Folic acid and autism: updated evidences.

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition that impairs communication, socialization, and behavior. The association of ASD with folic acid has been investigated due to the importance of this vitamin for neurological health. This study is an update of the publication 'Folic acid and autism: What do we know?' and aims to systematically review studies examining the relationship between folic acid and ASD. The search resulted in 2,389 studies on folic acid and ASD, which were selected by two reviewers based on their titles and abstracts. Studies meeting the inclusion criteria were fully read. The 52 included studies involved 10,429 individuals diagnosed with ASD and assessed the intake of vitamin B6, folic acid, and vitamin B12; serum levels of these vitamins, homocysteine, and methionine; therapeutic interventions using folic acid; and the association between maternal exposure to this vitamin and the risk of ASD. The evidence of insufficient folic acid intake in most individuals with ASD remains consistent in this update. No association was found between maternal exposure to folic acid and the risk of ASD in their children. Despite observed improvements in communication, socialization, and behavior in individuals with ASD following folic acid interventions, it is crucial to consider the individuality and complexity of ASD. Given the relevance of the topic, there remains a need for more high-quality research and clinical trials characterized by rigorous methodological designs.

Astroglial S100B Secretion Is Mediated by Ca2+ Mobilization from Endoplasmic Reticulum: A Study Using Forskolin and DMSO as Secretagogues.

S100B, a homodimeric Ca2+-binding protein, is produced and secreted by astrocytes, and its extracellular levels have been used as a glial marker in brain damage and neurodegenerative and psychiatric diseases; however, its mechanism of secretion is elusive. We used primary astrocyte cultures and calcium measurements from real-time fluorescence microscopy to investigate the role of intracellular calcium in S100B secretion. In addition, the dimethyl sulfoxide (DMSO) effect on S100B was investigated in vitro and in vivo using Wistar rats. We found that DMSO, a widely used vehicle in biological assays, is a powerful S100B secretagogue, which caused a biphasic response of Ca2+ mobilization. Our data show that astroglial S100B secretion is triggered by the increase in intracellular Ca2+ and indicate that this increase is due to Ca2+ mobilization from the endoplasmic reticulum. Also, blocking plasma membrane Ca2+ channels involved in the Ca2+ replenishment of internal stores decreased S100B secretion. The DMSO-induced S100B secretion was confirmed in vivo and in ex vivo hippocampal slices. Our data support a nonclassic vesicular export of S100B modulated by Ca2+, and the results might contribute to understanding the mechanism underlying the astroglial release of S100B.

Preventive effects of resveratrol against early-life impairments in the animal model of autism induced by valproic acid.

Background: Autism Spectrum Disorder (ASD) is a neurodevelopmental condition characterized by social interaction deficits and repetitive/stereotyped behaviors. Its prevalence is increasing, affecting one in 36 children in the United States. The valproic acid (VPA) induced animal model of ASD is a reliable method for investigating cellular, molecular, and behavioral aspects related to the disorder. Trans-Resveratrol (RSV), a polyphenol with anti-inflammatory and antioxidant effects studied in various diseases, has recently demonstrated the ability to prevent cellular, molecular, sensory, and social deficits in the VPA model. In this study, we examined the effects of prenatal exposure to VPA and the potential preventive effects of RSV on the offspring. Method: We monitored gestational weight from embryonic day 6.5 until 18.5 and assessed the onset of developmental milestones and morphometric parameters in litters. The generalized estimating equations (GEE) were used to analyze longitudinal data. Results: Exposure to VPA during rat pregnancy resulted in abnormal weight gain fold-changes on embryonic days 13.5 and 18.5, followed by fewer animals per litter. Additionally, we discovered a positive correlation between weight variation during E15.5-E18.5 and the number of rat pups in the VPA group. Conclusion: VPA exposure led to slight length deficiencies and delays in the onset of developmental milestones. Interestingly, the prenatal RSV treatment not only prevented most of these delays but also led to the early onset of certain milestones and improved morphometric characteristics in the offspring. In summary, our findings suggest that RSV may have potential as a therapeutic intervention to protect against the negative effects of prenatal VPA exposure, highlighting its importance in future studies of prenatal neurodevelopmental disorders.

GABAergic synaptic transmission and cortical oscillation patterns in the primary somatosensory area of a valproic acid rat model of autism spectrum disorder.

Autism spectrum disorder (ASD) is characterized by impaired social communication and interaction associated with repetitive or stereotyped behaviour. Prenatal valproic acid (VPA) exposure in rodents is a commonly used model of ASD. Resveratrol (RSV) has been shown to prevent interneuronal and behavioural impairments in the VPA model. We investigated the effects of prenatal VPA exposure and RSV on the GABAergic synaptic transmission, brain oscillations and on the genic expression of interneuron-associated transcription factor LHX6 in the primary somatosensory area (PSSA). Prenatal VPA exposure decreased the sIPSC and mIPSC frequencies and the sIPSC decay kinetics onto layers 4/5 pyramidal cells of PSSA. About 40% of VPA animals exhibited absence-like spike-wave discharge (SWD) events associated with behaviour arrest and increased power spectrum density of delta, beta and gamma cortical oscillations. VPA animals had reduced LHX6 expression in PSSA, but VPA animals treated with RSV had no changes on synaptic inhibition or LHX6 expression in the PSSA. SWD events associated with behaviour arrest and the abnormal increment of cortical oscillations were also absent in VPA animals treated with RSV. These findings provide new venues to investigate the role of both RSV and VPA in the pathophysiology of ASD and highlight the VPA animal model as an interesting tool to investigate pathways related to the aetiology and possible future therapies to this neuropsychiatric disorder.

Resveratrol Treatment of Autism Spectrum Disorder-A Pilot Study.

OBJECTIVES: Considering autism spectrum disorder (ASD) as a neurodevelopmental condition associated with immune system impairments, we aimed to evaluate the potential benefits, efficacy, tolerability, and safety of the anti-inflammatory, antioxidant, and neuroprotective trans -resveratrol (RSV) in behavioral impairments and in a set of 8 microRNAs (miR) related to the immune system in pediatric subjects with ASD. METHODS: This is an open-label pilot trial over a 3 months (90 days) study follow-up period designed to assess the effect of 200 mg/d RSV on 5 boys aged 10 to 13 (11.8 ± 1.1) years diagnosed with ASD according to Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition . RESULTS: The RSV treatment significantly reduced the Aberrant Behavior Checklist total score ( P = 0.042) and Irritability ( P = 0.041), with no alteration in Stereotypical Behavior ( P = 0.066), Hyperactivity ( P = 0.068), and Lethargy/Social Withdrawal ( P = 0.078) subscales. On the Clinical Global Impression scale, 3 individuals showed marked improvement in behavior; one showed mild improvement, and the other had no changes. The RSV treatment increased the miR-195-5p ( P = 0.043), an important modulator of targets related to inflammatory and immunological pathways. RSV administration did not present adverse effects and did not alter clinical laboratory results. CONCLUSIONS: RSV is a safe molecule for administrating in the pediatric population, able to modulate behavior alterations and molecules associated with the immune system, becoming a promising therapeutic strategy for large-scale studies in ASD, to investigate both behavioral and molecular approaches.

Usability evaluation of circRNA identification tools: Development of a heuristic-based framework and analysis.

BACKGROUND AND OBJECTIVE: Circular RNAs (circRNAs) are endogenous molecules of non-coding RNA that form a covalently closed loop at the 3' and 5' ends. Recently, the role of these molecules in the regulation of gene expression and their involvement in several human pathologies has gained notoriety. The identification of circRNAs is highly dependent on computational methods for analyzing RNA sequencing data. However, bioinformatics software is known to be problematic in terms of usability. Evidence points out that tools for identifying circRNAs can have such problems, negatively impacting researchers in this field. Here we present a heuristic-based framework for evaluating the usability of command-line circRNA identification software. METHODS: We used heuristics evaluation to comprehensively identify the usability issues in a sample of circRNA identification tools. RESULTS: We identified 46 usability issues presented individually in four tools. Most of the issues had cosmetic or minor severity. These are unlikely to challenge experienced users but may cause inconvenience for novice users. We also identified severe issues with the potential to harm users regardless of their experience. The areas most affected were the documentation and the installability of the tools. CONCLUSIONS: With the proposed framework, we formally describe, for the first time, the usability problems that can affect users in this area of circRNA research. We hope that our framework can help researchers evaluate their software's usability during development.

Resveratrol Prevents Cytoarchitectural and Interneuronal Alterations in the Valproic Acid Rat Model of Autism.

Autism spectrum disorder (ASD) is a prevalent neurodevelopmental disorder characterized by several alterations, including disorganized brain cytoarchitecture and excitatory/inhibitory (E/I) imbalance. We aimed to analyze aspects associated with the inhibitory components in ASD, using bioinformatics to develop notions about embryonic life and tissue analysis for postnatal life. We analyzed microarray and RNAseq datasets of embryos from different ASD models, demonstrating that regions involved in neuronal development are affected. We evaluated the effect of prenatal treatment with resveratrol (RSV) on the neuronal organization and quantity of parvalbumin-positive (PV+), somatostatin-positive (SOM+), and calbindin-positive (CB+) GABAergic interneurons, besides the levels of synaptic proteins and GABA receptors in the medial prefrontal cortex (mPFC) and hippocampus (HC) of the ASD model induced by valproic acid (VPA). VPA increased the total number of neurons in the mPFC, while it reduced the number of SOM+ neurons, as well as the proportion of SOM+, PV+, and CB+ neurons (subregion-specific manner), with preventive effects of RSV. In summary, metabolic alterations or gene expression impairments could be induced by VPA, leading to extensive damage in the late developmental stages. By contrast, due to its antioxidant, neuroprotective, and opposite action on histone properties, RSV may avoid damages induced by VPA.

The role of T-cells in neurobehavioural development: Insights from the immunodeficient nude mice.

Mice homozygous for the nude mutation (Foxn1nu) are hairless and exhibit congenital dysgenesis of the thymic epithelium, resulting in a primary immunodeficiency of mature T-cells, and have been used for decades in research with tumour grafts. Early studies have already demonstrated social behaviour impairments and central nervous system (CNS) alterations in these animals, but did not address the complex interplay between CNS, immune system and behavioural alterations. Here we investigate the impact of T-cell immunodeficiency on behaviours relevant to the study of neurodevelopmental and neuropsychiatric disorders. Moreover, we aimed to characterise in a multidisciplinary manner the alterations related to those findings, through evaluation of the excitatory/inhibitory synaptic proteins, cytokines expression and biological spectrum signature of different biomolecules in nude mice CNS. We demonstrate that BALB/c nude mice display sociability impairments, a complex pattern of repetitive behaviours and higher sensitivity to thermal nociception. These animals also have a reduced IFN-γ gene expression in the prefrontal cortex and an absence of T-cells in meningeal tissue, both known modulators of social behaviour. Furthermore, excitatory synaptic protein PSD-95 immunoreactivity was also reduced in the prefrontal cortex, suggesting an intricate involvement of social behaviour related mechanisms. Lastly, employing biospectroscopy analysis, we have demonstrated that BALB/c nude mice have a different CNS spectrochemical signature compared to their heterozygous littermates. Altogether, our results show a comprehensive behavioural analysis of BALB/c nude mice and potential neuroimmunological influences involved with the observed alterations.

Transcription factors in neurodevelopmental and associated psychiatric disorders: A potential convergence for genetic and environmental risk factors.

Neurodevelopmental disorders (NDDs) are a heterogeneous and highly prevalent group of psychiatric conditions marked by impairments in the nervous system. Their onset occurs during gestation, and the alterations are observed throughout the postnatal life. Although many genetic and environmental risk factors have been described in this context, the interactions between them challenge the understanding of the pathways associated with NDDs. Transcription factors (TFs)-a group of over 1,600 proteins that can interact with DNA, regulating gene expression through modulation of RNA synthesis-represent a point of convergence for different risk factors. In addition, TFs organize critical processes like angiogenesis, blood-brain barrier formation, myelination, neuronal migration, immune activation, and many others in a time and location-dependent way. In this review, we summarize important TF alterations in NDD and associated disorders, along with specific impairments observed in animal models, and, finally, establish hypotheses to explain how these proteins may be critical mediators in the context of genome-environment interactions.

Resveratrol prevents brain edema, blood-brain barrier permeability, and altered aquaporin profile in autism animal model.

Autism spectrum disorder can present a plethora of clinical conditions associated with the disorder, such as greater brain volume in the first years of life in a significant percentage of patients. We aimed to evaluate the brain water content, the blood-brain barrier permeability, and the expression of aquaporin 1 and 4, and GFAP in a valproic acid-animal model, assessing the effect of resveratrol. On postnatal day 30, Wistar rats of the valproic acid group showed greater permeability of the blood-brain barrier to the Evans blue dye and a higher proportion of brain water volume, prevented both by resveratrol. Prenatal exposition to valproic acid diminished aquaporin 1 in the choroid plexus, in the primary somatosensory area, in the amygdala region, and in the medial prefrontal cortex, reduced aquaporin 4 in medial prefrontal cortex and increased aquaporin 4 levels in primary somatosensory area (with resveratrol prevention). Valproic acid exposition also increased the number of astrocytes and GFAP fluorescence in both primary somatosensory area and medial prefrontal cortex. In medial prefrontal cortex, resveratrol prevented the increased fluorescence. Finally, there was an effect of resveratrol per se on the number of astrocytes and GFAP fluorescence in the amygdala region and in the hippocampus. Thus, this work demonstrates significant changes in blood-brain barrier permeability, edema formation, distribution of aquaporin 1 and 4, in addition to astrocytes profile in the animal model of autism, as well as the use of resveratrol as a tool to investigate the mechanisms involved in the pathophysiology of autism spectrum disorder.

Resveratrol prevents long-term structural hippocampal alterations and modulates interneuron organization in an animal model of ASD.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by impairments in both communication and social interaction, besides repetitive or stereotyped behavior. Although the etiology is unknown, environmental factors such as valproic acid (VPA) increase the risk of ASD onset. Resveratrol (RSV), a neuroprotective molecule, has been shown to counteract the effects of intrauterine exposure to VPA. We aimed to evaluate histological parameters related to hippocampal morphology and to the distribution of parvalbumin- (PV), calbindin- (CB), and somatostatin-positive (SOM) interneurons sub-populations, in addition to evaluate the total/phosphorylation levels of PTEN, AKT, GSK3ß and total CK2 in the animal model of autism induced by VPA, as well as addressing the potential protective effect of RSV. On postnatal day 120, histological analysis showed a loss in total neurons in the dentate gyrus (DG) and decreased CB+ neurons in DG and CA1 in VPA animals, both prevented by RSV. In addition, PV+ neurons were diminished in CA1, CA2, and CA3, and SOM+ were interestingly increased in DG (prevented by RSV) and decreased in CA1 and CA2. A hippocampal lesion similar to sclerosis was also observed in the samples from the VPA group. Besides that, VPA reduced AKT and PTEN immunocontent, and VPA increased CK2 immunocontent. Thus, this work demonstrated long-term effects of prenatal exposure to ASD in different sub-populations of interneurons, structural damage of hippocampus, and also alteration in proteins associated with pivotal cell signaling pathways, highlighting the role of RSV as a tool for understanding the pathophysiology of ASD.

Effects of single-dose antipurinergic therapy on behavioral and molecular alterations in the valproic acid-induced animal model of autism.

Autism spectrum disorder (ASD) is characterized by deficits in communication and social interaction, restricted interests, and stereotyped behavior. Environmental factors, such as prenatal exposure to valproic acid (VPA), may contribute to the increased risk of ASD. Since disturbed functioning of the purinergic signaling system has been associated with the onset of ASD and used as a potential therapeutic target for ASD in both clinical and preclinical studies, we analyzed the effects of suramin, a non-selective purinergic antagonist, on behavioral, molecular and immunological in an animal model of autism induced by prenatal exposure to VPA. Treatment with suramin (20 mg/kg, intraperitoneal) restored sociability in the three-chamber apparatus and decreased anxiety measured by elevated plus maze apparatus, but had no impact on decreased reciprocal social interactions or higher nociceptive threshold in VPA rats. Suramin treatment did not affect VPA-induced upregulation of P2X4 and P2Y2 receptor expression in the hippocampus, and P2X4 receptor expression in the medial prefrontal cortex, but normalized an increased level of interleukin 6 (IL-6). Our results suggest an important role of purinergic signaling modulation in behavioral, molecular, and immunological aberrations described in VPA model, and indicate that the purinergic signaling system might be a potential target for pharmacotherapy in preclinical studies of ASD.

Abnormal empathy-like pro-social behaviour in the valproic acid model of autism spectrum disorder.

Impairments in social behaviour are a defining feature of autism spectrum disorder (ASD). Individuals with ASD also usually present some difficulty to recognize or understand another person's feelings. Therefore, it is possible that altered empathy processing could hinder typical social interaction in ASD. Recently, robust paradigms confirmed that rodents show primordial forms of empathy-like behaviour. Therefore, in this work, we used one of these new protocols to test pro-social behaviour in the rat model of autism induced by Valproic Acid (VPA). We also evaluated possible beneficial effects of Resveratrol, since it can prevent social deficits in the VPA model. Rats were tested on their ability to open a restrainer to release a trapped conspecific. Exposure to VPA precludes the timely manifestation of this empathy-like behaviour, but does not affect its continuation after its first expression. We also found a significant correlation between average speed during the first day of test and becoming an Opener. Similarly, rats able to open the restrainer on the first day had an increased likelihood of repeating this behaviour in the later days of the testing programme. We did not find any protective effects of Resveratrol. Further investigation of empathy-like behaviour in the VPA model and in other models of autism could help to clarify the behavioural and neural processes underpinning the basic aspects of empathy alterations in autistic individuals.

Reduced CD4 T Lymphocytes in Lymph Nodes of the Mouse Model of Autism Induced by Valproic Acid.

OBJECTIVE: Considering the potential role of lymphocytes in the pathophysiology of autism spectrum disorder (ASD), we aimed to evaluate possible alterations of T cell pools in the lymphoid organs of an animal model of autism induced by valproic acid (VPA). Pregnant Swiss mice received a single intraperitoneal injection of 600 mg/kg of VPA (VPA group) or saline (control group) on day 11 of gestation. Male offspring were euthanized on postnatal day 60 for removal of thy-muses, spleens, and a pool of inguinal, axillary and brachial lymph nodes. Cellularity was evaluated, and flow cytometry analysis was performed on cell suspensions incubated with the mouse antibodies anti-CD3-FITC, anti-CD4-PE, and anti-CD8-PE-Cy7. We observed that the prenatal exposure to VPA induced a reduction in the numbers of CD3+CD4+ T cells in their lymph nodes when compared to the control animals. This was specific since it was not seen in the thymus or spleen. The consistent decrease in the number of CD4+ T cells in subcutaneous lymph nodes of mice from the animal model of autism may be related to the allergic symptoms frequently observed in ASD. Further research is necessary to characterize the immunological patterns in ASD and the connection with the pathophysiology of this disorder.

Neuroimmune Alterations in Autism: A Translational Analysis Focusing on the Animal Model of Autism Induced by Prenatal Exposure to Valproic Acid.

Autism spectrum disorder (ASD) is a highly prevalent developmental disorder characterized by deficits in communication and social interaction and in stereotyped or repetitive behaviors. Besides the classical behavioral dyad, several comorbidities are frequently present in patients with ASD, such as anxiety, epilepsy, sleep disturbances, and gastrointestinal tract dysfunction. Although the etiology of ASD remains unclear, there is supporting evidence for the involvement of both genetic and environmental factors. Valproic acid (VPA) is an anticonvulsant and mood stabilizer that, when used during the gestational period, increases the risk of ASD in the offspring. The animal model of autism induced by prenatal exposure to VPA demonstrates important structural and behavioral features that can be observed in individuals with autism; it is thus an excellent tool for testing new drug targets and developing novel behavioral and drug therapies. In addition, immunological alterations during pregnancy could affect the developing embryo because immune molecules can pass through the placental barrier. In fact, exposure to pathogens during the pregnancy is a known risk factor for ASD, and maternal immune activation can lead to autistic-like features in animals. Interestingly, neuroimmune alterations are common in both autistic individuals and in animal models of ASD. We summarize here the important alterations in inflammatory markers, such as cytokines and chemokines, in patients with ASD and in the VPA animal model.

Intracellular Pathogen Infections and Immune Response in Autism.

BACKGROUND/AIMS: Perinatal exposure to infections during critical developmental periods is a promising area of study in autism spectrum disorder (ASD). Epidemiological data has highlighted this relationship, pointing out significant correlations between perinatal exposure to pathogens and the occurrence of ASD. The aim of this review is to critically examine the present state of the art on intracellular pathogenic infection during pregnancy and postnatally, pointing out possible correlations with the development of ASD. METHODS: We reviewed and collected studies concerning potential associations between intracellular pathogens like viral, bacterial, and parasite infection and the risk of ASD. RESULTS: We included 14 publications, considering bacterial and/or viral infection that demonstrated the potential to trigger ASD. Nine case-control studies were included and 5 of them reported an association between infections and ASD. One of the 2 cohorts investigated demonstrated that maternal infection increased the risk of ASD in the offspring. Three cross-sectional studies demonstrated that ASD patients presented with chronic infections and active neuroinflammatory processes. Most of the reports suggest inflammatory response as a common factor, and interleukin 6 appears to be a key-player in this process. CONCLUSION: The immune responses generated by organisms that cause perinatal maternal infection, i.e., bacteria, viruses, or parasites, have been associated with the development of autism in offspring. Physiological changes transmitted from the mother during chronic or acute inflammation should be further investigated so that modulatory preventive measures can be developed.

Data on social transmission of food preference in a model of autism induced by valproic acid and translational analysis of circulating microRNA.

This article contains data of Social Transmission of Food Preference in an animal model of autism and the evaluation of a set of microRNA analyzed in autistic patients and animal model of autism. The analyses of the absolute consumption of two flavored food by male rats prenatally exposed to valproic acid (VPA) and treated with resveratrol (RSV), showed that VPA animals show a trend to eat less of the flavored food presented by a demonstrator rat. We also identified 13 microRNA with similar levels among rodents' experimental groups, as well as 11 microRNA with no alterations between autistic and control subjects. Further evaluation of mechanisms of VPA and RSV actions on behavioral and molecular alterations can shed light in important biomarkers and etiological triggers of autistic spectrum disorders.

Resveratrol Prevents Cellular and Behavioral Sensory Alterations in the Animal Model of Autism Induced by Valproic Acid.

Autism spectrum disorder (ASD) is characterized by impairments in both social communication and interaction and repetitive or stereotyped behaviors. Although its etiology remains unknown, genetic and environmental risk factors have been associated with this disorder, including the exposure to valproic acid (VPA) during pregnancy. Resveratrol (RSV) is an anti-inflammatory and antioxidant molecule known to prevent social impairments in the VPA animal model of autism. This study aimed to analyze the effects of prenatal exposure to VPA, as well as possible preventive effects of RSV, on sensory behavior, the localization of GABAergic parvalbumin (PV+) neurons in sensory brain regions and the expression of proteins of excitatory and inhibitory synapses. Pregnant rats were treated daily with RSV (3.6 mg/kg) from E6.5 to E18.5 and injected with VPA (600 mg/kg) in the E12.5. Male pups were analyzed in Nest Seeking (NS) behavior and in whisker nuisance task (WNT). At P30, the tissues were removed and analyzed by immunofluorescence and western blotting. Our data showed for the first time an altered localization of PV+-neurons in primary sensory cortex and amygdala. We also showed a reduced level of gephyrin in the primary somatosensory area (PSSA) of VPA animals. The treatment with RSV prevented all the aforementioned alterations triggered by VPA. Our data shed light on the relevance of sensory component in ASD and highlights the interplay between RSV and VPA animal model as an important tool to investigate the pathophysiology of ASD.

Sex Differences and Estrous Cycle Changes in Synaptic Plasticity-related microRNA in the Rat Medial Amygdala.

The posterodorsal medial amygdala (MePD) is a sex steroid-sensitive and sexually dimorphic subcortical area that dynamically modulates social behaviors in rats. As different microRNA (miRNA) can act as post-transcriptional regulators of synaptic processing, we addressed changes that occur in miRNA expression in the MePD of males and females along the estrous cycle. The expression of miR25-3p, miR132-3p, miR138-5p, miR181a-5p, miR195-5p, and miR199a-5p, involved in neuronal cytoskeleton remodeling and synaptic plasticity, were evaluated by RT-qPCR. We found that the expression of miR138-5p was higher in males than in females along the different phases of the estrous cycle. Males also showed higher levels of miR-181a when compared to females in diestrus and estrus. On the other hand, when compared to females in proestrus, males presented lower levels of miR132-3p and miR199a-5p. The expression of miR25-3p was higher in diestrus females than in proestrus females. In addition, diestrus females showed higher values of miR25-3p, miR181a-5p, and miR195-5p when compared to estrus females. These miRNA expression profiles indicate a variable and fine-tuned protein regulation in the adult MePD. It is likely that these miRNA can be involved in structural and functional synaptic features and plasticity characteristic of males and cycling females and for the MePD regulation of mammalian reproduction.