Increased risk of developing schizophrenia in animals exposed to cigarette smoke during the gestational period

Accepted Manuscript Increased risk of developing schizophrenia in animals exposed to cigarette smoke during the gestational period

Alexandra I. Zugno, Mariana B. Oliveira, Gustavo A. Mastella, Alexandra S.A. Heylmann, Lara Canever, Felipe D. Pacheco, Louyse S. Damazio, Sullivan A. Citadin, Luiz Antonio de Lucca, Lutiana Roque Simões, Fernanda Malgarin, Josiane Budni, Tatiana Barichello, Patricia F. Schuck, João Quevedo PII: DOI: Reference:

S0278-5846(16)30249-4 doi: 10.1016/j.pnpbp.2017.02.010 PNP 9015

To appear in:

Progress in Neuropsychopharmacology & Biological Psychiatry

Received date: Revised date: Accepted date:

30 September 2016 12 December 2016 12 February 2017

Please cite this article as: Alexandra I. Zugno, Mariana B. Oliveira, Gustavo A. Mastella, Alexandra S.A. Heylmann, Lara Canever, Felipe D. Pacheco, Louyse S. Damazio, Sullivan A. Citadin, Luiz Antonio de Lucca, Lutiana Roque Simões, Fernanda Malgarin, Josiane Budni, Tatiana Barichello, Patricia F. Schuck, João Quevedo , Increased risk of developing schizophrenia in animals exposed to cigarette smoke during the gestational period. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Pnp(2017), doi: 10.1016/j.pnpbp.2017.02.010

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Increased Risk of Developing Schizophrenia in Animals Exposed to Cigarette Smoke during the Gestational Period

Alexandra I. Zugno1 , Mariana B. Oliveira1 , Gustavo A. Mastella1 , Alexandra S. A. Heylmann1 , Lara Canever1 , Felipe D. Pacheco1 , Louyse S. Damazio1 , Sullivan A. Citadin1 ,

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Tatiana Barichello1,3 , Patricia F. Schuck2 , João Quevedo1,3,4

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Luiz Antonio de Lucca1 , Lutiana Roque Simões1 , Fernanda Malgarin2 , Josiane Budni1 ,

1

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Laboratory of Neurosciences, Graduate Program in Health Sciences, Health Sciences Unit, University of Southern Santa Catarina (UNESC), Criciúma, SC, Brazil. 2

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Laboratory Inborn Errors of Metabolism, Graduate Program in Health Sciences, Health Sciences Unit, University of Southern Santa Catarina (UNESC), Criciúma, SC, Brazil. 3

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Translational Psychiatry Program, Department of Psychiatry and Behavioral Sciences, McGovern Medical School, The University of Texas Health Science Center at Houston (UTHealth), Houston, TX, USA. 4

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Neuroscience Graduate Program, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX, USA.

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Corresponding author: Alexandra Ioppi Zugno Pharm.D., Ph.D. Laboratório de Neurociências, Programa de Pós-Graduação em Ciências da Saúde - PPGCS, Unidade Acadêmica de Saúde - UNASAU, Universidade do Extremo Sul Catarina - UNESC, Tel: 55 48 34312618, Criciúma, SC, Brazil. E-mail: [email protected]

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ABSTRACT

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Cigarette smoking during the prenatal period has been investigated as a causative factor of obstetric abnormalities, which lead to cognitive and behavioural changes associated with schizophrenia. The aim of this study was to investigate behaviour and AChE activity in brain structures in adult rats exposed to cigarette smoke during the prenatal period. Pregnant rats were divided into non-PCSE (non-prenatal cigarette smoke exposure) and PCSE (prenatal cigarette smoke exposure) groups. On post-natal day 60, the rats received saline or ketamine for 7 days and were subjected to behavioural tasks. In the locomotor activity task, the nonPCSE+ketamine and PCSE+ketamine groups exhibited increased locomotor activity compared with the saline group. In the social interaction task, the non-PCSE+ketamine and PCSE+ketamine groups exhibited an increased latency compared with the control groups. However, the PCSE+ketamine group exhibited a decreased latency compared with the nonPCSE+ketamine group, which indicates that the cigarette exposure appeared to decrease, the social deficits generated by ketamine. In the inhibitory avoidance task, the nonPCSE+ketamine, PCSE, and PCSE+ketamine groups exhibited impairments in working memory, short-term memory, and long-term memory. In the pre-pulse inhibition (PPI) test, cigarette smoke associated with ketamine resulted in impaired PPI in 3 pre-pulse (PP) intensity groups compared with the control groups. In the biochemical analysis, the AChE activity in brain structures increased in the ketamine groups; however, the PCSE+ketamine group exhibited an exacerbated effect in all brain structures. The present study indicates that exposure to cigarette smoke during the prenatal period may affect behaviour and cerebral cholinergic structures during adulthood.

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Keywords: schizophrenia; maternal smoking; acetylcholinesterase; locomotor activity; social interaction; inhibitory avoidance.

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Abbreviations: ACh - acetylcholine; AChE - acetylcholinesterase; ANOVA - analysis of variance; BChE - butyrylcholinesterase; ChAT - choline acetyltranferase; CNS - central nervous system; DA - dopamine; DNA - deoxyribonucleic acid; DTNB - 5,5-dithiobis-2nitrobenzoic acid; EDTA - ethylenediamine tetraacetic acid; MK-801 - dizocilpine maleate; nAChR - nicotinic acetylcholine receptor; nAChRs - α7 - α7 nicotinic acetylcholine receptors; NIH - National Institutes of Health; NMDAR1 - NMDA receptor subunit 1; P - pulse; PCSE prenatal cigarette smoke exposure; PND - postnatal day; PP - pre-pulse; PPI - pre-pulse inhibition; SBNeC - Brazilian Society for Neuroscience and Behavior; UNESC Universidade do Extremo Sul Catarinense.

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1 INTRODUCTION

A novel hypothesis ranks schizophrenia as a neurodevelopmental disorder that affects 0.5 to 1.0% of the world’s population (Rapoport et al., 2005). Maternal smoking comprises an environmental event investigated as a risk factor for the onset of schizophrenia in adulthood, which affects cognitive and behavioural disorders (Baguelin-Pinaud et al., 2010). Preclinical

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studies have demonstrated that exposure to nicotine induces changes in the developmental neurochemical markers of dopamine (DA) and may cause hyperactivity in offspring (Azam et

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al., 2007). In addition, a study published in our laboratory demonstrated that exposure to

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cigarette smoke during gestation causes persistent changes in lipids and proteins, as well as deoxyribonucleic acid (DNA) damage, which leads to oxidative stress in cells (Fraga et al.,

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2011).

Acetylcholine (ACh) is an excitatory neurotransmitter synthesized in presynaptic neurons by choline acetyltransferase (ChAT); it acts on the neuromuscular junction and binds

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to muscarinic or nicotinic receptors. It has a main role in motor, cognitive and memory functions (Voss et al., 2008). Acetylcholinesterase (AChE) is a regulatory enzyme that

ACh

inactivation

(Soreq

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controls the transmission of a nerve pulse through a cholinergic synapse via hydrolysis and and

Seidman,

2001).

In

schizophrenic

patients,

cognitive

dysfunctions are often present and have been related to changes in the cholinergic system

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(Voss et al., 2008).

Evidence suggests there is a relationship between the cholinergic and glutamatergic systems. A study conducted by Colgin and colleagues (2003) indicated that cholinergic induce

excitatory

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signals

activity

in

glutamate,

which facilitates memory acquisition.

Furthermore, the involvement of the cholinergic system in the physiopathology of mental disorders has been well established. Changes in ACh receptors occur in schizophrenia. The habit of smoking is significantly increased in schizophrenics compared with the general population, and this difference appears to be significant even in patients with another psychiatric disorder (A et al., 2003; Aguilar et al., 2005; McCreadie, 2002). These patients also manifest more severe positive symptoms, which indicates the involvement of nicotinic receptors in this disorder (Aguilar et al., 2005; Kalman et al., 2005; Ziedonis et al., 1994). In this context, studies indicate that individuals chronically exposed to cigarette smoke in the prenatal period had an increased risk of developing cognitive deficits, hyperactivity and conduct disorder during adolescence (Abreu-Villaca et al., 2010; Liu and Zhao, 2004).

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Neurodevelopmental issues may affect children whose mothers are subjected to stress during pregnancy (Brixey et al., 1993; O'Donnell, 2011). Smoking during the gestational period may trigger obstetric abnormalities and low weight shortly after birth, which lead to cognitive and behavioural changes during childhood (Baguelin-Pinaud et al., 2010). This change in early neurodevelopment may modify the organization of axonal connections in the synaptic projections that affect neuronal cell proliferation, migration and apoptosis (Van de

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Berg et al., 2002), which are necessary for the development of the central nervous system (CNS) (Stathopoulou et al., 2013).

related

to

changes

in

neurodevelopment

and

consequently

related

to

the

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directly

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Based on evidence that exposure to cigarette smoke during the prenatal period is

pathophysiology of schizophrenia, data in the literature indicate that an inappropriate intrauterine

environment

favours

the

incidence

of psychiatric disorders in offspring;

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moreover, the synergism between glutamatergic and cholinergic systems may comprise a mechanism that underlies the symptoms of this disorder.

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Overall, these findings have not been completely explained, and additional studies are warranted. Thus, in this study, we hypothesized that adult rats exposed to cigarette smoke during the prenatal period will exhibit an increased susceptibility to trigger schizophrenia-like

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behaviour and changes in the cholinergic system during adulthood.

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2.1 Animals

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2 MATERIALS AND METHODS

All experimental procedures that involved animals were performed in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and the recommendations of the Brazilian Society for Neuroscience and Behavior (SBNeC). These procedures were approved by the local Ethics Committee of the Universidade do Extremo Sul Catarinense (UNESC) under protocol number 118/2012. Pregnant female Wistar rats (3 - 4 months old, 250 - 280 g weight) were obtained from the local breeding colony and were individually housed with sawdust bedding and ad libitum access to food and water.

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2.2 Prenatal cigarette smoke exposure (PCSE)

Dams (pregnant females, n = 25) were randomly assigned to one of two treatment groups (n = 15 per group: 1) non-PCSE (not exposed to cigarette smoke) or 2) PCSE (exposed to cigarette smoke throughout the pregnancy). Each rat was exposed to the smoke of 12 commercially filtered cigarettes per day (a total of 8.0 mg of tar and 0.6 mg of nicotine) for a period of 22

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consecutive days. The animals were placed in an inhalation chamber (dimensions of 40 x 30 x 25 cm) inside a fume extraction cabinet. Cigarettes were coupled to a 60 mL plastic syringe;

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thus, puffs could be drawn in, expelled, and aspirated with this syringe (20 puffs of 50 mL).

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Each puff was immediately injected into the chamber. The animals were maintained in this smoke-air condition (3%) for 6 min. The cover was subsequently removed from the inhalation chamber, and the smoke was evacuated within 1 min by activating the cabinet exhaust fan.

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The dams were immediately exposed to cigarette smoke from a second cigarette for 6 min. This process was repeated three times per treatment, and these four cigarette treatments were

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performed three times per day (morning, noon, and afternoon), which resulted in 72 min of

2.3 Ketamine treatment

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cigarette smoke that produced 300 mg/mm 3 of total particulate matter in the chamber.

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The rats were weaned on postnatal day (PND) 21. On PND 60, the rats were randomly assigned to one of the following treatment groups: 1) non-PCSE + saline; 2) non-PCSE + 25 mg/kg ketamine; 3) PCSE + saline; and 4) PCSE + 25 mg/kg ketamine. All animals received

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a single intraperitoneal injection, once a day, for 7 days, according to the treatment protocols, at a volume of 1 mL/100 mg of body weight per injection (de Oliveira et al., 2011). After the last injection of ketamine, the rats were subjected to behavioural tasks according to protocols described by de Oliveira and Hunt (de Oliveira et al., 2011; Hunt et al., 2006).

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Timeline of exposure to cigarette smoke, treatment with ketamine and behaviour tasks

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2.4 Open field task

The animals were assessed in the open field test 30 min after the final ketamine injection. The

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test was performed in an arena with dimensions of 50 x 25 x 50 cm. The locomotor activity was measured for 15 min using a computerized system (Activity Monitor, Insight Laboratory

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Equipment, Ribeirão Preto, SP). The locomotor activity was quantified as the distance covered by the animal (in cm) in blocks of five minutes. The distance covered was calculated

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as the sum of the position changes monitored by the system.

2.5 Social Interaction

Impaired social interaction is a characteristic behaviour of animal models of autism spectrum disorders and schizophrenia (Mohn et al., 1999; Schneidert and Przewlocki, 2005; DiciccoBloom et al., 2006; Moldin et al., 2006). The animals (n = 12) were tested in ambient light/dark and unfamiliar conditions in an open field apparatus. On the day of the experiment, the animalswere socially isolated in a plastic box that measured 43 x 28 x 15 cm for 3 h prior to the experiment. The task consisted of placing two animals in the same group randomized into cages for 15 min. The social behaviour was assessed for a pair of animals; thus, the behaviours of individual animals were not analysed (Schneidert and Przewlocki, 2005). The

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latency to the first interaction, the number of social contacts and the total time were measured (Niesink and Van Ree, 1989; Schneider and Przewlocki, 2005). 2.6 Inhibitory avoidance The assessment of inhibitory avoidance (n = 12 per group) was initiated 24 h after the final injection of ketamine. Avoidance consists of a perspex box in which the floor is composed of

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parallel metal bars. A platform is placed at the left wall of the device (Quevedo et al., 1997; Roesler et al., 2003). In the training session, the animals were placed on the platform to

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measure the time required for the animal down with four legs of this site (latency).

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Immediately after leaving the platform, the animal received a shock of 0.4 mA for 2 s. In the test session, the animal was again placed on the platform and the time required to fall (latency) was measured; however, no shock was administered. The latency is characterized by

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the classical parameter of memory retention. The intervals between the training and test were measured immediately after training to assess working memory, 1.5 h after training to

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measure short-term memory (Bevilaqua et al., 2003; Izquierdo et al., 1998) and 24 h after to evaluate long-term memory (Bevilaqua et al., 2003; de Lima et al., 2005).

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2.7 Pre-pulse inhibition test

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The quantification of inhibition, which was promoted by a pre-pulse startle response induced by a pulse, was performed based on a previous study (Levin et al., 2011). Boxes with seal sound were used to measure the startle (Insight, EUA - EP 175). The animals initially

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remained in the boxes for a 5 min period of habituation. Ten pulses were subsequently applied to the habituation of the animals (this number was not considered for the calculations). During the session, 3 types of stimuli were presented 10 times and were randomly distributed in intervals of 20 s: (1) pulses of 120 dB for 40 ms (able to produce a startle response), (2) prepulses of 65, 70 or 75 dB for 20 ms presented 80 ms prior to the pulse, and (3) the absence of a stimulus. The mean startle amplitude after the session pulse (P), as well as the mean amplitude of the startle response sessions after the pre-pulse - pulse (PP) are calculated for each animal.

2.8 Measurement of AChE activity

The AChE activity in brain regions and the BChE activity in serum were determined

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according to Ellman’s method (1961). Hydrolysis rates were measured in 1 mL of a 0.8 mM solution that contained 100 mM phosphate buffer (pH 7.5) and 1.0 mM 5,5-dithiobis-2nitrobenzoic acid (DTNB). A sample (50 mL) was added to the solution and pre-incubated for 3 min. Hydrolysis was spectrophotometrically monitored at 412 nm (thiolate dianion formation) at 25 ºC for 2 - 3 min in 30 s intervals. The samples were evaluated in duplicates.

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Data were expressed in mmol ACSCh/h mg protein.

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2.9 Measurement of ChAT activity

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Brain samples were weighed and homogenized (5%) in 50 mM phosphate buffer (pH 7.4) that contained 0.2% triton X-100 and 20 mM ethylene diamine tetraacetic acid (EDTA) (pH 7.4).

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The supernatant was used to determine the ChAT activity. The ChAT activity was spectrophotometrically determined via the absorbance at 324 nm. One unit of enzyme activity was defined as 1 mmol of reduced coenzyme formed per minute per milligram protein (Chao

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and Wolfgram, 1972).

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2.10 Protein assay

The protein levels were determined using the Lowry method (1951), with bovine serum

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albumin used as a standard (Lowry et al., 1951).

2.11 Statistical analysis

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The data from the AChE and ChAT activities and behavioural tests, such as social interaction, locomotor activity and pre-pulse inhibition (PPI), were reported as two-way analyses of variance (ANOVAs) followed by Newman-Keuls post hoc test and are expressed as the mean ± S.D. In all comparisons, p < 0.05 indicated statistical significance. The inhibitory avoidance test was expressed as the median and interquartile range followed by a Wilcoxon test.

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3 RESULTS

3.1 Behavioural Tests

Figure 1 indicates the effects of cigarette exposure in pregnancy and ketamine

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administration in offspring (25 mg/kg) on locomotor activity. Two-way ANOVAs indicated interactions significant between the variables: smoke cigarette and ketamine [F(1.41) = 5.26,

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p < 0.05]. The results indicated that the non-PCSE+ketamine and PCSE+ketamine groups

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exhibited increased locomotor activity compared with the saline group (p < 0.01). Figure 2 indicates that the social interaction of these animals was impaired by ketamine (dose of 25 mg/kg). A two-way ANOVA indicated differences between cigarette

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and ketamine [F(1.17) = 8.77, p<0.01] in the latency (figure 2A). There were no significant differences between the groups regarding the total number of contacts (figure 2B) or the total

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time of contact (figure 2C).

Figure 3 demonstrates the effect of cigarette smoke exposure in an inhibitory avoidance task. The results demonstrated that the non-PCSE group exhibited an increased

compared

with

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latency time in all tasks tested (p < 0.01) (immediate, short, and long-term memory) training in the same group.

The non-PCSE+ketamine,

PCSE and

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PCSE+ketamine groups exhibited alterations in short- and long-term memory. Moreover, the non-PCSE+ketamine group exhibited impaired long-term memory compared with the non-PCSE group (p < 0.01). The animals in the PCSE group demonstrated

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impaired working memory, as well as short- and long-term memory evaluation, which indicate a damage effect caused by prenatal stress. PCSE+ketamine also induced cognitive deficits in all memories tested compared with the control group (figure 3). Figure 4 indicates that Non-PCSE+ketamine diminished the PPI test compared with the control group in three intensities of pulse investigated (70, 75, and 80 dB) (p < 0.01). Moreover, there was an effect of ketamine in altering the sensory-motor profile in animals. The PCSE+ketamine group also exhibited this inhibition compared with the controls (p < 0.05).

3.2 Biochemical tests

3.2.1 AChE activity

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Biochemical analyses were performed in three brain structures (the prefrontal cortex, hippocampus, and striatum). Figure 5 demonstrates the effects of cigarette smoke exposure early in life and ketamine administration (25 mg/kg) on the activity of the enzyme AChE in these three structures in adult offspring. A two-way ANOVA indicated interactions between the following variables in the prefrontal PCSE+ketamine [F(1.21) = 19.09, p < 0.01].

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Figure 5A indicates the AChE activity was increased in the non-PCSE+ketamine group (p < 0.05) in the prefrontal cortex. In the groups in which the animals were exposed to

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cigarette smoke, these values were more substantially increased compared with the control

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group (p < 0.01).

Figure 5B indicates the hippocampus results and interactions between cigarette and ketamine [F(1.18) = 639.56, p<0.01]. Ketamine increased the AChE activity regardless of

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whether the animals were exposed to cigarette smoke. In the striatum (figure 5C), the same profile was identified. Interactions were identified between cigarette and ketamine [F(1.20) =

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23.48, p < 0.01].

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3.2.2 ChAT activity

In the prefrontal cortex, a two-way ANOVA of the ChAT activity indicated a

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significant effect of the ketamine group [F(1,16) = 11.92, p = 0.0033] but not the cigarette smoke group [F(1,16) = 1.959, p = 0.1807]. In the hippocampus, a two-way ANOVA of the ChAT activity indicated there was no

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significant effect of the ketamine group [F(1,16) = 0.1592, p = 0.6952]; however, there was a significant effect of the cigarette smoke group [F(1,16) = 5.125, p = 0.0378] with no significant interaction [F(1,16) = 3.503, p = 0.0797]. In the striatum, a two-way ANOVA regarding the ChAT activity indicated a significant effect of the ketamine group [F(1,12) = 8.1, p < 0.0147] and cigarette smoke group [F(1,12) = 6.362, p = 0.0268], as well as a significant interaction [F(1,12) = 5.942, p = 0.0313].

4 DISCUSSION

As a result of the association between environmental changes during the prenatal period and the susceptibility to the development of schizophrenia, the present study aimed to

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assess adult animals that were exposed to cigarette smoke during pregnancy via behavioural and biochemical parameters. Cigarette smoke contains bioactive compounds that cause alterations in brain development and irreversible neurochemical changes (Naeye, 1992; Slotkin, 1999). Several compounds, such as nicotine, are psychoactive (Rose, 2006) and are able to cross the placenta barrier, thereby leading to alterations in the growth and behaviour of offspring (Vaglenova et

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al., 2008).

Studies using nicotine administration in adolescence and adults indicate that the

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stimulation of alpha7-cholinergic receptors affects the serotoninergic and dopaminergic

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systems (Carr et al., 1989; Eddins et al., 2009; King et al., 1991; Xu et al., 2001). Nicotine induces hyperactivity by increasing dopamine release in the striatum and pre-frontal cortex (Azam et al., 2007; Blood-Siegfried and Rende, 2010; Muneoka et al., 1997). These studies

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are consistent with the current findings, which indicate cigarette smoke exposure and ketamine administration increase locomotor activity in rats during adulthood. According to

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several studies, nicotine has a direct action on nicotinic receptors, causes dopamine release in the striatum and alters behaviour in a long-term way (Clarke and Reuben, 1996; Gold et al., 2009; Grady et al., 1992; Rapier et al., 1990; Wonnacott, 1997).

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Studies using schizophrenia animal models indicate that dysfunctions in glutamatergic cortical neurons may be associated with the dopaminergic and cholinergic systems (Carlsson

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et al., 1999; Coyle et al., 2003; Grace, 1991; Jentsch and Roth, 1999). In the present study, ketamine was used to induce schizophrenia-like symptoms in animals (de Oliveira et al., 2011; Smith et al., 2011).

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Our results demonstrate that both cigarette exposure and ketamine administration alter locomotor activity in animals, and this effect is induced via NMDA receptor blockade. It was also demonstrated that the hyperlocomotion generated by the combination of two factors (cigarette + ketamine) is likely a result of a more complex interaction between the cholinergic, dopaminergic and glutamatergic systems. Preclinical studies have used the social interaction test to assess behaviour related to negative symptoms (Deroza et al., 2012; Sams-Dodd, 1995). Thus, based on the results presented in this study during social interaction, ketamine may induce negative symptoms, which are reflected by an increased latency in animals in the PCSE+Ketamine group and are consistent with the social changes present in schizophrenic patients (Neill et al., 2010). Interestingly, studies have demonstrated smoking as a protective factor (Zammit et al., 2003) as a result of the slowdown of cognitive changes generated by ketamine via the action

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of nicotine in humans (Knott et al., 2011). Thus, in some cases, the use of cigarettes may comprise a potential method of self-medication when related to the cognitive and affective symptoms and the side effects of therapeutic drugs (Levin et al., 1996), as well as negative symptoms (Chambers et al., 2001; Deroza et al., 2012). The findings regarding the latency test parameter of social interaction, in which the non-PCSE+ketamine group exhibited a significant difference compared

with PCSE+ketamine

group, support cigarette smoke

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exposure as a potential diminishing factor of the negative symptoms generated by ketamine. However, it is important to emphasize that this study aimed to assess the effects of

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prenatal exposure to cigarette smoke in relation to the presence of negative symptoms in

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offspring during adulthood, which differs from other studies that indicate an effect of negative symptom reduction when animals are directly exposed to smoking (Deroza et al., 2012). Nevertheless, these findings reinforce the idea that it is not clear whether nicotine is beneficial

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in schizophrenia; moreover, there are no theories that explain the association between a negative trend in long-term schizophrenia and smoking (Aguilar et al., 2005).

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This study also implemented the inhibitory avoidance test to analyse the cognitive profile of animals and verify that the exposure to smoke during pregnancy and ketamine in adulthood induces memory deficits. The test demonstrated that the non-PCSE+ketamine exhibits memory damage compared

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group

with the control group. Individuals with

schizophrenia exhibit cognitive changes before and immediately after a psychosis outbreak,

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and it may represent a trait marker for the beginning of the disorder (Zaytseva et al., 2015). Studies from the literature have

related

cognitive alterations with NMDA receptor

dysfunctions. A reduction in NMDAR1s (NR1s) in genetically modified rats diminishes

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social and cognitive functions (Mohn et al., 1999). Furthermore, the chronic administration of ketamine has been associated with an impairment in memory even following treatment termination (Chatterjee et al., 2011; Zugno et al., 2014). Nicotine interferes with multiple neurotransmitter functions, such as the acetylcholine and catecholamine systems. Long-term changes in these systems may be involved in the behavioural changes that occur following prenatal exposure (Ernst et al., 2001; Levin et al., 1993; Slotkin, 2004). Studies indicate that schizophrenic individuals exhibit a lower expression of type α7 nicotinic acetylcholine receptors (nAChRs α7) (Breese et al., 2000; Guan et al., 1999; Marutle et al., 2001). The α7-like receptors are predominately expressed during developmental phase therefore, prenatal cigarette exposure leads to activation and stunning, which unleashes their long-term dysfunction (Srinivasan et al., 2011).

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Furthermore, nicotine overexpresses cholinergic receptor development and alters neuronal migration, proliferation and cellular differentiation. These effects induce decreased neuronal production and abnormal synaptic function (Ernst et al., 2001; Slotkin, 1998). One study demonstrated that nicotine exposure during pre and postnatal phases triggers cognitive impairment and high angiogenic behaviour, as well as chronic changes in the cholinergic system in the long term (Eppolito et al., 2010). These findings may explain, at least in part,

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the behavioural results identified in this study and indicate the influence of the gestational period on cognitive mechanisms, even during adulthood.

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Pre-pulse inhibition (PPI) is related to symptoms of schizophrenia, such as thought

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disorder and distraction (Turetsky et al., 2007). In this study, we demonstrated that 25 mg/kg of ketamine altered this parameter. A likely explanation for these effects is suggested by evidence in which the infusion of dizocilpine maleate (MK-801), an antagonist of NMDA

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receptors, leads to the loss of PPI likely because of changes in the limbic system structures, such as the amygdala and hippocampus. It is believed that these receptors are expressed in

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GABAergic interneurons, and the blockade of these receptors triggers an interrupts the inhibition of neuronal activity in these regions (Bakshi and Geyer, 1998). The inhibition response to a sound stimulus has been considered a potential animal

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model that is consistent with the characteristics exhibited in schizophrenic patients. It is regulated by the cortico-limbic circuit-pale-striate, which exhibits prejudice by experimental

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particularities, such as the use of NMDA receptor antagonists (Gogos et al., 2012). Changes in cognitive function and processing of the auditory system have also been associated with smoking during the gestational period (Fried et al., 1998, 2003; Jacobsen et al., 2007;

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Jacobsen et al., 2006; McCartney et al., 1994; Picone et al., 1982; Saxton, 1978; Sexton et al., 1990). These data are consistent with our study, in which there was an unleashed PPI deficit compared with the non-PCSE group in all intensities of the pre-pulse. It is believed that the behavioural alterations

identified

are

linked

with

nicotine,

which

acts on nicotinic

acetylcholine receptors (nAChRs) during the gestational period (Navarro et al., 1989; Slotkin et al., 1987). Nicotine may delay the development of brain areas that are responsible for PPI and are rich in acetylcholine synapses (Beninato and Spencer, 1986; Broide et al., 1995; Broide et al., 1996; Happe and Morley, 2004). Our study also assessed the AChE activity. Our results demonstrated animals that received ketamine increased the activity of the enzyme in the three structures analysed (the prefrontal cortex, hippocampus and striatum), which may lead to a degradation of ACh in these structures. These data corroborate previous studies in which chronically administered

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NMDA-type receptor antagonists increased AChE activity (Zugno et al., 2015; Zugno et al., 2013b; Zugno et al., 2014). ACh is an excitatory neurotransmitter, which is essential for motor functions, memory, learning, the control of blood flow to the brain, perception, and selective attention (Schetinger et al., 1999; Yu and Dayan, 2002). Moreover, alterations in the ACh level are associated with delusions and hallucinations (Burt, 2000; Hasselmo, 2006).

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Ketamine has been demonstrated to be an antagonist of nAChRs (Chatterjee et al., 2012; Scheller et al., 1996); it increases the amount of ACh in the synaptic cleft, activates

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AChE activity and causes damage in memory formation (Chatterjee et al., 2012).

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Our results also indicated the lack of an effect on ChAT activity, which suggests that the increase in AChE may be caused by an increase in ACh release but not an increase in synthesis.

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Biochemical changes support the findings in the behavioural cognitive tests. The prejudice in the sensory motor filter caused by ketamine and the reduction of PPI

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accompanies the increase in AChE in the three regions assessed. Previous work has demonstrated that rivastigmine (an AChE antagonist) increased ACh and decreased PPI (Lysakowski et al., 1989; Wenk, 1997).

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Our results also indicated an interaction with AChE activity when ketamine and cigarette smoke were combined, which contributes to the evidence that cigarette smoke may

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lead to symptoms associated with schizophrenia (Zugno et al., 2013a). Nicotine stimulates nAChRs, which induces altered neurodevelopmental cholinergic functions because it acts similar to acetylcholine (Eriksson et al., 2000; Navarro et al., 1989).

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In this context, nicotine exposure may affect the development of the cerebellum, hippocampus and sensory cortex, as well as alter sensory, motor and memory functions (Roy and Sabherwal, 1998). The

neurochemical changes

and

behavioural mechanisms

are

not completely

understood. However, the current findings provide critical evidence with respect to elucidating the importance of the developmental period in determining the susceptibility to chronic diseases during adulthood.

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Figure

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administration

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Legends

Effects of Prenatal Cigarette Smoke Exposure (PCSE) and/or ketamine (25mg/kg)

in

total distance

covered.

Two-way analyses of variance

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(ANOVAs) followed by Newman-Keuls post hoc test were used. Values expressed in Mean +/- Standard Error of Mean, n = 12, *Different from Non-PCSE+Saline.

Figure

2.

Effects of Prenatal Cigarette Smoke Exposure (PCSE) and/or ketamine

administration (25mg/kg) in social interaction test.

Two-way analyses of variance

(ANOVAs) followed by Newman-Keuls post hoc test were used. Values expressed in Mean +/- Standard Error of Mean, n = 12, *Different from Non-PCSE+Saline.

Figure

3.

Effects of Prenatal Cigarette Smoke Exposure (PCSE) and/or ketamine

administration (25mg/kg) in prepulse inhibition.

Two-way analyses of variance (ANOVAs)

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followed by Newman-Keuls post hoc test were used. Values expressed in Mean +/- Standard Error of Mean, n = 12, *Different from Non-PCSE+Saline.

Figure

4.

Effects of Prenatal Cigarette Smoke Exposure (PCSE) and/or ketamine

administration (25mg/kg) in inhibitory avoidance task. The test was expressed as the median and interquartile range followed by a Wilcoxon test. Values expressed in Median +/-

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Interquartile Range, n = 12, *Different from Training (same treatment) # Different from Non-

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PCSE+Saline (same time after training).

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Figure 5. Acetylcholinesterase (AChE) activity in the prefrontal cortex, hippocampus and striatum of prenatal cigarette smoke exposure (PCSE) animals or non-PCSE animals, treated with saline or ketamine (25 mg/kg), in adulthood. Two-way analyses of variance (ANOVAs)

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followed by Newman-Keuls post hoc test were used. Values are expressed as mean +/- S.E.M.

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(n = 4 - 6), *p < 0.05 as compared to the non-PCSE/saline group.

Figure 6. Choline Acetyltransferase (ChAT) activity in the prefrontal cortex, hippocampus and striatum of prenatal cigarette smoke exposure (PCSE) animals or non-PCSE animals,

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treated with saline or ketamine (25 mg/kg), in adulthood. Two-way analyses of variance (ANOVAs) followed by Newman-Keuls post hoc test were used. Values are expressed as

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mean +/- S.E.M. (n = 4 - 6), *p < 0.05 as compared to the non-PCSE/saline group.

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ACCEPTED MANUSCRIPT

AC C

EP T

ED

MA

NU

SC

Figure 5

RI

PT

ACCEPTED MANUSCRIPT

AC C

EP T

ED

MA

NU

SC

Figure 6

ACCEPTED MANUSCRIPT

Highlights  Cigarrete smoke exposure in pregnant dams caused damage in offspring in adult age.  Administration of ketamine and ketamine plus cigarrete increased the locomotor activity; of

ketamine

reproduced

the

negative

PT

 Administration schizophrenia;

symptoms

of

RI

 Ketamine increased AchE activity in the brain structures;

SC

 Cigarrete smoke exposure increases the damage caused by ketamine

AC C

EP T

ED

MA

NU

administration alone.