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

L-type calcium channels (LTCCs) are the major calcium entry path into rod cells, which make up approximately 97% of photoreceptors in mice.1–3 In the dark, rod cell membranes are depolarized, resulting in the sustained opening of synaptic LTCCs of the Cav1.4 subtype and presumably Cav1.3 LTCCs in rod nuclei.4–6 Persistent opening of Cav1.4 channels triggers continuous release of the neurotransmitter glutamate.7 Exocytosis at the photoreceptor synapse is also modulated by the synaptic interaction between arrestin-1 and N-ethylmaleimide sensitive factor.8 Just anterior to the photoreceptor synapse, retinal interneuron horizontal cells (HCs), which express LTCCs, receive glutamatergic input from photoreceptors, resulting in depolarization of their plasma membrane.9 Horizontal cell depolarization produces, by as yet an unclear mechanism, inhibitory signaling whose target on photoreceptors are Cav1.4 channels.10 Our current understanding of proteins that regulate glutamatergic-evoked HC inhibitory signaling to photoreceptors (i.e., Cav1.4 and arrestin-1) is mainly based on light-based studies ex vivo. It is unclear if these proteins are also at play in mice in total darkness in vivo when HCs experience a maximum and sustained glutamate exposure. The degree to which rod LTCCs are open or closed in vivo can be objectively evaluated by measuring specific uptake of Mn2+, which is an LTCC probe, using manganese-enhanced MRI (MEMRI), the imaging modality of choice for noninvasively studying retinal ion homeostasis.11 Because Mn2+ efflux from cells is slow compared with influx,12 the extent of accumulation a few hours after injection is a useful measure of manganese influx. Manganese-enhanced MRI relies on the well-defined laminar structure of the retina to distinguish, for example, inner retinal uptake from outer retinal uptake.13–18 In the outer retina, the dominant photoreceptor cells in the mouse retina are the rods.1 Thus, uptake of manganese from 50% to 88% depth into the retina primarily reflects a single cell type. We recently directly demonstrated that MRI has the resolution to, for example, distinguish between inner segments and outer segments, based on the light-evoked expansion of the subretinal space in mice and rats.19,20 On the other hand, MEMRI cannot differentiate among MEMRI signals in the inner retina, where there is substantial overlap of several different cell types, including HCs. In any event, uptake is increased by rod cell membrane depolarization (i.e., opening of LTCCs with dark adaptation versus being closed in the light) and the LTCC-specific agonist Bay K 8644 in light-adapted animals.21–23 Rod cell uptake is strongly inhibited in the dark by LTCC-specific blockers, such as nifedipine,24 and D-cis-diltiazem (DIL).25 Importantly, MEMRI uniquely measures layer-specific retinal LTCC activity in complete darkness that was encoded while mice were awake and freely moving. Here, using MEMRI and genetically modified mice, we test the hypothesis that HC inhibitory signaling controls the degree to which rod cell membranes are depolarized as measured by the extent to which LTCCs are open in complete darkness in the mouse retina in vivo. In the dark, polarization of the rod cell membrane (and thus the degree to which associated LTCCs are open) reflects a balance of two competing signals: a depolarization signal generated from the outer segments (which will open LTCCs) and a hyperpolarization signal generated from HCs (which will close LTCCs). If, for example, HC inhibitory signaling were completely absent, the depolarization signal would dominate and LTCCs will be maximally open. In this study, we examined Cav1.4 and arrestin-1, two proteins that normally regulate the release of glutamate, an essential condition that engages HC inhibitory signaling in bright and dim light. Our prediction was that in each knockout mouse, HC inhibitory signaling would be impaired and LTCCs would be open to a greater extent than normal. We also predicted that the absence of Cav1.3, a channel not known to regulate glutamate release, would not produce a supernormal uptake phenotype.