Abstract
Purpose::
Intrinsically photosensitive retinal ganglion cells (ipRGCs) are mammalian ocular photoreceptors that synchronize circadian rhythms and regulate pupil size. Melanopsin is the photopigment of ipRGCs, but the signaling cascade coupling photoactivated melanopsin to the voltage response is unknown. Invertebrate-like features of melanopsin and ipRGCs suggest that these cells may use a phototransduction cascade like that in rhabdomeric photoreceptors. We tested this hypothesis.
Methods::
Adult rat ipRGCs were retrolabeled from the suprachiasmatic nucleus. After enzymatic dissociation, isolated ipRGC somata exhibited robust light-evoked currents in whole-cell recordings for >1h after break-in, even after 4d in culture. Excised outside-out patches were obtained by withdrawing the pipette from the cell.
Results::
Light-evoked whole-cell currents were totally blocked by drugs targeting key phosphoinositide signaling components. General G-protein antagonists (GDPßS), specific blockers of Gq/11-class G-proteins (GPAnt-2a), and inhibitors of the effector enzyme PLCß (U73122) all abolished photoresponses within 10 min. Drugs targeting Gi/o- or Gs-class G-proteins (GPAnt-2, pertussis toxin and cholera toxin) were without effect. We next asked whether PLCß is linked to the light-gated channel through cytosolic (IP3-based) or membrane-associated (DAG-based) messengers. Neither blocking IP3 receptors (heparin) nor depleting internal calcium stores (thapsigargin) eliminated the light response, though these agents slowed recovery and accelerated rundown. Thus, the alternative, membrane-delimited signaling pathway may be responsible. Indeed, excised outside-out patches of ipRGC membrane were autonomously photosensitive, generating robust light-evoked inward currents in voltage clamp and membrane depolarization and trains of action potentials in current-clamp.
Conclusions::
Phototransduction in ipRGCs uses a signaling cascade like that in rhabdomeric photoreceptors. Light absorption by the invertebrate-like photopigment melanopsin activates a Gq/11-class G-protein, which stimulates PLCß. A membrane-delimited second messenger links PLCß to the light-gated channel, which may be a TRPC channel. These findings reinforce the emerging view that vertebrate ipRGCs and invertebrate rhabdomeric photoreceptors evolved from a common ancestral cell type.
Keywords: photoreceptors • electrophysiology: non-clinical • ganglion cells