Effect of the M1 Muscarinic Acetylcholine Receptor on Retinal Neuron Number Studied with Gene-Targeted Mice

Abstract

Pharmacological activation of the M1 muscarinic receptor subtype was suggested to promote the survival of retinal neurons. We examined the hypothesis that the M1 receptor is crucial for retinal neuron survival in vivo by using mice devoid of the M1 receptor gene. Muscarinic receptor gene expression was determined in the retina using real-time PCR. The amount of neurons in the retinal ganglion cell layer and of axons in the optic nerve was determined in retinal wholemounts stained with cresyl blue and in optic nerve cross-sections stained with toluidine blue, respectively. mRNA of all five muscarinic receptor subtypes (M1-M5) was detected in the retina from wild-type mice. Remarkably, M2 and M3 receptor mRNA were most abundant. In retinas from M1 receptor-deficient mice, M4 receptor mRNA expression was increased compared to that of wild-type mice, while no marked changes in the mRNA expression levels of the other muscarinic receptor subtypes were observed. The amount of cells in the retinal ganglion cell layer and the amount of axons in the optic nerve did not differ between M1 receptor-deficient and wild-type mice. The present findings suggest that the M1 receptor is not essential for the survival of retinal neurons in vivo.

The M1 muscarinic acetylcholine receptor subtype is important for retinal neuron survival in aging mice

“…In a previous study from our laboratory conducted on 5 month-old M1R−/− mice of a mixed genetic background, we found that neuron number in the RGC layer and optic nerve axon number were not affected by M 1 receptor deficiency 31 . One possible explanation for these findings was that the M1R−/− mice used in the previous study might have been too young to develop neuronal loss.…”

“…In the retina, cholinergic amacrine cells are the source of the physiological muscarinic receptor agonist, acetylcholine, which was shown to transmit cholinergic signals to RGCs via muscarinic and nicotinic receptors 40–43 . However, although multiple muscarinic receptor subtypes, including the M 1 receptor, were detected on RGCs, amacrine cells and bipolar cells, the specific role of the individual subtypes in retinal cell-to-cell signaling is still poorly understood 31,42 . Overlapping receptor subtype expression patterns in individual cell types, limited selectivity of pharmacological ligands and low specificity of antibodies for individual muscarinic receptor subtypes are obstacles in this regard 28,44,45 .…”

Apolipoprotein E Deficiency Causes Endothelial Dysfunction in the Mouse Retina

“…Next, a retinal wholemount was prepared in cold PBS, transferred onto a glass slide, and stained with cresyl blue as previously described [29]. After the staining procedure, sixteen predefined areas per wholemount, eight central and eight peripheral, of 150 μ m × 200 μ m were photographed by a blinded investigator as reported in detail previously [25, 30, 31]. Per photograph, all cresyl blue-positive cells were counted manually by a blinded investigator using the cell counter plugin for ImageJ software (NIH, http://rsb.info.nih.gov/ij/) as previously described [25, 31].…”

“…After the staining procedure, sixteen predefined areas per wholemount, eight central and eight peripheral, of 150 μ m × 200 μ m were photographed by a blinded investigator as reported in detail previously [25, 30, 31]. Per photograph, all cresyl blue-positive cells were counted manually by a blinded investigator using the cell counter plugin for ImageJ software (NIH, http://rsb.info.nih.gov/ij/) as previously described [25, 31]. The mean cell density (cells/mm 2 ) was calculated and multiplied with the wholemounts' surface area to obtain the total number of cells per retina.…”

“…Axons were counted manually by a blinded investigator using ImageJ software. The mean axon density (axons/mm 2 ) was calculated and multiplied by the cross-sectional area to obtain the total number of axons per optic nerve as described recently in detail [25, 31].…”