Most of the known physiological effects of nitric oxide (NO) in the brain are mediated by activation of specialised guanylyl cyclase-coupled receptors, leading to a rise in intracellular cGMP. Apart from protein kinase activation little is known about subsequent cGMP signal transduction. In optic nerve axons, hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels, which bind cGMP (and cAMP) directly, appear to be a target. The objective was to test this possibility directly using electrophysiological methods. Studies were initially carried out by recording extracellularly from Schaffer collateral/commissural axons in hippocampal slices, where the NO-cGMP pathway contributes to synaptic plasticity. Pharmacological manipulation of the NO-cGMP pathway failed to affect significantly axonal conduction at 0.2 - 5 Hz, a frequency range in which HCN channels were found to influence conduction reliability. Raising cAMP levels were similarly ineffective suggesting that, unlike in optic nerve, the subunit composition is likely to render the HCN channels relatively cyclic nucleotide-insensitive. Next, I investigated two neuronal types known to express the cyclic nucleotide-sensitive HCN channel subunits (HCN2 and/or HCN4), namely the principal cells of the medial nucleus of the trapezoid body and of the deep cerebellar nuclei. Using whole-cell voltage clamp, I found no reproducible evidence of regulation of HCN channel function by NO, even though exogenous cGMP was effective routinely and the neurones expressed NO-activated guanylyl cyclase, as shown by immunohistochemistry. I then carried out a series of non-invasive sharp electrode current-clamp recordings in deep cerebellar nuclear neurones. Using the characteristic voltage sag as an index of HCN channel operation, exogenous NO was found to modulate the channels reproducibly. Attempts to refine the original whole-cell recording solution to optimise preservation of the NO-cGMP pathway failed to restore NO-sensitivity. Minimising cell dialysis by using the perforated-patch variant of the whole-cell method, however, was successful. The results provide direct evidence that HCN channels are potential downstream mediators of NO-cGMP signaling in the deep cerebellar nuclei and suggest that the importance of this transduction pathway may have been previously overlooked because of unsuitable recording methods. 3.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:498498 |
| Date | January 2008 |
| Creators | Wilson, Gary William |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1445186/ |
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