Characterization of the Rapid-Onset Type of Behavioral
Sensitization to Amphetamine in Mice: Role of
Cibele Cristina Chinen*,1, Rulian Ricardo Faria1and Roberto Frussa-Filho1
1Departamento de Farrmacologia, Escola Paulista de Medicina, Universidade Federal de Sa ˜o Paulo, Sa ˜o Paulo, SP, Brazil
A rapid-onset type of behavioral sensitization (ROBS) has been demonstrated in rats treated with a single ‘priming’ injection of
amphetamine (AMP). In that species, however, this phenomenon was restricted to AMP-induced stereotyped behavior (SB), not
occurring for the locomotor-stimulant effect (LSE) of AMP and not reflecting environment-specific sensitization. In the present study, the
ROBS was characterized in the mouse. Mice received a single ‘priming’ intraperitoneal injection of 5.0mg/kg AMP which was paired
or not with environment. At different intervals (3, 4 or 5h) subgroups were tested for AMP (1.5 or 5.0mg/kg)-induced SB or AMP
(1.5mg/kg)-induced open-field LSE. Results showed that: (1) in the absence of drug–environment association, a priming injection
of AMP increased the SB induced by a 1.5mg/kg AMP challenge injection given 3h (but not 4 or 5h) later; (2) when the dose of AMP
challenge injection was increased to 5.0mg/kg, an enhancement of SB was verified at all the intervals tested (3, 4, and 5h); (3) when
animals were tested in an open field, the priming injection of AMP produced an increase in the LSE of a 1.5mg/kg AMP challenge
injection, given 4h later; (4) drug–environment association increased both SB and locomotion after a saline challenge injection and
potentiated the rapid-onset sensitization of both behaviors in AMP-challenged mice. Collectively, these results demonstrate that the
ROBS phenomenon also occurs in mice, is extended to AMP-induced LSE, and is markedly potentiated by (but does not depend on)
Neuropsychopharmacology (2006) 31, 151–159. doi:10.1038/sj.npp.1300789; published online 8 June 2005
Keywords: amphetamine; conditioning; stereotypy; locomotion; behavioral sensitization
While there is tolerance to many of the effects of repeated
drug treatments, the psychomotor and positive reinforcing
effects of amphetamine (AMP) and other drugs of abuse
often become progressively greater with repeated adminis-
tration (Robinson and Becker, 1986; Piazza et al, 1990;
De Vries et al, 1998). This phenomenon, called behavioral
sensitization, is usually measured in terms of locomotion or
stereotypy in rodents (Robinson and Becker, 1986; Wise
et al, 1996; Camarini et al, 2000). Sensitization of AMP-
induced stereotyped behavior (SB) is considered to be an
important model of dopaminergic nigrostriatal plasticity
(Robinson and Becker, 1986). As regards sensitization of the
locomotor-stimulatory effect of AMP (and other drugs of
abuse), this model has been suggested to be useful for
studying mechanisms underlying both dopaminergic meso-
accumbens plasticity (Henry and White, 1991; Wolf et al,
1994) and drug craving in humans (Robinson and Berridge,
The extent to which behavioral sensitization is induced by
drug pre-exposure is highly dependent on the environmental
context in which the injections are given as well as on the
nature of the pretreatment regimen. Concerning the envir-
onmental context, it has been suggested that environmental
cues might be conditioned stimuli for drug-like conditioned
responses, potentiating the development of behavioral
sensitization (Hayashi et al, 1980; Pierce and Kalivas, 1997;
Costa et al, 2001; Frussa-Filho et al, 2004). Although
sensitization of the locomotor-activating effect of AMP and
other drugs of abuse has been also observed when drug
injections are not paired with the observation environment
(Bellot et al, 1996, 1997; Costa et al, 2001), this environmental
modulation of sensitization is especially interesting because
it is well known that environmental cues trigger craving and
drug-seeking behavior in humans (Childress et al, 1986;
Niaura et al, 1988; Carter and Tiffany, 1999).
With respect to the nature of the pretreatment regimen, it
has been demonstrated that it is not necessary to repeatedly
administer AMP for long periods of time to produce
Online publication: 3 May 2005 at http://www.acnp.org/citations/
Received 24 November 2004; revised 20 April 2005; accepted 21 April
*Correspondence: Dr CC Chinen, Escola Paulista de Medicina,
UNIFESP-Edifı ´cio Leal Prado 1 andar, Rua Botucatu, 862-CEP 04023-
062-Sa ˜o Paulo, SP, Brazil, Tel: þ55 11 5549 4122, Fax: 55 11 5542
4176, E-mail: email@example.com or firstname.lastname@example.org
Neuropsychopharmacology (2006) 31, 151–159
& 2006 Nature Publishing Group All rights reserved 0893-133X/06 $30.00
behavioral sensitization. Indeed, a single injection of AMP
has been reported to enhance both stereotypy (Browne and
Segal, 1977; Ellison and Morris, 1981) and locomotor
stimulation (Vanderschuren et al, 1999a) produced by a
subsequent injection of AMP given weeks later. Within this
context, a more important variable appears to be the
interval between AMP treatment (or AMP single injection)
and the challenge injection of the drug. Indeed, several
studies have found that the magnitude of AMP-induced
behavioral sensitization gradually increases with prolonged
withdrawal after AMP-repeated treatment (Kolta et al, 1985;
Paulson et al, 1991; Vanderschuren et al, 1999b) or after
AMP single exposure (Vanderschuren et al, 1999a).
The above-mentioned temporal profile of the behavioral
sensitization phenomenon was however questioned by
studies performed by Kuczenski and Segal (1999a,b). They
demonstrated that the behavioral response of rats to low,
nonstereotypy doses of AMP (0.5–1.5mg/kg) at very short
intervals (3–5h) after an acute, ‘priming’, injection with
4.0mg/kg AMP resulted in the emergence of intense, focused
stereotypies in the absence of an altered caudate-putamen
extracellular dopamine response. Such a finding might be
especially important within the context of binge patterns of
stimulant abuse involving frequent administration of the
drug at short intervals. However, its clinical implications
appear to be considerable attenuated by the experimental
suggestion that this rapid-onset behavioral sensitization
phenomenon did not occur for the locomotor-stimulant
effect (LSE) of AMP and did not reflect environment-specific
sensitization (Kuczenski and Segal, 1999a).
The present study had three purposes. These were to
determine (1) if this rapid-onset sensitization of AMP-
induced SB would also be verified in mice, (2) whether or
not the phenomenon would be also demonstrated for the
LSE of the drug, and (3) whether or not it would be
dependent on (or at least be influenced by) environmental
MATERIALS AND METHODS
Subjects. Male EPM-M1 mice weighing 30–40g were used.
The animals arrived at the experimental laboratory at least
10 days before the beginning of the experiments. They were
housed in plastic cages (32?42?18cm), 15 per cage, with
ad libitum access to food and water. Light/dark cycle (lights
on at 07:00h, off at 19:00h) and temperature (221C) were
kept constant. All experiments took place between 08:00 and
18:00h. The animals were maintained and used in
accordance with the guidelines of the Committee on Care
and Use of Laboratory Animal Resources, National
Research Council, USA.
Test agents. AMP (Sigma) was used. The drug was freshly
diluted in saline (SAL) solution and was given intraper-
itoneally in volumes not greater than 10ml/kg body weight.
SAL was used as control solution.
Stereotypy studies. The animals were observed for SB in
wire mesh cages (16?30?18cm) free of water and food.
Stereotypy was quantified every 5min for 150min after
AMP administration according to the scoring system
proposed by Setler et al (1976), with some modifications
validated in our laboratory for mice. Briefly, scores varying
from 0 to 4 were attributed to an animal’s behavior by an
observer who was unaware of the drug treatment. The
grading system was as follows: 0, asleep or stationary; 1,
active; 2, active with predominantly stereotyped sniffing
and rearing; 3, stereotyped sniffing with bursts of licking
and/or gnawing and biting; 4, continual licking and/or
gnawing of cage grids. Animals were used only once.
Open-field studies. At 15min after injection, the animals
were individually placed in the center of the open-field
arena for direct quantification of locomotion frequency
during 5min. The open-field apparatus used in the present
study was a circular wooden box (40cm in diameter and
50cm high) with an open top and a floor divided into 19
squares. Hand-operated counters were used to score
locomotion frequency (number of floor units entered)
during the 5-min sessions. This period of time has been
demonstrated to be effective in detecting AMP-induced
behavioral sensitization in mice (Bellot et al, 1997; Costa
et al, 2001; Frussa-Filho et al, 2004). All the observations
were conducted blind. The animals were used only once.
Experiment 1. Time–response curve to 5.0mg/kg AMP
acute administration in mice. In all, 20 mice were habituated
in individual polypropylene cages (20?30?12.5cm) (IC)
for 150min. After this time, 10 animals received an intra-
peritoneal injection of SAL (NaCl 0.9%) and the other 10
mice received an intraperitoneal injection of 5.0mg/kg AMP.
Immediately later, they were placed in stereotypy observation
cages for SB quantification.
Experiment 2. Validation of the phenomenon of rapid-
onset sensitization of AMP-induced SB in mice and
possible involvement of drug-environment association. In
total, 117 mice were allocated to two groups (57–60 animals
each). Animals of the first group were habituated for
150min in stereotypy observation cages (OC) and mice of
the second group were habituated in IC. After this period,
animals received an intraperitoneal injection of NaCl 0.9%
(SAL) or 5.0mg/kg AMP (a dose which is effective in
inducing intense and focused SB in this species, in our
laboratorial conditions). Immediately after the respective
injections, animals that had been habituated in IC
were placedin OC(PAIR)
been habituated in OC were placed in IC (NPAIR). The
animals were maintained in the respective environments for
150min. After this period, all animals were placed in their
home cages. Animals of the four resulting groups (SAL-
NPAIR, AMP-NPAIR, SAL-PAIR, AMP-PAIR) were sub-
divided into three groups and received a challenge injection
of 1.5mg/kg AMP (an ineffective dose to promote intense
and focused SB in mice, in our laboratorial conditions) 3, 4
or 5h after their respective ‘priming’ injections). Immedi-
ately afterwards, all animals were placed in stereotypy
observation cages and SB was quantified. All the animals
were observed simultaneously, varying the time of the
Rapid-onset sensitization to amphetamine in mice
CC Chinen et al
Kuczenski R, Segal DS (1999b). Sensitization of amphetamine-
induced stereotyped behaviors during the acute response:
role of D1 and D2 dopamine receptors. Brain Res 822:
Kuribara H (1994). Early post-treatment with haloperidol retards
induction of metamphetamine sensitization in mice. Eur J
Pharmacol 256: 295–299.
Kuribara H (1996). Effects of sulpiride and nemonapride,
benzamide derivatives having distinct potencies of antagonistic
action on dopamine D2 receptors, on sensitization to metam-
phetamine in mice. J Pharm Pharmacol 48: 292–296.
Niaura RS, Rohsenow DJ, Binkoff JA, Monti PM, Pedraza M,
Abrams DB (1988). Relevance of cue reactivity to understanding
alcohol and smoking relapse. J Abnorm Psychol 97: 133–152.
Paulson PE, Camp DM, Robinson TE (1991). Time course of
transient behavioral depression and persistent behavioral
sensitization in relation to regional brain monoamine concen-
trations during amphetamine withdrawal in rats. Psychophar-
macology (Berlin) 103: 480–492.
Piazza PV, Deminiere JM, le Moal M, Simon H (1990). Stress- and
pharmacologically-induced behavioral sensitization increases
vulnerability to acquisition of amphetamine self-administration.
Brain Res 514: 22–26.
Pierce RC, Kalivas PW (1997). A circuitry model of the expression
of behavioral sensitization to amphetamine-like psychostimu-
lants. Brain Res Rev 25: 192–216.
Pijnenburg AJ, Honig WM, Van Rossum JM (1975). Inhibition of
D-amphetamine-induced locomotor activity by injection of
haloperidol into the nucleus accumbens of the rat. Psycho-
pharmacologia 41: 87–95.
Robinson TE, Becker JB (1986). Enduring changes in brain and
behavior produced by chronic amphetamine administration: a
review and evaluation of animal models of amphetamine
psychosis. Brain Res Rev 11: 157–198.
Robinson TE, Berridge KC (1993). The neural basis of drug
craving: an incentive-sensitization theory of addiction. Brain Res
Rev 18: 247–291.
Self DW, Nestler EJ (1995). Molecular mechanisms of drug
reinforcement and addiction. Ann Rev Neurosci 18: 463–495.
Setler P, Sarau H, Mckenzie G (1976). Differential attenuation of
some effects of haloperidol in rats given scopolamine. Eur J
Pharmacol 39: 117–126.
Vanderschuren LJ, Schmidt ED, De Vries TJ, Van Moorsel CA,
Tilders FJ, Schoffelmeer AN (1999a). A single exposure to
amphetamine is sufficient to induce long-term behavioral,
neuroendocrine, and neurochemical sensitization in rats.
J Neurosci 19: 9579–9586.
Vanderschuren LJ, Schoffelmeer AN, Mulder AH, De Vries TJ
(1999b). Dopaminergic mechanisms mediating the long-term
expression of locomotor sensitization following pre-exposure to
morphine or amphetamine. Psychopharmacology (Berlin) 143:
Weiss F, Hurd YL, Ungerstedt U, Markou A, Plotsky PM, Koob GF
(1992). Neurochemical correlates of cocaine and ethanol self-
administration. Ann N Y Acad Sci 654: 220–241.
Wise RA, Bozarth MA (1987). A psychomotor stimulant theory of
addiction. Psychological Rev 94: 469–492.
Wise RA, Gingras MA, Amit Z (1996). Influence of novel and
habituated testing conditions on cocaine sensitization. Eur J
Pharmacol 307: 15–19.
Wolf ME, White FJ, Hu XT (1994). MK-801 prevents alterations
in the mesoaccumbens dopamine system associated with
behavioral sensitization to amphetamine. J Neurosci 14(Part 2):
Rapid-onset sensitization to amphetamine in mice
CC Chinen et al