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-Opa1Q285STOP 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

Investigating non-image forming photoreception in a mouse model of autosomal dominant optic atrophy

Autosomal Dominant Optic Atrophy (ADOA) is a progressive optic neuropathy affecting mainly the retinal ganglion cells (RGCs). It is associated with mutations in the Opa1 gene and is phenotypically characterized by decreased visual acuity, central field deficits and colour vision defects. Experimental work on Opa1 mutant mice (B6; C3-Opa1Q285STOP) has permitted further characterisation of the pathophysiology of the disease. A specific functional visual deficit in the photopic negative response of the electroretinogram has been described in these mice, possibly due to altered dendritic pruning of RGCs. However, non-image-forming (NIF) visual function, which is regulated by a subset of RGCs that express the photopigment melanopsin, has not yet been extensively investigated in Opa1 mutant mice. We were interested in whether RGC dysfunction in Opa1 mutants affects NIF behaviours. We evaluated circadian behaviour, sleep behaviour and melanopsin expression in Opa1 mutant mice (Opa1+/-) and littermate controls (Opa1+/+). Opa1 mutant mice were able to entrain their behaviour rhythm to a normal 12:12 hr light/dark cycle, confining their activity to the dark phase. The suppression of activity by acute light exposure at night (negative masking) was equivalent between genotypes. Circadian phase shift responses to 480 nm or 520 nm light pulses during the subjective night were preserved in Opa1+/- mice relative to wildtype controls. The acute induction of sleep by light exposure at night was also present in Opa1+/- mice and not significantly different to Opa1+/+ animals. Immunohistochemical characterisation of melanopsin cells in flatmount retinae revealed no significant differences in cell numbers betweeen genotypes. Melanopsin (Opn4) transcript levels were also equivalent between Opa1 wildtype and mutant mice. There was also no obvious difference in melanopsin cell stratification patterns. The data overwhelmingly support the preservation of the NIF visual system in Opa1 mutant mice. The findings are consistent with patient studies suggesting increased resistance of melanopsin-expressing RGCs in conditions of mitochondrial optic atrophy. Further work is needed to extend our understanding of the possible neuroprotective mechanism involved which could lead to exciting therapeutic strategies.This thesis is not currently available in OR

Phase shift behaviour in <i>Opa1^+/+</i> and <i>Opa1^+/−</i> mice.

<p>Representative actograms from (A) <i>Opa1</i>^+/+ and (B) <i>Opa1</i>^+/<i>−</i> mice in constant dark (DD) conditions. Animals were exposed to 15 min light pulses every ∼15 days. Photon matched pulses at 480 nm (black arrow) or 525 nm (white arrow; 1×10¹¹ photons/s/cm²) were applied at CT16. Animals were also exposed to a dark sham pulse condition (grey arrow). (C) The size of the phase shift response are plotted for the 525 nm, 480 nm and sham conditions for <i>Opa1^+/+</i> (n = 6) and <i>Opa1^+/−</i> (n = 7) mice. A two-way ANOVA with genotype and wavelength as factors was performed. There was no significant effect of wavelength (<i>p</i> = 0.66) or genotype (<i>p</i> = 0.17) and the interaction of genotype and wavelength was not significant (<i>p</i> = 0.91).</p

Masking response in <i>Opa1^+/+</i> and <i>Opa1^+/−</i> mice.

<p>(A) The average wheel running revolutions on the night (gray background) of the 3 h light pulse (white background) are plotted relative to the baseline levels (the night before the pulse) for <i>Opa1^+/+</i> (n = 6) and <i>Opa1^+/−</i> (n = 7) mice. The masking pulse completely suppressed activity in both genotypes immediately. ANOVA analysis found no significant effect of genotype on the baseline corrected activity levels (p = 0.468) (B) Hourly breakdown of activity during the masking pulse. A 2-way ANOVA using activity in each hour of light pulse and genotype as factors found a significant effect of hour of light pulse (p<0.005) but no significant effect of genotype (p = 0.143) and no interaction between genotype and light pulse hour (p = 0.359). All data are presented as mean ± SEM.</p

Circadian behaviour in <i>Opa1^+/+</i> and <i>Opa1^+/−</i> mice.

<p>Representative actograms from (A) <i>Opa1 </i>^+/+ and (B) <i>Opa1 </i>^+/− mice entrained to a 12/12 LD cycle and subsequently released into constant darkness (DD). Each horizontal line corresponds to one day and the data has been double plotted. The black vertical bars represent activity (i.e. wheel revolutions). The shaded region represents lights ON. (C) Table showing average period (τ), total activity levels and length of the active phase in LD and in DD for <i>Opa1^+/+</i> (n = 6) and <i>Opa1^+/−</i> (n = 7) mice. There were no significant differences between genotypes (unpaired students t-test; p values are shown). All data are presented as mean ± SEM.</p

Pupil light reflex in <i>Opa1^+/+</i> and <i>Opa1^+/−</i> mice.

<p>The average minimum pupil area expressed as a percentage of maximum dilation following illumination with various intensities of white light for <i>Opa1^+/+</i> (n = 5) and <i>Opa1^+/−</i> (n = 5) mice. All data are fitted with four term sigmoidal functions (solid lines) of the form y = y0+a/(1+exp(-(x-x0)/b)) (goodness of fit of fitted curve to actual data (R2): <i>Opa1^+/+</i> = 0.993 and <i>Opa1^+/−</i> = 0.995). A 2-way ANOVA using intensity and genotype as factors showed a significant effect of light intensity (p<0.0001) but no significant effect of genotype (p = 0.51) and no significant interaction between genotype and intensity (p = 0.99).</p

Melanopsin expression in <i>Opa1^+/+</i> and <i>Opa1^+/−</i> retinae.

<p>Overall distribution of melanopsin-positive RGCs in a flatmount retina from (A) <i>Opa1</i>^+/+ and (B) <i>Opa1</i>^+/<i>−</i> mice. The total number of melanopsin expressing cells was not significantly different between genotypes (<i>Opa1^+/+</i>: n = 3; <i>Opa1^+/</i>: n = 3). (C) Quantification of melanopsin (<i>Opn4</i>) and <i>Opa1</i> gene expression by real time quantitative PCR. Expression levels in <i>Opa1^+/−</i> animals are plotted relative to wildtype data. No significant difference in expression was detected for <i>Opn4</i> between genotypes. A significant reduction in <i>Opa1</i> expression was observed in <i>Opa1^+/−</i> mice relative to wildtype controls (student's t-test. * = p<0.005). (D) Representative confocal images of melanopsin cells in <i>Opa1^+/+</i> and <i>Opa1^+/−</i> retinae. A projected image of a confocal stack (from the inner plexiform layer to the ganglion cell layer) is shown for each genotype. An image at the plane of the outermost region of sublamina a and an image at the plane of the innermost region of sublamina b from the same image stacks is also shown.</p