Department of Anatomy and Neurobiology, Program in Neuroscience, University of Maryland School of Medicine, Baltimore, Maryland 21201
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ABSTRACT |
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Hayar, Abdallah,
Phillip M. Heyward,
Thomas Heinbockel,
Michael T. Shipley, and
Matthew Ennis.
Direct Excitation of Mitral Cells Via Activation of
1-Noradrenergic Receptors in Rat Olfactory Bulb Slices.
J. Neurophysiol. 86: 2173-2182, 2001.
The main olfactory bulb
receives a significant modulatory noradrenergic input from the locus
coeruleus. Previous in vivo and in vitro studies showed that
norepinephrine (NE) inputs increase the sensitivity of mitral cells to
weak olfactory inputs. The cellular basis for this action of NE
is not understood. The goal of this study was to investigate the effect
of NE and noradrenergic agonists on the excitability of mitral cells,
the main output cells of the olfactory bulb, using whole cell
patch-clamp recording in vitro. The noradrenergic agonists,
phenylephrine (PE, 10 µM), isoproterenol (Isop, 10 µM), and
clonidine (3 µM), were used to test for the functional presence of
1-,
-, and
2-receptors, respectively, on mitral cells. None of
these agonists affected olfactory nerve (ON)-evoked field potentials
recorded in the glomerular layer, or ON-evoked postsynaptic currents
recorded in mitral cells. In whole cell voltage-clamp recordings, NE
(30 µM) induced an inward current (54 ± 7 pA, n = 16) with an EC50 of 4.7 µM. Both PE and Isop
also produced inward currents (22 ± 4 pA, n = 19, and 29 ± 9 pA, n = 8, respectively), while
clonidine produced no effect (n = 6). In the presence
of TTX (1 µM), and blockers of excitatory and inhibitory fast
synaptic transmission [gabazine 5 µM,
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) 10 µM, and
(±)-2-amino-5-phosphonopentanoic acid (APV) 50 µM], the
inward current induced by PE persisted (EC50 = 9 µM), whereas that of Isop was absent. The effect of PE was also
observed in the presence of the Ca2+ channel
blockers, cadmium (100 µM) and nickel (100 µM). The inward current
caused by PE was blocked when the interior of the cell was perfused
with the nonhydrolyzable GDP analogue, GDP
S, indicating that the
1 effect is mediated by G-protein coupling. The current-voltage relationship in the absence and presence of PE indicated that the
current induced by PE decreased near the equilibrium potential for
potassium ions. In current-clamp recordings from bistable mitral cells,
PE shifted the membrane potential from the downstate (
52 mV) toward
the upstate (
40 mV), and significantly increased spike generation in
response to perithreshold ON input. These findings indicate that NE
excites mitral cells directly via
1 receptors, an effect that may
underlie, at least in part, increased mitral cell responses to weak ON
input during locus coeruleus activation in vivo.
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INTRODUCTION |
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The mammalian main olfactory
bulb (MOB) receives a significant noradrenergic input from the locus
coeruleus (LC) (Fallon and Moore 1978; McLean and
Shipley 1991
; McLean et al. 1989
; Shipley et al. 1985
). Noradrenergic inputs to the MOB play important
roles in olfactory function. Olfactory cues increase the discharge of LC neurons in behaving animals (Aston-Jones and Bloom
1981
) and trigger rapid increases in norepinephrine (NE) levels
in the olfactory bulb (Brennan et al. 1990
;
Rangel and Leon 1995
; Rosser and Keverne 1985
). LC-NE projections to the main and accessory olfactory
bulb are critical for the formation and/or recall of specific olfactory memories, pheromonal regulation of pregnancy, and postpartum maternal behavior (Brennan et al. 1990
; Dluzen and Ramirez
1989
; Kaba et al. 1989
; Rosser and
Keverne 1985
; Sullivan et al. 1989
,
1992
; Wilson and Leon 1988
).
Despite several decades of research, the postsynaptic targets and
neurophysiological actions of NE inputs to the MOB have remained
elusive. Based on anatomical considerations, both the mitral cells and
the granule cells are potential targets of NE inputs to MOB.
Noradrenergic fibers are localized exclusively in the subglomerular
layers where they terminate densely in the internal plexiform and the
granule cell layers, and moderately in the external plexiform and
mitral cell layers (McLean et al. 1989; Shipley
et al. 1985
). The glomerular layer is nearly devoid of
noradrenergic fibers (McLean et al. 1989
). In agreement
with the distribution of NE fibers, both mitral cells and granule cells express several noradrenergic receptor subtypes, namely
1 and
2
receptors (Day et al. 1997
; McCune et al.
1993
; Pieribone et al. 1994
;
Winzer-Serhan et al. 1997
).
Exogenous application of NE in the mammalian MOB has been reported to
produce a number of effects. Iontophoretic application of NE was found
to inhibit mitral cell spontaneous activity, presumably by excitation
of granule cells (McLennan 1971). Field potential studies in the rat suggested that NE, acting at
1 receptors, depolarized granule cells (Mouly et al. 1995
), an effect
that would also inhibit mitral cells. Alternatively, in the turtle and
dissociated rat MOB cultures, NE disinhibited mitral cells (Jahr
and Nicoll 1982
; Trombley 1992
,
1994
; Trombley and Shepherd 1992
). This
effect was attributed to
2 receptor-mediated presynaptic inhibition
of granule and/or mitral cell dendrites. More recent electrophysiological studies in vivo and in vitro have demonstrated a
consistent action of NE in the MOB. Endogenously released or exogenously applied NE increased the responses of mitral cells to weak
or perithreshold olfactory nerve (ON) input (Ciombor et al.
1999
; Jiang et al. 1996
). The specific site of
action of NE in these later studies was not determined.
Taken together, the findings above indicate that the net influence of endogenously released NE in the MOB circuit is likely to result from direct postsynaptic actions on mitral cell output neurons as well as on granule cell interneurons. At present, however, there is no information about the cellular effects of NE on mammalian MOB neurons in vivo or in slice preparations. The goal of the present study therefore was to investigate the cellular actions of NE on mitral cells using whole cell patch-clamp recordings in rat olfactory bulb slices.
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METHODS |
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Sprague-Dawley rats (18-22 days old), of either sex, were anesthetized with chloral hydrate and decapitated in accordance with Institutional Animal Care and Use Committee and National Institutes of Health guidelines. The olfactory bulbs were removed and immersed in sucrose-artificial cerebrospinal fluid (sucrose-ACSF) equilibrated with 95% O2-5% CO2 (pH 7.38) at 4-10°C. The sucrose-ACSF had the following composition (in mM): 26 NaHCO3, 1 NaH2PO4, 3 KCl, 5 MgSO4, 0.5 CaCl2, 10 glucose, and 248 sucrose. Horizontal slices (400 µm thick) were cut with a microslicer (Ted Pella, Redding, CA). After a period of recovery (15-20 min) at 30°C, the slices were incubated until used at room temperature (22°C) in ACSF equilibrated with 95% O2-5% CO2 and composed of (in mM) 124 NaCl, 26 NaHCO3, 3 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2, and 10 glucose. For recording, a single slice was placed in a recording chamber on an upright epifluorescent microscope (Olympus BX50WI, Tokyo) and submerged in normal ACSF equilibrated with 95% O2-5% CO2, perfused at the rate of 1.5-2.5 ml/min at 30°C.
Patch pipettes were pulled from borosilicate glass capillaries with an
inner filament (1.5 mm OD, Clark, Kent, UK) on a pipette puller
(Sutter P97) and were filled with a solution of the following composition (in mM): 114 K-gluconate, 17.5 KCl, 4 NaCl, 4 MgCl2, 10 HEPES, 0.2 EGTA, 3 Mg2ATP, and 0.3 Na2GTP; in
some experiments, 0.02% Lucifer yellow (Molecular Probes, Eugene, OR)
was included in the pipette solution. Osmolarity was adjusted to 270 mOsm and pH to 7.3. The pipette resistance was 5-8 M. Whole cell
voltage- and current-clamp recordings were made using an Axopatch-200B amplifier (Axon Instruments, Foster City, CA). Liquid junction potential was 9-10 mV, and all reported voltage measurements were not
corrected for this potential. Only recordings made with an access
resistance of <30 M
were included in this study.
Electrical stimulation (Grass S8800 stimulator, Astro-Med, West
Warwick, RI) was performed using two stainless steel wires (50 µm
diam, A-M Systems, Everett, WA), insulated except at their tips
positioned in the olfactory nerve (ON) layer. Stimulus pulses of
10-300 µA, were 100 µs duration and were applied at 0.05 Hz. Evoked field potentials were recorded in the glomerular layer using
glass pipettes (0.5-2 M) filled with 2 N NaCl.
Drugs and solutions of different ionic content were applied to the slice by switching the perfusion with a three-way electronic valve system (General Valve, Fairfield, NJ). Norepinephrine bitartrate, phenylephrine, clonidine, isoproterenol, prazosin, and propranolol were obtained from Sigma (St. Louis, MO). Tetrodotoxin (TTX), gabazine (SR95531), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and (±)-2-amino-5-phosphopentanoic acid (APV) were obtained from Research Biochemicals International (Natick, MA).
During the experiments, analog signals were low-pass Bessel filtered at 2 kHz (Axopatch 200B, Axon Instruments), digitized at 10 kHz (Instrutech, Long Island, NY), and stored on videotape for later analysis. They were also collected through a Digidata-1200A Interface (Axon Instruments), and digitized at 10-20 kHz. Group data, expressed as means ± SE, were statistically analyzed with paired t-tests unless otherwise stated.
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RESULTS |
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Effects of noradrenergic receptor agonists on ON-evoked synaptic responses
The previously reported NE-induced increase in sensitivity of
mitral cells to weak ON input (Ciombor et al. 1999;
Jiang et al. 1996
) could be due to enhanced postsynaptic
responses of mitral cell to glutamatergic input from ON terminals. To
test this hypothesis, we investigated the effect of noradrenergic
agonists on ON-evoked field potentials (fEPSPs) recorded in the
glomerular layer (Fig. 1, A
and B). The peak amplitude of the fEPSPs was not changed by
PE (10 µM, 0.72 ± 0.1 mV vs. 0.72 ± 0.1 mV, mean ± SE, P = 0.09, n = 4), isoproterenol
(Isop; 10 µM, 0.78 ± 0.11 mV vs. 0.78 ± 0.11 mV,
P = 0.52, n = 3), or clonidine (3 µM,
0.69 ± 0.1 mV vs. 0.67 ± 0.1 mV, P = 0.19, n = 3). However, NE (30 µM) slightly reduced the
fEPSPs by 17 ± 7% (1.1 ± 0.08 mV vs. 0.91 ± 0.09 mV, P = 0.007, n = 4). Since this action
was not mimicked by any of the selective noradrenergic receptor
agonists, we wondered whether NE could activate other inhibitory
receptors in the glomeruli. Dopamine, a transmitter present in
periglomerular interneurons, was recently reported to presynaptically
inhibit ON terminals via the D2 receptor subtype (Hsia et al.
1999
). Therefore we tested the ability of the D2 dopamine
receptor antagonist, sulpiride, to block the inhibitory effect of NE.
Sulpiride (100 µM) reversed the inhibitory effect of NE on fEPSPs in
four slices tested (NE: 17 ± 7% reduction, NE + sulpiride:
3 ± 1% reduction, P = 0.02). Moreover,
sulpiride, applied alone, produced no change by itself on the fEPSP,
but it prevented the effect of NE (sulpiride: 0.87 ± 0.16 mV;
sulpiride + NE: 0.85 ± 0.15 mV, n = 3, not
shown).
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We also tested the effects of noradrenergic agonists on ON-evoked
excitatory postsynaptic currents (EPSCs) recorded in mitral cells using
whole cell recordings (Fig. 1, C and D). NE (30 µM) reduced the amplitude of the evoked EPSCs in all cells tested by
an average of 46 ± 5% (188 ± 35 pA vs. 103 ± 73 pA,
n = 11, P = 0.004). In five of five
cells tested, the depressive effect of NE on the amplitude of the
evoked EPSCs was reversed by application of sulpiride (NE: 39 ± 5% reduction, NE + sulpiride: 5 ± 3% reduction, P = 0.003). In contrast, there was no effect of PE (10 µM, 144 ± 35 pA vs. 146 ± 37 pA, P = 0.62, n = 6), Isop (10 µM, 222 ± 32 pA vs.
208 ± 24 pA, P = 0.22, n = 4), or
clonidine (3 µM, 172 ± 35 pA vs. 177 ± 33 pA,
P = 0.35, n = 5) on the amplitude of evoked EPSCs in all cells tested. Taken together, these results indicate that EPSCs induced by ON stimulation are not discernibly modulated by noradrenergic receptors. The apparent depressive effects
of NE on ON-evoked responses are probably due to NE activation of
inhibitory D2 dopaminergic receptors located on ON terminals, consistent with similar pharmacological findings in the substantia nigra (Grenhoff et al. 1995).
Effect of noradrenergic agonists on mitral cell membrane currents
NE produced an inward current in all mitral cells tested (range
23-110 pA, 54 ± 7 pA, n = 16, Fig.
2) in voltage-clamp mode at the holding
potential of 60 mV. The magnitude of the response to NE was
concentration dependent (1-30 µM; Fig. 2, A and
B). The concentration of NE was increased consecutively at
4-min intervals in the same cells, and the EC50
of the NE response was 4.7 µM. Using this protocol, NE induced an
inward current that was not significantly different from when it was
applied at a single concentration of 30 µM (59 ± 13 pA,
n = 4 vs. 54 ± 7 pA, n = 16, P = 0.73, unpaired t-test), indicating that
there was no substantial desensitization of the response to NE with
prolonged application.
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The inward current caused by NE could be due to its interaction with
different noradrenergic receptor subtypes, namely 1 and
receptors, which are known to produce excitatory effects in many
neurons throughout the brain (for review, see Hein and Kobilka
1995
). In three cells, a second application of NE in the presence of the
1 and
receptor antagonists (prazosin 1 µM, and
propranolol 10 µM, respectively) produced no significant effect (<4
pA, Fig. 2C). The effect of NE recovered partially after
wash out of the antagonists (>30 min). Next, we investigated which specific noradrenergic agonists could mimic the effect of NE (Fig. 3). Although prazosin has been reported
to be an antagonist of
2B and
2C receptor subtypes (Hieble
and Ruffolo 1996
),
2 receptors do not appear to be involved
in the inward current induced by NE in mitral cells because clonidine
(3 µM) produced no detectable change in holding current (0.3 ± 1 pA, n = 6). Concentrations of clonidine higher than 3 µM were not tested because they can activate nonspecifically
1
receptors. In contrast, the
1 noradrenergic agonist, PE (10 µM),
produced an inward current in 15 of 19 mitral cells tested (range 5-55
pA, 22 ± 4 pA, n = 19). In the four remaining cells, PE produced no detectable effect (<4 pA). The effect of PE was
reproducible on a second application with no evidence of desensitization (n = 3). In three mitral cells that
responded significantly to PE (>25 pA), a second application of PE in
the presence of the
1 receptor antagonist, prazosin (1 µM),
produced no detectable current.
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The receptor agonist, Isop (10 µM) also induced an inward current
in all cells tested (range 8-80 pA, 29 ± 9 pA, n = 8). A second application of Isop in the presence of the
receptor antagonist propranolol (10 µM) produced no significant inward current
(n = 3, Fig. 3C). Moreover, Isop produced no
significant effect when applied for the first time in the presence of
propranolol (n = 4). Isop also increased the frequency
of spontaneous EPSCs (sEPSCs). The sEPSCs were similar to "the
long-lasting depolarizations" that have been described recently
(Carlson et al. 2000
). The inward current caused by Isop
could therefore result, in part, from a network effect due to an
increase in excitatory input to mitral cells. Alternatively, Isop might
reduce tonic inhibition to mitral cells (disinhibition) by inhibiting
inhibitory interneurons (namely, granule and periglomerular cells). To
investigate these two possibilities, the effect of Isop on the holding
current and on sEPSCs was examined in the presence of the
GABAA receptor antagonist gabazine (5 µM); the
N-methyl-D-aspartate (NMDA) receptor antagonist
APV (50 µM) was included in the bath to prevent epileptic activity
caused by application of gabazine alone (not shown). Under these
conditions, the inward currents produced by Isop (10 µM) persisted
(29 ± 6 pA, n = 5; Figs.
4A and 5), and the frequency
of sEPSCs increased in all cells tested by an average of 81 ± 12% (from 0.31 ± 0.07 Hz to 0.54 ± 0.09 Hz,
n = 5, P = 0.004, Fig.
5). All sEPSCs (recorded in APV and
gabazine) were
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA)/kainate receptor dependent and action potential dependent as
they were blocked by CNQX (n = 4) or TTX (1 µM,
n = 3, not shown) (see also Carlson et al.
2000
). Although PE induced an inward current in gabazine and
APV (32 ± 9 pA, n = 5; Fig. 4), it did not change
the frequency of sEPSCs.
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In the next experiment, we investigated whether the PE- and Isop-induced inward currents are mediated by direct activation of noradrenergic receptors on mitral cells. To eliminate possible indirect effects, we blocked action potential propagation by TTX (1 µM), and ionotropic glutamate and GABAA receptors were blocked by the antagonists APV (50 µM) and CNQX (10 µM), and gabazine (5 µM), respectively. Under these conditions, Isop (10 µM) had no detectable effect on the holding current of mitral cells (0.5 ± 1.3, n = 6; Figs. 3D and 4A), indicating that the effect of Isop is mediated by an indirect circuit action or requires TTX-sensitive sodium channels in mitral cells. However, in identical conditions, PE (10 µM) still induced an inward current (15-35 pA) in five of seven cells tested (18 ± 5 pA, n = 7; Figs. 3B and 4A). Because PE produced a direct effect on mitral cells, we examined the effect of PE at different concentrations (1-100 µM) independent of network interactions (in the presence of TTX, CNQX, APV, and gabazine). We measured the concentration-response relationship only in cells that responded with more than 15 pA of inward current to 10 µM of PE. In these cells, PE produced a small inward current (4.6 ± 0.8 pA) starting at 1 µM, and the estimated EC50 was 9.0 µM (n = 5, Fig. 4B).
Extracellular calcium influx could be one of several second-messenger
pathways activated by 1 receptor (Han et al. 1987
; Pan et al. 1994
; Vaughan et al. 1996
). To
test this possibility, we applied PE (10 µM) in the presence of
blockers of voltage-dependent Ca2+ channels,
cadmium (100 µM) and nickel (100 µM), in addition to TTX, CNQX,
APV, and gabazine. Under these conditions, mitral cells still responded
to PE with an inward current (18 ± 4 pA, n = 4) comparable to that observed in the absence of
Ca2+ channel blockers (Fig. 4). This result
indicates that the inward current produced by PE does not result from
calcium entry into mitral cells, and therefore voltage-dependent
Ca2+ channels play little or no role in the
1
receptor-mediated response.
1 receptor responses are mediated by G-protein-coupled signaling
pathways (for review, see Hein and Kobilka 1995
;
Zhong and Minneman 1999
). To determine whether the
PE-induced inward current in mitral cells is G-protein mediated, we
examined the effect of PE after inactivation of G-proteins by including
GDP
S (1 mM) in the pipette solution (Chu and Hablitz
2000
; Lin and Dun 1998
; Schneider et al.
1998
). In mitral cells perfused with an intracellular solution
containing GDP
S (1 mM), PE (10 µM) produced no detectable effect
on the holding current (n = 3, not shown). Therefore
G-proteins are involved in the mechanism of action of PE in mitral cells.
The preceding results indicate that the PE-induced current is not
carried primarily by influx of calcium through cadmium- and
nickel-sensitive calcium channels or by influx of sodium through TTX-sensitive sodium channels. Therefore we investigated whether the
response to PE could be explained by a modulation of a potassium conductance. The current-voltage relationship in the absence and presence of PE indicated that the current induced by PE tended to
decrease, but did not reverse in polarity, at the equilibrium potential
for potassium ions (Fig. 6A).
We assume that under blockade of fast synaptic transmission, as well as
sodium and calcium channel blockade, the major ion channels that
contribute to the conductance of the membrane are potassium channels.
In this case, the decrease in the slope of the current-voltage curve is
indicative of an increase in membrane input resistance due to closure
of potassium channels that were open at the range of holding potentials
tested (110 to
30 mV). The inability to obtain a reversal potential for the PE-induced current is probably due to an inadequate space clamp
of the mitral cells that have long lateral and apical dendrites. Similar results were also obtained in dorsal raphe neurons (Pan et al. 1994
) and ventrolateral rat periaqueductal gray neurons (Vaughan et al. 1996
).
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One possible mechanism for the action of PE is a reduction of the
transient outward potassium conductance
IA as has been described for dorsal
raphe serotonergic neurons (Aghajanian 1985). To
investigate this possibility, we activated A-like currents by holding
the membrane potential at
80 mV for 400 ms (to deinactivate
IA), followed by a depolarizing
voltage step to
45 mV (Aghajanian 1985
). This protocol
generated a transient outward current of 300-1,500 pA that decayed to
baseline level within 100-300 ms. PE had no effect on the amplitude
(from 636 ± 343 pA to 634 ± 330 pA, n = 4, P = 0.89) or the decay time constant (from 91 ± 24 ms to 89 ± 20 ms, n = 4, P = 0.82) of the current evoked by this protocol (Fig. 6B).
Taken together, these results suggest that the primary ionic mechanism
for the PE-induced response is a decrease in a potassium conductance
that is different from IA.
Effects of 1 receptor activation on membrane potential, and
spontaneous and ON-evoked discharge
A previous extracellular unit study showed that NE, acting through
1 receptors, increases responses of mitral cells to weak (i.e.,
perithreshold) ON shocks, by reducing the percentage of response
failures to ON stimulation (Ciombor et al. 1999
). The preceding results indicate that NE, via a direct
1
receptor-mediated effect, evokes an inward current in mitral cells.
This suggests that
1 receptor activation may depolarize mitral
cells, an action that could enhance mitral cell responsiveness to ON
input. In the next experiments, therefore, we investigated the effects
of PE on mitral cell membrane potential, and spontaneous and ON-evoked discharge.
Mitral cells recorded in vitro exhibit membrane potential bistability
(Heyward et al. 2001). As shown in Fig.
7, mitral cells generate two levels of
membrane potential separated by about 10 mV: a "down-state,"
subthreshold for spike generation, and a perithreshold "up-state,"
in which the cells are more responsive to ON input than the down-state
(Fig. 8). Generation of the up-state is
an active, voltage-dependent process, sensitive to membrane
depolarization (Heyward et al. 2001
). We hypothesized,
therefore that
1-receptor activation might depolarize mitral cells
and thereby increase the proportion of time spent by mitral cells in
the up-state, an effect that would enhance responsiveness to weak ON
input. This hypothesis was tested using current-clamp recording.
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Figure 7 shows mitral cell spontaneous activity, and the membrane
potential distributions of activity recorded before and during exposure
to PE (n = 9, 10 µM). The membrane potential
distributions show the proportion of time spent by the cell at each
membrane potential. The distributions are bimodal; the two peaks
corresponding to the down-state and up-state. PE application resulted
in an overall depolarizing shift in the membrane potential distribution of about 1-2 mV (control: 53.4 ± 0.2 mV; PE:
51.5 ± 0.2 mV, P < 10
7; control, PE:
1.9 mV, n = 9 cells), with a corresponding increase in
time spent by the cell in the up-state potentials (from 16 ± 2.1% to 34 ± 5.4%, P = 0.009; see Fig. 7). This
depolarization was not associated with a change in spontaneous firing
rate (control: 3.7 ± 1.8 Hz; PE: 3.6 ± 1.4 Hz,
P = 0.47).
The depolarization resulting from PE application was, however,
associated with increased responsiveness of mitral cells to ON
stimulation (Fig. 8). Single ON shocks were delivered at perithreshold intensity, sufficient to elicit short-latency action potentials in
about 50% of trials. As shown in Fig. 8, short-latency spikes (latency
<20 ms) were reliably elicited when ON shocks were delivered at
up-state potentials, while ON shocks in the down-state resulted in
either no response, or a spike at long latency (>20 ms). In the
presence of PE, membrane potentials were depolarized, the probability
of ON-evoked spikes increased, and the mean spike latency was reduced.
The proportion of trials in which perithreshold stimulation elicited
spikes was increased from 56 ± 9% to 82 ± 10% in the
presence of PE (P < 0.04, n = 217 trials in 7 cells). In the presence of PE, there was a 1.8-fold
increase in the proportion of trials in which short-latency spikes were
generated in response to perithreshold ON stimulation (control: 38%;
PE: 68%, P < 0.02, n = 7 cells), and a
smaller reduction in the proportion of trials in which long-latency
spikes were elicited (control: 18%, PE: 13%, P < 0.03, n = 7 cells). This change in the distribution of response latencies (Fig. 8) corresponded to a significant reduction in
overall response latency (P < 0.0005, n = 7). These data are consistent with the results of
earlier studies (Ciombor et al. 1999) and support the
hypothesis that
1 receptor activation results in mitral cell
depolarization, thereby increasing the probability of a short-latency
response to ON stimulation.
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DISCUSSION |
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The present results demonstrate that NE, acting at the 1
receptor, induces an apparent inward current that depolarizes mitral cells. This apparent inward current and the accompanying depolarization appear to be due to a decreased potassium conductance involving a
G-protein signaling pathway. Together, these changes significantly modulate the excitability state of mitral cells by 1)
biasing the membrane potential to the perithreshold up-state and
2) enhancing the generation of action potentials in response
ON input.
Olfactory nerve terminals are not modulated by noradrenergic receptors
Electrical stimulation of the ON layer evoked in the glomerular
layer fEPSPs that are produced by currents generated, for the most
part, within the glomeruli (Aroniadou-Anderjaska et al. 1997). It also evoked long-lasting PSCs in mitral cells
confirming previous results (Carlson et al. 2000
;
Chen and Shepherd 1997
; Desmaisons et al.
1999
; Ennis et al. 1996
; Keller
et al. 1998
; Nickell et al. 1996
). None of the
specific noradrenergic agonists used in this study were effective in
modulating these two types of ON-evoked excitatory responses. The lack
of noradrenergic modulation of excitatory inputs from ON terminals is
consistent with anatomical data showing that the glomerular layer,
where axons of ON terminals synapse with mitral cell apical dendrites,
is nearly devoid of NE fibers (McLean et al. 1989
).
Unexpectedly, NE (30 µM) reduced ON-evoked fEPSPs recorded in the
glomerular layer and ON-evoked EPSCs in mitral cells. This NE action
appears to be mediated by nonnoradrenergic receptors because the
depressive effects of NE on ON-evoked fEPSPs and EPSCs were prevented
or reversed by the D2-dopamine receptor antagonist sulpiride. In the
MOB, D2 receptors are localized exclusively in the ON and glomerular
layers (Coronas et al. 1997; Koster et al.
1999
; Nickell et al. 1991
). Recent studies
demonstrate that dopamine, a transmitter present in juxtaglomerular
neurons, inhibits glutamate release by activation of presynaptic D2
receptors on ON terminals (Berkowicz and Trombley 2000
;
Hsia et al. 1999
). Taken together, these findings
suggest that exogenously applied NE may suppress glutamate release from
ON terminals by activating inhibitory presynaptic dopamine D2 receptors
present on ON terminals. These actions of NE at dopamine receptors are
not without precedent. Interactions between NE and dopamine receptors
have been reported in binding studies (Newman-Tancredi et al.
1997
), and electrophysiological studies demonstrated that
NE-induced hyperpolarizations in substantia nigra neurons were
completely blocked by sulpiride (Grenhoff et al. 1995
).
However, because there are virtually no NE fibers in the glomerular
layer, it is unlikely that NE released in the "infraglomerular layers" gains access to D2 receptors. These results suggest that care
should be taken in interpreting the pharmacological effects of
exogenously applied monoamines in the MOB.
and
2-receptors
Low to moderate levels of receptors are located in the
glomerular and granule cell layers (Woo and Leon 1995
).
Application of the
receptor agonist Isop consistently caused an
inward current in mitral cells. This current, however, was abolished
when mitral cells were pharmacologically isolated from major circuit
effects by application of TTX and blockers of fast synaptic
transmission. This result suggests that the inward current induced in
mitral cells by
receptor activation is most likely a circuit
effect, perhaps resulting from increased glutamate release from
excitatory inputs, or a decrease in inhibitory GABA inputs to mitral
cells, or both. A decrease in GABA input is, however, an unlikely
explanation since the effect of Isop persisted in the presence of the
GABAA antagonist gabazine. This leaves increased
glutamate release as the remaining possibility. The mechanism for such
an effect is unclear as Isop did not alter responses evoked by
stimulation of the ON, the only known glutamatergic synaptic input to
mitral cells. Recent studies in vitro, however, have shown that in
normal physiological media, mitral cells exhibit spontaneous
depolarizing events that are mediated by glutamatergic recurrent
intraglomerular dendrodendritic interactions among mitral/tufted cells
(Carlson et al. 2000
). It is therefore conceivable that
Isop may enhance the release of glutamate from the apical or lateral
dendrites of mitral or tufted cells. Since Isop did not directly evoke
currents in mitral cells, this hypothesized effect of Isop may be
caused by excitation of tufted cells or of, as of yet undiscovered,
excitatory MOB interneurons. Direct excitation of tufted cells might
enhance dendrodendritic excitatory interactions among the tufts of both tufted cells and mitral cells within the glomeruli, leading to an
increase in recurrent excitatory glutamate release. An additional possibility is that Isop may increase glutamate release by enhancing calcium currents in the dendrites of mitral or tufted cells. Activation of
receptors has been reported to facilitate glutamate release in
the amygdala by increasing presynaptic calcium influx (Huang et
al. 1996
, 1998
). The Isop-induced increase in
the excitability of mitral cells observed here may play a role in
NE-induced,
receptor-mediated facilitation of olfactory learning
in neonatal animals (Sullivan et al. 1989
,
1992
, 2000
).
Receptor localization studies indicate that mitral cell express 2
receptors (Winzer-Serhan et al. 1997
). In the present
study, the selective
2 receptor agonist, clonidine, did not produce any detectable currents in mitral cells at the holding potential of
60 mV. Our results, however, do not exclude the presence of functional
2 receptors in mitral cells. Although clonidine did not
produce a detectable effect,
2 receptors could modulate
high-threshold, voltage-gated channels that are closed at the holding
potentials tested in the present study. In this regard, it is
noteworthy that Trombley (1992
, 1994
)
reported that clonidine decreased high-threshold calcium currents in
mitral cells in culture, an effect that reduced glutamate release from
these cells. It is also possible that potential actions of clonidine
may have been prevented by dialysis of intracellular messengers by the
patch pipette solution. Finally, it is possible that the effects of
2 receptors occur at dendritic sites too remote to be detected by
somatic recordings.
Activation of 1 receptor induces an inward current in mitral
cells
NE, the 1 receptor agonist PE and the
receptor agonist Isop
induced relatively similar inward currents in mitral cells. In
conditions that eliminate fast synaptic transmission (TTX, APV, CNQX,
and gabazine), the inward currents elicited by PE persisted, whereas
those elicited by Isop were abolished. This suggests that endogenously
released NE may directly modify the membrane conductance of mitral
cells via activation of the
1 receptor subtype. The calculated
EC50 for the PE-evoked inward current in the
present study was 9 µM. This value is higher than the
EC50 for the PE-induced depolarization in dorsal
raphe neurons (1.4 µM) (Pan et al. 1994
). It is
possible that the
1 receptor subtype in mitral cells has lower
affinity to the agonist PE. Indeed, at least three subtypes of
1
receptors have been cloned so far (for review, see Docherty 1998
).
Current-voltage curves generated in the presence and absence of PE
demonstrated that the inward currents evoked by PE decreased at
negative membrane potentials near the calculated equilibrium potential
for K+ ions (96 mV). Space-clamp limitations in
mitral cells, in addition to the relatively small magnitude of the PE
response (about 20 pA in TTX), precluded accurate determination of the
reversal potential of the conductance modulated by PE. Additionally,
the current-voltage curves in the presence and absence of PE suggest
that activation of
1 receptors is associated with increased input
resistance. Taken together, these finding suggest that the
-induced
currents in mitral cells are mediated by decreased
K+ conductance. In agreement with this, PE-evoked
currents persisted in the presence of the Ca2+
channel blockers cadmium and nickel, indicating that
Ca2+ or Ca2+-dependent
potassium channels were not involved. Chloride channels are probably
not involved in the inward current produced by PE because their
activation would produce an outward current at potentials more positive
than
65 mV, which is the equilibrium potential for chloride
ions in our conditions. However, we cannot rule out some other
possibilities, such as a contribution from sodium TTX-insensitive channels.
Although activation of 1 receptors has been reported to reduce
IA in serotonergic neurons by 34%
(Aghajanian 1985
), A-like currents were not discernibly
affected by PE in mitral cells. A reasonable candidate mechanism for
the
1 receptor-mediated excitation is a decrease of a leak
potassium conductance, which is decreased by activation of
1
receptors in several brain areas including the dorsal motor nucleus of
the vagus (Fukuda et al. 1987
), hypoglossal motoneurons
(Parkis et al. 1995
), dorsal raphe (Pan et
al. 1994
), thalamus, and cortex (Wang and McCormick
1993
). The present study showed that the PE-induced inward
current was prevented by intracellular dialysis with GDP
S, a
manipulation that blocks G-protein activation. This indicates that the
PE-induced inward current is mediated by an
1 receptor
G-protein-coupled mechanism. This is similar to the signaling pathway
mediating
1 receptor-dependent inhibition of the leak potassium
current in other cell types (Grenhoff et al. 1995
;
Pan et al. 1994
; Parkis et al. 1995
).
Noradrenergic axons are very dense in the granule cell, mitral cell,
and external plexiform (EPL) layers (Halasz et al. 1978; McLean et al. 1989
). The EPL has the highest level of
1 receptor binding sites in the MOB (Jones et al.
1985b
) and, indeed, the highest density of
1 receptors in
the brain (Young and Kuhar 1980
). The EPL contains the
lateral dendrites of mitral cells and the apical dendrites of the
granule cells. Thus both cell types are potential targets of NE fibers.
In agreement with this, both mitral and granule cells express
1
receptor mRNA (Day et al. 1997
; McCune et al.
1993
; Pieribone et al. 1994
). Previous studies
reported
1 receptor-mediated changes in ON-evoked discharge of
mitral cells or evoked field potential activity in the MOB (Ciombor et al. 1999
; Mouly et al.
1995
; Perez et al. 1987
), although the specific
site of the
1 receptor action was not determined. The present
results indicate that the
1 receptor-mediated responses are due, at
least in part, to direct
1 receptor-mediated modulation of mitral cells.
A previous study (Trombley and Shepherd 1992) of
dissociated cultured mitral cells did not detect any NE-evoked changes
in mitral cell holding currents over the range of voltages similar to
those examined in the present study. That study was performed on
immature MOB neurons harvested from 1- to 2-day-old rat pups. At birth,
however, the MOB exhibits very little
1 receptor binding; the levels
of
1 receptors subsequently increase during the second postnatal
week and remain stable thereafter (Jones et al. 1985a
). This postnatal developmental expression may explain, in part, why
NE-evoked inward currents comparable to those observed in the present
study were not detected in immature mitral cells.
Activation of 1 receptors depolarizes mitral cells and increases
responses to ON input
In current-clamp recordings, rat mitral cells in vitro exhibit
membrane potential bistability (Ennis et al. 1997;
Heyward et al. 2001
), generating two levels of membrane
potential separated by about 10 mV: a "down-state," subthreshold
for spike generation, and an "up-state," perithreshold for spike
generation. Mitral cell bistability is due to intrinsic membrane
properties and persists in the presence of blockers of ionotropic
glutamate receptors and GABAA receptors.
Application of PE consistently elicited a relatively small membrane
depolarization. This depolarization increased the amount of time spent
by the mitral cells at the relatively depolarized, up-state potentials.
This depolarization was not associated with a change in spontaneous
firing rate of mitral cells, consistent with the results of
bath-applied PE in previous studies (Ciombor et al.
1999
).
The sensitivity of mitral cells to ON input differs dramatically in the
up- and the down-states, such that mitral cells are more responsive to
ON stimuli, and ON-evoked spikes occur at shorter onset latencies, in
the up- versus the down-state (Heyward et al. 2001).
Based on these findings, the depolarization evoked by PE, and the
resulting increased time spent by mitral cells in the up- versus the
down-state, should increase the excitability of mitral cells in
response to ON input. In agreement with this, PE significantly
increased the probability of ON-evoked spiking and decreased the
overall latency of evoked spikes. Additionally, increased membrane
resistance caused by PE may improve the ability of weak or subthreshold
ON-evoked synaptic responses to initiate action potentials in mitral
cells. As these experiments could not be performed in the presence of
synaptic blockers, we cannot exclude that potential PE actions on other
cells in the MOB network may have contributed to the mitral cell
depolarization and increased responsivity to ON input. However, the
present voltage-clamp experiments demonstrate that PE directly
modulates mitral cells in a manner consistent with a depolarizing
action. This suggests that the increased responsivity to ON input is at
least partially due to direct effects of PE on mitral cells.
These results are in agreement with the effects of NE and PE on mitral
cell responses to ON stimulation reported in previous extracellular
recording studies. Activation of the pontine nucleus locus coeruleus
(LC), the sole source of noradrenergic projections to MOB
(Shipley et al. 1985), selectively and dramatically
enhanced responses of mitral cells to weak or perithreshold ON
stimulation in vivo (Jiang et al. 1996
). Application of
NE or PE similarly increased short-latency spikes in mitral cell
produced by perithreshold ON shocks in vitro (Ciombor et al.
1999
); these effects were prevented by
1 receptor
antagonists. The later study demonstrated that the NE- and PE-evoked
increase in ON-evoked responses was due to a reduction in response
failures to perithreshold ON shocks, a finding confirmed in the present experiments.
Functional implications
NE inputs to the bulb play important roles in olfactory function.
LC-NE projections to the main and accessory olfactory bulb are pivotal
to the formation of and/or recall of specific olfactory memories,
pheromonal regulation of pregnancy and postpartum maternal behavior
(Brennan et al. 1990; Dluzen and Ramirez
1989
; Kaba et al. 1989
; Rosser and
Keverne 1985
; Sullivan et al. 1989
,
1992
; Wilson and Leon 1988
). The present
results suggest that direct
1 receptor-mediated actions of NE
interact with intrinsic membrane properties (bistability) to increase
the excitability of mitral cells in response to relatively weak levels
of olfactory nerve input. While other actions of NE in the MOB network
are possible, the present results taken together with previous studies
(Ciombor et al. 1999
; Jiang et al. 1996
)
suggest that endogenously released NE may increase the sensitivity of
mitral cells to aid in the detection or discrimination of weak odors.
Overall, the behavioral and electrophysiological findings indicate that
NE plays a critical role in modulating olfactory function, including
formation and/or recall of specific olfactory memories.
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ACKNOWLEDGMENTS |
---|
We thank Dr. Asaf Keller for critical review of the manuscript.
This work was supported by National Institutes of Health Grants DC-03195, DC-02588, DC-00347, and NS-36940.
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FOOTNOTES |
---|
Address for reprint requests: A. Hayar, Dept. of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD 21201 (E-mail: ahaya001{at}umaryland.edu).
Received 12 February 2001; accepted in final form 2 July 2001.
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REFERENCES |
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