Ectopic Expression of Mouse Melanopsin in Drosophila Photoreceptors Reveals Fast Response Kinetics and Persistent Dark Excitation

Abstract

The intrinsically photosensitive M1 retinal ganglion cells (ipRGC) initiate non-image-forming light-dependent activities and express the melanopsin (OPN4) photopigment. Several features of ipRGC photosensitivity are characteristic of fly photoreceptors. However, the light response kinetics of ipRGC is much slower due to unknown reasons. Here we used transgenic Drosophila, in which the mouse OPN4 replaced the native Rh1 photopigment of Drosophila R1-6 photoreceptors, resulting in deformed rhabdomeric structure. Immunocytochemistry revealed OPN4 expression at the base of the rhabdomeres, mainly at the rhabdomeral stalk. Measurements of the early receptor current, a linear manifestation of photopigment activation, indicated large expression of OPN4 in the plasma membrane. Comparing the early receptor current amplitude and action spectra between WT and the Opn4-expressing Drosophila further indicated that large quantities of a blue absorbing photopigment were expressed, having a dark stable blue intermediate state. Strikingly, the light-induced current of the Opn4-expressing fly photoreceptors was ϳ40-fold faster than that of ipRGC. Furthermore, an intense white flash induced a small amplitude prolonged dark current composed of discrete unitary currents similar to the Drosophila single photon responses. The induction of prolonged dark currents by intense blue light could be suppressed by a following intense green light, suggesting induction and suppression of prolonged depolarizing afterpotential. This is the first demonstration of heterologous functional expression of mammalian OPN4 in the genetically emendable Drosophila photoreceptors. Moreover, the fast OPN4-activated ionic current of Drosophila photoreceptors relative to that of mouse ipRGC, indicates that the slow light response of ipRGC does not arise from an intrinsic property of melanopsin.The intrinsically photosensitive retinal ganglion cells (ipRGC) 2 are a subclass of retinal ganglion cells expressing the visual pigment, melanopsin (OPN4), which calibrates by direct photic input the circadian pacemaker of the master circadian clock and supports some non-image forming light-dependent functions (reviewed in Ref. 1). There are difficulties in advancing understanding of ipRGC phototransduction. The main obstacle is the scarcity of ipRGC and the low expression levels of phototransduction proteins in these cells. This difficulty makes it nearly impossible to investigate phototransduction of the ipRGC by employing the same set of biochemical and electrophysiological approaches that proved successful in characterizing rhodopsin signaling processes in image-forming rod photoreceptor cells. Therefore, at present, the knowledge of phototransduction of ipRGC is still fragmented (1). A promising way to characterize the OPN4 photopigment arises from the apparent similarity between phototransduction of ipRGC and invertebrates. It has been well established that several features of ipRGC photosensitivity are also characteristic of invertebrate photoreceptor cells. (i)...

Melanopsin and the Intrinsically Photosensitive Retinal Ganglion Cells: Biophysics to Behavior

“…If melanopsin couples to a cascade that exhibits amplification and negative feedback, the light response gains sensitivity and dynamic range, features that are lacking in responses driven by channelrhodopsins. In addition, melanopsin tends to produce responses that rise and decay slowly (Bailes and Lucas, 2013;Lin et al, 2008;Spoida et al, 2016; but see Yasin et al, 2017), making the level of cellular activation more robust to fluctuations in illumination. At the same time, light of a particular composition can be used to suppress melanopsin activity, providing a measure of temporal control (Emanuel and Do, 2015;Spoida et al, 2016).…”

Melanopsin phototransduction: beyond canonical cascades

“…Non-image forming behaviors operate across sustained timescales, often integrating light information over hours and days, whereas visual perception requires high spatial and temporal resolution. Surprisingly, disruption of the slow and sustained melanopsin phototransduction cascade results in deficits in both M1-dependent, non-image forming behaviors as well as M2-M6-dependent visual perception (Wong et al, 2005;Gamlin et al, 2007;Do et al, 2009;Kankipati et al, 2010;Spoida et al, 2016;Yasin et al, 2017). Despite this diversity in structure and function of ipRGC subtypes, the melanopsin phototransduction cascade was long assumed to be similar across subtypes (Do et al, 2009;Spoida et al, 2016;Yasin et al, 2017).…”

Knockdown of Dehydrodolichyl Diphosphate Synthase in the Drosophila Retina Leads to a Unique Pattern of Retinal Degeneration

“…morphogenesis (Kumar et al, 1997;Yasin et al, 2017). Thus, besides its function in light reception, rhodopsin plays an essential structural role during photoreceptors morphogenesis.…”

“…Furthermore, the results gave the first evidence directly indicating that preventing the attachment of the oligosaccharide chain markedly impedes rhodopsin maturation resulting in a pronounced rhodopsin reduction ( Katanosaka et al, 1998 ). Rh1 rhodopsin elimination leads to severe shrinkage of the rhabdomeres ( Kumar and Ready, 1995 ; Yasin et al, 2017 ). There is a critical developmental period in which rhodopsin plays its key role in photoreceptor morphogenesis ( Kumar et al, 1997 ; Yasin et al, 2017 ).…”

“…Rh1 rhodopsin elimination leads to severe shrinkage of the rhabdomeres ( Kumar and Ready, 1995 ; Yasin et al, 2017 ). There is a critical developmental period in which rhodopsin plays its key role in photoreceptor morphogenesis ( Kumar et al, 1997 ; Yasin et al, 2017 ). Thus, besides its function in light reception, rhodopsin plays an essential structural role during photoreceptors morphogenesis.…”