Light-dependent changes in the outer plexiform layer of the mouse retina

Abstract

The ability of the visual system to relay meaningful information over a wide range of lighting conditions is critical to functional vision, and relies on mechanisms of adaptation within the retina that adjust sensitivity and gain as ambient light changes. Photoreceptor synapses represent the first stage of image processing in the visual system, thus activity-driven changes at this site are a potentially powerful, yet under-studied means of adaptation. To gain insight into these mechanisms, the abundance and distribution of key synaptic proteins involved in photoreceptor to ON-bipolar cell transmission were compared between light-adapted mice and mice subjected to prolonged dark exposure (72 hours), by immunofluorescence confocal microscopy and immunoblotting. We also tested the effects on protein abundance and distribution of 0.5-4 hours of light exposure following prolonged darkness. Proteins examined included the synaptic ribbon protein, ribeye, and components of the ON-bipolar cell signal transduction pathway (mGluR6, TRPM1, RGS11, GPR179, Goα). The results indicate a reduction in immunoreactivity for ribeye, TRPM1, mGluR6, and RGS11 following prolonged dark exposure compared to the light-adapted state, but a rapid restoration of the light-adapted pattern upon light exposure. Electron microscopy revealed similar ultrastructure of light-adapted and dark-adapted photoreceptor terminals, with the exception of electron dense vesicles in dark-adapted but not light-adapted ON-bipolar cell dendrites. To assess synaptic transmission from photoreceptors to ON-bipolar cells, we recorded electroretinograms after different dark exposure times (2, 16, 24, 48, 72 hours) and measured the b-wave to a-wave ratios. Consistent with the reduction in synaptic proteins, the b/a ratios were smaller following prolonged dark exposure (48-72 hours) compared to 16 hours dark exposure (13-21%, depending on flash intensity). Overall, the results provide evidence of light/dark-dependent plasticity in photoreceptor synapses at the biochemical, morphological, and physiological levels.

Keywords: bipolar cell; electroretinogram (ERG); immunoblot (western blot); immunofluorescence; light adaptation; photoreceptor; retina; ribbon synapse.

Figure 1

Diagram of photoreceptor synaptic terminals. Rod spherules have one active zone that contacts two horizontal cell dendrites and two rod bipolar cell dendrites. A single cone pedicle contains multiple active zones, each of which contacts two horizontal cell dendrites and dendrites of multiple subtypes of ON and OFF cone bipolar cells. RBC, rod bipolar cell; ON-BC, ON-bipolar cell; OFF-BC, OFF-bipolar cell; SR, synaptic ribbon.

Figure 2

Immunofluorescent labeling of retinal proteins differs between the light-adapted state and prolonged dark adaptation. Retina sections from mice exposed to standard indoor light for 4-6 hours (light-adapted) or to 72 hours of darkness were double labeled with the following pairs of antibodies: PKCα - conformation-specific antibody (green) plus conformation-independent antibody (red), Goα (green) plus recoverin (red), ribeye (green) plus TRPM1 (red); and single labeled for calretinin (red). Confocal images were collected and processed using identical settings for each pair of light-adapted and dark-adapted retina sections. Scale bars represent 10 μm.

Figure 3

Light-dependent changes in proteins at photoreceptor to ON-BC synapses. (A) Immunofluorescent labeling of the different proteins was compared in retinas from mice following normal light adaptation (LA: 12 hr dark + 6-8 hr indoor light), or 72 hours of darkness followed by 0, 0.5, 1, or 4 hours of exposure to indoor light. The proteins examined included a presynaptic ribbon protein (ribeye) and postsynaptic components of the ON-BC signal transduction pathway (TRPM1, mGluR6, GPR179, RGS11, and Goα), as well as the RBC kinase, PKCα. To the right of each row of confocal images are fluorescence intensity profiles for the 0 hr (red) and light-adapted (LA, green) images. The scale bar represents 10 μm for the Goα images and 20 μm for all other images. Retina sections from 4 mice were labeled for each condition. For each antibody, labeling was similar across the quadruplicate sections and representative images are shown. (B) Immunoblots of ribeye, PKCα and ON-BC proteins (TRPM1, mGluR6, RGS11, and Goα) from mice that were either light-adapted or dark exposed for 72 hours followed by 0, 0.5, 1, 2, or 4 hours of exposure to indoor light. To compare changes in protein concentration between the different states, equal quantities of protein were loaded per lane. Each sample was prepared from four retinas from four mice (the other retina from each mouse was used for the immunofluorescence in (A)). (C) Immunoblot bands were quantified by densitometry and normalized to the light-adapted levels. Standard errors were derived from quadruplicate blots (except for ribeye, which used triplicate blots). Significant differences relative to the 72 hr dark-exposed state are indicated with asterisks above the error bars (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).

Figure 4

EM micrographs of light-adapted and dark-adapted photoreceptor active zones. (A) Rod spherules: Electron dense vesicles are present in rod bipolar cell dendrites in the dark-adapted state, but are rarely seen in the light-adapted state. Horizontal cell dendrites are indicated with pink asterisks. (B) Cone pedicles: electron dense vesicles are present in some post-synaptic processes in the dark-adapted state, but not in the light-adapted state. The scale bar in each image represents 200 nm.

Figure 5

ERGs from mice following different dark exposure times. (A) Representative ERG traces from mice that were dark exposed for 2, 16, or 72 hours. Flash strength = -0.06 log(cd*s/m2^). (B) Scotopic ERGs were recorded from mice that had been dark exposed for 2, 16, 24, 48, or 72 hours. The mean b-wave to a-wave ratios are plotted for light intensities that gave a measurable a-wave (-0.06 to 2.41 log(cd*s/m^2)). Values for individual eyes are indicated with red circles. Significant differences relative to the 16 hr dark-adapted state (our standard dark-adaptation time) are indicated with asterisks (*P ≤ 0.05, **P ≤ 0.01).