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l -Dopa treatment during perinatal development leads to different behavioral alterations in female vs. male juvenile Swiss mice
Accepted Manuscript l-Dopa treatment during perinatal development leads to different behavioral alterations in female vs. male juvenile Swiss mice
Lorena Oliveira de Matos, Ana Luiza de Araujo Lima Reis, Lorena Terene Lopes Guerra, Leonardo de Oliveira Guarnieri, Muiara Aparecida Moraes, Nayara Soares Sena Aquino, Raphael E. Szawka, Grace Schenatto Pereira, Bruno Rezende Souza PII: DOI: Reference:
S0091-3057(18)30269-7 doi:10.1016/j.pbb.2018.08.002 PBB 72626
To appear in:
Pharmacology, Biochemistry and Behavior
Received date: Revised date: Accepted date:
31 May 2018 25 July 2018 6 August 2018
Please cite this article as: Lorena Oliveira de Matos, Ana Luiza de Araujo Lima Reis, Lorena Terene Lopes Guerra, Leonardo de Oliveira Guarnieri, Muiara Aparecida Moraes, Nayara Soares Sena Aquino, Raphael E. Szawka, Grace Schenatto Pereira, Bruno Rezende Souza , l-Dopa treatment during perinatal development leads to different behavioral alterations in female vs. male juvenile Swiss mice. Pbb (2018), doi:10.1016/ j.pbb.2018.08.002
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Title: L-Dopa treatment during perinatal development leads to different behavioral alterations in female vs. male juvenile Swiss mice Author names: Lorena Oliveira de Matos, MSc1; Ana Luiza de Araujo Lima Reis, MSc1; Lorena Terene
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Lopes Guerra1, Leonardo de Oliveira Guarnieri, PhD1, Muiara Aparecida Moraes MSc1; Nayara Soares Sena Aquino, PhD 2; Prof. Raphael E. Szawka, PhD2, Prof. Grace Schenatto
1
Núcleo de Neurociências, Department of Physiology and
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Authors’ Affiliations:
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Pereira, PhD1, Prof. Bruno Rezende Souza, PhD1
901;
2
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Biophysics, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil, 31270Department of Physiology and Biophysics, Universidade Federal de Minas Gerais,
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Belo Horizonte, MG, Brazil, 31270-901.
Author:
Assistant
Professor,
Bruno
Rezende
Souza,
PhD,
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Corresponding
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Laboratory of Origin: Núcleo de Neurociências (NNC)
[email protected], Núcleo de Neurociências, Department of Physiology and
901
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Biophysics, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil, 31270-
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Running Title: Ontogenetic behavioral consequences of L-Dopa Keywords: Dopamine, neurodevelopment, behavior, sexual dimorphism, ontogeny, neonatal ORCID ID: 0000-0002-7064-7956 Color figures: No
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Abstract: Alterations in dopaminergic signaling and neurodevelopment are associated with many neuropsychiatric disorders, such as attention deficit and hyperactivity disorder (ADHD), autism, and schizophrenia. Imbalances in dopamine levels during prenatal development are associated with behavioral alterations later in life, like hyperactivity and addiction, and it is
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possible that dopaminergic imbalances may have diverse effects during different
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neurodevelopmental windows. In this study, we investigate whether an increase in
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dopamine levels during the perinatal developmental window affects behavior of juvenile male and female Swiss mice. In order to do so, we intraperitoneally administered daily
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doses of L-Dopa to mice pups beginning from postnatal day 1 (PD1) to PD5, which increased the levels of dopamine and its metabolite, 3,4-dihydroxyphenylacetic acid
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(DOPAC), in the striatum of the pups. At the age of 4 weeks, we submitted the juvenile males and females to the open field test, elevated plus maze, forced swimming test, and
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sucrose preference test. We observed that increase of dopamine levels during the perinatal
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developmental window increased exploratory behavior in juvenile females, but not males. We observed no changes in anxiety- and depressive-like behaviors. In contrast, we
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observed that increased dopamine levels during the perinatal period lead to hedonic
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alterations in juvenile males, but not females. Our results show that dopamine signaling is important for behavioral development and that transient imbalance of dopamine levels causes juvenile behavioral alterations, which are different in males than in females. These data may help in better understanding the spectrum of symptoms associated with different neuropsychiatric disorders.
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Introduction: Several neuropsychiatric disorders share both dopaminergic and neurodevelopmental hypotheses but few studies show a convergence of both (Bale et al., 2010; Robinson et al., 2001; Souza and Tropepe, 2011). For example, schizophrenic (SCZ) patients present alterations in volume and morphology of specific brain areas (Bähner and Meyer-
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Lindenberg, 2017; Hoftman et al., 2016; van Erp et al., 2015; Zhuo et al., 2017).
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Behavioral alterations and early symptoms of SCZ emerge during adolescence, usually
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before clinical diagnosis, and children from SCZ parents present alterations in frontolimbic activity (Cornblatt and Keilp, 1994; Erlenmeyer-Kimling et al., 2000; Hart et al.,
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2013).
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Additionally, several studies have demonstrated an association between polymorphism in dopaminergic genes and SCZ (González-Castro et al., 2016; Moskvina et al., 2009).
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There are also changes in dopaminergic signaling in SCZ patients, such as increase of
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dopaminergic D2 receptors and alterations of Neuronal Calcium Sensor-1 (NCS-1) and Dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa (DARPP-32) levels, which are independent of antipsychotic treatments (Albert et al., 2002; Brito-Melo et al., 2012;
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Koh et al., 2003; Souza et al., 2010, 2008, Torres et al., 2009b, 2009a; Wong et al., 1986).
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Furthermore, it is well known that SCZ patients present an increase of presynaptic dopamine (DA) levels and DA release (Abi-Dargham et al., 1998; Breier et al., 1997; Howes et al., 2009; Laruelle et al., 1996; Lindström et al., 1999; Reith et al., 1994). Dopaminergic signaling can also be transiently affected by perinatal exposure to drugs, such as cocaine and metamphetamine, causing cognitive alterations and mental health issues later in life (reviewed by Ross et al., 2015). Furthermore, not only stress but also viral, fungal or even parasitic infections can impair dopaminergic signaling and lead to
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behavioral abnormalities (Abercrombie et al., 1989; Kumar et al., 2011; Prandovszky et al., 2011; Rasheed et al., 2010; Severance et al., 2016; Simanjuntak et al., 2017) During development, dopaminergic signaling is involved in many aspects of cell cycle and functions, such as cell proliferation, cell differentiation, morphogenesis and neuronal migration (Crandall et al., 2007; Hedlund et al., 2016; Popolo et al., 2004; Schmidt et al.,
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1996; Souza et al., 2011). Because dopaminergic signaling molecules are expressed since
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early prenatal development, it is possible that dopaminergic alterations may be involved in
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some of the developmental abnormalities found in many neuropsychiatric disorders (Björklund et al., 1968; Coyle and Axelrod, 1972; Olson and Seiger, 1972; Schambra et al.,
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1994; Specht et al., 1981; Unis et al., 1998). Several animal models have been used to understand the developmental hypothesis for neurodevelopmental disorders. For example,
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knockout mice for the dopaminergic enzyme tyrosine hydroxylase (TH) and dopaminergic receptors show several behavioral alterations with respect to motor behavior, feeding
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behavior, reward behavior, and cognition (Baik et al., 1995; El-Ghundi et al., 2003;
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Maldonado et al., 1997; Smith et al., 1998; Szczypka et al., 1999; Xu et al., 1994). A common model for studying the consequences of DA increases is to use knockout
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(KO) mice for DA transporter (DAT), which shows persistent extracellular DA within the
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synaptic cleft, since diffusion is the only clearance mechanism (Giros et al., 1996). These animals present locomotor hyperactivity and alterations in exploratory and maternal behavior (Giros et al., 1996; Spielewoy et al., 2000). However, DAT KO mice do not express DAT throughout their entire lives, which makes it difficult to study the developmental dynamic over multiple stages of brain development (Andersen, 2003). For example, during prenatal development, phenomena like neurogenesis, neuronal migration, and cell differentiation are very pronounced (Bayer et al., 1993; McConnell, 1990;
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O’Rourke et al., 1992). During early postnatal development there is an increase in synaptogenesis and apoptosis (Aghajanian and Bloom, 1967; Blaschke et al., 1998). Furthermore, there are ontogenetic sex differences during brain development (Tobet et al., 2009). Female rat pups have more dopaminergic projections to striatum than do their male counterparts, with higher levels of DA and tyrosine hydroxylase (TH) (Ovtscharoff et
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al., 1992). Similarly, male pups show higher levels of testosterone and estradiol than
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females, which delays perinatal development of the dopaminergic system (Ovtscharoff et
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al., 1992; Stewart et al., 1991; Stewart and Rajabi, 1994). Since there exists a well-known sex-bias toward male animals and cells in research and it does not represent the prevalence
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of psychiatric disorders observed in humans, it is important to include both sexes to investigate the role of DA in neurodevelopment (Prendergast et al., 2014; Zucker and
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Beery, 2010).
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Taking all these data in consideration, it is not possible to understand whether the
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behavioral abnormalities shown by KO animals are developmental consequences or the consequence of synaptic dynamics at the moment the behavioral test was administered. Nor is it possible to know what phenomenon was affected by the increase of DA, or in
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which developmental window. Furthermore, since DAT KO females show alterations in
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maternal behavior, it is possible that the lack of maternal care is also affecting the development of pups (Chocyk et al., 2013; Teicher et al., 2004). Our hypothesis is that an increase of DA during specific developmental windows causes different alterations in brain and behavior development. TH and DA begin to be expressed during intrauterine embryonic development (Coyle and Axelrod, 1972; Kalsbeek et al., 1988; Olson and Seiger, 1972; Specht et al., 1981). However, there is a significant increase of dopaminergic synaptic formation in the first days after birth (Jung, 1996; Kalsbeek et al., 1988; LerouxNicollet et al., 1990; Schmidt et al., 1982; Verney et al., 1982). The first 5 PD of mice is an
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important developmental window with a significant increase in synaptogenesis and apoptosis, which is essential for adapting to a changing environment (Aghajanian and Bloom, 1967; Blaschke et al., 1998). On the other hand, these phenomena make this developmental window a time of increased vulnerability. It is therefore possible that an imbalance of dopaminergic signaling within this period might impair behavioral
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development (Ignacio et al., 1995; Rice and Barone, 2000). Because of this, we
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investigated whether a transient increase of brain DA levels during the first 5 PD would
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cause behavioral abnormalities in juvenile female and male mice.
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Materials: Animals Male and female Swiss mice (8–12 weeks of age) were purchased from the Animal Facility of the Universidade Federal de Minas Gerais (CEBIO, UFMG, Brazil) for mating.
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Mice were raised in polypropylene cages (27 cm × 17 cm × 12 cm), provided with wood shavings, housed three to five per cage, maintained in a climate-controlled room with 12
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h–12 h light/dark cycle (lights on from 8am to 8pm) and temperature at 22 ± 2 °C and 40-
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70% relative humidity. Free access to food (Nuvilab CR1-Nuvital) and water was provided
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throughout the study.
All protocols were performed during the light phase of the cycle. Behavioral protocols
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were performed in juvenile (30-33 day old) male and female Swiss mice offspring. The Animal Use Ethics Committee of the Universidade Federal de Minas Gerais (CEUA
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39/2015) approved all procedures and the experiments were carried out in accordance with
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NIH guidelines for the use and care of animals. Experimental design and drug administration
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Mating was stimulated by placing two females with one male. After 4 days, the male
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was removed from the cage and two females were housed per cage. At the 19th day of gestation, each female was housed individually. Cages were then inspected daily for the presence of pups. The day of birth was designated PD 0. All pups were randomly assigned to their treatment groups and, for identification, the distal phalanx (toe clipping) of PD 1 pups was removed. The pups were daily treated with L-Dopa (Sigma-Aldrich, D-1507) or saline as control from PD 1 to PD 5. The pups were treated with L-Dopa at concentrations of 10mg/kg, 25mg/kg or 50mg/kg body weight (Kashihara et al., 2002; Kim et al., 2002). They were intraperitoneally injected with a final volume of 20 µL using a 30G needle
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coupled to a 25 µl Hamilton. A Naïve non-manipulated group was also included. Weaning took place at PD 22 and animals were housed according to sex (2 to 3 per cage) and treatment. The animals underwent all behavioral testing at 28-33 days of age (FIGURE 1). During the first day of behavioral tests, animals were submitted to an open field test (OFT) and elevated plus maze test (EPM). On the following day, animals were submitted to a
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forced swimming test (FST), and two days later they were submitted to sucrose preference
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test (SPT).
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Open field test (OFT)
Spontaneous locomotor activity in a new environment was assessed using an
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automatic open field apparatus (LE 8811 IR Motor Activity Monitors PANLAB, Harvard
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Apparatus; Spain), with an acrylic box measuring 23 x 23 x 35 cm. Animals explored the open field for 5 minutes. Total distance traveled was recorded (Walsh and Cummins, 1976)
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and the percentage of time spent in the center of the box (10 x 10 cm) was used as an
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anxiety measure (Prut and Belzung, 2003). Increased locomotion and rearing was considered a stimulant effect (Prut and Belzung, 2003). The open field arena was cleaned
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with 70% ethanol between each trial. Elevated plus maze test (EPM)
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EPM is a simple method which induces an approach–avoidance conflict for assessing anxiety responses of rodents (Walf and Frye, 2007). The apparatus consists of two open arms (30 cm x 6 cm) and two closed arms (30 cm x 6 cm x 16 cm) crossed in the middle perpendicularly to each other and elevated 30 cm above the floor. Mice were placed in the central area and allowed to move freely for 5 minutes (Pádua-Reis et al., 2017). The number of entries into the open arms and the time spent in the open arms were used as indices of anxiety (Pellow et al., 1985).
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Forced swimming test (FST) The FST is a useful tool to assess depressive-like behavior in mice and was carried out as described by Porsolt et al. (1977), with minor modifications. Mice were individually transferred to a vertical glass cylinder (17 cm inner diameter × 27 cm height) filled with water. The water’s temperature was monitored throughout the test and maintained between
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25 and 27 ◦C. At the end of the test, the animal was removed and gently dried under a
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heated air jet. Merely floating, with no movement of the forelimbs or hind limbs, was
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considered immobility and was quantified.
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Sucrose preference test (SPT)
The sucrose preference was assessed using the two-bottle choice method. Animals
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were maintained alone in the home cage and were habituated for 8 h with two bottles containing water. Eighteen hours before the test, animals were water deprived. The test
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consisted of placing two bottles in the home cage: one containing water and the other
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containing a 3% sucrose solution. Both bottles were weighed immediately before the test, and then again 1, 2, 3, 12, 24 and 48 h afterwards. Sucrose preference was analyzed using
al., 2013).
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the equation: [sucrose consumption/sucrose consumption + water consumption] (Imai et
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High performance liquid chromatography with electrochemical detection (HPLC-ED) Because the nigrostriatal pathway is one of the major dopaminergic systems in the brain, we used the striatum as a reference brain area for changes in DA neurotransmission caused by perinatal treatment with L-DOPA. L-DOPA is a precursor of DA and was administered intraperitoneally. Thus, it is reasonable to expect changes in the striatum to reflect changes in dopaminergic activity in the brain. The striatum of PD 5 mice pups was dissected bilaterally 30 minutes after the last treatment and immediately after decapitation.
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Structures were homogenized in 400 μl of 0.15 M perchloric acid, 0.1 mM EDTA, and 1.7 µM 3,4- dihydroxybenzylamine as an internal standard. Homogenates were centrifuged at 4 ºC for 20 minutes at 12,000 g. Protein content was determined in the remaining pellet by the Bradford method (Bradford, 1976). Dopamine (DA) and 3,4- dihydroxyphenylacetic acid (DOPAC) were measured in supernatant as previously described (Aquino et al., 2017;
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Rabelo et al., 2017). Shortly afterwards, 20 µL of samples were injected into an HPLC-ED
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system equipped with a C-18 column (250 x 4 mm, Purospher, 5 µm; Merck, Darmstadt,
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Germany), preceded by a C-18 4 x 4-mm guard column. The mobile phase (100 mM sodium dihydrogen phosphate monohydrate, 10 mM sodium chloride, 0.1 mM EDTA, 0.38
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M sodium 1-octanesulfonic acid, and 10%; pH 3.5) was pumped at a flow rate of 1.0 mL/min. The potential in the electrochemical detector (Decade 2; Antec Scientific, The
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Netherlands) was set to 0.40 V vs. Ag/AgCl reference electrode and the filter at 0.01 Hz. The Class-VP software (Shimadzu, Kyoto, Japan) was used for chromatography plotting,
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and quantification was done using the internal standard method based on peak height.
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All samples were measured in one assay. The intraassay coefficient of variation was 1.4% for DA and 1.7% for DOPAC. DA levels were considered to reflect neurotransmitter
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content in synaptic vesicles, whereas DOPAC levels reflected the amount of DA release
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(Lookingland et al., 1987). Neurotransmitter to metabolite ratio was used as a measure of dopaminergic turnover (Rabelo et al., 2017). Statistical analysis Data were tested for normality using the Shapiro-Wilk test and tested for equal variance by computing the Spearman rank correlation between the absolute values of the residuals and the observed value of the dependent variable. The parametric data were analyzed by One Way ANOVA followed by Tukey’s post-hoc test. Tukey’s post-hoc test was used to control the experimentwise type I error rate. The non-parametric data were
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analyzed by Kruskal-Wallis One Way Analysis of Variance on Ranks followed by Dunn's Method post-hoc test. All tests were performed using SigmaPlot version 12.5.0.38. Parametric data are presented as mean and standard deviation (SD). Non-parametric data are presented as median and interquartile (Weissgerber et al., 2015). Significance was set at p < 0.05 for all tests. All statistical data comparing the L-Dopa treatments versus Saline
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and comparing Naïve versus Saline groups are described at the Tables 1 and 2 respectively.
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Results: Daily L-Dopa treatment in the first 5 PD increases DA levels and its metabolites in the striatum of Swiss mice. Previous studies showed an increase of DA levels and a slight increase of
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noradrenaline levels in the brains of rat pups treated with L-Dopa (Kellogg and Lundborg, 1972a, 1972b). To confirm that PD5 Swiss mice treated daily and intraperitoneally with L-
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Dopa experience an increase of DA within the brain, we separated the pups into three
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groups: non-handled (Naïve), saline-injected (Saline) and L-Dopa-injected (50mg/Kg) pups. Each day, the pups (except the for the naïve group) were treated intraperitoneally,
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from PD0 to PD5. One hour after the last injection, we collected the striatum of female and
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male PD5 pups for HPLC analysis. We observed that saline-treated females showed a decrease of 86% in their DA levels, as compared to the naïve group (F(2,12)= 11.878, One
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Way Anova, Tukey, p= 0.001), and L-Dopa-treated female pups showed an increase of
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426% in their DA levels as compared to the saline-treated group (F(2,12)= 11.878, One Way Anova, Tukey, p= 0.04) (Figure 2A). On the other hand, L-Dopa treatment increased levels of the DA metabolite DOPAC in female pups treated with L-Dopa. L-Dopa-treated
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females showed an increase of approximately 380% in DOPAC levels as compared to
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naïve pups (F(2,13)= 39.340, One Way Anova, Tukey, p< 0.001) and saline groups (F(2,13)= 39.340, One Way Anova, Tukey, p< 0.001). In order to evaluate DA turnover in the striatum of L-Dopa-treated pups, we analyzed the ratio of DOPAC/DA. We observed an increase of the DOPAC/DA ratio in females treated with L-Dopa. Both female pups treated with Saline (KW H= 9.396, 2 d.f., Dunn's Method, p<0.05) and L-Dopa (KW H= 9.396, 2 d.f., Dunn's Method, p<0.05) showed an increase of approximately 600% in DOPAC/DA ratio compared to naïve.
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We observed no differences in DA levels between male pups (Figure 2A) (KW H= 3.956, 2 d.f., p=0.138). On the other hand, L-Dopa-treated males showed an increase of approximately 500% in DOPAC levels compared to naïve (KW H= 9.906, 2 d.f., Tukey, p<0.05) and 400% compared to saline-treated pups (KW H= 9.906, 2 d.f., Tukey, p<0.05) (Figure 2B). L-Dopa-treated males also showed an increase of 123% in the ratio of
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DOPAC/DA compared to the naïve group (F(2,14)= 4.050, One Way Anova, Tukey, p=
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0.034) (Figure 2C). Taken together, these data suggest that L-Dopa treatment was efficient
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in increasing DA release in the striatum of female and male pups, as compared to their naïve and saline counterparts.
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An increase of DA during the first 5 PD induces hyperactivity in juvenile females,
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but not males.
Hyperactivity of dopaminergic signaling is associated with alterations in locomotor
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activity. Because of this, we investigated whether an increase of DA levels during the first
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days of life would affect the locomotor activity of juvenile mice. Each day, we treated Swiss mice pups with saline, L-Dopa 10mg/Kg, L-Dopa 25mg/Kg or L-Dopa 50mg/Kg, from PD0 to PD5, and we evaluated locomotor activity at PD28 by OFT (Figure 1). We
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observed no alterations in the locomotor activity of juvenile males (Figure 3). We also did
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not see any changes in the time spent in the center by juvenile females (Figure 3B). However, we observed an increase in the total distance and in the number of rearings of LDopa 25mg/Kg and L-Dopa 50mg/Kg treated females (Figure 3A,C). The L-Dopa 25mg/Kg treated females moved around approximately 40% more than the saline (F(3,49)= 4.296, One Way Anova, Tukey, p= 0.025) and L-Dopa 10mg/Kg (F(3,49)= 4.296, One Way Anova, Tukey, p= 0.036) groups (Figure 3A). There was also an increase of 44% in the number of rearings in the L-Dopa 25mg/Kg treated group, as compared to LDopa 10mg/Kg group (F(3,49)= 5.401, One Way Anova, Tukey, p= 0.035), as well as a
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slight increase as compared to the saline group (F(3,49)= 5.401, One Way Anova, Tukey, p< 0.082) (Figure 3C). The L-Dopa 50mg/Kg treated females showed an increase of approximately 50% in the number of rearings compared to saline (F(3,49)= 5.401, One Way Anova, Tukey, p= 0.032) and L-Dopa 10mg/Kg treated females (F(3,49)= 5.401, One Way Anova, Tukey, p= 0.013) (Figure 3C). Interestingly, we observed locomotor
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hyperactivity only in juvenile females that suffered an imbalance of dopaminergic
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signaling in the first 5 PD.
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Increased levels of DA in the first 5 PD did not affect anxiety-like behavior in juvenile Swiss mice.
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Female mice treated with L-Dopa in the first 5 PD showed hyperactivity, including an
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increase in the number of rearings. Since dopaminergic signaling is involved in anxietylike behavior, and the number of rearings can indicate this behavior in mice, we tested this
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behavioral phenotype by EPM (Calhoon and Tye, 2015; Prut and Belzung, 2003). We did
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not see any alterations in juvenile females’ behavior during the test, such as percentage of time in the open arms, total time in open arms, number of rearings, number of dippings, number of stretchings, time at center and time on edge of open arms (Figure 4). In juvenile
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males, we observed a decrease in time spent at the center in the L-Dopa 25mg/Kg treated
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group as compared to saline-treated mice (F(3,42)= 4.167, One Way Anova, Tukey, p= 0.013) and to L-Dopa 50mg/Kg treated mice (F(3,42)= 4.167, One Way Anova, Tukey, p= 0.021) (Figure 4F). However, we did not see any changes in the behavior of juvenile males or in the percentage of time in open arms, total time in open arms, number of rearings, number of dippings, number of stretchings, or time on edge of open arms (Figure 4A-E,G). Depressive-like behavior in juvenile Swiss mice is not affected by L-Dopa treatment in the first 5 PD.
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Several studies have shown involvement of DA in depressive behavior and dopaminergic signaling is one of the targets for antidepressants (Otte et al., 2016; Tye et al., 2012). Because of this, we used FST to investigate whether L-Dopa treatment in the first 5 PD would lead to depressive-like behavior in juvenile mice. We observed that neither female nor male juveniles showed alterations in the number of climbings, in total
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climbing-time, total swimming-time, latency immobility, number of immobility episodes,
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or total time of immobility (Figure 5).
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Increased levels of DA in the first 5 PD impacts hedonic behavior only in male juvenile Swiss mice.
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DA is highly involved in the reward system, and it is well known that pups exposed to
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cocaine show alterations in reward behavior. Here we tested whether an increase of DA levels in the first 5 PD would impact the hedonic behavior of juvenile Swiss mice. Firstly,
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we evaluated the total liquid consumption of juvenile females through time, and we
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observed an increase of consumption in the L-Dopa 10mg/Kg treated group compared to saline (F (26,130) = 7.319, Two Way Anova, Tukey, p= 0.019) and L-Dopa 50mg/Kg treated groups (F (26,130) = 7.319, Two Way Anova, Tukey, p= 0.024) at 48 hours (Figure
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6A). However, when we tested data limited to 48 h (One Way Anova), we did not see any
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differences in total liquid consumption between groups (Figure 6A’). Subsequently, we investigated sucrose preference over 48 h and we observed a difference in sucrose consumption between the groups (Figure 6B), as highlighted by the 48 h data (Figure 6B’). We observed significant alterations in total consumption and sucrose preference in juvenile males treated with L-Dopa in the first 5 PD. We observed a decrease of liquid consumption in the L-Dopa 50mg/Kg treated group compared to saline (F (30,150) = 8.486, Two Way Anova, Tukey, p= 0.003), L-Dopa 10mg/Kg (F (30,150) = 8.486, Two Way Anova, Tukey, p= 0.012) and L-Dopa 25mg/Kg treated groups (F (30,150) = 8.486,
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Two Way Anova, Tukey, p= 0.309) at 12 hours. At 24 hours, there was a difference between L-Dopa 50mg/Kg and L-Dopa 25mg/Kg treated group (F (30,150) = 8.486, Two Way Anova, Tukey, p= 0.015). We also observed a decrease of total consumption in LDopa 50mg/Kg treated group compared to Saline (F (30,150) = 8.486, Two Way Anova, Tukey, p< 0.001), L-Dopa 10mg/Kg (F (30,150) = 8.486, Two Way Anova, Tukey, p<
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0.001) and L-Dopa 25mg/Kg treated groups (F (30,150) = 8.486, Two Way Anova, Tukey,
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p< 0.001) at 48 hours (Figure 6C). When we tested only the data of 48 hours we confirmed
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a significant decrease of sucrose preference in juvenile males treated with L-Dopa 50mg/Kg treated group compared to Saline (F (3,30) = 7.494, One Way Anova, Tukey, p=
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0.012), L-Dopa 10mg/Kg (F (3,30) = 7.494, One Way Anova, Tukey, p= 0.005) and LDopa 25mg/Kg treated groups (F (3,30) = 7.494, One Way Anova, Tukey, p< 0.001) at 48
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hours (Figure 6C’).
We investigated the sucrose preference of juvenile males and observed a difference
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between L-Dopa 50mg/Kg treated group with L-Dopa 10mg/Kg (F (30,150) = 15.45, Two
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Way Anova, Tukey, p= 0.035) and L-Dopa 25mg/Kg treated groups (F (30,150) = 15.45, Two Way Anova, Tukey, p= 0.026) at 1 hour. At 2 hours, we observed a difference
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between L-Dopa 50mg/Kg treated group with L-Dopa 10mg/Kg (F (30,150) = 15.45, Two
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Way Anova, Tukey, p= 0.019) and L-Dopa 25mg/Kg treated groups (F (30,150) = 15.45, Two Way Anova, Tukey, p= 0.038) (Figure 6D). When we tested only the data of 2 hours we confirmed a significant difference in sucrose preference in juvenile males treated with L-Dopa 50mg/Kg treated group compared to L-Dopa 10mg/Kg (F (3,30) = 4.371, One Way Anova, Tukey, p= 0.016) and L-Dopa 25mg/Kg treated groups (F (3,30) = 4.371, One Way Anova, Tukey, p< 0.031) at 2 hours (Figure 6D’).
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Discussion: Ontogenetic disturbances of dopaminergic signaling are associated with permanent alterations in juvenile and adult behavior (Brown et al., 2002; Brus et al., 2003; Kostrzewa et al., 2016). In this study, we showed that a transient increase of DA levels during the perinatal developmental window can cause behavioral alterations in juvenile Swiss mice,
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and, additionally, that these changes are different between males and females.
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Dopaminergic enzymes are expressed in the perinatal brain and DA levels are stable in
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the first 5 days of postnatal development in rats (Agrawal et al., 1966; Karki et al., 1962; Kellogg and Lundborg, 1973; Nachmias, 1960). Previous studies have shown that L-Dopa
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acute administration in rat pups (PD1 and PD4) significantly increases DA levels 30
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minutes post-injection, but there is only a very slight increase in noradrenaline levels (Kellogg and Lundborg, 1972b, 1972a; Van Hartesveldt et al., 1991). They also observed
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an increase of motor activity 30 minutes after L-Dopa administration. The difference in
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motor behavior was observed when the rat pups were acutely treated with at least 50mg/Kg L-Dopa (Camp and Rudy, 1987; Stehouwer et al., 1994). Here, we observed an increase of DA, DOPAC and the DOPAC/DA ratio in the female striatum 1 hour after acute
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administration of 50mg/Kg L-Dopa in PD5 mice pups. Male pups displayed an increase in
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DOPAC and DOPAC/DA ratio, but not DA levels, subsequent to L-Dopa administration (Figure 2A). Differently from most of the studies, we decided to include the Naïve group as additional experimental control. Interestingly, the Saline injected pups showed an increase DOPAC/DA ratio compared to Naïve. This increase might be cause by the minor stress of manipulation and injection (Abercrombie et al., 1989). It is known that female rat pups have higher TH levels and dopaminergic projections to striatum than do males (Ovtscharoff et al., 1992). Also, the higher neonatal levels of testosterone and estradiol in males compared to females delay perinatal dopaminergic system development in male
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pups. Because of this, DA levels increase earlier in female rat pups than in males (Stewart et al., 1991; Stewart and Rajabi, 1994). Several studies have shown that dopaminergic imbalances during prenatal and postnatal development can cause behavioral alterations in juvenile and adult rodents. For example, adult CD1 male mice (PD60) exposed to L-Dopa during prenatal development,
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from E11 until birth, display alterations in cocaine-induced behavior (Ren et al., 2011).
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Furthermore, treatment with the DA transporter inhibitor bupropion from E6 to PD20
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affects male and female adult rats’ (PD60) behavior in different ways (Sprowles et al., 2017). In addition, adult rats (PD60) treated postnatally with D2 agonist (Quinpirole) from
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PD1 until PD11 show higher active avoidance and deficits in spatial memory (Brown et al., 2002; Brus et al., 2003, 1997). Some of these behavioral alterations can be detected even in
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juvenile animals. Juvenile rats (PD22-28) treated daily with quinpirole, from PD1 until PD21, display behavioral alterations like vertical jumping, spatial memory, and quinpirole-
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induced yawning (Brown et al., 2004; Kostrzewa et al., 2004, 1993b, 1993a). However, as
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mentioned, DA levels are very stable in the first 5 days of postnatal development (Agrawal et al., 1966). Thus, we wondered whether an increase of DA levels within this stable
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developmental window could permanently affect juvenile behavior in Swiss mouse.
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In this study, we observed an increase in the total distance travelled (Figure 3A) and in the number of rearings (Figure 3C) of only female juvenile Swiss mice treated with LDopa from PD1 to PD5. Increased number of rearings and total distance travelled in the OFT is correlated with increased levels of anxiety-like behavior in mice (Borta and Schwarting, 2005; Seibenhener and Wooten, 2015). These data suggest that an increase of DA levels in the first 5 PD amplifies anxiety-like behavior in juvenile females, but not males (Figure 3). However, we did not observe any behavioral alterations in the EPM test, including in the number of rearings, which is commonly used to test anxiety-like behavior
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by exploiting the conflict between preference for protected areas and the contrary drive to explore new environments (File et al., 1994; Rodgers et al., 1999; Walf and Frye, 2007) (Figure 4). Since we did not observe changes in the number of rearings in the EPM test, and the number of rearings in the OFT can also be interpreted as information-gathering routine, it is possible that the L-Dopa-treated juvenile females have an increase of
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exploratory behavior, rather than an increase of anxiety-like behavior (Lever et al., 2006;
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Mansour et al., 2003).
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On the other hand, only male juvenile Swiss mice treated with L-Dopa from PD1 to PD5 showed a decrease of total consumption (Figure 6C and C’) and alterations in sucrose
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preference (Figure 6D and D’) in the SPT. Decrease of sucrose consumption is associated with anhedonia and depressive-like behavior (Overstreet, 2012a; Willner et al., 1992;
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Zhang et al., 2017). Our results suggest that an increase of DA in the first 5 PD impairs hedonic behavior in juvenile males but not females (Figure 6). However, we did not detect
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any changes in behavior during the FST, which is commonly used to test rodents’ escape-
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directed behavior in water. A pronounced lack of this behavior is associated with depressive-like behavior (Overstreet, 2012b; Slattery and Cryan, 2012) (Figure 5).
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Therefore, it is more likely that L-Dopa-treated juvenile males have impairment of
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motivation-like behavior than depressive like-behavior. The sexual dimorphism of the dopaminergic systems in adult rodents is well known. DA release and uptake is greater in the striatum of adult female rats than in males, and basal DA tone is chronically higher in the striatum of adult males than in females (Becker, 2009; Walker et al., 1999). There are also differences in the number of dopaminergic cells and projections in the mesocortical pathway between adult males and females (Kritzer and Creutz, 2008). In addition, sex hormones influence DA turnover, and the estrous cycle influences dopaminergic signaling in adult rats (Becker et al., 1982; Becker and Cha, 1989;
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Castner et al., 1993; Di Paolo et al., 1985; Jori et al., 1976; Joyce and Van Hartesveldt, 1984; Parvizi and Wuttke, 1983). Here we studied the permanent consequences of perinatal DA increase in the behavior of prepubertal females and males. Some studies have shown that the levels of dopaminergic receptors are different between prepubertal male and female rats. At the age of PD25, female rats have less D1 receptors (but more D2
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receptors) than do males (Andersen et al., 1997; Andersen and Teicher, 2000). In the
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present study, we observed distinct behavioral consequences of perinatal DA increases
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between male and female mice. Dopaminergic signaling, which is involved in many aspects of cell cycle and functions, is different in the brains of perinatal male and female
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rodents (Crandall et al., 2007; Hedlund et al., 2016; Ovtscharoff et al., 1992; Popolo et al., 2004; Schmidt et al., 1996; Souza et al., 2011; Stewart et al., 1991; Stewart and Rajabi,
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1994). It is possible that increased DA levels during perinatal development can lead to different permanent morphological changes in females than in males. In addition, it is
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possible that the distinct behavioral consequences in prepubertal female and male mice is
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due to the supersensitization of dopaminergic receptors, also known as receptor-priming, described in previous studies (Kostrzewa et al., 2016, 1993a).
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Although we did not investigate molecular mechanisms involved in perinatal
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developmental reprogramming by increase of DA levels within the pups’ brains, nor the biochemical consequences in juvenile mice, there are putative epigenetic mechanisms and long-term effects that should be explored in future studies. For example, adult DAT-KO mice show higher levels of extracellular DA but decreased levels of TH (Jones et al., 1998). DAT-KO mice also show a decrease of D2 autoreceptor levels and functions, as well as a downregulation of D1 and D2 postsynaptic receptors within the striatum (Fauchey et al., 2000; Jones et al., 1999). In a recent study using site-specific knockout mice lacking the dopaminergic D2 autoreceptors in dopaminergic neurons (DA-D2RKO),
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in which there is less inhibition of DA synthesis, it was observed that adult mutant mice generate specific alterations of gene expression in the prefrontal cortex (Brami-Cherrier et al., 2014). They observed an increase of the repressive histone mark H3K9me2/3 and a massive decrease of gene expression, such as Akt1, Nrg1, and genes for glutamatergic receptors (Brami-Cherrier et al., 2014). However, these studies used KO mice that did not
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express DAT or D2 autoreceptors throughout life. Thus, it is not possible to know whether
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these alterations are a consequence of the lack of DAT or D2 autoreceptors in the adult
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brain, or whether they stem from the lack of these proteins in a specific developmental window. On the other hand, L-Dopa treatment showed increased deacetylation of histone
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H4K5, as well as K8, K12 and K16 within the striatum of mice previously treated with dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Nicholas
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et al., 2008). Furthermore, other pharmacological experiments have shown that an imbalance of dopaminergic signaling during the first 2 or 3 weeks of life can affect
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dopaminergic receptor levels or sensitivity in young adults (Gelbard et al., 1990;
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Kostrzewa et al., 2016, 1993a, 1993b; Kostrzewa and Saleh, 1989). Thus, it is possible that increased DA levels in mice pups can change histone acetylation and/or methylation,
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which can affect dopaminergic signaling machinery in prepubertal or adult mice.
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One of the limitations identified in our study is the stress effect of saline intraperitoneal injection. Saline injection can be used as mild stress procedure, and a recent study has shown that a single intraperitoneal injection of saline solution is enough to change levels of corticosterone and proinflammatory cytokines in the blood and brains of adult male rats (Advani et al., 2009; Freiman et al., 2016). This stress response might be caused by novel-situation exposure, as suggested by Ryabinin et al., (1999). They compared the effects of two weeks of handling, sham and saline injection stress in adolescent male DBA/2J and C57BL/6J inbred mice. In their study, they showed that a
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single injection increased the expression of c-Fos and Fos-related antigens in the brain, but repeated saline injections led to experimental habituation in C57BL/6J adolescent mice (Ryabinin et al., 1999). In our study, we observed that DA levels and the DOPAC/DA ratio in saline injected pups were different than in naïve pups, which might be caused by the stress of injection. Acute stress induces DA release within the brain, including in the
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striatum, which is similar to our DOPAC/DA results in Saline injected mice (Abercrombie
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et al., 1989). Since the striatum was dissected 30 minutes after saline administration, we
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postulate that this dopaminergic response is caused by the stress of intraperitoneal injection. In contrast, chronic stress decreases the total level of DA within the striatum,
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which is similar to our total DA results, suggesting that the daily administration of saline from PD1 to PD5 was a mild stress to the pups (Rasheed et al., 2010). Interestingly, this
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difference was mainly observed in female pups. On the other hand, after the neonatal treatments, all mice remained in their home cages and received minimal handling (only
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during bedding changes) until PD28. At the age of PD28, all animals were submitted to
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behavioral tests. Since exposure to a novel experimental situation induces stress, and repeated saline injection stress can lead to habituation, the behavioral differences between
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naïve and saline-treated juvenile mice might be caused by the stress of naïve animals being intensely handled for the first time in their lives (Ryabinin et al., 1999). So, it is possible
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that L-Dopa treatment had a protective or an additional effect to the stress of saline injection. Because of this, we chose to show naïve data in separate statistical analyses (Table 2). However, as far as we know, none of the developmental studies using pharmacological tools included naïve animals as a control (Blažević and Hranilović, 2013; Brown et al., 2004, 2002; Hranilovic et al., 2011; Levav et al., 2004; Sprowles et al., 2017). Therefore, our study emphasizes the importance of naïve group inclusion in
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experimental designs and the need to investigate these variables commonly used in developmental studies. Finally, many neuropsychiatric disorders, such as SCZ, autism, and ADHD, share both dopaminergic and neurodevelopmental hypotheses (Bale et al., 2010; Pavăl, 2017; Robinson et al., 2001; Souza and Tropepe, 2011). For example, there is an increase of
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presynaptic DA level within the striatum of SCZ patients (Howes et al., 2009; Lindström et
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al., 1999). Since dopaminergic signaling is involved in many aspects of neurodevelopment,
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it is possible that imbalance in dopaminergic signaling in different developmental windows could be involved in various symptoms in mental disorders of women and men (Crandall et
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al., 2007; Hedlund et al., 2016; Popolo et al., 2004; Schmidt et al., 1996; Souza et al., 2011). Also, several studies have shown that almost 20% of worldwide children and
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adolescents present at least one mental disorder, and this high frequency of mental disorders in youth is very concerning (Costello and Maughan, 2015; Polanczyk et al.,
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2015). Because of this, it is crucial to understand the spectrum of behavioral alterations in
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young subjects for further development of better interventions (Patel et al., 2007). Thus, future studies are necessary in order to understand the ontogenetic molecular mechanisms
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increases of DA.
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related to dopaminergic signaling and the morphophysiological consequences of perinatal
Conclusion:
Perinatal L-Dopa treatment showed ontogenetically different dopaminergic signaling in male than in female Swiss mice, consequently leading to different behavioral alterations in female versus male juvenile Swiss mice.
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Conflict of Interest: The authors declare no competing financial interests Authors Contributions: LOM, ALALR, LTLG, LOG, MAM and NSSA designed the research, performed the experiments and data collection, and analyzed the data; RES, GSP and BRS designed the research, supervised the study, drafted, revised and edited the
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manuscript. All authors have approved the final article Funding: This work was supported by CNPq [grant number 480260/2012-5, and MAM
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scholarship]; FAPEMIG [grant numbers APQ-01896-13, APQ-03109-16, and LTLG
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scholarship); CAPES (LOM and ALALR scholarship); Recém Contratado Grant from
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PRPq-UFMG; International Society for Neurochemistry (ISN) Return Home Grant.
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https://doi.org/10.1038/465690a
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Zucker, I., Beery, A.K., 2010. Males still dominate animal studies. Nature 465, 690–690.
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Figure Legends: Figure 1 – Experimental design. Male and female swiss mice with 6-8 weeks old were housed together for mating for four days. The pregnant females were housed in pair and at the 19th day of pregnancy they were
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separated for birth. From the first day of birth (PD0) until the 5th day of age (PD5), the pups were weighted and intraperitoneally injected once a day with Saline, L-Dopa
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10mg/Kg, L-Dopa 25mg/Kg or L-Dopa 50mg/Kg, in a final volume of 20µL. Each
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manipulation lasted an average of 1 to 2 minutes and the pups were returned to the cage with the mother immediately after the manipulation. The striatum of some of the PD5 pups
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were collected 1 hour after the last treatment and processed to evaluate DA and DOPAC
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levels after treatment. The other group of treated pups were left together with their mothers until PD22, when they were separated by sex and treatment. Both PD28 female and male
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mice were subjected to Open Field Test in the morning and to Elevated Plus Maze Test in
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the afternoon. At the PD29, the mice were subjected to Forced Swimming Test. From PD30 to PD33, mice were subjected to Sucrose Preference Test.
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Figure 2 – Levels of DA and DOPAC within striatum of PD5 swiss mouse. Swiss mice pups were separated in three groups: Naïve (non-handled), Saline injected and
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L-Dopa 50mg/Kg injected pups. The pups were daily treated (except the Naïve group) from PD0 to PD5. Striatum were collected 1 hour after the treatment at PD5. (A) Saline treated females showed a decrease in DA levels compared to Naïve and L-Dopa treated mice. There were no differences between males. (B) L-Dopa treatment increased the levels of DOPAC in both females and males treated with L-Dopa. (C) L-Dopa treatment also increased the ratio of DOPAC/DA in both females and males compared to Naïve. Number of animals is between parenthesis. DOPAC data from females are parametric data and are
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represented as mean ± SD. All the other data is nonparametric and are represented as median with interquartile range. For parametric data it was used One Way ANOVA test and Tukey Test. For nonparametric data it was used Kruskal-Wallis One Way ANOVA on Ranks and Tukey Test. *p < 0.05.
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Figure 3 – Increased levels of DA in the first 5 PD increases locomotor activity of juvenile females but not males.
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Locomotor activity of juvenile Swiss mice (PD28) daily treated with Saline, L-Dopa
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10mg/Kg, L-Dopa 25mg/Kg or L-Dopa 50mg/Kg from PD0 to PD5 were tested by OFT. (A) Only L-Dopa 25mg/Kg treated females showed an increase of total distance. (B) L-
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Dopa treatment did not affect the time spent in center on females or males. (C) L-Dopa
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50mg/Kg treated females showed an increase in the number of rearings compared to Saline and L-Dopa 10mg/Kg treated females. L-Dopa 25mg/Kg treated females also showed an
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increase in the number of rearings compared to L-Dopa 10mg/Kg. There were no
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differences between males. Number of animals is between parenthesis. Total distance data from males are nonparametric data and are represented as median with interquartile range. All the other data is parametric and are represented as mean ± SD. For parametric data it
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was used One Way ANOVA test and Tukey Test. For nonparametric data it was used
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Kruskal-Wallis One Way ANOVA on Ranks and Tukey Test. *p < 0.05. Dashed lines are the mean of Naïve groups. Figure 4 – Increased levels of DA in the first 5 PD did not affect anxiety-like behavior of juvenile Swiss mice. Anxiety-like behavior of juvenile Swiss mice (PD28) daily treated with Saline, L-Dopa 10mg/Kg, L-Dopa 25mg/Kg or L-Dopa 50mg/Kg from PD0 to PD5 were tested by EPM. We did not observe any alterations in the (A) percentage of entries in open arms, (B) time
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in open arms, (C) number of rearings, (D) number of dippings, (E) number of stretchings and (G) time on edge of open arms. (F) Only females treated with L-Dopa 25mg/Kg showed a decrease of time spent on the center. Number of animals is between parenthesis. Number of dippings and time on edges data of females and number of stretchings and time on edges of males are nonparametric data and are represented as median with interquartile
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range. All the other data is parametric and are represented as mean ± SD. For parametric
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data it was used One Way ANOVA test and Tukey Test. For nonparametric data it was
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used Kruskal-Wallis One Way ANOVA on Ranks and Tukey Test. *p < 0.05. Dashed lines are the mean of Naïve groups.
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Figure 5 – Increased levels of DA in the first 5 PD did not affect depressive-like
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behavior of juvenile Swiss mice.
Depressive-like behavior of juvenile Swiss mice (PD29) daily treated with Saline, L-Dopa
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10mg/Kg, L-Dopa 25mg/Kg or L-Dopa 50mg/Kg from PD0 to PD5 were tested by FST.
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We did not observe any alterations in the (A) number of climbings, (B) total time of climbing, (C) total time of swimming, (D) latency immobility, (E) immobility episodes and (G) total time of immobility. Number of animals is between parenthesis. Total time of
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climbing, total time of swimming and number of immobility episodes in females, and
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latency immobility in both sexes, are nonparametric data and are represented as median with interquartile range. All the other data is parametric and are represented as mean ± SD. For parametric data it was used One Way ANOVA test and Tukey Test. For nonparametric data it was used Kruskal-Wallis One Way ANOVA on Ranks and Tukey Test. *p < 0.05. Dashed lines are the mean of Naïve groups. Figure 6 – Increased levels of DA in the first 5 PD affects only anhedonia behavior of male juvenile Swiss mice.
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Anhedonia behavior of juvenile Swiss mice (PD30-33) daily treated with Saline, L-Dopa 10mg/Kg, L-Dopa 25mg/Kg or L-Dopa 50mg/Kg from PD0 to PD5 were tested by Sucrose Preference test. (A) Both Two Way ANOVA and (A’) One Way ANOVA did not detect any difference on total consumption between groups. (B) Juvenile females showed no differences in sucrose preference at any times, (B’) as highlighted at the time of 48 hour.
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On the other hand, juvenile males showed alterations in total consumption. (C) The L-
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Dopa 50mg/Kg treated group showed a decrease in total consumption at 12, 24 and 48
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hours. (C’) The decrease of total consumption by L-Dopa 50mg/Kg treated males at 48 hours is highlighted. (D) It was also observed differences in sucrose preference between
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the L-Dopa 10mg/Kg and L-Dopa 25mg/Kg treated males with L-Dopa 50mg/Kg treated group, as it is highlighted at D’. Number of animals is between parenthesis. The data is
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parametric and is represented as mean ± SD. To analyze the differences between the groups throughout time, we used Two Way ANOVA and Tukey Test (A-D). To analyze
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differences between groups in one timepoint it was used One Way ANOVA test and Tukey
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Test (A’-D’). *p < 0.05. Dashed lines are the mean of Naïve groups.
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Table 1 - Saline versus L-Dopa treatments groups
Figure 3B
Male Fema le
Figure 3C
Male Fema le
Figure 4A
Male Fema le
Figure 4B
Male Fema le
Figure 4C
Male Fema le
Figure 4D
Male Fema le
Figure 4E
Male Fema le
Figure 4F
Male Fema le Male
0.001
0.969 Not applicable
0.138 <0.00 1
0.041
0.490
One-Way ANOVA Kruskal-Wallis One-Way ANOVA on Ranks
0.003 0.083
0.847 Not applicable
One-Way ANOVA
0.030
0.530
One-Way ANOVA
0.042 <0.00 1
0.469
One-Way ANOVA Kruskal-Wallis One-Way ANOVA on Ranks
0.028
0.545 Not applicable
One-Way ANOVA Kruskal-Wallis One-Way ANOVA on Ranks
0.349 0.250
0.076 Not applicable
One-Way ANOVA
0.003
0.856
One-Way ANOVA
0.366
0.068
One-Way ANOVA Kruskal-Wallis One-Way ANOVA on Ranks
0.153 0.220
0.221 Not applicable
One-Way ANOVA
0.006
0.780
One-Way ANOVA
0.022
0.585
One-Way ANOVA Kruskal-Wallis One-Way ANOVA on Ranks One-Way ANOVA
0.020
0.603 Not applicable 0.689
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Male Fema le
One-Way ANOVA
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Figure 3A
Power (α = 0.05)
0.009
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Male Fema le
p value
1.000 Not applicable Not applicable
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Figure 2C
One-Way ANOVA Kruskal-Wallis One-Way ANOVA on Ranks Kruskal-Wallis One-Way ANOVA on Ranks
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Male Fema le
One-Way ANOVA Kruskal-Wallis One-Way ANOVA on Ranks
One-Way ANOVA
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Figure 2B
Normally distributed Non-normally distributed Normally distributed Non-normally distributed Non-normally distributed Normally distributed Normally distributed Non-normally distributed Normally distributed Normally distributed Normally distributed Normally distributed Non-normally distributed Normally distributed Non-normally distributed Normally distributed Normally distributed Normally distributed Non-normally distributed Normally distributed Normally distributed Normally distributed Non-normally distributed Normally
Type of test
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Fema le
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Figure 2A
Data Structure
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Sex
0.007
0.500
0.496 0.012
0.977
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Male Fema le
Figure 5E
Male Fema le
Figure 5F
Male Fema le
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Figure 6A Figure 6A' Figure 6B Figure 6B' Figure 6C Figure 6C' Figure 6D Figure 6D'
Male Fema le Fema le Fema le Fema le Male Male Male Male
One-Way ANOVA
0.221
0.145
One-Way ANOVA Kruskal-Wallis One-Way ANOVA on Ranks Kruskal-Wallis One-Way ANOVA on Ranks Kruskal-Wallis One-Way ANOVA on Ranks Kruskal-Wallis One-Way ANOVA on Ranks Kruskal-Wallis One-Way ANOVA on Ranks Kruskal-Wallis One-Way ANOVA on Ranks Kruskal-Wallis One-Way ANOVA on Ranks
0.956
0.963
0.049 Not applicable Not applicable Not applicable Not applicable Not applicable Not applicable Not applicable
One-Way ANOVA
0.580
0.049
One-Way ANOVA
0.349
0.070
0.941
0.049
N/A
N/A
0.251
0.125
N/A
N/A Not applicable
0.367
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Figure 5D
0.617
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Male Fema le
0.019
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Figure 5C
One-Way ANOVA
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Figure 5B
Male Fema le
Not applicable
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Male Fema le
0.008
D
Figure 5A
Kruskal-Wallis One-Way ANOVA on Ranks
One-Way ANOVA
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Fema le
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Figure 4G
distributed Non-normally distributed Normally distributed Normally distributed Normally distributed Non-normally distributed Non-normally distributed Non-normally distributed Non-normally distributed Non-normally distributed Non-normally distributed Non-normally distributed Normally distributed Normally distributed Normally distributed Normally distributed Normally distributed Normally distributed Normally distributed Normally distributed Normally distributed Normally distributed Normally distributed
Two-Way RM ANOVA One-Way ANOVA Two-Way RM ANOVA Kruskal-Wallis One-Way ANOVA on Ranks Two-Way RM ANOVA One-Way ANOVA Two-Way RM ANOVA One-Way ANOVA
0.464 0.367 0.464 0.367 0.110
0.357 N/A <0.00 1
N/A 0.957
N/A
N/A
0.011
0.705
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Table 2 - Naïve versus Saline treatment Sex
Figure 4D
Figure 4E
Figure 4F Figure 4G Figure 5A
Figure 5B
Male Femal Figure 5C e Male Figure Femal 5D e
Figure 5E
t-test t-test
0.007 0.006
0.782 0.794
Normally distributed Normally distributed
t-test t-test
Normally distributed Normally distributed
t-test t-test
Normally distributed Normally distributed
t-test t-test
Normally distributed Normally distributed Non-normally distributed Normally distributed
Male Femal e
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Normally distributed Normally distributed
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0.002 0.018
0.932 0.613 0.233 0.050
0.120 <0.001
0.217 0.975
t-test t-test Mann-Whitney U Statistic t-test
0.660 0.027
0.050 0.530
0.126 0.005
Not applicable 0.811
t-test t-test
0.016 0.062
0.638 0.531
Normally distributed Normally distributed Non-normally distributed Normally distributed
t-test t-test Mann-Whitney U Statistic t-test
0.087 0.258
0.284 0.082
<0.001 0.012
Not applicable 0.688
Normally distributed Normally distributed
t-test t-test
0.147 0.003
0.179 0.886
Normally distributed Non-normally distributed Non-normally distributed Normally distributed
t-test Mann-Whitney U Statistic Mann-Whitney U Statistic t-test
0.789
0.050
0.066
Not applicable
0.128 0.194
Not applicable 0.128
Normally distributed Non-normally distributed
t-test Mann-Whitney U Statistic
0.010
0.738
0.103
Not applicable
Normally distributed
t-test
0.715
0.050
Normally distributed Normally distributed
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0.111 0.397
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Figure 4C
0.777 0.761
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Figure 4B
Power (α = 0.05)
0.007 0.007
D
Figure 4A
p value
t-test t-test
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Figure 3C
Type of test
Normally distributed Normally distributed
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Figure 3B
Femal e Male Femal e Male Femal e Male Femal e Male Femal e Male Femal e Male Femal e Male Femal e Male Femal e Male Femal e Male Femal e Male Femal e
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Figure 3A
Data Structure
ACCEPTED MANUSCRIPT 56 t-test
0.017
0.617
Normally distributed Normally distributed
t-test t-test
0.261 0.264
0.082 0.079
Normally distributed
t-test
0.026
0.563
Normally distributed
t-test
0.581
0.050
Male
Normally distributed
t-test
0.325
0.052
Male
Normally distributed
t-test
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Figure 6A' Figure 6B' Figure 6C' Figure 6D'
Normally distributed
0.606
0.050
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Figure 5F
Male Femal e Male Femal e Femal e
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Highlights:
1 – Perinatal L-Dopa treatment increases juvenile females’ exploratory behavior
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3 – Perinatal L-Dopa treatment impairs juvenile males’ hedonic behavior
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4 – Perinatal L-Dopa treatment did not affect anxiety- and depressive-like behaviors
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Lorena Oliveira de Matos, Ana Luiza de Araujo Lima Reis, Lorena Terene Lopes Guerra, Leonardo de Oliveira Guarnieri, Muiara Aparecida Moraes, Nayara Soares Sena Aquino, Raphael E. Szawka, Grace Schenatto Pereira, Bruno Rezende Souza PII: DOI: Reference:
S0091-3057(18)30269-7 doi:10.1016/j.pbb.2018.08.002 PBB 72626
To appear in:
Pharmacology, Biochemistry and Behavior
Received date: Revised date: Accepted date:
31 May 2018 25 July 2018 6 August 2018
Please cite this article as: Lorena Oliveira de Matos, Ana Luiza de Araujo Lima Reis, Lorena Terene Lopes Guerra, Leonardo de Oliveira Guarnieri, Muiara Aparecida Moraes, Nayara Soares Sena Aquino, Raphael E. Szawka, Grace Schenatto Pereira, Bruno Rezende Souza , l-Dopa treatment during perinatal development leads to different behavioral alterations in female vs. male juvenile Swiss mice. Pbb (2018), doi:10.1016/ j.pbb.2018.08.002
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Title: L-Dopa treatment during perinatal development leads to different behavioral alterations in female vs. male juvenile Swiss mice Author names: Lorena Oliveira de Matos, MSc1; Ana Luiza de Araujo Lima Reis, MSc1; Lorena Terene
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Lopes Guerra1, Leonardo de Oliveira Guarnieri, PhD1, Muiara Aparecida Moraes MSc1; Nayara Soares Sena Aquino, PhD 2; Prof. Raphael E. Szawka, PhD2, Prof. Grace Schenatto
1
Núcleo de Neurociências, Department of Physiology and
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Authors’ Affiliations:
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Pereira, PhD1, Prof. Bruno Rezende Souza, PhD1
901;
2
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Biophysics, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil, 31270Department of Physiology and Biophysics, Universidade Federal de Minas Gerais,
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Belo Horizonte, MG, Brazil, 31270-901.
Author:
Assistant
Professor,
Bruno
Rezende
Souza,
PhD,
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Corresponding
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Laboratory of Origin: Núcleo de Neurociências (NNC)
[email protected], Núcleo de Neurociências, Department of Physiology and
901
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Biophysics, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil, 31270-
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Running Title: Ontogenetic behavioral consequences of L-Dopa Keywords: Dopamine, neurodevelopment, behavior, sexual dimorphism, ontogeny, neonatal ORCID ID: 0000-0002-7064-7956 Color figures: No
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Abstract: Alterations in dopaminergic signaling and neurodevelopment are associated with many neuropsychiatric disorders, such as attention deficit and hyperactivity disorder (ADHD), autism, and schizophrenia. Imbalances in dopamine levels during prenatal development are associated with behavioral alterations later in life, like hyperactivity and addiction, and it is
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possible that dopaminergic imbalances may have diverse effects during different
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neurodevelopmental windows. In this study, we investigate whether an increase in
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dopamine levels during the perinatal developmental window affects behavior of juvenile male and female Swiss mice. In order to do so, we intraperitoneally administered daily
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doses of L-Dopa to mice pups beginning from postnatal day 1 (PD1) to PD5, which increased the levels of dopamine and its metabolite, 3,4-dihydroxyphenylacetic acid
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(DOPAC), in the striatum of the pups. At the age of 4 weeks, we submitted the juvenile males and females to the open field test, elevated plus maze, forced swimming test, and
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sucrose preference test. We observed that increase of dopamine levels during the perinatal
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developmental window increased exploratory behavior in juvenile females, but not males. We observed no changes in anxiety- and depressive-like behaviors. In contrast, we
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observed that increased dopamine levels during the perinatal period lead to hedonic
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alterations in juvenile males, but not females. Our results show that dopamine signaling is important for behavioral development and that transient imbalance of dopamine levels causes juvenile behavioral alterations, which are different in males than in females. These data may help in better understanding the spectrum of symptoms associated with different neuropsychiatric disorders.
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Introduction: Several neuropsychiatric disorders share both dopaminergic and neurodevelopmental hypotheses but few studies show a convergence of both (Bale et al., 2010; Robinson et al., 2001; Souza and Tropepe, 2011). For example, schizophrenic (SCZ) patients present alterations in volume and morphology of specific brain areas (Bähner and Meyer-
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Lindenberg, 2017; Hoftman et al., 2016; van Erp et al., 2015; Zhuo et al., 2017).
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Behavioral alterations and early symptoms of SCZ emerge during adolescence, usually
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before clinical diagnosis, and children from SCZ parents present alterations in frontolimbic activity (Cornblatt and Keilp, 1994; Erlenmeyer-Kimling et al., 2000; Hart et al.,
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2013).
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Additionally, several studies have demonstrated an association between polymorphism in dopaminergic genes and SCZ (González-Castro et al., 2016; Moskvina et al., 2009).
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There are also changes in dopaminergic signaling in SCZ patients, such as increase of
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dopaminergic D2 receptors and alterations of Neuronal Calcium Sensor-1 (NCS-1) and Dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa (DARPP-32) levels, which are independent of antipsychotic treatments (Albert et al., 2002; Brito-Melo et al., 2012;
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Koh et al., 2003; Souza et al., 2010, 2008, Torres et al., 2009b, 2009a; Wong et al., 1986).
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Furthermore, it is well known that SCZ patients present an increase of presynaptic dopamine (DA) levels and DA release (Abi-Dargham et al., 1998; Breier et al., 1997; Howes et al., 2009; Laruelle et al., 1996; Lindström et al., 1999; Reith et al., 1994). Dopaminergic signaling can also be transiently affected by perinatal exposure to drugs, such as cocaine and metamphetamine, causing cognitive alterations and mental health issues later in life (reviewed by Ross et al., 2015). Furthermore, not only stress but also viral, fungal or even parasitic infections can impair dopaminergic signaling and lead to
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behavioral abnormalities (Abercrombie et al., 1989; Kumar et al., 2011; Prandovszky et al., 2011; Rasheed et al., 2010; Severance et al., 2016; Simanjuntak et al., 2017) During development, dopaminergic signaling is involved in many aspects of cell cycle and functions, such as cell proliferation, cell differentiation, morphogenesis and neuronal migration (Crandall et al., 2007; Hedlund et al., 2016; Popolo et al., 2004; Schmidt et al.,
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1996; Souza et al., 2011). Because dopaminergic signaling molecules are expressed since
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early prenatal development, it is possible that dopaminergic alterations may be involved in
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some of the developmental abnormalities found in many neuropsychiatric disorders (Björklund et al., 1968; Coyle and Axelrod, 1972; Olson and Seiger, 1972; Schambra et al.,
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1994; Specht et al., 1981; Unis et al., 1998). Several animal models have been used to understand the developmental hypothesis for neurodevelopmental disorders. For example,
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knockout mice for the dopaminergic enzyme tyrosine hydroxylase (TH) and dopaminergic receptors show several behavioral alterations with respect to motor behavior, feeding
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behavior, reward behavior, and cognition (Baik et al., 1995; El-Ghundi et al., 2003;
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Maldonado et al., 1997; Smith et al., 1998; Szczypka et al., 1999; Xu et al., 1994). A common model for studying the consequences of DA increases is to use knockout
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(KO) mice for DA transporter (DAT), which shows persistent extracellular DA within the
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synaptic cleft, since diffusion is the only clearance mechanism (Giros et al., 1996). These animals present locomotor hyperactivity and alterations in exploratory and maternal behavior (Giros et al., 1996; Spielewoy et al., 2000). However, DAT KO mice do not express DAT throughout their entire lives, which makes it difficult to study the developmental dynamic over multiple stages of brain development (Andersen, 2003). For example, during prenatal development, phenomena like neurogenesis, neuronal migration, and cell differentiation are very pronounced (Bayer et al., 1993; McConnell, 1990;
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O’Rourke et al., 1992). During early postnatal development there is an increase in synaptogenesis and apoptosis (Aghajanian and Bloom, 1967; Blaschke et al., 1998). Furthermore, there are ontogenetic sex differences during brain development (Tobet et al., 2009). Female rat pups have more dopaminergic projections to striatum than do their male counterparts, with higher levels of DA and tyrosine hydroxylase (TH) (Ovtscharoff et
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al., 1992). Similarly, male pups show higher levels of testosterone and estradiol than
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females, which delays perinatal development of the dopaminergic system (Ovtscharoff et
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al., 1992; Stewart et al., 1991; Stewart and Rajabi, 1994). Since there exists a well-known sex-bias toward male animals and cells in research and it does not represent the prevalence
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of psychiatric disorders observed in humans, it is important to include both sexes to investigate the role of DA in neurodevelopment (Prendergast et al., 2014; Zucker and
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Beery, 2010).
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Taking all these data in consideration, it is not possible to understand whether the
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behavioral abnormalities shown by KO animals are developmental consequences or the consequence of synaptic dynamics at the moment the behavioral test was administered. Nor is it possible to know what phenomenon was affected by the increase of DA, or in
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which developmental window. Furthermore, since DAT KO females show alterations in
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maternal behavior, it is possible that the lack of maternal care is also affecting the development of pups (Chocyk et al., 2013; Teicher et al., 2004). Our hypothesis is that an increase of DA during specific developmental windows causes different alterations in brain and behavior development. TH and DA begin to be expressed during intrauterine embryonic development (Coyle and Axelrod, 1972; Kalsbeek et al., 1988; Olson and Seiger, 1972; Specht et al., 1981). However, there is a significant increase of dopaminergic synaptic formation in the first days after birth (Jung, 1996; Kalsbeek et al., 1988; LerouxNicollet et al., 1990; Schmidt et al., 1982; Verney et al., 1982). The first 5 PD of mice is an
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important developmental window with a significant increase in synaptogenesis and apoptosis, which is essential for adapting to a changing environment (Aghajanian and Bloom, 1967; Blaschke et al., 1998). On the other hand, these phenomena make this developmental window a time of increased vulnerability. It is therefore possible that an imbalance of dopaminergic signaling within this period might impair behavioral
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development (Ignacio et al., 1995; Rice and Barone, 2000). Because of this, we
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investigated whether a transient increase of brain DA levels during the first 5 PD would
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cause behavioral abnormalities in juvenile female and male mice.
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Materials: Animals Male and female Swiss mice (8–12 weeks of age) were purchased from the Animal Facility of the Universidade Federal de Minas Gerais (CEBIO, UFMG, Brazil) for mating.
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Mice were raised in polypropylene cages (27 cm × 17 cm × 12 cm), provided with wood shavings, housed three to five per cage, maintained in a climate-controlled room with 12
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h–12 h light/dark cycle (lights on from 8am to 8pm) and temperature at 22 ± 2 °C and 40-
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70% relative humidity. Free access to food (Nuvilab CR1-Nuvital) and water was provided
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throughout the study.
All protocols were performed during the light phase of the cycle. Behavioral protocols
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were performed in juvenile (30-33 day old) male and female Swiss mice offspring. The Animal Use Ethics Committee of the Universidade Federal de Minas Gerais (CEUA
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39/2015) approved all procedures and the experiments were carried out in accordance with
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NIH guidelines for the use and care of animals. Experimental design and drug administration
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Mating was stimulated by placing two females with one male. After 4 days, the male
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was removed from the cage and two females were housed per cage. At the 19th day of gestation, each female was housed individually. Cages were then inspected daily for the presence of pups. The day of birth was designated PD 0. All pups were randomly assigned to their treatment groups and, for identification, the distal phalanx (toe clipping) of PD 1 pups was removed. The pups were daily treated with L-Dopa (Sigma-Aldrich, D-1507) or saline as control from PD 1 to PD 5. The pups were treated with L-Dopa at concentrations of 10mg/kg, 25mg/kg or 50mg/kg body weight (Kashihara et al., 2002; Kim et al., 2002). They were intraperitoneally injected with a final volume of 20 µL using a 30G needle
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coupled to a 25 µl Hamilton. A Naïve non-manipulated group was also included. Weaning took place at PD 22 and animals were housed according to sex (2 to 3 per cage) and treatment. The animals underwent all behavioral testing at 28-33 days of age (FIGURE 1). During the first day of behavioral tests, animals were submitted to an open field test (OFT) and elevated plus maze test (EPM). On the following day, animals were submitted to a
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forced swimming test (FST), and two days later they were submitted to sucrose preference
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test (SPT).
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Open field test (OFT)
Spontaneous locomotor activity in a new environment was assessed using an
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automatic open field apparatus (LE 8811 IR Motor Activity Monitors PANLAB, Harvard
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Apparatus; Spain), with an acrylic box measuring 23 x 23 x 35 cm. Animals explored the open field for 5 minutes. Total distance traveled was recorded (Walsh and Cummins, 1976)
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and the percentage of time spent in the center of the box (10 x 10 cm) was used as an
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anxiety measure (Prut and Belzung, 2003). Increased locomotion and rearing was considered a stimulant effect (Prut and Belzung, 2003). The open field arena was cleaned
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with 70% ethanol between each trial. Elevated plus maze test (EPM)
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EPM is a simple method which induces an approach–avoidance conflict for assessing anxiety responses of rodents (Walf and Frye, 2007). The apparatus consists of two open arms (30 cm x 6 cm) and two closed arms (30 cm x 6 cm x 16 cm) crossed in the middle perpendicularly to each other and elevated 30 cm above the floor. Mice were placed in the central area and allowed to move freely for 5 minutes (Pádua-Reis et al., 2017). The number of entries into the open arms and the time spent in the open arms were used as indices of anxiety (Pellow et al., 1985).
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Forced swimming test (FST) The FST is a useful tool to assess depressive-like behavior in mice and was carried out as described by Porsolt et al. (1977), with minor modifications. Mice were individually transferred to a vertical glass cylinder (17 cm inner diameter × 27 cm height) filled with water. The water’s temperature was monitored throughout the test and maintained between
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25 and 27 ◦C. At the end of the test, the animal was removed and gently dried under a
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heated air jet. Merely floating, with no movement of the forelimbs or hind limbs, was
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considered immobility and was quantified.
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Sucrose preference test (SPT)
The sucrose preference was assessed using the two-bottle choice method. Animals
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were maintained alone in the home cage and were habituated for 8 h with two bottles containing water. Eighteen hours before the test, animals were water deprived. The test
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consisted of placing two bottles in the home cage: one containing water and the other
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containing a 3% sucrose solution. Both bottles were weighed immediately before the test, and then again 1, 2, 3, 12, 24 and 48 h afterwards. Sucrose preference was analyzed using
al., 2013).
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the equation: [sucrose consumption/sucrose consumption + water consumption] (Imai et
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High performance liquid chromatography with electrochemical detection (HPLC-ED) Because the nigrostriatal pathway is one of the major dopaminergic systems in the brain, we used the striatum as a reference brain area for changes in DA neurotransmission caused by perinatal treatment with L-DOPA. L-DOPA is a precursor of DA and was administered intraperitoneally. Thus, it is reasonable to expect changes in the striatum to reflect changes in dopaminergic activity in the brain. The striatum of PD 5 mice pups was dissected bilaterally 30 minutes after the last treatment and immediately after decapitation.
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Structures were homogenized in 400 μl of 0.15 M perchloric acid, 0.1 mM EDTA, and 1.7 µM 3,4- dihydroxybenzylamine as an internal standard. Homogenates were centrifuged at 4 ºC for 20 minutes at 12,000 g. Protein content was determined in the remaining pellet by the Bradford method (Bradford, 1976). Dopamine (DA) and 3,4- dihydroxyphenylacetic acid (DOPAC) were measured in supernatant as previously described (Aquino et al., 2017;
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Rabelo et al., 2017). Shortly afterwards, 20 µL of samples were injected into an HPLC-ED
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system equipped with a C-18 column (250 x 4 mm, Purospher, 5 µm; Merck, Darmstadt,
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Germany), preceded by a C-18 4 x 4-mm guard column. The mobile phase (100 mM sodium dihydrogen phosphate monohydrate, 10 mM sodium chloride, 0.1 mM EDTA, 0.38
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M sodium 1-octanesulfonic acid, and 10%; pH 3.5) was pumped at a flow rate of 1.0 mL/min. The potential in the electrochemical detector (Decade 2; Antec Scientific, The
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Netherlands) was set to 0.40 V vs. Ag/AgCl reference electrode and the filter at 0.01 Hz. The Class-VP software (Shimadzu, Kyoto, Japan) was used for chromatography plotting,
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and quantification was done using the internal standard method based on peak height.
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All samples were measured in one assay. The intraassay coefficient of variation was 1.4% for DA and 1.7% for DOPAC. DA levels were considered to reflect neurotransmitter
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content in synaptic vesicles, whereas DOPAC levels reflected the amount of DA release
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(Lookingland et al., 1987). Neurotransmitter to metabolite ratio was used as a measure of dopaminergic turnover (Rabelo et al., 2017). Statistical analysis Data were tested for normality using the Shapiro-Wilk test and tested for equal variance by computing the Spearman rank correlation between the absolute values of the residuals and the observed value of the dependent variable. The parametric data were analyzed by One Way ANOVA followed by Tukey’s post-hoc test. Tukey’s post-hoc test was used to control the experimentwise type I error rate. The non-parametric data were
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analyzed by Kruskal-Wallis One Way Analysis of Variance on Ranks followed by Dunn's Method post-hoc test. All tests were performed using SigmaPlot version 12.5.0.38. Parametric data are presented as mean and standard deviation (SD). Non-parametric data are presented as median and interquartile (Weissgerber et al., 2015). Significance was set at p < 0.05 for all tests. All statistical data comparing the L-Dopa treatments versus Saline
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and comparing Naïve versus Saline groups are described at the Tables 1 and 2 respectively.
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Results: Daily L-Dopa treatment in the first 5 PD increases DA levels and its metabolites in the striatum of Swiss mice. Previous studies showed an increase of DA levels and a slight increase of
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noradrenaline levels in the brains of rat pups treated with L-Dopa (Kellogg and Lundborg, 1972a, 1972b). To confirm that PD5 Swiss mice treated daily and intraperitoneally with L-
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Dopa experience an increase of DA within the brain, we separated the pups into three
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groups: non-handled (Naïve), saline-injected (Saline) and L-Dopa-injected (50mg/Kg) pups. Each day, the pups (except the for the naïve group) were treated intraperitoneally,
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from PD0 to PD5. One hour after the last injection, we collected the striatum of female and
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male PD5 pups for HPLC analysis. We observed that saline-treated females showed a decrease of 86% in their DA levels, as compared to the naïve group (F(2,12)= 11.878, One
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Way Anova, Tukey, p= 0.001), and L-Dopa-treated female pups showed an increase of
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426% in their DA levels as compared to the saline-treated group (F(2,12)= 11.878, One Way Anova, Tukey, p= 0.04) (Figure 2A). On the other hand, L-Dopa treatment increased levels of the DA metabolite DOPAC in female pups treated with L-Dopa. L-Dopa-treated
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females showed an increase of approximately 380% in DOPAC levels as compared to
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naïve pups (F(2,13)= 39.340, One Way Anova, Tukey, p< 0.001) and saline groups (F(2,13)= 39.340, One Way Anova, Tukey, p< 0.001). In order to evaluate DA turnover in the striatum of L-Dopa-treated pups, we analyzed the ratio of DOPAC/DA. We observed an increase of the DOPAC/DA ratio in females treated with L-Dopa. Both female pups treated with Saline (KW H= 9.396, 2 d.f., Dunn's Method, p<0.05) and L-Dopa (KW H= 9.396, 2 d.f., Dunn's Method, p<0.05) showed an increase of approximately 600% in DOPAC/DA ratio compared to naïve.
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We observed no differences in DA levels between male pups (Figure 2A) (KW H= 3.956, 2 d.f., p=0.138). On the other hand, L-Dopa-treated males showed an increase of approximately 500% in DOPAC levels compared to naïve (KW H= 9.906, 2 d.f., Tukey, p<0.05) and 400% compared to saline-treated pups (KW H= 9.906, 2 d.f., Tukey, p<0.05) (Figure 2B). L-Dopa-treated males also showed an increase of 123% in the ratio of
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DOPAC/DA compared to the naïve group (F(2,14)= 4.050, One Way Anova, Tukey, p=
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0.034) (Figure 2C). Taken together, these data suggest that L-Dopa treatment was efficient
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in increasing DA release in the striatum of female and male pups, as compared to their naïve and saline counterparts.
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An increase of DA during the first 5 PD induces hyperactivity in juvenile females,
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but not males.
Hyperactivity of dopaminergic signaling is associated with alterations in locomotor
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activity. Because of this, we investigated whether an increase of DA levels during the first
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days of life would affect the locomotor activity of juvenile mice. Each day, we treated Swiss mice pups with saline, L-Dopa 10mg/Kg, L-Dopa 25mg/Kg or L-Dopa 50mg/Kg, from PD0 to PD5, and we evaluated locomotor activity at PD28 by OFT (Figure 1). We
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observed no alterations in the locomotor activity of juvenile males (Figure 3). We also did
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not see any changes in the time spent in the center by juvenile females (Figure 3B). However, we observed an increase in the total distance and in the number of rearings of LDopa 25mg/Kg and L-Dopa 50mg/Kg treated females (Figure 3A,C). The L-Dopa 25mg/Kg treated females moved around approximately 40% more than the saline (F(3,49)= 4.296, One Way Anova, Tukey, p= 0.025) and L-Dopa 10mg/Kg (F(3,49)= 4.296, One Way Anova, Tukey, p= 0.036) groups (Figure 3A). There was also an increase of 44% in the number of rearings in the L-Dopa 25mg/Kg treated group, as compared to LDopa 10mg/Kg group (F(3,49)= 5.401, One Way Anova, Tukey, p= 0.035), as well as a
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slight increase as compared to the saline group (F(3,49)= 5.401, One Way Anova, Tukey, p< 0.082) (Figure 3C). The L-Dopa 50mg/Kg treated females showed an increase of approximately 50% in the number of rearings compared to saline (F(3,49)= 5.401, One Way Anova, Tukey, p= 0.032) and L-Dopa 10mg/Kg treated females (F(3,49)= 5.401, One Way Anova, Tukey, p= 0.013) (Figure 3C). Interestingly, we observed locomotor
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hyperactivity only in juvenile females that suffered an imbalance of dopaminergic
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signaling in the first 5 PD.
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Increased levels of DA in the first 5 PD did not affect anxiety-like behavior in juvenile Swiss mice.
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Female mice treated with L-Dopa in the first 5 PD showed hyperactivity, including an
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increase in the number of rearings. Since dopaminergic signaling is involved in anxietylike behavior, and the number of rearings can indicate this behavior in mice, we tested this
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behavioral phenotype by EPM (Calhoon and Tye, 2015; Prut and Belzung, 2003). We did
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not see any alterations in juvenile females’ behavior during the test, such as percentage of time in the open arms, total time in open arms, number of rearings, number of dippings, number of stretchings, time at center and time on edge of open arms (Figure 4). In juvenile
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males, we observed a decrease in time spent at the center in the L-Dopa 25mg/Kg treated
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group as compared to saline-treated mice (F(3,42)= 4.167, One Way Anova, Tukey, p= 0.013) and to L-Dopa 50mg/Kg treated mice (F(3,42)= 4.167, One Way Anova, Tukey, p= 0.021) (Figure 4F). However, we did not see any changes in the behavior of juvenile males or in the percentage of time in open arms, total time in open arms, number of rearings, number of dippings, number of stretchings, or time on edge of open arms (Figure 4A-E,G). Depressive-like behavior in juvenile Swiss mice is not affected by L-Dopa treatment in the first 5 PD.
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Several studies have shown involvement of DA in depressive behavior and dopaminergic signaling is one of the targets for antidepressants (Otte et al., 2016; Tye et al., 2012). Because of this, we used FST to investigate whether L-Dopa treatment in the first 5 PD would lead to depressive-like behavior in juvenile mice. We observed that neither female nor male juveniles showed alterations in the number of climbings, in total
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climbing-time, total swimming-time, latency immobility, number of immobility episodes,
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or total time of immobility (Figure 5).
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Increased levels of DA in the first 5 PD impacts hedonic behavior only in male juvenile Swiss mice.
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DA is highly involved in the reward system, and it is well known that pups exposed to
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cocaine show alterations in reward behavior. Here we tested whether an increase of DA levels in the first 5 PD would impact the hedonic behavior of juvenile Swiss mice. Firstly,
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we evaluated the total liquid consumption of juvenile females through time, and we
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observed an increase of consumption in the L-Dopa 10mg/Kg treated group compared to saline (F (26,130) = 7.319, Two Way Anova, Tukey, p= 0.019) and L-Dopa 50mg/Kg treated groups (F (26,130) = 7.319, Two Way Anova, Tukey, p= 0.024) at 48 hours (Figure
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6A). However, when we tested data limited to 48 h (One Way Anova), we did not see any
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differences in total liquid consumption between groups (Figure 6A’). Subsequently, we investigated sucrose preference over 48 h and we observed a difference in sucrose consumption between the groups (Figure 6B), as highlighted by the 48 h data (Figure 6B’). We observed significant alterations in total consumption and sucrose preference in juvenile males treated with L-Dopa in the first 5 PD. We observed a decrease of liquid consumption in the L-Dopa 50mg/Kg treated group compared to saline (F (30,150) = 8.486, Two Way Anova, Tukey, p= 0.003), L-Dopa 10mg/Kg (F (30,150) = 8.486, Two Way Anova, Tukey, p= 0.012) and L-Dopa 25mg/Kg treated groups (F (30,150) = 8.486,
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Two Way Anova, Tukey, p= 0.309) at 12 hours. At 24 hours, there was a difference between L-Dopa 50mg/Kg and L-Dopa 25mg/Kg treated group (F (30,150) = 8.486, Two Way Anova, Tukey, p= 0.015). We also observed a decrease of total consumption in LDopa 50mg/Kg treated group compared to Saline (F (30,150) = 8.486, Two Way Anova, Tukey, p< 0.001), L-Dopa 10mg/Kg (F (30,150) = 8.486, Two Way Anova, Tukey, p<
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0.001) and L-Dopa 25mg/Kg treated groups (F (30,150) = 8.486, Two Way Anova, Tukey,
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p< 0.001) at 48 hours (Figure 6C). When we tested only the data of 48 hours we confirmed
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a significant decrease of sucrose preference in juvenile males treated with L-Dopa 50mg/Kg treated group compared to Saline (F (3,30) = 7.494, One Way Anova, Tukey, p=
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0.012), L-Dopa 10mg/Kg (F (3,30) = 7.494, One Way Anova, Tukey, p= 0.005) and LDopa 25mg/Kg treated groups (F (3,30) = 7.494, One Way Anova, Tukey, p< 0.001) at 48
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hours (Figure 6C’).
We investigated the sucrose preference of juvenile males and observed a difference
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between L-Dopa 50mg/Kg treated group with L-Dopa 10mg/Kg (F (30,150) = 15.45, Two
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Way Anova, Tukey, p= 0.035) and L-Dopa 25mg/Kg treated groups (F (30,150) = 15.45, Two Way Anova, Tukey, p= 0.026) at 1 hour. At 2 hours, we observed a difference
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between L-Dopa 50mg/Kg treated group with L-Dopa 10mg/Kg (F (30,150) = 15.45, Two
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Way Anova, Tukey, p= 0.019) and L-Dopa 25mg/Kg treated groups (F (30,150) = 15.45, Two Way Anova, Tukey, p= 0.038) (Figure 6D). When we tested only the data of 2 hours we confirmed a significant difference in sucrose preference in juvenile males treated with L-Dopa 50mg/Kg treated group compared to L-Dopa 10mg/Kg (F (3,30) = 4.371, One Way Anova, Tukey, p= 0.016) and L-Dopa 25mg/Kg treated groups (F (3,30) = 4.371, One Way Anova, Tukey, p< 0.031) at 2 hours (Figure 6D’).
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Discussion: Ontogenetic disturbances of dopaminergic signaling are associated with permanent alterations in juvenile and adult behavior (Brown et al., 2002; Brus et al., 2003; Kostrzewa et al., 2016). In this study, we showed that a transient increase of DA levels during the perinatal developmental window can cause behavioral alterations in juvenile Swiss mice,
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and, additionally, that these changes are different between males and females.
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Dopaminergic enzymes are expressed in the perinatal brain and DA levels are stable in
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the first 5 days of postnatal development in rats (Agrawal et al., 1966; Karki et al., 1962; Kellogg and Lundborg, 1973; Nachmias, 1960). Previous studies have shown that L-Dopa
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acute administration in rat pups (PD1 and PD4) significantly increases DA levels 30
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minutes post-injection, but there is only a very slight increase in noradrenaline levels (Kellogg and Lundborg, 1972b, 1972a; Van Hartesveldt et al., 1991). They also observed
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an increase of motor activity 30 minutes after L-Dopa administration. The difference in
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motor behavior was observed when the rat pups were acutely treated with at least 50mg/Kg L-Dopa (Camp and Rudy, 1987; Stehouwer et al., 1994). Here, we observed an increase of DA, DOPAC and the DOPAC/DA ratio in the female striatum 1 hour after acute
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administration of 50mg/Kg L-Dopa in PD5 mice pups. Male pups displayed an increase in
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DOPAC and DOPAC/DA ratio, but not DA levels, subsequent to L-Dopa administration (Figure 2A). Differently from most of the studies, we decided to include the Naïve group as additional experimental control. Interestingly, the Saline injected pups showed an increase DOPAC/DA ratio compared to Naïve. This increase might be cause by the minor stress of manipulation and injection (Abercrombie et al., 1989). It is known that female rat pups have higher TH levels and dopaminergic projections to striatum than do males (Ovtscharoff et al., 1992). Also, the higher neonatal levels of testosterone and estradiol in males compared to females delay perinatal dopaminergic system development in male
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pups. Because of this, DA levels increase earlier in female rat pups than in males (Stewart et al., 1991; Stewart and Rajabi, 1994). Several studies have shown that dopaminergic imbalances during prenatal and postnatal development can cause behavioral alterations in juvenile and adult rodents. For example, adult CD1 male mice (PD60) exposed to L-Dopa during prenatal development,
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from E11 until birth, display alterations in cocaine-induced behavior (Ren et al., 2011).
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Furthermore, treatment with the DA transporter inhibitor bupropion from E6 to PD20
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affects male and female adult rats’ (PD60) behavior in different ways (Sprowles et al., 2017). In addition, adult rats (PD60) treated postnatally with D2 agonist (Quinpirole) from
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PD1 until PD11 show higher active avoidance and deficits in spatial memory (Brown et al., 2002; Brus et al., 2003, 1997). Some of these behavioral alterations can be detected even in
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juvenile animals. Juvenile rats (PD22-28) treated daily with quinpirole, from PD1 until PD21, display behavioral alterations like vertical jumping, spatial memory, and quinpirole-
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induced yawning (Brown et al., 2004; Kostrzewa et al., 2004, 1993b, 1993a). However, as
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mentioned, DA levels are very stable in the first 5 days of postnatal development (Agrawal et al., 1966). Thus, we wondered whether an increase of DA levels within this stable
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developmental window could permanently affect juvenile behavior in Swiss mouse.
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In this study, we observed an increase in the total distance travelled (Figure 3A) and in the number of rearings (Figure 3C) of only female juvenile Swiss mice treated with LDopa from PD1 to PD5. Increased number of rearings and total distance travelled in the OFT is correlated with increased levels of anxiety-like behavior in mice (Borta and Schwarting, 2005; Seibenhener and Wooten, 2015). These data suggest that an increase of DA levels in the first 5 PD amplifies anxiety-like behavior in juvenile females, but not males (Figure 3). However, we did not observe any behavioral alterations in the EPM test, including in the number of rearings, which is commonly used to test anxiety-like behavior
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by exploiting the conflict between preference for protected areas and the contrary drive to explore new environments (File et al., 1994; Rodgers et al., 1999; Walf and Frye, 2007) (Figure 4). Since we did not observe changes in the number of rearings in the EPM test, and the number of rearings in the OFT can also be interpreted as information-gathering routine, it is possible that the L-Dopa-treated juvenile females have an increase of
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exploratory behavior, rather than an increase of anxiety-like behavior (Lever et al., 2006;
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Mansour et al., 2003).
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On the other hand, only male juvenile Swiss mice treated with L-Dopa from PD1 to PD5 showed a decrease of total consumption (Figure 6C and C’) and alterations in sucrose
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preference (Figure 6D and D’) in the SPT. Decrease of sucrose consumption is associated with anhedonia and depressive-like behavior (Overstreet, 2012a; Willner et al., 1992;
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Zhang et al., 2017). Our results suggest that an increase of DA in the first 5 PD impairs hedonic behavior in juvenile males but not females (Figure 6). However, we did not detect
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any changes in behavior during the FST, which is commonly used to test rodents’ escape-
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directed behavior in water. A pronounced lack of this behavior is associated with depressive-like behavior (Overstreet, 2012b; Slattery and Cryan, 2012) (Figure 5).
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Therefore, it is more likely that L-Dopa-treated juvenile males have impairment of
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motivation-like behavior than depressive like-behavior. The sexual dimorphism of the dopaminergic systems in adult rodents is well known. DA release and uptake is greater in the striatum of adult female rats than in males, and basal DA tone is chronically higher in the striatum of adult males than in females (Becker, 2009; Walker et al., 1999). There are also differences in the number of dopaminergic cells and projections in the mesocortical pathway between adult males and females (Kritzer and Creutz, 2008). In addition, sex hormones influence DA turnover, and the estrous cycle influences dopaminergic signaling in adult rats (Becker et al., 1982; Becker and Cha, 1989;
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Castner et al., 1993; Di Paolo et al., 1985; Jori et al., 1976; Joyce and Van Hartesveldt, 1984; Parvizi and Wuttke, 1983). Here we studied the permanent consequences of perinatal DA increase in the behavior of prepubertal females and males. Some studies have shown that the levels of dopaminergic receptors are different between prepubertal male and female rats. At the age of PD25, female rats have less D1 receptors (but more D2
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receptors) than do males (Andersen et al., 1997; Andersen and Teicher, 2000). In the
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present study, we observed distinct behavioral consequences of perinatal DA increases
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between male and female mice. Dopaminergic signaling, which is involved in many aspects of cell cycle and functions, is different in the brains of perinatal male and female
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rodents (Crandall et al., 2007; Hedlund et al., 2016; Ovtscharoff et al., 1992; Popolo et al., 2004; Schmidt et al., 1996; Souza et al., 2011; Stewart et al., 1991; Stewart and Rajabi,
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1994). It is possible that increased DA levels during perinatal development can lead to different permanent morphological changes in females than in males. In addition, it is
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possible that the distinct behavioral consequences in prepubertal female and male mice is
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due to the supersensitization of dopaminergic receptors, also known as receptor-priming, described in previous studies (Kostrzewa et al., 2016, 1993a).
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Although we did not investigate molecular mechanisms involved in perinatal
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developmental reprogramming by increase of DA levels within the pups’ brains, nor the biochemical consequences in juvenile mice, there are putative epigenetic mechanisms and long-term effects that should be explored in future studies. For example, adult DAT-KO mice show higher levels of extracellular DA but decreased levels of TH (Jones et al., 1998). DAT-KO mice also show a decrease of D2 autoreceptor levels and functions, as well as a downregulation of D1 and D2 postsynaptic receptors within the striatum (Fauchey et al., 2000; Jones et al., 1999). In a recent study using site-specific knockout mice lacking the dopaminergic D2 autoreceptors in dopaminergic neurons (DA-D2RKO),
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in which there is less inhibition of DA synthesis, it was observed that adult mutant mice generate specific alterations of gene expression in the prefrontal cortex (Brami-Cherrier et al., 2014). They observed an increase of the repressive histone mark H3K9me2/3 and a massive decrease of gene expression, such as Akt1, Nrg1, and genes for glutamatergic receptors (Brami-Cherrier et al., 2014). However, these studies used KO mice that did not
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express DAT or D2 autoreceptors throughout life. Thus, it is not possible to know whether
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these alterations are a consequence of the lack of DAT or D2 autoreceptors in the adult
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brain, or whether they stem from the lack of these proteins in a specific developmental window. On the other hand, L-Dopa treatment showed increased deacetylation of histone
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H4K5, as well as K8, K12 and K16 within the striatum of mice previously treated with dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Nicholas
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et al., 2008). Furthermore, other pharmacological experiments have shown that an imbalance of dopaminergic signaling during the first 2 or 3 weeks of life can affect
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dopaminergic receptor levels or sensitivity in young adults (Gelbard et al., 1990;
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Kostrzewa et al., 2016, 1993a, 1993b; Kostrzewa and Saleh, 1989). Thus, it is possible that increased DA levels in mice pups can change histone acetylation and/or methylation,
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which can affect dopaminergic signaling machinery in prepubertal or adult mice.
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One of the limitations identified in our study is the stress effect of saline intraperitoneal injection. Saline injection can be used as mild stress procedure, and a recent study has shown that a single intraperitoneal injection of saline solution is enough to change levels of corticosterone and proinflammatory cytokines in the blood and brains of adult male rats (Advani et al., 2009; Freiman et al., 2016). This stress response might be caused by novel-situation exposure, as suggested by Ryabinin et al., (1999). They compared the effects of two weeks of handling, sham and saline injection stress in adolescent male DBA/2J and C57BL/6J inbred mice. In their study, they showed that a
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single injection increased the expression of c-Fos and Fos-related antigens in the brain, but repeated saline injections led to experimental habituation in C57BL/6J adolescent mice (Ryabinin et al., 1999). In our study, we observed that DA levels and the DOPAC/DA ratio in saline injected pups were different than in naïve pups, which might be caused by the stress of injection. Acute stress induces DA release within the brain, including in the
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striatum, which is similar to our DOPAC/DA results in Saline injected mice (Abercrombie
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et al., 1989). Since the striatum was dissected 30 minutes after saline administration, we
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postulate that this dopaminergic response is caused by the stress of intraperitoneal injection. In contrast, chronic stress decreases the total level of DA within the striatum,
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which is similar to our total DA results, suggesting that the daily administration of saline from PD1 to PD5 was a mild stress to the pups (Rasheed et al., 2010). Interestingly, this
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difference was mainly observed in female pups. On the other hand, after the neonatal treatments, all mice remained in their home cages and received minimal handling (only
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during bedding changes) until PD28. At the age of PD28, all animals were submitted to
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behavioral tests. Since exposure to a novel experimental situation induces stress, and repeated saline injection stress can lead to habituation, the behavioral differences between
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naïve and saline-treated juvenile mice might be caused by the stress of naïve animals being intensely handled for the first time in their lives (Ryabinin et al., 1999). So, it is possible
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that L-Dopa treatment had a protective or an additional effect to the stress of saline injection. Because of this, we chose to show naïve data in separate statistical analyses (Table 2). However, as far as we know, none of the developmental studies using pharmacological tools included naïve animals as a control (Blažević and Hranilović, 2013; Brown et al., 2004, 2002; Hranilovic et al., 2011; Levav et al., 2004; Sprowles et al., 2017). Therefore, our study emphasizes the importance of naïve group inclusion in
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experimental designs and the need to investigate these variables commonly used in developmental studies. Finally, many neuropsychiatric disorders, such as SCZ, autism, and ADHD, share both dopaminergic and neurodevelopmental hypotheses (Bale et al., 2010; Pavăl, 2017; Robinson et al., 2001; Souza and Tropepe, 2011). For example, there is an increase of
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presynaptic DA level within the striatum of SCZ patients (Howes et al., 2009; Lindström et
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al., 1999). Since dopaminergic signaling is involved in many aspects of neurodevelopment,
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it is possible that imbalance in dopaminergic signaling in different developmental windows could be involved in various symptoms in mental disorders of women and men (Crandall et
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al., 2007; Hedlund et al., 2016; Popolo et al., 2004; Schmidt et al., 1996; Souza et al., 2011). Also, several studies have shown that almost 20% of worldwide children and
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adolescents present at least one mental disorder, and this high frequency of mental disorders in youth is very concerning (Costello and Maughan, 2015; Polanczyk et al.,
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2015). Because of this, it is crucial to understand the spectrum of behavioral alterations in
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young subjects for further development of better interventions (Patel et al., 2007). Thus, future studies are necessary in order to understand the ontogenetic molecular mechanisms
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increases of DA.
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related to dopaminergic signaling and the morphophysiological consequences of perinatal
Conclusion:
Perinatal L-Dopa treatment showed ontogenetically different dopaminergic signaling in male than in female Swiss mice, consequently leading to different behavioral alterations in female versus male juvenile Swiss mice.
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Conflict of Interest: The authors declare no competing financial interests Authors Contributions: LOM, ALALR, LTLG, LOG, MAM and NSSA designed the research, performed the experiments and data collection, and analyzed the data; RES, GSP and BRS designed the research, supervised the study, drafted, revised and edited the
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manuscript. All authors have approved the final article Funding: This work was supported by CNPq [grant number 480260/2012-5, and MAM
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scholarship]; FAPEMIG [grant numbers APQ-01896-13, APQ-03109-16, and LTLG
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scholarship); CAPES (LOM and ALALR scholarship); Recém Contratado Grant from
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PRPq-UFMG; International Society for Neurochemistry (ISN) Return Home Grant.
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Xu, M., Hu, X.-T., Cooper, D.C., Moratalla, R., Graybiel, A.M., White, F.J., Tonegawa, S., 1994. Elimination of cocaine-induced hyperactivity and dopamine-mediated neurophysiological effects in dopamine D1 receptor mutant mice. Cell 79, 945–955. https://doi.org/10.1016/0092-8674(94)90026-4 Zhang, Y., Wang, Y., Lei, H., Wang, L., Xue, L., Wang, X., Zhu, X., 2017. Optimized
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https://doi.org/10.1038/465690a
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Zucker, I., Beery, A.K., 2010. Males still dominate animal studies. Nature 465, 690–690.
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Figure Legends: Figure 1 – Experimental design. Male and female swiss mice with 6-8 weeks old were housed together for mating for four days. The pregnant females were housed in pair and at the 19th day of pregnancy they were
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separated for birth. From the first day of birth (PD0) until the 5th day of age (PD5), the pups were weighted and intraperitoneally injected once a day with Saline, L-Dopa
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10mg/Kg, L-Dopa 25mg/Kg or L-Dopa 50mg/Kg, in a final volume of 20µL. Each
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manipulation lasted an average of 1 to 2 minutes and the pups were returned to the cage with the mother immediately after the manipulation. The striatum of some of the PD5 pups
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were collected 1 hour after the last treatment and processed to evaluate DA and DOPAC
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levels after treatment. The other group of treated pups were left together with their mothers until PD22, when they were separated by sex and treatment. Both PD28 female and male
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mice were subjected to Open Field Test in the morning and to Elevated Plus Maze Test in
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the afternoon. At the PD29, the mice were subjected to Forced Swimming Test. From PD30 to PD33, mice were subjected to Sucrose Preference Test.
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Figure 2 – Levels of DA and DOPAC within striatum of PD5 swiss mouse. Swiss mice pups were separated in three groups: Naïve (non-handled), Saline injected and
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L-Dopa 50mg/Kg injected pups. The pups were daily treated (except the Naïve group) from PD0 to PD5. Striatum were collected 1 hour after the treatment at PD5. (A) Saline treated females showed a decrease in DA levels compared to Naïve and L-Dopa treated mice. There were no differences between males. (B) L-Dopa treatment increased the levels of DOPAC in both females and males treated with L-Dopa. (C) L-Dopa treatment also increased the ratio of DOPAC/DA in both females and males compared to Naïve. Number of animals is between parenthesis. DOPAC data from females are parametric data and are
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represented as mean ± SD. All the other data is nonparametric and are represented as median with interquartile range. For parametric data it was used One Way ANOVA test and Tukey Test. For nonparametric data it was used Kruskal-Wallis One Way ANOVA on Ranks and Tukey Test. *p < 0.05.
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Figure 3 – Increased levels of DA in the first 5 PD increases locomotor activity of juvenile females but not males.
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Locomotor activity of juvenile Swiss mice (PD28) daily treated with Saline, L-Dopa
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10mg/Kg, L-Dopa 25mg/Kg or L-Dopa 50mg/Kg from PD0 to PD5 were tested by OFT. (A) Only L-Dopa 25mg/Kg treated females showed an increase of total distance. (B) L-
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Dopa treatment did not affect the time spent in center on females or males. (C) L-Dopa
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50mg/Kg treated females showed an increase in the number of rearings compared to Saline and L-Dopa 10mg/Kg treated females. L-Dopa 25mg/Kg treated females also showed an
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increase in the number of rearings compared to L-Dopa 10mg/Kg. There were no
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differences between males. Number of animals is between parenthesis. Total distance data from males are nonparametric data and are represented as median with interquartile range. All the other data is parametric and are represented as mean ± SD. For parametric data it
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was used One Way ANOVA test and Tukey Test. For nonparametric data it was used
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Kruskal-Wallis One Way ANOVA on Ranks and Tukey Test. *p < 0.05. Dashed lines are the mean of Naïve groups. Figure 4 – Increased levels of DA in the first 5 PD did not affect anxiety-like behavior of juvenile Swiss mice. Anxiety-like behavior of juvenile Swiss mice (PD28) daily treated with Saline, L-Dopa 10mg/Kg, L-Dopa 25mg/Kg or L-Dopa 50mg/Kg from PD0 to PD5 were tested by EPM. We did not observe any alterations in the (A) percentage of entries in open arms, (B) time
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in open arms, (C) number of rearings, (D) number of dippings, (E) number of stretchings and (G) time on edge of open arms. (F) Only females treated with L-Dopa 25mg/Kg showed a decrease of time spent on the center. Number of animals is between parenthesis. Number of dippings and time on edges data of females and number of stretchings and time on edges of males are nonparametric data and are represented as median with interquartile
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range. All the other data is parametric and are represented as mean ± SD. For parametric
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data it was used One Way ANOVA test and Tukey Test. For nonparametric data it was
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used Kruskal-Wallis One Way ANOVA on Ranks and Tukey Test. *p < 0.05. Dashed lines are the mean of Naïve groups.
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Figure 5 – Increased levels of DA in the first 5 PD did not affect depressive-like
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behavior of juvenile Swiss mice.
Depressive-like behavior of juvenile Swiss mice (PD29) daily treated with Saline, L-Dopa
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10mg/Kg, L-Dopa 25mg/Kg or L-Dopa 50mg/Kg from PD0 to PD5 were tested by FST.
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We did not observe any alterations in the (A) number of climbings, (B) total time of climbing, (C) total time of swimming, (D) latency immobility, (E) immobility episodes and (G) total time of immobility. Number of animals is between parenthesis. Total time of
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climbing, total time of swimming and number of immobility episodes in females, and
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latency immobility in both sexes, are nonparametric data and are represented as median with interquartile range. All the other data is parametric and are represented as mean ± SD. For parametric data it was used One Way ANOVA test and Tukey Test. For nonparametric data it was used Kruskal-Wallis One Way ANOVA on Ranks and Tukey Test. *p < 0.05. Dashed lines are the mean of Naïve groups. Figure 6 – Increased levels of DA in the first 5 PD affects only anhedonia behavior of male juvenile Swiss mice.
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Anhedonia behavior of juvenile Swiss mice (PD30-33) daily treated with Saline, L-Dopa 10mg/Kg, L-Dopa 25mg/Kg or L-Dopa 50mg/Kg from PD0 to PD5 were tested by Sucrose Preference test. (A) Both Two Way ANOVA and (A’) One Way ANOVA did not detect any difference on total consumption between groups. (B) Juvenile females showed no differences in sucrose preference at any times, (B’) as highlighted at the time of 48 hour.
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On the other hand, juvenile males showed alterations in total consumption. (C) The L-
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Dopa 50mg/Kg treated group showed a decrease in total consumption at 12, 24 and 48
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hours. (C’) The decrease of total consumption by L-Dopa 50mg/Kg treated males at 48 hours is highlighted. (D) It was also observed differences in sucrose preference between
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the L-Dopa 10mg/Kg and L-Dopa 25mg/Kg treated males with L-Dopa 50mg/Kg treated group, as it is highlighted at D’. Number of animals is between parenthesis. The data is
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parametric and is represented as mean ± SD. To analyze the differences between the groups throughout time, we used Two Way ANOVA and Tukey Test (A-D). To analyze
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differences between groups in one timepoint it was used One Way ANOVA test and Tukey
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Test (A’-D’). *p < 0.05. Dashed lines are the mean of Naïve groups.
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Table 1 - Saline versus L-Dopa treatments groups
Figure 3B
Male Fema le
Figure 3C
Male Fema le
Figure 4A
Male Fema le
Figure 4B
Male Fema le
Figure 4C
Male Fema le
Figure 4D
Male Fema le
Figure 4E
Male Fema le
Figure 4F
Male Fema le Male
0.001
0.969 Not applicable
0.138 <0.00 1
0.041
0.490
One-Way ANOVA Kruskal-Wallis One-Way ANOVA on Ranks
0.003 0.083
0.847 Not applicable
One-Way ANOVA
0.030
0.530
One-Way ANOVA
0.042 <0.00 1
0.469
One-Way ANOVA Kruskal-Wallis One-Way ANOVA on Ranks
0.028
0.545 Not applicable
One-Way ANOVA Kruskal-Wallis One-Way ANOVA on Ranks
0.349 0.250
0.076 Not applicable
One-Way ANOVA
0.003
0.856
One-Way ANOVA
0.366
0.068
One-Way ANOVA Kruskal-Wallis One-Way ANOVA on Ranks
0.153 0.220
0.221 Not applicable
One-Way ANOVA
0.006
0.780
One-Way ANOVA
0.022
0.585
One-Way ANOVA Kruskal-Wallis One-Way ANOVA on Ranks One-Way ANOVA
0.020
0.603 Not applicable 0.689
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Male Fema le
One-Way ANOVA
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Figure 3A
Power (α = 0.05)
0.009
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Male Fema le
p value
1.000 Not applicable Not applicable
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Figure 2C
One-Way ANOVA Kruskal-Wallis One-Way ANOVA on Ranks Kruskal-Wallis One-Way ANOVA on Ranks
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Male Fema le
One-Way ANOVA Kruskal-Wallis One-Way ANOVA on Ranks
One-Way ANOVA
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Figure 2B
Normally distributed Non-normally distributed Normally distributed Non-normally distributed Non-normally distributed Normally distributed Normally distributed Non-normally distributed Normally distributed Normally distributed Normally distributed Normally distributed Non-normally distributed Normally distributed Non-normally distributed Normally distributed Normally distributed Normally distributed Non-normally distributed Normally distributed Normally distributed Normally distributed Non-normally distributed Normally
Type of test
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Fema le
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Figure 2A
Data Structure
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Sex
0.007
0.500
0.496 0.012
0.977
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Male Fema le
Figure 5E
Male Fema le
Figure 5F
Male Fema le
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Figure 6A Figure 6A' Figure 6B Figure 6B' Figure 6C Figure 6C' Figure 6D Figure 6D'
Male Fema le Fema le Fema le Fema le Male Male Male Male
One-Way ANOVA
0.221
0.145
One-Way ANOVA Kruskal-Wallis One-Way ANOVA on Ranks Kruskal-Wallis One-Way ANOVA on Ranks Kruskal-Wallis One-Way ANOVA on Ranks Kruskal-Wallis One-Way ANOVA on Ranks Kruskal-Wallis One-Way ANOVA on Ranks Kruskal-Wallis One-Way ANOVA on Ranks Kruskal-Wallis One-Way ANOVA on Ranks
0.956
0.963
0.049 Not applicable Not applicable Not applicable Not applicable Not applicable Not applicable Not applicable
One-Way ANOVA
0.580
0.049
One-Way ANOVA
0.349
0.070
0.941
0.049
N/A
N/A
0.251
0.125
N/A
N/A Not applicable
0.367
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Figure 5D
0.617
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Male Fema le
0.019
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Figure 5C
One-Way ANOVA
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Figure 5B
Male Fema le
Not applicable
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Male Fema le
0.008
D
Figure 5A
Kruskal-Wallis One-Way ANOVA on Ranks
One-Way ANOVA
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Fema le
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Figure 4G
distributed Non-normally distributed Normally distributed Normally distributed Normally distributed Non-normally distributed Non-normally distributed Non-normally distributed Non-normally distributed Non-normally distributed Non-normally distributed Non-normally distributed Normally distributed Normally distributed Normally distributed Normally distributed Normally distributed Normally distributed Normally distributed Normally distributed Normally distributed Normally distributed Normally distributed
Two-Way RM ANOVA One-Way ANOVA Two-Way RM ANOVA Kruskal-Wallis One-Way ANOVA on Ranks Two-Way RM ANOVA One-Way ANOVA Two-Way RM ANOVA One-Way ANOVA
0.464 0.367 0.464 0.367 0.110
0.357 N/A <0.00 1
N/A 0.957
N/A
N/A
0.011
0.705
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Table 2 - Naïve versus Saline treatment Sex
Figure 4D
Figure 4E
Figure 4F Figure 4G Figure 5A
Figure 5B
Male Femal Figure 5C e Male Figure Femal 5D e
Figure 5E
t-test t-test
0.007 0.006
0.782 0.794
Normally distributed Normally distributed
t-test t-test
Normally distributed Normally distributed
t-test t-test
Normally distributed Normally distributed
t-test t-test
Normally distributed Normally distributed Non-normally distributed Normally distributed
Male Femal e
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Normally distributed Normally distributed
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0.002 0.018
0.932 0.613 0.233 0.050
0.120 <0.001
0.217 0.975
t-test t-test Mann-Whitney U Statistic t-test
0.660 0.027
0.050 0.530
0.126 0.005
Not applicable 0.811
t-test t-test
0.016 0.062
0.638 0.531
Normally distributed Normally distributed Non-normally distributed Normally distributed
t-test t-test Mann-Whitney U Statistic t-test
0.087 0.258
0.284 0.082
<0.001 0.012
Not applicable 0.688
Normally distributed Normally distributed
t-test t-test
0.147 0.003
0.179 0.886
Normally distributed Non-normally distributed Non-normally distributed Normally distributed
t-test Mann-Whitney U Statistic Mann-Whitney U Statistic t-test
0.789
0.050
0.066
Not applicable
0.128 0.194
Not applicable 0.128
Normally distributed Non-normally distributed
t-test Mann-Whitney U Statistic
0.010
0.738
0.103
Not applicable
Normally distributed
t-test
0.715
0.050
Normally distributed Normally distributed
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0.111 0.397
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Figure 4C
0.777 0.761
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Figure 4B
Power (α = 0.05)
0.007 0.007
D
Figure 4A
p value
t-test t-test
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Figure 3C
Type of test
Normally distributed Normally distributed
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Figure 3B
Femal e Male Femal e Male Femal e Male Femal e Male Femal e Male Femal e Male Femal e Male Femal e Male Femal e Male Femal e Male Femal e Male Femal e
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Figure 3A
Data Structure
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0.017
0.617
Normally distributed Normally distributed
t-test t-test
0.261 0.264
0.082 0.079
Normally distributed
t-test
0.026
0.563
Normally distributed
t-test
0.581
0.050
Male
Normally distributed
t-test
0.325
0.052
Male
Normally distributed
t-test
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Figure 6A' Figure 6B' Figure 6C' Figure 6D'
Normally distributed
0.606
0.050
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Figure 5F
Male Femal e Male Femal e Femal e
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Highlights:
1 – Perinatal L-Dopa treatment increases juvenile females’ exploratory behavior
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3 – Perinatal L-Dopa treatment impairs juvenile males’ hedonic behavior
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4 – Perinatal L-Dopa treatment did not affect anxiety- and depressive-like behaviors
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6