The overall goal of this program is to better understand visual image processing in the mammalian inner retina by defining and characterizing its transmitter systems, cell morphology and circuitry. The focus of these studies is on somatostatin (SRIF), a potent inhibitory transmitter localized to wide-field amacrine cells and its five G protein-coupled receptors, SSTR1-5. The proposed studies are focused on determining the presence of the SSTRs, their cellular expression patterns and relationship to SRIF-containing amacrine cell processes to address the hypothesis that SRIF has a paracrine mode of action and a modulatory influence at both the cellular and circuitry levels by acting at distinct SSTRs expressed by multiple cell populations. Investigations will also study the inhibitory actions of SRIF on intracellular Ca2+ levels in acutely isolated rodent rod bipolar cells and salamander photoreceptors, which strongly express SSTR2, to better define the modulatory action of SRIF at the cellular level.
Specific aim 1 will determine and characterize SSTR1-SSTR5 mRNA tissue and cellular distribution using RT-PCR, RNA blot hybridization and in situ hybridization immunohistochemistry using the rodent retina.
Specific aim 2 will investigate the sites of action of SRIF by defining the expression of SSTR1-SSTR5 and their relationship to SRIF fibers using immunohistochemistry in the rodent and rabbit retina.
Specific aim 3 will investigate the inhibitory action of SRIF at the cellular level using acutely isolated rodent rod bipolar cells and salamander photoreceptors with fluorometric Ca2+ imaging techniques. Studies will characterize SRIF inhibition of a K+ stimulated intracellular Ca2+ increase in these cells, and SRIF's modulation of the Go/Gi G proteins that have been implicated in the cellular action of this peptide. Finally, studies will evaluate SRIF action utilizing SRRF-1 and SRRF-2 receptor agonists. These studies will use rod bipolar cells from normal and Go- or Gi-deficient mice with a gene disruption of the alpha-o, alpha-i1, alpha-i2 or alpha-i3 subunits, and from SSTR-2-deficient mice with a gene disruption of SSTR-2. Experiments will utilize sequence-specific RNA probes, antibodies, SRIF-1 and -2 receptor agonists, and a highly specific SSTR2 antagonist to accomplish the experimental objectives of this application. Proposed studies will provide new information about the role of peptides in the modulation of retinal cells and circuitry in the inner retina, thus providing the basis for a better understanding of visual image processing in the retina. These objectives are consistent with the health-related goals of the National Eye Institute to develop more effective treatments and ultimately to prevent retinal diseases.
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