Characterization and Expression of Nicotinic ACh Receptors in the Mammalian Retina

Abstract

Abstract: : Purpose: Epidemiological and clinical studies suggest that nicotinic agonists can act as neuroprotective agents. However, before investigating the possible neuroprotective properties of RGCs via nAChRs, the presence and subunit composition of nAChRs in the retina needs to be confirmed. Methods: Radioligand binding assays with pig retinal homogenates, functional imaging with rat retinal slices and RT-PCR analysis with human and knockout mouse retina. Results: Binding studies with ₃H-cytisine, ₃H-epibatidine and ₁₂₅I-α-bungarotoxin (BTX) in porcine retinal membranes gave high affinity binding with specificity that was consistent with typical α4 and α3 subunits & suggestive of α7 nicotinic binding sites, respectively. Retinal studies with ₁₂₅I-α-BTX autoradiography in the pig and image analysis with deconvoluting microscopy using tagged α-BTX in the rat supported these findings. Time-series analysis of intracellular calcium dynamics, in response to nicotine, showed concentration-dependent increases in fluorescence. Similar responses to epibatidine and the α7 selective agonist AR-R 17779, were observed with low nanomolar and micromolar concentrations, respectively. A sample of human retina and RPE were screened for α7 nAChR message using human whole brain and hippocampal cDNAs as positive controls. Bands with the proper MW were only detected in brain, hippocampal & retinal cDNA, and not in the negative controls, human RPE and and dH₂O. Sequencing of the 450bp PCR product from human retinal cDNA, confirmed that it was α7. In a parallel study, retinal tissue from transgenic knock-out and wild-type α7 mice were screened for α7 nAChR message. Eyes from wild-type and knock-out mice were cryosectioned and processed through a dehydration series. The inner portion of each retinal section (corresponding to the RGC layer) was dissected by laser capture microdissection from the wild-type and knock-out mice. A strong band in the PCR assay for the wild-type sample was found with no corresponding band for the knockout. Mouse hippocampal cDNA served as a positive control with dH₂O as a negative. Furthermore, when the PCR fragment from the wild-type inner retina was sequenced from both ends of the PCR product, the sequence was confirmed as α7. This demonstrates that α7 message is present in wild-type mouse inner retina & missing in the knock-out. Conclusion: Collectively, these results indicate the presence of multiple nicotinic receptors in the mammalian retina and suggest the presence of specific subtypes including the α7 nAChR.

Keywords: 554 retina • 557 retina: proximal(bipolar, amacrine, and ganglion cells) • 490 neurotransmitters/neurotransmitter systems