The amphibian disease chytridiomycosis in amphibians is caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd) and has resulted in dramatic declines and extinctions of amphibian populations worldwide. A hypervirulent, globally-dispersed pandemic lineage (Bd-GPL) is thought to be largely responsible for population declines and extinctions, although numerous endemic lineages have also been found. Recent reports of amphibian declines have been linked to the emergence of Bd in Cameroon, a major hotspot of African amphibian diversity. However, it is not known whether Bd-GPL or other lineages have been found in this region. This study therefore aims to examine Bd lineage diversity in the region and predict the distribution of this pathogen under current and future climate conditions using data from this study and from historical records. Almost 15% (52/360) of individuals tested positive for Bd using a standard quantitative PCR diagnostic. Infected amphibians were found at all eight sites sampled in this study. Species distribution models generated in BIOMOD2 indicate that areas with highest predicted environmental suitability occur in the Cameroon highlands and several protected areas throughout the country. These areas of high environmental suitability for Bd are projected to shift or decrease in size under future climate change. However, montane regions with high amphibian diversity are predicted to remain highly suitable. Phylogenetic analysis of the ITS sequences obtained from a set of positive Bd samples indicate that most fall within the Bd-GPL lineage while the remainder group with isolates from either Brazil or South Korea. Although more in depth phylogenetic analyses are needed, identification of Bd-GPL lineages in areas of high amphibian diversity emphasizes the need to continue to monitor for Bd and develop appropriate conservation strategies to prevent its further spread.

Memories associated with drug use can trigger strong motivation for the drug, which increases relapse vulnerability in substance use disorder (SUD). Currently there are no treatments for relapse to abuse of psychostimulants, such as methamphetamine (METH). We previously reported that storage of memories associated with METH, but not those for fear or food reward, and the concomitant spine density increase are disrupted in a retrieval-independent manner by depolymerizing actin in the basolateral amygdala complex (BLC) of adult male rats and mice. Similar results are achieved in males through intra-BLC or systemic inhibition of nonmuscle myosin II (NMII), a molecular motor that directly drives actin polymerization. Given the substantial differences in physiology between genders, we sought to determine if this immediate and selective disruption of METH-associated memory extends to adult females. A single intra-BLC infusion of the NMII inhibitor Blebbistatin (Blebb) produced a long-lasting disruption of context-induced drug seeking for at least 30days in female rats that mirrored our prior results in males. Furthermore, a single systemic injection of Blebb prior to testing disrupted METH-associated memory and the concomitant increase in BLC spine density in females. Importantly, as in males, the same manipulation had no effect on an auditory fear memory or associated BLC spine density. In addition, we established that the NMII-based disruption of METH-associated memory extends to both male and female adolescents. These findings provide further support that small molecular inhibitors of NMII have strong therapeutic potential for the prevention of relapse to METH abuse triggered by associative memories.

Epigenetic and transcriptional mechanisms have been shown to contribute to long-lasting functional changes in adult neurons. The purpose of this study was to identify any such modifications in diseased retinal tissues from a mouse model of rhodopsin mutation-associated autosomal dominant retinitis pigmentosa (ADRP), Q344X, relative to age-matched wild-type (WT) controls.

In addition to cognitive impairments, neurodevelopmental disorders often result in sensory processing deficits. However, the biological mechanisms that underlie impaired sensory processing associated with neurodevelopmental disorders are generally understudied and poorly understood. We found that SYNGAP1 haploinsufficiency in humans, which causes a sporadic neurodevelopmental disorder defined by cognitive impairment, autistic features, and epilepsy, also leads to deficits in tactile-related sensory processing. In vivo neurophysiological analysis in Syngap1 mouse models revealed that upper-lamina neurons in somatosensory cortex weakly encode information related to touch. This was caused by reduced synaptic connectivity and impaired intrinsic excitability within upper-lamina somatosensory cortex neurons. These results were unexpected, given that Syngap1 heterozygosity is known to cause circuit hyperexcitability in brain areas more directly linked to cognitive functions. Thus, Syngap1 heterozygosity causes a range of circuit-specific pathologies, including reduced activity within cortical neurons required for touch processing, which may contribute to sensory phenotypes observed in patients.

Cytokinesis is the last step of mitotic cell division that separates the cytoplasm of dividing cells. Small molecule inhibitors targeting either the elements of the regulatory pathways controlling cytokinesis, or the terminal effectors have been of interest as potential drug candidates for the treatment of various diseases. Here we present a detailed protocol for a cell-based cytokinesis assay that can be used for the discovery of novel cytokinesis inhibitors. The assay is performed in a 96-well plate format in 48 h. Living cells, nuclei and nuclei of dead cells are identified by a single staining step using three fluorescent dyes, followed by rapid live cell imaging. The primary signal is the nuclei-to-cell ratio (NCR). In the presence of cytokinesis inhibitors, this ratio increases over time, as the ratio of multinucleated cells increases in the population. The ratio of dead nuclei to total nuclei provides a simultaneous measure of cytotoxicity. A screening window coefficient (Z`) of 0.65 indicates that the assay is suitable for screening purposes, as the positive and negative controls are well-separated. EC50 values can be reliably determined in a single 96-well plate by using only six different compound concentrations, enabling the testing of 4 compounds per plate. An excellent test-retest reliability (R 2 = 0.998) was found for EC50 values covering a ~1500-fold range of potencies. Established small molecule inhibitors of cytokinesis operating via direct action on actin dynamics or nonmuscle myosin II are used to demonstrate the robustness, simplicity and flexibility of the assay.

It remains unclear to what extent neurodevelopmental disorder (NDD) risk genes retain functions into adulthood and how they may influence disease phenotypes. SYNGAP1 haploinsufficiency causes a severe NDD defined by autistic traits, cognitive impairment, and epilepsy. To determine if this gene retains therapeutically-relevant biological functions into adulthood, we performed a gene restoration technique in a mouse model for SYNGAP1 haploinsufficiency. Adult restoration of SynGAP protein improved behavioral and electrophysiological measures of memory and seizure. This included the elimination of interictal events that worsened during sleep. These events may be a biomarker for generalized cortical dysfunction in SYNGAP1 disorders because they also worsened during sleep in the human patient population. We conclude that SynGAP protein retains biological functions throughout adulthood and that non-developmental functions may contribute to disease phenotypes. Thus, treatments that target debilitating aspects of severe NDDs, such as medically-refractory seizures and cognitive impairment, may be effective in adult patients.

Mounting evidence has established that diet-induced obesity (DIO) is associated with deficits in hippocampus-dependent memory. The bulk of research studies dealing with this topic have utilized rats fed a high-fat diet as an experimental model. To date, there has been a paucity of research studies that have established whether the memory deficits exhibited in DIO rats can be recapitulated in mice. Moreover, the majority of experiments that have evaluated memory performance in rodent models of DIO have utilized memory tests that are essentially aversive in nature (i.e., Morris water maze). The current study sought to fill an empirical void by determining if mice maintained on a high-fat diet exhibit deficits in two non-aversive memory paradigms: novel object recognition (NOR) and object location memory (OLM). Here we report that mice fed a high-fat diet over 23 weeks exhibit intact NOR, albeit a marked impairment in hippocampus-dependent OLM. We also determined the existence of corresponding aberrations in gene expression within the hippocampus of DIO mice. DIO mice exhibited significant reductions in both SIRT1 and PP1 mRNA within the hippocampus. Our data suggest that mice maintained on a high-fat diet present with impaired hippocampus-dependent spatial memory and a corresponding alteration in the expression of genes that have been implicated in memory consolidation.

Protease-activated receptor-1 (PAR1) is an unusual G-protein coupled receptor (GPCR) that is activated through proteolytic cleavage by extracellular serine proteases. Although previous work has shown that inhibiting PAR1 activation is neuroprotective in models of ischemia, traumatic injury, and neurotoxicity, surprisingly little is known about PAR1's contribution to normal brain function. Here, we used PAR1-/- mice to investigate the contribution of PAR1 function to memory formation and synaptic function. We demonstrate that PAR1-/- mice have deficits in hippocampus-dependent memory. We also show that while PAR1-/- mice have normal baseline synaptic transmission at Schaffer collateral-CA1 synapses, they exhibit severe deficits in N-methyl-d-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP). Mounting evidence indicates that activation of PAR1 leads to potentiation of NMDAR-mediated responses in CA1 pyramidal cells. Taken together, this evidence and our data suggest an important role for PAR1 function in NMDAR-dependent processes subserving memory formation and synaptic plasticity.

Previous studies have shown that beta amyloid (Abeta) peptide triggers the activation of several signal transduction cascades in the hippocampus, including the extracellular signal-regulated kinase (ERK) cascade. In this study we sought to characterize the cellular localization of phosphorylated, active ERK in organotypic hippocampal cultures after acute exposure to either Abeta (1-42) or nicotine.

DNA methylation is a covalent chemical modification of DNA catalyzed by DNA methyltransferases (DNMTs). DNA methylation is associated with transcriptional silencing and has been studied extensively as a lifelong molecular information storage mechanism put in place during development. Here we report that DNMT gene expression is upregulated in the adult rat hippocampus following contextual fear conditioning and that DNMT inhibition blocks memory formation. In addition, fear conditioning is associated with rapid methylation and transcriptional silencing of the memory suppressor gene PP1 and demethylation and transcriptional activation of the synaptic plasticity gene reelin, indicating both methyltransferase and demethylase activity during consolidation. DNMT inhibition prevents the PP1 methylation increase, resulting in aberrant transcription of the gene during the memory-consolidation period. These results demonstrate that DNA methylation is dynamically regulated in the adult nervous system and that this cellular mechanism is a crucial step in memory formation.

Using a hippocampus-dependent contextual threat learning and memory task, we report widespread, coordinated DNA methylation changes in CA1 hippocampus of Sprague-Dawley rats specific to threat learning at genes involved in synaptic transmission. Experience-dependent alternations in gene expression and DNA methylation were observed as early as 1 h following memory acquisition and became more pronounced after 24 h. Gene ontology analysis revealed significant enrichment of functional categories related to synaptic transmission in genes that were hypomethylated at 24 h following threat learning. Integration of these data sets with previously characterized epigenetic and transcriptional changes in brain disease states suggested significant overlap between genes regulated by memory formation and genes altered in memory-related neurological and neuropsychiatric diseases. These findings provide a comprehensive resource to aid in the identification of memory-relevant therapeutic targets. Our results shed new light on the gene expression and DNA methylation changes involved in memory formation, confirming that these processes are dynamic and experience-dependent. Finally, this work provides a roadmap for future studies to identify linkage of memory-associated genes to altered disease states.

The increased prevalence of neurodevelopmental disorders during the last half-century led us to investigate the potential for intergenerational detrimental neurodevelopmental effects of synthetic female gonadal hormones, typically used in contraceptive pills. We examined 3 separate cohorts of mice over the span of 2 years, a total of 150 female F0 mice and over 300 male and female rodents from their F1 progeny. We demonstrate that F1 male offsprings of female mice previously exposed to the synthetic estrogen 17α-ethinylestradiol (EE2) in combination with the synthetic progestin Norethindrone, exhibit neurodevelopmental and behavioral differences compared to control mice. Because the EE2 + Norethindrone administration resulted in gene expression changes in the exposed F0 mice ovaries persisting after the end of treatment, it is likely that the synthetic hormone treatment caused changes in the germline cells and that led to altered neurodevelopment in the offsprings. An altered gene expression pattern was discovered in the frontal cortex of male mice from the first offspring (F1.1) at infancy and an ADHD-like hyperactive locomotor behavior was exhibited in young male mice from the second offspring (F1.2) of female mice treated with contraceptive pill doses of EE2 + Norethindrone prior to pregnancy. The intergenerational neurodevelopmental effects of EE2 + Norethindrone treatment were sex specific, predominantly affecting males. Our observations in mice support the hypothesis that the use of synthetic contraceptive hormones is a potential environmental factor impacting the prevalence of human neurodevelopmental disorders. Additionally, our results indicate that contraceptive hormone drug safety assessments may need to be extended to F1 offspring.

Loss-of-function variants in SYNGAP1 cause a developmental encephalopathy defined by cognitive impairment, autistic features, and epilepsy. SYNGAP1 splicing leads to expression of distinct functional protein isoforms. Splicing imparts multiple cellular functions of SynGAP proteins through coding of distinct C-terminal motifs. However, it remains unknown how these different splice sequences function in vivo to regulate neuronal function and behavior. Reduced expression of SynGAP-α1/2 C-terminal splice variants in mice caused severe phenotypes, including reduced survival, impaired learning, and reduced seizure latency. In contrast, upregulation of α1/2 expression improved learning and increased seizure latency. Mice expressing α1-specific mutations, which disrupted SynGAP cellular functions without altering protein expression, promoted seizure, disrupted synapse plasticity, and impaired learning. These findings demonstrate that endogenous SynGAP isoforms with α1/2 spliced sequences promote cognitive function and impart seizure protection. Regulation of SynGAP-αexpression or function may be a viable therapeutic strategy to broadly improve cognitive function and mitigate seizure.

Stress profoundly impacts the brain and increases the risk of developing a psychiatric disorder. The brain's response to stress is mediated by a number of pathways that affect gene expression and protein function throughout the cell. Understanding how stress achieves such dramatic effects on the brain requires an understanding of the brain's stress response pathways. The majority of studies focused on molecular changes have employed repeated or chronic stress paradigms to assess the long-term consequences of stress and have not taken an integrative genomic and/or proteomic approach. Here, we determined the lasting impact of a single stressful event (restraint) on the broad molecular profile of the basolateral amygdala complex (BLC), a key brain region mediating emotion, memory and stress. Molecular profiling performed thirty days post-restraint consisted of small RNA sequencing, RNA sequencing and quantitative mass spectrometry and identified long-lasting changes in microRNA (miRNA), messenger RNA (mRNA) and proteins. Alignment of the three datasets further delineated the regulation of stress-specific pathways which were validated by qPCR and Western Blot analysis. From this analysis, mir-29a-5p was identified as a putative regulator of stress-induced adaptations in the BLC. Further, a number of predicted mir-29a-5p targets are regulated at the mRNA and protein level. The concerted and long-lasting disruption of multiple molecular pathways in the amygdala by a single stress event is expected to be sufficient to alter behavioral responses to a wide array of future experiences, including exposure to additional stressors.

Appetitive responses to weight loss are mediated by a nutrient-sensing neural network comprised of melanocortin neurons. The role of neural melanocortin-3 receptors (MC3R) in mediating these responses is enigmatic. Mc3r knockout mice exhibit a paradoxical phenotype of obesity and reduced feeding-related behaviors in situations of nutrient scarcity. Here we examined whether MC3Rs expressed in mesolimbic neurons regulate feeding-related motivational responses.

Human haploinsufficiency of the transcription factor Tcf4 leads to a rare autism spectrum disorder called Pitt-Hopkins syndrome (PTHS), which is associated with severe language impairment and development delay. Here, we demonstrate that Tcf4 haploinsufficient mice have deficits in social interaction, ultrasonic vocalization, prepulse inhibition, and spatial and associative learning and memory. Despite learning deficits, Tcf4(+/-) mice have enhanced long-term potentiation in the CA1 area of the hippocampus. In translationally oriented studies, we found that small-molecule HDAC inhibitors normalized hippocampal LTP and memory recall. A comprehensive set of next-generation sequencing experiments of hippocampal mRNA and methylated DNA isolated from Tcf4-deficient and WT mice before or shortly after experiential learning, with or without administration of vorinostat, identified "memory-associated" genes modulated by HDAC inhibition and dysregulated by Tcf4 haploinsufficiency. Finally, we observed that Hdac2 isoform-selective knockdown was sufficient to rescue memory deficits in Tcf4(+/-) mice.

Epigenetic mechanisms such as DNA methylation are essential regulators of the function and information storage capacity of neurons. DNA methylation is highly dynamic in the developing and adult brain, and is actively regulated by neuronal activity and behavioural experiences. However, it is presently unclear how methylation status at individual genes is targeted for modification. Here, we report that extra-coding RNAs (ecRNAs) interact with DNA methyltransferases and regulate neuronal DNA methylation. Expression of ecRNA species is associated with gene promoter hypomethylation, is altered by neuronal activity, and is overrepresented at genes involved in neuronal function. Knockdown of the Fos ecRNA locus results in gene hypermethylation and mRNA silencing, and hippocampal expression of Fos ecRNA is required for long-term fear memory formation in rats. These results suggest that ecRNAs are fundamental regulators of DNA methylation patterns in neuronal systems, and reveal a promising avenue for therapeutic targeting in neuropsychiatric disease states.

Apolipoprotein E receptor 2 (Apoer2), a member of the LDL receptor gene family, and its ligand Reelin control neuronal migration during brain development. Apoer2 is also essential for induction of long-term potentiation (LTP) in the adult brain. Here we show that Apoer2 is present in the postsynaptic densities of excitatory synapses where it forms a functional complex with NMDA receptors. Reelin signaling through Apoer2 markedly enhances LTP through a mechanism that requires the presence of amino acids encoded by an exon in the intracellular domain of Apoer2. This exon is alternatively spliced in an activity-dependent manner and is required for Reelin-induced tyrosine phosphorylation of NMDA receptor subunits. Mice constitutively lacking the exon perform poorly in learning and memory tasks. Thus, alternative splicing of Apoer2, a novel component of the NMDA receptor complex, controls the modulation of NMDA receptor activity, synaptic neurotransmission, and memory by Reelin.

Reward-related memories are essential for adaptive behavior and evolutionary fitness, but they are also a core component of maladaptive brain diseases such as addiction. Reward learning requires dopamine neurons located in the ventral tegmental area (VTA), which encode relationships between predictive cues and future rewards. Recent evidence suggests that epigenetic mechanisms, including DNA methylation, are essential regulators of neuronal plasticity and experience-driven behavioral change. However, the role of epigenetic mechanisms in reward learning is poorly understood. Here we show that the formation of reward-related associative memories in rats upregulates key plasticity genes in the VTA, which are correlated with memory strength and associated with gene-specific changes in DNA methylation. Moreover, DNA methylation in the VTA is required for the formation of stimulus-reward associations. These results provide the first evidence that that activity-dependent methylation and demethylation of DNA is an essential substrate for the behavioral and neuronal plasticity driven by reward-related experiences.

Transcriptional changes in the hippocampus are required for memory formation, and these changes are regulated by numerous post-translational modifications of chromatin-associated proteins. One of the epigenetic marks that has been implicated in memory formation is histone 3 lysine 4 trimethylation (H3K4me3), and this modification is found at the promoters of actively transcribed genes. The total levels of H3K4me3 are increased in the CA1 region of the hippocampus during memory formation, and genetic perturbation of the K4 methyltransferases and demethylases interferes with forming memories. Previous chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) analyses failed to detect changes in H3K4me3 levels at the promoters of memory-linked genes. Since the breadth of H3K4me3 marks was recently reported to be associated with the transcriptional outcome of a gene, we re-analyzed H3K4me3 ChIP-seq data sets to identify the role of H3K4me3 broad domains in CA1 neurons, as well as identify differences in breadth that occur during contextual fear conditioning. We found that, under baseline conditions, broad H3K4me3 peaks mark important learning and memory genes and are often regulated by super-enhancers. The peaks at many learning-associated genes become broader during novel environment exposure and memory formation. Furthermore, the important learning- and memory-associated lysine methyltransferases, Kmt2a and Kmt2b, are involved in maintaining H3K4me3 peak width. Our findings highlight the importance of analyzing H3K4me3 peak shape, and demonstrate that breadth of H3K4me3 marks in neurons of the hippocampus is regulated during memory formation.