Abstract
Purpose: :
In the mammalian retina, cone opsin is susceptible to chemical bleaching with hydroxylamine, and rhodopsin is susceptible to bleaching with a combination of light and hydroxylamine. Hydroxylamine forms a Schiff base with retinal, replacing the Schiff base formed by retinal and opsin, and creates an oxime that is not usable by the opsin. The susceptibility of melanopsin to bleaching in situ with hydroxylamine alone or in combination with light has not been characterized.
Methods: :
Ex vivo multielectrode array recordings from live murine retina were used to monitor melanopsin-dependent (ipRGC) and cone-dependent retinal ganglion cell light responses to to 480 nm light at 3.98x1013 photons/cm2/s before and after 15 minute hydroxylamine treatments. Neutralized hydroxylamine in AMES buffer was used at concentrations of 10 mM, 30 mM and 60 mM. Light co-treatment consisted of 480 nm light at 3.98x1013 photons/cm2/s during the 15 minutes of hydroxylamine treatment.
Results: :
Treatment of retinas with 10 mM hydroxylamine had little effect on ipRGC responses and caused a transient decrease in cone-driven activity. At 30 mM hydroxylamine treatment, cones were completely chemically bleached, while ipRGC activity was decreased by only 20%. With the addition of light to the 30 mM hydroxylamine treatment, a condition under which both rod and cone activity should be eliminated, ipRGC activity remained unchanged from hydroxylamine treatment alone. At 60 mM with or without light co-treatment, ipRGC activity is reduced to 50-60%of baseline.
Conclusions: :
Melanopsin-driven ipRGCs are resistant in situ to bleaching conditions sufficient to fully bleach cone photopigment. Such data provide further support for the hypothesis that the melanopsin photocycle is independent of both rod and cone photocycle mechanisms.
Keywords: retinoids/retinoid binding proteins • photoreceptors • circadian rhythms