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Translatome Analyses Using Conditional Ribosomal Tagging in GABAergic Interneurons and Other Sparse Cell Types.

ABSTRACT: GABAergic interneurons comprise a small but diverse subset of neurons in the mammalian brain that tightly regulate neuronal circuit maturation and information flow and, ultimately, behavior. Because of their centrality in the etiology of numerous neurological disorders, examining the molecular architecture of these neurons under different physiological scenarios has piqued the interest of the broader neuroscience community. The last few years have seen an explosion in next-generation sequencing (NGS) approaches aimed at identifying genetic and state-dependent subtypes in neuronal diversity. Although several approaches are employed to address neuronal molecular diversity, ribosomal tagging has emerged at the forefront of identifying the translatomes of neuronal subtypes. This approach primarily relies on Cre recombinase-driven expression of hemagglutinin A (HA)-tagged RiboTag mice exclusively in the neuronal subtype of interest. This allows the immunoprecipitation of cell-type-specific, ribosome-engaged mRNA, expressed both in the soma and the neuronal processes, for targeted quantitative real-time PCR (qRT-PCR) or high-throughput RNA sequencing analyses. Here we detail the typical technical caveats associated with successful application of the RiboTag technique for analyzing GABAergic interneurons, and in theory other sparse cell types, in the central nervous system. Published 2020. U.S. Government. Basic Protocol 1: Breeding mice to obtain RiboTag homozygosity Support Protocol 1: Detection of ectopic Cre recombinase expression Basic Protocol 2: The RiboTag assay Support Protocol 2: Real-time quantitative PCR (qRT-PCR) assay of RiboTag-derived cell-type-specific RNA Support Protocol 3: Construction of cell-type-specific RNA-seq library Support Protocol 4: Secondary analyses of RiboTag-derived RNA-seq dataset.

SUBMITTER: Mahadevan V

PROVIDER: S-EPMC7317066 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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Translatome Analyses Using Conditional Ribosomal Tagging in GABAergic Interneurons and Other Sparse Cell Types.

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