Subregion-specific rules govern the distribution of neuronal immediate-early gene induction.
ABSTRACT: The induction of immediate-early gene (IEG) expression in brain nuclei in response to an experience is necessary for the formation of long-term memories. Additionally, the rapid dynamics of IEG induction and decay motivates the common use of IEG expression as markers for identification of neuronal assemblies ("ensembles") encoding recent experience. However, major gaps remain in understanding the rules governing the distribution of IEGs within neuronal assemblies. Thus, the extent of correlation between coexpressed IEGs, the cell specificity of IEG expression, and the spatial distribution of IEG expression have not been comprehensively studied. To address these gaps, we utilized quantitative multiplexed single-molecule fluorescence in situ hybridization (smFISH) and measured the expression of IEGs (Arc, Egr2, and Nr4a1) within spiny projection neurons (SPNs) in the dorsal striatum of mice following acute exposure to cocaine. Exploring the relevance of our observations to other brain structures and stimuli, we also analyzed data from a study of single-cell RNA sequencing of mouse cortical neurons. We found that while IEG expression is graded, the expression of multiple IEGs is tightly correlated at the level of individual neurons. Interestingly, we observed that region-specific rules govern the induction of IEGs in SPN subtypes within striatal subdomains. We further observed that IEG-expressing assemblies form spatially defined clusters within which the extent of IEG expression correlates with cluster size. Together, our results suggest the existence of IEG-expressing neuronal "superensembles," which are associated in spatial clusters and characterized by coherent and robust expression of multiple IEGs.
Project description:Sensitive detection of sensory-evoked neuronal activation is a key to mechanistic understanding of brain functions. Since immediate early genes (IEGs) are readily induced in the brain by environmental changes, tracing IEG expression provides a convenient tool to identify brain activity. In this study we used in situ hybridization to detect odor-evoked induction of ten IEGs in the mouse olfactory system. We then analyzed IEG induction in the cyclic nucleotide-gated channel subunit A2 (Cnga2)-null mice to visualize residual neuronal activity following odorant exposure since CNGA2 is a key component of the olfactory signal transduction pathway in the main olfactory system.We observed rapid induction of as many as ten IEGs in the mouse olfactory bulb (OB) after olfactory stimulation by a non-biological odorant amyl acetate. A robust increase in expression of several IEGs like c-fos and Egr1 was evident in the glomerular layer, the mitral/tufted cell layer and the granule cell layer. Additionally, the neuronal IEG Npas4 showed steep induction from a very low basal expression level predominantly in the granule cell layer. In Cnga2-null mice, which are usually anosmic and sexually unresponsive, glomerular activation was insignificant in response to either ambient odorants or female stimuli. However, a subtle induction of c-fos took place in the OB of a few Cnga2-mutants which exhibited sexual arousal. Interestingly, very strong glomerular activation was observed in the OB of Cnga2-null male mice after stimulation with either the neutral odor amyl acetate or the predator odor 2, 3, 5-trimethyl-3-thiazoline (TMT).This study shows for the first time that in vivo olfactory stimulation can robustly induce the neuronal IEG Npas4 in the mouse OB and confirms the odor-evoked induction of a number of IEGs. As shown in previous studies, our results indicate that a CNGA2-independent signaling pathway(s) may activate the olfactory circuit in Cnga2-null mice and that neuronal activation which correlates to behavioral difference in individual mice is detectable by in situ hybridization of IEGs. Thus, the in situ hybridization probe set we established for IEG tracing can be very useful to visualize neuronal activity at the cellular level.
Project description:Three Homer genes regulate the activity of metabotropic glutamate receptors mGluR1a and mGluR5 and their coupling to releasable intracellular Ca2+ pools and ion channels. Only the Homer 1 gene evolved bimodal expression of constitutive (Homer 1b and c) and immediate early gene (IEG) products (Homer 1a and Ania 3). The IEG forms compete functionally with the constitutive Homer proteins. The complex expression of the Homer 1 gene, unique for IEGs, focused our attention on the gene organization. In contrast to most IEGs, which have genes that are <5 kb, the Homer 1 gene was found to span approximately 100 kb. The constitutive Homer 1b/c forms are encoded by exons 1-10, whereas the IEG forms are encoded by exons 1-5 and parts of intron 5. RNase protection demonstrated a >10-fold activity-dependent increase in mRNA levels exclusively for the IEG forms. Moreover, fluorescent in situ hybridization documented that new primary Homer 1 transcripts are induced in neuronal nuclei within a few minutes after seizure, typical of IEGs, and that Homer 1b-specific exons are excluded from the activity-induced transcripts. Thus, at the resting state of the neurons, the entire gene is constitutively transcribed at low levels to yield Homer 1b/c transcripts. Neuronal activity sharply increases the rate of transcription initiation, with most transcripts now ending within the central intron. These coordinate transcriptional events rapidly convert a constitutive gene to an IEG and regulate the expression of functionally different Homer 1 proteins.
Project description:Optogenetics is a powerful neuromodulatory tool with many unique advantages to explore functions of neuronal circuits in physiology and diseases. Yet, interpretation of cellular and behavioral responses following in vivo optogenetic manipulation of brain activities in experimental animals often necessitates identification of photoactivated neurons with high spatial resolution. Although tracing expression of immediate early genes (IEGs) provides a convenient approach, neuronal activation is not always followed by specific induction of widely used neuronal activity markers like c-fos, Egr1 and Arc. In this study we performed unilateral optogenetic stimulation of the striatum in freely moving transgenic mice that expressed a channelrhodopsin-2 (ChR2) variant ChR2(C128S) in striatal medium spiny neurons (MSNs). We found that in vivo blue light stimulation significantly altered electrophysiological activity of striatal neurons and animal behaviors. To identify photoactivated neurons we then analyzed IEG expression patterns using in situ hybridization. Upon light illumination an induction of c-fos was not apparent whereas another neuronal IEG Npas4 was robustly induced in MSNs ipsilaterally. Our results demonstrate that tracing Npas4 mRNA expression following in vivo optogenetic modulation can be an effective tool for reliable and sensitive identification of activated MSNs in the mouse striatum.
Project description:Profound induction of immediate early genes (IEGs) by neural activation is a critical determinant for plasticity in the brain, but intervening molecular signals are not well characterized. We demonstrate that inositol polyphosphate multikinase (IPMK) acts noncatalytically as a transcriptional coactivator to mediate induction of numerous IEGs. IEG induction by electroconvulsive stimulation is virtually abolished in the brains of IPMK-deleted mice, which also display deficits in spatial memory. Neural activity stimulates binding of IPMK to the histone acetyltransferase CBP and enhances its recruitment to IEG promoters. Interestingly, IPMK regulation of CBP recruitment and IEG induction does not require its catalytic activities. Dominant-negative constructs, which prevent IPMK-CBP binding, substantially decrease IEG induction. As IPMK is ubiquitously expressed, its epigenetic regulation of IEGs may influence diverse nonneural and neural biologic processes.
Project description:A hallmark of temporal lobe epilepsy (TLE) is hippocampal neuronal demise and aberrant mossy fiber sprouting. In addition, unrestrained neuronal activity in TLE patients induces gene expression including immediate early genes (IEGs) such as Fos and Egr1.We employed the mouse pilocarpine model to analyze the transcription factor (TF) serum response factor (SRF) in epileptogenesis, seizure induced histopathology and IEG induction. SRF is a neuronal activity regulated TF stimulating IEG expression as well as nerve fiber growth and guidance. Adult conditional SRF deficient mice (Srf CaMKCreERT2 ) were more refractory to initial status epilepticus (SE) acquisition. Further, SRF deficient mice developed more spontaneous recurrent seizures (SRS). Genome-wide transcriptomic analysis uncovered a requirement of SRF for SE and SRS induced IEG induction (e.g. Fos, Egr1, Arc, Npas4, Btg2, Atf3). SRF was required for epilepsy associated neurodegeneration, mossy fiber sprouting and inflammation. We uncovered MAP kinase signaling as SRF target during epilepsy. Upon SRF ablation, seizure evoked induction of dual specific phosphatases (Dusp5 and Dusp6) was reduced. Lower expression of these negative ERK kinase regulators correlated with altered P-ERK levels in epileptic Srf mutant animals.Overall, this study uncovered an SRF contribution to several processes of epileptogenesis in the pilocarpine model.
Project description:In the mammalian hippocampus, changes in the expression of immediate early genes (IEGs) is thought to contribute to long term plastic changes in neurons brought about by learning tasks and high frequency stimulation of synapses. The phosphatase calcineurin has emerged as an important negative regulator of hippocampus-dependent learning and long term potentiation. Here we investigated the possibility that the constraining action of calcineurin on hippocampal plasticity is mediated in part by regulation of gene expression through negative control of transcription factors, such as cAMP-response element (CRE)-binding protein (CREB). We assessed the effect of calcineurin inhibitors on CREB activation by neuronal activity and show that calcineurin activity is in fact required for CREB-mediated gene expression. However, inhibition of calcineurin had disparate effects on the transcriptional induction of CREB-dependent IEGs. We find that the IEG c-fos is unaffected by suppression of calcineurin activity, the plasticity-related genes Egr1/Zif268 and Egr2/Krox-20 are up-regulated, and genes encoding the orphan nuclear hormone receptors Nor1 and Nur77 are down-regulated. We further show that the up-regulation of particular IEGs is probably due to the presence of serum response elements (SREs) in their promoters, because SRE-mediated gene expression is enhanced by calcineurin blockers. Moreover, expression of the c-fos gene, which is unaffected by calcineurin inhibitors, could be down-regulated by mutating the SRE. Conversely, SRE-mediated c-fos induction in the absence of a functional CRE was enhanced by calcineurin inhibitors. Our experiments thus implicate calcineurin as a negative regulator of SRE-dependent neuronal genes.
Project description:Gene transcription and translation in the hippocampus is of critical importance in hippocampus-dependent memory formation, including during Morris water maze (MWM) learning. Previous work using gene deletion models has shown that the immediate-early genes (IEGs) c-Fos, Egr-1, and Arc are crucial for such learning. Recently, we reported that induction of IEGs in sparse dentate gyrus neurons requires ERK MAPK signaling and downstream formation of a distinct epigenetic histone mark (i.e., phospho-acetylated histone H3). Until now, this signaling, epigenetic and gene transcriptional pathway has not been comprehensively studied in the MWM model. Therefore, we conducted a detailed study of the phosphorylation of ERK1/2 and serine10 in histone H3 (H3S10p) and induction of IEGs in the hippocampus of MWM trained rats and matched controls. MWM training evoked consecutive waves of ERK1/2 phosphorylation and H3S10 phosphorylation, as well as c-Fos, Egr-1, and Arc induction in sparse hippocampal neurons. The observed effects were most pronounced in the dentate gyrus. A positive correlation was found between the average latency to find the platform and the number of H3S10p-positive dentate gyrus neurons. Furthermore, chromatin immuno-precipitation (ChIP) revealed a significantly increased association of phospho-acetylated histone H3 (H3K9ac-S10p) with the gene promoters of c-Fos and Egr-1, but not Arc, after MWM exposure compared with controls. Surprisingly, however, we found very little difference between IEG responses (regarding both protein and mRNA) in MWM-trained rats compared with matched swim controls. We conclude that exposure to the water maze evokes ERK MAPK activation, distinct epigenetic changes and IEG induction predominantly in sparse dentate gyrus neurons. It appears, however, that a specific role for IEGs in the learning aspect of MWM training may become apparent in downstream AP-1- and Egr-1-regulated (second wave) genes and Arc-dependent effector mechanisms.
Project description:Transcription of immediate early genes (IEGs) in neurons is exquisitely sensitive to neuronal activity, but the mechanism underlying the earliest of these transcription events is largely unknown. Here we demonstrate that very fast IEGs (VF-IEGs) such as arc/arg3.1 are poised for rapid transcription by the stalling of RNA Polymerase II (Pol II) just downstream of the transcription start site. RNAi-depletion of two subunits of a mediator of Pol II stalling, Negative Elongation Factor, reduces Pol II occupancy of the arc promoter and compromises rapid induction of arc and other VF-IEGs. In contrast, reduction of Pol II stalling did not prevent expression of other fast IEGs (F-IEGs). These F-IEGs are expressed with comparatively slower kinetics and largely lack promoter proximal Pol II stalling. Taken together, our data strongly indicate that very fast kinetics of neuronal IEG expression require poised Pol II and suggest a role for this mechanism in transcription-dependent learning and memory. TTX withdrawal induced neuronal activity. To study activity-induced gene expression, neurons were treated with TTX for 48 hours and then TTX was washed out either for 15 minutes (W15) or for 45 minutes (W45). Gene expression was measured in these two groups in comparison to TTX treated neurons.
Project description:Transcription of immediate early genes (IEGs) in neurons is exquisitely sensitive to neuronal activity, but the mechanism underlying the earliest of these transcription events is largely unknown. Here we demonstrate that very fast IEGs (VF-IEGs) such as arc/arg3.1 are poised for rapid transcription by the stalling of RNA Polymerase II (Pol II) just downstream of the transcription start site. RNAi-depletion of two subunits of Negative Elongation Factor, a mediator of Pol II stalling, reduces the Pol II occupancy of the arc promoter and compromises the rapid induction of arc and other VF-IEGs. In contrast, reduction of Pol II stalling did not prevent expression of fast IEGs (F-IEGs). These F-IEGs are expressed with comparatively slower kinetics and largely lack promoter proximal Pol II stalling. Taken together, our data strongly indicate that very fast kinetics of neuronal IEG expression require poised Pol II and suggest a role for this mechanism in transcription-dependent learning and memory. Examination of Pol II bindnig genome-wide in rat neurons
Project description:Neuronal immediate-early gene (IEG) expression is regulated by synaptic activity and plays an important role in the neuroplastic mechanisms critical to memory consolidation. IEGs can be divided into two functional classes: (1) regulatory transcription factors (RTFs), which can broadly influence cell function depending on the "downstream" genes they regulate, and (2) "effector" proteins, which may directly modulate specific cellular functions. The objective of the current study was to determine whether the expression of an effector IEG (Arc) was similar to, or different from, that of two well characterized RTF IEGs (c-fos and zif268) after learning. IEG RNA levels from rats trained in spatial and nonspatial water tasks were determined using RNase protection assays and in situ hybridization. Overall, the regulation of the three IEGs was similar in the hippocampus and the entorhinal and primary visual cortices. Consequently, IEG RNA levels were positively correlated within a structure. By contrast, Arc and zif268 RNA levels were not correlated or only weakly correlated across structures, although c-fos RNA levels were moderately correlated across structures. Arc RNA expression differed from that of zif268 and c-fos in two regards: (1) hippocampal Arc RNA levels were correlated with learning of the hippocampal-dependent spatial, but not hippocampal-independent cued response, water task, and (2) Arc RNA levels in the hippocampus and entorhinal cortex increased after spatial reversal learning relative to an asymptotic performance group. Thus, although the expression of Arc, zif268, and c-fos exhibited many similarities, Arc was most responsive to differences in behavioral task demands.