Genomic Decoding of Neuronal Depolarization by Stimulus-Specific NPAS4 Heterodimers.

Cells regulate gene expression in response to salient external stimuli. In neurons, depolarization leads to the expression of inducible transcription factors (ITFs) that direct subsequent gene regulation. Depolarization encodes both a neuron's action potential (AP) output and synaptic inputs, via excitatory postsynaptic potentials (EPSPs). However, it is unclear if distinct types of electrical activity can be transformed by an ITF into distinct modes of genomic regulation. Here, we show that APs and EPSPs in mouse hippocampal neurons trigger two spatially segregated and molecularly distinct induction mechanisms that lead to the expression of the ITF NPAS4. These two pathways culminate in the formation of stimulus-specific NPAS4 heterodimers that exhibit distinct DNA binding patterns. Thus, NPAS4 differentially communicates increases in a neuron's spiking output and synaptic inputs to the nucleus, enabling gene regulation to be tailored to the type of depolarizing activity along the somato-dendritic axis of a neuron.

Stimulus-specific remodeling of the neuronal transcriptome through nuclear intron-retaining transcripts.

The nuclear envelope has long been considered primarily a physical barrier separating nuclear and cytosolic contents. More recently, nuclear compartmentalization has been shown to have additional regulatory functions in controlling gene expression. A sizeable proportion of protein-coding mRNAs is more prevalent in the nucleus than in the cytosol, suggesting regulated mRNA trafficking to the cytosol, but the mechanisms underlying controlled nuclear mRNA retention remain unclear. Here, we provide a comprehensive map of the subcellular localization of mRNAs in mature mouse cortical neurons, and reveal that transcripts retained in the nucleus comprise the majority of stable intron-retaining mRNAs. Systematically probing the fate of nuclear transcripts upon neuronal stimulation, we found opposite effects on sub-populations of transcripts: while some are targeted for degradation, others complete splicing to generate fully mature mRNAs that are exported to the cytosol and mediate rapid increases in protein levels. Finally, different forms of stimulation mobilize distinct groups of intron-retaining transcripts, with this selectivity arising from the activation of specific signaling pathways. Overall, our findings uncover a cue-specific control of intron retention as a major regulator of acute remodeling of the neuronal transcriptome.

Activity-Dependent Degradation of the Nascentome by the Neuronal Membrane Proteasome.

Activity-dependent changes in neuronal function require coordinated regulation of the protein synthesis and protein degradation machinery to maintain protein homeostasis, critical for proper neuronal function. However, the biochemical evidence for this balance and coordination is largely lacking. Leveraging our recent discovery of a neuronal-specific 20S membrane proteasome complex (NMP), we began exploring how neuronal activity regulates its function. Here, we found that the NMP degrades exclusively a large fraction of ribosome-associated nascent polypeptides that are being newly synthesized during neuronal stimulation. Using deep-coverage and global mass spectrometry, we identified the nascent protein substrates of the NMP, which included products encoding immediate-early genes, such as c-Fos and Npas4. Intriguingly, we found that turnover of nascent polypeptides and not full-length proteins through the NMP occurred independent of canonical ubiquitylation pathways. We propose that these findings generally define a neuronal activity-induced protein homeostasis program of coordinated protein synthesis and degradation through the NMP.

Cell assembly analysis of neural circuits for innate behavior in Drosophila melanogaster using an immediate early gene stripe/egr-1.

Innate behavior, such as courtship behavior, is controlled by a genetically defined set of neurons. To date, it remains challenging to visualize and artificially control the neural population that is active during innate behavior in a whole-brain scale. Immediate early genes (IEGs), whose expression is induced by neural activity, can serve as powerful tools to map neural activity in the animal brain. We screened for IEGs in vinegar fly Drosophila melanogaster and identified stripe/egr-1 as a potent neural activity marker. Focusing on male courtship as a model of innate behavior, we demonstrate that stripe-GAL4-mediated reporter expression can label fruitless (fru)-expressing neurons involved in courtship in an activity (experience)-dependent manner. Optogenetic reactivation of the labeled neurons elicited sexual behavior in males, whereas silencing of the labeled neurons suppressed courtship and copulation. Further, by combining stripe-GAL4-mediated reporter expression and detection of endogenous Stripe expression, we established methods that can label neurons activated under different contexts in separate time windows in the same animal. The cell assembly analysis of fru neural population in males revealed that distinct groups of neurons are activated during interactions with a female or another male. These methods will contribute to building a deeper understanding of neural circuit mechanisms underlying innate insect behavior.

The guanine nucleotide exchange factor RapGEF2 is required for ERK-dependent immediate-early gene (Egr1) activation during fear memory formation.

The MAP kinase ERK is important for neuronal plasticity underlying associative learning, yet specific molecular pathways for neuronal ERK activation are undetermined. RapGEF2 is a neuron-specific cAMP sensor that mediates ERK activation. We investigated whether it is required for cAMP-dependent ERK activation leading to other downstream neuronal signaling events occurring during associative learning, and if RapGEF2-dependent signaling impairments affect learned behavior. Camk2α-cre+/-::RapGEF2fl/fl mice with depletion of RapGEF2 in hippocampus and amygdala exhibit impairments in context- and cue-dependent fear conditioning linked to corresponding impairment in Egr1 induction in these two brain regions. Camk2α-cre+/-::RapGEF2fl/fl mice show decreased RapGEF2 expression in CA1 and dentate gyrus associated with abolition of pERK and Egr1, but not of c-Fos induction, following fear conditioning, impaired freezing to context after fear conditioning, and impaired cAMP-dependent long-term potentiation at perforant pathway and Schaffer collateral synapses in hippocampal slices ex vivo. RapGEF2 expression is largely eliminated in basolateral amygdala, also involved in fear memory, in Camk2α-cre+/-::RapGEF2fl/fl mice. Neither Egr1 nor c-fos induction in BLA after fear conditioning, nor cue-dependent fear learning, are affected by ablation of RapGEF2 in BLA. However, Egr1 induction (but not that of c-fos) in BLA is reduced after restraint stress-augmented fear conditioning, as is freezing to cue after restraint stress-augmented fear conditioning, in Camk2α-cre+/-::RapGEF2fl/fl mice. Cyclic AMP-dependent GEFs have been genetically associated as risk factors for schizophrenia, a disorder associated with cognitive deficits. Here we show a functional link between one of them, RapGEF2, and cognitive processes involved in associative learning in amygdala and hippocampus.

Real-time imaging of Arc/Arg3.1 transcription ex vivo reveals input-specific immediate early gene dynamics.

The ability of neurons to process and store salient environmental features underlies information processing in the brain. Long-term information storage requires synaptic plasticity and regulation of gene expression. While distinct patterns of activity have been linked to synaptic plasticity, their impact on immediate early gene (IEG) expression remains poorly understood. The activity regulated cytoskeleton associated (Arc) gene has received wide attention as an IEG critical for long-term synaptic plasticity and memory. Yet, to date, the transcriptional dynamics of Arc in response to compartment and input-specific activity is unclear. By developing a knock-in mouse to fluorescently tag Arc alleles, we studied real-time transcription dynamics after stimulation of dentate granule cells (GCs) in acute hippocampal slices. To our surprise, we found that Arc transcription displayed distinct temporal kinetics depending on the activation of excitatory inputs that convey functionally distinct information, i.e., medial and lateral perforant paths (MPP and LPP, respectively). Moreover, the transcriptional dynamics of Arc after synaptic stimulation was similar to direct activation of GCs, although the contribution of ionotropic glutamate receptors, L-type voltage-gated calcium channel, and the endoplasmic reticulum (ER) differed. Specifically, we observed an ER-mediated synapse-to-nucleus signal that supported elevations in nuclear calcium and, thereby, rapid induction of Arc transcription following MPP stimulation. By delving into the complex excitation-transcription coupling for Arc, our findings highlight how different synaptic inputs may encode information by modulating transcription dynamics of an IEG linked to learning and memory.

The Integrator Complex in Transcription and Development.

The Integrator complex is conserved across metazoans and controls the fate of many nascent RNAs transcribed by RNA polymerase II (RNAPII). Among the 14 subunits of Integrator is an RNA endonuclease that is crucial for the biogenesis of small nuclear RNAs and enhancer RNAs. Integrator is further employed to trigger premature transcription termination at many protein-coding genes, thereby attenuating gene expression. Integrator thus helps to shape the transcriptome and ensure that genes can be robustly induced when needed. The molecular functions of Integrator subunits beyond the RNA endonuclease remain poorly understood, but some can act independently of the multisubunit complex. We highlight recent molecular insights into Integrator and propose how misregulation of this complex may lead to developmental defects and disease.

A whole-brain analysis of functional connectivity and immediate early gene expression reveals functional network shifts after operant learning.

Previous studies of operant learning have addressed neuronal activities and network changes in specific brain areas, such as the striatum, sensorimotor cortex, prefrontal/orbitofrontal cortices, and hippocampus. However, how changes in the whole-brain network are caused by cellular-level changes remains unclear. We, therefore, combined resting-state functional magnetic resonance imaging (rsfMRI) and whole-brain immunohistochemical analysis of early growth response 1 (EGR1), a marker of neural plasticity, to elucidate the temporal and spatial changes in functional networks and underlying cellular processes during operant learning. We used an 11.7-Tesla MRI scanner and whole-brain immunohistochemical analysis of EGR1 in mice during the early and late stages of operant learning. In the operant training, mice received a reward when they pressed left and right buttons alternately, and were punished with a bright light when they made a mistake. A group of mice (n = 22) underwent the first rsfMRI acquisition before behavioral sessions, the second acquisition after 3 training-session-days (early stage), and the third after 21 training-session-days (late stage). Another group of mice (n = 40) was subjected to histological analysis 15 min after the early or late stages of behavioral sessions. Functional connectivity increased between the limbic areas and thalamus or auditory cortex after the early stage of training, and between the motor cortex, sensory cortex, and striatum after the late stage of training. The density of EGR1-immunopositive cells in the motor and sensory cortices increased in both the early and late stages of training, whereas the density in the amygdala increased only in the early stage of training. The subcortical networks centered around the limbic areas that emerged in the early stage have been implicated in rewards, pleasures, and fears. The connectivities between the motor cortex, somatosensory cortex, and striatum that consolidated in the late stage have been implicated in motor learning. Our multimodal longitudinal study successfully revealed temporal shifts in brain regions involved in behavioral learning together with the underlying cellular-level plasticity between these regions. Our study represents a first step towards establishing a new experimental paradigm that combines rsfMRI and immunohistochemistry to link macroscopic and microscopic mechanisms involved in learning.

Acute stress modulates the outcome of traumatic brain injury-associated gene expression and behavioral responses.

Psychological stress and traumatic brain injury (TBI) result in long-lasting emotional and behavioral impairments in patients. So far, the interaction of psychological stress with TBI not only in the brain but also in peripheral organs is poorly understood. Herein, the impact of acute stress (AS) occurring immediately before TBI is investigated. For this, a mouse model of restraint stress and TBI was employed, and their influence on behavior and gene expression in brain regions, the hypothalamic-pituitary-adrenal (HPA) axis, and peripheral organs was analyzed. Results demonstrate that, compared to single AS or TBI exposure, mice treated with AS prior to TBI showed sex-specific alterations in body weight, memory function, and locomotion. The induction of immediate early genes (IEGs, e.g., c-Fos) by TBI was modulated by previous AS in several brain regions. Furthermore, IEG upregulation along the HPA axis (e.g., pituitary, adrenal glands) and other peripheral organs (e.g., heart) was modulated by AS-TBI interaction. Proteomics of plasma samples revealed proteins potentially mediating this interaction. Finally, the deletion of Atf3 diminished the TBI-induced induction of IEGs in peripheral organs but left them largely unaltered in the brain. In summary, AS immediately before brain injury affects the brain and, to a strong degree, also responses in peripheral organs.

Characterization of the Ictalurid herpesvirus 1 immediate-early gene ORF24 and its potential role in transcriptional regulation in yeast.

Herpesviruses adhere to a precise temporal expression model in which immediate-early (IE) genes play a crucial role in regulating the viral life cycle. However, there is a lack of functional research on the IE genes in Ictalurid herpesvirus 1 (IcHV-1). In this study, we identified the IcHV-1 ORF24 as an IE gene via a metabolic inhibition assay, and subcellular analysis indicated its predominant localisation in the nucleus. To investigate its function, we performed yeast reporter assays using an ORF24 fusion protein containing the Gal4-BD domain and found that BD-ORF24 was able to activate HIS3/lacZ reporter genes without the Gal4-AD domain. Our findings provide concrete evidence that ORF24 is indeed an IE gene that likely functions as a transcriptional regulator during IcHV-1 infection. This work contributes to our understanding of the molecular mechanisms underlying fish herpesvirus IE gene expression.

Comprehensive analysis of transcription factors involved in odontoblast differentiation mechanism.

Primary cultured odontoblasts rapidly lose their tissue-specific phenotype. To identify transcription factors (TF) that are important for the maintenance of the odontoblast phenotype, primary cultures of C57BL/6 J mouse dental mesenchymal cells (DMC) were isolated, and expression of TF and odontoblast marker genes in cells immediately after isolation and 2 days after culture were comprehensively evaluated and compared using RNA-sequencing (RNA-seq). The expression of odontoblast markers in mouse dental mesenchymal cells decreased rapidly after isolation. In addition, the expression of Hedgehog-related, Notch-related, and immediate- early gene (IEG)-related transcription factors significantly decreased. Forced expression of these genes in lentiviral vectors, together with fibroblast growth factor 4 (FGF4), fibroblast growth factor 9 (FGF9), and the Wnt pathway activator CHIR99021, significantly induced the expression of odontogenic marker genes. These results indicate, for the first time, that Notch signaling and early genes may be important for maintaining odontoblast cultures. Furthermore, simultaneous stimulation of FGF, Wnt, Hedgehog, Notch pathways, and IEG transcription factors cooperatively promoted the maintenance of the odontoblast phenotype. These results suggest that the Hedgehog and Notch signaling pathways may play an important role in maintaining odontoblast phenotypes, in addition to FGF and Wnt signaling.

Neuronal activity-related transcription is blunted in immature compared to mature dentate granule cells.

Immature dentate granule cells (DGCs) generated in the hippocampus during adulthood are believed to play a unique role in dentate gyrus (DG) function. Although immature DGCs have hyperexcitable membrane properties in vitro, the consequences of this hyperexcitability in vivo remain unclear. In particular, the relationship between experiences that activate the DG, such as exploration of a novel environment (NE), and downstream molecular processes that modify DG circuitry in response to cellular activation is unknown in this cell population. We first performed quantification of immediate early gene (IEG) proteins in immature (5-week-old) and mature (13-week-old) DGCs from mice exposed to a NE. Paradoxically, we observed lower IEG protein expression in hyperexcitable immature DGCs. We then isolated nuclei from active and inactive immature DGCs and performed single-nuclei RNA-Sequencing. Compared to mature nuclei collected from the same animal, immature DGC nuclei showed less activity-induced transcriptional change, even though they were classified as active based on expression of ARC protein. These results demonstrate that the coupling of spatial exploration, cellular activation, and transcriptional change differs between immature and mature DGCs, with blunted activity-induced changes in immature cells.

Adult-born neurons inhibit developmentally-born neurons during spatial learning.

Ongoing neurogenesis in the dentate gyrus (DG) subregion of the hippocampus results in a heterogenous population of neurons. Immature adult-born neurons (ABNs) have physiological and anatomical properties that may give them a unique role in learning. For example, compared to older granule neurons, they have greater somatic excitability, which could facilitate their recruitment into memory traces. However, recruitment is also likely to depend on interactions with other DG neurons through processes such as lateral inhibition. Immature ABNs target inhibitory interneurons and, compared to older neurons, they receive less GABAergic inhibition. Thus, they may induce lateral inhibition of mature DG neurons while being less susceptible to inhibition themselves. To test this we used a chemogenetic approach to silence immature ABNs as rats learned a spatial water maze task, and measured activity (Fos expression) in ABNs and developmentally-born neurons (DBNs). A retrovirus expressing the inhibitory DREADD receptor, hM4Di, was injected into the dorsal DG of male rats at 6w to infect neurons born in adulthood. Animals were also injected with BrdU to label DBNs or ABNs. DBNs were significantly more active than immature 4-week-old ABNs. Silencing 4-week-old ABNs did not alter learning but it increased activity in DBNs. However, silencing ABNs did not affect activation in other ABNs within the DG. Silencing ABNs also did not alter Fos expression in parvalbumin- and somatostatin-expressing interneurons. Collectively, these results suggest that ABNs may directly inhibit DBN activity during hippocampal-dependent learning, which may be relevant for maintaining sparse hippocampal representations of experienced events.

Route-dependent spatial engram tagging in mouse dentate gyrus.

The dentate gyrus (DG) of hippocampus is hypothesized to act as a pattern separator that distinguishes between similar input patterns during memory formation and retrieval. Sparse ensembles of DG cells associated with learning and memory, i.e. engrams, have been labeled and manipulated to recall novel context memories. Functional studies of DG cell activity have demonstrated the spatial specificity and stability of DG cells during navigation. To reconcile how the DG contributes to separating global context as well as individual navigational routes, we trained mice to perform a delayed-non-match-to-position (DNMP) T-maze task and labeled DG neurons during performance of this task on a novel T-maze. The following day, mice navigated a second environment: the same T-maze, the same T-maze with one route permanently blocked but still visible, or a novel open field. We found that the degree of engram reactivation across days differed based on the traversal of maze routes, such that mice traversing only one arm had higher ensemble overlap than chance but less overlap than mice running the full two-route task. Mice experiencing the open field had similar ensemble sizes to the other groups but only chance-level ensemble reactivation. Ensemble overlap differences could not be explained by behavioral variability across groups, nor did behavioral metrics correlate to degree of ensemble reactivation. Together, these results support the hypothesis that DG contributes to spatial navigation memory and that partially non-overlapping ensembles encode different routes within the context of an environment.

Urban and rural male song sparrows (Melospiza melodia) differ in territorial aggression and activation of vasotocin neurons in response to song challenge.

When living in urban habitats, 'urban adapter' species often show greater aggression toward conspecifics, yet we do not understand the mechanisms underlying this behavioral shift. The neuroendocrine system regulates socio-sexual behaviors including aggression and thus could mediate behavioral responses to urbanization. Indeed, urban male song sparrows (Melospiza melodia), which are more territorially aggressive, also have greater abundance of the neuropeptide arginine vasotocin (AVT) in nodes of the brain social behavior network. Higher abundance of AVT could reflect long-term synthesis that underlies baseline territoriality or short-term changes that regulate aggression in response to social challenge. To begin to resolve the timeframe over which the AVT system contributes to habitat differences in aggression we used immediate early gene co-expression as a measure of the activation of AVT neurons. We compared Fos induction in AVT-immunoreactive neurons of the bed nucleus of the stria terminalis (BSTm) and paraventricular nucleus of the hypothalamus (PVN) between urban and rural male song sparrows in response to a short (< 5 min.) or long (> 30 min.) song playback to simulate territorial intrusion by another male. We found that urban males had a higher proportion of Fos-positive AVT neurons in both brain regions compared to rural males, regardless of the duration of song playback. Our results suggest that AVT neurons remain activated in urban males, independently of the duration of social challenge. These findings that Fos induction in AVT neurons differs between rural and urban male song sparrows further implicate this system in regulating behavioral responses to urbanization.

High estradiol reduces adult neurogenesis but strengthens functional connectivity within the hippocampus during spatial pattern separation in adult female rats.

Adult neurogenesis in the dentate gyrus plays an important role for pattern separation, the process of separating similar inputs and forming distinct neural representations. Estradiol modulates neurogenesis and hippocampus function, but to date no examination of estradiol's effects on pattern separation have been conducted. Here, we examined estrogenic regulation of adult neurogenesis and functional connectivity in the hippocampus after the spatial pattern separation task in female rats. Ovariectomized Sprague-Dawley rats received daily injections of vehicle, 0.32 µg (Low) or 5 µg (High) of estradiol benzoate until the end of experiment. A single bromodeoxyuridine (BrdU) was injected one day after initiation of hormone or vehicle treatment and rats were tested in the delayed nonmatching to position spatial pattern separation task in the 8-arm radial maze for 12 days beginning two weeks after BrdU injection. Rats were perfused 90 min after the final trial and brain sections were immunohistochemically stained for BrdU/neuronal nuclei (NeuN) (new neurons), Ki67 (cell proliferation), and the immediate early gene, zif268 (activation). Results showed that high, but not low, estradiol reduced the density of BrdU/NeuN-ir cells and had significant inter-regional correlations of zif268-ir cell density in the hippocampus following pattern separation. Estradiol treatment did not influence pattern separation performance or strategy use. These results show that higher doses of estradiol can reduce neurogenesis but at the same time increases correlations of activity of neurons within the hippocampus during spatial pattern separation.

The transcription factor CEBP homolog of Penaeus vannamei contributes to WSSV replication.

The cellular transcription factors are known to play important roles in virus infection. The present study cloned and characterized a transcription factor CCAAT/Enhancer-binding protein homolog from the shrimp Penaeus vannamei (designates as PvCEBP), and explored its potential functions in white spot syndrome virus (WSSV) infection. PvCEBP has an open reading frame (ORF) of 864 bp encoding a putative protein of 287 amino acids, which contained a highly C-terminal conserved bZIP domain. Phylogenetic tree analysis showed that PvCEBP was evolutionarily clustered with invertebrate CEBPs and closely related to the CEBP of Homarus americanus. Quantitative real-time PCR (qPCR) analysis revealed that PvCEBP was expressed in all examined shrimp tissues, with transcript levels increased in shrimp hemocytes and gill upon WSSV challenge. Furthermore, knockdown of PvCEBP mediated by RNA interference significantly decreased the expression of WSSV genes and viral loads, while enhanced the shrimp survival rate under WSSV challenge. In silico prediction and reporter gene assays demonstrated that PvCEBP could activate the promoter activity of the viral immediate-early gene ie1. Collectively, our findings suggest that PvCEBP is annexed by WSSV to promote its propagation by enhancing the expression of viral immediate-early genes.

Olfactory Information Storage Engages Subcortical and Cortical Brain Regions That Support Valence Determination.

The olfactory bulb (OB) delivers sensory information to the piriform cortex (PC) and other components of the olfactory system. OB-PC synapses have been reported to express short-lasting forms of synaptic plasticity, whereas long-term potentiation (LTP) of the anterior PC (aPC) occurs predominantly by activating inputs from the prefrontal cortex. This suggests that brain regions outside the olfactory system may contribute to olfactory information processing and storage. Here, we compared functional magnetic resonance imaging BOLD responses triggered during 20 or 100 Hz stimulation of the OB. We detected BOLD signal increases in the anterior olfactory nucleus (AON), PC and entorhinal cortex, nucleus accumbens, dorsal striatum, ventral diagonal band of Broca, prelimbic-infralimbic cortex (PrL-IL), dorsal medial prefrontal cortex, and basolateral amygdala. Significantly stronger BOLD responses occurred in the PrL-IL, PC, and AON during 100 Hz compared with 20 Hz OB stimulation. LTP in the aPC was concomitantly induced by 100 Hz stimulation. Furthermore, 100 Hz stimulation triggered significant nuclear immediate early gene expression in aPC, AON, and PrL-IL. The involvement of the PrL-IL in this process is consistent with its putative involvement in modulating behavioral responses to odor experience. Furthermore, these results indicate that OB-mediated information storage by the aPC is embedded in a connectome that supports valence evaluation.

Endogenous SOLOIST/MRTFB i4, a Neuronal Isoform of MKL2/MRTFB, Positively and Negatively Regulates SRF Target Immediate Early Genes in Neuro-2a Cells.

Megakaryoblastic leukemia 2 (MKL2)/myocardin-related transcription factor-B (MRTFB) is a serum response factor (SRF) cofactor that is enriched in the brain and controls SRF target genes and neuronal morphology. There are at least four isoforms of MKL2/MRTFB. Among these, MKL2/MRTFB isoform 1 and spliced neuronal long isoform of SRF transcriptional coactivator (SOLOIST)/MRTFB isoform 4 (MRTFB i4) are highly expressed in neurons. Although, when overexpressed in neurons, isoform 1 and SOLOIST/MRTFB i4 have opposing effects on dendritic morphology and differentially regulate SRF target genes, it is unknown how endogenous SOLOIST/MRTFB i4 regulates gene expression. Using isoform-specific knockdown, we investigated the role of endogenous SOLOST/MRTFB i4 in regulating the expression of other MKL2/MRTFB isoforms and SRF-target genes in Neuro-2a cells. Knockdown of SOLOIST/MRTFB i4 downregulated SOLOIST/MRTFB i4, while it upregulated isoform 1 without affecting isoform 3. Knockdown of SOLOIST/MRTFB i4 downregulated the SRF target immediate early genes egr1 and Arc, while it upregulated c-fos. Double knockdown of isoform 1 and SOLOIST/MRTFB i4 inhibited c-fos expression. Taken together, our findings in Neuro-2a cells suggest that endogenous SOLOIST/MRTFB i4 positively regulates egr1 and Arc expression. In addition, endogenous SOLOIST/MRTFB i4 may negatively regulate c-fos expression, possibly by downregulating isoform 1 in Neuro-2a cells.

Loss of Arc attenuates the behavioral and molecular responses for sleep homeostasis in mice.

The activity-regulated cytoskeleton-associated protein (Arc) gene is a neural immediate early gene that is involved in synaptic downscaling and is robustly induced by prolonged wakefulness in rodent brains. Converging evidence has led to the hypothesis that wakefulness potentiates, and sleep reduces, synaptic strengthening. This suggests a potential role for Arc in these and other sleep-related processes. However, the role of Arc in sleep remains unknown. Here, we demonstrated that Arc is important for the induction of multiple behavioral and molecular responses associated with sleep homeostasis. Arc knockout (KO) mice displayed increased time spent in rapid eye movement (REM) sleep under baseline conditions and marked attenuation of sleep rebound to both 4 h of total sleep deprivation (SD) and selective REM deprivation. At the molecular level, the following homeostatic sleep responses to 4-h SD were all blunted in Arc KO mice: increase of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluA1 and its phosphorylation in synaptoneurosomes; induction of a subset of SD-response genes; and suppression of the GluA1 messenger RNA in the cortex. In wild-type brains, SD increased Arc protein expression in multiple subcellular locations, including the nucleus, cytoplasm, and synapse, which is reversed in part by recovery sleep. Arc is critical for these behavioral and multiple molecular responses to SD, thus providing a multifunctional role for Arc in the maintenance of sleep homeostasis, which may be attributed by the sleep/wake-associated changes in subcellular location of Arc.