Genetic dissection of horizontal cell inhibitory signaling in mice in complete darkness in vivo

Abstract

Purpose: To test the hypothesis that horizontal cell (HC) inhibitory signaling controls the degree to which rod cell membranes are depolarized as measured by the extent to which L-type calcium channels (LTCCs) are open in complete darkness in the mouse retina in vivo.

Methods: Dark-adapted wild-type (wt), CACNA1F (Ca(v)1.4(-/-)), arrestin-1 (Arr1(-/-)), and CACNA1D (Ca(v)1.3(-/-)) C57Bl/6 mice were studied. Manganese-enhanced MRI (MEMRI) evaluated the extent that rod LTCCs are open as an index of loss of HC inhibitory signaling. Subgroups were pretreated with D-cis-diltiazem (DIL) at a dose that specifically antagonizes Ca(v)1.2 channels in vivo.

Results: Knockout mice predicted to have impaired HC inhibitory signaling (Ca(v)1.4(-/-) or Arr1(-/-)) exhibited greater than normal rod manganese uptake; inner retinal uptake was also supernormal. Genetically knocking out a closely associated gene not expected to impact HC inhibitory signaling (CACNA1D) did not generate this phenotype. The Arr1(-/-) mice exhibited the largest rod uptake of manganese. Manganese-enhanced MRI of DIL-treated Arr1(-/-) mice suggested a greater number of operant LTCC subtypes (i.e., Ca(v)1.2, 1.3, and 1.4) in rods and inner retina than that in DIL-treated Ca(v)1.4(-/-) mice (i.e., Ca(v)1.3). The Ca(v)1.3(-/-) + DIL-treated mice exhibited evidence for a compensatory contribution from Ca(v)1.2 LTCCs.

Conclusions: The data suggest that loss of HC inhibitory signaling is the proximate cause leading to maximally open LTCCs in rods, and possibly inner retinal cells, in mice in total darkness in vivo, regardless of compensatory changes in LTCC subtype manifested in the mutant mice.

Figure 1

Degree of retinal LTCC opening as measured by extent of manganese uptake in vivo and impact of DIL: central (±0.4–1.0 mm from the optic nerve head, illustrated by the white boxes in the inset image) retinal manganese uptake (as evaluated via 1/T₁) profiles of (A) dark-adapted wt mice (closed black circles, n = 19), Ca_v1.4^−/− mice (closed gold circles, n = 5), Arr1^−/− mice (closed magenta circles, n = 5), and (B) DIL-treated wt, Ca_v1.4^−/−, and Arr1^−/− mice (color scheme as in [A]). Data are shown as a function of distance from the retina/nonretina borders, where 0% is the vitreous/retina border and 100% is the retina/choroid border. Regions near borders are not shown because these regions likely include some signal from outside of the retina (i.e., partial volume averaging with vitreous or choroid/sclera). Bicolored lines above profiles indicate retinal regions with significant (P < 0.05) differences in manganese uptake between control and experimental mice indicated by the color. Above graph: Simplified schematic of retina and support circulations.^– The retina has a well-defined laminar structure that allows us to reasonably label, on high-resolution MEMRI (21.9-μm axial resolution), regions of uptake at 24% to 50% depth as the inner nuclear layer, at 50% to 68% depth as rod nuclei, at 68% to 88% depth as the rod inner segment region, and >88% as the rod outer segment region.

Figure 2

Extent of LTCC opening as measured by MEMRI in Ca_v1.3^−/− mice and impact of DIL in vivo. Retinal manganese uptake profiles of dark-adapted wt mice (closed black circles), Ca_v1.3^−/− mice (closed green circles), or Ca_v1.3^−/− mice + DIL treatment (closed blue circles) on either a C57Bl/6J background (top row) or C57BL/6/SVEV129 F2 hybrid background (bottom row). (A) Wild-type (n = 19) versus Ca_v1.3^−/− (n = 5) mice, (B) Ca_v1.3^−/− (n = 5) versus Ca_v1.3^−/− mice + DIL treatment (n = 5), (C) wt (n = 5) versus Ca_v1.3^−/− (n = 5) mice, and (D) Ca_v1.3^−/− (n = 5) versus Ca_v1.3^−/− mice + DIL treatment (n = 3). Graphs are presented using the conventions in Figure 1. Red lines above profiles indicate retinal regions with significant (P < 0.05) differences in manganese uptake between control and experimental mice indicated.

Figure 3

Simplified schematic representation highlighting the targets studied. Illustration of the retina and support circulations (using conventions in Fig. 1), including the HCs.^– In the dark, rod cell membranes are depolarized, resulting in the sustained opening of synaptic LTCCs of the Ca_v1.4 subtype and presumably Ca_v1.3 LTCCs in rod nuclei.^– Persistent opening of Ca_v1.4 channels (but not, apparently, Ca_v1.3 LTCCs) triggers continuous release of the neurotransmitter glutamate, a process regulated by arrestin-1.^, When HCs receive glutamatergic input, their membranes depolarize and send (by a yet unclear process) inhibitory signals back to the photoreceptors.^, The present data suggest that the target proteins chosen (based on light-based studies ex vivo) also participate in inhibitory signaling in mice in total darkness in vivo when HCs experience a maximum and sustained glutamate exposure.