Proceedings of The Physiological Society

Sleep Sleep and Circadian Rhythms (London, UK) (2018) Proc Physiol Soc 42, C13

Poster Communications

Exploring the molecular clock in sympathetic preganglionic neurons

C. Nathan1, J. Aspden1, S. Deuchars1, J. Deuchars1

1. Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom.

Cardiovascular physiology exhibits a diurnal rhythm e.g. blood pressure dips at night and increases in the morning. Loss of diurnal rhythm of blood pressure is correlated to an increased risk of developing cardiovascular diseases. Blood pressure is to a large part controlled by sympathetic nervous system activity, which exhibits diurnal activity. Since sympathetic preganglionic neurons (SPNs) are the final common pathway the central nervous system influences blood pressure, this project aims to determine if SPN function could be regulated by diurnal expression of genes. The diurnal expression of genes encoding proteins involved in determining neuronal activity were investigated in RNA extracted from the whole spinal cord and from micro-punches that included the location of the majority of SPNs, the intermediolateral cell column (IML) at 7:30 AM and 7:30 PM of C57/Bl6 mice (N= 10) that were terminally anaesthetised with 80mg/Kg intraperitoneal sodium pentobarbitone and had their spinal cords removed and RNA extracted using an RNA extraction kit. qPCR revealed mRNA levels of Bmal1 and Per2 varied with time of day in the punch and spinal cord samples (N=10); Bmal1 mRNA was higher in the morning while Per2 mRNA was higher during the evening. Diurnal rhythm of Bmal1 and Per2 protein levels in SPNs were then examined using immunofluorescence. C57/Bl6 mice (N= 15) were terminally anaesthetised as described above at morning and evening time points, perfused with 4% paraformaldehyde and the spinal cords removed and sectioned at 50 µm on a vibrating microtome. Bmal1 and Per2 protein levels within the SPN nucleus vary with time of day with Bmal1 levels being higher in the morning (N=5 animals, n=15 sections). To investigate a broader sample of genes, RNAseq was performed on micropunches obtained as above. Diurnal rhythm of expression was observed in potassium channel subunits (e.g. Voltage-gated potassium channel subunit beta-2, Kcnab2), sodium channel subunits (e.g. Sodium channel subunit beta-1, Scn1B) and glutamate subunits (e.g. Glutamate [NMDA] receptor subunit 3B, Grin3B) consistent with increased neuronal activity. Single cell patch-Seq is currently underway to examine if genes identified in micropunches are present in single SPNs.

Where applicable, experiments conform with Society ethical requirements