Melanopsin Triggers the Release of Internal Calcium Stores in Response to Light† (original) (raw)
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Light-Evoked Calcium Responses of Isolated Melanopsin-Expressing Retinal Ganglion Cells
Journal of Neuroscience, 2007
A small number (Ͻ2%) of mammalian retinal ganglion cells express the photopigment melanopsin and are intrinsically photosensitive (ipRGCs). Light depolarizes ipRGCs and increases intracellular calcium levels ([Ca 2ϩ ] i) but the signaling cascades underlying these responses have yet to be elucidated. To facilitate physiological studies on these rare photoreceptors, highly enriched ipRGC cultures from neonatal rats were generated using anti-melanopsin-mediated plate adhesion (immunopanning). This novel approach enabled experiments on isolated ipRGCs, eliminating the potential confounding influence of rod/cone-driven input. Light induced a rise in [Ca 2ϩ ] i (monitored using fura-2 imaging) in the immunopanned ipRGCs and the source of this Ca 2ϩ signal was investigated. The Ca 2ϩ responses were inhibited by 2-aminoethoxydiphenyl borate, SKF-96365 (1-2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1Himidazole), flufenamic acid, lanthanum, and gadolinium, consistent with the involvement of canonical transient receptor potential (TRP) channels in ipRGC phototransduction. However, the contribution of direct Ca 2ϩ flux through a putative TRP channel to ipRGC [Ca 2ϩ ] i was relatively small, as most (ϳ90%) of the light-induced Ca 2ϩ responses could be blocked by preventing action potential firing with tetrodotoxin. The L-type voltage-gated Ca 2ϩ channel (VGCC) blockers verapamil and (ϩ)-cis-diltiazem significantly reduced the lightevoked Ca 2ϩ responses, while the internal Ca 2ϩ stores depleting agent thapsigargin had negligible effect. These results indicate that Ca 2ϩ influx through VGCCs, activated after action potential firing, was the primary source for light-evoked elevations in ipRGC [Ca 2ϩ ] i. Furthermore, concurrent Ca 2ϩ imaging and cell-attached electrophysiological recordings demonstrated that the Ca 2ϩ responses were highly correlated to spike frequency, thereby establishing a direct link between action potential firing and somatic [Ca 2ϩ ] i in lightstimulated ipRGCs.
Melanopsin-expressing ganglion cells in primate retina signal colour and irradiance and project to
Nature, 2005
Light stimuli from a 100-W tungsten source were filtered (neutral density and narrowband interference filters;10 nm width; Oriel), gated by a shutter (Uniblitz VS35;Vincent Associates) and calibrated by a radiometer (S370, UDT Instruments). The irradiance of the unfiltered ('white') stimulus was (in photons s 21 cm 22 ): 4 £ 10 12 at 400 nm, 6 £ 10 13 at 500 nm and 1 £ 10 14 at 600 nm.
Journal of General Physiology, 2013
In mammals, the discovery of melanopsin and intrinsic light responsiveness in a small population of retinal ganglion cells (ipRGCs) paved the way for understanding light regulation of nonvisual functions, like the pupillary reflex or the entrainment of circadian rhythms. The scarcity of ipRGCs, however, is a hurdle for investigating melanopsin signaling mechanisms. In the avian retina, melanopsin expresses abundantly in the outer nuclear layer too, but its functionality is unknown. We used the chicken embryo retina as a model system to investigate possible physiological roles of melanopsin in non-ganglion cells. A polyclonal antibody targeting both chicken melanopsin isoforms was validated by Western blot and used to corroborate the distribution pattern in fixed retina sections; strong immunoreactivity occurred in areas that likely include horizontal, bipolar, and some amacrine cells. Retinas were enzymatically dissociated to yield morphologically well-preserved isolated neurons; their physiological viability was tested with whole cell recording. Although voltage-gated currents were found in the different cell types, initial recordings failed to reveal direct changes in membrane current by photostimulation. However, light could reversibly modulate voltage-gated currents. Fluorescence imaging in cells loaded with calcium indicators demonstrated a Ca fluorescence increase in selected bipolar cells and small neurons, likely to comprise a subtype of amacrine cells. Higher sensitivity measurements, using a photomultiplier and pulsed light to extend the recording period, showed that in a minority of bipolar cells, light evoked Ca responses with a long latency and a slow time course, spanning minutes. A reassessment of electrical responses occurring on such a long time scale revealed a small inward current (tens of pA) with a similar time course. These observations indicate that intrinsic photosensitivity is not confined to rods, cones, and some ganglion cells, but extends to additional retinal cell types. Its physiological role remains to be investigated. Supported by Colciencias grant 222852128276.
The Journal of biological chemistry, 2014
The G protein-coupled light-sensitive receptor melanopsin is involved in non-image-forming light responses including circadian timing. The predicted secondary structure of melanopsin indicates a long cytoplasmic tail with many potential phosphorylation sites. Using bioinformatics, we identified a number of amino acids with a high probability of being phosphorylated. We generated antibodies against melanopsin phosphorylated at Ser-381 and Ser-398, respectively. The antibody specificity was verified by immunoblotting and immunohistochemical staining of HEK-293 cells expressing rat melanopsin mutated in Ser-381 or Ser-398. Using the antibody recognizing phospho-Ser-381 melanopsin, we demonstrated by immunoblotting and immunohistochemical staining in HEK-293 cells expressing rat melanopsin that the receptor is phosphorylated in this position during the dark and dephosphorylated when light is turned on. On the contrary, we found that melanopsin at Ser-398 was unphosphorylated in the dark...
Control of intracellular calcium in vertebrate photoreceptors
Neuroscience Research Supplements, 1989
Calcium seemed for several years to be the most likely candidate for the intraeellular messenger which is responsible for linking the absorption of light by rhodopsin to the first electrical event in l~hototransduction, the suppression of current flowing across the outer segment membrane a , 3o
Photon capture and signalling by melanopsin retinal ganglion cells
Nature, 2009
A subset of retinal ganglion cells has recently been discovered to be intrinsically photosensitive, with melanopsin as the pigment. These cells project primarily to brain centers for non-imageforming visual functions such as the pupillary light reflex and circadian photoentrainment. How well they signal intrinsic light absorption to drive behavior remains unclear. Here we report fundamental parameters governing their intrinsic light responses and associated spike generation. The membrane density of melanopsin is 10 4 -fold lower than that of rod and cone pigments, resulting in a very low photon-catch and a phototransducing role only in relatively bright light. Nonetheless, each captured photon elicits a large and extraordinarily prolonged response, with a unique shape among known photoreceptors. Remarkably, like rods, these cells are capable of signalling single-photon absorption. A flash causing a few hundred isomerized melanopsin molecules in a retina is sufficient for reaching threshold for the pupillary light reflex.
VA Opsin, Melanopsin, and an Inherent Light Response within Retinal Interneurons
Current Biology, 2003
and resembled amacrine cells. These data provided the first indication that light detection within the retina is not Molecular Neuroscience Division of Neuroscience and confined to the rods and cones [2]. This retinal distribution of VA opsin now appears to be a general feature in Psychological Medicine Imperial College Faculty of Medicine a number of teleost species, including the zebrafish [3] and smelt (Plecoglossus altivelis) [4]. However, VA opsin Charing Cross Hospital Fulham Palace Road may not be the only non-rod, non-cone photopigment within the teleost retina, as very recently homologs of London W6 8RF United Kingdom the melanopsin gene family have been isolated from zebrafish [5] and Atlantic cod [6]. Melanopsin (Opn4) was first identified as the candidate photopigment responsible for light-induced mela-Summary nosome aggregation in Xenopus dermal melanophores [7]. More detailed in situ studies then demonstrated that Background: Although photoreception is best under-Xenopus melanopsin expression was not restricted to stood in rods and cones, it is increasingly clear that the melanophores, but also occurred in cells of the horithese are not the only photoreceptive cells of the vertezontal cell layer of the retina, the retinal pigment epithebrate retina. While considerable attention has been paid lium (RPE), and the iris. Mammalian orthologs of Xenoto the role of melanopsin in the generation of intrinsic pus melanopsin were subsequently isolated from light sensitivity in the retinal ganglion cells of mammals, humans and mice, and expression was found to be renothing is known about the photoreceptive capacity of stricted to a subset of cells in the retinal ganglion cell the horizontal cells of the fish retina in which both VA (RGC) layer and other cells in the inner nuclear layer of opsin and melanopsin are expressed. As yet, there has the retina [8-10]. Most recently, orthologs of melanopsin been little more than speculation as to the physiological have been isolated from the chicken pineal [11] and from function of these opsins within local retinal circuit neuhorizontal cells in the zebrafish [5] and cod [6] retina. rons. To date, there is no biochemical analysis on melanop-Results: VA opsin and melanopsin have been isolated sin, and the role of this protein within the vertebrate and localized within the well-characterized cyprinid retretina remains unclear. All the attempts to place melaina of the roach (Rutilus rutilus). Parallel electrophysionopsin into a functional context have been confined to logical studies identified a novel subtype of horizontal mammals. For example, melanopsin is expressed in a cell (HC-RSD) characterized by a depolarizing response subset of intrinsically photosensitive RGCs [12], and that fits an opsin photopigment with a max of 477 nm. melanopsin knockout studies have shown that this opsin The HC-RSD cells mediate responses to light that are plays a role in both circadian and pupillary responses characterized by long integration times, well beyond to light [13-15]. Whatever the function of melanopsin in those observed for rods and cones. Significantly, HCmammals, it is clearly associated with non-rod, non-RSD responses persist when the conventional photorecone photoreception within the retina. ceptor inputs are saturated by background light. While considerable attention has been paid to the Conclusions: The syncytium of coupled horizontal cells role of novel opsins in the generation of intrinsic light has long been considered to provide a signal of overall sensitivity in the RGCs of mammals, nothing is known retinal irradiance. Our data suggest that this light inforabout the photoreceptive capacity of the horizontal cells mation is, at least in part, derived from a population of of the fish retina in which VA opsin and melanopsin intrinsically photosensitive VA opsin and/or melanopsin are expressed. As yet, there has been little more than horizontal cells. speculation as to the physiological function of the expressed opsins within these local retinal circuit neurons.