Date Approved

7-2009

Graduate Degree Type

Thesis

Degree Name

Cell and Molecular Biology (M.S.)

Degree Program

Cell and Molecular Biology

Abstract

Glaucoma is associated with excitotoxicity in which increased glutamate release leads to apoptotic death of retinal ganglion cells (RGCs). Acetylcholine (ACh) has shown neuroprotection of RGCs through the stimulation of RGCs’ nicotinic acetylcholine receptors (nAChRs). The cholinergic amacrine cells are the only cells in retina which synthesize and release ACh. They get excitatory inputs from bipolar cells. The presence of α7nicotinic acetylcholine receptors (α7nAChRs) on the cholinergic amacrines, bipolar cells and RGCs is documented. Recently, stimulation of presynaptic nAChRs of cholinergic cells was shown to enhance ACh release in the rat superior cervical ganglion. Therefore, we hypothesized that α7nAChRs stimulation by tropisetron and PNU282987 (α7nAChR agonists) could induce ACh release through either direct stimulation of cholinergic amacrine α7nAChRs or indirect stimulation of bipolar α7nAChRs. For ACh release studies, pig eyes were dissected and cholinergic amacrine cells were labeled with 40μCi of 3H-choline in which the retina was flashed with light (3Hz) for 30 minutes to maximize 3H-choline uptake. Then, the eyecup was transferred to a perfusion chamber, washed for 20 minutes. 1 minute output fractions were collected into vials and prepared for liquid scintillation counting. To assess the viability of the preparation, light and kainate were applied. Light (2-3 fold increase), kainate (3-4 fold; 10-100μM), α7nAChR agonists (2-4 fold; 0.01-100nM) evoked ACh release greater than the baseline in the absence of DNQX (a glutamate receptor antagonist). In the presence of DNQX, which blocked bipolar input to cholinergic cells, α7nAChRs stimulation did not increase ACh release from baseline. Hence, the possibility of indirect input of bipolar α7nAChRs for ACh release was supported. Our results indicate that ACh release through α7nAChRs stimulation is possible and specifically the bipolar α7nAChRs release of ACh via an indirect positive feedback mechanism. During excitotoxicity, ACh released by amacrine cells, might feedback on bipolar nAChRs to increase ACh release. The neuroprotective effect of tropisetron on α7nAChRs on isolated RGCs is documented. Our study suggests that tropisetron might also protect RGCs through increased ACh release by possible indirect modulation. This study indicates the possibility of dual therapeutic targets of α7nAChRs in the retina for neuroprotection against RGC excitotoxicity.

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