Genetic Dissection of Retinal Pathways Using the Electroretinogram (ERG)

Abstract

Purpose:: To gain a greater understanding of the rod and cone pathways in mice using pathway-specific knockouts.

Methods:: Electroretinography (ERG) was used to record rod- and cone-driven signals from mice constitutively lacking connexin36 (Cx36-/-), a gap junction protein present in AII amacrine cell (AIIAC)and photoreceptor gap junctions and mice lacking the basic helix-loop-helix transcription factor (bhlhb4-/-), which exhibit greatly diminished numbers of rod bipolar cells (DBCr). We also examined mice in which Cx36 was conditionally deleted only in cone photoreceptors (Cx36-/fl;RGP/BPCre). Dark-adapted ERG responses to brief flashes over a wide range of stimulus energies, sufficient to evoke rod-driven positive and negative scotopic threshold responses (pSTR, nSTR) to weak stimuli (<0.3 rh*/rod), and cone-driven contributions to a- and b-waves with strong stimuli.

Results:: All mice had a pSTR. The nSTR was absent in Cx36-/- and Bhlhb4-/- mice and present in WT, WT mice with ganglion cell lesions and Cx36-/fl;RGP/BPCre mice. Cx36-/- and Cx36-/fl;RGP/BPCre mice had normal b-wave amplitudes for stimuli up to those producing 100 rh*/rod. Cx36-/- and Cx36-/fl;RGP/BPCre mice had decreased b-wave amplitudes in response to stimuli producing 100 to 1x10⁴ rh*/rod, but normal b-waves for stimuli that produced >1x10⁴ rh*/rod.

Conclusions:: Our data suggests that the nSTR originates from the AIIAC-AIIAC or AIIAC-depolarizing cone bipolar cell (DBCc) electrical synapse pathway, because neither Cx36-/- mice whichlack AIIAC gap junctions, nor Bhlhb4-/- mice, which have diminished DBCr input to AIIACs, exhibited nSTRs. However, the nSTR was present in the conditional Cx36 KO which is presumed to have normal AIIAC-AIIAC and AIIAC-DBCc electrical synapses Moreover, our results suggest that rod-cone coupling via Cx36 plays an important role in visual signaling for the midrange stimulus energy producing between 100 rh*/rod -1x10⁴ rh*/rod. Both the constitutive and conditional Cx36KOs had decreased b-wave amplitudes for these mid-range stimuli, but normal b-waves in response to weaker stimuli likely to produce only rod-driven signals, and stronger stimuli that would also produce cone-driven signals.

Keywords: electroretinography: non-clinical • retinal connections, networks, circuitry • gap junctions/coupling