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INTRODUCTION |
A neuron can synaptically
modulate its own output through several mechanisms. First, the axon can
establish autaptic contacts on its somato-dendritic compartment (see
review by Bekkers 1998
; Pouzat and Marty
1998
). A second and more common mechanism involves receptors
located at the axon terminal. On calcium entry, transmitter is
released, activating autoreceptors as well as postsynaptic receptors.
In most cases, activation of autoreceptors results in inhibition of
transmitter release, although in some cases activation of autoreceptors
located at terminals enhances transmitter release (see review by
Langer 1997
). Finally, a neuron may regulate its firing
through the activation of autoreceptors located on the soma and
dendrites. These receptors are coupled to G-proteins and mediate a slow
autoinhibition. However, recent studies have suggested that in the
olfactory bulb, activation of mitral cell dendritic receptors could
involve excitatory ligand-gated channels (Aroniadou-Anderjaska
et al. 1999
; Chen et al. 1998
; Isaacson 1999
; Salin and Charpak 1998
).
Mitral cells (MC) receive olfactory information from sensory neurons
and relay it to the cortex after a local processing with olfactory bulb
interneurons. MC dendrites are divided into two distinct compartments
that are involved in segregated circuits: 1) the primary
dendrite that receives hundreds of axonal excitatory terminals from
sensory neurons within a given glomerulus and make dendrodendritic
synapses with inhibitory periglomerular cells; 2) the
secondary dendrites that make dendrodendritic synapses with inhibitory
granular cells (Rall et al. 1966
) in the external plexiform layer. Both types of dendrites release glutamate
and possess a high-density of
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA),
N-methyl-D-aspartate (NMDA), and metabotropic
glutamate receptors (Montague and Greer 1999
;
Petralia et al. 1994a
,b
; van den Pol
1995
). However, electron microscopy studies have suggested that
excitatory afferent synapses are located exclusively in the distal
region of the primary dendrite (Price and Powell 1970
). Thus the major part of the mitral cell dendritic arbor is entirely devoid of glutamatergic inputs, although its membrane is provided with
glutamatergic receptors. This mismatch has raised the following question: are these dendritic receptors synaptically activated when
glutamate is released at and diffuses from the dendrodendritic synapses? It has been shown that the NMDA glutamate autoreceptors of MC
dendrites are indeed activated by glutamate released at the
dendrodendritic synapses (Aroniadou-Anderjaska et al.
1999
; Chen et al. 1998
; Isaacson
1999
; Salin and Charpak 1998
). We show here
that, in addition to NMDA receptors, non-NMDA autoreceptors are
activated by sodium or calcium spikes, and we analyze the role of
self-excitation in the control of MC discharge.
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METHODS |
Horizontal olfactory bulb slices (300-400 µm) were obtained
from 12- to 22-day-old Sprague-Dawley rats. Self-excitation was also
observed in MCs of adult rats (2-5 mo, not shown). Rats were deeply
anesthetized with pentobarbital sodium and the brain dissected out in
ice cold saline solution (in mM: 124 NaCl, 26 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 2 Ca2Cl, 1 MgCl2, and 10 D-glucose
saturated with 95% O2-5%
CO2, pH 7.4). Recordings from mitral cells were performed in an extracellular medium of the same composition as above,
using the whole-cell recording configuration with the Axopatch 200A
amplifier (Axon Instruments, Burlingame, CA) in voltage- or
current-clamp modes. Recording electrodes were filled with two
different types of solution: potassium gluconate solution (120 mM
K-gluconate, 10 mM KCl, 2 mM MgCl2, 8 mM NaCl, and 10 mM
HEPES) and cesium gluconate (in experiments where calcium currents were
evoked by a voltage pulse; in mM: 120 Cs-gluconate, 10 CsCl, 2 MgCl2, 8 NaCl, and 10 HEPES). Recordings were done at room
temperature (22-24°C) and in some cases at 32-34°C. Biocytin
(0.5%) or Lucifer yellow (1%) was routinely added to the recording
electrode solution to allow morphological identification of the
recorded cells. Patch pipettes filled with extracellular solution were
used for electrical stimulations (0.1 ms, 10- to 100-µA pulses) of
neurons and fibers in the glomerular and external plexiform
layers. Inter-trial intervals were 10-20 s. In some experiments, a
fine patch pipette was used to selectively section and remove mitral
cell axons and dendrites. The following drugs were used: picrotoxin
(PTX, Sigma), D-2-amino-5-phosphonovalerate (APV,
Tocris), 6-cyano-7-nitroquinolaxine2,3-dione (CNQX, Tocris), MK-801
hydrogen maleate (MK-801, RBI),
3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP,
Tocris), tetrodotoxin (TTX, Sigma),
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA, Sigma).
 |
RESULTS |
MC action potentials, when recorded in control conditions, are
followed by a profound synaptic inhibition due to the activation of
reciprocal dendrodendritic synapses (Fig.
1A, left)
(Isaacson and Strowbridge 1998
; Jahr and Nicoll
1982
; Nowycky et al. 1981a
; Rall and
Shepherd 1968
; Schoppa et al. 1998
). In
the presence of GABAA receptor antagonists, a
slow depolarizing afterpotential (DAP) was unmasked (Fig. 1A,
middle) (Aroniadou-Anderjaska et al. 1999
;
Isaacson 1999
; Nicoll and Jahr 1982
;
Nowycky et al. 1981b
). DAP was abolished by kynurenate,
a non-selective ionotropic glutamate receptor antagonist (Fig.
1A, right), suggesting that it did not result
from the activation of intrinsic membrane properties but rather that it
was a slow glutamate-mediated excitatory postsynaptic potential
(slow-EPSP). In voltage-clamp conditions (Fig. 1B1), voltage
steps evoking partially clamped sodium spikes evoked slow inward
synaptic currents underlying the slow-EPSP. In the absence of
extracellular Mg2+, the slow current was strongly
enhanced (Fig. 1B1). In the presence of 1 mM
Mg2+, the current underlying the slow-EPSP
displayed a rectification at hyperpolarized potentials typical of an
NMDA current blocked by magnesium (Fig. 1, B1 and
B2).

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Fig. 1.
Blockade of GABAA receptors unmasks a slow
glutamate-mediated excitatory postsynaptic potential (EPSP) in mitral
cells. A: in control conditions (left,
current-clamp recording, extracellular magnesium: 1 mM,
Vh: 62 mV), an action potential triggered
a strong recurrent synaptic inhibition (arrow) that was blocked during
bath application of the GABAA receptor antagonist
picrotoxin (PTX, 100 µM, middle). Note that the
blockade of inhibition unmasked a slow depolarizing afterpotential
(DAP, double arrow) during which the cell fires. Subsequent application
of the nonselective ionotropic glutamate receptor antagonist kynurenate
(KYN, 10 mM) strongly reduced the slow DAP, which thus corresponds to a
slow-EPSP (right). The bottom lines indicate the
depolarizing current pulses. B1: voltage-clamp
recordings (averages of 5) of the
N-methyl-D-aspartate (NMDA) current
underlying the slow-EPSP. A single sodium spike induced an inward
current that was reduced in the presence of magnesium [100 µM PTX
and 20 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX); averages of
5]. The D-APV-sensitive component of the slow excitatory
postsynaptic current was voltage dependent, being reduced at
hyperpolarized potentials when the recording was done in the presence
of magnesium (1 mM). The evoked Na currents were digitally removed. The
bottom lines indicate the voltage steps. B2: summary
graph illustrating the voltage dependency of the
D-APV-sensitive component of the slow synaptic current
recorded in the presence of magnesium (100 µM PTX and 10-20 µM
CNQX). The abscissa shows the holding potential of the cell and the
ordinate the maximum peak amplitude. The error bars indicate SE.
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In contrast to a recent study (Isaacson 1999
), we found
that the slow current presented two components: a major one sensitive to the NMDA receptor antagonists D-APV (25-100 µM) and
CPP (10-20 µM) and a second one sensitive to the non-NMDA receptor
antagonists CNQX (10-20 µM) and
2,3dioxo-6-nitro-1,2,3,4-tetrahydrobenzoquinoxaline-7-sulphonamide (NBQX) (10 µM; Fig. 2, A and
B1). About 20% of the total current recorded in absence of
Mg2+ was blocked by CNQX (Fig. 2B1;
28.5 ± 7 pA vs. 136.8 ± 36.4 pA, mean ± SE,
n = 10, P = 0.01, Student's
t-test). The broad spectrum metabotropic glutamate receptor
antagonist (S)-
-methyl-4-carboxyphenylglycine (MCPG)
(0.5-1.5 mM) had no effect (Fig. 2B1). The
non-NMDA-mediated current, although slow, was faster than the decay of
the NMDA-mediated component (AMPA/kainate-R component: 58.6 ± 11.3 ms, n = 6 vs. NMDA-R component: 230.1 ± 25.1 ms, n = 7, P = 0.0001; Fig.
2B2).

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Fig. 2.
The slow glutamate-mediated excitatory postsynaptic current has 2 components. A: voltage-clamp recordings
(Vh = 70 mV) of the current
underlying the slow-EPSP. Time course amplitude of the fast ( ,
see inset) and of the slow ( ) component
of the inward current recorded in the absence of extracellular
magnesium and with PTX. A single sodium spike induced an inward current
that was reduced by the successive application of D-APV
(100 µM) and of D-APV (100 µM)-2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzoquinoxaline-7-sulphonamide
(NBQX) (10 µM). B1: summary graph of each
glutamatergic receptor-mediated component contributing to the slow
excitatory postsynaptic current. Peak values were obtained after
subtraction of the currents recorded in the presence of 10-20 µM
CNQX (7 cells or 10-20 µM NBQX, in 3 cells), 50-100 µM
D-APV [6 cells or 10-20 µM
3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP) in 4 cells] and 0.5-1.5 mM (S)- -methyl-4-carboxyphenylglycine
(MCPG) (4 cells), respectively, from the control current peak
values. After the subtraction, the maximum peak currents represented in
the bar graph were calculated as in A (see and
the 1st vertical bar). Calculations of the peak currents were done at
least 10 min after the beginning of application of the antagonists
(average of 5-10 traces). Note that in average the NMDA-R component
represents more than 80% of the total current. MCPG exerted no action
on the slow excitatory EPSC, suggesting that mGLU-R-mediated response
does not contribute to the slow-EPSP. B2: summary graph
of the decay time constant of the NMDA-R and non-NMDA-R currents
(recordings in 0 mM magnesium and with PTX).
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To test whether this phenomenon was a self-excitation due to the
activation of MC glutamatergic autoreceptors, we considered and
eliminated a variety of other potential synaptic mechanisms. The
slow-EPSP could be due to a recurrent excitation since excitatory synaptic contacts impinging on MCs have been observed (Martinez and Freeman 1984
; Nicoll 1971
; Nowycky et
al. 1981b
). Action potentials (Bischofberger and
Jonas 1997
; Chen et al. 1997
; Isaacson
and Strowbridge 1998
) initiated or backpropagating in dendrites
could thus activate periglomerular and/or external plexiform excitatory cells via dendritic release sites (Fig.
3, top). We thus used focal
electrical stimulations to activate neurons or fibers in the glomerular
and external plexiform layers. Extracellular electrical stimulations
generated large EPSCs in mitral cells that were reduced in the presence
of (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCGIV),
an agonist of group II metabotropic glutamate receptors (Fig.
3A1). As in other regions of the brain (Kamiya and
Ozawa 1999
; Macek et al. 1996
; see for review:
Conn and Pin 1997
), DCGIV acted presynaptically by
inhibiting glutamate release as suggested by the switch from
paired-pulse depression to paired-pulse facilitation of the response
(Fig. 3, A1 and A2). While DCGIV (10 µM)
strongly decreased the size of the extracellularly evoked EPSP (Fig.
3B1, 79 ± 5.7% of decrease, n = 12 cells in 7 slices, 13 sites of electrical stimulation located in the
glomerular layer and 12 sites of electrical stimulation located in the
external plexiform layer), it did not reduce the amplitude of the
slow-EPSP (Fig. 3B2) generated in the same mitral cells.
Thus it is unlikely that olfactory bulb excitatory fibers, which
are inhibited by DCGIV, play a role in the generation of the
slow-EPSP.

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Fig. 3.
Excitatory connections sensitive to group II metabotropic glutamate
receptor agonists and autapses do not play a role in the genesis of the
slow EPSP. Top: schematic diagram illustrating 2 hypotheses that may explain self-excitation: 1) the
action potential backpropagating in the primary dendrite activates a
disynaptic excitatory circuit in the olfactory bulb; 2)
the action potential activates an axo-dendritic autapse.
A1: (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine
(DCGIV), a group II metabotropic glutamate receptor agonist,
inhibited excitatory responses evoked by focal electrical stimulation
of the olfactory bulb superficial layers. Left:
voltage-clamp recording (averages of 10 traces, K-gluconate-filled
patch pipette) of an EPSC evoked by an extracellular stimulation of the
glomerular layer with a patch pipette filled with the extracellular
solution (PTX and Mg2+, 1 mM). Middle: low
concentration of DCGIV (1 µM) reduced the size of the evoked inward
current. No steady-state current was observed during the application of
DCGIV. Right: the application of the ionotropic
glutamate receptor antagonists D-APV (50 µM) and NBQX (10 µM) totally abolished the evoked EPSC. A2: the average
traces of the recording made in the presence of picrotoxin (thin line,
1) and with the addition of DCGIV (thick line, 2) are scaled with the
1st response. The change from paired-pulse depression to paired-pulse
facilitation suggests that DCGIV acts presynaptically.
B1: an EPSP (averages of 10 traces) was evoked in a
mitral cell in response to an electrical stimulation applied focally in
the external plexiform layer. DCGIV (10 µM) deeply reduced the
amplitude of the excitatory response. B2: in contrast,
DCGIV had no effect on the slow-EPSP recorded in the same cell,
indicating the involvement of 2 different mechanisms in the generation
of these EPSPs (the extracellular solution contained PTX and
Mg2+, 1 mM). The evoked sodium current was digitally
removed. C: the suppression of sodium spikes does not
block the slow-EPSC. Voltage-clamp recordings were performed with a
patch pipette containing cesium gluconate
(Vh = 70 mV). In control (PTX), a
60-mV voltage step evoked a sodium current associated with a calcium
current (truncated) followed by a slow-EPSC. Tetrodotoxin (1 µM) and
then the non-NMDA receptor antagonist NBQX (10 µM) reduced the size
of the inward current, which was then completely abolished by
D-APV (50 µM); Mg = 0 mM.
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Another possibility was that MCs establish autaptic excitatory synapses
since they send axonal collaterals in the external plexiform layer,
which also contains their secondary dendrites (Fig. 3, top).
Autapses have recently been shown to be functional in the CNS
(Pouzat and Marty 1998
). We found that when MCs were recorded in voltage-clamp conditions with a cesium-gluconate containing pipette, the inward current following a mixed sodium/calcium spike (partially clamped) decreased by only 6.5 ± 2.6%
(n = 6) in the presence of tetrodotoxin (TTX, Fig.
3C, 1 µM), a finding previously reported (Isaacson
1999
; Nicoll and Jahr 1982
), suggesting that sodium spikes are not a prerequisite for the induction of the slow
synaptic response. The role of an autaptic connection mediated by a
calcium spike propagation was further excluded by experiments where the
MC axon was sectioned from the soma before the recording (n = 5, data not shown) without blocking the slow
synaptic response. Both NMDA-R and AMPA/kainate-R currents persisted in
these recording conditions. Altogether, these results exclude autaptic
connections as a mechanism for the genesis of the slow-EPSP.
In MCs, self-excitation could occur either at dendritic release sites
located on the glomerular tuft or on secondary dendrites since
backpropagating action potentials reach both levels of the dendritic
tree (Bischofberger and Jonas 1997
; Chen et al.
1997
; Isaacson and Strowbridge 1998
). The
glomerular layer was completely removed (before recording) in a first
set of experiments. Figure 4 indicates
that, in the absence of the distal primary dendrite, MCs still
presented a prolong evoked inward current due to the activation of
ionotropic glutamate receptors. Additional experiments showed that MCs
without distal primary dendrites consistently displayed slow-EPSPs with
large amplitudes (4.8 ± 1.2 mV, n = 6 vs.
5.7 ± 0.7 mV in control, n = 7, Mg2+ = 0 mM).

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Fig. 4.
The slow-EPSP persists in the absence of the apical tuft. Top
left: picture of a mitral cell labeled by Lucifer yellow. Note
the absence of the distal part of the primary dendrite. Top
right: location of the labeled mitral cell in the olfactory
bulb. The section of the superficial external plexiform layer was made
immediately after cutting the slice with a small piece of razor blade
and under the control of a microscope. The single arrow shows the end
of the primary dendrite. Bottom: most of the slow-EPSC
recorded in the illustrated mitral cell was blocked in the presence of
D-APV (voltage clamp, K-gluconate, average of 15 traces,
Mg2+= 1 mM).
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In another series of experiments, we quantified the contribution of
both dendritic compartments by comparing the amplitude of the slow-EPSP
before and after section (with a gentle suction) of the apical primary
dendrite. The dendritic section abolished the olfactory nerve-evoked
EPSPs (n = 3). We analyzed the effect of dendritic
removal on the amplitude of both the NMDA- and non-NMDA components of
the slow-EPSP. We found that the amplitude of the non-NMDA component
significantly decreased after suppression of the primary dendrite (Fig.
5, A1 and A2;
44.2 ± 10.1%, n = 6). However, the major
component of the slow-EPSP (i.e., the NMDA one) was almost unaffected
(an average decrease of 9.3 ± 1.6%, n = 6). As
shown on Fig. 5B2, the section of the apical primary dendrite did not modify significantly the input resistance and the
resting membrane potential in the same MCs. This rules out that a
significant decrease of the NMDA response was covered by an increase in
the space constant. Altogether, these results suggest that action
potentials backpropagating in the primary dendrite activate primarily
non-NMDA autoreceptors while those backpropagating in secondary
dendrites activate mainly NMDA autoreceptors.

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Fig. 5.
NMDA receptors of the secondary dendrites mediate the major
component of the slow-EPSP. A1: respective contribution
of both dendritic compartments to the slow-EPSP. The slow-EPSP was
recorded in control conditions (Mg2+ = 1 mM, PTX, 100 µM), then in the presence of D-APV (50 µM),
D-APV, and CNQX (5 µM), and finally back in control
conditions (top, 3 average traces of 10 responses). In a
2nd step, the apical primary dendrite was sectioned with a gentle
suction under visual control, and the slow-EPSP was again recorded in
the 3 conditions [bottom, 3 dark traces that are the
average of 10 responses; light traces are the average responses with
the primary dendrite (in 1 and 2)]. The dendritic sectioning of the
recorded neuron was relatively easy because the primary dendrites were
very well defined under the microscope. The sectioning of the primary
dendrite had a small effect on the slow-EPSP (see 4 + 1; bottom
left traces). Note that this small decrease in the slow-EPSP is
mainly due to a decrease in the non-NMDA-R-mediated component (see
the 2 superimposed traces 5 + 2, bottom middle traces).
Inset: superimposed average traces (20 traces) of the
cell membrane potential before (thin line) and after (thick line)
sectioning to illustrate the absence of input resistance change.
B1: summary graph showing the percentage of the NMDA and
the AMPA components of the slow-EPSP before and after section of the
apical dendrite. Note that the NMDA component was only slightly
modified by the sectioning. The measurements of the slow-EPSP were done
10 min after the beginning of the application of the glutamate receptor
antagonists. The following concentrations of the receptor antagonists
were used in these series of experiments: D-APV (25-50
µM), CNQX (5-10 µM). B2: the sectioning of the
apical primary dendrite has no significant effect on the input
resistance (RN) and the resting membrane
potential (RMP). The quantification of the input resistance immediately
before and after the section of the apical dendrite does not show a
significant increase (141.6 ± 12.7 vs. 147 ± 13.9 M ,
P = 0.17, paired t-test,
n = 6, analysis done with the cells illustrated in
B1). No modification of the membrane resting potential
was observed during this manipulation (60.5 ± 0.8 vs. 60.5 ± 0.5 mV, n = 6).
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In secondary dendrites, the slow-EPSP could result from the activation
of dendrodendritic synapses established between a MC and another
excitatory connection insensitive to DCGIV but also from the activation
of MC ionotropic glutamate autoreceptors by glutamate released from the
dendrites. If the latter hypothesis were true, the slow-EPSP should be
observed in conditions where the entire olfactory bulb network is
uncoupled. In the presence of PTX, CNQX, TTX, and extracellular
Mg2+ (2 mM), there were no synaptic interactions
between cells at rest (the resting membrane potential was about
80
mV). Evoking a calcium current in a MC in these conditions did not
induce any synaptic component (Fig.
6A, left). However,
when the membrane potential of the stimulated cell was depolarized to
40 mV, to relieve the Mg2+ block, the NMDA
current was revealed (Fig. 6A, right). These results strongly suggest that NMDA autoreceptors mediate
self-excitation in secondary dendrites.

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Fig. 6.
NMDA autoreceptors mediate MC's self-excitation. A:
glutamate released by secondary dendrites activates NMDA autoreceptors
in an uncoupled network. In the presence of Mg2+ (2 mM),
PTX (100 µM), TTX (1 µM), and CNQX (20 µM), evoking a calcium
current did not induce any slow inward current when the membrane
potential was voltage clamped at 80 mV (left). In
contrast, when the membrane was depolarized to 40 mV, to relieve the
magnesium block, the NMDA component of the slow excitatory postsynaptic
current was revealed (right). B: NMDA
autoreceptors mediate miniature synaptic currents. At a holding
potential of +40 mV and in the presence of tetrodotoxin (1 µM),
outward synaptic events were recorded (left) and could
be blocked by 50 µM D-APV (right). Note
the absence of D-APV-insensitive spontaneous outward
currents. C1: in another cell, NMDA miniature synaptic
currents were 1st recorded in the same conditions (bottom
left). After 10 min, an additional patch pipette containing the
calcium chelator BAPTA (20 mM) was used to record the same cell.
Fifteen minutes after the intracellular application of BAPTA, the slow
inward current evoked by a calcium current totally disappeared
(top right, Vh = 70 mV). In the same
cell, the frequency of spontaneous synaptic events also decreased
(bottom right) after this manipulation, suggesting that
they were generated, at least in part, by a direct activation of NMDA
autoreceptors by glutamate released from the recorded MC
(Vh = +40 mV). C2:
cumulative distribution of NMDA miniature events recorded in a MC in
control (thin line) and in the presence of BAPTA (thick line) after 15 min (2.379 ± 2.41 s vs. 4.2 ± 0.624 s,
P = 0.0002; nonparametric Kolmogoroff-Smirnoff
test). C3: summary graph of the inter-event interval in
control and in BAPTA for 4 MCs (means of 2.03 ± 0.26 s and
5.1 ± 0.64 s, respectively, P = 0.004, paired
t-test). Picrotoxin (100 µM) was applied in all
experiments.
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The NMDA autoreceptors are also spontaneously activated in conditions
of minimal glutamate release. In the presence of PTX, TTX, and in the
absence of extracellular Mg2+, the membrane
potential of MCs was clamped at +40 mV. NMDA-mediated miniature
synaptic potentials could then be recorded (Fig. 6B). These
events did not result from the activation of NMDA receptors by ambient
glutamate. Indeed, when a second patch pipette was then used to inject
the calcium chelator BAPTA (Fig. 6, C1 and C2,
bottom) in the same cell, the frequency of spontaneous NMDA synaptic currents progressively diminished after breaking the membrane
seal. Concurrent with the decrease in spontaneous miniature synaptic
potentials, the current underlying the evoked slow-EPSP at
70 mV was
progressively blocked (Fig. 6C1, top right) as BAPTA slowly
diffused to dendritic release sites (Adler et al. 1991
; Borst and Sakmann 1996
). Fifteen minutes after the seal
break, the inter-event interval had increased from 2.03 ± 0.26 s to 5.1 ± 0.64 s (P = 0.004, n = 4, paired t-test, Fig. 6C3)
while the amplitude of NMDA miniature currents had not changed (13 ± 2.4 pA vs. 10.9 ± 1.1 pA, n = 4, P > 0.5).
To understand the functional role of the slow-EPSP, we examined its
dynamic properties. Figure 7A
illustrates that there was a temporal summation of the slow-EPSP with
an increase in cell firing. Paired-pulse facilitation occurred when two
sodium action potentials were separated by <300 ms, and was maximal at
an inter-spike interval of approximately 50 ms (Fig. 7, B
and C). Given that, during odor stimulation, MCs fire in a
range close to this interval (Wellis et al. 1989
), it is
likely that self-excitation significantly contributes to the mitral
cell's response.

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Fig. 7.
Short-term facilitation of self-excitation by mitral cell's action
potentials. A: the size of the slow-EPSP is modulated in
an activity-dependent manner. Top: the slow-EPSP (arrow)
increases with the number of spikes (1 on the left, 4 on
the right; thick lines, Vh:
59 mV). Application of glutamate receptor antagonists blocked the
slow-EPSP (thin lines) and revealed the afterhyperpolarization (star)
entirely. Bottom: the traces (averages of 10) represent
the difference between traces obtained in control (100 µM PTX, 20 µM CNQX, Mg2+ = 1 mM) and with the addition of the
NMDA antagonist D-APV (50 µM). The sodium spikes are
digitally removed. B: paired-pulse facilitation of the
current underlying the slow-EPSP. Top: a mitral cell was
voltage-clamped at 70 mV (K-gluconate) and 2 voltage steps (2 ms, 60 mV) were applied successively, separated by either 30 ms (thick line)
or 1 s (thin line). The 2nd slow-EPSC clearly increased when the
inter-pulse interval was brief (30 ms). The extracellular solution
contained 100 µM PTX and 20 µM CNQX (Mg2+ = 0).
The evoked sodium currents were digitally removed. C:
summary graph showing that paired-pulse facilitation lasts up to 300 ms
(n = 5 cells). The paired-pulse ratio was
calculated as the ratio of the amplitudes of the 2nd slow-EPSC over the
1st slow-EPSC. The dash line indicates a paired-pulse facilitation
ratio of 100%.
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To demonstrate this, we tested the role of NMDA autoreceptors on
MC discharge. Given that the dendrodendritic inhibition is entirely
dependent on the glutamate ionotropic receptor activation (Isaacson and Strowbridge 1998
; Schoppa et al.
1998
), the use of extracellularly applied glutamate receptor
antagonists may result also in an important effect on MC firing by a
blockade of the disynaptic inhibition. Thus to address the issue of the physiological role of self-excitation, it was necessary to block synaptic inhibition prior to test the effect of NMDA receptor antagonists. In the presence of PTX, CNQX, and extracellular
Mg2+ (and in 3 cases after a cut of the primary
dendrite), a train of action potentials was regularly evoked with a
current depolarizing step (Fig.
8A1). During the mitral cell
discharge, NMDA autoreceptors were activated. After having established
a stable baseline of the cell discharge, we applied the NMDA receptor
antagonists MK801, D-APV, or CPP. The NMDA open channel
blocker MK801 modestly, but significantly, increased the average
inter-spike interval (19.8 ± 1.8 ms vs. 23.3 ± 2.3 ms for
the 3rd inter-spike interval, n = 6, P = 0.0125, paired t-test, 4 cases in 1 mM
Mg2+ and 2 cases with 0.8 mM
Mg2+) in a use-dependent manner (Fig.
8A2). Application of the competitive NMDA receptor
antagonists D-APV (50 µM) and CPP (10 µM) had also similar effects (20.3 ± 1.3 ms vs. 25.1 ± 2.1 ms,
P = 0.008, n = 4). The protocol used
here evoked trains of several action potentials that may activate
numerous surrounding mitral cells (i.e., Isaacson 1999
).
Thus the application of competitive NMDA antagonists may overestimate
the real contribution of NMDA autoreceptors on the MC's excitability.
In contrast, the activity-dependent blocker MK801 inhibited the NMDA
autoreceptors activated by the glutamate release from the recorded cell
and not all NMDA receptors present in the network. As shown in Fig.
8A1, the first spike of the discharge was exactly
superimposed in control (with PTX) and in MK801 conditions, indicating
that the blockade of the effect of ambient glutamate (Sah et al.
1989
) did not produce a large change in the membrane resting
potential (Fig. 8B1) and in the spike threshold (Fig. 8B2). For different values of depolarizing current, the
latency of the first spike was also the same in control conditions and in the presence of MK801. Moreover, in the entire population of cells
analyzed here, the latency of the first spike after an application of
the NMDA-R antagonists was not modified. As a result, the average spike
frequency for each cell decreased after application of MK801, although
the spike threshold was not modified (Fig. 8A3, left). Figure 8A3 (right) illustrates that MK801
also modified MCs' firing pattern, flattening the discharge rate as a
function of time. These results indicate that the NMDA-mediated
self-excitation acts as an excitatory feedback that shapes MC's own
activity.

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Fig. 8.
Self-excitation exerts a positive feedback on MC activity.
A1: self-excitation facilitates MC firing. Application
of MK801 (40 µM) for 20 min decreased the number of spikes evoked by
a depolarizing current step (100 µM PTX, 20 µM CNQX, 0.8 mM
Mg2+). The 1st spikes were superimposed in both conditions
(resting membrane potential: 59.7 vs. 59.5 mV; spike threshold:
47 vs. 46.8 mV). Experiments during which the membrane potential or
the bridge balance compensation varied were discarded.
A2: time course of the effects of MK801 (same cell as in
A1). Subsequent application of 50 µM D-APV
had no effect. A3: the current-average frequency curve
(left) during application of MK801 demonstrates that
activation of NMDA autoreceptors increases cell excitability.
Right: MK801 shapes MC's firing pattern. The
distribution of the inter-spike interval was plotted as a function of
the spike rank during a MC's discharge evoked by 2 depolarizing
current pulses (0.15 nA in control; 0.25 nA in the presence of MK801).
Current values were chosen (arrows on the left graph) to
induce similar average firing frequency. In control condition (PTX,
CNQX, and Mg2+), self-excitation caused an acceleration of
cell firing that was blocked by MK801. B1: the resting
membrane potential (RMP) was not modified by the application of MK801
(quantification done in control conditions and 25 min after the
beginning of the application of MK801; 60 ± 0.6 mV vs.
60.2 ± 0.8 mV, n = 6, P = 0.29, paired t-test). B2: MK801 did not
change the spike threshold (1st spike: 49.4 ± 0.9 mV vs.
49.3 ± 1 mV (n = 6, P = 0.78, paired t-test).
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DISCUSSION |
The goal of the present paper was to examine in more detail the
mechanisms of self-excitation and its physiological role. We first
demonstrate that olfactory bulb excitatory connections are not
responsible for the slow-EPSP. The fact that intracellular blockade of
the spontaneous glutamate release deeply reduced the frequency of
miniature NMDA-R-mediated synaptic currents in the same neuron
directly implicates NMDA autoreceptors. However, in the experiments
shown here, we cannot totally exclude the possibility of the
contribution of glutamate release from neighboring MCs (Isaacson
1999
). The paper also shows that, during self-excitation, there
is a modest but significant activation of AMPA/kainate autoreceptors. These autoreceptors are located in part in the glomerular region of the
primary dendrite while the major component of the slow-EPSP results
from the activation of NMDA autoreceptors located on secondary dendrites. Finally, the study indicates that there is a frequency facilitation of self-excitation and that it may control the MC's firing in an activity-dependent manner.
Given the presence of intrinsic excitatory connections in olfactory
bulb (see RESULTS), it was important to discard the
possibility that the slow-EPSP was generated by conventional synaptic
mechanisms. We were able to separate the two synaptic mechanisms by
using an agonist of presynaptic metabotropic glutamate receptors and by
examining the frequency of miniature NMDA currents after blockade of
glutamate release in the recorded neuron. We considered also the
respective contribution of primary and secondary dendrites in the
generation of self-excitation. Indeed, the major component of the
self-excitation results from an activation of glutamate receptors
localized in MC secondary dendrites, indicating the important role of
backpropagating action potentials in the MC integration of the signal.
The coincidence of bursts of axonal action potentials and the
self-excitation may, in turn, facilitate the generation of calcium and
sodium action potentials in secondary dendrites depending on the level
of dendrodendritic inhibition (Larkum et al. 1999
).
Finally, it has been recently shown that the distal primary dendrite
can initiate mitral cell action potentials (Chen et al.
1997
). In this context, it will be important to determine the
potential role of dendritic action potentials evoked by activation of
the olfactory nerve in the generation of self-excitation and, reciprocally, to examine the contribution of self-excitation to the
regulation of distal dendritic impulses.
Here, we have shown that self-excitation is mediated in part by
AMPA/kainate receptors. Given that one previous report (Isaacson 1999
) did not mention this result, we discard, by using high
doses of different NMDA-R antagonists, the possibility of an incomplete blockade of NMDA receptors. In the work of Isaacson, self-excitation was evoked with Ca currents, using cesium-gluconate-filled pipettes and tetrodotoxin. In our experiments, the size of the NMDA-R-mediated currents when evoked by Ca spikes in the absence of extracellular magnesium is very large in comparison with the amplitude of the AMPA-R-mediated currents. In that case, it was possible that the non-NMDA component was partially masked by the Ca currents/spikes. Indeed, when Na spikes evoked the slow current (with potassium gluconate-filled patch pipette), the rise time of the AMPA/kainate-R response was so fast that it was difficult to distinguish it from the
end of the sodium current (partially clamped). Our result indicates
that AMPA receptor subunits localized on MC dendrites (Montague
and Greer 1999
) have a physiological role. Thus self-excitation is not silent at the resting membrane potential since AMPA/kainate receptor-mediated EPSPs may unblock NMDA receptors. It
will be important to determine whether AMPA and NMDA autoreceptors are co-localized in the neighborhood of the dendrodendritic synapses.
The duration of the CNQX/NBQX-sensitive component is long in
comparison to the known kinetics of the AMPA receptors. Several reasons
may explain this result. First, it is possible that there is also a
contribution of kainate receptors in self-excitation since it has been
shown that kainate receptor-mediated response present a prolong decay
(Castillo et al. 1997
). Second, dendrodendritic synapses
are located on several sites of secondary dendrites, and
backpropagating action potentials may induce a prolonged glutamate release by an activation of several release sites. Finally, we cannot
exclude an imperfect voltage and space clamp of the glutamate receptor-mediated responses since MCs possess very long processes (the
site of recording was in the soma).
The presence of an AMPA receptor-mediated response is surprising
given the low affinity of the receptor for glutamate. It has been
demonstrated that glutamate spillover may activate NMDA or metabotropic
glutamate receptors that have a much higher affinity for glutamate than
AMPA receptors (Kullmann and Asztely 1998
). Given the
results of models of glutamate diffusion (Clements
1996
), the localization of AMPA receptors should be very close
to the release sites (i.e., a distance smaller than 400 nm) (see
Holmes 1995
). Increase in glutamate concentration by
release of glutamate from neighboring sites (see, for example,
Scanziani et al. 1997
) located on the MC dendrites or a
delayed clearance of the neurotransmitter caused by some obstacle to
the neurotransmitter diffusion in the extracellular space could also
contribute to the AMPA receptor activation. In support of these latter
possibilities is the relatively slow time course of the decay of the
AMPA receptor component that could be in part due to a prolonged
presence of glutamate (Barbour et al. 1994
).
We found a modulation of MC excitability by self-excitation,
suggesting a precise role of this phenomenon for the temporal coding of
odor signals. It is now well established that sensory neurons lack
response selectivity for odor ligands and present a relatively high
level of spontaneous activity (Duchamp-Viret et al.
1999
; Malnic et al. 1999
). In contrast, MCs have
a much narrower tuning curve than sensory neurons (Duchamp-Viret
and Duchamp 1997
; Mori and Yoshihara
1995
), and the cellular interactions underlying the increase in
selectivity in the olfactory bulb have yet to be determined.
Self-excitation may contribute to an increase in the signal-to-noise
(S/N) ratio by amplifying active inputs from sensory neurons with an
excitatory feedback mechanism. The behavior of such an amplifier has
been extensively analyzed in models of recurrent excitation in cerebral
cortex (Douglas et al. 1995
; Somers et al.
1995
), and it has been shown that this could be a powerful
mechanism for improving the response selectivity of cortical neurons to
noisy inputs. Indeed, the effect of NMDA autoreceptor activation on the
slope of the average frequency curve (see Fig. 8A3) suggests
an increase in the S/N ratio for the specific set of MCs that are most
activated, as in these models. Together with the disynaptic inhibition,
self-excitation exerts a push-pull regulation of the spike discharge
that may contribute to the coding of olfactory inputs.
Address for reprint requests: P.-A. Salin, CSG-CNRS, Campus
Universitaire, 15 rue Hugues Picardet, 21000 Dijon, France (E-mail: salin{at}cesg.cnrs.fr).