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The Journal of Neuroscience, July 1, 2000, 20(13):5124-5134
Calcium Influx through NMDA Receptors Directly Evokes GABA
Release in Olfactory Bulb Granule Cells
Brian
Halabisky,
Daniel
Friedman,
Milan
Radojicic, and
Ben W.
Strowbridge
Department of Neurosciences, Case Western Reserve University,
Cleveland, Ohio 44106-4975
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ABSTRACT |
Recurrent inhibition in olfactory bulb mitral cells is mediated via
reciprocal dendrodendritic synapses with granule cells. Although
GABAergic granule cells express both NMDA and non-NMDA glutamate
receptors, dendrodendritic inhibition (DDI) relies on the activation of
NMDA receptors. Using whole-cell recordings from rat olfactory bulb
slices, we now show that olfactory NMDA receptors have a dual role;
they depolarize granule cell spines, and they provide a source of
calcium that can evoke GABA exocytosis. We demonstrate that exogenous
NMDA can trigger GABA release after blockade of voltage-dependent
calcium channels (VDCCs) with Cd. We also find that postsynaptic
depolarization alone can evoke GABA release via a separate mechanism
that relies on calcium influx through Cd-sensitive VDCCs. By
selectively manipulating postsynaptic responses in granule cells with
high-K or low-Na extracellular solutions, we show that endogenous
glutamate can elicit GABA release via both NMDA receptor- and
VDCC-dependent pathways. Finally, we find that blockade of Na
channels in granule cells with tetrodotoxin enhances DDI, presumably by
reducing the depolarization of granule cells during DDI and thereby
increasing the driving force for Ca entry through NMDA receptors. These
results provide evidence of a novel mechanism for evoked transmitter
release that depends on Ca influx through ionotropic receptors and
provides a new potential site for synaptic plasticity in the olfactory bulb.
Key words:
olfactory bulb; mitral cell; glutamate receptor; EPSP; NMDA receptor; recurrent inhibition
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INTRODUCTION |
Release of the neurotransmitter
contained in presynaptic vesicles is triggered by a rise of
intracellular Ca
([Ca2+]i) within
the presynaptic terminal (Katz and Miledi, 1967 ; Augustine et al.,
1985; Mintz et al., 1995). Abundant evidence (Adler et al., 1991; Mintz
et al., 1995; Borst and Sakmann, 1996; Neher, 1998) indicates that
influx through voltage-dependent Ca channels (VDCCs) located
near vesicle-docking sites can provide the Ca influx needed to evoke
synaptic transmission. However, activation of presynaptic
neurotransmitter receptors that are permeable to Ca may also enable a
sufficient rise in intracellular Ca to trigger transmitter release.
This hypothesis is supported by studies of axon terminals that contain
presynaptic nicotinic ACh receptors (nAChRs) that are highly permeable
to Ca (Vernino et al., 1992; Rathouz and Berg, 1994; Gray et al.,
1996). Application of exogenous nicotine enhanced the frequency of
glutamatergic spontaneous EPSC by acting on presynaptic nAChRs in chick
medial habenula and interpeduncular nuclei (McGehee et al., 1995).
Similar results were found in rat when nicotine applied to mossy fiber
synapses in the hippocampus increased the frequency of miniature
glutamatergic EPSPs (Gray et al., 1996), an effect that persists after
blockade of VDCCs with cadmium.
In the olfactory bulb presynaptic granule cell spines contain
functional NMDA receptors (Isaacson and Strowbridge, 1998; Schoppa et
al., 1998) that are critically involved in triggering exocytosis of
GABA (Isaacson and Strowbridge, 1998). These dendritic spines form
reciprocal synapses with the secondary dendrites and somata of
glutamatergic mitral cells (Rall et al., 1966; Price and Powell, 1970a-c), the principal cell type in the olfactory bulb. This dendrodendritic microcircuit supports both recurrent feedback (Jahr and
Nicoll, 1980, 1982; Nowycky et al., 1981a,b, Isaacson and Strowbridge,
1998; Schoppa et al., 1998) and lateral inhibition of mitral cells
(Isaacson and Strowbridge, 1998). The close spatial relationship
between the postsynaptic active zone within the spine presumably containing glutamate receptorsand docked vesicles containing GABA
raises the possibility that the influx of Ca through NMDA receptors can
directly trigger exocytosis. Alternatively, the depolarization caused
by NMDA and AMPA receptor activation could open VDCCs near docked
vesicles. In the latter model, the Ca influx through VDCCs triggers
exocytosis, whereas influx of Ca through NMDA receptors plays only a
modulatory role by elevating residual Ca within the spine. Isaacson and
Strowbridge (1998) showed that direct depolarization (with KCl) evoked
GABA release from granule cell processes that could be blocked by Cd,
supporting the hypothesis that Ca influx through VDCCs can trigger
exocytosis. However, it is not clear from these studies whether Ca
influx through NMDA receptors present on the same spine also could
evoke transmitter release and which mechanism is dominant under
physiological conditions (i.e., when receptors on the spine are
activated by endogenous glutamate).
To address these questions, we explored the mechanisms controlling GABA
release from granule cell spines using whole-cell recordings from rat
olfactory bulb slices. We initially confirm that Ca influx through
VDCCs can trigger GABA release from granule cells. Unexpectedly, we
also find that exogenous NMDA can effectively trigger GABA release from
granule cell spines when all VDCCs capable of supporting transmitter
release are blocked with Cd. These results support a model in which
GABA release can be stimulated by Ca influx through either NMDA
receptors or VDCCs. By varying the amount of glutamate released by a
mitral cell, we show that Ca influx through VDCCs triggers GABA release
in response to relatively small EPSPs, whereas most GABA release in
response to large-amplitude EPSPs is triggered directly by Ca influx
through NMDA receptors.
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MATERIALS AND METHODS |
Olfactory bulb slices (300 µm) from 14- to 21-d-old Sprague
Dawley rats were made using a Leica (Nussloch, Germany) VT1000S vibratome. Slices were incubated in a 30°C water bath for 30 min and
then maintained submerged at room temperature. Whole-cell patch-clamp
recordings were made in mitral or granule cells visualized under
infrared-differential interference contrast optics (Zeiss Axioskop FS) using a Axopatch 1D amplifier (Axon Instruments). Mitral
and granule cells were distinguished by their location and the size of
their somata. Olfactory bulb slices were superfused with artificial
CSF (ACSF) that contained (in mM): NaCl 124, KCl 5, NaH2PO4 1.23, NaHCO3 26, dextrose 10, CaCl2 2.5, and MgSO4 1.2, equilibrated with 95% O2/5%
CO2 and warmed to 30°C (flow rate, 1-2
ml/min). As indicated in the figure legends, Mg was omitted from the
ACSF in some experiments. Except where noted, tetrodotoxin (TTX; 1 µM) was routinely added to the ACSF to block fast Na
currents. In some experiments (indicated in the figure legends)
N-methyl-D-glucamine (NMG; 124 mM) was substituted for 124 mM NaCl in the ACSF (pH adjusted to 7.4 by
titrating with HCl). This "low-Na" ACSF contained 27 rather than
151 mM Na.
Patch-clamp electrodes used for voltage-clamp recording (2-4 M
resistance) typically contained (in mM): CsCl 115, HEPES
10, EGTA 1, TEA-Cl 25, MgATP 4, Na3GTP 0.3, and
phosphocreatine 10. For voltage-clamp recordings during focal
application of NMDA and KCl, the internal solution contained (in
mM): Cs-methanesulfonate 140, NaCl 8, HEPES 10, EGTA 0.2, MgATP 4, Na3GTP 0.3, and phosphocreatine 10. In
some experiments (indicated in the figure legends), QX-314 (5 mM) was added to the internal solution to block Na currents in the mitral cell. The internal solution used for current-clamp recordings of granule cells contained (in mM):
K-methylsulfate 140, NaCl 8, HEPES 10, EGTA 0.2, MgATP 4, Na3GTP 0.3, and phosphocreatine 10. To evoke
glutamate release from mitral cells, steps to +10 mV were used from a
holding potential of  70 mV, unless noted otherwise in the figure legends.
Voltage and current records were low-pass filtered at 2 kHz and sampled
at 5 kHz using an ITC-18 16-bit analog-to-digital converter
(Instrutech). Series resistance, which typically was <10 M, was
routinely compensated by >80%. Data were streamed to a hard disk
using a custom Visual Basic program or acquired directly into IgorPro
(WaveMetrics). IgorPro and Origin 5.0 (Microcal) were used to analyze
data. Dendrodendritic inhibition was quantified using custom macros in
IgorPro that calculated the postsynaptic current integral (reported in
nanoamperes second) over a 2 sec period beginning 50 msec after
the end of the presynaptic voltage step. Action potential amplitude was
measured from the resting membrane potential. Example traces are
averages of five to six responses. In most experiments, drugs were
applied by switching the perfusion media. A Picospritzer (General
Valve, Fairfield, NJ) was used to focally apply receptor agonists and
KCl. TTX was obtained from Calbiochem (La Jolla, CA), the glutamate
receptor agonists and antagonists were obtained from Research
Biochemicals (Natick, MA), and all other chemicals were obtained from
Sigma (St. Louis, MO). Data are shown as the mean ± SEM.
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RESULTS |
We first examined the postsynaptic glutamate receptors that
mediate excitation of granule cells during dendrodendritic inhibition (DDI). Dendrodendritic inhibition in the olfactory bulb is thought to
result from glutamate released from the secondary dendrites and somata
of mitral cells exciting dendritic spines of granule cells and causing
the subsequent release of GABA and feedback inhibition in mitral cells
(see Fig. 1A). Previous
work (Isaacson and Strowbridge, 1998; Schoppa et al., 1998) suggested
that DDI relied on the activation of both NMDA and non-NMDA receptors
on granule cells. We investigated the roles of non-NMDA and NMDA receptors in DDI by recording from mitral cells with Mg-free ACSF (to
relieve the voltage-dependent Mg block of NMDA receptors.) Mitral cells
were recorded under whole-cell patch clamp with a CsCl-based internal
solution that resulted in inward GABAA
receptor-mediated currents at a holding potential of 70 mV. We
applied a depolarizing voltage step to +10 mV (for 50 msec) to evoke
DDI in mitral cells. Under these conditions, the selective non-NMDA
receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium (NBQX) did not affect the DDI response (93.0 ± 6.6% of control in 5 µM NBQX; n = 9;
105.2 ± 6.3% of control in 20 µM NBQX;
n = 5.) However, the subsequent application of the NMDA receptor antagonist D-2-amino-5-phosphopentanoic
acid (D-APV; 50 µM) caused a marked reduction of the
dendrodendritic response to 4.9 ± 0.8% of control
(n = 9). No further reduction was observed with the
combination of NBQX (5 µM) and
D-APV (4.5 ± 0.5% of control; 50 µM D-APV;
n = 10; see Fig. 1B,C). By contrast,
6,7-dinitroquinoxalone-2,3-dione (DNQX; 20 µM),
a less selective AMPA receptor antagonist (Randle et al., 1992),
reduced the DDI response to 50.7 ± 6.7% of control (n = 3), consistent with previous work (Isaacson and
Strowbridge, 1998). Although we find no requirement for activation of
non-NMDA receptors when eliciting DDI in Mg-free ACSF, AMPA receptors
may mediate the initial depolarization of granule cells when
physiological concentrations of Mg are present (see Isaacson and
Strowbridge, 1998; Schoppa et al., 1998, Schoppa and Westbrook,
1999).
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Figure 1.
NMDA receptors are required for dendrodendritic
inhibition. A, Schematic diagram of the reciprocal
synaptic connection between mitral cells and granule cell dendritic
spines. B, Bottom, Plot of the reduction
in dendrodendritic inhibition by antagonists of NMDA and non-NMDA
glutamate receptors. Although the non-NMDA receptor antagonist
NBQX (5 µM) did not affect DDI, the subsequent
application of D-APV (50 µM) nearly abolished
DDI. Top, Sample responses showing control responses
(left) and responses in NBQX (middle) and
in both NBQX and D-APV (right).
C, Summary of the actions of glutamate receptor
antagonists. All experiments were performed in Mg-free TTX ACSF using a
CsCl-based internal solution. The number of cells tested
is shown in parentheses. GC,
Granule cell; MC, mitral cell; Rec,
recording electrode.
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To examine the role of Ca currents through NMDA receptors in triggering
dendrodendritic inhibition, we first blocked VDCCs with Cd. We had
shown previously (Isaacson and Strowbridge, 1998) that Ca influx
through VDCCs in granule cell spines can evoke GABA release. We tested
if focal application of NMDA along the secondary dendrites and somata
of mitral cells elicits GABAergic IPSCs in mitral cells. In the
presence of 100 µM Cd, focal applications of NMDA (0.5 mM; 25-75 msec pressure pulse duration; see Fig. 2A) near the cell body
region evoked an outward current that persisted for several seconds
(Fig. 2B). Most responses to NMDA application in the
external plexiform layer (EPL) and mitral cell layer (MCL) were
biphasic; an initial IPSC was triggered with very short latency and was
followed by a slower, longer-duration IPSC. Both phases were blocked
completely by the GABAA receptor antagonist
picrotoxin (PTX; 50 µM; Fig.
2B-D). Mitral cells were held at +10 mV under voltage clamp, near the reversal potential of glutamate receptors, to
minimize currents through NMDA autoreceptors on the mitral cell
(Isaacson, 1999; Friedman and Strowbridge, 2000). Synaptic responses to
NMDA were not significantly diminished by increasing the concentration
of Cd to 200 µM, suggesting that the blockade of VDCCs in granule cells was complete (response integral, 648 ± 218 pA sec in 100 µM Cd and 560 ± 176 pA
sec in 200 µM Cd; n = 8;
p > 0.05 in paired t test; Fig.
2B-D). We also verified that Ca influx through NMDA
receptors leads to GABA release in physiological concentrations of Mg
by coapplying AMPA and NMDA (both 0.5 mM; 10 msec
puff durations) in the EPL in the presence of 1.2 mM Mg. Coapplication of AMPA and NMDA evoked a
response in 100 µM Cd that persisted for
several seconds (response integral, 1017 ± 384 pA sec;
n = 3) and that was abolished with the addition of 50 µM PTX (response integral, 135 ± 20 pA
sec; n = 3; Fig. 2E). These findings
suggest that the calcium influx through NMDA receptors on dendrites of
granule cells can trigger GABA release in the absence of calcium influx
through intrinsic voltage-gated calcium channels. Alternatively, it is
possible that NMDA receptors are not directly coupled to GABA release
but that instead NMDA receptor activation leads to a release of calcium
from internal stores in the granule cell, triggering exocytosis. This
explanation seems unlikely because depletion of internal stores with
thapsigargin (500 nM) had no significant effect
on IPSCs evoked by NMDA application near the soma in the presence of
200 µM Cd (115 ± 37% of control DDI
response; n = 3; data not shown).
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Figure 2.
Mitral cell IPSCs evoked by Ca influx through NMDA
receptors on granule cells. A, Diagram of the
experimental setup illustrates the location of the puffer pipette used
to activate NMDA receptors on granule cell spines. The pipette was
filled with 0.5 mM NMDA and positioned in the EPL, close to
the mitral cell body. B, Focal NMDA application in the
EPL evokes GABA release in the presence of the Ca channel antagonist
Cd. Left, Responses of a mitral cell held at +10 mV to
NMDA with 100 µM Cd in the bath (bottom
trace is the average of 5 consecutive responses) are
shown. Middle, Increasing the Cd concentration to 200 µM did not reduce the IPSC evoked by NMDA.
Right, The response to NMDA was almost completely
abolished by the addition of PTX (50 µM).
Asterisks indicate onset of focal drug application.
C, Plot of NMDA-evoked IPSC versus time in the same
mitral cell shown in B is presented.
Dashed line indicates steady state level of DDI in
100 µM Cd. D, Summary plot of IPSCs evoked
by NMDA in the EPL in 100 µM Cd, 200 µM Cd,
and 200 µM Cd + 50 µM PTX is shown. There
was no significant difference in the integral of the NMDA-evoked IPSC
in 100 and 200 µM Cd. All experiments were performed in
Mg-free TTX ACSF using a Cs-methanesulfonate-based internal solution.
E, Coapplication of AMPA and NMDA in the EPL evokes GABA
release in the presence of Mg (1.2 mM) and Cd (100 µM; left; average of 5 consecutive
traces) that is completely abolished by PTX (50 µM; right).
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Although the activation of NMDA receptors on granule cell spines in the
EPL leads to a large inhibitory current in mitral cells that is
resistant to blockade with Cd, depolarization of granule cell processes
outside the EPL and therefore not associated with reciprocal
dendrodendritic synapses (Price and Powell, 1970a,b) with either
NMDA or KCl always evoked GABA currents that were highly sensitive to
Cd. Focal application of NMDA in the granule cell layer (GCL) elicited
monophasic outward GABAergic currents in mitral cells (Fig.
3A,B) that were markedly
slower in onset than were responses to NMDA in the EPL and could be
blocked completely by PTX (50 µM; data not
shown). Bath application of 100 µM Cd strongly
reduced these inhibitory responses (to 14 ± 6% of control; n = 4; Fig. 3F), suggesting that
synaptic depolarization of granule cells via NMDA receptors can trigger
GABA release through two distinct pathways. When NMDA receptors are
activated near sites of reciprocal dendrodendritic synapses with mitral
cells (in the EPL or MCL), the Ca influx through these receptors
appears to trigger GABA exocytosis directly. However, NMDA application
in the GCL, where there are no reciprocal synapses, causes a
depolarization of the granule cell that activated VDCCs at GABA release
sites in the EPL. These findings suggest that both voltage-gated Ca channels and NMDA receptors are coupled to GABA release from granule cells.
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Figure 3.
Role of Ca channels in granule cells during
dendrodendritic inhibition. A, Schematic diagram of the
experimental setup is shown. B, Left Column, Responses
of a mitral cell held at +10 mV to brief puffs of NMDA under control
conditions (bottom trace is average of 5 responses) are shown. Middle Column, Bath application of
Cd (100 µM) nearly abolished the IPSC evoked by focal
NMDA application in the GCL. Right Column, The effect of
Cd was reversible on washout. C, Diagram of the
experimental setup illustrates the location of the puffer pipette
filled with KCl (90 mM) in the EPL. D,
Left Column, Synaptic responses of a mitral cell to the
focal application of KCl under control conditions are shown.
Middle Columns, Bath application of 100 µM Cd completely and reversibly blocked the KCl-evoked
IPSC. Right Column, The response to KCl also was blocked
by PTX (50 µM). E, Plot of the integral of
the KCl-evoked IPSC versus time in the same mitral cell shown in
D is presented. Top dashed line indicates
basal DDI; bottom dashed line indicates 0 nA sec.
F, Summary of the effect of Cd (100 µM) on
NMDA-, KCl-, or AMPA + NMDA-evoked IPSCs is shown. All
experiments in A-E were performed with Mg-free TTX ACSF
using a Cs-methanesulfonate-based internal solution. Experiments with
AMPA + NMDA application were performed in TTX ACSF containing 1.2 mM Mg.
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Besides blocking VDCCs, this concentration of Cd may also attenuate
NMDA responses (Mayer et al., 1989), thereby inhibiting part of the DDI
response. We examined this possibility by activating NMDA autoreceptors
present on the soma of mitral cells with focal NMDA application in the
presence of PTX to block GABAergic inhibition. Cadmium (100 µM) only slightly reduced the EPSC recorded in mitral cells (86.2 ± 10.2% of control; n = 3; data not
shown). These results suggest that there is only a minimal reduction of
the postsynaptic response to NMDA by the concentration of Cd used to
block VDCCs in granule cells.
Although Cd greatly reduced the IPSC evoked by distal NMDA puffs (in
the GCL), it is possible that the remaining response was mediated by
some Cd-resistant VDCCs. Alternatively, the residual IPSC could be
mediated by diffusion of NMDA to the EPL or MCL. To differentiate
between these hypotheses, we tested the effect of 100 µM
Cd on the IPSC evoked by focal application of KCl in the EPL (Fig.
3C). Under these conditions, GABA release relies entirely on
Ca influx through VDCCs; the KCl-evoked IPSC can be used, therefore, as
a sensitive test to determine whether all VDCCs that are coupled to
GABA release sites are blocked by 100 µM Cd.
Brief KCl puffs evoked a PTX-sensitive response that was rapid in onset
and shorter in duration compared with either of the NMDA-evoked
responses (Fig. 3D). In three mitral cells, bath application
of Cd (100 µM) completely abolished the
KCl-evoked IPSC (mean relative response, 33 ± 16% of control;
n = 3; Fig. 3D-F), revealing a small
inward current deflection that was also observed in PTX. These results
demonstrate that GABA can be released from granule cell spines by
direct depolarization and activation of VDCCs. Furthermore, VDCCs in
granule cells that can trigger GABA release appear to be completely
blocked by 100 µM Cd, suggesting that the
NMDA-evoked IPSCs we recorded in Cd (Fig. 2B) are
elicited by Ca influx through NMDA receptors on granule cell spines.
By using focal depolarization with NMDA and KCl, we have shown that Ca
influx through either VDCCs or NMDA receptors can evoke GABA release
from granule cells. We next addressed the question of which source of
Ca influx is dominant under different physiological conditions by
selectively blocking Na or K currents in granule cells. Membrane
depolarization in the granule cell spine is likely to activate VDCCs.
If GABA release relies primarily on Ca influx through VDCCs, then Na
channels may further depolarize granule cells (in addition to the
glutamatergic EPSP) and would be expected to enhance GABA release.
Blockade of these channels with TTX would be expected to reduce DDI
recorded in mitral cells. By contrast, if GABA release normally depends
on the Ca influx through NMDA receptors, depolarization provided by
nearby Na channels would be expected to reduce the driving force for Ca
entry and thereby reduce GABA release. Under these conditions, blockade
of Na channels would increase Ca influx through NMDA receptors and
enhance DDI.
We examined the effects of blockade of Na channels in granule cells
with TTX (1 µM) on the DDI response recorded in mitral cells held at 70 mV and filled with a CsCl-based internal solution containing QX-314 (to block Na channels in the mitral cell; 5 mM). We found that TTX had opposite effects on DDI
depending on the amount of glutamate released by the mitral cell.
Glutamate release was modulated by altering the duration of the
depolarizing voltage step in the mitral cell. Responses to brief steps
(2-3 msec; Fig. 4A)
were consistently decreased by TTX, with many voltage steps failing to
evoke DDI. Even under control conditions, we found that DDI responses
evoked by short-duration voltage steps were highly variable. By
contrast, TTX enhanced DDI evoked by longer-duration voltage steps
(e.g., 25-50 msec; Fig. 4C). Responses to these steps
showed less random variability but typically were depressed during
stimulus trains, reaching steady-state levels after 10-15 stimuli.
Blockade of Na channels in granule cells did not appear to affect this
depression (mean change in  = 8.8 ± 7.5%;
n = 8) but rather enhanced the steady-state DDI
response. Results from experiments with bath application of TTX are
summarized in Figure 4E. We found that TTX
significantly decreased DDI responses evoked by voltage steps of 2 or 3 msec duration to 17.9 ± 5.6 and 48.4 ± 2.1% of control,
respectively (p < 0.03; n = 9).
TTX significantly increased responses to 25 and 50 msec steps to
140 ± 12 and 137 ± 10% of control, respectively
(p < 0.03; n = 7 and 9). There
was no significant effect of TTX on DDI evoked by 5 msec pulses
(90.0 ± 28.0% of control; n = 9). We also
confirmed that this concentration of TTX was sufficient to block action potentials in granule cells. When granule cells were recorded under
current-clamp conditions, we found that 1 µM
TTX completely blocked granule cell action potential firing even after
current injection steps that had elicited multiple spikes in control
ACSF (n = 2; Fig. 4F).
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Figure 4.
Role of Na channels in granule cells during
dendrodendritic inhibition. A, Plot of DDI versus
stimulus number before (left) and after
(right) bath application of TTX (1 µM).
Dendrodendritic IPSCs were recorded in mitral cells and evoked at 0.067 Hz by 3 msec voltage steps from 70 to +10 mV. Short depolarizing
pulses often failed to elicit a DDI response in TTX, and the amplitude
of successful responses was decreased. Inset, Average
successful responses before and after TTX. Calibration: 1 nA, 0.5 sec. B, Schematic diagram of a granule cell spine
illustrating the proposed role for Na channels in augmenting the DDI
response. AP, Action potential.
C, Plot of DDI evoked by longer steps (50 msec) versus
stimulus number before (left) and after
(right) TTX (different mitral cell from that shown in
A). Under control conditions, the DDI response decreases
exponentially until a steady-state level is reached
(dashed line; = 0.038 sec 1). TTX increases the DDI
responses to long depolarizing pulses while not significantly affecting
the rate of depression during the train ( = 0.039 sec1). Inset,
Average DDI responses before and after TTX. Calibration: 1 nA, 0.5 sec.
D, Schematic diagram illustrating the proposed mechanism
by which blockade of Na channels increases the amount of GABA released
after NMDA receptor activation. E, Summary plot of the
effect of TTX on DDI at different step durations. F,
Current-clamp recording of a granule cell response to a brief (100 msec) depolarizing current injection. TTX abolished the action
potential firing (right) at a current (60 pA) that
evoked multiple action potentials under control conditions
(middle). Experiments in A-E were
performed in Mg-free ACSF using a CsCl-based internal solution
containing QX-314 (5 mM). The experiment shown in
F was performed in normal ACSF using a
K-methylsulfate-based internal solution.
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Although the enhancement of DDI with TTX is likely caused by increased
Ca influx through NMDA receptors (attributable to a reduction in the
depolarization of the granule cells), DDI may also increase because
granule cells become disinhibited. Recordings from mitral cells (Fig.
5A) consistently show frequent
spontaneous synaptic potentials that are blocked by PTX (50 µM), suggesting that they are mediated by
spontaneous spiking in granule cells. We also find that the frequency
of spontaneous IPSCs is increased by the bath application of K channel
blockers such as Cs (Fig. 5A; 5 mM)
and 4-AP (100 µM; data not shown). Because
these spontaneous synaptic inputs may modulate DDI, we simultaneously
recorded the effect of TTX on DDI and spontaneous synaptic activity
(estimated by the current variance in 1 sec epochs) in another
population of mitral cells. As shown in Figure 5B, TTX
markedly depressed spontaneous synaptic activity (to 23 ± 6% of
control; n = 6) at approximately the same time that DDI
responses were enhanced (to 140 ± 11% of control; 50 msec
voltage-step duration). The two effects of TTX were separable after
washout of TTX. Enhanced DDI responses recovered rapidly after washout
of TTX, whereas the rate of spontaneous synaptic activity remained
depressed for >5 min after DDI responses had returned to control
levels. These results suggest that the enhancement of DDI by TTX is not
caused by disinhibition of granule cells.
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Figure 5.
Increase in dendrodendritic inhibition with TTX is
not caused by a reduction in spontaneous synaptic inputs.
A, Bath application of Cs (5 mM) increases
the frequency and amplitude of spontaneous IPSCs in voltage-clamped
mitral cells. Most spontaneous synaptic inputs are inhibitory and are
blocked by PTX (50 µM). Three consecutive sweeps are
shown from the same mitral cell held at 70 mV. B,
Summary plots of the effects of TTX (1 µM) on
dendrodendritic inhibition (top) and spontaneous
synaptic activity (current variance; bottom) from six
mitral cells are shown. Tetrodotoxin increases dendrodendritic
inhibition (to 140 ± 11% of control; n = 6)
while reducing spontaneous synaptic activity (current variance
decreases to 23 ± 6% of control; n = 6). The
two effects of TTX are independent because they recover with different
time courses after washout of TTX. Experiments were performed in
Mg-free ACSF using a CsCl-based internal solution containing QX-314 (5 mM).
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We next examined whether depolarization of granule cells with elevated
extracellular K modulates DDI. If DDI responses evoked by long-duration
voltage steps depend on Ca influx through NMDA receptors, KCl-induced
depolarization should reduce this Ca influx by reducing the driving
force for Ca entry. In six mitral cells tested, increasing the
extracellular K concentration from 5 to 12 mM consistently
decreased the decay time constant to 59.7 ± 5.9% of control.
Primarily as a consequence of the shortened response, the integral of
the DDI response decreased to 31 ± 4.9% of control (n = 6; Fig.
6A). The effects of
elevated K were rapidly reversible, suggesting that the modulation of
DDI was caused by direct depolarization of granule cell processes. We
never observed an initial enhancement of the DDI response after
switching to high-K ACSF, as might be expected if a large component of
the GABA release was triggered by Ca influx through VDCCs. Instead,
these results are consistent with a direct coupling between Ca influx
through NMDA receptors and GABA release from granule cell spines.
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Figure 6.
Depolarization of granule cells decreases
dendrodendritic inhibition. A, Bottom,
Plot of the decrease in DDI (to 10.8% of control) after elevation of
extracellular [K+] from 5 to 12 mM is
shown. The response in 12 mM
[K+]o is reduced nearly to the level
seen during subsequent blockade of NMDA receptors with 50 µM D-APV (to 2.4% of control). Top,
Left, Middle, Example records show the reduction in peak
amplitude and the faster decay rate of the DDI response in elevated
[K+]o. Right, Example
records are normalized to the peak amplitude. B,
Middle, Blockade of K channels by 4-AP (100 µM) does not affect DDI recorded in mitral cells.
Bottom, Both 4-AP and Cs (data not shown) increase the
spontaneous synaptic activity recorded in mitral cells, quantified by
current variance. Dashed lines indicate
the control DDI integral and current variance. Top,
Example records show typical DDI responses under control conditions and
in 4-AP and Cs (5 mM). C, Summary of the
effects of increasing [K+]o and
blocking K channels on the dendrodendritic response in mitral cells is
shown. D, Summary of the effects of K channel blockade
on the current variance recorded in mitral cells is shown. Experiments
were performed in Mg-free TTX ACSF using a CsCl-based internal
solution.
|
|
In another test of the NMDA Ca influx model, we examined whether
blockade of K channels in granule cells modulated DDI. If GABA release
depends on Ca influx through VDCCs, then increasing the excitability of
granule cells with 4-aminopyridine (4-AP) or Cs would be expected to
enhance DDI. We found, however, that moderate concentrations of 4-AP
(100 µM) or CsCl (5 mM) had little effect on
DDI evoked by long-duration voltage steps (94.3 ± 13.2%; n = 11; 93.3 ± 4.5; n = 4, respectively; 50 msec voltage steps; Fig. 6B,C).
These agents consistently increased spontaneous PTX-sensitive synaptic
inputs recorded in mitral cells, suggesting that they were effective in
reducing K currents in granule cells. Current variance increased to
187 ± 12.4% of control with 4-AP (n = 10) and to 399 ± 160.0% of control with Cs (n = 6;
Fig. 6B,D). Results from experiments with K channel
blockers and elevated extracellular K are summarized in Figure 6,
C and D. Both the strong inhibition of DDI by
high K and the lack of modulation of DDI by 4-AP and Cs are consistent
with VDCC-independent GABA release triggered by Ca influx through NMDA
receptors on granule cell spines.
Finally, we examined whether substituting NMG, a nonpermeant
monovalent cation, for most of the Na normally present in the ACSF
would enhance DDI. This manipulation represents a strong test of the
hypothesized dependence of GABA release on Ca influx through NMDA
receptors. This model predicts that NMG substitution will enhance Ca
influx by reducing the postsynaptic depolarization of granule cells
during DDI. If GABA release relies instead on activation of VDCCs, the
reduction of EPSPs in the granule cell should cause a decrease in DDI.
We found that low-Na ACSF consistently increased DDI evoked by
long-duration voltage steps (from 0.73 ± 0.17 to 1.32 ± 0.12 nA sec; n = 11; Fig.
7A,D). As illustrated in
Figure 7A, the enhancement of the DDI integral in low-Na
ACSF was caused primarily by a prolongation in the response (DDI decay time constant increased to 183.4 ± 17.8% of control;
n = 11). Although the holding current did not change
when DDI response increased, we often observed a dramatic increase in
holding current after prolonged exposure to low-Na ACSF, presumably
reflecting secondary nonspecific effects of the altered extracellular
solution. These periods of increased holding current were always
accompanied by a dramatic reduction in DDI; the time
axis is interrupted, and DDI responses are blanked during
this period in Figure 7. Both the initial, presumably direct, effect of
low-Na ACSF on DDI and the subsequent increase in holding current were
always reversible after switching to normal ACSF (see Fig.
7A).
View larger version (30K):
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|
Figure 7.
Increasing the driving force for Ca through NMDA
receptor channels enhances GABA release. A,
Middle, Plot of the enhancement of dendrodendritic
inhibition after replacement of 124 mM Na with equimolar
NMG. Top, Sample records showing dendrodendritic
responses before (Control) and after
(NMG) switching to low-Na ACSF. Bottom,
NMG did not change the holding current during the time that DDI is
elevated. The effect of NMG was reversible after washout of NMG.
However, after a period of enhanced DDI responses, NMG often caused a
pronounced reduction in DDI and a concomitant increase in holding
current (indicated by the broken time
axis; data not shown). B, Plot of the
decrease in granule cell action potential amplitude during NMG
substitution. Inset, Representative action potentials
before and after NMG substitution. Action potential amplitudes are
reduced by NMG substitution at the time that DDI responses were
enhanced in the experiment shown in A. C,
Bottom, Plot of the elevation of DDI responses by NMG
substitution after partial blockade of NMDA receptors on granule cells
with 10 µM D-APV. Top, Sample
control records, responses in D-APV, and responses after
NMG substitution in D-APV (+NMG).
D, Summary of the results with NMG substitution. Note
the greater enhancement of DDI by NMG after partial blockade of NMDA
receptors with D-APV. Experiments in A and
C were performed in Mg-free TTX ACSF using a CsCl-based
internal solution. The experiment in B was performed in
normal ACSF using a K-methylsulfate-based internal solution.
|
|
We performed several control experiments with low-Na ACSF. We first
determined the time course of the reduction in Na gradient after
switching to low-Na ACSF. We recorded action potentials evoked in
granule cells by direct current injection (Fig. 7B) in
control and low-Na ACSF. The amplitude of these action potentials decreased at approximately the same time that DDI responses were increased (Fig. 7, compare A, B), suggesting that
the enhancement of DDI was caused by a reduction in the postsynaptic
depolarization of granule cells. Finally, we determined whether NMG
substitution also enhanced DDI responses after partial blockade of NMDA
receptors. A more prominent role for Ca influx through VDCCs could be
masked if the EPSP maximally activated VDCCs in granule cell spines. By
using a competitive receptor antagonist to reduce the postsynaptic depolarization, activation of VDCCs should be reduced, and any involvement of Ca influx through VDCCs should be revealed as a decrease
in DDI with NMG substitution. We found, however, that even in the
presence of 10 µM D-APV,
NMG still enhanced DDI responses to long-duration voltage steps
(490 ± 142% of DDI response in D-APV,
n = 4; Fig. 7C). Results from experiments
with NMG substitution are summarized in Figure 7D and are
consistent with a direct coupling between Ca influx through NMDA
receptors and GABA release from granule cell spines.
| |
DISCUSSION |
We find that Ca influx through two distinct pathways can trigger
GABA exocytosis from dendrites of olfactory granule cells. In
accordance with our previous findings, we show that GABA release can be
triggered by Ca influx through VDCCs, mostly likely high-threshold P/Q
channels (Isaacson and Strowbridge, 1998). We now report that Ca influx
through NMDA receptors on the spines of granule cells can evoke GABA
release. This pathway is effective even after blockade of all VDCCs
coupled to GABA release by Cd. By selectively altering the
postsynaptic depolarization produced by NMDA receptor activation of
granule cells, we provide evidence that most of the GABA release evoked
by brief EPSPs is caused by Ca influx through VDCCs. However, longer-duration EPSPs evoke additional GABA release, primarily via
direct actions of NMDA receptor Ca influx on exocytosis. These data
suggest a new role of Ca-permeable ligand-gated channels in directly
evoking neurotransmitter release. This pathway also provides a new,
potentially important site of synaptic plasticity in the olfactory bulb.
GABA release evoked by Ca influx through NMDA receptors
and VDCCs
Dendritic spines of olfactory granule cells function both as
postsynaptic elements, with receptors activated by glutamate released
from nearby dendritic and somatic processes of mitral cells (Wellis and
Kauer, 1993; Isaacson and Strowbridge, 1998; Schoppa et al., 1998), and
as presynaptic terminals capable of releasing GABA in a Ca-dependent
manner (Isaacson and Strowbridge, 1998). Although granule cells express
NMDA and non-NMDA glutamate receptors (Isaacson and Strowbridge 1998;
Schoppa et al., 1998; Sassoè-Pognetto and Otterson, 2000),
GABA release appears to depend primarily on the activation of
postsynaptic NMDA receptors. Electron microscopic studies reveal
relatively close spacing between the postsynaptic active zone in
granule cell spines, presumably containing most of the postsynaptic
glutamate receptors, and sites of docked vesicles containing GABA
(Price and Powell, 1970b; Sassoè-Pognetto and Otterson,
2000). The relatively close proximity between presynaptic and
postsynaptic sites within the spine raises the possibility that the
critical requirement for activation of the NMDA receptor in DDI
reflects a direct role of Ca entering through NMDA receptors in
triggering GABA exocytosis. Although in some synapses, including the
squid giant synapse, VDCCs are thought to be intimatelyassociated with
proteins that control exocytosis (Adler et al., 1991), there is
evidence from other synapses that transmitter can be released as a
consequence of Ca influx from relatively distant sites, potentially including Ca-permeable receptors. For example, the inhibition of
glutamate release in the auditory brainstem by the relatively slow-acting Ca chelator EGTA (Borst and Sakmann, 1996) has been interpreted as evidence of distant coupling between sites of Ca entry
and vesicle-docking sites. Although we have demonstrated previously
(Isaacson and Strowbridge, 1998) that Ca entry through VDCCs can evoke
GABA release from granule cells depolarized with KCl, it is unclear
from previous studies whether endogenous glutamate released from mitral
cells elicits GABA release by activating VDCCs and what role the Ca
entering through NMDA receptors plays in exocytosis.
The large Cd-insensitive IPSCs evoked by NMDA observed in this study
strongly suggest that Ca entry through postsynaptic NMDA receptors can
trigger GABA release from granule cell spines. However, this
interpretation depends on the ability of Cd to block all VDCCs that
could support GABA release from granule cells. To address this issue we
showed that Cd dramatically reduced IPSCs evoked by NMDA application to
distal sites on the granule cell. In addition, we showed that Cd
completely and reversibly blocked DDI evoked by focal depolarization of
spines with KCl. Together, these experiments support a model in which
GABA release from granule cell spines can be controlled directly by Ca
influx through NMDA receptors. We also observed very different time
courses in the responses to NMDA and KCl. Direct activation of NMDA
receptors on the granule cell spines evoked biphasic IPSCs. The rapid
onset and short duration of the early phase of this response may
reflect release from a small, readily releasable pool of vesicles
located close to the NMDA receptors. The prolonged second phase may
reflect the release of more distant vesicles located closer to VDCCs as
well as the repopulation of the proximal release sites. Direct
depolarization of the spine evoked an IPSC with very rapid onset
kinetics, consistent with relatively close coupling between VDCCs.
Although the inability of thapsigargin to modify the biphasic time
course of responses to NMDA in the EPL suggests that release of Ca from
internal stores does not directly trigger GABA exocytosis, additional
experiments will be required to address this issue completely.
Opposite effects of Na channel blockade on
dendrodendritic inhibition
Can endogenous glutamate released by mitral cells also evoke GABA
release using similar mechanisms? Although this question is difficult
to answer directly, we obtained several lines of evidence that suggest
endogenous glutamate can trigger recurrent IPSCs via both VDCC- and
NMDA receptor-dependent mechanisms. The high degree of response
variability when relatively small amounts of glutamate are released is
consistent with amplification of the glutamatergic EPSP by intrinsic Na
channels in the granule cell. Presumably the glutamate EPSP triggers a
variable number of action potentials, thus enhancing the variability
normally associated with transmitter release. These granule action
potentials, in turn, would provide a larger stimulus for activating
VDCCs coupled to GABA release. This model explains why blocking granule cell Na channels with TTX reduces DDI evoked by short-duration voltage
steps. Blockade of persistent Na current in granule cells also could
contribute to the reduction in DDI. By contrast, when larger amounts of
glutamate are released (by using longer-duration voltage steps), TTX
has the opposite effect, and DDI responses are enhanced. In addition,
control responses evoked by long-duration steps show much less
variability than the TTX-sensitive responses elicited by short-duration
steps. One potential explanation for the decreased variability is that
the larger control EPSP evoked now fully activates VDCCs in granule
cells, reducing the variability associated with spike generation.
Alternatively, the Ca influx through NMDA receptors activated during
long-duration EPSPs causes additional GABA secretion. In both models,
activation of Na channels in granule cells functions to decrease Ca
entry (and DDI) by reducing the driving force for Ca. A pronounced
depression of DDI responses also is apparent after trains of
long-duration voltage steps that are not affected by Na channel
blockade. Although we did not explore the mechanism underlying this
effect, it is possible that the larger concentration of glutamate
released by mitral cells activates metabotropic receptors on the mitral
or granule cells that function to inhibit DDI. The lack of correlation
we found between the time course of enhancement of DDI by TTX and the
reduction in spontaneous synaptic activity suggests that the effects of
TTX are not caused by disinhibition of granule cells.
Modulation of GABA release by altering the driving force for
Ca entry
The results using elevated-K ACSF to depolarize granule cells
suggest that GABA release from granule cell spines is limited by the Ca
driving force. A dependence on driving force would be expected if Ca
entry through NMDA receptors governs GABA release. Postsynaptic
depolarization would not be expected to alter the conductance
underlying the glutamatergic EPSPonly the inward synaptic current,
mediated in part by Ca (Mayer and Westbrook, 1987; Iino et al., 1990).
Alternatively, the depolarization provided by the control EPSP may
fully activate VDCCs coupled to GABA release; additional depolarization
(because of the opening of intrinsic Na channels) would then function
to reduce Ca influx through VDCCs. We found that NMG substitution
consistently increased DDI in control conditions as well as after the
reduction of granule cell EPSPs with 10 µM
D-APV. This consistent enhancement of DDI strongly suggests
that granule cell depolarization can inhibit GABA release by reducing
Ca influx through NMDA receptors. These experiments are complicated by
secondary effects of the low-Na ACSF that appear to affect both mitral
and granule cell intrinsic properties. We do show, however, that the
time course of DDI enhancement is well matched by an indicator of the
Na gradient in granule cells (action potential amplitude).
We also found no effect of two K channel blockers on DDI. Such an
effect might be expected if GABA release strongly depended on VDCC
activation because K channels are likely to regulate granule cell
excitability. Modulation of DDI responses by high concentrations of
4-AP was reported recently by Schoppa and Westbrook (1999). The
apparent absence of DDI modulation by Cs and 4-AP in our study is
consistent with a dominant role for NMDA receptors as the source of the
Ca influx triggering GABA release evoked by prolonged granule cell EPSPs.
Our findings support the hypothesis that Ca influx through both NMDA
receptors and VDCCs can evoke GABA release from granule cell spines,
with VDCCs playing a preferential role when GABA release is triggered
by small-amplitude EPSPs and Ca influx through NMDA receptors governing
release in response to large EPSPs. The ability of Ca influx through
NMDA receptors to evoke transmitter release may account for the
critical role of NMDA receptor activation in DDI reported in this and
previous studies (Isaacson and Strowbridge, 1998; Schoppa et al.,
1998). On the basis of our results, it seems that activation of
non-NMDA receptors evokes GABA release exclusively via the
VDCC-dependent pathway, whereas NMDA receptor activation seems to
trigger GABA release both by activating VDCCs and via direct action of
Ca influx through the receptor itself. Although in these experiments
glutamate release from the mitral cell was controlled by voltage-clamp
command steps, our results suggest that Ca influx through NMDA
receptors may govern DDI evoked by high-frequency trains of
back-propagating action potentials in mitral cell secondary dendrites.
By contrast, we would predict that the feedback inhibition triggered by
unitary back-propagating action potentials relies on VDCCs (and perhaps
on Na channels) in granule cell spines. The dual role of NMDA receptors
on granule cells, both to depolarize the cell and to act as a source of
Ca capable of triggering GABA exocytosis, may also lead to new
mechanisms for plasticity in the DDI response. Depolarization of
granule cells, because of remote EPSPs, for example, may function to
shift the mechanisms triggering GABA release and alter the time course of recurrent inhibition.
Our findings provide evidence for direct coupling between Ca entry
through presynaptic ligand-gated receptors and evoked transmitter release. This function for NMDA receptors is consistent with the relatively close spacing between presynaptic and postsynaptic sites in
granule cell spines in the olfactory bulb (Price and Powell, 1970b;
Sassoè-Pognetto and Otterson, 2000). The additional Ca influx
provided by NMDA receptors appears to enhance both the amplitude and
reliability of reciprocal dendrodendritic inhibition. The dual role of
NMDA receptors that have been found in the olfactory bulb may also
occur elsewhere in the CNS with other types of Ca-permeable presynaptic
receptors. For example, activation of nicotinic ACh receptors leads to
Ca accumulation in axon terminals in the spinal cord (McGehee et al.,
1995) and increases the frequency of Cd-resistant spontaneous miniature
synaptic potentials in both the hippocampus (Gray et al., 1996) and
thalamus (Léna and Changeux, 1997).
| |
FOOTNOTES |
Received Feb. 7, 2000; revised March 28, 2000; accepted April 11, 2000.
This study was supported by National Institutes of Health Grant NS
33590. Dr. Strowbridge is a Mount Sinai Health Care Foundation Scholar.
We thank Drs. R. Traub and D. Kunze for helpful comments on this manuscript.
Correspondence should be addressed to Dr. Ben W. Strowbridge,
Department of Neurosciences, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4975. E-mail: bxs48{at}po.cwru.edu.
| |
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V. Kapoor and N. N. Urban
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[Abstract]
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A. S. Ghatpande, K. Sivaraaman, and S. Vijayaraghavan
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[Abstract]
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L. J. Blakemore, M. Resasco, M. A. Mercado, and P. Q. Trombley
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[Abstract]
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T. Zelles, J. D. Boyd, A. B. Hardy, and K. R. Delaney
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S. B. Dietz and V. N. Murthy
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[Abstract]
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A. Hayar, M. T. Shipley, and M. Ennis
Olfactory Bulb External Tufted Cells Are Synchronized by Multiple Intraglomerular Mechanisms
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[Abstract]
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V. Egger, K. Svoboda, and Z. F. Mainen
Dendrodendritic Synaptic Signals in Olfactory Bulb Granule Cells: Local Spine Boost and Global Low-Threshold Spike
J. Neurosci.,
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[Abstract]
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C. P. J de Kock, N. Burnashev, J. C Lodder, H. D Mansvelder, and A. B Brussaard
NMDA receptors induce somatodendritic secretion in hypothalamic neurones of lactating female rats
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November 15, 2004;
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[Abstract]
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R. Balu, P. Larimer, and B. W. Strowbridge
Phasic Stimuli Evoke Precisely Timed Spikes in Intermittently Discharging Mitral Cells
J Neurophysiol,
August 1, 2004;
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[Abstract]
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P.-M. Lledo, A. Saghatelyan, and M. Lemasson
Inhibitory Interneurons in the Olfactory Bulb: From Development to Function
Neuroscientist,
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[Abstract]
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S. Lagier, A. Carleton, and P.-M. Lledo
Interplay between Local GABAergic Interneurons and Relay Neurons Generates {gamma} Oscillations in the Rat Olfactory Bulb
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A. P. Davison, J. Feng, and D. Brown
Dendrodendritic Inhibition and Simulated Odor Responses in a Detailed Olfactory Bulb Network Model
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[Abstract]
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V. Egger, K. Svoboda, and Z. F. Mainen
Mechanisms of Lateral Inhibition in the Olfactory Bulb: Efficiency and Modulation of Spike-Evoked Calcium Influx into Granule Cells
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B. Halabisky and B. W. Strowbridge
{gamma}-Frequency Excitatory Input to Granule Cells Facilitates Dendrodendritic Inhibition in the Rat Olfactory Bulb
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D. Friedman and B. W. Strowbridge
Both Electrical and Chemical Synapses Mediate Fast Network Oscillations in the Olfactory Bulb
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J. S. Isaacson and H. Vitten
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B. J Hall and K. R Delaney
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N. N Urban and B. Sakmann
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D. LIM, K. KYOZUKA, G. GRAGNANIELLO, E. CARAFOLI, and L. SANTELLA
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D. G Placantonakis and J. P Welsh
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U. Scheidweiler, L. Nezlin, J. Rabba, B. Muller, and D. Schild
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J. M. Christie, N. E. Schoppa, and G. L. Westbrook
Tufted Cell Dendrodendritic Inhibition in the Olfactory Bulb Is Dependent on NMDA Receptor Activity
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T. W. Margrie, B. Sakmann, and N. N. Urban
Action potential propagation in mitral cell lateral dendrites is decremental and controls recurrent and lateral inhibition in the mammalian olfactory bulb
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J. S. Isaacson
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[Abstract]
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T. W. Margrie, B. Sakmann, and N. N. Urban
Action potential propagation in mitral cell lateral dendrites is decremental and controls recurrent and lateral inhibition in the mammalian olfactory bulb
PNAS,
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J. S. Isaacson
Mechanisms governing dendritic gamma -aminobutyric acid (GABA) release in the rat olfactory bulb
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A. Didier, A. Carleton, J. G. Bjaalie, J.-D. Vincent, O. P. Ottersen, J. Storm-Mathisen, and P.-M. Lledo
A dendrodendritic reciprocal synapse provides a recurrent excitatory connection in the olfactory bulb
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TOP
ABSTRACT
INTRODUCTION
