A low dose of nicotine is sufficient to produce nicotine withdrawal in mice

doi:10.4236/health.2010.21001

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Vol.2, No.1, 1-7 (2010

doi:10.4236/health.2010.21001

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A low dose of nicotine is sufficient to produce nicotine

withdrawal in mice

Besson Morgane1,2, Suarez Sandra1, Changeux Jean-Pierre1, Granon Sylvie1,3*

1Département de Neuroscience, Institut Pasteur, Paris, France; sylvie.granon@u-psud.fr

2Department of Experiment Psychology, Downing Site, Cambridge, UK

3Centre de Neuroscience Paris Sud, Université d’Orsay, Orsay, France

Received 12 October 2009; revised 12 November 2009; accepted 13 November 2009.

ABSTRACT

The objective of our study was to investigate

whether the chronic administration of a low

dose of nicotine can be followed by a withdrawal

syndrome at cessation of nicotine delivery. Pre-

vious studies showed various results, depend-

ing in the doses of nicotine, species, ways of

administration and behavioural paradigms, but

all emphasized a withdrawal effect on some or

all of the following spontaneous behaviours:

grooming, rearing, body shake or tremor, body

scratching, abdominal constriction, jumping.

However, it is not clear which behaviour is ex-

actly altered, as a global behavioural index is

most frequently used. This is not clear either if

anxiety modulates the behavioral expression of

withdrawal or which factors contribute to its

locomotors effect, if any. To distant-angle these

processes, we scored each of these behaviours

individually before nicotine exposure, during

continuous nicotine delivery and at cessation of

nicotine delivery after precipitated withdrawal

by mecamylamine injection. We also measured

locomotor activity and anxiety levels in the

same animals. We used a low dose of nicotine

(2.4 mg/kg/day as free base) that has been pre-

viously shown to produce nicotinic receptors

up-regulation, both in the brain and in blood

cells. With such a low dose, nicotine withdrawal

didn’t affect locomotion nor anxiety levels but

increased the number of rearing, jumping, and

marginally, body-scratching. Other behaviours,

classically considered to contribute to with-

drawal syndrome, were unaffected, e.g., groom-

ing, body or forelimb shakes. Our results show

that anxiety may be dissociated from the be-

havioural withdrawal syndrome. Also, the se-

verity of the syndrome produced by nicotine

withdrawal is qualitatively and quantitatively

different from the one induced by other drugs of

abuse and also by the one produced by nicotine

at higher doses.

Keywords: Withdrawal; Anxiety; Locomotor Activity;

Nicotinic Receptors

1. INTRODUCTION

Nicotine is known to be an addictive substance in hu-

mans as well as in other mammals [1-3]. As such, be-

havioral studies in animal models show that nicotine

induces a withdrawal syndrome [4,5], associated with

the increased exhibition of multiple heterogeneous be-

haviors, many of which being shared by other drugs of

abuse [2,6]. These findings, however, are confounded by

two major factors: 1) in most cases, nicotine withdrawal

is not associated with the same severity of symptoms as

other drugs of abuse [6]; 2) global scores alone are not

highly informative as they may reflect the grouping of

different processes and different origins. Indeed, with-

drawal signs are constituted of exaggerated levels of

behaviours that are part of the normal behavioural rep-

ertoire of the animal, and as any behaviour, they may be

influenced by multiple parameters such as genetic strain,

individual and group level of stress, experimenter ma-

nipulation, lightning level or noise.

Authors of previous studies targeted several behav-

iours that are affected by nicotine as well as by other

addictive drugs, i.e., rearing, abdominal constriction,

grooming, scratching, chewing, cage scratching, and head

nodding and jumping [7-10], and established a global

abstinence score. In order to match with what others

previously established, we scored the same behaviours

but individually. Further, we scored the frequency of

these behaviours in the same animals, before, during and

after nicotine administration, in order to question whether

their frequency varies within and between individuals

and/or represents robust indicators of withdrawal. We

therefore expected to provide evidence regarding which

of these behaviours are specifically affected by nicotine

B. Morgane et al. / HEALTH 2 (2010) 1-7

withdrawal. We chose to administer continuously a low

dose of nicotine (2.4 mg/kg/day, as free-base), via os-

motic mini-pumps, as it was previously shown that it is

sufficient to produce nicotinic receptor up-regulation in

the mouse [11] and to induce withdrawal syndrome in the

rat [12]. In accordance with previous studies [7,8,10,12,13],

we then used 1 mg/kg of mecamylamine to precipitate

homogeneously the withdrawal the last day of mini-pump

theoretical depletion.

Withdrawal from chronic use of nicotine also results in

increased anxiety [14,15], and reduced locomotor activity

[8,12,16-20], although these last processes were altered

either after higher doses of nicotine than the one we chose,

or in rats instead of mice. We have therefore tested the

effects of nicotine withdrawal, after the low dose admin-

istered, on locomotor activity and anxiety behaviours of

the mouse.

Experiments have been carried out following the

guidelines of the European Communities Council

(86/609/EEC). Twenty-two C57BL6/J male mice (3 to 5

months) supplied by Charles River Laboratories (France)

were housed individually upon arrival under a 12h

light-dark cycle in rooms at a controlled temperature

(21°C). All experiments were carried out during the light

phase, from 10 am to 6 pm. They were handled during

approximately ten days before the beginning of the tests.

They were slightly food deprived one week before the

start of the experiments with their weight adjusted at 90%

of their normal body weight, as the same animals were

subsequently used in a learning experiment, in order to

minimize the use of animals. However, in order to reduce

anxiety potentially induced by food deprivation, animals

were fed before the experiments.

After handling, mice were habituated 15 minutes/day

for three days to the environment (a large 50 cm X 30 cm

transparent box, containing clean bedding) in which the

behaviours were scored. Two experimenters, blind to the

drug condition of the animals, scored the behaviours on

line. Before mini-pump implantation, we first established

the baseline level of each of the following behaviours:

rearing, grooming, body shakes, body tremor and ab-

dominal constriction, jumping, scratching, forelimb

shake, chewing and head shakes. After the baseline

measurement, mini-pumps were implanted subcutane-

ously under general anesthesia (a combination of xy-

lazine, 2.5%, and ketamine, 15% in 82.5% of PBS).

Mini-pumps (Alzet) were filled with either nicotine (2.4

mg/kg/day free base) or saline delivered at a constant rate

(0.25 l/hour) for 28 days. In order to measure the effect

of chronic nicotine administration, we scored the same

behaviours a second time two weeks after mini-pump

implantation. At the last day of mini-pump delivery, i.e.,

the 28th day, mecamylamine was injected 15-20 minutes

before the beginning of the withdrawal test at a dose of 1

mg/kg, IP. The same behaviours were then scored sub-

sequently to evaluate the effects of nicotine withdrawal.

Locomotor activity was measured for 30 minutes in an

empty large open field (1m in diameter) made of white

plastic. It was situated in a room containing distal visual

cues with light level set between 80 and 100 Lux and was

placed under a camera connected to a video-track system

that allows the discrimination of navigatory -speed >

11.8cm/sec- and exploratory movements -speed <

6.8cm/sec- [17]. It has been shown that rodents sponta-

neously avoid the center of a large open area which is more

brightly illuminated than the periphery [21]. We therefore

used the number of visits and time spent in the center as an

index of anxiety during free exploratory activity. The

baseline level of anxiety is altered by novelty [22]. There-

fore, in order to avoid the alteration of the anxiety level

generated by the first visit of a new environment, we

conducted exploratory experiments in distinct groups of

mice under the chronic administration of nicotine or saline

and during withdrawal of nicotine or saline.

Each behavior was analyzed using repeated measures

analysis of variance (ANOVAs) with “Drug condition” as

the main within-subject factor, with 3 levels (before im-

plantation, under chronic treatment and withdrawal), and

“treatment” (with 2 levels, saline and nicotine) as the

main between-subjects factors. Upon significant main

effect (i.e., for p<0.05), data was further analyzed with

paired t-tests.

Statistical analysis revealed a significant effect of the

main within-subject factor, drug condition, for rearing

(F(2,40)=15.38, p<0.0001), jumping (F(2,40)=3.98, p<0.026),

body-scratching (F(2,40)=5.05, p=0.01), grooming (F(2,40)=

3.23, p=0.05) and forelimb shakes (F(2,40)=12.23, p<

0.0001), suggesting that, individually, each of these pa-

rameters were altered either by the treatment and/or the

repetition of the test. We also observed a significant in-

teraction drug condition X treatment for rearing (F(2,40)

=3.96, p=0.027), suggesting that nicotine and saline

treatment produced a different effect on rearing. By con-

trast, no significant effect of drug condition and no in-

teraction drug condition x treatment were observed for

abdominal constriction (respectively, F<1 and F(2,40)=1.61,

NS) suggesting that this variable was not altered by the

drug condition and, therefore, may not contribute sig-

nificantly to the withdrawal syndrome.

When measured under chronic treatment, rearing

(t=-0.86, p=0.4, df=20) and jumping (t=0.34, p=0.74,

df=20) levels were similar in animals receiving saline and

those receiving nicotine, making both variables unaf-

fected by the chronic administration of nicotine, as illus-

trated in Figure 1. Grooming was the only behaviour that

showed a significant main effect of group (F(1,20)=7.44,

p=0.013) and a significant main effect of drug condition

(F(2,40)=3.23, p=0.05) with no significant interaction (F<1,

NS), suggesting that the drug condition was identical for

the group receiving saline and the one receiving nicotine.

It further suggests that the group effect (i.e. nicotine effect)

was not dependent of the drug condition, i.e., both groups

B. Morgane et al. / HEALTH 2 (2010) 1-7

Openly accessible at/HEALH

Figure 1. Quantification of spontaneous behaviours affected by chronic administration of nicotine and nicotine with-

drawal precipitated by injection of mecamylamine. Rearing, jumping, scratching, grooming and forelimb shakes were

scored before mini-pump implantation (baseline), under chronic nicotine (white) or saline (black) treatment, and the last

day of mini-pump delivery, 20 min after mecamylamine injection (withdrawal). * indicates a significant difference be-

tween animals under saline and those under nicotine.

may show different levels of grooming before mini-pump

implantation. Post-hoc tests confirmed that the level of

grooming was marginally different between the saline and

the nicotine groups at baseline condition (t=-1.95,

p=0.065, df=20) and was significantly different under

chronic treatment (t=-2.38, p=0.027, df=20). These results

suggest that nicotine had no effect by itself on grooming

but that both groups, analysed by experimenters blind to

the drug condition of the animals, were different regard-

ing this parameter from the beginning of the experiment.

Body-scratching was marginally affected by the chronic

drug treatment (t=-2.05, p=0.054, df=20). As illustrated in

Figure 1, and confirmed by paired t-tests, animals re-

ceiving saline showed significantly less body- scratching

levels when tested on baseline than when tested under

mini-pumps (p=0.026) whereas animals receiving nico-

tine did not show different level of body- scratching be-

tween the two testing conditions (p=1.0). This suggests

that the repetition of the test affected only animals that

were not under nicotine treatment.

Forelimb shakes were unaffected by nicotine admini-

stration as the number of paw tremor recorded was not

significantly different between animals receiving saline

and those receiving nicotine under chronic treatment

(t=0.17, p=0.87, df=20).

Post-hoc tests confirmed that the levels of rearing

(t=-7.03, p<0.0001, df=10), jumping (t=-2.33, p=0.042,

df=10), body-scratching (t=-2.14, p=0.058, df=10) and

forelimb shakes (t=-2.58, p=0.027, df=10) were signifi-

cantly different under nicotine administration and during

withdrawal. The levels of rearing, jumping and body

scratching didn’t change from chronic treatment to with-

drawal condition in saline animals (respectively: t=0.4,

p=0.7, NS; t=0.25, p=0.81, NS and t=-0.99, p=0.35, NS),

suggesting that these behaviours were altered specifically

by nicotine withdrawal, as illustrated in Figure 1 Fore-

limb shakes were significantly altered in mice under

chronic administration of saline (paired t-test conducted

for chronic condition versus withdrawal condition, t=-2.7,

p=0.022, df=10), suggesting that this behaviour was

affected by mecamylamine administration but not by

nicotine withdrawal. Post-hoc tests showed that nicotine

withdrawal did not affect grooming (t=-1.43, p=0.18,

df=10).

B. Morgane et al. / HEALTH 2 (2010) 1-7

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Figure 2. Exploratory and navigatory activity recorded in the circular open field for 30min during chronic treatment

(A) and during withdrawal (B). No significant difference was found between animals under saline (black) and those

under nicotine (white).

Navigation and exploration were not affected by the

chronic administration of nicotine, as illustrated in Fig-

ure 2A ANOVAs confirmed that there was no treatment

effect on navigation nor on exploration (both Fs<1, NS).

For both types of behaviour, there was a significant effect

of habituation (for navigation: F(5,85)=44.6, p<0.0001; for

exploration: F(5,85)=31.85, p<0.0001) and no significant

interaction treatment X habituation (both Fs<1, NS)

suggesting that subjects receiving saline as well as those

receiving nicotine showed normal habituation.

Navigation and exploration (Figure 2) were not statis-

tically different after withdrawal in saline and in nicotine

animals, as there was no treatment effect neither for

navigation (F(1,12)=1.05, NS) nor for exploration (F<1,

NS). Within 30 minutes, there was a significant habitua-

tion for both navigation (F(5,60)=57.96, p<0.0001) and

exploration (F(5,60)=25.02, p<0.0001) but no significant

interaction treatment X habituation for either navigation

(F(5,60)=1.1, p=0.37, NS) or exploration (F<1, NS). These

results showed that the levels of exploratory and navi-

gatory activity as well as habituation were not affected by

nicotine withdrawal.

The chronic administration of nicotine didn’t affect the

number of entries in the centre (data not shown, ANOVAs

for drug effect: F(1,17)=2.45, NS) but produced a trend to

reduce the time spent in the centre of the arena (Figure

3A, ANOVAs for drug effect: F(1,17)=4.33, p=0.053),

suggesting that, in an unfamiliar environment, chronic

nicotine administration tends to increase anxiety level.

The total number of entries in the centre (data not

shown) and the time spent in the centre of the open field

in 30 minutes (Figure 3B) were not different after with-

drawal between saline and nicotine animals, as confirmed

by ANOVAs (no drug effect for number of entries: F<1,

NS; no drug effect for time in the centre: F<1, NS). These

results showed that the level of anxiety was not affected

by nicotine withdrawal in an unfamiliar environment.

Our present results demonstrate that only rearing,

jumping and body-scratching reflect specifically nicotine

withdrawal syndrome in mice, when nicotine dependence

is produced by the continuous administration of a low

dose i.e., 2.4mg/kg/day. This dose has been shown to

induce a significant up-regulation of nicotinic receptors

both in the blood and in the brain [11]. We find that in

these conditions nicotine withdrawal affects neither

anxiety nor locomotion. Previous studies have used sig-

nificantly higher doses, i.e., up to ten times higher, that

induced withdrawal signs of different nature than the one

we observed in the present study but that also increased

anxiety level [9,14,15]. Interestingly, when using a dose

B. Morgane et al. / HEALTH 2 (2010) 1-7

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Figure 3. Time spent in the centre of the brightly illuminated open field over one single 30-minute session during chronic

treatment (A) and during nicotine withdrawal (B). Under chronic nicotine, mice tended to decrease the time spent in the

centre of the open field, as compared to animals receiving saline (A). No significant difference was found between ani-

mals receiving saline (black) and those receiving nicotine (white) during withdrawal (B).

in a range similar to our, but excluding rearing and

jumping from their scores, Damaj and collaborators [14]

did not find any withdrawal signs or anxiety alteration.

Therefore, in agreement with previous studies using a

large range of nicotine doses, our present results provide

evidence that a low dose of nicotine given continuously

also induces an increase in the frequency of some specific

behaviour. Using a low dose of nicotine, we were able to

show dissociation between anxiety and other behavioural

processes that may influence the general withdrawal

syndrome. This dissociation suggests that withdrawal

signs may not be exclusively due to an increase in anxiety

levels after nicotine abstinence. It must be noticed that

our animals were under a slight food deprivation schedule

and, although they were fed before behavioural recording,

we cannot exclude that chronic food deprivation has

modified the effects of nicotine withdrawal. We con-

ducted experiments (unpublished results) showing that a

slight chronic food deprivation doesn’t alter by itself

navigatory and exploratory processes. These experiments

do not provide evidence that food deprivation does not

interfere with the nicotine exposure, for example by in-

creasing anxiety levels. Such data are not yet available, to

our knowledge.

Rearing behaviours have been shown to be sensitive to

manipulations of the dopaminergic system [23]. Hilde-

brand and collaborators [13] also showed in nicotine-

dependent rats that intra-Ventral Tegmental Area injec-

B. Morgane et al. / HEALTH 2 (2010) 1-7

Openly accessible at/HEALH

tion of mecamylamine induced a rearing frequency modi-

fication with a dose of nicotine similar to our dose range.

Therefore, it is possible that chronic nicotine administra-

tion followed by precipitated withdrawal created a deficit

in cholinergic transmission which, in turn, reduced do-

pamine (DA) release, leading to an increased level of

rearing. Microdialysis studies indeed showed that in

nicotine-dependent rats, precipitated withdrawal leads to

a decrease in DA release in the nucleus accumbency and

in the prefrontal cortex [24,25]. Considering the role of

the DA system in addiction [1], it is therefore not sur-

prising that behaviours that are influenced by its modu-

lation are particularly affected during withdrawal. Fore-

limb shakes are generally considered as a behavioural

manifestation associated with drug withdrawal [8,26].

Our results showed that their frequency was affected by

mecamylamine administration but not by nicotine with-

drawal, as it was observable also in mice that were ad-

ministered saline chronically. Previous studies [14,27]

showed that this measure as well as some other signs are

affected by hexamethonium or chlorisondamine, nico-

tinic antagonists which penetrate poorly the blood-brain

barrier. Therefore, our data, along with previous ones [19]

suggests that forelimb shakes are from peripheral origin

and cannot be considered as a centrally-mediated with-

drawal sign. We further show here that some of the be-

haviours usually recorded as part of a withdrawal syn-

drome, i.e., grooming, may be affected by the repetition

of testing, but not by the nicotine treatment or withdrawal.

It is furthermore noticeable that a baseline difference

between groups (Figure 1), uncorrected as the experi-

menters were blind to the animals’ treatment, remains

observable for weeks, despite the habituation of the

animals. This suggests that grooming is a behaviour that

varies strongly between individuals, reinforcing the in-

terest of longitudinal studies that allow baseline meas-

urements. We also showed that body scratching was af-

fected by nicotine administration and, only in animals

that received nicotine, by withdrawal. Although this ef-

fect is statistically evidenced, it must be noticed that in

absolute numbers, it is not highly represented in our data

and should therefore be confirmed, despite the fact that it

is classically associated with drug withdrawal [8,10,20,27].

Together with grooming data, scratching data emphasized

the necessity to give baseline levels of such spontaneous

behaviours when scoring drug effects.

In the open field, our data showed that mice under

chronic administration of nicotine tend to spend less time

in the centre of the arena, suggesting that, in an unfamiliar

environment, chronic nicotine administration tends to

increase anxiety. Our results also show that nicotine

withdrawal, at a low dose, had no effect on anxiety when

the anxiogenic environment was not familiar. This result

is compatible with the one of other authors who compared

the withdrawal effect after different doses of nicotine on

anxiety [14].

Locomotor activity was affected neither by chronic

nicotine exposure, nor by withdrawal. It is noticeable that,

in rats, a similar dose of nicotine induced a temporary

decrease of locomotor activity that was observable be-

tween 16 to 24 hours after mini-pump removal [6,12], but

not in mice after pharmacological precipitation [28]. It is

not possible to conclude firmly from experimental data

obtained in different species (rats versus mice) and with

two different procedures (mini-pump removal versus

precipitation). Indeed, it is known that the sensitivity to

nicotine is different in rats and mice [29]. Also, it is pos-

sible that mini-pump removal, that allows enough time

for producing alteration in striatal DA concentration [12],

produces a higher impact on locomotor activity than

pharmacological precipitation, via DA mechanisms.

In conclusion, our present results support the idea that a

continuous administration of low dose of nicotine, fol-

lowed by pharmacological precipitation of withdrawal, is

sufficient to produce a withdrawal syndrome in the mouse

represented by increased levels of rearing and jumping.

With such a low dose, nicotine withdrawal didn’t affect

locomotion nor anxiety levels. Other behaviours, con-

tributing to nicotine withdrawal after higher doses of

nicotine, were unaffected. Therefore, in spite of the fact

that anxiety may produce some of the behaviours associ-

ated with nicotine withdrawal, other behaviours, unre-

lated with anxiety can also be affected. These results can

be discussed in terms of a relatively dissociable role of

nicotinic receptors in the reward versus somatic de-

pendence systems.

The future determination of whether these behaviours

rely on specific neurotransmitter systems or brain struc-

tures is expected to complement the current therapeutical

proposal of the withdrawal syndrome [30, for review].

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