Dataset Information

Subcellular sequencing of single neurons reveals the dendritic transcriptome of GABAergic interneurons.

ABSTRACT: Although mRNAs are localized in the processes of excitatory neurons, it is still unclear whether interneurons also localize a large population of mRNAs. In addition, the variability in the localized mRNA population within and between cell types is unknown. Here we describe the unbiased transcriptomic characterization of the subcellular compartments of hundreds of single neurons. We separately profiled the dendritic and somatic transcriptomes of individual rat hippocampal neurons and investigated mRNA abundances in the soma and dendrites of single glutamatergic and GABAergic neurons. We found that, like their excitatory counterparts, interneurons contain a rich repertoire of ~4000 mRNAs. We observed more cell type-specific features among somatic transcriptomes than their associated dendritic transcriptomes. Finally, using celltype-specific metabolic labeling of isolated neurites, we demonstrated that the processes of glutamatergic and, notably, GABAergic neurons were capable of local translation, suggesting mRNA localization and local translation are general properties of neurons.

SUBMITTER: Perez JD

PROVIDER: S-EPMC7819707 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Subcellular sequencing of single neurons reveals the dendritic transcriptome of GABAergic interneurons.

Perez Julio D JD Dieck Susanne Tom ST Alvarez-Castelao Beatriz B Tushev Georgi G Chan Ivy Cw IC Schuman Erin M EM

eLife 20210106

Although mRNAs are localized in the processes of excitatory neurons, it is still unclear whether interneurons also localize a large population of mRNAs. In addition, the variability in the localized mRNA population within and between cell types is unknown. Here we describe the unbiased transcriptomic characterization of the subcellular compartments of hundreds of single neurons. We separately profiled the dendritic and somatic transcriptomes of individual rat hippocampal neurons and investigated ...[more]

PMID: 33404500

Similar Datasets

Project description:Fast-spiking (FS) interneurons are important elements of neocortical circuitry that constitute the primary source of synaptic inhibition in adult cortex and impart temporal organization on ongoing cortical activity. The highly specialized intrinsic membrane and firing properties that allow cortical FS interneurons to perform these functions are attributable to equally specialized gene expression, which is ultimately coordinated by cell-type-specific transcriptional regulation. Although embryonic transcriptional events govern the initial steps of cell-type specification in most cortical interneurons, including FS cells, the electrophysiological properties that distinguish adult cortical cell types emerge relatively late in postnatal development, and the transcriptional events that drive this maturational process are not known. To address this, we used mouse whole-genome microarrays and whole-cell patch clamp to characterize the transcriptional and electrophysiological maturation of cortical FS interneurons between postnatal day 7 (P7) and P40. We found that the intrinsic and synaptic physiology of FS cells undergoes profound regulation over the first 4 postnatal weeks and that these changes are correlated with primarily monotonic but bidirectional transcriptional regulation of thousands of genes belonging to multiple functional classes. Using our microarray screen as a guide, we discovered that upregulation of two-pore K leak channels between P10 and P25 contributes to one of the major differences between the intrinsic membrane properties of immature and adult FS cells and found a number of other candidate genes that likely confer cell-type specificity on mature FS cells. Experiment Overall Design: We characterized the transcriptional maturation of genetically labeled FS interneurons in mouse S1 cortex using whole-genome microarrays.mRNA was harvested from manually sorted GFPexpressing neurons dissociated from acutely prepared brain slices at a range of developmental time points (P7, P10, P15, P25, and P40) and subsequently reverse transcribed, amplified, labeled, and hybridized to Affymetrix 430 2.0 whole-genome microarrays (three microarrays from three different animals per condition). We used the G42 transgenic mice described by Chattopadhyaya et al. (2004) for all experiments.

Project description:Recent findings have revealed the complexity of the transcriptional landscape in mammalian cells. One recently described class of novel transcripts are the Cytoplasmic Intron-sequence Retaining Transcripts (CIRTs), hypothesized to confer post-transcriptional regulatory function. For instance, the neuronal CIRT KCNMA1i16 contributes to the firing properties of hippocampal neurons. Intronic sub-sequence retention within IL1-? mRNA in anucleate platelets has been implicated in activity-dependent splicing and translation. In a recent study, we showed CIRTs harbor functional SINE ID elements which are hypothesized to mediate dendritic localization in neurons. Based on these studies and others, we hypothesized that CIRTs may be present in a broad set of transcripts and comprise novel signals for post-transcriptional regulation. We carried out a transcriptome-wide survey of CIRTs by sequencing micro-dissected subcellular RNA fractions. We sequenced two batches of 150-300 individually dissected dendrites from primary cultures of hippocampal neurons in rat and three batches from mouse hippocampal neurons. After statistical processing to minimize artifacts, we found a broad prevalence of CIRTs in the neurons in both species (44-60% of the expressed transcripts). The sequence patterns, including stereotypical length, biased inclusion of specific introns, and intron-intron junctions, suggested CIRT-specific nuclear processing. Our analysis also suggested that these cytoplasmic intron-sequence retaining transcripts may serve as a primary transcript for ncRNAs. Our results show that retaining intronic sequences is not isolated to a few loci but may be a genome-wide phenomenon for embedding functional signals within certain mRNA. The results hypothesize a novel source of cis-sequences for post-transcriptional regulation. Our results hypothesize two potentially novel splicing pathways: one, within the nucleus for CIRT biogenesis; and another, within the cytoplasm for removing CIRT sequences before translation. We also speculate that release of CIRT sequences prior to translation may form RNA-based signals within the cell potentially comprising a novel class of signaling pathways.

Project description:BackgroundThe PAM/Highwire/RPM-1 (PHR) proteins are conserved signaling proteins that regulate axon length and synapse formation during development. Loss of function in Caenorhabditis elegans rpm-1 results in axon termination and synapse formation defects in the mechanosensory neurons. An explanation for why these two phenotypes are observed in a single neuronal cell has remained absent. Further, it is uncertain whether the axon termination phenotypes observed in the mechanosensory neurons of rpm-1 mutants are unique to this specific type of neuron, or more widespread defects that occur with loss of function in rpm-1.ResultsHere, we show that RPM-1 is localized to both the mature axon tip and the presynaptic terminals of individual motor neurons and individual mechanosensory neurons. Genetic analysis indicated that GABAergic motor neurons, like the mechanosensory neurons, have both synapse formation and axon termination defects in rpm-1 mutants. RPM-1 functions in parallel with the active zone component SYD-2 (Liprin) to regulate not only synapse formation, but also axon termination in motor neurons. Our analysis of rpm-1-/-; syd-2-/- double mutants also revealed a role for RPM-1 in axon extension. The MAP3K DLK-1 partly mediated RPM-1 function in both axon termination and axon extension, and the relative role of DLK-1 was dictated by the anatomical location of the neuron in question.ConclusionsOur findings show that axon termination defects are a core phenotype caused by loss of function in rpm-1, and not unique to the mechanosensory neurons. We show in motor neurons and in mechanosensory neurons that RPM-1 is localized to multiple, distinct subcellular compartments in a single cell. Thus, RPM-1 might be differentially regulated or RPM-1 might differentially control signals in distinct subcellular compartments to regulate multiple developmental outcomes in a single neuron. Our findings provide further support for the previously proposed model that PHR proteins function to coordinate axon outgrowth and termination with synapse formation.

Dataset Information

Subcellular sequencing of single neurons reveals the dendritic transcriptome of GABAergic interneurons.

Publications

Subcellular sequencing of single neurons reveals the dendritic transcriptome of GABAergic interneurons.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets