Exploring the molecular clock in sympathetic preganglionic neurons

Sympathetic preganglionic neurons (SPNs) are the final point of influence the central nervous system has on sympathetic nerve activity. These cells are predominantly located in the intermediolateral cell column (IML) of the thoracic spinal cord. Sympathetic nerve activity exhibits a diurnal rhythm which is evident in blood pressure variations. This project examines if there is evidence of a molecular clock in SPNs and aims to characterize SPN gene expression profile in mice.
The first aim was to identify a marker to isolate SPNs for flow cytometry so gene expression could be studied. Data mining from on-line resources identified potential candidates, immunofluorescence (IF) was applied to test selectivity of identified SPN markers. IF identified Galectin-3, previously undescribed in mouse spinal cord tissue, as a selective label for IML SPNs. Galectin-3 and other candidate markers did not label all SPNs.
The presence of a molecular clock with the potential to directly influence sympathetic activity was examined. Quantitative polymerase chain reaction (qPCR) revealed molecular clock genes Bmal1 and Per2 were diurnally expressed in IML samples. Immunofluorescence revealed SPNs expressed Per2 and Bmal1 proteins which displayed diurnal rhythmicity, suggesting SPNs possess a molecular clock.
Gene expression in IML samples and SPNs was examined with RNAseq was performed. A new method to obtain single SPNs and IML samples from frozen tissue was devised. RNA-seq from IML and single cells revealed gene expression profiles detailing receptor/channel subtypes and subunits present. RNA-seq identified a potential selective marker for SPNs which was confirmed with immunofluorescence as the GABAA θ subunit. qPCR of genes that influence SPN activity selected from RNAseq results revealed diurnal rhythm of expression levels in the IML.
These studies indicate that SPNs contain molecular clock machinery which may control expression of genes/proteins that influence SPN and sympathetic nerve activity.