Activity-regulated Gene Research Articles

Single-nucleus RNA-seq identifies one galanin neuronal subtype in mouse preoptic hypothalamus activated during recovery from sleep deprivation

The preoptic area of the hypothalamus (POA) is essential for sleep regulation. However, the cellular makeup of the POA is heterogeneous, and the molecular identities of the sleep-promoting cells remain elusive. To address this question, this study compares mice during recovery sleep following sleep deprivation to mice allowed extended sleep. Single-nucleus RNA sequencing (single-nucleus RNA-seq) identifies one galanin inhibitory neuronal subtype that shows upregulation of rapid and delayed activity-regulated genes during recovery sleep. This cell type expresses higher levels of growth hormone receptor and lower levels of estrogen receptor compared to other galanin subtypes. single-nucleus RNA-seq also reveals cell-type-specific upregulation of purinergic receptor (P2ry14) and serotonin receptor (Htr2a) during recovery sleep in this neuronal subtype, suggesting possible mechanisms for sleep regulation. Studies with RNAscope validate the single-nucleus RNA-seq findings. Thus, the combined use of single-nucleus RNA-seq and activity-regulated genes identifies a neuronal subtype functionally involved in sleep regulation.

Neurodegenerative diseases reflect the reciprocal roles played by retroelements in regulating memory and immunity.

Tetrapod endogenous retroelements (ERE) encode proteins that have been exapted to perform many roles in development and also in innate immunity, including GAG (group specific antigen) proteins from the ERE long terminal repeat (LTR) family, some of which can assemble into viral-like capsids (VLCs) and transmit mRNA across synapses. The best characterized member of this family is ARC (activity-regulated cytoskeletal gene), that is involved in memory formation. Other types of EREs, such as LINES and SINES (long and short interspersed elements), have instead been exapted for immune defenses against infectious agents. These immune EREs identify host transcripts by forming the unusual left-handed Z-DNA and Z-RNA conformations to enable self/nonself discrimination. Elevated levels of immune EREs in the brain are associated with neurodegenerative disease. Here I address the question of how pathways based on immune EREs are relate to the memory EREs that mediate neural plasticity. I propose that during infection and in other inflammatory states, ERE encoded GAG capsids deliver interferon-induced immune EREs that rapidly inhibit translation of viral RNAs in the dendritic splines by activation of protein kinase R (PKR). The response limits transmission of viruses and autonomously replicating elements, while protecting bystander cells from stress-induced cell death. Further, the PKR-dependent phosphorylation of proteins, like tau, disrupts the endocytic pathways exploited by viruses to spread to other cells. The responses come at a cost. They impair memory formation and can contribute to pathology by increasing the deposition of amyloid beta.

The molecular cytoarchitecture of the adult mouse brain

The function of the mammalian brain relies upon the specification and spatial positioning of diversely specialized cell types. Yet, the molecular identities of the cell types and their positions within individual anatomical structures remain incompletely known. To construct a comprehensive atlas of cell types in each brain structure, we paired high-throughput single-nucleus RNA sequencing with Slide-seq1,2—a recently developed spatial transcriptomics method with near-cellular resolution—across the entire mouse brain. Integration of these datasets revealed the cell type composition of each neuroanatomical structure. Cell type diversity was found to be remarkably high in the midbrain, hindbrain and hypothalamus, with most clusters requiring a combination of at least three discrete gene expression markers to uniquely define them. Using these data, we developed a framework for genetically accessing each cell type, comprehensively characterized neuropeptide and neurotransmitter signalling, elucidated region-specific specializations in activity-regulated gene expression and ascertained the heritability enrichment of neurological and psychiatric phenotypes. These data, available as an online resource (www.BrainCellData.org), should find diverse applications across neuroscience, including the construction of new genetic tools and the prioritization of specific cell types and circuits in the study of brain diseases.

General anesthesia alters CNS and astrocyte expression of activity-dependent and activity-independent genes.

General anesthesia represents a common clinical intervention and yet can result in long-term adverse CNS effects particularly in the elderly or dementia patients. Suppression of cortical activity is a key feature of the anesthetic-induced unconscious state, with activity being a well-described regulator of pathways important for brain health. However, the extent to which the effects of anesthesia go beyond simple suppression of neuronal activity is incompletely understood. We found that general anesthesia lowered cortical expression of genes induced by physiological activity in vivo, and recapitulated additional patterns of gene regulation induced by total blockade of firing activity in vitro, including repression of neuroprotective genes and induction of pro-apoptotic genes. However, the influence of anesthesia extended beyond that which could be accounted for by activity modulation, including the induction of non activity-regulated genes associated with inflammation and cell death. We next focused on astrocytes, important integrators of both neuronal activity and inflammatory signaling. General anesthesia triggered gene expression changes consistent with astrocytes being in a low-activity environment, but additionally caused induction of a reactive profile, with transcriptional changes enriched in those triggered by stroke, neuroinflammation, and Aß/tau pathology. Thus, while the effects of general anesthesia on cortical gene expression are consistent with the strong repression of brain activity, further deleterious effects are apparent including a reactive astrocyte profile.

Activity-regulated gene expression across cell types of the mouse hippocampus.

Activity-regulated gene (ARG) expression patterns in the hippocampus (HPC) regulate synaptic plasticity, learning, and memory, and are linked to both risk and treatment responses for many neuropsychiatric disorders. The HPC contains discrete classes of neurons with specialized functions, but cell type-specific activity-regulated transcriptional programs are not well characterized. Here, we used single-nucleus RNA-sequencing (snRNA-seq) in a mouse model of acute electroconvulsive seizures (ECS) to identify cell type-specific molecular signatures associated with induced activity in HPC neurons. We used unsupervised clustering and a priori marker genes to computationally annotate 15,990 high-quality HPC neuronal nuclei from N = 4 mice across all major HPC subregions and neuron types. Activity-induced transcriptomic responses were divergent across neuron populations, with dentate granule cells being particularly responsive to activity. Differential expression analysis identified both upregulated and downregulated cell type-specific gene sets in neurons following ECS. Within these gene sets, we identified enrichment of pathways associated with varying biological processes such as synapse organization, cellular signaling, and transcriptional regulation. Finally, we used matrix factorization to reveal continuous gene expression patterns differentially associated with cell type, ECS, and biological processes. This work provides a rich resource for interrogating activity-regulated transcriptional responses in HPC neurons at single-nuclei resolution in the context of ECS, which can provide biological insight into the roles of defined neuronal subtypes in HPC function.

Development of a platform to investigate long-term potentiation in human iPSC-derived neuronal networks.

SummaryImpairment of long-term potentiation (LTP) is a common feature of many pre-clinical models of neurological disorders; however, studies in humans are limited by the inaccessibility of the brain. Human induced pluripotent stem cells (hiPSCs) provide a unique opportunity to study LTP in disease-specific genetic backgrounds. Here we describe a multi-electrode array (MEA)-based assay to investigate chemically induced LTP (cLTP) across entire networks of hiPSC-derived midbrain dopaminergic (DA) and cortical neuronal populations that lasts for days, allowing studies of the late phases of LTP and enabling detection of associated molecular changes. We show that cLTP on midbrain DA neuronal networks is largely independent of the N-methyl-D-aspartate receptor (NMDAR) and partially dependent on brain-derived neurotrophic factor (BDNF). Finally, we describe activity-regulated gene expression induced by cLTP. This cLTP-MEA assay platform will enable phenotype discovery and higher-throughput analyses of synaptic plasticity on hiPSC-derived neurons.

Mapping cis-regulatory elements in human neurons links psychiatric disease heritability and activity-regulated transcriptional programs.

SUMMARYGenome-wide association studies (GWASs) have identified hundreds of loci associated with psychiatric diseases, yet there is a lack of understanding of disease pathophysiology. Common risk variants can shed light on the underlying molecular mechanisms; however, identifying causal variants remains challenging. We map cis-regulatory elements in human neurons derived from pluripotent stem cells. This system allows us to determine enhancers that activate the transcription of neuronal activity-regulated gene programs, which are thought to be critical for synaptic plasticity and are not possible to identify from postmortem tissues. Using the activity-by-contact model, we create variant-to-gene maps to interpret the function of GWAS variants. Our work nominates a subset of variants to elucidate the molecular mechanisms involving GWAS-significant loci. It also highlights that in vitro human cellular models are a powerful platform for identifying and mechanistic studies of human trait-associated genetic variants in cell states that are inaccessible from other types of human samples.

Cohesin-dependence of neuronal gene expression relates to chromatin loop length.

Cohesin and CTCF are major drivers of 3D genome organization, but their role in neurons is still emerging. Here, we show a prominent role for cohesin in the expression of genes that facilitate neuronal maturation and homeostasis. Unexpectedly, we observed two major classes of activity-regulated genes with distinct reliance on cohesin in mouse primary cortical neurons. Immediate early genes (IEGs) remained fully inducible by KCl and BDNF, and short-range enhancer-promoter contacts at the IEGs Fos formed robustly in the absence of cohesin. In contrast, cohesin was required for full expression of a subset of secondary response genes characterized by long-range chromatin contacts. Cohesin-dependence of constitutive neuronal genes with key functions in synaptic transmission and neurotransmitter signaling also scaled with chromatin loop length. Our data demonstrate that key genes required for the maturation and activation of primary cortical neurons depend on cohesin for their full expression, and that the degree to which these genes rely on cohesin scales with the genomic distance traversed by their chromatin contacts.

Investigation of the correlation between immune thrombocytopenia and T cell activity-regulated gene polymorphism using functional study

Thrombocytopenia is a condition where the platelet count is under 100 × 109/L, which is caused by various disorders. However, the mechanism of thrombocytopenia is still unclear. Hence, we tried to investigate the correlation between immune thrombocytopenia (ITP) and single nucleotide polymorphisms (SNPs) of genes related to T cell activation. There were 32 ITP patients and 30 healthy controls enrolled in this study. PCR and sequencing were used to find out the significant SNPs, which we focused on the promoter region of CTLA4 and CD28. In this study, the ITP cases were divided into primary ITP group, secondary ITP group, and the combination of the two to the follow-up analysis. Moreover, dual-luciferase reporter assay was used to evaluate the transcription activity of the significant SNP. We found the − 1765_rs11571315 of CTLA4 gene was associated with primary ITP (p = 0.006), secondary ITP (p = 0.008), and the combination of the two (p = 0.003). Moreover, the −318_rs5742909 also had statistical significance in secondary ITP group that was only caused by autoimmune disease (p = 0.019). In functional study, the rs5742909 would decrease 19% of the transcription activity when it carried a T-allele at this position (p = 0.040). It was noted that CTLA4 gene polymorphism was related to ITP but not CD28. According to our results, we surmised that CTLA4 is involved in the pathogenesis of ITP, and the secondary ITP result from the lower CTLA4 expression that leads to T cell over-activation.

Outcome-Locked Cholinergic Signaling Suppresses Prefrontal Encoding of Stimulus Associations.

Acetylcholine (ACh) is thought to control arousal, attention, and learning by slowly modulating cortical excitability and plasticity. Recent studies, however, discovered that cholinergic neurons emit precisely timed signals about the aversive outcome at millisecond precision. To investigate the functional relevance of such phasic cholinergic signaling, we manipulated and monitored cholinergic terminals in the mPFC while male mice associated a neutral conditioned stimulus (CS) with mildly aversive eyelid shock (US) over a short temporal gap. Optogenetic inhibition of cholinergic terminals during the US promoted the formation of the CS-US association. On the contrary, optogenetic excitation of cholinergic terminals during the US blocked the association formation. The bidirectional behavioral effects paralleled the corresponding change in the expression of an activity-regulated gene, c-Fos in the mPFC. In contrast, optogenetic inhibition of cholinergic terminals during the CS impaired associative learning, whereas their excitation had marginal effects. In parallel, photometric recording from cholinergic terminals in the mPFC revealed strong innate phasic responses to the US. With subsequent CS-US pairings, cholinergic terminals weakened the responses to the US while developing strong responses to the CS. The across-session changes in the CS- and US-evoked terminal responses were correlated with associative memory strength. These findings suggest that phasic cholinergic signaling in the mPFC exerts opposite effects on aversive associative learning depending on whether it is emitted by the outcome or the cue.SIGNIFICANCE STATEMENT Drugs compensating for the decline of acetylcholine (ACh) are used for cognitive impairment, such as Alzheimer's disease. However, their beneficial effects are limited, demanding new strategies based on better understandings of how ACh modulates cognition. Here, we report that by manipulating ACh signals in the mPFC, we can control the strength of aversive associative learning in mice. Specifically, the suppression of ACh signals during an aversive outcome facilitated its association with a preceding cue. In contrast, the suppression of ACh signals during the cue impaired learning. Considering that this paradigm depends on the brain regions affected in Alzheimer's disease, our findings indicate that precisely timed control of ACh signals is essential to refine ACh-based strategies for cognitive enhancement.

Pharmacological profiles of 5‐HT 2A Receptor (5‐HT 2A R):5‐HT 2C R Interactions In Vitro

Aims The serotonin 5-HT2A receptor (5-HT2AR) and 5-HT2CR have garnered intense interest from drug discovery programs due to their role in metabolic (e.g., obesity) and neuropsychiatric disorders (e.g., addiction, depression, psychosis). Functional and physical interactions between the 5-HT2AR and 5-HT2CR occur in living cells (Felsing et al., PLoS One 13(8):e0203137, 2018) and rat brain (Price et al., ACS Chem Neurosci 10:3241, 2019). Intriguingly, when the two receptors are in complex, the 5-HT2CR is functionally dominant, i.e., silences signaling from the 5-HT2AR in vitro (Moutkine et al., J Biol Chem 292:6352, 2017). In the present study, we have extended these observations to uncover additional cellular signaling output associated with the 5-HT2AR:5-HT2CR complex and explore disruption of the receptor interfaces important for their interaction and signaling. Methods . A split luciferase complementation assay (LCA) was employed to assess the direct coupling of 5-HT2AR and 5-HT2CR in live HEK293 cells. The cDNA for the human 5-HT2AR and 5-HT2CR fused with complementary N-terminal (NLuc) and C-terminal (CLuc) inactive fragments, respectively, were transfected into HEK293 cells using template mammalian expression vectors and standard cloning techniques. Association of the receptor constructs in vitro within ~50 nm reconstitutes luciferase activity and light is released in the presence of D-luciferin. The expression of the activity-regulated immediate early gene early growth response 1 (EGR1) was used to trace cellular responsivity to 5-HT in cells. A truncated version of the 5-HT2CR transmembrane helix IV (TM4) domain conjugated to a polyethylene glycol chain (PEG-TM4 5-HT2CR) was synthesized as a tool to disrupt formation of the 5-HT2AR:5-HT2CR complex. Results . Serotonin (5-HT) readily induced robust expression of EGR1 mRNA in cells solely expressing 5-HT2AR, but not 5-HT2CR; 5-HT-evoked EGR1 expression was blunted in cells co-expressing the 5-HT2CR plus 5-HT2AR. In the LCA, vehicle (0.1% DMSO) treated cells exhibit robust complementation (i.e., increased luminescence) between the 5-HT2AR and 5-HT2CR. The PEG-TM4 5-HT2CR peptide significantly decreased complementation (p<0.05) in a concentration-dependent manner indicating an inhibition of association between the 5-HT2AR and 5-HT2CR. Conclusions Blunted 5-HT-evoked EGR1 expression in cells expressing both receptors supports the “master regulator” role for the 5-HT2CR when in complex with the 5-HT2AR. Secondly, the formation of the 5-HT2AR:5-HT2CR complex in vitro is dependent on an interaction interface located at the 5-HT2CR TM4. Ongoing studies are testing the hypothesis that the PEG-TM4 5-HT2CR peptide restores a critical signaling output of 5-HT2AR function, which will indicate the importance of allosteric regulation by the heteromer complex. Future studies with our novel TM domain peptides will help elucidate determinants of receptor complex formation and signaling and the physiological relevance of the 5-HT2AR:5-HT2CR complex in vivo.

The WD40-Repeat Protein WDR-20 and the Deubiquitinating Enzyme USP-46 Promote Cell Surface Levels of Glutamate Receptors.

Reversible modification of AMPA receptors (AMPARs) with ubiquitin regulates receptor levels at synapses and controls synaptic strength. The conserved deubiquitinating enzyme (DUB) ubiquitin-specific protease-46 (USP-46) removes ubiquitin from AMPARs and protects them from degradation in both Caenorhabditis elegans and mammals. Although DUBs are critical for diverse physiological processes, the mechanisms that regulate DUBs, especially in the nervous system, are not well understood. We and others previously showed that the WD40-repeat proteins WDR-48 and WDR-20 bind to and stimulate the catalytic activity of USP-46. Here, we identify an activity-dependent mechanism that regulates WDR-20 expression and show that WDR-20 works together with USP-46 and WDR-48 to promote surface levels of the C. elegans AMPAR GLR-1. usp-46, wdr-48, and wdr-20 loss-of-function mutants exhibit reduced levels of GLR-1 at the neuronal surface and corresponding defects in GLR-1-mediated behavior. Increased expression of WDR-20, but not WDR-48, is sufficient to increase GLR-1 surface levels in an usp-46-dependent manner. Loss of usp-46, wdr-48, and wdr-20 function reduces the rate of local GLR-1 insertion in neurites, whereas overexpression of wdr-20 is sufficient to increase the rate of GLR-1 insertion. Genetic manipulations that chronically reduce or increase glutamate signaling result in reciprocal alterations in wdr-20 transcription and homeostatic compensatory changes in surface GLR-1 levels that are dependent on wdr-20 This study identifies wdr-20 as a novel activity-regulated gene that couples chronic changes in synaptic activity with increased local insertion and surface levels of GLR-1 via the DUB USP-46.SIGNIFICANCE STATEMENT Deubiquitinating enzymes (DUBs) are critical regulators of synapse development and function; however, the regulatory mechanisms that control their various physiological functions are not well understood. This study identifies a novel role for the DUB ubiquitin-specific protease-46 (USP-46) and its associated regulatory protein WD40-repeat protein-20 (WDR-20) in regulating local insertion of glutamate receptors into the neuronal cell surface. This work also identifies WDR-20 as an activity-regulated gene that couples chronic changes in synaptic activity with homeostatic compensatory increases in surface levels of GLR-1 via USP-46. Given that 35% of USP family DUBs associate with WDR proteins, understanding the mechanisms by which WDR proteins regulate USP-46 could have implications for a large number of DUBs in other cell types.

Neuronal activity regulates alternative exon usage

Neuronal activity-regulated gene transcription underlies plasticity-dependent changes in the molecular composition and structure of neurons. A large number of genes regulated by different neuronal plasticity inducing pathways have been identified, but altered gene expression levels represent only part of the complexity of the activity-regulated transcriptional program. Alternative splicing, the differential inclusion and exclusion of exonic sequence in mRNA, is an additional mechanism that is thought to define the activity-dependent transcriptome. Here, we present a genome wide microarray-based survey to identify exons with increased expression levels at 1, 4 or 8 h following neuronal activity in the murine hippocampus provoked by generalized seizures. We used two different bioinformatics approaches to identify alternative activity-induced exon usage and to predict alternative splicing, ANOSVA (ANalysis Of Splicing VAriation) which we here adjusted to accommodate data from different time points and FIRMA (Finding Isoforms using Robust Multichip Analysis). RNA sequencing, in situ hybridization and reverse transcription PCR validate selected activity-dependent splicing events of previously described and so far undescribed activity-regulated transcripts, including Homer1a, Homer1d, Ania3, Errfi1, Inhba, Dclk1, Rcan1, Cda, Tpm1 and Krt75. Taken together, our survey significantly adds to the comprehensive understanding of the complex activity-dependent neuronal transcriptomic signature. In addition, we provide data sets that will serve as rich resources for future comparative expression analyses.

The Rab5-Rab11 Endosomal Pathway is Required for BDNF-Induced CREB Transcriptional Regulation in Hippocampal Neurons.

Brain-derived neurotrophic factor (BDNF) is a key regulator of the morphology and connectivity of central neurons. We have previously shown that BDNF/TrkB signaling regulates the activity and mobility of the GTPases Rab5 and Rab11, which in turn determine the postendocytic sorting of signaling TrkB receptors. Moreover, decreased Rab5 or Rab11 activity inhibits BDNF-induced dendritic branching. Whether Rab5 or Rab11 activity is important for local events only or for regulating nuclear signaling and gene expression is unknown. Here, we investigated, in rat hippocampal neuronal cultures derived from embryos of unknown sex, whether BDNF-induced signaling cascades are altered when early and recycling endosomes are disrupted by the expression of dominant-negative mutants of Rab5 and Rab11. The activity of both Rab5 and Rab11 was required for sustained activity of Erk1/2 and nuclear CREB phosphorylation, and increased transcription of a BDNF-dependent program of gene expression containing CRE binding sites, which includes activity-regulated genes such as Arc, Dusp1, c-fos, Egr1, and Egr2, and growth and survival genes such as Atf3 and Gem Based on our results, we propose that early and recycling endosomes provide a platform for the integration of neurotrophic signaling from the plasma membrane to the nucleus in neurons, and that this mechanism is likely to regulate neuronal plasticity and survival.SIGNIFICANCE STATEMENT BDNF is a neurotrophic factor that regulates plastic changes in the brain, including dendritic growth. The cellular and molecular mechanisms underlying this process are not completely understood. Our results uncover the cellular requirements that central neurons possess to integrate the plasma membrane into nuclear signaling in neurons. Our results indicate that the endosomal pathway is required for the signaling cascade initiated by BDNF and its receptors at the plasma membrane to modulate BDNF-dependent gene expression and neuronal dendritic growth mediated by the CREB transcription factor. CREB is a key transcription factor regulating circuit development and learning and memory.

Modeling human age-associated increase in Gadd45γ expression leads to spatial recognition memory impairments in young adult mice

Aging is associated with the progressive decay of cognitive function. Hippocampus-dependent processes, such as the formation of spatial memory, are particularly vulnerable to aging. Currently, the molecular mechanisms responsible for age-dependent cognitive decline are largely unknown. Here, we investigated the expression and function of the growth arrest DNA damage gamma (Gadd45γ) during aging and cognition. We report that Gadd45γ expression is increased in the hippocampus of aged humans and that Gadd45γ overexpression in the young adult mouse hippocampus compromises cognition. Moreover, Gadd45γ overexpression in hippocampal neurons disrupted cAMP response element-binding protein signaling and the expression of well-established activity-regulated genes. This work shows that Gadd45γ expression is tightly controlled in the hippocampus and its disruption may be a mechanism contributing to age-related cognitive impairments observed in humans.

The NMDA receptor subunit GluN3A regulates synaptic activity-induced and myocyte enhancer factor 2C (MEF2C)-dependent transcription

N-Methyl-d-aspartate type glutamate receptors (NMDARs) are key mediators of synaptic activity-regulated gene transcription in neurons, both during development and in the adult brain. Developmental differences in the glutamate receptor ionotropic NMDA 2 (GluN2) subunit composition of NMDARs determines whether they activate the transcription factor cAMP-responsive element-binding protein 1 (CREB). However, whether the developmentally regulated GluN3A subunit also modulates NMDAR-induced transcription is unknown. Here, using an array of techniques, including quantitative real-time PCR, immunostaining, reporter gene assays, RNA-Seq, and two-photon glutamate uncaging with calcium imaging, we show that knocking down GluN3A in rat hippocampal neurons promotes the inducible transcription of a subset of NMDAR-sensitive genes. We found that this enhancement is mediated by the accumulation of phosphorylated p38 mitogen-activated protein kinase in the nucleus, which drives the activation of the transcription factor myocyte enhancer factor 2C (MEF2C) and promotes the transcription of a subset of synaptic activity-induced genes, including brain-derived neurotrophic factor (Bdnf) and activity-regulated cytoskeleton-associated protein (Arc). Our evidence that GluN3A regulates MEF2C-dependent transcription reveals a novel mechanism by which NMDAR subunit composition confers specificity to the program of synaptic activity-regulated gene transcription in developing neurons.

Dotdotdot: an automated approach to quantify multiplex single molecule fluorescent in situ hybridization (smFISH) images in complex tissues.

Multiplex single-molecule fluorescent in situ hybridization (smFISH) is a powerful method for validating RNA sequencing and emerging spatial transcriptomic data, but quantification remains a computational challenge. We present a framework for generating and analyzing smFISH data in complex tissues while overcoming autofluorescence and increasing multiplexing capacity. We developed dotdotdot (https://github.com/LieberInstitute/dotdotdot) as a corresponding software package to quantify RNA transcripts in single nuclei and perform differential expression analysis. We first demonstrate robustness of our platform in single mouse neurons by quantifying differential expression of activity-regulated genes. We then quantify spatial gene expression in human dorsolateral prefrontal cortex (DLPFC) using spectral imaging and dotdotdot to mask lipofuscin autofluorescence. We lastly apply machine learning to predict cell types and perform downstream cell type-specific expression analysis. In summary, we provide experimental workflows, imaging acquisition and analytic strategies for quantification and biological interpretation of smFISH data in complex tissues.

Sox11 is an Activity-Regulated Gene with Dentate-Gyrus-Specific Expression Upon General Neural Activation.

Neuronal activity initiates transcriptional programs that shape long-term changes in plasticity. Although neuron subtypes differ in their plasticity response, most activity-dependent transcription factors (TFs) are broadly expressed across neuron subtypes and brain regions. Thus, how region- and neuronal subtype-specific plasticity are established on the transcriptional level remains poorly understood. We report that in young adult (i.e., 6-8weeks old) mice, the developmental TF SOX11 is induced in neurons within 6h either by electroconvulsive stimulation or by exploration of a novel environment. Strikingly, SOX11 induction was restricted to the dentate gyrus (DG) of the hippocampus. In the novel environment paradigm, SOX11 was observed in a subset of c-FOS expressing neurons (ca. 15%); whereas around 75% of SOX11+ DG granule neurons were c-FOS+, indicating that SOX11 was induced in an activity-dependent fashion in a subset of neurons. Environmental enrichment or virus-mediated overexpression of SOX11 enhanced the excitability of DG granule cells and downregulated the expression of different potassium channel subunits, whereas conditional Sox11/4 knock-out mice presented the opposite phenotype. We propose that Sox11 is regulated in an activity-dependent fashion, which is specific to the DG, and speculate that activity-dependent Sox11 expression may participate in the modulation of DG neuron plasticity.

The atypical cadherin flamingo determines the competence of neurons for activity-dependent fine-scale topography

The topographic projection of afferent terminals into two-dimensional maps is essential for sensory systems to encode the locations of sensory stimuli. In vertebrates, guidance cues are critical for establishing a coarse topographic map, while neuronal activity directs fine-scale topography between adjacent afferent terminals. However, the molecular mechanism underlying activity-dependent fine-scale topography is not well known. Studies in the Drosophila visual system have demonstrated that cell-adhesion molecules direct fine-scale topography, but whether or not these molecules are involved in activity-dependent fine-scale topography remains to be determined. We previously reported that the nociceptors in Drosophila larvae form an activity-dependent fine-scale topographic system. The establishment of this system is instructed by the level of neuronal activity in individual nociceptors. Here, we show that the atypical cadherin Flamingo (Fmi) is required for establishing the nociceptor topographic map. We found that the topographic defect caused by loss of fmi was epistatic to the inhibition of neuronal activity and the overexpression of the activity-regulated gene Trim9. These results suggest that Fmi and neuronal activity interact to regulate fine-scale topography. This study provides a link between neuronal activity and the cell-adhesion molecule in the establishment of fine-scale topography.

Firing Rate Homeostasis Can Occur in the Absence of Neuronal Activity-Regulated Transcription.

Despite dynamic inputs, neuronal circuits maintain relatively stable firing rates over long periods. This maintenance of firing rate, or firing rate homeostasis, is likely mediated by homeostatic mechanisms such as synaptic scaling and regulation of intrinsic excitability. Because some of these homeostatic mechanisms depend on transcription of activity-regulated genes, including Arc and Homer1a, we hypothesized that activity-regulated transcription would be required for firing rate homeostasis. Surprisingly, however, we found that cultured mouse cortical neurons from both sexes grown on multi-electrode arrays homeostatically adapt their firing rates to persistent pharmacological stimulation even when activity-regulated transcription is disrupted. Specifically, we observed firing rate homeostasis in Arc knock-out neurons, as well as knock-out neurons lacking the activity-regulated transcription factors AP1 and SRF. Firing rate homeostasis also occurred normally during acute pharmacological blockade of transcription. Thus, firing rate homeostasis in response to increased neuronal activity can occur in the absence of neuronal-activity-regulated transcription.SIGNIFICANCE STATEMENT Neuronal circuits maintain relatively stable firing rates even in the face of dynamic circuit inputs. Understanding the molecular mechanisms that enable this firing rate homeostasis could potentially provide insight into neuronal diseases that present with an imbalance of excitation and inhibition. It has long been proposed that activity-regulated transcription could underlie firing rate homeostasis because activity-regulated genes turn on when neurons are above their target firing rates and include many genes that could regulate firing rate. Surprisingly, despite this prediction, we found that cortical neurons can undergo firing rate homeostasis in the absence of activity-regulated transcription, indicating that firing rate homeostasis can be controlled by non-transcriptional mechanisms.