- Email: [email protected]
Maternal exercise during pregnancy ameliorates the postnatal neuronal impairments induced by prenatal restraint stress in mice
Int. J. Devl Neuroscience 31 (2013) 267–273
Contents lists available at SciVerse ScienceDirect
International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Maternal exercise during pregnancy ameliorates the postnatal neuronal impairments induced by prenatal restraint stress in mice Carlos Bustamante ∗ , Ricardo Henríquez, Felipe Medina, Carmen Reinoso, Ronald Vargas, Rodrigo Pascual Laboratorio de Neurociencias, Escuela de Kinesiología, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, Chile
a r t i c l e
i n f o
Article history: Received 12 November 2012 Received in revised form 18 February 2013 Accepted 18 February 2013 Keywords: Prenatal stress Maternal voluntary wheel running Apical dendrites Parietal cortex Locomotor behavior
a b s t r a c t Clinical and preclinical studies have demonstrated that prenatal stress (PS) induces neuronal and behavioral disturbances in the offspring. In the present study, we determined whether maternal voluntary wheel running (VWR) during pregnancy could reverse the putative deleterious effects of PS on the neurodevelopment and behavior of the offspring. Pregnant CF-1 mice were randomly assigned to control, restraint stressed or restraint stressed + VWR groups. Dams of the stressed group were subjected to restraint stress between gestational days 14 and delivery, while control pregnant dams remained undisturbed in their home cages. Dams of the restraint stressed + VWR group were subjected to exercise between gestational days 1 and 17. On postnatal day 23 (P23), male pups were assigned to one of the following experimental groups: mice born from control dams, stressed dams or stressed + VWR dams. Locomotor behavior and pyramidal neuronal morphology were evaluated at P23. Animals were then sacrificed, and Golgi-impregnated pyramidal neurons of the parietal cortex were morphometrically analyzed. Here, we present two major findings: first, PS produced significantly diminished dendritic growth of parietal neurons without altered locomotor behavior of the offspring; and second, maternal VWR significantly offset morphological impairments. © 2013 ISDN. Published by Elsevier Ltd. All rights reserved.
Clinical studies have demonstrated that maternal stress during pregnancy is associated with physiological (Tollenaar et al., 2011) and behavioral (de Weerth et al., 2003) disturbances in the offspring. For example, it has been demonstrated that children born from mothers that have suffered from emotional adversity in late pregnancy demonstrate behavioral alterations such as hyperactivity disorder (O’Connor et al., 2002; Huizink et al., 2003). Consistent with these clinical findings, experimental studies have revealed links between prenatal stress (PS) and behavioral disturbances in juvenile offspring, such as increased locomotor activity (Emack and Matthews, 2011) and anxiety-like behaviors (Miyagawa et al., 2011). These abnormalities are likely due to morphological and functional changes in key areas of the brain that control motor and emotional behaviors, such as the prefrontal cortex (Muhammad et al., 2012) and amygdala (Salm et al., 2004; Kraszpulski et al., 2006). In this regard, it has been demonstrated that prenatally stressed animals show altered dendritic outgrowth of pyramidal neurons of the cingulate anterior area of the PFC (Murmu et al.,
∗ Corresponding author at: Escuela de Kinesiología, Pontificia Universidad Católica de Valparaíso, Avenida Universidad 330, Curauma, Valparaiso, Chile. Tel.: +56 32 2274022; fax: +56 32 2274044. E-mail address: [email protected] (C. Bustamante). 0736-5748/$36.00 © 2013 ISDN. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijdevneu.2013.02.007
2006). These findings are not surprising because the PFC is a highly plastic brain area in which neuronal maturation occurs primarily over a critical period in postnatal life (Altman and Bayer, 1990; Kolb, 1976). In addition, the parietal cortex has also demonstrated plastic changes associated with early life stress. Bock et al. (2005) found that the pups of rats subjected the stress of maternal separation during the pre-weaning period exhibit significant changes in the morphology of pyramidal neurons in the somatosensory region of the brain. However, at present, it is still unclear whether pyramidal neurons from this cortical area are vulnerable to the effects of stressful experiences suffered during prenatal life. Thus, our first aim was to determine the effects of PS on the apical dendritic outgrowth of pyramidal neurons in layer II/III of the parietal cortex in offspring evaluated during the post-weaning period (P23). We selected the apical domain of pyramidal neurons for two reasons: first, it has previously been reported that this dendritic domain is more vulnerable to the effects of PS than the basal domain (Murmu et al., 2006; Cerqueira et al., 2007); and second, because the apical dendrites of layer II/III are the targets of the feedback projections coming from the same and other cortical areas (Spratling, 2002). Because the mice exhibit dense topographically organized projections from the somatosensory cortex to the motor cortex (Bayer and Altman, 1991; Porter, 1996), we evaluated their locomotor behaviors using an open field (OF) test, a test that is widely used to
268
C. Bustamante et al. / Int. J. Devl Neuroscience 31 (2013) 267–273
assess locomotor and exploratory behaviors in rodents (Walsh and Cummings, 1976). On the other hand, it has been shown that exercise during pregnancy may prevent or limit adverse maternal and fetal morbidities (Gavard and Artal, 2008; Weissgerber et al., 2006). For instance, controlled exercise in pregnant women results in beneficial effects on their blood pressure parameters (Yeo et al., 2000) and prevents maternal–fetal diseases, such as type 2 diabetes (Dempsey et al., 2004) and preeclampsia (Weissgerber et al., 2004). Thus, several putative mechanisms that may account for the beneficial effects of exercise on maternal/fetal health have been proposed, including the enhancement of placental growth and vascularity, the reduction of oxidative stress and amelioration of endothelial dysfunction (Weissgerber et al., 2004; Sankaralingam et al., 2011). Furthermore, it has been shown that the children born from mothers who exercised regularly during pregnancy exhibit enhanced oral language scores at the age of five compared with control groups (Clapp, 1996). At present, the mechanisms that account for these cognitive effects are still largely unknown. Interestingly, these findings are, at least partially, similar to the changes observed in rodent studies. Moreover, maternal exercise during pregnancy has been associated with improvements in the spatial learning and short-term memory of juvenile offspring (Parnpiansil et al., 2003; Kim et al., 2007). These changes appear to be related to modifications in the structure and function of specific hippocampal regions, as shown by studies demonstrating that the offspring born from dams subjected to exercise during pregnancy show enhanced hippocampal neurogenesis (Lee et al., 2006). Furthermore, it has been hypothesized that enhancement of the expression of neurotrophins, such as brain-derived neurotrophic factor (BDNF), could, at least partially explain these beneficial effects (Kim et al., 2007). Based on these findings of the effects of maternal exercise, the second aim of this study was to determine whether maternal wheel running exercise during pregnancy ameliorates or reverses the putative deleterious effects of PS on the dendritic morphology of pyramidal neurons in layer II/III of the parietal cortex and locomotor behavior in prenatally stressed mice. Considering the above-mentioned aims, two hypotheses were tested: (i) PS induces a significant decrement in apical dendritic outgrowth of the pyramidal neurons concomitant with an altered locomotor activity in the prenatally stressed mice; and (ii) maternal exercise during pregnancy ameliorates the deleterious effects associated with PS. 1. Experimental procedures 1.1. Animals and experimental conditions Twenty-four virgin female mice (CF-1) were mated with twenty-four adult male mice and housed for 1 day under standard laboratory conditions; i.e., a 12-h inverted light–dark cycle (lights on at 11:00 p.m.), 18 ± 2 ◦ C, and food and water ad libitum (one male/one female per cage). The day of mating was considered “day zero of pregnancy” (G0), and this assumption was corroborated by the following observations: (i) the daily weight gain of the pregnant dams and (ii) the length of gestation. A cohort of ten female mice fulfilled the above-mentioned criteria and were included in the current study. All of the pregnant females were socially housed in transparent Plexiglas cages (30 cm × 19 cm × 13 cm; 2–3 dams per cage) with sawdust bedding and were randomly assigned to the one of the following three experimental groups: control (C, n = 3), restraint stressed (RS, n = 3) or RS + VWR (n = 4). All pregnant dams remained socially housed until the day before parturition. 1.2. Prenatal stress procedure The restraint stress procedure was performed according to a previously described protocol (Bustamante et al., 2010). Briefly, from gestational day 14 (G14) until delivery (see Fig. 1B), the pregnant females were subjected to three daily stress sessions at 9.00 a.m., 2.00 p.m. and 6.00 p.m. during which they were placed in plastic cylinders (11 cm long and 4 cm diameter) for 45 min. The control pregnant females were left undisturbed in their home cages (see Fig. 1A) and were handled only when the cages of all groups (C, RS and RS + VWR) were cleaned three times a week.
Fig. 1. Timeline showing a summary of the experimental design. G: gestational age; P: postnatal age (days). RS: restraint stress; VWR: voluntary wheel running.
1.3. Voluntary wheel running (VWR) protocol Between G1 and G17, each female mouse from the RS + VWR group was subjected to the VWR protocol (see Fig. 1C) during the dark phase for 4 h per day (2.00–6.00 p.m.) through housing in a cage that contained a running wheel (diameter, 14 cm; width, 6 cm). Under this condition, the mice had free access to voluntary exercise and food and water ad libitum. The number of daily wheel turns was registered by a magnetic sensor that was placed on the roof of the cage and connected to a computer. After weaning (P23), male pups only were randomly selected from each of the following three groups (to prevent possible litter effects, we used only 1–2 pups/litter/experiment): (i) mice born from a control mother (C), (ii) mice born from a stressed mother (RS) and (iii) mice born from a stressed mother subjected to VWR (RS + VWR). 1.4. Open field (OF) test On P23, the male mice from each group (C, n = 5; RS, n = 5 and RS + VWR, n = 5) were evaluated using the OF test (Walsh and Cummings, 1976). Briefly, this test was conducted in a square arena (40 cm × 40 cm) that was enclosed by continuous 21 cmhigh walls made of black wood. The arena was divided by Anymaze software into 25 equal sized squares containing one central zone (8 cm × 8 cm) and 24 surrounding squares (peripheral zone). The animals were examined during their dark cycle (between 2.00 and 6.00 p.m.), and testing was conducted under dim light (approximately 80 lx). Each mouse was placed in the center of the OF and allowed to freely explore the arena for 90 s. The animals’ behaviors were recorded using a camera that was located 40 cm above the apparatus (Logitech Quick cam version 9.5.0), and the data were analyzed using the Anymaze software. The number of lines crossed by each animal (with all four limbs), the total distance traversed and the total time spent in the central zone (a square of 8 cm × 8 cm in area) of the apparatus within 90 s were quantified. The open field apparatus was wiped between each test with a clean alcohol-dipped cloth to eliminate any olfactory clues left by previously tested animals. 1.5. Histological procedures and dendritic analysis To study the effect of prenatal stress and the potential “therapeutic” effects of VWR on the dendritic outgrowth of pyramidal neurons, mice were sacrificed under deep ether anesthesia, and their brains were immediately dissected out, fixed and stained with the Golgi-Cox-Sholl procedure (Sholl, 1953). After 45 days of slow metallic mercury impregnation, the brains were dehydrated in a graded alcohol series (25%–50%–75%–90%–95%–100%, v/v; Merck) and fixed in Paraplast. Coronal sections (thickness, 120 m) were cut using a sledge microtome, re-hydrated by in a graded alcohol series (100%–95%–90%–75%–50%–25%, v/v; Merck), treated with potassium disulfide/oxalic acid (5% dilution; Merck) and coverslipped. All slides were coded to avoid experimental bias and to maximize reliability. To qualify for dendritic morphometric evaluation, the pyramidal neurons were required to fulfill the following criteria: (a) have a well-defined somata shape; (b) show adequate staining of the soma and dendrites; (c) exhibit uninterrupted apical dendritic processes; (d) have no extensive dendrites that overlapped with neighboring neurons; and (e) be localized to layer II/III of the parietal cortex (approximately 150–350 m from the pial surface), which was carefully delimited by coordinates described in the Rat Stereotaxic Atlas (Paxinos and Watson, 1998). A total of 299 pyramidal neurons cells (approximately 20 neurons/animal) were analyzed using an Olympus CX-3 light microscope (400×). The neurons were imaged using a digital camera (5.0 CCD Camera) and analyzed using Micrometrics SE Premium V-2.8 software, which measured the following: (i) the total apical dendritic length per neuron (m) and (ii) the number of apical branches per neuron. At respect, the branching was determined
C. Bustamante et al. / Int. J. Devl Neuroscience 31 (2013) 267–273
Fig. 2. (A) Body weight gains of control (C), prenatal stress (RS) and prenatal stress + voluntary wheel running (RS + VWR) dams between G1 until G21. (B) Kilometers ran per session by RS + VWR dams between G1 until G17. Data are presented as the means ± S.E.M.
by counting the total number of dendritic branch points emerging from the main trunk of the apical dendrite. All behavioral and anatomical data were obtained from the same animals. All experimental procedures were performed in accordance with protocols that were approved by the Bioethics Committee of the Pontificia Universidad Católica de Valparaíso and international guidelines. This study was conducted using a minimal number of animals (5 for each group). 1.6. Statistical analysis Total dendritic length/neuron, number of branching dendrites, behavioral and reproductive/developmental data were analyzed using the one way analysis of variance (ANOVA) followed by Tukey’s HSD post hoc test (STATA 9.1 software). The results are expressed as the mean ± S.E.M. values. p < 0.05 was considered statistically significant.
2. Results As expected, the mice subjected to prenatal stress showed a significant decrease in the total apical dendritic length of the pyramidal neurons compared to the control group (a p < 0.05, F(2, 296) ; Fig. 3A). Furthermore, RS animals showed a significant reduction in the number of dendritic branches compared to age-matched controls (a p < 0.05, F(2, 296) ; Fig. 3B). In contrast, prenatally stressed mice subjected to maternal exercise during pregnancy showed a significant increase in both the total apical dendritic length and the number of dendritic branches (b p < 0.05, F(2, 296) ; Fig. 3A and B) compared to the RS group, suggesting that maternal exercise induced a significant recovery effect. Representative microphotographs of the pyramidal neurons in C, RS and RS + VWR animals are shown in Fig. 5. Importantly, neither the PS nor the VWR protocol affected the following: (i) the length of the gestation, (ii) the litter size, (iii) the male/female ratio, or (iv) the body weight gains of the pups during lactation (see Table 1). The RS mice showed a significant decrease in body weight compared with the C group at the age of P23 (a p < 0.05, F(2, 12) ; Table 1). The body weight gains of the three groups of the pregnant dams during pregnancy did not show significant differences (Fig. 2A). The pregnant dams subjected to VWR ran 2.79 ± 0.17 km with a minimum of 1.57 ± 0.17 km (G17) and a maximum of 3.65 ± 0.21 km (G10). The average kilometers ran
269
Fig. 3. Effect of prenatal stress and maternal voluntary wheel running on total apical dendritic length/per neuron (A) and total number of dendritic branching/per neuron (B) from selected pyramidal neurons. Data are presented as the means ± S.E.M. C: control group (n = 5); RS: prenatal stress group (n = 5); RS + VWR: prenatal stress + maternal exercise group (n = 5); a p < 0.05 compared with C group; b p < 0.05 compared with RS + VWR group (ANOVA test).
per session (4 h daily) for the pregnant dams (RS + VWR group) are shown in Fig. 2B. Contrary to expected, no significant differences were found in locomotor parameters between the C and RS group. Prenatally stressed mice exhibited an increase in the number of lines crossed and in the total distance traversed in the OF test compared to agematched control mice (Fig. 4A and B), but this difference was not significant. In addition, although the RS animals spent less total time in the central zone of the apparatus compared to the C group, this difference was not significant (Fig. 4C). 3. Discussion Although PS has been shown to induce a variety of deleterious effects in behavioral and neuronal function, knowledge of the underlying cytoarchitectural alterations and how these PS-induced changes may be prevented is still relatively sparse. We addressed these issues in the present study, which resulted in two central findings: first, exposure of pregnant dams to restraint stress during the third week of pregnancy induced morphological changes in the cytoarchitecture of apical dendrites in pyramidal neurons of the parietal cortex without significant disturbances in the locomotor behavior of their offspring at the post-weaning period; second, these PS-induced neuronal alterations were ameliorated by maternal VWR during pregnancy. In the current study, prenatally stressed mice exhibited a significant diminishment in dendritic length and branching of the apical dendrites in layer II/III pyramidal neurons, consistent with data reported by Jia et al. (2010) that showed that the total length of the apical dendrites of pyramidal neurons in hippocampal area CA3 are significantly shorter in prenatally stressed rats compared to control animals. Moreover, our morphological data are consistent with the results of Murmu et al. (2006), who demonstrated that maternal stress during the prenatal period is associated with a
270
C. Bustamante et al. / Int. J. Devl Neuroscience 31 (2013) 267–273
Table 1 Values of some reproductive and developmental parameters (mean ± S.E.M.).
C RS RS + VWR
Length of gestation
Litter size
Male/female ratio
Body weight (P1)
Body weight (P7)
Body weight (P14)
Body weight (P23)
20.7 ± 0.3 20.3 ± 0.3 20.3 ± 0.3
11.7 ± 1 13.0 ± 1.2 12.0 ± 0.3
0.5 ± 0.07 0.7 ± 0.05 0.6 ± 0.07
2 ± 0.9 1.7 ± 0.7 1.9 ± 0.6
3.4 ± 0.7 3 ± 0.6 3.3 ± 0.6
7 ± 0.4 5.7 ± 0.3 7.2 ± 1
12.7 ± 1.7 9.4 ± 0.7a 10 ± 0.9
Values of some reproductive and developmental parameters. The results are displayed as the mean ± S.E.M. for: (i) mice born from a control mother (C), (ii) mice born from a stressed mother (RS) and (iii) mice born from a stressed mother subjected to VWR (RS + VWR). The body weights at P1 considered the whole litter (male and female pups). The body weight at P7, P14 and P23 considered the body weights of male mice only. a p < 0.05 compared to C group (ANOVA test).
Fig. 4. Effect of prenatal stress and maternal voluntary wheel running on the number of crossed lines (A), total distance traversed (B) and time in central zone in the open field test (C). Data are expressed as the means ± S.E.M. C: control group (n = 5); RS: prenatal stress group (n = 5); RS + VWR: prenatal stress + maternal exercise group (n = 5).
significant dendritic retraction or remodeling of pyramidal neurons in layer II/III of the anterior cingulate cortex in offspring evaluated during the post-weaning period. In this study, pregnant female mice were subjected to restraint stress three times a day from G14 until delivery; this method is commonly employed to induce an increase in maternal plasma corticosterone (CORT) levels (Barros et al., 2006). The dendritic impairment exhibited by the prenatally stressed mice may be, at least partially, due to the toxic effects of excess circulating glucocorticoids (GCs) on neuronal development (Takahashi, 1998; Seckl and Meaney, 2004). Many studies in rodents have demonstrated that pups born from pregnant rats subjected to stress during the last week of pregnancy show alterations in the circadian rhythm of pituitary–adrenocortical activity during the postnatal period (Koehl et al., 1999). Similarly, it has been demonstrated that PS is associated with a prolonged HPAaxis response in the offspring (Koenig et al., 2005) and unregulated neurotransmission in noradrenergic, serotoninergic and dopaminergic systems (Takahashi et al., 1992; Hayashi et al., 1998; Berger et al., 2002; Miyagawa et al., 2011; Wyrwoll and Holmes, 2012). Several mechanisms have been proposed to contribute to the neuronal damage associated with chronic hypercortisolemia. First, an excess of GCs is associated with disturbances in glucose utilization, which compromises the energy resources of the cells (Horner et al., 1990). Second, GCs inhibit glutamate uptake by glial cells (Virgin et al., 1991), resulting in a persistent influx of Ca2+ , which results in neurotoxicity and neuronal death (Ankarcrona et al., 1995). Third, it has been demonstrated that hypercortisolemia mediates stress-induced decreases in neurotrophins, particularly BDNF, in many brain areas (Smith et al., 1995; Uysal et al., 2011); BDNF is a well-known factor associated with neuronal growth and branching (Murakami et al., 2005). Fourth, it has been demonstrated that PS is associated with impaired MAP-2 (microtubule-associated protein 2) synthesis in the brain (Barros et al., 2006) and thus, decreases in MAP-2 levels in developing stressed mice could potentially impair
Fig. 5. Representative photomicrograph of Golgi-Cox-stained pyramidal neurons from control (A), prenatal stress (B) and prenatal stress + maternal exercise (C) groups. Bar: 20 m.
C. Bustamante et al. / Int. J. Devl Neuroscience 31 (2013) 267–273
synaptogenesis and neurite outgrowth. This impairment may be directly associated with the reduced dendritic arborization in the apical domain observed in this study. Importantly, the PS protocol was applied during a time window when parietal neurons are still very immature. Thus, another potential mechanism that may contribute to the altered dendritic morphology exhibited by the prenatally stressed animals may involve PS-induced delays of dendritic and synaptic development, which may already be morphologically detectable at early post-weaning ages. It is well known that, during development, the dendritic structure of parietal neurons, at least partially, depends on the appropriate establishment of highly specific inputs from afferent thalamic fibers (Wise et al., 1979), and thus the effects of PS may potentially alter patterns of thalamocortical connectivity. In addition, it is important to note that aside from having a short-term effect during intrauterine life, PS might render many structures vulnerable to subsequent brain challenges (Diaz et al., 1997). Thus, it has been suggested that PS might affect regressive events such as dendritic and synaptic pruning, which normally occur during the post-weaning period (Huttenlocher, 1979). Moreover, as our prenatally stressed animals were raised by their biological mothers, another important variable that may account for the reported dendritic changes is the quality of preweaning mother–pup interactions. In this regard, it has been demonstrated that stressful experiences during pregnancy affect mother–offspring relationships (Moore and Power, 1986), which alter postpartum maternal care (Champagne and Meaney, 2006) and result in reductions in maternal attention (Maccari et al., 1995). In this regard, it has been shown that the deleterious effect of prenatal stress on the activity of the HPA axis may be minimized when the animals are reared by non-stressed mothers (Maccari et al., 1995). To address this interesting question, further studies should analyze the impact of PS on neuronal development in mice reared by either stressed or control surrogate mothers (i.e., utilizing cross-fostering procedures). On the basis of these aforementioned findings, it is possible that disturbances in the dendritic morphology observed in the RS group might be related not only to prenatal events but also to differential maternal care given to the offspring by stressed dams during the lactation period (Yaka et al., 2007). Furthermore, Burton et al. (2007) postulated that prenatal stress provides the background for subsequent postnatal environmental effects such as the diminishment of somatosensory/affective stimulation associated with maternal neglect. Importantly, neither the PS nor the VWR protocols affected the following: (i) the duration of the pregnancy, (ii) the body weight gains in the three groups of the pregnant dams, and (iii) the size of the litters and the body weight gains of the pups during lactation. In addition, litter compositions were not significantly different between the three groups. The only difference observed was that the RS mice showed a significant diminishment in body weight compared with the control group at P23. It is important to note that body weight outcomes related to maternal stress during pregnancy remain controversial because some studies have reported enhanced body weights in prenatally stressed rats during adulthood (Schulz et al., 2011), but other reports have found significant decreases in fetal body weight throughout development in prenatally stressed rats and decreased body weights and adiposity in mice (Pankevich et al., 2009). In the current study, the prenatally stressed mice showed a significant reduction in body weight compared with the control group evaluated at P23. Our results are similar to those found by Aziz et al. (2012) in mice exposed to prenatal stress and evaluated in the early postweaning period. In this regard, it has been suggested that altered weight gain in prenatally stressed mice could be related to long-term impacts of PS on feeding behavior and energy metabolism in the offspring (Pankevich et al., 2009). Additionally, it is possible that this reduction in body weight gain in the PS group may be associated with altered
271
lactation and maternal neglect during the preweaning period (Yaka et al., 2007). Regarding of the results in OF test and contrary to expected, mice subjected to PS did not exhibit altered locomotor behavior compared to the control animals. The behavioral data reported here are in agreement with the study of Green et al. (2011) that showed that prenatally stressed rats exhibit no differences in the number of line crossings or time spent in the central zone of the OF compared to control rats. However, some studies have reported contradictory results. On one hand, Deminière et al. (1992) found that prenatally stressed rats showed hyperactive-like behaviors in the OF; similar results have been reported by Burton et al. (2006) and Martínez-Téllez et al. (2009). On the other hand, some reports have shown decrements in exploratory behavior exhibited by prenatally stressed mice in the OF (Sternberg and Ridgway, 2003; Miyagawa et al., 2011). These inconsistencies may be due to variability in the paradigms used in different laboratories that include variability in the rodent strain used, types of stressors, periods or durations of stressors, methods of measuring locomotion and the postnatal period in which the locomotor behavior was evaluated. Another important factor to consider is the duration of the test; in this respect, in our study the mice were evaluated over 90 s. In many studies locomotor behavior of prenatally stressed animals has been evaluated in over ranges of 5 min (Ordyan and Pivina, 2004) or more (Peters, 1986). All these factors may affect locomotor behavior, and for that reason, we do not rule out the possibility that altered behavior may have been apparent if the animals were evaluated in a longer test. On the other hand, our data demonstrate that maternal exercise during pregnancy significantly reversed the deleterious effects of PS on neuronal development because prenatally stressed animals born from dams subjected to VWR during pregnancy showed a significant increase in apical dendritic length and branching of pyramidal neurons in the parietal cortex compared to the nonexercised stressed mice. Moreover, these mice showed no signs of hyperactivity/anxiety. The fact that VWR significantly ameliorated or reversed the deleterious effects associated with PS confirms the widespread beneficial effects of maternal exercise on offspring development (Rosa et al., 2011). These findings are similar to our previous results (Bustamante et al., 2010) that demonstrated that voluntary exercise significantly benefits the neurobehavioral development of prenatally stressed mice. To our knowledge, this is the first report to analyze the effects of maternal exercise during pregnancy on the dendritic morphology of parietal neurons in prenatally stressed animals. The mechanism controlling the reported dendritic changes is, at present, unknown; however, we propose several hypotheses. First, the expression of several neurotrophins, including nerve growth factor (NGF), fibroblast growth factor 2 (FGF-2), insulin-like growth factor-1 (IGF-1) and BDNF, are increased in the rodent brain after a few days of exercise (Oliff et al., 1998; Vaynman et al., 2004; Berchtold et al., 2005; Ding et al., 2006; Uysal et al., 2011). Recently, Aksu et al. (2012) found that maternal treadmill exercise during pregnancy is associated with an increase in vascular endothelial growth factor (VEGF) and BDNF levels in the postnatal rat cortex. Similarly, Parnpiansil et al. (2003) found that maternal exercise increased hippocampal BDNF expression and neurogenesis in postnatal rat brains. The exerciseinduced up-regulation of BDNF expression has been proposed to correlate with increases in dendritic complexity in several brain areas, including the hippocampus (Redila and Christie, 2006). Thus, VWR may potentially, at least partially, counteract the deleterious effects of PS on neurotrophin expression in the brain and thus enhance dendritic development in pyramidal neurons of the parietal cortex. Second, it has been demonstrated that maternal stress is associated with placental vasoconstriction via abnormal augmentation in maternal catecholamine levels; these phenomena may
272
C. Bustamante et al. / Int. J. Devl Neuroscience 31 (2013) 267–273
lead to fetal hypoxia, which would enhance fetal HPA-axis reactivity (Matthews and Phillips, 2010). In contrast, it was recently demonstrated that prenatal maternal exercise protects postnatal rat brains from hypoxia via increases in blood vessel density in the fetal brain (Akhavan et al., 2012). Thus, it is feasible that VWR may counteract the constriction of placental blood vessels associated with increased maternal catecholamine levels. Finally, VWR may affect the maternal HPA-axis itself by contributing to the regulation of stress responses of the dams during gestation. Preclinical studies have demonstrated that exercise in the form of wheel running is associated with a number of adaptive behavioral and physiological effects, including a reduction in stress-associated behaviors, neurogenesis, angiogenesis and increases in neurotrophic factors (Salam et al., 2009; Duman et al., 2008). We believe that all of these effects on the exercised dams are clinically relevant because it has been postulated that maternal behavior serves to “program” hypothalamic-pituitary-adrenal responses to stress in offspring (Liu et al., 1997). These putative mechanisms may contribute to explain the changes observed in dendritic development in the RS + VWR group and could potentially be related to the neuroendocrine and structural changes associated with prenatal maternal exercise. In summary, PS significantly altered dendritic development during the post-weaning period in offspring but did not produce significant alterations in locomotor behavior. This neuronal impairment was rescued to a remarkable degree by maternal exercise during gestation. These results confirm the beneficial effects of VWR during pregnancy as a preventive agent against chronic stressful events. Acknowledgment This research was supported by Grant PUCV – VRIEA 127.705/2008. References Aksu, I., Baykara, B., Ozbal, S., Cetin, F., Sisman, A.R., Dayi, A., Gencoglu, C., Tas, A., Büyük, E., Gonenc-Arda, S., Uysal, N., 2012. Maternal treadmill exercise during pregnancy decreases anxiety and increases prefrontal cortex VEGF and BDNF levels of rat pups in early and late periods of life. Neuroscience Letters 516, 221–225. Altman, J., Bayer, S.A., 1990. Migration and distribution of two populations of hippocampal granule cell precursors during the perinatal and postnatal periods. Journal of Comparative Neurology 301, 365–381. Ankarcrona, M., Dypbukt, J.M., Bonfoco, E., Zhivotovsky, B., Orrenius, S., Lipton, S.A., Nicotera, P., 1995. Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 15, 961–973. Akhavan, M.M., Foroutan, T., Safari, M., Sadighi-Moghaddam, B., Emami-Abarghoie, M., Rashidy-Pour, A., 2012. Prenatal exposure to maternal voluntary exercise during pregnancy provides protection against mild chronic postnatal hypoxia in rat offspring. Pakistan Journal of Pharmaceutical Sciences 25, 233–238. Aziz, N.H.K.A., Kendall, D.A., Pardon, M.C., 2012. Prenatal exposure to chronic mild stress increases corticosterone levels in the amniotic fluid and induces cognitive deficits in female offspring, improved by treatment with the antidepressant drug amitriptyline. Behavioural Brain Research 231, 29–39. Barros, V.G., Duhalde-Vega, M., Caltana, L., Brusco, A., Antonelli, M.C., 2006. Astrocyte-neuron vulnerability to prenatal stress in the adult rat brain. Journal of Neuroscience Research 83, 787–800. Bayer, S.A., Altman, J., 1991. Neocortical Development. Raven Press, New York. Berchtold, N.C., Chinn, G., Chou, M., Kesslak, J.P., Cotman, C.W., 2005. Exercise primes a molecular memory for brain-derived neurotrophic factor protein induction in the rat hippocampus. Neuroscience 133, 853–861. Berger, M.A., Barros, V.G., Sarchi, M.I., Tarazi, F.I., Antonelli, M.C., 2002. Long-term effects of prenatal stress on dopamine and glutamate receptors in adult rat brain. Neurochemical Research 27, 1525–1533. Bock, J., Gruss, M., Becker, S., Braun, K., 2005. Experience-induced changes of dendritic spine densities in the prefrontal and sensory cortex: correlation with developmental time windows. Cerebral Cortex 15, 802–808. Burton, C., Lovic, V., Fleming, A.S., 2006. Early adversity alters attention and locomotion in adult Sprague-Dawley rats. Behavioral Neuroscience 120, 665–675. Burton, C.L., Chatterjee, D., Chatterjee-Chakraborty, M., Lovic, V., Grella, S.L., Steiner, M., Fleming, A.S., 2007. Prenatal restraint stress and motherless rearing disrupts expression of plasticity markers and stress-induced corticosterone release in adult female Sprague-Dawley rats. Brain Research 1158, 28–38.
Bustamante, C., Bilbao, P., Contreras, W., Martínez, M., Mendoza, A., Reyes, A., Pascual, R., 2010. Effects of prenatal stress and exercise on dentate granule cells maturation and spatial memory in adolescent mice. International Journal of Developmental Neuroscience 28, 605–609. Cerqueira, J.J., Taipa, R., Uylings, H.B., Almeida, O.F., Sousa, N., 2007. Specific configuration of dendritic degeneration in pyramidal neurons of the medial prefrontal cortex induced by differing corticosteroid regimens. Cerebral Cortex 17, 1998–2006. Champagne, F.A., Meaney, M.J., 2006. Stress during gestation alters postpartum maternal care and the development of the offspring in a rodent model. Biological Psychiatry 59, 1227–1235. Clapp 3rd, J.F., 1996. Morphometric and neurodevelopmental outcome at age five years of the offspring of women who continued to exercise regularly throughout pregnancy. Journal of Pediatrics 129, 856–863. Deminière, J.M., Piazza, P.V., Guegan, G., Abrous, N., Maccari, S., Le Moal, M., Simon, H., 1992. Increased locomotor response to novelty and propensity to intravenous amphetamine self-administration in adult offspring of stressed mothers. Brain Research 586, 135–139. Dempsey, J.C., Butler, C.L., Sorensen, T.K., Lee, I.M., Thompson, M.L., Miller, R.S., Frederick, I.O., Williams, M.A., 2004. A case–control study of maternal recreational physical activity and risk of gestational diabetes mellitus. Diabetes Research and Clinical Practice 66, 203–215. de Weerth, C., van Hees, Y., Buitelaar, J.K., 2003. Prenatal maternal cortisol levels and infant behavior during the first 5 months. Early Human Development 74, 139–151. Diaz, R., Fuxe, K., Ogren, S.O., 1997. Prenatal corticosterone treatment induces longterm changes in spontaneous and apomorphine-mediated motor activity in male and female rats. Neuroscience 81, 129–140. Ding, Q., Vaynman, S., Akhavan, M., Ying, Z., Gomez-Pinilla, F., 2006. Insulin-like growth factor I interfaces with brain-derived neurotrophic factor-mediated synaptic plasticity to modulate aspects of exercise-induced cognitive function. Neuroscience 140, 823–833. Duman, C.H., Schlesinger, L., Russell, D.S., Duman, R.S., 2008. Voluntary exercise produces antidepressant and anxiolytic behavioral effects in mice. Brain Research 1199, 148–158. Emack, J., Matthews, S.G., 2011. Effects of chronic maternal stress on hypothalamopituitary-adrenal (HPA) function and behavior: no reversal by environmental enrichment. Hormones and Behavior 60, 589–598. Gavard, J.A., Artal, R., 2008. Effect of exercise on pregnancy outcome. Clinical Obstetrics and Gynecology 51, 467–480. ˜ A.E., Martinez, P.A., Frazer, A., Green, M.K., Rani, C.S., Joshi, A., Soto-Pina, Strong, R., Morilak, D.A., 2011. Prenatal stress induces long term stress vulnerability, compromising stress response systems in the brain and impairing extinction of conditioned fear after adult stress. Neuroscience 192, 438–451. Hayashi, A., Nagaoka, M., Yamada, K., Ichitani, Y., Miake, Y., Okado, N., 1998. Maternal stress induces synaptic loss and developmental disabilities of offspring. International Journal of Developmental Neuroscience 16, 209–216. Horner, H., Packan, D.A., Sapolsky, R., 1990. Glucocorticoids inhibit glucose transport in cultured hippocampal neurons and glia. Neuroendocrinology 52, 57–64. Huizink, A.C., Robles de Medina, P.G., Mulder, E.J., Visser, G.H., Buitelaar, J.K., 2003. Stress during pregnancy is associated with developmental outcome in infancy. Journal of Child Psychology and Psychiatry and Allied Disciplines 44, 810–818. Huttenlocher, P.R., 1979. Synaptic density in human frontal cortex—developmental changes and effects of aging. Brain Research 163, 195–205. Jia, N., Yang, K., Sun, Q., Cai, Q., Li, H., Cheng, D., Fan, X., Zhu, Z., 2010. Prenatal stress causes dendritic atrophy of pyramidal neurons in hippocampal CA3 region by glutamate in offspring rats. Developmental Neurobiology 70, 114–125. Kim, H., Lee, S.H., Kim, S.S., Yoo, J.H., Kim, C.J., 2007. The influence of maternal treadmill running during pregnancy on short-term memory and hippocampal cell survival in rat pups. International Journal of Developmental Neuroscience 25, 243–249. Koehl, M., Darnaudéry, M., Dulluc, J., Van Reeth, O., Le Moal, M., Maccari, S., 1999. Prenatal stress alters circadian activity of hypothalamo-pituitary-adrenal axis and hippocampal corticosteroid receptors in adult rats of both gender. Journal of Neurobiology 40, 302–315. Koenig, J.I., Elmer, G.I., Shepard, P.D., Lee, P.R., Mayo, C., Joy, B., Hercher, E., Brady, D.L., 2005. Prenatal exposure to a repeated variable stress paradigm elicits behavioral and neuroendocrinological changes in the adult offspring: potential relevance to schizophrenia. Behavioural Brain Research 156, 251–261. Kolb, B., 1976. Functional development of prefrontal cortex in rats continues into adolescence. Science 193, 335–336. Kraszpulski, M., Dickerson, P.A., Salm, A.K., 2006. Prenatal stress affects the developmental trajectory of the rat amygdala. Stress 9, 85–95. Lee, H.H., Kim, H., Lee, J.W., Kim, Y.S., Yang, H.Y., Chang, H.K., Lee, T.H., Shin, M.C., Lee, M.H., Shin, M.S., Park, S., Baek, S., Kim, C.J., 2006. Maternal swimming during pregnancy enhances short-term memory and neurogenesis in the hippocampus of rat pups. Brain and Development 28, 147–154. Liu, D., Diorio, J., Tannenbaum, B., Caldji, C., Francis, D., Freedman, A., Sharma, S., Pearson, D., Plotsky, P.M., Meaney, M.J., 1997. Maternal care, hippocampal glucocorticoid receptors, and hypothalamic-pituitary-adrenal responses to stress. Science 277, 1659–1662. Maccari, S., Piazza, P.V., Kabbaj, M., Barbazanges, A., Simon, H., Le Moal, M., 1995. Adoption reverses the long-term impairment in glucocorticoid feedback induced by prenatal stress. Journal of Neuroscience 15, 110–116.
C. Bustamante et al. / Int. J. Devl Neuroscience 31 (2013) 267–273 Martínez-Téllez, R.I., Hernández-Torres, E., Gamboa, C., Flores, G., 2009. Prenatal stress alters spine density and dendritic length of nucleus accumbens and hippocampus neurons in rat offspring. Synapse 63, 794–804. Matthews, S.G., Phillips, D.I., 2010. Minireview: transgenerational inheritance of the stress response: a new frontier in stress research. Endocrinology 151, 7–13. Miyagawa, K., Tsuji, M., Fujimori, K., Saito, Y., Takeda, H., 2011. Prenatal stress induces anxiety-like behavior together with the disruption of central serotonin neurons in mice. Neuroscience Research 70, 111–117. Moore, C.L., Power, K.L., 1986. Prenatal stress affects mother–infant interaction in Norway rats. Developmental Psychobiology 19, 235–245. Muhammad, A., Carroll, C., Kolb, B., 2012. Stress during development alters dendritic morphology in the nucleus accumbens and prefrontal cortex. Neuroscience 216, 103–109. Murakami, S., Imbe, H., Morikawa, Y., Kubo, C., Senba, E., 2005. Chronic stress, as well as acute stress, reduces BDNF mRNA expression in the rat hippocampus but less robustly. Neuroscience Research 53, 129–139. Murmu, M.S., Salomon, S., Biala, Y., Weinstock, M., Braun, K., Bock, J., 2006. Changes of spine density and dendritic complexity in the prefrontal cortex in offspring of mothers exposed to stress during pregnancy. European Journal of Neuroscience 24, 1477–1487. O’Connor, T.G., Heron, J., Golding, J., Beveridge, M., Glover, V., 2002. Maternal antenatal anxiety and children’s behavioral/emotional problems at 4 years. Report from the Avon Longitudinal Study of Parents and Children. British Journal of Psychiatry 180, 502–508. Oliff, H.S., Berchtold, N.C., Isackson, P., Cotman, C.W., 1998. Exercise-induced regulation of brain-derived neurotrophic factor (BDNF) transcripts in the rat hippocampus. Molecular Brain Research 61, 147–153. Ordyan, N.E., Pivina, S.G., 2004. Characteristics of the behavior and stress-reactivity of the hypophyseal–adrenal system in prenatally stressed rats. Neuroscience and Behavioral Physiology 34, 569–574. Pankevich, D.E., Mueller, B.R., Brockel, B., Bale, T.L., 2009. Prenatal stress programming of offspring feeding behavior and energy balance begins early in pregnancy. Physiology and Behavior 98, 94–102. Parnpiansil, P., Jutapakdeegul, N., Chentanez, T., Kotchabhakdi, N., 2003. Exercise during pregnancy increases hippocampal brain-derived neurotrophic factor mRNA expression and spatial learning in neonatal rat pup. Neuroscience Letters 352, 45–48. Paxinos, G., Watson, C., 1998. The Rat Brain in Stereotaxic Coordinates, fourth ed. Academic Press, New York. Peters, D.A.V., 1986. Prenatal stress increases the behavioural response to serotonin agonists and alters open field behavior in the rat. Pharmacology Biochemistry and Behavior 25, 873–877. Porter, L.L., 1996. Somatosensory input onto pyramidal tract neurons in rodent motor cortex. Neuroreport 7, 2309–2315. Redila, V.A., Christie, B.R., 2006. Exercise-induced changes in dendritic structure and complexity in the adult hippocampal dentate gyrus. Neuroscience 37, 1299–1307. Rosa, B.V., Firth, E.C., Blair, H.T., Vickers, M.H., Morel, P.C., 2011. Voluntary exercise in pregnant rats positively influences fetal growth without initiating a maternal physiological stress response. American Journal of Physiology: Regulatory Integrative and Comparative Physiology 300, R1134–R1141. Salam, J.N., Fox, J.H., Detroy, E.M., Guignon, M.H., Wohl, D.F., Falls, W.A., 2009. Voluntary exercise in C57 mice is anxiolytic across several measures of anxiety. Behavioural Brain Research 197, 31–40. Salm, A.K., Pavelko, M., Krouse, E.M., Webster, W., Kraszpulski, M., Birkle, D.L., 2004. Lateral amygdaloid nucleus expansion in adult rats is associated with exposure to prenatal stress. Brain Research: Developmental Brain Research 148, 159–167.
273
Sankaralingam, S., Jiang, Y., Davidge, S.T., Yeo, S., 2011. Effect of exercise on vascular superoxide dismutase expression in high-risk pregnancy. American Journal of Perinatology 28, 803–810. Schulz, K.M., Pearson, J.N., Neeley, E.W., Berger, R., Leonard, S., Adams, C.E., Stevens, K.E., 2011. Maternal stress during pregnancy causes sex-specific alterations in offspring memory performance, social interactions, indices of anxiety, and body mass. Physiology and Behavior 104, 340–347. Seckl, J.R., Meaney, M.J., 2004. Glucocorticoid programming. Annals of the New York Academy of Sciences 1032, 63–84. Sholl, D., 1953. Dendritic organization in the neurons of the visual and motor cortices of the cat. Journal of Anatomy 87, 387–407. Smith, M.A., Makino, S., Kvetnansky, R., Post, R.M., 1995. Stress and glucocorticoids affect the expression of brain-derived neurotrophic factor and neurotrophin-3 mRNAs in the hippocampus. Journal of Neuroscience 15, 1768–1777. Spratling, M.W., 2002. Cortical region interactions and the functional role of apical dendrites. Behavioral and Cognitive Neuroscience Reviews 1, 219–228. Sternberg, W.F., Ridgway, C.G., 2003. Effects of gestational stress and neonatal handling on pain, analgesia, and stress behavior of adult mice. Physiology and Behavior 78, 375–383. Takahashi, L.K., Turner, J.G., Kalin, N.H., 1992. Prenatal stress alters brain catecholaminergic activity and potentiates stress-induced behavior in adult rats. Brain Research 574, 131–137. Takahashi, L., 1998. Prenatal stress: consequences of glucocorticoids on hippocampal development and function. International Journal of Developmental Neuroscience 16, 199–207. Tollenaar, M.S., Beijers, R., Jansen, J., Riksen-Walraven, J.M., de Weerth, C., 2011. Maternal prenatal stress and cortisol reactivity to stressors in human infants. Stress 14, 53–65. Uysal, N., Sisman, A.R., Dayi, A., Aksu, I., Cetin, F., Gencoglu, C., Tas, A., Buyuk, E., 2011. Maternal exercise decreases maternal deprivation induced anxiety of pups and correlates to increased prefrontal cortex BDNF and VEGF. Neuroscience Letters 505, 273–278. Vaynman, S., Ying, Z., Gómez-Pinilla, F., 2004. Exercise induces BDNF and synapsin I to specific hippocampal subfields. Journal of Neuroscience Research 76, 356–362. Virgin Jr., C.E., Ha, T.P., Packan, D.R., Tombaugh, G.C., Yang, S.H., Horner, H.C., Sapolsky, R.M., 1991. Glucocorticoids inhibit glucose transport and glutamate uptake in hippocampal astrocytes: implications for glucocorticoid neurotoxicity. Journal of Neurochemistry 57, 1422–1428. Walsh, R.N., Cummings, R.A., 1976. The open-field test: a critical review. Psychological Bulletin 83, 482–504. Weissgerber, T.L., Wolfe, L.A., Davies, G.A., 2004. The role of regular physical activity in preeclampsia prevention. Medicine and Science in Sports and Exercise 36, 2024–2031. Weissgerber, T.L., Wolfe, L.A., Davies, G.A., Mottola, M.F., 2006. Exercise in the prevention and treatment of maternal–fetal disease: a review of the literature. Applied Physiology, Nutrition, and Metabolism 31, 661–674. Wise, S.P., Fleshman Jr., J.W., Jones, E.G., 1979. Maturation of pyramidal cell form in relation to developing afferent and efferent connections of rat somatic sensory cortex. Neuroscience 4, 1275–1297. Wyrwoll, C.S., Holmes, M.C., 2012. Prenatal excess glucocorticoid exposure and adult affective disorders: a role for serotonergic and catecholamine pathways. Neuroendocrinology 95, 47–55. Yaka, R., Salomon, S., Matzner, H., Weinstock, M., 2007. Effect of varied gestational stress on acquisition of spatial memory, hippocampal LTP and synaptic proteins in juvenile male rats. Behavioural Brain Research 179, 126–132. Yeo, S., Steele, N.M., Chang, M.C., Leclaire, S.M., Ronis, D.L., Hayashi, R., 2000. Effect of exercise on blood pressure in pregnant women with a high risk of gestational hypertensive disorders. Journal of Reproductive Medicine 45, 293–298.
Contents lists available at SciVerse ScienceDirect
International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Maternal exercise during pregnancy ameliorates the postnatal neuronal impairments induced by prenatal restraint stress in mice Carlos Bustamante ∗ , Ricardo Henríquez, Felipe Medina, Carmen Reinoso, Ronald Vargas, Rodrigo Pascual Laboratorio de Neurociencias, Escuela de Kinesiología, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, Chile
a r t i c l e
i n f o
Article history: Received 12 November 2012 Received in revised form 18 February 2013 Accepted 18 February 2013 Keywords: Prenatal stress Maternal voluntary wheel running Apical dendrites Parietal cortex Locomotor behavior
a b s t r a c t Clinical and preclinical studies have demonstrated that prenatal stress (PS) induces neuronal and behavioral disturbances in the offspring. In the present study, we determined whether maternal voluntary wheel running (VWR) during pregnancy could reverse the putative deleterious effects of PS on the neurodevelopment and behavior of the offspring. Pregnant CF-1 mice were randomly assigned to control, restraint stressed or restraint stressed + VWR groups. Dams of the stressed group were subjected to restraint stress between gestational days 14 and delivery, while control pregnant dams remained undisturbed in their home cages. Dams of the restraint stressed + VWR group were subjected to exercise between gestational days 1 and 17. On postnatal day 23 (P23), male pups were assigned to one of the following experimental groups: mice born from control dams, stressed dams or stressed + VWR dams. Locomotor behavior and pyramidal neuronal morphology were evaluated at P23. Animals were then sacrificed, and Golgi-impregnated pyramidal neurons of the parietal cortex were morphometrically analyzed. Here, we present two major findings: first, PS produced significantly diminished dendritic growth of parietal neurons without altered locomotor behavior of the offspring; and second, maternal VWR significantly offset morphological impairments. © 2013 ISDN. Published by Elsevier Ltd. All rights reserved.
Clinical studies have demonstrated that maternal stress during pregnancy is associated with physiological (Tollenaar et al., 2011) and behavioral (de Weerth et al., 2003) disturbances in the offspring. For example, it has been demonstrated that children born from mothers that have suffered from emotional adversity in late pregnancy demonstrate behavioral alterations such as hyperactivity disorder (O’Connor et al., 2002; Huizink et al., 2003). Consistent with these clinical findings, experimental studies have revealed links between prenatal stress (PS) and behavioral disturbances in juvenile offspring, such as increased locomotor activity (Emack and Matthews, 2011) and anxiety-like behaviors (Miyagawa et al., 2011). These abnormalities are likely due to morphological and functional changes in key areas of the brain that control motor and emotional behaviors, such as the prefrontal cortex (Muhammad et al., 2012) and amygdala (Salm et al., 2004; Kraszpulski et al., 2006). In this regard, it has been demonstrated that prenatally stressed animals show altered dendritic outgrowth of pyramidal neurons of the cingulate anterior area of the PFC (Murmu et al.,
∗ Corresponding author at: Escuela de Kinesiología, Pontificia Universidad Católica de Valparaíso, Avenida Universidad 330, Curauma, Valparaiso, Chile. Tel.: +56 32 2274022; fax: +56 32 2274044. E-mail address: [email protected] (C. Bustamante). 0736-5748/$36.00 © 2013 ISDN. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijdevneu.2013.02.007
2006). These findings are not surprising because the PFC is a highly plastic brain area in which neuronal maturation occurs primarily over a critical period in postnatal life (Altman and Bayer, 1990; Kolb, 1976). In addition, the parietal cortex has also demonstrated plastic changes associated with early life stress. Bock et al. (2005) found that the pups of rats subjected the stress of maternal separation during the pre-weaning period exhibit significant changes in the morphology of pyramidal neurons in the somatosensory region of the brain. However, at present, it is still unclear whether pyramidal neurons from this cortical area are vulnerable to the effects of stressful experiences suffered during prenatal life. Thus, our first aim was to determine the effects of PS on the apical dendritic outgrowth of pyramidal neurons in layer II/III of the parietal cortex in offspring evaluated during the post-weaning period (P23). We selected the apical domain of pyramidal neurons for two reasons: first, it has previously been reported that this dendritic domain is more vulnerable to the effects of PS than the basal domain (Murmu et al., 2006; Cerqueira et al., 2007); and second, because the apical dendrites of layer II/III are the targets of the feedback projections coming from the same and other cortical areas (Spratling, 2002). Because the mice exhibit dense topographically organized projections from the somatosensory cortex to the motor cortex (Bayer and Altman, 1991; Porter, 1996), we evaluated their locomotor behaviors using an open field (OF) test, a test that is widely used to
268
C. Bustamante et al. / Int. J. Devl Neuroscience 31 (2013) 267–273
assess locomotor and exploratory behaviors in rodents (Walsh and Cummings, 1976). On the other hand, it has been shown that exercise during pregnancy may prevent or limit adverse maternal and fetal morbidities (Gavard and Artal, 2008; Weissgerber et al., 2006). For instance, controlled exercise in pregnant women results in beneficial effects on their blood pressure parameters (Yeo et al., 2000) and prevents maternal–fetal diseases, such as type 2 diabetes (Dempsey et al., 2004) and preeclampsia (Weissgerber et al., 2004). Thus, several putative mechanisms that may account for the beneficial effects of exercise on maternal/fetal health have been proposed, including the enhancement of placental growth and vascularity, the reduction of oxidative stress and amelioration of endothelial dysfunction (Weissgerber et al., 2004; Sankaralingam et al., 2011). Furthermore, it has been shown that the children born from mothers who exercised regularly during pregnancy exhibit enhanced oral language scores at the age of five compared with control groups (Clapp, 1996). At present, the mechanisms that account for these cognitive effects are still largely unknown. Interestingly, these findings are, at least partially, similar to the changes observed in rodent studies. Moreover, maternal exercise during pregnancy has been associated with improvements in the spatial learning and short-term memory of juvenile offspring (Parnpiansil et al., 2003; Kim et al., 2007). These changes appear to be related to modifications in the structure and function of specific hippocampal regions, as shown by studies demonstrating that the offspring born from dams subjected to exercise during pregnancy show enhanced hippocampal neurogenesis (Lee et al., 2006). Furthermore, it has been hypothesized that enhancement of the expression of neurotrophins, such as brain-derived neurotrophic factor (BDNF), could, at least partially explain these beneficial effects (Kim et al., 2007). Based on these findings of the effects of maternal exercise, the second aim of this study was to determine whether maternal wheel running exercise during pregnancy ameliorates or reverses the putative deleterious effects of PS on the dendritic morphology of pyramidal neurons in layer II/III of the parietal cortex and locomotor behavior in prenatally stressed mice. Considering the above-mentioned aims, two hypotheses were tested: (i) PS induces a significant decrement in apical dendritic outgrowth of the pyramidal neurons concomitant with an altered locomotor activity in the prenatally stressed mice; and (ii) maternal exercise during pregnancy ameliorates the deleterious effects associated with PS. 1. Experimental procedures 1.1. Animals and experimental conditions Twenty-four virgin female mice (CF-1) were mated with twenty-four adult male mice and housed for 1 day under standard laboratory conditions; i.e., a 12-h inverted light–dark cycle (lights on at 11:00 p.m.), 18 ± 2 ◦ C, and food and water ad libitum (one male/one female per cage). The day of mating was considered “day zero of pregnancy” (G0), and this assumption was corroborated by the following observations: (i) the daily weight gain of the pregnant dams and (ii) the length of gestation. A cohort of ten female mice fulfilled the above-mentioned criteria and were included in the current study. All of the pregnant females were socially housed in transparent Plexiglas cages (30 cm × 19 cm × 13 cm; 2–3 dams per cage) with sawdust bedding and were randomly assigned to the one of the following three experimental groups: control (C, n = 3), restraint stressed (RS, n = 3) or RS + VWR (n = 4). All pregnant dams remained socially housed until the day before parturition. 1.2. Prenatal stress procedure The restraint stress procedure was performed according to a previously described protocol (Bustamante et al., 2010). Briefly, from gestational day 14 (G14) until delivery (see Fig. 1B), the pregnant females were subjected to three daily stress sessions at 9.00 a.m., 2.00 p.m. and 6.00 p.m. during which they were placed in plastic cylinders (11 cm long and 4 cm diameter) for 45 min. The control pregnant females were left undisturbed in their home cages (see Fig. 1A) and were handled only when the cages of all groups (C, RS and RS + VWR) were cleaned three times a week.
Fig. 1. Timeline showing a summary of the experimental design. G: gestational age; P: postnatal age (days). RS: restraint stress; VWR: voluntary wheel running.
1.3. Voluntary wheel running (VWR) protocol Between G1 and G17, each female mouse from the RS + VWR group was subjected to the VWR protocol (see Fig. 1C) during the dark phase for 4 h per day (2.00–6.00 p.m.) through housing in a cage that contained a running wheel (diameter, 14 cm; width, 6 cm). Under this condition, the mice had free access to voluntary exercise and food and water ad libitum. The number of daily wheel turns was registered by a magnetic sensor that was placed on the roof of the cage and connected to a computer. After weaning (P23), male pups only were randomly selected from each of the following three groups (to prevent possible litter effects, we used only 1–2 pups/litter/experiment): (i) mice born from a control mother (C), (ii) mice born from a stressed mother (RS) and (iii) mice born from a stressed mother subjected to VWR (RS + VWR). 1.4. Open field (OF) test On P23, the male mice from each group (C, n = 5; RS, n = 5 and RS + VWR, n = 5) were evaluated using the OF test (Walsh and Cummings, 1976). Briefly, this test was conducted in a square arena (40 cm × 40 cm) that was enclosed by continuous 21 cmhigh walls made of black wood. The arena was divided by Anymaze software into 25 equal sized squares containing one central zone (8 cm × 8 cm) and 24 surrounding squares (peripheral zone). The animals were examined during their dark cycle (between 2.00 and 6.00 p.m.), and testing was conducted under dim light (approximately 80 lx). Each mouse was placed in the center of the OF and allowed to freely explore the arena for 90 s. The animals’ behaviors were recorded using a camera that was located 40 cm above the apparatus (Logitech Quick cam version 9.5.0), and the data were analyzed using the Anymaze software. The number of lines crossed by each animal (with all four limbs), the total distance traversed and the total time spent in the central zone (a square of 8 cm × 8 cm in area) of the apparatus within 90 s were quantified. The open field apparatus was wiped between each test with a clean alcohol-dipped cloth to eliminate any olfactory clues left by previously tested animals. 1.5. Histological procedures and dendritic analysis To study the effect of prenatal stress and the potential “therapeutic” effects of VWR on the dendritic outgrowth of pyramidal neurons, mice were sacrificed under deep ether anesthesia, and their brains were immediately dissected out, fixed and stained with the Golgi-Cox-Sholl procedure (Sholl, 1953). After 45 days of slow metallic mercury impregnation, the brains were dehydrated in a graded alcohol series (25%–50%–75%–90%–95%–100%, v/v; Merck) and fixed in Paraplast. Coronal sections (thickness, 120 m) were cut using a sledge microtome, re-hydrated by in a graded alcohol series (100%–95%–90%–75%–50%–25%, v/v; Merck), treated with potassium disulfide/oxalic acid (5% dilution; Merck) and coverslipped. All slides were coded to avoid experimental bias and to maximize reliability. To qualify for dendritic morphometric evaluation, the pyramidal neurons were required to fulfill the following criteria: (a) have a well-defined somata shape; (b) show adequate staining of the soma and dendrites; (c) exhibit uninterrupted apical dendritic processes; (d) have no extensive dendrites that overlapped with neighboring neurons; and (e) be localized to layer II/III of the parietal cortex (approximately 150–350 m from the pial surface), which was carefully delimited by coordinates described in the Rat Stereotaxic Atlas (Paxinos and Watson, 1998). A total of 299 pyramidal neurons cells (approximately 20 neurons/animal) were analyzed using an Olympus CX-3 light microscope (400×). The neurons were imaged using a digital camera (5.0 CCD Camera) and analyzed using Micrometrics SE Premium V-2.8 software, which measured the following: (i) the total apical dendritic length per neuron (m) and (ii) the number of apical branches per neuron. At respect, the branching was determined
C. Bustamante et al. / Int. J. Devl Neuroscience 31 (2013) 267–273
Fig. 2. (A) Body weight gains of control (C), prenatal stress (RS) and prenatal stress + voluntary wheel running (RS + VWR) dams between G1 until G21. (B) Kilometers ran per session by RS + VWR dams between G1 until G17. Data are presented as the means ± S.E.M.
by counting the total number of dendritic branch points emerging from the main trunk of the apical dendrite. All behavioral and anatomical data were obtained from the same animals. All experimental procedures were performed in accordance with protocols that were approved by the Bioethics Committee of the Pontificia Universidad Católica de Valparaíso and international guidelines. This study was conducted using a minimal number of animals (5 for each group). 1.6. Statistical analysis Total dendritic length/neuron, number of branching dendrites, behavioral and reproductive/developmental data were analyzed using the one way analysis of variance (ANOVA) followed by Tukey’s HSD post hoc test (STATA 9.1 software). The results are expressed as the mean ± S.E.M. values. p < 0.05 was considered statistically significant.
2. Results As expected, the mice subjected to prenatal stress showed a significant decrease in the total apical dendritic length of the pyramidal neurons compared to the control group (a p < 0.05, F(2, 296) ; Fig. 3A). Furthermore, RS animals showed a significant reduction in the number of dendritic branches compared to age-matched controls (a p < 0.05, F(2, 296) ; Fig. 3B). In contrast, prenatally stressed mice subjected to maternal exercise during pregnancy showed a significant increase in both the total apical dendritic length and the number of dendritic branches (b p < 0.05, F(2, 296) ; Fig. 3A and B) compared to the RS group, suggesting that maternal exercise induced a significant recovery effect. Representative microphotographs of the pyramidal neurons in C, RS and RS + VWR animals are shown in Fig. 5. Importantly, neither the PS nor the VWR protocol affected the following: (i) the length of the gestation, (ii) the litter size, (iii) the male/female ratio, or (iv) the body weight gains of the pups during lactation (see Table 1). The RS mice showed a significant decrease in body weight compared with the C group at the age of P23 (a p < 0.05, F(2, 12) ; Table 1). The body weight gains of the three groups of the pregnant dams during pregnancy did not show significant differences (Fig. 2A). The pregnant dams subjected to VWR ran 2.79 ± 0.17 km with a minimum of 1.57 ± 0.17 km (G17) and a maximum of 3.65 ± 0.21 km (G10). The average kilometers ran
269
Fig. 3. Effect of prenatal stress and maternal voluntary wheel running on total apical dendritic length/per neuron (A) and total number of dendritic branching/per neuron (B) from selected pyramidal neurons. Data are presented as the means ± S.E.M. C: control group (n = 5); RS: prenatal stress group (n = 5); RS + VWR: prenatal stress + maternal exercise group (n = 5); a p < 0.05 compared with C group; b p < 0.05 compared with RS + VWR group (ANOVA test).
per session (4 h daily) for the pregnant dams (RS + VWR group) are shown in Fig. 2B. Contrary to expected, no significant differences were found in locomotor parameters between the C and RS group. Prenatally stressed mice exhibited an increase in the number of lines crossed and in the total distance traversed in the OF test compared to agematched control mice (Fig. 4A and B), but this difference was not significant. In addition, although the RS animals spent less total time in the central zone of the apparatus compared to the C group, this difference was not significant (Fig. 4C). 3. Discussion Although PS has been shown to induce a variety of deleterious effects in behavioral and neuronal function, knowledge of the underlying cytoarchitectural alterations and how these PS-induced changes may be prevented is still relatively sparse. We addressed these issues in the present study, which resulted in two central findings: first, exposure of pregnant dams to restraint stress during the third week of pregnancy induced morphological changes in the cytoarchitecture of apical dendrites in pyramidal neurons of the parietal cortex without significant disturbances in the locomotor behavior of their offspring at the post-weaning period; second, these PS-induced neuronal alterations were ameliorated by maternal VWR during pregnancy. In the current study, prenatally stressed mice exhibited a significant diminishment in dendritic length and branching of the apical dendrites in layer II/III pyramidal neurons, consistent with data reported by Jia et al. (2010) that showed that the total length of the apical dendrites of pyramidal neurons in hippocampal area CA3 are significantly shorter in prenatally stressed rats compared to control animals. Moreover, our morphological data are consistent with the results of Murmu et al. (2006), who demonstrated that maternal stress during the prenatal period is associated with a
270
C. Bustamante et al. / Int. J. Devl Neuroscience 31 (2013) 267–273
Table 1 Values of some reproductive and developmental parameters (mean ± S.E.M.).
C RS RS + VWR
Length of gestation
Litter size
Male/female ratio
Body weight (P1)
Body weight (P7)
Body weight (P14)
Body weight (P23)
20.7 ± 0.3 20.3 ± 0.3 20.3 ± 0.3
11.7 ± 1 13.0 ± 1.2 12.0 ± 0.3
0.5 ± 0.07 0.7 ± 0.05 0.6 ± 0.07
2 ± 0.9 1.7 ± 0.7 1.9 ± 0.6
3.4 ± 0.7 3 ± 0.6 3.3 ± 0.6
7 ± 0.4 5.7 ± 0.3 7.2 ± 1
12.7 ± 1.7 9.4 ± 0.7a 10 ± 0.9
Values of some reproductive and developmental parameters. The results are displayed as the mean ± S.E.M. for: (i) mice born from a control mother (C), (ii) mice born from a stressed mother (RS) and (iii) mice born from a stressed mother subjected to VWR (RS + VWR). The body weights at P1 considered the whole litter (male and female pups). The body weight at P7, P14 and P23 considered the body weights of male mice only. a p < 0.05 compared to C group (ANOVA test).
Fig. 4. Effect of prenatal stress and maternal voluntary wheel running on the number of crossed lines (A), total distance traversed (B) and time in central zone in the open field test (C). Data are expressed as the means ± S.E.M. C: control group (n = 5); RS: prenatal stress group (n = 5); RS + VWR: prenatal stress + maternal exercise group (n = 5).
significant dendritic retraction or remodeling of pyramidal neurons in layer II/III of the anterior cingulate cortex in offspring evaluated during the post-weaning period. In this study, pregnant female mice were subjected to restraint stress three times a day from G14 until delivery; this method is commonly employed to induce an increase in maternal plasma corticosterone (CORT) levels (Barros et al., 2006). The dendritic impairment exhibited by the prenatally stressed mice may be, at least partially, due to the toxic effects of excess circulating glucocorticoids (GCs) on neuronal development (Takahashi, 1998; Seckl and Meaney, 2004). Many studies in rodents have demonstrated that pups born from pregnant rats subjected to stress during the last week of pregnancy show alterations in the circadian rhythm of pituitary–adrenocortical activity during the postnatal period (Koehl et al., 1999). Similarly, it has been demonstrated that PS is associated with a prolonged HPAaxis response in the offspring (Koenig et al., 2005) and unregulated neurotransmission in noradrenergic, serotoninergic and dopaminergic systems (Takahashi et al., 1992; Hayashi et al., 1998; Berger et al., 2002; Miyagawa et al., 2011; Wyrwoll and Holmes, 2012). Several mechanisms have been proposed to contribute to the neuronal damage associated with chronic hypercortisolemia. First, an excess of GCs is associated with disturbances in glucose utilization, which compromises the energy resources of the cells (Horner et al., 1990). Second, GCs inhibit glutamate uptake by glial cells (Virgin et al., 1991), resulting in a persistent influx of Ca2+ , which results in neurotoxicity and neuronal death (Ankarcrona et al., 1995). Third, it has been demonstrated that hypercortisolemia mediates stress-induced decreases in neurotrophins, particularly BDNF, in many brain areas (Smith et al., 1995; Uysal et al., 2011); BDNF is a well-known factor associated with neuronal growth and branching (Murakami et al., 2005). Fourth, it has been demonstrated that PS is associated with impaired MAP-2 (microtubule-associated protein 2) synthesis in the brain (Barros et al., 2006) and thus, decreases in MAP-2 levels in developing stressed mice could potentially impair
Fig. 5. Representative photomicrograph of Golgi-Cox-stained pyramidal neurons from control (A), prenatal stress (B) and prenatal stress + maternal exercise (C) groups. Bar: 20 m.
C. Bustamante et al. / Int. J. Devl Neuroscience 31 (2013) 267–273
synaptogenesis and neurite outgrowth. This impairment may be directly associated with the reduced dendritic arborization in the apical domain observed in this study. Importantly, the PS protocol was applied during a time window when parietal neurons are still very immature. Thus, another potential mechanism that may contribute to the altered dendritic morphology exhibited by the prenatally stressed animals may involve PS-induced delays of dendritic and synaptic development, which may already be morphologically detectable at early post-weaning ages. It is well known that, during development, the dendritic structure of parietal neurons, at least partially, depends on the appropriate establishment of highly specific inputs from afferent thalamic fibers (Wise et al., 1979), and thus the effects of PS may potentially alter patterns of thalamocortical connectivity. In addition, it is important to note that aside from having a short-term effect during intrauterine life, PS might render many structures vulnerable to subsequent brain challenges (Diaz et al., 1997). Thus, it has been suggested that PS might affect regressive events such as dendritic and synaptic pruning, which normally occur during the post-weaning period (Huttenlocher, 1979). Moreover, as our prenatally stressed animals were raised by their biological mothers, another important variable that may account for the reported dendritic changes is the quality of preweaning mother–pup interactions. In this regard, it has been demonstrated that stressful experiences during pregnancy affect mother–offspring relationships (Moore and Power, 1986), which alter postpartum maternal care (Champagne and Meaney, 2006) and result in reductions in maternal attention (Maccari et al., 1995). In this regard, it has been shown that the deleterious effect of prenatal stress on the activity of the HPA axis may be minimized when the animals are reared by non-stressed mothers (Maccari et al., 1995). To address this interesting question, further studies should analyze the impact of PS on neuronal development in mice reared by either stressed or control surrogate mothers (i.e., utilizing cross-fostering procedures). On the basis of these aforementioned findings, it is possible that disturbances in the dendritic morphology observed in the RS group might be related not only to prenatal events but also to differential maternal care given to the offspring by stressed dams during the lactation period (Yaka et al., 2007). Furthermore, Burton et al. (2007) postulated that prenatal stress provides the background for subsequent postnatal environmental effects such as the diminishment of somatosensory/affective stimulation associated with maternal neglect. Importantly, neither the PS nor the VWR protocols affected the following: (i) the duration of the pregnancy, (ii) the body weight gains in the three groups of the pregnant dams, and (iii) the size of the litters and the body weight gains of the pups during lactation. In addition, litter compositions were not significantly different between the three groups. The only difference observed was that the RS mice showed a significant diminishment in body weight compared with the control group at P23. It is important to note that body weight outcomes related to maternal stress during pregnancy remain controversial because some studies have reported enhanced body weights in prenatally stressed rats during adulthood (Schulz et al., 2011), but other reports have found significant decreases in fetal body weight throughout development in prenatally stressed rats and decreased body weights and adiposity in mice (Pankevich et al., 2009). In the current study, the prenatally stressed mice showed a significant reduction in body weight compared with the control group evaluated at P23. Our results are similar to those found by Aziz et al. (2012) in mice exposed to prenatal stress and evaluated in the early postweaning period. In this regard, it has been suggested that altered weight gain in prenatally stressed mice could be related to long-term impacts of PS on feeding behavior and energy metabolism in the offspring (Pankevich et al., 2009). Additionally, it is possible that this reduction in body weight gain in the PS group may be associated with altered
271
lactation and maternal neglect during the preweaning period (Yaka et al., 2007). Regarding of the results in OF test and contrary to expected, mice subjected to PS did not exhibit altered locomotor behavior compared to the control animals. The behavioral data reported here are in agreement with the study of Green et al. (2011) that showed that prenatally stressed rats exhibit no differences in the number of line crossings or time spent in the central zone of the OF compared to control rats. However, some studies have reported contradictory results. On one hand, Deminière et al. (1992) found that prenatally stressed rats showed hyperactive-like behaviors in the OF; similar results have been reported by Burton et al. (2006) and Martínez-Téllez et al. (2009). On the other hand, some reports have shown decrements in exploratory behavior exhibited by prenatally stressed mice in the OF (Sternberg and Ridgway, 2003; Miyagawa et al., 2011). These inconsistencies may be due to variability in the paradigms used in different laboratories that include variability in the rodent strain used, types of stressors, periods or durations of stressors, methods of measuring locomotion and the postnatal period in which the locomotor behavior was evaluated. Another important factor to consider is the duration of the test; in this respect, in our study the mice were evaluated over 90 s. In many studies locomotor behavior of prenatally stressed animals has been evaluated in over ranges of 5 min (Ordyan and Pivina, 2004) or more (Peters, 1986). All these factors may affect locomotor behavior, and for that reason, we do not rule out the possibility that altered behavior may have been apparent if the animals were evaluated in a longer test. On the other hand, our data demonstrate that maternal exercise during pregnancy significantly reversed the deleterious effects of PS on neuronal development because prenatally stressed animals born from dams subjected to VWR during pregnancy showed a significant increase in apical dendritic length and branching of pyramidal neurons in the parietal cortex compared to the nonexercised stressed mice. Moreover, these mice showed no signs of hyperactivity/anxiety. The fact that VWR significantly ameliorated or reversed the deleterious effects associated with PS confirms the widespread beneficial effects of maternal exercise on offspring development (Rosa et al., 2011). These findings are similar to our previous results (Bustamante et al., 2010) that demonstrated that voluntary exercise significantly benefits the neurobehavioral development of prenatally stressed mice. To our knowledge, this is the first report to analyze the effects of maternal exercise during pregnancy on the dendritic morphology of parietal neurons in prenatally stressed animals. The mechanism controlling the reported dendritic changes is, at present, unknown; however, we propose several hypotheses. First, the expression of several neurotrophins, including nerve growth factor (NGF), fibroblast growth factor 2 (FGF-2), insulin-like growth factor-1 (IGF-1) and BDNF, are increased in the rodent brain after a few days of exercise (Oliff et al., 1998; Vaynman et al., 2004; Berchtold et al., 2005; Ding et al., 2006; Uysal et al., 2011). Recently, Aksu et al. (2012) found that maternal treadmill exercise during pregnancy is associated with an increase in vascular endothelial growth factor (VEGF) and BDNF levels in the postnatal rat cortex. Similarly, Parnpiansil et al. (2003) found that maternal exercise increased hippocampal BDNF expression and neurogenesis in postnatal rat brains. The exerciseinduced up-regulation of BDNF expression has been proposed to correlate with increases in dendritic complexity in several brain areas, including the hippocampus (Redila and Christie, 2006). Thus, VWR may potentially, at least partially, counteract the deleterious effects of PS on neurotrophin expression in the brain and thus enhance dendritic development in pyramidal neurons of the parietal cortex. Second, it has been demonstrated that maternal stress is associated with placental vasoconstriction via abnormal augmentation in maternal catecholamine levels; these phenomena may
272
C. Bustamante et al. / Int. J. Devl Neuroscience 31 (2013) 267–273
lead to fetal hypoxia, which would enhance fetal HPA-axis reactivity (Matthews and Phillips, 2010). In contrast, it was recently demonstrated that prenatal maternal exercise protects postnatal rat brains from hypoxia via increases in blood vessel density in the fetal brain (Akhavan et al., 2012). Thus, it is feasible that VWR may counteract the constriction of placental blood vessels associated with increased maternal catecholamine levels. Finally, VWR may affect the maternal HPA-axis itself by contributing to the regulation of stress responses of the dams during gestation. Preclinical studies have demonstrated that exercise in the form of wheel running is associated with a number of adaptive behavioral and physiological effects, including a reduction in stress-associated behaviors, neurogenesis, angiogenesis and increases in neurotrophic factors (Salam et al., 2009; Duman et al., 2008). We believe that all of these effects on the exercised dams are clinically relevant because it has been postulated that maternal behavior serves to “program” hypothalamic-pituitary-adrenal responses to stress in offspring (Liu et al., 1997). These putative mechanisms may contribute to explain the changes observed in dendritic development in the RS + VWR group and could potentially be related to the neuroendocrine and structural changes associated with prenatal maternal exercise. In summary, PS significantly altered dendritic development during the post-weaning period in offspring but did not produce significant alterations in locomotor behavior. This neuronal impairment was rescued to a remarkable degree by maternal exercise during gestation. These results confirm the beneficial effects of VWR during pregnancy as a preventive agent against chronic stressful events. Acknowledgment This research was supported by Grant PUCV – VRIEA 127.705/2008. References Aksu, I., Baykara, B., Ozbal, S., Cetin, F., Sisman, A.R., Dayi, A., Gencoglu, C., Tas, A., Büyük, E., Gonenc-Arda, S., Uysal, N., 2012. Maternal treadmill exercise during pregnancy decreases anxiety and increases prefrontal cortex VEGF and BDNF levels of rat pups in early and late periods of life. Neuroscience Letters 516, 221–225. Altman, J., Bayer, S.A., 1990. Migration and distribution of two populations of hippocampal granule cell precursors during the perinatal and postnatal periods. Journal of Comparative Neurology 301, 365–381. Ankarcrona, M., Dypbukt, J.M., Bonfoco, E., Zhivotovsky, B., Orrenius, S., Lipton, S.A., Nicotera, P., 1995. Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 15, 961–973. Akhavan, M.M., Foroutan, T., Safari, M., Sadighi-Moghaddam, B., Emami-Abarghoie, M., Rashidy-Pour, A., 2012. Prenatal exposure to maternal voluntary exercise during pregnancy provides protection against mild chronic postnatal hypoxia in rat offspring. Pakistan Journal of Pharmaceutical Sciences 25, 233–238. Aziz, N.H.K.A., Kendall, D.A., Pardon, M.C., 2012. Prenatal exposure to chronic mild stress increases corticosterone levels in the amniotic fluid and induces cognitive deficits in female offspring, improved by treatment with the antidepressant drug amitriptyline. Behavioural Brain Research 231, 29–39. Barros, V.G., Duhalde-Vega, M., Caltana, L., Brusco, A., Antonelli, M.C., 2006. Astrocyte-neuron vulnerability to prenatal stress in the adult rat brain. Journal of Neuroscience Research 83, 787–800. Bayer, S.A., Altman, J., 1991. Neocortical Development. Raven Press, New York. Berchtold, N.C., Chinn, G., Chou, M., Kesslak, J.P., Cotman, C.W., 2005. Exercise primes a molecular memory for brain-derived neurotrophic factor protein induction in the rat hippocampus. Neuroscience 133, 853–861. Berger, M.A., Barros, V.G., Sarchi, M.I., Tarazi, F.I., Antonelli, M.C., 2002. Long-term effects of prenatal stress on dopamine and glutamate receptors in adult rat brain. Neurochemical Research 27, 1525–1533. Bock, J., Gruss, M., Becker, S., Braun, K., 2005. Experience-induced changes of dendritic spine densities in the prefrontal and sensory cortex: correlation with developmental time windows. Cerebral Cortex 15, 802–808. Burton, C., Lovic, V., Fleming, A.S., 2006. Early adversity alters attention and locomotion in adult Sprague-Dawley rats. Behavioral Neuroscience 120, 665–675. Burton, C.L., Chatterjee, D., Chatterjee-Chakraborty, M., Lovic, V., Grella, S.L., Steiner, M., Fleming, A.S., 2007. Prenatal restraint stress and motherless rearing disrupts expression of plasticity markers and stress-induced corticosterone release in adult female Sprague-Dawley rats. Brain Research 1158, 28–38.
Bustamante, C., Bilbao, P., Contreras, W., Martínez, M., Mendoza, A., Reyes, A., Pascual, R., 2010. Effects of prenatal stress and exercise on dentate granule cells maturation and spatial memory in adolescent mice. International Journal of Developmental Neuroscience 28, 605–609. Cerqueira, J.J., Taipa, R., Uylings, H.B., Almeida, O.F., Sousa, N., 2007. Specific configuration of dendritic degeneration in pyramidal neurons of the medial prefrontal cortex induced by differing corticosteroid regimens. Cerebral Cortex 17, 1998–2006. Champagne, F.A., Meaney, M.J., 2006. Stress during gestation alters postpartum maternal care and the development of the offspring in a rodent model. Biological Psychiatry 59, 1227–1235. Clapp 3rd, J.F., 1996. Morphometric and neurodevelopmental outcome at age five years of the offspring of women who continued to exercise regularly throughout pregnancy. Journal of Pediatrics 129, 856–863. Deminière, J.M., Piazza, P.V., Guegan, G., Abrous, N., Maccari, S., Le Moal, M., Simon, H., 1992. Increased locomotor response to novelty and propensity to intravenous amphetamine self-administration in adult offspring of stressed mothers. Brain Research 586, 135–139. Dempsey, J.C., Butler, C.L., Sorensen, T.K., Lee, I.M., Thompson, M.L., Miller, R.S., Frederick, I.O., Williams, M.A., 2004. A case–control study of maternal recreational physical activity and risk of gestational diabetes mellitus. Diabetes Research and Clinical Practice 66, 203–215. de Weerth, C., van Hees, Y., Buitelaar, J.K., 2003. Prenatal maternal cortisol levels and infant behavior during the first 5 months. Early Human Development 74, 139–151. Diaz, R., Fuxe, K., Ogren, S.O., 1997. Prenatal corticosterone treatment induces longterm changes in spontaneous and apomorphine-mediated motor activity in male and female rats. Neuroscience 81, 129–140. Ding, Q., Vaynman, S., Akhavan, M., Ying, Z., Gomez-Pinilla, F., 2006. Insulin-like growth factor I interfaces with brain-derived neurotrophic factor-mediated synaptic plasticity to modulate aspects of exercise-induced cognitive function. Neuroscience 140, 823–833. Duman, C.H., Schlesinger, L., Russell, D.S., Duman, R.S., 2008. Voluntary exercise produces antidepressant and anxiolytic behavioral effects in mice. Brain Research 1199, 148–158. Emack, J., Matthews, S.G., 2011. Effects of chronic maternal stress on hypothalamopituitary-adrenal (HPA) function and behavior: no reversal by environmental enrichment. Hormones and Behavior 60, 589–598. Gavard, J.A., Artal, R., 2008. Effect of exercise on pregnancy outcome. Clinical Obstetrics and Gynecology 51, 467–480. ˜ A.E., Martinez, P.A., Frazer, A., Green, M.K., Rani, C.S., Joshi, A., Soto-Pina, Strong, R., Morilak, D.A., 2011. Prenatal stress induces long term stress vulnerability, compromising stress response systems in the brain and impairing extinction of conditioned fear after adult stress. Neuroscience 192, 438–451. Hayashi, A., Nagaoka, M., Yamada, K., Ichitani, Y., Miake, Y., Okado, N., 1998. Maternal stress induces synaptic loss and developmental disabilities of offspring. International Journal of Developmental Neuroscience 16, 209–216. Horner, H., Packan, D.A., Sapolsky, R., 1990. Glucocorticoids inhibit glucose transport in cultured hippocampal neurons and glia. Neuroendocrinology 52, 57–64. Huizink, A.C., Robles de Medina, P.G., Mulder, E.J., Visser, G.H., Buitelaar, J.K., 2003. Stress during pregnancy is associated with developmental outcome in infancy. Journal of Child Psychology and Psychiatry and Allied Disciplines 44, 810–818. Huttenlocher, P.R., 1979. Synaptic density in human frontal cortex—developmental changes and effects of aging. Brain Research 163, 195–205. Jia, N., Yang, K., Sun, Q., Cai, Q., Li, H., Cheng, D., Fan, X., Zhu, Z., 2010. Prenatal stress causes dendritic atrophy of pyramidal neurons in hippocampal CA3 region by glutamate in offspring rats. Developmental Neurobiology 70, 114–125. Kim, H., Lee, S.H., Kim, S.S., Yoo, J.H., Kim, C.J., 2007. The influence of maternal treadmill running during pregnancy on short-term memory and hippocampal cell survival in rat pups. International Journal of Developmental Neuroscience 25, 243–249. Koehl, M., Darnaudéry, M., Dulluc, J., Van Reeth, O., Le Moal, M., Maccari, S., 1999. Prenatal stress alters circadian activity of hypothalamo-pituitary-adrenal axis and hippocampal corticosteroid receptors in adult rats of both gender. Journal of Neurobiology 40, 302–315. Koenig, J.I., Elmer, G.I., Shepard, P.D., Lee, P.R., Mayo, C., Joy, B., Hercher, E., Brady, D.L., 2005. Prenatal exposure to a repeated variable stress paradigm elicits behavioral and neuroendocrinological changes in the adult offspring: potential relevance to schizophrenia. Behavioural Brain Research 156, 251–261. Kolb, B., 1976. Functional development of prefrontal cortex in rats continues into adolescence. Science 193, 335–336. Kraszpulski, M., Dickerson, P.A., Salm, A.K., 2006. Prenatal stress affects the developmental trajectory of the rat amygdala. Stress 9, 85–95. Lee, H.H., Kim, H., Lee, J.W., Kim, Y.S., Yang, H.Y., Chang, H.K., Lee, T.H., Shin, M.C., Lee, M.H., Shin, M.S., Park, S., Baek, S., Kim, C.J., 2006. Maternal swimming during pregnancy enhances short-term memory and neurogenesis in the hippocampus of rat pups. Brain and Development 28, 147–154. Liu, D., Diorio, J., Tannenbaum, B., Caldji, C., Francis, D., Freedman, A., Sharma, S., Pearson, D., Plotsky, P.M., Meaney, M.J., 1997. Maternal care, hippocampal glucocorticoid receptors, and hypothalamic-pituitary-adrenal responses to stress. Science 277, 1659–1662. Maccari, S., Piazza, P.V., Kabbaj, M., Barbazanges, A., Simon, H., Le Moal, M., 1995. Adoption reverses the long-term impairment in glucocorticoid feedback induced by prenatal stress. Journal of Neuroscience 15, 110–116.
C. Bustamante et al. / Int. J. Devl Neuroscience 31 (2013) 267–273 Martínez-Téllez, R.I., Hernández-Torres, E., Gamboa, C., Flores, G., 2009. Prenatal stress alters spine density and dendritic length of nucleus accumbens and hippocampus neurons in rat offspring. Synapse 63, 794–804. Matthews, S.G., Phillips, D.I., 2010. Minireview: transgenerational inheritance of the stress response: a new frontier in stress research. Endocrinology 151, 7–13. Miyagawa, K., Tsuji, M., Fujimori, K., Saito, Y., Takeda, H., 2011. Prenatal stress induces anxiety-like behavior together with the disruption of central serotonin neurons in mice. Neuroscience Research 70, 111–117. Moore, C.L., Power, K.L., 1986. Prenatal stress affects mother–infant interaction in Norway rats. Developmental Psychobiology 19, 235–245. Muhammad, A., Carroll, C., Kolb, B., 2012. Stress during development alters dendritic morphology in the nucleus accumbens and prefrontal cortex. Neuroscience 216, 103–109. Murakami, S., Imbe, H., Morikawa, Y., Kubo, C., Senba, E., 2005. Chronic stress, as well as acute stress, reduces BDNF mRNA expression in the rat hippocampus but less robustly. Neuroscience Research 53, 129–139. Murmu, M.S., Salomon, S., Biala, Y., Weinstock, M., Braun, K., Bock, J., 2006. Changes of spine density and dendritic complexity in the prefrontal cortex in offspring of mothers exposed to stress during pregnancy. European Journal of Neuroscience 24, 1477–1487. O’Connor, T.G., Heron, J., Golding, J., Beveridge, M., Glover, V., 2002. Maternal antenatal anxiety and children’s behavioral/emotional problems at 4 years. Report from the Avon Longitudinal Study of Parents and Children. British Journal of Psychiatry 180, 502–508. Oliff, H.S., Berchtold, N.C., Isackson, P., Cotman, C.W., 1998. Exercise-induced regulation of brain-derived neurotrophic factor (BDNF) transcripts in the rat hippocampus. Molecular Brain Research 61, 147–153. Ordyan, N.E., Pivina, S.G., 2004. Characteristics of the behavior and stress-reactivity of the hypophyseal–adrenal system in prenatally stressed rats. Neuroscience and Behavioral Physiology 34, 569–574. Pankevich, D.E., Mueller, B.R., Brockel, B., Bale, T.L., 2009. Prenatal stress programming of offspring feeding behavior and energy balance begins early in pregnancy. Physiology and Behavior 98, 94–102. Parnpiansil, P., Jutapakdeegul, N., Chentanez, T., Kotchabhakdi, N., 2003. Exercise during pregnancy increases hippocampal brain-derived neurotrophic factor mRNA expression and spatial learning in neonatal rat pup. Neuroscience Letters 352, 45–48. Paxinos, G., Watson, C., 1998. The Rat Brain in Stereotaxic Coordinates, fourth ed. Academic Press, New York. Peters, D.A.V., 1986. Prenatal stress increases the behavioural response to serotonin agonists and alters open field behavior in the rat. Pharmacology Biochemistry and Behavior 25, 873–877. Porter, L.L., 1996. Somatosensory input onto pyramidal tract neurons in rodent motor cortex. Neuroreport 7, 2309–2315. Redila, V.A., Christie, B.R., 2006. Exercise-induced changes in dendritic structure and complexity in the adult hippocampal dentate gyrus. Neuroscience 37, 1299–1307. Rosa, B.V., Firth, E.C., Blair, H.T., Vickers, M.H., Morel, P.C., 2011. Voluntary exercise in pregnant rats positively influences fetal growth without initiating a maternal physiological stress response. American Journal of Physiology: Regulatory Integrative and Comparative Physiology 300, R1134–R1141. Salam, J.N., Fox, J.H., Detroy, E.M., Guignon, M.H., Wohl, D.F., Falls, W.A., 2009. Voluntary exercise in C57 mice is anxiolytic across several measures of anxiety. Behavioural Brain Research 197, 31–40. Salm, A.K., Pavelko, M., Krouse, E.M., Webster, W., Kraszpulski, M., Birkle, D.L., 2004. Lateral amygdaloid nucleus expansion in adult rats is associated with exposure to prenatal stress. Brain Research: Developmental Brain Research 148, 159–167.
273
Sankaralingam, S., Jiang, Y., Davidge, S.T., Yeo, S., 2011. Effect of exercise on vascular superoxide dismutase expression in high-risk pregnancy. American Journal of Perinatology 28, 803–810. Schulz, K.M., Pearson, J.N., Neeley, E.W., Berger, R., Leonard, S., Adams, C.E., Stevens, K.E., 2011. Maternal stress during pregnancy causes sex-specific alterations in offspring memory performance, social interactions, indices of anxiety, and body mass. Physiology and Behavior 104, 340–347. Seckl, J.R., Meaney, M.J., 2004. Glucocorticoid programming. Annals of the New York Academy of Sciences 1032, 63–84. Sholl, D., 1953. Dendritic organization in the neurons of the visual and motor cortices of the cat. Journal of Anatomy 87, 387–407. Smith, M.A., Makino, S., Kvetnansky, R., Post, R.M., 1995. Stress and glucocorticoids affect the expression of brain-derived neurotrophic factor and neurotrophin-3 mRNAs in the hippocampus. Journal of Neuroscience 15, 1768–1777. Spratling, M.W., 2002. Cortical region interactions and the functional role of apical dendrites. Behavioral and Cognitive Neuroscience Reviews 1, 219–228. Sternberg, W.F., Ridgway, C.G., 2003. Effects of gestational stress and neonatal handling on pain, analgesia, and stress behavior of adult mice. Physiology and Behavior 78, 375–383. Takahashi, L.K., Turner, J.G., Kalin, N.H., 1992. Prenatal stress alters brain catecholaminergic activity and potentiates stress-induced behavior in adult rats. Brain Research 574, 131–137. Takahashi, L., 1998. Prenatal stress: consequences of glucocorticoids on hippocampal development and function. International Journal of Developmental Neuroscience 16, 199–207. Tollenaar, M.S., Beijers, R., Jansen, J., Riksen-Walraven, J.M., de Weerth, C., 2011. Maternal prenatal stress and cortisol reactivity to stressors in human infants. Stress 14, 53–65. Uysal, N., Sisman, A.R., Dayi, A., Aksu, I., Cetin, F., Gencoglu, C., Tas, A., Buyuk, E., 2011. Maternal exercise decreases maternal deprivation induced anxiety of pups and correlates to increased prefrontal cortex BDNF and VEGF. Neuroscience Letters 505, 273–278. Vaynman, S., Ying, Z., Gómez-Pinilla, F., 2004. Exercise induces BDNF and synapsin I to specific hippocampal subfields. Journal of Neuroscience Research 76, 356–362. Virgin Jr., C.E., Ha, T.P., Packan, D.R., Tombaugh, G.C., Yang, S.H., Horner, H.C., Sapolsky, R.M., 1991. Glucocorticoids inhibit glucose transport and glutamate uptake in hippocampal astrocytes: implications for glucocorticoid neurotoxicity. Journal of Neurochemistry 57, 1422–1428. Walsh, R.N., Cummings, R.A., 1976. The open-field test: a critical review. Psychological Bulletin 83, 482–504. Weissgerber, T.L., Wolfe, L.A., Davies, G.A., 2004. The role of regular physical activity in preeclampsia prevention. Medicine and Science in Sports and Exercise 36, 2024–2031. Weissgerber, T.L., Wolfe, L.A., Davies, G.A., Mottola, M.F., 2006. Exercise in the prevention and treatment of maternal–fetal disease: a review of the literature. Applied Physiology, Nutrition, and Metabolism 31, 661–674. Wise, S.P., Fleshman Jr., J.W., Jones, E.G., 1979. Maturation of pyramidal cell form in relation to developing afferent and efferent connections of rat somatic sensory cortex. Neuroscience 4, 1275–1297. Wyrwoll, C.S., Holmes, M.C., 2012. Prenatal excess glucocorticoid exposure and adult affective disorders: a role for serotonergic and catecholamine pathways. Neuroendocrinology 95, 47–55. Yaka, R., Salomon, S., Matzner, H., Weinstock, M., 2007. Effect of varied gestational stress on acquisition of spatial memory, hippocampal LTP and synaptic proteins in juvenile male rats. Behavioural Brain Research 179, 126–132. Yeo, S., Steele, N.M., Chang, M.C., Leclaire, S.M., Ronis, D.L., Hayashi, R., 2000. Effect of exercise on blood pressure in pregnant women with a high risk of gestational hypertensive disorders. Journal of Reproductive Medicine 45, 293–298.