Secretin: Expression, Endogenous Release and Multiple Neuroactive Actions in the Cerebellum

This dissertation, "Secretin: Expression, Endogenous Release and Multiple Neuroactive Actions in the Cerebellum" by Man-yan, Lee, 李敏茵, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled SECRETIN: EXPRESSION, ENDOGENOUS RELEASE AND MULTIPLE NEUROACTIVE ACTIONS IN THE CEREBELLUM Submitted by Lee Man Yan for the degree of Doctor of Philosophy at The University of Hong Kong in May 2005 Secretin, a 27 amino acid peptide, was the first hormone discovered. The gastrointestinal functions of secretin are well-established, although its functional role in the brain is still uncertain. It has been suggested that secretin offers therapeutic benefit in autism, and the cerebellum was the most frequently reported brain area to have abnormality in autistic patients. Taken together, these observations urge an in-depth investigation on the actions of secretin and its role as a neuropeptide in the cerebellum. In this study, the expression, release characteristics, electrophysiological effects and other neuroactive actions of secretin were investigated using various approaches. Secretin and secretin receptor mRNAs and peptides were found to be expressed prominently in distinct neurons in the rodent cerebellum, including the Purkinje cells and the deep cerebellar nuclear neurons. Electrophysiological study using whole-cell patch-clamp recordings from deep cerebellar nuclear neurons demonstrated that secretin consistently increased the frequency of the miniature inhibitory postsynaptic currents and suggested that secretin facilitates GABAergic inputs onto deep cerebellar nuclear neurons via a presynaptic mechanism. Taken together, it is proposed that secretin is released from the deep cerebellar nuclear neurons and acts as a retrograde messenger to potentiate GABA release from the Purkinje cells in the cerebellum. The role of secretin as a neuropeptide in the cerebellum was further confirmed by a release study using enzyme immuno-assay. Secretin was found to be released endogenously, involving voltage-gated sodium and calcium channels. The regulatory action of secretin in the cerebellum was demonstrated by monitoring the activation of regional Fos expression in response to intracerebroventricular injection of secretin in mice. Secretin-induced Fos expression was confined to the Purkinje cells and deep cerebellar nuclear neurons, consistent with the hypothesis that the action of secretin involves the activation of specific neurons in the cerebellum. In addition, microarray analysis was performed to investigate the effects of secretin in the gene expression profiles in the cerebellum. The data indicated that secretin is involved in many biological functions via the activation/silencing of genes in the process of neuroregulation. Further investigation on the expressions of secretin and secretin receptor in the human cerebellum was performed using in situ hybridization, immunohistochemistry, confocal laser scanning microscopy and real time RT-PCR. Secretin and secretin receptor mRNAs were confined to the Purkinje cells of the human cerebellum. Immunoreactivity of secretin was localized in the Purkinje cells. On detailed examination, secretin peptides were found to be expressed in both the somata and dendrites of the Purkinje cells. Therefore, the expression patterns of secretin and its receptor are similar in both human and rodent cerebellums. The role of secretin as a neuropeptide was clearly demonstrated in the present study. Based on this novel and comprehensive study showing the neuroactive functions of secretin i