A comparison of optical and electrophysiological methods for recording retinal ganglion cells during electrical stimulation

Abstract

Purpose/aim: To compare the efficacy of optical techniques with electrophysiological recordings for mapping retinal activity in response to electrical stimulation.

Materials and methods: Whole cell patch clamp, Ca(2+) imaging (Fluo-4-AM), and Na(+) imaging (CoroNa Green-AM) techniques were used to detect responses of neurons from mouse and salamander retina to electrical stimulation.

Results: Synaptic currents were observed in ≥23% of retinal ganglion cells (RGCs), indicating presynaptic Ca(2+) increases in the inner plexiform layer (IPL). Modest depolarization with 20-30 mM K(+) consistently evoked Ca(2+) responses measured with Fluo4, but Ca(2+) responses were almost never evoked by epiretinal stimulation. In salamander retina, responses were seen in the inner nuclear layer (INL) and IPL. In mouse retina, responses were also sometimes seen in the outer pexiform layer (OPL). OPL responses showed a longer latency than IPL responses, suggesting that outer retinal circuits do not trigger synaptic responses of RGCs. Simultaneous Ca(2+) imaging and electrophysiological recording of synaptic currents confirmed that Fluo4-loaded retinas remained responsive to stimulation. Epiretinal stimulation evoked action potentials in ≥67% of RGCs. CoroNa Green detected Na(+) changes stimulated by 20 mM K(+), but epiretinal stimulation did not evoke detectable Na(+) responses. Simultaneous imaging and electrophysiological recording confirmed the health of CoroNa Green-loaded retinas. We confirmed stimulation efficacy by simultaneously recording Na(+) changes and electrophysiological responses.

Conclusions: These data demonstrate that electrophysiological recordings show greater sensitivity than Na(+) or Ca(2+) imaging in response to electrical stimulation. The paucity of Ca(2+) responses is consistent with limited risk for Ca(2+)-mediated cell damage during electrical stimulation.

FIGURE 1

Fast action potential currents (spikes) and synaptic currents evoked from mice and salamander retinal ganglion cells by epireti-nal stimulation. (A) Spike (arrow) evoked in a mouse ON ganglion cell from a flatmount retina preparation by injection of cathodic current (1 ms, 12 µA). (B) Synaptic current (arrow) evoked in a different ON ganglion cell from a flatmount mouse retina preparation (25 ms, 18 µA). (C) Spike (arrow) evoked in retinal ganglion cell from a salamander retinal slice (25 ms, 10 µA). (D) Synaptic current (arrow) evoked in retinal ganglion cell from a salamander retinal slice (30 ms, 15 µA).

FIGURE 2

Intracellular Ca²⁺ changes evoked by modest depolarizing stimulation. (A) A confocal stack (fifteen 1 µm sections) of a salamander retinal slice loaded with the Ca²⁺-sensitive dye, Fluo4. (B) Fluo4 fluorescence changes evoked by bath application of 20 mM K⁺ which is sufficient to stimulate ~10 mV depolarization in rod photoreceptors. Ca²⁺ changes are plotted as a function of time for a rod soma (region 1 in panel A), an amacrine cell soma (region 2), and synaptic puncta within the IPL (regions 3 and 4). (C) A confocal stack (15 sections) of a mouse retinal slice loaded with Fluo4. (D) Fluo4 fluorescence changes evoked by bath application of 30 mM K⁺ which is sufficient to stimulate ~10 mV depolarization in rod photoreceptors. Ca²⁺ changes are shown for a rod soma (region 1 in panel A), an amacrine cell soma (region 2), and synaptic puncta in the IPL (regions 3–5).

FIGURE 3

Detection of Ca²⁺ changes in response to epiretinal stimulation in the inner retina of a mouse retinal slice. (A) Single confocal section of a Fluo4-loaded retinal slice showing the IPL and a portion of the INL. (B) Difference image showing Ca²⁺ changes created by subtracting the control image from a post-stimulus image obtained immediately after stimulation (100 ms, 14 µA). (C) Ca²⁺ changes in three cell bodies from the INL and two puncta from the IPL plotted as a function of time. The corresponding regions of interest are denoted in panels A and B.

FIGURE 4

Latency from initiation of the cathodic stimulus pulse to the beginning of the Ca²⁺ response in the IPL and OPL. Responses in the OPL showed a significantly longer latency than those observed in the IPL (p < 0.0019).

FIGURE 5

Detection of Ca²⁺ changes in response to epiretinal stimulation in a salamander retinal slice. (A) Single confocal section of a Fluo4-loaded retinal slice. (B) Difference image showing Ca²⁺ changes in the INL and IPL created by subtracting the control image from the post-stimulus image (100 ms, 7 µA). (C) Ca²⁺ changes in the amacrine cell from the INL (region 1) and IPL (region 2) plotted as a function of time.

FIGURE 6

Simultaneous imaging of Ca²⁺ changes and elec-trophysiological recording of a synaptic current in response to epiretinal electrical stimulation in salamander retinal slice. (A) Epiretinal stimulation (100 ms, 17 µA) evoked a large, sustained synaptic current in a voltage-clamped RGC. Note the presence of transient miniature synaptic currents throughout the response. (B) Single confocal section of the retinal slice loaded with Fluo4. (C) Ca²⁺ change from a presumptive amacrine cell from the INL plotted as a function of time. The region of interest within which these Ca²⁺ changes were measured is indicated by the circle in panel B.

FIGURE 7

Sensitivity of CoroNa Green was examined by bath application of varying concentrations of high K⁺. Panel A shows a confocal stack of a retinal slice stained with CoroNa Green (fif-teen 1 µm sections). Application of 20 mM K⁺ evoked a detectable increase in fluorescence in cells throughout the retina. Panel B shows fluorescence changes plotted for a presumptive bipolar cell in the distal portion of the INL, an amacrine cell in the proximal INL, and an RGC. The corresponding regions of interest are indicated by the circles in (A). The fluorescence increase evoked by bath application of high K⁺ was superimposed on a slow decrease in fluorescence due to dye bleaching.

FIGURE 8

Simultaneous imaging of Na⁺ changes and electro-physiological recording of a fast Na⁺ spike evoked by epiretinal electrical stimulation in salamander retinal slice. (A) Single confocal section of the retinal slice loaded with CoroNa Green showing a portion of the IPL and GC layer. (B) Difference image subtracting a control image from the post-stimulus image reveals no detectable Na⁺ changes. (C) Epiretinal stimulation (50 ms, 15 µA) evoked a fast Na⁺ spike (arrow) in a voltage-clamped RGC.