The objectives of this proposal are to provide training in the techniques of immunocytochemistry, confocal microscopy and electrophysiological recording as means to study the function of gap junctions in retinal physiology and neural transmission. Specifically, this proposal is designed to identify and characterize the connexins forming the homologous gap junctions between All amacrine cells (Ails) and the heterologous gap junctions between Ails and cone bipolar cells (CBs). It is unknown exactly how many different types of CBs exist in rabbit and how many of these types are coupled to Ails.
Specific Aim I of this proposal will address this issue through injection of biotinylated tracers into Ails in flat mount retina. Subsequent confocal examination of the labeled CBs will be used to classify them into different groups based on immunoreactivity and morphology.
Specific Aim 2 combines molecular and immunocytochemical approaches to define the connexins that comprise the AII-CB gap junctions. Known retinal connexins will be localized to specific cell types in rabbit retina by confocal microscopic examination of retinas immunoreacted with connexin antibodies and well described cell type markers. Antisense oligonucleotides will be used to ?knockdown? expression of these connexins, and loss of tracer coupling to particular cell types will indicate that the uncoupled cells normally express the knocked down connexin.
Specific Aim 3 will develop a slice preparation using dual whole cell patch clamp to determine the electrical conductance and pharmacological regulation of All coupling, progressing our understanding of how these gap junctions function physiologically.
| Trexler, E Brady; Li, Wei; Massey, Stephen C (2005) Simultaneous contribution of two rod pathways to AII amacrine and cone bipolar cell light responses. J Neurophysiol 93:1476-85 |
| Massey, Stephen C; O'Brien, Jennifer J; Trexler, E Brady et al. (2003) Multiple neuronal connexins in the mammalian retina. Cell Commun Adhes 10:425-30 |
