Role of connexin channels in the retinal light response of a diurnal rodent

Abstract

Several studies have shown that connexin channels play an important role in retinal neural coding in nocturnal rodents. However, the contribution of these channels to signal processing in the retina of diurnal rodents remains unclear. To gain insight into this problem, we studied connexin expression and the contribution of connexin channels to the retinal light response in the diurnal rodent Octodon degus (degu) compared to rat, using in vivo ERG recording under scotopic and photopic light adaptation. Analysis of the degu genome showed that the common retinal connexins present a high degree of homology to orthologs expressed in other mammals, and expression of Cx36 and Cx43 was confirmed in degu retina. Cx36 localized mainly to the outer and inner plexiform layers (IPLs), while Cx43 was expressed mostly in cells of the retinal pigment epithelium. Under scotopic conditions, the b-wave response amplitude was strongly reduced by 18-β-glycyrrhetinic acid (β-GA) (-45.1% in degu, compared to -52.2% in rat), suggesting that connexins are modulating this response. Remarkably, under photopic adaptation, β-GA increased the ERG b-wave amplitude in degu (+107.2%) while reducing it in rat (-62.3%). Moreover, β-GA diminished the spontaneous action potential firing rate in ganglion cells (GCs) and increased the response latency of ON and OFF GCs. Our results support the notion that connexins exert a fine-tuning control of the retinal light response and have an important role in retinal neural coding.

Keywords: connexins; multi-electrode array (MEA); neural coding; physiology; retina.

Figure 1

Phylogenetic tree of gap junctions present in the retina of different mammals. Multiple alignments with subsequent neighbor-joining and bootstrap analysis were performed on protein sequences of human, guinea pig, degu, and rat. Connexin orthologs tend to group together whereas the paralog sequences are further apart.

Figure 2

Expression and immunolocalization of Cx36 and Cx43 in the retina of degu. (A) Immunodetection of Cx36 (arrowhead) in retinal extracts of degu and rat, and brain cortex as positive control. No immunostaining was visible in heart extracts of rat. (B,C) Fluorescent micrograph showing Cx35/36 immunolabeling in degu and rat retina. Immunolocalization of Cx36 along the OPL and in the IPL was observed as intensely fluorescent puncta. Less frequently, Cx36 labeling was observed in the GCL, where puncta were localized to the margins of cell bodies. The arrowhead points to a blood vessel with strong non-specific labeling (D) Immunodetection of Cx43 in retinal extracts of degu and rat. Cx43 shows the expected electrophoretic mobility around 43 kDa. (E) Fluorescent micrograph of retinal Cx43 immunoreactivity in degu. The immunoreactive puncta of Cx43 were clearly visible between pigment epithelial cells (top) and possibly in glial cells present in the inner retinal layers. (F,G) Fluorescence micrographs of pigment epithelium layer cells showing abundant Cx43 gap junction plaques in cellular appositions. The cell nuclei were counterstained with propidium iodide staining. PE, pigment epithelial cells; OS, photoreceptor outer segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars: 20 μm (B,C,F,G) and 50 μm (E).

Figure 3

Differential effect of connexin channel inhibition on the amplitude of the ERG b-wave in the retina of degu and rat during dark and light adapted conditions. Left: representative ERG tracers obtained from degu (A–C) and rat (B–D) eyes under control conditions or after treatment with β-GA (150 μM), at the maximum intensity used. Bars indicate the stimulus duration (λ = 500 nm). Dotted lines indicate baseline level. Recordings were realized under dark (A,B) and light (C,D) adapted conditions. Right: intensity-response functions under dark and light conditions before and after treatments with β-GA. Asterisks represent the statistical significance with respect to control (Paired t-test, ^*p < 0.05; ^**p < 0.01; ^***p < 0.001).

Figure 4

Spike-triggered average (left) and temporal profile (right) under control conditions. The left panel shows the result of the STA algorithm on the response of GCs to checkerboard stimuli. Each frame has a number indicating the order before the spike time (zero represents the spike occurrence time point). The complete sequence covers −250 ms before the spike time with a time resolution of 16.67 ms. Blue/red represents low/high intensities, respectively. Top, example of an ON GC (STA estimated from n = 2882 spikes); bottom, example of an OFF GC (STA estimated from n = 9263 spikes). In both cases, the light intensity before the spike occurrence is either a decrease (top) or an increase of the mean intensity of the stimuli. The right panel shows the temporal profile of the zone of the receptive field with the highest response. Time zero represents the spike occurrence. Time-to-peak represents the time between the spike occurrence and the maximum peak of the stimulus. Each blue point corresponds to a time panel figure on the left. The red line is a spline interpolation of the curve defined by the blue dots.

Figure 5

Functional ganglion cell characterization under control and β-GA conditions. Using STA analysis, ganglion cells were classified and clustered into five different groups according to their ON or OFF preference and response timing. (A) The dynamics of the response timing is represented for both control (left) and β-GA conditions (right). β-GA decreased the number of ganglion cells with a valid RF. (B) Distribution of time-to-peak responses under β-GA and control conditions for ON and OFF ganglion cells. Under β-GA treatment, the response latency in the temporal response profile increased compared to control. For both ON and OFF cells, the latencies observed in the β-GA condition presented a larger standard deviation compared to controls. ON cells were more affected, allowing to fit a bimodal distribution (right). (C) Comparison of time-to-peak vs. time-to-zero-cross for the same conditions described in (B). Comparison of time-to-peak for control and β-GA treatment for the same cluster allows concluding that every cell type was affected by β-GA, in particular the ON type labeled as C3. The strait line indicates zero difference between β-GA and control.