Chronically high stress hormone levels dysregulate sperm long noncoding RNAs and their embryonic microinjection alters development and affective behaviours.

Previous studies on paternal epigenetic inheritance have shown that sperm RNAs play a role in this type of inheritance. The microinjection of sperm small noncoding RNAs into fertilised mouse oocytes induces reprogramming of the early embryo, which is thought to be responsible for the differences observed in adult phenotype. While sperm long noncoding RNAs (lncRNAs) have also been investigated in a previous study, their microinjection into fertilised oocytes did not yield conclusive results regarding their role in modulating brain development and adult behavioural phenotypes. Therefore, in the current study we sought to investigate this further. We used our previously established paternal corticosterone (stress hormone) model to assess sperm lncRNA expression using CaptureSeq, a sequencing technique that is more sensitive than the ones used in other studies in the field. Paternal corticosterone exposure led to dysregulation of sperm long noncoding RNA expression, which encompassed lncRNAs, circular RNAs and transposable element transcripts. Although they have limited functional annotation, bioinformatic approaches indicated the potential of these lncRNAs in regulating brain development and function. We then separated and isolated the sperm lncRNAs and performed microinjections into fertilised oocytes, to generate embryos with modulated lncRNA populations. We observed that the resulting adult offspring had lower body weight and altered anxiety and affective behavioural responses, demonstrating roles for lncRNAs in modulating development and brain function. This study provides novel insights into the roles of lncRNAs in epigenetic inheritance, including impacts on brain development and behaviours of relevance to affective disorders.

Experience-dependent neural plasticity, learning, and memory in the era of epitranscriptomics.

In this short review, we highlight recent findings in the emerging field of epitranscriptomic mechanisms and discuss their potential role in neural plasticity, learning and memory. These include the influence of RNA modifications on activity-induced RNA structure states, RNA editing and RNA localization, and how qualitative state changes in RNA increase the functional diversity and information-carrying capacity of RNA molecules. We predict that RNA modifications may be just as important for synaptic plasticity and memory as quantitative changes in transcript and protein abundance, but with the added advantage of not being required to signal back to the nucleus, and therefore better suited to be coordinated with the temporal dynamics of learning.

Persistent histone modifications at the BDNF and Cdk-5 promoters following extinction of nicotine-seeking in rats.

Drugs of addiction lead to a wide range of epigenetic changes at the promoter regions of genes directly implicated in learning and memory processes. We have previously shown that the histone deactylase inhibitor, sodium butyrate (NaB), accelerates the extinction of nicotine-seeking and provides resistance to relapse. Here, we explore the potential molecular mechanisms underlying this effect. Rats received intravenous nicotine or saline self-administration, followed by 6 days of extinction training, with each extinction session followed immediately by treatment with NaB or vehicle. On the last day of extinction, rats were killed and the medial ventral prefrontal cortex retained for chromatin immunoprecipitation and quantitative polymerase chain reaction (qPCR). A history of nicotine exposure significantly decreased H3K14 acetylation at the brain-derived neurotrophic factor (BDNF) exon IV promoter, and this effect was abolished with NaB treatment. In contrast, nicotine self-administration alone, resulted in a significant decrease in histone methylation at the H3K27me3 and H3K9me2 marks in the promoter regions of BDNF exon IV and cyclin-dependent kinase 5 (Cdk-5). Quantitative PCR-identified changes in several genes associated with NaB treatment that were independent of nicotine exposure; however, an interaction of nicotine history and NaB treatment was detected only in the expression of BDNF IV and BDNF IX. Together these results suggest that nicotine self-administration leads to a number of epigenetic changes at both the BDNF and Cdk-5 promoters, and that these changes may contribute to the enhanced extinction of nicotine-seeking by NaB.

Elevated paternal glucocorticoid exposure alters the small noncoding RNA profile in sperm and modifies anxiety and depressive phenotypes in the offspring.

Recent studies have suggested that physiological and behavioral traits may be transgenerationally inherited through the paternal lineage, possibly via non-genomic signals derived from the sperm. To investigate how paternal stress might influence offspring behavioral phenotypes, a model of hypothalamic-pituitary-adrenal (HPA) axis dysregulation was used. Male breeders were administered water supplemented with corticosterone (CORT) for 4 weeks before mating with untreated female mice. Female, but not male, F1 offspring of CORT-treated fathers displayed altered fear extinction at 2 weeks of age. Only male F1 offspring exhibited altered patterns of ultrasonic vocalization at postnatal day 3 and, as adults, showed decreased time in open on the elevated-plus maze and time in light on the light-dark apparatus, suggesting a hyperanxiety-like behavioral phenotype due to paternal CORT treatment. Interestingly, expression of the paternally imprinted gene Igf2 was increased in the hippocampus of F1 male offspring but downregulated in female offspring. Male and female F2 offspring displayed increased time spent in the open arm of the elevated-plus maze, suggesting lower levels of anxiety compared with control animals. Only male F2 offspring showed increased immobility time on the forced-swim test and increased latency to feed on the novelty-supressed feeding test, suggesting a depression-like phenotype in these animals. Collectively, these data provide evidence that paternal CORT treatment alters anxiety and depression-related behaviors across multiple generations. Analysis of the small RNA profile in sperm from CORT-treated males revealed marked effects on the expression of small noncoding RNAs. Sperm from CORT-treated males contained elevated levels of three microRNAs, miR-98, miR-144 and miR-190b, which are predicted to interact with multiple growth factors, including Igf2 and Bdnf. Sustained elevation of glucocorticoids is therefore involved in the transmission of paternal stress-induced traits across generations in a process involving small noncoding RNA signals transmitted by the male germline.

Methyl CpG binding domain ultra-sequencing: a novel method for identifying inter-individual and cell-type-specific variation in DNA methylation.

Experience-dependent changes in DNA methylation can exert profound effects on neuronal function and behaviour. A single learning event can induce a variety of DNA modifications within the neuronal genome, some of which may be common to all individuals experiencing the event, whereas others may occur in a subset of individuals. Variations in experience-induced DNA methylation may subsequently confer increased vulnerability or resilience to the development of neuropsychiatric disorders. However, the detection of experience-dependent changes in DNA methylation in the brain has been hindered by the interrogation of heterogeneous cell populations, regional differences in epigenetic states and the use of pooled tissue obtained from multiple individuals. Methyl CpG Binding Domain Ultra-Sequencing (MBD Ultra-Seq) overcomes current limitations on genome-wide epigenetic profiling by incorporating fluorescence-activated cell sorting and sample-specific barcoding to examine cell-type-specific CpG methylation in discrete brain regions of individuals. We demonstrate the value of this method by characterizing differences in 5-methylcytosine (5mC) in neurons and non-neurons of the ventromedial prefrontal cortex of individual adult C57BL/6 mice, using as little as 50 ng of genomic DNA per sample. We find that the neuronal methylome is characterized by greater CpG methylation as well as the enrichment of 5mC within intergenic loci. In conclusion, MBD Ultra-Seq is a robust method for detecting DNA methylation in neurons derived from discrete brain regions of individual animals. This protocol will facilitate the detection of experience-dependent changes in DNA methylation in a variety of behavioural paradigms and help identify aberrant experience-induced DNA methylation that may underlie risk and resiliency to neuropsychiatric disease.

The long non-coding RNA Gomafu is acutely regulated in response to neuronal activation and involved in schizophrenia-associated alternative splicing.

Schizophrenia (SZ) is a complex disease characterized by impaired neuronal functioning. Although defective alternative splicing has been linked to SZ, the molecular mechanisms responsible are unknown. Additionally, there is limited understanding of the early transcriptomic responses to neuronal activation. Here, we profile these transcriptomic responses and show that long non-coding RNAs (lncRNAs) are dynamically regulated by neuronal activation, including acute downregulation of the lncRNA Gomafu, previously implicated in brain and retinal development. Moreover, we demonstrate that Gomafu binds directly to the splicing factors QKI and SRSF1 (serine/arginine-rich splicing factor 1) and dysregulation of Gomafu leads to alternative splicing patterns that resemble those observed in SZ for the archetypal SZ-associated genes DISC1 and ERBB4. Finally, we show that Gomafu is downregulated in post-mortem cortical gray matter from the superior temporal gyrus in SZ. These results functionally link activity-regulated lncRNAs and alternative splicing in neuronal function and suggest that their dysregulation may contribute to neurological disorders.

Paternal deprivation during infancy results in dendrite- and time-specific changes of dendritic development and spine formation in the orbitofrontal cortex of the biparental rodent Octodon degus.

The aim of this study in the biparental rodent Octodon degus was to assess the impact of paternal deprivation on neuronal and synaptic development in the orbitofrontal cortex, a prefrontal region which is essential for emotional and cognitive function. On the behavioral level the quantitative comparison of parental behaviors in biparental and single-mother families revealed that (i) degu fathers significantly participate in parental care and (ii) single-mothers do not increase their maternal care to compensate the lack of paternal care. On the brain structural level we show in three-week-old father-deprived animals that layer II/III pyramidal neurons in the orbitofrontal cortex displayed significantly lower spine densities on apical and basal dendrites. Whereas biparentally raised animals have reached adult spine density values at postnatal day 21, fatherless animals seem "to catch up" by a delayed increase of spine density until reaching similar values as biparentally raised animals in adulthood. However, in adulthood reduced apical spine numbers together with shorter apical dendrites were observed in father-deprived animals, which indicates that dendritic growth and synapse formation (seen in biparental animals between postnatal day 21 and adulthood) were significantly suppressed. These results demonstrate that paternal deprivation delays and partly suppresses the development of orbitofrontal circuits. The retarded dendritic and synaptic development of the apical dendrites of layer II/III pyramidal neurons in the orbitofrontal cortex of adult fatherless animals may reflect a reduced excitatory connectivity of this cortical subregion.

Partial reversal of the effect of maternal care on cognitive function through environmental enrichment.

Maternal care influences hippocampal development in the rat. The offspring of mothers that exhibit increased levels of pup licking/grooming and arched-back nursing (High LG-ABN mothers) show increased hippocampal N-methyl-D-aspartate (NMDA) receptor binding and enhanced hippocampal-dependent spatial learning. In these studies we examined whether environmental enrichment from days 22-70 of life might reverse the effects of low maternal care. Environmental enrichment eliminated the differences between the offspring of High and Low LG-ABN mothers in both Morris water maze learning and object recognition. However, enrichment did not reverse the effect of maternal care on long-term potentiation in the dentate gyrus or on hippocampal NMDA receptor binding. In contrast, peripubertal enrichment did reverse the effects of maternal care on hippocampal alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor binding. These findings provide evidence for the reversal of the effects of reduced maternal investment in early life on cognitive function in adulthood. Such effects might involve compensatory changes associated with peripubertal enrichment.