Complex housing in adulthood state-dependently affects the excitation-inhibition balance in the infralimbic prefrontal cortex of male C57Bl/6 mice

The prefrontal cortex (PFC) plays an important role in social behavior and is sensitive to stressful circumstances. Challenging life conditions might change PFC function and put individuals at risk for maladaptive social behavior. The excitation-inhibition (EI) balance of prefrontal neurons appears to play a crucial role in this process. Here, we examined how a challenging life condition in C57BL/6JolaHsd mice, i.e. group-housing 6 mice in a complex environment for 10 days in adulthood, changes the EI-balance of infralimbic prefrontal neurons in layer 2/3, compared to standard pair-housing. Slices were prepared from “ undisturbed ” mice, i.e. the first mouse taken from the cage, or mice taken ~15 min later, who were mildly aroused after removal of the first mouse. We observed a housing-condition by arousal-state interaction, with in the complex housing group an elevated EI-balance in undisturbed and reduced EI-balance in mildly aroused animals, while no differences were observed in standard housed animals. The change was explained by a shift in mIPSC and mEPSC frequency, while amplitudes remained unaffected. Female mice showed no housing-by-state interaction, but a main effect of housing was found for mIPSCs, with a higher frequency in complex-versus standard-housed females. No effects were observed in males who were complex-housed from a young age onwards. Explorative investigations support a potential mediating role of corticosterone in housing effects on the EI-balance of males. We argue that taking the arousal state of individuals into account is necessary to better understand the consequences of exposure to challenging life conditions for prefrontal function.


INTRODUCTION
Housing animals in an enriched environment from weaning onwards allows them to show more of their natural behavior, having increased space to move around, including access to running wheels, cognitive stimulation by changing configurations and living with more peers (Fares et al., 2013).Such an environment stimulates plasticity (van Praag et al., 2000) and would lead to results that are meaningful within a social network in a context that is more stimulating compared to the relatively poor environment of standard lab cages (Kempermann, 2019).Changing the housing of male mice in adulthood however, from standard housing in pairs to group-housing in a complex environment could prove challenging and would need a period of adaptation, where a new hierarchy is formed and territorial possibilities explored (Olsson & Dahlborn, 2002).This is reminiscent of the complex social society we live in and could represent a model for the challenges that exposure to the complexity of human environments like cities bring (Kempermann, 2019).Such experiences can shape brain and behaviour, indeed, individual differences in (social) stress coping and thriving in life are partly defined by such life experiences, that can shape both brain and behavior and can play a part in the vulnerability or resilience to mental health problems (Costa e Silva & Steffen, 2019;Holz et al., 2020).
A crucial brain region for the cognitive and social aspects of an enriched environment is the prefrontal cortex (PFC), that, in connection with sub-cortical areas like the ventral striatum and amygdala, plays an important role in social behavior, including motivation to interact, social recognition, and dominance hierarchy in both mice and men (Bicks et al., 2015;Huang et al., 2020;Murugan et al., 2017;Park et al., 2021).For instance, it was shown that the connection between the IL-PFC and the nucleus accumbens shell is sensitive to social isolation and important for social recognition (Park et al., 2021) .A higher firing rate of prefrontal pyramidal neurons has been shown to correspond to increased social motivation in mice and this firing rate can be modulated by different interneurons and influenced by different neuromodulators (Bicks et al., 2015).An optimal balance of excitation and inhibition (EI-balance) in cortical circuits is important for information J o u r n a l P r e -p r o o f processing and changes in the EI-balance of pyramidal neurons regulate its activity during specific social behaviors.In particular, GABAergic interneurons in the PFC play an important role in the EIbalance, affecting cognitive performance and social interaction (Ferguson & Gao, 2018).
Alterations in PFC functioning have been linked to neurodevelopmental disorders involving social deficits, like autism spectrum disorder, schizophrenia and depression (Mohapatra & Wagner, 2023;Yan & Rein, 2021).More specifically, an EI-imbalance has been proposed to account for various behavioral and electrophysiological phenotypes in autism and schizophrenia (Dienel et al., 2022;Rojas et al., 2014).This is supported by findings in mouse models.For instance, an elevation of the EIbalance in the mouse medial PFC was found to be associated with an impairment in cellular information processing and social deficits as seen in autism (Yizhar et al., 2011) and most schizophrenia mouse models replicate the endophenotype of altered EI-balance (Rosen et al., 2015).
The EI-balance in the infralimbic (IL-)PFC is also targeted by acute and chronic stress in rodents as well as humans, which could facilitate the onset of psychiatric disorders in predisposed individuals (Anderson et al., 2021;Arnsten, 2011;McKlveen et al., 2019).We found that in mice, acute exposure of adult infralimbic IL-PFC layer 2/3 cells to corticosteroids suppresses excitability within minutes (Karst & Joëls, 2023), while others have shown that later on (when examined after >1 hr, allowing genomic effects to take place), GABA release onto principal neurons in layer 5 of the prelimbic region is suppressed (Hill et al., 2011).More prolonged changes in the stress such as occur after adverse early postnatal conditions in mice, accelerate the maturation of the EI-balance in IL-PFC layer 2/3 pyramidal cells (Karst et al., 2020(Karst et al., , 2023) ) and reduce the ratio between AMPA-and NMDAreceptor-mediated excitatory responses later in life (Karst et al., 2020).Chronic unpredictable stress in adulthood downregulates glucocorticoid receptor (GR) expression specifically in a subset of IL-PFC interneurons, thereby increasing inhibition and decreasing the EI-balance in IL-PFC layer 5 cells, associated with impaired prefrontal-mediated behavior (McKlveen et al., 2016).
Housing groups of male mice in a complex environment during adulthood is an example of a challenging condition that affects the PFC.In recent years, several models have been developed to J o u r n a l P r e -p r o o f study the effect of a complex environment on physiology and behavior, such as the Visible Burrow System (VBS) (Bove et al., 2018;Herman & Tamashiro, 2017) and the Marlau cage (Fares et al., 2013;Kalamari et al., 2021;Rabadán et al., 2019), in which animals are exposed to more complexity and have more space to interact compared to the standard housing condition.In a recent study by Bove et al. (Bove et al., 2018), using the HPLC technique to determine amino acid concentrations in the PFC, group-housing of adult C57Bl/6J and BTBR mice for 8 days in the visible borrow system was reported to drastically change extracellular GABA and glutamate concentrations.It is presently unclear, though, whether these changes in extracellular amino acid level align with or differ from changes in IL-PFC synaptic function.
We presently aimed to determine to what extent a challenging, complex-housing condition (CH) of groups of male mice during adulthood affects the EI-balance in layer 2/3 pyramidal IL-PFC cells compared to that of standard pair-housed (SH) animals.To that purpose, adult male mice were group-housed (with 6 peers) for 9-11 days in a Marlau cage (CHadult group) and brain slices were prepared from animals directly taken out of the complex or standard housed condition.The frequency and amplitude of miniature excitatory and inhibitory postsynaptic currents (mEPSC and mIPSC respectively) were recorded from layer 2/3 pyramidal cells, yielding a reliable measure for the EI-balance.Earlier data in the hippocampus have shown that the consequences of prolonged periods of challenging conditions for excitability may depend on the organism's current (corticosteroid) state (e.g Karst & Joëls, 2003, 2007;Van Gemert & Joëls, 2006).Therefore, we examined cells from both undisturbed and mildly aroused mice, thus taking state into account.Furthermore, we examined putative housing (condition) and arousal (state) effects in female mice, for which adult complex housing might be a less stressful condition, since female hierarchy establishment involves less aggression (Beery et al., 2020).In the same line of reasoning, we tested male mice, group-housed in a Marlau cage from adolescence (postnatal day (PND)28) onward (CHyoung), allowing stable social hierarchies to be established (Beery et al., 2020).Finally, in an explorative attempt, we investigated a potential mediating role of corticosterone in the changes observed for EI-balance in males.
J o u r n a l P r e -p r o o f

Animals
Wildtype female and male C57BL/6JolaHsd mice were obtained from Envigo (Inotiv, The Netherlands).They underwent an acclimatization period of at least 2 weeks in the animal facility after arrival.All mice were housed in a temperature (21°C)-and humidity (40-60%)-controlled environment, with a 12-hour light-dark cycle (lights on at 8:00 am), and had ad libitum access to food and water.Unless otherwise stated animals were housed pair-wise in standard type II-L (long) Macrolon cages (31x16 cm).
All experiments were performed following the EU directive (2010/63/EU) for animal experiments and approved by the Central Authority for Scientific Procedures on Animals in the Netherlands (CCD approval AVD1110020186004).
Breeding involved housing two females with one male for 7 days, after which the male was removed.After these 7 days, the two female mice remained together for an additional 7 days before being individually housed in type II Makrolon breeding cages (21.5 x 16 cm) with sawdust bedding and a cotton Nestlet (5×5 cm, Technilab-BMI, Someren, The Netherlands) for nesting material.The cages were checked daily at 10:00 am to monitor the appearance of a litter.The birth discovery day was designated as PND1.On the following day (PND2), the litters were weighed and checked for sex balance.Only litters with 5-8 pups and at least 2 male and 2 female pups were used.For the results described in this study, we used (part of) the litters of 11 dams (offspring used for this study: n=42 mice, i.e. 30 males and 12 females, the remaining offspring was used in other experiments).The pups were weaned at 21 days of age.

Complex housing using Marlau cages
After weaning, pups stayed together with their littermates for another week, and were then assigned to the different experimental groups.No more than two pups from the same dam were assigned to a particular condition.
J o u r n a l P r e -p r o o f SH animals were housed in standard type II-L (long) Macrolon cages (31x16 cm) in pairs from PD26-28 onwards, and remained in these housing conditions until they were used for the electrophysiological experiment.During complex housing, 6 animals were housed in a MarlauTM cage (Viewpoint, Lyon, France, see paragraph below).For our main research question, regarding group housing in adult male mice, the group-housing started in adulthood (PND 90), leaving the mice in the Marlau cage for 10 days (CHadult) until they were killed for the electrophysiological experiment.To examine the generalizability and specificity of the effects we observed in male mice placed in a CH environment during adulthood, we introduced two extra comparisons: I) We compared female mice, housed for 10 days in a complex envrionment during adulthood (CHadult) with controls pair-housed in standard cages (SH).And II) we compared male mice that were complex housed starting at adolescent age (P26-28), i.e. 1 week following weaning and staying in the Marlau cage until the electrophysiological experiments in adulthood (CHyoung) with the control SH group.During the CH period, all mice were weighed and carefully examined daily for any potential injuries, while the control SH mice underwent these assessments every other day.
Marlau cages are large, enriched cages (length: 580 mm × Width:400 mm x Height: 320 mm) that have 2 floors and provide a complex and challenging environment for the mice.The first floor includes a small food compartment and a larger compartment, containing three running wheels, a shelter, unrestricted access to water, two woodblocks, ample nesting material and climbing stairs leading to the second floor.One-way doors are positioned between the compartments on the first floor, allowing mice to move from the small compartment to the larger one.To access the small compartment, mice are required to climb to the second floor and navigate through a maze, which leads to a tube connecting to the food-compartment on the first floor.The maze was changed every three days to ensure novel and sustained cognitive stimulation.

J o u r n a l P r e -p r o o f
For electrophysiological experiments, two animals per day were taken from their home cage between 8:30 and 9:00 AM (around the circadian nadir of corticosterone release), with an interval of ~15 minutes between the first and the second mouse.The experimenter performing the electrophysiology was blind to the experimental condition.
Control mice from the SH condition were housed 2 per cage and both were decapitated on the same day to prevent isolated housing of the remaining mouse, which is known to be stressful (Ferland & Schrader, 2011).The interval between decapitation of the first and second mouse was ~15 min, a period that is known to cause arousal or mild stress (see e.g.van Campen et al., 2018).
The limitation of our electrophysiological approach is that no more than 2 mice can be recorded on a single day by one investigator.Therefore, the six mice from the CH groups housed in a Marlau cage were recorded on three consecutive days.Of note, particularly in the CHadult group, this introduced a slight variation: The first two mice were decapitated after 9 days of CH, the 3 rd and 4 th after 10 days CH, and the 5 th and 6 th after 11 days CH.On all three days, a 15-minute interval was allowed between decapitation of the first and second mouse of the pair.
The experiments were performed in two series: the male SH and male CHadult were tested in both series (2 x n=6 per group), while the female SH, female CHadult and the male CHyoung groups were only tested in one series (n=6 per group).As a control group for the male CHyoung group, we only used the male SH animals (n=6) that were tested in the same series.
After decapitation, the brain was quickly removed from the skull and stored in ice cold slicing medium containing: 120 mM choline chloride, 3.5 mM KCl, 0.5 mM CaCl 2 , 6 mM MgSO 4 , 1.25 mMNaH 2 PO 4 , 25 mM D-glucose and 25 mM NaHCO 3 .Coronal slices of 350 μm thickness were made with a vibratome (Leica VT 1000S, Germany) and placed in artificial cerebrospinal fluid (ACSF) containing: 120 mM NaCl, 3.5 mM KCl, 1.3 mM MgSO 4 , 1.25 mM NaH 2 PO 4 , 2.5 mM CaCl 2 , 25 mM Dglucose and 25 mM NaHCO 3 and heat shocked at 32 o C for 20 min.Slices were then transferred to a storage bath at room temperature, and after staying there for at least 1 hr, one slice at a time was used for the recordings.

J o u r n a l P r e -p r o o f Exploration of a potentially mediating role by corticosterone
After decapitation, trunk blood was collected to later determine blood corticosterone levels using an ELISA kit (RE52211, IBL International, Germany).
In a follow up experiment, using a limited number of naïve adult male mice (10-12 weeks, n=20), we treated brain slices with either corticosterone (CORT 100 nM), the MR-agonist aldosterone (ALDO 10 nM), the GR-agonist RU28362 (RU283 100 nM, Roussel-Uclaf) or vehicle (VEH, saline with 0.09% ethanol), to study the genomic effect of corticosteroid receptor activation on glutamatergic and GABAergic transmission; these concentrations are based on earlier studies in the hippocampus and IL-PFC (Karst & Joëls, 2005, 2023).To that purpose, each of the compounds was added to the slice for 20 minutes, and properties of mEPSCs an mIPSCs were determined >1 hr after treatment, to allow enough time for genomic effects to develop.

mEPSC and mIPSC recordings
For recording, one slice at a time was transferred to the recording bath, and continuously perfused with ACSF at 32 o C. Cells were visualized with an upright microscope (Zeiss Aksioskop) with infrared DIC, a 40x water immersion objective, a 10× video lens, and a microscopy camera (Qimaging, Rolera bolt).All electrophysiological recordings were made from pyramidal-shaped neurons located in the infralimbic (IL) mPFC layer 2 or 3.All chemicals described below were obtained from Sigma-Aldrich (USA) unless otherwise specified.

J o u r n a l P r e -p r o o f
After the whole cell patch configuration was established, recordings of the mEPSCs and /or mIPSCs were started as soon as the recording remained stable for at least 5 minutes.Spontaneous electrical activity was filtered at 5 kHz and digitized at 10 kHz (Digidata 1322A; Axon Instruments).
First, mEPSCs were recorded for 5 minutes, followed by a 5-minute recording of mIPSCs (for examples see Fig 1).The mEPSCs (AMPA receptor-mediated currents) were recorded at a holding potential (Vh) of -65 mV, i.e. the reversal potential for chloride so that the mIPSC amplitude is negligible.
Subsequently, in the same neuron, the GABAergic mIPSCs was recorded at Vh of +10 mV, which is the reversal potential for glutamate, assuming that the mEPSC amplitude was negligible.All data was stored and afterwards analyzed with Clampfit 10.7.The events were detected with a template search and then analyzed for frequency and amplitude.

Statistics
Statistical analysis was performed with IBM SPSS Statistics 23.0.Data is presented as mean ± SEM.Normality of data distribution was tested with the Shapiro-Wilk test.
To assess whether complex housing and acute arousal state affected the electrophysiological measurements (EI-balance, mEPSCs and mIPSCs), we applied a two-way ANOVA, with housing condition and arousal state as main factors, thereby also analyzing interaction effects.Bonferroni corrected pairwise comparisons were performed to investigate post-hoc differences.We primarily focused on differences in the male CHadult versus standard housed (SH) groups, followed up by effects in female CHadult versus SH (sex effect) and males complex housed from adolescence onwards (CHyoung) versus SH.The same analyses were used for CORT plasma levels measured in the SH and CHadult mice.Due to technical difficulties, we were unable to retrieve data from 2 females.
When analyzing the acute corticosteroid effects (CORT, ALDO and RU283) on the electrophysiological properties of IL-PFC neurons in slices from naïve male mice, we employed a univariate ANOVA, using contrast testing for differences of the compounds compared to the vehicle (VEH) medium.

J o u r n a l P r e -p r o o f
For all comparisons, statistical significance was assumed with p<0.05.We also report on differences at trend level (0.05<p<0.08).
Analysis per cell is a common approach in electrophysiological experiments, if however many cells per animal are recorded in a limited amount of animals, this might create bias.In our experiments, only 2-3 cells were recorded per animal and group sizes in our main focus groups (i.e.male CHadult and SH groups) were relatively large.However, to control for a possible bias, we ran a repeated measures design for our main outcomes, including only the first two recordings obtained for each animal.

EI-balance in adult male mice exposed to 10 days of complex housing
The excitation-inhibition balance in IL-PFC layer 2/3 neurons of males that were exposed to 10 days of complex housing (Fig 2A, frequency measures) was different from the standard housed males, depending on arousal state (interaction effect: housing condition*arousal state F(1,67)=10.52,p<0.01, ⴄ 2 =.14).This is a robust effect: If we performed the statistical analysis with the animal (instead of cell) as experimental unit, a comparable interaction was observed (housing condition*arousal F(1,20)=5.04,p<0.05, ⴄ 2 =.20).We then also found an effect of the repeated measure within an animal (F(1,20)=6.16,p<0.05, ⴄ 2 =.24), yielding slightly lower values in the second recordings, but this effect was not different over the different conditions (repeat*housing condition F(1,20)=0.15,p=0.71 and repeat*arousal: F(1,20)=1.20,p=0.66) and did not influence the interaction effect we observed (repeat*housing condition*arousal: F(1,20)=1.86,p=0.19).

Specificity of housing effects on EI balance
Group-housing is known to be more stressful in male compared female rodents, and social housing can enhance stress coping in female rats, while it can increase adverse effects of stress in male mice (Tzeng et al., 2017;Westenbroek et al., 2003) Also, compared to group housing in adulthood, group housing from weaning onwards might be less stressful in males since this allows for a stable hierarchy to develop (Beery et al., 2020).Therefore, we also examined if housing condition -in interaction with the acute (arousal) state-affects excitability in IL-PFC layer 2/3 pyramidal cells of i) female mice or ii) male mice complex housed from adolescence onwards (Figure 3).We observed no significant effects of housing or arousal state in EI-balance or mEPSC frequency in females.We did observe a main effect of housing condition in female mIPSCs (F1,21)=5.95,p<0.05, partial ⴄ 2 =.25), irrespective of arousal state, with slightly higher frequencies of mIPSCs in CHadult compared to SH animals (Fig 3C).No effects were observed with respect to mEPSC or mIPSC amplitude in female mice (data not shown).

J o u r n a l P r e -p r o o f
The electrophysiological characteristics of IL-PFC layer 2/3 pyramidal cells in males housed together in complex cages from adolescence onwards were not different from standard housed animals; also the arousal state did not influence results (Fig 3D -F).Like in all other groups, the amplitude was not affected in male CHyoung animals (data not shown).
Overall, the effects of housing and arousal state on EI balance observed in male CHadult animals seem specific for this group, since we did not find similar effects in female or CHyoung animals.

Exploration of a potentially mediating role by corticosterone
High levels of corticosteroids, circulating in vivo due to a stressor or stressful circumstances, can induce genomic actions that persist after slice preparation, as has been described e.g. for hippocampal (Karst & Joëls, 2003) and prelimbic layer 5 pyramidal neurons (Yuen et al., 2009).The observed electrophysiological findings in the male CHadult mice could therefore be explained if i) the present condition and/or acute state alters the CORT level and ii) such altered CORT levels affect the EI-balance of IL-PFC neurons.
In agreement with the first part of this assumption, we found an increase in CORT plasma level in the CHadult compared to SH group (Fig 4, housing condition F(1,23)=10.2, p<0.01, partial ⴄ 2 =.34).In the SH condition, the second subject tested showed increased CORT levels compared to the first subject (F(1,11) = 18.4,p<0.01, partial ⴄ 2 =.65).This was not seen in the CH condition, where the variation in CORT was quite strong, possibly reflecting prior circumstances in the cage.
To test the second part of the assumption, i.e. the notion that higher CORT levels can influence the EI-balance of layer 2/3 IL-PFC neurons, we perfused 100 nM CORT -as well as the MR agonist aldosterone and the selective GR agonist RU28362-in vitro to slices prepared from (naïve) male mice at the nadir of the circadian rhythm.As shown in Figure 5C Overall i) CORT levels are increased by the challenging housing conditions and ii) high concentrations of CORT in vitro can -with a delay of >1 hr-decrease mIPSC frequency in IL-PFC layer 2/3 pyramidal neurons.This is compatible with -though no proof for-the motion that the effects on electrophysiology observed in the CHadult animals, might occur secondary to changed levels of CORT at the moment of slice preparation.

DISCUSSION
We here report a significant interaction between the complex housing of adult male mice in a Marlau cage on the one hand and acute (arousal) state on the other hand, concerning the EI-balance of IL-PFC layer 2/3 pyramidal neurons.The effect is explained by a state-dependent modulation of mIPSC and mEPSC frequency rather than amplitude.Complex housing of males from weaning onward did not change the EI-balance, nor did the balance of female mice that were complex housed in adulthood change compared to standard housed animals, although the relatively small group sizes in these control experiments might hamper generalization of the findings.Corticosterone plasma levels were higher in male complex housed versus standard housed animals and in vitro exposure of slices to corticosterone and a specific GR agonist could partly replicate the findings.The data emphasizes that taking the (acute) state of individuals into account is necessary to better understand the consequences of prolonged exposure to challenging life conditions for prefrontal function and its possible effects on important PFC related behaviors like learning, memory and social behavior, necessary for successful coping (resilience).

J o u r n a l P r e -p r o o f
Why study both condition and state?
In earlier studies on dentate granule cells, where GRs are abundantly present, we observed a stress condition-by-corticosterone interaction effect in AMPA and Ca-currents (Karst & Joëls, 2003;van Gemert et al., 2009;Van Gemert & Joëls, 2006).That is, the AMPA-and Ca-currents in granule cells from chronically stressed rats recorded at the nadir of the circadian CORT rhythm did not differ from those in the control unstressed group.However, when cells of the stressed group were recorded >1 hr after in vitro administration of CORT, both types of currents were increased compared to vehicletreated cells, while CORT treatment did not affect slices from unstressed animals.In CA1 pyramidal neurons, chronic stress by itself did increase the amplitude of Ca-currents (Karst & Joëls, 2007).In the CA1 area, adding 100 nM CORT in vitro increased Ca-current amplitude in slices prepared from control rats and mice (Chameau et al., 2007;Karst et al., 1994), but decreased it in chronically stressed rats (Karst & Joëls, 2007).Collectively, these studies support that the (acute) "state" of tissues -in this case of CORT-might be important to fully appreciate the influence of the animal's life history on their excitability.
We, therefore, examined IL-PFC cells from complex-and standard-housed mice both under undisturbed (i.e. the mouse was taken directly from the home cage at the nadir of the circadian cycle) and (acute) aroused conditions, the latter referring to mice taken ~15 minutes later from the home cage, a procedure that might actually be quite common in laboratories.Manipulating the (acute) state of the animal instead of applying CORT in vitro (as in our earlier studies) has the advantage of a higher ecological value.However, the interpretation of the findings in terms of hormonal contribution is less straightforward (see also below).Nonetheless, when considering the control standard housed group, we found that CORT levels were indeed very low, indicating that these mice were not aroused or stressed, while the second animal of each pair (taken ~15 min later) showed slightly higherthough still quite low-CORT levels, supporting that the earlier manipulation in the home cage indeed imposed some form of arousal (van Campen et al., 2018).Moreover, complex housing in adulthood increased CORT levels to a higher level independent of arousal condition, which is in line with the J o u r n a l P r e -p r o o f assumption that adult complex housing might cause a chronically stressful condition in mice, which can affect HPA-axis responsivity, as seen e.g. after chronic variable stress (Heck et al., 2020).The variation in CORT level in the complex-housed group was found to be quite high, which might depend on hierarchy status (Beery et al., 2020;Fulenwider et al., 2024).Higher CORT levels might change plasticity in the IL-PFC and pave the way to direct arousal effects to take place.Although group sizes for corticosterone level in the female and male CHyoung animals did not allow for statistical analysis, we have added preliminary data to the supplementary information (Fig S2).In female animals, we observed a low corticosterone level in the animals taken directly from the cage in both the SH and CHadult group, while corticosterone levels were higher in the mice that were subject to the arousal effect of being taken out ~15min later.Most CHyoung male animals showed low CORT levels, irrespective of the order of testing.These data can, however, only be viewed as preliminary and should be interpreted with the utmost caution.
Interestingly, in CHadult males, the CORT level of the second mouse of each pair tested for electrophysiology did not differ from the first.One could argue that, for mice, the manipulation of removing the only cage mate is more arousing than that of removing one of several cage mates.
However, earlier observations (van Campen et al., 2018) show that the rise in CORT level is comparable for each mouse taken from the home cage, irrespective of how many animals are left.
Alternatively, the data indicates that HPA-axis responsivity itself may have been affected by the housing condition, as was e.g.demonstrated for chronic variable stress (Heck et al., 2020).Of note, our CORT measurements were based on a single time-point observation, which is a very limited reflection of potential changes in HPA activity over the entire 24 hours of the day.

A potential mediating role of CORT
We performed an exploratory analysis to probe if changes in circulating CORT levels might contribute to the observed electrophysiological phenomena.Strong stressors induce the release of corticosteroid-binding globulin from the liver, hampering rapid access of CORT into the brain, but this J o u r n a l P r e -p r o o f does not pertain to the current situation of mild arousal (Qian et al., 2011).Therefore, we assume that steroids secreted from the adrenal cortex did enter the brain during the 15-minute delay between the intervention and preparation of the slices.
We showed in naive male mice brain slices that high CORT levels (100 nM), presumably via a gene-mediated and GR-dependent mechanism, reduce the mIPSC frequency of IL-PFC layer 2/3 pyramidal neurons.None of the mEPSC properties were affected, which is in line with earlier studies showing that stress (or CORT application) increases the readily releasable pool of glutamatecontaining vesicles in the PFC for a prolonged period (Musazzi et al., 2017) but this does not result in detectable changes in glutamatergic synaptic responses (Treccani et al., 2014).A reduced mIPSC frequency by CORT was earlier also described for layer 5 neurons (Hill et al., 2011).This was accompanied by enhanced paired-pulse responsiveness, which points to a reduced release probability of GABA-containing synaptic vesicles.Interestingly, we too noticed -in a side projectenhanced paired-pulse responsiveness in the CHadult compared to SH groups (unpublished observation).If complex housing increases the overall circulating levels of CORT, the reduced mIPSC frequency (and enhanced EI-balance) seen in the undisturbed complex-housed animals may reflect a property adjusted to more permanently elevated CORT levels, as has been described for many parameters after chronic unpredictable stress (see for overview Joëls et al., 2012).Importantly, while arousal state affected EI-balance in the CH adult group, it did not significantly affect CORT levels.This in itself already indicates that the currently observed effects on EI-balance are unlikely to be solely explained by altered circulating CORT levels.Instead, our experimental manipulations (arousal state and housing condition) likely caused the release of a myriad of stress signals, including monoamines, and peptides, next to corticosteroids (Barsegyan et al., 2019;Joëls & Baram, 2009;Schwabe et al., 2022).Changes in one or more of these stress signals may have contributed to the altered EI-balance.Clearly, future studies would need to explore the potential role of all of these molecules to gain more insight.

Functional relevance of change in EI-balance
Neurons in the PFC -next to other areas like the amygdala and hippocampus-play a role in HPA-axis regulation (McKlveen et al., 2019).Particularly manipulation of GR expression in the prelimbic cortex was found to be effective in terms of CORT release in combination with chronic variable stress (McKlveen et al., 2013).The same group also reported that chronic variable stress decreases the EIbalance in IL-PFC layer 5 neurons (McKlveen et al., 2016), comparable to our aroused complex housed animals.Possibly, these animals tested after chronic variable stress were indeed slightly aroused when slices were prepared.Of course, we cannot exclude that the observed discrepancies are caused by differences in species (rat versus mouse), layer (5 versus 2/3), or chronic condition (variable stress versus complex housing).In line with the latter, repeated stress in juvenile male rats was also reported to decrease glutamatergic transmission in prelimbic layer 5 neurons (Yuen et al., 2012) while chronic isolation stress caused sex-dependent functional changes (Wang et al., 2022).
The EI-balance in the mouse IL-PFC is also important for social behavior (Yizhar et al., 2011).
Thus, an optogenetically achieved increase in the IL-PFC E/I balance caused impaired cellular information processing and was associated with specific behavioral impairments and altered highfrequency power activity similar to those seen in some clinical conditions in humans.Selectively increasing GABAergic transmission in the IL-PFC restored the deficits, which is in line with the observation that particularly GABAergic interneurons in the PFC play an important role in the EIbalance and cognitive performance (Ferguson & Gao, 2018;Lewis et al., 2012).Our female complex housed animals showed increased mIPSC frequencies, irrespective of arousal state, which could be indicative of the beneficial (social supportive) effects of social housing in females.Indeed, social housing can enhance stress coping in female rats, while it can increase adverse effects of stress in male mice (Tzeng et al., 2017;Westenbroek et al., 2003).Although we did not include a female group that was CH from weaning onwards, we would hypothesize to see similar results, but this would need to be confirmed in future studies.In a recent study with (mixed-sex) complex-housed litters, BTBR and C57Bl/6J male mice were exposed to a visible burrow system for 5 days in adulthood (Bove et al., J o u r n a l P r e -p r o o f 2018).The C57Bl/6J mice showed increased GABA and reduced glutamate in whole mPFC as measured by HPLC.The latter might rather reflect the available pool of neurotransmitter than the synaptic properties of individual cells.Still, if these data are compared with the present, they would more resemble the aroused complex housed animals.
The fact that complex housing of male mice from the adolescent age onwards did not influence any of the electrophysiology measures, might be a result of the fact that a stable hierarchy had time to develop.Indeed, social hierarchy in mice entails quite some inter-male aggression and an unstable hierarchy might be stressful for both subordinate as well as dominant animals (Beery et al., 2020).The data observed in the CHyoung animals strengthens the idea that stress-related processes are involved in the complex housing effects found in CH adult animals.Inherent to using the complex housing paradigm is that we cannot attribute the changes we observed to one specific factor.It might be that changing one aspect, like a bigger cage, pair-versus group-housing, or introducing a running wheel, might have already caused changes.It would be practically very challenging to control all of these aspects.This was also not the main goal of this approach.The rationale for using enriched environments is to expose animals to meaningful contexts in which more species-related behavior can be expressed.Our data shows that a meaningful and challenging social environment, reminiscent of socially complex human situations like cities, induces changes in brain structures important for social functioning.
Overall, in this study we have found that exposure to a challenging social environment can influence EI-balance in the IL-PFC, which is known to be crucial for coping in social situations.It has been hypothesized that a disturbed, neuropsychiatric disease-related social interaction may occur when the PFC cellular EI-balance is elevated (Kehrer et al., 2008;Rubenstein & Merzenich, 2003).We propose that, at least in male mice, challenging housing conditions can contribute to a phenotype of changed PFC EI-balance, which might underlie subsequent individual differences in social stress coping, which might explain thriving or perishing.Interestingly, in humans, living in complex urban circumstances can be a risk factor for neuropsychiatric disorders such as schizophrenia and  & Steffen, 2019;Gasse et al., 2019;Kahn et al., 2015), among other factors (Paquin et al., 2021;Pignon et al., 2023).In light of the present findings in an animal model, it might be of interest for future studies in humans to not only take the (cumulative) stress exposure into account but also the acute (stress) state in which subjects are examined.J o u r n a l P r e -p r o o f

Follow
-up investigations revealed a clear difference in the CHadult group between the EI-balance measured in the first (non-aroused) compared to the second (aroused) mouse of each pair recorded on the same day (p<0.001),which was not seen in the SH group.Compared to the SH animals, the CH exposure gave a higher EI-balance in the non-aroused S1 subjects (p<0.01) and a slightly lower EI-J o u r n a l P r e -p r o o f balance in the aroused S2 subjects, (trend level, p=0.074).When assessing the underlying excitatory and inhibitory components, we also found an interaction effect for mEPSC frequency (Fig 2B) (housing condition*arousal state F(1,67)=4.901,p<0.05, partial ⴄ 2 =.071).Here, compared to the aroused SH animals, the aroused CH subjects showed a lower mEPSC frequency (p<0.01).For the mIPSC frequency (Fig 2C) we found a housing condition effect (F(1,67)= 5.21, p<0.05, partial ⴄ 2 =.075), with a trend towards an interaction effect (housing condition*arousal (F(1,67)=3.21,p=0.078, partial ⴄ 2 =.048).Like for the EI-balance, the non-aroused and aroused subjects of the CHadult group differed in mIPSC frequency (p<0.05), and the non-aroused CH animals had a lower mIPSC frequency compared to the non-aroused SH animals (p<0.01).No effects were observed in amplitude measures (Fig 2D-F).
, exposure to corticosteroids clearly impacted the mIPSC frequency recorded >1 hr later (F(3,37)=4.20,p <0.05, partial ⴄ 2 = .27),while the mEPSC frequency (Fig 5B) was unaltered.Contrast testing using the vehicle as the reference group, revealed a decrease in mIPSCs by both 100 nM CORT (p<0.01) and RU283 (p<0.01).The J o u r n a l P r e -p r o o f change in EI-balance based on mEPSC:mIPSC frequency (Fig 5A) was not significantly affected by the change in mIPSCs, although still explaining some of the variance (F(3,37)=1.76,p=0.17, partial ⴄ 2 =.14).Contrast testing revealed an increased EI-balance at trend level for CORT (p=0.064), but not RU (p=0.10).No effect of corticosteroids was observed in any of the amplitude measures (Fig 5D-F).
J o u r n a l P r e -p r o o f 21 depression (Costa e Silva

Figure 1 (
Figure 1 (A) Location of recordings of layer 2/3 IL-PFC neurons depicted with arrow and red color in the Nissl (left) and anatomical annotations (right) pictures from the Allen Mouse Brain Atlas and Allen Reference Atlas -Mouse Brain (atlas.brain-map.org).(B) Typical examples of mIPSC and mEPSC recordings of IL-PFC layer 2/3 pyramidal cells (right).

Figure 2 SHFigure 4
Figure 2 Electrophysiology measures in IL-PFC layer 2/3 pyramidal cells of males (M) that were either