Single cell q-PCR derived expression profiles of identified sensory neurons

Abstract

Sensory neurons are chemically and functionally heterogeneous and this heterogeneity has been examined extensively over the last several decades. These studies have employed a variety of different methodologies, including anatomical, electrophysiological and molecular approaches. Recent studies using next generation sequencing techniques have examined the transcriptome of single sensory neurons. Although, these reports have provided a wealth of exciting new information on the heterogeneity of sensory neurons, correlation with functional types is lacking. Here, we employed retrograde tracing of cutaneous and muscle afferents to examine the variety of mRNA expression profiles of individual, target-specific sensory neurons. In addition, we used an ex vivo skin/nerve/DRG/ spinal cord preparation to record and characterize the functional response properties of individual cutaneous sensory neurons that were then intracellularly labeled with fluorescent dyes, recovered from dissociated cultures and analyzed for gene expression. We found that by using single cell qPCR techniques and a limited set of genes, we can identify transcriptionally distinct groups. We have also used calcium imaging and single cell qPCR to determine the correlation between levels of mRNA expression and functional protein expression and how functional properties correlated with the different transcriptional groups. These studies show that although transcriptomics does map to functional types, within any one functional subgroup, there are highly variable patterns of gene expression. Thus, studies that rely on the expression pattern of one or a few genes as a stand in for physiological experiments, runs a high risk of data misinterpretation with respect to function. Significance statementExpression profiles of unidentified sensory neurons have been recently studied using RNASeq techniques. Here, we utilize a multifactorial approach to target identified 3 cutaneous and muscle afferents to examine expression and functional levels of specific high priority candidate genes using ex vivo electrophysiology, Ca2+ imaging, and single cell qPCR. Using this methodology, we were able to identify specific groups of neurons with distinct functional properties that corresponded to unique transcriptional profiles.This represents the first attempt to relate neuronal phenotype with levels of gene expression in single identified afferents and highlights the importance of combining functional analysis with transcriptomics.

Postnatal Arx transcriptional activity regulates functional properties of PV interneurons

“…Although our RNAseq data support an ECM-dependent mechanism in the alterations of PVI membrane properties, we acknowledge that the excitable properties of neurons, which can be influenced by localization or phosphorylation, may not always align to the transcriptomic level of a particular ion channel. Although RNA-Seq transcriptomic profiling provides a comprehensive analysis of actively regulated genes, many studies have found no correlation or seemingly inverse relationships between the expression of various membrane ( Adelman et al., 2019 ; Bomkamp et al., 2019 ; Földy et al., 2016 ; Larson et al., 2016 ; Tripathy et al., 2017 ) and synaptic ( Fazel Darbandi et al., 2018 ; Harrington et al., 2016 ; Yook et al., 2019 ) genes with their electrophysiological properties. Thus, the relationship between gene expression and cellular/behavioral phenotypes is complex, and this complexity must be taken into account in future studies into the ion channel mechanisms mediating hypoexcitability of PVIs in Arx CKO mice.…”

Unique Molecular Characteristics of Visceral Afferents Arising from Different Levels of the Neuraxis: Location of Afferent Somata Predicts Function and Stimulus Detection Modalities

“…The methods for single cell RT-qPCR and clustering and validation of these techniques have been previously described (Adelman et al, 2019). Briefly, the RNA collected from each cell was reverse transcribed and amplified using T7 linear amplification (Epicentre, MessageBOOSTER kit for cell lysate), cleaned with RNA Cleaner & Concentrator-5 columns (Zymo Research), and assessed using qPCR with optimized primers and SsoAdvanced SYBR Green Master Mix (BIO-RAD).…”

“…To classify afferent subtypes we used single-cell RT-qPCR to analyze transcripts of 28 genes previously used in a study for classification of identified cutaneous afferents (Adelman et al, 2019). These genes were shown to identify clusters that largely replicated the clusters produced by bulk, single cell RNAseq of DRG neurons unidentified with respect to innervation target or functional phenotype (Usoskin et al, 2015;Zeisel et al, 2018).…”

Early life nociception is influenced by peripheral growth hormone signaling

“…Throughout development distinct neurotrophic factors regulate neuronal subtype formation and innervation; however, these signaling growth factors also display non-canonical roles (Moqrich et al, 2004) (Shelton and Reichardt, 1984) (Mendell, 1996). Recent investigations also demonstrate that developing sensory neurons display changing transcriptional identities and functional maturation from the neonatal period through adulthood (Sharma et al, 2020) (Jankowski et al, 2014) (Adelman et al, 2019). While growth factors clearly alter subtype survival, we detect that modulation of neuronal GH signaling results in functional changes to normal somatosensory processing in both the skin (Ford et al, 2019) and the muscle.…”