Interactions of cone abundancies, opsin expression, and environmental lighting with emmetropization in chickens.

We had previously found that M to L cone abundancy ratios in the chicken retina are correlated with vitreous chamber depth and refractive state in chickens eyes, when they have normal visual exposure but not when they develop deprivation myopia. The finding suggests an interaction between cone abundancies and emmetropization. In the current study, we analyzed how stable this correlation was against changes in environmental variables and strain differences. We found that the correlation was preserved in two chicken strains, as long as they were raised in the laboratory facilities and not in the animal facilities of the institute. To determine the reasons for this difference, spectral and temporal lighting parameters were better adjusted in both places, whereas temperature, humidity, food, diurnal lighting cycles and illuminance were already matched. It was also verified that both strains of chickens had the same cone opsin amino acid sequences. The correlation between M to L cone abundancy and ocular biometry is highly susceptible to changes in environmental variables. Yet undetermined differences in lighting parameters were the most likely reasons. Other striking findings were that green cone opsin mRNA expression was downregulated when deprivation myopia developed. Similarly, red opsin mRNA was downregulated when chicks wore red spectacles, which made them more hyperopic. In summary, our experiments show that photoreceptor abundancies, opsin expression, and the responses to deprivation, and therefore emmetropization, are surprisingly dependent on subtle differences in lighting parameters.

Hydrogen/Deuterium Exchange Mass Spectrometry of Human Green Opsin Reveals a Conserved Pro-Pro Motif in Extracellular Loop 2 of Monostable Visual G Protein-Coupled Receptors.

Opsins comprise the protein component of light sensitive G protein-coupled receptors (GPCRs) in the retina of the eye that are responsible for the transduction of light into a biochemical signal. Here, we used hydrogen/deuterium (H/D) exchange coupled with mass spectrometry to map conformational changes in green cone opsin upon light activation. We then compared these findings with those reported for rhodopsin. The extent of H/D exchange in green cone opsin was greater than in rhodopsin in the dark and bleached states, suggesting a higher structural heterogeneity for green cone opsin. Further analysis revealed that green cone opsin exists as a dimer in both dark (inactive) and bleached (active) states, and that the predicted glycosylation sites at N32 and N34 are indeed glycosylated. Comparison of deuterium uptake between inactive and active states of green cone opsin also disclosed a reduced solvent accessibility of the extracellular N-terminal region and an increased accessibility of the chromophore binding site. Increased H/D exchange at the extracellular side of transmembrane helix four (TM4) combined with an analysis of sequence alignments revealed a conserved Pro-Pro motif in extracellular loop 2 (EL2) of monostable visual GPCRs. These data present new insights into the locus of chromophore release at the extracellular side of TM4 and TM5 and provide a foundation for future functional evaluation.

Simultaneous recording of multifocal VEP responses to short-wavelength and achromatic stimuli.

A paradigm is introduced that allows for simultaneous recording of the pattern-onset multifocal visual evoked potentials (mfVEP) to both short-wavelength (SW) and achromatic (A) stimuli. There were 5 sets of stimulus conditions, each of which is defined by two semi-concurrently presented stimuli, A64/SW (a 64% contrast achromatic stimulus and a short-wavelength stimulus), A64/A8 (64% achromatic/8% achromatic), A0/A8 (0% (gray) achromatic/8% achromatic), A64/A0 and A0/SW. When paired with A64 as part of A64/SW, the SW stimulus yielded mfVEP responses (SWmfVEP) with diminished amplitude in the fovea, consistent with the known sensitivity of the S-cone system. In addition, when A8, which is approximately equal to the L and M cone contribution of the SW stimulus, was recorded alone, the response to A8 was small, but significantly larger than noise. However, when A8 was paired with A64, the response to A8 was reduced to close to noise level, suggesting that the LM cone contribution of the SWmfVEP can be suppressed by A64. When A64 was recorded alone, the response to A64 was about 32% larger than the mfVEP for A64 when paired with the SW. Likewise, the presence of A64 stimulus also reduces the response of SWmfVEP by 35%. Finally, an intense narrow-band yellow background prolonged the latency of SW response for the A0/SW stimulus but not the latency of SW response for the A64/SW stimulus. These results indicate that it is possible to simultaneously record an SWmfVEP with little LM cone contribution along with an achromatic mfVEP.