Effects of prenatal immune activation on amphetamine-induced addictive behaviors: Contributions from animal models Aline R. Borc¸oi, Camilla L. Patti, Karina A. Zanin, Andr´e W. Hollais, Renan Santos-Baldaia, Liliane M.B. Ceccon, La´ıs F. Berro, Raphael WuoSilva, Stephanie B. Grapiglia, Luciana T.C. Ribeiro, Leonardo B. Lopes-Silva, Roberto Frussa-Filho PII: DOI: Reference:
S0278-5846(15)00125-6 doi: 10.1016/j.pnpbp.2015.05.015 PNP 8782
To appear in:
Progress in Neuropsychopharmacology & Biological Psychiatry
Received date: Revised date: Accepted date:
9 April 2015 30 May 2015 31 May 2015
Please cite this article as: Bor¸coi Aline R., Patti Camilla L., Zanin Karina A., Hollais Andr´e W., Santos-Baldaia Renan, Ceccon Liliane M.B., Berro La´ıs F., Wuo-Silva Raphael, Grapiglia Stephanie B., Ribeiro Luciana T.C., Lopes-Silva Leonardo B., FrussaFilho Roberto, Effects of prenatal immune activation on amphetamine-induced addictive behaviors: Contributions from animal models, Progress in Neuropsychopharmacology & Biological Psychiatry (2015), doi: 10.1016/j.pnpbp.2015.05.015
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT EFFECTS OF PRENATAL IMMUNE ACTIVATION ON AMPHETAMINE-INDUCED
SC RI
PT
ADDICTIVE BEHAVIORS: CONTRIBUTIONS FROM ANIMAL MODELS
Aline R. Borçoi1; Camilla L. Patti1; Karina A. Zanin1, 2; André W. Hollais1; Renan Santos-Baldaia1; Liliane M. B. Ceccon1; Laís F. Berro1,2; Raphael
NU
Wuo-Silva1; Stephanie B. Grapiglia1; Luciana T. C. Ribeiro1; Leonardo B. Lopes-Silva1,2; Roberto Frussa-Filho1† In memoriam
MA
†
1
ED
Departamento de Farmacologia, Universidade Federal de São Paulo, R.
PT
Botucatu, 862, Ed. Leal Prado, 1º andar, 04023062, São Paulo, SP, Brazil. 2
Departamento de Psicobiologia, Universidade Federal de São Paulo, R.
AC
CE
Napoleão de Barros, 925, 04021002, São Paulo, SP, Brazil.
*Correspondence: Camilla L. Patti Departamento de Farmacologia – UNIFESP Rua Botucatu, 862 – Ed. Leal Prado, 1º andar – 04023062 São Paulo, SP – Brazil. Fax (55-11) 5549 4122 r. 222 - Phone – (55-11) 5549 4122 e-mail: [email protected] or [email protected]
ACCEPTED MANUSCRIPT EFFECTS OF PRENATAL IMMUNE ACTIVATION ON AMPHETAMINE-INDUCED
PT
ADDICTIVE BEHAVIORS: CONTRIBUTIONS FROM ANIMAL MODELS
SC RI
Aline R. Borçoi1; Camilla L. Patti1; Karina A. Zanin1, 2; André W. Hollais1; Renan Santos-Baldaia1; Liliane M. B. Ceccon1; Laís F. Berro1,2; Raphael Wuo-Silva1; Stephanie B. Grapiglia1; Luciana T. C. Ribeiro1; Leonardo B.
In memoriam
MA
†
NU
Lopes-Silva1,2; Roberto Frussa-Filho1†
1
Departamento de Farmacologia, Universidade Federal de São Paulo, R.
ED
Botucatu, 862, Ed. Leal Prado, 1º andar, 04023062, São Paulo, SP, Brazil. 2
PT
Departamento de Psicobiologia, Universidade Federal de São Paulo, R.
AC
CE
Napoleão de Barros, 925, 04021002, São Paulo, SP, Brazil.
*Correspondence: Camilla L. Patti Departamento de Farmacologia – UNIFESP Rua Botucatu, 862 – Ed. Leal Prado, 1º andar – 04023062 São Paulo, SP – Brazil. Fax (55-11) 5549 4122 r. 222 - Phone – (55-11) 5549 4122 e-mail: [email protected] or [email protected]
ACCEPTED MANUSCRIPT ABSTRACT Background:
Prenatal
environmental
adversities
may
affect
brain
PT
development and are associated with increased risk for schizophrenia, an illness with 50% comorbidity with addiction. Maternal immune activation by
SC RI
poly-inosinic-citidilic acid (Poly(I:C)) exposure can promote behavioral alterations consistent with schizophrenia symptoms in rodents. Objectives: Considering the vulnerability to addiction in patients with schizophrenia, we
NU
evaluated the interactions between prenatal Poly(I:C) administration and
MA
addiction in two animal models (behavioral sensitization and conditioned place preference – CPP) in mice repeatedly treated with amphetamine (AMP).
ED
Additionally, stereotyped behavior and cross-sensitization with cocaine (COC) were also investigated. Methods: Swiss male mice offspring were submitted
PT
to prenatal administration of 5 mg/kg Poly(I:C) in the 9th day of pregnancy. At the age of 90 days, mice were treated with 2.5 mg/kg AMP for 9 days to
CE
evaluate behavioral sensitization or stereotyped behavior. Cross-sensitization
AC
with 10 mg/kg COC was evaluated 24 h after the last treatment day. For AMPinduced CPP evaluation, mice were treated during 8 consecutive days. Results: Prenatal Poly(I:C) administration potentiated both AMP-induced behavioral sensitization and CPP. Furthermore, Poly(I:C) increased crosssensitization with COC. Conclusions: Prenatal administration of Poly(I:C) is able to potentiate vulnerability to addiction in two animal models, without however modulating stereotyped behavior.
Keywords: schizophrenia; Poly(I:C); amphetamine; addiction; mice. 1. INTRODUCTION
ACCEPTED MANUSCRIPT
Schizophrenia is a neuropsychiatric illness characterized by paranoid
PT
disillusions, hallucinations, motivational impairment and affective and cognitive dysfunctions. It is supposed to affect approximately 1% of general
SC RI
population (Insel, 2010; Nagai et al., 2011; Van Os et al., 2010). Although the etiology of schizophrenia remains controversial, it is considered multifactorial and influenced by genetic and environmental factors (Jones et al., 2011; Van
NU
Os et al., 2010).
MA
Several hypotheses have been postulated to clarify the etiology of schizophrenia. In particular, the neurodevelopmental hypothesis is a
ED
fundamental theory that does not contradict other hypothesis. It proposes that genes involved in neurodevelopment are influenced by environmental insults
PT
in early life, including prenatal infections (Insel, 2010; Van Os et al., 2010), able to impair brain development. Epidemiologic, clinical, and preclinical
CE
investigations have provided evidence that maternal infection during
AC
pregnancy, like influenza, rubella, toxoplasmosis and herpes simplex type 2, contributes to the etiology of schizophrenia (Brown and Derkits, 2010; Ozawa et al., 2006; Rapoport, et al., 2005). Therefore, maternal immune activation has been suggested as an animal model of schizophrenia. The administration in the pregnant mother of a substance like the bacterial mimic lipopolysaccharide (LPS) or polyinosinicpolicytidylic acid (Poly(I:C)), that mimics a infectious agent, promotes the immune response (Meyer et al., 2005, 2009; Nagai et al., 2011; Vorhees et al., 2012). More specifically, Poly(I:C) is used to mimic the acute phase of a viral
ACCEPTED MANUSCRIPT infection, eliciting pro-inflammatory cytokine expression and stimulating innate immune response (Boksa, 2010; Fortier et al., 2004).
PT
A substantial number of patients with schizophrenia is addicted to drugs. Studies have demonstrated that drug abuse among these individuals is
SC RI
significantly higher than in the general population, assessed around 50% (Blanchard et al., 2000, Gregg et al., 2007; Kosten and Ziedonis, 1997; Pettersen et al., 2013). Although the basis of schizophrenia and addiction
NU
comorbidity is unclear, a number of theories have been suggested, such as
MA
the use of drugs to alleviate extrapyramidal symptoms induced by neuroleptics (Blanchard et al., 2000, Green, 2005; Pettersen et al., 2013).
ED
Another explanation includes the interaction between the brain circuitry involved both in schizophrenia and drug reward, the mesoaccumbens
PT
dopaminergic system (Bellack et al., 2006; Green, 2005; Gregg et al., 2007; Kalivas and O'brien, 2008; Koob, 1992).
CE
Within this context, the present study investigated the effects of
AC
amphetamine (AMP)-induced conditioned place preference (CPP) and behavioral sensitization in adult mice offspring after maternal immune activation induced by Poly(I:C) administration.
ACCEPTED MANUSCRIPT 2. MATERIAL AND METHODS
PT
2.1. Subjects Two-month-old female and 3-month-old male Swiss mice (40-45g,
SC RI
outbred, raised, and maintained in the Centre for Development of Experimental Models in Medicine and Biology of Universidade Federal de São Paulo) were used. Animals were housed under conditions of controlled
NU
temperature (22-23°C) and lighting (12h light, 12h dark; lights on at 6:45 a.m.).
MA
Food and water were available throughout the experiments. Animals used in this study were maintained in accordance with the National Institute of Health
ED
Guide for the Care and Use of Laboratory Animals (NIH Publications Nº 8023, revised 2011). The experimental procedures were approved by the
CE
2.2. Drugs
PT
Institutional Animal Care and Use Committee under the protocol #0164/12.
AC
Polyinosinic:polycytidylic potassium salt (Poly(I:C), 5 mg/kg; Sigma®), d-amphetamine (AMP, 2.5 mg/kg; Sigma®) and cocaine–HCl (COC, 10 mg/kg; Sigma®) were freshly diluted in 0.9% saline solution (SAL). Drugs were administered intraperitoneally (i.p.) at a volume of 10 ml/kg body weight. Control groups received an equivalent volume of SAL. The doses of AMP and COC used in this study were chosen based on previous data from our laboratory (Berro et al., 2014; Carvalho et al., 2009; Fukushiro et al., 2007, 2008; Saito et al., 2014). The dose of Poly(I:C) was determined from dosage ranges used by other investigators (Boksa, 2010).
ACCEPTED MANUSCRIPT 2.3. POLY(I:C) prenatal administration Females and males (2:1) were co-housed overnight and allowed to
PT
mate. Males were removed from the cage in the subsequent morning. Successful mating was confirmed by the presence of vaginal plug and the day
SC RI
was referred to as the gestational day (GD) 0 (Meyer et al., 2005). At GD 9 (Meyer et al., 2008b), pregnant mice received a single injection of SAL or Poly(I:C).
NU
All pregnant mice were housed individually after the injection and left
MA
undisturbed with the exception of regular cage cleaning until weaning. The day of birth was defined as the postnatal day (PD) 0. Pups were weaned on
ED
PD 21 and housed 2-3 per cage according to sex and litter. Offspring male mice born from females treated with SAL were allocated to control group
PT
(CTRL). Similarly, males born from females treated with Poly(I:C) composed
CE
the Poly(I:C) group.
AC
2.4. Open-field
The open-field was a circular wooden box (40 cm in diameter and 50
cm high) with an open top and a floor divided into 19 squares as previously described (Chinen et al., 2006). During a 10-min session, hand-operated counters were used to quantify locomotion frequency (number of entries into any floor unit with the four paws). The observer was unaware of the experimental design.
2.5. Stereotyped behavior
ACCEPTED MANUSCRIPT Mice were individually placed in wire mesh cages (16 x 30 x 18 cm) free of water and food for habituation during 60 min before the beginning of
PT
observation. Immediately after, animals received an injection of SAL or AMP and had their stereotyped behavior quantified after 20 min. This procedure
SC RI
was repeated during 9 alternated days. The stereotyped behavior was quantified for 15 s, every 5 min during 100 consecutive min. The stereotypy notes were attributed as proposed by Setler et al., 1976 and Chinen et al.,
NU
2006. Scores from 0 to 4 were attributed to each animal, as follows: 0) asleep
MA
or stationary; 1) active; 2) active with predominantly stereotyped sniffing and rearing; 3) stereotyped sniffing with bursts of licking and/or gnawing and
ED
biting; 4) continual licking and/or gnawing of cage grids.
PT
2.6 Conditioned place preference (CPP) The apparatus consisted of two conditioning compartments (one black
CE
with a white grid floor and other white with a black smooth floor) of equal size
AC
(40 x 18 x 16 cm) that were both accessible from a central choice compartment (40 x 10 x 10 cm) and the procedure consisted of two phases: conditioning and test. Conditioning: an unbiased design was used because mice have shown no preference for either of the compartments in pilot studies. Animals were randomly
assigned
to
an
experimental
group
and
a
“drug-paired
compartment” in a counterbalanced fashion, with the black compartment for half of the animals and the white compartment for the other half. One “drugpaired compartment” and one “non-drug-paired compartment” were defined for all animals. The conditioning trials were performed during 8 consecutive
ACCEPTED MANUSCRIPT days. Animals received SAL in the even days and AMP in the odd days. Twenty min after injection, mice were confined to the assigned drug- or non-
PT
drug-paired compartment for 10 min. Test: animals were placed in the central compartment with free access
SC RI
to each compartment during 15 min and without previous injections. Expression of drug-induced CPP was evidenced by the CPP score (difference between the time spent in the drug-paired and in the non-drug-paired
NU
compartments). Thus, positive score values are able to show preference for
2.7 Experimental design
MA
the conditioned compartment.
ED
2.7.1 Experiment 1: Effects of prenatal Poly(I:C) treatment on AMP-induced
PT
behavioral sensitization and cross-sensitization to COC Pregnant mice were treated with Poly(I:C) as detailed in item 2.3. Then,
CE
40 offspring male mice at PD 90 were randomly allocated to 4 (n=10/group):
AC
CTRL-SAL, CTRL-AMP, Poly(I:C)-SAL and Poly(I:C)-AMP. Animals were exposed to habituation sessions for 10 min in the open-field for 3 consecutive days, 20 min after a SAL injection. During the induction phase of behavioral sensitization, animals were treated with SAL or AMP for 9 days, every other day. Twenty min after the injection animals were exposed to the open-field arena for 10 min in the days 1, 5 and 9, which corresponded to the 1st, 3rd and 5th AMP injections. To evaluate cross-sensitization, animals received a COC challenge injection 10 days after the beginning of the AMP treatment. Five min after COC injection, mice were again exposed to the open-field.
ACCEPTED MANUSCRIPT
2.7.2 Experiment 2: Effects of repeated administration of AMP on stereotyped
PT
behavior in mice prenatally treated with Poly(I:C) Forty offspring male mice at PD 90 days were randomly allocated to 4
SC RI
groups (n=10 per group): CTRL-SAL, CTRL-AMP, Poly(I:C)-SAL and Poly(I:C)-AMP. The procedure was the same as described in item 2.7.1 replacing the open-field model with stereotyped behavior as described in item
MA
NU
2.5.
2.7.3 Experiment 3: Effects of prenatal Poly(I:C) treatment on AMP-induced
ED
CPP
Twenty offspring male mice at PD 90 were randomly allocated to 2
PT
groups (n=10 per group): CTRL-AMP and Poly(I:C)-AMP. Mice were conditioned as described in item 2.6. Thus, daily sessions were performed
CE
during 8 days and each animal underwent 4 conditioning sessions after SAL
AC
injection and 4 conditioning sessions after AMP. Post-conditioning test was performed 24h after the last conditioning day, in which animal was allowed to choose between the two compartments of CPP apparatus during 15 min in the absence of drug.
2.8 Statistical analysis Locomotors responses in different days of drug treatment were analyzed by ANOVA with repeated measures. Multiple comparisons were performed using two-ANOVA (with prenatal and postnatal treatment as factors) and Duncan's post hoc test when necessary. Within-group
ACCEPTED MANUSCRIPT comparisons were performed using the paired samples t-test. CPP score was analyzed using the Student’s t-test. A probability of p
S0278-5846(15)00125-6 doi: 10.1016/j.pnpbp.2015.05.015 PNP 8782
To appear in:
Progress in Neuropsychopharmacology & Biological Psychiatry
Received date: Revised date: Accepted date:
9 April 2015 30 May 2015 31 May 2015
Please cite this article as: Bor¸coi Aline R., Patti Camilla L., Zanin Karina A., Hollais Andr´e W., Santos-Baldaia Renan, Ceccon Liliane M.B., Berro La´ıs F., Wuo-Silva Raphael, Grapiglia Stephanie B., Ribeiro Luciana T.C., Lopes-Silva Leonardo B., FrussaFilho Roberto, Effects of prenatal immune activation on amphetamine-induced addictive behaviors: Contributions from animal models, Progress in Neuropsychopharmacology & Biological Psychiatry (2015), doi: 10.1016/j.pnpbp.2015.05.015
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT EFFECTS OF PRENATAL IMMUNE ACTIVATION ON AMPHETAMINE-INDUCED
SC RI
PT
ADDICTIVE BEHAVIORS: CONTRIBUTIONS FROM ANIMAL MODELS
Aline R. Borçoi1; Camilla L. Patti1; Karina A. Zanin1, 2; André W. Hollais1; Renan Santos-Baldaia1; Liliane M. B. Ceccon1; Laís F. Berro1,2; Raphael
NU
Wuo-Silva1; Stephanie B. Grapiglia1; Luciana T. C. Ribeiro1; Leonardo B. Lopes-Silva1,2; Roberto Frussa-Filho1† In memoriam
MA
†
1
ED
Departamento de Farmacologia, Universidade Federal de São Paulo, R.
PT
Botucatu, 862, Ed. Leal Prado, 1º andar, 04023062, São Paulo, SP, Brazil. 2
Departamento de Psicobiologia, Universidade Federal de São Paulo, R.
AC
CE
Napoleão de Barros, 925, 04021002, São Paulo, SP, Brazil.
*Correspondence: Camilla L. Patti Departamento de Farmacologia – UNIFESP Rua Botucatu, 862 – Ed. Leal Prado, 1º andar – 04023062 São Paulo, SP – Brazil. Fax (55-11) 5549 4122 r. 222 - Phone – (55-11) 5549 4122 e-mail: [email protected] or [email protected]
ACCEPTED MANUSCRIPT EFFECTS OF PRENATAL IMMUNE ACTIVATION ON AMPHETAMINE-INDUCED
PT
ADDICTIVE BEHAVIORS: CONTRIBUTIONS FROM ANIMAL MODELS
SC RI
Aline R. Borçoi1; Camilla L. Patti1; Karina A. Zanin1, 2; André W. Hollais1; Renan Santos-Baldaia1; Liliane M. B. Ceccon1; Laís F. Berro1,2; Raphael Wuo-Silva1; Stephanie B. Grapiglia1; Luciana T. C. Ribeiro1; Leonardo B.
In memoriam
MA
†
NU
Lopes-Silva1,2; Roberto Frussa-Filho1†
1
Departamento de Farmacologia, Universidade Federal de São Paulo, R.
ED
Botucatu, 862, Ed. Leal Prado, 1º andar, 04023062, São Paulo, SP, Brazil. 2
PT
Departamento de Psicobiologia, Universidade Federal de São Paulo, R.
AC
CE
Napoleão de Barros, 925, 04021002, São Paulo, SP, Brazil.
*Correspondence: Camilla L. Patti Departamento de Farmacologia – UNIFESP Rua Botucatu, 862 – Ed. Leal Prado, 1º andar – 04023062 São Paulo, SP – Brazil. Fax (55-11) 5549 4122 r. 222 - Phone – (55-11) 5549 4122 e-mail: [email protected] or [email protected]
ACCEPTED MANUSCRIPT ABSTRACT Background:
Prenatal
environmental
adversities
may
affect
brain
PT
development and are associated with increased risk for schizophrenia, an illness with 50% comorbidity with addiction. Maternal immune activation by
SC RI
poly-inosinic-citidilic acid (Poly(I:C)) exposure can promote behavioral alterations consistent with schizophrenia symptoms in rodents. Objectives: Considering the vulnerability to addiction in patients with schizophrenia, we
NU
evaluated the interactions between prenatal Poly(I:C) administration and
MA
addiction in two animal models (behavioral sensitization and conditioned place preference – CPP) in mice repeatedly treated with amphetamine (AMP).
ED
Additionally, stereotyped behavior and cross-sensitization with cocaine (COC) were also investigated. Methods: Swiss male mice offspring were submitted
PT
to prenatal administration of 5 mg/kg Poly(I:C) in the 9th day of pregnancy. At the age of 90 days, mice were treated with 2.5 mg/kg AMP for 9 days to
CE
evaluate behavioral sensitization or stereotyped behavior. Cross-sensitization
AC
with 10 mg/kg COC was evaluated 24 h after the last treatment day. For AMPinduced CPP evaluation, mice were treated during 8 consecutive days. Results: Prenatal Poly(I:C) administration potentiated both AMP-induced behavioral sensitization and CPP. Furthermore, Poly(I:C) increased crosssensitization with COC. Conclusions: Prenatal administration of Poly(I:C) is able to potentiate vulnerability to addiction in two animal models, without however modulating stereotyped behavior.
Keywords: schizophrenia; Poly(I:C); amphetamine; addiction; mice. 1. INTRODUCTION
ACCEPTED MANUSCRIPT
Schizophrenia is a neuropsychiatric illness characterized by paranoid
PT
disillusions, hallucinations, motivational impairment and affective and cognitive dysfunctions. It is supposed to affect approximately 1% of general
SC RI
population (Insel, 2010; Nagai et al., 2011; Van Os et al., 2010). Although the etiology of schizophrenia remains controversial, it is considered multifactorial and influenced by genetic and environmental factors (Jones et al., 2011; Van
NU
Os et al., 2010).
MA
Several hypotheses have been postulated to clarify the etiology of schizophrenia. In particular, the neurodevelopmental hypothesis is a
ED
fundamental theory that does not contradict other hypothesis. It proposes that genes involved in neurodevelopment are influenced by environmental insults
PT
in early life, including prenatal infections (Insel, 2010; Van Os et al., 2010), able to impair brain development. Epidemiologic, clinical, and preclinical
CE
investigations have provided evidence that maternal infection during
AC
pregnancy, like influenza, rubella, toxoplasmosis and herpes simplex type 2, contributes to the etiology of schizophrenia (Brown and Derkits, 2010; Ozawa et al., 2006; Rapoport, et al., 2005). Therefore, maternal immune activation has been suggested as an animal model of schizophrenia. The administration in the pregnant mother of a substance like the bacterial mimic lipopolysaccharide (LPS) or polyinosinicpolicytidylic acid (Poly(I:C)), that mimics a infectious agent, promotes the immune response (Meyer et al., 2005, 2009; Nagai et al., 2011; Vorhees et al., 2012). More specifically, Poly(I:C) is used to mimic the acute phase of a viral
ACCEPTED MANUSCRIPT infection, eliciting pro-inflammatory cytokine expression and stimulating innate immune response (Boksa, 2010; Fortier et al., 2004).
PT
A substantial number of patients with schizophrenia is addicted to drugs. Studies have demonstrated that drug abuse among these individuals is
SC RI
significantly higher than in the general population, assessed around 50% (Blanchard et al., 2000, Gregg et al., 2007; Kosten and Ziedonis, 1997; Pettersen et al., 2013). Although the basis of schizophrenia and addiction
NU
comorbidity is unclear, a number of theories have been suggested, such as
MA
the use of drugs to alleviate extrapyramidal symptoms induced by neuroleptics (Blanchard et al., 2000, Green, 2005; Pettersen et al., 2013).
ED
Another explanation includes the interaction between the brain circuitry involved both in schizophrenia and drug reward, the mesoaccumbens
PT
dopaminergic system (Bellack et al., 2006; Green, 2005; Gregg et al., 2007; Kalivas and O'brien, 2008; Koob, 1992).
CE
Within this context, the present study investigated the effects of
AC
amphetamine (AMP)-induced conditioned place preference (CPP) and behavioral sensitization in adult mice offspring after maternal immune activation induced by Poly(I:C) administration.
ACCEPTED MANUSCRIPT 2. MATERIAL AND METHODS
PT
2.1. Subjects Two-month-old female and 3-month-old male Swiss mice (40-45g,
SC RI
outbred, raised, and maintained in the Centre for Development of Experimental Models in Medicine and Biology of Universidade Federal de São Paulo) were used. Animals were housed under conditions of controlled
NU
temperature (22-23°C) and lighting (12h light, 12h dark; lights on at 6:45 a.m.).
MA
Food and water were available throughout the experiments. Animals used in this study were maintained in accordance with the National Institute of Health
ED
Guide for the Care and Use of Laboratory Animals (NIH Publications Nº 8023, revised 2011). The experimental procedures were approved by the
CE
2.2. Drugs
PT
Institutional Animal Care and Use Committee under the protocol #0164/12.
AC
Polyinosinic:polycytidylic potassium salt (Poly(I:C), 5 mg/kg; Sigma®), d-amphetamine (AMP, 2.5 mg/kg; Sigma®) and cocaine–HCl (COC, 10 mg/kg; Sigma®) were freshly diluted in 0.9% saline solution (SAL). Drugs were administered intraperitoneally (i.p.) at a volume of 10 ml/kg body weight. Control groups received an equivalent volume of SAL. The doses of AMP and COC used in this study were chosen based on previous data from our laboratory (Berro et al., 2014; Carvalho et al., 2009; Fukushiro et al., 2007, 2008; Saito et al., 2014). The dose of Poly(I:C) was determined from dosage ranges used by other investigators (Boksa, 2010).
ACCEPTED MANUSCRIPT 2.3. POLY(I:C) prenatal administration Females and males (2:1) were co-housed overnight and allowed to
PT
mate. Males were removed from the cage in the subsequent morning. Successful mating was confirmed by the presence of vaginal plug and the day
SC RI
was referred to as the gestational day (GD) 0 (Meyer et al., 2005). At GD 9 (Meyer et al., 2008b), pregnant mice received a single injection of SAL or Poly(I:C).
NU
All pregnant mice were housed individually after the injection and left
MA
undisturbed with the exception of regular cage cleaning until weaning. The day of birth was defined as the postnatal day (PD) 0. Pups were weaned on
ED
PD 21 and housed 2-3 per cage according to sex and litter. Offspring male mice born from females treated with SAL were allocated to control group
PT
(CTRL). Similarly, males born from females treated with Poly(I:C) composed
CE
the Poly(I:C) group.
AC
2.4. Open-field
The open-field was a circular wooden box (40 cm in diameter and 50
cm high) with an open top and a floor divided into 19 squares as previously described (Chinen et al., 2006). During a 10-min session, hand-operated counters were used to quantify locomotion frequency (number of entries into any floor unit with the four paws). The observer was unaware of the experimental design.
2.5. Stereotyped behavior
ACCEPTED MANUSCRIPT Mice were individually placed in wire mesh cages (16 x 30 x 18 cm) free of water and food for habituation during 60 min before the beginning of
PT
observation. Immediately after, animals received an injection of SAL or AMP and had their stereotyped behavior quantified after 20 min. This procedure
SC RI
was repeated during 9 alternated days. The stereotyped behavior was quantified for 15 s, every 5 min during 100 consecutive min. The stereotypy notes were attributed as proposed by Setler et al., 1976 and Chinen et al.,
NU
2006. Scores from 0 to 4 were attributed to each animal, as follows: 0) asleep
MA
or stationary; 1) active; 2) active with predominantly stereotyped sniffing and rearing; 3) stereotyped sniffing with bursts of licking and/or gnawing and
ED
biting; 4) continual licking and/or gnawing of cage grids.
PT
2.6 Conditioned place preference (CPP) The apparatus consisted of two conditioning compartments (one black
CE
with a white grid floor and other white with a black smooth floor) of equal size
AC
(40 x 18 x 16 cm) that were both accessible from a central choice compartment (40 x 10 x 10 cm) and the procedure consisted of two phases: conditioning and test. Conditioning: an unbiased design was used because mice have shown no preference for either of the compartments in pilot studies. Animals were randomly
assigned
to
an
experimental
group
and
a
“drug-paired
compartment” in a counterbalanced fashion, with the black compartment for half of the animals and the white compartment for the other half. One “drugpaired compartment” and one “non-drug-paired compartment” were defined for all animals. The conditioning trials were performed during 8 consecutive
ACCEPTED MANUSCRIPT days. Animals received SAL in the even days and AMP in the odd days. Twenty min after injection, mice were confined to the assigned drug- or non-
PT
drug-paired compartment for 10 min. Test: animals were placed in the central compartment with free access
SC RI
to each compartment during 15 min and without previous injections. Expression of drug-induced CPP was evidenced by the CPP score (difference between the time spent in the drug-paired and in the non-drug-paired
NU
compartments). Thus, positive score values are able to show preference for
2.7 Experimental design
MA
the conditioned compartment.
ED
2.7.1 Experiment 1: Effects of prenatal Poly(I:C) treatment on AMP-induced
PT
behavioral sensitization and cross-sensitization to COC Pregnant mice were treated with Poly(I:C) as detailed in item 2.3. Then,
CE
40 offspring male mice at PD 90 were randomly allocated to 4 (n=10/group):
AC
CTRL-SAL, CTRL-AMP, Poly(I:C)-SAL and Poly(I:C)-AMP. Animals were exposed to habituation sessions for 10 min in the open-field for 3 consecutive days, 20 min after a SAL injection. During the induction phase of behavioral sensitization, animals were treated with SAL or AMP for 9 days, every other day. Twenty min after the injection animals were exposed to the open-field arena for 10 min in the days 1, 5 and 9, which corresponded to the 1st, 3rd and 5th AMP injections. To evaluate cross-sensitization, animals received a COC challenge injection 10 days after the beginning of the AMP treatment. Five min after COC injection, mice were again exposed to the open-field.
ACCEPTED MANUSCRIPT
2.7.2 Experiment 2: Effects of repeated administration of AMP on stereotyped
PT
behavior in mice prenatally treated with Poly(I:C) Forty offspring male mice at PD 90 days were randomly allocated to 4
SC RI
groups (n=10 per group): CTRL-SAL, CTRL-AMP, Poly(I:C)-SAL and Poly(I:C)-AMP. The procedure was the same as described in item 2.7.1 replacing the open-field model with stereotyped behavior as described in item
MA
NU
2.5.
2.7.3 Experiment 3: Effects of prenatal Poly(I:C) treatment on AMP-induced
ED
CPP
Twenty offspring male mice at PD 90 were randomly allocated to 2
PT
groups (n=10 per group): CTRL-AMP and Poly(I:C)-AMP. Mice were conditioned as described in item 2.6. Thus, daily sessions were performed
CE
during 8 days and each animal underwent 4 conditioning sessions after SAL
AC
injection and 4 conditioning sessions after AMP. Post-conditioning test was performed 24h after the last conditioning day, in which animal was allowed to choose between the two compartments of CPP apparatus during 15 min in the absence of drug.
2.8 Statistical analysis Locomotors responses in different days of drug treatment were analyzed by ANOVA with repeated measures. Multiple comparisons were performed using two-ANOVA (with prenatal and postnatal treatment as factors) and Duncan's post hoc test when necessary. Within-group
ACCEPTED MANUSCRIPT comparisons were performed using the paired samples t-test. CPP score was analyzed using the Student’s t-test. A probability of p
