Table 1.
Experimental design showing the primary variables manipulated.
The Experimental Light Exposure refers to the light level during cut-loading at a wavelength of 850nm and was approximately equivalent to total darkness.
Fig 1.
Flow chart illustrating the key steps in the cut-loading procedure.
Figure made using the online application BioRender.com.
Table 2.
Both primary and secondary antibodies were diluted in 1% NDS and 0.5% Triton-x in PBS. RT, room temperature.
Fig 2.
Geometric model of dye-transfer through coupled horizontal cells.
(a) Cells located in C1 (*) receive dye (qr) from two cells in C0 and individually feed two cells (qs) in C2. (b) Cells located in C1 either receive input from three cells in C0 and feed one in C2 (cell a), or they receive input from one cell in C0 and feed three cells in C2 (cell b).
Fig 3.
The diffusion of molecular tracer from the cut location, modelled according to Eq (9).
At t = 0, all molecular tracer is located within the region–l < x < l surrounding the x-axis. Concentration of NeurobiotinTM is represented as relative fluorescent intensity along the z-axis.
Fig 4.
(a) Example image taken in outer portion of the inner nuclear layer of a Guinea pig retina cut-loaded with NeurobiotinTM. (b) The fluorescent intensity of the whole image measured along the horizontal axis extended away from the cut. (c) Close up of morphologically distinct horizontal cell subtypes: axon-bearing (‘B-type’, open arrow) and axon-less (‘A-type’, closed arrow) HCs. Image created from cut-loaded retina using the temporal colour-code function in Fiji for optical slices spanning the inner nuclear layer (see methods). (d) Cut-loaded aHCs and bHCs located adjacent to the cut, loaded with NeurobiotinTM (green) and co-labelled with Calbindin (red). (e) Cut-loaded aHCs located 500 μm from the cut, loaded with NeurobiotinTM (green) and co-labelled with Calbindin (red). (f) Semi-log plot of fluorescent intensity data from (b) (dashed red line) and fluorescent intensity of individual aHCs (grey markers, dashed blue line) and bHCs (purple markers, dashed purple line)), each fitted with an exponential decay function, Eq (1). (g) Fluorescent intensity of individual aHCs adjusted for the mean soma-soma cell distance.
Fig 5.
(a-e) The extent of dye-transfer through coupled a-type horizontal cells (aHCs) after different incubation times between 1 and 20 min. Images represent the average of 5, 1μm thick optical slices through the outer inner nuclear layer created using the z-project function in Fiji (see methods). The cut-edge is located on the left-hand side of each image. (f, g) aHCs cut-loaded with Neurobiotin (green) and the gap-junction impermeable dye Rhodamine dextran (red, aHCs identified with red arrows) after 3 and 10 min incubation respectively. (h) Relative mean cell fluorescence of aHCs per cell from the cut. Curves produced by fitting 3-paramenter polynomial fits to mean aHC data for each incubation time, fluorescence at the cut was excluded when creating the fit. The peak cell fluorescence for each curve is indicated by black stars. Scale bars signify 100μm.
Fig 6.
Comparison of three analytical techniques for assessing the extent of a-type horizontal (aHCs) cell coupling in cut-loaded Guinea pig retinas with increasing incubation times.
Column 1 (a, d, g) contains cell fluorescence data analysed using Method 1. Column 2 (b, e, h) shows the same data analysed using Method 2 and column 3 (c, f, i) shows the same data analysed using Method 3. The first row (a, b, c) contains example fits for cell fluorescence data collected per each cell separation following four incubation times, fitted with (a) Eq (1), (b) Eq (6) and (c) Eq (9) corresponding to Methods 1, 2 and 3 respectively. Open markers indicate cells adjacent to the cut with reduced fluorescence. Row 2 (d, e, f) contains the mean ± SE (black marker, black error bars) and individual measurements (grey markers) for each measure of cell-coupling obtained via: (d) Method 1 (e) Method 2 and (f) Method 3. Row 3 (g, h, i) contains the mean ± SE (black marker, black error bars) and individual measurements (grey markers) for each measure of cell-coupling normalised to the mean cell-cell spacing between adjacent aHCs, obtained via: (g) Method 1 (h) Method 2 and (i) Method 3. Crosses indicate outliers defined as data points greater than 1.5 standard deviations from the mean and are not included in calculated mean.
Fig 7.
Comparison of three analytical methods for describing dose-response data for a-type horizontal cell (aHC) coupling and meclofenamic acid (MFA) concentration in vitro.
The relative diffusion of NeurobiotinTM through HCs in tissue incubated in (a) 50μM MFA and (b) 250μM MFA. Column 1 (c, f) contains data analysed using Method 1, column 2 (d, g) shows the same data analysed using Method 2 and column 3 (c, f, i) shows the same data analysed using Method 3. The first row of plots (c, d, e) contains the mean ± SE (black marker, black error bars) and individual measurements (grey markers) for each measure of cell-coupling obtained via Methods: (c) 1, (d) 2 and (e) 3. Row 2 (f, g, h) contains the mean ± SE (black marker, black error bars) and individual measurements (grey markers) for each measure of cell-coupling normalised to the mean cell-cell spacing between adjacent aHCs, obtained via Methods (f)1, (g) 2 and (h) 3.
Table 3.
Reduction in cell coupling induced by the coupling inhibitor MFA, presented as a percentage reduction from a low-dose baseline concentration.
p-values indicate the statistical difference in calculated normalised effect sizes compared to Method 1.
Fig 8.
Identification of amacrine cells expressing neuronal nitric oxide synthase loaded with molecular tracer via cut-loading.
(a) Image created from z-stack slices spanning from the inner nuclear layer to the ganglion cell layer, flattened using the z-project function in Fiji. Open arrows, type-1 nNOS amacrine cells; solid arrows, displaced nNOS amacrine cells. (b, c) Vertical retinal sections labelled for nNOS (red) and DAPI (blue). (b) Open arrows highlight type-1 nNOS amacrine cells located in the inner nuclear layer (INL), (c) solid white arrows highlight displaced nNOS amacrine cells located in the ganglion cell layer (GCL). (d, e) Fluorescent intensity of NeurobiotinTM in cut-loaded type -1 and displaced nNOS amacrine cells analysed using (d) Method 2 (fitted with Eq 6); or (e) Method 3 (fitted with Eq 9). (f-g) Comparison of nNOS type-1 and nNOS displaced amacrine cells in their mean: (f) cell density, (g) cell-coupling coefficients kj, and (h) relative diffusion coefficients. Error bars are SEM. * p < 0.05.