Nicotine & Tobacco Research, Volume 12, Number 6 (June 2010) 589–597
Advance Access published on April 15, 2010
© The Author 2010. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco.
All rights reserved. For permissions, please e-mail: email@example.com
During abstinence, smokers experience deficits in cognition and
sensorimotor processing that may contribute to relapse (Hughes,
2007; Jacobsen et al., 2005; Mendrek et al., 2006; Powell, Pickering,
Dawkins, West, & Powell, 2004; Rissling, Dawson, Schell, &
Nuechterlein, 2007; Ward, Swan, & Jack, 2001). Pharmacother-
apies for nicotine dependence (ND) that target these symptoms
may enhance quitting success (Markou & Paterson, 2009). Food
and Drug Administration-approved smoking cessation thera-
pies including nicotine replacement (NRT), bupropion, and
varenicline reduce withdrawal symptoms and smoking urges
while improving mood and cognitive function (Patterson et al.,
2009; Rahman, Lopez-Hernandez, Corrigall, & Papke, 2008;
Shiffman, Ferguson, Gwaltney, Balabanis, & Shadel, 2006;
Shiffman et al., 2000; West, Baker, Cappelleri, & Bushmakin,
2008). Additionally, there is evidence that bupropion and va-
renicline can reduce cognitive deficits in animal models of nico-
tine withdrawal (Paterson, Balfour, & Markou, 2008; Portugal
& Gould, 2007; Raybuck, Portugal, Lerman, & Gould, 2008).
Varenicline is a newer medication that has superior efficacy
relative to NRT, bupropion, and placebo in clinical trials (Aubin
et al., 2008; Gonzales et al., 2006; Jorenby et al., 2006; Oncken
et al., 2006). Although varenicline is a potent partial agonist at
a4b2 receptors, it also has 25-fold lower affinity as a partial ago-
nist at a3b4, a3b2, and a6 nAChRs and 8-fold lower affinity as
full agonist at a7 receptors (Mihalak, Carroll, & Luetje, 2006).
However, less is known about the effects of varenicline on elec-
trophysiological measures of sensory processing.
In humans, the P50 is a positive voltage deflection in the
electroencephalogram (EEG) that occurs approximately 50 ms
after onset of an auditory stimulus. When paired auditory stim-
uli are presented at short interstimulus intervals, the first stimu-
lus (S1) elicits a larger response than the second stimulus (S2).
Background: Nicotine alters auditory event-related potentials
(ERPs) in rodents and humans and is an effective treatment for
smoking cessation. Less is known about the effects of the partial
nicotine agonist varenicline on ERPs.
Methods: We measured the effects of varenicline and nicotine on
the mouse P20 and varenicline and smoking on the human P50 in
a paired-click task. Eighteen mice were tested following nicotine,
varenicline, and their combination. One hundred and fourteen
current smokers enrolled in a placebo-controlled within-subject
crossover study to test the effects of varenicline during smoking and
abstinence. Thirty-two subjects participated in the ERP study, with
half receiving placebo first and half varenicline first (VP).
Results: Nicotine and varenicline enhanced mouse P20 ampli-
tude, while nicotine improved P20 habituation by selectively
increasing the first-click response. Similar to mice, abstinence
reduced P50 habituation relative to smoking by reducing the
first-click response. There was no effect of varenicline on P50
amplitude during abstinence across subjects. However, there
was a significant effect of medication order on P50 amplitude
during abstinence. Subjects in the PV group displayed reduced
P50 during abstinence, which was blocked by varenicline.
However, subjects in the VP group did not display abstinence-
induced P50 reduction.
Conclusions: Data suggest that smoking improves sensory
processing. Varenicline mimics amplitude changes associated
with nicotine and smoking but fails to alter habituation. The
effect of medication order suggests a possible carryover effect
from the previous arm. This study supports the predictive va-
lidity of ERPs in mice as a marker of drug effects in human
Mouse model predicts effects of smoking
and varenicline on event-related
potentials in humans
Noam D. Rudnick, B.S.,1 Andrew A. Strasser, Ph.D.,1,2 Jennifer M. Phillips, Ph.D.,1 Christopher Jepson, Ph.D.,1
Freda Patterson, Ph.D.,2 Joseph M. Frey, Ph.D.,3 Bruce I. Turetsky, M.D.,1 Caryn Lerman, Ph.D.,1,2 &
Steven J. Siegel, M.D., Ph.D.1,2
1 Department of Psychiatry, University of Pennsylvania, Philadelphia, PA
2 Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA
3 Global Clinical Development, Neuroscience, AstraZeneca, Wilmington, DE
Corresponding Author: Steven J. Siegel, M.D., Ph.D., Translational Neuroscience Program, Translational Research Laboratory,
125 South 31st Street, University of Pennsylvania, Philadelphia, PA 19104, USA. Telephone: 215-573-0278; Fax: 215-662-7903;
Present address: Jennifer M. Phillips, Ph.D., Department of Psychology, Mount St. Mary’s University, Emmitsburg, MD
Received July 20, 2009; accepted March 9, 2010
Mouse model predicts effects of smoking and varenicline
This phenomenon represents habituation of the response to
repeated stimuli and is sometimes referred to as habituation in
this context (Stevens, Kem, & Freedman, 1999; Stevens, Kem,
Mahnir, & Freedman, 1998). Several studies have investigated
the effects of nicotine on P50 habituation. Additionally, studies
have suggested a role of a7 nicotinic receptors in this function,
possibly by influencing cholinergic input to gamma amino bu-
tyric acid ergic interneurons (Stevens et al., 1998). Smoking im-
proves P50 habituation in schizophrenic individuals, and
nicotine gum is sufficient to achieve similar enhancements in
their relatives (Adler, Hoffer, Griffith, Waldo, & Freedman,
1992; Adler, Hoffer, Wiser, & Freedman, 1993). Changes in the
ratio of response for the S2:S1 are sometimes used as a measure
of habituation. Several studies demonstrate that the S2:S1 ratio
is sensitive to changes in S1 rather than S2, suggesting a change
in the ability to mount the initial response rather than the abil-
ity to habituate the second (Adler, Pang, Gerhardt, & Rose,
1988; Crawford, McClain-Furmanski, Castagnoli, & Castagnoli,
2002; Halene & Siegel, 2008; Maxwell, Kanes, Abel, & Siegel,
2004; Maxwell et al., 2006; Metzger, Maxwell, Liang, & Siegel,
2007; Phillips, Ehrlichman, & Siegel, 2007). For example,
nonpsychiatric heavy smokers exhibit greater P50 amplitude
and S2:S1 ratio compared with never-smokers (Crawford et al.).
However, studies of the acute effects of smoking versus absti-
nence on P50 responses in normal smokers have yielded mixed
results, with some studies demonstrating that the effects
of smoking are mediated by S1 response (Adler et al., 1993;
Crawford et al.; Domino, 2003; Kishimoto & Domino, 1998).
The topology and habituation of auditory event-related po-
tentials (ERPs) in the mouse closely resemble the human
responses but follow a faster time course. The mouse P20,
a positive deflection that occurs 20 ms after stimulus onset, is
thought to be analogous to the human P50 (Maxwell, Liang,
et al., 2004; Siegel et al., 2003; Umbricht et al., 2004). Acute
nicotine increases amplitude of the mouse P20 in response to
S1 and, as a result, reduces the apparent habituation ratio
(S2:S1; Metzger et al., 2007; Phillips et al., 2007). However, it is
important to note that such changes reflect altered registration
of the initial stimulus rather than improved suppression of the
second. Additionally, nicotine has been shown to reverse audi-
tory habituation deficits in mouse models of psychosis (Siegel
et al., 2005; Stevens, Meltzer, & Rose, 1995). Previous studies
indicate that dihydro-beta-erythroidine (DHbE) blocks nico-
tine’s effect on amplitude but not habituation of the P20 and
P20/N40 auditory ERPs (Siegel, S. et al., Annual Meeting of the
American College of Neuropsychopharmacology 2006 and
Phillips et al., in review; Kawai, Lazar, & Metherate, 2007;
Radek et al., 2006). DHbE blocks a4b2 and a4b4 and, to a
lesser extent, a2b2 and a3b2 as well as a2b4 nAChRs (Chavez-
Noriega et al., 1997). Therefore, previous studies with DHbE
suggest involvement of b2 and/or b4 containing nicotinic
receptors in generation of the P20.
In this study, we examined the effects of nicotine and
varenicline in mice. In mice, we predicted that nicotine would
enhance P20 habituation by increasing S1 response amplitude.
Based on the effect of DHbE and varenicline’s relative selectivity
for a4b2 nAChRs, we hypothesized that varenicline would in-
crease P20 amplitude without affecting habituation. We also
tested the effects of smoking versus abstinence and varenicline
versus placebo in human chronic smokers. We hypothesized
that smoking would enhance P50 habituation relative to absti-
nence by increasing S1 response amplitude. Furthermore, we
hypothesized that varenicline would attenuate the effects of ab-
stinence on P50 amplitude in human smokers.
Materials and methods
Eighteen male wild-type C57BL/6J mice (Jackson Labs, Bar
Harbor, ME) between 9 and 11 weeks of age were used for audi-
tory testing. Mice were housed in light- and temperature-
controlled animal facility accredited by the Association for the
Assessment and Accreditation of Laboratory Animal Care.
Animals were acclimated to the housing facility for 1 week prior
to testing and provided with food and water ad libitum. All pro-
tocols were conducted in accordance with University Labora-
tory Animal Resources guidelines and were approved by the
Institutional Animal Care and Use Committee.
Mice underwent stereotaxic implantation of electrode assemblies
(Plastic Products, Roanoke, VA) for later nonanesthetized record-
ing of auditory ERPs. Mice were anesthetized with isoflurane for
the duration of the implantation procedure. Unipolar recording
electrodes were placed unilaterally in the CA3 hippocampal region
(1.4 mm posterior, 2.65 mm lateral, and 2.75 mm deep relative to
bregma) and referenced to the ipsilateral frontal sinus to reflect
whole brain electrical activity from these two perspectives. The
electrode pedestal was secured to the skull using dental cement
(Ortho Jet; Lang Dental, Wheeling, IL) and ethyl cyanoacrylate
(Loctite; Henkel KGaA, Duesseldorf, Germany).
Mice received subcutaneous injections of 1.0 mg/kg nicotine
tartrate and 1.2 mg/kg varenicline tartrate (Pfizer, Groton, CT).
All drug concentrations are reported as freebase. The mouse
study was designed to mimic the human design such that each
mouse received each of four conditions as follows: nicotine
(similar to smoking), saline (similar to abstinence), nicotine
and varenicline (similar to taking varenicline while smoking),
and varenicline (similar to taking varenicline during absti-
nence). These conditions were separated by 48 hr. Animals were
divided into two groups and drug conditions were counterbal-
anced across recording sessions to control for any potential order
effects, similar to the human study as noted below (Table 1).
Electrophysiological testing was conducted for 4 days with a
washout period of 48 hr between recording sessions and three
stimuli presentations per session. The first presentation involved
no injection to acclimate animals to the stimuli, the second pre-
sentation followed a saline injection, and the third presentation
occurred 5 min after injection of the test compound(s). This
timing allows for recording of ERPs within 1 serum half-life for
nicotine in mouse. Stimuli were generated by Micro1401 hard-
ware and Spike 6 software (Cambridge Electronic Design,
Cambridge, UK) and were delivered through speakers attached
to the cage top. All recordings were performed in a home cage
environment, which was placed in a Faraday cage 15 min before
stimulus onset. White noise stimuli were presented at 85-dB
Nicotine & Tobacco Research, Volume 12, Number 6 (June 2010)
intensity, 10-ms duration, and 500-ms interstimulus interval.
Stimulus pairs were separated by 8 s, and a total of 50 paired
stimuli were presented.
EEG data were inline filtered between 1 and 500 Hz and baseline
corrected at stimulus onset. Using Spike 6, individual sweeps
were rejected for movement artifact based on a criterion of two
times the root mean squared amplitude per mouse. Based on
this criterion, 2% of individual sweeps were rejected. The P20
component was selected from each subject’s average ERP by de-
termining the maximum positive deflection between 10 and 30 ms.
Data from test sessions were analyzed using repeated measures
analyses of variance (ANOVAs) to determine the effects of nico-
tine, varenicline, stimulus (S1 vs. S2), and treatment order on
P20 amplitude. Effects on P20 habituation were assessed as
a significant interaction between pharmacological treatment
(varenicline or nicotine) and the responses to S1 and S2. We
performed repeated measures ANOVAs on baseline daily saline
data to control for effects of repeated testing. Significant effects
were followed by Fisher’s least significant difference (LSD) post
hoc comparisons using Statistica 6.0 (Statsoft, Inc., Tulsa, OK).
Thirty-two healthy smokers were recruited for a randomized,
double-blind placebo-controlled study of the effects of vareni-
cline on the P50 ERP (Supplemental Figure 1). Smokers re-
sponding to local advertisements for a smoking cessation
program were screened for eligibility in September 2006 to
August 2007. Eligible smokers were ≥18 years of age and had
smoked ≥10 cigarettes/day for the previous 12 months. In order
to increase the generalizability of results to the clinical setting,
we enrolled treatment-seeking smokers (those planning to quit
in the next 3 months; K. Perkins et al., 2008; K. A. Perkins,
Stitzer, & Lerman, 2006). Exclusion criteria included: history of
seizures, pregnancy, lactation or planning pregnancy, unstable
angina, history of heart attack or stroke in previous 6 months,
insulin dependent diabetes, current diagnosis or history of
DSM-IV Axis I psychiatric disorders or substance abuse, and
current use of smoking cessation or contraindicated medica-
tions. All subjects provided informed consent in accordance
with Institutional Review Board guidelines at the University of
Pennsylvania. Participants reported smoking between 10 and 50
cigarettes/day at study onset, with an average of 21.63 (SD =
10.05). The mean score on the Fagerström Test for Nicotine
Dependence (FTND) was 5.28 (SD = 2.44). The average age of
participants was 41.06 years (SD = 11.75), and they had been
smoking for an average of 24.78 years (SD = 12.20). Of the 32
participants, 50% were female, 56.25% were White, 40.63% were
Black, and 3.13% were Asian. Carbon monoxide (CO) was mea-
sured on the day of testing to confirm abstinence (CO ≤ 10 ppm).
There were two drug treatment phases during the study and
each participant received varenicline in one phase and placebo
in the other. Subjects receiving varenicline first and placebo sec-
ond comprised Group 1; subjects who received placebo first and
varenicline second comprised Group 2. Treatment order was
counterbalanced between subjects and phases were separated by
a nonmedicated 5- to 7-day washout period during which sub-
jects were instructed to smoke as usual. Participants began each
phase by smoking as usual for 10 days, which was followed by
3 days of mandatory abstinence. ERPs were obtained on Day 10
(smoking as usual) and Day 12 (second day of abstinence) for
both placebo and varenicline phases. This paradigm yielded a
total of four possible experimental conditions (placebo + smok-
ing, placebo + abstinence, varenicline + smoking, and vareni-
cline + abstinence). On Day 10 of each phase, subjects smoked
one of their own preferred brand cigarettes approximately
35 min before ERP testing. During each day of the mandatory
abstinence period, abstinence was biochemically verified by
breath CO samples less than 10 parts per million. Varenicline
was administered in a manner consistent with clinical titration
guidelines: 0.5 mg po Days 1–3, 0.5 mg po bid Days 4–7, and 1.0 mg
po bid Days 8–13 (Pfizer, 2007).
Participants wore a NeuroScan QuickCap (Compumedics,
Charlotte, NC) with ground sensors over the mastoid bones and
a recording sensor at Cz. Stimuli were presented at 85-dB inten-
sity, 0.1-ms duration, and 580 ms apart and were delivered bin-
aurally. Stimulus pairs were separated by 8 s, and a total of 100
paired stimuli were presented.
EEG data were digitally filtered between 10 and 80 Hz and base-
line corrected at stimulus onset using Vision Analyzer (Brain
Products Ltd., Gilching, Germany). Individual sweeps were
rejected as movement artifact if they exceeded an absolute value
of 100 mV. Based on this criterion, 8% of individual sweeps were
rejected. The P50 component was selected from each subject’s
average ERP by determining the maximum positive deflection
between 40 and 75 ms. We analyzed the data using repeated
measures ANOVAs to determine the effects of smoking, vareni-
cline, stimulus, and treatment order on P50 amplitude and
latency as well as any interaction effects. Effects on P50 habituation
Table 1. The mouse study was designed to mimic the human design such that each ani-
mal received four conditions as follows: nicotine (similar to smoking), saline (similar to
abstinence), nicotine and varenicline (similar to taking varenicline while smoking), and
varenicline (similar to taking varenicline during abstinence)
Nicotine & varenicline
Nicotine & varenicline
Note. Sessions were separated by 48 hr. Animals were divided into two groups and drug order were counterbalanced across recording sessions to
control for any potential order effects, similar to the human study.
Mouse model predicts effects of smoking and varenicline
were assessed as a significant interaction between pharmaco-
logical treatment (varenicline or smoking) and the responses to
S1 and S2. We included self-reported baseline cigarette con-
sumption as a covariate in our analysis. Significant effects were
followed by Fisher LSD post hoc comparisons using Statistica
For the mouse study, the second P20 response was significantly
reduced relative to the first (S1 = 104.68 mV ± 24.43, S2 = 27.53 ±
6.61, p < .001; Figure 1A, Table 2). Nicotine increased P20 am-
plitude (p = .009), and an interaction between nicotine and
stimulus (p < .001) indicated that nicotine enhanced habitua-
tion (Figure 2A). Post hoc analyses revealed an increased re-
sponse to S1 (p < .001) without a change in the response to S2
(p = .702). Figure 2B shows that varenicline increased overall
P20 amplitude (p = .019), but there was no significant interac-
tion with stimulus (p = .088). It is possible that increased statis-
tical power could reveal a significant effect of varenicline on P20
habituation, but it is important to note that the study size was
sufficient to reveal a highly significant effect of nicotine on P20
habituation. Acute drug exposure did not alter P20 responses
on subsequent testing days since there were no effects of treat-
ment order and baseline saline data revealed no differences
across treatment conditions prior to drug exposure.
For the human study, there was significant habituation of
the second P50 response relative to the first (S1 = 2.91 mV ±
0.39, S2 = 1.83 ± 0.29, p = .010; Figure 1B). There was no main
effect of abstinence on P50 amplitude (p = .584), but an interac-
tion with stimulus (p = .041) indicated that abstinence reduced
habituation relative to smoking. Post hoc analyses showed that
abstinence decreased S1 response amplitude (p = .004) but had
no effect on S2 response amplitude (p = .308; Figure 3A).
Neither baseline number of daily cigarettes (p = .296) nor FTND
(p = .751) were significantly associated with the effect of smoking
on auditory habituation.
There was no effect of varenicline on P50 amplitude (p =
.579) or habituation (p = .191) when averaged across treatment
orders (Group 1 and Group 2). However, there was a significant
effect of treatment order on P50 amplitude (p = .043). Subjects
who received placebo first and varenicline second (Group 2;
Figure 3B) had higher P50 amplitude than subjects receiving va-
renicline first and placebo second (Group 1; Figure 3B). Fur-
thermore, there was a significant interaction between varenicline,
smoking, and treatment order (p = .037), indicating that phar-
macological effects differed based on treatment order. Subjects
receiving placebo first exhibited decreased P50 amplitude dur-
ing abstinence (p = .004), which was attenuated by varenicline.
Subjects receiving varenicline first followed by placebo did
not have a similar effect (p = .862). P50 amplitude during absti-
nence was significantly higher on varenicline than on placebo
(p = .003) for subjects who received placebo first (p = .818)
without similar effect in subjects who received varenicline first
There was no significant main effect of varenicline on la-
tency when averaged across smoking conditions (smoking or
abstinent) and stimulus type (S1 or S2; p = .414). Abstinence
caused a significant increase in P50 latency relative to smoking
across all other conditions (p < .001). S2 response latency was
significantly higher than that for S1 (p < .001). Varenicline did
not modify the effects of smoking or stimulus type (no interac-
tion between varenicline and smoking or varenicline and stimu-
lus p > .05 for both comparisons). However, there was a
significant interaction between smoking and stimulus condition
Table 2. Mean and SD for the P20 ampli-
tude in mice and P50 amplitude in humans
for both S1 and S2 responses
Species Drug condition
Mouse Saline + salineS1
Nicotine + saline
Saline + varenicline
Nicotine + varenicline
HumanAbstinence + placebo
Smoking + placebo
Abstinence + varenicline S1
Smoking + varenicline
Figure 1. Grand averages of mouse (A) and human (B) event-related
potentials showing the responses to S1 (black) and S2 (gray). Maximum
positive deflections in (A) and (B) represent the P20 and P50 compo-
nents, respectively. Dotted lines indicate stimulus onset and arrows
indicate P20 and P50 components.
Nicotine & Tobacco Research, Volume 12, Number 6 (June 2010)
such that abstinence caused an increase in S2 latency (p < .01)
without significant changes on S1.
There are two main novel findings in the current study.
Although varenicline did not increase P50 across all subjects and
conditions, data indicate that it acts electrophysiologically as a
functional agonist to replace nicotine during periods of smok-
ing cessation. Second, acute nicotine has the same effects on the
mouse P20 as smoking does on the P50 in humans. This is a
crucial translational link for studies that make such an assump-
tion without direct data. Similarly, varenicline has the same ef-
fect on the mouse P20 as it does on the human P50. There has
been much debate regarding how ERP components in mouse
and human align. This study provides very strong support that
the mouse P20 is the appropriate correlate of the human P50.
We found that nicotine in mice and smoking (vs. absti-
nence) in humans enhanced habituation of the P20 and P50,
respectively. In both species, enhancement of habituation in-
volved an increased response to S1 without a change in the re-
sponse to S2. This finding is consistent with previous reports
that S1 amplitude changes, in the absence of S2 amplitude
changes, can be observed following cholinergic modulation of
auditory habituation (Crawford et al., 2002; Metzger et al., 2007;
Rudnick, Koehler, Picciotto, & Siegel, 2009). Varenicline in-
creased P20 amplitude in mice without an effect on habituation.
In the human crossover study, subjects receiving placebo during
Phase 1 exhibited decreased P50 amplitude during abstinence,
which was attenuated by varenicline during Phase 2. Subjects
receiving varenicline during Phase 1 followed by placebo during
Phase 2 exhibited no change in P50 amplitude during either
treatment phase. Although we cannot offer a definitive explana-
tion for the effect of treatment order, a pharmacologic carryover
effect cannot be ruled out. In summary, these findings support
the hypothesis that varenicline can modulate amplitude of the
P20 and P50 components (Table 3).
We show an acute effect of smoking status on P50 habitua-
tion in healthy current smokers. While previous studies employed
brief periods of abstinence (6–15 hr), we employed a longer pe-
riod of abstinence (3 days), which was confirmed by exhaled CO
(Adler et al., 1993; Crawford et al., 2002; Domino, 2003; Domino &
Kishimoto, 2002). Therefore, our paradigm may have been more
sensitive to effects of smoking and abstinence compared with
Figure 3. Effects of smoking and varenicline on human P50. (A)
Smoking enhances P50 habituation by selectively increasing the re-
sponse to S1. Amplitudes are averaged across varenicline conditions.
This shows the main effect of smoking when collapsing across other
variables. (B) Drug treatments were counterbalanced across study phases.
Group 1 received varenicline during Phase 1 and placebo during
Phase 2, while Group 2 received placebo during Phase 1 and varenicline
during Phase 2. Group 1 did not show any changes in P50 amplitude
across drug conditions, but Group 2 exhibited reduced P50 amplitude
during abstinence on placebo. Amplitudes are averaged across S1 and S2
because there was no statistical interaction that included stimulus con-
dition. Data are presented as mean ± SEM, and collapsed across vari-
ables for which no statistically significant interaction effects were found.
*p < .050 (Fisher’s LSD post hoc test).
Figure 2. Effects of nicotine and varenicline on the mouse P20.
(A) Nicotine enhances P20 habituation by selectively increasing the
response to S1. Amplitudes are averaged across varenicline conditions.
This means that nicotine increases the S1 response of the P20 regardless
of varenicline condition. (B) There was a main effect of varenicline, re-
gardless of nicotine condition (nicotine or saline) or stimulus condition
(S1 and S2). Varenicline increases P20 amplitude. Amplitudes are aver-
aged across nicotine and stimulus conditions (S1 and S2). Data are pre-
sented as mean ± SEM, and collapsed across variables for which no
statistically significant interaction effects were found. *p < .050 (Fisher’s
LSD post hoc test).
Mouse model predicts effects of smoking and varenicline
studies with a shorter duration of abstinence. In vivo radiotracer
experiments suggest that nicotine can take several days to clear
high-affinity binding sites (i.e., a4b2 nAChRs) in humans and
nonhuman primates (Staley et al., 2006). Therefore, the effects of
abstinence may follow a protracted time course. One potential
drawback of our approach is that each subject started from a dif-
ferent level of baseline smoking. We controlled for this possibility
by including self-reported baseline cigarette consumption as a
covariate in our analyses.
Auditory habituation depends on the responses to S1 and
S2, and frequently, these amplitudes are condensed into a single
ratio. This approach can obscure the mechanism of habituation
enhancements, which may depend on an increased response to
S1, decreased response to S2, or both. It has been proposed that
nicotine inhibits the response to S2 by activating a7 nAChRs
interneurons in the CA3 region of the hippocampus (Adler
et al., 1998; Stevens et al., 1998). However, this mechanism
alone is not sufficient to explain our data because nicotine and
smoking increased the amplitude of the S1 response without
significantly affecting the amplitude of the S2 response. Previ-
ous studies in rodents and humans show similar changes in the
response to S1 but not S2 (Crawford et al., 2002; Cromwell &
Woodward, 2007; Metzger et al., 2007; Phillips et al., 2007).
Varenicline, a relatively selective a4b2 nAChRs partial agonist
(Mihalak et al., 2006), increased amplitude but not habituation
of auditory ERPs. Therefore, it is likely that brain regions rich in
a4b2 nAChRs, the target of varenicline, contribute to the am-
plifying effect of nicotine on the S1 response. Consistent with
this hypothesis, nicotine enhances action potential propagation
and synaptic release in thalamocortical circuits via DHbE-
sensitive nAChRs (Kawai et al., 2007; Lambe, Picciotto, &
Aghajanian, 2003). These circuits are an obligate stage of audi-
tory processing and participate in generation of the midlatency
ERP components (Hinman & Buchwald, 1983; McGee, Kraus,
Comperatore, & Nicol, 1991). Even if nicotine increases the re-
sponse to S1 by enhancing thalamic transmission, it must also
activate inhibitory networks in order to prevent the response to
S2 from increasing in amplitude as well. Therefore, it is likely
that both a7 nAChRs- and a4b2 nAChRs-expressing brain re-
gions are involved in auditory habituation. Our initial hypoth-
esis was that varenicline would attenuate the effects of nicotine
in the presence of nicotine but mimic the effects of a full agonist
when given alone, consistent with activity as a partial agonist.
Data support that varenicline acted as a functional agonist when
given alone. The combination of varenicline and smoking were
similar to either smoking or varenicline alone.
Varenicline increased P20 amplitude in mice and P50 ampli-
tude during abstinence in humans. Changes in EEG power and
ERP amplitude are thought to reflect changes in arousal
(Kishimoto & Domino, 1998; Pickworth, Herning, & Henningfield,
1989). Because decreased arousal is a symptom of nicotine with-
drawal, varenicline’s effects on sensory habituation may con-
tribute to its therapeutic efficacy. However, nonspecific increases in
arousal may also contribute to sleep disturbances observed in some
clinical studies (Gonzales et al., 2006; Oncken et al., 2006).
Nonetheless, the aforementioned connection between ERP am-
plitude and arousal remains speculative because stimulants such
as amphetamine can decrease amplitude (Maxwell, Kanes, et al.,
2004). Interestingly, the smoking cessation medication bupro-
pion reduces the amplitude of ERPs in mice, similar to amphet-
amine (Siegel et al., 2005). To the best of our knowledge, the
effects of bupropion on human ERPs are not known. Although
alpha-7 nAChRs agonists such as 3-(2,4)-dimethoxybenzylidine
anabaseine and tropisetron reverse the effects of cocaine or am-
phetamine on ERPs, their effects on P50 or smoking status in
humans are not known (Hashimoto, Iyo, Freedman, & Stevens,
2005; Stevens et al., 1999). The most direct interpretation of
increased ERP amplitude may simply be that it reflects a greater
degree of phase synchrony in ongoing EEG rhythms (Jansen,
Agarwal, Hegde, & Boutros, 2003; Makeig et al., 2002).
Our human data reveal that treatment order significantly
modulated the effects of abstinence and varenicline on P50 am-
plitude. It is possible that subjects receiving varenicline prior to
placebo failed to undergo a reduction in P50 amplitude during
the placebo phase because varenicline had long-lasting effects in
the brain, despite the 5- to 7-day washout period and 17-hr half-
life of varenicline (Obach et al., 2006). An alternative explanation
for the order effect may be that abstinence failed to reduce P50
amplitude because of a floor effect. Overall P50 amplitudes also
differed by treatment order, suggesting that there may have been
baseline asymmetries in sensory processing between subjects
randomized to different treatment orders; however, in the
absence of baseline ERP data, we cannot examine this directly.
To simplify interpretation of results, future within-subject designs
may benefit from extending the washout period to 2 weeks.
Previous studies indicate that nicotine in rodents produces
similar biological and behavioral effects as smoking in humans,
and this study further supports the face validity of mouse mod-
els (Blendy et al., 2005; Corrigall, 1999; Lerman et al., 2007; Liu
et al., 2003; Slawecki, Gilder, Roth, & Ehlers, 2003). However,
there are limitations to this approach. Besides nicotine, ciga-
rettes contain psychoactive compounds such as monoamine
oxidase inhibitors that may play a role in dependence and audi-
tory habituation (Crawford et al., 2002; Guillem et al., 2005;
Siegel et al., 2005). Furthermore, our mouse study used acute
doses of nicotine, which likely fail to produce the types
of nAChRs upregulation associated with chronic smoking.
Although previous studies in mice have shown biological effects
at the dose of varenicline used in the present study, the lack of a
dose–response relationship for ERPs is a potential limitation
since we cannot determine the effects of higher doses, which
may have increased P50 amplitude in the VP group (Raybuck
Table 3. Consolidated results from mouse
and human experiments demonstrate
translational validity of event-related
Mouse P20 Human P50
S1 > S2
S1 > S2
Note. In both species, habituation of the second stimulus relative to the
first can be observed. Nicotine in mice selectively increases P20
response to S1, just as smoking in human selectively increases P50
response to S1. Last, varenicline increases overall ERP amplitude in
aNote that some subjects did not show differences between placebo
and varenicline due to a possible carryover effect.
Nicotine & Tobacco Research, Volume 12, Number 6 (June 2010)
et al., 2008; Rollema et al., 2009). Despite these caveats, the ex-
tensive interspecies overlap in our results suggests that acute
doses of cholinergic agents approximate chronic exposure in
humans. Thus, EEG in mice allows for rapid screening of novel
treatments for ND that may ameliorate sensory deficits associ-
ated with abstinence (Lerman et al.).
Supplementary Figure 1 can be found at Nicotine and
Tobacco Research online (http://www.ntr.oxfordjournals.org/).
This research was supported by the AstraZeneca, the National
Cancer Institute and National Institutes on Drug Abuse
(P50084718, CL, Principal Investigator), and a fellowship from
the Irene and Eric Simon Brain Research Foundation to NDR and
SJS; Pfizer generously provided varenicline for the mouse study.
Declaration of Interests
NDR has no potential conflicts of interest to disclose. AAS has no
potential conflicts of interest to disclose. JMP has no potential con-
flicts of interest to disclose. CJ has no potential conflicts of interest
to disclose. FP has no potential conflicts of interest to disclose. JMF
is an employee of AstraZeneca. BIT receives unrelated research
grant support from AstraZeneca and Pfizer pharmaceutical com-
panies. CL has received compensation and research funding from
Pfizer, AstraZeneca, and GlaxoSmith Kline. SJS has received unre-
lated research funding from AstraZeneca, unrelated research fund-
ing and compensation from NuPathe, and compensation from the
Network for Continuing Medical Education.
Adler, L. E., Hoffer, L. D., Wiser, A., & Freedman, R. (1993).
Normalization of auditory physiology by cigarette smoking in
schizophrenic patients. American Journal of Psychiatry, 150,
Adler, L. E., Hoffer, L. J., Griffith, J., Waldo, M. C., & Freedman, R.
(1992). Normalization by nicotine of deficient auditory sensory
gating in the relatives of schizophrenics. Biological Psychiatry,
Adler, L. E., Olincy, A., Waldo, M., Harris, J. G., Griffith, J.,
Stevens, K., et al. (1998). Schizophrenia, sensory gating, and
nicotinic receptors. Schizophrenia Bulletin, 24, 189–202.
Adler, L. E., Pang, K., Gerhardt, G., & Rose, G. M. (1988). Mod-
ulation of the gating of auditory evoked potentials by norepi-
nephrine: Pharmacological evidence obtained using a selective
neurotoxin. Biological Psychiatry, 24, 179–190.
Aubin, H. J., Bobak, A., Britton, J. R., Oncken, C., Billing, C. B., Jr.,
Gong, J., et al. (2008). Varenicline versus transdermal nicotine
patch for smoking cessation: Results from a randomised, open-
label trial. Thorax, 63, 717–724.
Blendy, J. A., Strasser, A., Walters, C. L., Perkins, K. A.,
Patterson, F., Berkowitz, R., et al. (2005). Reduced nicotine
reward in obesity: Cross-comparison in human and mouse. Psy-
chopharmacology, 180, 306–315.
Chavez-Noriega, L. E., Crona, J. H., Washburn, M. S., Urrutia, A.,
Elliott, K. J., & Johnson, E. C. (1997). Pharmacological charac-
terization of recombinant human neuronal nicotinic acetylcho-
line receptors h alpha 2 beta 2, h alpha 2 beta 4, h alpha 3 beta 2,
h alpha 3 beta 4, h alpha 4 beta 2, h alpha 4 beta 4 and h alpha 7
expressed in Xenopus oocytes. Journal of Pharmacology and
Experimental Therapeutics, 280, 346–356.
Corrigall, W. A. (1999). Nicotine self-administration in animals
as a dependence model. Nicotine & Tobacco Research, 1, 11–20.
Crawford, H. J., McClain-Furmanski, D., Castagnoli, N., Jr., &
Castagnoli, K. (2002). Enhancement of auditory sensory gating
and stimulus-bound gamma band (40 Hz) oscillations in heavy
tobacco smokers. Neuroscience Letters, 317, 151–155.
Cromwell, H. C., & Woodward, D. J. (2007). Inhibitory gating
of single unit activity in amygdala: Effects of ketamine, haloperi-
dol, or nicotine. Biological Psychiatry, 61, 880–889.
Domino, E. F. (2003). Effects of tobacco smoking on electroen-
cephalographic, auditory evoked and event related potentials.
Brain and Cognition, 53, 66–74.
Domino, E. F., & Kishimoto, T. (2002). Tobacco smoking in-
creases gating of irrelevant and enhances attention to relevant
tones. Nicotine & Tobacco Research, 4, 71–78.
Gonzales, D., Rennard, S. I., Nides, M., Oncken, C., Azoulay, S.,
Billing, C. B., et al. (2006). Varenicline, an alpha4beta2 nico-
tinic acetylcholine receptor partial agonist, vs sustained-release
bupropion and placebo for smoking cessation: A randomized
controlled trial. Journal of the American Medical Association,
Guillem, K., Vouillac, C., Azar, M. R., Parsons, L. H., Koob, G. F.,
Cador, M., et al. (2005). Monoamine oxidase inhibition dra-
matically increases the motivation to self-administer nicotine in
rats. Journal of Neuroscience, 25, 8593–8600.
Halene, T. B., & Siegel, S. J. (2008). Antipsychotic-like proper-
ties of phosphodiesterase 4 inhibitors: Evaluation of 4-(3-
with auditory event-related potentials and prepulse inhibition
of startle. Journal of Pharmacology and Experimental Therapeutics,
Hashimoto, K., Iyo, M., Freedman, R., & Stevens, K. E. (2005).
Tropisetron improves deficient inhibitory auditory processing
in DBA/2 mice: Role of alpha 7 nicotinic acetylcholine recep-
tors. Psychopharmacology, 183, 13–19.
Hinman, C. L., & Buchwald, J. S. (1983). Depth evoked poten-
tial and single unit correlates of vertex midlatency auditory
evoked responses. Brain Research, 264, 57–67.
Hughes, J. R. (2007). Effects of abstinence from tobacco: Valid
symptoms and time course. Nicotine & Tobacco Research, 9,
Mouse model predicts effects of smoking and varenicline
Jacobsen, L. K., Krystal, J. H., Mencl, W. E., Westerveld, M.,
Frost, S. J., & Pugh, K. R. (2005). Effects of smoking and smok-
ing abstinence on cognition in adolescent tobacco smokers. Bio-
logical Psychiatry, 57, 56–66.
Jansen, B. H., Agarwal, G., Hegde, A., & Boutros, N. N. (2003).
Phase synchronization of the ongoing EEG and auditory EP
generation. Clinical Neurophysiology, 114, 79–85.
Jorenby, D. E., Hays, J. T., Rigotti, N. A., Azoulay, S., Watsky, E. J.,
Williams, K. E., et al. (2006). Efficacy of varenicline, an
alpha4beta2 nicotinic acetylcholine receptor partial agonist, vs
placebo or sustained-release bupropion for smoking cessation:
A randomized controlled trial. Journal of the American Medical
Association, 296, 56–63.
Kawai, H., Lazar, R., & Metherate, R. (2007). Nicotinic control
of axon excitability regulates thalamocortical transmission.
Nature Neuroscience, 10, 1168–1175.
Kishimoto, T., & Domino, E. F. (1998). Effects of tobacco smok-
ing and abstinence on middle latency auditory evoked poten-
tials. Clinical Pharmacology and Therapeutics, 63, 571–579.
Lambe, E. K., Picciotto, M. R., & Aghajanian, G. K. (2003). Nic-
otine induces glutamate release from thalamocortical terminals
in prefrontal cortex. Neuropsychopharmacology, 28, 216–225.
Lerman, C., LeSage, M. G., Perkins, K. A., O’Malley, S. S., Siegel, S. J.,
Benowitz, N. L., et al. (2007). Translational research in medica-
tion development for nicotine dependence. Nature Reviews
Drug Discovery, 6, 746–762.
Liu, X., Koren, A. O., Yee, S. K., Pechnick, R. N., Poland, R. E., &
London, E. D. (2003). Self-administration of 5-iodo-A-85380, a
beta2-selective nicotinic receptor ligand, by operantly trained
rats. Neuroreport, 14, 1503–1505.
Makeig, S., Westerfield, M., Jung, T. P., Enghoff, S., Townsend, J.,
Courchesne, E., et al. (2002). Dynamic brain sources of visual
evoked responses. Science, 295, 690–694.
Markou, A., & Paterson, N. E. (2009). Multiple motivational
forces contribute to nicotine dependence. Nebraska Symposium
of Motivation, 55, 65–89.
Maxwell, C. R., Ehrlichman, R. S., Liang, Y., Trief, D., Kanes, S. J.,
Karp, J., et al. (2006). Ketamine produces lasting disruptions
in encoding of sensory stimuli. Journal of Pharmacology and
Experimental Therapeutics, 316, 315–324.
Maxwell, C. R., Kanes, S. J., Abel, T., & Siegel, S. J. (2004).
Phosphodiesterase inhibitors: A novel mechanism for receptor-
independent antipsychotic medications. Neuroscience, 129,
Maxwell, C. R., Liang, Y., Weightman, B. D., Kanes, S. J., Abel, T.,
Gur, R. E., et al. (2004). Effects of chronic olanzapine and
haloperidol differ on the mouse N1 auditory evoked potential.
Neuropsychopharmacology, 29, 739–746.
McGee, T., Kraus, N., Comperatore, C., & Nicol, T. (1991).
Subcortical and cortical components of the MLR generating
system. Brain Research, 544, 211–220.
Mendrek, A., Monterosso, J., Simon, S. L., Jarvik, M., Brody, A.,
Olmstead, R., et al. (2006). Working memory in cigarette smok-
ers: Comparison to non-smokers and effects of abstinence.
Addictive Behaviors, 31, 833–844.
Metzger, K. L., Maxwell, C. R., Liang, Y., & Siegel, S. J. (2007).
Effects of nicotine vary across two auditory evoked potentials in
the mouse. Biological Psychiatry, 61, 23–30.
Mihalak, K. B., Carroll, F. I., & Luetje, C. W. (2006). Varenicline
is a partial agonist at alpha4beta2 and a full agonist at alpha7 neu-
ronal nicotinic receptors. Molecular Pharmacology, 70, 801–805.
Obach, R. S., Reed-Hagen, A. E., Krueger, S. S., Obach, B. J.,
O’Connell, T. N., Zandi, K. S., et al. (2006). Metabolism and
disposition of varenicline, a selective alpha4beta2 acetylcholine
receptor partial agonist, in vivo and in vitro. Drug Metababolism
and Disposition, 34, 121–130.
Oncken, C., Gonzales, D., Nides, M., Rennard, S., Watsky, E.,
Billing, C. B., et al. (2006). Efficacy and safety of the novel selective
nicotinic acetylcholine receptor partial agonist, varenicline, for
smoking cessation. Archives of Internal Medicine, 166, 1571–1577.
Paterson, N. E., Balfour, D. J., & Markou, A. (2008). Chronic
bupropion differentially alters the reinforcing, reward-enhancing
and conditioned motivational properties of nicotine in rats.
Nicotine & Tobacco Research, 10, 995–1008.
Patterson, F., Jepson, C., Strasser, A. A., Loughead, J.,
Perkins, K. A., Gur, R. C., et al. (2009). Varenicline improves
mood and cognition during smoking abstinence. Biological
Psychiatry, 65, 144–149.
Perkins, K., Lerman, C., Stitzer, M., Fonte, C., Briski, J., Scott, J.,
et al. (2008). Development of procedures for early screening of
smoking cessation medications in humans. Clinical Pharmacol-
ogy and Therapeutics, 84, 216–221.
Perkins, K. A., Stitzer, M., & Lerman, C. (2006). Medication
screening for smoking cessation: A proposal for new method-
ologies. Psychopharmacology, 184, 628–636.
Pfizer. (2007). Chantix prescribing information [Investigator
Brochure]. Retrieved from http://www.pfizer.com/files/products
Phillips, J. M., Ehrlichman, R. S., & Siegel, S. J. (2007). Mecam-
ylamine blocks nicotine-induced enhancement of the P20 audi-
tory event-related potential and evoked gamma. Neuroscience,
Pickworth, W. B., Herning, R. I., & Henningfield, J. E. (1989).
Spontaneous EEG changes during tobacco abstinence and nico-
tine substitution in human volunteers. Journal of Pharmacology
and Experimental Therapeutics, 251, 976–982.
Portugal, G. S., & Gould, T. J. (2007). Bupropion dose-
dependently reverses nicotine withdrawal deficits in contextual
fear conditioning. Pharmacology Biochemistry and Behavior, 88,
Powell, J. H., Pickering, A. D., Dawkins, L., West, R., & Powell, J. F.
(2004). Cognitive and psychological correlates of smoking
597 Download full-text
Nicotine & Tobacco Research, Volume 12, Number 6 (June 2010)
abstinence, and predictors of successful cessation. Addictive
Behaviors, 29, 1407–1426.
Radek, R. J., Miner, H. M., Bratcher, N. A., Decker, M. W.,
Gopalakrishnan, M., & Bitner, R. S. (2006). Alpha4beta2 nicotinic
receptor stimulation contributes to the effects of nicotine in the
DBA/2 mouse model of sensory gating. Psychopharmacology, 187,
Rahman, S., Lopez-Hernandez, G. Y., Corrigall, W. A., &
Papke, R. L. (2008). Neuronal nicotinic receptors as brain tar-
gets for pharmacotherapy of drug addiction. CNS & Neurologi-
cal Disorders Drug Targets, 7, 422–441.
Raybuck, J. D., Portugal, G. S., Lerman, C., & Gould, T. J.
(2008). Varenicline ameliorates nicotine withdrawal-induced
learning deficits in C57BL/6 mice. Behavioral Neuroscience, 122,
Rissling, A. J., Dawson, M. E., Schell, A. M., & Nuechterlein, K. H.
(2007). Effects of cigarette smoking on prepulse inhibition, its
attentional modulation, and vigilance performance. Psychophys-
iology, 44, 627–634.
Rollema, H., Guanowsky, V., Mineur, Y. S., Shrikhande, A.,
Coe, J. W., Seymour, P. A., et al. (2009). Varenicline has
antidepressant-like activity in the forced swim test and aug-
ments sertraline’s effect. European Journal of Pharmacology, 605,
Rudnick, N. D., Koehler, C., Picciotto, M. R., & Siegel, S. J.
(2009). Role of beta2-containing nicotinic acetylcholine recep-
tors in auditory event-related potentials. Psychopharmacology,
Shiffman, S., Ferguson, S. G., Gwaltney, C. J., Balabanis, M. H., &
Shadel, W. G. (2006). Reduction of abstinence-induced with-
drawal and craving using high-dose nicotine replacement ther-
apy. Psychopharmacology, 184, 637–644.
Shiffman, S., Johnston, J. A., Khayrallah, M., Elash, C. A.,
Gwaltney, C. J., Paty, J. A., et al. (2000). The effect of bupropion
on nicotine craving and withdrawal. Psychopharmacology, 148,
Siegel, S. J., Connolly, P., Liang, Y., Lenox, R. H., Gur, R. E.,
Bilker, W. B., et al. (2003). Effects of strain, novelty, and NMDA
blockade on auditory-evoked potentials in mice. Neuropsychop-
harmacology, 28, 675–682.
Siegel, S. J., Maxwell, C. R., Majumdar, S., Trief, D. F., Lerman, C.,
Gur, R. E., et al. (2005). Monoamine reuptake inhibition and
nicotine receptor antagonism reduce amplitude and gating of
auditory evoked potentials. Neuroscience, 133, 729–738.
Slawecki, C. J., Gilder, A., Roth, J., & Ehlers, C. L. (2003). In-
creased anxiety-like behavior in adult rats exposed to nicotine
as adolescents. Pharmacology Biochemistry and Behavior, 75,
Staley, J. K., Krishnan-Sarin, S., Cosgrove, K. P., Krantzler, E.,
Frohlich, E., Perry, E., et al. (2006). Human tobacco smokers in
early abstinence have higher levels of beta2* nicotinic acetylcho-
line receptors than nonsmokers. Journal of Neuroscience, 26,
Stevens, K. E., Kem, W. R., & Freedman, R. (1999). Selective
alpha 7 nicotinic receptor stimulation normalizes chronic
cocaine-induced loss of hippocampal sensory inhibition in C3H
mice. Biological Psychiatry, 46, 1443–1450.
Stevens, K. E., Kem, W. R., Mahnir, V. M., & Freedman, R.
(1998). Selective alpha7-nicotinic agonists normalize inhibition
of auditory response in DBA mice. Psychopharmacology, 136,
Stevens, K. E., Meltzer, J., & Rose, G. M. (1995). Nicotinic cho-
linergic normalization of amphetamine-induced loss of audito-
ry gating in freely moving rats. Psychopharmacology, 119,
Umbricht, D., Vyssotky, D., Latanov, A., Nitsch, R., Brambilla, R.,
D’Adamo, P., et al. (2004). Midlatency auditory event-related
potentials in mice: Comparison to midlatency auditory ERPs in
humans. Brain Research, 1019, 189–200.
Ward, M. M., Swan, G. E., & Jack, L. M. (2001). Self-reported
abstinence effects in the first month after smoking cessation.
Addictive Behaviors, 26, 311–327.
West, R., Baker, C. L., Cappelleri, J. C., & Bushmakin, A. G.
(2008). Effect of varenicline and bupropion SR on craving, nic-
otine withdrawal symptoms, and rewarding effects of smoking
during a quit attempt. Psychopharmacology, 197, 371–377.