Impact of enriched environment on hippocampal-related behavioral changes induced by extended voluntary ethanol intake and noise exposure in male and female adolescent Wistar rats.

Impact of enriched environment on hippocampal-related behavioral changes induced by extended voluntary ethanol intake and noise exposure in male and female adolescent Wistar rats. | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL European Journal of Neuroscience This is a preprint and has not been peer reviewed. Data may be preliminary. 13 March 2025 V1 Latest version Share on Impact of enriched environment on hippocampal-related behavioral changes induced by extended voluntary ethanol intake and noise exposure in male and female adolescent Wistar rats. Authors : Gustavo Buján , Luciana D´Alessio , Héctor Serra , Gonzalo Corsi , Sonia Molina , and Laura Guelman 0000-0002-5251-3006 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174185672.20648684/v1 Published European Journal of Neuroscience Version of record Peer review timeline 219 views 176 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Human adolescents are frequently exposed to ethanol and/or to noise, agents that put brain development at risk. As the use of animal models has been demonstrated to reproduce human findings, the aim of the present study was to investigate if two weeks of ethanol intake, with an intermediate noise exposure, can affect different hippocampal-related behaviors in adolescent rats of both sexes. In addition, the enriched environment was used as an environmental housing strategy to prevent hippocampal-related behavioral changes. Importantly, although partial neuroprotection has been found in an animal model of brief ethanol intake, little is known about longer intake paradigms. Adolescent Wistar rats of both sexes were subjected to voluntary intermittent two-bottle choice paradigm of ethanol intake in for 2 weeks (6 sessions). A subgroup was exposed to noise for two hours after the third session. Some rats from both groups were housed in enriched environment cages. Finally, an assessment of hippocampal-related behaviors was performed. Data show different alterations in hippocampal-related behaviors, some of which were sex-specific and differ from those observed after a brief ethanol consumption. Most behavioral changes were prevented, at least partially, by enriched environment. These findings suggest that common environmental factors present in human adolescent venues may influence behavior that differs among sexes, as observed in the present animal model of extended ethanol intake. Additionally, an enriched environment proved to be a partially effective neuroprotective strategy in both sexes. Thus, implementation of non-pharmacological approaches may provide benefits against various challenges. Impact of enriched environment on hippocampal-related behavioral changes induced by extended voluntary ethanol intake and noise exposure in male and female adolescent Wistar rats. Buján, GE 1 ; D’Alessio, L 1,2 ; Serra, HA 1 ; Corsi, GN 3 ; Molina, SJ 1,3* ; Guelman, LR 1,3*# 1 Universidad de Buenos Aires. Facultad de Medicina. Departamento de Toxicología y Farmacología. 1ª Cátedra de Farmacología. Buenos Aires, Argentina. 2 CONICET – Universidad de Buenos Aires. Instituto de Biología Celular y Neurociencias (IBCN). Buenos Aires, Argentina. 3 CONICET – Universidad de Buenos Aires. Centro de Estudios Farmacológicos y Botánicos (CEFyBO). Buenos Aires, Argentina. *Contributed equally # Corresponding autor: Dr Laura Ruth Guelman, Universidad de Buenos Aires. Facultad de Medicina. Departamento de Toxicología y Farmacología. 1ª Cátedra de Farmacología, Paraguay 2155, piso 15, 1121, Ciudad Autónoma de Buenos Aires, Argentina. e-mail address: [email protected] ORCID: https://orcid.org/0000-0002-5251-3006 Acknowledgements We thank Eduardo Nieves for his helpful assistance in the care of laboratory animals and Patricia Fernandez, María Cristina Lincon and Alejandra Verón for their administrative support. Abstract Human adolescents are frequently exposed to ethanol and/or to noise, agents that put brain development at risk. As the use of animal models has been demonstrated to reproduce human findings, the aim of the present study was to investigate if two weeks of ethanol intake, with an intermediate noise exposure, can affect different hippocampal-related behaviors in adolescent rats of both sexes. In addition, the enriched environment was used as an environmental housing strategy to prevent hippocampal-related behavioral changes. Importantly, although partial neuroprotection has been found in an animal model of brief ethanol intake, little is known about longer intake paradigms. Adolescent Wistar rats of both sexes were subjected to voluntary intermittent two-bottle choice paradigm of ethanol intake for 2 weeks (6 sessions). A subgroup was exposed to noise for two hours after the third session. Some rats from both groups were housed in enriched environment cages. Finally, an assessment of hippocampal-related behaviors was performed. Data show different alterations in hippocampal-related behaviors, some of which were sex-specific and differ from those observed after a brief ethanol consumption. Most behavioral changes were prevented, at least partially, by enriched environment. These findings suggest that common environmental factors present in human adolescent venues may influence behavior that differs among sexes, as observed in the present animal model of extended ethanol intake. Additionally, an enriched environment proved to be a partially effective neuroprotective strategy in both sexes. Thus, implementation of non-pharmacological approaches may provide benefits against various challenges. Keywords : Noise, Ethanol, Behavior, Enriched Environment 1- Introduction Adolescence is a period of extensive neurodevelopmental and behavioral changes and has been associated with heightened levels of risk-taking (Spear, 2015). In humans, adolescent use and abuse of alcohol beverages have been linked with high incidence of different pathologies, including altered stress reactivity, anxiety and depression (Gamble & Diaz, 2020). Interestingly, adverse situations derived from alcohol use during adolescence have been strongly associated with the onset of mental illness in adulthood, as well as with substance use disorders. Furthermore, various studies have shown that during this period, exposure to different physical or chemical agents can interfere with the normal development of the Central Nervous System (CNS), (Wille-Bille et al. , 2017; Baliño et al. , 2021; Belhorma et al. , 2021; Molina et al. , 2024), generating changes that could be long-lasting. In particular, many studies have examined the consequences of heavy ethanol (EtOH) exposure in adolescence (i.e., intoxication), both in humans and experimental animals, but little is known about the effect of lower levels of plasma EtOH. Early life stages represent critical periods for an individual’s development and unfavorable experiences during this period can alter sensitivity to challenges later in life, potentially contributing to an individual’s vulnerability or resilience to psychopathologies such as anxiety, depression and/or addiction (Erickson et al. , 2024). Stress exposure is strongly associated with both anxiety and alcohol intake, and it has been suggested that stressful events may act as moderators that activate or amplify underlying vulnerability for anxiety and depression. In fact, individuals under stressful conditions tend to engage in problematic drinking behavior. Exposure to stress during childhood and adolescence seems to be a risk factor for alcohol use disorder and comorbid conditions, including posttraumatic stress disorders. In animals, stress exposure might increase anxiety-like behavior, locomotor activity and EtOH consumption in male and female rats (More et al. , 2024; Morys et al. , 2024). On the other hand, noise is a potentially stressful environmental stimulus that can be defined as an unpleasant sound, typically of moderate to high intensity that can be harmful when exceeding 80 dB (Berglund et al. , 2000). Although there has been a recent rise in excessive noise levels in large cities, their detrimental impact is frequently disregarded. Estimates from the World Health Organization and European agency (European Environment Agency, 2020), based on scientific evidence, showed that noise above an intensity of 65 dB -a level close to the minimum considered harmful (80 dB) - affects approximately 20% of the population and can cause various disorders (Berglund et al. , 2000; Li et al. , 2023) that can affect both auditory structures (Cappaert et al. , 2000; Hu & Zheng, 2008) and extra-auditory areas in the nervous, endocrine, and/or cardiovascular systems (Turner et al. , 2005; Basner et al. , 2014). In consequence, the widespread consumption of EtOH-containing beverages among adolescents worldwide, coupled with their simultaneous exposure to high-level noise in various entertainment settings, underscores the importance of studying the effects of these agents using animal models (Salling et al. , 2018; Wallas et al. , 2020; Barney et al. , 2022). Despite the limited data on the effects of EtOH intake in both sexes -both in human and animal studies, where males have been more frequently studied-recent research has increasingly focused on females, highlighting their greater vulnerability to stress-induced drinking. As reported previously by our group (Buján et al. , 2024), voluntary intermittent EtOH intake for one week, followed by 2 hours noise exposure, was able to induce disturbances in both behavioral and emotional aspects, that are known to involve the function of the hippocampus (HC), a structure known to participate in various types of memory as well as anxiety-related behaviors (Tatu & Vuillier, 2014). In addition to sex-dependent effects, housing in an enriched environment (EE) for one week effectively prevented most of the behavioral changes induced by both agents in this paradigm. However, there is a scarce number of studies in the literature that use self-administration of EtOH compared to forced exposure (Spear, 2000, 2015) and novel findings from voluntary access paradigms would be highly valuable. The extended paradigm used in the present paper might add new insight about the continuous EtOH drinking even if noise is only briefly present. In the present paradigm, extended EtOH intake was implemented to compare with previously published data of a short-duration paradigm. In particular, prolonged EtOH intake could be either more disruptive, due to prolonged exposure, or more beneficial, as it allows the organism to adapt to excess EtOH. Furthermore, given the well-established scientific value of EE housing as a preventive tool across various animal models, the limited research on the effects of intermittent voluntary EtOH intake combined with noise exposure in adolescent animals as well as the potential for prolonged EtOH intake to exacerbate behavioral outcomes, this study aimed to evaluate behavioral effects in both sexes and determine whether EE housing could mitigate most of them. 2- Materials and Methods 2.1. Animals Wistar rats of both sexes were purchased in the certified vivarium of the Facultad de Farmacia y Bioquímica at Universidad de Buenos Aires, Argentina. A total of nine female and three male rats were selected for breeding purposes and housed in the animal facility of the 1 a Cátedra de Farmacología (Facultad de Medicina, Universidad de Buenos Aires), with each cage containing three females and one male. Each female was used several times for mating purposes. When pregnancy was confirmed, female rats were housed individually in cages until the day of delivery, which was designated as postnatal day 0 (PND 0) for the litter. On average, each rat gave birth to 3-4 litters. Therefore, as the mean number of animals per litter was 10, 30-40 animals were born from each female. For the experiments, rats of both sexes of each litter were used. At weaning (PND21), two rats of the same sex were housed together in standard cages (SC, measures: 40 × 25 × 20 cm). Rats were randomly assigned to one of the following experimental groups: control (sham), noise, EtOH, and EtOH+noise. The experiments began when the animals reached the age of 28 days. A subgroup of animals of each experimental group was transferred to EE, in which 4-5 rats of the same sex were housed together. In SC, rats were housed in same-sex pairs in order to mitigate any potential additional stress stemming from isolation, as recommended by several authors, which reported that stressful situations, such as social isolation during early development, may constitute a significant risk factor for future EtOH consumption (Lopez & Laber, 2015) and could have lasting effects on behavioral and biological responses in rodents (Leussis & Andersen, 2008). For each group, 14 animals were assigned to behavioral tasks: 7 subjected to Open Field and Elevated Plus Maze tasks (using the same rat for both tests) and 7 for Inhibitory Avoidance task, using a total of 112 animals per sex. To minimize potential confounding factors related to the litter, only one male and one female from each litter were assigned to each experimental condition and behavioral task. All cages were equipped with ad libitum access to food and water. The lighting conditions followed a 12-hour light/dark cycle, with the light cycle starting at 7 AM. Mashed cornflower was used as bedding material and the temperature was maintained at 21 ± 2°C. To minimize disruption of the circadian rhythm, noise exposures and the subsequent behavioral tests were conducted between 8 AM and 12 PM. The animals were handled and euthanized in accordance with the guidelines provided by the Institutional Committee for the Use and Care of Laboratory Animals (CICUAL) at the Facultad de Medicina, Universidad de Buenos Aires. The experimental protocol employed was approved by this committee (#1396/18). CICUAL follows the regulations outlined in the ’Guidelines for the Care and Use of Laboratory Animals’ (NIH, 2011 revision) and the EC Directive 86/609/EEC (2010 revision) for conducting animal experiments. To minimize pain and discomfort, a CO 2 euthanasia chamber was utilized for animal sacrifice at the conclusion of the behavioral experiments for final disposal. 2.2. Noise exposure To prevent any unnecessary handling and minimize potential stress during the noise exposure procedure, animals of SC were maintained in their original cages. These cages were then placed inside a custom-built wooden sound chamber, which measured 1m x 1m x 1m and was equipped with a sound attenuation system made with Celotex™ and featured a ventilated top. Those animals housed in EE cages were transferred to SC for noise exposure purposes. To ensure that the animals’ circadian rhythm remained undisturbed, appropriate illumination (a 20-W lamp) was provided as reported previously (Uran et al. 2012). For sound amplification, a two-way active monitor (SKP, SK150A, 40 W RMS per channel) was used, placed 30 cm above the animal cage on the top of the sound chamber. Furthermore, white noise intensity was measured using an omnidirectional measuring condenser microphone (Behringer ECM8000) placed at different locations in the chamber, by taking an average of the different readings. TrueRTA computer software was chosen to generate white noise using a bandwidth of 20–20,000 Hz in octave bands. PND33 male and female rats were exposed to a 2-hour session of white noise with a sound pressure level (SPL) ranging from 95 to 97 decibels (dB) and a frequency range of 20-20,000 Hz. Conversely, control (sham) and EtOH groups were placed in the same chamber for the same duration but did not receive white noise exposure. Background level of environmental noise in the rat facilities ranged from 50 to 55 dB SPL, which falls within the safe range recommended by the World Health Organization (WHO) and EU guidelines (European Environment Agency, 2020) as well as is stated in several studies (Campeau et al. , 2002; Sasse et al. , 2008). 2.3. Intermittent voluntary EtOH intake (two-bottle choice paradigm) This paradigm was chosen to obtain an animal model that can more accurately replicate voluntary, not forced, EtOH intake in humans (Acevedo et al. , 2016; Miceli et al. , 2018; Fernández et al. , 2019) and consisted of a repeated and intermittent voluntary intake protocol. Briefly, animals housed in pairs (except for those animals housed in EE, in which 4-5 animals were placed together) were given 24-hour access to self-administered EtOH. Two bottles were placed onto cage lids, one with 5% EtOH dissolved in 1% sucrose -similar to what can be found in alcoholic beverages preferred by human adolescents (Acevedo et al. , 2016)- and another with 1% sucrose in tap water, from PND28 (i.e., 1 week after weaning) to PND39, three days a week, designated as “sessions” (S1: PND28; S2: PND30; S3:PND32; S4: PND34; S5:PND36; S6:PND38). After 24 h, EtOH-containing bottles were replaced with tap water bottles, which were available for the next 24 h (Segal et al. , 2022). This period of consumption was selected to allow EtOH exposure during the early adolescent development. At the end of each session, bottles and animals were weighed to record the grams of EtOH consumed (g/kg, grams of EtOH consumed per kg of body weight) for each rat in each 24-hour session, a method validated by Acevedo et al. (2016) and Wille-Bille et al. (2017) and has been already published in a previous paper of our laboratory (Buján et al. , 2024) Animals of the sham and noise only groups were given two bottles of 1% sucrose dissolved in tap water. Animals of all groups had the possibility to choose drinking from any of the available bottles (Carnicella et al. , 2014; Miranda-Morales et al. , 2016). Figure 1. Diagram of the experimental design used. 2.4. Behavioral tasks At PND39, after the end of the last EtOH intake session, animals were behaviorally assessed (see figure 1 for the timeline). Habituation: Before each test, the rats were individually placed in a transfer cage for 3 minutes so that they could become familiar with the compartment. Subsequently, the transfer cage was placed inside the behavioral room for 5 minutes to allow habituation to the surrounding space, thereby reducing the impact of various environmental factors that could potentially influence physiological and behavioral stress indicators (Walf & Frye, 2007). To reduce contamination from external noise, ambient sound was activated throughout the assessment; to eliminate olfactory stimuli, each device was cleaned with a 10% EtOH solution between sessions. 2.4.1. Inhibitory avoidance task This task was used to measure the memory of an aversive experience (e.g., an electric shock) through the simple avoidance of a preferred location (e.g., a dark place) in which it occurred. Once the associative memory is formed, rats learn to avoid access to the context where they received the discharge, although initially this site was considered “safer” or “pleasant”. This task is thought to depend on the dorsal HC and is a reliable index of associative memory (Izquierdo & Medina, 1997; Izquierdo et al. , 2016). Inhibitory avoidance procedures were carried out as previously reported (Molina, Capani, et al. , 2016; Molina et al. , 2019). – Apparatus: The apparatus consisted of a box (60 × 60 × 40 cm), divided into two compartments: one was illuminated with a lamp and had transparent acrylic walls, while the other was surrounded by black walls to allow it to be dark, as described by Roozendaal (2002). A removable door divides the two compartments. The floor of the dark compartment consisted of a stainless-steel grid at the bottom, through which a continuous current could be delivered. – Habituation session: the rat was placed into the lit box and allowed to freely explore the apparatus. Either after passing through the dark side three times or after remaining in it for 3 minutes, the rat was removed from the apparatus. After 10 minutes, the rat was reintroduced to the illuminated side. When it entered the dark side, the dividing door was closed, and the rat was confined to the dark side for 10 seconds. – Training session (T1): after one hour in its home cage, each rat was placed in the lit compartment, facing away from the dark compartment; the latency to move into the dark compartment was recorded. When the rat stepped with all four paws in this side, a foot shock (1.2 mA, 2 s) was delivered. The rat was quickly removed from the apparatus and returned to its home cage. – Retention session (T2): One hour following the training session, the rat was placed into the device using the same procedure, with the exception that no foot shock was delivered. The ratio between the latency to enter into the dark compartment in the retention and the training sessions (T2 and T1, respectively) was taken as a measure of associative memory retention (T2/T1 ratio). 2.4.2. Elevated plus maze task This task was used to evaluate anxiety-like and risk assessment behaviors that depend on the integrity of the ventral HC (Kjelstrup et al. , 2002; Carobrez & Bertoglio, 2005). Elevated plus maze validity is based on the natural conflict between the urge to explore a new environment and the tendency to avoid a potentially dangerous area (Pellow et al. , 1985). Anxiety-like behaviors were calculated as the latency to enter the open arms as well as the entries to those arms. When an increase in those parameters is observed, it could be stated that a reduction in anxiety-like behaviors could have occurred. Additionally, some ethological parameters can be evaluated using this task, designated as risk assessment behaviors, because they have been associated with detection and analysis of threatening situations (Rodgers & Cole, 1993; Carobrez & Bertoglio, 2005). One of these parameters is called head-dipping and describes the action of the animal when it stretches the head over the ledge of an open arm and bends down. This behavior can be performed both from the “protected” areas (e.g., closed arms and center platform), which provide the rat with a relative safety feeling, and in the “unprotected” areas (open arms) of the device. Elevated plus maze procedures were carried out as previously reported (Molina, Miceli, et al. , 2016; Molina et al. , 2019, 2021). – Apparatus: the wooden apparatus consisted of four arms of equal dimensions (50 × 10 cm), elevated to a height of 50 cm above the ground. Two enclosed arms, with walls measuring 40 cm in height, are positioned perpendicular to the other two open arms. The device was softly lit with a light located 2 m high. – Session: The rat was placed in one of the closed arms, facing the center of the maze, and allowed to freely explore it for 5 minutes. The latency to open arms, the percentage of number of entries to those arms, as well as the percentage of head dipping from closed arms (% HD-CA), were calculated. The percentage of entries to the open arms was calculated as the values of entries to open arms/(entries to open arms + entries to closed arms) × 100. The % HD-CA was calculated as HD-CA/(HD- open arms + HD- closed arms) × 100. The animal’s activity was recorded using a camcorder (Handycam DCR-DVD810, Sony). 2.4.3. Open field task An open field device was used to analyze rats’ exploratory activity and novelty reaction, behaviors known to depend on the HC (Barros et al., 2006; Leussis and Bolivar, 2006; Vianna et al., 2000). In this task, the activity can be used to assess changes induced by exposure to a novel environment. In consequence, vertical exploratory activity can be quantified by recording the number of rearing and climbing events, i.e., when rats stand on their hind legs, and incursions to the central quadrants can be also considered as exploratory behavior or, as measure of emotional reaction (Leussis & Bolivar, 2006; Abuhamdah et al. , 2012). Procedures were carried out as previously reported (Molina, Miceli, et al. , 2016; Molina et al. , 2021, 2024; Buján et al. , 2022) and the animal’s activity was recorded using a camcorder (Handycam DCR-DVD810, Sony). – Apparatus: OF device consisted of a 50 × 50 × 50 cm wooden box, with a floor divided into 25 equal squares by black lines. The open field was illuminated with a 20-W lamp located above. -Session: rats were placed on the center rear quadrant of the open field box and allowed to freely explore it for 5 minutes. The incursions to the central quadrants, as well as the number of rearing and climbing events, were recorded over the session. 2.5. Environmental enrichment (EE): This type of housing represents an experimental condition that can be implemented by exposing experimental animals to improved habitats (Novkovic et al. , 2015). Briefly, EE cages consisted of plastic cages, with larger dimensions when compared to SC (54 x 40 x 41 cm), with two levels containing two connecting ramps, a tunnel of PVC plastic connecting the second level to the lower floor, a wheel to run, built-in blocks and a feeder. Different toys were placed in each cage, which varied over time. Animals had balanced feed and Granix® sugar rings were regularly added for taste stimulation. In each cage, 4-5 animals from the same sex were housed. A subgroup of each group of experimental animals remained in the EE cages from PND28 until the end of the experiments and were cleaned every two days. 2.9. Statistical analyses: a normality test for each group (KS test) was performed. Significant differences between the groups were analyzed using a three-way ANOVA test with post-hoc comparisons (LSD), using Infostat/L software. In consequence, the factors used for the analyses were sex (male or female), treatment (sham, noise, EtOH, EtOH+noise), and housing (SC or EE). When interactions were significant, simple effects analysis were conducted. Results were expressed as mean values ± SEM, and graphs were created using Prism GraphPad software (version 8.4.3). A probability < 0,05 was accepted as significant. Statistically significant comparisons between different groups were indicated with a line with an asterisk (*), with a # for comparisons between SC and EE and with a ¥ for comparisons between males and females. For sessions 1-3, values of EtOH and EtOH+noise groups were unified in each sex and housing condition. Repeated measures statistical analysis was used to assess differences among sessions. 3- Results Figure 2 shows that noise exposure and EtOH intake, whether presented individually or together, led to a reduction in the T2/T1 ratio in the inhibitory avoidance task in animals of both sexes housed in SC, when compared to the respective sham group. In contrast, no significant differences between the groups were observed in animals housed in an EE. However, the T2/T1 ratio of animals housed in an EE was significantly different from that of the respective group of animals housed in SC in males, whereas females animals housed in EE exposed to noise did not differ from the ratio observed in SC (three-way ANOVA, F 15,104 = 11.16, p< 0.01; between-subjects factors: treatment (sham, noise, EtOH, EtOH+noise), F 3,104 = 26.26, p< 0.01; sex (male or female), F 1,104 = 1.52, NS; housing (SC or EE), F 1,104 = 17.94, p< 0.01). As some interactions were significant (between treatment and housing, F 3,104 = 16.42, sex and housing, F 1,104 = 5.35, p< 0.05), the corresponding simple effects analysis were performed. Figure 3 a and b showed that the latency to open arms increased in all groups only in males when compared with the respective sham. When animals were housed in an EE, females decreased EtOH intake when compared to EE-sham animals, whereas no changes were observed among male groups. Finally, females that drank EtOH, with or without being exposed to noise and housed in EE differed from the respective SC group. In contrast, male animals differed from the respective SC group in noise, EtOH and EtOH+noise groups (three-way ANOVA, F 15,99 =10.53, p< 0.01; between-subjects factors: treatment (sham, noise, EtOH, EtOH+noise), F 3,99 = 3.79, p< 0.05; sex (male or female), F 1,99 = 46.83, p< 0.01; housing (SC or EE), F 1,99 = 24.65, p< 0.01). As some interactions were significant (between treatment and sex, F 3,99 = 8.91, p< 0.01; treatment and housing, F 1,99 =7.93, p< 0.01), the corresponding simple effects analysis were performed. Figure 3 c and d show that noise exposure led to a reduction in the percentage of incursions into the open arms of the elevated plus maze in animals of both sexes housed in SC, alone or combined with EtOH intake. However, when EtOH was consumed alone, it caused a significant increase in this parameter in females, while a decrease was observed in males. In animals housed in an EE, no differences were found between any group and the sham group. Lastly, sham, noise and EtOH-treated females housed in EE showed significant differences from the corresponding SC group, while in males, the noise, EtOH, and EtOH+noise groups differed from their SC counterparts (three-way ANOVA, F 15,83 = 16.1 p< 0.01; between-subjects factors: treatment (sham, noise, EtOH, EtOH+noise), F 3,83 = 8.61, p< 0.01; sex (male or female), F 1,83 = 9.27, p< 0.01; housing (SC or EE), F 1,83 = NS). As some interactions were significant (between treatment and sex, F 1,83 = 11.62, p< 0.01; treatment and housing, F 3,83 = 12.33, sex and housing, F 1,83 = 107.86, p< 0.01), the corresponding simple effects analysis were performed. Figure 4 shows that, when presented individually, noise exposure and EtOH intake increased the percent of head dipping in the closed arms measured in the elevated plus maze device in females housed in SC compared to the sham group, whereas no changes were observed when both agents were present together. In contrast, males showed a decrease in this parameter in all groups housed in SC, when compared to sham. When females were housed in EE, a significant decrease was observed after noise exposure. In addition, all EE housed groups differed from the corresponding SC group. Finally, a significant increase was observed only in males that drank EtOH when compared with the sham group (three-way ANOVA, F 15,82 =34.71 p< 0.01; between-subjects factors: treatment (sham, noise, EtOH, EtOH+noise), F 3,82 =12.91; sex (male or female), F 1,82 =128.07; housing (SC or EE), F 1,82 = 97.93p= 0.05). As some interactions were significant (between treatment and sex, F 3,82 = 8.3, p< 0.01; treatment and housing, F 3,82 = 17.09, p< 0.01, housing and sex, F 1,82 = 45.4, p< 0.01), the corresponding simple effects analysis were performed. Figure 5 shows that EtOH and noise, when presented individually, increase the time spent in center in female animals housed in SC when compared to sham counterparts, whereas in males, time in center decreased in all groups when compared with the sham group. When animals were housed in EE, there were no differences among groups in females, whereas a decrease in this parameter was observed in males that drank EtOH or were exposed to noise individually. In addition, EE housing induced a decrease in time in center in the EtOH and noise groups when compared to the respective SC group in females, whereas an increase was observed in sham and EtOH+noise male groups when compared with the respective SC group (three-way ANOVA, F 15,84 = 18.37, p< 0.01; between-subjects factors: treatment (sham, noise, EtOH, EtOH+noise), F 3,84 = 6.23, p< 0.01; sex (male or female), F 1,84 = 16.97, p< 0.01; housing (SC or EE), F 1,84 = 5.87, p< 0.05). As some interactions were significant (between treatment and sex, F 3,84 = 36.52, p< 0.01; treatment and housing, F 3,84 = 23.43, p< 0.01; housing and sex, F 1,84 = 30.29, p< 0.01), the corresponding simple effects analysis were performed. In figure 6 it can be observed that noise exposure decreased the time of rearing and climbing in females, whereas an increase was observed in animals subjected to voluntary EtOH intake, alone or in combination with noise exposure. In contrast, males exposed to noise increased the time of rearing and climbing. When females were housed in EE, no difference among groups were found, whereas in males an increase is still observed in noise-exposed animals, alone or in combination with EtOH intake. Finally, all EE-housed females differed from the corresponding SC group, whereas in males this was observed only noise-exposed animals (three-way ANOVA, F 15,89 = 11.56, p< 0.01; between-subjects factors: treatment (sham, noise, EtOH, EtOH+noise), F 3,89 = 5.18, p< 0.01; sex (male or female), F 1,89 = 5.83, p< 0.05; housing (SC or EE), F 1,89 = 0.09, NS). As some interactions were significant (between treatment and sex, F 3,89 = 18.45, p< 0.01; treatment and housing, F 1,89 = 2.72, p< 0.05), the corresponding simple effects analysis were performed. Table 1 shows results of EtOH intake (g EtOH/kg/day) in males (a) and females (b), both in SC and EE housing (three-way ANOVA, F 47,687 = 4.63, p< 0.01; between-subjects factors: Housing: F 1,687 = 10.92, p< 0,01; Session: F 5,687 = 9.89, p< 0.01; Sex 1,687 = 0.29, NS). Data show that from S4 and until S6, males and females housed in SC drank significantly less EtOH (g/kg/day) than in S1-S3, both in animals exposed or not to noise. In contrast, when animals were housed in EE, different results were found in animals of each sex: in males, whereas a decrease in EtOH intake was observed from S2 and remained decreased until S6 only in animals that only drank EtOH, when compared with S1, when an intermediate noise exposure was included, animals drank more ETOH in S4-S6 when compared with the EtOH group counterparts, although it is not statistically significant. In females, EtOH intake in S4 decreased when compared with S1. In S5 and S6, animals that drank EtOH increased the amount of EtOH drank when compared to S4, reaching levels of S1-S3. In addition, in S5 and S6, noise-exposed females increased the amount of EtOH ingested when compared with S2 and S3. As some interactions were significant (between session and sex, F 5,687 = 2.56, p< 0.05; session and housing, F 5,687 = 14.8 p< 0.01), the corresponding simple effects analysis were performed. When total EtOH was calculated (i.e., the sum of EtOH consumed over all sessions), a significant decrease was observed in the EE-EtOH group when compared with the SC-EtOH group (t 280 =2.014, p< 0.05) only in males. 4- Discussion This study investigated the effects of voluntary intermittent EtOH intake combined with moderate noise exposure on various behaviors in male and female rats, including contextual associative memory, response to novelty, risk assessment, anxiety-like behavior, and exploratory activity. Moreover, EtOH consumption pattern was also analyzed. Identification of potential differences among sexes and comparison with the effects observed in rats subjected to a brief EtOH exposure (published in Bujan et al, 2024) were the main goals of this investigation. In addition, housing in an enriched environment has been tested as a non-pharmacological tool to prevent EtOH and noise-induced alterations. In particular, a significant decrease in associative memory (evaluated in the inhibitory avoidance device) was observed in all groups of animals of both sexes when housed in SC, when compared with sham animals, that were prevented if animals were housed in EE. These findings suggest that whereas EtOH and noise disrupted this type of memory in animals housed under SC conditions -likely due to their impact on hippocampal integrity-, two weeks of EE housing may provide a protective effect on this structure. When anxiety-like parameters were assessed (i.e., latency and number of incursions to open arms of the elevated plus maze device), an increase was observed in noise groups (with or without EtOH) of both sexes. In contrast, although EtOH alone increase anxiety-like behavior in males, it decreases it in females. Most changes were prevented if animals were housed in EE, except from males that drank EtOH, in which anxiolysis was observed. These results suggest that EE housing primarily counteracts aversive emotional behaviors, such as anxiety-like behavior. However, when the induced change leads to a more adaptive response -such as a reduction in anxiety-like behavior- EE would seem not to operate. Present data support previous works of our laboratory using different paradigms, in which we showed that excessive anxious behavior could lead to associative memory impairments (Molina et al., 2021, Buján et al., 2024). As noise is considered a stressor, it can promote anxiety-related responses, as observed in other models (Borodovitsyna et al., 2018; Tillage et al., 2021) and produce detrimental behavioral effects. However, the benefits of lower anxiety-like behavior levels remain controversial. It is important to note that higher levels of anxiety-like behavior can be advantageous, as they enhance an animal’s responsiveness to threats (Izquierdo et al., 2016). Conversely, reduced anxiety-like behavior can be maladaptive if it results in diminished environmental exploration and responsiveness to novelty. Interestingly, when animals were housed in SC, an increase in risk assessment was found in the noise alone and EtOH alone groups in females, whereas a decrease in all groups was observed in males. EE housing induced opposite changes to most of the parameters that were altered in the corresponding SC group, being like the EE sham group. Therefore, it could be suggested that whereas females seemed to be more cautious than males when exposed to noise or EtOH, EE appeared to compensate for these opposite changes and achieve the most adaptive behavior. When novelty reaction was evaluated, an increase was observed in the noise alone and EtOH alone groups in females, whereas a decrease was found in all groups of males. Interestingly, whereas prevention was found in females after housing in EE, no prevention was observed in males. Therefore, it could be suggested that when novelty responsiveness is increased, EE was able to restore this maladaptive behavior. In contrast, a decrease in novelty reaction, that could mean a minimum interest on the environment, seemed not to be dangerous for survival and for this reason, EE did not operate on this behavior. When the exploratory activity was recorded after housing in SC, a decrease was observed in females of the noise group, whereas an increase was found in males. In addition, intake of EtOH (with or without exposure to noise) induced an increase only in females. Housing in EE promoted a prevention in females, whereas in males the prevention was partial. These data suggest that the decrease in this parameter may be more effectively counteracted by EE housing, likely due to the sensory stimulation provided during the two-week period. In males, although the noise group decreased the exploratory activity when housed in EE, it did not match the corresponding sham group, probably because a little increase in this parameter should not be maladaptive. Finally, when EtOH intake (with or without exposure to noise) was assessed in animals housed in SC, a significant decrease was observed in both sexes over sessions S4-S6, when compared with the respective S1 session. EE housing led to an early reduction in EtOH intake in animals from the EtOH-alone group (S3-S6 in females and S2-S6 in males when compared with the corresponding S1), whereas if an intermediate noise exposure is included, EtOH intake in S4-S6 did not differ from S1-S3 sessions in either sex. This data means that noise might interfere with the potential prevention mediated by EE on EtOH intake, suggesting an impairment when both agents are present together. Interestingly, whereas rats housed in SC maintained intake values significantly decreased when compared with the respective S1 value, in EE intake values did not differ from the S1 value. Therefore, it could be suggested that whereas EE may serve as a positive stimulus to encourage adaptive behaviors, the recreational appeal of ramps, sweet food, and socializing in groups could also enhance EtOH intake as a hedonic stimulus. More research is needed to confirm this hypothesis. When present results of extended EtOH intake were compared with the previously published data of a brief EtOH intake (Buján et al., 2024), it could be concluded that in SC a brief intake is less harmful than extended consumption in males on most parameters, suggesting that the extended drinking period might impair performances because of the increase in the total EtOH amount ingested. In contrast, more detrimental effects were found in exploratory activity and associative memory parameters in females after a brief intake when compared with the extended intake period, probably because an extended period of EtOH intake may help compensate for the impairments caused by the first three sessions, resulting in fewer changes in the same parameters, possibly by activating adaptive mechanisms. Therefore, as it seems that three sessions of EtOH intake might be less detrimental than six sessions in males and more damaging in females, it could be proposed that females have a greater vulnerability, mainly in contextual memories that heavily depend on the HC. After housing in EE, extended EtOH intake seemed to be more harmful on risk assessment than brief intake in both sexes, in contrast to the increased anxiety-like behavior observed in some groups under this paradigm that could be interpreted as a more damaging effect. In consequence, the present data supports the notion that only three sessions of voluntary 5% EtOH intake are enough to induce anxiety-like behavior alterations in non-intoxicated animals during behavioral assessment when animals were environmentally stimulated, whereas six sessions might compensate for these alterations. When the differential effect on both sexes was evaluated, significant disparities were found between males and females in risk assessment, novelty reaction and exploratory activity. These data suggest that, although associative memory, a cognitive parameter, was equally affected in all groups of both sexes, an extended period of EtOH intake might induce a different response in males and females, that could probably be related to the hormonal status. Nevertheless, most alterations were prevented after EE housing. Other authors have found that EE was able to prevent EtOH-induced changes in different models. For example, Wille-Bille et al. (2020) analyzed the neuroprotective effect of EE on prenatal EtOH exposure in both male and female rats, focusing on gene expression modulation, consumption and anxiety responses. They found that housing in an EE tended to normalize the observed alterations. Additionally, previous results from our laboratory (Molina et al., 2022, 2021) showed that the behavioral, biochemical and amino acid neurotransmission system alterations found in male Wistar rats exposed in the infancy to different noise exposure schemes were mostly restored after housing in an EE for one or two weeks. In summary, voluntary EtOH consumption using an intermittent paradigm of two weeks with an intermediate acute noise exposure can lead to hippocampus-dependent behavioral changes, that appear to be sex-dependent, and most can be prevented by housing animals in an EE. Furthermore, these alterations differed from those previously found by our group after a brief EtOH intake. After noise exposure and EtOH intake separately, females displayed an increase and males a decrease in novelty reaction and risk assessment, together with associative memory deficit. In addition, whereas after noise exposure an increase in anxiety-like behavior was observed in both sexes, a decrease was observed in females and an increase only in males after EtOH intake. Finally, noise exposure induced an increase in exploratory activity in males and a decrease in females, whereas EtOH intake induced an increase in exploratory activity. Overall, these findings suggest that a cognitive parameter like associative memory seems to be a key target that can be altered, regardless of sex, after exposure to various environmental agents. This effect may be explained by increased anxiety-like behavior, a major factor that could predispose noise-exposed individuals to impaired associative memory. The lack of effect of the anxiolytic action of EtOH in females that could have improved the associative memory in the inhibitory avoidance task, could be related to the increases in novelty reaction, risk assessment and exploratory activity that might turn the animal more curious and increase the likelihood of entering a risky area, such as the inhibitory avoidance device. Finally, these data support the hypothesis that females are more vulnerable to alterations in awareness parameters that could compromise their safety survival. These results emphasize the importance of using animal models that replicate the environment of EtOH consumption in noisy settings during human adolescence, in order to better understand the behavioral impairments induced by this agent and the underlying mechanisms. 5- Conclusions Present results show that different hippocampal-dependent behavioral changes might be induced in animals of both sexes after an extended voluntary intermittent EtOH intake with an intermediate noise exposure, which differed among sexes. Furthermore, a comparison with previous results of a brief EtOH intake paradigm demonstrated that total EtOH intake may differentially affect behavioral outcomes. In addition, the significant differences observed when comparing animals housed in SC vs. EE cages might suggest that implementation of a non-pharmacological strategy could offer neuroprotective benefits against these challenges. Some of the differences observed in the groups subjected to voluntary EtOH intake alone may be attributed to the reduced amount of EtOH consumption. Future research might consider studying the effect of EE in a more chronic EtOH exposure to achieve a therapeutic value. Funding sources This work was supported by a grant of the Universidad de Buenos Aires (UBA), Argentina, awarded to LR Guelman (UBACYT 20020190100222BA) and a grant of the Florencio Fiorini Foundation to SJ Molina. GNC is an ANPCYT fellowship. GE Bujan was a postgraduate fellowship of UBA. Author statement Conceptualization and experimental design: LR Guelman and SJ Molina Writing - review & editing: LR Guelman and SJ Molina Critical review: L D’Alessio and HA Serra Funding acquisition: LR Guelman and SJ Molina Experiments and data analysis: GE Buján and LR Guelman Declaration of competing interests All authors declare that they have no conflicts of interest. References Abuhamdah, R.M.A., van Rensburg, R., Lethbridge, N.L., Ennaceur, A., Chazot, P.L., 2012. Effects of methimepip and JNJ-5207852 in Wistar rats exposed to an open-field with and without object and in Balb/c mice exposed to a radial-arm maze. Front Syst Neurosci. https://doi.org/10.3389/fnsys.2012.00054 Acevedo, M.B., Fabio, M.C., Fernández, M.S., Pautassi, R.M., 2016. Anxiety response and restraint-induced stress differentially affect ethanol intake in female adolescent rats. Neuroscience 334, 259–274. https://doi.org/10.1016/j.neuroscience.2016.08.011 Alfonso-Prieto, M., Biarnés, X., Vidossich, P., Rovira, C., 2009. The Molecular Mechanism of the Catalase Reaction. J Am Chem Soc 131, 11751–11761. https://doi.org/10.1021/ja9018572 Amodeo, L.R., Kneiber, D., Wills, D.N., Ehlers, C.L., 2017. Alcohol drinking during adolescence increases consumptive responses to alcohol in adulthood in Wistar rats. Alcohol 59, 43–51. https://doi.org/10.1016/j.alcohol.2016.12.002 Atucha, E., Roozendaal, B., 2015. The inhibitory avoidance discrimination task to investigate accuracy of memory. Front Behav Neurosci. https://doi.org/10.3389/fnbeh.2015.00060 Bahi, A., 2024. Gestational environmental enrichment prevents chronic social stress induced anxiety- and ethanol-related behaviors in offspring. Pharmacol Biochem Behav 234, 173679. https://doi.org/10.1016/j.pbb.2023.173679 Bahi, A., 2017. Environmental enrichment reduces chronic psychosocial stress-induced anxiety and ethanol-related behaviors in mice. Prog Neuropsychopharmacol Biol Psychiatry 77, 65–74. https://doi.org/10.1016/j.pnpbp.2017.04.001 Baliño, P., Romero-Cano, R., Muriach, M., 2021. Biochemical and behavioral consequences of ethanol intake in a mouse model of metabolic syndrome. Int J Mol Sci 22. https://doi.org/10.3390/ijms22020807 Baradaran, Z., Vakilian, A., Zare, M., Hashemzehi, M., Hosseini, M., Dinpanah, H., Beheshti, F., 2021. Metformin improved memory impairment caused by chronic ethanol consumption during adolescent to adult period of rats: Role of oxidative stress and neuroinflammation. Behavioural Brain Research 411, 113399. https://doi.org/10.1016/j.bbr.2021.113399 Barney, T.M., Vore, A.S., Deak, T., 2022. Acute Ethanol Challenge Differentially Regulates Expression of Growth Factors and miRNA Expression Profile of Whole Tissue of the Dorsal Hippocampus. Front Neurosci 16. https://doi.org/10.3389/fnins.2022.884197 Barros, D., Amaral, O.B., Izquierdo, I., Geracitano, L., do Carmo Bassols Raseira, M., Henriques, A.T., Ramirez, M.R., 2006. Behavioral and genoprotective effects of Vaccinium berries intake in mice. Pharmacol Biochem Behav 84, 229–234. https://doi.org/10.1016/j.pbb.2006.05.001 Bartsch, T., Wulff, P., 2015. The hippocampus in aging and disease: From plasticity to vulnerability. Neuroscience. https://doi.org/10.1016/j.neuroscience.2015.07.084 Basner, M., Babisch, W., Davis, A., Brink, M., Clark, C., Janssen, S., Stansfeld, S., 2014. Auditory and non-auditory effects of noise on health. The Lancet 383, 1325–1332. https://doi.org/10.1016/S0140-6736(13)61613-X Beers, R., Sizer, I., 1952. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem. 195, 133–140. Belhorma, K., Darwish, N., Benn-Hirsch, E., Duenas, A., Gates, H., Sanghera, N., Wu, J., French, R.L., 2021. Developmental ethanol exposure causes central nervous system dysfunction and may slow the aging process in a Drosophila model of fetal alcohol spectrum disorder. Alcohol 94. https://doi.org/10.1016/j.alcohol.2021.03.006 Berglund, B., Lindvall, T., Schwela, D.H., 2000. New Who Guidelines for Community Noise. Noise & Vibration Worldwide 31. https://doi.org/10.1260/0957456001497535 Borodovitsyna, O., Flamini, M.D., Chandler, D.J., 2018. Acute Stress Persistently Alters Locus Coeruleus Function and Anxiety-like Behavior in Adolescent Rats. Neuroscience 373. https://doi.org/10.1016/j.neuroscience.2018.01.020 Brown, S.A., McGue, M., Maggs, J., Schulenberg, J., Hingson, R., Swartzwelder, S., Martin, C., Chung, T., Tapert, S.F., Sher, K., Winters, K.C., Lowman, C., Murphy, S., 2008. A developmental perspective on alcohol and youths 16 to 20 years of age. Pediatrics 121, S290–S310. https://doi.org/10.1542/peds.2007-2243D Buján, G.E., D’Alessio, L., Serra, H.A., Molina, S.J., Guelman, L.R., 2022. Behavioural alterations induced by intermittent ethanol intake and noise exposure in adolescent rats. European Journal of Neuroscience 55, 1756–1773. https://doi.org/10.1111/ejn.15657 Campeau, S., Dolan, D., Akil, H., Watson, S.J., 2002. c-fos mRNA induction in acute and chronic audiogenic stress: Possible role of the orbitofrontal cortex in habituation. Stress 5, 121–130. https://doi.org/10.1080/10253890290027895 Cappaert, N.L.M., Klis, S.F.L., Muijser, H., Kulig, B.M., Smoorenburg, G.F., 2000. Noise-induced hearing loss in rats. Noise Health 3, 23–32. Carnicella, S., Ron, D., Barak, S., 2014. Intermittent ethanol access schedule in rats as a preclinical model of alcohol abuse. Alcohol 48, 243–252. https://doi.org/10.1016/j.alcohol.2014.01.006 Carobrez, A.P., Bertoglio, L.J., 2005. Ethological and temporal analyses of anxiety-like behavior: The elevated plus-maze model 20 years on. Neurosci Biobehav Rev 29, 1193–1205. https://doi.org/10.1016/j.neubiorev.2005.04.017 Chen, Z., Zhong, C., 2014. Oxidative stress in Alzheimer’s disease. Neurosci Bull 30, 271–281. https://doi.org/10.1007/s12264-013-1423-y Choi, M.R., Han, J.S., Chai, Y.G., Jin, Y., Lee, S., Kim, D., 2020. Gene expression profiling in the hippocampus of adolescent rats after chronic alcohol administration. Basic Clin Pharmacol Toxicol 126, 389–398. https://doi.org/10.1111/bcpt.13342 Cosgrove, J.A., Kelly, L.K., Kiffmeyer, E.A., Kloth, A.D., 2022. Sex-dependent influence of postweaning environmental enrichment in Angelman syndrome model mice. Brain Behav 12. https://doi.org/10.1002/brb3.2468 Dandi, E., Spandou, E., Tata, D.A., 2022. Investigating the role of environmental enrichment initiated in adolescence against the detrimental effects of chronic unpredictable stress in adulthood: Sex-specific differences in behavioral and neuroendocrinological findings. Behavioural Processes 200. https://doi.org/10.1016/j.beproc.2022.104707 de Souza, T.C.F., Périssé, A.R.S., Moura, M., 2015. Noise exposure and hypertension: investigation of a silent relationship. BMC Public Health 15, 328. https://doi.org/10.1186/s12889-015-1671-z DeWit, D.J., Adlaf, E.M., Offord, D.R., Ogborne, A.C., 2000. Age at first alcohol use: A risk factor for the development of alcohol disorders. American Journal of Psychiatry 157, 745–750. https://doi.org/10.1176/appi.ajp.157.5.745 Driver, A.S., Kodavanti, P.R.S., Mundy, W.R., 2000. Age-related changes in reactive oxygen species production in rat brain homogenates. Neurotoxicol Teratol 22, 175–181. https://doi.org/10.1016/S0892-0362(99)00069-0 Ehlers, C.L., Gizer, I.R., Vieten, C., Gilder, A., Gilder, D.A., Stouffer, G.M., Lau, P., Wilhelmsen, K.C., 2010. Age at regular drinking, clinical course, and heritability of alcohol dependence in the San Francisco family study: A gender analysis. American Journal on Addictions 19, 101–110. https://doi.org/10.1111/j.1521-0391.2009.00021.x Ennaceur, A., Michalikova, S., Chazot, P.L., 2009. Do rats really express neophobia towards novel objects? Experimental evidence from exposure to novelty and to an object recognition task in an open space and an enclosed space. Behavioural brain research 197, 417–434. https://doi.org/10.1016/j.bbr.2008.10.007 European Environment Agency, 2020. Environmental noise in Europe - 2020, European Environment Agency. Fanselow, M.S., Dong, H.W., 2010. Are the dorsal and ventral hippocampus functionally distinct structures? Neuron 65, 7–19. https://doi.org/10.1016/J.NEURON.2009.11.031 Fernández, M.S., Carrizo, J., Plaza, W., Haeger, P., Pautassi, R.M., 2019. Prenatal ethanol exposure potentiates isolation-induced ethanol consumption in young adult rats. Alcohol 75, 39–46. https://doi.org/10.1016/j.alcohol.2018.05.006 Fuentes-Santamaría, V., Alvarado, J.C., Mellado, S., Melgar-Rojas, P., Gabaldón-Ull, M.C., Cabanes-Sanchis, J.J., Juiz, J.M., 2022. Age-Related Inflammation and Oxidative Stress in the Cochlea Are Exacerbated by Long-Term, Short-Duration Noise Stimulation. Front Aging Neurosci 14. https://doi.org/10.3389/fnagi.2022.853320 Gogokhia, N., Japaridze, N., Tizabi, Y., Pataraya, L., Zhvania, M.G., 2021. Gender differences in anxiety response to high intensity white noise in rats. Neurosci Lett 742. https://doi.org/10.1016/j.neulet.2020.135543 Hahad, O., Prochaska, J.H., Daiber, A., Muenzel, T., 2019. Environmental Noise-Induced Effects on Stress Hormones, Oxidative Stress, and Vascular Dysfunction: Key Factors in the Relationship between Cerebrocardiovascular and Psychological Disorders. Oxid Med Cell Longev 2019, 4623109. https://doi.org/10.1155/2019/4623109 Haider, S., Sajid, I., Batool, Z., Madiha, S., Sadir, S., Kamil, N., Liaquat, L., Ahmad, S., Tabassum, S., Khaliq, S., 2020. Supplementation of Taurine Insulates Against Oxidative Stress, Confers Neuroprotection and Attenuates Memory Impairment in Noise Stress Exposed Male Wistar Rats. Neurochem Res 45. https://doi.org/10.1007/s11064-020-03127-7 Han, Y., Yuan, M., Guo, Y.S., Shen, X.Y., Gao, Z.K., Bi, X., 2022. The role of enriched environment in neural development and repair. Front Cell Neurosci. https://doi.org/10.3389/fncel.2022.890666 Healey, K.L., Kibble, S.A., Bell, A., Kramer, G., Maldonado-Devincci, A., Swartzwelder, H.S., 2022. Sex differences in the effects of adolescent intermittent ethanol exposure on exploratory and anxiety-like behavior in adult rats. Alcohol 98. https://doi.org/10.1016/j.alcohol.2021.11.002 Heim, C., Nemeroff, C.B., 1999. The impact of early adverse experiences on brain systems involved in the pathophysiology of anxiety and affective disorders, in: Biological Psychiatry. pp. 1509–1522. https://doi.org/10.1016/S0006-3223(99)00224-3 Hu, B.H., Zheng, G.L., 2008. Membrane disruption: An early event of hair cell apoptosis induced by exposure to intense noise. Brain Res 1239, 107–118. https://doi.org/10.1016/j.brainres.2008.08.043 Hunt, P.S., Barnet, R.C., 2016. Adolescent and adult rats differ in the amnesic effects of acute ethanol in two hippocampus-dependent tasks: Trace and contextual fear conditioning. Behavioural Brain Research 298. https://doi.org/10.1016/j.bbr.2015.06.046 Ibáñez, C., Acuña, T., Quintanilla, M.E., Pérez-Reytor, D., Morales, P., Karahanian, E., 2023. Fenofibrate Decreases Ethanol-Induced Neuroinflammation and Oxidative Stress and Reduces Alcohol Relapse in Rats by a PPAR-α-Dependent Mechanism. Antioxidants 12. https://doi.org/10.3390/antiox12091758 Izquierdo, I., Furini, C.R.G., Myskiw, J.C., 2016. Fear Memory. Physiol Rev. https://doi.org/10.1152/physrev.00018.2015 Izquierdo, I., Medina, J.H., 1997. Memory formation: the sequence of biochemical events in the hippocampus and its connection to activity in other brain structures. Neurobiol Learn Mem 68, 285–316. https://doi.org/10.1006/nlme.1997.3799 Jiang, S., Wang, Y.Q., Tang, Y.F., Lu, X., Guo, D., 2022. Pre-Exposure to Environmental Enrichment Protects against Learning and Memory Deficits Caused by Infrasound Exposure. Oxid Med Cell Longev 2022. https://doi.org/10.1155/2022/6208872 Kim, S.Y., Jeong, S.H., Park, E.C., 2023. Age at onset of alcohol consumption and its association with alcohol misuse in adulthood. Neuropsychopharmacol Rep 43. https://doi.org/10.1002/npr2.12302 Kjelstrup, K.G., Tuvnes, F.A., Steffenach, H.-A., Murison, R., Moser, E.I., Moser, M.-B., 2002. Reduced fear expression after lesions of the ventral hippocampus. Proceedings of the National Academy of Sciences. https://doi.org/10.1073/pnas.152112399 Kołota, A., Głąbska, D., Oczkowski, M., Gromadzka-Ostrowska, J., 2019. Influence of Alcohol Consumption on Body Mass Gain and Liver Antioxidant Defense in Adolescent Growing Male Rats. Int J Environ Res Public Health 16, 2320. https://doi.org/10.3390/ijerph16132320 Lees, B., Meredith, L.R., Kirkland, A.E., Bryant, B.E., Squeglia, L.M., 2020. Effect of alcohol use on the adolescent brain and behavior. Pharmacol Biochem Behav. https://doi.org/10.1016/j.pbb.2020.172906 Leussis, M.P., Andersen, S.L., 2008. Is adolescence a sensitive period for depression? Behavioral and neuroanatomical findings from a social stress model. Synapse 62, 22–30. https://doi.org/10.1002/syn.20462 Leussis, M.P., Bolivar, V.J., 2006. Habituation in rodents: A review of behavior, neurobiology, and genetics. Neurosci Biobehav Rev. https://doi.org/10.1016/j.neubiorev.2006.03.006 Li, X., Fu, B., Zhao, C., Hu, J., Zhang, X., Fu, Yiming, She, X., Gu, C., Cheng, M., Wang, F., Song, X., Dai, J., Yin, J., Fu, Yu, Zheng, P., Wu, F., Zhu, Y., Ma, K., Gao, X., Wang, M., Zeng, Q., Cui, B., 2023. Early-life noise exposure causes cognitive impairment in a sex-dependent manner by disrupting homeostasis of the microbiota–gut–brain axis. Brain Behav Immun 114, 221–239. https://doi.org/10.1016/j.bbi.2023.08.021 Li, Y., Zhang, Z., Wang, J., Liu, C., Liu, Y., Jiang, X., Chen, Q., Ao, L., Cao, J., Sun, L., Han, F., Liu, J., 2023. Effects and possible mechanisms of combined exposure to noise and carbon monoxide on male reproductive system in rats. Environ Toxicol. https://doi.org/10.1002/tox.23927 Lopez, M.F., Laber, K., 2015. Impact of social isolation and enriched environment during adolescence on voluntary ethanol intake and anxiety in C57BL/6J mice. Physiol Behav. https://doi.org/10.1016/j.physbeh.2014.11.012 Lu, C.Q., Zhong, L., Yan, C.H., Tian, Y., Shen, X.M., 2017. Effects of preweaning environmental enrichment on hippocampus-dependent learning and memory in developing rats. Neurosci Lett 640. https://doi.org/10.1016/j.neulet.2016.12.053 Miceli, M., Molina, S.J., Forcada, A., Acosta, G.B., Guelman, L.R., 2018. Voluntary alcohol intake after noise exposure in adolescent rats: Hippocampal-related behavioral alterations. Brain Res 1679, 10–18. https://doi.org/10.1016/j.brainres.2017.11.001 Mira, R.G., Lira, M., Quintanilla, R.A., Cerpa, W., 2020. Alcohol consumption during adolescence alters the hippocampal response to traumatic brain injury. Biochem Biophys Res Commun 528, 514–519. https://doi.org/10.1016/j.bbrc.2020.05.160 Miranda-Morales, R.S., Haymal, B., Pautassi, R.M., 2016. Effects of ethanol exposure in a familiar or isolated context during infancy on ethanol intake during adolescence. Dev Psychobiol 58, 968–979. https://doi.org/10.1002/dev.21427 Molina, S.J., Buján, G.E., Guelman, L.R., 2021. Noise-induced hippocampal oxidative imbalance and aminoacidergic neurotransmitters alterations in developing male rats: Influence of enriched environment during adolescence. Dev Neurobiol 81. https://doi.org/10.1002/dneu.22806 Molina, S.J., Buján, G.E., Rodriguez Gonzalez, M., Capani, F., Gómez-Casati, M.E., Guelman, L.R., 2019. Exposure of Developing Male Rats to One or Multiple Noise Sessions and Different Housing Conditions: Hippocampal Thioredoxin Changes and Behavioral Alterations. Front Behav Neurosci 13, 182. https://doi.org/10.3389/fnbeh.2019.00182 Molina, S.J., Capani, F., Guelman, L.R., 2016. Noise exposure of immature rats can induce different age-dependent extra-auditory alterations that can be partially restored by rearing animals in an enriched environment. Brain Res 1636, 52–61. https://doi.org/10.1016/j.brainres.2016.01.050 Mooney-Leber, S.M., Gould, T.J., 2018. The long-term cognitive consequences of adolescent exposure to recreational drugs of abuse, in: Learning and Memory. https://doi.org/10.1101/lm.046672.117 Mundt, M.P., Zakletskaia, L.I., Brown, D.D., Fleming, M.F., 2012. Alcohol-induced memory blackouts as an indicator of injury risk among college drinkers. Injury Prevention. https://doi.org/10.1136/ip.2011.031724 Novkovic, T., Mittmann, T., Manahan‐Vaughan, D., 2015. BDNF contributes to the facilitation of hippocampal synaptic plasticity and learning enabled by environmental enrichment. Hippocampus 25, 1–15. https://doi.org/10.1002/hipo.22342 Pellow, S., Chopin, P., File, S.E., Briley, M., 1985. Validation of open : closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Methods 14, 149–167. https://doi.org/10.1016/0165-0270(85)90031-7 Pervin, Z., Stephen, J.M., 2021. Effect of Alcohol on the Central Nervous System to Develop Neurological Disorder: Pathophysiological and Lifestyle Modulation can be Potential Therapeutic Options for Alcohol-Induced Neurotoxication. AIMS Neurosci 8, 390–413. https://doi.org/10.3934/NEUROSCIENCE.2021021 Phillips, S.A., Osborn, K., Hwang, C.-L., Sabbahi, A., Piano, M.R., 2021. Ethanol Induced Oxidative Stress in the Vasculature: Friend or Foe. Curr Hypertens Rev 16, 181–191. https://doi.org/10.2174/1573402115666190325124622 Popović, N., Caballero-Bleda, M., Popović, M., 2014. Post-Training Scopolamine Treatment Induced Maladaptive Behavior in Open Field Habituation Task in Rats. PLoS One 9, e100348. https://doi.org/10.1371/journal.pone.0100348 Prado Spalm, F.H., Cuervo Sánchez, M.L., Furland, N.E., Vallés, A.S., 2023. Lipid peroxidation and neuroinflammation: A possible link between maternal fructose intake and delay of acquisition of neonatal reflexes in Wistar female rats. Dev Neurobiol 83. https://doi.org/10.1002/dneu.22921 Recio, A., Linares, C., Banegas, J.R., Díaz, J., 2016. Road traffic noise effects on cardiovascular, respiratory, and metabolic health: An integrative model of biological mechanisms. Environ Res 146, 359–370. https://doi.org/10.1016/j.envres.2015.12.036 Rodgers, R.J., Cole, J.C., 1993. Influence of social isolation, gender, strain, and prior novelty on plus-maze behaviour in mice. Physiol Behav 54, 729–736. https://doi.org/10.1016/0031-9384(93)90084-S Roozendaal, B., 2002. Stress and Memory: Opposing Effects of Glucocorticoids on Memory Consolidation and Memory Retrieval GLUCOCORTICOIDS AND MEMORY FUNCTION. Neurobiol Learn Mem 78, 578–595. https://doi.org/10.1006/nlme.2002.4080 Salling, M.C., Skelly, M.J., Avegno, E., Regan, S., Zeric, T., Nichols, E., Harrison, N.L., 2018. Alcohol consumption during adolescence in a mouse model of binge drinking alters the intrinsic excitability and function of the prefrontal cortex through a reduction in the hyperpolarization-activated cation current. Journal of Neuroscience 38. https://doi.org/10.1523/JNEUROSCI.0550-18.2018 Sanchez-Marin, L., Gavito, A.L., Decara, J., Pastor, A., Castilla-Ortega, E., Suarez, J., de la Torre, R., Pavon, F.J., Rodriguez de Fonseca, F., Serrano, A., 2020. Impact of intermittent voluntary ethanol consumption during adolescence on the expression of endocannabinoid system and neuroinflammatory mediators. European Neuropsychopharmacology 33. https://doi.org/10.1016/j.euroneuro.2020.01.012 Sasse, S.K., Greenwood, B.N., Masini, C. V, Nyhuis, T.J., Fleshner, M., Day, H.E.W., Campeau, S., 2008. Chronic voluntary wheel running facilitates corticosterone response habituation to repeated audiogenic stress exposure in male rats. Stress 11, 425–437. https://doi.org/10.1080/10253890801887453 Shukla, M., Mani, K.V., Deepshikha, Shukla, S., Kapoor, N., 2020. Moderate noise associated oxidative stress with concomitant memory impairment, neuro-inflammation and neurodegeneration. Brain Behav Immun Health 5, 100089. https://doi.org/10.1016/j.bbih.2020.100089 Sircar, R., 2019. Estrogen Modulates Ethanol-Induced Memory Deficit in Postpubertal Adolescent Rats. Alcohol Clin Exp Res 43. https://doi.org/10.1111/acer.13921 Spear, L., 2000. Modeling adolescent development and alcohol use in animals. Alcohol Res Health 24, 115–123. Spear, L.P., 2015. Adolescent alcohol exposure: Are there separable vulnerable periods within adolescence? Physiol Behav 148, 122–30. https://doi.org/10.1016/j.physbeh.2015.01.027 Spreng, M., 2000. Central nervous system activation by noise. Noise Health 2, 49–58. Tatu, L., Vuillier, F., 2014. Structure and vascularization of the human hippocampus, in: The Hippocampus in Clinical Neuroscience. https://doi.org/10.1159/000356440 Teixeira, F.B., Santana, L.N.D.S., Bezerra, F.R., De Carvalho, S., Fontes, E.A., Prediger, R.D., Crespo-López, M.E., Maia, C.S.F., Lima, R.R., 2014. Chronic ethanol exposure during adolescence in rats induces motor impairments and cerebral cortex damage associated with oxidative stress. PLoS One. https://doi.org/10.1371/journal.pone.0101074 Terry-McElrath, Y.M., O’Malley, P.M., Johnston, L.D., 2014. Alcohol and marijuana use patterns associated with unsafe driving among U.S. high school seniors: high use frequency, concurrent use, and simultaneous use. J Stud Alcohol Drugs 75, 378–389. https://doi.org/10.15288/jsad.2014.75.378 Tillage, R.P., Foster, S.L., Lustberg, D., Liles, L.C., McCann, K.E., Weinshenker, D., 2021. Co-released norepinephrine and galanin act on different timescales to promote stress-induced anxiety-like behavior. Neuropsychopharmacology 46. https://doi.org/10.1038/s41386-021-01011-8 Toniazzo, A.P., Arcego, D.M., Lazzaretti, C., Mota, C., Schnorr, C.E., Pettenuzzo, L.F., Krolow, R., Fonseca Moreira, J.C., Dalmaz, C., 2019. Sex-dependent effect on mitochondrial and oxidative stress parameters in the hypothalamus induced by prepubertal stress and access to high fat diet. Neurochem Int 124, 114–122. https://doi.org/10.1016/j.neuint.2019.01.008 Townshend, J.M., Kambouropoulos, N., Griffin, A., Hunt, F.J., Milani, R.M., 2014. Binge Drinking, Reflection Impulsivity, and Unplanned Sexual Behavior: Impaired Decision-Making in Young Social Drinkers. Alcohol Clin Exp Res 38. https://doi.org/10.1111/acer.12333 Turner, J.G., Parrish, J.L., Hughes, L.F., Toth, L.A., Caspary, D.M., 2005. Hearing in laboratory animals: Strain differences and nonauditory effects of noise. Comp Med 55, 12–23. https://doi.org/10.1016/j.biotechadv.2011.08.021.Secreted Uran, S.L., Aon-Bertolino, M.L., Caceres, L.G., Capani, F., Guelman, L.R., 2012. Rat hippocampal alterations could underlie behavioral abnormalities induced by exposure to moderate noise levels. Brain Res 1471, 1–12. https://doi.org/10.1016/j.brainres.2012.06.022 Uran, S.L., Gómez-Casati, M.E., Guelman, L.R., 2014. Long-term recovery from hippocampal-related behavioral and biochemical abnormalities induced by noise exposure during brain development. Evaluation of auditory pathway integrity. International Journal of Developmental Neuroscience 37, 41–51. https://doi.org/10.1016/j.ijdevneu.2014.06.002 Varlinskaya, E.I., Hosová, D., Towner, T., Werner, D.F., Spear, L.P., 2020. Effects of chronic intermittent ethanol exposure during early and late adolescence on anxiety-like behaviors and behavioral flexibility in adulthood. Behavioural Brain Research 378. https://doi.org/10.1016/j.bbr.2019.112292 Varlinskaya, E.I., Kim, E.U., Spear, L.P., 2017. Chronic intermittent ethanol exposure during adolescence: Effects on stress-induced social alterations and social drinking in adulthood. Brain Res 1654, 145–156. https://doi.org/10.1016/j.brainres.2016.03.050 Vianna, M.R.M., Alonso, M., Viola, H., Quevedo, J., De Paris, F., Furman, M., De Stein, M.L., Medina, J.H., Izquierdo, I., 2000. Role of hippocampal signaling pathways in long-term memory formation of a nonassociative learning task in the rat. Learning and Memory 7, 333–340. https://doi.org/10.1101/lm.34600 Walf, A.A., Frye, C.A., 2007. The use of the elevated plus maze as an assay of anxiety-related behavior in rodents. Nat Protoc 2, 322–8. https://doi.org/10.1038/nprot.2007.44 Wallas, A.E., Eriksson, C., Ögren, M., Pyko, A., Sjöström, M., Melén, E., Pershagen, G., Gruzieva, O., 2020. Noise exposure and childhood asthma up to adolescence. Environ Res 185. https://doi.org/10.1016/j.envres.2020.109404 Weydert, C.J., Cullen, J.J., 2010. Measurement of superoxide dismutase, catalase and glutathione peroxidase in cultured cells and tissue. Nat Protoc 5, 51–66. https://doi.org/10.1038/NPROT.2009.197 White, B.A., Ivey, J.T., Velazquez-Cruz, R., Oliverio, R., Whitehead, B., Pinti, M., Hollander, J., Ma, L., Hu, G., Weil, Z.M., Karelina, K., 2023. Exercise intensity and sex alter neurometabolic, transcriptional, and functional recovery following traumatic brain injury. Exp Neurol 368. https://doi.org/10.1016/j.expneurol.2023.114483 WHO, 1999. WHO guidelines for community noise. Noise & Vibration Worldwide 31, 161. https://doi.org/10.1260/0957456001497535 Wille-Bille, A., de Olmos, S., Marengo, L., Chiner, F., Pautassi, R.M., 2017. Long-term ethanol self-administration induces DeltaFosB in male and female adolescent, but not in adult, Wistar rats. Prog Neuropsychopharmacol Biol Psychiatry 74, 15–30. https://doi.org/10.1016/j.pnpbp.2016.11.008 Xie, Q., Buck, L.A., Bryant, K.G., Barker, J.M., 2019. Sex Differences in Ethanol Reward Seeking Under Conflict in Mice. Alcohol Clin Exp Res 43. https://doi.org/10.1111/acer.14070 Xu, H., Li, H., Liu, D., Wen, W., Xu, M., Frank, J.A., Chen, J., Zhu, H., Grahame, N.J., Luo, J., 2021. Chronic Voluntary Alcohol Drinking Causes Anxiety-like Behavior, Thiamine Deficiency, and Brain Damage of Female Crossed High Alcohol Preferring Mice. Front Pharmacol 12. https://doi.org/10.3389/fphar.2021.614396 Zhang, Y., Zhu, M., Sun, Y., Tang, B., Zhang, G., An, P., Cheng, Y., Shan, Y., Merzenich, M.M., Zhou, X., 2021. Environmental noise degrades hippocampus-related learning and memory. Proceedings of the National Academy of Sciences 118. https://doi.org/10.1073/pnas.2017841117 Legends to figures Figure 1. Summary of the experimental design. Sham: control, non-exposed animals; PND: postnatal day; S1- S6: Sessions 1-6; EtOH: ethanol intake. Noise: noise exposure. SC: Standard Cage; EE: Enriched environment cage. Figure 2. Ratio between the latency to enter the dark compartment in the retention and the training session (T2/T1) in the IA task. (a) Females; (b) Males. *: indicate significant differences between groups denoted with a line (p < 0.05). #: indicate significant differences between SC and EE of the corresponding group. Φ: indicate significant differences between males and females of the corresponding group. Data are expressed as mean±SEM, n = 6–8 for each group. SC: standard cage; EE: Enriched environment cage. Figure 3. Percent of incursions and latency to open arms in the EPM task. (a) and (c) Females; (b) and (d): Males. *: indicate significant differences between groups denoted with a line (p < 0.05). #: indicate significant differences between SC and EE of the corresponding group. Φ: indicate significant differences between males and females of the corresponding group. Data are expressed as mean±SEM, n = 6–8 for each group. SC: standard cage; EE: Enriched environment cage. Figure 4. Percent number of head dipping in the closed arms (% HD-CA). (a) Females; (b) Males. *: indicate significant differences between groups denoted with a line (p < 0.05). #: indicate significant differences between SC and EE of the corresponding group. Φ: indicate significant differences between males and females of the corresponding group. Data are expressed as mean±SEM, n = 6–8 for each group. SC: standard cage; EE: Enriched environment cage. Figure 5. Time in the center of the OF task. a) Females; (b) Males. *: indicate significant differences between groups denoted with a line (p < 0.05). #: indicate significant differences between SC and EE of the corresponding group. Φ: indicate significant differences between males and females of the corresponding group. Data are expressed as mean±SEM, n = 6–8 for each group. SC: standard cage; EE: Enriched environment cage. Figure 6. Time of climbing + rearings in the OF task (seconds). (a) Males; (b) Females. *: indicate significant differences between groups denoted with a line (p < 0.05). #: indicate significant differences between SC and EE of the corresponding group. Φ: indicate significant differences between males and females of the corresponding group. Data are expressed as mean±SEM, n = 6–8 for each group. SC: standard cage; EE: Enriched environment cage. Supplementary Material File (table 1.docx) Download 16.53 KB Information & Authors Information Version history V1 Version 1 13 March 2025 Peer review timeline Published European Journal of Neuroscience Version of Record 20 Oct 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection European Journal of Neuroscience Authors Affiliations Gustavo Buján Universidad de Buenos Aires Facultad de Medicina View all articles by this author Luciana D´Alessio Universidad de Buenos Aires Facultad de Medicina View all articles by this author Héctor Serra Universidad de Buenos Aires Facultad de Medicina View all articles by this author Gonzalo Corsi CONICET View all articles by this author Sonia Molina CONICET View all articles by this author Laura Guelman 0000-0002-5251-3006 [email protected] Universidad de Buenos Aires View all articles by this author Metrics & Citations Metrics Article Usage 219 views 176 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Gustavo Buján, Luciana D´Alessio, Héctor Serra, et al. Impact of enriched environment on hippocampal-related behavioral changes induced by extended voluntary ethanol intake and noise exposure in male and female adolescent Wistar rats.. Authorea . 13 March 2025. DOI: https://doi.org/10.22541/au.174185672.20648684/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . Format Please select one from the list RIS (ProCite, Reference Manager) EndNote BibTex Medlars RefWorks Direct import Tips for downloading citations document.getElementById('citMgrHelpLink').addEventListener('click', function() { popupHelp(this.href); return false; }); $(".js__slcInclude").on("change", function(e){ if ($(this).val() == 'refworks') $('#direct').prop("checked", false); $('#direct').prop("disabled", ($(this).val() == 'refworks')); }); View Options View options PDF View PDF Figures Tables Media Share Share Share article link Copy Link Copied! Copying failed. Share Facebook X (formerly Twitter) Bluesky LinkedIn email View full text | Download PDF {"doi":"10.22541/au.174185672.20648684/v1","type":"Article"} Now Reading: Share Figures Tables Close figure viewer Back to article Figure title goes here Change zoom level Go to figure location within the article Download figure Toggle share panel Toggle share panel Share Toggle information panel Toggle information panel Go to previous graphic Go to next graphic Go to previous table Go to next table All figures All tables View all material View all material xrefBack.goTo xrefBack.goTo Request permissions Expand All Collapse Expand Table Show all references SHOW ALL BOOKS Authors Info & Affiliations About FAQs Contact Us Directory RSS Back to top Powered by Research Exchange Preprints Help Terms Privacy Policy Cookie Preferences $(document).ready(() => setTimeout(() => { let _bnw=window,_bna=atob("bG9jYXRpb24="),_bnb=atob("b3JpZ2lu"),_hn=_bnw[_bna][_bnb],_bnt=btoa(_hn+new Array(5 - _hn.length % 4).join(" ")); $.get("/resource/lodash?t="+_bnt); },4000)); (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'9ff4a7ae1c88300f',t:'MTc3OTM3Nzc2OA=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();