Gene-environmental regulation of the postnatal post-mitotic neuronal maturation.

Embryonic neurodevelopment, particularly neural progenitor differentiation into post-mitotic neurons, has been extensively studied. While the number and composition of post-mitotic neurons remain relatively constant from birth to adulthood, the brain undergoes significant postnatal maturation marked by major property changes frequently disrupted in neural diseases. This review first summarizes recent characterizations of the functional and molecular maturation of the postnatal nervous system. We then review regulatory mechanisms controlling the precise gene expression changes crucial for the intricate sequence of maturation events, highlighting experience-dependent versus cell-intrinsic genetic timer mechanisms. Despite significant advances in understanding of the gene-environmental regulation of postnatal neuronal maturation, many aspects remain unknown. The review concludes with our perspective on exciting future research directions in the next decade.

Comparing engineered nuclear-localized reporter cassettes.

Recent single-cell transcriptome analysis has revealed a tremendous breadth and specificity of neuropeptide-encoding gene expression in the nervous system of C. elegans. To analyze the dynamics of neuropeptide gene expression, as well as to dissect the regulatory mechanism by which their expression is controlled, reporter genes remain an important tool. Using CRISPR/Cas9 genome-engineering, we generate here reporter alleles for 6 different neuropeptide encoding genes (3 flp genes, 1 nlp and 2 insulin genes). We find that different reporter cassettes result in different levels of reporter expression and recommend usage of an SL2::GFP::H2B or GFP::H2B::SL2 cassette.

The neuropeptidergic connectome of C. elegans.

Efforts are ongoing to map synaptic wiring diagrams, or connectomes, to understand the neural basis of brain function. However, chemical synapses represent only one type of functionally important neuronal connection; in particular, extrasynaptic, "wireless" signaling by neuropeptides is widespread and plays essential roles in all nervous systems. By integrating single-cell anatomical and gene-expression datasets with biochemical analysis of receptor-ligand interactions, we have generated a draft connectome of neuropeptide signaling in the C. elegans nervous system. This network is characterized by high connection density, extended signaling cascades, autocrine foci, and a decentralized topology, with a large, highly interconnected core containing three constituent communities sharing similar patterns of input connectivity. Intriguingly, several key network hubs are little-studied neurons that appear specialized for peptidergic neuromodulation. We anticipate that the C. elegans neuropeptidergic connectome will serve as a prototype to understand how networks of neuromodulatory signaling are organized.

The heterochronic LIN-14 protein is a BEN domain transcription factor.

Heterochrony is a foundational concept in animal development and evolution, first introduced by Ernst Haeckel in 1875 and later popularized by Stephen J. Gould1. A molecular understanding of heterochrony was first established by genetic mutant analysis in the nematode C. elegans, revealing a genetic pathway that controls the proper timing of cellular patterning events executed during distinct postembryonic juvenile and adult stages2. This genetic pathway is composed of a complex temporal cascade of multiple regulatory factors, including the first-ever discovered miRNA, lin-4, and its target gene, lin-14, which encodes a nuclear, DNA-binding protein2,3,4. While all core members of the pathway have homologs based on primary sequences in other organisms, homologs for LIN-14 have never been identified by sequence homology. We report that the AlphaFold-predicted structure of the LIN-14 DNA binding domain is homologous to the BEN domain, found in a family of DNA binding proteins previously thought to have no nematode homologs5. We confirmed this prediction through targeted mutations of predicted DNA-contacting residues, which disrupt in vitro DNA binding and in vivo function. Our findings shed new light on potential mechanisms of LIN-14 function and suggest that BEN domain-containing proteins may have a conserved role in developmental timing.

Temporal transitions in the postembryonic nervous system of the nematode Caenorhabditis elegans: Recent insights and open questions.

After the generation, differentiation and integration into functional circuitry, post-mitotic neurons continue to change certain phenotypic properties throughout postnatal juvenile stages until an animal has reached a fully mature state in adulthood. We will discuss such changes in the context of the nervous system of the nematode C. elegans, focusing on recent descriptions of anatomical and molecular changes that accompany postembryonic maturation of neurons. We summarize the characterization of genetic timer mechanisms that control these temporal transitions or maturational changes, and discuss that many but not all of these transitions relate to sexual maturation of the animal. We describe how temporal, spatial and sex-determination pathways are intertwined to sculpt the emergence of cell-type specific maturation events. Finally, we lay out several unresolved questions that should be addressed to move the field forward, both in C. elegans and in vertebrates.

Two intrinsic timing mechanisms set start and end times for dendritic arborization of a nociceptive neuron.

Choreographic dendritic arborization takes place within a defined time frame, but the timing mechanism is currently not known. Here, we report that the precisely timed lin-4-lin-14 regulatory circuit triggers an initial dendritic growth activity, whereas the precisely timed lin-28-let-7-lin-41 regulatory circuit signals a subsequent developmental decline in dendritic growth ability, hence restricting dendritic arborization within a set time frame. Loss-of-function mutations in the lin-4 microRNA gene cause limited dendritic outgrowth, whereas loss-of-function mutations in its direct target, the lin-14 transcription factor gene, cause precocious and excessive outgrowth. In contrast, loss-of-function mutations in the let-7 microRNA gene prevent a developmental decline in dendritic growth ability, whereas loss-of-function mutations in its direct target, the lin-41 tripartite motif protein gene, cause further decline. lin-4 and let-7 regulatory circuits are expressed in the right place at the right time to set start and end times for dendritic arborization. Replacing the lin-4 upstream cis-regulatory sequence at the lin-4 locus with a late-onset let-7 upstream cis-regulatory sequence delays dendrite arborization, whereas replacing the let-7 upstream cis-regulatory sequence at the let-7 locus with an early-onset lin-4 upstream cis-regulatory sequence causes a precocious decline in dendritic growth ability. Our results indicate that the lin-4-lin-14 and the lin-28-let-7-lin-41 regulatory circuits control the timing of dendrite arborization through antagonistic regulation of the DMA-1 receptor level on dendrites. The LIN-14 transcription factor likely directly represses dma-1 gene expression through a transcriptional means, whereas the LIN-41 tripartite motif protein likely indirectly promotes dma-1 gene expression through a posttranscriptional means.

Temporal transitions in the post-mitotic nervous system of Caenorhabditis elegans.

In most animals, the majority of the nervous system is generated and assembled into neuronal circuits during embryonic development1. However, during juvenile stages, nervous systems still undergo extensive anatomical and functional changes to eventually form a fully mature nervous system by the adult stage2,3. The molecular changes in post-mitotic neurons across post-embryonic development and the genetic programs that control these temporal transitions are not well understood4,5. Here, using the model system Caenorhabditis elegans, we comprehensively characterized the distinct functional states (locomotor behaviour) and the corresponding distinct molecular states (transcriptome) of the post-mitotic nervous system across temporal transitions during post-embryonic development. We observed pervasive, neuron-type-specific changes in gene expression, many of which are controlled by the developmental upregulation of the conserved heterochronic microRNA LIN-4 and the subsequent promotion of a mature neuronal transcriptional program through the repression of its target, the transcription factor lin-14. The functional relevance of these molecular transitions are exemplified by a temporally regulated target gene of the LIN-14 transcription factor, nlp-45, a neuropeptide-encoding gene, which we find is required for several distinct temporal transitions in exploratory activity during post-embryonic development. Our study provides insights into regulatory strategies that control neuron-type-specific gene batteries to modulate distinct behavioural states across temporal, sexual and environmental dimensions of post-embryonic development.

The Prop1-like homeobox gene unc-42 specifies the identity of synaptically connected neurons.

Many neuronal identity regulators are expressed in distinct populations of cells in the nervous system, but their function is often analyzed only in specific isolated cellular contexts, thereby potentially leaving overarching themes in gene function undiscovered. We show here that the Caenorhabditis elegans Prop1-like homeobox gene unc-42 is expressed in 15 distinct sensory, inter- and motor neuron classes throughout the entire C. elegans nervous system. Strikingly, all 15 neuron classes expressing unc-42 are synaptically interconnected, prompting us to investigate whether unc-42 controls the functional properties of this circuit and perhaps also the assembly of these neurons into functional circuitry. We found that unc-42 defines the routes of communication between these interconnected neurons by controlling the expression of neurotransmitter pathway genes, neurotransmitter receptors, neuropeptides, and neuropeptide receptors. Anatomical analysis of unc-42 mutant animals reveals defects in axon pathfinding and synaptic connectivity, paralleled by expression defects of molecules involved in axon pathfinding, cell-cell recognition, and synaptic connectivity. We conclude that unc-42 establishes functional circuitry by acting as a terminal selector of functionally connected neuron types. We identify a number of additional transcription factors that are also expressed in synaptically connected neurons and propose that terminal selectors may also function as 'circuit organizer transcription factors' to control the assembly of functional circuitry throughout the nervous system. We hypothesize that such organizational properties of transcription factors may be reflective of not only ontogenetic, but perhaps also phylogenetic trajectories of neuronal circuit establishment.

Temporal, Spatial, Sexual and Environmental Regulation of the Master Regulator of Sexual Differentiation in C. elegans.

Sexual differentiation is controlled by diverse master regulatory factors across the animal kingdom. The transcription factor TRA-1 is the master regulator of somatic sexual differentiation in the nematode C. elegans, where it was reported to be expressed sex-specifically in the non-gonadal soma of hermaphrodites. Using a gfp-tagged allele of tra-1, we reveal unanticipated dynamics of TRA-1 protein expression in five dimensions: space, time, sex, environment, and subcellular localization. We show temporal regulation of TRA-1 protein accumulation in somatic tissues with different onsets of expression in different tissue types, indicating that sexual identity is not uniformly imposed. In hermaphrodites, neuronal expression is initially highly restricted and then increases variably between individuals during larval development until reaching panneuronal expression in the fourth larval stage. Unexpectedly, TRA-1 also accumulates in a subset of sex-shared neurons in the male. Additionally, a food signal is required to turn on TRA-1 expression in the intestine, and environmental stressors shut off TRA-1 expression in the entire non-gonadal soma, suggesting that somatic sexual differentiation may be affected by external conditions. We show that, in contrast to the protein degradation mechanisms that control TRA-1 accumulation in the adult, the temporal, sexual, and spatial specificities of TRA-1 accumulation during development are regulated transcriptionally. A nuclear hormone receptor, daf-12, previously implicated in developmental timing in C. elegans, contributes to temporal accumulation of TRA-1 in the nervous system. Our studies reveal a mosaic and dynamic nature of sexual identity acquisition and uncover hormonal control mechanisms for sexual differentiation of the brain.

Knockdown of the histone di-methyltransferase G9a in nucleus accumbens shell decreases cocaine self-administration, stress-induced reinstatement, and anxiety.

Comorbid neuropsychiatric disorders such as addiction and anxiety could involve common underlying mechanisms. One potential mechanism involves epigenetic regulation of histone 3 dimethylation at lysine 9 residues (H3K9me2) by the histone dimethyltransferase G9a. Here we provide evidence that local AAV-RNAi-mediated knockdown of G9a expression in nucleus accumbens shell (NAcSh) of male rats reduces both addictive-related and anxiety-related behaviors. Specifically, G9a knockdown reduces sensitivity to low dose cocaine reinforcement when cocaine is freely available (fixed ratio schedule). Similarly, G9a knockdown reduces motivation for cocaine under higher effort demands (progressive ratio schedule). Following several weeks of forced abstinence, G9a knockdown attenuates extinction responding and reinstatement triggered by either cocaine-priming injections or footshock stress. This decrease in addictive behavior is associated with a long-term reduction in anxiety-like behavior as measured by the elevated plus maze (EPM). G9a knockdown also reduces basal anxiety-like behavior in EPM and marble burying tests in drug-naïve rats. These results complement our previous work showing that increased G9a expression in NAcSh enhances addictive-related and anxiety-related behaviors, indicating that G9a bi-directionally controls these responses. These results also suggest that regulation of G9a-influenced gene expression could be a common epigenetic mechanism for co-morbid anxiety and psychostimulant addiction.

An atlas of Caenorhabditis elegans chemoreceptor expression.

One goal of modern day neuroscience is the establishment of molecular maps that assign unique features to individual neuron types. Such maps provide important starting points for neuron classification, for functional analysis, and for developmental studies aimed at defining the molecular mechanisms of neuron identity acquisition and neuron identity diversification. In this resource paper, we describe a nervous system-wide map of the potential expression sites of 244 members of the largest gene family in the C. elegans genome, rhodopsin-like (class A) G-protein-coupled receptor (GPCR) chemoreceptors, using classic gfp reporter gene technology. We cover representatives of all sequence families of chemoreceptor GPCRs, some of which were previously entirely uncharacterized. Most reporters are expressed in a very restricted number of cells, often just in single cells. We assign GPCR reporter expression to all but two of the 37 sensory neuron classes of the sex-shared, core nervous system. Some sensory neurons express a very small number of receptors, while others, particularly nociceptive neurons, coexpress several dozen GPCR reporter genes. GPCR reporters are also expressed in a wide range of inter- and motorneurons, as well as non-neuronal cells, suggesting that GPCRs may constitute receptors not just for environmental signals, but also for internal cues. We observe only one notable, frequent association of coexpression patterns, namely in one nociceptive amphid (ASH) and two nociceptive phasmid sensory neurons (PHA, PHB). We identified GPCRs with sexually dimorphic expression and several GPCR reporters that are expressed in a left/right asymmetric manner. We identified a substantial degree of GPCR expression plasticity; particularly in the context of the environmentally-induced dauer diapause stage when one third of all tested GPCRs alter the cellular specificity of their expression within and outside the nervous system. Intriguingly, in a number of cases, the dauer-specific alterations of GPCR reporter expression in specific neuron classes are maintained during postdauer life and in some case new patterns are induced post-dauer, demonstrating that GPCR gene expression may serve as traits of life history. Taken together, our resource provides an entry point for functional studies and also offers a host of molecular markers for studying molecular patterning and plasticity of the nervous system.

Regulation of BAZ1A and nucleosome positioning in the nucleus accumbens in response to cocaine.

Chromatin regulation, in particular ATP-dependent chromatin remodelers, have previously been shown to be important in the regulation of reward-related behaviors in animal models of mental illnesses. Here we demonstrate that BAZ1A, an accessory subunit of the ISWI family of chromatin remodeling complexes, is downregulated in the nucleus accumbens (NAc) of mice exposed repeatedly to cocaine and of cocaine-addicted humans. Viral-mediated overexpression of BAZ1A in mouse NAc reduces cocaine reward as assessed by conditioned place preference (CPP), but increases cocaine-induced locomotor activation. Furthermore, we investigate nucleosome repositioning genome-wide by conducting chromatin immunoprecipitation (ChIP)-sequencing for total H3 in NAc of control mice and after repeated cocaine administration, and find extensive nucleosome occupancy and shift changes across the genome in response to cocaine exposure. These findings implicate BAZ1A in molecular and behavioral plasticity to cocaine and offer new insight into the pathophysiology of cocaine addiction.

Histone arginine methylation in cocaine action in the nucleus accumbens.

Repeated cocaine exposure regulates transcriptional regulation within the nucleus accumbens (NAc), and epigenetic mechanisms-such as histone acetylation and methylation on Lys residues-have been linked to these lasting actions of cocaine. In contrast to Lys methylation, the role of histone Arg (R) methylation remains underexplored in addiction models. Here we show that protein-R-methyltransferase-6 (PRMT6) and its associated histone mark, asymmetric dimethylation of R2 on histone H3 (H3R2me2a), are decreased in the NAc of mice and rats after repeated cocaine exposure, including self-administration, and in the NAc of cocaine-addicted humans. Such PRMT6 down-regulation occurs selectively in NAc medium spiny neurons (MSNs) expressing dopamine D2 receptors (D2-MSNs), with opposite regulation occurring in D1-MSNs, and serves to protect against cocaine-induced addictive-like behavioral abnormalities. Using ChIP-seq, we identified Src kinase signaling inhibitor 1 (Srcin1; also referred to as p140Cap) as a key gene target for reduced H3R2me2a binding, and found that consequent Srcin1 induction in the NAc decreases Src signaling, cocaine reward, and the motivation to self-administer cocaine. Taken together, these findings suggest that suppression of Src signaling in NAc D2-MSNs, via PRMT6 and H3R2me2a down-regulation, functions as a homeostatic brake to restrain cocaine action, and provide novel candidates for the development of treatments for cocaine addiction.

BAZ1B in Nucleus Accumbens Regulates Reward-Related Behaviors in Response to Distinct Emotional Stimuli.

ATP-dependent chromatin remodeling proteins are being implicated increasingly in the regulation of complex behaviors, including models of several psychiatric disorders. Here, we demonstrate that Baz1b, an accessory subunit of the ISWI family of chromatin remodeling complexes, is upregulated in the nucleus accumbens (NAc), a key brain reward region, in both chronic cocaine-treated mice and mice that are resilient to chronic social defeat stress. In contrast, no regulation is seen in mice that are susceptible to this chronic stress. Viral-mediated overexpression of Baz1b, along with its associated subunit Smarca5, in mouse NAc is sufficient to potentiate both rewarding responses to cocaine, including cocaine self-administration, and resilience to chronic social defeat stress. However, despite these similar, proreward behavioral effects, genome-wide mapping of BAZ1B in NAc revealed mostly distinct subsets of genes regulated by these chromatin remodeling proteins after chronic exposure to either cocaine or social stress. Together, these findings suggest important roles for BAZ1B and its associated chromatin remodeling complexes in NAc in the regulation of reward behaviors to distinct emotional stimuli and highlight the stimulus-specific nature of the actions of these regulatory proteins. SIGNIFICANCE STATEMENT: We show that BAZ1B, a component of chromatin remodeling complexes, in the nucleus accumbens regulates reward-related behaviors in response to chronic exposure to both rewarding and aversive stimuli by regulating largely distinct subsets of genes.

Bidirectional Synaptic Structural Plasticity after Chronic Cocaine Administration Occurs through Rap1 Small GTPase Signaling.

Dendritic spines are the sites of most excitatory synapses in the CNS, and opposing alterations in the synaptic structure of medium spiny neurons (MSNs) of the nucleus accumbens (NAc), a primary brain reward region, are seen at early versus late time points after cocaine administration. Here we investigate the time-dependent molecular and biochemical processes that regulate this bidirectional synaptic structural plasticity of NAc MSNs and associated changes in cocaine reward in response to chronic cocaine exposure. Our findings reveal key roles for the bidirectional synaptic expression of the Rap1b small GTPase and an associated local synaptic protein translation network in this process. The transcriptional mechanisms and pathway-specific inputs to NAc that regulate Rap1b expression are also characterized. Collectively, these findings provide a precise mechanism by which nuclear to synaptic interactions induce "metaplasticity" in NAc MSNs, and we reveal the specific effects of this plasticity on reward behavior in a brain circuit-specific manner.

ACF chromatin-remodeling complex mediates stress-induced depressive-like behavior.

Improved treatment for major depressive disorder (MDD) remains elusive because of the limited understanding of its underlying biological mechanisms. It is likely that stress-induced maladaptive transcriptional regulation in limbic neural circuits contributes to the development of MDD, possibly through epigenetic factors that regulate chromatin structure. We establish that persistent upregulation of the ACF (ATP-utilizing chromatin assembly and remodeling factor) ATP-dependent chromatin-remodeling complex, occurring in the nucleus accumbens of stress-susceptible mice and depressed humans, is necessary for stress-induced depressive-like behaviors. We found that altered ACF binding after chronic stress was correlated with altered nucleosome positioning, particularly around the transcription start sites of affected genes. These alterations in ACF binding and nucleosome positioning were associated with repressed expression of genes implicated in susceptibility to stress. Together, our findings identify the ACF chromatin-remodeling complex as a critical component in the development of susceptibility to depression and in regulating stress-related behaviors.

Dishevelled-2 regulates cocaine-induced structural plasticity and Rac1 activity in the nucleus accumbens.

Chronic cocaine exposure increases the density of dendritic spines on medium spiny neurons (MSNs), the predominant neuronal cell type of the nucleus accumbens (NAc), a key brain reward region. We recently showed that suppression of Rac1, a small GTPase, is a critical mediator of this structural plasticity, but the upstream determinants of Rac1 activity in this context remain to be elucidated. In this study we examined whether isoforms of Dishevelled, a key hub protein of multiple branches of Wnt signaling, including Rac1, are regulated in the NAc by chronic cocaine, and whether these Dishevelled isoforms control Rac1 activity in this brain region in vivo. We found that chronic cocaine administration decreased expression of Dishevelled-2, and several other Wnt signaling components, in the NAc, and that overexpression of Dishevelled-2, but not Dishevelled-1, conversely upregulated Rac1 activity and prevented the cocaine induction of dendritic spines on NAc MSNs. We posit that the cocaine-induced downregulation of Dishevelled-2 in the NAc is an upstream regulator of Rac1 activity and plays an important role in the dynamic structural plasticity of NAc MSNs seen in response to chronic cocaine exposure.

Epigenetic basis of opiate suppression of Bdnf gene expression in the ventral tegmental area.

Brain-derived neurotrophic factor (BDNF) has a crucial role in modulating neural and behavioral plasticity to drugs of abuse. We found a persistent downregulation of exon-specific Bdnf expression in the ventral tegmental area (VTA) in response to chronic opiate exposure, which was mediated by specific epigenetic modifications at the corresponding Bdnf gene promoters. Exposure to chronic morphine increased stalling of RNA polymerase II at these Bdnf promoters in VTA and altered permissive and repressive histone modifications and occupancy of their regulatory proteins at the specific promoters. Furthermore, we found that morphine suppressed binding of phospho-CREB (cAMP response element binding protein) to Bdnf promoters in VTA, which resulted from enrichment of trimethylated H3K27 at the promoters, and that decreased NURR1 (nuclear receptor related-1) expression also contributed to Bdnf repression and associated behavioral plasticity to morphine. Our findings suggest previously unknown epigenetic mechanisms of morphine-induced molecular and behavioral neuroadaptations.