BDNF expression in hippocampus following early life stress

Accepted Manuscript Title: Beneficial effects of environmental enrichment on behavior, stress reactivity and synaptophysin/BDNF expression in hippocampus following early life stress Authors: nullvgenia Dandi, Aikaterini Kalamari, Olga Touloumi, Rosa Lagoudaki, Evangelia Nousiopoulou, Constantina Simeonidou, Evangelia Spandou, Despina null. Tata PII: DOI: Reference:

S0736-5748(17)30320-9 https://doi.org/10.1016/j.ijdevneu.2018.03.003 DN 2242

To appear in:

Int. J. Devl Neuroscience

Received date: Revised date: Accepted date:

30-11-2017 7-3-2018 8-3-2018

Please cite this article as: Dandi, x395;vgenia, Kalamari, Aikaterini, Touloumi, Olga, Lagoudaki, Rosa, Nousiopoulou, Evangelia, Simeonidou, Constantina, Spandou, Evangelia, Tata, Despina x391;., Beneficial effects of environmental enrichment on behavior, stress reactivity and synaptophysin/BDNF expression in hippocampus following early life stress.International Journal of Developmental Neuroscience https://doi.org/10.1016/j.ijdevneu.2018.03.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Beneficial effects of environmental enrichment on behavior, stress reactivity and synaptophysin / BDNF expression in hippocampus following early life stress

Εvgenia Dandi1, Aikaterini Kalamari1, Olga Touloumi3, Rosa Lagoudaki3, Evangelia Nousiopoulou3, Constantina Simeonidou2, Evangelia Spandou2*, Despina Α. Tata1* 1

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Laboratory of Cognitive Neuroscience, School of Psychology, Aristotle University of Thessaloniki, Thessaloniki, Greece 2 Laboratory of Experimental Physiology, School of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece 3 Laboratory of Neuroimmunology, School of Medicine, Aristotle University of Thessaloniki, Greece

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*Contact Information: 1 Despina A. Tata, Ph.D., Laboratory of Cognitive Neuroscience, School of Psychology, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece. Tel.: +302310 997369, Fax: +302310 997384, E-mail: [email protected] 2

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Evangelia Spandou, MD, Ph.D., Laboratory of Experimental Physiology, School of Medicine, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece. Tel.: +302310 999049, Fax: +302310 999079, E-mail: [email protected]

Highlights

We examined the interaction between MS and EE on behavior,

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

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neuroendocrine stress response and BDNF/SYN expression. EE protected against the MS-related increased anxiety and spatial

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memory impairments. 

EE decreased corticosterone levels in MS rats following exposure to an

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acute stressor.

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EE restored the downregulation of BDNF and SYN expression in MS rats.

Abstract Exposure to environmental enrichment can beneficially influence the behavior and enhance synaptic plasticity. The aim of the present study was to investigate the

mediated effects of environmental enrichment on postnatal stress-associated impact with regard to behavior, stress reactivity as well as synaptic plasticity changes in the dorsal hippocampus. Wistar rat pups were submitted to a 3-hour maternal separation (MS) protocol during postnatal days 1 – 21, while another group was left undisturbed. On postnatal day 23, a subgroup from each rearing condition (maternal separation, no-

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maternal separation) was housed in enriched environmental conditions until postnatal day 65 (6 weeks duration). At approximately three months of age, adult rats

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underwent behavioral testing to evaluate anxiety (Elevated Plus Maze), locomotion (Open Field Test), spatial learning and memory (Morris Water Maze) as well as non-

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spatial recognition memory (Novel Object Recognition Test). After completion of

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behavioral testing, blood samples were taken for evaluation of stress-induced plasma

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corticosterone using an enzyme-linked immunosorbent assay (ELISA), while

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immunofluorescence was applied to evaluate hippocampal BDNF and synaptophysin expression in dorsal hippocampus. We found that environmental enrichment protected

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against the effects of maternal separation as indicated by the lower anxiety levels and the reversal of spatial memory deficits compared to animals housed in standard

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conditions. These changes were associated with increased BDNF and synaptophysin expression in the hippocampus. Regarding the neuroendocrine response to stress,

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while exposure to an acute stressor potentiated corticosterone increases in maternallyseparated rats, environmental enrichment of these rats prevented this effect. The

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current study aimed at investigating the compensatory role of enriched environment against the negative outcomes of adverse experiences early in life concurrently on emotional and cognitive behaviors, HPA function and neuroplasticity markers.1

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Abbreviations

Keywords: early stress, enriched environment, visuospatial learning, spatial memory, recognition memory, corticosterone.

1. Introduction

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It is well established that environmental factors during the postnatal period have long term effects on behavior, neuroendocrine function and neuronal plasticity

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(Bock and Braun, 2011; Stiles, 2011). Epidemiological and clinical studies support the idea that stress early in life may lead to psychopathology in adulthood (Anda et al., 2009; Felitti et al., 1998; Heim and Binder, 2012; Heim and Nemeroff, 2001;

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Palagini et al., 2015). A well-established animal model of early stress that is widely

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used over the last decades is the maternal separation (MS) paradigm. During the first

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postnatal days, survival of rat pups depends exclusively on maternal care. In addition,

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the role of tactile stimulation, nourishing and passive contact is considered crucial for

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the regulation of Hypothalamic-Pituitary-Adrenal (HPA) axis (Levine, 2002, 2001). Prolonged MS disrupts the normal interaction between mother and pups and

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affects brain and behavior. Reduction in neurogenesis (Korosi et al., 2012; Lajud et al., 2012) and glucocorticoid receptors’ density (Aisa et al., 2008; Enthoven et al.,

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2008a, 2008b) has been reported as a result of MS. Early life stress also reduces synaptic formation and enhances neuroendocrine stress response (Bock and Braun, 2011). At behavioral level, maternally separated rats show increased anxiety and

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depressive-like behaviors in adulthood (Aisa et al., 2007; Veenema et al., 2007; Vetulani, 2013; Wang et al., 2015a; Wigger and Neumann, 1999). Several studies BDNF: Brain-derived neurotrophic factor; CORT: Corticosterone; EPM: Elevated plus maze; EE: Environmental Enrichment; MS: Maternal separation; NMS: No maternal separation; OFT: Open field test; PND: Postnatal day; SYN: Synaptophysin

have also reported impairments in various cognitive functions (e.g., learning and memory), following early life stress (Chocyk et al., 2014; Conrad, 2010; Cui et al., 2006; Mcewen, 1997; Tata et al., 2015; Xiong et al., 2015). Based on existing evidence, these effects have been associated with alterations in the expression of synaptic plasticity markers, such as the brain-derived neurotrophic factor (BDNF) and

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synaptophysin (SYN). BDNF, a main neurotrophin in mammals’ central nervous system, is essential for cell survival and regulation of dendritic and synaptic plasticity,

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mainly in the hippocampus (Fumagalli et al., 2007; Lu et al., 2005), while synaptophysin, an indirect indicator of the synapse number, is a synaptic vesicle

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protein involved in neurotransmission (Valtorta et al., 2004). It has been shown that

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adult rats exposed to MS condition expressed reduced expression of BDNF and SYN

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in various brain regions (Andersen and Teicher, 2004; Choy et al., 2008; Lippmann et

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al., 2007; Roth et al., 2009).

In contrast to the effects of early stress, it is well established that specific

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housing conditions, such as environmental enrichment (EE), can exert a neuroprotective role (Baroncelli et al., 2010; Gelfo et al., 2011; Jha et al., 2011).

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Environmental enrichment (EΕ) promotes neurogenesis (Monteiro et al., 2014) and synaptogenesis (Rampon et al., 2000), enhances hippocampal LTP (Cortese et al.,

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2018) and increases dendritic arborization as well as spine density, thus facilitating neuronal communication (Leggio et al., 2005). In addition, it improves learning and

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memory (Frick and Fernandez, 2003; Harburger et al., 2007; Leggio et al., 2005; Rampon and Tsien, 2000) and protects from cognitive deficits caused by brain damage or exposure to stress (Hralová et al., 2013; Nozari et al., 2014; Schreiber et al., 2014; Wright and Conrad, 2008) or aging (Cortese et al., 2018). Furthermore, enriched housing promotes social interaction, locomotor activity and exploratory

behavior (Brenes Sáenz et al., 2006) and regulates emotional behavior. Rodents housed in EE show decreased anxiety and depressive-like behaviors in various behavioral tasks (Chapillon et al., 1999; Friske and Gammie, 2005; Ishihama et al., 2010; Nicolas et al., 2015). These effects seem to be associated with a number of neuronal and neuroendocrine changes that may, in turn, result in greater resistance to

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stress later in life (Connors et al., 2014; Crofton et al., 2015; Fox et al., 2006).

These findings support the hypothesis that EE could compensate for the

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detrimental effects of stress. In fact, existing evidence indicates that EE attenuates the chronic stress impact on behavior and hippocampal integrity, thus protecting against

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the stress-associated cognitive deficits, emotional dysregulation, dendritic atrophy and

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reduced neurogenesis (Hutchinson et al., 2012; Veena et al., 2009; Wright and

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Conrad, 2008). Similar to adult stress, restorative effects of EE have been reported in

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rodents submitted to prenatal (Laviola et al., 2008; Pascual et al., 2015; Yang et al., 2007) or juvenile stress (Ilin and Richter-Levin, 2009). To the best of our knowledge,

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there is limited information regarding the compensatory action of EE following early stress (do Prado et al., 2016; Francis et al., 2002; Koe et al., 2016; Vivinetto et al.,

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2013) and so far, no study has looked at the interaction of maternal separation and subsequent exposure to EE conditions concurrently at cognitive and emotional

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behavior, HPA function and markers of synaptic plasticity (BDNF, SYN) during adulthood. The critical importance of the first postnatal weeks to normal development

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(Rice and Barone, 2000), further supports the significance of exploring the hypothesis that EE experience may overcome behavioral and neurobiological deficits induced by early postnatal stress. The purpose of the current study was to explore the hypothesis that EE during adolescence may protect against the negative outcomes of maternal separation on

spatial and non-spatial learning and memory, anxiety, as well as neuroendocrine stress response during adulthood. Furthermore, given the essential role of BDNF and SYN in cognitive functions and emotional behavior, we aimed at exploring the expression of these two proteins in the hippocampus, a structure particularly vulnerable to stress.

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2. Materials and Method 2.1. Animals

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Female Wistar rats on the second gestational week were individually housed until delivery. The day of birth was designated as postnatal day 0 (PND0). Totally, 47

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neonates were included in the experiment and all animals participated in behavioral

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testing, as well as in corticosterone and BDNF/synaptophysin measurements. In order to ensure normal growth and development, body weight measurements of neonates

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were taken on the 3rd, 8th, 14th and 21st postnatal days. All animals were maintained on a 12:12 light/dark cycle (08:00 light / 20:00 dark) and standard temperature (22±2⁰C),

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with food and water available ad libitum. Handling of the pups and behavioral testing were performed by the same personnel since familiarity with the experimenter

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increases consistency in animal testing (van Driel and Talling, 2005). Εxperimental

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procedures were conducted in accordance to the Institutional Animal Ethics EL 54 BIO 20.

2.2. Rearing conditions

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On PND1 litters were assigned randomly to no-maternal separation (NMS)

(NS= 23) or maternal separation (MS) (N = 24) conditions. The pups of the NMS condition remained undisturbed in their cages with their dams until weaning (PND 23). The MS condition involved a daily 3 hr separation of the pups from their dams during the three postnatal weeks (PND1-21) (Huot et al., 2001). The maternal

separation procedure took place between 0900 and 1400 h and was performed as previously described (Tata et al., 2015). Briefly, dams were first removed from their cages, followed by the pups, which were placed, as a litter, in plastic containers. Upon completion of the 3hr separation period, pups were returned to their home cage, followed by their dam. Litter size ranged from 6-10 rats.

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2.3. Post-weaning housing conditions

On PND 23 rats from each of the two rearing conditions were randomly

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assigned to either standard housing (SH) (N = 22) or enriched environment (EE)

conditions (N = 25). In the SH condition, rats in same-gender pairs were housed in

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standard laboratory cages. In the EE condition (PND23-65), same-gender groups of 5-

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7 rats were housed in large cages (60cm x 45cm x 76cm) containing various non

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chewable toys, climbing platforms, tunnels and running wheels (Griva et al., 2017).

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To promote exploratory and novelty-seeking behavior, every 3-4 days toys as well as food and water containers were moved to a new location in the cage. Once a week the

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cages were cleaned and platforms, tunnels and running wheels were rearranged, and toys were replaced by new ones. On PND 65, animals of the EE condition were

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transferred in pairs to standard laboratory cages, where they were kept until behavioral testing began.

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The final four groups that emerged after experimental manipulations were the

following: a) non-maternally separated rats housed in standard housing conditions

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(NMS/SH; N = 10), b) non-maternally separated rats housed in enriched environment condition (NMS/EE; N = 13) c) maternally-separated rats housed in standard housing conditions (MS/SH; N = 12), d) maternally-separated rats housed in enriched environment (MS/EE; N = 12). The animals of each experimental group were obtained from 2 to 3 litters.

2.4. Behavioral testing Behavioral testing took place at approximately 2.5 months of age in order to examine emotional and exploratory behavior as well as learning and memory. Anxiety-like behavior was assessed by the Elevated Plus Maze (EPM) (PND 67), locomotion and exploratory behavior was estimated by the Open Field Test (OFT)

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(PND 69), while non-spatial and spatial (visual) learning and memory were assessed

by the Novel Object Recognition (NORT) (PND 70) and the Morris Water Maze

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(MWM) (PND72-76) tests, respectively. Behavioral testing occurred during the light

cycle between 09:00 – 15:00. Rat behavior was recorded by means of a ceiling-

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mounted camera placed 160 cm above the experimental arena. Animals’ swimming

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behavior in the MWM was analyzed by a data acquisition system (Ethovision v.2.3,

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Noldus Information Technology), while in case of NORT and EPM testing recoded

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behavior was manually scored by two researchers blind to experimental conditions. Experimenters were blind to experimental conditions both during behavioral

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testing and data analysis. All experimental apparatuses were cleaned thoroughly with a 25% ethanol solution after each trial to eliminate odor cues.

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2.4.1 Elevated Plus Maze (EPM)

EPM is a behavioral test that is used to measure anxiety (Handley and

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Mithani, 1984) based on rodents’ innate preference of dark and enclosed spaces and their unconditioned fear of height /open areas. The apparatus was cross-shaped, with

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two open and two closed arms (50cm x 10cm); the height of the walls was 40cm and the maze was positioned 50cm from the floor. The animals were placed in the open central square (10cm x 10cm) formed at the four arms intersection, facing the open arm opposite to experimenter, and were left undisturbed for 5 minutes to explore the maze. Increased time and number of entries in the open arms (Walf and Frye, 2007)

as well as smaller occurrence of risk assessment behaviors (i.e., stretch attend posture, closed arms returns) (Cruz et al., 1994; Doremus et al., 2006; Rodgers and Dalvi, 1997) indicate lower levels of anxiety. In the present study assessment of anxiety was based on a) the ratio of open arms entries (open arms entries/total entries), b) the ratio of the open arms time (open arms time/total arms time), c) the number of stretch

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attend posture/SAP (the animal’s two hind legs remain in an arm while it elongates its head and shoulders out of the arm and then returns to its initial position) and d) the

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number of returns to closed arms (the animal’s half front body exits a closed arm followed by return to the closed arm). In addition, the total number of arm entries was

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estimated as an index of general activity.

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2.4.2. Open Field test (OFT)

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Open field test is used to measure locomotion and exploration (Lau et al., 2008). The apparatus used in the current study consisted of a wooden square arena

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(100cm x 100cm) surrounded by 40 cm high wall to prevent escape. The arena’s floor

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was divided into 16 equally sized sectors (25cm x 25cm). The four squares in the center formed the inner zone of the arena, while the twelve outer squares formed the

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outer zone (peripheral squares). Rats were placed facing the wall and they were allowed to freely explore the arena for 6 minutes.

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Two behavioral parameters were analyzed in order to estimate general activity

and exploration, the number a) of square visits (ambulation) (Bubser et al., 1992;

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Suchecki et al., 2000) and b) rearings (Gamberini et al., 2015; Kulesskaya and Voikar, 2014), respectively. A square visit was recorded if at least half of animal’s torso was in a sector, while rearing when animals kept hind paws on the floor with the front limbs off it. 2.4.3. Novel Object Recognition Task (NORT)

This task was administered in order to examine the non-spatial episodic memory (Ennaceur and Meliani, 1992). It took place in a wooden open field arena (see above, 2.4.2.), and it was preceded by two habituation sessions (6 min duration each). The testing consisted of two trials separated by a 70 min delay. In trial 1

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(sample phase; 4 min duration), the rats were allowed to freely explore two identical objects that were placed at the two adjacent back corners of the arena at a distance of

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25cm from the wall. The objects used were made of metal or glass and were heavy enough so that they could not be displaced by the rats. In addition, we ensured that the

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objects were of similar attractiveness for the rats. Object exploration was

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operationally defined as directly attending to the object with the head no more than 2

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cm from the object (Ennaceur and Delacour, 1988). In trial 2 (choice phase; 3 min

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duration) one of the two objects was replaced by a new one of similar height (Bevins and Besheer, 2006; Ennaceur and Delacour, 1988). Under normal conditions, rats tend

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to explore more the novel object, a preference that is indicative of non-spatial memory. The length of the specific inter-trial interval (ITI) was based on finding that

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under control conditions rats can still recognize the novel object and subsequently the danger of floor effects due to longer ITIs is eliminated (Ennaceur et al., 1997). In the

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current study, we estimated the discrimination ratio, defined as the exploration time of the novel object in trial 2 divided by the total time exploring both objects in the same

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trial. Exploration time of each object during the sample phase was also calculated in order to ensure that any preference shown for the new object would be due to its novelty and not to difference in the exploration during the sample phase. 2.4.4. Morris Water Maze (MWM)

Morris Water Maze is used to estimate spatial learning and memory (Morris, 1984). In the present study the maze consisted of a plastic circular tank (1.6 m/diameter x 50cm/height) filled with water in standard temperature (25±1⁰C) and was virtually divided into four quadrants: north-west (NW), north-east (NE), southwest (SW) and south-east (SE). An escape platform (10cm x 10cm) made of

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transparent Plexiglas was placed in the middle of one of the quadrants (SW), half-way between the center and the wall, and 1.5cm below the surface. Pictures on the walls

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and the furniture of the room were used as extra-maze cues. Animals entered the maze facing the wall of the pool and were allowed to swim for 60 sec in order to locate the

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submerged platform. In case an animal failed to locate the platform within the

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specified period, it was gently guided to the platform by the experimenter; once the

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animal had reached the platform it was allowed to remain there for 15 sec. The interval between trials of each day was 1 min (including the 15 sec period spent on the

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platform). The animals’ swimming behavior was recorded by a video camera

Technology).

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connected to a data acquisition system (Ethovision v.2.3, Noldus Information

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2.4.4.1. Spatial acquisition phase (spatial learning): In this phase animals had to learn

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to navigate to the platform based on external cues. Spatial acquisition phase lasted 4 days and animals were given 4 trials/day (totally 16 trials). The platform was permanently located in the SW quadrant of the maze and start position was different

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for each trial. A semi-random approach of start positions for each trial was chosen (Vorhees and Williams, 2006). Time to reach the platform (escape latency; sec) was measured to estimate visuospatial learning. Normally, animals record less time to locate the platform from day to day as a result of learning (Morris, 1984). Swim speed (velocity; cm/sec) was also estimated in order to ensure that any differences in time to

locate the platform was attribute to learning acquisition and not to differences in the swimming speed. 2.4.4.2. Probe trial: In order to assess spatial memory, a probe trial was administered 24 h after the last acquisition day. During this trial (60 sec), the escape platform was removed and each animal entered the maze from a novel position (180º from the

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platform position during the acquisition phase). The time spent to the target quadrant

(SW) as well as the frequency of entries in the area where the platform used to be

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located during the acquisition phase were used as indices of spatial memory (D’Hooge and De Deyn, 2001; Shinohara and Hata, 2014).

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2.5. Corticosterone assessment

One week after completion of behavioral testing blood was collected from the

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rats’ carotid artery in order to determine corticosterone levels before and after stress

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induction. Stress was induced by placing the rat cage on a rotating disk (78 revolutions per minute) for a 15 min period. According to existing evidence, cage

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rotation for durations of 10 or 20 min can produce significant increase in circulating corticosterone concentrations (Gein and Sharavieva, 2016; Riley, 1981). In order to

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avoid circadian variability, blood sampling was carried out between 0900 and 1100 for all control and experimental groups. Samples were centrifuged (12.000 r.p.m. for

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10 min at 4 ℃) and plasma was extracted and maintained at -80℃ until assay. Corticosterone concentrations were assessed using an enzyme-linked immunosorbent

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assay (ELISA) kit (MB Biomedicals, 07DE9922). 2.6. Tissue preparation and Immunofluorescence All animals aged approximately 3.5 months were euthanized following anaesthetization.

Brains

were

removed

immediately

and

post-fixed

(4%

paraformaldehyde in 0.1 M phosphate-buffer saline, 3 X 24 h at 4℃). Coronal blocks

were gradually hydrated and embedded in paraffin. Serial coronal sections of 5μm thick were taken at the level of dorsal hippocampus (-3.24mmto -3.36mm posterior to bregma) (Paxinos and Watson, 2007). Expression of BDNF and SYN on these sections was evaluated using immunofluorescence. Following deparaffinization and hydration, steamer was used for antigen retrieval (pH = 6, 1h). Next, sections were

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incubated to blocking buffer (10% Fetal Bovine Serum, 2% Normal Goat Serum, 30 min) and treated with a primary antibody against BDNF (1:400, rabbit polyclonal,

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Santa Cruz), or synaptophysin (1:100, mouse monoclonal, CloneSY38, DAKO) overnight (4οC). Depending on the primary antibody, sections were exposed to goat

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anti-rabbit IgC (Biotium 488) or goat Anti-MouseIgG (Biotium 555) for BDNF or

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SYN, respectively. Nuclei were counterstained with DAPI and mounted with the

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corresponding Biotium medium (Biotium 23004).

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Tissue images were captured from sections with a fluorescent microscope Zeiss Axioplan-2 using a CCD camera (Nikon DS-5M). Two sections per animal

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were chosen with care to allow comparison of similar regions across experimental conditions. Immunoreactivity for BDNF and SYN was estimated within the CA3,

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CA2 and CA1 stratum radiatum and the dentate gyrus (DG) molecular layer with 40x objective lens. Approximately sixteen microscopic fields per section/animal were

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analyzed. Expression of BDNF and SYN was measured using the ImageJ software (version1.45b, NIH) and is presented as mean Integrated Density (arbitrary density

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units) of all four hippocampal subregions. 2.7. Statistical analyses All statistical analyses were performed using the statistical program SPSS Statistics (v. 22). Effects of the two experimental conditions (rearing: NMS, MS and housing: SH, EE) on behavior (elevated plus maze, open field test, novel object

recognition,

MWM

retention

phase),

plasma

corticosterone

levels

and

immunofluorescence markers (BDNF, SYN) were tested using 2 X 2 ANOVAs with rearing and housing as the between-subjects factors. A three factor repeated-measures ANOVA (between-subjects factors: rearing, housing; within-subjects factor: day) was applied to analyze the latency to locate the platform (MWM; acquisition phase). Body

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weight measurement during the first three postnatal weeks (3rd, 8th, 14th, 21st postnatal days) were analyzed by a two factor repeated-measures ANOVA (between-subjects

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factors: rearing; within-subjects factor: postnatal day). Pairwise comparisons with Bonferroni correction were used in order to explore simple effects (i.e., effect of each

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factor within each level of the other factor). Data are presented as mean values

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(±SEM). Statistical significance was set at a < .05 for all measures.

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3. Results 3.1. Body weight

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A repeated-measures analysis showed a significant increase of body weight over the three postnatal weeks [F (3, 126) = 1593.8, p < .001, partial η² = .974; 3rd day:

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9.19 (.255), 8th day: 17.43 (.481), 14th day: 30.65 (.783), 21st day: 47.1 (.974)]. Maternal separation did not affect the body weight [F (1, 42) = 1.602, partial η² =

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.037] neither interacted with postnatal day [F (3, 126) = 2.541, p > .05, partial η² = .057].

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3.2. Elevated Plus Maze Ratio of open arm entries: Analysis revealed a significant main effect of EE [F

(1, 43) = 11.68, p < .01, partial η² = .214; SH: .262(.021), EE: .361(.02)], but not of MS condition [F (1, 43) = .23, p > .05, partial η² = .005, NMS: .305(.021.), MS: .319(.02)] (Fig. 1A). Pairwise comparisons to explore the significant MS x EE

interaction [F (1, 43) =47.97, p < .001, partial η² = .527] showed that while maternal separation in standard-housed animals decreased the number of entries [F (1, 43) = 19.47, p < .001, partial η² = .312, NMS/SH: .355 (.031) vs. MS/SH: .169(.028)], this effect was completely reversed by EE housing [F (1, 43) = 55.13, p < .001, MS/SH: .169 (.028) vs. MS/EE: .468 (.028)].

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Ratio of time spent in open arms: Both MS and EE conditions affected time spent in open arms [MS: F (1, 43) = 6.90, p < .05, partial η² = .138, NMS: .226(.022.),

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MS: .306 (.021); EE: F (1, 43) = 15.87, p < .001, partial η² = .270, SH: .205(.022.), EE: .327(.021)] (Fig. 1B).The two manipulations significantly interacted with each

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other [MS x EE: F (1, 43) = 52.85, p < .001, partial η² = .551]. Maternal separation in standard-housed condition decreased the time spent in open arms [F (1, 43) = 19.47, p

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< .01, partial η² = .19, NMS/SH: .276 (.033) vs. MS/SH: .134 (.030)]. Housing of MS

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rats in EE condition significantly reversed the decreased time seen in MS animals that lived in SH conditions [F (1, 43) = 65.264, p < .001; MS/SH: .134 (.030) vs. MS/EE:

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.478 (.030)].

Closed arm returns: Housing conditions affected the specific behavior, with

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animals exposed to EE performing less returns compared to SH animals [F (1, 43) = 23.48, p <.001, partial η² = .353] (Fig. 1C). Results revealed a tendency of the effect

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of rearing on closed arm returns, which did not reach statistical significance [F (1, 43) = 3.73, p =.06, partial η² = .08]. The two factors interacted with each other [F (1, 43)

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= 8.23, p < .01, partial η² = .161]. Specifically, MS increased closed arms returns for SH animals [F (1, 43) = 10.793, p < .01; NMS/SH: 2.5 (.525) vs. MS/SH: 4.833 (.479)], but exposure of MS rats to EE condition totally reversed this effect [F (1, 43) = 16.898, p < .001; MS/SH: 4.833 (.479) vs. MS/EE: 1.083 (.479)]

Stretch Attend Posture (SAP): There were no significant effects of number of SAPs caused by either the rearing or housing manipulations [MS: F (1, 43) = .276, p > .05, partial η² = .006; EE: F (1, 43) = 2.28, p > .05, partial η² = .05]. However, the effect of EE differed as a function of rearing, as indicated by the significant interaction [F (1, 43) = 17.78, p < .001, partial η² = .292]. MS increased the SAPs in

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the standard-housed condition [F (1, 43) = 10.532, p < .01, partial η² = .197; NMS/SH: 2.8 (.785) vs. MS/SH: 6.25 (.717)]. While EE condition did not affect

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occurrence of this behavior in the NMS rats [F (1, 43) = 3.556, p > .05, partial η² = .076], it caused a significant decrease in the MS groups [F (1, 43) = 16.898, p < .001,

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partial η² = .282; MS/SH: 6.25 (.717), MS/EE: 2.083(.717)] (Fig. 1D).

Total arm entries: Neither MS nor EE conditions affected the total arm entries

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[MS: F (1, 43) = .110, p > .05, partial η² = .003, NMS: 16.45 (.641), MS: 16.75

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(.622); EE: F (1, 43) = .092, p > .05, partial η² = .002, SH: 16.47 (.653), EE: 16.74 (.61)].

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3.3 Open Field

Rearings (vertical activity): Analysis of rearings performed in the outer zone

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revealed a significant effect of EE [F (1, 43) = 4.943, p < .05, partial η² = .103]. EE treated animals performed more rearings than SH treated animals [EE: 12.18 (1.22),

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SH: 8.22 (1.3)]. MS condition did not affect vertical activity [F (1, 43) = 2.264, p > .005, partial η² = .050]. The effect of EE was not a function of rearing, as indicated by

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the non-significant interaction [F (1, 43) = .064, p > .05, partial η² = .001] (Fig. 2A). Ambulation: There were no significant effects for the total number of square

visits caused by either the MS [F (1, 43) = 1.373, p > .05, partial η² = .031] or EE manipulations [F (1, 43) = 2.103, p > .05, partial η² = .047], and the two factors did not interact with each other [F (1, 43) = .101, p > .05, partial η² = .002] (Fig. 2B).

3.4 Novel Object Recognition Task (NORT) MS or EE did not differentiate the groups in exploration time of the object A1 [MS: F (1, 43) = 3.102, p > .05, partial η2 = .067; EE: F (1, 43) = .188, p > .05, partial η2 = .004] or object A2 [MS: F ( 1, 43) = 2.431, p > .05, partial η2= .054; EE: F (1, 43) = .520, p > .05, partial η2 = .012] during the sample phase (trial 1). Analysis of

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the exploration time from the choice phase (trial 2) indicated no significant effect of MS [F (1, 40) = .494, p > .05, partial η2 = .012], EE [F (1, 40) = 1.635, p > .05, partial

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η2 = .039] or a significant interaction between the two factors [F(1, 40) = 1.48, p >

.05, partial η2 = .036] on discrimination ratio (Fig. 3). This finding indicates that all

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animals reacted similarly to novelty regardless of experimental conditions.

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3.5 Morris Water Maze

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Spatial learning: Spatial learning was significantly affected by EE, with EE treated animals being faster to locate the platform compared to the SH condition [F (1,

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43) = 62.47, p < .001, partial η² = .592] (Fig. 3). The rearing condition exerted no

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influence on latency [MS: F (1, 43) = .848, p > .05, partial η² = .019] neither interacted with EE [MS x EE: F (1, 43) = 1.419, p > .05, partial η² = .032]. The

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performance of rats was improved over the 4-day period [day: F (3, 129) = 174.309, p < .001, partial η² = .802]. Bonferroni’s pairwise comparisons revealed a gradual

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decrease in latency from the first to the third day (p < .001), but no significant difference was found between the third and the fourth day (p > .05). The “day” factor

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interacted with housing [day x EE: F (3, 129) = 6.42, p< .001, partial η² = .13]. In order to ensure that differences in latency to locate the platform were not

due to differences in swim speed, a two-way ANOVA (MS x EE) was conducted on mean velocity/day. No statistically significant difference was found as a result of MS [day 1: F (1, 43) = 3.72, p >.05, partial η² = .08, day 2: F (1, 43) = 4.01, p > .05,

partial η² = .085), day 3: F (1, 43) = 3.17, p >.05, partial η² = .07, day 4: F (1, 43) = .08, p = .77, partial η² = .002] or EE [day 1: F (1, 43) = 1.86, p > .05, partial η² = .041, day 2: F (1, 43) = .44, p > .05, partial η² = .011, day 3: F (1, 43) = .82, p > .05, partial η² = .019, day 4: F (1, 43) = 2.76, p > .05, partial η² = .06]. Probe trial: A two-way ANOVA revealed a significant effect for MS on time

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spent in the target quadrant (SW) [F (1, 43) = 6.74, p < .05, partial η² = .135] as well

as on frequency of entries into the platform area [F (1, 43) = 9.113 p < .01, partial η² =

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.175] (Fig. 4). MS animals spend less time and did fewer entries than the non-

maternally separated animals. The EE condition did not affect any of the two measures [time: F (1, 43) = .43, p > .05, partial η² = .01; frequency: F (1, 43) = .866, p

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> .05, partial η² = .020]. Interestingly, housing in EE seems to protect against stress-

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induced impairments, as indicated by the significant interaction between MS and ΕΕ

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for the duration [F (1, 43) = 7.57, p < .01, partial η² = .150] and frequency of entries

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[F (1, 43) = 4.474, p < .05, partial η² = .094]. Specifically, MS rats raised in standard

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housing conditions exhibited poorer performance compared to NMS rats as indicated by duration time [F (1, 43) = 13.395, p < .01; NMS/SH: 23.544 (1.183) vs. MS/SH:

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17.68 (1.08)] and frequency [F (1, 43) = 12.349, p < .01; NMS/SH: 3.30 (1.418) vs. MS/SH: 1.33 (1.154). Housing of maternally separated rats in EE reversed this effect

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[duration: F (1, 43) = 5.93, p < .05; MS/SH: 17.68 (1.08) vs. MS/EE: 21.42 (1.080); frequency: F (1, 43) = 4.78, p < .05, MS/SH: 1.33 (1.154) vs. MS/EE: 2.50 (1.243)].

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As far as the time spent in the remaining (non-target) quadrants (NE, NW, SE), there was no difference as a function of MS or EE in time spent in the a) NE quadrant [MS: F (1, 43) = 1.24, p > .05, partial η² = .028; EE: F (1,43) = .012, p > .05, partial η² = .00; NMS/SH = 11.15 (1.21), NMS/EE = 12.05 (1.18), MS/SH = 13.53 (1.36), MS/EE = 12.37 (1.02)], b) NW quadrant [MS: F(1, 43) = .057, p > .05, partial η² = .001; EE:

F (1, 43) = .417, p > .05, partial η² = .01; NMS/SH = 11.58 (0.65), NMS/EE = 13.41 (1.32), MS/SH = 14.57 (1.62), MS/EE = 11.04 (1.26)] and c) SE quadrant [MS: F (1, 43) = .819, p > .05, partial η² = .019; EE: F (1,43) =.006 , p > .05, partial η² = .00; NMS/SH = 12.34 (1.07), NMS/EE = 12.28 (1.51), MS/SH = 13.41 (1.32), MS/EE =

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13.68 (1.38)].

3.6. Plasma Corticosterone Analysis

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Basal levels: There were no significant effects for the basal corticosterone

levels by either MS [F (1, 38) = 1.04, p > .05, partial η² = .027] or EE manipulations [F (1, 38) = .028, p > .05, partial η² = .001], and the two factors did not interact with

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each other [F (1, 38) = .000, p > .05, partial η² = .000; NMS/SH:25.58 (6.695),

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NMS/EE:24.59 (5.316), MS/SH:19.73 (5.484), MS/EE:18.11 (4.854)].

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Stress-induced levels: Analysis revealed a significant effect of housing

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condition on corticosterone elevations in response to acute stress [F (1, 41) = 6.168, p < .017, partial η² = .131], with the SH rats expressing significantly higher levels than

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the animals of the EE manipulation (Fig. 5). The effect of EE was a function of MS as

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indicated by their significant interaction [MS x EE: F (1, 41) = 7.920, p < .01, partial η² = .162]. Pairwise comparisons revealed that maternal separation caused significant

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elevations in animals of the SH condition compared to NMS rats [F (1, 41) = 4.887, p < .05; NMS/SH: 261 (20.102) vs. MS/SH: 321.167 (18.35)]. On the contrary, exposure to EE blocked this effect [F (1, 41) = 13.788, p < .01, partial η² = .252;

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MS/SH: 321.167 (18.35) vs. MS/EE: 220.100 (20.102)].

3.7. Immunoreactivity of BDNF and Synaptophysin in the hippocampus BDNF: Analysis of variance revealed a significant main effect of MS on the expression of hippocampal BDNF [F (1, 19) = 9.134, p < .001, partial η² = .325], with

MS animals expressing lower levels compared to NMS (Fig.6). In addition, immunoreactivity was significantly higher in animals that were housed in enriched environment, as indicated by the main effect of the EE condition [F (1, 19) = 27.068, p < .001, partial η² = .588]. In NMS condition, BDNF expression was significantly higher in rats that were exposed to enriched conditions compared to standard housing

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[F (1, 19) = 7.470, p < .05, NMS/SH: 21.64 (1.91) vs NMS/EE: 28.72 (1.74), partial

η² = .282]. Pairwise comparisons revealed that maternal separation significantly

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reduced the BDNF levels in SH animals [F (1, 19) = 9.545, p < .001, partial η² = .334,

NMS/SH: 21.64 (1.91) vs.MS/SH: 13.9 (1.62)]. Environmental enrichment of maternally separated rats counteracted this MS-associated decrease [F (1, 19) =

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21.696, p < .001, partial η² = .533, MS/SH: 13.9 (1.62) vs MS/EE: 25.57 (1.91)].

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Synaptophysin (SYN): Both MS and EE conditions significantly affected the SYN immunoreactivity in the hippocampus [MS: F (1, 19) = 14.172, p < .01, partial

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η² = .427; EE: F (1, 19) = 25.848, p < .001, partial η² = .57], with MS decreasing and

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EE increasing its levels (Fig. 7). Additionally, environmental enrichment significantly increased SYN expression in the non-maternally separated rats [F (1, 19) = 12.769, p

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< .01, NMS/SH: 27.59 (2.21) vs NMS/EE: 38.31 (2.02), partial η² = .402]. In SH rats, maternal separation significantly decreased SYN expression [F (1, 19) = 7.125, p <

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.05, partial η² = .273; NMS/SH: 27.59 (2.85), MS/SH: 19.84 (1.74)]. Housing of maternally-separated rats in enriched conditions reversed this effect [F (1, 19) =

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13.093, p < .01, partial η² = .408; MS/SH: 19.84 (1.74) vs. MS/EE: 30.34 (2.80)].

4. Discussion It is well known that environmental conditions can exert a strong influence on brain development, behavior, and neuroplasticity. In particular, early adverse

experiences may lead to psychopathology and cognitive impairments that can persist until adulthood, while environmental enrichment (EE) appears to have a beneficial role on different aspects of brain function and behavior. Τhe aim of the present study was to explore whether Environmental Enrichment (EE) during adolescence can counterbalance the negative impact of early stress in the form of maternal separation

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(MS). In particular, we investigated the effects of these environmental manipulations

as well as their interaction on cognition, emotionality, stress reactivity and expression

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of BDNF and synaptophysin in the hippocampus.

Effect of maternal separation and environmental enrichment on anxiety and

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stress reactivity

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Emotionality was evaluated by the EPM task, a reliable behavioral test of

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anxiety. Analysis of four behavioral indices (open-arms time and entries, closed-arms

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returns and SAPs) showed that daily MS during the first three postnatal weeks increased anxiety-like behavior during adulthood. Specifically, our data suggest that

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MS potentiated the standardly housed (SH) rodent’s unconditioned fear towards open spaces and increased closed arm returns as well as SAPs, a behavioral profile

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indicative of increased anxiety. The reduced open-arms preference seems not to be

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associated with a general decrease in activity, as indicated by the comparable ambulation scores and arm entries in our Open Field and EPM task, respectively, among the MS and NMS groups. Specifically, there was no significant difference

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between NMS and MS groups with respect to the horizontal (ambulation) or vertical (rearing) activity, a finding also reported previously (Markostamou et al., 2016; Shalev and Kafkafi, 2002; Vivinetto et al., 2013). Interestingly, post-weaning housing of maternally separated rats in EE conditions acted beneficially, decreasing significantly the risk assessment behaviors

(i.e., stretch attend posture, closed arms returns) while increased open arms entries and time. These behavioral effects of MS and EE conditions appear to be mediated by alterations in the HPA axis reactivity. Indeed, maternally separated rats that were housed in standard post-weaning conditions (MS/SH group) expressed significantly higher levels of corticosterone in response to a stress challenge as adults. On the

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contrary, housing in EE condition (MS/EE group) counterbalanced the MS effects,

thus attenuating adrenocortical responses to levels similar to those of the NMS group.

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It should be also reported that basal corticosterone concentrations did not differ significantly as a function of rearing or housing.

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Our findings are in accordance with previous studies reporting the detrimental

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effects of early adversity on emotionality and stress reactivity. In fact, it has been

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shown that MS over the first 2-3 postnatal weeks significantly increases anxiety and

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depression-like behaviors and enhances HPA responses to stressors during adulthood (Aisa et al., 2007; Kalinichev et al., 2002; Koe et al., 2016; Liu et al., 2000; Veenema

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et al., 2007). In our experiment, the potentiated corticosterone increase of MS/SH rats in response to an acute stressor (i.e., cage rotation) was not associated with elevated

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basal corticosterone in MS/SH compared to NMS/SH groups. This finding is in accordance with studies reporting no differences in basal concentrations in animals

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previously exposed to early-life stress (Knuth and Etgen, 2007; Koe et al., 2016; Rüedi-Bettschen et al., 2005; Slotten et al., 2006; Wigger and Neumann, 1999). Given

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the significant role of the hippocampus, particularly its glucocorticoid receptor (GR) system, in the HPA negative feedback mechanism, it may be suggested that hippocampal alterations may be associated with the potentiated stress response. In fact, decreases in the GR density and mRNA expression have been reported following early stress (Aisa et al., 2008; Enthoven et al., 2008b). This downregulation appears to

dysregulate the negative feedback mechanism, thus leading to further elevations of plasma corticosterone and increased anxiety (Sampedro-Piquero et al., 2014). Existing data suggest that glucocorticoids elevations play an important role in the effects of MS. Specifically, both recognition memory deficits and depressive-like behavior are completely reversed after administration of a GR antagonist (Aisa et al., 2008, 2007),

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while treatment with the corticosterone synthesis inhibitor, metyrapone, reduces the increased vulnerability to kindling epileptogenesis seen in maternally separated rats

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(Koe et al., 2014).

Contrary to early stress, EE appears to have beneficial behavioral and

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neurobiological effects. A body of evidence suggests that EE housing attenuates

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anxiety and HPA-mediated endocrine responses evoked by stressors in adulthood

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(Chapillon et al., 1999; Fox et al., 2006), while it increases GR expression in

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hippocampal areas (Vivinetto et al., 2013). The regulatory impact of EE is also supported by studies of reduced anxiety in animal models of anxiety, depression or

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mental disorders (e.g., schizophrenia and ADHD) (Brenes Sáenz et al., 2006; Ishihama et al., 2010; Nicolas et al., 2015) as well as in gestational inflammation-

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induced HPA axis hyperactivity in young rats (Connors et al., 2014). The majority of existing data highlighting the compensatory role of EE mainly refer to chronic stress

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paradigms administered during adulthood. Specifically, EE housing prior to or following periods of adult chronic stress reduces emotionality and HPA activity to an

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acute stressor (Wright and Conrad, 2008). Recently, it has been shown that a short episode of EE during adulthood reduces anxiety-like behavior in MS rats (Koe et al., 2016). The increases in GR expression seen in EE-housed rats that were subsequently exposed to both chronic and acute stress as adults appear to contribute to a more adaptive response to chronic stress (Zanca et al., 2015). To the best of our knowledge,

so far only one study has investigated the role of EE on enhanced HPA reactivity in maternally-separated rats, reporting a total reversal of corticosterone to normal levels under conditions of EE (Francis et al., 2002). Effect of maternal separation and environmental enrichment on cognitive function

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Behavioral testing in the Morris Water Maze (MWM) revealed a beneficial effect of EE on visuospatial learning during adulthood. The groups that were housed

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in post-weaning EE conditions exhibited shorter latencies, compared to the SH rats, to locate the platform, an indication of improved spatial acquisition. Our findings are in

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line with previous studies demonstrating the beneficial effects of environmental

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enrichment on cognitive function. In fact, EE has been associated with improvement

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in spatial learning, memory, and orientation in adult and aged rodents (Frick and

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Fernandez, 2003; Harburger et al., 2007; Hullinger et al., 2015; Leggio et al., 2005) as well as protection against age-associated impairment in LTP induction (Stein et al.,

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2016).

MS did not impair spatial acquisition, a finding also reported by other studies

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following postnatal stress (Aisa et al., 2009; Akatsu et al., 2015; Grace et al., 2009). However, it did impair spatial memory, as indicated by our probe trial data. These

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long term negative consequences in memory (spatial or fear-associated) are in line with previous reports demonstrating spatial and fear-associated memory impairments

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following maternal separation (Chocyk et al., 2014; Sousa et al., 2014; Xiong et al., 2015). This finding may imply that spatial memory is more vulnerable than visuospatial learning to the effect of early adversity, as it has been also suggested regarding the effects of chronic stress (Conrad, 2010). In fact, it has been reported that the spatial learning deficits seen in juvenile rats following postnatal social isolation

are restored in adulthood, an over-time improvement possibly associated with compensatory changes that take place (i.e., damaged dendrites are repaired, new ones are sprouting) within the hippocampus (Frisone et al., 2002). Interestingly, post weaning EE offered total protection against the spatial memory deficits associated with MS. While MS rats spent less time in the target

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quadrant and made fewer frequencies of entries into the platform area, compared to NMS group, EE completely reversed this effect. As far as the impact of EE on spatial

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memory of MS rats, previous findings also support its compensatory role against

cognitive deficits following chronic adult (Hutchinson et al., 2012; Veena et al., 2009;

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Wright and Conrad, 2008), juvenile (Ilin and Richter-Levin, 2009) or early stress

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induced by MS or limited nesting/bedding material (Cui et al., 2006; do Prado et al.,

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2016; Vivinetto et al., 2013). However, the present study is the first to demonstrate

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the ameliorating role of EE against spatial memory impairments in rodents that experienced early stress in the form of maternal separation.

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Behavioral analysis of the NORT data revealed that neither the type of rearing nor housing manipulations affected non-spatial recognition memory. While a number

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of studies showed impairments in recognition memory following early adversity (Aisa et al., 2008; Daniels et al., 2009; Hulshof et al., 2011), other investigators report

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no effect of postnatal stress on this type of memory (Grace et al., 2009; Mourlon et al., 2010; Vivinetto et al., 2013). It is possible that differences in duration and type of

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postnatal stress as well as time of testing may account for this discrepancy. It should be mentioned that the absence of deficits in the non-spatial recognition memory reported here is in line with previous research conducted in our lab (Tata et al., 2015). The fact that MS rats were impaired in the probe trial of the MWM, but not in the NOR task, implies that our MS paradigm selectively affects spatial memory. In fact,

Leret and colleagues have also shown impairments in spatial memory but not object recognition both in adolescent and adult rats that had been exposed to MS (Leret et al., 2010). Regarding the role of housing, so far there is limited information concerning the effect of post-weaning EE on NORT performance. Although EE housing of 3-month old adult rats for 3 weeks (PND90-111) improves novel object

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recognition (Bechara and Kelly, 2013), EE exposure for a longer period of time

during an earlier developmental stage (PND21-60) does not seem to affect it

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(Vivinetto et al., 2013). These findings probably suggest that the effectiveness of EE

on recognition memory may be a function of time of exposure and duration of

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exposure.

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Effects of maternal separation and environmental enrichment on BDNF and

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synaptophysin expression in the hippocampus

synaptophysin (SYN) in

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Increasing evidence underlines the essential role of hippocampal BDNF and cognition as well as anxiety and depression-related

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behaviors (Heldt et al., 2007; Liu et al., 2005; Mitchelmore and Gede, 2014). In the present study, MS rats expressed behaviors indicative of impaired memory and

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increased anxiety, while EE exerted a beneficial effect in the MS group. In order to estimate whether these behavioral changes were mediated by alterations in neuronal

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plasticity, we evaluated the expression of these two markers (BDNF and SYN) in the dorsal hippocampus. BDNF plays an essential role in synapse formation, neuronal

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survival and growth and it has been suggested to be involved in transducing the effects of environmental manipulation on brain function during early developmental stages (Cirulli et al., 2003). In addition, BDNF expression in multiple brain regions is sensitive to adverse life experiences (Han et al., 2011; Meng et al., 2011).

According to our analysis, MS significantly decreased the expression of BDNF in in the dorsal hippocampus. This finding is in line with previous studies showing a downregulation in BDNF mRNA or protein expression following 2-3 weeks of MS (Aisa et al., 2009; Lippmann et al., 2007; MacinterplayQueen et al., 2003). However, the investigation of the MS impact on BDNF expression has yielded

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inconsistent results due to the involvement of various factors. The duration of adverse

postnatal manipulation appears to be a determining factor, since postnatal stress of

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shorter duration did not affect BDNF levels (i.e., a single episode of 24 h maternal deprivation, or 6-day MS) (Choy et al., 2008; Markostamou et al., 2016; Roceri et al.,

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2002). Furthermore, the developmental period of the animals as well as the brain

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region seem to influence MS effects. In fact, it has been shown that MS induces

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different patterns (up or down-regulation) of BDNF expression along with age in

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different brain regions (Pardon et al., 2009; Récamier-Carballo et al., 2017; Wang et al., 2015b). This differential impact of MS on BDNF expression reflects the complex

mechanisms.

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functional consequences of adverse early life experiences in synaptic plasticity

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In addition to the BDNF changes, we found that prolonged MS significantly decreased SYN in the dorsal hippocampus. SYN, a synaptic vesicle-associated

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protein, is a general synaptic marker and its presence indicates the efficiency of (Thiel, 1993; Valtorta et al., 2004). It has been previously reported that MS can cause

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down-regulation of SYN during adulthood, and this effect might be responsible for impaired hippocampal neurotransmission and behavioral changes (Aisa et al., 2009; Andersen and Teicher, 2004). The possibility that BDNF and SYN expression may have been affected by the acute stress (i.e., rotation) applied at the end of the experiments cannot be excluded.

Previous studies have shown that acute stress alters the expression of neurotrophic factors and synaptic regulatory proteins and these alterations may be responsible for some of the morphological and behavioral changes observed after stress exposure (Amin et al., 2015; Gao et al., 2006; Smith et al., 1995; Thome et al., 2001). Specifically, acute stress induces a brain region-dependent differential expression of

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BDNF (Lakshminarasimhan and Chattarji, 2012). Furthermore, the type of stressor,

the duration of stress or the age of the animals are factors that also affect the outcome

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(Badowska-Szalewska et al., 2017; Fuchikami et al., 2009; Molteni et al., 2009). However, the present study did not intend to explore the possible effect of acute stress

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on markers of synaptic plasticity or its functional consequences. Furthermore, it

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should be mentioned that all animals were subjected to the acute stress condition,

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which implies that all they would have been equally affected.

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Contrary to the effects of MS, exposure to EE increased the levels of both SYN and BDNF expression in the non-stressed group. Previous studies have also

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supported the beneficial effects of EE as indicated by the enhanced BDNF both at mRNA and protein levels (Cao et al., 2014; Griva et al., 2017; Ickes et al., 2000;

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Mosaferi et al., 2015). In addition, our analysis showed that EE upregulated SYN in control animals, an increase which is in agreement with previous studies (Birch et al.,

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2013; Lambert et al., 2005; Nithianantharajah et al., 2004) and indicates the involvement of this synaptic protein in experience-dependent neuroplasticity.

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In our study, the beneficial role of EE is mainly supported by its compensatory

effect against the MS-associated BDNF and SYN decreases. Specifically, the levels in the stressed rats exposed to EE were significantly higher than those in the MS animals housed in standard conditions, and did not differ from those in the non-stressed groups. Existing evidence supports the compensatory effect of EE on brain damage in

models of chronic stress or aging. In particular, EE protects from dendritic atrophy as well as reductions in neurogenesis and neurotrophin expression following adult stress (Hutchinson et al., 2012; Shilpa et al., 2017; Veena et al., 2009; Vega-Rivera et al., 2016). Previous studies have also shown that exposure to EE during a critical developmental period (0-2 postnatal months), is more effective at increasing BDNF

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levels (Jha et al., 2016). In this respect, our BDNF increases are in line with existing evidence, indicating a beneficial role of post-weaning EE (PND23-65) against MS-

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associated decreases in BDNF expression. Given that EE completely restored spatial

memory impairments in MS group and improved spatial acquisition, it could be

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suggested that EE-associated increases in SYN and BDNF may be a prerequisite for

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these behavioral effects. In fact, higher levels of BDNF and SYN have been

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correlated with cognitive improvement (Chen et al., 2013; Frick and Fernandez,

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et al., 2017; Pereira et al., 2009).

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2003), while according to others may not be a prerequisite (Bennett et al., 2006; Griva

Conclusion

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In the current study we explored the interaction of 3-week MS and subsequent exposure to enriched conditions on various behaviors, neuroendocrine stress response

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and two plasticity markers, BDNF and synaptophysin. To date, existing studies suggest the negative impact of MS or, on the contrary, the beneficial role of EE on

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emotional and cognitive behaviors, stress response and brain anatomy and neurochemistry. Besides the main effects of the two environmental conditions on learning and memory, corticosterone levels and BDNF and SYN expression, here we report that EE can compensate against stress-associated spatial memory deficits, increased anxiety and downregulation of BDNF and SYN. To the best of our

knowledge, this is the first study to show that MS and EE interact with each other, modulating adult’s behavior and neuroendocrine response to stress as well as neuroplasticity. Given the important role of early experiences, the need to better understand the underlying mechanisms that mediate behavioral changes is further

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emphasized. Acknowledgements: The authors are grateful to Alexandros Antonellos (Microanalysi

Medical Athens S.A) for his excellent technical assistance in corticosterone

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measurements (ELISA).

Funding: This research has been financially supported by the General Secretariat for

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Research and Technology (GSRT) and the Hellenic Foundation for Research and

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Innovation (HFRI) (Scholarship Code: 95144).

References

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and Protein Kinase M Zeta (PKMζ) Trafficking During Chronic Stress: A Protective Mechanism? Front. Behav. Neurosci. 9, 303. doi:10.3389/fnbeh.2015.00303

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Fig. 1. (A) Entries and (B) time spent in the open arms expressed as a ratio of the total entries and time spent in both open and closed arms of the EPM. Maternal separation significantly decreased number of entries and time spent in the open arms in SH animals (NMS/SH vs MS/SH, entries:**p < .001, time:*p < .01), but exposure to EE reversed this outcome (MS/SH vs. MS/EE, #p < .05). (C) Number of returns to closed arms of the EPM. MS animals reared in standard housing performed more closed arms returns, compared to NMS treated animals (NMS/SH vs. MS/SH: *p < .01). Exposure of maternally-separated animals to EE decreased the number of returns compared to MS animals of the SH condition (MS/SH vs. MS/EE: #p < .001). (D) Among animals raised in SH condition, MS rats exhibited increased number of Stretch-Attend Postures (SAPs) in relation to non-maternally separated animals (NMS/SH vs. MS/SH, *p < .01), but EE reduced occurrence of this behavior (MS/SH vs. MS/EE: #p < .001).

IP T SC R U N A M ED PT CC E A s Fig. 2. (A) Number of rearings in the outer zone of the open field during the 6-min period. Environmental enrichment significantly increased the number of rearings (#p <

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.05, EE main effect). (B) Total entries (ambulation). There were no significant effects for the total number of square visits caused by either the MS or EE manipulations (p > .05) and the two factors did not interact with each other (p > .05) (Fig. 2B).

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Fig. 3. Mean values of the discrimination ratio of all groups in the novel object recognition. Preference for the novel object did not differ among groups as a function of maternal separation or environmental enrichment (p > .05)

IP T SC R U N A M ED PT CC E A Fig. 4. Latency to find the platform of all groups in the spatial learning protocol of the Morris water maze. All animals improved their performance over the 4-day period (#p

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< .05). EE treated animals exhibited lower latencies compared to SH treated animals (*p < .001).

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Fig. 5. (A) Time spent in the target quadrant and (B) frequency of entries in the platform area during the probe trial of the Morris water maze. MS rats housed in standard conditions spent less time and did fewer entries, compared to NMS/SH rats (*p < .01), while exposure to EE increased the time and the number of entries for MS rats (MS/SH vs MS/EE, #p < .05).

IP T SC R U N A M ED PT CC E A Fig. 6. (A) MS animals housed in SH had higher plasma corticosterone levels after exposure to acute stress, compared to NMS animals (NMS/SH vs MS/SH,*p < .05).

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Rearing of maternally-separated animals in EE significantly reduced these corticosterone elevations (MS/SH vs MS/EE, #p < .05).

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Fig. 7. (A) BDNF expression in the dorsal hippocampus expressed as integrated density. MS animals raised in SH had lower BDNF levels compared to NMS animals NMS/SH of the same rearing condition (NMS/SH vs MS/SH, *p < .001). Environmental enrichment significantly increased protein expression in standard housed rats (NMS/SH vs NMS/EE, #p < .05) and counteracted the decreased levels in maternallyseparated rats (MS/SH vs MS/EE, ##p < .001). (B) Representative photomicrographs of BDNF immunofluorescence staining in the hippocampal CA1 region. Total magnification 400x, scale bar = 100μm.

IP T SC R U N A M ED PT CC E A Fig. 8. (A) Synaptophysin (SYN) expression in the dorsal hippocampus expressed as integrated density. Environmental enrichment significantly increased protein expression in standard housed rats (NMS/SH vs NMS/EE, #p < .01). Maternal

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separation significantly decreased SYN expression in rats of the SH condition (NMS/SH vs MS/SH, *p < .05). Enriched environment reversed the decreased SYN levels seen in maternally separated rats (MS/SH vs MS/EE, #p < .01). Representative photomicrographs of SYN immunofluorescence staining in the hippocampal CA1 region. Total magnification 400x, scale bar = 100μm.

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