GABAA overactivation potentiates the effects of NMDA blockade during the brain growth spurt in eliciting locomotor hyperactivity in juvenile mice

Neurotoxicology and Teratology 50 (2015) 43–52

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GABAA overactivation potentiates the effects of NMDA blockade during the brain growth spurt in eliciting locomotor hyperactivity in juvenile mice Juliana Oliveira-Pinto, Danielle Paes-Branco, Fabiana Cristina-Rodrigues, Thomas E. Krahe, Alex C. Manhães, Yael Abreu-Villaça, Cláudio C. Filgueiras ⁎ Laboratório de Neurofisiologia, Departamento de Ciências Fisiológicas, Instituto de Biologia Roberto Alcantara Gomes, Centro Biomédico, Universidade do Estado do Rio de Janeiro, Av. Prof. Manoel de Abreu 444, 5 andar, Vila Isabel, Rio de Janeiro, RJ, 20550-170, Brazil

a r t i c l e

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Article history: Received 22 December 2014 Received in revised form 25 May 2015 Accepted 31 May 2015 Available online 6 June 2015 Keywords: ADHD model Ambulation GABA mimetic NMDA blockade

a b s t r a c t Both NMDA receptor blockade and GABAA receptor overactivation during the brain growth spurt may contribute to the hyperactivity phenotype reminiscent of attention-deficit/hyperactivity disorder. Here, we evaluated the effects of exposure to MK801 (a NMDA antagonist) and/or to muscimol (a GABAA agonist) during the brain growth spurt on locomotor activity of juvenile Swiss mice. This study was carried out in two separate experiments. In the first experiment, pups received a single i.p. injection of either saline solution (SAL), MK801 (MK, 0.1, 0.3 or 0.5 mg/kg) or muscimol (MU, 0.02, 0.1 or 0.5 mg/kg) at the second postnatal day (PND2), and PNDs 4, 6 and 8. In the second experiment, we investigated the effects of a combined injection of MK (0.1 mg/kg) and MU (doses: 0.02, 0.1 or 0.5 mg/kg) following the same injection schedule of the first experiment. In both experiments, locomotor activity was assessed for 15 min at PND25. While MK promoted a dose-dependent increase in locomotor activity, exposure to MU failed to elicit significant effects. The combined exposure to the highest dose of MU and the lowest dose of MK induced marked hyperactivity. Moreover, the combination of the low dose of MK and the high dose of MU resulted in a reduced activity in the center of the open field, suggesting an increased anxietylike behavior. These findings suggest that, during the brain growth spurt, the blockade of NMDA receptors induces juvenile locomotor hyperactivity whereas hyperactivation of GABAA receptors does not. However, GABAA overactivation during this period potentiates the effects of NMDA blockade in inducing locomotor hyperactivity. © 2015 Elsevier Inc. All rights reserved.

1. Introduction The attention-deficit/hyperactivity disorder (ADHD) is a neurobehavioral disorder mainly characterized by inattentiveness, impulsiveness and hyperactivity that typically begins in childhood and often persists into adolescence and adulthood (Dopheide and Pliszka, 2009). Epidemiological data have shown that ADHD is the most prevalent neurobehavioral disorder of childhood, affecting 2 to 17% of school-aged children worldwide (Centers for Disease Control and Prevention—CDC, 2010; Dopheide and Pliszka, 2009; Faraone et al., 2003; Skounti et al., 2007). While ADHD appears to be one of the most heritable psychiatric disorders, with genetic studies suggesting a heritability ranging from 60% to 80% in children (Chang et al., 2013; Merwood et al., 2013), several environmental risk factors, including prenatal stress (Rodriguez and Bohlin, 2005) and gestational exposure to neurotoxic substances such as lead (Eubig et al., 2010) or ethanol (Bhatara et al., 2006; Burd et al.,

⁎ Corresponding author. E-mail address: ccfi[email protected] (C.C. Filgueiras).

http://dx.doi.org/10.1016/j.ntt.2015.05.011 0892-0362/© 2015 Elsevier Inc. All rights reserved.

2003; Doig et al., 2008), have been frequently associated with this neurodevelopmental disorder. The three main symptoms of ADHD have been modeled in rodents (Sagvolden et al., 2005). However, locomotor hyperactivity, usually assessed by ambulation in open field tests, is the most frequently studied. Increased locomotor activity is a behavioral trait of several inbred strains, knockouts, and transgenic rodents used as models of ADHD (Mill, 2007; Russell, 2007; van der Kooij and Glennon, 2007; Yan et al., 2009). Hyperactivity has been consistently described in rats and mice developmentally exposed to neurotoxic substances such as ethanol (Kelly et al., 1987; Nunes et al., 2011; Riley et al., 1993) and lead (Ma et al., 1999; Moreira et al., 2001; Silbergeld and Goldberg, 1974), or endocrine disruptors such as bisphenol (Kiguchi et al., 2008; Zhou et al., 2011). Long-lasting locomotor hyperactivity has also been described in rodents submitted to early maternal isolation (Niwa et al., 2011; Sterley et al., 2013) or perinatal hypoxia (Delcour et al., 2012; Gramatte and Schmidt, 1986). It is important to note that in most non-genetic rodent models of ADHD, locomotor hyperactivity emerges as a consequence of a developmental perturbation during the first 10 days of postnatal life. During this period, which overlaps

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substantially with the brain growth spurt (Dobbing and Sands, 1979), the brain rapidly increases in size and intense neurogenesis, neural cell migration and synaptogenesis take place (Bandeira et al., 2009; Brazel et al., 2003; Kolb and Whishaw, 1989). Moreover, during the brain growth spurt, neuronal circuits are refined by pruning of connections and neuronal apoptosis (Olney et al., 2000). It is well known that these events, which are essential to the establishment of neural circuits, are under modulatory influence of neurotransmission, especially that mediated by glutamate and GABA (Huang, 2009; Jiang et al., 2005; Lujan et al., 2005; Zhang and Sun, 2011). For instance, both glutamate and GABA, acting in an excitatory manner on their respective receptors, generate calcium currents which provide trophic functions in the developing brain, influencing synaptic plasticity, dendritic branching and promoting proliferation and migration of neuronal progenitors (Ben-Ari et al., 2007; Dehorter et al., 2012; Nguyen et al., 2001). Therefore, any perturbation of either glutamatergic or GABAergic signaling during the brain growth spurt can disrupt the proper wiring of the brain and lead to long-lasting neurobehavioral problems. Consistent with this idea, ethanol, an agent strongly associated with locomotor hyperactivity in rodents as well as with ADHD in children, exhibits both NMDA antagonist and GABA mimetic properties (Dopico and Lovinger, 2009; Proctor et al., 2006). Similarly, drugs utilized frequently in obstetric and pediatric medicine, such as anesthetics and anticonvulsant agents that reduce neuronal excitability via NMDA receptors (NMDAR) antagonism and/or GABAA receptors (GABAAR) agonism, have also been associated with inattention and impulsivity/hyperactivity (DiMaggio et al., 2011; Domizio et al., 1993; Fredriksson and Archer, 2004; Moore et al., 2000). Both the blockade of NMDAR and the overactivation of GABAAR during the brain growth spurt trigger widespread apoptotic neurodegeneration (Ikonomidou, 2009), reduce neurogenesis and cell proliferation in the infant rodent brain (Stefovska et al., 2008) and promote acute and long-lasting dysregulation in proteins associated with cell proliferation and neuronal circuit formation, effects that may have a central role in generating the hyperactive phenotype (Kaindl et al., 2008). Interestingly, NMDA antagonists and GABA agonists produce their own distinct brain damage pattern (Ikonomidou et al., 2000, 2001), probably due to marked differences between NMDAR and GABAAR regarding their spatiotemporal distribution during development. Therefore, simultaneous NMDAR blockade and GABAAR activation may promote a more robust and widespread neurodegenerative response than that caused by the single administration of a NMDA antagonist or a GABA mimetic drug (Jevtovic-Todorovic et al., 2003). Finally, the GABAAR is mainly depolarizing during the rodent neonatal period (Ben-Ari et al., 2007; Kirmse et al., 2011), so that, regarding electrical activity, the potentiation of currents through GABAAR and the inhibition of currents through NMDAR have opposite effects. It is possible that the inactivation of NMDAR and/or the hyperactivation of GABAAR during the brain growth spurt have distinct neurobehavioral outcomes. However, to date, there are few studies exploring the late neurobehavioral consequences of early manipulations of the GABAAR and NMDAR systems and the relative contributions of NMDA antagonism and augmentation of GABAergic activity to the manifestation of locomotor hyperactivity. In this sense, here we study the locomotor activity of juvenile Swiss mice exposed to MK801 (NMDAR antagonist) and/or to muscimol (GABAAR agonist) during the brain growth spurt. 2. Methods 2.1. Animal treatment This study was conducted under the approval of the Universidade do Estado do Rio de Janeiro. All experiments were carried out in compliance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Subjects

were Swiss mice that were bred and maintained in our laboratory on a 12:12 h light/dark cycle (lights on: 2:00, lights off: 14:00) in a temperature-controlled room (22 ± 1 °C). Access to food and water was unrestricted. Original breeding stock was obtained from Instituto Vital Brazil (Rio de Janeiro, RJ, Brazil). The day of birth was considered postnatal day 1 (PND1). This study was carried out in two separate experiments. In the first experiment, pups within the same litter were assigned to receive a single injection (26 μL/g i.p.) of either MK801 (MK) or muscimol (MU) every other day from PND2 to PND8 (four injections in total). The following MK doses were used: 1) LOW — 0.1 mg/kg; 2) INT — 0.3 mg/kg and 3) HIGH — 0.5 mg/kg. The following MU doses were used: 1) LOW — 0.02 mg/kg; 2) INT — 0.1 mg/kg and 3) HIGH — 0.5 mg/kg. A given pup received only one drug/dose combination. Control pups were i.p. injected with saline solution (SAL). Litters were not culled and pups were pseudo-randomly distributed into 7 treatment groups: SAL (31 mice from 19 litters), MKLOW (33 mice from 20 litters), MKINT (25 mice from 10 litters), MUHIGH (35 mice from 17 litters), MULOW (32 mice from 11 litters), MUINT (27 mice from 14 litters), and MUHIGH (21 mice from 18 litters). In order to minimize the risk of injury to internal organs, a 28-gauge needle was carefully inserted to just penetrate the abdominal wall and reach the peritoneal cavity. Leakage from the injection site was minimized by slowly withdrawing the needle from the abdominal cavity. The doses of MK and MU were chosen based on the range of doses usually adopted in studies that investigated neurobehavioral consequences of i.p. administration of these drugs in rodents (Ouagazzal et al., 1993; Stefovska et al., 2008; Yoon et al., 2002; Zarrindast et al., 2006). Treatment on alternate days was adopted to ensure that the animals were exposed for the majority of the brain growth spurt period and at the same time minimize stress and injury associated with drug administration. Of note, young infants subjected to general anesthesia multiple times (two or more) have an increased risk of developing neurobehavioral disorders such as ADHD (Sprung et al., 2012). In addition, our drug administration protocol mimics the ‘binge’ consumption of ethanol in humans, which is associated with severe neurobehavioral deficits (Maier and West, 2001). In the second experiment, we investigated the effects of the combined injection of MK and MU. Thus, an additional sample of mice (143 pups from 17 litters) was assigned to receive MK and MU simultaneously. Pups were i.p injected every other day from PND2 to PND8 (similar to the first experiment). Siblings were distributed into four treatments groups. Three of the groups received a fixed 0.1 mg/kg dose of MK (MK LOW ) and one of the different MU doses used in the first experiment. Control pups were i.p. injected with saline solution (SAL). Thus, the pups were pseudo-randomly distributed into the following groups: SAL (n = 28), MK LOW +MU LOW (n = 31), MKLOW+MUINT (n = 34) and MKLOW+MUHIGH (n = 50). At weaning (PND21), animals from the same litter were separated by sex and housed in groups of 2–5 mice per cage. The mortality rate was calculated separately for each group as follows: number of animals that died until PND25 divided by the total number of animals injected at PND2. At PND25, the spontaneous locomotor activity of the animals was evaluated in the open field test. 2.2. Open field test The open field arena (Insight, Ribeirão Preto, SP, Brazil) consisted of a transparent acrylic box (46 × 46 × 43 cm) that was equipped with two arrays of 16 infrared beams each, positioned at 1.5 cm above the floor to measure horizontal spontaneous locomotor activity. Interruptions of photocell beams were detected by a computer system, and the location of the animal was calculated by the equipment software (Insight Equipamentos Científicos, Ribeirão Preto, SP, Brazil). Behavioral analysis was performed under red dim light illumination between 14:30 and 16:00 h in a room with an ambient temperature of 22 ± 1 °C. Each mouse was individually placed in the center of the

J. Oliveira-Pinto et al. / Neurotoxicology and Teratology 50 (2015) 43–52

open field, we counted the number of pixels in the center and periphery of images obtained from the computer-assisted video tracking system. The 8 bit images (404 × 404 pixels) of the total tracked activity of 261 animals (first experiment: 91 males and 71 females; second experiment: 56 males and 43 females) were transformed to black and white, where black pixels represent the animal's activity in the open field. Measurements of the number of black pixels in the center (200 × 200 pixels) and periphery (351 × 351 pixels) were conducted blind using the Histogram function of Image J software. Of note, the activity measured by interruptions of photocell beams was highly correlated with the total number of pixels (Pearson correlation, R = 0.84, d.f. = 207, P b 0.001), indicating that the evaluation of locomotor activity by means of the quantification of the number of pixels provides a good estimate of the locomotor activity of the animal. Thus, the number of pixels in the center divided by the total distance traveled was used to assess anxiety-like behavioral changes.

Table 1 Mortality rate in the first experiment. Group

SAL MKLOW MKINT MKHIGH MULOW MUINT MUHIGH

Number of animals injected at PND2

Mortality rate

a

31 33 25 35 32 27 21

12.9% 12.1%b 24.0% 42.9%a,b,c,d 12.5%c 14.8%d 19.0%

Animals tested at PND25 Females

Males

9 11 11 7 12 13 9

18 18 8 13 16 10 8

Similar letters represent a pairwise comparison. Fisher's Exact Test: a, dP b 0.05; b, cP b 0.01.

arena. At the end of each session, the animal was returned to its home cage and, before another animal was placed in the open field arena, the floor was cleaned with a 40% ethanol solution and dried. Cumulative horizontal (ambulation) spontaneous locomotor activity was determined at 3-min time-intervals for a 15-min period. Rodents spontaneously avoid the central part of the open field, and increases of the ratio between the activity in the center and the total locomotor activity are indicators of an increased anxiety-like behavior (Prut and Belzung, 2003). To quantify the activity in the center of the

A

6

Body mass (g)

5

4

45

2.3. Statistical analyses Fisher's Exact Tests were performed for the analyses of mortality rate. For all other analyses, data were compiled as means and standard errors of the mean. Pups from a given litter were assigned to different experimental groups. Despite that, most groups had more than one

SAL MK LOW MK INT MK HIGH MU LOW

**

** ***

MU INT MU HIGH

*

** ***

***

3

**

2

1

0

PND2

PND4

PND6

PND8

B 40

35

Body mass (g)

30

25

20

15

10

5

0 SAL

MK LOW

MK INT

MK HIGH

MU LOW

MU INT

MU HIGH

Fig. 1. Mean body mass (±SEM) of mice injected, from PND2 to PND8 (PND1 = birth day) with MK801 (MKLOW: 0.1 mg/kg, MKINT: 0.3 mg/kg or MKHIGH: 0.5 mg/kg) muscimol (MULOW: 0.02 mg/kg, MUINT: 0.1 mg/kg or MUHIGH: 0.5 mg/kg) or saline (SAL) during PND2-PND8 (A) and at PND25 (B). Note that both MK and MU exposure resulted in a significant body mass reduction throughout the treatment period (A). FPLSD: *P b 0.05, **P b 0.01 and ***P b 0.01 comparisons with the SAL group.

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male and one female from the same litter. Thus, data from males or females of the same litter were averaged within each group to minimize litter effects and avoid over-sampling (Wainwright, 1998). Separate univariate analysis of variance (uANOVA) was performed for weight data at PND25. Repeated measures analyses of variance (rANOVAs) were performed for both body weight data during the treatment period (postnatal day as the within-subjects factor) and open field data (timeinterval as the within-subjects factor). Neonatal treatment and sex were used as between-subject factors for both uANOVAs and rANOVAs. Regarding rANOVAs, for simplicity, we reported results based only on the averaged univariate F tests. Whenever the sphericity assumption was violated, we used the Greenhouse–Geisser correction, which adjusts the degrees of freedom, in order to avoid Type I errors. Significance was assumed at the level of P b 0.05 (two-tailed). Individual group differences were evaluated post-hoc by Fisher's Protected Least Significant Difference (FPLSD). 3. Results 3.1. First experiment 3.1.1. Mortality rate and body mass gain The majority of the animals survived the i.p. injections (Table 1). In the group of animals injected with MK, there was a dose-dependent increase in the mortality rate. The mortality rate of the MKHIGH group was significantly higher than those observed for the SAL and MKLOW groups. The pairwise comparisons involving the muscimol-injected and SAL groups did not reach statistical significance. Offspring body masses during the treatment period are shown in Fig. 1A. The mean litter body masses increased significantly from PND2 to PND8 [age: F(1.7,117.9) = 1278.9; P b 0.001]. However, the MKINT, MKHIGH and MUHIGH groups exhibited growth slower than that of the SAL group, as demonstrated by a significant neonatal treatment × age interaction [F(9.5,117.9) = 9.2, P b 0.001] as well as a main effect of neonatal treatment [F(6,71) = 10.4, P b 0.001]. At PND8, the mean pup body masses of the MKINT, MKHIGH and MUHIGH groups were, respectively, 15%, 31% and 16% smaller than those of the SAL group (Fig. 1A). No differences were observed between the SAL, MKLOW, MULOW and MUINT groups. At PND25 (Fig. 1B), no significant differences in body weight were observed among groups [F(6,74) =

4000

3.1.2. Open field data The distance traveled decreased significantly from the first (731.5 ± 23.3) to the fifth (369.9 ± 13.7) time-interval [time-interval: F(2.6,189.2) = 74.1; P b 0.001]. The decrease in ambulatory activity observed throughout the testing session did not differ among groups [time-interval × neonatal treatment: F(15.8,189.2) = 0.7; P N 0.84]. However, regarding the total distance traveled (Fig. 2), the treatment with MK increased ambulatory activity in a dose-dependent way [neonatal treatment: F(6,72) = 3.0; P b 0.05]. The total distances traveled by the MKINT and MKHIGH groups were, respectively, 21% and 33% higher than that of the SAL group (Fig. 2). No differences were observed between the SAL, MKLOW, MULOW, MUINT and MUHIGH groups. The total distance traveled by males (3008.6 ± 76.7 cm) was significantly higher than that of females (2700.4 ± 88.4 cm) [sex: F(1,72) = 5.6; P b 0.05]. However, there were no significant interactions involving gender and other factors. For all animals collapsed across sex and neonatal treatment, the activity in the periphery (16,110 ± 271 pixels) was significantly greater than that in the center (5638 ± 271 pixels) [uANOVA, F(1,191) = 746.4; P b 0.001]. The activity in the center of the arena corrected by the total distance traveled was not affected by sex or treatment with MK or MU. 3.2. Second experiment Considering that the MK treatment caused a dose-dependent increase in mortality rate as well as a reduction in body mass gain, it seemed likely that the highest doses of MK combined with MU would cause a significant increase in mortality and/or delay in pup development. Therefore, only the lowest dose of MK was combined with different doses of muscimol. 3.2.1. Mortality rate and body mass gain The majority of the animals survived the i.p. injections. While the mortality rate of the MKLOW+MUHIGH group was significantly higher than those observed for the other groups (Table 2), there were no differences between the SAL, MKLOW+MULOW and MKLOW+MUINT groups.

*** **

3500

Total distance traveled (cm)

1.1; P = 0.36]. There was no sex effect or interaction between sex and neonatal treatment.

3000 2500 2000 1500 1000 500 0 SAL

MK LOW

MK INT

MK HIGH

MU LOW

MU INT

MU HIGH

Fig. 2. Mean (±SEM) distance traveled in the open field for PND25 (PND1 = birth day) mice injected with MK801 (MKLOW: 0.1 mg/kg, MKINT: 0.3 mg/kg or MKHIGH: 0.5 mg/kg), muscimol (MULOW: 0.02 mg/kg, MUINT: 0.1 mg/kg or MUHIGH: 0.5 mg/kg) or saline (SAL) from PND2 to PND8. Note that, as compared to the SAL group, the treatment with MK801 increased the ambulatory activity in a dose-dependent way. FPLSD: **P b 0.01 and ***P b 0.01 comparisons with the SAL group.

J. Oliveira-Pinto et al. / Neurotoxicology and Teratology 50 (2015) 43–52 Table 2 Mortality rate in the second experiment. Group

SAL MKLOW+MULOW MKLOW+MUINT MKLOW+MUHIGH

Number of animals injected at PND2

Mortality rate

a

27 31 34 50

11.1% 9.7%b 8.8%c 44.4%a,b,c

Animals tested at PND25 Females

Males

10 8 11 18

11 18 17 10

Similar letters represent a pairwise comparison. Fisher's Exact Test: a, b, and cP b 0.01.

Offspring body masses during the treatment period are shown in Fig. 3A. The mean litter body masses increased significantly from PND2 to PND8 [age: F(1.5,77.0) = 657.0; P b 0.001]. The MKLOW+MUHIGH group exhibited growth slower than that of the SAL group [neonatal treatment × age: F(4.5,77.0) = 4.3, P b 0.01]. At PND8, the mean pup body mass of the MKLOW+MUHIGH group was 18.6% smaller than that of pups in the SAL group (Fig. 3A). No differences were observed between the SAL, MKLOW+MULOW and MKLOW+MUINT groups. At PND25 (Fig. 3B), pups in the MKLOW+MUHIGH group were, respectively, 18% and 14% lighter than pups in the SAL and MKLOW+MUINT groups [neonatal treatment: F(1,73) = 2.9; P b 0.05]. No significant differences in body mass were observed between the

A

47

SAL, MKLOW+MULOW and MKLOW+MUINT groups. The mean weight of males (26.9 ± 0.8 g) was significantly higher than that of females (24.3 ± 0.8 g) [sex: F(1,73) = 4.7; P b 0.05], however, there was no interaction between gender and neonatal treatment. 3.2.2. Open field data The distance traveled reduced significantly from the first to the fifth time-intervals [time-interval: F(2.1,117.1) = 53.3; P b 0.001]. The decrease in ambulatory activity observed along the testing session did not differ among groups [time-interval × neonatal treatment: F(6.3,117.1) = 0.7; P N 0.10]. However, the total distance traveled by the MKLOW+MUHIGH group was, respectively, 26%, 20% and 22% higher than that of the SAL, MKLOW+MULOW and MKLOW+MUINT groups [neonatal treatment: F(3,55) = 3.0; P b 0.05] (Fig. 4). No differences were observed between the SAL, MKLOW+MULOW and MKLOW+MUINT groups. There was no sex effect or significant interactions involving sex and the other factors. For all animals collapsed across sex and neonatal treatment, the activity in the periphery (15,406 ± 495 pixels) was significantly greater than that in the center (5997 ± 495 pixels) [uANOVA, F(1,97) = 180.6; P b 0.001]. While no significant differences were observed in males, females of the MKLOW+MUHIGH group exhibited significantly lower activity in the center than that of the SAL, MKLOW+MULOW and MKLOW+MUINT groups [neonatal treatment × sex: F(3,46) = 4.0; P b 0.01] (Fig. 5).

SAL

6

MKLOW +MULOW MKLOW +MUINT

Body mass (g)

5

#

MKLOW +MUHIGH

4

*

3

2

1

0

PND2

PND4

PND8

PND6

B 35

#

Body mass (g)

30

**

25 20 15 10 5 0 SAL

MKLOW +MULOW

MKLOW +MUINT

MKLOW +MUHIGH

Fig. 3. Mean body mass (±SEM) of mice injected, from PND2 (PND1 = birth day) to PND8 with saline (SAL) or with 0.1 mg/kg of MK801 (MKLOW) combined with one of three doses of muscimol (MULOW: 0.02 mg/kg, MUINT: 0.1 mg/kg or MUHIGH: 0.5 mg/kg) during PND2–PND8 (A) and at PND25 (B). Note that the combination of MKLOW+MUHIGH resulted in a significant body weight reduction. FPLSD: *P b 0.05 and **P b 0.01, comparisons with SAL group; #P b 0.05 comparisons with the MKLOW+MUINT group.

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**

Total distance traveled (cm)

3500 3000 2500 2000 1500 1000 500 0 SAL

MKLOW +MULOW

MKLOW +MUINT

MKLOW +MUHIGH

Fig. 4. Mean (±SEM) distance traveled in the open field for PND25 (PND1 = birth day) mice injected with saline (SAL) or with 0.1 mg/kg of MK801 (MKLOW) combined with one of three doses of muscimol (MULOW: 0.02 mg/kg, MUINT: 0.1 mg/kg or MUHIGH: 0.5 mg/kg) from PND2 to PND8. Note that the combination of MKLOW+MUHIGH increased ambulatory activity. FPLSD: **P b 0.01 in comparisons with all other groups.

4. Discussion This study was carried out to investigate the effects of exposure to MK801 (NMDA antagonist) and/or to muscimol (GABAA agonist) during the brain growth spurt on the locomotor activity of juvenile mice. Two major observations were made: 1) while the exposure solely to MK promoted a dose-dependent increase in locomotor activity, the exposure solely to increasing doses of MU failed to affect it (first experiment); 2) the combined exposure to the highest dose of MU and the lowest dose of MK (neither of which affected locomotor activity when administered separately) induced a marked hyperactivity (second experiment). These findings suggest that during the brain growth spurt the blockade of NMDA receptors induces juvenile locomotor hyperactivity whereas overactivation of GABAAR does not. Interestingly, our results also indicate that GABAA overactivation during the brain growth spurt potentiates the effects of NMDA blockade in inducing locomotor hyperactivity. In addition, the fact that females exposed to both the 4.5

% of activity in center

4.0 3.5 3.0 2.5

**

2.0 1.5 1.0 0.5 0.0 SAL

MKLOW +MULOW

MKLOW +MUINT

MKLOW +MUHIGH

Fig. 5. Mean (±SEM) of percentage of activity in the center distance traveled in the open field for PND25 (PND1 = birth day) female mice injected with saline (SAL) or with 0.1 mg/kg of MK801 (MKLOW) combined with one of three doses of muscimol (MULOW: 0.02 mg/kg, MUINT: 0.1 mg/kg or MUHIGH: 0.5 mg/kg) from PND2 to PND8. Note that the combination of MKLOW+MUHIGH increased anxiety-like behavior. FPLSD: **P b 0.01 in comparisons with all other groups.

highest dose of MU and the lowest dose of MK concomitantly displayed a marked reduction in the percentage of activity in the center of the arena suggests that the combined blockade of NMDAR and GABAAR overactivation during brain growth spurt may increase anxiety in a sex-dependent way. The electrical activity mediated by the GABAAR or NMDAR has been implicated in regulating many particular aspects of brain development (Huang, 2009; Jiang et al., 2005; Lujan et al., 2005; Zhang and Sun, 2011). The depolarization caused by the activation of GABAA or NMDA receptors promotes a transient rise in intracellular Ca2+ concentration that activates a wide range of cytosolic Ca2+-sensitive proteins that, in turn, activate several signaling pathways mediating cell survival, differentiation and formation of neuronal circuits (Ben-Ari et al., 1997; Hardingham and Bading, 2003; Lu et al., 2013; Markova and Lenne, 2012). Considering that, during the brain growth spurt, the potentiation of currents through the GABAAR and the inhibition of currents through the NMDAR have opposite effects regarding electrical activity and modulation of intracellular Ca2+ levels, it is reasonable to suppose that the administration of muscimol and MK-801 would have opposite effects on Ca2+-mediated developmental processes. Of note, the Ca2+ influx caused by excessive activation of GABAA receptors may overload the intracellular buffering capacity, leading to toxic levels of this ion (Zhao et al., 2011). In addition, the signaling ability of Ca2+ relies on the presence of signaling proteins anchored at specific locations within the cell, and on the duration and frequency of Ca2 + concentration changes (Aumann and Horne, 2012; Markova and Lenne, 2012). Therefore, both the NMDAR blockade and the GABAAR overactivation, by reducing the amplitude and frequency of the neuronal Ca2+-oscillations (Sinner et al., 2011a,b), share several deleterious effects on brain development. The blockade of NMDA receptors and/or the overactivation of GABAA receptors during the brain growth spurt promote an increase in neuroapoptosis (Ikonomidou, 2009). Some authors have suggested that increased apoptosis during this developmental period may underlie the development of locomotor hyperactivity as well as other neurobehavioral disturbances (Han et al., 2005; Ieraci and Herrera, 2006; Medina, 2011; Wozniak et al., 2004). This idea is consistent with findings showing that children with ADHD exhibit reductions in cortical thickness and smaller volumes in the prefrontal cortex, striatum and dorsal anterior cingulate cortex, and that these smaller volumes are associated with greater ADHD symptom severity (Dopheide and Pliszka, 2009; Krain and Castellanos, 2006). Besides the reductions in neuronal number, early exposure to muscimol or to MK801 has several other actions that disrupt the normal sequence of events involved in the formation of brain connections that may lead to dysfunctional corticostriatal and corticocerebellar circuits observed in ADHD (Arnsten, 2011; Durston et al., 2011; Liston et al., 2011). For instance, the blockade of NMDAR or the overactivation of GABAAR in immature neurons, even at concentrations that are too low to induce apoptosis, impairs neuronal differentiation and reduces synapse integrity (Sinner et al., 2011a,b); dysregulates several proteins involved in neuronal migration, axon growth and guidance (Kaindl et al., 2008) and promotes alterations in lamination and heterotopic cell clusters (Heck et al., 2007; Reiprich et al., 2005) in the rodent cerebral cortex. Most of these effects have been directly or indirectly associated with an increase in locomotor activity (Casanova, 2014; Drerup et al., 2010; Komada et al., 2014; Shin et al., 2004). All of the above suggests that both the blockade of NMDAR and the overactivation of GABAAR during the brain growth spurt may perturb neuronal circuits in the brain and, consequently, the animal's behavior. However, the fact that locomotor hyperactivity was observed only in MK-801-treated mice suggests that, during the brain growth spurt, the blockade of NMDA receptors is more important than the hyperactivation of GABAA receptors in eliciting juvenile locomotor hyperactivity. Accordingly, increased locomotor activity has been described in juvenile rats after the treatment with MK-801 from PND6 to PND21 (Schiffelholz et al., 2004), from PND7 to PND10 (Kocahan et al., 2013) and from PND1

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to PND20 (Facchinetti et al., 1993), as well as after the administration of other NMDAR antagonists during the brain growth spurt (Boctor and Ferguson, 2010; Fredriksson and Archer, 2003). The absence of locomotor hyperactivity in mice treated with muscimol is in line with the finding that muscimol exposure at PND1 and PND2 did not affect spontaneous locomotor activity of juvenile rats in an open field test (Nunez et al., 2003). Considering that both the reduction of the population of cells expressing NMDAR and that perturbation of the NMDAR-mediated processes involved with the formation of neuronal circuits may have persistent consequences, our data suggest that a reduction in NMDAR transmission is one of the underlying mechanisms leading to symptoms associated with ADHD and hyperactive behavior (Dramsdahl et al., 2011; Elia et al., 2012; Jensen et al., 2009; Zhang et al., 2012). Accordingly, a reduction in NMDA receptor-mediated function has been demonstrated in rats of the SHR strain (Spontaneously Hypertensive Rat), which are widely used as a model for the study of ADHD (Jensen et al., 2009). Mice lacking the NMDAR 2A subunit gene GluRe1 exhibit spontaneous locomotor hyperactivity (Miyamoto et al., 2001). Genetic polymorphism of both the NR2A and the NR2B subunits of the NMDA receptors have been associated with ADHD in children (Dorval et al., 2009; Turic et al., 2004). Of note, impaired NMDA-mediated function may contribute to dysfunction in catecholaminergic systems since the activity of midbrain dopaminergic neurons is regulated by corticofugal glutamatergic activation (Sagvolden et al., 2005). In adult rats, the blockade of NMDA receptors leads to a secondary increase in cortical glutamate release which in turn disrupts dopaminergic neurotransmission in the prefrontal cortex, in part, by stimulating AMPA/kainate receptors (Moghaddam et al., 1997). Hyperactive/impulsive behavior is associated with reduced post-synaptic efficacy of dopaminergic and noradrenergic modulation of neuronal circuits of the prefrontal and anterior cingulate cortices (Arnsten and Pliszka, 2011; Sagvolden et al., 2005). The fact that exposure only to muscimol did not affect locomotor activity is, to some extent, surprising since some studies have suggested that dysfunctional GABAergic transmission may also contribute to ADHD (Edden et al., 2012; Lou, 2012) as well as to the emergence of locomotor hyperactivity in rodents (Viggiano, 2008; Yee et al., 2005; Zhou et al., 2011). This observation suggests that the period from PND2 to PND8 may not cover the time window during which GABAAR overactivation is critical to the establishment of a hyperactive phenotype. Considering that during brain development, GABAergic synapses are established before glutamatergic synapses (Ben-Ari et al., 2007), it is possible that the critical period of GABAA-mediated processes precedes that of the NMDAR. Accordingly, mice lacking the subunit α3 of GABAA receptors exhibit a slight but significant elevation in locomotor activity (Yee et al., 2005). Locomotor hyperactivity was observed in rats whose dams were injected daily with phenobarbital or pentobarbital from gestational days 9 to 21 (Martin et al., 1985). The fact that the combined exposure of MKLOW and MUHIGH promotes hyperactivity, while the exposure to either substance separately does not, is suggestive that the blockade of NMDAR and the overactivation of the GABAAR during the brain growth spurt have an additive effect. Accordingly, the simultaneous administration of an anesthetic cocktail containing a NMDAR antagonist and two GABAAR agonists in 7-day old rats promotes a more robust and widespread apoptotic response than that caused by each drug administered separately (Jevtovic-Todorovic et al., 2003). The administration of ethanol, which has both NMDA antagonistic and GABAA agonistic properties, to 7-day-old infant rats triggers a neurodegenerative response that is more robust than the response to MK801 or GABA mimetics alone (Ikonomidou et al., 2000). The additive detrimental effect of the combination of NMDAR blockade and overactivation of GABAAR may underlie the strong association between developmental exposure to ethanol and the expression of ADHD. Approximately 41% of children whose mothers drank alcoholic beverages during gestation have an ADHD diagnosis

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(Bhatara et al., 2006). In studies considering children with fetal alcohol syndrome (FAS), which represents the most severe outcome of prenatal ethanol exposure (Goodlett et al., 2005; Riley and McGee, 2005), this percentage ranges from 73% (Burd et al., 2003) to 95% (Fryer et al., 2007). Both the MKHIGH and MKLOW+MUHIGH groups presented high mortality rates. The majority of deaths observed in these groups occurred between injections suggesting an acute toxic effect of MK-801 and muscimol. It has been demonstrated that treatment with NMDA blockers and GABAA agonists can lead to respiratory depression in neonates (Fregosi et al., 2004; Poon et al., 2000), which in turn, is commonly associated with hypoxia, a lack of adequate oxygen supply that can cause death or severely affect brain development (Nyakas et al., 1996). Moreover, it is well known in humans that the number of miscarriages (Chiodo et al., 2012) as well as the severity of pathological phenotypes in the newborn (Riley and McGee, 2005) increase as the amount of ethanol consumed increases. Thus, it may be unclear as to whether the behavioral changes observed in the MKHIGH and MKLOW+MUHIGH groups were a result of the diminished health of the animal, a direct effect of the drug on the brain, or some combination of these factors. Studies that suggest that illness associated with systemic drug toxicity does not necessarily alter locomotor activity and that drugs can affect motor activity in the absence of systemic side effects (Cory-Slechta et al., 2001; Stanton, 1994). A poor health state or malaise is generally associated with motor incoordination, abnormal posture, tremors, motor stereotypy and decreased body mass in rodents (Cory-Slechta et al., 2001; Henck, 2002; Tilson, 1987). Despite a the significant weight reduction in the MKLOW+MUHIGH group, here we found: 1) no apparent abnormal movement or posture, tremor and stereotypic movements during animal care and handling; 2) locomotor hyperactivity in mice treated with 0.3 mg/kg of MK-801, a dose at which the mortality rate and body mass did not differ from that of the control group; and 3) that the body mass of the MKHIGH group, which presented a high mortality rate and locomotor hyperactivity, did not differ from controls. Our data showing that the number of pixels in the periphery was higher than that in the center of the arena corroborates the idea that mice avoid open areas (Prut and Belzung, 2003). This behavior has been largely used to assess anxiety in rodents. An increase in central locomotion or in the time spent in the central part of the arena is usually interpreted as an anxiolytic-like effect while the decrease of these measures is associated with an anxiogenic effect (Prut and Belzung, 2003). Interestingly, our data indicate that the combination of the high dose of MU and the low dose of MK has an anxiogenic effect only in females. Similarly, Sprague–Dawley adult females, but not males, prenatally exposed to ethanol presented an increase in anxiety-like behaviors as compared to controls (Osborn et al., 1998; Vaglenova et al., 2008). A gender difference was also observed in humans prenatally exposed to ethanol, with females showing higher rates of anxiety than males (Famy et al., 1998). Sex differences in anxiety-like behaviors have been associated with changes in the hypothalamic–pituitary–adrenal axis activity, as evidenced by increased corticosterone levels after elevated plus maze or open field testing of prenatal alcohol-exposed females, but not males (Hellemans et al., 2008; Osborn et al., 1998). 5. Conclusions Although both GABA and NMDA neurotransmitter systems appear to play important roles in activity-dependent mechanisms of synaptogenesis as well as in the establishment of neural circuitry, the blockade of NMDAR during the brain growth spurt was more effective in eliciting juvenile locomotor hyperactivity than the overactivation of GABAAR. These results suggest that, at least during the brain growth spurt, the reduction of NMDA-mediated transmission can play a significant role in the etiology of locomotor hyperactivity. The fact that the combined exposure to the highest dose of MU and the lowest dose of MK, which did not affect locomotor activity when administered separately, induced a

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marked hyperactivity suggest that GABAAR overactivation may potentiate the actions of NMDAR blockade during the brain growth spurt. Taken together, these results may have important implications since several xenobiotics and pharmacological agents utilized frequently in obstetric and pediatric medicine exhibit NMDA antagonist and/or GABAA mimetic properties. Financial support This work was supported by grants from Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro — FAPERJ (# E-26/111.640/2010 and E-26/102.288/2013); Conselho Nacional de Desenvolvimento Científico e Tecnológico — CNPq (# 308040/2009-0 and # 306594/2012-9); and Sub-reitoria de Pós-graduação e Pesquisa da Universidade do Estado do Rio de Janeiro (SR2-UERJ). Conflicts of interest The authors report no conflicts of interest. Acknowledgment The authors are thankful to Ulisses Rizzo for animal care and to the Instituto Vital Brazil (Rio de Janeiro, RJ, Brazil) for the donation of the original breeding stock of Swiss mice. References Arnsten, A.F., 2011. Catecholamine influences on dorsolateral prefrontal cortical networks. Biol. Psychiatry 69, e89–e99. Arnsten, A.F., Pliszka, S.R., 2011. Catecholamine influences on prefrontal cortical function: relevance to treatment of attention deficit/hyperactivity disorder and related disorders. Pharmacol. Biochem. Behav. 99, 211–216. Aumann, T., Horne, M., 2012. Activity-dependent regulation of the dopamine phenotype in substantia nigra neurons. J. Neurochem. 121, 497–515. Bandeira, F., Lent, R., Herculano-Houzel, S., 2009. Changing numbers of neuronal and nonneuronal cells underlie postnatal brain growth in the rat. Proc. Natl. Acad. Sci. U. S. A. 106, 14108–14113. Ben-Ari, Y., Khazipov, R., Leinekugel, X., Caillard, O., Gaiarsa, J.L., 1997. GABAA, NMDA and AMPA receptors: a developmentally regulated ‘menage a trois’. Trends Neurosci. 20, 523–529. Ben-Ari, Y., Gaiarsa, J.L., Tyzio, R., Khazipov, R., 2007. GABA: a pioneer transmitter that excites immature neurons and generates primitive oscillations. Physiol. Rev. 87, 1215–1284. Bhatara, V., Loudenberg, R., Ellis, R., 2006. Association of attention deficit hyperactivity disorder and gestational alcohol exposure: an exploratory study. J. Atten. Disord. 9, 515–522. Boctor, S.Y., Ferguson, S.A., 2010. Altered adult locomotor activity in rats from phencyclidine treatment on postnatal days 7, 9 and 11, but not repeated ketamine treatment on postnatal day 7. Neurotoxicology 31, 42–54. Brazel, C.Y., Romanko, M.J., Rothstein, R.P., Levison, S.W., 2003. Roles of the mammalian subventricular zone in brain development. Prog. Neurobiol. 69, 49–69. Burd, L., Klug, M.G., Martsolf, J.T., Kerbeshian, J., 2003. Fetal alcohol syndrome: neuropsychiatric phenomics. Neurotoxicol. Teratol. 25, 697–705. Casanova, M.F., 2014. Autism as a sequence: from heterochronic germinal cell divisions to abnormalities of cell migration and cortical dysplasias. Med. Hypotheses 83, 32–38. Centers for Disease Control and Prevention—CDC, 2010. Increasing prevalence of parentreported attention-deficit/hyperactivity disorder among children — United States, 2003 and 2007. MMWR Morb. Mortal. Wkly Rep. 59, 1439–1443. Chang, Z., Lichtenstein, P., Asherson, P.J., Larsson, H., 2013. Developmental twin study of attention problems: high heritabilities throughout development. JAMA Psychiatry 70, 311–318. Chiodo, L.M., Bailey, B.A., Sokol, R.J., Janisse, J., Delaney-Black, V., Hannigan, J.H., 2012. Recognized spontaneous abortion in mid-pregnancy and patterns of pregnancy alcohol use. Alcohol 46, 261–267. Cory-Slechta, D.A., Crofton, K.M., Foran, J.A., Ross, J.F., Sheets, L.P., Weiss, B., et al., 2001. Methods to identify and characterize developmental neurotoxicity for human health risk assessment. I: behavioral effects. Environ. Health Perspect. 109 (Suppl. 1), 79–91. Dehorter, N., Vinay, L., Hammond, C., Ben-Ari, Y., 2012. Timing of developmental sequences in different brain structures: physiological and pathological implications. Eur. J. Neurosci. 35, 1846–1856. Delcour, M., Russier, M., Amin, M., Baud, O., Paban, V., Barbe, M.F., et al., 2012. Impact of prenatal ischemia on behavior, cognitive abilities and neuroanatomy in adult rats with white matter damage. Behav. Brain Res. 232, 233–244. DiMaggio, C., Sun, L.S., Li, G., 2011. Early childhood exposure to anesthesia and risk of developmental and behavioral disorders in a sibling birth cohort. Anesth. Analg. 113, 1143–1151.

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