CRF-CRF System Activation Mediates Withdrawal-Induced Increases in Nicotine SelfAdministration in Nicotine-Dependent Rats Author(s): Olivier George, Sandy Ghozland,

Marc R. Azar, Pietro Cottone, Eric P. Zorrilla, Loren H. Parsons, Laura E. O'Dell, Heather N. Richardson, George F. Koob Reviewed work(s): Source: Proceedings of the National Academy of Sciences of the United States of America, Vol. 104, No. 43 (Oct. 23, 2007), pp. 17198-17203 Published by: National Academy of Sciences Stable URL: http://www.jstor.org/stable/25450205 . Accessed: 23/04/2012 01:19
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mediates withdrawal CRF-CRF? system activation in nicotine increases induced self-administration rats in nicotine-dependent
Olivier George**, Sandy Ghozland*, Marc R. Azar*, Heather N. Richardson*, and George F. Koob*
on the Neurobiology *Committee Texas, El Paso, TX 79968 Communicated Nicotine, of Addictive Disorders,

Pietro Cottone*,

Eric P. Zorrilla*,
La Jolla, CA 92037;

Loren H. Parsons*,

Laura E. O'Dell*,

The Scripps Research

Institute,

and ^Department

of Psychology,

University

of

by Floyd E. Bloom, The Scripps Research

Institute,

La Jolla, CA, August central

13, 2007 nucleus

(received of and

for review May the amygdala CRF

1, 2007) measured were in vivo by using and assessed before

the main of tobacco, induces psychoactive ingredient emotional that contribute abstinence symptoms negative during to a profound for nicotine. the neurobiological However, craving re nicotine how mechanisms produces dependence underlying mains both one mechanism We understood. demonstrate for poorly the anxiety-like of withdrawal and excessive symptoms intake observed nicotine after abstinence, recruitment of through stress the extrahypothalamic fac peptide corticotropin-releasing tor the may bility (CRF) system CRF-CRFi represent to tobacco and activation may system a prominent addiction. | stress of CRFi contribute target receptors. to nicotine Overactivation of and dependence the vulnera investigating

were levels

microdialysis

RIA.

after precipitated withdrawal by administering mecamylamine to block nicotine receptors in rats with chronic administration of nicotine (nicotine-dependent rats) or saline (nondependent delivered by osmotic minipumps rats), (9). In dependent rats,
mecamylamine robustly increased CRF-like immunoreactivity

(CRF-L-IR) in the central amygdala (by >500% compared with baseline, Fig. IB), with levels returning to baseline after 2 h (Fig.
1A). between This increase was not observed in saline-treated rats,

for

injected with mecamylamine,

the two groups.

and baseline

levels did not differ

abstinence

| addiction

| amygdala

| deprivation

Precipitated Activation

Withdrawal of CRF<| Receptors.

Increases To test

addiction is the leading avoidable cause of disease and premature death in the U.S., responsible for >400,000 Tobacco deaths annually (1,2). The main psychoactive ingredient respon sible for tobacco addiction has long been hypothesized to be
nicotine. Nicotine acutely

Anxiety-Like the hypothesis

Behavior

Through that withdraw

al-induced increases in CRF activity, through activation of the CRFi receptor, might be a mechanism responsible for the
appearance of a negative emotional state, we measured anxiety

like behavior
dependent rats

during
and

precipitated
nondependent

withdrawal
rats, using

in nicotine
the defensive

effects (3, 4) by activating reward systems, including the me solimbic dopamine system (5, 6). However, the transition from nicotine use to nicotine dependence has been hypothesized to
result from powerful tation may neuroadaptative to continue need involve changes tobacco in the brain use (7, 8). that produce for neuroadap the negative a Such

produces

modest

positive

reinforcing

the mechanisms The

injection burying test (16,17). In dependent rats, mecamylamine increased the time spent burying (+243%), and decreased the two markers of active anxiety-like latency to bury (-70%), behavior (17), compared with vehicle injection (Fig. 1C) without affecting general activity (rearing), nonanxiety behaviors (rest
ing, grooming), or a passive not form of anxiety-like behavior

responsible negative

emotional
dependent powerful drug

states observed
individuals source (9,

during abstinence
10).

from nicotine
emotional state

in

(freezing)
Mecamylamine

[see also

supporting
with

information
alter

(SI) Table
behavior effects

2].
in of

produced by nicotine withdrawal

of negative intake.

is hypothesized
leading nicotinic

to represent a
to excessive an

reinforcement

Spontaneous such tagonists cally decreases

and precipitated (using as mecamylamine) nicotine reward function and brain effect of nicotine,

withdrawal the efficacy suggesting

receptor dramati

nondependent on extracellular mecamylamine 1A and B). Pretreatment (Fig. CRFi receptor nonpeptide

did injection consistent rats,

anxiety-like the differential

of CRF-L-IR levels amygdala a selective small-molecule with antagonist (Af,7V-bis(2-methoxy

of natural

ethyl)-3-(4-methoxy-2-methylphenyl)-2,5-dimethyl-pyrazolo

reinforcers
initial, weak

in rodents (9, 11). These

reinforcing

effects occur despite

the

[1,5a] pyrimidin-7-amine

(MPZP), 4 mg/kg,

s.c.)?11blocked

the

there must Author contributions: O.G. and S.G. contributed equally to thiswork; O.G., S.G., P.C., E.P.Z., L.H.P., L.E.O.,H.N.R., and G.F.K. designed research; O.G., S.G.,M.R.A., P.C., E.P.Z.,and L.H.P. performed research; O.G. and S.G. analyzed data; and O.G., S.G., and G.F.K.wrote the paper. The authors declare no conflict of interest. Abbreviations: CRF, corticotropin-releasing factor; MPZP, (A/,/V-bis(2-methoxyethyl)-3-(4 CRF-L-IR,CRF methoxy-2-methylphenyl)-2,5-dimethyl-pyrazolo[1,5a]pyrimidin-7-amine; like-immunoreactivity. +To whom correspondence should be addressed. E-mail: ogeorge@scripps.edu. ^Richardson, H. N., Funk, C. K., Grant, Y., Zorrilla, E. P., Koob, G. F. (2006) Program No. 783 4 Neuroscience Meeting Planner, Atlanta, GA (Soc Neurosci, Washington, DC), www. sfn.org/am2006/. TSpecio, S. E., Zorrilla, E. P.,O'Dell, L. E., Boutrel, B., Smith, R.T., Grant, Y., Koob, G. F. (2004) Program No. 77711 Neuroscience Meeting Planner (Soc Neurosci, Washington, DC), http://sfn.scholarone.com/itin2004/. This article contains supporting information online at www.pnas.org/cgi/content/full/ 0707585104/DC1. ? 2007 by The National Academy of Sciences of the USA

be other mechanisms driving the development of nicotine de pendence. The general hypothesis tested here is that chronic
nicotine use recruits a major brain stress system, the extrahy

factor (CRF) system (7, 12 pothalamic corticotropin-releasing 15), which contributes critically to the motivation to continue tobacco use. To this end, we tested whether (/) nicotine with drawal activates the CRF system in the central nucleus of the amygdala, (ii) CRF overactivity, via CRF type 1 receptors
(CRFi), and induces an anxiety-like abstinence of state, a component the motivation mechanism. of the to

negative emotional

state hypothesized

to drive nicotine depen

increases

dence, (Hi) nicotine take nicotine, by means

a CRFi-dependent

Results
Precipitated Withdrawal Increases CRF Levels in the Central Nucleus of

the Amygdala. To test the hypothesis that nicotine withdrawal activates the extrahypothalamic CRF system, CRF levels in the
17198-17203 I PNAS | October 23,2007 | vol.104

no.

43 www.pnas.org/cgi/doi/10.1073/pnas.0707585104

B
-O- Non dependent .-#- Nicotine dependent]

800

ep ??
o*

CO

s| _j
?

60?
CD

iNon dependent iNicotine dependent

400 O) 300 c
>* -Q

CZ3 IZ2 I ?&

Vehicle + Vehicle CRF1 antagonist Mecamylamine Mecamylamine + CRF1 antagonist

ul c? 400
rrc? 200

200

c O O

? Vehicle
oooooooooooo

Mecamylamine Vehicle Mecamylamine

C 0 Q. C/5 100 CD E

P
Treatment

\ ill
saline

pump

nicotine

pump

Time (min)

Treatment

on extracellular levels of CRF-L-IR in the central nucleus of the amygdala and CRF antagonist Effects of mecamylamine-precipitated nicotine withdrawal Fig. 1. in rats by using the defensive behavior blockade of precipitated withdrawal-induced (1.5 mg/kg, i.p.) burying test. (A) Effect of mecamylamine anxiety-like as measured in chronic nicotine on extracellular levels of CRF-L-IR in the central nucleus of the amygdala by in vivo microdialysis -precipitated withdrawal = = n n 6) rats (*, P < 0.05 vs. nondependent). (B) CRF-L-IR levels 7) and chronic saline pump-treated (nondependent, pump-treated (nicotine-dependent, of baseline (first three samples) during the first four samples after vehicle or mecamylamine injections (*, P < 0.05 vs. vehicle). (0 CRF? expressed as percentage in rats by using the defensive behavior blockade of precipitated withdrawal-induced (1.5 mg/kg, i.p.) anxiety-like burying test. Mecamylamine antagonist with the CRF? antagonist rats increased the time spent burying (*, P < 0.05 vs. vehicle), an effect blocked by pretreatment (MPZP, injection in nicotine-dependent = 7-9 4 mg/kg s.c, -1 h) (n Data represent mean ? SEM. per group, #, P < 0.05 vs. mecamylamine).

anxiogenic-like

effect

of mecamylamine

to

increase

burying

in

abstinence

from

chronic

nicotine

administration

increases

anx

rats (Fig. 1C). nicotine-dependent To confirm that increases of CRF in the central nucleus of the
amygdala elicit anxiety-like behavior, we measured anxiety-like

iety-like behavior in rats (21, 22). Nicotine intake significantly increased during the first session after each cycle of abstinence (Fig. 2A) and returned to baseline by the 4th day of nicotine
self-administration. The "nicotine-deprivation effect" reflected

behavior using the defensive burying test, after bilateral infusion of CRF (30 pmol total dose) in the central nucleus of the
amygdala of naive rats. CRF administered directly into the amyg

mainly increased drug intake during the active (dark) period, which represents ^80% of daily nicotine intake, but a significant
(data albeit increase, smaller, not shown). Rats amount 6.4 ? of 1.2 was also observed during the exhibited "drug-loading" requiring of prethan light period dur behavior attained

dala increased the time spent burying and decreased the latency to bury during the first 5 min after infusion (Table 1), without
affecting other behaviors (rearing, grooming, resting, and freez

ing the beginning of the active period such that the nondeprived
baseline in only intake h. normally Scatter plot higher 12 h was vs. postabstinence

ing). The low burying baseline observed in these animals can be explained by the extensive handling, and the higher body weight of the rats (^600g) in this experiment, two factors known to decrease baseline level of burying (18, 19). Such a low baseline allows for anxiogenic-like effects to be detected more easily and has been reported previously in young rats under different
conditions tor activation (20). Thus, increased abstinence CRF appears during and CRFi release, recep to mediate anxiety-like

z u 0

nicotine
nence

intake shows that the majority

intakes were

(>93%)
preabstinence

of postabsti
nicotine

nicotine

intakes, demonstrating the robustness of the phenomenon (Fig. 273). The fact that postabstinence nicotine intakes measured during the four successive cycles were (i) highly correlated with
each other

behavior during precipitated withdrawal in nicotine-dependent rats. This hypothesis predicts that, in dependent rats, a period of
abstinence subsequent lead to an increase may access to nicotine and in nicotine that blocking intake the during action the of

= 2C) and (ii) evenly distributed around they x line, and (Hi) that the coefficient of variation between subjects was three times higher than the coefficient of variation within subjects (Fig. 2D) demonstrate the existence of reliable interindividual differences
in the We effect then of abstinence the effect in nicotine after on nicotine course exposing intake 3 days of was intake. of rats appearance to different of the dura evaluated time by

(mean

r =

0.81,

range:

0.72-0.92,

all P <

0.05;

Fig.

CRF
increase

using

a CRFi

receptor
intake.

antagonist

could prevent

this

in nicotine Increases we

nicotine-deprivation Abstinence nicotine Nicotine To extended access at a used Intake an access consisted constant in Rats Given the animal of unit effect model four Extended of of Access to on Self-Administration. intake, to 23-h evaluate abstinence intermittent of per induced reached relevance dence, a we increase a maximum of the

tions of abstinence, from 1 h to ^2 months

(1,201 h). Abstinence

48 h, after significant and remained abstinence,

exposure The intermittent self-administration

to nicotine dose

self-administration. consecutive (0.03 days mg/kg

elevated even after 2 months of abstinence

tested nicotine-deprivation the effect of 3 days not to induce effect

(Fig. 2E). To test the

to nicotine depen in rats given of

of abstinence

injection), followed by 3 days of abstinence,

Table 1. Effect on of CRF defensive infusion burying Latency to bury, s 341 ? 78 165 ? 91* in the central

because

3 days of
the

limited access to nicotine

condition known

self-administration
any

(1 h per session),
signs

spontaneous

nucleus

of

withdrawal (23). We found that abstinence had no effects in rats with limited access (Fig. 3B), whereas, as observed in the
previous experiments, lever abstinence were markedly not affected increased nicotine

amygdala

responding
Grooming, 21 ? 17 s Rearing, 103 ? 91 ? s 15 15 3A). Inactive

in rats with extended access (23 h per session) (Fig.

responses by abstinence. Increases system

Treatment Vehicle CRF (30 pmol)

Burying, 0.1 ? 0.2 7.1 ? 4.7*

30 ? 27

Antagonism in Nicotine

Intake. To

of CRFi Receptor evaluate

Prevents the

Abstinence-Induced role of the CRF-CRFi

CRF (30 pmol) or vehicle (PBS) were infused in the central nucleus of the 1min before the beginning of the defensive burying test. Behaviors amygdala were recorded during the first 5 min of the test. Resting and freezing did not differ between groups. *, P < 0.05 vs. vehicle.

in the nicotine-deprivation
CRFi with receptor intermittent antagonist access

effect, we
MPZP

tested the effect of the

in rats responding self-administration

on nicotine nicotine

to extended

(23 h, 4 d/week). After

George et al PNAS

abstinence, pretreatment with the CRFi

| October 23,2007 | vol.104 | no. 43 | 17199

<45.8% 45678 910 12341234123 Baseline ND-, ND2 ND3 Sessions 0.9 1.2 1.5 1.8 2.1 2.4 2.7 NDE. 1.8 2.1 2.4 2.7

Pre-ND intake (mg/Kg) 0.5,

1.8 1.6

(mg/Kg)

1-4

1-2 '%% O Q_ 0.8

Between Within 0.6 L 0.1 1 ri 10 100 1000 10000

Duration of Abstinence

(h)

Characterization of the nicotine-deprivation effect. (A) Total (23-h) active and inactive responses after repeated cycles of 72 h of nicotine deprivation Fig. 2. effect. Scatter plot of nicotine intakes observed (ND), followed by 4 days of self-administration (*, P< 0.05 vs. baseline). (B) Robustness of the nicotine-deprivation The numbers represent the percentage of measures during the first session before (pre-ND) and after (post-ND) each of the four cycles of nicotine deprivation. above and below the y = x line. (Q Reliability of the nicotine-deprivation effect. Correlation of post-ND nicotine intakes between each of the four cycles (ND(-d vs. ND(o) = NDi vs. ND2, ND2 vs. ND3, and ND3 vs. ND4). (D) Coefficient of variation of post-ND intakes between subjects vs. within subjects (*, P < 0.05). (?) Effect of duration of abstinence 12-h period of nicotine access. (*, P < 0.05 vs. 1 h). Note logarithmic time scale. Dotted (h) on active responses during the subsequent lines represent mean ? SEM of the 1-h time point (*, P < 0.05 vs. 1 h). Data represent mean ? SEM.

antagonist dose-dependently
compared deprivation the CRFi induced with effect receptor escalation vehicle-treated compared

decreased nicotine
rats, and with baseline blocked levels.

intake (Fig. 44)

the As nicotine nicotine expected, self the

robust

increase

in nicotine

intake

in rats allowed Finally, the

extended increased

access nicotine

to nicotine

self-administration.

intake can be blocked

receptor Nicotine rats antagonist. withdrawal,

by pretreatment
precipitated to nicotine. a maximum

with
by

a specific CRFi
in

antagonist of nicotine intake

decreased occurred

administration

during the active (dark) period when abstinence

but not of during

mecamylamine,

creased CRF release

chronically concentration exposed reached

inactive (light) period

receptor antagonist

(data not shown). Efficacy

with the magnitude

in the central nucleus of the amygdala

Interstitial 30 min after amygdalar mecamylamine CRF

in

of the CRFi
the nico

correlated

tine-deprivation effect observed in any given subject (Fig. AB). CRFi receptor antagonist efficacy did not correlate with the = magnitude of baseline responding (r 0.05, not significant) and had no effect in rats given limited access to nicotine (1 h) (Fig.
a 4C), supporting specific nicotine dependence. relation to abstinence responding and

injection, with levels returning to baseline after 2 h. This pattern half-life of may be explained by the short pharmacokinetic mecamylamine (?4 h) (24), and the constant exposure to
nicotine. dependent were both Withdrawal-induced rats and associated CRF release in nicotine rats, and infusion of CRF intraamygdala an increase with in time spent in na?ve burying,

Discussion
This demonstrates that precipitated report tine dependent CRF release rats, increases of the amygdala and increases anxiety-like a CRFi-dependent mechanism. Nicotine withdrawal, in the central behavior abstinence in nico nucleus of a

a decreased latency to bury in the defensive burying test, whereas the CRFi receptor antagonist MPZP reversed the increase in
defensive nicotine ses face observed burying withdrawal. The and predictive during mecamylamine-precipitated test defensive burying as an animal model of posses normal

by means produces

validity

and pathological
reflects and an active is respectively

anxiety;

in particular,

time spent burying

environment, anxiolyticand

to an anxiogenic strategy coping and decreased increased by

B * i=i Baseline Post Abstinence 40 30 i Baseline Post Abstinence

anxiogenic-like mecamylamine,
or This nonanxiety argues

of compounds (16, 17, 25). Administration MPZP, or CRF did not change general activity
behaviors such as rearing, effects, and resting, and nonspecific confirming grooming. an earlier

against

? 20 or

report showing that CRF infusion in the central nucleus of the amygdala does not alter feeding or grooming behavior (26).
Also, the increase or CRF in may and the time of defensive administration mecamylamine an increase conditions to passive observed burying was not associated after with

Active Inactive Lever Press

La

Active Inactive Lever Press

effect. Total responses during Fig. 3. Specificity of the nicotine-deprivation = = the entire session in rats given extended (23 h, n 7) (A), or limited (1 h, n 6) (B) access to nicotine before and after 72 h of abstinence (*, P < 0.05 vs. baseline). Data represent mean ? SEM.

under these spent freezing. Freezing a different measure of anxiety related represent can be dissociated not active and avoidance from the active form of anxiety measured by

pharmacologically

the time spent burying or the latency to bury (17). Perhaps the CRF-CRFi system is not involved in all aspects of negative

17200

I www.pnas.org/cgi/doi/10.1073/pnas.0707585104

George

et

al.

veh

veh Nicotine (Active

20

40

60

80

100

veh

CR^ antagonist (mg/kg)

Effect Deprivation after vehicle) responses

CR^ antagonist (mg/kg)

Abstinence-induced escalation of nicotine intake isblocked by a CRF? receptor antagonist. (A) Effect of a CRFt antagonist (MPZP, s.c, -1 h) on nicotine Fig. 4. access to nicotine (*, P < 0.05 vs. baseline; #, P < 0.05 vs. after-abstinence vehicle treatment, self-administration during the active period in rats given extended n = 8). (B) Correlation between magnitude in the nicotine-deprivation of the nicotine-deprivation effect and percentage effect after CRFi antagonist. changes of nicotine (r= -0.71, P< 0.05). Thexaxis The higherthe the antagonist blocked self-administration effect, the more effectively nicotine-deprivation represents in active responses after the highest dose of MPZP (20 mg/kg), in percentage active responses after vehicle injection, and the y axis represents the reduction (MPZP, s.c, -1 h) on baseline nicotine injection. (Q Lack of effect of the CRFi receptor antagonist changes compared with active responses after vehicle = in rats given limited access to nicotine self-administration (n 10). Data represent mean ? SEM. responding

emotions,

and

some

forms

of

anxiety-like

behavior

may

be

The

unchanged
been receptor

during nicotine withdrawal.

The CRF

system has

results present self-administration leads In to this

demonstrate

that

the potential

for nicotine

implicated antagonists increased following

in anxiety-like and studies of CRFi behavior, for anxiolytic have promising drug potential

and access. more

progressively increased nicotine regard, the the

withdrawal during develops intake renewed drug during effect be may nicotine-deprivation

development
showing behaviors

(13). Our

findings with nicotine

CRF release, from other and the

add to reports
and

amygdalar withdrawal

ethanol, cocaine, including and suggest that overactivation CRFi vational system may constitute of drug

opiates, of

anxiety-like of abuse, drugs cannabinoids (27-32), CRF of moti of with nucleus the a

to comparable reliable Moreover, of may fact

effect alcohol-deprivation (42). were interindividual differences observed nicotine-deprivation a relevant effect, suggesting of individual marker

that

in the magnitude this measure the

the

represent the

a common withdrawal.

extrahypothalamic denominator Overactivation

vulnerability
ported known by not

to nicotine
that

dependence.

This hypothesis
effect

is sup
was not

nicotine-deprivation

aspects

observed

in ratswith limited (1 h) access to nicotine, a condition

to induce spontaneous incubation is unlikely or a loss of signs effect, to result tolerance of withdrawal, see from above. The a central

is also associated withdrawal CRF-CRFi system during of the dopaminergic in the central system hypo activation to mediate whether anxiety-like the increase behavior

aspect of nicotine dependence

produce the reward effect deprivation state for nicotine

of the amygdala (33), suggesting that both systems may interact

withdrawal. However, during are in CRF and the decrease in dopamine and needs further is unknown investigation. that an with intermittent escalating-dose abstinence suggesting intake (34). induces regimen of nic

(23), but a condition

sufficient to
nicotine reward of nicotine

LU zLU ? O OC 3 LU Z

a sensitized effect

to the

linked causally We recently otine associated

showed

during withdrawal (both of which would have led to a decrease in nicotine intake compared with baseline) but may be better
a construct. reinforcement Here, explained by negative depen rats may dent to escalate be hypothesized their nicotine intake a to obtain after abstinence relief from CRF-CRFj resulting mediated state. anxiety-like The effect creased dependently reinforced more role was the CRF-CRFi in the nicotine-deprivation system confirmed that the in by the experiment showing was nicotine intake observed dose after abstinence blocked the with MPZP. This is result by pretreatment was fact that the CRF] receptor antagonist at reducing in individual intake nicotine animals again effect suggesting be may a of

of nicotine levels intake, high increase nicotine subsequent three nicotine of intake. to forced The the abstinence

periods produces that abstinence may New data presented increase was mainly is fol period of intake of

herein extend this finding by showing that at a constant unit dose,

days a marked effect where the nicotine-deprivation human condition, during

observed during the early active period (dark). This situation is

similar very lowed by an The time abstinence early active levels increase of in smoking, recovery

(light), followed by a titration period of nicotine

course to the original basal

intake (35, 36).

by effective

if deprivation

is not initiated has not been fully investigated, but

on the results that recovery time will depend suggest preliminary duration of withdrawal, the magnitude of the deprivation effect, the number of self-administration and the period of sessions, nicotine was found intake vs. (light dark). to be a long-lasting The to the an time effect nicotine-deprivation that progressively phenomenon course of the nicotine-deprivation of incubation of reward The

a high effect, exhibiting nicotine-deprivation that the magnitude of the nicotine-deprivation marker of individual to nicotine vulnerability potential unknown inactivation stress-like role of and

in these CRF2 receptors would further require investigation. of CRF2 to is more receptor likely an than response effect, antianxiety-like

The dependence. effects is currently However, produce based a on

develops during the first week of abstinence and remains robust

for at least effect craving drug drugs with 2 months. is similar (37), where after delivery (cocaine, the phenomenon in responding to increase for cues related across has been withdrawal observed several methamphetamine) effect but to not is observed after and natural rewards

pharmacological and knockout studies (43, 44). Antagonism of CRFi receptors prevents deficits in brain reward function (15)
and increases in anxiety-like behavior (present report) associ

ated with precipitated nicotine withdrawal. Thus, MPZP

istration creases and state be may in nicotine to block abstinence-induced hypothesized a reduction intake of negative through states with to contributing nicotine abstinence. results suggest iswithdrawal-induced system, which the that negative

admin
in reward

heroin,

(sucrose) as well
incubation reinstatement incubation

(37-41). However,

the increase in responding

under extinction to the drug self-administration. and itself reexposure

anxiety-like associated together,

emotional in the

sessions effect leads

Taken nicotine CRF-CRFi

these

(37), and there is little evidence

increased

to date suggesting
drug

that the

dependence receptor

a key mechanism of overactivation to the

contributes

increased

George

e? al

PNAS

| October

23,2007

| vol.104

| no. 43

| 17201

emotional negative The recruitment explain use to nonnicotine one site nicotine

state of such

that drives a

of vulnerability dependence

negative for the and for

nicotine intake. subsequent emotional may system transition from nicotine a new target for addiction.

New England Nuclear, Boston, Tyr?]r/hCRF (-4,000 cpm/50 ?A; MA) were added to each well and incubated for an additional
24 h at 4?C. Wells residual well. radioactivity of Sensitivity were was blotted rinsed, counted by dry, and a y-counter fmol per well, separated, for 5 min and dose interrange and per and from

pharmacotherapy

suggests tobacco

Materials
All search Effort

and Methods
procedures Animal to reduce were Care by The approved and Use Committee Re Scripps in and were

intraassay 7-11%. Defensive

the assay is ?0.1 coefficients of variation

at the ED50

animal-use Institute's was made

accordance with

the National

Institutes of Health
of animals

guidelines.
both

the number

by using

between- and within-subject design studies. Adult male Wistar rats (Charles River Laboratories, Wilmington, MA) were housed cycle. Tests were performed
(10:00 a.m.). in a temperature-controlled vivarium with a 12-h/12-h light/dark

at the beginning of the dark cycle

= = minipumps delivering either saline (n 33) or nicotine (n 31) as described above. After 14 days (3.16 mg/kg/day, free base, s.c.) of pump exposure, testing was performed 5-8 h into the dark cycle in a standard cage with 2 in of bedding (wood shavings) along the bottom and a small hole centered in one side 1 inch
above the test the bedding to accommodate or the its vehicle shock were probe. Rats were

Burying

Behavior.

Rats

were

s.c.

implanted

with

osmotic

habituated hydrogen tartrate salt (Sigma, Natick, MA) was

at pH 7.4 self-administered as free and via experimenter-administered via catheter. indwelling jugular was base. Mecamylamine (Sigma)

(45 min) to the test cage for 2 days before testing. On

administered 30

day, mecamylamine

Drugs. Nicotine
dissolved minipump are Doses or

in saline

min before behavioral testing, and the CRFi antagonist or its vehicle were administered 45 min before behavioral testing (n = 7-9 per group). On contact with the probe and shock delivery
(using verified latency grooming, condition Intracerebral tized with above from the an a Coulbourn by and 1.5 mA, shocker, AC, precision was the probe deactivated. response, duration of probe-directed rearing, burying, were and freezing measured from videotape a startle using a computer and program. CRF Infusions. Rats and were anesthe stainless were <1 s), The

expressed

dissolved
antagonist

in saline and administered

i.p. (1 ml/kg). The CRFi

(N, A^bis(2-methoxyethyl)-3-(4-methoxy-2-methyl

or phenyl)-2,5-dimethyl-pyrazolo [1,5a] pyrimidin-7-amine, MPZP) was synthesized at The Scripps Research Institute by P. Wirshing, dissolved in 20% hydroxypropyl ?-cyclodextrin (Cav itron; Cargill, Wayzata, MN) in isotonic saline at pH 4.5 and administered s.c. (2ml/kg, 45-60 min before testing). Rat/human CRF was supplied by Jean Rivier (The Salk Institute, La Jolla, CA). CRF was dissolved in lx PBS (pH 7.4) and prepared fresh a few minutes before intracerebral injection. The doses and time
of injections were selected based on previous studies?\

resting, a over

10-min period by an experimenter blind to the subject treatment

and

Cannulations

isof lurane-oxygen nucleus flat skull of the

mixture,

26-gauge

steel guide cannulas (Plastics One, Roanoke, VA)

central with were amygdala guide

aimed 2 mm

stereotaxically cannulas Intracerebral were

implanted bilaterally: AP -2.6 mm; ML ?4.2 mm; V -5.2 mm,

dura, (46). with The secured

Microdialysis. The changes

the central nucleus of the

in interstitial levels of CRF-L-IR

amygdala were examined during

in

to the skull with dental cement and anchor screws, and guide
cannulas maintained stylets. injections

withdrawal (1.5 mg/kg, i.p.). Rats mecamylamine-precipitated were s.c. implanted with osmotic minipumps (model 2ml2, 14 days, 5 jul/h;Durect, Palo Alto, CA) delivering either saline
(nondependent, n = or nicotine 5) (nicotine-dependent, n = 1)

were administered with the use of injectors (33-gauge; Plastics One) that projected 2 mm past the guide cannula to the central
nucleus of the amygdala. The injectors were attached to 70 cm

free base, s.c.) and a microdialysis guide (3.16 mg/kg/day, cannula (SciPro, Sanborn NY) stereotaxically positioned 1mm above the central nucleus of the amygdala by using the following
coordinates After 14 days

of calibrated polyethylene-20 tubing preloaded with drug solu tion. This cohort of rats had been extensively handled previously in the context of a food intake study, in which they received
administration central washout with were nucleus of s.c. and a CRFi into the receptor antagonist in a Latin-square the amygdala A design. before the present of 7 days was study imposed on chow, maintained time, animals during which of Rats were randomly assigned to CRF vs.

(ML) ? 4.2 mm; ventral (V) -6.5 mm, from dura with flat skull].
of pump exposure, polyestersulfone membrane, microdialysis 15-kDa molecular a probe mass (1-mm cutoff;

[anteroposterior

(AP)

?3.3

mm;

mediolateral

SciPro) was lowered into the guide cannula and allowed to equilibrate for 12 h (1 jud/minflow rate, artificial cerebrospinal fluid). Subsequently dialysate samples (30-min fractions) were
collected for a period Sample of baseline challenge tubes were and after saline sampling a by using within-subjects injections on wet ice during collection kept and and

vehicle conditions balanced for previous diet history, which was statistically unrelated to performance in the defensive burying test. The CRF group (n = 5) was infused bilaterally (30 pmol
a volume total dose) with Hamilton microsyringes same cannulae volume 1 min of PBS. after the of 0.25 and two u.1 per infusion were the side over 30 s by using Ap

period all subjects handled daily.

mecamylamine design.

= paratus, Holliston, MA). The control group (n 5) received the

Injectors end of removed infusions, from and guide rats were

pumps

(Harvard

were
CRF

then frozen on dry ice until later analysis by RIA.

Immunoassay. Dialysate CRF-like immunoreactivity was

quantified with a sensitive and specific solid-phase RIA adapted from Zorrilla et al. (45) to increase sensitivity. Immulon-4 96-well plates (Dynatech, Chantilly, VA) were coated with protein A/G (1 u.g/100 /xl, 1 M NaHC03 per well, pH 9.0; Calbiochem, La Jolla, CA) overnight. Plates were rinsed with wash buffer (0.15 M K2HPO4 supplemented with 0.2 mM ascorbic acid and 0.1% Tween-20, pH 7.5) to dislodge loose Protein A/G. Wells were incubated 48 h at 4?C with 50 ?Aof anti-CRF serum (rC68, generously provided by W. Vale, The Salk Institute) at a titer of 1:300,000 in gelatin assay buffer. After of three rinses to dislodge loose antibody, 50 ?x\ dilute sample (in duplicate) or standard (3-1,000 pg/ml, in quadruplicate) were incubated overnight at 4?C. After incubation, 50 ?A of [125I
17202 I wvvw.pnas.org/cgi/doi/10.1073/pnas.0707585104 George et al.

returned to the home cage for 1min before being tested in the defensive burying test.
Nicotine dures Self-Administration. for both i.v. The apparatus and and detailed proce of

catheterization

self-administration

nicotine
in 23-h tation

rats have been described (34). Adult male Wistar g) were first allowed to nose-poke for food and water (280-330
sessions of before and after recovery from of After catheters. jugular to rats were allowed acquisition self-administer surgical these nicotine implan op?rant (0.03

= 1 lever-response, mg/kg/100 ui/1 s, free base, fixed ratio out = 20 s) under different paradigms.
Experiment nicotine A: Effect of MPZP self-administration in ShA rats. Rats during were allowed 1 h, daily

responses,

time

to acquire "short-access"

sessions (ShA, n = 10) for at least 10 days. The CRFi antagonist MPZP (0, 5, 10, 20 mg/kg) was then administered by using a
Latin

3-5 days). The 72-h cycle period was repeated four times to analyze the reproducibility of the results (Fig. 2A). Statistical Analysis. Results were analyzed with SPSS software using ANO VA (SPSS, Chicago, IL. In all cases, a normality test and an equal variance test were performed before the ANO VA to ensure its validity. The following variables (dependent/
nondependent: of nicotine or four access: sham/abstinence: levels; two levels; pharmacological two active/inactive response: factor. Depending sessions as on the two two levels; duration two treatments: were the used condi as

= 7) were allowed to self-administer (n 6) and LgA rats (n nicotine during daily sessions during at least 10 days. Then, they
were followed submitted by one of left to session in their 3 days of abstinence of nicotine self-administration effect. vivarium for home cage, to assess

Experiment

1-2 intervening with square design, B: Effect of nicotine deprivation

treatment-free in ShA and

days. LgA rats. ShA

the magnitude rats were then period by using catheter and an used

the nicotine-deprivation in the undisturbed experiment

ShA

and LgA a 1-month

levels;

levels) analysis,

for

C. Catheter barbiturate,

ultrashort-acting used.

was tested patency Brevital (methohexital

between-subjects of self-administration were tests used and

tion (baseline/postabstinence:
ples) Keuls When are We

two levels) and the time (number

or number factors. were sam of microdialysis Post hoc Newman

sodium, 10mg/ml, 2 mg per rat), and only rats with a fully patent
were C: Effect of MPZP on the nicotine-deprivation effect Experiment of experiment allowed After B, rats were completion nicotine administer 23-h, "long-access" during daily in LgA rats. to self sessions

within-subjects Pearson correlations of ANO test was ? SEM. VA used,

assumptions ric Kruskal-Wallis shown as mean

were

used when necessary. the nonparamet violated, t test. Data followed by Welch's

= (LgA, n 8) for at least 10 days. Then,

on a lever for nicotine

they were allowed

in four

to

respond

self-administration

4-day

cycles, each separated by three intervening days of abstinence in their home cage. MPZP was administered before the first session
a Latin each of abstinence square cycle by using D: Further characterization of the nicotine-deprivation Experiment of experiment After submitted C, rats were completion after successive abstinence cycles periods. of nicotine The different rats were self-administration durations of to design. effect. to nine and were

periods abstinence

tested in the following order 72 h, 48 h, 265 h, 12 h, 1,201 h. After

each abstinence period, allowed self-administer

nicotine until they reached their predeprivation

baseline

(range

thank Katy Rahmani, Robert Lintz, Yanabel Grant, Thomas Green and Molly Brennan for technical assistance; and Frederic Ambroggi for helpful discussions; and Michael Arends for editorial Luigi Pulvirenti on the assistance. This is publication number 18662 of the Committee of Addictive from The Scripps Research Insti Disorders Neurobiology tute. We also thank the Tobacco of the Research Network Etiology Robert Wood Johnson Foundation for discussion and support. This work was Related Disease Research supported Program by Tobacco of the State of California the National Grant 12RT-0099, (TRDRP) of Diabetes Institute and Digestive Diseases and Kidney Grant and the Pearson Center for Alcoholism and Addiction DK26741, Research. well,

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| October

23,2007

| vol.104

| no. 43

| 17203