# Auditory Stimulation Dishabituates Olfactory Responses via Noradrenergic Cortical Modulation

Authors: Jonathan J. Smith; Kiseko Shionoya; Regina M. Sullivan; Donald A. Wilson
Journal: Neural Plasticity
 (2009)

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## Abstract

Dishabituation is a return of a habituated response if context or contingency changes. In the mammalian olfactory system, metabotropic glutamate receptor mediated synaptic depression of cortical afferents underlies short-term habituation to odors. It was hypothesized that a known antagonistic interaction between these receptors and norepinephrineß-receptors provides a mechanism for dishabituation. The results demonstrate that a
108 dB siren induces a two-fold increase in norepinephrine content in the piriform cortex.
The same auditory stimulus induces dishabituation of odor-evoked heart rate orienting bradycardia
responses in awake rats.  Finally, blockade of piriform cortical norepinephrine ß-receptors with bilateral intracortical infusions of propranolol
(100 μM) disrupts auditory-induced dishabituation of odor-evoked bradycardia responses. These results provide a cortical mechanism for a return of habituated sensory responses following a
cross-modal alerting stimulus.


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## Body

## 1. Introduction

Dishabituation
is a rapid recovery from  habituation, often (though not necessarily) following
an intense stimulus potentially in a different modality from that of the
habituated stimulus [1].  Dishabituation allows for a return of
responsiveness to filtered stimuli if context or contingency changes and thus
can be highly adaptive in dynamic environments. Though habituation and
dishabituation are relatively “simple” forms of memory, disruptions in these
basic processes are associated with and/or could lead to higher-order
cognitive, emotional, or information processing impairments
[2–4].In
invertebrates, synaptic mechanisms of dishabituation include both a reversal of
habituation, and in some situations, an additional sensitization of the
response [5, 6].
Invertebrate dishabituation is dependent on modulatory inputs such as serotonin
to the sensorimotor circuit that reverse or counteract the synaptic changes
underlying habituation [5].  For example, habituation in Aplysia is mediated by a decrease in
presynaptic neurotransmitter release from the sensory neuron onto motor
neurons [7, 8].
Serotonin can reverse this depression and in nondepressed synapses induce
facilitation of neurotransmitter release
[9–11].In
the mammalian olfactory system, short-term habituation to odors is mediated by
a metabotropic glutamate receptor (mGluR)-dependent depression of cortical
afferent synapses. Both odor stimulation in
vivo [12] and patterned electrical
stimulation of cortical afferents in
vitro [13]
induce depression of the glutamatergic afferent synaptic input to piriform
cortex pyramidal cells. Blockade of presynaptic mGluR group III receptors
within the piriform cortex prevents this synaptic depression in vitro [13],
impairs cortical adaptation to odors [13],
and reduces behavioral odor habituation
[14–16].  Infusion of the mGluR group III agonist AP4
into the piriform cortex induces afferent synaptic depression
[14] and depresses odor-evoked reflexes (Wilson,
unpublished observations). Together, these results suggest a necessary and
sufficient role of piriform cortical mGluR group III receptors in short-term
odor habituation.Interestingly,
Cai et al. have demonstrated that mGluR group III receptor-mediated second
messenger cascades are modulated by
noradrenergic ß-receptors [17].
Activation of noradrenergic ß-receptors can disrupt the effects of mGluR group
III receptors.  In fact, the
noradrenergic ß-receptor agonist isoproterenol can block synaptic depression at in vitro piriform cortical afferents [13].  The piriform cortex, and olfactory system in
general, receives a strong noradrenergic input from the locus coeruleus [18].These
results lead to the hypothesis that events which activate the locus coeruleus
(e.g., novel, unexpected, or intense stimuli) could block piriform cortical
afferent synaptic depression, leading to a reinstatement of odor-evoked
responses dishabituation.  The present experiments test this hypothesis
by demonstrating that an auditory stimulus can enhance NE release in the
piriform cortex and induce dishabituation of odor-evoked reflexes. Finally, the
dishabituation can be blocked with infusions of a noradrenergic ß-receptor
antagonist into the piriform cortex.


## 2. Materials and Methods

Adult
male Long-Evans hooded rats were used as subjects. Animals were housed on a
12:12 light-dark cycle, with testing occurring during the light portion. Food
and water were available ad lib, and housing, care, and experimental protocols
conformed to NIH and University of Oklahoma IACUC standards.Electrocardiograms
were recorded with subcutaneous telemetry devices (Data Sciences, Intl.).  Animals were anesthetized with isoflurane and
the telemetry device was implanted under the dorsal skin near the shoulders,
with electrode leads passed subcutaneously to ventral muscles at anterior and
posterior locations providing strong electrocardiogram signals.  Wounds were closed with sutures and animals
allowed to recover approximately one week before testing.  For analysis and manipulation of piriform
cortical norepinephrine, in some animals guide cannulas (plastics one, 26
gauge) were also implanted into the anterior piriform cortex (unilateral for
microdialysis, bilateral for drug infusions; 1.0 mm anterior to Bregma).For
testing  of habituation/dishabituation, animals were placed in a darkened test
chamber (either a 16 cm × 22 cm × 15 cm Plexiglas box or a 20 cm diameter × 20 cm high glass beaker. No difference was noted between results in the two
chambers). The animals were allowed to acclimate for 10 minutes. Following
acclimation, odor pulses were delivered (4 pulses, 2-second duration, 60-second
interstimulus interval (ISI)) to determine mean baseline response magnitude
(average of the response to the 4 spaced stimuli).  Odorants were either isoamyl acetate or
eugenol and were delivered by passing clean air over an odorant saturated
(250–400μL) Kimwipe through Teflon tubing into the test chamber.  The odor pulses were then continued at 20-second
interstimulus intervals for 100
repetitions [19]
to induce habituation (uninfused rats) or 60 trials (infused rats). The reduced
number of habituation trials in the infused rats was sufficient to induce
habituation (see Figure 2) but was short enough to accommodate the time required
for the 20-minute drug infusions. The extent of habituation was then monitored by
4 additional odor pulses with a 60-second ISI, with the mean response magnitude to
the four stimuli serving as the habituated response magnitude for each animal.  Finally, experimental rats (noninfused, aCSF
and propranolol infused) then  received 4 final odor pulses (60-second ISI) that
were each  preceded by a 1–2-second auditory  stimulus (siren, 108 dB, 3 kHz,
RadioShack).   Control  animals received
the same final  set of odors, but no auditory stimulus. In two  animals,  one of
the  postacoustic responses was dropped due to large artifacts in the
recording, thus, their mean responses were based on three stimuli rather than
four. The mean response to these four stimuli was used as the measure of
postacoustic dishabituation in each animal. Odor-evoked heart rate orienting
response magnitude was calculated as the maximal change in instantaneous
beats/minute within 10 seconds of the odor stimulus.  Measures for each time point were the average
of the 4 evoked responses (baseline, posthabituation, postacoustic).During
pharmacological manipulations, 5 minutes prior to behavioral testing onset (25 minutes
prior to dishabituation induction), bilateral infusions were begun (0.15μL/min, 20 minutes). Five minutes after infusion onset, the 20-minute habituation
protocol was begun which allowed the full 3 μL infusion to be completed at
least 5 minutes prior to the dishabituation induction. Drugs were infused with a
syringe pump through an internal cannula (33 gauge) placed in  the guide
cannula. Animals were infused with either artificial cerebrospinal fluid (124 mM NaCl, 5 mM KCl, 1.24 mM KH2PO4, 2.4 mM CaCl2, 1.3 mM MgSO4,
26 mM NaHCO3 and 10 mM glucose) or 100 μM propranolol in artificial
cerebrospinal fluid.  Each animal was
tested with both perfusates in a counterbalanced order, with at least 4 days
between consecutive tests. Following data collection, rats implanted with
bilateral cannulas were transcardially perfused with saline and 10% formalin.
Brains were sectioned coronally at 40 μm and cannula tips localized in cresyl
violet stained sections.For
microdialysis/HPLC analysis of piriform cortex NE content, on the day of
testing a microdialysis probe (EICOM corp., Kyoto, Japan; A-I-8-02, 8 mm long,
2 mm membrane, 220μm diameter) was placed through the guide cannula with the
tip in the left anterior piriform cortex. Microdialysis probes were inserted
into the guide tube of the animal 60 minutes before data collection. During
microdialysis experiments, rats were placed in a 27 cm diameter acrylic
circular cage that was able to move freely with rotary bearings (EICOM corp.,
Kyoto, Japan), or in a 21 cm × 28 cm foot shock chamber. Microdialysis
probes were continually perfused with an artificial cerebrospinal fluid (aCSF)
containing 147 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl2, and  0.85 mM MgCl2 in deionized water at a flow rate of
2.0 μL/min using a microinjection pump
(EICOM, ESP-64). Dialysates were  collected and held at 4°C
automatically in 10 minutes intervals into plastic microvials preloaded with 2 μL of 12.5 mM perchloric acid/250 μM EDTA using refrigerated fraction collector
(EICOM, EFC-82). Samples (20 μL) were collected during each 10 minutes, with the
first six samples collected for baseline. After 60 minutes, rats were stimulated by
0.5 mA foot shock or acoustic startle that were presented at
1-minute interval for
10 minutes. Footshock was included as a condition to allow comparison with the less
noxious auditory stimulus used in the behavioral tests. Following 10 minutes of
stimulation, samples were collected for an additional 60 minutes. On completion of
the experiment, dialysate samples were immediately capped and stored at −80°C
until HPLC (EICOM, ECD-300) analysis.Dyalysate
norepinephrine (NE) was determined by HPLC with electrochemical detection.
Mobile phase (pH 6.0, 0.1 M sodium phosphate buffer with 5% HPLC grade
methanol, 400 mg/L sodium 1-octanesulfonato, 50 mg/L EDTA) were pumped through
a reversed-phase column (SC-5ODS, Eicom, 2.1 diameter × 150 mm) at a flow rate
of 0.23 mL/min using an ECD detector (450 mV). All substances were identified
and quantified by comparing retention times and peak areas with those of
external standards. At the beginning of each day, standard mixtures (4
different concentrations, 3 times each) were injected to check and adjust
calibration curves. During the course of autoinjection of dialysate fractions,
a standard mixture was injected as every fifth sample to monitor and correct
calibration curves.


## 3. Results

We first examined whether an intense
nonolfactory sensory experience could elevate norepinephrine levels within the
piriform cortex. To test this, we implanted guide cannulas into the anterior
piriform cortex to allow microdialysis-HPLC analysis of norepinephrine release
in awake rats exposed to an auditory stimulus or to 0.5 mA footshock. Samples
were taken every 10 minutes. As shown in Figure1, mean basal norepinephrine release
was stable over approximately 1 hour until the animal was stimulated with
either a moderate (0.5 mA) footshock (n=3) or the acoustic stimulus (n=3).  With either stimulus, norepinephrine
content was significantly elevated 2-fold and stayed elevated for 20 minutes
(repeated measures ANOVA, main effect of time: shock, F(13,26)  =  50.41, P<0.001; acoustic, F(13,26) = 202.42, P<0.001).  Post hoc Fisher tests revealed samples at 10
and 20 minutes poststimulation were significantly more different than all other points
(P<0.01).Figure 1
HPLC analysis of microdialysis samples taken from the anterior piriform cortex showed
a significant increase in NE content following either siren stimulation as used
here, or for comparison, footshock. This increase lasted at least 20 minutes.(a) Representative raw recordings of
odor-evoked heart rate orienting responses (instantaneous beats/min) in
response to odor stimulation before and after habituation, and after
presentation of a 108 dB siren. (b) Schematic of experimental design.
Odor test pulses (3-4 repeats) were presented with at least 60-second
interstimulus intervals to determine mean baseline response magnitude. Then
repeated stimuli (20-second ISI) were presented to induce habituation which was
tested with 4 spaced stimuli similar to that during baseline (60-second ISI).  To induce dishabituation, a 1–2-second siren
(upward arrows) was played 10–30 seconds prior to spaced odor stimuli.
(a)(b)Given that activation of
noradrenergic ß-receptors has been shown to block the synaptic depression that
mediates short-term odor habituation [13],
we hypothesized that the same auditory stimulus that elevates norepinephrine in
the piriform cortex may induce odor dishabituation. The experimental design is
described in the Methods and shown in Figure 2. Four groups of rats (noninfused:
experimental, n=4, control, n=5; infused: artificial cerebrospinal fluid
(aCSF), n=6, propranolol (100 μM), n=6) were tested for odor-evoked
bradycardia reflexes. There was no significant difference in initial response
magnitude between groups (experimental 22 ± 4.5 beats per min [BPM] decrease;
control 18.4±3.5 BPM, aCSF, mean = 18.4±3.5 BPM; propranolol, mean = 22.0±4.5 BPM; ANOVA, N.S.); thus responses are presented as percent of baseline. All
groups showed strong habituation to repeated stimulation  at
20-second
interstimulus intervals (Figure 3).  The
experimental animals (noninfused, aCSF, and propranolol infused) were then
exposed to a loud auditory stimulus, and odor stimulation resumed. Noninfused
control animals received no auditory stimulus.
Both the noninfused and the aCSF infused experimental animals showed a
significant increase to near baseline levels in odor-evoked bradycardia
response compared to immediately after habituation training. noninfused
control animals which had no auditory stimulation and acoustically stimulated
animals whose piriform cortex was bilaterally infused with the ß-receptor
antagonist propranolol showed no significant change from habituation levels
(Figure 3).  A 2 (time: habituated
response, postacoustic response) × 4 (group) repeated measures ANOVA on response magnitude across groups
showed a significant time X group interaction (F(3,17) = 6.06, P<0.01).
Post hoc Fisher tests revealed that the postacoustic response magnitude in the
noninfused experimental animals and the aCSF infused animals was significantly
different (P<0.05) from the habituated response magnitude. No significant
dishabituation was observed in the noninfused control or propranolol infused
rats.Figure 3
Auditory stimulation induces dishabituation of odor-evoked autonomic
reflexes.  All groups showed significant
habituation of the heart rate orienting reflex (HROR). Initial mean raw
response magnitude did not differ between groups.  Experimental animals were then presented with
the siren and odor responses re-tested.
Odor-evoked responses in noninfused and aCSF-infused experimental
animals showed a significant increase in postacoustic response magnitude
compared to habituation levels (asterisk).
Control animals, which received no siren stimulus, and
propranolol-infused rats that did receive the acoustic stimulus showed no
dishabituation. There was no significant change in basal heart rate across time
points in any group.Importantly,
there was no significant change in basal heart rate (immediately prior to odor
stimulation) between groups across the different time points (ANOVA, time X
condition interaction, N.S.). Thus, a sound-induced change in basal heart rate
cannot account for the increased bradycardia response.  Furthermore, there was no difference in
initial basal heart rate between experimental and control conditions.Histological
analysis confirmed bilateral cannula tip placements within the anterior
piriform cortex (Figure4).Figure 4
Reconstruction of experiment 3 cannula tip locations in infused rats (black
dots) on stereotaxic plates from
[20].  All tips were within the anterior piriform
cortex, ranging from 1.0 anterior to 1.60 mm posterior to Bregma.  Gray line represents layer II of the piriform
cortex.


## 4. Discussion

To our knowledge, this is the first
demonstration of dishabituation in mammalian olfaction.  The term dishabituation is often used
incorrectly to describe responses to test stimuli that differ from the
habituation stimulus (e.g., habituate to odor A, test response to odor B).  However, dishabituation
[1]
refers to a recovery of habituation to single stimulus following some other
events (e.g., habituate to odor A, stimulus B occurs, and response to odor A is no
longer habituated).  The present results
demonstrate that an auditory stimulus capable of elevating norepinephrine
levels in the anterior piriform cortex can produce dishabituation of response
to odor. Blockade of noradrenergic ß-receptors within the anterior piriform
cortex disrupts this dishabituation.
Given previous work demonstrating the necessary and sufficient role of
piriform cortical mGluR group III receptors in short-term odor habituation [14–16]
and the interaction between noradrenergic ß-receptors and mGluR group III
receptor function
[13, 17],
these results imply that dishabituation of odor-evoked reflexes may be mediated
by a reversal of synaptic mechanisms underlying habituation.Historically, there is a debate over
whether dishabituation reflects a reversal of habituation mechanisms, or rather
reflects an additional process (e.g., sensitization) superimposed upon
habituation.  Recent work suggests that
both processes may be involved inAplysia [5]. Similarly, although in vitro synaptic physiology [13]
and the present results are consistent with reversal of cortical synaptic
adaptation contributing to dishabituation of odor responses, it is probable
that some sensitization also occurs. For example, activation of the locus
coeruleus enhances olfactory bulb mitral cell responsiveness to olfactory nerve
input [21]
and increases respiratory entrainment of piriform cortical single-unit
activity [22], either of which could
enhance odor-evoked reflexive responses.
Therefore, although noradrenergic ß-receptor blockade within the
anterior piriform cortex prevented significant dishabituation here, other,
noncortical mechanisms may also contribute to the overall response
recovery.  These same mechanisms may also
contribute to heighten olfactory system responses to novel or important odors
given that the locus coeruleus responds most strongly to novel stimuli, showing
rapid adaptation with stimulus repetition
[23].Finally, habituation and sensory
gating are critical for normal perception, cognition, and emotion, and
disruptions are associated with disorders such as schizophrenia and autism
spectrum disorder
[3, 4].  Together with previous work, the present
findings suggest that at least in mammalian olfaction, cortical circuits play a
crucial role in these very basic memory phenomena. Understanding how
information flow through cortical circuits is modulated during habituation and
dishabituation will have important implications for understanding these
disorders.



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