THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics
J Pharmacol Exp Ther 349:348–354, May 2014
Activation of a4b2*/a6b2* Nicotinic Receptors Alleviates
Anxiety during Nicotine Withdrawal Without Upregulating
Nicole L. Yohn, Jill R. Turner,1and Julie A. Blendy
Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
Received November 26, 2013; accepted March 11, 2014
Although nicotine mediates its effects through several nicotinic
acetylcholine receptor (nAChR) subtypes, it remains to be
determined which nAChR subtypes directly mediate heightened
anxiety during withdrawal. Relative success in abstinence
has been found with the nAChR partial agonist varenicline
(Chantix; Pfizer, Groton, CT); however, treatment with this
drug fails to alleviate anxiety in individuals during nicotine
withdrawal. Therefore, it is hypothesized that success can be
found by the repurposing of other nAChR partial agonists for
cessation therapies that target anxiety. It is noteworthy that the
selective partial agonists for a4b2, ABT-089 [2-methyl-3-[2(S)-
pyrrolidinylmethoxy]pyridine], and a7, ABT-107 [5-(6-[(3R)-1-
azabicyclo[2.2.2]oct-3-yloxy] pyridazin-3-yl)-1H-indole] (AbbVie,
North Chicago, IL), have not been evaluated as possible
therapeutics for nicotine cessation. Therefore, we examined the
effect of ABT-089 and ABT-107 on anxiety during withdrawal
from nicotine in the novelty-induced hypophagia (NIH) para-
digm. We found that short-term administration of ABT-089 and
ABT-107 alleviate anxiety-like behavior during withdrawal from
nicotine while long-term administration of ABT-089 but not
ABT-107 reduces anxiety-like behavior during withdrawal. After
behavioral testing, brains were harvested and b2-containing
nAChRs were measured using [3H]epibaditine. ABT-089 and
ABT-107 do not upregulate nAChRs, which is in contrast to the
upregulation of nAChRs observed after nicotine. Furthermore,
ABT-089 is anxiogenic in nicotine naive animals, suggesting that
the effects on anxiety are specifically related to the nicotine-
dependent state. Together, these studies identify additional
nAChR partial agonists that may aid in the rational development
of smoking cessation aids.
Cigarette smoking isthe leading cause of death in the United
States each year [Centers for Disease Control and Prevention
(CDC), 2002]. However, approximately 20% of the American
population smokes despite the acknowledged health risks and
socioeconomic costs [Centers for Disease Control and Pre-
vention (CDC), 2011]. In addition, maintenance of smoking
cessation is at best modest, with 80% of smokers relapsing
within the first year of quitting (Polosa and Benowitz, 2011). It
is projected that if prevalence of use does not decrease from
present rates, cigarette smoking and tobacco use will result in
identification of effective smoking cessation therapies is ur-
Nicotine, which is the major addictive component in to-
bacco, plays a critical role in initial tobacco reinforcement and
dependence (Le Foll and Goldberg, 2006). Although many
factors influence ongoing nicotine dependence, relapse to
smoking is highly correlated with the severity of withdrawal
symptoms present during abstinence (Ockene et al., 2000;
Krall et al., 2002). These symptoms include difficulty con-
centrating, increased craving, depressed mood, and increased
anxiety (Hughes, 1992, 2007). Varenicline (Chantix; Pfizer,
Groton, CT), a partial agonist at a4b2 nicotinic acetylcholine
receptors (nAChRs) and a full agonist at a7 and a3b4 nAChRs
is currently the best in class treatment of smoking cessation
(Coe et al., 2005; Garrison and Dugan, 2009). However, al-
though varenicline has been shown to improve both concen-
tration and depressed mood and mitigate craving, recent
studies in mice and human subjects have shown treatment
does not improve nicotine withdrawal-induced anxiety
(Turner et al., 2013b; Cinciripini et al., 2013). This may be
of special importance because anxiety arising due to nicotine
withdrawal has been correlated with relapse rates (Zhou
et al., 2009).
Nicotine acts at multiple nAChR subtypes and understand-
ing which subtypes contribute to the detrimental side effects
experienced during withdrawal is critical for identification
of novel and improved therapeutics. More specifically, it is
This work was supported by the National Institutes of Health National
Cancer Institute [Grant P50-CA143187]; and the National Institutes of Health
National Institute on Drug Abuse [Grants K99-DA032681, T32-DA28874].
This work was presented at the following meeting: Yohn NL, Turner JR,
Klehm J, and Blendy JA (2013) Nicotinic acetylcholine receptor regulation
during nicotine withdrawal in mice. Society for Research on Nicotine and
Tobacco 19th Annual International Conference; 2013 Mar 16; Abstract No.
P054-99, Boston, MA.
1Current affiliation: Department of Drug Discovery and Biomedical Sciences,
South Carolina College of Pharmacy, University of South Carolina, Columbia,
ABBREVIATIONS: ABT-089, 2-methyl-3-[2(S)-pyrrolidinylmethoxy]pyridine; ABT-107, 5-(6-[(3R)-1-azabicyclo[2.2.2]oct-3-yloxy] pyridazin-3-yl)-
1H-indole; ANOVA, analysis of variance; [3H]EB, [3H]epibaditine; nAChR, nicotinic acetylcholine receptor; NIH, novelty-induced hypophagia.
at ASPET Journals on November 3, 2015
important to examine how targeting of the cholinergic system
can promote abstinence by reducing withdrawal-induced
anxiety. The various subtypes of nAChRs play different roles
in anxiety and nicotine withdrawal. For example, alterations
in the activation of the a4 nAChR subunit result in height-
ened anxiety (Ross et al., 2000; Labarca et al., 2001). Fur-
thermore, although a7 nAChRs have not been implicated
directly in anxiety (Paylor et al., 1998; Vicens et al., 2011) they
are necessary in the ventral tegmental area for the expression
of withdrawal from nicotine, suggesting that targeting of the
a7 subtype may also relieve nicotine withdrawal symptoms
(Nomikos et al., 1999).
Long-term nicotine exposure produces a region-specific up-
regulation of b2-containing nAChRs. This phenomenon is
thought to contribute to nicotine addiction (Wonnacott, 1990;
Marks et al., 1992; Buisson and Bertrand, 2002) and has been
confirmed in several systems, including cultured cells and
rodent and human tissues (Marks et al., 1983; Schwartz and
Kellar, 1985; Benwell et al., 1988; Peng et al., 1994; Breese
et al., 1997; Perry et al., 1999; Staley et al., 2006). Long-term
exposure to varenicline upregulates nAChRs, which parallels
its anxiolytic effects during cessation from nicotine (Turner
et al., 2011). However, the more selective a4b2 nAChR com-
pound, sazetidine-A, does not increase nAChRs despite
behavioral anxiolytic effects (Turner et al., 2010, 2013b;
Hussmann et al., 2012). Therefore, the role of nAChR up-
regulation in mediating withdrawal-induced anxiety is as yet
structure in Lin et al., 1997] is a selective partial agonist for
a4b2* receptors with high selectivity for a4a5b2 and activity
at a6b2* receptors (Rueter et al., 2004; Marks et al., 2009).
ABT-089 has been rigorously studied as a treatment of
cognitive disorders and has been used successfully in patients
with Alzheimer’s disease and attention deficit disorder, with
few unintended side effects (Lin et al., 1997; Rueter et al.,
2004). ABT-107 [5-(6-[(3R)-1-azabicyclo[2.2.2]oct-3-yloxy] pyr-
idazin-3-yl)-1H-indole; structure in Bitner et al., 2010] is
a selective agonist with high affinity at a7 nAChRs that has
been characterized as a cognitive enhancer in animal models
of Alzheimer’s disease and has low incidence of side effects at
varying doses in patients (Bitner et al., 2010; Malysz et al.,
2010; Othman et al., 2011). Both ABT-089 and ABT-107
enhance learning in naive animals (Decker et al., 1997; Bitner
et al., 2010). However, ABT-089 and ABT-107 have not been
evaluated for their role in reducing anxiety after nicotine
withdrawal or regulation of nAChRs. Therefore, we tested
ABT-089 and ABT-107 and the selective targeting of distinct
nAChR subtypes to identify which subtype may mediate
anxiety during withdrawal from nicotine.
Materials and Methods
Animals. Male 129SvJ;C57Bl/6J F1 hybrid mice (6–12 weeks of
age, 20–30 g) were purchased from Taconic Farms (Hudson, NY),
double-housed, and maintained on a 12-hour light/dark cycle with
food and water ad libitum in accordance with the University of
Pennsylvania Animal Care and Use Committee. All experimental
testing sessions were conducted between 9:00 AM and 1:00 PM, with
animals randomly assigned to treatment conditions and tested in
counterbalanced order. For all studies, mice were acclimated to the
behavioral testing facility for 1 hour prior to testing.
Drugs. Doses of nicotine tartrate (Sigma-Aldrich, St. Louis, MO),
ABT-089, and ABT-107 (synthesized by AbbVie) are reported as free
base weight. For short-term studies, all drugs were prepared imme-
diately before use in 0.9% saline and injected intraperitoneally. Dose
response curves indicated that 1.2 mg/kg for ABT-089 and 0.03 mg/kg
for ABT-107 (data not shown) do not alter locomotor activity in male
129SvJ;C57Bl/6J F1 hybrid mice.
For long-term treatment studies, nicotine (18 mg/kg per day), ABT-
089 (0.769 mg/kg per day), and ABT-107 (0.32 mg/kg per day) were
dissolved in 0.9% saline solution and administered for 14 days
subcutaneously via osmotic minipump (model 2002; Alzet, Cupertino,
CA). Mice were anesthetized with an isoflurane/oxygen mixture
(1–3%), and osmotic minipumps were inserted subcutaneously using
aseptic surgery techniques. Minipumps were placed parallel to the
spine at shoulder level with the flow moderator directed away from
the surgical incision. The wound was closed with 7-mm stainless steel
wound clips (Reflex; Cellpoint Scientific, Gaithersburg, MD).
Novelty-Induced Hypophagia. One week prior to the start of
training, mice were housed in groups of two. Training days consisted
of daily sessionsin which mice were exposed to a highly palatable food
(peanut butter chips; Nestle, Glendale, CA) presented in a clear
plastic dish. During training and home cage-testing sessions, a plastic
insert (dividing the standard cage lengthwise) was used to separate
mice in each cage. Mice were acclimated to the barriers 1 hour prior to
presentation of the food. Food was placed in the cage for 15 minutes,
and latency to consume was measured (seconds). Training criterion
wasmet oncea latencyunder20secondstoapproachandconsumethe
food with ,20% variability existed between mice.
Experiment 1: Short-Term Administration. For short-term
ABT-089/ABT-107 drug treatment studies, mice were implanted with
14-day osmotic minipumps filled with nicotine or saline (for experi-
mental design schematic, see Fig. 1A). On the last day of minipump
viability, animals were tested in the home cage environment 10 minutes
after an injection of saline. After the home test occurred, the nicotine
minipump was removed in three-fourths of the nicotine-treated
animals and half of the saline-treated animals. Twenty-four hours
later, animals were acclimated for a novel testing day and given an
intraperitoneal injection of ABT-089/ABT-107 or saline 10 minutes
prior to testing in the novel environment. The novel environment
consisted of an empty standard cage with no bedding that was wiped
with cleanser (1:10 Pine-Sol dilution; Clorox Co., Oakland, CA) to
supply a novel odor, and placed in a white box with white light illumi-
nation (2150 lux). Latency to consume was recorded over 15 minutes.
On both test days, the amount consumed (grams) of peanut butter
chips was recorded.
Experiment 2: Long-Term Administration. For long-term
drug treatment studies, mice were implanted with 14-day osmotic
minipumps filled with nicotine or saline. After 14 days, nicotine mini-
pumps were removed and replaced with a 14-day osmotic minipump
containing either nicotine, ABT-089, or ABT-107. After 7 days of the
second treatment, osmotic minipumps were removed from half of the
nicotine, ABT-089, and ABT-107 treatment groups to initiate spon-
taneous withdrawal. The remaining animals continued drug treat-
ment for an additional 7 days or underwent 7 days of withdrawal from
nicotine, ABT-089, or ABT-107. A home day was conducted, and novel
day testing occurred 24 hours later. Animals were killed, and brains
were harvested for receptor binding experiments (for experimental
design schematic, see Fig. 2A).
Receptor Binding. Mice used in the long-term ABT-089/ABT-
107 experiment were killed and used for the receptor binding
experiment. Brain regions examined were constrained by a minimal
tissue amount required for homogenate-binding assays. Tissues were
harvested from animals immediately after behavioral testing. The
samples were homogenized in 50 mM Tris-HCl (Sigma-Aldrich)
buffer, pH 7.4 at 24°C, and centrifuged twice at 35,000g for 10
minutes in fresh buffer. The membrane pellets were resuspended in
fresh buffer and added to tubes containing a saturating concentration
(2 nM) of [3H]epibaditine ([3H]EB; PerkinElmer Life and Analytical
Anxiolytic Effects and nAChR Regulation of ABT-089
at ASPET Journals on November 3, 2015
Sciences, Boston, MA). [3H]EB is a high-affinity ligand for all
heteromeric nAChRs with low nonspecific binding (Badio and Daly,
1994). [3H]EB was incubated with tissue in Tris buffer, pH 7.4, for
2 hours at 24°C with [3H]EB. Bound receptors were separated from
free ligand by vacuum filtration over GF/C glass fiber filters (Brandel,
Gaithersburg, MD)that werepretreated with0.5% polyethyleneimine
(Sigma-Aldrich). The filters were then counted in a liquid scintillation
counter.Nonspecificbinding wasdetermined inthe presence of 300mM
nicotine, and specific binding was defined as the difference between
total binding and nonspecific binding.
Statistical Analysis. All data are presented as mean 6 S.E.M.
For experiment 1, latency served as a dependent variable in two-way
analysis of variance (ANOVA) followed post hoc by Bonferonni
multiple comparisons test to detect differences. In experiment 2
(Fig. 2), a repeated measures two-way ANOVA was used to determine
significant differences between treatment groups with time (Home
Day, Novel Day) as a repeated-measure (within) factor. A planned
comparison (Bonferonni multiple comparison) was performed to test
the hypothesis that long-term ABT-089 or ABT-107 administration
during nicotine withdrawal shows decreased latency to consume in
a novel environment, comparing the Nic/ABT-089 and Nic/ABT-107
group with all other groups. For receptor binding studies, an ANOVA
followed post hoc by Bonferonni multiple comparisons was used to
detect differences between treatment groups. Statistical analyses
were carried out using the GraphPad Prism 5.0 software package
(GraphPad Software, San Diego, CA).
Short-Term Administration of ABT-089 and ABT-107
Is Anxiolytic during Nicotine Withdrawal In the NIH
Test. Withdrawal from long-term nicotine increases latency
to consume in a novel environment and short-term adminis-
tration of ABT-089 and ABT-107 significantly reduces this
latency (Fig. 1B). There are significant differences between
treatment groups on novel day [main effect of day, F(1,35) 5
82.34, P , 0.0001; main effect of treatment, F(4,35) 5 9.736,
P , 0.0001; interaction, F(4,35) 5 10.25, P , 0.0001].
Bonferonni post hoc analysis indicated that nicotine-treated
mice had significantly lower latency to consume (P , 0.05),
whereas mice experiencing 24-hour withdrawal from nicotine
Fig. 1. The effect of short-term ABT-089 and ABT-107 on the behavior of
mice during nicotine withdrawal in the NIH. (A) Mice were implanted
with osmotic minipumps containing nicotine or saline. After 2 weeks of
treatment, minipumps were removed from three-fourths of the nicotine
animals to induce spontaneous nicotine withdrawal. ABT-089 and ABT-
107 were administered intraperitoneally 10 minutes before NIH testing.
(B) Latency to consume in the novel environment was measured over 15
minutes and is reported as mean latency 6 S.E.M. *P , 0.05, **P , 0.01,
***P , 0.001 compared with saline on Novel Day. †P , 0.0001 compared
with 24-hour withdrawal (WD) on Novel Day (n = 8–12).
Fig. 2. The effect of long-term ABT-089 and ABT-107 on the behavior of
mice during nicotine withdrawal in the NIH. Data are mean (6 S.E.M.) of
treatment groups composed of four to nine mice per group. (A) Mice were
implanted with osmotic minipumps containing nicotine (Nic) or saline
(Sal). After 2 weeks of treatment, minipumps were removed from half of
the nicotine animals and replaced with a minipump containing ABT-089
or ABT-107. One week after minipump switch, minipumps were removed
from half of the nicotine, ABT-089, and ABT-107 treatment groups to
induce spontaneous withdrawal. (B) Animals were tested in the NIH and
latency to consume in the novel environment 2 weeks after minipump
switch and 1 week after removal of the second minipump was measured.
Latency to consume in the novel environment 2 weeks after minipump
exchange and 7-day withdrawal from nicotine is reported as mean latency 6
S.E.M. †P , 0.05compared with nicotine withdrawal on Novel Day. (n = 4–9).
Yohn et al.
at ASPET Journals on November 3, 2015
had significantly greater latency to consume (P , 0.001)
compared with saline-treated controls. Short-term adminis-
tration of ABT-107 significantly reduced latency to consume
the food in the novel environment during 24-hour withdrawal
from nicotine (P , 0.01) compared with saline controls.
Administration of ABT-107 and ABT-089 significantly re-
duced latency to consume at 24-hour withdrawal compared
with mice undergoing 24-hour withdrawal from nicotine (P ,
0.0001). Likewise, animals maintained on nicotine showed
a reduced latency to consume in the novel environment com-
pared with animals experiencing 24-hour withdrawal from
between home day and novel test day (data not shown).
Long-Term Administration of ABT-089 Is Anxiolytic
during Nicotine Withdrawal In the NIH Test. To better
model a therapeutic administration paradigm, we tested the
impact of ABT-089 and ABT-107 on withdrawal-induced
anxiety after long-term exposure of the drug. A schematic of
the long-term administration paradigm is shown in Fig. 2A.
Data in Fig. 2B demonstrate that 14-day administration of
ABT-089 and ABT-107 during nicotine withdrawal reduced
latency to consume compared with saline controls, although
not significantly [main effect of day, F(1,24) 5 18.22, P 5
0.0003]. Planned comparison of Nic/ABT-089 and Nic/ABT-
107 to other treatment groups revealed that compared with
nicotine withdrawal (7 days), ABT-089 significantly reduced
latency to consume (P , 0.05), whereas ABT-107 did not.
Animals in which minipumps were removed to induce
spontaneous withdrawal from ABT-089 or ABT-107 did not
show reduced latency to consume in the novel environment
compared with saline controls or nicotine withdrawal.
ABT-089 Does Not Upregulate nAChRs during Nico-
tine Withdrawal. As previously demonstrated, nicotine
upregulates nAChRs, and receptors return to basal levels as
early as 24 hour into withdrawal from nicotine (Fig. 3 and
Turner et al., 2011). Long-term nicotine increases nAChRs in
the hippocampus, cortex, striatum, and thalamus (Fig. 3;
main effect of treatment, hippocampus: [F(4,27) 5 8.462;
P 5 0.0001], cortex: [F(4,29) 5 15.91; P , 0.0001], striatum:
[F(4,29) 5 8.017; P 5 0.0002], thalamus: [F(4,28) 5 6.774; P 5
0.0006]. However, after 7 days of withdrawal from nicotine,
nAChRs are no longer upregulated compared with saline
control animals. ABT-089 (Nic/ABT-089) and ABT-107 (Nic/
ABT-107) administration during withdrawal from nicotine
does not maintain upregulated nAChRs compared with saline
ABT-089 Alone Does Not Upregulate nAChRs. To
determine whether long-term administration of ABT-089
alone can upregulate nAChRs, brain regions of interest were
harvested from animals exposed to ABT-089 for 14 days.
Long-term administration of ABT-089 in nicotine naive mice
did not significantly upregulate nAChRs in the hippocampus,
cortex, striatum, or thalamus of treated animals compared
with saline controls: hippocampus: [F(2,21) 5 0.3893 ;P 5
0.6820), cortex: [F(2,19) 5 0.3749; P 5 0.6923], striatum: [F
(2,21) 5 0.9581; P 5 0.3998], thalamus: [F(2,20) 5 2.985; P 5
0.0734]. Additionally, there is no up- or downregulation of
receptors after 24-hour withdrawal from ABT-089 in brain
regions chosen for analysis (Fig. 4).
ABT-089 Alone Is Anxiogenic in Naive Animals. Be-
cause ABT-089 blocked nicotine-induced anxiety long term in
nicotine-dependent mice, we tested the effects of long-term
administration of ABT-089 in nicotine naive mice. An effect of
day was revealed using two-way ANOVA [F(1,21) 5 61.06;
P , 0.0001]; however, there was no effect of treatment on
latency to consume (Fig. 5). Although latency to consume in the
novel environment appears greater in animals treated with
long-term administration of ABT-089 as well as in animals
undergoing 24-hour withdrawal from long-term ABT-089 at
the time of testing, this increase in latency is nonsignificant
Fig. 3. Nicotinic receptor regulation after nicotine withdrawal in the
presence or absence of ABT-089 or ABT-107. Homogenate-binding
experiments with a saturating concentration of [3H]EB were performed
on hippocampal, cortical, striatal, and thalamic tissues from animals
treated long term with nicotine for 2 weeks and then long term with ABT-
089 or ABT-107 for 2 weeks during withdrawal from nicotine. Additional
treatment groups include animals treated with long-term saline (4 weeks),
long-term nicotine (4 weeks), and animals treated with nicotine for 3
weeks and then withdrawn from nicotine for 7 days (via nicotine minipump
groups. *P , 0.01, **P , 0.001, ***P , 0.0001 compared with saline
(n = 5–10).
Fig. 4. Nicotinic receptor regulation after long-term treatment with ABT-
089. Homogenate-binding experiments with a saturating concentration of
[3H]EB were performed on hippocampal, cortical, striatal, and thalamic
tissues from animals treated long term with ABT-089 or saline for 2
weeks. Minipumps were removed from half of the ABT-089 animals to
induce spontaneous withdrawal for 24 hours. Data are mean (6S.E.M.) of
treatment groups (n = 7–8).
Anxiolytic Effects and nAChR Regulation of ABT-089
at ASPET Journals on November 3, 2015
Short-term administrations of ABT-089 and ABT-107
alleviate anxiety during nicotine withdrawal; however, only
ABT-089 is effective in alleviating anxiety after long-term
administration. Additionally, ABT-089 mediates its effects
without upregulating nAChRs, a hallmark of sustainednAChR
activation with nicotine. Therefore, ABT-089 may be an ef-
fective compound in treating individuals with heightened
anxiety during nicotine abstinence through a different cellu-
lar mechanism than other cessation therapies.
Although administration of varenicline during smoking
cessation results in the improvement of both positive affect
and cognitive function (Patterson et al., 2009), efficacy in
alleviating anxiety during nicotine withdrawal is more com-
plicated (Cinciripini et al., 2013). Specifically, short-term but
not long-term administration of varenicline reduces anxiety
during withdrawal. For example, in naive animals, short-
term and long-term varenicline administration is anxiolytic in
the NIH test (Turner et al., 2010). However, in nicotine-
experienced animals, short-term administration of vareni-
cline during nicotine withdrawal fails to alleviate anxiety-like
behavior (Turner et al., 2013b), suggesting a differential effect
of varenicline based on the drug experienced state of the
subject. Additionally, a recent study in smokers identified
anxiety as one of the symptom domains in which varenicline
treatment was ineffective (Cinciripini et al., 2013). Therefore,
this lack of anxiolytic activity during nicotine withdrawal may
underlie the low success rate of varenicline in a subset of
smokers (Garrison and Dugan, 2009; Moore et al., 2010).
Because varenicline acts at a number of different nAChR
subtypes, identifying those subtypes that underlie the bene-
ficial effects of the drug while avoiding subtypes responsible
for negative side effects is necessary. Additionally, using a
more selective ligand to produce desirable effects during nic-
otine withdrawal could provide tailored therapies to suit the
individual needs of quitters.
Nicotine exerts its biologic effects through activation of
central nAChRs that exist as subtypes determined by a and b
subunit compositions (Gotti and Clementi, 2004). The hetero-
meric a4b2* subtype and the homomeric a7 are the most
prevalent receptor subtypes in the central nervous system
(Court et al., 2000). Varenicline, the best in class medication
for smoking cessation, acts at both of these subtypes (Coe
et al., 2005). ABT-089 is selective for the a4b2* and a6b2*
(Marks et al., 2009) subtypes that have been implicated in
anxiety (Ross et al., 2000; Labarca et al., 2001). ABT-107 is
selective for the a7 subtype (Malysz et al., 2010) that has been
implicated in generalized nicotine withdrawal syndrome
(Nomikos et al., 1999). Therefore, we used these highly se-
lective nicotinic compounds to determine the contribution of
specific subtypes during nicotine withdrawal on anxiety using
the NIH paradigm and further examined the effects of these
drugs on receptor regulation with [3H]EB binding assay.
The NIH paradigm is a sensitive measure for potential
anxiolytic drugs and anxiogenic effects of withdrawal on both
acute and extended drug administration paradigms (Dulawa
et al., 2004; Dulawa and Hen, 2005). The NIH test is sensitive
to the anxiolytic effect of long-term nicotine (Turner et al.,
2010, 2013b) and the anxiogenic effect of 24-hour withdrawal
from nicotine (as shown in Fig. 1). Short-term administration
of ABT-089 and ABT-107 during 24-hour withdrawal from
long-term nicotine treatment reduces anxiety-like behavior in
animals (Fig. 1B). However, long-term administration of ABT-
089, but not ABT-107, during nicotine withdrawal showed
a significant decrease in latency to consume compared with
animals undergoing 7-day withdrawal from nicotine (Fig. 2B).
Nicotine withdrawal typically causes an increased latency
to consume food in a novel environment above that of saline-
treated animals (Fig. 1B and Turner et al., 2010, 2013a,b).
However, these observations are generally evident during the
first 24 to 72 hours of nicotine withdrawal. In Fig. 2B, the
long-term effects of nicotine withdrawal are observed, and
data demonstrate that increased anxiety occurs compared
with long-term nicotine administration during nicotine
withdrawal that persists beyond a 72-hour time point. Thus,
long-term exposure of ABT-089 throughout the withdrawal
period when individuals may be particularly vulnerable to
withdrawal-induced anxiety is an important aspect of our
study design. In humans, although most reports of anxiety
occur during the first 24 hours, a return to baseline can take
up to 4 weeks (Hughes, 1992). Therefore, the efficacy of ABT-
089 to reduce anxiety within an extended time period
suggests that it would be an effective treatment of the
alleviation of both the initial anxiety experienced during
withdrawal (Fig. 1) as well as anxiety over the course of
abstinence. However, it should be noted that the anxiolytic
effect of ABT-089 is only evident when drug is on board,
because latency to consume in a novel environment is in-
creased 7 days after withdrawal from ABT-089 (Fig. 2B).
Previous studies suggest that the upregulated pool of
nAChRs arising from long-term exposure to nicotine may
drive elements of the nicotine withdrawal syndrome (Turner
et al., 2011; Gould et al., 2012), and this protracted up-
regulation of nAChRs has been correlated with reduced
ability to maintain abstinence in the clinical population
(Staley et al., 2006). Therefore, to determine whether ABT-
089 reduced anxiety during nicotine withdrawal in the NIH
via maintenance of upregulated nAChRs, we quantified
Fig. 5. The effect of long-term ABT-089 and withdrawal from ABT-089 on
the behavior of mice in the NIH. Data are mean (6S.E.M.) of treatment
groups composed of eight mice per group. Mice were implanted with
osmotic minipumps containing ABT-089 or saline. After 2 weeks of
treatment, minipumps were removed from half of the ABT-089 animals to
induce spontaneous nicotine withdrawal. Twenty-four hours later, latency
to consume in a novel environment over 15 minutes was measured in all
treatment groups. Data are presented as mean latency (6S.E.M.) of
treatment groups (n = 8).
Yohn et al.
at ASPET Journals on November 3, 2015
[3H]EB binding density after long-term ABT-089 administra-
tion during nicotine withdrawal. Long-term ABT-089 does not
upregulate heteromeric nAChRs during nicotine withdrawal
(Fig. 3). However, it is effective in alleviating anxiety during
withdrawal (Fig. 2B). Likewise, long-term ABT-107 does not
upregulate heteromeric nAChRs (Fig. 3). This outcome was
expected, because a7 nAChRs do not upregulate to the same
degree as heteromeric nAChRs after long-term nicotine
treatment (Mugnaini et al., 2002). These findings suggest
that ABT-089 functionally reduces anxiety during nicotine
withdrawal without maintaining upregulation of heteromeric
nAChRs. Therefore, in contrast to varenicline, ABT-089 al-
lows nAChRs to downregulate back to saline levels while pro-
viding an anxiolytic effect during withdrawal from nicotine.
Because of the efficacy of ABT-089 in alleviating nicotine
withdrawal-induced anxiety, we sought to determine whether
long-term ABT-089 alone could reduce anxiety in the NIH,
thereby broadening the clinical applications of this compound.
In addition, we evaluated whether abstinence from long-term
administration of ABT-089 produces a withdrawal state that
may affect anxiety-like behavior. We found that administra-
tion of ABT-089 for 2 weeks in drug-naive animals did not
decrease anxiety in the NIH. In addition, 24-hour withdrawal
from ABT-089 did not significantly increase latency compared
with saline-treated animals (Fig. 5). Although a previous
study showed ABT-089 decreased anxiety, this was after
short-term rather than long-term administration (Lin et al.,
1997). In addition, unlike the decrease in latency observed
with long-term nicotine administration (Fig. 1B), ABT-089
administration fails to produce an anxiolytic effect. The vastly
different behavioral effects of ABT-089 compared with nico-
tine suggest that the partial agonist activity produces dif-
ferent biochemical effects compared with the promiscuous
activity of nicotine. Additionally, our findings from [3H]EB
binding assays demonstrate that long-term ABT-089 admin-
istration in drug-naive animals does not upregulate nAChRs
(Fig. 4), similar to its effects in nicotine-dependent animals.
Our findings suggest that partial activation of a4b2*/a6*
and a7 nAChRs may have an effect on the initial anxiety
experienced during nicotine withdrawal, and sustained
activation of a4b2*/a6* nAChRs through long-term exposure
to ABT-089 during nicotine withdrawal alleviates anxiety.
This finding supports previous research implicating a4 in
anxiety (Ross et al., 2000; Labarca et al., 2001). Additionally,
b2 knockout mice and, therefore, bereft of a4b2* heterodimers
have reduced anxiety during withdrawal from long-term nico-
tine and after mecamylamine precipitated withdrawal from
nicotine (Jackson et al., 2008). Finally, a subset of a6-containing
nAChRs (a6b2b3*) regulates sustained release on dopamine
nerve terminals and, therefore, may provide the efficacy through
which both short-term and long-term ABT-089 alleviate with-
drawal symptoms (Marks et al., 2009). To date, a7 nAChRs
have not been implicated in anxiety during nicotine with-
drawal (Vicens et al., 2011), and they do not appear to be nec-
essary for somatic signs of withdrawal (Markou and Paterson,
2001). However, as short-term ABT-107 reduced latency to
consume during nicotine withdrawal, the targeting of a7 nAChRs
may relieve withdrawal symptoms by activating nAChRs in
the absence of nicotine.
Finally, findings from this study suggest a complex associ-
ation between nAChR regulation and behavior. This study is
the first to demonstrate that the selective partial nAChR
agonist ABT-089 reduces anxiety during nicotine withdrawal
without maintaining the upregulated pool of heteromeric
nAChRs. However, although the ligand binding results are
similar in drug-naive and drug-experienced animals exposed
to long-term administration of ABT-089, their behavioral
profiles are strikingly different. These findings highlight the
importance of drug screening in nicotine-experienced ani-
mals. Interestingly, ABT-107, a selective partial agonist at
a7 nAChRs is capable of alleviating anxiety at 24-hour
withdrawal from nicotine, but not after extended administra-
tion, suggesting the role of several types of nAChRs in the
initial withdrawal symptoms after nicotine cessation that
may differ when withdrawal is extended. Therefore, the
temporal specificity of the roles of discrete nAChR subtypes
after the onset of nicotine withdrawal needs to be further
studied to better identify improved therapeutics for nicotine
The authors thank AbbVie for generously providing ABT-089 and
ABT-107 and Dr. Lynne Rueter (Associate Director II, Neuroscience
Discovery, AbbVie) for helpful discussions. They also thank Emily
Fernandez for experimental assistance.
Participated in research design: Yohn, Turner, and Blendy.
Conducted experiments: Yohn.
Performed data analysis: Yohn and Turner.
Wrote or contributed to the writing of the manuscript: Yohn,
Turner, and Blendy.
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Address correspondence to: Julie A. Blendy, Department of Pharmacology,
Perelman School of Medicine, University of Pennsylvania, 125 S. 31st Street,
Philadelphia, PA 19104. E-mail: firstname.lastname@example.org
Yohn et al.
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