Electrophysiological Changes During Early Steps of Retinitis Pigmentosa

Abstract

PURPOSE. The rhodopsin mutation P23H is responsible for a significant portion of autosomaldominant retinitis pigmentosa, a disorder characterized by rod photoreceptor death. The mechanisms of toxicity remain unclear; previous studies implicate destabilization of P23H rhodopsin during light exposure, causing decreased endoplasmic reticulum (ER) exit and ER stress responses. Here, we probed phototransduction in Xenopus laevis rods expressing bovine P23H rhodopsin, in which retinal degeneration is inducible by light exposure, in order to examine early physiological changes that occur during retinal degeneration. METHODS. We recorded single-cell and whole-retina responses to light stimuli using electrophysiology. Moreover, we monitored morphologic changes in rods after different periods of light exposure. RESULTS. Initially, P23H rods had almost normal photoresponses, but following a brief light exposure varying from 4 to 32 photoisomerizations per disc, photoresponses became irreversibly prolonged. In intact retinas, rods began to shed OS fragments after a rodsaturating exposure of 12 minutes, corresponding to approximately 10 to 100 times more photoisomerizations. CONCLUSIONS. Our results indicate that in P23H rods light-induced degeneration occurs in at least two stages, the first involving impairment of phototransduction and the second involving initiation of morphologic changes.

Mechanosensitivity is an essential component of phototransduction in vertebrate rods

“…laevis frogs were enucleated and hemisected under a stereotactic microscope with an IR 820 nm illumination. Dissociated rods were obtained as reported previously [ 29 ]. Briefly, the intact rods obtained by mechanical dissociation were immersed in Ringer solution containing 110 mM NaCl, 2.5 mM KCl, 1 mM CaCl2, 1.6 mM MgCl2, 3 mM HEPES-NaOH, 0.01 mM EDTA, and 10 mM glucose (pH 7.7–7.8, buffered with NaOH).…”

“…We delivered GsMTx-4 using a second similar pipette connected to a picospritzer and positioned 50-100 μm from the OS of the recorded rod ( Fig 5A). Prior to drug exposure, presentation of bright flashes of about 2,500 R � /rod [29] triggered suppression of the rod circulating current for 5 s (Fig 5B and 5C). When the inhibitor was gently injected into the bath, using around 4 psi of pressure, the OS of the recorded rod was displaced from its original position (compare upper and lower panels in Fig 5A), signaling the arrival of GsMTx-4.…”

Calcium flares and compartmentalization in rod photoreceptors

“…Male and female frogs of the species X. laevis were dark adapted overnight, anesthetized with tricaine, and euthanized under dim red light. Their eyes were dissected as previously reported (45). Single rods consisting of an IS+OS were obtained by gentle mechanical dissociation of a small piece of retina, cut from the whole with surgical scissors.…”

“…The recording of rod photoreceptors light response (SI Appendix, Fig. S2) were obtained as previously described (45). The Axopatch 200A (Molecular Devices, LLC) was used in voltage clamp mode to record the sensory neurons currents, which were digitized at 10 kHz and low-pass filtered at 20 Hz.…”

Redefining the role of Ca2+-permeable channels in photoreceptor degeneration using diltiazem

“…To date electrophysiological recordings from single photoreceptors of retinal disease models are very rare [42]. Still, we may expect rd1 rod photoreceptors to be in a permanently depolarized state due to the high CNGC activity triggered by high cGMP.…”