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Department of Biological Science, Program in Neuroscience, Florida State University, Tallahassee, Florida 32306-4340
Submitted 22 November 2002; accepted in final form 23 February 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Other investigators have characterized the laminar and
cellular expression of both D1 and D2 dopamine receptor subtypes in the OB
(Coronas et al. 1997;
Koster et al. 1999;
Levey et al. 1993).
Olfactory experience has a significant effect on the expression of TH, OB
dopamine content, and D2 receptors. After unilateral naris occlusion, TH and
dopamine content decreases sharply (Baker
et al. 1983) and D2 receptor expression dramatically increases
(Guthrie et al. 1991).
Behavioral experiments have shown that D1 receptor activation improved odor
detection performance (Doty et al.
1998). Conversely, D2 receptor activation diminished odor
detection performance in another series of experiments
(Doty and Risser 1989).
Using electrophysiological techniques, other investigators have shown that
dopamine reduces evoked potentials in mitral cells from the turtle OB
(Nowycky et al. 1983) and the
rat OB (Ennis et al. 2001).
Furthermore, the reduction in endogenous dopamine caused by naris closure
augments both the number of mitral cells that respond to an individual odorant
and the number of odorants to which an individual mitral cell will respond
upon odor presentation to the reopened naris
(Wilson and Sullivan 1995).
Recent electrophysiological studies have shed some light on the synaptic
mechanisms underlying these effects. D2 receptor activation on the presynaptic
terminal of olfactory receptor neurons (ORNs) decreases the probability of
glutamate release, and hence, excitability of mitral/tufted (M/T) cells is
diminished (Berkowicz and Trombley
2000; Ennis et al.
2001; Hsia et al.
1999). Also, activation of different dopamine receptor subtypes on
M/T cells (D2) and interneurons (D1) mediates differential yet complementary
transduction cascades with the overall effect of increasing GABAergic
inhibition of M/T cells (Brunig et al.
1999).
In this study, we combined patch-clamp electrophysiological recording with primary culture techniques to further identify the synaptic effects of dopamine. Our results suggest that dopamine may contribute to odor information processing through presynaptic inhibition of excitatory synaptic transmission between M/T cells and interneurons.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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The procedure for preparing primary cultures of OB neurons is described in
detail elsewhere (Trombley and Blakemore
1999). Briefly, P1–P5 Sprague-Dawley rat pups were
decapitated in accordance with the institutional guidelines of The Care and
Use of Laboratory Animals approved by the National Institutes of Health and
The Florida State University's Animal Care and Use Committee. OBs were
removed, cut into 1-mm cubes, and enzymatically treated in a calcium-buffered
papain solution for 1 h at 37°C. The OBs were triturated with a
fire-polished pipette until a single-cell suspension was achieved. The cells
(250,000 cells/dish) were plated in 35-mm culture dishes on a confluent
monolayer of previously prepared OB astrocytes. The neuronal media was
comprised of 95% minimal essential medium (MEM, Gibco), 5% horse serum
(Gibco), 6 g/l glucose, and a nutrient supplement (Serum Extender,
Collaborative Research). Astrocyte layers were obtained by plating a
suspension of OB cells in a 75-cm2 flask containing 90% MEM, 10%
fetal calf serum, and 6 g/l glucose. Once confluent, the cells were
resuspended enzymatically with 0.125% trypsin and plated onto 35-mm dishes
coated with poly-L-lysine (30,000–70,000 MW, 10 µg/ml,
Sigma). Addition of 10–5 M
cytosine-
-D-arabinofuranoside (Sigma) to the media 1 day
after plating the neurons prevented the overgrowth of astrocytes.
Neuronal Identity
Presumptive M/T cells and interneurons were identified based on the
morphological, physiological, and immunohistochemical criteria established by
Trombley and Westbrook (1990).
Briefly, the cultures contained morphologically distinct populations of
neurons: a small number of large-diameter pyramidal-shaped neurons
(20–40 µm soma) and a much larger population of small-diameter
bipolar neurons (5–10 µm soma). These characteristics correlate with
M/T and granule/periglomerular cells, respectively. Electrophysiological
analyses of these two morphologically distinct populations further support the
notion that they reflect M/T cells and interneurons. Intracellular stimulation
of neurons with morphology reflecting M/T cells in vivo invariably evoked
glutamate-mediated EPSPs in monosynaptically coupled interneurons. In
contrast, intracellular stimulation of the small bipolar neurons evoked
GABA-mediated IPSPs, consistent with their identity as interneurons. We have
previously shown that these morphologically and physiologically distinct
populations can also be identified by immunohistochemical markers. The large
pyramidal neurons, the presumptive M/T cells, were
N-acetylaspartylglutamate immunoreactive; in contrast, the small
bipolar neurons, presumptive granule/periglomerular neurons, were glutamic
acid decarboxylase immunoreactive
(Trombley and Westbrook
1990).
Electrophysiology
Electrophysiological recordings were obtained at room temperature from OB neurons after 6–14 days in culture. The acquisition software (AxoData and AxoGraph, Axon Instruments) was run on a Macintosh G3 computer and used to control an AxoClamp 2B amplifier (Axon Instruments).
The 35-mm culture dishes functioned as the recording chambers and were perfused at 0.5–2.0 ml/min with a bath solution consisting of (in mM) 162.5 NaCl, 2.5 KCl, 2 CaCl2, 10 HEPES, 10 glucose, 0 MgCl2, and 1 µM glycine (pH 7.3, osmolarity 325 mOsm). Patch electrodes were pulled from borosilicate glass to a final tip resistance of 4–6 MOhm and filled with a solution containing (in mM) 145 KMeSO4 or CsCl, 1 MgCl2, 10 HEPES, 5 Mg-ATP, 0.5 Mg-GTP, and 1.1 EGTA (pH 7.2, osmolarity 310 mOsm).
Drugs were diluted in bath solution and applied via a gravity-fed flow-pipe perfusion system, comprised of a row of 600 µm OD, square glass barrels. An electronic manipulator (Warner Instruments) controlled the position of the flow pipes, and pinch clamps regulated drug delivery. The solution delivery system produced complete drug exchange within 100 ms. In the text and figures, "control" data represent cells perfused with bath solution. The drugs used in these experiments were dopamine, glutamate, tetrodotoxin (TTX), SKF38393 (all from Sigma), and bromocriptine mesylate (from Tocris).
Spontaneous synaptic activity was recorded from interneurons in current-clamp mode. Quantitative analyses of the effects of dopamine and dopamine receptor agonists were done by comparing the number of excitatory postsynaptic potentials (EPSPs) during a 20-s interval that exceeded 5 mV before, during, and after drug application. Evoked potentials were obtained by simultaneous whole cell recording from monosynaptically coupled M/T cells and interneurons. Electrical stimulation of a presynaptic M/T cell evoked an action potential, which was recorded as an excitatory postsynaptic potential in the postsynaptic interneuron. Membrane input resistance was determined in current-clamp mode as well, using a current injection varying from 30–55 pA. To examine membrane currents evoked by glutamate-receptor agonists, whole cell recordings were made from interneurons in discontinuous, single-electrode, voltage-clamp mode at a switch frequency of 8–12 kHz. Membrane currents were filtered at 1–3 kHz and digitized at 5–10 kHz. Calcium current data were gathered in continuous, single-electrode, voltage-clamp mode to help reduce electrical noise. To more easily identify and quantify an effect on calcium channels, 10 mM BaCl2 was substituted for CaCl2 in the experimental drug solutions. Tetrodotoxin (1 µM) was added to block sodium currents, and Cs-based intracellular solutions were used to block potassium currents.
Immunocytochemistry
Our immunocytochemical procedures were modified from previous protocols
(Trombley and Westbrook 1990).
Primary neuronal cultures were rinsed three times for 10 min with
phosphate-buffered saline (PBS) and fixed for 30 min in 4% buffered
formaldehyde, which was osmotically adjusted to 320 mOsm with sucrose. The
cultures were then rinsed three times for 10 min with PBS. To prevent
nonspecific antibody binding, a blocking solution containing 10% normal donkey
serum (NDS) or normal rabbit serum in PBS was added to each fixed culture for
10 min. The blocking solution was removed by rinsing three times for 10 min
with PBS. The primary antibody was diluted in PBS then added to the cultures
to incubate for 24 h at room temperature. The next day, the cultures were
rinsed three times for 10 min with PBS to remove the primary antibody. The
secondary antibody also was diluted in PBS and added to the cultures for 1 h.
After incubation, the secondary antibody was removed by washing three times
for 10 min with PBS. All PBS was then removed, and coverslips were applied
inside the culture dish using Aquamount (Fisher Scientific, Fairlawn, NJ). The
primaries used were a mouse anti-tyrosine hydroxylase monoclonal antibody
(Chemicon International, Temecula, CA) at a 1:10,000 dilution and a goat
anti-D2 receptor polyclonal antibody (Santa Cruz Biotech, Santa Cruz, CA) at a
1:1,000 dilution. The corresponding secondaries were a goat anti-mouse IgG
conjugated to Cy3 (Jackson Immunoresearch, West Grove, PA) used at a 1:200
dilution for TH and a rabbit anti-goat IgG conjugated to AlexaFluor 568
(Molecular Probes, Eugene, OR) at a 1:3,000 dilution for D2 receptors. The
fluorescent images were acquired on a Leica DM-LFS microscope using OpenLab
3.0 software.
Statistics
All statistics were performed using GraphPad Prism version 3.00 (San Diego, CA). Data were analyzed using a one-way ANOVA with Newman-Keuls multiple comparison post-test. All data are presented as the mean ± SE.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Several studies have shown that innervation by olfactory sensory neurons is
necessary to maintain both dopamine content
(Kawano and Margolis 1982) and
TH expression in OB juxtaglomerular cells
(Baker et al. 1983;
Brunjes et al. 1985;
Cho et al. 1996). To determine
whether dopamine could be synthesized in our cultures in the absence of
olfactory sensory neurons, immunocytochemical techniques were used to examine
TH expression. TH was expressed in every culture dish examined (n =
7; Fig. 1A),
independent of the age of the animal at dissection (P1–P5) or time in
vitro (7–12 days). Approximately 1% of the neurons were immunoreactive
for TH. Immunocytochemical controls were prepared by omitting addition of the
primary antibody in some cultures and by substituting NDS for primary antibody
in others. No significant amount of secondary binding occurred in either
situation. These results suggest that dopamine is likely synthesized in
primary culture without innervation from the olfactory epithelium.
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Although it has been suggested that the majority of OB dopamine D2
receptors are expressed in olfactory sensory terminals
(Guthrie et al. 1991;
Koster et al. 1999), other
studies support the notion that D2 receptors are expressed by OB neurons
(Coronas et al. 1997;
Levey et al. 1993;
Mansour et al. 1990) and are
functional (Brunig et al. 1999;
Coronas et al. 1999). To
address this issue further, we used immunocytochemical techniques to determine
whether D2 receptors are expressed by the cultures we examined
electrophysiologically. The majority of OB neurons, including M/T cells
(Fig. 1B), were
immunoreactive for D2 receptors. Neurons from control cultures, which were
exposed to the secondary antibody but not the primary, were not
immunoreactive. These results are consistent with our electrophysiological
results in which the majority of OB neurons were sensitive to dopamine or the
D2 receptor agonist, bromocriptine.
Dopamine inhibits spontaneous excitatory synaptic activity
To determine whether dopamine has any effect on excitatory synaptic transmission between OB neurons, whole cell current-clamp recordings were obtained from interneurons before, during, and after dopamine (30 µM) application (Fig. 2). In 13 of the 14 cells tested, spontaneous excitatory activity in the interneuron was substantially inhibited during dopamine application. Dopamine reduced the number of excitatory synaptic events >5 mV during a 20-s interval to 24.2 ± 7.2% of control. The latency of recovery to baseline activity ranged from 10 s to 3 min. Figure 2 shows an example of the recovery latency. That the recovery was not instantaneous was likely due to the metabotropic nature of dopamine receptors and the duration necessary to inactivate the effects of second messengers.
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To determine which dopamine receptor subtype was mediating the inhibitory effect of dopamine, spontaneous activity in interneurons was examined in the presence of dopamine receptor subtype-selective agonists. Application of the D2 receptor agonist bromocriptine mesylate (1 µM) dramatically inhibited spontaneous excitatory activity in 9 of 13 neurons (Fig. 3, 2nd trace). Bromocriptine reduced the number of excitatory synaptic events >5 mV in a 20-s interval to 21.8 ± 6.3% of control. Application of 30 µM SKF38393, a D1 receptor agonist, inhibited spontaneous excitatory transmission in 12 of 14 neurons tested (Fig. 3, 4th trace). SKF38393 reduced the number of excitatory synaptic events >5 mV during a 20-s interval to 36.1 ± 8.8% of control. The effects of dopamine, bromocriptine, and SKF38393 on the excitatory synaptic event number were not statistically different from one another (P > 0.05). Like dopamine, both SKF38393 and bromocriptine exerted effects on both individual and compound EPSPs.
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Dopamine attenuates evoked EPSPs in interneurons
Dopamine could reduce spontaneous excitatory transmission as an indirect consequence of increasing spontaneous inhibitory transmission. That is, increasing inhibition onto excitatory neurons could reduce the probability of glutamate release from excitatory neurons. To eliminate this type of network effect as the basis of dopamine's actions on excitatory synaptic transmission, identified pre- and postsynaptic neurons were isolated by recording from monosynaptically coupled neuron pairs. After both a M/T cell and an interneuron were whole cell patch-clamped, a suprathreshold positive current pulse was injected to evoke an action potential in the M/T cell. The amplitude and the kinetics of the subsequent EPSP evoked in the interneuron were then analyzed.
The group of traces in Fig.
4 are representative data, consisting of reliable, time-locked
EPSPs from an interneuron following an evoked action potential in a
presynaptic M/T cell. In the presence of 30 µM dopamine, the peak amplitude
of the EPSP decreased to 68 ± 7% of the control amplitude (n =
6, P < 0.001). Neither the 10–90% rise time of the EPSP nor
the presynaptic action potential shape or amplitude were modified by dopamine
(P > 0.05). Following dopamine removal, the amplitude of the EPSP
recovered to control values (Fig.
4A). As shown in Fig.
4B, application of 1 µM bromocriptine, a D2 receptor
agonist, mimicked the dopamine-mediated attenuation of EPSP amplitude (62
± 4% of control, n = 5, P < 0.02). As with
dopamine, the 10–90% rise time of the EPSP was not affected (P
> 0.05). However, the neurons did not recover well from the effects of
bromocriptine after washout, perhaps due to the very high binding affinity of
bromocriptine for the D2 receptor (Coronas
et al. 1999). In contrast to the effects of D2 receptor
activation, D1 receptor activation with the selective D1 receptor agonist
SKF38393 (30 µM) did not significantly affect monosynaptic transmission
(n = 8, P = 0.67), as shown in
Fig. 4C. SKF38393 also
did not alter the 10–90% rise time of the EPSP (P >
0.05).
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Dopamine exerts no direct effect on the postsynaptic interneuron
We also wanted to determine whether the effects of dopamine on excitatory transmission were presynaptic, postsynaptic, or both. First, we examined the effects of dopamine on membrane resistance in the interneuron, since a decrease in postsynaptic membrane resistance would reduce the EPSP amplitude. Hyperpolarizing current injections (250 ms, 30–55 pA) were made in interneurons in current-clamp mode from a membrane potential of –60 mV. Flow-pipe application of 30 µM dopamine had no effect on the amplitude or kinetics of the resulting voltage deflection (n = 4, Fig. 5A). This result suggests that dopamine neither directly opens nor closes ion channels in the postsynaptic cell membrane sufficiently to cause a measurable change in resistance.
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Dopamine has no direct effects on glutamate receptors
A second hypothesis is that dopamine reduces EPSP amplitude via inhibition of postsynaptic glutamate receptors. To test this hypothesis, interneurons were whole cell voltage-clamped at –65 mV, and a glutamate receptor-mediated current was evoked by flow-pipe application of 500 µM glutamate, a concentration that activates all subtypes of ionotropic glutamate receptors. Dopamine (30 µM) was then coapplied during the glutamate-evoked current. Dopamine had no effect on either the kinetics or amplitude of the glutamate-mediated current (n = 8, Fig. 5B). Potential effects of dopamine on glutamate receptors were further examined by comparing currents evoked by glutamate alone with currents evoked by the coapplication of glutamate and dopamine (Fig. 5C). (These traces are in contrast to the trace in 5B, in which dopamine was coapplied during the middle of a glutamate-evoked current). Under these conditions too, dopamine did not alter the glutamate-evoked current (Fig. 5C, n = 6).
The possibility that prolonged exposure is required for dopamine to exert its effects was also tested. The initial current evoked by 500 µM glutamate served as a control. The neurons were incubated in 100 µM dopamine for 15–20 min, and glutamate was reapplied. Preincubation with dopamine did not affect the amplitude of the current evoked by glutamate (data not shown). This lack of evidence for postsynaptic modulation of glutamate-mediated excitation by dopamine suggests that the presynaptic M/T cell is the likely target of dopamine's action.
Dopamine inhibits calcium channel currents in M/T cells
Because calcium channel modulation is a common mechanism of action for presynaptic inhibition, the effect of dopamine on calcium channel currents in M/T cells was examined. M/T cells were voltage clamped at –60 mV and stepped to 0 mV for 50 ms. For ease of analysis, 10 mM Ba was substituted for Ca to enhance the amplitude of the current. Sodium currents were blocked with 1 µM TTX, and potassium currents were blocked with intracellular Cs. As shown in Fig. 6A, calcium channel currents were diminished to 30 ± 7% of control amplitudes (n = 8 of 11, P < 0.001) in the presence of 30 µM dopamine. Due to a combination of the rundown of calcium currents over time (which normally occurs with whole cell recording) and possible lingering dopaminergic effects, the currents in the cells tested only recovered to 67% of their original amplitudes. Application of 1 µM bromocriptine also attenuated calcium currents in M/T cells (Fig. 6B). Bromocriptine diminished calcium currents to 66 ± 5% of the original amplitude, with subsequent recovery to 83% of the original current amplitude (n = 8 of 12, P < 0.001). Although dopamine and bromocriptine had similar effects on evoked EPSPs, dopamine had a greater effect on calcium channel currents. A potential explanation for this discrepancy is that dopamine activates additional dopamine receptors that also attenuate calcium channel currents. In contrast to dopamine and bromocriptine, the reduction in calcium channel currents in response to application of 30 µM SKF38393 was no greater than that expected from normal current rundown (n = 7; Fig. 6C).
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To further identify the type of calcium channels affected by dopamine, 30
µM dopamine was applied after the L-type channels had been blocked by
nifedipine (10–100 µM). Nifedipine alone reduced the calcium channel
current to 64 ± 5% of control (n = 9, P < 0.01).
Application of dopamine blocked the nifedipine-resistant calcium channel
currents by 29 ± 7% (Fig.
6D, n = 9, P < 0.001). These results
suggest that dopamine can inhibit the dihydropyridine-resistant high-voltage
calcium channels (N- and P/Q-type) previously implicated in transmitter
release in the OB (Isaacson
2001; Isaacson and Strowbridge
1998). The effects of nifedipine at 10 µM were near saturating
and not significantly different those at 100 µM (data not shown). The
degree of calcium channel current block at either 10 or 100 µM nifedipine
was similar to the 40% block with 10 µM nifedipine previously observed in
these neurons (Trombley
1992).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Ennis et al.
2001; Hsia et al.
1999). This study suggests that activation of presynaptic D2
receptors on M/T cells can also attenuate glutamatergic transmission between
OB neurons and that the effect may be mediated by inhibition of calcium
channels. Synaptic organization and dopamine neurons in the OB
The axons of the olfactory sensory neurons enter the OB and innervate
periglomerular (PG) neurons, superficial tufted cells, and the primary
dendrites of mitral cells within glomeruli. Each glomerulus is thought to
operate as a functional unit, and each responds best to a specific group of
odors (Mombaerts 1999). The
GABAergic PG neurons that circumscribe the glomeruli mediate feedback
inhibition within glomeruli and lateral (feedforward) inhibition between
glomeruli (Shepherd 1972). A
large subset of PG cells is also dopaminergic
(Gall et al. 1987;
Kosaka et al. 1985), as is a
subpopulation of superficial tufted cells
(Halasz et al. 1981). The
latter neurons make reciprocal dendrodendritic synapses with PG cells and
often project their axon collaterals to synapse on PG and short axon cell
bodies (Pinching and Powell
1971). Their axons and axon collaterals may not project outside
the bulb, and the function of these neurons is not yet understood.
Although it has been reported that olfactory nerve transection reduces TH
and dopamine expression in the OB (Brunjes
et al. 1985), the present study provides immunocytochemical
evidence that there is significant expression of TH (and perhaps, dopaminergic
transmission) in cultured neurons in the absence of olfactory sensory neuron
input. The proportion of TH-immunoreactive neurons in our cultures is
consistent with recent findings in cultured neurons
(Cigola et al. 1998;
Puche et al. 1999) and the
intact bulb (McLean and Shipley 1998). Cigola and colleagues reported that a
48-h treatment with high external potassium concentrations, used to maintain
membrane depolarization, increased baseline TH expression in mouse OB cultures
2.4-fold. Application of an L-type calcium channel antagonist blocked this
increase in expression (Cigola et al.
1998). Puche and colleagues reported that co-culturing rat OB
cells with olfactory epithelium also caused a 2.4-fold increase in TH
expression. Interestingly, they found that application of an
N-methyl-D-aspartate (NMDA) receptor antagonist could
block this effect. These data suggest that calcium influx is an important
modulator of TH expression and that the route of calcium entry may be either
through NMDA receptors or voltage-gated calcium channels.
Our use of astrocyte feeder layers promotes high neuronal survival and rapid formation of synaptic contacts. Given this, our data may suggest that ongoing spontaneous synaptic activation of NMDA receptors and/or depolarization sufficient to activate voltage-gated calcium channels are adequate to maintain levels of TH expression in culture similar to those observed in the intact bulb (McLean and Shipley 1998). Alternatively, some expression of TH may be independent of these mechanisms. Differentiation between these hypotheses awaits further experimentation.
Dopamine modulates neurotransmission
Previous reports have shown that dopamine can modulate neurotransmission in
the OB. In the turtle OB, exogenous dopamine application both delayed the
onset and decreased the amplitude of field potentials recorded from mitral
cells (Nowycky et al. 1983).
More recently, in the rat and turtle OB, olfactory sensory neuron activation
of mitral cells was reduced in the presence of dopamine or D2 receptor
agonists (Berkowicz and Trombley
2000; Ennis et al.
2001; Hsia et al.
1999). Ennis et al.
(2001) also reported that
dopamine inhibits olfactory sensory neuron activation of M/T cells in both rat
and mouse and further demonstrated that dopamine inhibits olfactory sensory
neuron activation of juxtaglomerular cells. Significantly, these effects were
eliminated in D2 receptor knockout mice.
The present results are consistent with these previous reports. They also
provide additional understanding of the role of dopamine by demonstrating that
dopamine can modulate excitatory transmission between OB M/T cells and
interneurons. Our results, as well as the results of Brunig et al.
(1999) described in the next
paragraph, support the notion of functional D2 receptors on M/T cells.
However, in contrast to our results, Ennis et al.
(2001) reported that dopamine
had no effect on mitral cell-evoked excitatory postsynaptic currents (EPSCs)
in juxtaglomerular cells. Although the reason for this discrepancy is unclear,
there are at least several possible explanations. The experiments by Ennis and
colleagues were conducted in mice, whereas ours were in rats; there may be
species differences that explain the different findings. In addition, their
protocol (stimulation of the mitral cell layer or lateral olfactory tract)
generated long-lasting bursts of EPSCs. Under these conditions, initial
monosynaptic events are difficult to resolve. In some of our experiments, we
used synaptically coupled pairs of neurons; this allowed us to focus the
effects of dopamine on the initial monosynaptic component of transmission
between M/T cells and interneurons, while also eliminating polysynaptic or
network effects. It is possible that these initial monosynaptic events are
more sensitive to the effects of dopamine than are the mechanisms that
underlie bursting behavior. Another possible explanation for these disparate
findings is that mitral and tufted cells may differ in their sensitivity to
dopamine. It is possible that many of our experiments included tufted
cell-to-interneuron transmission. In contrast, the experiments by Ennis and
colleagues may have included only mitral cell transmission, since five of the
six cells examined involved direct stimulation of the mitral cell layer.
Collectively, these results are consistent with observations in other brain
regions in the rat, including the parabrachial region
(Chen et al. 1999), nucleus
accumbens (Kalivas and Duffy
1997), and ventral tegmental area
(Koga and Momiyama 2000),
where dopamine also decreases glutamatergic neurotransmission. However, the
effects of dopamine on neurotransmission are not limited to excitatory
transmission. For example, GABAergic inputs to the rat striatum are also
sensitive to dopamine receptor-mediated inhibition
(Delgado et al. 2000).
Dopamine also modulates inhibitory transmission of OB neurons in culture.
Brunig et al. (1999) reported
that D1 receptor activation attenuates GABA-gated chloride currents in
cultured interneurons and that stimulation of D2 receptors on cultured M/T
cells potentiates GABA-gated chloride currents. These findings by Brunig and
colleagues may explain why the D1 receptor agonist SKF38393 appeared to affect
spontaneous activity in our experiments while not affecting other aspects of
excitatory neurotransmission (e.g., evoked monosynaptic potentials,
voltage-gated calcium currents, membrane resistance). That is, a
SKF38393-mediated reduction in inhibition of GABAergic interneurons, which
could potentiate inhibition of M/T cells, could secondarily reduce spontaneous
excitatory transmission.
Dopamine's effects may be mediated via inhibition of calcium channel currents
In the present experiments, as in most of the cited examples, the effects
of dopamine appear to be presynaptic. This interpretation is based on our
finding that dopamine did not directly affect postsynaptic membrane resistance
nor directly inhibit postsynaptic glutamate-mediated currents. It is also
supported by previous OB studies demonstrating that dopamine has no effect on
M/T cell NMDA, AMPA, or kainate receptors
(Berkowicz and Trombley 2000),
juxtaglomerular cell input resistance, or the amplitude of miniature
excitatory postsynaptic currents (Ennis et
al. 2001; Hsia et al.
1999). Furthermore, the possibility that postsynaptic effects on
network behavior were indirectly mediating dopamine's effects on excitatory
transmission (as might occur with recording of spontaneous activity) was
eliminated by recording from monosynaptically coupled pairs of identified M/T
cells and interneurons.
It has been demonstrated in the turtle that dopamine reduces calcium influx
in olfactory sensory neuron terminals
(Wachowiak and Cohen 1999) and
that presynaptic D2 receptor activation inhibits glutamate release from these
terminals (Berkowicz and Trombley
2000). The fact that dopamine, or D2 receptor agonists, can reduce
calcium channel currents evoked from presynaptic M/T cells lends further
support to the notion that dopamine reduces excitatory transmission via
reductions in glutamate release. Dopamine may have had a greater effect on
calcium channel currents than bromocriptine because dopamine activates all
dopamine receptors (more than 1 of which may affect calcium channels), whereas
bromocriptine is selective for D2 receptors. That dopamine and bromocriptine
had similar effects on monosynaptic transmission suggests that dopamine's D2
effects on calcium channels is all that is necessary to account for dopamine's
effects on EPSPs. Our results on the effects of dopamine and D2 receptor
agonists on calcium currents and glutamate release are consistent with recent
results from experiments on ventral tegmental neurons
(Koga and Momiyama 2000).
This is not the first examination of calcium channel currents in cultured
OB neurons. From a holding potential of –60 mV, we previously observed
calcium currents in cultured M/T cells that activated near –40 mV,
peaked at 0 mV, and reversed near +55
(Trombley 1992;
Trombley and Westbrook 1992).
Transient or T-type currents were small and rarely observed, even from a
holding potential of –100 mV where most T-type channels would be
available for activation (Trombley
1992; Trombley and Westbrook
1992).
Dopamine receptors
The recently cloned dopamine receptors are typically grouped into either
the D1 or D2 subfamily according to sequence homology and pharmacological
attributes (Neve and Neve
1997; Sidhu and Niznik
2000). These receptors have a distinct laminar and cellular
distribution throughout the OB. In the rat, D1 receptors are found in the
glomerular, external plexiform, mitral cell, internal plexiform, and granule
cell layers, whereas D2 receptors are located in the olfactory nerve,
glomerular, and external plexiform layers
(Coronas et al. 1997;
Levey et al. 1993;
Mansour et al. 1990;
Nickell et al. 1991).
Although there is some controversy, several studies support the notion that
M/T cells express D2 receptors. Although not specifically addressed in the
text, autoradiographic in situ hybridization data presented by Mansour et al.
(1990) suggest that D2
receptor mRNA is expressed in the mitral cell layer. Levey et al.
(1993) reported D2 receptor
immunoreactivity in the external plexiform layer as well as the glomerular and
olfactory nerve layers. In a functional study, Brunig et al.
(1999) demonstrated a D2
receptor-mediated action on GABAA receptor-mediated currents in M/T
cells. In the present study, we provide immunocytochemical evidence of D2
receptors on M/T cells. Furthermore, our electrophysiological data showing
that dopamine and the D2 receptor agonist bromocriptine modulate calcium
channels and excitatory transmission between M/T cells and interneurons
indicate that these receptors are functional.
Dopamine receptors are metabotropic. Therefore each receptor subtype may
interact with G-proteins specific to a certain cell type or location
(Sidhu and Niznik 2000),
resulting in cell type-specific effects. Brunig et al.
(1999) have reported that
dopamine modulates GABAA receptors in rat OB in a cell-specific
manner that involves differential effects at D1 and D2 receptors. Dopamine
reduced currents through GABA-gated Cl-channels in interneurons via activation
of D1 receptors and subsequent phosphorylation of GABAA receptors
by protein kinase A. In contrast, dopamine enhanced GABA-mediated responses in
M/T cells via activation of D2 receptors and phosphorylation of
GABAA receptors by protein kinase C.
The two receptor subtypes also tend to have a specific synaptic deposition
(Brunig et al. 1999;
Nickell et al. 1991), which
may allow dopamine to differentially modulate synaptic circuits. Results from
the OB and other brain regions indicate that D2 receptors tend to play a
presynaptic role and D1 receptors, a postsynaptic role, in neurotransmission
(Hsu et al. 1995). Our results
are consistent with the general notion that D2 receptor activation can reduce
transmitter release. In contrast to the effects of the D1 selective agonist
SKF38393, the D2 selective agonist bromocriptine mimicked dopamine's
inhibition of calcium channel currents and evoked EPSPs.
Significance to olfactory function
Olfactory experience has a significant effect on the concentration of both
TH and dopamine within the OB (Baker et al.
1983). Olfactory deprivation, from either unilateral olfactory
nerve transection or unilateral naris occlusion, reduces OB dopamine content
in the ispsilateral bulb by as much as 75%
(Baker et al. 1983) and
enhances bulb responsiveness to odors, as measured by single-unit recordings
and 2-deoxyglucose autoradiography
(Guthrie et al. 1990;
Wilson and Sullivan 1995).
This enhanced responsiveness, as well as a decrease in odor discrimination, is
mimicked by application of the D2 receptor antagonist, spiperone
(Wilson and Sullivan 1995).
Other behavioral experiments have shown that activation of D1 or D2 receptors
can increase or decrease the threshold for odor detection, respectively
(Doty and Risser 1989;
Doty et al. 1998). These
results suggest that dopamine may contribute to both odor detection
sensitivity and discrimination. Previous electrophysiological studies, along
with the present study, may provide synaptic bases for these observations;
these studies demonstrate that activation of presynaptic D2 receptors can
reduce both olfactory sensory neuron activation of M/T cells and
juxtaglomerular cells (Berkowicz and
Trombley 2000; Ennis et al.
2001; Hsia et al.
1999) as well as the excitation of inhibitory interneurons by M/T
cells. The schematic in Fig. 7
depicts where dopamine may attenuate glutamatergic transmission in the OB.
However, a complete understanding of how dopamine may influence the behavior
of synaptic circuits important to odor information processing requires further
knowledge of what patterns of activity evoke dopamine release (e.g., olfactory
sensory neuron and/or M/T cell stimulation of dopaminergic neurons) and
whether the conditions of dopamine release can differentially affect circuits
(e.g., synaptic transmission from olfactory sensory neuron to M/T cell,
olfactory sensory neuron to juxtaglomerular cell, or M/T to interneuron).
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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This work has been supported in part by the National Institute for Deafness and Other Communication Disorders. N. G. Davila was supported by a National Institutes of Health Joint Neuroscience Predoctoral Training Grant and a National Research Service Award DC-05354-01 from the National Institute for Deafness and Other Communication Disorders.
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FOOTNOTES |
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Address for reprint requests: N. G. Davila, Dept. of Biological Science, Program in Neuroscience, Florida State Univ., Tallahassee, FL 32306-4340 (E-mail: davila{at}neuro.fsu.edu).
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