Retinal photoreceptors are highly differentiated postmitotic neurons that transduce photons into electrical signals. While the functions of many photoreceptor-specific genes can be evaluated by direct gene targeting, here we facilitate the studies of nonphotoreceptor-specific genes in these cells by developing an Opsin-iCre transgenic mouse line, iCre-75, in which a 4-kb mouse rod opsin promoter drives the expression of bacteriophage P1 Cre recombinase. Immunohistochemical analysis demonstrated that Cre recombinase is present exclusively in the outer nuclear layer of iCre75 mouse retina. Cre expression is found only in rods and not in cones. The expression level reached 188+/-44 ng per retina at postnatal day (pnd) 11 and increased to 687+/-56 ng at 2 months and older. Cre-mediated excision of floxed genomic DNA was absent at pnd 4, became detectable at pnd 7, and was completed by pnd 18. Retinal morphology and electroretinograms were normal in 8-month-old transgenic animals. The iCre-75 transgenic mice are thus suitable for future genetic studies of essential genes in retinal rod photoreceptors.

It has been shown in rd1 and rd10 models of photoreceptor degeneration (PD) that inner retinal neurons display spontaneous and rhythmic activities. Furthermore, the rhythmic activity has been shown to require the gap junction protein connexin 36, which is likely located in AII amacrine cells (AII-ACs). In the present study, an autosomal dominant PD model called rhoΔCTA, whose rods overexpress a C-terminally truncated mutant rhodopsin and degenerate with a rate similar to that of rd1, was used to investigate the generality and mechanisms of heightened inner retinal activity following PD. To fluorescently identify cholinergic starburst amacrine cells (SACs), the rhoΔCTA mouse was introduced into a combined ChAT-IRES-Cre and Ai9 background. In this mouse, we observed excitatory postsynaptic current (EPSC) oscillation and non-rhythmic inhibitory postsynaptic current (IPSC) in both ON- and OFF-SACs. The IPSCs were more noticeable in OFF- than in ON-SACs. Similar to reported retinal ganglion cell (RGC) oscillation in rd1 mice, EPSC oscillation was synaptically driven by glutamate and sensitive to blockade of NaV channels and gap junctions. These data suggest that akin to rd1 mice, AII-AC is a prominent oscillator in rhoΔCTA mice. Surprisingly, OFF-SAC but not ON-SAC EPSC oscillation could readily be enhanced by GABAergic blockade. More importantly, weakening the AII-AC gap junction network by activating retinal dopamine receptors abolished oscillations in ON-SACs but not in OFF-SACs. Furthermore, the latter persisted in the presence of flupirtine, an M-type potassium channel activator recently reported to dampen intrinsic AII-AC bursting. These data suggest the existence of a novel oscillation mechanism in mice with PD.

SHORT ABSTRACT This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation. LONG ABSTRACT The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

There was a mistake in the x-axis label in Figure 5B as published. The corrected Figure 5 appears below. The authors apologize for the typographical mistake. This error does not change the scientific conclusions of the article in any way. Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.