Horizontal cells (HCs) form reciprocal synapses with rod and cone photoreceptors, an arrangement that underlies lateral inhibition in the retina. HCs send negative and positive feedback signals to photoreceptors, but how HCs initiate these signals remains unclear. Unfortunately, because HCs have no unique neurotransmitter receptors, there are no pharmacological treatments for perturbing membrane potential specifically in HCs. Here we use transgenic zebrafish whose HCs express alien receptors, enabling cell-type-specific control by cognate alien agonists. To depolarize HCs, we used the Phe-Met-Arg-Phe-amide (FMRFamide)-gated Na+ channel (FaNaC) activated by the invertebrate neuropeptide FMRFamide. To hyperpolarize HCs we used a pharmacologically selective actuator module (PSAM)-glycine receptor (GlyR), an engineered Cl- selective channel activated by a synthetic agonist. Expression of FaNaC or PSAM-GlyR was restricted to HCs with the cell-type selective promoter for connexin-55.5. We assessed HC-feedback control of photoreceptor synapses in three ways. First, we measured presynaptic exocytosis from photoreceptor terminals using the fluorescent dye FM1-43. Second, we measured the electroretinogram (ERG) b-wave, a signal generated by postsynaptic responses. Third, we used Ca2+ imaging in retinal ganglion cells (RGCs) expressing the Ca2+ indicator GCaMP6. Addition of FMRFamide significantly decreased FM1-43 destaining in darkness, whereas the addition of PSAM-GlyR significantly increased it. However, both agonists decreased the light-elicited ERG b-wave and eliminated surround inhibition of the Ca2+ response of RGCs. Taken together, our findings show that chemogenetic tools can selectively manipulate negative feedback from HCs, providing a platform for understanding its mechanism and helping to elucidate its functional roles in visual information processing at a succession of downstream stages.

Lateral inhibition in the vertebrate retina depends on a negative feedback synapse between horizontal cells (HCs) and rod and cone photoreceptors. A change in pH is thought to be the signal for negative feedback, but its spatial profile in the synaptic cleft is unknown. Here we use three different membrane proteins, each fused to the same genetically-encoded pH-sensitive Green Fluorescent Protein (GFP) (pHluorin), to probe synaptic pH in retina from transgenic zebrafish (Danio rerio) of either sex. We used the cone transducin promoter to express SynaptopHluorin (pHluorin on vesicle-associated membrane protein (VAMP2)) or CalipHluorin (pHluorin on an L-type Ca²⁺ channel) and the HC-specific connexin-55.5 promoter to express AMPApHluorin (pHluorin on an AMPA receptor). Stimulus light led to increased fluorescence of all three probes, consistent with alkalinization of the synaptic cleft. The receptive field size, sensitivity to surround illumination, and response to activation of an alien receptor expressed exclusively in HCs, are consistent with lateral inhibition as the trigger for alkalinization. However, SynaptopHluorin and AMPApHluorin, which are displaced farther from cone synaptic ribbons than CalipHluorin, reported a smaller pH change. Hence, unlike feedforward glutamatergic transmission, which spills over to allow cross talk between terminals in the cone network, the pH change underlying HC feedback is compartmentalized to individual synaptic invaginations within a cone terminal, consistent with private line communication.SIGNIFICANCE STATEMENT Lateral inhibition (LI) is a fundamental feature of information processing in sensory systems, enhancing contrast sensitivity and enabling edge discrimination. Horizontal cells (HCs) are the first cellular substrate of LI in the vertebrate retina, but the synaptic mechanisms underlying LI are not completely understood, despite decades of study. This paper makes a significant contribution to our understanding of LI, by showing that each HC-cone synapse is a "private-line" that operates independently from other HC-cone connections. Using transgenic zebrafish expressing pHluorin, a pH-sensitive GFP variant spliced onto three different protein platforms expressed either in cones or HCs we show that the feedback pH signal is constrained to individual cone terminals, and more stringently, to individual synaptic contact sites within each terminal.