Regulation of prefrontal patterning and connectivity by retinoic acid.

The prefrontal cortex (PFC) and its connections with the mediodorsal thalamus are crucial for cognitive flexibility and working memory1 and are thought to be altered in disorders such as autism2,3 and schizophrenia4,5. Although developmental mechanisms that govern the regional patterning of the cerebral cortex have been characterized in rodents6-9, the mechanisms that underlie the development of PFC-mediodorsal thalamus connectivity and the lateral expansion of the PFC with a distinct granular layer 4 in primates10,11 remain unknown. Here we report an anterior (frontal) to posterior (temporal), PFC-enriched gradient of retinoic acid, a signalling molecule that regulates neural development and function12-15, and we identify genes that are regulated by retinoic acid in the neocortex of humans and macaques at the early and middle stages of fetal development. We observed several potential sources of retinoic acid, including the expression and cortical expansion of retinoic-acid-synthesizing enzymes specifically in primates as compared to mice. Furthermore, retinoic acid signalling is largely confined to the prospective PFC by CYP26B1, a retinoic-acid-catabolizing enzyme, which is upregulated in the prospective motor cortex. Genetic deletions in mice revealed that retinoic acid signalling through the retinoic acid receptors RXRG and RARB, as well as CYP26B1-dependent catabolism, are involved in proper molecular patterning of prefrontal and motor areas, development of PFC-mediodorsal thalamus connectivity, intra-PFC dendritic spinogenesis and expression of the layer 4 marker RORB. Together, these findings show that retinoic acid signalling has a critical role in the development of the PFC and, potentially, in its evolutionary expansion.

Perinatal phthalate exposure increases developmental apoptosis in the rat medial prefrontal cortex.

Phthalates are a class of endocrine disruptors found in a variety of consumer goods, and offspring can be exposed to these compounds during gestation and lactation. Our laboratory has found that perinatal exposure to an environmentally relevant mixture of phthalates resulted in a decrease in cognitive flexibility and in neuron number in the adult rat medial prefrontal cortex (mPFC). Here, we examine effects of phthalate treatment on prenatal cellular proliferation and perinatal apoptosis in the mPFC. To examine the phthalate effects on cellular proliferation, dams consumed 0, 1, or 5 mg/kg of the phthalate mixture daily from embryonic day 2 (E2) through the day of birth (P0), and on E16 and E17, they were injected with BrdU. The mPFC of offspring was analyzed on P5 and showed a decrease in labelled cells in the phthalate exposed groups. To examine whether changes in BrdU density observed on P5 were due to altered cell survival, cell death was measured on E18, P0, and P5 using a TUNEL assay in a separate cohort of prenatally exposed offspring. There was an increase in TUNEL labelled cells at E18 in the phthalate exposed groups. In the final experiment, dams consumed the phthalate mixture from E2 through P10, at which time mPFC tissue was stained with TUNEL. Phthalate treated subjects showed a higher density of apoptotic cells at P10. These results indicate both pre- and postnatal phthalate exposure increases apoptosis in the male and female rat mPFC. While the impact of phthalates on proliferation cannot be ruled out, these data do not allow for definitive conclusions.

Comparative Transcriptomic Analysis of Cerebral Organoids and Cortical Neuron Cultures Derived from Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) can be differentiated along various neuronal lineages to generate two-dimensional neuronal cultures as well as three-dimensional brain organoids. Such iPSC-derived cellular models are being utilized to study the basic biology of human neuronal function and to interrogate the molecular underpinnings of disease biology. The different cellular models generated from iPSCs have varying properties in terms of the diversity and organization of the cells as well as the cellular functions that are present. To understand transcriptomic differences in iPSC-derived monolayer neuronal cultures and three-dimensional brain organoids, we differentiated eight human iPSC lines from healthy control subjects to generate cerebral organoids and cortical neuron monolayer cultures from the same set of iPSC lines. We undertook RNA-seq experiments in these model systems and analyzed the gene expression data to identify genes that are differentially expressed in cerebral organoids and two-dimensional cortical neuron cultures. In cerebral organoids, gene ontology analysis showed enrichment of genes involved in tissue development, response to stimuli, and the interferon-γ pathway, while two-dimensional cortical neuron cultures showed enrichment of genes involved in nervous system development and neurogenesis. We also undertook comparative analysis of these gene expression profiles with transcriptomic data from the human fetal prefrontal cortex (PFC). This analysis showed greater overlap of the fetal PFC transcriptome with cerebral organoid gene expression profiles compared to monolayer cortical neuron culture profiles. Our studies delineate the transcriptomic differences between cortical neuron monolayer cultures and three-dimensional cerebral organoids and can help inform the appropriate use of these model systems to address specific scientific questions.

Excess of RALGAPB de novo variants in neurodevelopmental disorders.

Autism spectrum disorder is a neurodevelopmental disorder (NDD) with complex genetic architecture marked primarily by social and communication impairments along with deficits in restrictive and repetitive behaviors. Due to the complex nature and genetic heterogeneity of the disease, genotype and phenotype correlation remains challenging. Prior studies have implicated RALGAPB as a candidate gene for ASD, but stringent analysis is required to determine the pathogenicity. By targeted sequencing, we identified a new de novo RALGAPB missense variant (c.1238C> T; p.T413M) in an ASD family. By leveraging published large-scale genome sequencing studies, we curated five de novo likely gene-disruptive (LGD) variants and 5 de novo missense variants in ASD and related NDDs and revealed a genome-wide significant excess of RALGAPB de novo LGD variants (P_adjust = 0.0053). Quantitative reverse transcription PCR revealed that the frameshift variant c.1927dupA; p.N643fs*3 reduced mRNA expression levels confirming the loss-of-function effect. Co-expression analysis using human brain transcriptome data provide the potential functional link of RALGAPB and 38 ASD and/or NDD genes. Our study suggests RALGAPB as a new NDD risk gene which should be considered in clinical diagnosis of ASD and related NDDs.

A Chromatin Accessibility Atlas of the Developing Human Telencephalon.

To discover regulatory elements driving the specificity of gene expression in different cell types and regions of the developing human brain, we generated an atlas of open chromatin from nine dissected regions of the mid-gestation human telencephalon, as well as microdissected upper and deep layers of the prefrontal cortex. We identified a subset of open chromatin regions (OCRs), termed predicted regulatory elements (pREs), that are likely to function as developmental brain enhancers. pREs showed temporal, regional, and laminar differences in chromatin accessibility and were correlated with gene expression differences across regions and gestational ages. We identified two functional de novo variants in a pRE for autism risk gene SLC6A1, and using CRISPRa, demonstrated that this pRE regulates SCL6A1. Additionally, mouse transgenic experiments validated enhancer activity for pREs proximal to FEZF2 and BCL11A. Thus, this atlas serves as a resource for decoding neurodevelopmental gene regulation in health and disease.

Protective Effect of Flax Seed on Brain Teratogenicity Induced by Lamotrigine in Rat Fetuses.

The objective of this study was to assess the effects of the hydroalcoholic extract of flax seed on the teratogenic activity of lamotrigine in the brain of fetuses of rats who had received the drug. In this experimental study, 40 female rats were assigned randomly into four groups and after mating and confirming the vaginal plug, the control animals (group 1) were kept with no intervention, and the other three experimental groups were intraperitoneally injected with respective lamotrigine (75 mg/kg), and 100 and 200 mg/kg of flax seed hydroalcoholic extract. The drug was administered during the organogenesis period. Rats were sacrificed at the 20th day of gestation (one day before term) and fetuses were macroscopically examined, weighed and crown-rump length measured. Fetal brain specimens were processed for H&E and for histological study, using the ImageJ software. Results showed that fetuses of the experimental groups that received lamotrigine had reduced body weight, prefrontal cortical and hippocampal thickness, and pyramidal neurons in the hip-pocampus; Nevertheless, these factors were improved by high-dose administration of flax seed in the experimental group 3 and 4. Our research concludes that lamotrigine negatively influences the development of brain in rats and flax seed has a protective impact on these complications.

Recovery of BDNF and CB1R in the Prefrontal Cortex Underlying Improvement of Working Memory in Prenatal DEHP-Exposed Male Rats after Aerobic Exercise.

Early-life exposure to di-(2-ethylhexyl)-phthalate (DEHP) has been suggested to relate to hyperactivity, lack of attention, and working memory deficits in school-age children. Brain-derived neurotrophic factor (BDNF) and endocannabinoids are induced by aerobic exercises to provide beneficial effects on brain functions. This study investigated the mechanisms underlying working memory impairment and the protective role of exercise in prenatal DEHP-exposed male rats. Sprague Dawley dams were fed with vehicle or DEHP during gestation. The male offspring were trained to exercise on a treadmill for 5 weeks, which was followed by an assessment of their working memory with a T-maze delayed non-match-to-sample task. The expressions of BDNF, dopamine D1 receptor (D1R), cannabinoid receptor 1 (CB1R), and fatty acid amide hydrolase (FAAH) in the prefrontal cortex were detected by Western blot. The results showed that DEHP-exposed rats exhibited working memory impairments without significant alterations in locomotor activities. The reduced expressions of prefrontal BDNF and CB1R were obtained in the DEHP-exposed rats, while D1R and FAAH were barely affected. Importantly, aerobic exercise during childhood-adolescence prevented the impairment of working memory in the DEHP-exposed rats by recovering the BDNF and CB1R expressions in the prefrontal cortex. These findings suggest that exercise may provide beneficial effects in ameliorating the impairment of working memory in the prenatal DEHP-exposed male rats at late adolescence.

Prenatal expression of D-aspartate oxidase causes early cerebral D-aspartate depletion and influences brain morphology and cognitive functions at adulthood.

The free D-amino acid, D-aspartate, is abundant in the embryonic brain but significantly decreases after birth. Besides its intracellular occurrence, D-aspartate is also present at extracellular level and acts as an endogenous agonist for NMDA and mGlu5 receptors. These findings suggest that D-aspartate is a candidate signaling molecule involved in neural development, influencing brain morphology and behaviors at adulthood. To address this issue, we generated a knockin mouse model in which the enzyme regulating D-aspartate catabolism, D-aspartate oxidase (DDO), is expressed starting from the zygotic stage, to enable the removal of D-aspartate in prenatal and postnatal life. In line with our strategy, we found a severe depletion of cerebral D-aspartate levels (up to 95%), since the early stages of mouse prenatal life. Despite the loss of D-aspartate content, Ddo knockin mice are viable, fertile, and show normal gross brain morphology at adulthood. Interestingly, early D-aspartate depletion is associated with a selective increase in the number of parvalbumin-positive interneurons in the prefrontal cortex and also with improved memory performance in Ddo knockin mice. In conclusion, the present data indicate for the first time a biological significance of precocious D-aspartate in regulating mouse brain formation and function at adulthood.

Decoding the development of the human hippocampus.

The hippocampus is an important part of the limbic system in the human brain that has essential roles in spatial navigation and the consolidation of information from short-term memory to long-term memory1,2. Here we use single-cell RNA sequencing and assay for transposase-accessible chromatin using sequencing (ATAC-seq) analysis to illustrate the cell types, cell linage, molecular features and transcriptional regulation of the developing human hippocampus. Using the transcriptomes of 30,416 cells from the human hippocampus at gestational weeks 16-27, we identify 47 cell subtypes and their developmental trajectories. We also identify the migrating paths and cell lineages of PAX6+ and HOPX+ hippocampal progenitors, and regional markers of CA1, CA3 and dentate gyrus neurons. Multiomic data have uncovered transcriptional regulatory networks of the dentate gyrus marker PROX1. We also illustrate spatially specific gene expression in the developing human prefrontal cortex and hippocampus. The molecular features of the human hippocampus at gestational weeks 16-20 are similar to those of the mouse at postnatal days 0-5 and reveal gene expression differences between the two species. Transient expression of the primate-specific gene NBPF1 leads to a marked increase in PROX1+ cells in the mouse hippocampus. These data provides a blueprint for understanding human hippocampal development and a tool for investigating related diseases.

Exposure to valproic acid during middle to late-stage corticogenesis induces learning and social behavioral abnormalities with attention deficit/hyperactivity in adult mice.

Single prenatal exposure to valproic acid (VPA) in rodents is a widely used preclinical model of autism spectrum disorder (ASD). Continuous prenatal VPA exposure has been recently proposed as a new ASD model that closely captures the neuropathological features of ASD, including increases in cerebral cortex volume and the number of cortical upper layer neurons. We investigated the influence of prenatal VPA exposure on the behavior of adult offspring of pregnant dams that received intraperitoneal injections of VPA twice on one day during the genesis of cortical upper layer neurons. Mice exposed to VPA at E14 (E14-VPA) showed typical behavior abnormalities including reduced social interaction, hyperactivity, and poor maze learning due to attention deficit/impulsivity relative to healthy controls. Histological analysis revealed that E14-VPA mice had significantly increased neuronal density and impaired neural activity in the prefrontal cortex, but not the somatosensory area, which is likely linked to the observed abnormalities in social behavior. These results suggest that this VPA exposure method is a good model for gaining new insights into the underlying neuropathology of ASD.

Temporal dynamics of miRNAs in human DLPFC and its association with miRNA dysregulation in schizophrenia.

Brain development is dependent on programmed gene expression, which is both genetically and epigenetically regulated. Post-transcriptional regulation of gene expression by microRNAs (miRNAs) is essential for brain development. As abnormal brain development is hypothesized to be associated with schizophrenia, miRNAs are an intriguing target for this disorder. The aims of this study were to determine the temporal dynamics of miRNA expression in the human dorsolateral prefrontal cortex (DLPFC), and the relationship between miRNA's temporal expression pattern and dysregulation in schizophrenia. This study used next-generation sequencing to characterize the temporal dynamics of miRNA expression in the DLPFC of 109 normal subjects (second trimester-74 years of age) and miRNA expression changes in 34 schizophrenia patients. Unlike mRNAs, the majority of which exhibits a wave of change in fetuses, most miRNAs are preferentially expressed during a certain period before puberty. It is noted that in schizophrenia patients, miRNAs normally enriched in infants tend to be upregulated, while those normally enriched in prepuberty tend to be downregulated, and the targets of these miRNAs are enriched for genes encoding synaptic proteins and those associated with schizophrenia. In addition, miR-936 and miR-3162 were found to be increased in the DLPFC of patients with schizophrenia. These findings reveal the temporal dynamics of miRNAs in the human DLPFC, implicate the importance of miRNAs in DLPFC development, and suggest a possible link between schizophrenia and dysregulation of miRNAs enriched in infancy and prepuberty.

Mechanisms for the Approach/Avoidance Decision Applied to Autism.

As a neurodevelopmental disorder with serious lifelong consequences, autism has received considerable attention from neuroscientists and geneticists. We present a hypothesis of mechanisms plausibly affected during brain development in autism, based on neural pathways that are associated with social behavior and connect the prefrontal cortex (PFC) to the basal ganglia (BG). We consider failure of social approach in autism as a special case of imbalance in the fundamental dichotomy between behavioral approach and avoidance. Differential combinations of genes mutated, differences in the timing of their impact during development, and graded degrees of hormonal influences may help explain the heterogeneity in symptomatology in autism and predominance in boys.

Acute and chronic neurobehavioral effects of the designer drug and bath salt constituent 3,4-methylenedioxypyrovalerone in the rat.

BACKGROUND: The substantial increase in use of 3,4-methylenedioxypyrovalerone (MDPV), a popular recreational synthetic cathinone, has raised legitimate questions about its behavioral consequences and abuse liability. AIMS: The aim of this study was to study MDPV-induced neurobehavioral effects in the rat, using different paradigms traditionally developed to study drug-attributed addictive properties. METHODS: Different patterns of intraperitoneal 3 mg/kg MDPV administration were investigated. Consequences on rat horizontal locomotion and behavior of acute, intermittent (once daily dosing over 10 days), and binge (three-time daily dosing for 3 days) MDPV administration as well as challenge after 10 day MDPV withdrawal were studied. The dopamine receptor-D1 antagonist, SCH23390, was bilaterally infused in the nucleus accumbens to determine the role of D1-receptors in MDPV-related effects on the associative memory recall using the conditioned place preference paradigm. In addition, in a separate experience using western blot, we investigated the effects of chronic MDPV administration (four injections during 24 h) on ΔFosB expression in the nucleus accumbens, caudate putamen, and prefrontal cortex. RESULTS: Acute MDPV administration increased stereotypies and open arm entries in the elevated plus maze while SCH23390 abolished MDPV-induced enhancing effects on memory consolidation. Intermittent MDPV administration resulted in sensitization of MDPV-induced locomotor effects and tolerance during the following challenge. With binge MDPV administration, locomotor activity was not altered despite tolerance onset after challenge. SCH23390 abolished MDPV-induced conditioned place preference. Chronic MDPV administration induced ΔFosB accumulation in the nucleus accumbens, caudate putamen, and prefrontal cortex. CONCLUSIONS: Our findings clearly show that MDPV produces profound behavioral alterations mediated by the activation of the dopaminergic system similarly to other amphetamines.

Prenatal stress and the developing brain: Risks for neurodevelopmental disorders.

The prenatal period is increasingly considered as a crucial target for the primary prevention of neurodevelopmental and psychiatric disorders. Understanding their pathophysiological mechanisms remains a great challenge. Our review reveals new insights from prenatal brain development research, involving (epi)genetic research, neuroscience, recent imaging techniques, physical modeling, and computational simulation studies. Studies examining the effect of prenatal exposure to maternal distress on offspring brain development, using brain imaging techniques, reveal effects at birth and up into adulthood. Structural and functional changes are observed in several brain regions including the prefrontal, parietal, and temporal lobes, as well as the cerebellum, hippocampus, and amygdala. Furthermore, alterations are seen in functional connectivity of amygdalar-thalamus networks and in intrinsic brain networks, including default mode and attentional networks. The observed changes underlie offspring behavioral, cognitive, emotional development, and susceptibility to neurodevelopmental and psychiatric disorders. It is concluded that used brain measures have not yet been validated with regard to sensitivity, specificity, accuracy, or robustness in predicting neurodevelopmental and psychiatric disorders. Therefore, more prospective long-term longitudinal follow-up studies starting early in pregnancy should be carried out, in order to examine brain developmental measures as mediators in mediating the link between prenatal stress and offspring behavioral, cognitive, and emotional problems and susceptibility for disorders.

Prenatal neural origins of infant motor development: Associations between fetal brain and infant motor development.

Functional circuits of the human brain emerge and change dramatically over the second half of gestation. It is possible that variation in neural functional system connectivity in utero predicts individual differences in infant behavioral development, but this possibility has yet to be examined. The current study examines the association between fetal sensorimotor brain system functional connectivity and infant postnatal motor ability. Resting-state functional connectivity data was obtained in 96 healthy human fetuses during the second and third trimesters of pregnancy. Infant motor ability was measured 7 months after birth using the Bayley Scales of Infant Development. Increased connectivity between the emerging motor network and regions of the prefrontal cortex, temporal lobes, posterior cingulate, and supplementary motor regions was observed in infants that showed more mature motor functions. In addition, females demonstrated stronger fetal-brain to infant-behavior associations. These observations extend prior longitudinal research back into prenatal brain development and raise exciting new ideas about the advent of risk and the ontogeny of early sex differences.

A single-cell RNA-seq survey of the developmental landscape of the human prefrontal cortex.

The mammalian prefrontal cortex comprises a set of highly specialized brain areas containing billions of cells and serves as the centre of the highest-order cognitive functions, such as memory, cognitive ability, decision-making and social behaviour. Although neural circuits are formed in the late stages of human embryonic development and even after birth, diverse classes of functional cells are generated and migrate to the appropriate locations earlier in development. Dysfunction of the prefrontal cortex contributes to cognitive deficits and the majority of neurodevelopmental disorders; there is therefore a need for detailed knowledge of the development of the prefrontal cortex. However, it is still difficult to identify cell types in the developing human prefrontal cortex and to distinguish their developmental features. Here we analyse more than 2,300 single cells in the developing human prefrontal cortex from gestational weeks 8 to 26 using RNA sequencing. We identify 35 subtypes of cells in six main classes and trace the developmental trajectories of these cells. Detailed analysis of neural progenitor cells highlights new marker genes and unique developmental features of intermediate progenitor cells. We also map the timeline of neurogenesis of excitatory neurons in the prefrontal cortex and detect the presence of interneuron progenitors in early developing prefrontal cortex. Moreover, we reveal the intrinsic development-dependent signals that regulate neuron generation and circuit formation using single-cell transcriptomic data analysis. Our screening and characterization approach provides a blueprint for understanding the development of the human prefrontal cortex in the early and mid-gestational stages in order to systematically dissect the cellular basis and molecular regulation of prefrontal cortex function in humans.

Global spectral clustering in dynamic networks.

Community detection is challenging when the network structure is estimated with uncertainty. Dynamic networks present additional challenges but also add information across time periods. We propose a global community detection method, persistent communities by eigenvector smoothing (PisCES), that combines information across a series of networks, longitudinally, to strengthen the inference for each period. Our method is derived from evolutionary spectral clustering and degree correction methods. Data-driven solutions to the problem of tuning parameter selection are provided. In simulations we find that PisCES performs better than competing methods designed for a low signal-to-noise ratio. Recently obtained gene expression data from rhesus monkey brains provide samples from finely partitioned brain regions over a broad time span including pre- and postnatal periods. Of interest is how gene communities develop over space and time; however, once the data are divided into homogeneous spatial and temporal periods, sample sizes are very small, making inference quite challenging. Applying PisCES to medial prefrontal cortex in monkey rhesus brains from near conception to adulthood reveals dense communities that persist, merge, and diverge over time and others that are loosely organized and short lived, illustrating how dynamic community detection can yield interesting insights into processes such as brain development.

Conservation, evolution, and regulation of splicing during prefrontal cortex development in humans, chimpanzees, and macaques.

Changes in splicing are known to affect the function and regulation of genes. We analyzed splicing events that take place during the postnatal development of the prefrontal cortex in humans, chimpanzees, and rhesus macaques based on data obtained from 168 individuals. Our study revealed that among the 38,822 quantified alternative exons, 15% are differentially spliced among species, and more than 6% splice differently at different ages. Mutations in splicing acceptor and/or donor sites might explain more than 14% of all splicing differences among species and up to 64% of high-amplitude differences. A reconstructed trans-regulatory network containing 21 RNA-binding proteins explains a further 4% of splicing variations within species. While most age-dependent splicing patterns are conserved among the three species, developmental changes in intron retention are substantially more pronounced in humans.

The schizophrenia- and autism-associated gene, transcription factor 4 regulates the columnar distribution of layer 2/3 prefrontal pyramidal neurons in an activity-dependent manner.

Disruption of the laminar and columnar organization of the brain is implicated in several psychiatric disorders. Here, we show in utero gain-of-function of the psychiatric risk gene transcription factor 4 (TCF4) severely disrupts the columnar organization of medial prefrontal cortex (mPFC) in a transcription- and activity-dependent manner. This morphological phenotype was rescued by co-expression of TCF4 plus calmodulin in a calcium-dependent manner and by dampening neuronal excitability through co-expression of an inwardly rectifying potassium channel (Kir2.1). For we believe the first time, we show that N-methyl-d-aspartate (NMDA) receptor-dependent Ca2+ transients are instructive to minicolumn organization because Crispr/Cas9-mediated mutation of NMDA receptors rescued TCF4-dependent morphological phenotypes. Furthermore, we demonstrate that the transcriptional regulation by the psychiatric risk gene TCF4 enhances NMDA receptor-dependent early network oscillations. Our novel findings indicate that TCF4-dependent transcription directs the proper formation of prefrontal cortical minicolumns by regulating the expression of genes involved in early spontaneous neuronal activity, and thus our results provides insights into potential pathophysiological mechanisms of TCF4-associated psychiatric disorders.