Adrenergic Receptor-Mediated Disinhibition of Mitral
Cells Triggers Long-Term Enhancement of Synchronized
Oscillations in the Olfactory Bulb
Sruthi Pandipati, David H. Gire and Nathan E. Schoppa
J Neurophysiol 104:665-674, 2010. First published 10 June 2010;
doi: 10.1152/jn.00328.2010
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J Neurophysiol 104: 665– 674, 2010.
First published June 10, 2010; doi:10.1152/jn.00328.2010.
Adrenergic Receptor-Mediated Disinhibition of Mitral Cells Triggers
Long-Term Enhancement of Synchronized Oscillations in the Olfactory Bulb
Sruthi Pandipati,1,2 David H. Gire,1 and Nathan E. Schoppa3
1
Neuroscience Program, 2Medical Scientist Training Program, and 3Department of Physiology and Biophysics, University of Colorado,
Anschutz Medical Campus, Aurora, Colorado
Submitted 9 April 2010; accepted in final form 3 June 2010
INTRODUCTION
Norepinephrine (NE) has been found to be critical for
various forms of olfactory learning in rodents, ranging from
early learning preference in neonates (McLean and Harley
2004; Sullivan et al. 1989, 2000) to enhanced two-odor discrimination in adults (Doucette et al. 2007). Many of the
actions of NE appear to be specific to the first processing
olfactory structure, the olfactory bulb, which receives ⬃40% of
the output from the noradrenergic nucleus, the locus coeruleus
(McLean et al. 1989). Also, pharmacological blockade of
adrenergic receptors (ARs) within the bulb can curtail learning
(Doucette et al. 2007; Sullivan et al. 1989, 2000). For early
learning preference, which is perhaps the best-characterized
form of olfactory learning, a basic model has emerged (Wilson
and Sullivan 1994) in which learned preference for a specific
odor results from the combined activation of the locus coerAddress for reprint requests and other correspondence: N. E. Schoppa,
University of Colorado, Anschutz Medical Campus, Mail Stop 8307, PO Box
6511, Aurora, CO 80045 (E-mail: [email protected]).
www.jn.org
uleus (LC), together with odor-evoked stimulation of a specific
population of bulb neurons. By this mechanism, behavioral
changes that are specific to an odor occur when LC activation
results in the release of NE throughout the bulb, which is then
permissive for changes that are localized to neurons activated
by that odor.
Despite evidence indicating that NE is essential for olfactory
learning, the cellular mechanisms within the bulb that could
underlie behavioral effects are not well understood. One strong
candidate for the cellular action of NE is disinhibition of output
mitral cells (MCs) due to a reduction in GABAergic signals
from granule cells (GCs) at dendrodendritic synapses. Disinhibition due to acute NE exposure has been observed in
recordings of inhibitory currents in turtle olfactory bulb slices
(Jahr and Nicoll 1982). There is also evidence in rats for
AR-mediated disinhibition based on in vivo extracellular measurements of paired-pulse inhibition (Okutani et al. 1998;
Wilson and Leon 1988). NE-induced disinhibition is a plausible mechanism for olfactory learning because the enhanced
depolarization caused by disinhibition could lead to short-term
increases in intracellular calcium of the type that can lead to
subsequent long-term synaptic modifications that underlie
learning. Also, disinhibition would have the required permissive role in inducing odor-specific cellular changes because the
resulting enhanced depolarization would only occur in neurons
activated by a specific odor. There is, however, some question
as to whether disinhibition is the dominant action of NE in the
rodent bulb (Nai et al. 2009). NE at some concentrations
appears to increase, not decrease, GC-to-MC GABAergic
transmission during direct recordings of inhibitory currents in
MCs. In addition, NE can have acute actions on bulb circuit
properties other than on inhibition (Hayar et al. 2001; Yuan et
al. 2000), which could also underlie its behavioral effects.
Our study tested the hypothesis that NE can lead to specific
long-term changes in the cellular properties of the olfactory
bulb through acute disinhibitory effects, using electrophysiological recordings in rat olfactory bulb slices. We first tested
whether NE can cause disinhibition at MC-GC synapses and
the subtype(s) of ARs that mediate this effect. Subsequently,
we examined whether NE-induced disinhibition underlies a
previously reported long-term increase in synchronized gamma
frequency (30 –70 Hz) oscillations in extracellular local field
potential (LFP) signals in the bulb (Gire and Schoppa 2008).
This effect was shown to result from a conditioning stimulus
that combined brief exposure to NE and local electrical stimulation of olfactory nerve (ON) afferents. Because synchronized oscillations may be important for integrating olfactory
information (Mori et al. 1999; Stopfer et al. 1997), our results
0022-3077/10 Copyright © 2010 The American Physiological Society
665
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Pandipathi S, Gire DH, Schoppa NE. Adrenergic receptor-mediated disinhibition of mitral cells triggers long-term enhancement of synchronized
oscillations in the olfactory bulb. J Neurophysiol 104: 665–674, 2010. First
published June 10, 2010; doi:10.1152/jn.00328.2010. Norepinephrine (NE)
is widely implicated in various forms of associative olfactory learning
in rodents, including early learning preference in neonates. Here we
used patch-clamp recordings in rat olfactory bulb slices to assess
cellular actions of NE, examining both acute, short-term effects of NE
as well as the relationship between these acute effects and long-term
cellular changes that could underlie learning. Our focus for long-term
effects was on synchronized gamma frequency (30 –70 Hz) oscillations, shown in prior studies to be enhanced for up to an hour after
brief exposure of a bulb slice to NE and neuronal stimulation. In terms
of acute effects, we found that a dominant action of NE was to reduce
inhibitory GABAergic transmission from granule cells (GCs) to output mitral cells (MCs). This disinhibition was also induced by
clonidine, an agonist specific for ␣2 adrenergic receptors (ARs). Acute
NE-induced disinhibition of MCs appeared to be linked to long-term
enhancement of gamma oscillations, based, first, on the fact that
clonidine, but not agonists specific for other AR subtypes, mimicked
NE’s long-term actions. In addition, the ␣2 AR-specific antagonist
yohimbine blocked the long-term enhancement of the oscillations due
to NE. Last, brief exposure of the slice to the GABAA receptor
antagonist gabazine, to block inhibitory synapses directly, also induced the long-term changes. Acute disinhibition is a plausible permissive effect of NE leading to olfactory learning, because, when
combined with exposure to a specific odor, it should lead to neuronspecific increases in intracellular calcium of the type generally associated with long-term synaptic modifications.
666
S. PANDIPATI, D. H. GIRE, AND N. E. SCHOPPA
examining the underlying causes of enhanced synchrony in the
bulb could have direct implications for understanding olfactory
learning mechanisms.
METHODS
Electrophysiology
RESULTS
The base extracellular solution for all recordings contained (in mM)
125 NaCl, 25 NaHCO3, 1.25 NaH2PO4, 25 glucose, 3 KCl, 2 CaCl2,
and 1 MgCl2 (pH 7.3), and was oxygenated (95% O2-5% CO2). In
experiments studying the long-term effects of NE on gamma oscillations, the bath solution included 100 ␮M sulpiride, a dopamine D2
receptor antagonist, to block possible inhibitory effects of NE on
presynaptic ON terminals through D2 receptors (Ennis et al. 2001;
Hayar et al. 2001). For whole cell patch-clamp recordings of MC
inhibitory postsynaptic currents (IPSCs), the intracellular pipette solution contained 125 KCl, 2 MgCl2, 0.025 CaCl2, 1 EGTA, 2 NaATP,
0.5 NaGTP, and 10 HEPES (pH ⫽ 7.3 with KOH). For recordings of
glutamatergic excitatory PSCs (EPSCs) in GCs, the KCl was replaced
with equimolar K-gluconate. LFPs were recorded using glass micropipettes filled with extracellular solution (resistance: 1–5 M⍀) placed in
the EPL within 50 ␮m of the mitral cell layer. All whole cell and
extracellular recordings were made with a multi-clamp 700B patchclamp amplifier (Molecular Devices, Sunnyvale, CA) and were lowpass filtered at 0.25–5 kHz using an eight-pole Bessel filter and
digitized at 0.5–10 kHz. Data were acquired using Axograph software
(Molecular Devices) on a Macintosh G4. For electrical stimulation in
the granule cell or external plexiform layers, a glass patch pipette
(0.5–2 M⍀) containing the extracellular solution was placed in these
layers. Brief pulses (100 ␮s; intensities as indicated) triggered by a
biphasic stimulus isolation unit (BSI-950; Dagan, Minneapolis, MN)
were applied that were separated by 15–20 s. For ON stimulation at
theta frequency, stimuli (100 ␮s; 10 –500 ␮A) were applied as a
pattern consisting of five short bursts (3 pulses at 100 Hz) separated
by 250 ms (4 Hz). Each stimulus pattern was applied every 30 s. In the
whole cell voltage-clamp recordings, the cell holding potential was
⫺70 mV. The membrane voltage Vm and series resistance Rs were
constantly monitored. Data acquisition was terminated when Vm was
more positive than – 40 mV or if Rs was ⬎20 M⍀. In all experiments,
test drugs were bath-applied through a constant flow perfusion system.
Data analysis
Analysis was done using Axograph, as well as custom-written
software in Matlab (release 7.1, The Mathworks). Statistical significance was determined via Student’s t-test. Data values are reported as
means ⫾ SE.
For the analysis of acute effects of NE and AR-specific agonists on
MC IPSCs and GC EPSCs, in most cases test measurements were
compared with control measurements that were taken both before
drug application and also after drug washout. This was done to help
alleviate possible problems with run-down in the whole cell current
measurements. Measurements of the current variance in Fig. 1D in
responses to theta frequency ON stimulation were done in 200 ms
sections following each three-pulse stimulus burst.
J Neurophysiol • VOL
NE causes an acute reduction in GABAergic transmission
from GCs to MCs
To test whether NE causes disinhibition at dendrodendritic
synapses in rat bulb slices, we first evaluated NE’s effects on
IPSCs in MCs that were evoked by local electrical stimulation
(100 ␮s; single shocks of 50 –200 ␮A) in the granule cell layer,
GCL, with a patch pipette (Fig. 1, A and B). Most MC/GC
synapses in the bulb are reciprocal synapses in which the site
of GABAergic transmission from GCs onto the MC is directly
adjacent to the site of glutamatergic transmission from MCs
onto GCs. To isolate GABAergic transmission, experiments
were done in the presence of glutamate receptor antagonists to
block excitatory transmission, either kynurenate (3 mM) or the
combination of the AMPA receptor antagonist 2,3-dihydroxy-6nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX, 20 ␮M)
and the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (D-AP5, 50 ␮M). Under these conditions, GCL
stimulation resulted in fast IPSC events that occurred with a
short onset-time (time to 50% maximum current ⫽ 2.3 ⫾ 0.3
ms, n ⫽ 6 cells) that were blocked by the GABAA receptor
antagonist gabazine (2–5 ␮M; 95 ⫾ 1% amplitude decrease,
n ⫽ 6; Fig. 1A). The peak amplitude of the IPSC evoked by
GCL stimulation was not significantly affected (P ⫽ 0.11) by
whether the MC had an intact or truncated apical dendrite (cut
fortuitously during the slice preparation; Supplementary
Fig. S1;1 60 ⫾ 10 pA for intact, n ⫽ 5; 114 ⫾ 21 pA for
truncated, n ⫽ 3), as expected if the IPSC originated from GCs
rather than GABAergic juxtaglomerular cells. As can be seen
by the raw current traces and the plot of the response time
course (Fig. 1, A and B), bath application of NE (20 ␮M)
caused a reversible reduction in the IPSCs (44 ⫾ 3% decrease,
n ⫽ 8, P ⬍ 0.00005; see Fig. 3E). These results suggest that a
dominant effect of NE at 20 ␮M is a reduction in GABAergic
transmission at GC-to-MC synapses.
As a second measure of NE effects on inhibitory transmission from MCs to GCs, we turned to recordings of inhibitory
synaptic activity evoked by patterned stimulation of ON afferent fibers at the theta frequency (done in the absence of
glutamate receptor blockers), which is a stimulus pattern designed to mimic the breathing cycle (see METHODS for details of
stimulus protocol). Under these conditions, inhibition appeared
1
The online version of this article contains supplemental data.
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The experiments described here were performed in 300 –330 ␮m thick
horizontal olfactory bulb slices that were prepared as previously described (Schoppa et al. 1998). Except where noted, slices were taken from
rats at postnatal day P9-13. Slices were viewed under differential interference contrast optics at 40x magnification (Axioskop; Carl Zeiss,
Thornwood, NY). All experiments were approved by the Institutional
Animal Care and Use Committee at the University of Colorado, Anschutz Medical Campus. All experiments were done at 31–34° C.
Analysis of spontaneous mIPSCs (Figs. 2 and 3, G and H) was done
using a standard event detection algorithm in the Axograph software. In
some of the mIPSC recordings, there was a modest run-down in the
frequency of mIPSCs just after the start of the recording. In those cases,
a stable control baseline in frequency was obtained prior to drug application.
For the analysis of the LFP oscillations evoked by theta frequency
stimulation, traces underwent a supplementary filtering off-line (8-pole
band-pass Butterworth filter at 10 –100 Hz) prior to further analysis. To
detect oscillatory activity, a fast Fourier transform (FFT) was performed
on filtered LFP recordings; data were analyzed in 200-ms sections following the end of each electrical stimulus burst. The resulting power
spectra were integrated from 30 to 70 Hz, and this value was used as a
measure of the magnitude of gamma frequency oscillatory activity. For
the LFP measurements in response to N-methyl-D-aspartate shown in Fig.
5A, data traces were filtered at 0.5–100 Hz.
DISINHIBITION TRIGGERS ENHANCED SYNCHRONY
667
C
A
Stim
Record
Record
Stim
250 ms
GCL Stim
GBZ
NE
NE
Wash
Wash
Control
B
ON Stim
20 ms
NE
GBZ
D
0
1.0
NE
-100
0.5
-200
-300
0
0
10
20
0
20
10
Time (min)
Time (min)
as a barrage of rapid IPSC events that persisted for the duration
of the stimulus pattern, reflecting on-going bulb network activity. As was seen for the IPSC evoked by GCL stimulation,
NE caused a marked reduction in the barrages of IPSCs evoked
by ON stimulation, as measured by the whole cell current
variance (Fig. 1, C and D; 50 ⫾ 12% decrease in variance,
n ⫽ 9, P ⫽ 0.002). These results indicate that NE can cause
disinhibition of MCs under stimulus conditions that resemble
the physiological situation. In addition, they further confirmed
that NE can have specific effects on inhibition onto MCs from
GCs rather than juxtaglomerular cells because it has been
determined that the large majority of IPSCs evoked by patterned ON stimulation reflect GC inputs (Schoppa 2006a).
NE has been reported to have differing effects on GC-to-MC
GABAergic transmission, depending on the NE concentration
applied (Nai et al. 2009). In our study, we limited our analysis
to NE effects at 20 ␮M because that concentration was previously shown to be effective at inducing long-term physiological changes in the bulb (Gire and Schoppa 2008). The events
underlying this long-term change are the main interest of the
present study.
Mechanisms of NE-induced acute disinhibition
In addition to recordings of IPSCs evoked by electrical
stimulation, we also assayed NE’s effect on spontaneous mIPSCs
recorded in the presence of the sodium channel blocker tetroJ Neurophysiol • VOL
dotoxin (TTX; 1 ␮M) and glutamate receptor antagonists
(NBQX plus D-APV). Recordings of mIPSCs are often done to
obtain specific information about the mechanisms of pre- and
postsynaptic actions of neuromodulators. In our recordings
(Fig. 2, A and B; from our standard animal age group), NE (20
␮M) had little effect on mIPSC amplitude (15 ⫾ 5% decrease
in amplitude of the average detected event, n ⫽ 4, P ⫽ 0.07)
or frequency (14 ⫾ 15% increase, n ⫽ 4, P ⫽ 0.40). The
absence of an effect of NE on the mIPSC amplitude suggests
that the NE-induced reduction in IPSCs due to electrical GCL
stimulation (Fig. 1, A and B) was independent of effects on
postsynaptic GABAA receptors on MCs. Meanwhile, the noneffect of NE on mIPSC frequency implies that NE does not
have a direct action on presynaptic synaptic vesicle release
machinery on GC dendrites. Interestingly, the noneffect of NE
on mIPSCs that we observed differs from the recent study by
Nai and co-workers (2009), which found that NE increased the
mIPSC frequency at a 20 ␮M concentration. This difference
appears to reflect at least in part the ages of the rats used in the
studies. Our standard animal age (P9-13) was younger than that
used by Nai and co-workers (P14-31). When we conducted
experiments in slices from rats at P18-19, NE (20 ␮M) significantly increased the mIPSC frequency (174 ⫾ 39% increase,
n ⫽ 4, P ⫽ 0.021; Fig. 2, C and D).
We also examined which AR subtype mediated the NEinduced reduction in GC-to-MC transmission, first, by testing
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FIG. 1. Norepinephrine (NE) acutely decreases GABAergic
transmission at granule (GC) to mitral cell (MC) synapses. A: an
example experiment in which GC-to-MC GABAergic transmission was isolated by recording inhibitory postsynaptic currents
(IPSCs) in a MC in response to electrical stimulation in the
granule cell layer (GCL). Glutamate receptor antagonists were
added to block MC-to-GC transmission. The 4 traces (averages
of 10 responses) show that NE caused a reversible decrease in
the amplitude of the gabazine (GBZ)-sensitive IPSC. A highchloride-containing patch-pipette solution was used, resulting
in inward-going IPSC events at the –70 mV holding potential.
Stimulus artifacts were deleted in the displayed traces. B: the
time plot for the experiment in A. C: a reversible reduction in
evoked MC IPSCs was also observed in response to patterned
olfactory nerve (ON) stimulation at the theta frequency (see
cartoon trace of stimulus pattern at bottom). D: time plot for the
experiment in C. IPSC activity was quantified from the current
variance.
Control
668
S. PANDIPATI, D. H. GIRE, AND N. E. SCHOPPA
A
C
MC mIPSCs in P13 Rat
MC mIPSCs in P19 Rat
Aver Detected
mIPSC
Control
Aver Detected
mIPSC
Control
NE
NE
5 ms
10 ms
2s
1s
5 ms
B
D
20
9
18
8
16
7
14
6
12
NE
10
5
BMI
NE
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8
3
6
2
4
1
2
0
0
0
10
20
Time (min)
30
40
0
5
10
15
20
25
30
Time (min)
the effect of different AR sub-type-specific agonists on
IPSCs evoked by GCL stimulation (Fig. 3; measured in the
presence of NBQX, 20 ␮M, and D-AP5, 50 ␮M). The ␣2
AR-specific agonist clonidine (5 ␮M) was found, like NE, to
be effective in reducing the IPSCs (40 ⫾ 7% decrease, n ⫽
8, P ⫽ 0.007; Fig. 3, A, B, and E), although no effect was
observed due to either the ␣1 AR-specific agonist phenylephrine (PE, 20 ␮M; 2 ⫾ 22% amplitude decrease, n ⫽ 5)
nor the ␤ AR-specific agonist isoproteronol (Isop, 100 ␮M;
4 ⫾ 6% amplitude decrease, n ⫽ 5; Fig. 3, C–E). The
average effect of clonidine on the IPSCs evoked by GCL
stimulation (40% decrease) was very similar to that due to
NE (average decrease of 44%; see preceding text), suggesting that ␣2 ARs mediate the large majority of the NEinduced reduction in evoked IPSCs. Consistent with this
interpretation, we also found that blockade of ␣2 ARs with
the ␣2 receptor-specific antagonist yohimbine (20 ␮M)
eliminated the decrease in evoked IPSCs due to NE (20 ␮M;
1 ⫾ 13% increase in IPSC amplitude due to NE plus
yohimbine, n ⫽ 5, P ⫽ 0.96; Fig. 3F). Finally, as with the
NE-induced reduction in inhibition in our standard youngeraged animals (P9-13), ␣2 AR activation with clonidine in
younger animals reduced the evoked IPSC without altering
spontaneous mIPSC events measured in TTX (Fig. 3, G and
H; 15 ⫾ 10% decrease in amplitude, n ⫽ 6, P ⫽ 0.20; 6 ⫾
9% increase in frequency, n ⫽ 6, P ⫽ 0.55). Unlike NE,
clonidine did not increase the mIPSC frequency in older
animals (at P23; 3 ⫾ 28% increase in frequency, n ⫽ 5, P ⫽
0.91; data not shown). The NE-induced increase in mIPSC
frequency observed in older animals (Fig. 2, C and D) was
likely due to activation of ␣1 or ␤ ARs as previously
described (Nai et al. 2009).
NE has no effect on MC-to-GC transmission at
dendrodendritic synapses
As discussed in the preceding text, MCs and GCs make
reciprocal connections. Thus NE could affect GABAergic
transmission from GCs onto MCs not only due to direct actions
J Neurophysiol • VOL
at GC-to-MC GABAergic synapses but also indirectly by affecting MC-to-GC excitatory glutamatergic transmission
(Trombley and Shepherd 1992). To examine NE actions on
MC-to-GC transmission at reciprocal synapses, we performed
voltage-clamp recordings from GCs while directly stimulating
the external plexiform layer (EPL; 100 ␮s, 100–500 ␮A; Fig. 4A).
This stimulation has been reported to elicit a class of excitatory
postsynaptic current (EPSC) events with moderate kinetics
(␶decay ⫽ 6 – 8 ms) (Balu et al. 2007; Schoppa 2006b) that
reflect glutamatergic transmission from MC lateral dendrites
onto the distal apical dendrites of GCs. Recordings of MCto-GC excitation were done in the presence of bicuculline
methiodide (BMI; 10 ␮M) to block GABAA receptors. NE (20
␮M) had no effect on the EPSCs evoked by stimulation in the
EPL (2 ⫾ 6% increase, n ⫽ 4; Fig. 4, A and B), indicating that
NE actions at reciprocal synapses are confined to GC-to-MC
transmission.
We also tested the effect of NE on EPSCs in GCs evoked
by electrical stimulation in the GCL. GCL stimulation
evokes a class of EPSCs in granule cells with faster decay
kinetics (␶decay ⫽ 1–2 ms) than EPSCs evoked by EPL
stimulation and that originate at a location relatively close to
the granule cell soma (Balu et al. 2007; Schoppa 2006b). We
found that NE (20 ␮M) reversibly inhibited these fast EPSC
events (42 ⫾ 6% decrease, n ⫽ 5, P ⬍ 0.05; Fig. 4, C and
D). We did not further explore NE effects at these synapses
in the present study. The net effect of an NE-induced
reduction in the very fast EPSCs in GCs would be disinhibition of MCs, similar to the NE-induced reduction in GCto-MC transmission characterized in the preceding text, but
this disinhibitory effect may not have been a major contributing factor in the previously-reported increase in synchronized gamma frequency oscillations caused by the combination of NE and ON stimulation (Gire and Schoppa 2008).
While some of the fast EPSCs could be derived from axon
collaterals of MCs (Schoppa 2006b), a significant fraction of
them appear to reflect centrifugal inputs from the piriform
cortex (Balu et al. 2007), which, in bulb slices, are likely not
activated by ON stimulation.
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10
FIG. 2. NE-effects on miniature IPSCs (mIPSCs) in MCs
from rats of different ages. A: an example recording of mIPSCs
in a bulb slice taken from a rat pup in our standard age group
(P9-13). Raw traces (left) and averages of detected mIPSCs
(right; averages of 36 events) are shown under control conditions and in the presence of NE. The amplitudes of the averaged
mIPSCs were not significantly altered by NE. Recordings were
done in the presence of glutamate receptor blockers (NBQX and
D-AP5) and TTX (1 ␮M). B: time plot of mIPSC frequency for
the experiment in A, showing that NE had no significant effect.
Note that the GABAA receptor blocker bicuculline methiodide
(BMI; 10 ␮M) blocked the mIPSCs. C and D: NE caused an
increase in the mIPSC frequency in a MC from an older P19 rat
pup. The effect can be clearly observed in the raw example data
traces (C, left) as well as in the time plot of frequency measurements in D. In this experiment, there was a ⬃2-fold increase in the size of the detected mIPSC after NE application
(C, right), though the effect likely reflected an inability to detect
small mIPSC events under the conditions of very high mIPSC
frequency in NE rather than an actual effect of NE on mIPSC
amplitude.
DISINHIBITION TRIGGERS ENHANCED SYNCHRONY
A
C
GCL Stim
669
F
Control
Isop
NE
+Yohimbine
Record
Control
10 ms
Stim
D
G
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0
-50
Wash
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20 ms
MC mIPSCs in P12 Rat
Control
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50 ms
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1 sec
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Clon
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PE
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Time (min)
FIG. 3. ␣2 adrenergic receptors (ARs) mediate disinhibition at GC-to-MC synapses. A: the ␣2 AR-specific agonist clonidine, like NE, caused a reversible
decrease in IPSCs in MCs evoked by GCL stimulation. Each trace from the displayed experiment reflects an average of 10 responses. B: time plot for the
experiment in A. C and D: isoproterenol (Isop), an agonist specific for ␤ ARs, had no effect on IPSCs evoked by GCL stimulation. Current records (C) and time
plot (D) are shown. E: summary of effects of various AR-specific agonists on MC IPSCs evoked by GCL stimulation. Significant decreases in IPSCs were caused
by NE and clonidine but not agonists specific for ␤ ARs (Isop) or ␣1 ARs (phenylephrine, PE). F: the NE-induced reduction in the IPSC evoked by GCL
stimulation was mainly eliminated when NE was co-applied with the ␣2 AR-specific antagonist yohimbine (20 ␮M), further supporting a role for ␣2 ARs. Each
trace reflects an average of 10 responses. G and H: recordings of mIPSCs in the presence of TTX, showing that clonidine had no effect on the frequency. Current
records (G) and time plot of mIPSC frequency (H) are shown.
Conditioning stimulus composed of NE and NMDA leads to
long-term changes in gamma oscillations
The main result of our studies thus far is that NE, through
activation of ␣2 ARs, can cause disinhibition of MCs,
specifically by reducing GABAergic transmission from GCs
onto MCs at reciprocal synapses. The objective of the rest of
this study was to test whether this acute disinhibition was
the key effect of the conditioning stimulus that combined
NE and ON stimulation, which resulted in long-term enhancement of synchronized gamma oscillations. As will be
described in the following text, the basic design of the
experiments to establish the link between NE-induced disinhibition and long-term effects was to test whether specific
drugs mimic NE’s effect in inducing long-term changes, but,
as a first step toward this analysis, we wanted to explore
altering the ON stimulation component of the conditioning
stimulus. The Gire and Schoppa (2008) study found that NE
plus ON stimulation led to significant long-term enhancement of the gamma oscillations (96 ⫾ 38% increase), yet the
J Neurophysiol • VOL
effects were somewhat variable, with large (greater than
twofold) increases in gamma oscillations being observed in
3 of 10 LFP recordings.
As an alternative to ON stimulation, we used bath application of a low-to-moderate concentration of NMDA (12.5 ␮M)
as part of the conditioning stimulus. It has previously been
shown that NMDA can elicit slow synchronized depolarizations in MCs that are qualitatively similar to those resulting
from electrical ON stimulation (Schoppa and Westbrook
2001). We reasoned that the NMDA-driven depolarizations
occur across broader regions of the bulb, and thus should
provide a stronger conditioning stimulus. In addition, it has
been shown that NMDA infusions into the olfactory bulb in
vivo can enhance odor discrimination in a manner similar to
prior odor exposure (Mandairon et al. 2006). Figure 5A shows
representative LFP records measured the EPL in response to
NMDA (band-pass filtered between 0.5 and 100 Hz), showing
the NMDA-evoked slow oscillatory LFP (1–2 Hz) that corresponds to the slow phasic depolarizations seen in published
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GCL Stim
GCL Stim
670
S. PANDIPATI, D. H. GIRE, AND N. E. SCHOPPA
A
Evidence that NE-induced disinhibition drives long-term
enhancement of gamma oscillations
C
Stim in EPL
Stim in GCL
Record
Record
Wash
NE
Control
10 ms
10 ms
B
D
0
0
-100
-50
-200
-100
-300
0
5
Time (min)
10
0
10
20
Time (min)
FIG. 4. NE effects on glutamatergic signaling onto GCs. A and B: NE
had no effect on excitatory postsynaptic currents (EPSCs) recorded in GCs
evoked by electrical stimulation in the external plexiform layer (EPL).
These EPSCs, with moderate ⬃10 ms decay kinetics, reflect dendrodendritic transmission from MC lateral dendrites (see arrow and “⫹” in the
cartoon in A). Recordings were done in the presence of the GABAA
receptor blocker BMI (10 ␮M). C and D: NE reversibly reduced EPSCs in
GCs evoked by stimulation in the granule cell layer. These EPSCs, with
rapid decay kinetics, appear to at least partly reflect glutamatergic centrifugal inputs from the piriform cortex (Balu et al. 2007). Each trace in A and
C reflects an average of 10 responses.
whole cell patch-clamp recordings from MCs (Schoppa and
Westbrook 2001). When a conditioning stimulus that combined NMDA (12.5 ␮M) with NE (20 ␮M) was tested on
gamma frequency oscillations (Fig. 5, B–E), there was a 196 ⫾
80% enhancement of the gamma oscillation power (integrated
power from 30 to 70 Hz) measured 20 –30 min after washout
of NE and NMDA (n ⫽ 8, P ⫽ 0.04). While the increase in
power was not specific to the gamma range (30 –70 Hz; see
power spectra in Fig. 5D), the peaks of the power spectra after
the NMDA-plus-NE conditioning stimulus were between 30
and 70 Hz in seven of eight experiments (the 1 outlier peaked
at 22 Hz), indicating that the enhancement effects were very
strong in the gamma range. NMDA, when applied alone, had
no long-term effect on the synchronized oscillations (41 ⫾
37% enhancement in integrated power from 30 to 70 Hz, n ⫽
5, P ⫽ 0.32; Fig. 5F) nor did NE alone (4 ⫾ 34% enhancement
in power, n ⫽ 4). NMDA as part of a conditioning stimulus
appeared to be somewhat more reliable than ON stimulation,
with greater than twofold increases in the LFP power being
observed in 5 of 8 recordings with NMDA application, as
compared with 3 of 10 for ON stimulation. We therefore
adopted NMDA application as part of the conditioning stimulus in the mechanistic experiments that followed.
J Neurophysiol • VOL
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NE
Control
Besides causing disinhibition at GC-to-MC synapses, acute
exposure of the bulb to NE can cause direct excitation of MCs
via ␣1 AR-mediated closure of potassium channels (Hayar et
al. 2001) as well as specific changes in glomeruli that are
mediated by ␤ ARs (enhanced excitatory transmission from
ON terminals and depression of juxtaglomerular cell inhibition) (Yuan 2009; Yuan et al. 2000). To address whether
disinhibition at GC-to-MC synapses accounts for the NEinduced long-term enhancement of the synchronized oscillations, we first examined whether the ␣2 AR-specific agonist
clonidine, which causes disinhibition, could mimic NE in causing the long-term effect. We indeed found this to be the case.
Clonidine (5 ␮M), when combined with NMDA as a conditioning stimulus, had somewhat variable effects on the oscillations, but across 23 experiments, the drug caused a significant
long-term increase in the oscillation power (Fig. 6, A–C; 105 ⫾
47% increase in integrated power between 30 and 70 Hz, P ⫽
0.030; measured 40 – 60 min after NE washout) with 8 of 23
showing a greater than twofold increase in LFP power. In
contrast, neither the ␣1 AR-specific agonist PE (20 ␮M; 4 ⫾
2% increase in LFP power, n ⫽ 4, P ⫽ 0.10) nor the ␤
AR-specific agonist Isop (100 ␮M; 0 ⫾ 17% change, n ⫽ 5,
P ⫽ 0.98), when applied in combination with NMDA,
altered the oscillations nor did the combination of PE, Isop,
and NMDA (25 ⫾ 18% decrease, n ⫽ 4, P ⫽ 0.25). The
average effect of a clonidine-plus-NMDA conditioning
stimulus on the oscillatory power in our experiments (105%
enhancement) was somewhat less than that of NE-plusNMDA (196% enhancement); this might suggest that ␣1 or
␤ ARs could facilitate ␣2 ARs in mediating the NE-induced
enhancement in the oscillations. However, the difference in
the clonidine and NE-effects was not statistically significant
(P ⫽ 0.33). In addition, we found that NE, when co-applied
with the ␣2 AR-specific antagonist yohimbine (20 ␮M) and
NMDA, failed to induce any long-term increase in the
synchronized oscillations (27 ⫾ 8% decrease in integrated
power between 30 –70 Hz, n ⫽ 7, P ⫽ 0.018), further
supporting a dominant role of ␣2 AR-mediated disinhibition
for enhancement of synchrony. The small decrease in the
oscillatory power following the conditioning stimulus composed of NE, NMDA, and yohimbine suggests that ARs
other than the ␣2 subtype may depress synchrony, though
this effect was quite weak.
A second prediction of a mechanism in which NE-induced
disinhibition mediates long-term enhancement of the
gamma oscillations is that direct pharmacological blockade
of inhibitory synapses should mimic the effect of NE. To
examine this possibility, we tested the effect of the GABAA
receptor antagonist gabazine, using concentrations (2–5
␮M) that were high enough to block the large majority of
the receptors (95% reduction in IPSC amplitude; see Fig. 1A
and preceding text), but sufficiently low that the drug could
be washed out completely after application of the conditioning stimulus. Washout of gabazine was important because
the drug would be expected to cause direct reduction of the
gamma oscillations, which depend on GABAA receptor activation for their inhibitory phase (Lledo et al. 2005). We
found that a conditioning stimulus that included gabazine
DISINHIBITION TRIGGERS ENHANCED SYNCHRONY
A
B
Test
Conditioning
Measurements
Stimulus
(ON Stim)
(No ON Stim)
Control
Measurements
(ON Stim)
10-20 min
Record
C
671
10 min
30-60 min
Control
100 ms
Base solution
NMDA
ON Stim
D
E
F
10
7
8
6
*
300
7
5
6
4
5
3
4
200
NE+NMDA
3
2
100
2
1
0
400
9
1
10 20 30 40 50 60 70 80 90 100
Frequency (Hz)
0
0
10
20
30
40
50
Time (min)
NE
+NMDA
No
Cond
Stimulus
NMDA
NE
FIG. 5. NE plus N-methyl-D-aspartate (NMDA) as a conditioning stimulus leads to a long-term increase in synchronized oscillations. A: LFPs recorded in the
EPL, showing slow oscillatory responses evoked by bath-applied NMDA (12.5 ␮M). Data were filtered at 0.5–100 Hz. B: protocol used to examine effects of
various conditioning stimuli on gamma oscillations in LFP signals in the EPL. Following a control period, a conditioning stimulus was applied for 10 min, which
was then followed by a test period initiated on washout of the conditioning drugs. In the control and test periods, test stimuli were applied that consisted of
patterned stimulation of the ON at the theta frequency. C: LFP responses to ON stimulation before and after application of a conditioning stimulus that included
NE (20 ␮M) and NMDA (12.5 ␮M). Note the enhancement in the oscillatory signal, seen also in the enlarged insets at right. Data were filtered at 10 –100 Hz.
D: power spectra (between 10 and 100 Hz) derived from the same experimental recordings as in C. Note that the NE-plus-NMDA conditioning stimulus increased
the power across the 10 –100 Hz range with a particularly strong effect around 40 –50 Hz. The spectrum for the postconditioning stimulus-condition reflects
recordings made 50 – 60 min following washout of NE-plus-NMDA. Only a limited frequency range in the power spectra, down to 10 Hz, is shown, because
LFP signals had to be analyzed in data segments constrained by the patterned ON stimulation protocol (see C and METHODS). No data point is shown at 60 Hz
to eliminate possible contamination from line-noise. E: time plot for the experiment in C, showing changes in the integrated power of gamma oscillations between
30 –70 Hz following removal of the conditioning stimulus. F: summary of the effects of different conditioning stimuli on gamma oscillations (NE-plus-NMDA,
NE alone, or NMDA alone). All test measurements for the summary were made 30 – 60 min following washout of the conditioning drugs. Also shown are the
results when no conditioning stimulus was applied. These data reflect the magnitude of the oscillations recorded 50 – 60 min after the start of the recording as
compared with the first 10 min.
(2–5 ␮M) along with NMDA (12.5 ␮M) was quite effective
in eliciting a long-term enhancement of gamma oscillations
(Fig. 7, A and B; 105 ⫾ 36% increase in integrated power
between 30 and 70 Hz measured 20 –30 min after gabazine
wash-out, n ⫽ 7, P ⫽ 0.028). This result, showing that
direct block of inhibition can enhance the oscillations, supports the hypothesis that NE works via disinhibition in
inducing long-term changes.
DISCUSSION
NE has been shown to be involved in olfactory learning
through actions in the main olfactory bulb. In this study, we
examined both acute effects of NE on dendrodendritic synapses in the bulb and also long-term circuit-level changes that
could underlie olfactory learning.
J Neurophysiol • VOL
Acute effects of NE at dendrodendritic synapses
In terms of acute actions, we found that NE caused a
reduction in GABA release from GCs onto MCs at dendrodendritic synapses through activation of ␣2 ARs. This conclusion
was based on the fact that both NE and the ␣2 AR-specific
agonist clonidine reduced GC-mediated IPSCs; moreover, the
␣2 AR-specific antagonist eliminated NE’s acute effect. This
effect, ␣2 AR-mediated reduction in GC-to-MC transmission,
fits with long-standing results showing NE-induced disinhibition of MCs (Jahr and Nicoll 1982; Wilson and Leon 1988),
although these prior studies did not determine whether the
reduced inhibition results from direct effects on GC-to-MC
transmission or is a secondary result of reduced MC-to-GC
excitatory transmission. We found that NE’s effects at reciprocal dendrodendritic synapses are specific to GC-to-MC trans-
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60 min after conditioning stimulus (NE+NMDA)
500 ms
672
A
S. PANDIPATI, D. H. GIRE, AND N. E. SCHOPPA
B
LFP recordings in EPL
PostClon + NMDA
16
200 ms
14
12
Control
10
8
6
Control
4
2
0
10 20 30 40 50 60 70 80 90 100
200 ms
60 min Post-Conditioning Stimulus (Clon+NMDA)
Frequency (Hz)
C
4
Clon + NMDA
2
1
0
ON Stim
D
0
10
20
30
40
50
60
70
80
Time (min)
300
E
*
4
200
NE + Yohimbine
+ NMDA
3
2
100
1
0
0
Clon
+NMDA
Isop
+NMDA
PE
+NMDA
Isop
+PE
+NMDA
0
10
20
30
40
50
60
Time (min)
mission (but see Trombley and Shepherd 1992). In our studies,
we focused on NE actions at a 20-␮M concentration; this was
done to fit with our prior results that showed long-lasting
cellular changes due to NE at that concentration (Gire and
Schoppa 2008; see following text). The actual concentration of
NE at the site of ARs under physiological conditions is not
known. Microdialysis studies have indicated that the bulk
concentration of NE in the bulb after stimulation of the locus
coeruleus is quite low, just below ⬃1 nM (El-Etri et al. 1999),
but the local concentration of NE at AR sites at dendrodendritic
synapses could of course be much higher.
The most likely mechanistic explanation for the ␣2 ARmediated reduction in GC-to-MC transmission is decreased
calcium channel activity in GCs. In cultured bulb GCs, it has
been shown that NE can reduce calcium channel activity
(Trombley 1992), while ␣2 ARs mediate reduced calcium
currents in many other neuron types (Boehm and Huck 1991;
Czesnik et al. 2001; Gollasch et al. 1991; Schofield 1990;
Timmons et al. 2004; Trombley 1992). In addition, we found in
our studies that neither the amplitude nor frequency of mIPSCs
was decreased by NE nor clonidine (Figs. 2 and 3), which
would argue against alternate mechanisms of reduced GC-MC
transmission, including postsynaptic actions of NE on GABA
receptors on MCs or presynaptic effects on vesicle release
machinery. Also clonidine did not affect other intrinsic membrane properties of GCs in a manner that could account for
decreased GABAergic transmission (2.0 ⫾ 0.5 mV depolarization in resting potential, n ⫽ 3, P ⫽ 0.042; 8 ⫾ 5% increase
in input resistance, n ⫽ 3, P ⫽ 0.25; 2.4 ⫾ 2.0 mV more
depolarized action potential threshold, n ⫽ 3, P ⫽ 0.29).
J Neurophysiol • VOL
There are a few differences in the results we obtained with
respect to acute AR-mediated effects on spontaneous mIPSCs
as compared with those reported in a recent study by Nai and
co-workers (2009). First, they found that the dominant effect of
the broad-spectrum agonist NE at a 20-␮M concentration was
enhanced mIPSC frequency, whereas we often found no effect
of NE on mIPSCs. This difference appeared to reflect an
animal-age issue, as an NE-induced enhancement of mIPSCs
was observed in a subset of our experiments done in slices
from older animals similar in age to those used by Nai and
co-workers. An additional difference was the action of the ␣2
AR-specific agonist clonidine on mIPSC frequency in MCs:
Nai and co-workers (2009) reported a decrease whereas we
found no effect. This discrepancy does not appear to be related
to animal-age as we observed noneffects in both young and
older rats and instead could reflect technical differences in
methods used to measure or analyze mIPSCs.
Link between NE-induced disinhibition and long-term effects
on bulbar function
In addition to showing that NE can cause acute disinhibition
at dendrodendritic synapses, this study also provided evidence
that linked the NE-induced disinhibition to long-term increases
in synchronized gamma frequency oscillations (Gire and
Schoppa 2008). The link was made based on the fact that
specific activation of the ␣2-AR subclass, which mediated
disinhibition, was able to mimic the effect of NE in causing
long-term enhancement of the oscillations. Also blocking ␣2ARs prevented NE from inducing the long-term enhancement.
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3
FIG. 6. The ␣2 AR-specific agonist clonidine induces a
long-term increase in gamma oscillations. A: consecutive traces
of LFP recordings of responses to ON stimulation made under
control conditions and ⬃60 min following washout of a conditioning stimulus comprised of clonidine (5 ␮M) and NMDA
(12.5 ␮M). Note the large increase in the oscillatory signal
following the conditioning stimulus, also shown in the boxed
insets at right. Data were filtered at 10 –100 Hz. B: power
spectra derived from the same experimental recordings as in A,
showing that the clonidine-plus-NMDA conditioning stimulus
increased the oscillatory power across the 10 –100 Hz range.
C: time plot of the experiment in A, showing a long-term
increase in the integrated power of gamma oscillations between
30 and 70 Hz due to a clonidine-plus-NMDA conditioning
stimulus. D: summary of the effects of various conditioning
stimuli on the integrated power of the LFP oscillations between
30 and 70 Hz. Results are shown for drug combinations that
included NMDA plus agonists specific for different AR subtypes
(Clon, clonidine; Isop, isoproteronol; PE, phenylephrine). E: results from another experiment showing that the NE plus NMDA
conditioning stimulus failed to enhance the LFP oscillations (integrated power between 30 and 70 Hz) when the drugs were coapplied with the ␣2 AR-specific antagonist yohimbine (20 ␮M).
DISINHIBITION TRIGGERS ENHANCED SYNCHRONY
A
LFP recordings in EPL
Control
30 min after conditioning stimulus (GBZ+NMDA)
100 ms
B
7
5
disinhibition-induced rises in intracellular calcium may have a
priming effect on LTP.
Among the issues to be resolved in future studies are the
specific steps in the biochemical path that lead to the longlasting enhancement of the synchronized oscillations, especially the role of calcium. The fact that a strong depolarization in the MC network appears to be required for the
long-lasting changes suggests that depolarization-induced
calcium rises may be important, happening either in the
MCs themselves or in GCs that are excited by MCs. The
source of the calcium could be voltage-gated calcium channels or NMDA receptors activated by depolarizationinduced removal of magnesium-blockade. Another mechanistic issue is which specific synapse(s) are undergoing the
long-term modifications, leading to changes in the gamma
oscillations. The relevant synapses could be the MC-GC
dendrodendritic synapses that underlie the gamma oscillations (Lledo et al. 2005) or, alternatively, mechanisms that
affect neuronal excitability that would secondarily impact activity
at MC-GC synapses (Gire and Schoppa 2008).
Relationship to olfactory learning
GBZ+NMDA
3
1
0
10
20
30
40
50
60
Time (min)
FIG. 7. Direct pharmacological block of GABAA receptors leads to longterm enhancement of gamma oscillations. A: consecutive traces of LFP
recordings of responses to ON stimulation, made under control conditions and
⬃30 min following washout of a conditioning stimulus comprised of gabazine
(GBZ; 5 ␮M) and NMDA (12.5 ␮M). Note the large increase in the oscillatory
signal following removal of the conditioning stimulus, also shown in the boxed
insets at right. Data were filtered at 10 –100 Hz. B: time plot of the experiment
in A, showing a long-term increase in the integrated power of gamma
oscillations between 30 and 70 Hz due to the GBZ-plus-NMDA conditioning
stimulus. Note also that, just following the conditioning stimulus, there was a
moderate decrease in oscillations that preceded the enhancement (66% decrease during minutes 30 –32 vs. control period). This decrease was presumably due to residual GBZ prior to washout. In this experiment, the first 50 ms
of data following each ON stimulus burst was ignored in the analysis to avoid
a lower-frequency component of the response (see traces in A).
In addition, pharmacological block of GABAA receptors to
reduce inhibition directly was also able to mimic NE’s effects.
Importantly, we found that NE-induced disinhibition by itself,
without concurrent neural stimulation (done here by application of
a low concentration of NMDA), was not sufficient for the longterm enhancement of the gamma oscillations (Fig. 5F). This
requirement for both disinhibition and neuronal stimulation as a
conditioning stimulus leads us to propose a mechanism for longterm changes that depends, most critically, on the generation of a
strong depolarization in the MC network. Under natural conditions, the impetus for the strong depolarization would be provided
by an odor, which would depolarize a select group of MCs to
some extent, but it is only when there is co-occurring disinhibition
(resulting from LC activation) that the depolarization would be
large enough to induce long-lasting changes. Such a scenario, in
which disinhibition acts as a facilitator of long-term cellular
changes, has been reported for long-term potentiation (LTP) at
hippocampal CA1 synapses (Hsu et al. 1999). In that system,
J Neurophysiol • VOL
From a functional perspective, an obvious comparison to be
made is between our cellular results and AR-dependent forms of
olfactory learning in which rodents learn to prefer, or discriminate
better, odors with which they associate a reward (Doucette et al.
2007; McLean and Harley 2004; Sullivan et al. 1989, 2000). Our
results could in principle provide a cellular basis for learning,
since an increase in gamma frequency synchronized oscillations
might increase the synaptic weights of specific odor signals in the
cortex (Beshel et al. 2007). The key cellular event that underlies
olfactory learning may also not be enhanced synchrony per se, but
another change in the bulb for which the gamma oscillations
provide a read-out. These could include enhancement in network
excitation or inhibition (Coopersmith and Leon 1984; Johnson
and Leon 1996; Okutani et al. 1998, 1999; Shea et al. 2008;
Wilson et al. 1987), either of which could be associated with
increased gamma oscillations.
Is there any evidence that could link the cellular changes
observed in our study to behavioral changes? One of our
findings, suggesting a link, is that NE and clonidine both
enhanced gamma oscillations only after the drugs had been
washed out (see time plots in Figs. 5E and 6C). Thus ␣2 ARs
appear to be involved in the induction but not maintenance of
long-term changes in gamma oscillations. This characteristic is
similar to associative olfactory learning, where NE appears to
be involved in the acquisition, but not expression, of learned
odor preference (Sullivan and Wilson 1991). In addition, there
are interesting cellular/behavioral comparisons to be made with
respect to animal age. At least certain types of associative
learning appear to be most pronounced during a “sensitive
period” in newborn rats (Moriceau and Sullivan 2004; Woo
and Leon 1987), which fits at least roughly with our data
suggesting that NE has disinhibitory acute effects of the type
required for long-lasting cellular changes mainly in younger
animals. In older animals, acute increases in inhibition dueanimals. In older animals, acute increases in inhibition due to NE
during a conditioning period (see Fig. 2D and (Nai and coworkers 2009)) may prevent learning. There are some mismatches
in our cellular effects as compared with reported behavioral ef-
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200 ms
ON Stim
673
674
S. PANDIPATI, D. H. GIRE, AND N. E. SCHOPPA
fects. For example, the age range of our younger rats (P9-13) was
slightly older than the newborn rats (⬍P10) in which associative
learning appears to be most pronounced. Also the evidence is
strongest for a function for ␤ ARs in associative olfactory learning
in newborn rats (Sullivan et al. 1989), whereas ␣2 ARs were most
important in our studies. Resolution to these issues will require
additional studies that more carefully examine the role of different
AR sub-types in rodents of varying age. Cellular studies could
reveal a stronger role for ␤ ARs at specific synapses in very young
animals (⬍P10) (Yuan et al. 2000), for example, while additional
behavioral studies that focus on ␣2 ARs could reveal novel roles
for these receptors in olfactory learning in young rodents.
We thank Dr. Diego Restrepo, University of Colorado, Anschutz Medical
Campus, for helpful discussions.
GRANTS
This work was supported by National Institute of Deafness and Other
Communication Disorders Grants F30 DC-010323 to S. Pandipati, F31 DC009118 to D. H. Gire, and RO1 DC-000566 to N. E. Schoppa.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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