Orexin-A potentiates intrinsic light responses of intrinsically photosensitive retinal ganglion cells in mouse retina

Abstract

Purpose : The neuropeptide orexin-A is widely expressed in mammalian retina and orexin receptor 1 (OX₁R) is localized to intrinsically photosensitive retinal ganglion cells (ipRGCs) that express the photopigment melanopsin and respond to light directly. However, the role of orexin-A in visual function is still largely unknown. Here we sought to investigate whether orexin-A modulates melanopsin-driven (intrinsic) light responses of ipRGC M2 subtype.

Methods : We used BAC transgenic mice in which ipRGCs were labeled with td-Tomato. Cell-attached and/or whole-cell patch-clamp recording techniques were used to record the light response and membrane potential of genetically-labeled ipRGCs in flat-mount mouse retinas. To record intrinsic light responses of ipRGCs, L-AP4, DNQX, D-AP5 were included in Ames’ medium to block rod/cone signaling.

Results : Exogenous orexin-A (500 nM) significantly increased intrinsic light-evoked spiking rates of M2 cells, and the effect was abolished by the OX₁R antagonist SB334867, but not by the OX₂R antagonist TCS OX229. In contrast, SB334867 significantly decreased the intrinsic light-evoked firing rates of M2 cells, indicating that endogenous orexins also potentiate the intrinsic light responses. In complete darkness with intact synapses, orexin-A directly depolarized M2 cells and increased their spontaneous spiking rates through activation of OX₁Rs. After applying L-AP4, D-AP5 and DNQX to block glutamatergic inputs to ipRGCs, along with TTX and carbenoxolone to block Na⁺-mediated action potentials and gap junction transmission, respectively, we found that the amplitudes of orexin-A-induced depolarization of M2 cells were not altered, suggesting that the orexin effect is independent on presynaptic inputs. Moreover, the K⁺ channel blocker BaCl₂ or the nonselective cation channel blocker FFA partly blocked the orexin-A-induced depolarization, and co-application of BaCl₂ and FFA totally eliminated the orexin-A effect on M2 cells. However, blockage of Ca²⁺ channel or Na⁺-Ca²⁺ exchanger did not change the depolarizing amplitudes of M2 cells.

Conclusions :
Orexin-A directly depolarizes M2 cells through activation of OX₁Rs. Dual ionic mechanisms including activation of the nonselective cation channels and block of an inward rectifier K⁺ channels mediate the effects of orexin-A on M2 cells.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.