Centre National de la Recherche Scientifique, Institut Alfred Fessard, 91198 Gif-sur-Yvette, France
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ABSTRACT |
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Hsia, Albert Y., Jean-Didier Vincent, and Pierre-Marie Lledo. Dopamine Depresses Synaptic Inputs Into the Olfactory Bulb. J. Neurophysiol. 82: 1082-1085, 1999. Both observations in humans with disorders of dopaminergic transmission and molecular studies point to an important role for dopamine in olfaction. In this study we found that dopamine receptor activation in the olfactory bulb causes a significant depression of synaptic transmission at the first relay between olfactory receptor neurons and mitral cells. This depression was found to be caused by activation of the D2 subtype of dopamine receptor and was reversible by a specific D2 receptor antagonist. A change in paired-pulse modulation during the depression suggests a presynaptic locus of action. The depression was found to occur independent of synaptic activity. These results provide the first evidence for dopaminergic control of inputs to the main olfactory bulb. The magnitude and locus of dopamine's modulatory capabilities in the bulb suggest important roles for dopamine in odorant processing.
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INTRODUCTION |
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Both behavioral and molecular studies point to a
potentially important role for dopamine in olfaction. Parkinson's
patients, for instance, have been found to have impaired odor
recognition (Hawkes and Shephard 1998). In addition,
systemic injection of dopamine analogues has been shown to result in
impaired odor detection (Doty and Risser 1989
). On the
molecular level, dopamine receptor expression has been found to be high
in the main olfactory bulb, as are levels of the rate-limiting
synthetic enzyme of dopamine, tyrosine hydroxylase (TH) (Coronas
et al. 1997
, Halasz et al. 1977
).
Together these observations motivated us to study at the cellular level
a possible modulatory role of dopamine in the mammalian olfactory bulb.
The bulb receives inputs from the olfactory epithelium via the
olfactory nerve, which forms excitatory, glutamatergic synapses in
regions of the bulb known as glomeruli. There, synapses are made onto
the dendrites of mitral cells, the primary output neurons of the bulb.
Glomerular borders are defined in part by periglomerular cells, which
are both GABAergic and dopaminergic. These periglomerular cells are
excited by dendrodendritic synapses from mitral cells, and possibly
also by axodendritic synapses from the olfactory nerve (Pinching
and Powell 1971). Because TH levels are highest in the
glomerular layer (Halasz et al. 1977
), and because
dopamine receptors have been localized to the olfactory nerve and
glomerular layers (Coronas et al. 1997
), we hypothesized that it is at the first synapse between the olfactory nerve and mitral
cells where dopamine might play a modulatory role.
Dopamine receptors are classified into two broad families: D1 and
D2 (Missale et al. 1998). D1 receptors are only sparsely expressed in the bulb and are absent in the olfactory nerve layer; in
fact, their presence had been doubtful until recently (Coronas et al. 1997
). It is the D2 receptors that show prominent
expression in the bulb, specifically in the olfactory nerve and
glomerular layers (Coronas et al. 1997
), and hence in
this study we used a specific D2 receptor agonist to probe for a
possible role of dopamine in modulating olfactory nerve input to the bulb.
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METHODS |
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Experiments were performed on olfactory bulb slices obtained
from 1- to 6-wk-old Wistar rats. Slices (300-400 µm) were prepared as described (Chen and Shepherd 1997). After at least a
1-h recovery period at 30°C, slices were transferred to a submersion
chamber, where they were continuously superfused (~2 ml/min) with a
22-25°C ACSF solution saturated with 95%
O2-5% CO2. The external
ACSF solution was composed of (in mM) 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 Mg2SO4, 1.0 NaH2PO4, 26.2 NaHCO3, and 10 D-glucose.
For field recording, bipolar, stainless-steel stimulating electrodes were placed in the olfactory nerve layer, and recording pipettes were placed in individual glomeruli (see Fig. 1A).
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Whole-cell recordings were performed under visual control with an
upright Zeiss Axioskop microscope and infrared differential interference contrast (IR-DIC) videomicroscopy. Mitral cells were easily identified by their location and morphology (see Fig.
2A) (Shepherd and Greer
1998). Microelectrodes had a resistance of ~8 M
. The
whole-cell pipette solution was composed of (in mM) 123 Cs-gluconate,
10 CsCl, 1 CaCl2, 10 Cs-EGTA, 10 HEPES-Na, 8 NaCP, 10 D-glucose, 0.3 GTP, 2 Mg-ATP, and 0.2 AMPc, pH
7.3. Cells were voltage clamped at
80 mV.
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Evoked synaptic responses were elicited at 0.05 Hz. For paired-pulse modulation experiments, paired pulses were delivered 40 ms apart, and the peak amplitude of the second response was divided by the first. On- and off-line data analysis was carried out with Acquis1 (G. Sadoc, CNRS-ANVAR, France).
Fast inhibitory transmission was blocked with picrotoxin (100 µM).
Excitatory responses were blocked through the addition of the
ionotropic glutamate receptor antagonist, kynurenic acid (10 mM). The
N-methyl-D-aspartate NMDA receptor antagonist,
D,L-2-amino-5-phosphonopentanoic acid (D,L-APV,
100 µM), was added in some experiments to isolate responses mediated
by -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors.
D,L-APV, quinpirole, and sulpiride were obtained from Tocris (Illkirch, France) and all other drugs and salts were purchased from Sigma (Strasbourg, France).
Results are presented as means ± SE. Data were compared statistically by the Student's t-test, and significance was defined as P < 0.05.
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RESULTS |
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D2 receptor agonist quinpirole inhibits transmission at the olfactory nerve-mitral cell synapse
We first performed extracellular field recordings in individual glomeruli (see Fig. 1A) and assessed the effect of D2 receptor activation on the strength of field excitatory postsynaptic potentials (fEPSPs) in response to olfactory nerve stimulation. We found that quinpirole, a specific D2 receptor agonist, significantly depressed fEPSP strength by 41 ± 5% (Fig. 1, B and C; P = 0.0023, n = 5).
Because a component of the glomerularly recorded fEPSP could be contributed by the depolarization of periglomerular cells in addition to mitral cells, we performed the analogous experiment in the whole-cell configuration, recording from visually identified mitral cells (see Fig. 2A). A depression of similar magnitude (47 ± 15%) was observed (Fig. 2, B-D; P = 0.047, n = 5), suggesting that quinpirole's depressive effect does indeed occur at olfactory nerve synapses onto mitral cells.
Depressive effect of quinpirole is D2 receptor specific and reversible
Pretreatment of slices with the specific D2 receptor antagonist, sulpiride, completely prevented the depressive effect of quinpirole (Fig. 1C; P = 0.0068, n = 4). In addition, the quinpirole-induced response was completely reversed by subsequent application of sulpiride (Fig. 2, C and D; n = 5). Sulpiride alone had a very small (+5 ± 1%) effect on baseline synaptic transmission in naive slices (Fig. 2, E and F; P = 0.012, n = 3).
D2 receptor-mediated depression causes a change in paired-pulse modulation and is activity independent
To determine the locus of D2 receptor-mediated depression, we
first tested whether quinpirole application changed the degree of
paired-pulse modulation, measured as the ratio of the strengths of two
closely-spaced EPSPs. The degree of paired-pulse modulation has been
found to correlate with the probability of transmitter release from
presynaptic terminals (Markram and Tsodyks 1996). Indeed, quinpirole caused a 71 ± 24% change in the degree of
paired-pulse modulation (Fig. 3,
A-C; P = 0.042, n = 5), suggesting a
presynaptic locus of inhibition.
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We next assessed whether the D2 receptor-mediated depression required olfactory nerve activity. Figure 3D illustrates a typical experiment. First, as a control, we stopped stimulation of the olfactory nerve for 10 min, and then resumed stimulation. Then, after reestablishing a baseline, we again stopped stimulation for 10 min, although this time applying quinpirole just after halting stimulation. On resuming stimulation, we found that from the very first response in the presence of quinpirole, synaptic transmission was depressed to a similar degree as during experiments with continual stimulation (32 ± 6% for 1st response after nonstimulation; Fig. 3, D and F; P = 0.023, n = 3). Figure 3E summarizes the effect of a period of nonstimulation alone and shows that, in contrast to results obtained with quinpirole, there is a small (18 ± 1%), transient potentiation following nonstimulation (P = 0.0019, n = 3).
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DISCUSSION |
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Dopamine has been shown to act as a neuromodulator in a variety of
systems, including the retina (Djamgoz and Wagner 1992), nucleus accumbens (Nicola and Malenka 1998
), and
hippocampus (Otmakhova and Lisman 1999
). Our
demonstration of a strong synaptic depression induced by dopamine
receptor activation at the first synaptic relay between the olfactory
epithelium and the olfactory bulb suggests an important role for
dopamine in mediating the entry of olfactory information into the brain.
D2 receptor activation most likely depresses transmitter release from olfactory nerve terminals
D2 receptors are the most abundant subtype of dopamine receptor in
the olfactory bulb (Coronas et al. 1997). We found that one functional effect of D2 receptor activation in the bulb is a
significant depression of synaptic transmission between olfactory receptor neurons and mitral cells. The finding of a concomitant change
in paired-pulse modulation suggests that this depression is due at
least in part to a presynaptic mechanism. To determine whether there
may also be a postsynaptic contribution to the depression (e.g.,
downregulation of glutamate receptors at individual synapses) would
require the analysis of miniature EPSCs (mEPSCs). However, we found
that mEPSC frequency was too low to permit analysis (<0.1 Hz). In
addition, attempts to stimulate asynchronous release through the
replacement of Ca2+ by Sr2+
were also unsuccessful, perhaps due to significant cable filtering between olfactory nerve-mitral cell synapses and the mitral cell soma
(~400 µm apart). Although we cannot rule out such additional depression postsynaptically, molecular evidence points to D2 receptors being expressed in olfactory receptor neurons. D2 mRNA transcripts are
abundant in olfactory receptor neurons (Shipley et al.
1991
), although undetectable in the mitral cell layer
(Coronas et al. 1997
). Certainly our inclusion of
Cs-gluconate in the whole-cell pipette solution does allow us to rule
out postsynaptic modification of K+ conductances.
Possible mechanisms leading to synaptic depression by D2 receptors
The fact that experiments were carried out in the presence of a blocker of fast inhibitory transmission rules out the possibility that D2 receptor activation led to inhibition via GABAergic transmission. We therefore suggest that D2 receptors have a direct depressive effect at presynaptic terminals.
We found that the D2 receptor-induced depression is independent of
activity (Fig. 3, D-F), ruling out a necessity for
concurrent calcium signaling. One possibility is that D2 receptor
activation leads to inhibition of adenylate cyclase (AC); decreased AC
activity has been shown in numerous systems to inhibit transmitter
release (e.g., Tzounopoulos et al. 1998). In the bulb,
D2 receptor activation both decreases cAMP levels (Mania-Farnell
et al. 1993
) and reduces AC activity (Coronas et al.
1999
). Another possible mechanism is a direct effect of D2
receptor activation on voltage-gated ionic channels (Missale et
al. 1998
).
Potential role of dopamine as a neuromodulator in the bulb
The fact that TH is found exclusively in periglomerular cells
(Halasz et al. 1977) suggests that endogenous dopamine
is released from these cells. Electron microscopic studies have not
found synapses between periglomerular cells and the presynaptic
terminals or axons of olfactory neurons (Pinching and Powell
1971
), necessitating that this dopamine activate receptors
extrinsic to synaptic specializations, perhaps in a diffuse fashion.
One possibility is that dopamine may set the olfactory detection
threshold by tonically suppressing all inputs into the bulb. The
circuit could then adapt to decreasing odorant concentrations by
decreasing dopamine release. Consistent with this model is the
observation that in response to inactivity resulting from unilateral
odor deprivation, TH activity in the glomerular layer markedly
decreases (Nadi et al. 1981
).
Dopamine might also act as a mediator of heterosynaptic depression,
fine-tuning the glomerular activation pattern in response to odors.
Dopamine levels have also been found to increase during odor learning
(Coopersmith et al. 1991), suggesting that dopamine modulation may have important roles in synaptic plasticity in the bulb.
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ACKNOWLEDGMENTS |
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We thank A. Carleton and G. Sadoc for invaluable technical assistance. We are grateful to G. Shepherd and H. McLean for comments on this manuscript.
This study was supported by the Institut Universitaire de France and the Fyssen Foundation.
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FOOTNOTES |
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Address for reprint requests: P.-M. Lledo, Centre National de la Recherche Scientifique, Institut Alfred Fessard, Ave. de la Terrasse, 91198 Gif-sur-Yvette, France.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 16 March 1999; accepted in final form 14 April 1999.
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REFERENCES |
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