Adhesion GPCR Latrophilin 3 regulates synaptic function of cone photoreceptors in a trans-synaptic manner

Cone photoreceptors mediate daylight vision in vertebrates. Changes in neurotransmitter release at cone synapses encode visual information and is subject to precise control by negative feedback from enigmatic horizontal cells. However, the mechanisms that orchestrate this modulation are poorly understood due to a virtually unknown landscape of molecular players. Here, we report a molecular player operating selectively at cone synapses that modulates effects of horizontal cells on synaptic release. Using an unbiased proteomic screen, we identified an adhesion GPCR Latrophilin3 (LPHN3) in horizontal cell dendrites that engages in transsynaptic control of cones. We detected and characterized a prominent splice isoform of LPHN3 that excludes a element with inhibitory influence on transsynaptic interactions. A gain-of-function mouse model specifically routing LPHN3 splicing to this isoform but not knockout of LPHN3 diminished Ca_V1.4 calcium channel activity profoundly disrupted synaptic release by cones and resulted in synaptic transmission deficits. These findings offer molecular insight into horizontal cell modulation on cone synaptic function and more broadly demonstrate the importance of alternative splicing in adhesion GPCRs for their physiological function.

Vision is a key sensory modality essential for the survival of most living organisms. In mammals, it is enabled by the retina: a neural structure composed of more than 60 distinct neurons each uniquely wired into the circuitry and with particular roles in image processing (1, 2). Vision begins with the detection of light by rod and cone photoreceptors. Rod photoreceptor cells are exquisitely sensitive to light and mediate vision at low light levels (3, 4). However, most vertebrates including humans rely on cone cells for daytime vision (5). Accordingly, cones have an extremely broad range of light sensitivity spanning 6 to 7 orders of magnitude (6), quickly adapting to changes in luminance and providing high spatial and temporal visual acuity (5, 7, 8). The molecular, cellular, and circuit mechanisms that allow cones to perform their tasks has been a subject of intense interest, providing groundbreaking discoveries that illuminate fundamental organizational principles that govern signal processing by neural circuits in general.The capture and processing of photons by the phototransduction cascade of cones generates graded changes in membrane potential: hyperpolarizing to light and depolarizing with darkness (7). These voltage signals alter the ongoing rate of neurotransmitter glutamate release at the cone synapse to relay information about light and dark to the retinal circuitry (9). The molecular entity that mediates this transformation is the L-type voltage-gated Ca²⁺ channel, Ca_V1.4 (10–12). It is located at specialized active zones containing synaptic ribbons and couples light-driven changes in voltage to changes in local Ca²⁺ levels thereby regulating the vesicular fusion machinery (13, 14). The Ca_V1.4 channel forms a macromolecular complex with a number of synaptic molecules and thus plays a pivotal role in both the structural and functional organization of the presynaptic active zone of photoreceptors (15). Accordingly, changes in Ca_V1.4 function imposed by binding partners or environment have a tremendous impact on the synaptic communication of cone photoreceptors and vision (16–18).Cones form synaptic contacts with three types of neurons. They synapse with postsynaptic ON- and OFF-type bipolar cells (BC) to relay visual information to the downstream neuronal circuitry (19, 20). Cones also contact lateral inhibitory neurons known as horizontal cells (HCs) that connect adjacent to BC dendrites, forming a tripartite synaptic triad (20). This elaborate synaptic arrangement of cones is a site of major influence on how visual information is processed contributing to unique cone physiology and adaptive capacity for daylight detection (21, 22).The function of HCs and their physiological mechanisms are particularly intriguing. HCs powerfully modulate synaptic transmission at cone synapses (23). Light-evoked hyperpolarization of HCs counteracts light-induced suppression of glutamate release from cone terminals, thereby providing strong negative feedback (23, 24). Because each HC contacts multiple cones, this negative influence on surrounding cones is a major mechanism for producing lateral inhibition, a classical feature of signal processing in the retina that enhances contrast and spatial resolution of vision (25). In addition, feedback from individual HC dendrites to specific cone terminals and ribbons can also act locally, fine-tuning synaptic output to local illumination gradients (26–29).While the role of HCs from a circuit perspective is well understood, the mechanisms that they use to provide negative feedback are subject to debate and controversy. At least three different explanations have been provided: direct ephaptic effects (30, 31), changes in synaptic pH (28, 32, 33), and modulation by GABA released from HCs (34, 35). These models are not necessarily mutually exclusive and unifying theories have been proposed (35, 36). Importantly, one of the central effects invariably observed in response to HC feedback is modulation of the Ca_V1.4 function at the active zones of cone terminals (32, 37). However, there is a significant void in our understanding of molecular mechanisms by which HCs modulate transmission of cone signals, mostly due to a paucity of players known to operate at this synapse. Identification and functional characterization of molecular elements involved in coordinating HC influence in cone synapses can transform our understanding of this enigmatic area of visual neuroscience.Here, we performed an unbiased proteomic profiling of proteins selectively enriched in cone synapses. This led to identification of an adhesion G protein–coupled receptor (aGPCR), latrophilin3 (LPHN3), whose role in retina physiology, photoreceptor synaptic development, and function was previously unexplored. We show that alternative splicing of LPHN3 in the retina generates unique isoforms with distinct properties. Using mouse models, we demonstrate that changes in LPHN3 splicing regulate cone synaptic transmission transsynaptically by affecting Ca_V1.4 function. These findings reveal a molecular player with a pivotal role in regulating synaptic function of cone photoreceptors.