Muscarinic acetylcholine receptor localization and activation effects on ganglion response properties

Abstract

Purpose: The activation and blockade of muscarinic acetylcholine receptors (mAChRs) affects retinal ganglion cell light responses and firing rates. This study was undertaken to identify the full complement of mAChRs expressed in the rabbit retina and to assess mAChR distribution and the functional effects of mAChR activation and blockade on retinal response properties.

Methods: RT-PCR, Western blot analysis, and immunohistochemistry were used to identify the complement and distribution of mAChRs in the rabbit retina. Extracellular electrophysiology was used to determine the effects of the activation or blockade of mAChRs on ganglion cell response properties.

Results: RT-PCR of whole neural retina resulted in the amplification of mRNA transcripts for the m1 to m5 mAChR subtypes. Western blot and immunohistochemical analyses confirmed that all five mAChR subtypes were expressed by subpopulations of bipolar, amacrine, and ganglion cells in the rabbit retina, including subsets of cells in cholinergic and glycinergic circuits. Nonspecific muscarinic activation and blockade resulted in the class-specific modulation of maintained ganglion cell firing rates and light responses.

Conclusions: The expression of mAChR subtypes on subsets of bipolar, amacrine, and ganglion cells provides a substrate for both enhancement and suppression of retinal responses via activation by cholinergic agents. Thus, the muscarinic cholinergic system in the retina may contribute to the modulation of complex stimuli. Understanding the distribution and function of mAChRs in the retina has the potential to provide important insights into the visual changes that are caused by decreased ACh in the retinas of Alzheimer's patients and the potential visual effects of anticholinergic treatments for ocular diseases.

Figure 1.

PST histograms (nine trials) of the responses of an RGC to puff application of 500 μM choline. Choline application (A, horizontal bar) suppressed the firing of the cell (P < 0.001) compared with the normal maintained firing rate (B). The time course of choline-induced suppression was reduced by bath application of 3 μM atropine (C, P < 0.01), and the maintained firing rate was decreased (D).

Figure 2.

PST histograms (15 trials) showing the responses of an OFF RGC to light flashes (A), light flashes paired with choline (C), and choline alone (B). In regular Ames medium, the cell responded to 1-second light stimuli with sustained OFF response (black bars). The response to a 1-second application of 1 mM choline (gray bars) was multiphasic and included both enhancement and suppression of firing. Bath application of 3 μM atropine (D–F) resulted in decreases across all conditions, but did not completely eliminate the choline-induced enhancement of the firing rate.

Figure 3.

RT-PCR of mRNA extracted from rabbit retina demonstrated the presence of m1 to m5: m1, 573 bp; m2, 469 bp; m3, 434 bp; m4, 592 bp; and m5, 451 bp. Product identity was confirmed by sequencing. Sequence homology to human ranged from 83% to 95%.

Figure 4.

Western blot analysis with antibodies to the five muscarinic mAChR subtypes resulted in single bands, indicating the presence of mAChR protein and showed that each antibody was specific for a single protein in retinal neural extracts.

Figure 5.

Comparison of labeling patterns of two antibodies against m2: mAb367 (green) and NLS 1333 (red). There was expected correspondence in the labeling of cell bodies (overlay). Confocal images of five optical sections taken at 0.5-μm intervals. Scale bar, 50 μm.

Figure 6.

Antibodies against muscarinic AChRs labeled specific subpopulations of horizontal, bipolar, amacrine, and ganglion cells, and immunoreactive processes were broadly distributed throughout the IPL. Confocal images of five optical sections were obtained in 0.5-μm intervals. Scale bar, 50 μm.

Figure 7.

The distribution of muscarinic AChR subtypes relative to ChAT. Maximum projection images of five optical confocal sections showed immunoreactivity to the mAChR subtypes (top row) and ChAT (middle row). Immunoreactivity for all subtypes predominated in the inner retina, with a broad distribution through the IPL, although m3 immunoreactivity was closely apposed to ChAT immunoreactivity in the IPL. Subsets of ChAT-immunoreactive cells express each of the mAChR subtypes. Confocal images of five optical sections were obtained in 0.5-μm intervals. Scale bar, 40 μm.

Figure 8.

The distribution of muscarinic subtypes relative to glyt-1. Maximum projection images of five optical confocal sections showed immunoreactivity to the mAChR subtypes (top row) and glyt-1 (middle row). Colocalization was limited. A small proportion of amacrine cells that expressed m1, m2, and m4 also expressed glyt-1. Cells expressing m3 and m5 did not express glyt-1. Confocal images of five optical sections were obtained in 0.5-μm intervals. Scale bar, 50 μm.

Figure 9.

Pair-wise comparisons of m1, m3, and m5 immunoreactivity relative to that of m2. Maximum projection images of five optical confocal sections shows immunoreactivity to m1, m3, and m5 (top row) relative to m2 immunoreactivity (middle row). There was very limited colocalization between m1 and m2 (left). Subsets of bipolar, amacrine, and ganglion cells expressed both m3 and m2 (middle), whereas only cells in the GCL expressed both m5 and m2 (right). Confocal images of five optical sections were obtained in 0.5-μm intervals. Scale bar, 32 μm.

Figure 10.

Pair-wise comparisons of m1, m3, and m5 immunoreactivity relative to m4. Maximum projection images of five optical confocal sections showed immunoreactivity to m1, m3, and m5 (top row) relative to m4 immunoreactivity (middle row). The labeling patterns of m1 and m4 (left) and m5 and m4 (right) were distinctly similar but limited to the IPL and GCL. There was no apparent colocalization of m3 and m4 in neural retina, but there was apparent colocalization in the NFL (middle). Confocal images of five optical sections were obtained in 0.5-μm intervals. Scale bar, 50 μm.

Figure 11.

Comparison of m2 mAChR expression relative to m4 mAChR expression. Maximum projection images of five optical confocal sections showed immunoreactivity to antibodies against m2 (top) and antibodies against m4 (middle). Most of the cells in the INL expressed m2 but not m4. Immunoreactivity for both subtypes was broadly distributed throughout the IPL. More detailed examination showed two narrow bands in the IPL that were immunoreactive for m4 but not m2. One band was located in the outer portion of the OFF sublamina, and the other band was located in the outer portion of the ON sublamina. Most of the cells in the GCL expressed m4 but not m2. A small subset of RGCs was immunoreactive to antibodies against both m2 and m4. Confocal images of five optical sections were obtained in 0.5-μm intervals. Scale bar, 50 μm.