ABSTRACT: The ?7 nicotinic acetylcholine receptor (nAChR) is widely expressed in the nervous system, including in the inner retinal neurons in all species studied to date. Although reductions in the expression of ?7 nAChRs are thought to contribute to the memory and visual deficits reported in Alzheimer's disease (AD) and schizophrenia , the ?7 nAChR knockout (KO) mouse is viable and has only slight visual dysfunction. The absence of a major phenotypic abnormality may be attributable to developmental mechanisms that serve to compensate for ?7 nAChR loss. We hypothesized that the upregulation of genes encoding other nAChR subunits or muscarinic acetylcholine receptor (mAChR) subtypes during development partially accounts for the absence of major deficiencies in the ?7 nAChR KO mouse. The purpose of this study was to determine whether the deletion of the ?7 nAChR subunit in a mouse model resulted in changes in the regulation of other cholinergic receptors or other ion channels in an ?7 nAChR KO mouse when compared to a wild-type (WT) mouse.To examine gene expression changes, we employed a quantitative real-time polymerase chain reaction (qPCR) using whole retina RNA extracts as well as RNA extracted from selected regions of the retina. These extracts were collected using laser capture microdissection (LCM). The presence of acetylcholine receptor (AChR) subunit and subtype proteins was determined via western blotting. To determine any differences in the number and distribution of choline acetyltransferase (ChAT) amacrine cells, we employed wholemount and vertical immunohistochemistry (IHC) and cell counting. Additionally, in both WT and ?7 nAChR KO mouse retinas, the distribution of the nAChR subunit and mAChR subtype proteins were determined via IHC for those KO mice that experienced mRNA changes.In the whole retina, there was a statistically significant upregulation of ?2, ?9, ?10, ?4, nAChR subunit, and m1 and m4 mAChR subtype transcripts in the ?7 nAChR KO mice. However, the retinal layers showed complex patterns of transcript expression. In the ganglion cell layer (GCL), m2 and m4 mAChR subtype transcripts were significantly upregulated, while ?3 and ?4 nAChR subunit transcripts were significantly downregulated. In the inner portion of the inner nuclear layer (iINL), ?2, ?9, ?4, nAChR subunit, and m3 and m4 mAChR subtype transcripts were significantly downregulated. In the outer portion of the inner nuclear layer (oINL), ?2, ?4, and m4 AChR subunit transcripts were significantly upregulated. Western blot experiments confirmed the protein expression of ?3-?5 and ?9-containing nAChR subunits and m1-m2 mAChR subtypes in mouse retinas. IHC results supported many of the mRNA changes observed. Finally, this is the first report of ?9 and ?10 nAChR subunit expressions in the retina of any species.Rather than a simple upregulation of a single AChR subunit or subtype, the absence of the ?7 nAChR in the KO mice was associated with complex layer-specific changes in the expression of AChR subunits and subtypes.
