Introduction: Epigenetic mechanisms involving microRNAs (miRNAs) play a fundamental role in many biological processes, particularly during prenatal and early postnatal development. Their role in adolescent brain development, however, has been poorly described. The present study aimed to explore miRNA expression in the hippocampus during adolescence compared to adulthood in rats. Method: The brains of female and male Wistar rats were extracted, and the hippocampus was freshly dissected at postnatal day 41 (adolescence) and postnatal day 98 (adulthood). An epigenome-wide analysis was conducted to identify the miRNAs significantly expressed in adolescence compared to adulthood. Additionally, target genes of such miRNAs were considered to perform an exploratory Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Results: We identified 16 differentially expressed miRNAs in adolescent male rats compared with adult male rats and 4 differentially expressed miRNAs in adolescent females compared with adult females. Enrichment analysis reinforced that the target genes found are related to neurodevelopmental processes such as cell proliferation, cell migration, and nervous system development. Conclusion: Our findings suggest a complex pattern of miRNA expression during adolescence, which differs from that in adulthood. The differential expression of miRNA in the hippocampus during adolescence may be associated with the late developmental changes occurring in this brain region. Furthermore, the observed sex differences in miRNA expression patterns indicate potential sexual differentiation in hippocampal development. Further comprehensive investigations are needed to elucidate the roles of miRNA in normal brain development.

The role of epigenetic mechanisms in nervous system development is an area of growing interest. Various epigenetic mechanisms, including DNA methylation, histone methylation and acetylation, and microRNA (miRNA), play essential roles in the formation of functional brain circuits [1]. miRNAs are small noncoding RNAs of 21 to 33 nucleotides of length that modulate messenger RNA (mRNA) translation. They bind to untranslated regions of their target mRNAs often resulting in reduced gene expression through transcript degradation or mRNA silencing [2]. A single miRNA can bind to multiple mRNA targets, thereby regulating the expression of numerous genes. Additionally, a single mRNA can also be the target of multiple miRNAs. Thus, miRNAs are important regulators of gene expression that play a fundamental role in many biological processes (BPs).

In mammals, miRNA has been involved in different phases of neuronal development such as cell differentiation, apoptosis, and synaptic plasticity [3‒5]. However, the specific roles during late postnatal developmental phases remain poorly understood. Brain development is a complex and prolonged process that occurs in a stepwise manner from gestation to late adolescence. In fact, adolescence represents a transitional stage from childhood dependence to adult independence and is associated with important changes in late-developing brain areas [1, 6]. The hippocampus and cerebral cortex [7, 8], in particular, undergo substantial reorganization during this stage, which is associated with the peculiar adolescent behavior [6, 9]. Notably this shaping of brain circuitry during adolescence is thought to be partly mediated by miRNA in response to environmental influences.

Previous research with rodent models has primarily focused on miRNA expression patterns and their alteration by environmental factors, such as stress or drugs, during adolescence [10]. Also, considerable efforts have been made to elucidate the potential role of miRNA as risk factors for psychiatric disorders [11]. However, it is equally important to investigate the expression pattern of miRNA in control groups without any intervention throughout life. For example, Prins et al. [12] investigated the expression patterns of specific miRNA during normal pubertal development, focusing on miRNAs that are potential regulators of development-related genes, such as brain-derived neurotrophic factor and sirtuin-1. They reported differential expression pattern of miR-10a-5p, miR-26a, miR-103, and miR-495 in the dorsal and ventral hippocampus of male rats at different postnatal days (PNDs 30, 44, and 73). Another study found a progressive decrease in the expression of miR-19a/b-3p, miR-34a, and miR-488-3p during adolescence in the ventral hippocampus [13]. Given that hippocampal reorganization plays a critical role in the characteristic adolescent behavior [6], the present study aimed to explore dorsal hippocampal miRNA expression during adolescence compared to adulthood in rats. To achieve this, we conducted an epigenome-wide analysis to contribute to a deeper understanding of normal late development processes.

Animals

Four pregnant Wistar rats were purchased from Charles River Laboratories (Wilmington, MA, USA) and housed individually. Pups from the four litters (10 males; 10 females) were weaned on PND 21 and group-housed, with males and females in separate cages. Rats were maintained on a 12-h light-dark cycle with ad libitum access to food and water. These animals served as a control for another experiment (to be published) so they went through an intermittent saline administration procedure. Rats were injected i.p. 2 g/kg saline on a 2-day on/off schedule from PND 28 to PND 41. Animals were humanely euthanized using pentobarbital (100 mg/kg) followed by rapid decapitation at PND 41 (adolescent group) and PND 98 (adult group).

Tissue Collection

Brains were immediately extracted, and the hippocampus was freshly dissected (−3.30 relative to Bregma, according to The Rat Brain in Stereotaxic Coordinates, Fourth Edition Atlas) [14]. The hippocampus was flash frozen in liquid nitrogen and stored at −80 C until processed.

Transcriptome Sequencing

RNA extraction from dorsal hippocampus was performed using the RNeasy Lipid Tissue Mini Kit (Qiagen). Total cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, USA). For reverse transcription, it was used 200 ng of total RNA from each sample. A solution-phase assay was carried out (Applied Biosystems). Small RNA extraction was done using the miRNA Micro Kit (Qiagen), and the checking of RNA purity and integrity was performed with NanoDrop (Thermo Fisher) and Bioanalyzer (Agilent), respectively. The preparation of the small RNA library required several enzymatic steps to include in the final library only the small RNA fragments. Sequencing of multiplexed libraries was performed in the NextSeq 500 equipment (Illumina).

Statistical Analysis of Differentially Expressed miRNA

FastQs were obtained using Illumina’s bcl2fastq software. The quality of the sequences was screened using FastQC and MultiQC [15]. First, raw sequence adapter was removed using the Cutadapt software [16]. Then, 4 bps and low-quality bps (<20 in Phred scale) in each end were trimmed. The expression of miRNA was obtained with miARma-Seq pipeline [17], and bowtie2 was used to align the sequences [18]. miRNA was annotated using the miRbase database [19]. Expression was normalized with NOISeq [20] following the approach of trimmed mean of M [21].

Principal component analysis (PCA) was conducted in order to visually represent the variance of data and to evaluate quality problems, such as contaminated samples, processing errors, or anomalous measurements. PCA plots were obtained with ggplot2 [22]. The differential expression analysis was performed following the default recommendations of DESeq2 [23]. DESeq2 performs an internal normalization for each gene, fits negative binomial generalized linear models for each gene, and uses the Wald test for significance testing [23]. As suggested and considered by many authors [24, 25], a miRNA was considered differentially expressed when the log2FC was >+/−1.5 and the p value-adj was <0.05.

Gene Ontology and Kyoto Encyclopedia of Genes and Genomes Enrichment Analysis

To our exploratory analysis, we used miRNA target gene prediction software miRTarBase and DianaTarBase to identify experimentally validated miRNA target genes and miRDB to identify predicted miRNA target genes. We combined the results of these databases and performed enrichment analysis using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) with the available resources of Gene Ontology (GO) [26] and Kyoto Encyclopedia of Genes and Genomes (KEGG) [27]. The GO analysis included three independent categories: BP, cellular component (CC), and molecular function (MF). Rattus norvegicus was selected as species. We used the Benjamini-Hochberg p value <0.05 to correct for multiple comparisons.

Differentially Expressed miRNA in Adolescent Male Rats Compared with Adult Male Rats

We took five samples for each group, one per animal (7H, 8H, 9H, 10H, and 11H for adolescence, and 28H, 29H, 30H, 31H, and 32H, for adulthood). PCA is shown in Figure 1.

Fig. 1.

PCA of the expressed miRNA in adolescent and adult male rats.

Fig. 1.

PCA of the expressed miRNA in adolescent and adult male rats.

Close modal

We identified 16 miRNAs that were differentially expressed between groups. Among these, the expression of 9 miRNAs (miR-449a-5p, miR-20a-3p, miR-298-3p, miR-296-5p, miR-501-5p, miR-135b-5p, miR-19a-3p, miR-144-3p, and miR-542-3p) was significantly increased, while the expression of 7 miRNAs (miR-3064-5p, miR-6324, miR-187-5p, let-7b-5p, miR-92b-5p, miR-664-2-5p, and miR-672-5p) was significantly decreased in adolescent male rats compared to adult male rats (Fig. 2; Table 1). In adolescent females, miR-190a-5p was under-expressed and miR-206-3p, miR-298-3p, miR-296-5p were over-expressed in comparison with adult females.

Fig. 2.

a Volcano plot. b Heatmap of differentially expressed miRNA in adolescent male rats compared with adult male rats.

Fig. 2.

a Volcano plot. b Heatmap of differentially expressed miRNA in adolescent male rats compared with adult male rats.

Close modal
Table 1.

Differentially expressed miRNA in adolescent male rats compared with adult male rats

miRNABase meanLog2 (fold change)Up-down regulationp-adj
miR-20a-3p 3.73 3.25 Up 0.011 
miR-144-3p 7.03 2.14 Up 0.023 
miR-19a-3p 8.66 1.96 Up 0.002 
miR-449a-5p 24.62 1.93 Up 0.014 
miR-296-5p 187.23 1.89 Up 0.0011 
miR-135b-5p 481.81 1.85 Up 1.57E-07 
miR-298-3p 35.59 1.86 Up 5.04E-05 
miR-501-5p 9.55 1.62 Up 0.0218 
miR-542-3p 124.27 1.53 Up 0.001 
miR-187-5p 70.08 −1.52 Down 0.001 
miR-664-2-5p 311.28 −1.64 Down 5.04E-05 
let-7b-5p 32,147.22 −1.67 Down 0.001 
miR-3064-5p 13.23 −1.68 Down 0.003 
miR-672-5p 185.24 −1.92 Down 1.38E-06 
miR-92b-5p 67.05 −2.40 Down 2.51E-07 
miR-6324 11.15 −3.17 Down 0.013 
miRNABase meanLog2 (fold change)Up-down regulationp-adj
miR-20a-3p 3.73 3.25 Up 0.011 
miR-144-3p 7.03 2.14 Up 0.023 
miR-19a-3p 8.66 1.96 Up 0.002 
miR-449a-5p 24.62 1.93 Up 0.014 
miR-296-5p 187.23 1.89 Up 0.0011 
miR-135b-5p 481.81 1.85 Up 1.57E-07 
miR-298-3p 35.59 1.86 Up 5.04E-05 
miR-501-5p 9.55 1.62 Up 0.0218 
miR-542-3p 124.27 1.53 Up 0.001 
miR-187-5p 70.08 −1.52 Down 0.001 
miR-664-2-5p 311.28 −1.64 Down 5.04E-05 
let-7b-5p 32,147.22 −1.67 Down 0.001 
miR-3064-5p 13.23 −1.68 Down 0.003 
miR-672-5p 185.24 −1.92 Down 1.38E-06 
miR-92b-5p 67.05 −2.40 Down 2.51E-07 
miR-6324 11.15 −3.17 Down 0.013 

Differentially Expressed miRNA in Adolescent Female Rats Compared with Adult Female Rats

We took five samples for each group, one per animal (17H, 18H, 19H, 20H, and 21H for adolescence, and 38H, 39H, 40H, 41H, and 42H for adulthood). PCA is shown in Figure 3. We identified 4 miRNAs that were differentially expressed between the two groups, with 3 miRNAs showing increased expression and 1 miRNA showing decreased expression in adolescent female rats compared to adult (Fig. 4; Table 2).

Fig. 3.

PCA of the expressed miRNA in adolescent and adult female rats.

Fig. 3.

PCA of the expressed miRNA in adolescent and adult female rats.

Close modal
Fig. 4.

a Volcano plot. b Heatmap of differentially expressed miRNA in adolescent female rats compared with adult female rats.

Fig. 4.

a Volcano plot. b Heatmap of differentially expressed miRNA in adolescent female rats compared with adult female rats.

Close modal
Table 2.

Differentially expressed miRNA in adolescent female rats compared with adult rats

miRNABase meanLog2 (fold change)Up-down regulationp-adj
miR-206-3p 48.97 2.04 Up 0.046 
miR-298-3p 35.59 1.74 Up 0.004 
miR-296-5p 187.23 1.64 Up 0.043 
miR-190a-5p 114.28 −1.58 Down 0.038 
miRNABase meanLog2 (fold change)Up-down regulationp-adj
miR-206-3p 48.97 2.04 Up 0.046 
miR-298-3p 35.59 1.74 Up 0.004 
miR-296-5p 187.23 1.64 Up 0.043 
miR-190a-5p 114.28 −1.58 Down 0.038 

GO and KEGG Pathway Enrichment Analyses Using Target Genes of Differentially Expressed miRNA in Adolescent Males

To gain insights into the functions and pathways associated with the differentially expressed miRNA, we performed an exploratory GO and KEGG enrichment analysis on these target genes. Figure 5 shows the top 12 significant GO terms obtained for each category. The genes were enriched in the BP category, including positive regulation of cell proliferation, cell migration, positive regulation of cell migration, and nervous system development. In terms of CC category, the target genes were related to neuronal cell body, glutamatergic synapse, dendrite, and axon. Moreover, they were enriched in the MF category, including protein binding, ATP binding, RNA polymerase II transcription factor activity sequence-specific DNA binding, chromatin binding, sequence-specific DNA binding among others.

Fig. 5.

Top 12 significant GO terms for each category in male rats.

Fig. 5.

Top 12 significant GO terms for each category in male rats.

Close modal

Regarding the KEGG pathway analysis, the top 15 pathways are shown in Figure 6. They included signaling pathways regulating pluripotency of stem cells, pathways related to cancer and to endocrine signaling, forkhead box O and cAMP signaling pathways, and Ras signaling pathway, among others (Fig. 6).

Fig. 6.

Top 15 significant KEGG pathways in males.

Fig. 6.

Top 15 significant KEGG pathways in males.

Close modal

GO and KEGG Pathway Enrichment Analyses Using Target Genes of Differentially Expressed miRNA in Adolescent Females

For adolescent females, GO analysis revealed enrichment of target genes in BP response to activity, CC cytoplasm, and cytosol and also for protein binding in the MF category (Fig. 7). KEGG pathway analysis did not detect significant enrichments in this group.

Fig. 7.

Significant GO terms for each category in female rats.

Fig. 7.

Significant GO terms for each category in female rats.

Close modal

This exploratory study provides valuable insights into the miRNA expression patterns in the dorsal hippocampus during adolescence compared to adulthood in rats. To our knowledge, it is the first study that focuses on miRNA expression changes related to normal development in rats. Our genome-wide approach revealed a complex pattern of miRNAs that were either under- or over-expressed in the hippocampus of adolescent compared to adult animals in both male and female rats. Regarding males, we found 7 under-expressed miRNAs (miR-3064-5p, miR-6324, miR-187-5p, let-7b-5p, miR-92b-5p, miR-664-2-5p, and miR-672-5p) and 9 over-expressed miRNAs (miR-449a-5p, miR-20a-3p, miR-298-3p, miR-296-5p, miR-501-5p, miR-135b-5p, miR-19a-3p, miR-144-3p, and miR-542-3p). As for females, we found that miR-190a-5p was under-expressed and miR-206-3p, miR-298-3p, miR-296-5p were over-expressed. It does not seem feasible a relevant contribution of the potential stressful effects of i.p. saline injections even though we cannot discard it. It has been previously reported that repeated i.p. saline injections can be a mild stressor only in the Fischer-344 strain but not in Sprague-Dawley rats [28]. Likewise, no effect of saline injections on body weight and plasma corticosterone levels has been reported even if they are repeatedly applied during early developmental stages from PND 2 to PND 12 [29]. Moreover, the miRNAs showing an adolescent peculiar pattern of expression in the present study are related to processes involved in development but not in stress regulation. In any case, given that adolescent and adult rats were subjected to the same i.p. saline injection protocol, the different patterns of miRNA expression can be attributed to the specific developmental processes taken place during adolescence.

The results showed that adolescent males seem to exhibit a higher number of differentially expressed miRNAs than females in comparison with their respective adult groups, suggesting potential sexual differentiation in hippocampal development. In fact, previous research has reported sex differences in the hippocampal development [28, 30]. Sex differences in brain-derived neurotrophic factor expression have been found in the hippocampus of neonatal rats [31], and epigenetic mechanisms of cell proliferation also differ between males and females in postnatal hippocampal development [32].

Although we cannot provide a specific explanation for the higher number of differentially expressed miRNAs in males than in females, this could reflect sex differences in either complexity or temporal course of the hippocampal maturation. A greater complexity of the hippocampal maturation in males than in females could be related to the described outperformance of male rats in hippocampal-dependent spatial learning and memory tasks [33]. It can be envisaged that a better performance would be associated with a more complex and protracted maturation of the hippocampus. Otherwise, the lower number of miRNAs differentially expressed in adolescent and adult females could indicate earlier hippocampal maturation in females than in males. Although adolescence cannot be identified with puberty [34], it can be proposed that pubertal hormonal changes may play a role in the observed variability. Taking into account vaginal opening and preputial separation as indicators of the puberty onset in Wistar rats, it has been reported earlier sexual maturity in females than in males. There is variability in the onset of puberty ranging from PND 33–35 in females to PND 37–40 [35], while females reach sexual maturity on PND 56 and male on PND 70 [36]. If changes of miRNA expression in the adolescent hippocampus are influenced by pubertal hormones at the early stages of puberty onset, it can be proposed that some of them had already taken place before PND 41 in which we extracted the brains. In fact, endogenous steroids can differentially affect cell proliferation in the hippocampus of male and female rats [28, 30]. It is remarkable that despite the differences in the number of differentially expressed miRNAs, we found that miR-298-3p and miR-296-5p were over-expressed during adolescence in both sexes. Considering that these miRNAs have been associated with cell apoptosis in different tissues, the regulation of this cellular process may not greatly differ between sexes [37, 38].

Given the limited availability of previous studies on miRNA expression during normal development, direct comparisons are hindered. Existing findings mainly come from control groups within research focused on the role of miRNA in different pathologies, and in many cases only using males, neglecting the role in non-pathological development. Nevertheless, some of the miRNAs found in this research, including let-7b, miR-17, miR-19, and miR-92, have been previously associated with important developmental processes. In particular, over-expression of let-7b has been reported to lead to reduced stem cell proliferation and increased cell differentiation, while inhibition by knockout animals leads to increased cell proliferation and less differentiation [39]. Moreover, let-7b promotes neuronal differentiation in the mammalian retina [40]. Likewise, miR-92, although combined with miR-17, increases axonal growth while its inhibition decreases it [41]. Furthermore, let-7b and miR-19 have been associated with the transition from quiescence to proliferation of adult neural stem cells [33] and miR-19 has been identified as a key regulator of new cell migration in the adult hippocampus [42]. We found under-expression of let-7b and over-expression of miR-135b and miR-19b in the adolescent hippocampus in comparison with the adult hippocampus. The rest of the miRNAs differentially expressed in adolescence have not been previously related to adult hippocampal neurogenesis. This indicates that even if adult hippocampal neurogenesis remains in the adult dentate gyrus the contribution of miRNAs to this process can be differentiated of those involved in the adolescent developmental stage.

Considering that these miRNAs are regulators of hundreds of genes, we performed an exhaustive search of their target genes by using different databases. We found that genes targeted by the differentially expressed miRNA between adolescent and adult males were involved in many BPs that are relevant to neurodevelopment such as positive regulation of cell proliferation, cell migration, positive regulation of cell migration, and nervous system development. In the KEGG pathway enrichment analysis, we additionally observed significant routes that are directly related to the nervous system development. One of the most significant pathways found is the TNF signaling pathway, which can induce different processes such as apoptosis, cell survival, and inflammation. It is important to note that it plays an important role in the development of the nervous system and more specifically in the hippocampus [43‒45]. Regarding the thyroid hormone pathway, the deficit of these hormones impairs cellular differentiation and delays the migration of neurons in the developing hippocampus [46, 47]. It is well known the role of pluripotent stem cells in the nervous system formation and the late neurogenesis during adolescence [48]. Also, the Ras signaling pathway potentiation of this pathway reduces adult neurogenesis by affecting generative cells in the hippocampus and impairing short-term and object recognition memory [49]. Likewise, growth hormone is involved in brain development through processes such as neuronal differentiation, neurogenesis, and plasticity [50] and promotes the proliferation of progenitor cells and the formation of new neurons in the hippocampus [51‒53]. Finally, the forkhead box O family regulates the expression of genes involved in apoptosis, cell cycle control, synaptic plasticity, and neurogenesis [54, 55]. In the hippocampus, the loss of these transcription factors is associated with altered dendrite morphology, spine defects, and an impaired ability for memorizing [56, 57]. Thereafter, the relevance of the reported pathways is consistent with its role in normal brain development. Even though the findings presented in this study are exploratory, they are particularly interesting as noteworthy epigenetic changes are identified during adolescence, a late developmental stage often overlooked.

Overall, our study highlights a complex pattern of miRNA expression during adolescence that differs from that found in adulthood. Consistent with previous studies that reported a delayed onset of hippocampal-dependent learning abilities during adolescence, our pathway analysis suggests that targeted genes of such miRNAs are associated with processes related to late hippocampal development. Finally, our results underscore the importance of studying sex differences during hippocampal development, further highlighting the need for comprehensive investigations in this area.

These experiments are part of the Ph.D. research conducted by AV-Á in the doctoral program of Psychology at the University of Granada.

The procedures were approved by the University of Granada Ethics Committee for Animal Research and by the Regional Ministry of Agriculture, Fisheries and Rural Development of Andalusia (1/06/2022/078).

The authors have no conflicts of interest to declare.

This study was funded by the research projects PID2020-114269GB-I00 (MCIN/AEI/10.13039/501100011033), BSEJ.514.UGR20 (FEDER, Junta de Andalucía, Spain), “Instituto de Salud Carlos III,” project PI18/00467 (co-funded by European Regional Development Fund/European Social Fund “A way to make Europe”/“Investing in your future”), and a predoctoral fellowship to AV-Á (FPU18/05012, MIU, Spain).

M.G., F.G., and A.V.-Á. were responsible for the study concept and design. A.V.-Á., F.G., and M.G. were responsible for the experimental execution. A.V.-Á. was responsible for the drafting of the manuscript. A.V.-Á., F.G., B.G., P.R., and M.G. were responsible for the data analysis and interpretation. B.G., P.R., F.G., and M.G. were responsible for the critical revision of the manuscript. M.G. and F.G. supervised the study. B.G., F.G., and M.G. were responsible for securing funding. All authors read and approved the final manuscript.

The datasets generated and/or analyzed during the current study are available in the Open Science Framework repository, doi:10.17605/OSF.IO/EB2HV. Further inquiries can be directed to the corresponding author.

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