Heppenstal_Ret_GFP
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Paul Heppenstall
PROVIDER: E-MTAB-1836 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Here we identified two populations of myelinated sensory neurons that display markedly different phenotypes in terms of their action potential characteristics and responses to mechanical stimuli based on their expression of Calcitonin Gene-Related Peptide (CGRP). Myelinated neurons that did not express CGRP responded to mechanical stimuli with significantly larger currents during whole-cell voltage clamp recordings than their CGRP-positive counterparts, regardless of whether these neurons projected to the dorsal hindpaw skin or the gastrocnemius muscle. Importantly, this discrepancy could not be explained by a differential expression of mechanosensitive or mechanically-gated proteins like Stoml3 or Piezo2. Following inflammation of the skin or muscle, myelinated neurons demonstrated a sensitization to mechanical stimuli characterized by increased current amplitudes. Interestingly, myelinated neurons expressing CGRP are sensitized to mechanical stimuli following cutaneous inflammation of the paw, while myelinated neurons that do not express CGRP are sensitized to mechanical stimuli following inflammation of the gastrocnemius muscle. Microarray data was obtained from these populations by first using Fluorescence-Activated Cell Sorting (FACS) to separate the populations of interest. 15 different samples were analyzed, 3 biological replicates for each group (5 groups: saline paw-injected, CFA paw-injected, saline muscle-injected, acid muscle-injected, and CFA muscle-injected). The saline injected groups (paw and muscle) are considered controls.
2015-11-06 | E-GEOD-65613 | biostudies-arrayexpress
Project description:The goal of this study was to analyze global gene expression in specific populations of somatosensory neurons in the periphery, including major, non-overlapping populations that include nociceptors, pruriceptors, and prorioceptors. The mammalian somatosensory nervous system encodes the perception of specific environmental stimuli. The dorsal root ganglion (DRG) contains distinct somatosensory neuron subtypes that innervate diverse peripheral tissues, mediating the detection of thermal, mechanical, proprioceptive, pruriceptive, and nociceptive stimuli. We purified discrete subtypes of mouse DRG somatosensory neurons by flow cytometry using fluorescently labeled mouse lines (SNS-Cre/TdTomato, Parv-Cre/TdTomato) in combination with Isolectin B4-FITC surface staining (IB4). This allowed identification of transcriptional differences between these major populations, revealing enrichment of voltage-gated ion channels, TRP channels, G-protein coupled receptors, transcription factors, and other functionally important classes of genes within specific somatosensory neuron subsets. SNS-Cre mice were bred with Rosa26-TdTomato mice to generate SNS-Cre/TdTomato reporter mice. Parv-Cre mice were bred with Rosa26-TdTomato mice to generate Parv-Cre/TdTomato mice. Isolectin B4-FITC was used to stain the surface of SNS-Cre/TdTomato reporter mice. We used these strategies of fluorescent labeling to purify distinct murine sensory neuron subsets from the dorsal root ganglia (DRG) by fluorescence activated cell sorting (FACS). Neurons were sorted directly in Qiazol for total RNA extraction and microarray analysis. Whole DRG tissue was also included for transcriptome analysis to compare with purified neuronal populations.
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Project description:Proprioception relies on two main classes of proprioceptive sensory neurons (pSNs). These neurons innervate two distinct peripheral receptors in muscle, muscle spindles (MSs) or Golgi tendon organs (GTOs), and synapse onto different sets of spinal targets, but the molecular basis of their distinct pSN subtype identity remains unknown. We used microarray analysis to compare gene expression profiles between MS- and GTO- innervating proprioceptors. We generated transgenic mice in which MS and GTO pSNs are labelled with different fluorescent proteins (see de Nooij et al., 2015 for details). We used Fluorescent Activated Cell Sorting (FACS) to isolate the MS and GTO pSN subsets from dissociated DRG from p7-10 transgenic mice. Neurons from multiple FACS experiments were pooled into three samples each for the MS and GTO pSN subset.
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Project description:Transcriptional analysis of identified DRG subpopulations. Cell-type specific intrinsic programs instruct neuronal subpopulations before target-derived factors influence later neuronal maturation. Retrograde neurotrophin signaling controls neuronal survival and maturation of dorsal root ganglion (DRG) sensory neurons, but how these potent signaling pathways intersect with transcriptional programs established at earlier developmental stages remains poorly understood. Here we determine the consequences of genetic alternation of NT3 signaling on genome-wide transcription programs in proprioceptors, an important sensory neuron subpopulation involved in motor reflex behavior. We find that the expression of many proprioceptor-enriched genes is dramatically altered by genetic NT3 elimination, independent of survival-related activities. Combinatorial analysis of gene expression profiles with proprioceptors isolated from mice expressing surplus muscular NT3 identifies an anticorrelated gene set with transcriptional levels scaled in opposite directions. Voluntary running experiments in adult mice further demonstrate the maintenance of transcriptional adjustability of genes expressed by DRG neurons, pointing to life-long gene expression plasticity in sensory neurons. We combined a mouse line expressing GFP under the control of the TrkC promoter (BAC transgene approach) with various NT3 signaling mutants in order to identify the transcriptional changes in identified subpopulations of dorsal root ganglia (DRG) neurons. Sorted cells were processed for RNA extraction and hybridization on Affymetrix microarrays. Analysis was performed a postnatal (p) day p0. Subsequent analysis focused on the transcriptional profile of DRG neuron subpopulations at specific lumbar levels. Additional work addressed the transcriptional changes in whole DRG in adult mice with and without physical exercise.
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