animals at an age when developmental
modifications are occurring in both glutamate receptors (Kew et
al. 1998
; Vallano 1998
) and VDCCs (McEvery et al. 1998
), these mechanisms also might
reflect a developmental phenomenon.
A possible explanation for the present findings is that the origin of
the spike is electronically distant from the soma, and there is a
difference between the characteristics of the distally occurring EPSPs
and EPSPs measured at the soma. EPSPs measured at the soma reflect the
summation of many distal synaptic inputs and do not necessarily mirror
EPSPs seen at single synaptic sites. These distally occurring
non-NMDA EPSPs could be of insufficient duration to evoke the
Ca2+ spikes not only due to the fast dissociation
rate and the greater desensitization of non-NMDA receptors but also due
to activation of K+ channels. Whereas unitary
synaptic events mediated by NMDA receptors can last for several hundred
milliseconds, non-NMDA-receptor-mediated currents are much briefer,
typically less than a few milliseconds (Jahr and Lester
1992; Lester et al. 1990
; Randall et al.
1990
). Given our mean observed latency for the evoked
Ca2+ spikes by
NMDA-receptor-mediated transmission of 19.7 ms, it is likely that our
distal non-NMDA EPSPs evoked by single stimulus pulses were too brief
to activate the observed Ca2+
spike. Additionally, the rapid activation of K+
channels enables them to be activated by non-NMDA EPSPs, thus reducing
the duration and amplitude of EPSPs. Such regulation of EPSPs by
K+ channels has been demonstrated in the
hippocampus (Hoffman et al. 1997
). The blockade of
K+ channels would be expected to prolong unitary
depolarizations by preventing the repolarization of the membrane by
voltage-dependent K+ currents and by increasing
input resistance, thereby increasing the decay constant of the
depolarization. This mechanism is supported by our observation that
non-NMDA EPSPs could elicit the Ca2+ spike under
the blockade of K+ channel with
Cs+. Although QX-314 has been reported to block
some K+ channels, it was clear that in our
preparation this effect was insignificant in comparison to the effect
of Cs+. In addition, we were able to elicit an
analogous Ca2+-mediated phenomenon in the absence
of QX-314, using instead superfused TTX and exogenously applied glutamate.
Colocalization of NMDA receptors (but not non-NMDA receptors) with
certain types of VDCCs also might provide a mechanism for preferential
activation of VDCCs by NMDA transmission. A recent immunolabeling study
examining the subcellular locations of glutamate receptor subunits in
lateral and basolateral amygdala found a different distribution pattern
between NMDA and non-NMDA-receptor subunits (Farb et al.
1995). Whereas most (~75%) of the labeled dendritic
NMDA-receptor subunits were found in spines, the majority (~59%) of
labeled non-NMDA-receptor subunits were localized to the shaft.
Although this distribution pattern does not preclude the possibility of
colocalization of NMDA and non-NMDA receptors within single spines as
has been shown in the hippocampus (Bekkers and Stevens
1989
), these findings suggest that the relative contribution of
NMDA and non-NMDA receptors in excitatory transmission is different across different regions of the dendritic membrane in these cells. As
of yet, no studies have examined the dendritic localization of VDCCs in
cells of the amygdala. However, given recent evidence of active
VDCCs in dendritic spines of other cells (Jaffe et al. 1994
; Mills et al. 1994
; Segal
1995
), it is plausible that NMDA receptors tightly colocalize
with VDCCs in dendritic spines, allowing only local NMDA EPSPs to
activate the VDCCs. Alternatively, non-NMDA receptors might colocalize
tightly with potassium channels, keeping prolonged depolarization from
activating VDCCs.
In spite of our inability to recruit the Ca2+
spike by isolated non-NMDA EPSPs, non-NMDA glutamate receptor
antagonists clearly increased the threshold stimulus intensity required
to recruit the Ca2+ spike (see Fig. 3). This may
be because the stimulation pathway is polysynaptic, whereas a major
component of excitatory neurotransmission in the amygdala is
accomplished via non-NMDA receptors (Gean and Chang
1992; Rannie et al. 1991
). Also,
non-NMDA-receptor-mediated EPSPs enhance NMDA-receptor-mediated EPSPs
by providing sufficient depolarization to relieve the
Mg2+ block of the NMDA receptor. An interesting
observation is that calcium spikes were evoked by trains of stimulation
in the presence of APV but not in the presence of MK801. There are
reports that LTP in the basolateral complex of the amygdala was blocked
partially by a moderate concentration (50 µM) of APV, whereas it was
fully blocked by higher concentration (100 µM) of APV (Huang
and Kandel 1998
; Maren 1995
). Together with
these reports, our result suggest that the action of APV on the NMDA
receptor is subject to competitive displacement under conditions of
high glutamate concentrations in the synapse.
Preferential activation of these channels by NMDA EPSPs is important because these Ca2+ currents may contribute to NMDA-dependent plasticity under normal conditions even though the Ca2+ spikes observed in this study may occur only in hyperexcitable states such as under blockade of GABAA receptors. It remains to be determined whether this Ca2+ current might be evoked locally in the postsynaptic membrane during LTP-inducing tetanic stimulation.
In addition, because we recorded whole cell events at the soma, we
cannot be certain whether non-NMDA EPSPs activate VDCCs locally in
distal dendrites that simply fail to propagate to the soma or whether
they might occur too temporally or spatially disparate to be integrated
into a somatic spike. However, we think it is most likely that the
whole cell events we recorded accurately represent local events in the
distal dendrites. Studies of hippocampal LTP show that
non-NMDA-dependent LTP can be elicited that is VDCC dependent and
typically requires blockade of K+ channels
(Huang and Malenka 1993; Petrozzino and Connor
1994
). This suggests that Ca2+ influx
through dendritic VDCCs is sufficient to induce synaptic potentiation
but that in the presence of NMDA-receptor blockade, even tetanic
stimulation fails to activate these VDCCs unless K+ channels are blocked. Moreover the observation
that Ca2+ spikes can be triggered by non-NMDA
EPSPs under these conditions might explain why certain types of
NMDA-independent, but VDCC-dependent, cellular events induced by
K+ channel blockers require non-NMDA EPSPs
(Aniksztejn and Ben-Ari 1991
; Johnston et al.
1992
).
In conclusion, we have found that NMDA-receptor-mediated
neurotransmission preferentially activates VDCCs in pyramidal cells of
the basolateral amygdala. This finding supports the possibility that
NMDA-receptor-dependent processes that require the influx of
Ca2+, such as neuroplasticity, could be mediated
by activation of VDCCs in addition to Ca2+ influx
through the NMDA-receptor complex. Although anatomic data suggest the
possibility of subcellular colocalization of NMDA receptors and VDCCs
in these cells, this remains to be verified. Our data show that simply
prolonging the non-NMDA-receptor-mediated EPSP depolarization with
stimulus trains or cyclothiazide is insufficient to activate these
spikes. This suggests that the duration of the unitary
non-NMDA-receptor-mediated EPSPs in the distal dendrites may be too
brief to activate distally located VDCCs in these cells. Previous
studies have indicated a critical role for K+
channels in regulating these dendritic Ca2+
conductances (Hoffman et al. 1997). These studies, along
with our data, suggest that increased input resistance in the dendrites and blockade of K+ channel-mediated
repolarization are the necessary conditions for activation of VDCCs in
the absence of NMDA-receptor stimulation. This model is supported by
our finding that prolonging non-NMDA-receptor-mediated depolarizations
by K+ channel blockade enables the triggering of
the Ca2+ spikes.
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FOOTNOTES |
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Address for reprint requests: S. D. Moore, Bldg. 16, Rm. 25, Veterans Administration Medical Center, 508 Fulton St., Durham, NC 27705.
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 1 March 1999; accepted in final form 6 October 1999.
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REFERENCES |
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