Abstract
Purpose :
To measure the RPE (c-wave) and Müller cell (slow P-III) response to subretinal changes in K+ concentration driven by rod or cone photoresponses in vivo.
Methods :
All mice were handled according to NIH and ARVO guidelines under a protocol approved by the UC Davis IACUC. Full-field ERGs were measured from dark-adapted animals in response to 510 nm flashes. Cone-driven ERGs were isolated with a rod-suppressing background or genetically by comparing recordings obtained from C57Bl/6J mice (WT), from mice with no functional cones (Gnat2-/-), and mice with no functional rods (Gnat1-/-). Pharmacological agents targeting Kir2.1, Kir4.1 and Kir7.1 channels were injected into the eye under dim red illumination. Responses of Müller cells to cone-driven changes in sub-retinal K+ concentration were isolated by injecting Kir4.1 inhibitors in combination with AP4 and PDA. Immunohistochemistry was used to assess the distribution of molecular targets of Kir channel inhibitors, and transmission electron microscopy was used to visualize the geometrical distribution of Müller cells, photoreceptors and RPE cells in the subretinal space.
Results :
The contribution of cone photoreceptors to the RPE-driven c-wave was negligible. The maximal amplitudes of the a-waves and c-waves were indistinguishable between WT and Gnat2-/- animals (a-wave: 1075 ± 15 µV vs 1010 ± 25 µV c-wave: 1828 ± 25 µV vs 1823 ± 27 µV), and no c-wave was observed in Gnat1-/- animals. Kir7.1 and Kir2.1 inhibitors were found to partially suppress the c-wave. Consistent with the close apposition of Müller cell processes to the inner segments of photoreceptors, Müller cell-driven slow-PIII responses triggered by cone stimulation were substantial. Complete flash families of slow-PIII responses driven by rods or cones were isolated with inhibitors of Kir4.1 channels.
Conclusions :
The RPE-driven c-wave response of WT mice is dominated by signals arising in rods. Cone-mediated changes in sub-retinal K+ concentration elicit responses from Müller cells that can be isolated in vivo by means of Kir4.1 channel inhibitors. Blocking these non-neuronal sources of signal allows fully isolated cone photoresponse families and Off-bipolar responses to be recorded. Through pharmacological and genetic dissection of the ERG, the functional state of cones, their downstream synaptic partners, and the supporting cast of RPE and Müller cells can be assessed in vivo.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.