# Early Amyloid-β Deposits and Astrogliosis Are Modulated by Housing Conditions in 5xFAD Mice

Christine Manrique, Martine Migliorati, Guy Escoffier, Stéphane D Girard,

Kévin Baranger, Santiago Rivera, François S Roman

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Christine Manrique, Martine Migliorati, Guy Escoffier, Stéphane D Girard, Kévin Baranger, et al. 2025. *hal-05409503*

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# Early Amyloid-β Deposits and Astrogliosis Are Modulated by Housing Conditions in 5xFAD Mice

**Christine Manrique**

Aix-Marseille Univ, CNRS, INP

**Martine Migliorati**

Aix-Marseille Univ, CNRS, INP

**Guy Escoffier**

Aix-Marseille Univ, CNRS, INP

**Stéphane D. Girard**

VECT-HORUS SAS

**Kévin Baranger**

La Rochelle Université, UMR 7266 CNRS LIENSs

**Santiago Rivera**

Aix-Marseille Univ, CNRS, INP

**François S. Roman**

francois.roman@univ-amu.fr

Aix-Marseille Univ, CNRS, INP

## Research Article

**Keywords:** Alzheimer's disease, 5xFAD mice, isolated environment, enriched environment, amyloid plaques, gliosis

**Posted Date:** May 28th, 2025

**DOI:** https://doi.org/10.21203/rs.3.rs-6704640/v1

**License:** © This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

**Additional Declarations:** No competing interests reported.
**Abstract**

Epigenetic mechanisms in Alzheimer's disease (AD) play a determining role in disease progression. To provide further insight on this matter, we evaluated whether early exposure of 5xFAD mice, a transgenic mouse model of AD, to either a standard, isolated or complex environment have a significant effect on the development of pathological hallmarks. Accordingly, we manipulated the animal environment with different housing conditions resulting in different cognitive and social stimuli. Amyloid plaques and gliosis were evaluated by immunohistochemistry at three months of age after two months of breeding under three different housing conditions. Our results demonstrated that both hallmarks were exacerbated in a complex environment and a slightly less so in an isolated condition, compared with a standard social housing condition. Unexpectedly, an enriched environment with high behavioural stimulation can exacerbate the progression of the disease at least in the early stages. These data cast doubt on the general assumption that an enriched environment conveys beneficial effects on disease progression and suggest that strong environmental stimuli may be counterproductive, at least in the early stages of the pathology.

**Introduction**

It has been known since the early 1960s that environmental enrichment or impoverishment of rat cerebral cortex results in larger or smaller and more or less extensive cortical depth [1-5]. Since then, living in an enriched environment that promotes sensorimotor, cognitive and social stimulation has been associated with significant positive changes in brain biochemistry, synaptic connectivity, neurogenesis and neural function. Conversely, living in an impoverished environment has been associated with the opposite (for a review [6]). A general decline in behaviour, memory and cognitive skills is known to occur during ageing. However, the exact molecular and/or cellular dysfunction mechanisms that promote and/or maintain neurogenesis to counteract the deleterious passing-year effect remain to be identified. As reported above, an enriched environment in rat increases neurogenesis, neuronal sprouting, cell size and improves learning and memory. Considering these beneficial effects, several studies have reported that environmental enrichment improves behaviour, cognition and brain functional markers in young mice in a well-characterized SAMP8 mouse model of accelerated senescence suitable for studying brain aging and neurodegeneration [7-9].

Degeneration in aging can be viewed as a normal process in contrast to AD, a neurodegenerative disorder resulting from complex interactions between genetic and environmental factors [10]. The initial asymptomatic phase involves multiple deregulated molecular pathways, characterized by transcriptional changes, protein processing, amyloid β (Aβ) aggregation, tau hyperphosphorylation, aberrant cell signalling and impaired metabolism. All these progressively lead to exacerbated inflammation/gliosis, oxidative stress, synapse loss and neural death characteristics of AD pathology [11].

5xFAD animals are a knock-in mouse model of AD carrying five familial mutations on two genes, *APP* and *PSEN1*, coding respectively for amyloid-β precursor protein (APP) and presenilin 1 (PS1), under the
control of the neuron-specific Thy1 promoter [12]. They recapitulate human pathology in a short period of time, and brain Aβ deposits and incipient gliosis can be observed as early as 2 months of age [12, 13]. We recently reported hippocampus-dependent long-term memory impairment using a novel Helico Maze (HM) in 3 month-old 5xFAD mice [14], which correlated with the number of amyloid plaques [15].

Although the role of epigenetic mechanisms in AD remains poorly understood, recent evidence shows that they are deregulated during AD progression in mouse models, particularly by manipulating the environment through housing enriched with cognitive and social stimulation [3, 16, 17]. The impact of an enriched environment on the pathological features of 5xFAD mice has never been studied. To this end, we housed 5xFAD mice from 1 to 3 months of age in a Marlau™ cage, which recreates a complex and enriched environment providing cognitive, physical and social stimulation [18]. We used immunohistochemical approaches to investigate the effect of different housing conditions on Aβ deposition and astrogliosis in five brain structures selected for their involvement in cognitive, motor and social behaviour, i.e. the frontal, motor and entorhinal cortices, the lateral septum and the dentate gyrus in the dorsal hippocampus.

The frontal cortex plays an important role in cognition in AD. The function of this area is impaired not only in humans [19–23] but also in 5xFAD mice [24, 25]. Voluntary exercise driven by the motor cortex improves cognitive performance, increases neurogenesis and is protective even in old aged rodents [26, 27]. The lateral septum controls kinship behaviour, but also mate choice, altruism and bonding [28]. The dorsal hippocampus is involved in learning and memory [29, 30] and social memory [28]. Although memory demands are not very high in the complex environment of a Marlau™ cage, the way to quickly find access to free water and food through the maze changes is likely to involve hippocampal pathways [18]. Finally, we studied the entorhinal cortex, as we have previously observed a spatio-temporal correlation between amyloidosis and gliosis in this region, along with cognitive deficits in 5xFAD mice [31]. Surprisingly, we show that gliosis and amyloidogenesis are enhanced in an enriched environment, suggesting that intense behavioural stimulation can exacerbate the progression of the pathology.

# Materials and Methods

## Animals

Animal experiments were approved by the Ethics Committee of the Medical Faculty of Marseille (research project: APAFIS#2021122207368432) and carried out in accordance with the guidelines published in the European Communities Council Directive of November 24, 1986 (86/609/EEC) and with the recommendations of the ARRIVE guide [32]. All efforts were made to minimize animal suffering and to reduce the number of mice used.

The generation of the 5xFAD mouse model has been described previously [12]. These transgenic mice overexpress the mutant human APP gene encoding the 695 amino-acid-isof orm of APP bearing the Swedish (K670N, M671L), Florida (I716V), and London (V717I) FAD mutations, and the human PSEN1
gene encoding the human presenilin 1 with two FAD mutations, M146L and L286V. The two transgenes are under the transcriptional control of the neuronal specific elements of the mouse Thy1 promoter. The 5xFAD mice used in this study are on the C57Bl6 background [33, 34]. Offspring were obtained by crossing 5xFAD males to WT females, and only 5xFAD male heterozygous mice and non-transgenic WT male littermates controls were used in this study. Prior to experiments, genomic DNA was extracted from the tail tips of all mice at postnatal day 21 to assess their genotype by PCR [13, 35]. All the mice were bred in our SOPF animal facility under a 12-hour light–dark cycle at 22–24°C with access to food and water *ad libitum*. At one month of age, male WT and 5XFAD mice have been divided into 3 groups and housed in 3 different conditions: standard (SC), isolated (IC) or enriched conditions (EC) for 2 months before sacrifice and brain area evaluation.

## Housing conditions

Pups were weaned and split by sex and genotype at post-natal day 30. Only males were kept for the experiment, then for the following 2 months, 3 groups were constituted and housed in 3 different conditions: standard, isolated or enriched conditions.

### Standard housing condition

Under standard condition (SC), 5xFAD mice were raised in standard cages of 425 x 266 x 185 mm in size equipped with 2 shelters (two red houses). Ten mice were kept in 2 cages of 5 animals (Fig. 1A).

### Isolated condition

Under isolated condition (IC), 5xFAD mice were housed in cages of 365 x 207 x 140 mm in size, solely equipped of 1 shelter (1 red house). The 10 mice were kept one per cage (Fig. 1B).

### Enriched condition

Under environmentally enriched condition (EC), 12 5xFAD were housed in the same Marlau™ cage (Marlau™, Viewpoint, Lyon, France) [18], which had a size of 580 x 400 x 320 mm, including 2 floors, providing an extensive exploration area (Fig. 1C). The ground floor of the Marlau™ cage consisted of two separated compartments: the smaller part contained food, and the larger part contained water, running wheels and a red house (Fig. 1D). To get food, the mice must climb a stairway to reach the second floor, where a maze was placed. Once through the maze, the mice slid through a tunnel back down to the ground floor, directly into the smaller compartment and had free access to food. By passing through a one-way door, they can enter most of the ground floor for water and free access to the running wheels. To both maximize novelty and cognitive stimulation, the maze was changed twice a week, with a total of 6 different configurations repeated each month (Fig. 1E).
# Immunohistochemistry

Immunostaining of brain sections was performed on 3-month-old 5xFAD mice (N = 32). The number of mice analyzed in this study was determined and validated using the resource equation method [36].

Mice were deeply anaesthetized with an intraperitoneal injection of xylazine (15 mg/kg) and ketamine (150 mg/kg) (Ceva Santé Animale, Libourne, France), and intracardially perfused first with 0.9% NaCl and then AntigenFix (Diapath, Voisins-Le-Bretonneux, France). Brains were removed from the skulls and postfixed overnight in cold AntigenFix at 4°C. Serial coronal brain sections (30 µm thick) were generated using a Leica CM3050 S cryostat (Leica biosystems, Nanterre, France), with a focus on the following interaural (IA) levels: frontal cortex (7 to 6.38 mm IA), the motor cortex and lateral septum (6.38 to 3.22 mm IA), the hippocampus and the entorhinal cortex (2,72 to 0,00 mm IA), according to the stereotaxic coordinates of the Paxinos and Franklin Atlas 2019 [37]. Sections were stored at -20°C in a cryoprotectant solution composed of 30% glycerol and 30% ethylene glycol (both purchased from Sigma-Aldrich, Saint Quentin-Fallavier, France) in 0.05 M phosphate-buffered saline (PBS, Thermo Fisher Scientific, Saint Aubin, France). After several washes in PBS, floating brain sections were incubated for 1 h at room temperature (RT) in blocking buffer containing 10% bovine serum albumin (BSA, Sigma-Aldrich), 0.5% Triton X-100 (Sigma-Aldrich) in 0.1M PBS, and then overnight at 4°C with the following primary antibodies rabbit polyclonal anti-GFAP (1/400, Dakocytomation, Trappes, France) and mouse monoclonal anti-Αβ 6E10 (1/1000, BioLegend, Ozyme, Saint-Cyr l'Ecole, France) in 0.1M PBS containing 2% BSA and 0.5% Triton X-100. Brain slices were then washed 3 x 5 min with PBS containing 2% BSA and then 3 x 5 min with PBS containing 5% BSA, followed by incubation with highly cross-adsorbed Alexa Fluor™ 488- or 594-conjugated anti-rabbit or anti-mouse secondary antibodies (1/800, Thermo Fisher Scientific) for 1 h 30 min at RT in the dark. After washing, as described above, slides were counterstained with 0.5 µg/mL Hoechst blue (#33342, Sigma-Aldrich) in 0.1M PBS containing 2% BSA for 30 min at RT and mounted with ProLong Gold Antifade reagent (Thermo Fisher Scientific) onto Superfrost glass slides (Dutscher, Bernolsheim, France).

# Image acquisition and analysis

Images were acquired using an AxioObserver Z1 microscope (Carl Zeiss S.A.S., Marly le Roi, France). Extracellular amyloid deposits in the five brain areas were counted manually in blind and the number of plaques per mm² of brain surface was calculated using the Zen software (Zeiss). ImageJ software (NIH) was used for the quantification of GFAP staining. Images were binarized to 16-bit black and white images, and a fixed intensity threshold was applied to define GFAP staining. The percentage of the area covered by fluorescent staining was calculated. The final data represent the average of 2 tissue sections from the same brain area per mouse. Representative images of large brain sections were obtained using the mosaic mode of the Zen software with a 10x objective.

# Statistical analysis
We first verified that all the parameters tested were normally distributed using the Shapiro-Wilk test for small sample sizes, before analysing them using one-way analysis of variance (ANOVA), followed, if significant, # p < 0.05 and ## p < 0.01, by Newman Keuls post-hoc (NKS) analysis using Graph Prism 5.02 software. In the figures, NKS * indicates p < 0.05, ** indicates p < 0.01.

# Results

## Amyloid plaques

Over a period of 2 months, i.e., from 1 to 3 months of age, immunohistochemical analyses (Fig. 2A) of the frontal (FrA), motor (M2) and entorhinal (Ect) cortex, lateral septum (LSI) and dorsal hippocampus (dentate gyrus and part of CA1) (DG), revealed an overall statistically significant difference between the three housing conditions [F(2,157) = 5.34; p < 0.01] (Fig. 2B, Total). 5xFAD mice from the EC group developed significantly more amyloid plaques than the SC and IC groups, as revealed by the NKS post-hoc test (p < 0.01 and p < 0.05, respectively). No significant difference was observed between SC and IC groups. We showed that this result did not originate from the frontal cortex area, as no difference was detected between groups in this region [F(2,29) = 0.94; NS] (Fig. 2B). Conversely, there was a substantial difference in motor cortex (M2) area between the three groups [F(2,29) = 6.37; p < 0.01] (Fig. 2B). NKS analysis revealed significant differences between SC, IC and EC housing conditions (p < 0.01 and p < 0.05 respectively). We measured 2.2 and 1.66 times more amyloid deposits in the EC group than in the SC and IC groups, respectively. No differences were observed between the SC and IC groups. Concerning the lateral septum (LSI) area (Fig. 2B), a significant difference was observed between the three groups [F(2,29) = 3.86; p < 0.05], which can be explained by the significant increases in the IC (27.8%) and EC (26.75%) groups compared with the SC group (NKS: p < 0.05). The IC and EC groups showed approximately the same number of amyloid plaques.

In the dentate gyrus (DG) of the dorsal hippocampus (Fig. 2B), a significant difference in the number of amyloid deposits was also observed between the three groups [F(2,29) = 5.04; p < 0.05]. The density of amyloid deposits in the EC group was 100% higher than in the SC group and 57% higher than in the IC group (both NKS: p < 0.05). A significant difference was found between the three groups in the entorhinal cortex (Fig. 4B); [F(2,29) = 3.68; p < 0.05], but it did not reach statistical significance when analysed by pairs of groups (NKS: NS).

## Astrogliosis

Analyses of astrogliosis with GFAP staining (Fig. 3A) revealed an overall significant difference between the three groups when all the brain structures were considered [F(2,157) = 4.54; p < 0.05] (Fig. 3B, Total), likely explained by the higher percentage of staining (60%) in the EC group, compared to the SC (NKS: p < 0.01). In the frontal cortex (FrA), dentate gyrus (DG) and entorhinal cortex (Ect), no significant differences were observed in GFAP staining (Fig. 3B), i.e., [F (2,29) = 3.68; NS] and [F(2,29) ≤ 0.59; NS], respectively. In contrast, a strong astrogliosis was observed in the motor cortex (M2) (Fig. 3B), with a significant difference between the three groups [F(2,29) = 5.62; p < 0.01], associated with higher staining
observed in the EC group compared to the SC (57%) and IC (46%) groups (NKS: p < 0.05). In the lateral septum (LSI) (Fig. 3B), as in the motor cortex (M2), a significant difference between the three groups was observed [F(2,29) = 5.52; p < 0.01]. However, only the SC group exhibited about three times less GFAP staining than the IC and EC groups (NKS: p < 0.05). No difference was observed between the IC and EC groups (NKS: NS).

Discussion

In this study, we investigated the consequences of different housing conditions of young male 5xFAD mice on amyloid deposition and astrogliosis. We and others have extensively characterized this transgenic mouse model of AD [12, 13, 15, 24, 25, 34, 38–40], one of the earliest and the most aggressive concerning the appearance of senile plaques, neuroinflammation and memory deficits, key hallmarks of AD. Our results were unexpected, given the substantial literature in the field conveying the idea that an enriched housing environment reduces both amyloidosis and astrogliosis [41, 42]. On the contrary, we observed a stimulatory effect of these pathological markers by the EC, compared with the SC. Under IC, only the number of amyloid deposits was significantly lower than in the EC.

Over the last twenty years, several research articles have highlighted the efficacy of EC against AD pathogenesis [43–48]. Only one study has reported the opposite, i.e., EC worsens amyloidosis in the APPswe/PS1dE9 transgenic mouse model [49]. Taken together, our results and those in the literature raise several points, particularly the importance of the brain regions studied, the nature of the enrichments and the different transgenic mouse models used. In our study, the levels of amyloid and astrogliosis in the frontal cortex were identical across the 3 groups of mice. This area has been implicated in a variety of cognitive and executive cognitive control, including learning, memory, decision-making and cognitive control in rats and humans [50];[51];[52]; [53]. Lesions in this brain area impaired learning and memory in mice, similar to what has been observed in monkeys and humans [54]. These cognitive functions are particularly affected in AD [19–23], but also in 5xFAD mice [33, 34, 39, 55]. The frontal cortex seems to be solicited in the same way in the three housing conditions, in terms of specific working memory or executive function, even in our enriched housing condition.

The most detrimental effects, in terms of Aβ deposition and astrogliosis were observed in the motor cortex in the EC compared to the SC and IC. While motor and physical activity in the IC is limited by the cage size, in the SC the ratio of the number of mice over the cage size is fairly equivalent to that in the EC. However, in the latter, physical activity is certainly stimulated by using the running wheels and exploring the maze to access food. Recently, positive effects of environmental factors (e.g., social stimulation and physical activity) on AD markers were found when these activities were maintained for 4 months in 4 month-old Tg-SwDI male/female mice [56]. Animals showed significant cognitive/behavioural improvements in several tasks involving motor function (rotarod) and exploratory behaviour (radial arm maze), as well as reduced cerebral vascular amyloid pathology in the thalamus. However, although thalamic vascular amyloid was specifically reduced, levels of insoluble Aβ were still
elevated [56]. In our conditions, none of these markers were detected in the striatum/thalamus (data not shown).

In the lateral septum, Aβ deposition and astrogliosis were significantly increased in the EC and in the IC compared to the SC. While no clear link between this brain area and AD pathogenesis has been described, the lateral septum it is known to be involved in social behaviour. It has previously been described that socially housed mice showed the greatest changes in physiological and cognitive/behavioural outcomes [56]. This is consistent with our observations in the SC compared to the IC groups. However, AD markers in mice housed at a rate of 12 per cage in the EC were similar to those observed in the IC. We cannot exclude the possibility that in the EC, mice can isolate themselves in a given compartment and regulate their own social environment to avoid unwanted social contact. In the DG of the dorsal hippocampal region, where Aβ deposits and inflammation are the most visible at 3 months of age in 5xFAD mice [15], environment complexity induced a significant increase in amyloid plaques compared to the SC and IC housing. Hippocampus-dependent memory is an important feature of AD, and the onset and progression of memory deficits are crucial, especially to assess the efficacy of new therapeutic strategies aimed at delaying the onset of the disease. In the Marlau™ cage, the memory performances are not particularly demanding per se. However, twice a week the set-up of the mazes was changed, and we could suspect the importance of the memory, day after day, in finding the way through the maze to reach the freely available water and food. The entorhinal cortex-hippocampus pathway could be much more solicited in the Marlau™ cage, therefore explaining the increase in Aβ deposition in the dentate gyrus and, to a lesser extent, in the entorhinal cortex, while no increase in astrogliosis is observed in either brain structure. The increase in amyloid deposition in the dentate gyrus and entorhinal cortex supports our previous observations showing an inverse correlation between the number of Aβ plaques and hippocampal dependent long-term memory performance [15]. Similar deficits in 5xFAD mice have been detected at 4 months of age in the olfactory tubing maze [39], the radial arm water maze [40], and the contextual fear conditioning task [57, 58]. AD studies have mainly focused on the entorhinal cortex and hippocampus due to their implication on memory formation and spatial navigation (for a review, [59]). Only one study has previously shown that environmental enrichment exacerbates amyloid plaque formation in a transgenic mouse model of AD [49], in line with our findings. Most commonly, improvements in behavioural performance and associated reductions in amyloidosis and neuroinflammation in the entorhinal cortex and hippocampus have generally been associated with environmental enrichment (for a review, see [60]), but none of these studies has been performed in the Marlau™ cage. The apparent contradiction in the literature could be explained by the diversity of elements that make up EC in the different studies, including toys, shelters, hideouts, mazes, and objects of different materials, sizes and shapes. This ultimately have a different impact on brain and physical activity, as well as social interactions, making it difficult to compare the effects of EC across studies. Mechanistically, it has been shown in hippocampal cultures that stimulation of neuronal activity increases the activity of the β-APP cleaving enzyme 1 (BACE1) and consequently the accumulation of the APPβ-C-terminal fragment, which is the precursor of Aβ [61]. Thus, an increase in synaptic activity under EC could directly influence the processing of APP to enhance Aβ production.
Conclusions

The present results show that the pathological changes associated with AD can be influenced by
environmental factors, but that these effects differently affect brain structures in function of cognitive,
physical and social demands. We found that isolated housing conditions increase early amyloid
deposition and associated inflammation, and that these effects are even more pronounced in the
complex environment provided by the Marlau™ cage. This can create conditions that have the effect of
combining increased locomotor activity, stimulating long-term memory to navigate periodically changing
mazes, and possibly reducing social interactions through preferential isolation. In the future, it will be
interesting to test 5xFAD mice housed in the Marlau™ cage in different behavioural tasks, in particular
the Helico-maze [15], to decrypt whether the aggravation of amyloid plaque deposition and inflammation
is correlated with impaired behavioural performance. In addition, it would be interesting to measure the
levels of circulating hormones involved in stress, such as cortisol, to determine a possible relationship
between housing conditions and stress, especially considering the prominent role of chronic stress in AD
(see for review[62]). An overly stressful cognitive and social environment could exacerbate rather than
slow or prevent the development of AD, and would explain why ordinary people with moderate or very
active lifestyles still develop AD.

Abbreviations

AD

Alzheimer's disease

EE

Environmental enrichment

APP

Amyloid precursor protein

PSEN1

Presenilin 1

HM

Helico maze

SC

Standard condition

IC

Isolated condition

EC

Enriched condition

Declarations

Ethics approval and consent to participate
Not applicable.

**Consent for publication**

Not applicable.

**Availability of data and materials**

The datasets during and/or analysed during the current study available from the corresponding author on reasonable request.

**Competing interests**

The authors declare that they have no competing interests.

**Funding**

Research supported by grants from Aix Marseille Université with the LABEX A*MIDEX "Pépinière d'excellence".

**Authors'contributions**

Christine Manrique, Martine Migliorati, Guy Escoffier and François S. Roman performed the experiments, collected and analyzed the data.

Stéphane D. Girard, Kévin Baranger, Santiago Rivera and François Roman interpreted and wrote the manuscript.

All the authors read and approved the manuscript

**Acknowledgements**

This work was supported by funding from the CNRS, and Aix Marseille Université.

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## Figures
<figure><img src="image_2.png" /><figcaption>**Figure 1**</figcaption></figure>

**Housing conditions.** A: Standard Condition (SC), B: Isolated Condition (IC) and C: Enriched Condition (EC). D: *MarlauTM* cage used in EC with tunnels, ladder, slider, nesting area and 2 running wheels. In black: 1 example of the maze positioned in the upper floor and its ventilated cover on the side. E: The different mazes used successively during the 2 months of housing in the *MarlauTM* cage (see EC in the housing conditions of the Materials and Methods section for more details). All the photographs and
schematics are not to the same scale (see also in the housing conditions of the Materials and Methods for the actual dimensions of the cages and mazes).

<figure><img src="image_3.png" /><figcaption>Figure 2</figcaption></figure>

**Density of amyloid plaques in the brain of 5xFAD mice housed in standard, individual and enriched conditions.** A: Amyloid deposits in the frontal association cortex (FrA), secondary motor cortex (M2), lateral septum nucleus, intermediate (LSI), dentate gyrus in dorsal hippocampus (DG) and entorhinal
cortex (Ect). Schemes on the left represent brain coronal sections from Paxinos and Franklin (2019)[37], where Aβ immunostaining was performed. The schematic insets represent enlarged portions of the brain areas, where amyloid plaques were counted. Panels on the right represent fluorescence microphotographs of these inset areas in coronal brain sections from 5xFAD mice housed in standard (SC), individual (IC) or enriched (EC) conditions, immunostained with anti-Aβ antibody (6E10) in green. Cell nuclei were stained blue with Hoechst. B: Mean number (± SEM) of amyloid plaques per mm² in SC N=10, IC N=10 or EC N=12. Total refers to the average number of amyloid plaques across the five brain structures considered. # or * p< 0.05; ## or ** p<0.01. # represents ANOVA statistics; * represents Newman Keuls multiple comparisons post-hoc test statistics. For all images scale bars: 200 µm. NS: Not significant.
<figure><img src="image_4.png" /><figcaption>Figure 3</figcaption></figure>

**Astrogliosis in the brain of 5xFAD mice housed in standard, individual and enriched conditions.** A: Gliosis in the frontal association cortex (FrA), secondary motor cortex (M2), lateral septum nucleus, intermediate (LSI), dentate gyrus in dorsal hippocampus (DG) and entorhinal cortex (Ect). Schemes on the left represent brain coronal sections from Paxinos and Franklin (2019)[37], where GFAP immunostaining was performed. The schematic insets represent enlarged portions of the brain areas,
where astrogliosis was evaluated. Panels on the right represent fluorescence microphotographs of these
inset areas in coronal brain sections from 5xFAD mice housed in standard (SC), individual (IC) or
enriched (EC) conditions, immunostained with anti-GFAP antibody in red. Cell nuclei were stained blue
with Hoechst. B: Mean percentage (± SEM) of GFAP staining coverage of the studied brain area in SC
N=10 IC N=10 or EC N=12. Total refers to the average percentage of GFAP staining across the five brain
structures considered. # or * p< 0.05; ## or ** p<0.01. # represents ANOVA statistics; * represents
Newman Keuls multiple comparisons post-hoc test statistics. For all images scale bars: 200 µm. NS: Not
significant.