Sex-specific effects of early life stress exposure on memory performance and medial prefrontal cortex transcriptomic pattern of adolescent mice

doi:10.21203/rs.3.rs-4852454/v1

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Sex-specific effects of early life stress exposure on memory performance and medial prefrontal cortex transcriptomic pattern of adolescent mice

https://doi.org/10.21203/rs.3.rs-4852454/v1

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Early life stress (ELS) is considered a risk factor for the development of cognitive and executive disfunctions throughout development. The medial portion of the prefrontal cortex (mPFC) is directly implicated with short-term and working memory. Furthermore, due to its late development compared to other brain regions, the mPFC is considered a vulnerable brain region to ELS exposure. Here, we investigated the effects of ELS on PFC-dependent memory as well as mPFC transcriptomic profile. From postnatal day (PND) 2 to PND 15 BALB/cJ mice were exposed to maternal separation for 3h per day combined with limited bedding (ELS group) or left undisturbed (CT group). During the period of stress, maternal behavior observations were recorded pre-MS and post-MS. During adolescence (PND45 to PND47), male and female were tested for working memory performance in the Y-maze and short-term recognition memory in the object in place task (OIP). Later, we assessed mRNA level alterations in the mPFC by RNA-seq. Here, we showed that ELS increases maternal care post-MS and the number of nest exits pre-MS and post-MS. Furthermore, males and females exposed to ELS showed impairments in the OIP, while only females performed worse in the Y-maze. Regarding the mPFC transcriptome, we identified 13 differently expressed genes in the females, with significant influence of chaperon-mediated protein folding processes, while 4 genes were altered in males. In conclusion, we showed that ELS alters maternal behavior and leads to more extensive impairments in memory function and transcriptomic alterations in females compared to males.

Maternal Separation

Limited Bedding

RNA-seq

Working memory

Short-term memory

Early life stress (ELS) is considered a risk factor for the development of several psychiatric conditions [1], as well as increased vulnerability to cognitive and executive disfunctions later in life [2]. Moreover, long-term alterations in the hypothalamic-pituitary-adrenal (HPA) axis response and impairments in the corticolimbic system circuitry have been strongly associated with adverse experiences during early development [3, 4]. The prefrontal cortex (PFC) is one of the brain regions included in the corticolimbic system, and mainly its medial portion (mPFC), is directly implicated with short-term and working memory, decision-making, and emotion regulation [5]. Due to its late development when compared to other brain regions, and high density of glucocorticoid receptors, the mPFC is considered a vulnerable brain region to the effects of ELS [6]. Past studies have reported that exposure to preclinical models of ELS can impair memory performance on PFC-dependent tasks [7–9]. Nevertheless, there are still gaps in the literature to fully unreal the molecular underpinnings associated with those long-term cognitive deficits. In addition, considering the polygenic characteristics of these impairments, a genome-wide approach is increasingly necessary to comprehend such conditions.

Recent unbiased transcriptome analyses of the mPFC in rodents exposed to ELS have yielded diverse findings. For instance, Shi et al. [10] identified a heightened vulnerability of the oxytocinergic gene pathway to postnatal maternal separation (MS) in rats, whereas Yang et al. [11] observed that MS adversely affected the dopamine signaling and neuronal development pathways in the mPFC of C57BL/6J mice. Other studies combined ELS paradigms with stress exposure during adulthood and found significant alterations in gene pathways such as extracellular matrix structure, collagen trimer, and ribosomal pathways in the mPFC [12, 13]. Furthermore, Peña et al. [14] demonstrated sex-specific effects within an ELS model that combined limited bedding (LB) and MS, occurring from postnatal day 10 to 17 in females and from postnatal day 10 to 20 in male C57BL/6J mice. Their findings indicated that in males, ELS predominantly impacted the myelination pathway in the mPFC, while in females, it primarily affected immune system processes. These findings underscore the influence of rodent strain, sex, and stress model on transcriptome experiments investigating the enduring consequences of ELS. As a result, differentially expressed genes and affected pathways in the mPFC can vary substantially across studies. Furthermore, an additional factor is the time of assessment. All the aforementioned studies analyzed transcriptome changes induced by ELS in adult animals. However, it is noteworthy that adolescent animals may exhibit distinct alterations, both in molecular and behavioral phenotypes, in response to ELS compared to adult animals [15]. This temporal aspect adds another layer of complexity to the understanding of the long-term effects of ELS in rodent models.

Therefore, in this study we sought to investigate how a model of ELS could impact working and short-term recognition memory performance of male and female adolescent mice. Additionally, we examined changes in the mPFC transcriptome using RNA-seq. In contrast to previous studies that performed transcriptomic analysis in the mPFC, we focused our investigation on BALB/cJ mice, a strain recognized for displaying increased susceptibility to stress [16].

2.1 Animals

This study was performed with male and female BALB/cJ mice obtained from the Center of Experimental Biological Models (CeMBE) at Pontifical Catholic University of Rio Grande do Sul (PUCRS), Porto Alegre, Brazil. All animals were housed in mouse Plexiglas cages (22 cm x 16 cm x 14 cm) with controlled ventilation, temperature (21°C ± 1°C) and humidity (55% ± 5%). A 12/12 h light-dark cycle was implemented (lights on at 7 a.m. to 7 p.m.), with mouse chow and water available ad libitum. All experiments were approved by the Ethics Committee on Animal Use (CEUA) of PUCRS under #8546 and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH).

To avoid stress associated with animal transportation, the animals were bred in-house. Breeding procedure consisted in housing two females and one male in the same cage for three days. Two weeks later the females were individually housed and daily inspected until the presence of pups was confirmed. The day of birth was designated as postnatal day 0 (PND 0). After birth, cross-fostering was performed, and the litters were culled to 6–8 pups per litter. The dam and pups were then randomly assigned to control (CT) or ELS conditions. For this experiment, we used 7 litters for CT group and 7 litters for ELS group. At PND 21 two to three animals per cage were group-housed with same-sex littermates. Each litter contributed with a maximum of two animals per sex for each experimental condition to avoid potential litter effects. A total of 13 male CT, 13 female CT, 14 male ELS and 14 female ELS animals were utilized in this study. Animals were briefly weight at PND 2, PND 16 and PND 45. Working and short-term recognition memory performance was assessed during adolescence at PND 45 and PND 47. Experimental timeline can be found in Fig. 1A.

2.2 Early life stress protocol

The ELS protocol consisted in simultaneously exposing the animals to MS and LB. Our combined ELS protocol was adapted from the model previously proposed by Peña et al. [17], and it is intended to impose multiple adverse experiences during early development. From PND 2 to PND 15, animals from the ELS group were reared in LB cages with reduced amount of nesting material (1 g of cotton). In addition, the home cage floor was covered using an aluminum mesh platform with wood shavings underneath. The dams could not reach the wood shavings in order to use as nesting material, but it was necessary to permit the mouse droppings to fall below the platform without trapping the pups. Dams and litters from the CT group were placed in cages with standard amount of wood shavings and 4 g of cotton to be used as nesting material. After PND 15, animals from the ELS group were returned to regular rearing conditions. Cage cleaning occurred once a week for both groups.

Simultaneously, the MS protocol was performed by separating the pups for 3 hours per day (9 a.m. to 12 p.m.) between PND 2 to PND 15. To start the protocol, the dams were removed from the home cage and kept in a different colony room to avoid any dam-pup ultrasonic vocalization. Pups were then individually separated into small plastic containers. A digital-regulated heating pad (32°C ± 3°C) was placed underneath the containers to control the temperature and prevent possible hypothermia. After MS period, pups were returned to the home cage followed by the dam. CT group was left undisturbed during the protocol.

2.3 Maternal behavior analysis

Maternal behavior during the first weeks of development has a direct impact on pup neurodevelopment [18]. For this reason, we conducted intercalated observations of maternal behavior (n = 7 dams per group) from PND 3 to PND 15 (7 days total). The observations were performed every 3 minutes over a period of 18 minutes (7 observations) before (8:30 a.m.) and after the MS period (2 p.m.). The maternal behaviors evaluated were nursing (N), licking (L), nest building (B) and contact with pups (C). Moreover, during the 18 minutes of observation, the frequency of exits from the nest performed by the dam was accounted. The sum of all maternal behaviors (MB = N + L + B + C) evaluated throughout the days was used for pre-MS and post-MS maternal behavior analysis.

2.4 Y-maze

Working memory performance was evaluated at PND 45 in the Y-maze test. The apparatus consisted of three identical arms (30 cm x 5 cm x 10 cm). This working memory test is performed in two phases (sample and test), and it is based on the premise that rodents are more prone to explore novel areas when compared to previously explored environments. In the sample phase, animals were allowed to explore two arms (start and familiar arm) for 5 minutes, while the third arm (novel arm) was blocked with a removable wall. After the sample phase, animals were kept in a holding cage for 1 minute while the wall that blocked the novel arm was removed. In the test phase, animals were allocated in the start arm, and could freely explore all three arms of the apparatus for 2 minutes. The apparatus was cleaned with 70% ethanol between animals to avoid potential odor cues. A working memory index was calculated using the respective formula: [time spent in novel arm / (time spent in the familiar arm + time spent in the novel arm)] x 100. The analysis of the time spent in each arm was performed using Any-Maze software version 4.9 (ANY-Maze, Inc., Greensburg, PA).

2.5 Object in Place

The object in place (OIP) task was performed at PND 47 in an open field arena (33 cm x 33 cm) to investigate for short-term recognition memory. The OIP is performed in two phases (acquisition and test) and requires the animal to associate the characteristics of an object and also the spatial location where this object was encountered. One day before the task, animals were habituated to the apparatus for 10 minutes. Total distance travelled was evaluated as a measure of locomotor activity during habituation. For the acquisition phase, animals were introduced in the center of the apparatus, which had 4 different objects in the corners, and were allowed to explore all objects for 5 minutes. After the acquisition phase, animals were placed in a holding cage for 5 minutes while the position of two objects of the four objects was inverted. In the test phase, animals were allowed to explore all objects for 3 minutes. A recognition index of the time exploring the objects that changed position in relation to the objects that were not changed was performed with the following formula: [time exploring inverted objects / (time exploring intact objects + time exploring inverted objects)] x 100. Object exploration was defined by actively touching with the head, sniffing, or pawing the objects. If an animal climbed on top of the objects, it was not considered exploratory behavior [19]. The apparatus and the objects were cleaned with 70% ethanol between each phase and animals to avoid any olfactory cues. Analysis of exploration time was performed using Any-Maze software version 4.9 (ANY-Maze, Inc., Greensburg, PA).

2.6 Total RNA extraction and RNA-seq library preparation

Animals were euthanized 2 hours after the last behavioral testing. Total RNA (n = 6/group) was extracted from mPFC tissue homogenate via Total RNA was extracted using QIAzol (Qiagen) according to the manufacturer's protocol and reconstituted in 15 ul of RNAse-free water. All RNA samples were quantified via Qubit HS RNA assays (Thermo Fischer) and had RNA integrity number (RIN) > 6 as assessed with Agilent 2100 Bioanalyzer total RNA assay (Agilent Technologies). Total RNA (1 ug) was used to construct 150bp paired-end (PE) RNA-seq libraries with the Illumina Truseq™ RNA sample prep kit (Illumina, USA) as per manufacturer's instructions. The size and concentration of the PE RNA-seq libraries were verified on an Agilent 2100 Bioanalyzer and sequenced on an Illumina Novaseq 6000.

2.7 Transcriptomic analysis

Quality control and adapter trimming of raw data were performed using the wrapper Trim Galore version 0.6.6 [20], FastQC version 0.11.5 [21], and Cutadapt version 3.4 [22]. Quality control statistics were summarized using MultiQC version 1.11 [23]. Mus musculus reference genome and annotation were obtained from the Ensembl Genome Browser (Mus_musculus - Ensembl Genome Browser 104, n.d.). The “fasta” file for the primary assembly of GRCm39 release 104 was used as the reference genome and the corresponding “gtf” file was used as the annotation. Trimmed paired-end Illumina reads were aligned utilizing the STAR aligner version 2.7.9a [24]. Aligned reads were quantified for using FeatureCounts version 2.0.1 [25]. Quality control, adapter trimming, alignment, and quantification steps were implemented as a part of a pipeline using the Nextflow programming language version 21.04.3 [26]. The Nextflow pipeline was implemented using a Singularity container [27]. Nextflow in combination with Singularity containers enables reproducible computational workflows across platforms. RNA-seq outlier samples were detected utilizing the PcaGrid function [28].

On average, 87% of reads were successfully aligned, resulting in an average of 19.7 million uniquely aligned reads per sample. The removal of 1 outlier per group from downstream analysis resulted in 5 biological replicates per condition. Differential gene expression (DGE) analysis was performed utilizing DeSeq2 version 1.34.0 [29]. Using a Benjamini–Hochberg false discovery rate correction, the threshold for significance in the DGE analysis was the adjusted p-value < 0.1 and 2logFC +/- 0.5. A Gene Ontology (GO) analysis was performed in the list of genes with unadjusted p-value < 0.01, using the gProfiler2 R package. Enriched pathways (adjusted p-value < 0.05) were selected for biological or molecular processes. Graphical presentation of transcriptomic findings was performed using ggplot2, enhancedvolcano, and enrichplot R packages.

The deconvolution of RNA-seq data to estimate cell-type proportions in each sample was conducted using the CIBERSORT R package. The reference dataset for this analysis was the Allen Brain Map Mouse Cortex and Hippocampus SMART-seq single-cell RNA sequencing data. This reference included profiles for astrocytes, microglia, oligodendrocytes, pyramidal neurons, and interneurons.

2.8 Validation of transcriptomic data

Real-time quantitative polymerase chain reaction (qPCR) was performed in a distinct cohort of animals exposed to the same ELS model to validate RNA-seq results. Total RNA (500 ng) from the mPFC was utilized for complementary DNA (cDNA) synthesis using the QuantiTect Reverse Transcription Kit (Qiagen) as per manufacturer's instructions. Real-Time qPCR was performed using Rotor Gene (Qiagen) and each SYBR Green PCR reaction was run in duplicate. The fold change relative expression was calculated using the ΔΔ Ct method with the control group of the same sex as reference and Pgk ct values as endogenous controls. Primers sequences are reported in table S1. To verify primer specificities, melting curve analyses and agarose gels were performed.

2.9 Statistical analysis

Two-way ANOVAs were used to analyze group differences with rearing condition (ELS and CT) and sex (Male and Female) as factors. When necessary, ANOVAs were followed by Tukey’s post-hoc tests to investigate specific effects in pairwise comparison. Normality of data distribution was analyzed for all variables, using the Shapiro-Wilk Test of Normality, and homogeneity of variances was tested via Levene’s Test of Equality of Error Variances. Results are expressed as group mean ± standard error of the mean (SEM) with individual subjects represented with dots, and statistical significance was considered as a p-value < 0.05. Statistical analyses were performed with SPSS software v.25.0 (SPSS, Chicago, IL, USA) and GraphPad Prism 9 (GraphPad Software Inc., La Jolla, CA, USA).

3.1 Body weight control

At PND 2, no statistical difference in weight was observed between groups before the start of the ELS protocol (Fig. 1B). One day after the end of ELS (PND 16), we observed a trend in which animals exposed to ELS had a reduction in weight [t (12) = 2.051, p = 0.062; Fig. 1C]. At PND 45, females showed an expected reduction in body weight compared to males independent of rearing condition [sex effect: F (1,50) = 44.61, p < 0.001; Fig. 1D].

3.2 Early life stress exposure disrupts quality and frequency of maternal care

Considering the importance of an adequate maternal behavior in the offspring neurodevelopment, we performed a pre-MS and post-MS maternal care analysis. In relation to the frequency of maternal care, no significant differences between groups were observed pre-MS (Fig. 1E), but post-MS maternal care was significantly increased in ELS dams [t (12) = 2.728, p = 0.018; Fig. 1F]. Moreover, ELS dams presented a fragmented pattern of maternal care, which was manifested by an increased frequency of nest exits pre-MS [t (12) = 4.494, p < 0.001; Fig. 1G] and post-MS [t (12) = 3.447, p = 0.004; Fig. 1H].

3.3 Early life stress impairs working and recognition memory of adolescent mice

Taken in account the effects of ELS exposure on PFC-dependent memory function, we investigated the performance of males and females using the Y-maze for working memory and the OIP for short-term recognition memory. Regarding the Y-maze task, we identified a significant interaction effect [F (1, 46) = 4.732, p = 0.034; Fig. 2A]. Post-hoc analysis revealed that females exposed to ELS presented a reduction in the working memory index compared to CT animals (p = 0.002), while this effect was not observed in male animals. In relation to the OIP task, we observed that animals from the ELS group interacted significantly less with the inverted set of objects compared to the CT animals independent of sex [F (1,50) = 51.23, p < 0.001; Fig. 2C]. In relation to the locomotor activity in the habituation phase of the OIP task, no difference between groups was observed (Fig. 2B).

3.4 Early life stress exposure leads to sex-specific alterations in the cortical transcriptomic pattern

To investigate the effects of early-life stress (ELS) exposure on the mPFC transcriptome, we conducted RNA-seq analysis on adolescent animals, analyzing males and females separately with postnatal treatment (control or ELS) as a between-subjects factor. Before performing differential gene expression analysis, we deconvoluted the RNA-seq data using publicly available single-cell RNA sequencing data from mouse brains as references. Cell-type proportions for astrocytes, pyramidal neurons, microglia, and interneurons were analyzed, with oligodendrocyte proportions found to be negligible. No significant group effects or group-by-sex interaction effects were observed in cell-type proportions (Figure S1).

For males, the analysis revealed 4 differently expressed genes (DEGs) between CT and ELS with three downregulated (Serpina3h, Fkbp5, and Gm19439), and one (Gm18422) upregulated (Fig. 3A). For females, the analysis revealed 13 DEGs with two downregulated (Elk1 and Gm29100), and eleven (Hspa1b, Hspa5, Sdf2l1, Hspb1, Anxa3, Manf, Ccn2, Creld2, Gm3294, Phf21b and Homer1) upregulated (Fig. 3B). Heatmaps illustrate the expression data of DEGs for individual samples (Fig. 3C and 3D). In order to comprehensively evaluate the broader impact of ELS exposure on the regulation of the mPFC transcriptome, we conducted GO analysis of genes exhibiting differential expression using a less stringent significance threshold (unadjusted p-value < 0.01). This analysis unveiled enriched pathways associated with the chaperone-mediated protein folding in females (Fig. 3F), whereas in males, the enriched pathways were linked to responses to stress and external stimuli (Fig. 3E). To validate the RNA-seq findings, qPCR analyses were performed on five selected DEGs identified in females exposed to ELS compared to those not exposed. The findings confirmed the upregulation effect of ELS on the following genes: Hspa1b, Hspb1, Sdf2l1, Anxa3, and Cnn2 (Figure S2).

This study aimed to investigate the effects of a combined model of ELS using MS and LB on working and short-term recognition memory performance of adolescent male and female mice, as well as mPFC transcriptomic pattern. The main findings of the present study are: (1) Dams exposed to ELS show increased frequency of nest exits pre-MS and post-MS and overall maternal care behavior post-MS. (2) Exposure to ELS impaired working memory in females and short-term recognition memory of both male and female adolescent mice. (3) sex-specific transcriptomic alterations were identified in animals exposed to ELS, where the majority of DEGs were observed in females. (4) chaperone-mediated protein folding was the top GO term for females, while response to stimulus was the top term observed for males. These findings indicate that females may experience more pronounced memory impairments following ELS exposure, and that alterations in chaperone-mediated protein folding processes might be involved in such memory deficits.

It is well known that maternal care during the first weeks of development plays a key role in pup neurodevelopment [30, 31]. However, exposure to ELS may disrupt this relationship, which can have a long-term impact on the development of the offspring [18]. Here, we showed that ELS increased the frequency of maternal care post-MS, suggesting a potential adaptative response as an attempt to mitigate the effects of MS. Indeed, previous studies have shown that this increase in maternal care may attenuate the long-term impairments provoked by MS [32–34]. Furthermore, previous studies have demonstrated that LB exposure causes a disruption in the quality of maternal care, which is mainly due to an increase in the number of nest exits [35, 8]. This fragmented behavior was observed in our study in both pre-MS and post-MS analysis. This increase in nest exits may not only prolong the time that the dams are away from the pups, but also interrupt important behaviors, such as nursing and licking [36].

Regarding the performance in the memory tasks evaluated, we identified sex-specific alterations induced by ELS exposure. Although both males and females showed impairments in the short-term recognition memory task, only females exhibited deficits in working memory. These findings align with previous clinical and preclinical studies, which indicate that females are at greater risk to the effects of ELS [37–39]. Despite highly significant, the underlying mechanisms contributing to the heightened vulnerability of females to ELS remain insufficiently understood. Moreover, this could also be attributed to the distinct susceptibility of certain brain regions to ELS. Working memory, which was affected only in females, relies heavily on PFC function [40], while object recognition is also dependent on a connectivity between PFC and hippocampus [41]. Although both regions are known to be stress-sensitive, it is plausible that PFC-dependent functions experienced sex-specific effects due to ELS exposure.

Considering the key role of the PFC in working and short-term memory function, we focused our transcriptomic analysis in this region. The majority of DEGs (13) were identified in females, while 4 DEGs were identified in males. This goes in line with our behavior data, which showed greater phenotypic deficits in females. While few studies have performed transcriptomic analysis in the PFC following ELS in both sexes [14, 13], it is possible to hypothesize that the greater number of DEGs in females could be attributed to an increased vulnerability to ELS exposure.

70-kDa Heat shock proteins (HSP70s) related genes (Hspa1b, Hspb1, Hspa5) were among the top upregulated candidates in our RNA-seq results for the females. HSP70s are molecular chaperones that play a crucial role in maintaining cellular homeostasis and are known to be upregulated after exposure to various stressors [42]. In accordance with our sequencing data, it has been previously reported that exposure to MS increases the expression of Hspa1b and Hspb1 in the mPFC of rats [43]. Our GO analysis indicated an enrichment of chaperone-mediate protein folding and refolding processes, which are one of the main functions of HSP70s. An upregulation of HSP70s occurs when there is an accumulation of misfolding of proteins, which is a hallmark of diseases associated with memory/cognitive impairments [44, 45]. This data may suggest that the increased expression of HSP70s may serve as a protective mechanism against long-term cognitive impairments caused by ELS exposure. Furthermore, Sdf2l1, another gene observed in our analysis, acts by increasing the time that misfolded proteins have to achieve a correctly folded conformation, which facilitates chaperone-mediated protein refolding processes [46].

Regarding the RNA-seq analysis of males, we identified a downregulation of Fkbp5, a well-known modulator of the HPA-axis. Fkbp5 is a negative regulator of glucocorticoid receptor (GR) signaling, directly influencing cortisol/corticosterone levels [47]. While proper HPA-axis function is essential for physiological homeostasis, increased or decreased reactivity of this system can lead to an altered stress response [48]. In accordance, we have previously shown that male mice exposed to the same ELS model exhibited a blunted corticosterone response and increased anxiety-like behavior [49]. Although the males in our study were less affected in the memory tasks evaluated and showed less DEGs than the females, it is possible to suggest that these animals exposed to ELS remain vulnerable to certain HPA-related phenotypic alterations.

It is important to interpret our data with some limitations. Our RNA-seq analysis was concentrated on a single brain region. While exposure to stressors during early development can lead to changes across multiple brain regions, we chose to focus on the mPFC due to its direct involvement in short-term and working memory functions. Additionally, to minimize the number of animals utilized in our study, we exclusively investigated adolescent animals. This decision may limit the ability of this experiment to generalize the effects of ELS across different developmental stages.

In conclusion, our study showed that ELS induces impairs PFC-dependent memory performance, with more pronounced effects observed in female animals. These phenotypic alterations may be associated with inadequate maternal care during early development. Additionally, RNA-seq analysis revealed that females exhibited greater differences following ELS exposure, particularly influencing chaperone-mediated protein folding processes. Nevertheless, male animals exposed to ELS showed an alteration in Fkbp5, a key regulator of HPA-axis function.

Conflicts of Interest

The authors have no relevant financial or non-financial interests to disclose.

Funding

This work was supported by the Brazilian funding agencies: Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) [grant numbers: 306580/2022-5, 402503/2023-6, 442776/2018-7], Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul [grant number: 17/2551-0001146-5], Lundbeck Foundation [grant number: R434-2023-382].

Author Contribution

R.O: Conceptualization, Investigation, Formal analysis, and Writing - Original Draft; T.W.V: Conceptualization; Formal analysis, Writing – Original Draft; B.A.H: Formal analysis; K.C.C: Investigation; Writing - Review & Editing; F.S.L: Investigation; R.G.O Conceptualization, Project Administration, Funding acquisition, Writing - Review & Editing.

Data Availability

All RNA-seq code and data are available at: (https://github.com/bernardo-heberle/dcnl_rna_seq_els_vs_ct). Data for behavior and Real-time PCR will be made available upon request to the corresponding author R.G.O ([email protected]).

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Sex-specific effects of early life stress exposure on memory performance and medial prefrontal cortex transcriptomic pattern of adolescent mice

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Abstract

Figures

1. Introduction

2. Methods

2.1 Animals

2.2 Early life stress protocol

2.3 Maternal behavior analysis

2.4 Y-maze

2.5 Object in Place

2.6 Total RNA extraction and RNA-seq library preparation

2.7 Transcriptomic analysis

2.8 Validation of transcriptomic data

2.9 Statistical analysis

3. Results

3.1 Body weight control

3.2 Early life stress exposure disrupts quality and frequency of maternal care

3.3 Early life stress impairs working and recognition memory of adolescent mice

3.4 Early life stress exposure leads to sex-specific alterations in the cortical transcriptomic pattern

4. Discussion

Declarations

Conflicts of Interest

Funding

Author Contribution

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1