Article Figures & Data
Figures
- Figure 1.
Genetic engineering of Cx57-iCre knock-in transgenic mice. A, The Cx57 coding region was removed and replaced with an in-frame, codon-optimized, improved iCre gene to produce the mouse Cx57-iCre targeting construct pCx57.6. B–D, In Cx57-iCre:: ACTB-mT/mG retinae, non-iCre-expressing cells express membrane-bound tdTomato (mTomato; B), the expression of membrane-bound eGFP (mGFP) appears only in horizontal cells (C) within the retina, suggesting that recombination occurred specifically in horizontal cells within the retina (D, merge). Note that the overall morphology of the retina appeared normal. E–G, Calbindin immunoreactivity identified horizontal cells (E, blue), and Cre immunolabeling (F, red) occurred exclusively in horizontal cell bodies (G, merge). Scale bar, 10 µm. H–J, Cx57-iCre crossed with a Cre-dependent reporter line (Ai14, R26R-tdTomato) produced tdTomato expression (I, red) in horizontal cells (H) overlapping perfectly with calbindin immunolabeling (J, white). Scale bar, 10 µm. K, Cre expression occurred in horizontal cells in all regions of the retina. Confocal micrograph of a whole-mount retina from a homozygous Cx57-iCre mouse labeled with antibodies to calbindin (blue) and Cre recombinase (red). Arrow points to a horizontal cell body containing Cre. Red looping bands are nonspecific labeling of blood vessels by the 2° antibody. Projection of four confocal images; z-steps = 0.5 µm. Scale bar, 20 µm. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
- Figure 2.
Targeted knockout of VGAT from horizontal cells in Cx57-VGAT−/− retinae. A, Crosses of Cx57-iCre+/- and VGATflox/flox mouse lines resulted in a conditional knockout of VGAT in horizontal cells in Cx57-VGAT−/− retinae. B, Vertical section of a Cx57-VGAT+/+ mouse immunolabeled for VGAT in the OPL and IPL. C, VGAT immunolabeling in Cx57-VGAT−/− retinae is missing from horizontal cell tips, but remains unchanged in the IPL. The remaining VGAT-immunoreactive fibers and puncta in the OPL likely correspond to processes of GABAergic interplexiform cells. Note that both B and C show these labeled fibers and puncta. The overall morphology of the retina is unchanged. INL, Inner nuclear layer. Projection of 12 optical sections, z-steps = 0.5 µm. Scale bar, 20 µm.
- Figure 3.
Voltage-gated K+ and Ca2+ channel currents remain normal in horizontal cells from Cx57-VGAT−/− retinae. A–H, Whole-cell voltage-clamp recordings from isolated horizontal cells from Cx57-tdTomato VGAT+/+ (A–D) and Cx57-VGAT−/− (E–H) mice show that the membrane currents of horizontal cells are unaffected by the deletion of VGAT. Examples of inward and outward K+ currents from normal (A) and VGAT knock-out (E) horizontal cells are similar, and their peak current–voltage (I–V) relations (B, F) are comparable. Ca channel currents recorded with 10 mm Ba2+ in wild-type (C) and knockout (G) are similar, with indistinguishable I–V relations (D, H).
- Figure 4.
Excitatory and inhibitory ligand-gated currents are present in horizontal cells from Cx57-VGAT−/− retinae. A, B, Whole-cell voltage-clamp recordings, with CsCl-filled pipettes, of isolated horizontal cells from control Cx57-tdTomato VGAT+/+ (A) and knock-out Cx57-VGAT−/− mice (B) show that ionotropic GABA receptors in horizontal cells are unaffected by the deletion of VGAT. Currents in response to ramps (−80 to +40 mV, 240 ms) are shown before (control) and during bath superfusion of 50 μm muscimol, a GABAAR agonist. The slope conductance of the current–voltage (I–V) relations increased in both in Cx57-VGAT+/+ and Cx57-VGAT−/− mice during muscimol superfusion (values summarized in text), although the muscimol-induced conductance was larger in the knock out animals. C, D, Glutamate receptor function was tested with 50 μm kainate superfused on Cx57-VGAT+/+ (C) and Cx57-VGAT−/− horizontal cells (D). Currents in response to ramps are shown before and during bath superfusion of 50 μm kainate. The increases in slope conductance of the I–V relations during kainate superfusion were not significantly different in horizontal cells from VGAT+/+ and VGAT−/− mice (values summarized in text).
- Figure 5.
Deletion of VGAT in horizontal cells results in the loss of inhibitory feedback modulation of photoreceptor [Ca2+]i. Feedback inhibition to photoreceptors was eliminated in retinal slices from Cx57-VGAT−/− mice. A, In Cx57-VGAT+/+ mice, 50 μm kainate (KA), which depolarizes horizontal cells, inhibited high [K+]-evoked calcium signals in photoreceptors (timing shown in bars below traces), suggesting an increase in inhibitory feedback. The high [K+] stimulus was always superfused for 30 s, the timing of which is shown by the horizontal bar below the traces. C, 50 μm NBQX, which hyperpolarizes horizontal cells, enhanced calcium signals in photoreceptors, suggesting a decrease in inhibitory feedback. B, D, In Cx57-VGAT−/− retinal slices, kainate did not increase inhibitory feedback to photoreceptors (B), and NBQX did not reduce inhibitory feedback (D). Fluorescence traces are shown normalized to the first of each paired response to high [K+] application. Calibration: 60 s. E, Confocal image of a Cx57-tdTomato retinal slice loaded with fluo-4 (green). The expression of tdTomato (red) identifies the horizontal cells within the slice. Photoreceptor cell bodies are in the outer nuclear layer and their axon terminals are in the OPL. F, Summary of photoreceptor calcium signal amplitudes in retinal slices from VGAT+/+ and VGAT−/− mice treated with kainate and NBQX. Graph shows normalized mean values ± SDs from multiple retinal slices with one data point (filled circle) per slice, each of which was averaged from 5 to 20 individual photoreceptors from three to five different eyes. ONL, Outer nuclear layer; INL, inner nuclear layer.
Tables
Measurement/group Data structure Type of test Confidence interval a Kir Normal Welch’s t test −257 to 389 (pA) b Kv Normal Welch’s t test −1822 to 2328 (pA) c Cav Normal Welch’s t test −139 to 97 (pA) d V½ Normal Welch’s t test −14.6 to 22.8 (mV) e Muscimol WT Unknown Wilcoxon signed rank test 1.35–0.14 (nS) f Muscimol KO Normal Paired t test 4.58–0.85 (nS) g Muscimol WT vs KO Normal Welch’s t test 0.57–3.60 (nS) h Kainate WT Normal Paired t test 2.19–0.78 (nS) i Kainate KO Normal Paired t test 2.39–0.21 (nS) j Kainate WT vs KO Normal Welch’s t test −1.28 to 0.91 (nA) k [Ca]i kainate WT Normal Welch’s t test −28.1 to −13.5 (%) l [Ca]i NBQX WT Normal Welch’s t test 8.0–44.6 (%) m [Ca]i kainate KO Unknown Wilcoxon rank sum test −8.2 to 24.0 (%) n [Ca]i NBQX KO Normal Welch’s t test −9.3 to 12.8 (%)
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