Assessment of hippocampal-related behavioral changes in adolescent rats of both sexes following voluntary intermittent ethanol intake and noise exposure: a putative underlying mechanism and implementation of a non-pharmacological preventive strategy

Ethanol (EtOH) intake and noise exposure are particularly concerning among human adolescents because the potential to harm brain. Unfortunately, putative underlying mechanisms remain to be elucidated. Moreover, although neuroprotection tools could aid to prevent individuals from injury, limited data are available. Using an adolescent animal model, present study aims to explore a possible mechanism that could underlie the altered hippocampus-dependent behaviors induced by EtOH intake and/or noise exposure, as well as the neuroprotective impact of an environmental challenge. Adolescent Wistar rats of both sexes were subjected to an intermittent voluntary EtOH intake paradigm for one week. A subgroup of animals was exposed to white noise for two hours after the last session of EtOH intake. Some animals of both groups were housed in EE cages. Hippocampal-dependent behavioral assessment and hippocampal oxidative state evaluation were performed. Present results show that different behavioral alterations might be induced in animals of both sexes after EtOH intake, with or without sequential exposure to noise, that in some cases are sex-specic. Moreover, hippocampal oxidative imbalance seems to be one of the potential underlying mechanisms. Additionally, most behavioral and oxidative alterations were prevented by EE. These �ndings suggest that two recreational agents may impact behavior via oxidative pathways, in both sexes, in an animal model. In addition, improved housing resulted an effective neuroprotective strategy. Therefore, it could be suggested that a non-pharmacological approach might potentially provide neuroprotective advantages against other challenges. Finally, it might be worth considering its potential for translational human bene�t.


Introduction
Adolescence constitutes a pivotal phase marked by profound changes in the Central Nervous System (CNS).During this period, multiple connections are being established, giving rise to the development of diverse nervous processes.This phenomenon has been extensively studied and documented by several authors (see Spear(2015) for a detailed review).Moreover, different investigations have indicated that during this period, the presence of different physical or chemical agents can disrupt the normal Excessive alcohol consumption during adolescence is closely associated with an elevated risk of accidents, injuries, and engaging in risky behaviors such as unprotected sex and substance abuse.Furthermore, it can lead to long-term health consequences, including the development of alcohol addiction, impaired brain development, cognitive de cits, and an increased susceptibility to mental disorders (Mundt et al. 2012;Townshend et al. 2014).Furthermore, animal studies have demonstrated that early consumption of EtOH may be associated with a progression towards alcohol dependence in adulthood (Ehlers et al. 2010;Terry-McElrath et al. 2014;Amodeo et al. 2017).Considering the scarcity of reports in the literature utilizing self-administration of EtOH, compared to a forced exposure, novel ndings using voluntary access paradigms would be highly valuable.Lastly, unraveling the cellular and molecular mechanisms involved in EtOH-induced damage, especially within the developing CNS, could facilitate the development of experimental tools with the speci c goal of mitigating its effects.
On the other hand, noise is a physical agent that can also likewise pose risks to health (Recio et al. 2016;Li et al. 2023a).It is a potentially stressful environmental stimulus that can be de ned as an unpleasant sound, typically of moderate to high intensity, and can be harmful when exceeding 80 dB (Berglund et al. 2000).Despite the recent surge in high noise levels observed in major cities, its negative impact has frequently been overlooked.Estimates from the World Health Organization and European agency (European Environment Agency, 2020; WHO, 1999), based on scienti c 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. 2023b) that can affect both auditory structures (Cappaert et al., 2000;Hu and Zheng, 2008) and extra-auditory areas in the nervous, endocrine, and/or cardiovascular systems (Basner et al., 2014;de Souza et al., 2015;Spreng, 2000;Turner et al., 2005).
Among CNS structures, dysfunction or damage to the hippocampus (HC) by physical o chemical agents can result in disturbances in behavioral and emotional aspects.In particular, the HC has been shown to be involved in various types of memory (episodic, semantic, declarative), novelty detection, spatial navigation, and the binding of temporally and spatially distributed representations (Bartsch and Wulff, 2015;Fanselow and Dong, 2010).In addition to these cognitive functions, the HC also participates in the regulation of emotions (such as anxiety) and stress response (Tatu and Vuillier, 2014).Moreover, changes in HC oxidative state can induce misfunctioning of this structure (Haider et  It is known that oxidative stress can lead to neuronal damage because it disrupts the balance between reactive oxygen species (ROS) and antioxidants that is particularly relevant in the brain, considering the high vulnerability of CNS attributed to the high oxygen consumption rates and abundant lipid membranes, proteins and DNA that are susceptible to oxidative damage.Among antioxidants, catalase (CAT) is an antioxidant enzyme that prevents cellular oxidative damage by degrading hydrogen peroxide into water and oxygen with high e ciency (Alfonso-Prieto et al. 2009).Although previous studies from our laboratory have demonstrated that noise exposure can cause hippocampal damage in animals exposed during infancy, particularly affecting the oxidative state (Uran et al. 2014;Molina et al. 2021), we have not explored this aspect in animals exposed during adolescence.
Finally, the implementation of non-pharmacological neuroprotective strategies, such as the use of an enriched environment (EE), is achieving signi cant importance in basic research and its possible application in human beings.Environmental parameters have attracted far more attention in neurodevelopment and repairing neurological dysfunction due to the limited therapeutic effect of drugs and the relatively uncontrollable genetic background (Han et al., 2022).The EE includes more housing space when compared with standard cages (SC), with various toys and tunnels.In particular, it has proven to enhance performance in several learning and memory tasks.It has been shown to overcome learning defects caused by different genetic and neurological alterations (Lu et al., 2017).Given the proven e cacy of EE housing across diverse animal models, and the shortage of studies examining the effects of EtOH intake using an intermittent voluntary intake paradigm in combination with noise exposure, it would be of interest to investigate whether housing in an EE can prevent the effects produced by both agents in adolescent animals housed in SC.
Interestingly, in medical and scienti c research it is important to consider sex as a biological variable because biological differences between males and females can in uence susceptibility to diseases, treatment responses, and overall health outcomes, having broad effects on physiological processes in the body and being an essential modulator of brain function and behavior (Gogokhia et al. 2021).As there are few reports that demonstrate differences between sexes in EtOH consumption and noise exposure (Xie et al. 2019), examining the effects of these agents, both separately and in combination, as well as the possibility of protection through an environmental intervention in both sexes in a model of voluntary and intermittent EtOH intake and noise exposure, could produce unequal results that might allow the development of different prevention strategies personalized according to sexes differences.
For these reasons, the aims of the present study were to investigate a potential underlying mechanism of HC-dependent altered behaviors triggered after EtOH intake and/or noise exposure observed in adolescent animals of both sexes as well as the implementation of an environmental challenge used as a neuroprotective tool.

| Animals
Wistar rats of both sexes were purchased in the certi ed 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.When pregnancy was con rmed, female rats were housed individually in cages until the day of delivery, which was designated as postnatal day 0 (PND0) for the litter.
For the experiments, rats of both sexes were utilized.To minimize potential confounding factors related to the litter, only one male and one female from each litter were assigned to each experimental condition, behavioral task and oxidative state determination.
At weaning (PND21), two rats of the same sex were housed together in standard cages (SC, measures: 40 × 25 × 16 cm).A subgroup of animals of each experimental group was transferred to enriched cages (EE), in which same sex 4-5 rats were housed together (see below).
The experiments began when the animals reached the age of 28 days.Rats were randomly assigned to one of the following experimental groups: control (sham), noise, EtOH, and EtOH + noise.On average, seven rats were assigned to each group for each 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 corn ower was used as bedding material and the temperature was maintained at 21 ± 2°C.To minimize disruption of the circadian rhythm, both noise exposures and behavioral tests were conducted between 8 AM and 12 PM on PND33.
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 sacri ce at the conclusion of the experiments.For oxidative state experiments, animals were decapitated to obtain the HC.

| Noise exposure
To prevent any unnecessary handling and minimize potential stress to the animals during the noise exposure procedure, we maintained them in their original cages.These cages were then placed inside a custom-built wooden sound chamber, which measured 1 × 1 x 1 m 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 (Uran et al. 2012).
For sound ampli cation, 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.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, a group of animals was placed in the same chamber for the same duration but did not receive white noise exposure, and they were designated as the "sham" or "control" group.Furthermore, animals that consumed ethanol (EtOH) but were not exposed to noise were also sham-exposed to noise.Background level of white 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 (WHO 1999; European Environment Agency 2020) as well as is stated in several studies (Campeau et al. 2002;Sasse et al. 2008).
The intensity and duration of the white noise exposure in this study were carefully selected to ensure its potential translational value.The chosen parameters aimed to simulate and approximate the levels of noise experienced in various real-life settings, such as workplaces, recreational venues, and noisy urban areas.This decision was made considering the ndings reported by the WHO, which provided insights into the typical white noise levels present in such environments.

| Intermittent voluntary EtOH intake (two-bottle choice paradigm)
This paradigm was chosen in order 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.Brie y, animals were subjected to self-administered EtOH intake, from PND28 (i.e., 1 week after weaning) on three alternate days, designated as "sessions" (S1: PND28; S2: PND30; S3:PND32).This period of consumption was selected to allow exposure during the early adolescent development.According to the paradigm used, two bottles were placed on the top of each cage, in which two animals were housed together (except from those animals housed in EE, in which 4-5 animals were placed together): at the beginning of each session, animals of the EtOH and EtOH + noise groups were given two bottles, one with 5% ethanol 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.Animals of the sham and noise 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 bottles (Carnicella et al. 2014; Miranda-Morales et al. 2016).After 24 h, the bottles in all cages were replaced with a bottle containing tap water, which was available for the next 24 h.This procedure was repeated in the following sessions.
It should be highlighted that sucrose was used as a sweetener to motivate and stabilize daily EtOH intake, as different studies indicate that uninitiated adolescent Wistar rats drink low amounts of EtOH (concentrations of less than 5-6%), unless mixed with sucrose or deprived of water.In addition, the intermittent access procedure generally results in more pronounced intake acquisition curves than continuous access procedures (Acevedo et al. 2016;Miranda-Morales et al. 2016).
Extra bottles were placed in an empty cage that were used to correct for possible accidental leaks (i.e., spill control).The bottles were weighed before and after each session to provide a measure of uid intake, a method validated by Acevedo et al. (2016) and Wille-Bille et al. (2017).The amount of EtOH intake was calculated by subtracting the amount remaining after each 24-h period from the volume measured in each bottle at the beginning of the session and corrected by the % EtOH present in the solution (i.e., 5%).The animals were weighed, and EtOH intake was expressed per kilogram animal weight (g/kg body weight).The mean value of the total volume of ingested liquid (ml ingested liquid /kg body weight) and the volume of 5% EtOH solution consumed were also calculated, and an average value was estimated, as detailed in Bahi (2017), to obtain the percentage of EtOH preference ([ml EtOH intake/ml ingested liquid/kg body weight]*100).

Behavioral tasks
Behavioral tasks were performed at the end of the third day of voluntary EtOH intake, (PND33), after exposure to noise or sham groups (see Fig. 1 to view the timeline).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 signi cant risk factor for future EtOH consumption (Lopez and Laber 2015) and could have lasting effects on behavioral and biological responses in rodents (Leussis and Andersen 2008).In EE cages, animals were housed in groups of 4-5 animals throughout the experiment.
Habituation: Before each test, the rats were individually placed in a transfer cage for 3 minutes so that they could become familiar to 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 in uence physiological and behavioral stress indicators (Walf and 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% ethanol solution between sessions.

Inhibitory avoidance (IA) 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 an unpleasant experience occurred.Once the associative memory (AM) 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 and Medina 1997;Izquierdo et al. 2016).IA procedures were carried out as previously reported (Molina 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 oor 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 con ned 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).

Elevated plus maze (EPM) 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 and Bertoglio 2005).EPM validity is based on the natural con ict between the urge to explore a new environment and the tendency to avoid a potentially dangerous area (Pellow et al., 1985) (Pellow et al., 1985) (Pellow et al., 1985) (Pellow et al., 1985) (Pellow et al., 1985) (Pellow et al., 1985) (Pellow et al., 1985) (Pellow et al., 1985).Anxiety-related behaviors were calculated as the time spent in 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-related 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 threats or threatening situations (Rodgers and Cole 1993;Carobrez and Bertoglio 2005).One of these parameters is called head-dipping (HD) 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.EPM procedures were carried out as previously reported (Molina et al. 2016(Molina et al. , 2019(Molina et al. , 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 during 5 min.The percentage of time spent in 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 time and entries to the open arms was calculated as the values of open arms/(open arms + closed arms) × 100.The % HD-CA was calculated as HD-CA/(HD-CAarm + HD-OA) × 100.Only few rats randomly distributed across experimental groups fell when they walked along the open arms; these animals were excluded from the study.The animal's activity was recorded using a camcorder (Handycam DCR-DVD810, Sony).

Open eld (OF) task
An open eld device was used to analyze rats' exploratory activity and habituation memory, behaviors known to depend on the HC (Vianna et al. 2000; Barros et al. 2006; Leussis and Bolivar 2006).In this task, the activity in the rst session can be used to assess changes induced by exposure to a novel environment.In consequence, vertical exploratory activity can be quanti ed by recording the number of rearing and climbing events, i.e., rats 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 (Abuhamdah et al., 2012;Leussis and Bolivar, 2006).In addition, locomotor activity can be recorded by quantifying the number of crossed lines in the rst session (Popović et al. 2014).Furthermore, when performing a second session on the device, rats' habituation memory can be measured.In fact, a reduction in locomotor activity is expected to be found in naive animals because repeated exposure to the same environment turns it into a familiar place.Therefore, a signi cant difference between crossed lines in the rst and second sessions (i.e., more lines crossed in the rst than in the second session) can be considered as an index of preservation of habituation memory, as is usually observed in sham, nonexposed and non-EtOH-drinking animals.On the contrary, a similar locomotor activity in both sessions would indicate an alteration in habituation memory.Procedures were carried out as previously reported (Molina et al. 2016(Molina et al. , 2019(Molina et al. , 2021;;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 oor divided into 25 equal squares by black lines.The OF was illuminated with a 20-W lamp located above.
-First session: rats were placed on the center rear quadrant of the OF box and allowed to freely explore it during 5 min.The number of crossed lines, incursions to the central quadrants, as well as the number of rearing and climbing events, were recorded over the session.
-Second session: after 1 h in their home cages, animals were acclimatized to the behavioral room and allowed to explore the apparatus for another 5 min.The number of crossed lines was recorded and compared with the number crossed in the rst session to evaluate habituation to the device.

| 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).Brie y, 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 oor, a wheel to run, built-in blocks and a feeder.Different toys were placed in each cage, which varied along 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.The animals remained in the EE cages from PND28 until the end of the experiments and were cleaned every two days.
2.6 | Tissue collection: At PND33, a subgroup of animals was decapitated, the brain was exposed, and HC was rapidly dissected on an ice frozen petri dish.Then, the tissue was weighed and immediately stored in a −80°C freezer until the experiment, for a period no longer than a month.Finally, once approximately 20 samples were collected, ROS determination and CAT activity measurement were run.
2.7 | Reactive oxygen species determination: The levels of hippocampal ROS were determined by a method described by Driver et al. (2000), with modi cations.Brie y, tissues were homogenized in ice-cold with Locke's solution (0.5 mg of tissue/ml).Then, homogenates were pipetted into 24-well plates (0.45 ml/well) and left to warm at room temperature for 5 min.After that, 5 μl of dichloro-uorescein diacetate (0.97 mg of DCFH/ml in DSMO, 10 μM nal concentration) was added to each well and the mixture was incubated at room temperature for 15 min.Finally, the uorescence was measured at 485 nm (excitation) and 530 nm (emission).A standard curve was performed using oxidized dichloro-uorescein (DCF).The results were calculated as pmol DCF/mg of protein and expressed as average ± SEM.Protein concentration in homogenates was determined using a commercial kit based on the Bradford method, using bovine serum albumin (BSA) as standard.
2.8 | Catalase activity: CAT activity was determined according to Beers and Sizer (1952) and Weydert and Cullen (2010).Brie y, tissue homogenates 10% (w/v) were prepared in 50 mM phosphate buffer using a sonicator.Then, 50 μl of homogenate were placed in glass tubes and 4 ml of phosphate buffer was added.Tubes were mixed by inversion and each sample was divided into two parts.After that, 1 ml of phosphate buffer was added to one of the portions and 1 ml of 0.06 M hydrogen peroxide (H 2 O 2 ) solution was added to the other.The samples were measured immediately in a quartz cuvette using a spectrophotometer and the value of the rst sample (used as reference blank) was subtracted to that of the second tube.The absorbance was recorded every 15 s for a total of 150 s at a wavelength of 240 nm.A standard curve was performed using CAT from bovine liver.The results were calculated as units of CAT/mg of protein and expressed as average ± SEM.The concentration of proteins was determined using the Bradford method, as described in the previous item.
2.9 | Statistical analyses: a normality test for each group (KS test) was performed.Signi cant differences between the groups were analyzed using a three-way ANOVA test with post-hoc comparisons (LSD), using Infostat/L software.The factors were sex (male or female), treatment (sham, noise, EtOH, EtOH+Noise), and housing (SC or EE).When interactions were signi cant, 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 signi cant.Statistically signi cant 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.

Results
Figure 2 shows that in male animals housed in SC, noise exposure decreased the T2/T1 ratio in the inhibitory avoidance task in both sexes when compared to the respective sham group, as well as in females subjected to either noise, voluntary EtOH intake alone or sequentially exposed to noise.Furthermore, male and females differed in the T2/T1 ratio in all groups (three-way ANOVA, F 15.114 = 14,67, p < 0.01; between-subjects factors: treatment (sham, noise, EtOH, noise + EtOH), F 3,114 = 12,5, p < 0.01; sex (male or female), F 1,114 = 21,38, p < 0.01; housing (SC or EE), F 1,114 = 6,26, p = 0.014).As some interactions were signi cant (between treatment and sex, F 3,114 = 3,91, p = 0.011; treatment and housing, In Fig. 3 is shown that noise exposure decreased the percentage of time spent in the open arms of the EPM device only when it was preceded by EtOH intake in males housed in SC compared to the sham group.In contrast, in females housed in SC, only the intake of EtOH alone increased the percentage of this parameter, compared to their sham group.Finally, in animals housed in EE, no signi cant differences were observed in either group of both sexes, when compared to the respective SC group (three-way ANOVA, F 15,109 = 6,86, p < 0.01; between-subjects factors: treatment (sham, noise, EtOH, noise + EtOH), In Fig. 4 is shown that noise exposure decreased the percentage of incursions to the open arms of the EPM device only in males housed in SC, of all groups compared to the sham group.In contrast, the intake of EtOH, alone or sequentially to noise exposure, increased the percentage of this parameter in females housed in SC, compared to their sham group.Finally, in animals housed in EE, no signi cant differences were observed in either group of both sexes, when compared to the respective SC group (three-way ANOVA, F15,107 = 8,01, p < 0.01; between-subjects factors: treatment (sham, noise, EtOH, noise + EtOH), F 3,107 = 7,42, p < 0.01; sex (male or female), F 1,107 = 8,30, p < 0.01; housing (SC or EE), F 1,107 = 2,37, NS).
Figure 5 shows that EtOH intake increased the % HD-CA measured in the EPM device in males housed in SC, compared to the sham group, whereas in females an increase was observed when animals ingested EtOH and were sequentially exposed to noise.An increase in the % HD-CA was observed only in male animals that were exposed to noise and housed in EE, when compared to the corresponding SC group (three-way ANOVA, F15,106 = 3,28, p < 0.01; between-subjects factors: treatment (sham, noise, EtOH, noise + EtOH), F 3,106 = 2,53, NS; sex (male or female), F 1,106 = 1,98, NS; housing (SC or EE), F 1,106 = 3,93, p = 0,05).As some interactions were signi cant (between treatment and sex, F 1,106 , = 2,78, p = 0.045; treatment and housing, F 1,106 = 5,08, p < 0.01), the corresponding simple effects analysis were performed.
In Fig. 7, both noise exposure and EtOH intake -alone or in the presence of noise-decreased the incursions to the center recorded in the OF device in animals of both sexes housed in SC, when compared with the respective sham group.Interestingly, no signi cant differences in male animals housed in EE were observed in either group when compared to the respective SC group, whereas an increase when compared to the sham group was observed in females that were subjected to EtOH intake and sequentially exposed to noise (three-way ANOVA, F15,110 = 5,04, p < 0.01; between-subjects factors: treatment (sham, noise, EtOH, noise + EtOH), F 3,110 = 9,09, p < 0.01; sex (male or female), F 1,110 = 0,73, NS; housing (SC or EE), F 1,110 = 2,94, NS).As some interactions were signi cant (between treatment and sex, F 1,110 , = 4,24, p = 0.0074; treatment and housing, F 1,110 = 9,81, p < 0.01), the corresponding simple effects analysis were performed.In consequence, the following post-hoc comparisons were made (denoted with an asterisk, *, In Fig. 8 it can be observed that the intake of EtOH followed by exposure to noise increased the total time of rearing + climbing measured in the OF device only in males housed in SC, compared to the sham group, whereas in females a decrease in this parameter was observed in all groups when compared with the respective sham group.In addition, in animals housed in EE no signi cant differences were observed among groups (three-way ANOVA, F15,116 = 5,04, p < 0.01; between-subjects factors: treatment (sham, noise, EtOH, noise + EtOH), F 3,116 = 3,14, p = 0.0285; sex (male or female), F 1,116 = 1,46, NS; housing (SC or EE), F 1,116 = 0,46, NS).As some interactions were signi cant (between treatment and sex, Figure 9 showed that EtOH intake increased hippocampal ROS levels in both sexes housed in SC, when compared to the respective sham group.In addition, female animals exposed to noise showed a signi cant decrease in hippocampal ROS levels.Finally, in animals housed in EE, no signi cant differences were observed among either group (three-way ANOVA, F15,163 = 10,25, p < 0.01; betweensubjects factors: treatment (sham, noise, EtOH, noise + EtOH), F 3,163 = 12,78, p < 0.01; sex (male or female), F 1,163 = 0,55, NS; housing (SC or EE), F 1,163 = 85,66, p < 0.01).As one interaction was signi cant (between treatment and housing, F 1,163 = 8,86, p < 0.01), the corresponding simple effects analysis was performed.In consequence, the following post-hoc comparisons were made (denoted with an asterisk, *, When Catalase activity was measured (Fig. 10), an increase was observed in animals from both sexes that ingested EtOH and were housed in SC, compared to the respective sham group.No signi cant differences were observed among either group after housing in EE (three-way ANOVA, F15,81 = 6,88, p < 0.01; between-subjects factors: treatment (sham, noise, EtOH, noise + EtOH), F 3,81 = 11,27, p < 0.01; sex (male or female), F 1,81 = 4,81, p = 0,0318; housing (SC or EE), F 1,163 = 85,66, p < 0.01).As one interaction was signi cant (between treatment and housing, F 1,81 = 21,15, p < 0.01), the corresponding simple effects analysis was performed.In consequence, the following post-hoc comparisons were made (denoted with an asterisk, *, # or ¥): EtOH + noise: SC, p < 0,05; EE, NS; e) SC vs EE: all groups, p < 0,05; 3) Males vs. Females: SC: all groups, NS; EE: EtOH + Noise, p < 0,05; Sham, noise or EtOH, NS.

Discussion
Whereas previous studies of our laboratory have found that rats exposed to noise and/or subjected to EtOH intake experienced behavioral changes in parameters related to associative memory, exploratory activity, and anxiety-related behaviors (Buján et al. 2022), in the present paper we found that that these alterations were prevented by housing in EE.Importantly, present results show that an oxidative pathway seems to be one potential mechanism that could underlie the behavioral alterations.Finally, oxidative changes were also prevented by enrichment of the living environment of the animals, suggesting it as an effective neuroprotection strategy.
4.1 |Sex-dependent behavioral alterations: housing in an enriched environment is an effective neuroprotective tool in both sexes.
A large body of literature suggested that positive environmental stimuli such as EE had anxiolytic-and antidepressant-like properties in various animal models of different pathologies and injuries (Córneo et al., 2022; Wukitsch et al., 2020; X. Zhang et al., 2021).In the present study, hippocampal-dependent behaviors have been assessed after voluntary intermittent EtOH intake, with or without subsequent noise exposure in male and female adolescent rats and sex-dependent alterations were detected.Interestingly, EE, used as a non-pharmacological neuroprotective tool in those animals, was able to prevent most of them in both sexes.
The ndings revealed a notable decrease in the T2/T1 ratio in both male and female rats exposed to noise compared to their respective control groups in the IA test.Additionally, voluntary EtOH consumption, either alone or in conjunction with noise exposure, notably lowered this parameter in female animals.This decrease in the T2/T1 ratio indicates an impairment in associative contextual memory due to exposure to these stimuli (Atucha and Roozendaal, 2015).Furthermore, these results demonstrate that while noise was able to generate memory impairment in both sexes, females would be more vulnerable to the effects of EtOH intake.One week of EE housing was su cient to improve the de cit in AM observed in animals housed in SC.Supporting our results, different authors have demonstrated that the acute consumption of EtOH during adolescence can signi cantly alter contextual memory, particularly in a sexspeci c manner, with females exhibiting heightened vulnerability, as evidenced by research conducted by Hunt and Barnet (2016) and further supported by Sircar (2019).Sircar's ndings suggest that the increased susceptibility among females could be attributed to the in uence of estrogen, a key gonadal hormone in females, supporting the sex-dependence observed in the present manuscript.Interestingly, our research group (Molina et al., 2021) had reported an impairment of contextual memory in male rats induced by a single exposure to noise of infant rats.However, no prevention was observed after 2 weeks of EE housing.In contrast, our current ndings show a total improvement of this parameter.This difference could be mainly attributed to the earlier developmental age of animals reported in the mentioned paper (i.e., 15 vs. 28 PND), suggesting that the stage of development is crucial for an environmental intervention to be effective.
On the other hand, sex-speci c alterations in anxiety-related behavior, which were increased in males and decreased in females, were completely prevented by housing in an EE.Research of other authors has established that intermittent and enforced EtOH intake in adolescence induces anxiety-related behavioral changes, like those observed in our study (Varlinskaya et al. 2020;Healey et al. 2022).These studies utilized extended paradigms involving a minimum of 10 sessions of forced EtOH consumption, administering doses leading to alcohol intoxication.Our ndings support the notion that merely three sessions of voluntary 5% EtOH intake are enough to induce anxiety-related behavioral alterations in nonintoxicated animals during behavioral assessment.This emphasizes the critical need for utilizing voluntary consumption models that closely simulate the drinking habits of adolescent humans.It also underscores the concerning potency of EtOH, capable of inducing notable alterations in the HC and disturbing its oxidative balance after only a few sessions (see below), consequently instigating behavioral changes.Whereas exposure to noise resulted in heightened anxiety-related behavior in both sexes, a signi cant difference was found only among male rats.However, all changes were prevented after EE housing.As mentioned earlier, moderate to high-intensity noise is considered a stressor, thus likely to in uence and promote anxiety-related responses, as observed in other models (Borodovitsyna et al. 2018;Tillage et al. 2021).An escalation in these behaviors due to stressor exposure might serve as an adaptive response, aiding the animal in reacting suitably to threats (Izquierdo et al. 2016).However, excessive anxious behavior could lead to cognitive impairments (Molina et al. 2021), such as decreased associative memory, as observed in male rats.Furthermore, reduced anxiety-like behavior can also be maladaptive if it leads to decreased environmental exploration and novelty responsiveness, as observed in female rats that consumed EtOH, causing the animal to be less alert to potential dangers.Moreover, this reduced environmental exploration might also have impacted contextual associative memory, indicating that female rats may exhibit de cits in learning aversive situations crucial for survival.
A parameter that was signi cantly sensitive to the effects of both EtOH and noise, as well as to the combination of both, is entries to central quadrants of the OF device, which were found decreased in all groups of both sexes.There is controversy regarding the meaning of this parameter, as some authors interpret it as a measure of anxiety-related behavior, while others see it as a measure of environmental (Ennaceur et al. 2009;Abuhamdah et al. 2012).In this research, although this parameter was affected similarly in both sexes, it may be interpreted differently when compared to the results found in the other evaluated parameters.For instance, in males, entries to the central quadrants could be considered an indicator of anxiety-related behavior, as they decrease similarly to the percentage of entries and time spent in the open arm of the EPM.On the contrary, in females, this parameter seems to be related to exploratory behavior, as an overall decrease is observed.As was found in the other parameters, EE housing was able to prevent all these changes, normalizing anxiety parameters and suggesting that EE might also be able to operate on emotional behaviors.
Finally, risk assessment and habituation memory were barely affected by the aversive stimuli used.According to Leussis and Bolivar (2006), animals explore the novel environment and form an internal representation with spatial information.When this map is 'complete', the animal reduces exploration and is considered habituated to the context.Consequently, in the present work, the preservation of habituation memory is striking considering that associative memory (formed by acquiring information from the context) and exploratory activity were affected, especially in female rats.On the other hand, it would have also been expected to nd an increase in risk assessment in males showing heightened anxiety-related behavior, as these defensive behaviors entail greater caution and a search for potential escape strategies in the face of danger (Carobrez and Bertoglio 2005).However, this increase was only observed in the group of animals that consumed EtOH.Future research will be necessary to evaluate these discrepancies.
When animals were housed in EE, this parameter was increased in males exposed to noise, suggesting that a defensive behavior might act to compensate for a decrease in anxiety-like behavior induced in animals housed in SC.
These results are supported by several authors that have studied the effects of EE in rodents of both sexes and demonstrated positive effects on various behavioral parameters (Gabriel et al., 2020; Sakhaie et al., 2020).Interestingly, EE during the gestation signi cantly and consistently reduced social stressinduced anxiety-like behavior in the adulthood, that increased EtOH intake through a two-bottle choice drinking paradigm such as this used in our experiments (Bahi, 2024).In addition, Rico-Barrio et al. (2019) reported that young adult mice exposed to EE signi cantly recovered recognition, spatial and associative memory as well as motor coordination skills and balance that were signi cantly impaired after adolescent EtOH drinking with respect to controls, exerting positive effects on the long-term negative cognitive de cits induced by EtOH consumption during adolescence.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(Molina et al., , 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.
Unfortunately, relatively few preclinical studies have explored sex-speci c effects of different types of stressors or chemical agents on somatic, neuroendocrine and behavioral aspects, as well as their prevention by environmental non-pharmacological strategies.However, the ndings are not always consistent.Certain studies support that males are more vulnerable to somatic effects of chronic stress (e.g., adrenal hypertrophy, decreased body weight), while females are more susceptible to emotion-related behavioral and neuroendocrine changes, whereas there are studies reporting decreased anxiety-related behavior in female rodents, with males being more sensitive to the negative behavioral effects of stress (Dandi et al., 2022).
To highlight, although EE bene cial effects are thought to be sex-speci c, present results show no difference between sexes.Nevertheless, there is also some controversy.One reason is the insu cient number of studies that were conducted with animals of both sexes.Thus, a direct comparison regarding the EE impact on brain and behavior may not have been possible.In addition, when both sexes were represented, differences in age, strain, species or EE duration make it di cult to draw conclusions.In the present work, no sex-dependent differences were found in neither parameter when housed in EE, suggesting that the mild impairments observed in all of them are passible to be modi ed by an environmental intervention, being an effective route to correcting both modi ed phenotypes, as suggested by Cosgrove et al. (2022) in a mice model of Angelman syndrome.
In summary, voluntary EtOH consumption and 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.Females seem to be more sensitive to EtOH, displaying reduced anxiety-related behavior, exploratory activity, novelty response, and associative memory de cits.Conversely, a predominantly anxiogenic effect was observed in males.On the other hand, while associative memory de cits were induced in both sexes by noise, a notable anxiogenic effect was speci cally observed in males.
4.2 |Hippocampal oxidative imbalance (OI): one potential underlying mechanism that can be prevented by housing in an EE.
As widely reported, oxidative stress plays an important role in the pathogenesis of several diseases, leading to cellular damage and death (Chen and Zhong 2014; Phillips et al. 2021).In our laboratory, we have previously reported that exposure of developing infant male rats to white noise resulted in alterations of the oxidative state of the HC that persisted until adolescence and varied depending on the age and scheme of exposure (Molina et al. 2021).In addition, studies of other authors have demonstrated that noise exposure can increase stress hormone levels, leading to elevated OI (Hahad et al. 2019), as well as induce a variety of CNS injuries including neuroin ammation, neurodegeneration, and alterations in various behavioral parameters (Shukla et al. 2020).On the other hand, it has been shown that EtOH consumption during adolescence can lead to alterations in different structures of the CNS, with OI being proposed as a possible mechanism involved in the induced damage (Teixeira et  Present manuscript results show an increase in ROS levels in the group exposed to intermittent voluntary EtOH intake, both in males and females housed in SC.In contrast, in the group exposed to noise, a decrease in ROS levels was observed only in female animals, suggesting that its effect seems to be sexdependent.Supporting these results, authors such as White et al. (2023) reported sex differences in ROS after traumatic brain injury.Therefore, it could be hypothesized that a single exposure to noise may be su cient to generate an oxidative imbalance in females, that could be the consequence of an insu cient endogenous antioxidant system, in comparison with males.Conversely, although it could have been induced an initial increase in hippocampal ROS following noise exposure of males, it might be possible that a rapid counteraction directed by some endogenous antioxidant defenses, succeeded in maintaining the redox balance and suggesting that males have more defensive tools for coping with this environmental agent.Other authors also found differences among sexes: in an animal model, Prado Spalm et al. (2023) reported that only female progeny presented an increase in oxidative stress and antioxidant enzymes activities (SOD and CAT) in response to an oxidative damage-induced diet, suggesting that these enzymes might constitute an early putative protection mechanism that seem not to be active in males.
Concerning voluntary EtOH intake, it appears that three intermittent intake sessions would be su cient to induce hippocampal OI in both sexes, suggesting that EtOH could be a stronger stressor.Interestingly, after 7 months of EtOH voluntary drinking, OI is also found by other authors and it is not sex-speci c (Xu et al. 2021).Further experiments might con rm if in our experimental model, more intake sessions will worsen the OI.Importantly, processes such as neuroin ammation are thought to be associated with EtOH consumption that can be induced by two mechanisms, as suggested by Ibáñez et al (2023): one of them involves the oxidation of EtOH, generating ROS that, in turn can activate nuclear factor kappa-light-chainenhancer of activated B cells (NF-κB), leading to pro-in ammatory responses; through another mechanism, EtOH can increase the expression of NADPH oxidase, which generates even more ROS, perpetuating the in ammatory response.Studying EtOH-induced neuroin ammation will be the focus of our future investigations.
On the other hand, results show that hippocampal CAT activity increased both in males and females exposed to intermittent voluntary EtOH intake compared to their respective sham groups.Therefore, it could be suggested that the chosen paradigm for EtOH consumption seems to be effective in eliciting a response of the antioxidant CAT enzymatic activity as a compensation for the observed increase in ROS levels, as reported by other authors (Kołota et  ).On the contrary, no changes in CAT activity were found in any other group after exposure to noise.Interestingly, exposure to noise after the end of the EtOH intake paradigm seems to counteract EtOH effect, suggesting that last stressor could trigger compensatory mechanisms capable to achieve a new oxidative balance.Although these results differ from those reported by our laboratory (Uran et al. 2014), where an increase in CAT activity was found after a single exposure to noise on postnatal day 15, it could be attributed to the different neurodevelopmental stage of the experimental subjects used, that might account for differences in oxidative stress markers.
Given that a decrease in hippocampal ROS levels following exposure to noise observed in females was not accompanied by changes in CAT activity, it could be suggested that other components of the endogenous antioxidant system may be operating to reduce OI.Supporting these results, Toniazzo et al.
(2019) studied the effects of social isolation, a well-known stressor, during the prepubertal period, along with a high-fat diet, on oxidative status and mitochondrial function in various brain structures of male and female rats.They found sexual dimorphism in antioxidant enzymes, with higher superoxide dismutase (SOD) activity in females compared to males, and females also exhibited lower activity of complex I-III, the main mediators of ROS production, compared to males.Additional experiments could help in clarifying this matter.
When levels of ROS and CAT activity were evaluated after housing a subgroup of animals in EE cages, the de cits observed in those animals housed in SC were completely prevented.These ndings support those reported by Zhang et al. (2021), who suggest that EE was an effective tool for preventing hippocampal neuroin ammation and oxidative stress as well as promoting HC-related cognitive enhancement.In summary, the short duration of the environmental stimulation used in the present study appears to be su cient to generate improvements, in part due to the youth of the animals, as they were still in a growth stage with greater vulnerability and resilience to environmental stimuli, as reported by other authors (Heim and Nemeroff 1999; Spear 2000).

Conclusions
Present results show that different hippocampal-dependent behavioral changes might be differentially induced in animals of both sexes after voluntary intermittent EtOH intake, with or without sequential exposure to noise, being hippocampal oxidative imbalance one of the potential underlying mechanisms.
In addition, the signi cant differences observed when comparing animals housed in SC vs. EE cages might suggest that implementation of a non-pharmacological strategy could offer neuroprotective bene ts against these challenges.
Hence, these ndings suggest that two frequently found environmental and recreational agents may impact behavior via oxidative pathways in both sexes in an animal model that could be avoided through an environmental strategy.Finally, it might be worth considering its future application to achieve translational bene ts.

Declarations
development of the CNS (Acevedo et al. 2016; Varlinskaya et al. 2017; Wille-Bille et al. 2017; Mooney-Leber and Gould 2018; Lees et al. 2020; Baliño et al. 2021; Belhorma et al. 2021; Pervin and Stephen 2021), generating changes that could be long-lasting.In particular, the widespread consumption of drinks containing ethanol (EtOH) among adolescents worldwide, as well as their exposure to high-level noise in diverse entertainment settings(Brown et al. 2008; Salling et al. 2018) demonstrate the relevance of studying the effect of these agents using animal models (DeWit et al. 2000; Sanchez-Marin et al. 2020; Wallas et al. 2020; Barney et al. 2022).In fact, EtOH is one of the most used recreational abuse substances among adolescents.Therefore, chronic consumption during adolescence should constitute a major public health concern (Kim et al. 2023).

Figure 2 Ratio
Figure 2

Figure 3 Percent
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Figure 4 Percent
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Figure 5 Percent
Figure 5