It is generally accepted that neuropeptides have transmitter-related roles in the central nervous system. However, despite detailed knowledge of their distribution, much remains to be learned about their postsynaptic effects and about the properties of peptidergic neurons. Because the peptidergic neurons in situ are relatively inaccessible, cultures from CNS regions known to contain peptidergic neurons offer a potentially useful approach for their study, and it is on this premise that the present proposal is based. This proposal will focus on examining isolated, identified peptidergic neurons of the rat retina. For the purpose of cell culture, the retina offers the unique advantage of being able to be separated into uniform layers so that enriched retinal cell populations can be separately maintained in culture.
The aims of this proposal are: 1) To identify and characterize VIP-, substance P-, somatostatin-, TRH- and CRF- containing neurons in culture. Using immunohistochemistry, the morphological features of each class of peptidergic neurons will be described and an attempt will be made to establish criteria which can be used to distinguish them. A correlative study will also examine the distribution and three-dimensional profiles of these neurons in the intact retina. 2) To investigate the excitable properties of identified peptidergic neurons in culture. Neurons displaying certain morphological features will be preselected for intracellular recording, their responses to electrical and chemical stimuli will be examined with microelectrodes, and their neuropeptide phenotype will be confirmed subsequently by immunohistochemistry. 3) To examine factors which may regulate neuronal development and peptide expression in culture. Experimental focus will be placed on identifying and investigating a trophic influence of VIP on embryonic neurons developing in culture and on a possible role of electrical activity in regulating the expression of VIP. Overall, the research will contribute to a basic understanding of peptide-mediated neuronal function both in the mature and developing CNS.
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