Analytical methods for assessing retinal cell coupling using cut-loading

Electrical coupling between retinal neurons contributes to the functional complexity of visual circuits. "Cut-loading" methods allow simultaneous assessment of cell-coupling between multiple retinal cell-types, but existing analysis methods impede direct comparison with gold standard direct dye injection techniques. In the current study, we both improved an existing method and developed two new approaches to address observed limitations. Each method of analysis was applied to cut-loaded dark-adapted Guinea pig retinae (n = 29) to assess coupling strength in the axonless horizontal cell type ('a-type', aHCs). Method 1 was an improved version of the standard protocol and described the distance of dye-diffusion (space constant). Method 2 adjusted for the geometric path of dye-transfer through cut-loaded cells and extracted the rate of dye-transfer across gap-junctions in terms of the coupling coefficient (kj). Method 3 measured the diffusion coefficient (De) perpendicular to the cut-axis. Dye transfer was measured after one of five diffusion times (1-20 mins), or with a coupling inhibitor, meclofenamic acid (MFA) (50-500μM after 20 mins diffusion). The standard protocol fits an exponential decay function to the fluorescence profile of a specified retina layer but includes non-specific background fluorescence. This was improved by measuring the fluorescence of individual cell soma and excluding from the fit non-horizontal cells located at the cut-edge (p<0.001) (Method 1). The space constant (Method 1) increased with diffusion time (p<0.01), whereas Methods 2 (p = 0.54) and 3 (p = 0.63) produced consistent results across all diffusion times. Adjusting distance by the mean cell-cell spacing within each tissue reduced the incidence of outliers across all three methods. Method 1 was less sensitive to detecting changes induced by MFA than Methods 2 (p<0.01) and 3 (p<0.01). Although the standard protocol was easily improved (Method 1), Methods 2 and 3 proved more sensitive and generalisable; allowing for detailed assessment of the tracer kinetics between different populations of gap-junction linked cell networks and direct comparison to dye-injection techniques.