Clozapine pre-treatment has a protracted hypolocomotor effect on the

induction and expression of amphetamine sensitization

Andrea Susana Herrera
1
, Jos Patricio Casanova
1
, Rafael Ignacio Gatica,
Fabiola Escobar, Jos Antonio Fuentealba
Department of Pharmacy, Faculty of Chemistry, Ponticia Universidad Catlica de Chile, Santiago, Chile
Millennium Science Nucleus in Stress and Addiction, Ponticia Universidad Catlica de Chile, Santiago, Chile
a b s t r a c t a r t i c l e i n f o
Article history:
Received 10 June 2013
Received in revised form 12 July 2013
Accepted 31 July 2013
Available online 13 August 2013
Keywords:
Amphetamine
Clozapine
Locomotor sensitization
Amphetamine locomotor sensitization is an animal model for the study of addiction and schizophrenia. The an-
tipsychotic clozapine blocks the hyperlocomotion induced by an acute injection of amphetamine, but its effect on
locomotor sensitization after repeated amphetamine administration remains unknown. In the present study we
investigate the effect of repeated administration of clozapine on the induction and expression of amphetamine
locomotor sensitization. We propose that repeated administration of clozapine blocks the induction and expres-
sion of amphetamine sensitization. Male SpragueDawley rats were classied according to their locomotor
response to an acute saline injection in high responder saline (HRS) or low responder saline (LRS). Rats from
both groups were injected once daily with amphetamine for 5 consecutive days. Horizontal locomotor activity
was measured during 40 min. Four days after the last injection, an acute dose of amphetamine was administered
to assess the expression of sensitization. Clozapine was injected once daily for 4 consecutive days before (pre-
treatment) or after (treatment) induction of sensitization. Pre-treatment with clozapine signicantly decreases
both acute amphetamine-induced hyperlocomotion and the induction and expression of amphetamine sensiti-
zation only in LRS rats, showing a protracted hypolocomotor effect. On the other hand, clozapine treatment
had no effect over locomotor response on the expression of amphetamine sensitization in either LRS or HRS
rats. These data suggest that clozapine effect on amphetamine locomotor response depends on individual differ-
ences. Also, our results suggest that clozapine pre-treatment attenuates the neuroplasticity underlying amphet-
amine sensitization, but clozapine treatment is unable to reverse these changes once amphetamine sensitization
has been induced.
2013 Elsevier Inc. All rights reserved.
1. Introduction
The repeated treatment with psychostimulants leads to locomotor
sensitization. This is evidenced as a progressive and persistent increase
in locomotor activity in rodents and is accompanied by neuroplastic
changes in the nuclei that are part of the motivation circuit, such as
medial prefrontal cortex (mPFC) (Casanova et al., 2013), the nucleus
accumbens (NAc) (Escobar et al., 2012) and the ventral tegmental
area (VTA) (for review see Pierce and Kalivas, 1997). This long-
term behavioral, neurochemical and molecular adaptive responses
in the motive circuit are thought to underlie the pathological drug
craving that leads to relapse to drug seeking even after long period
of withdrawal (Robinson and Berridge, 1993). For example, it has
been shown that behavioral sensitization after amphetamine (AMPH)
repeated treatment enhances the motivation to drug intake, measured
as an acceleration in the scaling cocaine self-administration, when ex-
tended access to drugs procedures are used (Ferrario and Robinson,
2007). On the other hand, locomotor sensitization after repeated treat-
ment with AMPH is also used as a model of schizophrenia (for review,
see Featherstone et al., 2007). In fact, AMPH repeated administration
is associated with psychosis that resembles that of paranoid-
schizophrenia (Sato et al., 1983) and clear evidence shows that a
large enough dose of AMPH can produce a brief psychotic reaction
(Curran et al., 2004). Interestingly, Tenn et al. (2005) have proposed
a partial sensitization to AMPH as a putative model of the prodro-
mal state for schizophrenia.
Clozapine, a dibenzodiazepine with dopamine (DA), serotonin,
histamine and adrenergic receptor antagonist properties (Bruhwyler
et al., 1990) is an atypical antipsychotic licensed for the treatment of
schizophrenia and related schizo-affective disorders in patients refrac-
tory or intolerant to other antipsychotics drugs (Campbell et al.,
1999). Clozapine has also shown efcacy in the treatment of patients
Progress in Neuro-Psychopharmacology & Biological Psychiatry 47 (2013) 16
Abbreviations: ANOVA, analysis of variance; DA, dopamine; HR, high responder; HRS,
high responder saline; i.p, intraperitoneal; LR, low responder; LRS, low responder saline;
mPFC, medial prefrontal cortex; NAc, nucleus accumbens; PCP, phenylciclidine; SUD, sub-
stance use disorder; VTA, ventral tegmental area.
Corresponding author at: Department of Pharmacy, Faculty of Chemistry, Ponticia
Universidad Catolica de Chile, Chile. Tel.: +56 2 2354 5908; fax: +56 2 2354 4744.
E-mail address: jfuentea@uc.cl (J.A. Fuentealba).
1
These authors have equal contribution to this work.
0278-5846/$ see front matter 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.pnpbp.2013.07.023
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with schizophrenia and co-occurring substance use disorder (SUD).
SUD is common in patients with schizophrenia and is associated
with an earlier age onset of schizophrenia and a poor response to
acute treatment compared with patients who do not use substances
(Green, 2006). This particularly prevalent comorbidity has led to
propose that addiction and schizophrenia could share common neu-
robiological substrates related to positive reinforcement, incentive
motivation and behavioral inhibition, particularly in cortical and
temporal-limbic structures (Chambers et al., 2001).
AMPHlocomotor sensitization can be examined in two distinct tem-
poral domains, termed induction and expression. Induction of locomo-
tor sensitization is dened as the transient sequence of neuroadaptive
changes produced by psychostimulant repeated administration re-
sponsible for the increase in locomotor activity. The expression is de-
ned as the permanent establishment of these neuroadaptive changes
responsible for the increased locomotor activity observed after a
washout period (Pierce and Kalivas, 1997). Although there is evidence
showing that clozapine blocks acute AMPH-induced hyperlocomotion
(O'Neill and Shaw, 1999), its effects on locomotor sensitization after
AMPH repeated administration remain unknown. Meng et al. (1998)
showed that co-adminstration of clozapine and AMPH during the in-
duction of sensitization process attenuates the expression of vertical lo-
comotor sensitization. However, the effect of clozapine over horizontal
locomotor activity, an AMPH-induced behavior dependent on dopami-
nergic neurotransmission in the motivational circuit (Sharp et al.,
1987), could not be assessed because after AMPH repeated administra-
tiononly vertical locomotor sensitizationwas observed. Consistent with
this previous study, it has been reported that co-adminstration of cloza-
pine and AMPH attenuates the progress in the locomotor response in-
duced by the repeated treatment with AMPH, in a model of partial
sensitization that resembles the prodromal state of schizophrenia
(Tenn et al., 2005). Although both studies strongly suggest that cloza-
pine can block the effects of acute administration of AMPH during the
induction of locomotor sensitization, there is no evidence that clozapine
can reverse the changes permanently established after AMPH repeated
administration. In this sense, there is evidence that both acute and re-
peated administration of clozapine modulate gene expression in the
mPFC (Kontkanen et al., 2002), suggesting permanent neuroplastic
changes in the motivational circuit following antipsychotic treatment.
Taking into the account this research background, we propose that re-
peated administration of clozapine blocks the induction and expression
of locomotor sensitization.
Given that differential response to a mild stress is associated with
susceptibility to develop AMPH locomotor sensitization (Piazza et al.,
1989), we classied rats according to their locomotor response to
an acute injection of saline in high responder saline (HRS) and low
responder saline (LRS). We then tested the effect of clozapine on
AMPH-induced hyperlocomotion and sensitization in both groups.
In order to study whether repeated administration of clozapine
may prevent AMPH-induced locomotor sensitization, experiments
assessing the induction of horizontal locomotor sensitization in rats
pretreated with a clinically used low dose of clozapine (Kapur
et al., 2003) were carried out. Moreover, to study whether repeated
treatment with clozapine may reverse AMPH sensitization, experi-
ments assessing the expression of horizontal locomotor sensitization
in rats treated with a low dose of clozapine during the washout peri-
od were also conducted.
2. Materials and methods
2.1. Animals
Male SpragueDawley rats, weighing about 250280 g were grown
and maintained during the drug administration in the Animal Care
Facility of the Faculty of Chemistry, Ponticia Universidad Catlica
de Chile. Rats were housed in a colony room in groups of three or four
per cage, where they were kept at room temperature of 20 3 C on
a 12 h light/dark cycle with a food regimen and water ad libitum. All
procedures were in strict accordance with the guidelines published in
the NIH Guide for the Care and Use of Laboratory Animals and with
the principles presented in the Guidelines for the Use of Animals in
Neuroscience Research by the Society for Neuroscience. Rats were han-
dled for three days and before starting the experiments.
2.2. Drugs
D-Amphetamine (Laboratorios Chile, Santiago, Chile) and clozapine
hydrochloride (Laboratorio Recalcine, Santiago, Chile) were dissolved
in saline to be administered intraperitoneally (i.p.).
2.3. Behavioral assessment
Horizontal locomotor activity was measured in a 15 47 26 cm
test box, equipped with two pairs of infrared lights located lengthwise,
separated by 25 cmand 5 cmabove the oor. Crossovers in the test box
were monitored every 1 min during 40 min using a counting device
programmed to count only when both infrared light beams were
interrupted consecutively. Animals were allowed habituating to the
test box for 10 min before any experimental protocol.
2.4. Experimental design and protocol
Basal locomotor activity. Before starting the locomotor sensitization
protocol we evaluated the individual locomotor reactivity to an acute
injection of saline (day 0, Fig. 1), considered as a mild stress (Barrot
et al., 1999). Rats were injected with 500 L i.p. of saline and horizontal
locomotor activity was measured for 40 min. Two groups of rats were
classied on the basis of their level of locomotor activity, either below
or above the median of the group as: high responder saline (HRS) rats
and low responder saline (LRS) rats. Both groups were used to study
the effect of clozapine over AMPH locomotor sensitization.
Amphetamine locomotor sensitization protocol. The process of AMPH
locomotor sensitization was developed according to the schedule de-
scribed by Hedou et al. (2001).
Control groups. Before starting the induction of AMPH sensitization
process, rats were divided in two groups: Saline/Saline/Saline (S/S/S)
and Saline/Amphetamine/Saline (S/A/S). Rats from both groups were
pre-treated with saline once daily for four days (days 1 to 4; Fig. 1).
Rats were returned to their home cages immediately after the injection.
The locomotor activity was not measured during these days. Twenty-
four hours after the last injection, rats were administered with AMPH
(1.5 mg/kg i.p.) or saline once daily between 10:00 and 12:00 h, for
ve consecutive days (days 5 to 9, Fig. 1). Horizontal locomotor activity
was measured in both groups for 40 min after each injection. Twenty-
four hours after completion of the induction of the locomotor sensitiza-
tion process, rats from both groups were treated with saline once daily
for four days (washout period, days 10 to 13). Rats were returned to
their home cages immediately after the injection. During these days,
locomotor activity was not measured. On day 14, rats from both
groups were injected with AMPH (1.5 mg/kg i.p.), and horizontal
Fig. 1. Amphetamine sensitization protocol: pretreatment period (day 1 to day 4), induc-
tion process (day 5 to day 9), washout period (day 10 to day 13) and expression (day 14).
2 A.S. Herrera et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 47 (2013) 16
locomotor activity was measured for 40 min to assess expression of
locomotor sensitization.
Clozapine pre-treatment groups. Before starting the induction of
AMPH sensitization process, rats were divided in two groups: Cloza-
pine/Saline/Saline (C/S/S) and Clozapine/Amphetamine/Saline (C/A/S).
Rats from both groups were injected once daily for four days (days
1 to 4, Fig. 1) with a low dose of clozapine (5 mg/kg i.p., Kapur
et al., 2003). Rats were returned to their home cages immediately
after the injection. The locomotor activity was not measured during
these days. Twenty-four hours after the last injection of clozapine,
rats were administered with AMPH (1.5 mg/kg i.p.) or saline once
daily between 10:00 and 12:00 h, for ve consecutive days (days 5
to 9, Fig. 1). Horizontal locomotor activity was measured in both
groups for 40 min after each injection. Twenty-four hours after com-
pletion the induction of the locomotor sensitization process, rats
from both groups were treated with saline solution once daily for
four days (washout period, days 10 to 13). Rats were returned to
their home cages immediately after the injection. During these
days locomotor activity was not measured. On day 14, rats from
both groups were injected with AMPH (1.5 mg/kg i.p.) and horizon-
tal locomotor activity was measured for 40 min to assess expression
of locomotor sensitization.
Clozapine treatment protocol. Before starting the induction of AMPH
sensitization process, rats were divided in two groups: Saline/Saline/
Clozapine (S/S/C) and Saline/Amphetamine/Clozapine (S/A/C). Rats
from both groups were injected once daily for four days (days 1 to 4,
Fig. 1) with saline. Rats were returned to their home cages immediately
after the injection. The locomotor activity was not measured during
these days. Twenty-four hours after the last injection of saline, rats
were administered with an AMPH (1.5 mg/kg i.p.) or saline once daily
between 10:00 and 12:00 h, for ve consecutive days (days 5 to 9,
Fig. 1). Horizontal locomotor activity was measured in both groups for
40 min after each injection. In this protocol only rats that developed
AMPH locomotor sensitization were used. We consider induction of lo-
comotor sensitization when the rat has at least doubled the crossovers/
40 min on day 9 compared to day 5 (Fig. 1). Twenty-four hours after the
last injection of the locomotor sensitization protocol (day 10), rats from
both groups were injected once daily for four days (days 10 to 13) with
clozapine (5 mg/kg i.p.). Rats from both groups were returned to their
home cages immediately after the injection. During these days loco-
motor activity was not measured. On day 14, rats from both groups
were injected with AMPH (1.5 mg/kg i.p.) and horizontal locomotor
activity was measured for 40 min to assess expression of locomotor
sensitization.
2.5. Statistical analyses
All statistical analyses were performed using Prism 4.0 GraphPad
Software. Resultant data were analyzed by two-way ANOVA and
Bonferroni post-test; one-way ANOVA and NewmanKeuls post test;
unpaired and paired t-test when appropriate. All data are expressed as
mean SEM.
3. Results
3.1. Basal locomotor activity
Rats were classied according to their locomotor response to an
acute saline injection. Rats with a locomotor response below the me-
dian (7 crossings in 40 min) were classied as LRS (2.79 0.32
crossings in 40 min; n = 34), whereas rats with a locomotor re-
sponse above the median were classied as HRS (12.25 1.01
crossings in 40 min; n = 28). Furthermore, a positive correlation
was observed between the locomotor response to an acute saline in-
jection and an acute AMPH injection (Pearson r = 0.5458; p =
0.0004). About 75% of LRS rats developed AMPH-induced locomotor
sensitization, while only 28% of HRS rats developed AMPH-induced
locomotor sensitization.
3.2. High responder saline rats
Fig. 2 shows the mean horizontal locomotor activity of control,
clozapine pre-treatment and clozapine treatment groups during
the 40 min period test after saline or AMPH injection. A repeated two-
way ANOVA, with treatment conditions as the between-subjects var-
iable and days as the within-subjects variable, indicated a signicant
effect of days (F
2,44
= 26.31, p b 0.0001). The effect of treatment
(F
5,44
= 2.55, p = 0.0578) and interaction (F
10,44
= 1.27, p = 0.2727)
was considered not signicant.
Saline-injected rats. ABonferroni post hoc test revealed that the acute
injection of AMPH signicantly increased horizontal locomotor activity
in S/S/S rats (day 5, 11.60 2.32 crossings in 40 min vs day 14,
44.80 8.55 crossings in 40 min; p b 0.05), C/S/S rats (day 5,
12.00 3.72 crossings in 40 min vs day 14, 63.75 11.00 crossings
in 40 min; p b 0.001) and S/S/C rats (day 5, 7.75 1.65 crossings in
40 min vs day 14, 47.50 5.81 crossings in 40 min; p b 0.05).
Amphetamine-injected rats. An unpaired t-test revealed that the
acute injection of AMPHsignicantly increased horizontal locomotor
activity on day 5 in AMPH-injected rats compared to day 5 in saline-
injected rats (day 5 S/S/S: 11.60 2.32 crossings in 40 min vs day 5
S/A/S: 34.86 7.99 crossings in 40 min, p b 0.05; day 5 C/S/S:
12.00 3.72 crossings in 40 min vs day 5 C/A/S: 38.25 8.23 cross-
ings in 40 min, p b 0.05; day 5 S/S/C: 7.75 1.65 crossings in 40 min
vs day 5 S/A/C: 42.00 11.68 crossings in 40 min; p b 0.05). However,
AMPH repeated treatment failed to elicit locomotor sensitization.
3.3. Low responder rats
Fig. 3 shows the mean horizontal locomotor activity of control,
clozapine-pre-treatment and clozapine-treatment groups during
the 40 min period test after saline or AMPH injection. A repeated
two-way ANOVA, with treatment conditions as the between-subjects
variable and days as the within-subjects variable, revealed a signicant
effect of treatment (F
5,58
= 13.92, p b 0.0001), days (F
2,58
= 38.24,
p b 0.0001), and interaction (F
10,58
= 3.94, p b 0.001).
Saline-injected rats. A Bonferroni post hoc test demonstrated that the
acute injection of AMPH signicantly increased horizontal locomotor
activity in S/S/S rats (day 5, 2.14 0.88 crossings in 40 min vs day 14,
25.00 5.30 crossings in 40 min; p b 0.01) and C/S/S rats (day 5,
5.60 1.47 crossings in 40 min vs day 14, 27.40 7.73 crossings in
40 min; p b 0.05). No signicant differences were observed in S/S/C
rats (day 5, 1.50 0.96 crossings in 40 min vs day 14, 4.50 2.63
crossings in 40 min). Further analysis shows that in clozapine-treated
rats the horizontal locomotor activity after an acute dose of AMPHis sig-
nicantly lower compared to the response in saline-treated rats (S/S/S
day 14, 25.00 5.30 crossings in 40 min vs S/S/C day 14, 4.50 2.63
crossings in 40 min; p b 0.05, according to unpaired t-test), demon-
strating that the treatment with clozapine during washout period sig-
nicantly attenuated the hyperlocomotor effect of an acute injection
of AMPH (see Fig. 3).
Amphetamine-injected rats. Unlike what was observed in HRS rats, in
LRS rats repeated AMPH treatment induced locomotor sensitization. A
Bonferroni post-test revealed the induction of locomotor sensitization
after 5 daily injections of AMPHin S/A/S rats (day 5, 21.00 5.23 cross-
ings in 40 min vs day 9, 49.50 8.55 crossings in 40 min; p b 0.001),
C/A/S (day 5, 4.00 2.10 crossings in 40 min vs day 9, 27.80 4.18
crossings in 40 min; p b 0.05) and S/A/C rats (day 5, 15.33 3.16
crossings in 40 min vs day 9, 51.67 4.52 crossings in 40 min;
p b 0.001). Moreover, expressionof locomotor sensitizationwas evident
as an increased response to an AMPH challenge on day 14 in S/A/S (day
5, 21.00 5.23 crossings in 40 min vs day 14, 65.5 11.54 crossings in
40 min; p b 0.001), C/A/S (day 5, 4.00 2.10 crossings in 40 min vs
3 A.S. Herrera et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 47 (2013) 16
day 14, 26.80 8.55 crossings in 40 min; p b 0.05) and S/A/C rats (day
5, 15.33 3.36 crossings in 40 min vs day 14, 60.67 9.60 crossings
in 40 min; p b 0.001). A one-way ANOVA showed that in clozapine-
pretreated rats the horizontal locomotor activity after an acute dose of
AMPH is signicantly lower compared to the response in saline-
pretreated rats (S/A/S day 5, 21.0 5.23 crossings in 40 min vs C/A/S
day 5, 4.00 2.10 crossings in 40 min; p b 0.05, according to New-
manKeuls post test), demonstrating a signicant attenuation of
the hyperlocomotor effect of an acute AMPH injection in clozapine
pre-treated rats. Interestingly, clozapine pre-treatment attenuated
both the induction (S/A/S day 9, 49.50 8.50 crossings in 40 min
vs C/A/S day 9, 27.80 4.18 crossings in 40 min; p b 0.05, according
to NewmanKeuls post test) and the expression of horizontal loco-
motor activity (S/A/S day 14, 65.50 11.54 crossings in 40 min vs
C/A/S day 14, 26.80 8.55 crossings in 40 min; p b 0.05, according
to NewmanKeuls post test).
Fig. 2. Effect of clozapine on induction and expression of locomotor sensitization in HRS rats after amphetamine (AMPH) repeated treatment. Horizontal locomotor activity was measured
during 40 min after an acute injection of AMPH (1.5 mg/kg, i.p.) or saline. Prior to the induction process of AMPH locomotor sensitization, rats were pretreated once daily with clozapine
(5 mg/kg, i.p.) or saline during 4 days. Bars show the horizontal locomotor activity for the rst day (day 5) and last day (day 9) of the repeated treatment with AMPH or saline. During
withdrawal (washout) period, rats were injectedonce daily with clozapine (5 mg/kg, i.p.) or saline. Twenty-four hours after the last injection, a challenge dose of AMPHwas administered
to assess the expression of locomotor sensitization (day 14). Data corresponds to the mean SEMof four to seven independent experiments. * p b 0.05; ** p b 0.001, according to paired
two-way ANOVA and Bonferroni post hoc test; # p b 0.05 according to unpaired t-test.
Fig. 3. Effect of clozapine on induction and expression of locomotor sensitization in LRS rats after amphetamine (AMPH) repeated treatment. Horizontal locomotor activity was measured
during 40 min after an acute injection of AMPH(1.5 mg/kg, i.p.) or saline. Prior to the induction process of AMPHsensitization, rats were pretreated once daily with clozapine (5 mg/kg,
i.p.) or saline during 4 days. Bars showthe horizontal locomotor activity for the rst day (day 5) and last day (day 9) of the repeatedtreatment withAMPHor saline. During the withdrawal
period, rats were injectedonce daily with clozapine (5 mg/kg, i.p.) or saline. Twenty four hours after the last injection, a challenge dose of AMPHwas administered to assess the expression
of locomotor sensitization (day 14). Data corresponds to the mean SEM of ve to eight independent experiments. * p b 0.01, ** p b 0.001, # p b 0.05 according to paired two-way
ANOVA and Bonferroni post hoc test; & p b 0.05 according to unpaired one-way ANOVA and NewmanKeuls post hoc test ; % p b 0.05 according to unpaired t-test.
4 A.S. Herrera et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 47 (2013) 16
4. Discussion
The behavioral sensitization after AMPH repeated treatment is
commonly used as an animal model for addiction and schizophrenia
(Featherstone et al., 2007; Pierce and Kalivas, 1997). In this study, we
showthat pre-treatment with a lowdose of the atypical antipsychot-
ic clozapine has a protracted hypolocomotor effect that blunts the
expression of AMPH-induced locomotor sensitization in LRS rats.
However, clozapine treatment does not modify the expression of
amphetamine sensitization in either LRS or HRS rats. These results
allowus to partially accept our proposal, since the repeated adminis-
tration with clozapine modied the sensitized locomotor response to
amphetamine only in pre-treatment regimen in LRS rats. Our results
suggest that clozapine effect depends on the locomotor response to
stress; and also, that repeated administration of clozapine attenuates
the neuroplasticity induced by repeated treatment with AMPH,
underlying AMPH-induced locomotor sensitization.
We previously showed that approximately half of the animals treated
develop sensitization after AMPH repeated treatment (Casanova
et al., 2013), which contributes to a bulk of evidence showing a dif-
ferential susceptibility among individuals to develop addiction-like
behaviors (for review, see Blanchard et al., 2009). It has been
shown that the locomotor response to a novel environment, as well
as to an acute AMPHinjection, predicts this differential susceptibility
both associated with differences in response to stress (Piazza et al.,
1989, 1990). Given that exposure to a novel environment is as stress-
ful as a saline injection (Adell et al., 1997), we classied rats
according to their locomotor response to an acute saline injection,
considered as a mild stress (Barrot et al., 1999), in HRS rats and LRS
rats. Although, differences in motivational arousal may certainly ac-
count for this differential locomotor response to mild stress, comple-
mentary behavioral paradigms are necessary to assert this proposition
(Pfaff et al., 2002). We observed that the locomotor response to an
acute injection of saline correlates with the locomotor response to an
acute injection of AMPH. Interestingly, LRS rats develop and express
locomotor sensitization in higher percentage than HRS rats. These
results support the use of the locomotor response to an acute saline
injection as a predictor of susceptibility to develop locomotor sensi-
tization to AMPH.
The most studied neurobiological substrate regarding the individual
vulnerability to develop addiction-like behavior is the mesocorticolimbic
pathway (Blanchard et al., 2009). In regard to dopaminergic neuro-
transmission, it has been described that high responder (HR) novelty
rats have fewer D2 DA receptor binding sites, fewer D2 mRNA and
higher D1 binding sites in the NAc compared to low responder (LR)
novelty rats (Hooks et al., 1994). Moreover, it has been shown that
HR novelty rats have higher basal ring rate and bursting activity
in midbrain dopaminergic neurons and a decreased sensitivity to
the inhibitory effect of the D2 agonist quinpirole compared to LR
novelty rats (Marinelli and White, 2000). Regarding to serotoniner-
gic neurotransmission, HR novelty rats show lower activity of sero-
tonin receptors and lower serotonin extracellular levels than LR
novelty rats (Piazza et al., 1991; Verheij et al., 2009). These neurobi-
ological differences that accompany the locomotor response to stress
might also underlie the differential effect of clozapine observed in
the present research.
Several studies have shown that antipsychotic drugs, like halo-
peridol and clozapine, block AMPH and phenylciclidine (PCP)-
induced hyperlocomotion (Arnt, 1995; Meng et al., 1998; Sun et al.,
2009). In the present study, we also showthat clozapine signicantly
reduces the acute AMPH-induced hyperlocomotion using a similar
clozapine dose to the studies above-mentioned, which are represen-
tative of the clozapine doses used in clinical conditions (Kapur et al.,
2003). However, our experimental design has two important differ-
ences with the above-mentioned studies: 1-Unlike to an acute injec-
tion, clozapine was administered for four consecutive days; 2-In
contrast to a concomitant administration, the AMPH challenge took
place 24 h after the last clozapine injection. Therefore, neuroplastic
changes induced by repeated clozapine administration are likely to
underlie the blockade of AMPH-induced hyperlocomotion. Indeed,
Chiodo and Bunney (1985) showed that a chronic clozapine admin-
istration (10 mg/kg during 21 days) reduces the neuronal activity
in the VTA through a depolarization inactivation mechanism. Given
that the activity of mesolimbic dopaminergic neurons is necessary
for the locomotor activating effect of AMPH (Kelly and Iversen,
1976), the depolarization inactivation is a plausible mechanism to
account for the blockade of AMPH-induced hyperlocomotion observed
24 h after repeated administration of clozapine. However, changes in
the expression of dopaminergic and serotoninergic receptors, which
are important for the hyperlocomotor effect of psychostimulants (Filip
et al., 2001; Vezina and Stewart, 1989), may also be involved. In fact, a
downregulation of D1 and 5HT2 receptors has been described after
chronic clozapine treatment (Doat-Meyerhoefer et al., 2005; Lidow
et al., 1997). Notably, these clozapine-induced neuroadaptations seem
to be transient because the blockade of AMPH-inducedhyperlocomotion
is lost in clozapine pre-treated rats (compare S/S/S vs C/S/S, Fig. 3). Inter-
estingly, whereas the hyperlocomotor response to acute AMPH after
repeated clozapine administration was totally blocked in LRS rats, this
response was unaffected in HRS rats. Given that a higher expression of
D1 receptor has been described in the NAc of HR rats (Hooks et al.,
1994) and that the chronic administration of clozapine decreases the
mRNA D1 receptor levels in the NAc (Damask et al., 1996), a longer
period of clozapine administration might be needed to block the
hyperlocomotor effect of AMPH in HRS rats.
In the present study, the inductionand expression of AMPH-induced
locomotor sensitization were only observed in LRS rats. The higher loco-
motor response to AMPH observed in HRS relative to LRS, which may
underlie the above-mentioned neurobiological differences, generated
a ceiling effect on horizontal locomotor behavior, incompatible with
the induction of AMPH sensitization. We propose that a lower dose of
AMPH might be effective to induce locomotor sensitization in HRS rats.
Our results showthat clozapine repeated administration prior to the
AMPHsensitizationprotocol affected both the induction and expression
of locomotor sensitization. Although clozapine pre-treated rats still
develop and express locomotor sensitization to AMPH after a wash-
out period, the magnitude of the locomotor response is blunted,
when compared to the control group. These data suggest that after
clozapine pre-treatment, the modications in the neuroanatomical
substrates involved in locomotor sensitization (Pierce and Kalivas,
1997) are still taking place but in a lesser extent. The induction and
expression of locomotor sensitization have been associated with a
decrease in dopamine neurotransmission in the mPFC (Bjijou et al.,
2002; Banks and Gratton 1995). Also, it has been suggested that
the mPFC is one of the main loci for the pharmacological effects of
clozapine (Chou et al., 2006; Moghaddam and Bunney, 1990). In
this nucleus, a number of neuroplastic changes have been observed
after clozapine repeated administration: Lidow et al. (1997) showed
a downregulation of D1 receptor, Moran-Gates et al. (2006) reports
an increase in the expression of D2 receptors and Kontkanen et al.
(2002) showed a modulation of the expression of genes involved in
exocytosis of synaptic vesicles and in the regulation of intracellular
calcium. Therefore, clozapine pre-treatment might modify the func-
tioning of DA neurotransmission in the mPFC, attenuating the effects
of AMPH repeated administration and therefore, contributing to the
blunted sensitized response to AMPH.
In contrast to what was observed whit clozapine pre-treatment, clo-
zapine repeated administration after the induction of AMPH sensitiza-
tion did not affect the expression of locomotor sensitization. Although,
there is evidence for a pharmacological reversal of horizontal locomotor
sensitization to cocaine (Li et al., 2000) and methamphetamine (Shuto
et al., 2006), and for AMPH-induced sensitized stereotyped behavior
(Moro et al., 2007), evidence related to reversal of AMPH-induced
5 A.S. Herrera et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 47 (2013) 16
horizontal locomotor sensitization is still lacking. Given that the clo-
zapine treatment used in our study was unable to attenuate the expres-
sion of locomotor sensitization, an increase in clozapine dose and/or in
the length of treatment (Chiodo and Bunney, 1985) might reverse
the enduring neuroadaptations accompanying locomotor sensitiza-
tion (Pierce and Kalivas, 1997).
5. Conclusions
Taken together, our data show that clozapine effect on the
hyperlocomotor response to AMPH depends on the locomotor re-
sponse to stress; clozapine pre-treatment blunts the expression of
AMPH-induced locomotor sensitization in LRS rats, whereas cloza-
pine treatment does not modify it in either LRS or HRS rats. These re-
sults suggest that clozapine attenuates but do not reverse AMPH-
induced changes underlying locomotor sensitization.
Contributors
Jos Antonio Fuentealba designed the study. Andrea Herrera clas-
sied rats in HRS and LRS and carried out clozapine-treatment stud-
ies. Fabiola Escobar and Rafael Gatica performed the clozapine-
pretreatment experiments. Jos Patricio Casanova performed the
statistical analysis. Jos Antonio Fuentealba and Jos Patricio Casanova
wrote the manuscript.
Acknowledgments
This work was supported by FONDECYT Project # 11075068 (J.A.F);
MSI P06/008-F. We thank David Pessoa, PhD. for his technical assistance
and Laboratorios Recalcine S.A. (Santiago, Chile) for the generous dona-
tionof clozapine. We alsothankGabriela Velis for the Englishcorrection.
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