Cardiotrophin-l (CT-1) and Urocortin (UCN) are peptides which have previously been shown to have protective effects in cardiac myocytes and which induce heat shock protein expression. CT-1 is a member of the interleukin-6 family of cytokines. UCN is a peptide expressed in the brain that binds to the corticotrophin releasing hormone receptors. In this investigation the ability of CT-1 and UCN to protect cells against a lethal stress was tested in neonatal and adult sensory neurons. CT-1 has a protective effect against hypoxic ischaemia in neonatal sensory neurons; in contrast although CT-1 induces the expression of HSP 70 in adult sensory neurons it does not have a protective effect. Results from studies using the kinase inhibitors PD 98059, SB 203580 and LY 294002 suggest that CT-1 is activating the P42/44 MAPK pathway in neonatal sensory neurons. The western blots also reveal a higher level of phosphorylated P42 MAPK in the adult sensory neurons than in the neonatal sensory neurons suggesting that CT-1 does not have a protective effect in the adult neurons because this pathway is already activated. Surprisingly, UCN does not have a protective effect in sensory neurons. In the neonatal cells this could be explained by a lack of receptor expression since RT-PCR revealed an absence of mRNA for both receptors 1 and 2, however; in the adult neurons mRNA was present for both receptors. In conclusion CT-1 but not UCN has an age dependent protective effect in sensory neurons. CT-1 is not the only treatment known to have a protective effect that is lost with age, for instance ischaemic preconditioning is protective in young hearts but not in senescent hearts. The induction of heat shock proteins by mild stress is protective against further stress in young animals but the induction of HSPs is impaired with age. Investigations into the possible therapeutic benefit of increasing heat shock protein expression have previously been performed but this work was carried out using young animals and cells from young animals. This investigation therefore progressed to explore whether increasing heat shock protein expression in neurons from aged animals could protect them from lethal stresses. An HSV-1 based viral vector was used to individually overexpress three members of the heat shock family, HSP 27, HSP 70 and HSP 56, in sensory neurons of aged and neonatal Sprague Dawley rats in vitro. Transcription of the HSP genes in this vector is controlled by a CMV promoter, producing high levels of protein expression. The protective effect of the three proteins against heat shock and hypoxic ischaemia was tested in sensory neurons from aged and neonatal rats. As has been previously shown, in neonatal sensory neurons HSP 27 and HSP 70 protect against cell death due to heat shock. Encouragingly, both HSP 27 and HSP 70 protect the sensory neurons of aged animals against heat shock, although only HSP 27 gave a significant level of protection against hypoxic ischaemia in aged sensory neurons. In conclusion, it is possible to protect neuronal cells of aged animals against stress if the levels of heat shock proteins can be restored.

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels of the nicotinic superfamily. They are found widely in the autonomic nervous system and in selected regions of the central nervous system and are thought to be of importance in the progression and/or treatment of a number of disorders. Despite all this the exact role of cholinergic neurotransmission remains unclear and current cholinergic drug treatments leave a lot to be desired. As such nAChRs remain areas of immense interest and potential. One of the problems with nAChR research has been that the simple receptors obtained in heterologous expression systems probably bear little resemblance to the more complex stoichiometrics seen in vivo. Therefore my PhD dealt with the characterisation of some more complex nAChR compositions and the development of research tools to aid in the research of not just nAChRs but other ligand-gated and voltage-gated ion channels with complex compositions. In this I primarily used two-electrode voltage clamp methods in Xenopus laevis oocytes. My initial research investigated the role of P3 subunit incorporation into a range of nAChRs surprisingly this subunit, which produced only subtle effects when incorporated into a3p4 receptors, completely abolished the currents produced by all other pair and homomer receptors tested. Similar findings were obtained when hippocampal neurones in primary culture (which have a7-like responses) were transfected with the P3 subunit. This suggests a possible major role for P3 in nAChR regulation. One widely used method of constraining receptor stoichiometry is the use of "tandem" subunits. My research however uncovered serious flaws in this method which may render it worse than useless. Subsequent work showed that expression of "pentamer" constructs has greater potential, as it allows much greater control over receptor composition yet avoids the problems seen with the tandem approach.

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