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Maternal exercise during pregnancy prevents neurocognitive impairments in the juvenile offspring induced by prenatal stress
Neurology, Psychiatry and Brain Research 36 (2020) 1–7
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Maternal exercise during pregnancy prevents neurocognitive impairments in the juvenile offspring induced by prenatal stress
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Carlos Bustamante*, Carlos Ancatén, Cristian Gutiérrez-Rojas, Rodrigo Pascual Laboratorio de Neurociencias, Escuela de Kinesiología, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, Avenida Universidad N° 330, Curauma, Valparaíso, Chile
A R T I C LE I N FO
A B S T R A C T
Keywords: Prenatal stress Exercise Spatial learning Memory Hippocampus Dendrites
Purpose: Maternal exercise has shown beneficial effects on maternal/foetal health; however, the effects of maternal exercise on neurocognitive development in prenatally stressed offspring are unknown. The aim of the current study was to determine if maternal exercise during pregnancy prevents the effects of stress on spatial memory and learning as well as on the dendritic outgrowth of hippocampal neurons in prenatally stressed offspring. Methods: Ten pregnant mice were divided into three groups: control (C), restraint stress (RS) and restraint stress + voluntary wheel running (RS + VWR). Between gestational day 1 and 14 the dams from the RS + VWR group were subjected to the VWR protocol for 4 h per day. Moreover, from gestational day 14 until delivery the pregnant females from RS and RS + VWR group were subjected to three daily stress sessions. Between postnatal day 52 and 56, the male mice born of the three groups of dams were evaluated in the Morris Water Maze, and then, their neuronal morphology was analysed. Results: The stressed mice showed higher escape latencies and a significant reduction in both the number of entries to and in the time spent in the quadrant target of the maze, compared to controls, along with a reduction in the dendritic outgrowth of the hippocampal neurons. Moreover, stressed mice born from exercised mothers showed an improvement in spatial learning and memory, along with an increase in the dendritic length of dentate granule cells. Conclusions: Maternal exercise during pregnancy may be a beneficial factor to prevent the cognitive impairments and to ameliorate partially the impairments in the hippocampal dendritic outgrowth exhibited by prenatally stressed mice.
1. Introduction A large number of both experimental and clinical evidence show that early adverse experiences during pregnancy, such as maternal stress, are able to alter brain development and progeny behavior in short and long periods, increasing the risk of neurological, psychiatric and cognitive impairments (Babenko, Kovalchuk, & Metz, 2014). For instance, human studies (Tollenaar, Beijers, Jansen, Riksen-Walraven, & de Weerth, 2011) have shown that the progeny of mothers stressed during pregnancy exhibit decreased cognitive functions during infancy (Laplante et al., 2004; Polanska et al., 2017) or even in adulthood (Schwabe, Bohbot, & Wolf, 2012). These findings are consistent with the results found in rodent studies, showing that the offspring of pregnant dams, subjected to restraint stress (RS), exhibit both short and long-term spatial memory and learning disturbances (Hosseini-
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Sharifabad & Hadinedoushan, 2007; Sun et al., 2017; Yaka, Salomon, Matzner, & Weinstock, 2007). These cognitive impairments are likely related to functional and structural abnormalities in the hippocampus, a structure that shows high vulnerability to prenatal stress (PS) (Takahashi, 1998). Experimental studies in prenatally stressed rodents have shown that some hippocampal areas exhibit morphological disturbances, such as, shorter apical dendrites in the neurons belonging to the Cornu Amonis 3 area (CA3) at postnatal day 30 (Jia et al., 2010) and a diminishment in the dendritic development of granule dentate cells in adolescence (Bustamante et al., 2010) and adulthood, compared to control animals (Hosseini-Sharifabad & Hadinedoushan, 2007). On the other hand, clinical studies have shown that women who engage in physical exercise during pregnancy exhibit beneficial changes in their health status, such as a reduced risk of preeclampsia (Weissgerber, Wolfe, & Davies, 2004) and a decrease of symptoms of
Corresponding author. E-mail address: [email protected] (C. Bustamante).
https://doi.org/10.1016/j.npbr.2020.02.001 Received 15 July 2019; Received in revised form 11 January 2020; Accepted 5 February 2020 0941-9500/ © 2020 Elsevier GmbH. All rights reserved.
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2.3. Voluntary wheel running (VWR) protocol
anxiety and depression (Daley, Macarthur, & Winter, 2007). Moreover, maternal exercise during gestation not only benefits the mother but also improves foetal/infant health (Weissgerber, Wolfe, Davies, & Mottola, 2006) and neonatal outcomes (Murtezani, Pacarada, Ibraimi, Nevzati, & Abazi, 2014). Maternal exercise can also have a positive impact on the cognitive development of the offspring. For instance, “prenatally exercised” children exhibit enhanced oral language scores at the age of five compared to the control group (Clapp, 1996). These results seem to be associated with cerebral changes, such as an improvement in the maturation of the foetal brain, as has been reported in newborns (Labonte-Lemoyne, Curnier, & Ellemberg, 2017; Murtezani et al., 2014). Similarly, experimental studies have confirmed that maternal exercise during pregnancy is associated both with improvements in the spatial learning and short-term memory of juvenile rodent offspring (Parnpiansil, Jutapakdeegul, Chentanez, & Kotchabhakdi, 2003) with specific modifications in the structure of hippocampal regions, enhancements in hippocampal neurogenesis (Lee et al., 2006) and cell survival (Kim, Lee, Kim, Yoo, & Kim, 2007). These previously mentioned findings are very interesting because suggest the beneficial effect of maternal exercise on the neurobehavioural development of healthy offspring, however, according to our knowledge, the preventive role of maternal exercise on the cognitive deficit of the progeny, associated with prenatal stress, has not been widely studied. Thus, we firstly aimed to determine whether maternal exercise during pregnancy is able to prevent the deleterious effect of PS on spatial memory and learning evaluated at long term (juvenile age or “adolescence”). In addition, the second aim of the current study was to determine whether maternal exercise could prevent the deleterious effect of prenatal stress on hippocampal dendritic outgrowth in offspring, since this beneficial impact has been found on other brain areas, such as, parietal cortex (Bustamante et al., 2013) and amygdala (Ancatén, Gutiérrez-Rojas & Bustamante, 2017).
Between G1 and G14, the dams from the RS + VWR group were subjected to the VWR protocol (Bustamante et al., 2013) for 4 h per day (2.00–6.00 p.m.) by being housed in a cage that contained a running wheel. The animals had free access to voluntary exercise, as well as to food and water. A magnetic sensor placed on the roof of the cage and connected to a computer registered the number of daily wheel turns. The transfer of the pregnant dams to a new cage was carried out very carefully and minimally handled in order to avoid increased stress levels and other molecular changes (for example enhanced serum corticosterone levels). This method of transfer does not alter significantly the behaviour of the animals as have been demonstrated by the study of Rasmussen, Miller, Filipski, and Tolwani (2011) showing that the serum corticosterone levels of the minimally handled group did not differ statistically from that of the unmanipulated control group. After delivery (P0), all the dams were housed individually in Plexiglass cages with their litters. Male and female offspring were weaned at P21, and only male offspring from litters with similar numbers and mixes of gender were used for the experiments. In this regard two control litters were not included in the current study because they had a gender inequality (a higher proportion of females than males). Furthermore, only male mice were included because males may be more impaired by prenatal stress than females (Robinson & Bucci, 2012). At P22, the male pups were randomly selected from each of the following groups: (i) five mice born from a control mother (C); (ii) four mice born from a stressed mother (RS); and (iii) five mice born from a stressed mother subjected to VWR (RS + VWR). To prevent possible litter effects, we used only 1–2 male pups/litter/experiment. 2.4. Morris water maze test To evaluate spatial learning and memory, the Morris water maze test was adapted from a previous study (Bustamante et al., 2010). At P52, all animals from each group were evaluated in the maze. It is important to point out that, in the current study, the animals did not receive any pre training before starting the trials in the water maze, as indicated by Vorhees and Williams (2006). Briefly, the test consisted of a circular pool (diameter, 122 cm; height, 25 cm) filled to 20 cm with opaque water at room temperature (20 ± 1 °C). A 10 cm × 10 cm Plexiglass platform, onto which the mice could escape from the water, was positioned in the centre of one fixed quadrant 1 cm below the water surface. The challenge was to find the platform within 90 s. If the animals failed, they were gently guided to the platform. Each mouse performed four trials per day for four days (starting at 8:00 a.m., with a 10−15 min rest between trials). The entry position into the maze was changed each day, while the platform remained in the same position. The escape latency for each trial was recorded (i.e., the length of time each animal took to find the platform after being placed in the water). After swimming, each mouse was allowed to stay on the platform for 30 s. The hidden platform was removed on day 5 (P56), and memory retrieval was examined using a probe trial that lasted for 90 s. A computerized video-tracking system (ANY-Maze®, San Diego Instruments) was used to record the latency data from the learning trials, as well as the number of entries and the time spent inside the quadrant where the hidden platform had been placed. The entry and time data came from probe trials performed by the mice on day 5. Extra-maze cues for reference around the pool remained constant throughout the duration of the experiment.
2. Methods 2.1. Animals and experimental conditions Ten young female mice (CF-1) were mated with sexually experienced male mice (2:1 ratio per cage) and were housed in standard laboratory conditions, including a 12-h light-dark inverted cycle (lights on at 11:00 p.m.), an ambient temperature of 18 ± 2 °C and food and water ad libitum. Pregnancy was detected by the presence of semen in vaginal smears (gestational day 0, G0). All of the pregnant females were socially housed (until one-day prior to parturition) in transparent Plexiglass cages (30 × 19 × 13 cm; 2 dams per cage) with sawdust bedding, and they were randomly assigned to one of three experimental groups: control (C, n = 4), restraint stress (RS, n = 3) or restraint stress + voluntary wheel running (RS + VWR, n = 3). All experimental procedures were performed in accordance with protocols approved by the Bioethics Committee of the Pontificia Universidad Católica de Valparaíso and with international guidelines. This study was conducted using a minimal number of animals.
2.2. Prenatal restraint 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, 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 and were only handled when the cages of all the groups were cleaned, three times a week.
2.5. Histological procedures and dendritic analysis Mice were sacrificed under deep ether anaesthesia on P56. The brains were immediately dissected, fixed and stained using the GolgiCox-Sholl procedure (Sholl, 1953). Coronal sections (thickness, 120 μm) were cut using a sledge microtome, were treated with potassium 2
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dendritic length of the granule dentate cells (p < 0.005; Fig. 3C) compared to the RS group. In addition, the RS + VWR group showed a significant decrease in the total apical dendritic length of pyramidal CA3 neurons compared to the control group (p < 0,0001; Fig. 3A) but not in the basal dendritic domain of the CA3 neurons (Fig. 3B). Representative microphotographs of the pyramidal neurons and dentate granule cells of each group are shown in Fig. 4.
disulphide/oxalic acid (5 % dilution; Merck), and then coverslipped. All slides were coded to avoid experimental bias and to maximize reliability. To qualify for the dendritic morphometrical evaluation, the neurons needed to fulfill the following criteria: (a) have a well-defined somata shape; (b) show adequate staining of the soma and dendrites; (c) have uninterrupted dendritic processes; (d) have no extensive dendrites overlapping neighbouring neurons; and (e) be located in the outer region of the granule cell zone in the dentate gyrus or in the dorsal zone of hippocampus, carefully delimited by the coordinates described by Paxinos and Watson (2014). Three hundred fifteen dentate granule cells and 345 pyramidal neurons were analysed under an AM Scope light microscope (400X; Mainland, China). The neurons were imaged using a digital camera (Canon 5D Mark II; Shenzhen, China) and analysed using Micrometrics SE Premium V-2.8 software, which measured the total apical dendritic length of each pyramidal neuron (μm), the total basal dendritic length of each pyramidal neuron (μm) and the total dendritic length of each granule cell (μm). All of the behavioural and neuronal data were obtained from the same animals.
3.3. Body weight and number of pups No significant differences were observed in terms of body weight among the three groups of pregnant dams during pregnancy or during the weaning period, nor were there any differences found between the male offspring from the three groups evaluated at P52 (data not shown; p > 0.1). In addition, RS had no effect on the number of live pups born (12.0 ± 1.5; 11.8 ± 1.3 and 12.8 ± 1.5 pups/litter for the C, RS and RS + VWR groups, respectively; p > 0.1). 3.4. Kilometers ran per session
2.6. Statistical analysis The pregnant dams subjected to VWR ran 3,02 km ± 0,28 with a minimum of 2,30 ± 0,28 km (G14) and a maximum of 3,66 ± 0,29 km (G10). The average kilometers ran per session (4 h daily) for the pregnant dams (RS + VWR group) are shown in Fig. 5.
Repeated measures of variance were used to analyse the latency data from the learning trials of the Morris water maze. The total dendritic length/neuron and probe trial parameters (number of entries and time data) were analysed using a one-way ANOVA. All analyses were complemented post hoc with Tukey´s test (STATA 9.1 software) when significant differences (p < 0.05) were detected. The results were expressed as the means ± SEM.
4. Discussion The main purpose of the current study was to determine whether maternal exercise prevents the deleterious effect of neurocognitive disturbances caused by PS in offspring evaluated during adolescence. The results were the following: (i) maternal exercise during pregnancy prevents the spatial memory and learning disturbances exhibited by the prenatally stressed offspring evaluated during adolescence; (ii) maternal exercise partially prevented the dendritic impairments found in the hippocampal neurons, as only the granule dentate cells of the RS + VWR group showed beneficial changes; and finally, (iii) neither PS nor VWR changed the morphology of the dendritic basal domain in the CA3 pyramidal neurons. As we found previously (Bustamante et al., 2010), prenatally stressed mice showed memory and spatial learning impairments during the Morris water maze test (D’Hooge & De Deyn, 2001). Our results showed that the latency of the RS group during the test was greater than that of the control mice, a significant difference that was observed during the four acquisition days; similarly, the RS mice showed fewer entries into and spent less time exploring the target quadrant (probe trials) than the control mice did. These results are consistent with the impairment in hippocampal-dependent cognitive functions seen in juvenile animals stressed during the last week of gestation (Bustamante et al., 2010; Hosseini-Sharifabad & Hadinedoushan, 2007). On the other hand, maternal exercise ameliorated the adverse effect of PS on cognitive function in the adolescent offspring, as animals from the RS + VWR group showed a greater number of entries into and more time spent in the target quadrant than the RS group did. Moreover, the RS + VWR mice located the target platform more quickly than the mice born from non-exercised mothers. This significant difference was observed during the third and fourth days of the learning trials. These findings demonstrated by the adolescent offspring were in accordance with the results of some reports in which maternal exercise was found to be capable of improving spatial learning (Parnpiansil et al., 2003) and memory (Lee et al., 2006) in healthy rat pups. Recently, the study of Yau et al. (2019) found that male rats born from exercised mothers exhibit an improvement in temporal order memory at adult age, suggesting that the beneficial changes associated to maternal exercise are at long term. At present, there are no conclusive mechanisms that account for the aforementioned neurobehavioural results; however, here, we propose
3. Results 3.1. Behavioural analysis A significant interaction was found in the latency [F (6,33) = 9,877, p < 0,0001] together with a significant effect of both day [F (3,33) = 85,40, p < 0,0001] and group (F (2,11) = 14,89, p < 0,005). Post hoc comparisons revealed the following: (i) the RS group showed a higher escape latencies compared with the control mice over the four acquisition days (learning trials) (p < 0.005; Fig. 1A); (ii) on the other hand, the RS + VWR mice showed a lower escape latency compared with the RS group on the third and fourth days of the learning trials (p < 0.0005; see Fig. 1A). Moreover, RS animals showed a significant reduction in both the number of entries to [F (2,11) = 16,52, p < 0,005] and in the time spent in the quadrant target [F (2,11) = 14,25, p < 0,005] compared to controls (p < 0.005; Fig. 2A and B, respectively). However, the RS + VWR group showed significant improvements in memory and spatial learning due to a greater number of entries and more time spent in the target quadrant than those of the RS group (p < 0.005; Fig. 2A and B, respectively). Representative examples of the swim paths of each group are shown in Fig. 1B. 3.2. Histological analysis ANOVAs were statistically significant for the apical [F 2,154 = 22,34, p < 0,0001] and dendritic lengths [F 2,113 = 6,936, p < 0,005] of the dentate granule cells. Post hoc comparisons revealed that the RS animals showed a significant decrease in the total apical dendritic length of pyramidal CA3 neurons compared with the control group p < 0,0005; in addition, the RS mice showed a significant decrease in the dendritic length of the dentate granule cells compared with the controls p < 0.05; Fig. 3A and C. However, in the basal dendritic domain of the CA3 area, no differences were observed between the groups [F 2,209 = 0,1921, p = 0,82] see Fig. 3B). On the other hand, prenatally stressed mice subjected to maternal exercise during pregnancy showed a significant increase in the 3
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Fig. 1. (A) Effect of prenatal stress and maternal exercise on escape latency in the Morris water maze (learning trials). Data are represented as the means ± SEM. C: control group; RS: restraint stress group; RS + VWR: restraint stress + voluntary wheel running group; ap < 0.005 compared with C; bp < 0.0005 compared with RS + VWR (repeated measures analysis of variance, post hoc Tukey´s test). (B) Representative swim paths of mice during the behavioural test from the three groups. The platform is indicated by the black arrow, and the starting location of the mouse is indicated by the white arrow.
some explanations. First, VWR has demonstrated a protective effect against stress-related behaviours, such as maternal depression and anxiety (Burghardt, Fulk, Hand, & Wilson, 2004). In rodents, this effect may be mediated by the exercise-related regulatory impact on the hypothalamic–pituitary–adrenocortical (HPA) axis (Droste, Chandramohan, Hill, Linthorst, & Reul, 2007; Salam et al., 2009). Although we did not evaluate maternal behaviour during lactation, it is possible that the favourable effect of exercise on maternal behaviour may be indirectly beneficial for the behavioural development of the stressed offspring. Mother-pup interactions can be disturbed when the pregnant mother has been subjected to stress (Salam et al., 2009). In contrast, it is possible that exercised mothers can exhibit better or at least more, “normal” interactions with their pups, improving the sensory stimulation of the offspring. This phenomenon may contribute to “shape”, that is, more regulated emotional responses and better cognitive functions in the progeny (Liu et al., 1997). Second, VWR during gestation enhances the expression of several neurotrophins in the progeny’s brains, including brain-derived neurotrophic factor (BDNF) (Aksu et al., 2012). Gomes da Silva et al. (2016) also found that maternal exercise during pregnancy increases both BDNF levels and cell numbers in the progeny’s hippocampi, along with an enhancement in their spatial learning but in the long term. Although the mechanism that accounts for how maternal exercise increases BDNF levels in the offspring remains unclear, some researchers have postulated that maternal BDNF would cross the utero- placenta barrier reaching the fetal brain; this phenomenon may explain, at least partially, the improvement in the cognitive function of the offspring (Robinson & Bucci, 2012). Together with the above-mentioned data, it has been reported that maternal voluntary exercise could ameliorate cognitive deficits in the offspring associated to prenatal exposure to valproic acid. In this regard, the favorable effect of exercise during pregnancy on the cognitive development of the progeny would be mediated by the increase in the expression of vascular endothelial growth factor (VEGF) in rat pups (Rahimi, Akhavan, Kamyab, & Ebrahimi, 2018). Furthermore, maternal exercise during pregnancy prevents Alzheimer-like cognitive impairments in the offspring
Fig. 2. Effect of prenatal stress and maternal exercise on the number of entries to the target quadrant (A) and time spent in the target quadrant (B) during the Morris water maze (probe trials). Data are represented as the means ± SEM. C: control group; RS: restraint stress group; RS + VWR: restraint stress + voluntary wheel running group; ap < 0.005 compared with C; bp < 0.005 compared with RS + VWR (one-way ANOVA and post hoc Tukey´s test). 4
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cognitive function in the exercised animals (Collins et al., 2009). But, in addition it has been postulated that maternal exercise induces epigenetic changes that can be transferred to the offspring and affect the genetic expression in the progeny (Chalk & Brown, 2014). Some experimental studies have found that these epigenetic changes, after exercise, may be associated with improved cognitive function (Collins et al., 2009). Fourth, studies have confirmed the association between maternal exercise and structural changes in the hippocampi of offspring, such as increased neurogenesis (Lee et al., 2006) and cell survival (Kim et al., 2007). In this respect, an enhancement of neurogenesis has been correlated with an improvement in the performance of the Morris water maze in rodents (Kempermann, Kuhn, & Gage, 1997). Although, in the current study, we did not evaluated neither hippocampal cell survival nor the rate of neurogenesis in the RS + VWR group, the changes exhibited in terms of the dendritic length of the granule dentate cells, as we discuss below, may be a contributing factor in the improved spatial memory and learning observed in those mice. Concerning the second aim of the current study, our histological results showed that the RS mice exhibited an altered dendritic outgrowth in both dentate granule cells and in the apical domain of the pyramidal CA3 neurons. These morphological abnormalities may contribute, at least partially, to an explanation of the cognitive disturbances exhibited by the RS group, as has been postulated by Grigoryan and Segal (2013) who found that PS disturbs the network properties of hippocampal neurons. The granule dentate cells are involved in receiving, filtering and transmitting neural inputs from the entorhinal cortex to the hippocampal pyramidal cells (Tamura, Sajo, Kakita, Matsuki, & Koyama, 2011); for this reason, any perturbation that alters the hippocampal network properties will impair hippocampal-dependent functions. On the other hand, the basal dendritic length of the CA3 pyramidal neurons did not differ between the stressed and the control mice. A similar result in the basal dendritic domain of pyramidal CA3 neurons has been found in offspring rats subjected to prenatal (Suenaga, Yukie, Gao, & Nakaahara, 2012) and postnatal (Watanabe, Gould, & McEwen, 1992) stress. In this regard, the differences exhibited between the apical and basal dendrites revealed the functional significance of the input organization along the dendrites (Martinez-Tellez et al., 2009) and thus, it is possible that the apical domain appears to be more vulnerable to the impact of PS than the basal domain. The results of the current study show that maternal exercise exerts a selectively beneficial effect only in the dendritic length of the granule dentate cells. Although we do not have an explanation for these results, it is most likely that these differential effects may be related to the period in which the granule dentate cells or CA3 pyramidal neurons are born and develop. An important process in neurogenesis occurs between G16 to G19 (Bayer, Altman, Russo, & Zhang, 1993), but the neurogenesis of dentate granule cells begins in prenatal life and continues during the postnatal period (Altman & Bayer, 1990). The late development of the dentate gyrus makes it particularly sensitive to environmental and experience-dependent structural changes, such as maternal exercise (Kim et al., 2007; Lee et al., 2006). Moreover, although the apical domain of the pyramidal neurons in the CA3 area did not change, it is possible that the significant increase in the dendritic length of the granule dentate cells, and possibly in their complexity, may compensate for the prenatal stress-related impairments in the hippocampal network properties.
Fig. 3. Effect of prenatal stress and maternal exercise on the total dendritic length of apical (A) and basal domains of pyramidal neurons (B) and dentate granule cells (C). Data are represented as the means ± SEM. C: control group; RS: restraint stress group; RS + VWR: restraint stress + voluntary wheel running group; ap < 0.0005 compared with C; bp < 0.05 compared with C; cp < 0.005 compared with RS + VWR; dp < 0.0001 compared with C (one-way ANOVA and post hoc Tukey´s test).
associated with postnatal injection of amyloid-β oligomers (AβOs), as shown by Klein et al. (2019). In their report, it was found that the offspring born from dams subjected to gestational exercise (swimming) show, at adult age, an augmentation of functional mitochondria and the expression of synaptophysin together with an intact object recognition memory and spatial learning. Third, there is experimental evidence showing that exercise is able to generate epigenetic changes and modify the gene expression in the dentate gyrus of the rodent hippocampus, together with an increase in
4.1. Conclusions The main findings of the current study are (i) maternal exercise during pregnancy prevents the spatial learning/memory impairments induced by PS in adolescent mice; (ii) maternal exercise during pregnancy only prevents the impairments on dendritic outgrowth in dentate granule cells of the prenatally stressed offspring. Considering the 5
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Fig. 4. Representative photomicrographs of Golgi-cox-stained pyramidal neurons (A) and dentate granule cells (B) from each group (C: control group, RS: restraint stress group, and RS + VWR: restraint stress + voluntary wheel running group). Bar: 50 μm.
and interpreted data. Cristian Gutierrez-Rojas participated in the critical revision of the manuscript for important intellectual content. Carlos Ancatén contributed to the acquisition of data, technical and material support. Rodrigo Pascual developed the original idea, the design and the protocol. Ethical statement All experimental procedures were performed in accordance with protocols approved by the Bioethics Committee of the Pontificia Universidad Católica de Valparaíso and with international guidelines. According with these statements, this study was conducted using a minimal number of animals.
Fig. 5. Kilometers ran per session by RS + VWR dams between G1 until G17. Data are means ± SEM.
aforementioned conclusions, further studies are needed to expand the current knowledge of the preventive effects of maternal exercise on the neurodevelopment of prenatally stressed offspring. In addition, we recognize that the small number of mothers undergoing stress and exercise may be a limitation of the present study; for that reason, we recommend that future studies may increase the number of subjects considering an adequate balance with bioethics and experimentation with animals.
Financial disclosure This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of Competing Interest
Author´s contribution
The authors declare no conflict of interest.
Please clarify and write who was responsible for: Carlos Bustamante wrote the manuscript, and is guarantor. In addition, he prepared the manuscript for re submission and the response to the reviewers. Carlos Bustamante and Cristian Gutiérrez-Rojas abstracted analyzed
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.npbr.2020.02.001. 6
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Maternal exercise during pregnancy prevents neurocognitive impairments in the juvenile offspring induced by prenatal stress
T
Carlos Bustamante*, Carlos Ancatén, Cristian Gutiérrez-Rojas, Rodrigo Pascual Laboratorio de Neurociencias, Escuela de Kinesiología, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, Avenida Universidad N° 330, Curauma, Valparaíso, Chile
A R T I C LE I N FO
A B S T R A C T
Keywords: Prenatal stress Exercise Spatial learning Memory Hippocampus Dendrites
Purpose: Maternal exercise has shown beneficial effects on maternal/foetal health; however, the effects of maternal exercise on neurocognitive development in prenatally stressed offspring are unknown. The aim of the current study was to determine if maternal exercise during pregnancy prevents the effects of stress on spatial memory and learning as well as on the dendritic outgrowth of hippocampal neurons in prenatally stressed offspring. Methods: Ten pregnant mice were divided into three groups: control (C), restraint stress (RS) and restraint stress + voluntary wheel running (RS + VWR). Between gestational day 1 and 14 the dams from the RS + VWR group were subjected to the VWR protocol for 4 h per day. Moreover, from gestational day 14 until delivery the pregnant females from RS and RS + VWR group were subjected to three daily stress sessions. Between postnatal day 52 and 56, the male mice born of the three groups of dams were evaluated in the Morris Water Maze, and then, their neuronal morphology was analysed. Results: The stressed mice showed higher escape latencies and a significant reduction in both the number of entries to and in the time spent in the quadrant target of the maze, compared to controls, along with a reduction in the dendritic outgrowth of the hippocampal neurons. Moreover, stressed mice born from exercised mothers showed an improvement in spatial learning and memory, along with an increase in the dendritic length of dentate granule cells. Conclusions: Maternal exercise during pregnancy may be a beneficial factor to prevent the cognitive impairments and to ameliorate partially the impairments in the hippocampal dendritic outgrowth exhibited by prenatally stressed mice.
1. Introduction A large number of both experimental and clinical evidence show that early adverse experiences during pregnancy, such as maternal stress, are able to alter brain development and progeny behavior in short and long periods, increasing the risk of neurological, psychiatric and cognitive impairments (Babenko, Kovalchuk, & Metz, 2014). For instance, human studies (Tollenaar, Beijers, Jansen, Riksen-Walraven, & de Weerth, 2011) have shown that the progeny of mothers stressed during pregnancy exhibit decreased cognitive functions during infancy (Laplante et al., 2004; Polanska et al., 2017) or even in adulthood (Schwabe, Bohbot, & Wolf, 2012). These findings are consistent with the results found in rodent studies, showing that the offspring of pregnant dams, subjected to restraint stress (RS), exhibit both short and long-term spatial memory and learning disturbances (Hosseini-
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Sharifabad & Hadinedoushan, 2007; Sun et al., 2017; Yaka, Salomon, Matzner, & Weinstock, 2007). These cognitive impairments are likely related to functional and structural abnormalities in the hippocampus, a structure that shows high vulnerability to prenatal stress (PS) (Takahashi, 1998). Experimental studies in prenatally stressed rodents have shown that some hippocampal areas exhibit morphological disturbances, such as, shorter apical dendrites in the neurons belonging to the Cornu Amonis 3 area (CA3) at postnatal day 30 (Jia et al., 2010) and a diminishment in the dendritic development of granule dentate cells in adolescence (Bustamante et al., 2010) and adulthood, compared to control animals (Hosseini-Sharifabad & Hadinedoushan, 2007). On the other hand, clinical studies have shown that women who engage in physical exercise during pregnancy exhibit beneficial changes in their health status, such as a reduced risk of preeclampsia (Weissgerber, Wolfe, & Davies, 2004) and a decrease of symptoms of
Corresponding author. E-mail address: [email protected] (C. Bustamante).
https://doi.org/10.1016/j.npbr.2020.02.001 Received 15 July 2019; Received in revised form 11 January 2020; Accepted 5 February 2020 0941-9500/ © 2020 Elsevier GmbH. All rights reserved.
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2.3. Voluntary wheel running (VWR) protocol
anxiety and depression (Daley, Macarthur, & Winter, 2007). Moreover, maternal exercise during gestation not only benefits the mother but also improves foetal/infant health (Weissgerber, Wolfe, Davies, & Mottola, 2006) and neonatal outcomes (Murtezani, Pacarada, Ibraimi, Nevzati, & Abazi, 2014). Maternal exercise can also have a positive impact on the cognitive development of the offspring. For instance, “prenatally exercised” children exhibit enhanced oral language scores at the age of five compared to the control group (Clapp, 1996). These results seem to be associated with cerebral changes, such as an improvement in the maturation of the foetal brain, as has been reported in newborns (Labonte-Lemoyne, Curnier, & Ellemberg, 2017; Murtezani et al., 2014). Similarly, experimental studies have confirmed that maternal exercise during pregnancy is associated both with improvements in the spatial learning and short-term memory of juvenile rodent offspring (Parnpiansil, Jutapakdeegul, Chentanez, & Kotchabhakdi, 2003) with specific modifications in the structure of hippocampal regions, enhancements in hippocampal neurogenesis (Lee et al., 2006) and cell survival (Kim, Lee, Kim, Yoo, & Kim, 2007). These previously mentioned findings are very interesting because suggest the beneficial effect of maternal exercise on the neurobehavioural development of healthy offspring, however, according to our knowledge, the preventive role of maternal exercise on the cognitive deficit of the progeny, associated with prenatal stress, has not been widely studied. Thus, we firstly aimed to determine whether maternal exercise during pregnancy is able to prevent the deleterious effect of PS on spatial memory and learning evaluated at long term (juvenile age or “adolescence”). In addition, the second aim of the current study was to determine whether maternal exercise could prevent the deleterious effect of prenatal stress on hippocampal dendritic outgrowth in offspring, since this beneficial impact has been found on other brain areas, such as, parietal cortex (Bustamante et al., 2013) and amygdala (Ancatén, Gutiérrez-Rojas & Bustamante, 2017).
Between G1 and G14, the dams from the RS + VWR group were subjected to the VWR protocol (Bustamante et al., 2013) for 4 h per day (2.00–6.00 p.m.) by being housed in a cage that contained a running wheel. The animals had free access to voluntary exercise, as well as to food and water. A magnetic sensor placed on the roof of the cage and connected to a computer registered the number of daily wheel turns. The transfer of the pregnant dams to a new cage was carried out very carefully and minimally handled in order to avoid increased stress levels and other molecular changes (for example enhanced serum corticosterone levels). This method of transfer does not alter significantly the behaviour of the animals as have been demonstrated by the study of Rasmussen, Miller, Filipski, and Tolwani (2011) showing that the serum corticosterone levels of the minimally handled group did not differ statistically from that of the unmanipulated control group. After delivery (P0), all the dams were housed individually in Plexiglass cages with their litters. Male and female offspring were weaned at P21, and only male offspring from litters with similar numbers and mixes of gender were used for the experiments. In this regard two control litters were not included in the current study because they had a gender inequality (a higher proportion of females than males). Furthermore, only male mice were included because males may be more impaired by prenatal stress than females (Robinson & Bucci, 2012). At P22, the male pups were randomly selected from each of the following groups: (i) five mice born from a control mother (C); (ii) four mice born from a stressed mother (RS); and (iii) five mice born from a stressed mother subjected to VWR (RS + VWR). To prevent possible litter effects, we used only 1–2 male pups/litter/experiment. 2.4. Morris water maze test To evaluate spatial learning and memory, the Morris water maze test was adapted from a previous study (Bustamante et al., 2010). At P52, all animals from each group were evaluated in the maze. It is important to point out that, in the current study, the animals did not receive any pre training before starting the trials in the water maze, as indicated by Vorhees and Williams (2006). Briefly, the test consisted of a circular pool (diameter, 122 cm; height, 25 cm) filled to 20 cm with opaque water at room temperature (20 ± 1 °C). A 10 cm × 10 cm Plexiglass platform, onto which the mice could escape from the water, was positioned in the centre of one fixed quadrant 1 cm below the water surface. The challenge was to find the platform within 90 s. If the animals failed, they were gently guided to the platform. Each mouse performed four trials per day for four days (starting at 8:00 a.m., with a 10−15 min rest between trials). The entry position into the maze was changed each day, while the platform remained in the same position. The escape latency for each trial was recorded (i.e., the length of time each animal took to find the platform after being placed in the water). After swimming, each mouse was allowed to stay on the platform for 30 s. The hidden platform was removed on day 5 (P56), and memory retrieval was examined using a probe trial that lasted for 90 s. A computerized video-tracking system (ANY-Maze®, San Diego Instruments) was used to record the latency data from the learning trials, as well as the number of entries and the time spent inside the quadrant where the hidden platform had been placed. The entry and time data came from probe trials performed by the mice on day 5. Extra-maze cues for reference around the pool remained constant throughout the duration of the experiment.
2. Methods 2.1. Animals and experimental conditions Ten young female mice (CF-1) were mated with sexually experienced male mice (2:1 ratio per cage) and were housed in standard laboratory conditions, including a 12-h light-dark inverted cycle (lights on at 11:00 p.m.), an ambient temperature of 18 ± 2 °C and food and water ad libitum. Pregnancy was detected by the presence of semen in vaginal smears (gestational day 0, G0). All of the pregnant females were socially housed (until one-day prior to parturition) in transparent Plexiglass cages (30 × 19 × 13 cm; 2 dams per cage) with sawdust bedding, and they were randomly assigned to one of three experimental groups: control (C, n = 4), restraint stress (RS, n = 3) or restraint stress + voluntary wheel running (RS + VWR, n = 3). All experimental procedures were performed in accordance with protocols approved by the Bioethics Committee of the Pontificia Universidad Católica de Valparaíso and with international guidelines. This study was conducted using a minimal number of animals.
2.2. Prenatal restraint 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, 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 and were only handled when the cages of all the groups were cleaned, three times a week.
2.5. Histological procedures and dendritic analysis Mice were sacrificed under deep ether anaesthesia on P56. The brains were immediately dissected, fixed and stained using the GolgiCox-Sholl procedure (Sholl, 1953). Coronal sections (thickness, 120 μm) were cut using a sledge microtome, were treated with potassium 2
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dendritic length of the granule dentate cells (p < 0.005; Fig. 3C) compared to the RS group. In addition, the RS + VWR group showed a significant decrease in the total apical dendritic length of pyramidal CA3 neurons compared to the control group (p < 0,0001; Fig. 3A) but not in the basal dendritic domain of the CA3 neurons (Fig. 3B). Representative microphotographs of the pyramidal neurons and dentate granule cells of each group are shown in Fig. 4.
disulphide/oxalic acid (5 % dilution; Merck), and then coverslipped. All slides were coded to avoid experimental bias and to maximize reliability. To qualify for the dendritic morphometrical evaluation, the neurons needed to fulfill the following criteria: (a) have a well-defined somata shape; (b) show adequate staining of the soma and dendrites; (c) have uninterrupted dendritic processes; (d) have no extensive dendrites overlapping neighbouring neurons; and (e) be located in the outer region of the granule cell zone in the dentate gyrus or in the dorsal zone of hippocampus, carefully delimited by the coordinates described by Paxinos and Watson (2014). Three hundred fifteen dentate granule cells and 345 pyramidal neurons were analysed under an AM Scope light microscope (400X; Mainland, China). The neurons were imaged using a digital camera (Canon 5D Mark II; Shenzhen, China) and analysed using Micrometrics SE Premium V-2.8 software, which measured the total apical dendritic length of each pyramidal neuron (μm), the total basal dendritic length of each pyramidal neuron (μm) and the total dendritic length of each granule cell (μm). All of the behavioural and neuronal data were obtained from the same animals.
3.3. Body weight and number of pups No significant differences were observed in terms of body weight among the three groups of pregnant dams during pregnancy or during the weaning period, nor were there any differences found between the male offspring from the three groups evaluated at P52 (data not shown; p > 0.1). In addition, RS had no effect on the number of live pups born (12.0 ± 1.5; 11.8 ± 1.3 and 12.8 ± 1.5 pups/litter for the C, RS and RS + VWR groups, respectively; p > 0.1). 3.4. Kilometers ran per session
2.6. Statistical analysis The pregnant dams subjected to VWR ran 3,02 km ± 0,28 with a minimum of 2,30 ± 0,28 km (G14) and a maximum of 3,66 ± 0,29 km (G10). The average kilometers ran per session (4 h daily) for the pregnant dams (RS + VWR group) are shown in Fig. 5.
Repeated measures of variance were used to analyse the latency data from the learning trials of the Morris water maze. The total dendritic length/neuron and probe trial parameters (number of entries and time data) were analysed using a one-way ANOVA. All analyses were complemented post hoc with Tukey´s test (STATA 9.1 software) when significant differences (p < 0.05) were detected. The results were expressed as the means ± SEM.
4. Discussion The main purpose of the current study was to determine whether maternal exercise prevents the deleterious effect of neurocognitive disturbances caused by PS in offspring evaluated during adolescence. The results were the following: (i) maternal exercise during pregnancy prevents the spatial memory and learning disturbances exhibited by the prenatally stressed offspring evaluated during adolescence; (ii) maternal exercise partially prevented the dendritic impairments found in the hippocampal neurons, as only the granule dentate cells of the RS + VWR group showed beneficial changes; and finally, (iii) neither PS nor VWR changed the morphology of the dendritic basal domain in the CA3 pyramidal neurons. As we found previously (Bustamante et al., 2010), prenatally stressed mice showed memory and spatial learning impairments during the Morris water maze test (D’Hooge & De Deyn, 2001). Our results showed that the latency of the RS group during the test was greater than that of the control mice, a significant difference that was observed during the four acquisition days; similarly, the RS mice showed fewer entries into and spent less time exploring the target quadrant (probe trials) than the control mice did. These results are consistent with the impairment in hippocampal-dependent cognitive functions seen in juvenile animals stressed during the last week of gestation (Bustamante et al., 2010; Hosseini-Sharifabad & Hadinedoushan, 2007). On the other hand, maternal exercise ameliorated the adverse effect of PS on cognitive function in the adolescent offspring, as animals from the RS + VWR group showed a greater number of entries into and more time spent in the target quadrant than the RS group did. Moreover, the RS + VWR mice located the target platform more quickly than the mice born from non-exercised mothers. This significant difference was observed during the third and fourth days of the learning trials. These findings demonstrated by the adolescent offspring were in accordance with the results of some reports in which maternal exercise was found to be capable of improving spatial learning (Parnpiansil et al., 2003) and memory (Lee et al., 2006) in healthy rat pups. Recently, the study of Yau et al. (2019) found that male rats born from exercised mothers exhibit an improvement in temporal order memory at adult age, suggesting that the beneficial changes associated to maternal exercise are at long term. At present, there are no conclusive mechanisms that account for the aforementioned neurobehavioural results; however, here, we propose
3. Results 3.1. Behavioural analysis A significant interaction was found in the latency [F (6,33) = 9,877, p < 0,0001] together with a significant effect of both day [F (3,33) = 85,40, p < 0,0001] and group (F (2,11) = 14,89, p < 0,005). Post hoc comparisons revealed the following: (i) the RS group showed a higher escape latencies compared with the control mice over the four acquisition days (learning trials) (p < 0.005; Fig. 1A); (ii) on the other hand, the RS + VWR mice showed a lower escape latency compared with the RS group on the third and fourth days of the learning trials (p < 0.0005; see Fig. 1A). Moreover, RS animals showed a significant reduction in both the number of entries to [F (2,11) = 16,52, p < 0,005] and in the time spent in the quadrant target [F (2,11) = 14,25, p < 0,005] compared to controls (p < 0.005; Fig. 2A and B, respectively). However, the RS + VWR group showed significant improvements in memory and spatial learning due to a greater number of entries and more time spent in the target quadrant than those of the RS group (p < 0.005; Fig. 2A and B, respectively). Representative examples of the swim paths of each group are shown in Fig. 1B. 3.2. Histological analysis ANOVAs were statistically significant for the apical [F 2,154 = 22,34, p < 0,0001] and dendritic lengths [F 2,113 = 6,936, p < 0,005] of the dentate granule cells. Post hoc comparisons revealed that the RS animals showed a significant decrease in the total apical dendritic length of pyramidal CA3 neurons compared with the control group p < 0,0005; in addition, the RS mice showed a significant decrease in the dendritic length of the dentate granule cells compared with the controls p < 0.05; Fig. 3A and C. However, in the basal dendritic domain of the CA3 area, no differences were observed between the groups [F 2,209 = 0,1921, p = 0,82] see Fig. 3B). On the other hand, prenatally stressed mice subjected to maternal exercise during pregnancy showed a significant increase in the 3
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Fig. 1. (A) Effect of prenatal stress and maternal exercise on escape latency in the Morris water maze (learning trials). Data are represented as the means ± SEM. C: control group; RS: restraint stress group; RS + VWR: restraint stress + voluntary wheel running group; ap < 0.005 compared with C; bp < 0.0005 compared with RS + VWR (repeated measures analysis of variance, post hoc Tukey´s test). (B) Representative swim paths of mice during the behavioural test from the three groups. The platform is indicated by the black arrow, and the starting location of the mouse is indicated by the white arrow.
some explanations. First, VWR has demonstrated a protective effect against stress-related behaviours, such as maternal depression and anxiety (Burghardt, Fulk, Hand, & Wilson, 2004). In rodents, this effect may be mediated by the exercise-related regulatory impact on the hypothalamic–pituitary–adrenocortical (HPA) axis (Droste, Chandramohan, Hill, Linthorst, & Reul, 2007; Salam et al., 2009). Although we did not evaluate maternal behaviour during lactation, it is possible that the favourable effect of exercise on maternal behaviour may be indirectly beneficial for the behavioural development of the stressed offspring. Mother-pup interactions can be disturbed when the pregnant mother has been subjected to stress (Salam et al., 2009). In contrast, it is possible that exercised mothers can exhibit better or at least more, “normal” interactions with their pups, improving the sensory stimulation of the offspring. This phenomenon may contribute to “shape”, that is, more regulated emotional responses and better cognitive functions in the progeny (Liu et al., 1997). Second, VWR during gestation enhances the expression of several neurotrophins in the progeny’s brains, including brain-derived neurotrophic factor (BDNF) (Aksu et al., 2012). Gomes da Silva et al. (2016) also found that maternal exercise during pregnancy increases both BDNF levels and cell numbers in the progeny’s hippocampi, along with an enhancement in their spatial learning but in the long term. Although the mechanism that accounts for how maternal exercise increases BDNF levels in the offspring remains unclear, some researchers have postulated that maternal BDNF would cross the utero- placenta barrier reaching the fetal brain; this phenomenon may explain, at least partially, the improvement in the cognitive function of the offspring (Robinson & Bucci, 2012). Together with the above-mentioned data, it has been reported that maternal voluntary exercise could ameliorate cognitive deficits in the offspring associated to prenatal exposure to valproic acid. In this regard, the favorable effect of exercise during pregnancy on the cognitive development of the progeny would be mediated by the increase in the expression of vascular endothelial growth factor (VEGF) in rat pups (Rahimi, Akhavan, Kamyab, & Ebrahimi, 2018). Furthermore, maternal exercise during pregnancy prevents Alzheimer-like cognitive impairments in the offspring
Fig. 2. Effect of prenatal stress and maternal exercise on the number of entries to the target quadrant (A) and time spent in the target quadrant (B) during the Morris water maze (probe trials). Data are represented as the means ± SEM. C: control group; RS: restraint stress group; RS + VWR: restraint stress + voluntary wheel running group; ap < 0.005 compared with C; bp < 0.005 compared with RS + VWR (one-way ANOVA and post hoc Tukey´s test). 4
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cognitive function in the exercised animals (Collins et al., 2009). But, in addition it has been postulated that maternal exercise induces epigenetic changes that can be transferred to the offspring and affect the genetic expression in the progeny (Chalk & Brown, 2014). Some experimental studies have found that these epigenetic changes, after exercise, may be associated with improved cognitive function (Collins et al., 2009). Fourth, studies have confirmed the association between maternal exercise and structural changes in the hippocampi of offspring, such as increased neurogenesis (Lee et al., 2006) and cell survival (Kim et al., 2007). In this respect, an enhancement of neurogenesis has been correlated with an improvement in the performance of the Morris water maze in rodents (Kempermann, Kuhn, & Gage, 1997). Although, in the current study, we did not evaluated neither hippocampal cell survival nor the rate of neurogenesis in the RS + VWR group, the changes exhibited in terms of the dendritic length of the granule dentate cells, as we discuss below, may be a contributing factor in the improved spatial memory and learning observed in those mice. Concerning the second aim of the current study, our histological results showed that the RS mice exhibited an altered dendritic outgrowth in both dentate granule cells and in the apical domain of the pyramidal CA3 neurons. These morphological abnormalities may contribute, at least partially, to an explanation of the cognitive disturbances exhibited by the RS group, as has been postulated by Grigoryan and Segal (2013) who found that PS disturbs the network properties of hippocampal neurons. The granule dentate cells are involved in receiving, filtering and transmitting neural inputs from the entorhinal cortex to the hippocampal pyramidal cells (Tamura, Sajo, Kakita, Matsuki, & Koyama, 2011); for this reason, any perturbation that alters the hippocampal network properties will impair hippocampal-dependent functions. On the other hand, the basal dendritic length of the CA3 pyramidal neurons did not differ between the stressed and the control mice. A similar result in the basal dendritic domain of pyramidal CA3 neurons has been found in offspring rats subjected to prenatal (Suenaga, Yukie, Gao, & Nakaahara, 2012) and postnatal (Watanabe, Gould, & McEwen, 1992) stress. In this regard, the differences exhibited between the apical and basal dendrites revealed the functional significance of the input organization along the dendrites (Martinez-Tellez et al., 2009) and thus, it is possible that the apical domain appears to be more vulnerable to the impact of PS than the basal domain. The results of the current study show that maternal exercise exerts a selectively beneficial effect only in the dendritic length of the granule dentate cells. Although we do not have an explanation for these results, it is most likely that these differential effects may be related to the period in which the granule dentate cells or CA3 pyramidal neurons are born and develop. An important process in neurogenesis occurs between G16 to G19 (Bayer, Altman, Russo, & Zhang, 1993), but the neurogenesis of dentate granule cells begins in prenatal life and continues during the postnatal period (Altman & Bayer, 1990). The late development of the dentate gyrus makes it particularly sensitive to environmental and experience-dependent structural changes, such as maternal exercise (Kim et al., 2007; Lee et al., 2006). Moreover, although the apical domain of the pyramidal neurons in the CA3 area did not change, it is possible that the significant increase in the dendritic length of the granule dentate cells, and possibly in their complexity, may compensate for the prenatal stress-related impairments in the hippocampal network properties.
Fig. 3. Effect of prenatal stress and maternal exercise on the total dendritic length of apical (A) and basal domains of pyramidal neurons (B) and dentate granule cells (C). Data are represented as the means ± SEM. C: control group; RS: restraint stress group; RS + VWR: restraint stress + voluntary wheel running group; ap < 0.0005 compared with C; bp < 0.05 compared with C; cp < 0.005 compared with RS + VWR; dp < 0.0001 compared with C (one-way ANOVA and post hoc Tukey´s test).
associated with postnatal injection of amyloid-β oligomers (AβOs), as shown by Klein et al. (2019). In their report, it was found that the offspring born from dams subjected to gestational exercise (swimming) show, at adult age, an augmentation of functional mitochondria and the expression of synaptophysin together with an intact object recognition memory and spatial learning. Third, there is experimental evidence showing that exercise is able to generate epigenetic changes and modify the gene expression in the dentate gyrus of the rodent hippocampus, together with an increase in
4.1. Conclusions The main findings of the current study are (i) maternal exercise during pregnancy prevents the spatial learning/memory impairments induced by PS in adolescent mice; (ii) maternal exercise during pregnancy only prevents the impairments on dendritic outgrowth in dentate granule cells of the prenatally stressed offspring. Considering the 5
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Fig. 4. Representative photomicrographs of Golgi-cox-stained pyramidal neurons (A) and dentate granule cells (B) from each group (C: control group, RS: restraint stress group, and RS + VWR: restraint stress + voluntary wheel running group). Bar: 50 μm.
and interpreted data. Cristian Gutierrez-Rojas participated in the critical revision of the manuscript for important intellectual content. Carlos Ancatén contributed to the acquisition of data, technical and material support. Rodrigo Pascual developed the original idea, the design and the protocol. Ethical statement All experimental procedures were performed in accordance with protocols approved by the Bioethics Committee of the Pontificia Universidad Católica de Valparaíso and with international guidelines. According with these statements, this study was conducted using a minimal number of animals.
Fig. 5. Kilometers ran per session by RS + VWR dams between G1 until G17. Data are means ± SEM.
aforementioned conclusions, further studies are needed to expand the current knowledge of the preventive effects of maternal exercise on the neurodevelopment of prenatally stressed offspring. In addition, we recognize that the small number of mothers undergoing stress and exercise may be a limitation of the present study; for that reason, we recommend that future studies may increase the number of subjects considering an adequate balance with bioethics and experimentation with animals.
Financial disclosure This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of Competing Interest
Author´s contribution
The authors declare no conflict of interest.
Please clarify and write who was responsible for: Carlos Bustamante wrote the manuscript, and is guarantor. In addition, he prepared the manuscript for re submission and the response to the reviewers. Carlos Bustamante and Cristian Gutiérrez-Rojas abstracted analyzed
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.npbr.2020.02.001. 6
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