Genetic elimination of rod/cone coupling reveals the contribution of the secondary rod pathway to the retinal output

Abstract

In the retina, signals originating from rod and cone photoreceptors can reach retinal ganglion cells (RGCs)-the output neurons-through different pathways. However, little is known about the exact sensitivities and operating ranges of these pathways. Previously, we created rod- or cone-specific Cx36 knockout (KO) mouse lines. Both lines are deficient in rod/cone electrical coupling and therefore provide a way to selectively remove the secondary rod pathway. We measured the threshold of the primary rod pathway in RGCs of wild-type mice. Under pharmacological blockade of the primary rod pathway, the threshold was elevated. This secondary component was removed in the Cx36 KOs to unmask the threshold of the third rod pathway, still below cone threshold. In turn, the cone threshold was estimated by several independent methods. Our work defines the functionality of the secondary rod pathway and describes an additive contribution of the different pathways to the retinal output.

Fig. 1.. Schematic representation of the rod and cone pathways of the wildtype mammalian retina and of the rod- or cone-Cx36 KO retinas.

(A) Architecture of rod and cone circuits in the wild-type retina. Rod signals can reach RGCs—the output neurons of the retina— through three different pathways. The primary rod pathway (1), where rod signals are transmitted to RBCs, is of high sensitivity and plays a prominent role in the transmission of scarce light signals under very dim light (low scotopic) conditions near absolute visual threshold. In the secondary rod pathway or rod/cone pathway (2), rod signals may enter directly into the cone pathway through rod/cone gap junctions. In the tertiary rod pathway (3), rod signals may enter directly into the cone pathway through synaptic contacts between rods and some types of OFF CBCs. Cones contact both ON CBCs and OFF CBCs, thereby forming the entry of the cone pathways (C). AII, AII amacrine cell. (B) The secondary rod pathway is missing in the rod-Cx36 KO and cone-Cx36 KO mutant lines. The primary rod pathway can be blocked by APB. Therefore, the light responses of OFF RGCs are driven by the tertiary rod pathway and the cones in the mutant lines in the presence of APB. Because APB blocks the ON cone pathway as well, all light responses in ON RGCs are abolished in the presence of APB. Red lettering indicates circuits sensitive to APB.

Fig. 2.. Targeting, dendritic morphologies, and light response characteristics of the tOFF αRGC.

(A) αRGCs have the largest soma size among cells in the ganglion cell layer. Scale bar, 25 μm. (B) Dendritic processes and axon (white arrow) are revealed following neurobiotin injection and processing of the tissue. Scale bar, 50 μm. (C) Volume projection of z-stack images that include the inner plexiform layer showing the location of the dendrites of a tOFF αRGC (white arrow) between the two choline acetyltransferase (ChAT) bands. Two ChAT-positive starburst amacrine cell bodies (C1 and C2) also appear in the image. (D) Loose-patch recording of a tOFF αRGC showing a family of 25 consecutive recordings (spike raster sweeps) and the calculated mean firing rate. Note the transient nature of its light responses at lights OFF.

Fig. 3.. Contribution of the rod/cone pathway to the retinal output.

(A to F) Loose-patch recordings of the light responses of tOFF αRGCs in wild-type (A to C) and mutant Cx36 retinas (D to F). (A and D) Examples of raster plots that include 20 consecutive light responses and averaged firing rates. Recordings were obtained in the dark-adapted retina. A 0.5-s stimulus was presented at three different light intensities (0.06 R*/rod/s, top row [1]; 2 R*/rod/s, middle row [2]; and 20 R*/rod/s, bottom row [3]), before (left column, control), in the presence of 25 μM APB (middle column, APB), or after APB washout (> 30 min, right column, washout). In the wild type, APB eliminated the responses to the dimmest light stimulus, most of the responses at the intermediate intensity but had limited effect on responses to the brightest stimuli. In the mutant, APB eliminated the responses to both dim and intermediate light stimuli and greatly attenuated responses to the brightest stimuli. (B, C, E, and F) Averaged data from tOFF αRGCs obtained in cone-Cx36 KO ctl (n = 5) (B), rod-Cx36 KO ctl (n = 13) (C), cone-Cx36 KO (n = 8) (E), and rod-Cx36 KO retinas (n = 9) (F). The results are consistent with the primary (APB-sensitive) rod pathway being the most sensitive pathway and with the threshold of the secondary rod pathway being ~1 R*/rod/s and the elimination of this pathway in the mutant lines (also see Table 1 and fig. S2 for details). White arrows in (B) and (E) show the three intensities tested in (A) and (D).

Fig. 4.. Establishing the contribution of pure cone input to αRGCs – recording from ON αRGCs in the pan-Cx36 KO mouse.

(A) In the pan-Cx36 KO mouse, Cx36 gap junctions between photoreceptors and between AII amacrine cells and ON CBCs are eliminated. Consequently, rod signals cannot reach ON RGCs through the primary or the secondary rod pathways, and, therefore, cone signals only drive ON RGC light responses. Red lettering indicates circuits sensitive to APB. (B) Raw data. A 0.5-s stimulus was presented at three different light intensities (0.06 R*/rod/s, top row [1]; 20 R*/rod/s, middle row [2]; and 1000 R*/rod/s, bottom row [3]), before (left column, control), or in the presence of 25 μM APB (right column, +APB). As expected, the cone ON pathway was sensitive to APB. Inset shows averaged normalized responses of seven cells (one cell per retina) to intensities ranging from 0.002 to 100,000 R*/rod/s. Recording from ON αRGCs in the pan-Cx36 KO retina enabled us to determine the cone threshold in αRGCs (~175 R*/rod/s, gray arrowhead).

Fig. 5.. Establishing the contribution of pure cone input to αRGCs—recording from tOFF αRGCs in the Gnat1^irdr mouse.

(A) In the GNAT1^irdr mouse, rods are present but do not respond to light. Consequently, cone signals only drive tOFF αRGC light responses. Red lettering indicates circuits sensitive to APB; only the cone ON pathway is sensitive to APB. (B) Raw data. A 0.5-s stimulus was presented at three different light intensities (0.06 R*/rod/s, top row [1]; 20 R*/rod/s, middle row [2]; and 1000 R*/rod/s, bottom row [3]), before (left column, control), in the presence of 25 μM APB (middle column, +APB), or after APB washout (> 30 min, right column, washout). As expected, the cone OFF pathway was insensitive to APB. Inset shows averaged data from three cells (one cell per retina). Recording from tOFF RGCs in the GNAT1^irdr retina enabled us to determine the cone threshold (~172 R*/rod/s, colored arrowheads).

Fig. 6.. Contribution of the cone signals to the ON αRGC.

(A to C) The responses of an ON αRGC to a stimulus of intensity 25 R*/rod/s (A), 83 R*/rod/s (B), or 250 R*/rod/s (C). The stimulus was delivered for 1 s, twice, first at 1 Hz and 2 s later at 10 Hz (top trace). The bottom trace shows the raw spiking data. (D to F) Raster plots from 25 consecutive recordings under each of the conditions depicted in (A) to (C) (top trace). The bottom trace is a plot of the averaged spike rate (binned over 10 ms). (G to I) Nonlinear regression analysis of the data illustrated in D (G), E (H), and F (I) and collected during the presence of the 1-s 10-Hz stimulus (i.e., from time = 3.0 s to time = 4.0 s). Data were fit to a sine equation of frequency exactly 10.00 Hz. Regression curve and goodness of fit (R²) are shown in red. (J) Goodness of fit as a function of stimulus intensity at 10 Hz. Results are from 12 cells. The red curve was the fit of all the data to the Hill equation. (K) Standardized R² values as a function of stimulus intensity. R² is standardized for each cell to its highest value. The red curve is as in (J). The intensity at threshold (5% maximum response) equals 20 ± 3 R*/rod/s. The data suggest that cones contribute to the light responses of ON αRGCs at intensities greater than 20 R*/rod/s.

Fig. 7.. Establishing the contribution of all rod and cone inputs to tOFF αRGCs.

(A and B) Combining the various intensity-response curves allows us to establish the threshold (arrowheads) and contribution of the different rod pathways to tOFF αRGCs. From left to right, the intensity-response profiles are from wild-type littermate control (ctl) (control + APB) and from the mutant lines in the presence of APB. The cone intensity-response profile is generated from tOFF αRGCs recordings in the GNAT1^irdr (see Fig. 5). Note the similarity of the results obtained in cone-Cx36 KO mice (A) and in rod-Cx36 KO mice (B). Thresholds calculated as intensity to elicit 5% maximum response (see Table 1 for details).