Nicotine Receptor Subtype-Specific Effects on Auditory
Evoked Oscillations and Potentials
Robert E. Featherstone1, Jennifer M. Phillips2, Tony Thieu1, Richard S. Ehrlichman1, Tobias B. Halene1,
Steven C. Leiser3, Edward Christian3, Edwin Johnson3, Caryn Lerman1, Steven J. Siegel1*
1Department of Psychiatry, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 2Department of Psychology, Mount St. Mary’s University,
Emmitsburg, Maryland, United States of America, 3AstraZeneca, Wilmington, Delaware, United States of America
Background: Individuals with schizophrenia show increased smoking rates which may be due to a beneficial effect of
nicotine on cognition and information processing. Decreased amplitude of the P50 and N100 auditory event-related
potentials (ERPs) is observed in patients. Both measures show normalization following administration of nicotine. Recent
studies identified an association between deficits in auditory evoked gamma oscillations and impaired information
processing in schizophrenia, and there is evidence that nicotine normalizes gamma oscillations. Although the role of
nicotine receptor subtypes in augmentation of ERPs has received some attention, less is known about how these receptor
subtypes regulate the effect of nicotine on evoked gamma activity.
Methodology/Principal Findings: We examined the effects of nicotine, the a7 nicotine receptor antagonist
methyllycaconitine (MLA) the a4b4/a4b2 nicotine receptor antagonist dihydro-beta-erythroidine (DHbE), and the a4b2
agonist AZD3480 on P20 and N40 amplitude as well as baseline and event-related gamma oscillations in mice, using
electrodes in hippocampal CA3. Nicotine increased P20 amplitude, while DHbE blocked nicotine-induced enhancements in
P20 amplitude. Conversely, MLA did not alter P20 amplitude either when presented alone or with nicotine. Administration
of the a4b2 specific agonist AZD3480 did not alter any aspect of P20 response, suggesting that DHbE blocks the effects of
nicotine through a non-a4b2 receptor specific mechanism. Nicotine and AZD3480 reduced N40 amplitude, which was
blocked by both DHbE and MLA. Finally, nicotine significantly increased event-related gamma, as did AZD3480, while DHbE
but not MLA blocked the effect of nicotine on event-related gamma.
Conclusions/Significance: These results support findings showing that nicotine-induced augmentation of P20 amplitude
occurs via a DHbE sensitive mechanism, but suggests that this does not occur through activation of a4b2 receptors. Event-
related gamma is strongly influenced by activation of a4b2, but not a7, receptor subtypes, while disruption of N40
amplitude requires the activation of multiple receptor subtypes.
Citation: Featherstone RE, Phillips JM, Thieu T, Ehrlichman RS, Halene TB, et al. (2012) Nicotine Receptor Subtype-Specific Effects on Auditory Evoked Oscillations
and Potentials. PLoS ONE 7(7): e39775. doi:10.1371/journal.pone.0039775
Editor: Kenji Hashimoto, Chiba University Center for Forensic Mental Health, Japan
Received March 5, 2012; Accepted May 25, 2012; Published July 20, 2012
Copyright: ? 2012 Featherstone et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by P50-CA/DA-084718, (National Cancer Institute and National Institute of Drug Abuse, Caryn Lerman, PI) and R01DA023210
(National Institute of Drug Abuse, Steven Siegel, PI). No additional external funding received for this study. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: We have the following interests. Steven Leiser, Edward Christian and Edwin Johnson are former employees of AstraZeneca who provided
a pilot grant for this study. Steven Siegel has been a consultant and/or has received grant support from the following companies that develop and/or market
medications: NuPathe, AstraZeneca, and Merck, all unrelated to the current manuscript. Caryn Lerman has been a consultant and/or has received grant support
from the following companies that develop and/or market smoking cessation medications: AstraZeneca, Glaxo SmithKline, Novartis and Pfizer, all unrelated to the
current manuscript. AstraZeneca supplied AZD3480 for use in this study. There are no other patents, products in development or other marketed products to
declare. This does not alter our adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors.
* E-mail: firstname.lastname@example.org
Individuals with schizophrenia display changes in auditory
event-related potentials (ERPs) including decreased amplitude of
the P50 and N100 components and disrupted gating of the P50
[1,2,3,4], and these are assumed to reflect deficits in elementary
information processing. Nicotine has been shown to enhance P50
gating in people with schizophrenia and their first degree relatives
[4,5,6], suggesting that nicotinic agents could be useful for the
treatment of schizophrenia. Evidence from studies assessing
pharmacological response, changes in response to parametric
manipulations and response to novelty suggest that the mouse P20
and N40 are analogous to the human P50 and N100, respectively
[7,8,9,10,11,12,13,14,15]. In particular numerous studies have
demonstrated increases in mouse P20 amplitude following nicotine
administration, as well as nicotine-induced decreases in mouse
N40 amplitude [10,11,15]. Thus, rodent ERP methodologies have
great potential for translational drug discovery in schizophrenia.
In addition to changes in the ERP, nicotine has been shown to
enhance power in the gamma frequency range of the EEG
[15,16]. Gamma oscillations are thought to be generated in part
by parvalbumin expressing GABAergic interneurons, a cell
population that is disrupted in schizophrenia . As such,
gamma oscillations have been proposed as an important
PLoS ONE | www.plosone.org1July 2012 | Volume 7 | Issue 7 | e39775
biomarker of the integrity of this cell population . Numerous
studies have demonstrated reduced or altered gamma power in
schizophrenia and in physiologically relevant animal models of
schizophrenia [17,18,19]. Increases in gamma power have been
demonstrated during performance of cognitive tasks in control
subjects, especially during attention and working memory ,
suggesting that enhanced gamma activity may serve as a
mechanism through which nicotine influences schizophrenia
symptomology and cognition.
Recent studies have attempted to identify the specific nicotinic
acetylcholinergic receptor subtypes responsible for regulating the
effects of nicotine on ERPs, and more recently, evoked gamma
activity. At present the mechanism by which nicotine enhances
P20 amplitude is not entirely clear, although both a7 and a4b2
nicotinic receptors have been implicated [21,22,23,24,25,26,27].
Transgenic mice lacking the b2 subunit show a typical nicotine-
induced enhancement in P20 response to the first stimulus (S1) of a
paired stimulus presentation but fail to show the normal nicotine-
induced decrement in N40 S1 response , suggesting that the
role of the a4b2 receptor in sensory gating primarily involves the
b2 subunit and is limited to regulation of the N40, but not P20,
ERP component. While the a7 receptor has been shown to
influence P20 response, this appears to occur primarily through
decreased amplitude of response to the second stimulus (S2) of a
stimulus pair , with S1 being relatively unchanged. Much less
is presently known about how nicotine influences gamma EEG.
To resolve these questions, the present study examined the effects
of nicotine, the a7 nicotinic acetylcholine receptor antagonist
methyllycaconitine (MLA), the a4b4/a4b2 nicotinic acetylcholine
receptor antagonist dihydro-beta-erythroidine (DHBE) and the
a4b2 receptor agonist AZD3480 on amplitude and gating of the
P20 and N40 ERP components as well as baseline and evoked
gamma oscillations in mice.
Thirteen male C57BL/6J mice were used for experiment 1
(nicotine antagonist study) and 9 additional mice were used for
experiment 2 (AZD3480 study). All mice were obtained at 8 weeks
of age from Jackson Laboratories (Bar Harbor, ME), and were
assessed at 10 to 12 weeks of age. Mice were housed four to five
per cage until electrode implantation and single-housed thereafter
in a light- and temperature-controlled Association for Assessment
and Accreditation of Laboratory Animal Care-accredited animal
facility. Water and standard rodent chow were available ad libitum.
Experiments were conducted at the University of Pennsylvania
during the light phase between 9:00 AM and 3:00 PM. Mice were
allowed at least one week to acclimate to the housing facility before
starting any procedure. All protocols were performed in accor-
dance with University Laboratory Animal Resources guidelines
and were approved by the Institutional Animal Care and Use
Committee at the University of Pennsylvania (803887).
Nicotine hydrogen tartrate salt (1.0 mg/kg), methyllycaconitine
(MLA –10.0 mg/kg), and dihydro-beta-erythroidine (DHBE -
2.0 mg/kg) (Sigma-Aldrich, St. Louis, MO, USA) were dissolved
in 0.09% saline. Nicotine was administered intraperitoneally (I.P)
at a volume of 0.1 ml. MLA and DHBE were administered
subcutaneously, also at a volume of 0.1 ml. All drug doses are
expressed as drug base and were selected based on previous
research on nicotine’s effects on ERPs [10,11] and MLA and
DHBE’s abilityto block behavioral effectsof nicotine
[28,29,30,31]. AZD3480 (TC-1734) was obtained from AstraZe-
neca and was administered I.P. in a volume of 0.1 ml at a dose of 1
and 10 mg/kg. AZD3480 is a potent agonist of the nicotinic a4b2
receptor [32,33,34] that has shown efficacy for treatment of
cognitive impairments across a broad class of disease conditions.
Unipolar recording electrodes were unilaterally placed in the
CA3 hippocampal region under isoflurane anesthesia, (1.8 mm
posterior, 2.65 mm lateral, and 2.75 mm deep relative to bregma)
and referenced to the surface of the ipsilateral frontal hemisphere
(0.8 mm anterior, 2.65 mm lateral and 1 mm deep, relative to
bregma). Since the recording and reference electrodes are located
far apart from one another, it is likely that activity recorded using
this configuration extends far beyond the localized field generated
within the CA3 region, and, therefore, as in human EEG
recordings, reflects brain activity across a widespread area. For
this reason, histological verification of electrode placement was not
performed in the current study, although this has been reported
elsewhere using the same surgical procedures . ERPs recorded
using this configuration are characteristically similar to human
recordings from the Cz scalp location, as illustrated in a prior
publication by our group , and show numerous pharmaco-
logical (amphetamine, ketamine, nicotine, etc.) and parametric
responses (effects of ISI), similar to those observed in human EEG
studies from Cz [11,25,35,36,37,38]. Dental cement and ethyl
cyanoacrylate (Elmers, Columbus, OH) were used to secure the
electrode pedestal to the skull. Procedures were consistent with
descriptions published elsewhere [8,9,10,11,13].
For experiment 1, five days of ERP testing was conducted over a
two week period with a minimum of 24 hours washout period
between drug exposures and three recording sessions per test day.
The first recording session of each test day was an acclimation trial
followed by a baseline saline (vehicle) trial. The third testing
session consisted of the injection of the test compound(s). On the
first testing day, animals were acclimated to all handling and
testing procedures and received nicotine (1 mg/kg). On test day
two, animals received MLA (10 mg/kg), on test day three a
combination treatment of MLA (10 mg/kg) followed 5 minutes
later by nicotine (1 mg/kg). On the fourth day of testing, animals
received an injection of DHbE (2 mg/kg) and on the fifth and final
testing day animal received a combination treatment of DHbE
(2 mg/kg) followed 5 minutes later by nicotine (1 mg/kg). For
experiment 2 (AZD3480 study, ERPs were assessed over three
successive days, with each day being separated by a 72 hour
period. Mice were tested following saline vehicle, 1 mg/kg and
10 mg/kg on days 1, 4 and 7, respectively. Additionally, in order
to assess potential carry over effects of drug treatment, baseline
response was assessed prior to each test session.
In both experiments ERPs were recorded
following 50 stimulus presentations. Testing commenced five
minutes after the last injection. Stimuli were generated by
Micro1401 hardware and Spike2 software (Cambridge Electronic
Design, Cambridge, UK) and were delivered through speakers
attached to the cage top. Recordings were performed in 8 standard
mouse cages, all of which were located within a Faraday cage. 50
pairs of white noise clicks (85 db, 10 ms in duration) were
presented with a 9-s interstimulus interval against 70 dB of
background noise. Waveforms for the ERP and gamma analyses
were filtered between 1 and 500 Hz and baseline corrected at
stimulus onset. Individual sweeps were rejected for movement
artifact based on a criterion of 2 times the root mean squared
Nicotine effects on P20 and Gamma Oscillations
PLoS ONE | www.plosone.org2July 2012 | Volume 7 | Issue 7 | e39775
amplitude per mouse. Average waves were created from 50-ms
pre-stimulus to 200-ms post-stimulus. The P20 component was
selected for each ERP by determining the maximum positive
deflection between 10 and 30 ms (S1) and between 510 and
530 ms (S2). The N40 was selected by determining the maximum
negative deflection between 25 to 60 ms (S1) and between 525 and
560 ms (S2) post-stimulus. Since the latency of the maximum
deflection can differ greatly between animals, this method of
quantification will produce values for P20 and N40 that are
oftentimes higher than what appears in the grand average
waveform, which tends to smooth out maximal differences.
Sensory gating was analyzed by comparing the amplitude of S1
to the amplitude of S2, with successful sensory gating being
defined as a statistically significant difference between S1 relative
to S2, with S1 being greater than S2, and sensory gating failure
being defined as a lack of statistically significant difference between
S1 and S2. This was done separately for P20 and N40.
Event-related gamma (30 to 80 Hz) data were processed using
EEGLAB (Schwartz Center for Computational Neuroscience).
Epochs were extracted from between 2199 and 399 msec relative
to tone onset. Power was calculated using Morlet wavelets in 116
logarithmically spaced frequency bins between 4 and 120 Hz, with
wavelet cycle numbers ranging from 2 to 10 . Average post-
stimulus (0 to 60 milliseconds) gamma power was calculated for
each animal, and this served as the primary measure of gamma
power for statistical analysis. Gamma was defined as being
between the frequencies of 30 and 80 Hz.
Both the antagonist and AZD3480 experiments used a repeated
within-subjects design in which all animals were exposed to each
level of DRUG and each level of STIM (S1 and S2 components).
Separate analyses were conducted for the P20, N40 and for event-
related gamma The P20 and N40 data were analyzed using a 2
factor repeated measures ANOVA (DRUG by STIM), while
gamma data were analyzed using a single factor repeated measures
ANOVA (DRUG). In both experiments, repeated measures
analyses of variance (ANOVAs) were performed on baseline data
for each ERP component to rule out possible confounding effects
of time and repeated testing. In the case of a significant main effect
or interaction, Fisher’s LSD post-hoc tests were conducted. All
tests were two-tailed. Results are significant at p#0.05 unless
Event Related Potentials
Nicotine significantly increased amplitude of the P20 compo-
nent and reduced amplitude of the N40, consistent with previous
studies (Figure 1) [10,11,15,40].
pretreatment with DHbE, but not by MLA. Assessment of the effects of
nicotine antagonists was carried out using a within subjects design
in which each mouse was assessed on each drug, with sessions
being separated by a 24 hour washout period. N=13. A repeated
measures ANOVA on data from the pre-drug baseline trial of each
session for the antagonist study revealed no significant differences
over time [F(4,48)=2.42, p.0.05], indicating that there were no
significant lasting effects of any drug treatment. The mean
amplitude and standard error of the mean (in parentheses) for
each drug condition for S1 was; saline: 42.27 (6.84); nicotine:
1 (Nicotineantagonists DhbE and
Increases in P20 amplitude following nicotine are blocked by
67.98 (7.04); MLA: 30.65 (7.42); DHbE: 53.69 (7.01); MLA+ni-
cotine: 78.96 (14.55) and DHbE+nicotine: 28.83 (3.55). Amplitude
of the P20 component was significantly affected by drug treatment
[F(5,60)=6.33]. There was also a significant main effect for STIM
[F(1,12)=27.2], suggesting that significant sensory gating oc-
curred, and for the STIM6DRUG interaction [F(5,60)=11.38],
suggesting that drug effects on P20 amplitude differed for the first
and second stimuli. Post hoc testing on responses following S1
revealed that nicotine alone significantly increased P20 amplitude
while neither MLA nor DHbE alone had a significant effect on
P20 amplitude. The effect of Nicotine on P20 amplitude was
blocked by pre-treatment with DHbE but not by pre-treatment
with the a7 antagonist MLA. There were no significant treatment
effects on P20 amplitude following S2 (Figure 1 and 2). Comparison
of S1 and S2: The results of the statistical analyses were also used to
draw inferences about sensory gating. Since sensory gating
involves a reduction in S2 response relative to S1, a significant
difference between S1 and S2 is suggestive of intact sensory gating.
Post hoc comparisons from the analysis above revealed that
nicotine, MLA- and DHbE-treated animals displayed normal P20
gating relative to saline vehicle treated controls. However, P20
gating was disrupted in animals treated with DHbE plus nicotine,
(Figure 2), suggesting that this treatment combination disrupted
Amplitude of the P20 component was not
affected following treatment with the a4b2 agonist AZD3480. The mice
used to assess AZD3480 were a separate cohort from those used to
assess nicotine antagonists. N=9. Assessment of the effects of
AZD3480 was carried out using a within subjects design in which
each mouse was assessed on each drug, with sessions being
separated by a 72 hour washout period. A repeated measures
ANOVA on data from the pre-drug baseline trial of each session
no significant differences over time [F(2,16)=0.06, p.0.05],
indicating that there were no significant lasting effects of AZD3480
treatment across the duration of testing. Mean values for P20
amplitude were; Saline: 23.37 (3.7); 1 mg/kg AZD3480; 25.24
(3.8) and 10 mg/kg AZD3480:30.31 (9.2). No significant effects
were observed for either DRUG or for the STIM x DRUG
interaction following AZD3480 treatment, although there was a
significant main effect for STIM [F(1,8)=56.6]. Comparison of S1
and S2: Post-hoc tests showed increased S1 amplitude relative to S2
in all treatment conditions, suggesting that AZD3480 did not
affect P20 gating.
pretreatment with MLA or DhbE. A repeated measures ANOVA on
data from the pre-drug trial of each session for the N40
[F(4,48)=1.0, p.0.05], indicating that the baseline N40 at each
session was not affected by repeated testing or previous drug
exposures. Amplitude of the N40 component was significantly
affected by drug treatment [F(5,60)=3.20] and by STIM
[F(1,12)=7.2]. There was no significant STIM6DRUG interac-
tion, suggesting that drug effects on N40 amplitude did not differ
for the first and second stimuli. Post hoc testing for the main effect
of drug treatments revealed that nicotine significantly decreased
N40 amplitude. A significant reduction in N40 amplitude was
observed following MLA by itself. In contrast, DHbE alone did
not alter N40 amplitude and neither antagonist blocked nicotine-
induced decrement of N40 amplitude. The mean amplitude and
standard error of the mean (in parentheses) for each drug
condition for S1 was; saline: 95.87 (17.6); nicotine: 47.47 (18.9);
1 (Nicotine antagonistsDhbE and
Decreases in N40 amplitude following nicotine are not blocked by
Nicotine effects on P20 and Gamma Oscillations
PLoS ONE | www.plosone.org3July 2012 | Volume 7 | Issue 7 | e39775
MLA: 62.08 (11.9); DHbE: 95.46 (30.6); MLA+nicotine: 81.89
(31.2) and DHbE+nicotine: 83.84 (26.0). Comparison of S1 versus S2:
A significant effect was observed for STIM [F(1,12)=7.17], with
N40 amplitude being significantly greater following S1 relative to
S2. A lack of a significant STIM x DRUG interaction suggests that
the difference between S1 and S2 was consistent across treatment
conditions (Figure 3).
Treatment with the a4b2 antagonist
AZD3480 significantly reduced N40 amplitude. A repeated measures
ANOVA on data from the pre-drug trial of each session for the
N40 component revealed no significant differences over time
[F(2,16)=1.88, p.0.05], indicating that the baseline N40 at each
session was not affected by repeated AZD3480 treatment. Mean
values for N40 amplitude following drug treatment were; Saline:
53.02 (9.9); 1 mg/kg AZD3480; 57.9 (9.9) and 10 mg/kg
AZD3480:15.04 (10.9). A significant effect was seen for DRUG
[F(2,16)=8.02], STIM [F(1,8)=13.66], and the STIM x DRUG
interaction [F(2,16)=24.0]. Post hoc tests revealed significantly
reduced S1 amplitude following treatment with the high (10 mg/
kg) dose a4b2 agonist, relative to saline vehicle (Figure 3).
Comparison of S1 versus S2: The a4b2 agonist produced a significant
effect on gating of the N40 component, as revealed by a significant
effect of STIM [F(1,8)=13.66], as well as a significant interaction
between STIM x DRUG [F(2,16)=24.0]. Post hoc tests compar-
ing S1 and S2 response as a function of drug condition revealed
significant differences within the vehicle (p=0.0001) and low dose
(p=0.0001) groups. In contrast, no difference was observed
between S1 and S2 in the high dose group, suggesting disrupted
gating. Further, significant differences in N40 amplitude between
the high dose and vehicle groups was only seen for the S1
component (p=0.0001), demonstrating that reduced gating was
due solely to reduced S1 response (Figure 3).
Baseline and Evoked Gamma
Pretreatment with DHbE, but not MLA, blocked the ability for
nicotine to enhance evoked gamma oscillations. A repeated measures
ANOVA on pre-drug baseline data for evoked gamma revealed no
significant differences over time [F(4,48)=0.11, p,0.05], indicat-
ing that the evoked gamma activity was not affected by repeated
testing or previous drug exposure. Event-related gamma was
significantly affected by drug treatment [F(5,60)=6.31, p,0.05].
Post hoc tests showed enhanced gamma power following nicotine
(p=0.037) and following MLA + nicotine (p=0.0002). No change
was observed following DHbE treatment alone, while pretreat-
ment with DHbE blocked the effect of nicotine on gamma power.
MLA treatment by itself had no effect on gamma. Mean values for
evoked-gamma were: Saline: 1.31 (0.28); Nicotine; 1.82 (0.29);
MLA: 0.83 (0.33); DHbE: 1.7 (0.45); MLA+Nicotine: 2.39 (0.31)
and DHbE+Nicotine: 1.41 (0.57). This pattern of results suggests
that the effects of nicotine on event-related gamma are due to
activation of the a4b2 receptor.
Experiment 2 AZD3480.
The a4b2 agonist AZD3480 signifi-
cantly enhanced evoked-gamma oscillations. A significant effect of
AZD3480 was observed on event-related gamma [F(2,16)=3.7].
Post hoc tests showed a significant increase in gamma power
following low dose (p=0.016), but not high dose (Figure 4). Mean
values and standard error of the mean (in parentheses) for event-
related gamma were; Saline: 0.86 (0.21); 1 mg/kg: 1.8 (0.24) and
10 mg/kg: 1.43 (0.26).
1 (Nicotine antagonistsDhbE and
One limitation of this study was the within-subject design.
Minimum washout periods of 24 hours were provided between
each drug treatment and the half-lives in rodents of the drugs used
in this study did not exceed 30 minutes [41,42,43]. In addition, we
performed analyses of the pre-treatment data for each testing day
Figure 1. Effects of nicotine on the grand average waveform of the ERP. A) Raw EEG trace from an individual animal showing 4 stimulus pair
averagedERPwaveformfollowing10tonepairsD)20 tonepairs E)30tonepairs andF) 40tonepairsandG) 50tonepairs.
Nicotine effects on P20 and Gamma Oscillations
PLoS ONE | www.plosone.org4 July 2012 | Volume 7 | Issue 7 | e39775
Figure 2. Nicotine receptor subtype-specific effects on the P20 component of the ERP. A) P20 amplitude following nicotine and nicotine
receptor antagonists. Assessment of the effects of nicotine antagonists was carried out using a within subjects design in which each mouse was
assessed on each drug, with sessions being separated by a 24 hour washout period. N=13 for each condition. Nicotine (1 mg/kg) produced a
significant increase in P20 S1 (black) amplitude without concomitant changes in S2 (gray). The a4b4/a4b2 nicotinic receptor antagonist DHbE (2 mg/
kg) blocked the effect of nicotine on P20 amplitude, while the a7 antagonist MLA (10 mg/kg) had no effect on P20. S1 was significantly higher than
S2 in all conditions except nicotine plus DHbE. B) P20 amplitude following the a4b2 specific agonist AZD3480. Assessment of the effects of
AZD3480 was carried out using a within subjects design in which each mouse was assessed on each drug, with sessions being separated by a 72 hour
washout period. The mice used to assess AZD3480 were a separate cohort from those shown in Figure 2A used to assess nicotine antagonists. N=9
for each condition. No significant changes were observed on P20 amplitude following either a low (1 mg/kg) or high (10 mg/kg) dose of the a4b2
AZD3480 antagonist. # indicates significant difference from saline vehicle (all p,0.05).
Nicotine effects on P20 and Gamma Oscillations
PLoS ONE | www.plosone.org5 July 2012 | Volume 7 | Issue 7 | e39775
to address the potential effects of time and repeated drug exposure
for each dependent variable. As reported, there were no significant
effects of repeated testing for any variables, suggesting that neither
time nor exposure to multiple drugs affected baseline responses in
Consistent with previous reports [10,11,15], nicotine signifi-
cantly increased amplitude of the P20 response to the first click of
a paired-click stimulus (S1), but failed to alter response to the
second (S2) click. Administration of the a4b4/a4b2 antagonist
DHbE by itself did not affect P20 amplitude, but blocked nicotine-
Figure 3. Nicotine receptor subtype-specific effects on the N40 component of the ERP. A) N40 amplitude following nicotine and
nicotine receptor antagonists. Assessment of the effects of nicotine antagonists was carried out using a within subjects design in which each
mouse was assessed on each drug, with sessions being separated by a 24 hour washout period. N=13 for each condition. Nicotine (1 mg/kg) caused
a significant reduction in the N40 ERP which was blocked by both DHbE (2 mg/kg) and MLA (10 mg/kg). These data suggest that activation of both
a7 and a4b2 receptor systems are required for diminution of the N40 component. B) N40 amplitude following the a4b2 specific agonist
AZD3480. Assessment of the effects of AZD3480 was carried out using a within subjects design in which each mouse was assessed on each drug,
with sessions being separated by a 72 hour washout period. The mice used to assess AZD3480 were a separate cohort from those shown in Figure 2A
used to assess nicotine antagonists. N=9 for each condition. A significant reduction in N40 amplitude was seen following administration of the high
dose (10 mg/kg) of AZD3480, suggesting that stimulation of the a4b2 receptor is sufficient to reproduce the effect of nicotine on this component. No
significance was observed for the low (1 mg/kg) dose. No drugs tested showed any effect on S2 response - all significant effects were confined to
changes in S1 amplitude. # - indicates significant difference from saline vehicle (all p,0.05).
Nicotine effects on P20 and Gamma Oscillations
PLoS ONE | www.plosone.org6 July 2012 | Volume 7 | Issue 7 | e39775
induced increases in P20 amplitude. In contrast, administration of
the highly selective a7 antagonist MLA did not affect the P20
response when presented alone and failed to block nicotine
induced enhancements of P20 amplitude. Taken as a whole, this
pattern of data suggests that the ability for nicotine to enhance P20
amplitude occurs primarily through activation of a DHbE
sensitive, and not a7 receptor subtype sensitive mechanism. In
contrast to the effects of receptor specific antagonists, administra-
tion of the a4b2 agonist AZD3480 had no effect on P20
amplitude, consistent with previous findings in which the effect
of nicotine on P20 amplitude was not disrupted in b2 knockout
mice. DHbE has approximately 10 fold higher affinity at a4b4
receptors than at a4b2 , suggesting that the enhancing effects
of nicotine on the mouse P20 are mediated by a4b4 receptors.
The b4 receptor subunit plays a key role in mediating the
rewarding and addicting effects of nicotine  and is expressed in
brain regions that are likely important for the P50 response, such
as the medial habenula. It should be noted that the effect of DHbE
was not simply to block the effect of nicotine on P20 but rather
produced a significant decrease in amplitude relative to saline
vehicle or to DHbE treatment alone. This suggests that DHbE
actually reversed the direction of the effect of nicotine on P20
amplitude and that this likely occurred through a mechanism
other than that activated by DHbE treatment alone. While
previous reports have suggested a role for a7 in regulating both
P20 amplitude and gating in rodents [21,22,23,46], the current
study failed to provide evidence consistent with this notion. It is
likely that there are multiple nicotinic receptor subtypes that
mediate the effect of nicotine on P20 amplitude, including the a7
and b4 subtypes, and that inactivation of a7 activity alone is not
sufficient to fully block the response to nicotine. Interestingly, a
non-significant (p=0.08) trend towards reduced P20 amplitude
was observed following MLA treatment alone, suggesting that
blockade of the a7 receptor in the absence of nicotine produced an
effect opposite to that seen following agonist treatment, which
would be consistent with the notion of a limited regulatory role of
Figure 4. Nicotine receptor subtype-specific effects on auditory evoked gamma oscillations. (Top) Event-related Gamma (30 to 80 Hz)
activity following nicotine and nicotine receptor antagonists. Assessment of the effects of nicotine antagonists was carried out using a within subjects
design in which each mouse was assessed on each drug, with sessions being separated by a 24 hour washout period. N=13 for each condition.
Depicted top left A) is the average event-related gamma power (in dB) for the period between 0 and 60 msec following stimulus onset, across all 50
stimulus presentations. This was done by calculating the time-frequency response for each wavelet cycle within the 0 to 60 msec period and
averaging the resulting values to create a single number. All statistical analyses were conducted on these values. Nicotine (1 mg/kg) caused an
increase in evoked gamma activity, which was blocked by DHbE (2 mg/kg) but not MLA (10 mg/kg), indicating that the positive effect of nicotine on
gamma activity is mediated through the a4b2 receptor subtype. # indicates p,0.05. Top center B) depicts event-related gamma power across each
individual wavelet cycle between 100 msec pre-stimulus and 200 msec post-stimulus across all 50 stimulus presentations. Depicted top left C) is a
heat map showing event-related power in decibels (event-related spectral perturbation, i.e. the power following the stimulus expressed as a change
from baseline) from 200 msec prior to stimulus onset to 200 msec post onset (0=stimulus onset) across all 50 stimulus presentations following
vehicle and nicotine treatment. (Bottom) Event-related Gamma activity following administration of the a4b2 specific agonist AZD3480. Assessment of
the effects of AZD3480 was carried out using a within subjects design in which each mouse was assessed on each drug, with sessions being
separated by a 72 hour washout period. The mice used to assess AZD3480 were a separate cohort from those shown in Figure 2A used to assess
nicotine antagonists. N=9 for each condition. Bottom left D) shows a significant increase in event-related gamma following low (1 mg/kg), but not
high (10 mg/kg) dose AZD3480. The methodology used to create bottom left D), center E) and right F) figures are the same used to create figures A,
B and C, respectively. # - indicates significant differences from saline vehicle (all p,0.05).
Nicotine effects on P20 and Gamma Oscillations
PLoS ONE | www.plosone.org7 July 2012 | Volume 7 | Issue 7 | e39775
a7 in P20 amplitude. Also consistent with the findings on P20
amplitude, MLA did not disrupt any aspect of P20 gating, either
when administered alone or prior to nicotine treatment. In
contrast, DHbE + nicotine produced a significant disruption of
P20 gating, primarily due to a reduction in S1 response. This
suggests that the effect of nicotine on P20/P50 gating may occur
through a DHbE sensitive mechanism.
Similar to previous reports, nicotine significantly decreased N40
amplitude [10,11,15]. Administration of the a4b2 agonist
AZD3480 significantly reduced N40 amplitude in a manner
consistent with that seen following nicotine treatment. Likewise
pretreatment with DHbE blocked the ability for nicotine to
attenuate N40 amplitude. This pattern of results is consistent with
evidence that nicotine alters N40 response through activation of
the b2 subunit . A significant reduction in N40 was observed
following MLA treatment alone, suggesting a possible role for a7
in mediating the N40 response. While this result is also consistent
with the notion that blockade of the a7 receptor may have
subsequently led to increased activation of the a4b2 receptor,
MLA pretreatment was also sufficient to block the effect of
nicotine on N40 response. Thus, stimulation of a7 receptor may
play some role in regulating N40 amplitude. The N40, like the
P20, displayed gating such that responses to S1 were significantly
larger than responses to S2. However, unlike the P20, there was no
interaction between stimulus and drug treatment, suggesting that
N40 gating was not significantly affected by treatment with any of
the antagonists used here. In contrast, AZD3480 significantly
reduced gating, largely by reducing amplitude of the S1
component. These results suggest that the mechanisms that
govern N40 gating are largely consistent with those that govern
N40 amplitude and primarily involve stimulation of the b2
Gamma activity has been associated with perceptual and
cognitive processes as well as positive and negative symptoms in
schizophrenia [47,48,49,50,51,52]. In the present study, nicotine
increased event-related gamma oscillations, replicating a previous
study in our laboratory and a previous report regarding the effects
of smoking on gamma [15,16]. The nicotine-induced increases in
evoked gamma were blocked by DHbE but not by MLA,
suggesting a role for the a4b2 or a4b4 receptor in mediating
these effects. Consistent with this interpretation, treatment with
AZD3480 significantly increased both baseline FFT and event-
related power within the gamma range, further suggesting that
a4b2 receptors are critical to this effect. While there is much
evidence to suggest therapeutic effects of nicotine on schizophrenia
symptomology and cognitive function, few studies have assessed
the effect of nicotine on gamma oscillations. The findings reported
here raise the possibility that nicotine could produce some of its
therapeutic effects through enhancement of gamma activity, and
that this likely occurs primarily through stimulation of the b2
Conceived and designed the experiments: RF JP RE TH SL EC EJ JS.
Analyzed the data: RF JP TT TH EJ EC JS. Wrote the paper: RF JP TT
JS. Obtained funding for the experiments: SS CL.
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