Functional and proteomic changes which underlie differences in neuronal function in the rostral ventrolateral medulla (RVLM) of Spontaneously Hypertensive rats (SHR) compared to normotensive rats

Activity of presympathetic neurons in the RVLM contribute to sympathetic hyperactivity seen in hypertension. Voltage gated calcium channels (VGCC) underpin essential neuronal functions including excitability, neurotransmitter release and synaptic plasticity. LVA currents activate at hyperpolarized potentials and inactivate rapidly so can generate spontaneous neuronal firing and pacemaking activity, "burst firing" patterns and window currents. VGCC conductances are critical determinants of the ongoing activity of RVLM neurons. For example, Ni2+-sensitive Ca2+ currents drive bursting of RVLM neurons in response to focal hypoxia and in the absence of synaptic inputs in neonatal brain slice. We have previously shown that LVA, but not HVA conductance, in adult RVLM, is critical to maintaining sympathetic vasomotor tone in normotensive Sprague Dawley (SD) rats. The first aim was to determine functionally the role of the LVA conductance in the RVLM of SHR and normotensive Wistar Kyoto (WKY). Urethane anaesthetised (1.3 g/kg ip) ventilated animals were used. Bilateral microinjection of NiCl2 (50 nl, 50mM) were made into the RVLM of SD, WKY and SHR. In both SD (n=4) and WKY (n=5) SNA decreased immediately (peak change -56 ± 8% and -63 ± 4.5 %) as did BP (peak change -31 ± 5 and -39 ± 2 and mmHg) and HR (-33 ± 13 bpm and -57 ± 6 bpm). Vehicle had no effect. Ni2+ evoked effects were not significantly different between the two strains. In contrast injections of NiCl2 in SHR (n=6) elicited sustained increases in SNA (+54 ± 6 %), AP (+42 ± 8 mmHg) and HR (+41 ± 9 bpm). The quandary is to explain how Ni2+ could evoke this response in SHR. T-type channels can be modulated by various endogenous ligands and a number of functional and chemical differences have been individually described in the RVLM of SHR compared to WKY. In order to identify all possible candidates that could modify T-type channels we next sought to compare the protein profile in the RVLM of SHR (n=3) and WKY (n=3) by shot gun proteomics. Brains were rapidly removed under pentobarbitone anaesthesia (100mg/kg ip) and frozen before RVLM region dissection (~1mm x 1.5mm). Conventional protein preparation and digestion strategies preceded LC-MS/MS. In total 1438 proteins were identified with 168 significantly differentially expressed:70 up regulated and 98 down regulated in SHR vs WKY. Key pathways affected include: increased glutamatergic signalling (VGLUT2, EAAT, GNAO); synaptic vesicle related activity (SYN1, SYN2, SYT, SNAP25, SYNGR3, NRXN3, FLOT1, FLOT2), remodelling (MAP6, CAPZB, SEPT6, GSN, TUBB3, TUBB4, TUBB5, TUBB4B, EMS1), decreased GABAergic signalling (GAD, ABAT, GPHN), oxidative phosphorylation (ND1, NDUF6, CYTB, CYTC, ATP5G1) and altered glutathione signalling (GSS, GSTT1).