Sumathi Sekaran

Calcium imaging reveals a network of intrinsically light-sensitive inner-retinal neurons.

BACKGROUND Mice lacking rod and cone photoreceptors (rd/rd cl) are still able to regulate a range of responses to light, including circadian photoentrainment, the pupillary light reflex, and suppression of pineal melatonin by light. These data are consistent with the presence of a novel inner-retinal photoreceptor mediating non-image-forming irradiance detection. RESULTS We have examined the nature and extent of intrinsic light sensitivity in rd/rd cl retinae by monitoring the effect of light stimulation (470 nm) on intracellular Ca(2+) via FURA-2 imaging. Using this approach, which does not rely on pharmacological or surgical isolation of ganglion cells from the rod and cone photoreceptors, we identified a population of light-sensitive neurons in the ganglion cell layer (GCL). Retinal illumination induced an increase of intracellular Ca(2+) in approximately 2.7% of the neurons. The light-evoked Ca(2+) fluxes were dependent on the intensity and duration of the light stimulus. The light-responsive units formed an extensive network that could be uncoupled by application of the gap junction blocker carbenoxolone. Three types of light-evoked Ca(2+) influx were observed: sustained, transient, and repetitive, which are suggestive of distinct functional classes of GCL photoreceptors. CONCLUSIONS Collectively, our data reveal a heterogeneous syncytium of intrinsically photosensitive neurons in the GCL coupled to a secondary population of light-driven cells, in the absence of rod and cone inputs.

Melanopsin-Dependent Photoreception Provides Earliest Light Detection in the Mammalian Retina

BACKGROUND The visual system is now known to be composed of image-forming and non-image-forming pathways. Photoreception for the image-forming pathway begins at the rods and cones, whereas that for the non-image-forming pathway also involves intrinsically photosensitive retinal ganglion cells (ipRGCs), which express the photopigment melanopsin. In the mouse retina, the rod and cone photoreceptors become light responsive from postnatal day 10 (P10); however, the development of photosensitivity of the ipRGCs remains largely unexplored. RESULTS Here, we provide direct physiological evidence that the ipRGCs are light responsive from birth (P0) and that this photosensitivity requires melanopsin expression. Interestingly, the number of ipRGCs at P0 is over five times that in the adult retina, reflecting an initial overproduction of melanopsin-expressing cells during development. Even at P0, the ipRGCs form functional connections with the suprachiasmatic nucleus, as assessed by light-induced Fos expression. CONCLUSIONS The findings suggest that the non-image-forming pathway is functional long before the mainstream image-forming pathway during development.

Reversal of end-stage retinal degeneration and restoration of visual function by photoreceptor transplantation

One strategy to restore vision in retinitis pigmentosa and age-related macular degeneration is cell replacement. Typically, patients lose vision when the outer retinal photoreceptor layer is lost, and so the therapeutic goal would be to restore vision at this stage of disease. It is not currently known if a degenerate retina lacking the outer nuclear layer of photoreceptor cells would allow the survival, maturation, and reconnection of replacement photoreceptors, as prior studies used hosts with a preexisting outer nuclear layer at the time of treatment. Here, using a murine model of severe human retinitis pigmentosa at a stage when no host rod cells remain, we show that transplanted rod precursors can reform an anatomically distinct and appropriately polarized outer nuclear layer. A trilaminar organization was returned to rd1 hosts that had only two retinal layers before treatment. The newly introduced precursors were able to resume their developmental program in the degenerate host niche to become mature rods with light-sensitive outer segments, reconnecting with host neurons downstream. Visual function, assayed in the same animals before and after transplantation, was restored in animals with zero rod function at baseline. These observations suggest that a cell therapy approach may reconstitute a light-sensitive cell layer de novo and hence repair a structurally damaged visual circuit. Rather than placing discrete photoreceptors among preexisting host outer retinal cells, total photoreceptor layer reconstruction may provide a clinically relevant model to investigate cell-based strategies for retinal repair.

2-Aminoethoxydiphenylborane is an acute inhibitor of directly photosensitive retinal ganglion cell activity in vitro and in vivo.

The mammalian retina contains directly photosensitive retinal ganglion cells (RGCs), which use the photopigment melanopsin. The generation of mice lacking melanopsin has been invaluable in elucidating the function of these cells. These animals display deficiencies in circadian photoentrainment, the pupil light reflex, and the circadian regulation of the cone pathway. Interpreting the results from such gene knock-out models is always complicated by neuronal plasticity and the potential for restructuring of neuronal networks. Until now, the study of photosensitive RGCs has lacked an acute inhibitor. 2-Aminoethoxydiphenylborane (2-APB) is an antagonist at IP3 receptors and an inhibitor of canonical transient receptor potential ion channels (TRPCs). Here, we show that 2-APB is an extremely potent in vitro inhibitor of the photosensitive RGCs and that its effect is independent of store-dependent Ca2+ release. The identification of canonical TRPC6 and TRPC7 ion channels in melanopsin-expressing ganglion cells suggests that 2-APB may act directly on a TRPC ion channel. Importantly, using the pupil light reflex as a functional assay, we show that 2-APB inhibits photosensitive RGC activity in vivo. Collectively, our data further elucidate the phototransduction pathway in the photosensitive RGCs and demonstrate that 2-APB can be used to silence activity in these cells both in vitro and in vivo.

Nitric oxide release is induced by dopamine during illumination of the carp retina: serial neurochemical control of light adaptation.

Several lines of indirect evidence have suggested that nitric oxide may play an important role during light adaptation of the vertebrate retina. We aimed to verify directly the effect of light on nitric oxide release in the isolated carp retina and to investigate the relationship between nitric oxide and dopamine, an established neuromodulator of retinal light adaptation. Using a biochemical nitric oxide assay, we found that steady or flicker light stimulation enhanced retinal nitric oxide production from a basal level. The metabotropic glutamate receptor agonist l‐amino‐4‐phosphonobutyric acid, inhibited the light adaptation‐induced nitric oxide production suggesting that the underlying cellular pathway involved centre‐depolarizing bipolar cell activity. Application of exogenous dopamine to retinas in the dark significantly enhanced the basal production of nitric oxide and importantly, inhibition of endogenous dopaminergic activity completely suppressed the light‐evoked nitric oxide release. The effect of dopamine was mediated through the D1 receptor subtype. Imaging of the nitric oxide‐sensitive fluorescent indicator 4,5‐diaminofluorescein di‐acetate in retinal slices revealed that activation of D1 receptors resulted in nitric oxide production from two main spatial sources corresponding to the photoreceptor inner segment region and the inner nuclear layer. The results taken together would suggest that during the progression of retinal light adaptation there is a switch from dopaminergic to nitrergic control, probably to induce further neuromodulatory effects at higher levels of illumination and to enable more efficient spreading of the light adaptive signal.

Light‐evoked FOS induction within the suprachiasmatic nuclei (SCN) of melanopsin knockout (Opn4−/−) mice: A developmental study

The aims of this study were to address three related questions: (1) Do the photosensitive ganglion cells of the mouse convey light information to the suprachiasmatic nuclei (SCN) at P0? (2) Do the differentiating rods and cones contribute to light‐evoked FOS induction within the murine SCN at P4? (3) How does light‐evoked FOS induction within the SCN of melanopsin knockout (Opn4−/−) mice differ at P4 and P14? Our approaches took advantage of the published descriptions of murine ocular development, melanopsin knockout (Opn4−/−) mouse, and light‐induced expression of FOS (the phosphoprotein product of immediate early gene c‐fos) within the SCN as a marker of retinohypothalamic tract competence. Collectively, our results show that photosensitive melanopsin‐dependent retinal ganglion cells provide light information to the murine SCN on the day of birth, and possibly beforehand, and that developing rods and cones fail to provide light information to the SCN during early postnatal life. On the basis of previous publications and data presented here, we suggest that at ages around P14 the rods and cones might be capable of fully compensating for the loss of melanopsin‐photosensitive ganglion cells if exposure to light is of sufficiently long duration. These results are related to the broader context of recent findings and the potential role(s) of a neonatal photoreceptor.

Residual photosensitivity in mice lacking both rod opsin and cone photoreceptor cyclic nucleotide gated channel 3 alpha subunit.

The mammalian retina contains three classes of photoreceptor. In addition to the rods and cones, a subset of retinal ganglion cells that express the putative sensory photopigment melanopsin are intrinsically photosensitive. Functional and anatomical studies suggest that these inner retinal photoreceptors provide light information for a number of non-image-forming light responses including photoentrainment of the circadian clock and the pupil light reflex. Here, we employ a newly developed mouse model bearing lesions of both rod and cone phototransduction cascades (Rho(-/-) Cnga3(-/-)) to further examine the function of these non-rod non-cone photoreceptors. Calcium imaging confirms the presence of inner retinal photoreceptors in Rho(-/-) Cnga3(-/-) mice. Moreover, these animals retain a pupil light reflex, photoentrainment, and light induction of the immediate early gene c-fos in the suprachiasmatic nuclei, consistent with previous findings that pupillary and circadian responses can employ inner retinal photoreceptors. Rho(-/-) Cnga3(-/-) mice also show a light-dependent increase in the number of FOS-positive cells in both the ganglion cell and (particularly) inner nuclear layers of the retina. The average number of cells affected is several times greater than the number of melanopsin-positive cells in the mouse retina, suggesting functional intercellular connections from these inner retinal photoreceptors within the retina. Finally, however, while we show that wild types exhibit an increase in heart rate upon light exposure, this response is absent in Rho(-/-) Cnga3(-/-) mice. Thus, it seems that non-rod non-cone photoreceptors can drive many, but not all, non-image-forming light responses.

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.

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.