Non-image-forming light driven functions are preserved in a mouse model of autosomal dominant optic atrophy.

Autosomal dominant optic atrophy (ADOA) is a slowly progressive optic neuropathy that has been associated with mutations of the OPA1 gene. In patients, the disease primarily affects the retinal ganglion cells (RGCs) and causes optic nerve atrophy and visual loss. A subset of RGCs are intrinsically photosensitive, express the photopigment melanopsin and drive non-image-forming (NIF) visual functions including light driven circadian and sleep behaviours and the pupil light reflex. Given the RGC pathology in ADOA, disruption of NIF functions might be predicted. Interestingly in ADOA patients the pupil light reflex was preserved, although NIF behavioural outputs were not examined. The B6; C3-Opa1(Q285STOP) mouse model of ADOA displays optic nerve abnormalities, RGC dendropathy and functional visual disruption. We performed a comprehensive assessment of light driven NIF functions in this mouse model using wheel running activity monitoring, videotracking and pupillometry. Opa1 mutant mice entrained their activity rhythm to the external light/dark cycle, suppressed their activity in response to acute light exposure at night, generated circadian phase shift responses to 480 nm and 525 nm pulses, demonstrated immobility-defined sleep induction following exposure to a brief light pulse at night and exhibited an intensity dependent pupil light reflex. There were no significant differences in any parameter tested relative to wildtype littermate controls. Furthermore, there was no significant difference in the number of melanopsin-expressing RGCs, cell morphology or melanopsin transcript levels between genotypes. Taken together, these findings suggest the preservation of NIF functions in Opa1 mutants. The results provide support to growing evidence that the melanopsin-expressing RGCs are protected in mitochondrial optic neuropathies.

Diurnal and circadian regulation of connexin 36 transcript and protein in the mammalian retina.

PURPOSE: Gap junctional coupling between rod and cone photoreceptor cells is regulated by light and the circadian clock, and contributes to retinal light adaptation. Phosphorylation of connexin 36 (Cx36) has been proposed as the mechanism involved. We investigated whether retinal Cx36 is also regulated at the level of transcript and protein expression. METHODS: At specific time points in a diurnal or circadian cycle, Cx36 protein was assessed by Western blotting and immunohistochemistry, and Cx36 transcript by quantitative real time PCR in a melatonin-deficient (C57BL6/FVB) and two melatonin-proficient (C3H(+/+) and C3H(rd/rd)) mouse strains. RESULTS: In C57BL6/FVB mice during a diurnal cycle, Cx36 protein expression was rhythmic, peaking at approximately zeitgeber time (ZT) 20. However, this rhythm was not maintained in the circadian cycle. In C3H(+/+) mice levels of Cx36 protein were higher at night and subjective night relative to day and subjective day, respectively. These patterns of Cx36 expression were localized primarily to the outer plexiform layer in both strains. Cx36 transcript expression was higher at night and subjective night relative to day and subjective day in C57BL6/FVB and C3H(+/+) mice. Rhythmic expression of Cx36 transcript was lost in retinally degenerate C3H(rd/rd) mice. CONCLUSIONS: The results suggested the circadian control of Cx36 protein expression is dependent on melatonin, whereas the circadian regulation of Cx36 transcript expression may be controlled directly by the circadian clock. In addition to post-translational modification, regulation of Cx36 transcript and protein expression may be important during retinal light adaptation.

Differential expression of melanopsin isoforms Opn4L and Opn4S during postnatal development of the mouse retina.

Photosensitive retinal ganglion cells (pRGCs) respond to light from birth and represent the earliest known light detection system to develop in the mouse retina. A number of morphologically and functionally distinct subtypes of pRGCs have been described in the adult retina, and have been linked to different physiological roles. We have previously identified two distinct isoforms of mouse melanopsin, Opn4L and Opn4S, which are generated by alternate splicing of the Opn4 locus. These isoforms are differentially expressed in pRGC subtypes of the adult mouse retina, with both Opn4L and Opn4S detected in M1 type pRGCs, and only Opn4L detected in M2 type pRGCs. Here we investigate the developmental expression of Opn4L and Opn4S and show a differential profile of expression during postnatal development. Opn4S mRNA is detected at relatively constant levels throughout postnatal development, with levels of Opn4S protein showing a marked increase between P0 and P3, and then increasing progressively over time until adult levels are reached by P10. By contrast, levels of Opn4L mRNA and protein are low at birth and show a marked increase at P14 and P30 compared to earlier time points. We suggest that these differing profiles of expression are associated with the functional maturation of M1 and M2 subtypes of pRGCs. Based upon our data, Opn4S expressing M1 type pRGCs mature first and are the dominant pRGC subtype in the neonate retina, whereas increased expression of Opn4L and the maturation of M2 type pRGCs occurs later, between P10 and P14, at a similar time to the maturation of rod and cone photoreceptors. We suggest that the distinct functions associated with these cell types will develop at different times during postnatal development.

Cloning and distribution of myosin 3B in the mouse retina: differential distribution in cone outer segments.

Class III myosins are important for the function and survival of photoreceptors and ciliary hair cells. Although vertebrates possess two class III myosin genes, myo3A and myo3B, recent studies have focused on Myo3A because mutations in the human gene are implicated in progressive hearing loss. Myo3B may compensate for defects in Myo3A, yet little is known about its distribution and function. This study focuses on Myo3B expression in the mouse retina. We cloned two variants of myo3B from mouse retina and determined that they are expressed early in retinal development. In this study we show for the first time in a mammal that both Myo3B and Myo3A proteins are present in inner segments of all photoreceptors. Myo3B is also present in outer segments of S opsin-immunoreactive cones but not M opsin dominant cones. Myo3B is also detected in rare cells of the inner nuclear layer and some ganglion cells. Myo3B may have diverse roles in retinal neurons. In photoreceptor inner segments Myo3B is positioned appropriately to prevent photoreceptor loss of function caused by Myo3A defects.