Synaptotagmin-9 in mouse retina.

Synaptotagmin-9 (Syt9) is a Ca2+ sensor mediating fast synaptic release expressed in various parts of the brain. The presence and role of Syt9 in retina is unknown. We found evidence for Syt9 expression throughout the retina and created mice to conditionally eliminate Syt9 in a cre-dependent manner. We crossed Syt9fl/fl mice with Rho-iCre, HRGP-Cre, and CMV-Cre mice to generate mice in which Syt9 was eliminated from rods (rodSyt9CKO), cones (coneSyt9CKO), or whole animals (CMVSyt9). CMVSyt9 mice showed an increase in scotopic electroretinogram (ERG) b-waves evoked by bright flashes with no change in a-waves. Cone-driven photopic ERG b-waves were not significantly different in CMVSyt9 knockout mice and selective elimination of Syt9 from cones had no effect on ERGs. However, selective elimination from rods decreased scotopic and photopic b-waves as well as oscillatory potentials. These changes occurred only with bright flashes where cone responses contribute. Synaptic release was measured in individual rods by recording anion currents activated by glutamate binding to presynaptic glutamate transporters. Loss of Syt9 from rods had no effect on spontaneous or depolarization-evoked release. Our data show that Syt9 acts at multiple sites in the retina and suggest that it may play a role in regulating transmission of cone signals by rods.

Presynaptic Proteins and Their Roles in Visual Processing by the Retina.

The sense of vision begins in the retina, where light is detected and processed through a complex series of synaptic connections into meaningful information relayed to the brain via retinal ganglion cells. Light responses begin as tonic and graded signals in photoreceptors, later emerging from the retina as a series of spikes from ganglion cells. Processing by the retina extracts critical features of the visual world, including spatial frequency, temporal frequency, motion direction, color, contrast, and luminance. To achieve this, the retina has evolved specialized and unique synapse types. These include the ribbon synapses of photoreceptors and bipolar cells, the dendritic synapses of amacrine and horizontal cells, and unconventional synaptic feedback from horizontal cells to photoreceptors. We review these unique synapses in the retina with a focus on the presynaptic molecules and physiological properties that shape their capabilities.

SYNAPTOTAGMIN-9 IN MOUSE RETINA.

Synaptotagmin-9 (Syt9) is a Ca2+ sensor mediating fast synaptic release expressed in various parts of the brain. The presence and role of Syt9 in retina is unknown. We found evidence for Syt9 expression throughout the retina and created mice to conditionally eliminate Syt9 in a cre-dependent manner. We crossed Syt9fl/fl mice with Rho-iCre, HRGP-Cre, and CMV-cre mice to generate mice in which Syt9 was eliminated from rods (rodSyt9CKO), cones (coneSyt9CKO), or whole animals (CMVSyt9). CMVSyt9 mice showed an increase in scotopic electroretinogram (ERG) b-waves evoked by bright flashes with no change in a-waves. Cone-driven photopic ERG b-waves were not significantly different in CMVSyt9 knockout mice and selective elimination of Syt9 from cones had no effect on ERGs. However, selective elimination from rods decreased scotopic and photopic b-waves as well as oscillatory potentials. These changes occurred only with bright flashes where cone responses contribute. Synaptic release was measured in individual rods by recording anion currents activated by glutamate binding to presynaptic glutamate transporters. Loss of Syt9 from rods had no effect on spontaneous or depolarization-evoked release. Our data show that Syt9 is acts at multiple sites in the retina and suggest that it may play a role in regulating transmission of cone signals by rods.

EAAT5 glutamate transporter rapidly binds glutamate with micromolar affinity in mouse rods.

Light responses of rod photoreceptor cells in the retina are encoded by changes in synaptic glutamate release that is in turn shaped by reuptake involving EAAT5 plasma membrane glutamate transporters. Heterologously expressed EAAT5 activates too slowly upon glutamate binding to support significant uptake. We tested EAAT5 activation in mouse rods in vivo by stimulating glutamate transporter anion currents (IA(glu)) with UV flash photolysis of MNI-glutamate, varying flash intensity to vary glutamate levels. Responses to uncaging rose rapidly with time constants of 2-3 ms, similar to IA(glu) events arising from spontaneous release. Spontaneous release events and IA(glu) evoked by weak flashes also declined with similar time constants of 40-50 ms. Stronger flashes evoked responses that decayed more slowly. Time constants were twofold faster at 35°C, suggesting that they reflect transporter kinetics, not diffusion. Selective EAAT1 and EAAT2 inhibitors had no significant effect, suggesting IA(glu) in rods arises solely from EAAT5. We calibrated glutamate levels attained during flash photolysis by expressing a fluorescent glutamate sensor iGluSnFr in cultured epithelial cells. We compared fluorescence at different glutamate concentrations to fluorescence evoked by photolytic uncaging of MNI-glutamate. The relationship between flash intensity and glutamate yielded EC50 values for EAAT5 amplitude, decay time, and rise time of ∼10 µM. Micromolar affinity and rapid activation of EAAT5 in rods show it can rapidly bind synaptic glutamate. However, we also found that EAAT5 currents are saturated by the synchronous release of only a few vesicles, suggesting limited capacity and a role for glial uptake at higher release rates.

Using optogenetics to dissect rod inputs to OFF ganglion cells in the mouse retina.

Introduction: Light responses of rod photoreceptor cells traverse the retina through three pathways. The primary pathway involves synapses from rods to ON-type rod bipolar cells with OFF signals reaching retinal ganglion cells (RGCs) via sign-inverting glycinergic synapses. Secondly, rod signals can enter cones through gap junctions. Finally, rods can synapse directly onto cone OFF bipolar cells. Methods: To analyze these pathways, we obtained whole cell recordings from OFF-type α RGCs in mouse retinas while expressing channelrhodopsin-2 in rods and/or cones. Results: Optogenetic stimulation of rods or cones evoked large fast currents in OFF RGCs. Blocking the primary rod pathway with L-AP4 and/or strychnine reduced rod-driven optogenetic currents in OFF RGCs by ~1/3. Blocking kainate receptors of OFF cone bipolar cells suppressed both rod- and cone-driven optogenetic currents in OFF RGCs. Inhibiting gap junctions between rods and cones with mecloflenamic acid or quinpirole reduced rod-driven responses in OFF RGCs. Eliminating the exocytotic Ca2+ sensor, synaptotagmin 1 (Syt1), from cones abolished cone-driven optogenetic responses in RGCs. Rod-driven currents were not significantly reduced after isolating the secondary pathway by eliminating Syt1 and synaptotagmin 7 (Syt7) to block synaptic release from rods. Eliminating Syt1 from both rods and cones abolished responses to optogenetic stimulation. In Cx36 KO retinas lacking rod-cone gap junctions, optogenetic activation of rods evoked small and slow responses in most OFF RGCs suggesting rod signals reached them through an indirect pathway. Two OFF cells showed faster responses consistent with more direct input from cone OFF bipolar cells. Discussion: These data show that the secondary rod pathway supports robust inputs into OFF α RGCs and suggests the tertiary pathway recruits both direct and indirect inputs.

EHD2 overexpression promotes tumorigenesis and metastasis in triple-negative breast cancer by regulating store-operated calcium entry.

With nearly all cancer deaths a result of metastasis, elucidating novel pro-metastatic cellular adaptations could provide new therapeutic targets. Here, we show that overexpression of the EPS15-Homology Domain-containing 2 (EHD2) protein in a large subset of breast cancers (BCs), especially the triple-negative (TNBC) and HER2+ subtypes, correlates with shorter patient survival. The mRNAs for EHD2 and Caveolin-1/2, structural components of caveolae, show co-overexpression across breast tumors, predicting shorter survival in basal-like BC. EHD2 shRNA knockdown and CRISPR-Cas9 knockout with mouse Ehd2 rescue, in TNBC cell line models demonstrate a major positive role of EHD2 in promoting tumorigenesis and metastasis. Mechanistically, we link these roles of EHD2 to store-operated calcium entry (SOCE), with EHD2-dependent stabilization of plasma membrane caveolae ensuring high cell surface expression of the SOCE-linked calcium channel Orai1. The novel EHD2-SOCE oncogenic axis represents a potential therapeutic target in EHD2- and CAV1/2-overexpressing BC.

Correction: Mesnard et al. Eliminating Synaptic Ribbons from Rods and Cones Halves the Releasable Vesicle Pool and Slows Down Replenishment. Int. J. Mol. Sci. 2022, 23, 6429

The authors wish to make the following corrections to this paper [...].

Eliminating Synaptic Ribbons from Rods and Cones Halves the Releasable Vesicle Pool and Slows Down Replenishment.

Glutamate release from rod and cone photoreceptor cells involves presynaptic ribbons composed largely of the protein RIBEYE. To examine roles of ribbons in rods and cones, we studied mice in which GCamP3 replaced the B-domain of RIBEYE. We discovered that ribbons were absent from rods and cones of both knock-in mice possessing GCamP3 and conditional RIBEYE knockout mice. The mice lacking ribbons showed reduced temporal resolution and contrast sensitivity assessed with optomotor reflexes. ERG recordings showed 50% reduction in scotopic and photopic b-waves. The readily releasable pool (RRP) of vesicles in rods and cones measured using glutamate transporter anion currents (IA(glu)) was also halved. We also studied the release from cones by stimulating them optogenetically with ChannelRhodopsin2 (ChR2) while recording postsynaptic currents in horizontal cells. Recovery of the release from paired pulse depression was twofold slower in the rods and cones lacking ribbons. The release from rods at -40 mV in darkness involves regularly spaced multivesicular fusion events. While the regular pattern of release remained in the rods lacking ribbons, the number of vesicles comprising each multivesicular event was halved. Our results support conclusions that synaptic ribbons in rods and cones expand the RRP, speed up vesicle replenishment, and augment some forms of multivesicular release. Slower replenishment and a smaller RRP in photoreceptors lacking ribbons may contribute to diminished temporal frequency responses and weaker contrast sensitivity.

Exploring the Vitreoretinal Interface: A Key Instigator of Unique Retinal Hemorrhage Patterns in Pediatric Head Trauma.

PURPOSE: Various types of trauma can cause retinal hemorrhages in children, including accidental and nonaccidental head trauma. We used animal eyes and a finite element model of the eye to examine stress patterns produced during purely linear and angular accelerations, along with stresses attained during simulated repetitive shaking of an infant. METHODS: Using sheep and primate eyes, sclerotomy windows were created by removing the sclera, choroid, and retinal pigment epithelium to expose the retina. A nanofiber square was glued to a 5 mm2 area of retina. The square was pulled and separated from vitreous while force was measured. A finite element model of the pediatric eye was used to computationally measure tension stresses during shaking. RESULTS: In both sheep and primate eyes, tension stress required for separation of retina from vitreous range from 1 to 5 kPa. Tension stress generated at the vitreoretinal interface predicted by the computer simulation ranged from 3 to 16 kPa during a cycle of shaking. Linear acceleration generated lower tension stress than angular acceleration. Angular acceleration generated maximal tension stress along the retinal vasculature. Linear acceleration produced more diffuse force distribution centered at the poster pole. CONCLUSIONS: The finite element model predicted that tension stress attained at the retina during forcible shaking of an eye can exceed the minimum threshold needed to produce vitreoretinal separation as measured in animal eyes. Furthermore, the results show that movements that involve significant angular acceleration produce strong stresses localized along the vasculature, whereas linear acceleration produces weaker, more diffuse stress centered towards the posterior pole of the eye.

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 CaV1.4 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.

Photoreceptor Cell Calcium Dysregulation and Calpain Activation Promote Pathogenic Photoreceptor Oxidative Stress and Inflammation in Prodromal Diabetic Retinopathy.

This study tested the hypothesis that diabetes promotes a greater than normal cytosolic calcium level in rod cells that activates a Ca2+-sensitive protease, calpain, resulting in oxidative stress and inflammation, two pathogenic factors of early diabetic retinopathy. Nondiabetic and 2-month diabetic C57Bl/6J and calpain1 knockout (Capn1-/-) mice were studied; subgroups were treated with a calpain inhibitor (CI). Ca2+ content was measured in photoreceptors using Fura-2. Retinal calpain expression was studied by quantitative RT-PCR and immunohistochemistry. Superoxide and expression of inflammatory proteins were measured using published methods. Proteomic analysis was conducted on photoreceptors isolated from untreated diabetic mice or treated daily with CI for 2 months. Cytosolic Ca2+ content was increased twofold in photoreceptors of diabetic mice as compared with nondiabetic mice. Capn1 expression increased fivefold in photoreceptor outer segments of diabetic mice. Pharmacologic inhibition or genetic deletion of Capn1 significantly suppressed diabetes-induced oxidative stress and expression of proinflammatory proteins in retina. Proteomics identified a protein (WW domain-containing oxidoreductase [WWOX]) whose expression was significantly increased in photoreceptors from mice diabetic for 2 months and was inhibited with CI. Knockdown of Wwox using specific siRNA in vitro inhibited increase in superoxide caused by the high glucose. These results suggest that reducing Ca2+ accumulation, suppressing calpain activation, and/or reducing Wwox up-regulation are novel targets for treating early diabetic retinopathy.

Transmission at rod and cone ribbon synapses in the retina.

Light-evoked voltage responses of rod and cone photoreceptor cells in the vertebrate retina must be converted to a train of synaptic vesicle release events for transmission to downstream neurons. This review discusses the processes, proteins, and structures that shape this critical early step in vision, focusing on studies from salamander retina with comparisons to other experimental animals. Many mechanisms are conserved across species. In cones, glutamate release is confined to ribbon release sites although rods are also capable of release at non-ribbon sites. The role of non-ribbon release in rods remains unclear. Release from synaptic ribbons in rods and cones involves at least three vesicle pools: a readily releasable pool (RRP) matching the number of membrane-associated vesicles along the ribbon base, a ribbon reserve pool matching the number of additional vesicles on the ribbon, and an enormous cytoplasmic reserve. Vesicle release increases in parallel with Ca2+ channel activity. While the opening of only a few Ca2+ channels beneath each ribbon can trigger fusion of a single vesicle, sustained release rates in darkness are governed by the rate at which the RRP can be replenished. The number of vacant release sites, their functional status, and the rate of vesicle delivery in turn govern replenishment. Along with an overview of the mechanisms of exocytosis and endocytosis, we consider specific properties of ribbon-associated proteins and pose a number of remaining questions about this first synapse in the visual system.

Properties of multivesicular release from mouse rod photoreceptors support transmission of single-photon responses.

Vision under starlight requires rod photoreceptors to transduce and transmit single-photon responses to the visual system. Small single-photon voltage changes must therefore cause detectable reductions in glutamate release. We found that rods achieve this by employing mechanisms that enhance release regularity and its sensitivity to small voltage changes. At the resting membrane potential in darkness, mouse rods exhibit coordinated and regularly timed multivesicular release events, each consisting of ~17 vesicles and occurring two to three times more regularly than predicted by Poisson statistics. Hyperpolarizing rods to mimic the voltage change produced by a single photon abruptly reduced the probability of multivesicular release nearly to zero with a rebound increase at stimulus offset. Simulations of these release dynamics indicate that this regularly timed, multivesicular release promotes transmission of single-photon responses to post-synaptic rod-bipolar cells. Furthermore, the mechanism is efficient, requiring lower overall release rates than uniquantal release governed by Poisson statistics.

Determining the Tractional Forces on Vitreoretinal Interface Using a Computer Simulation Model in Abusive Head Trauma.

PURPOSE: Abusive head trauma (AHT) is the leading cause of infant death and long-term morbidity from injury. The ocular consequences of AHT are controversial, and the pathophysiology of retinal research findings is still not clearly understood. It has been postulated that vitreoretinal traction plays a major role in the retinal findings. A computer simulation model was developed to evaluate the vitreoretinal traction and determine whether the distribution of forces in different layers and locations of the retina can explain the patterns of retinal hemorrhage (RH) seen in AHT. DESIGN: Computer simulation model study. METHODS: A computer simulation model of the pediatric eye was developed to evaluate preretinal, intraretinal, and subretinal stresses during repetitive shaking. This model was also used to examine the forces applied to various segments along blood vessels. RESULTS: Calculated stress values from the computer simulation ranged from 3-16 kPa at the vitreoretinal interface through a cycle of shaking. Maximal stress was observed at the periphery of the retina, corresponding to areas of multiple vessel bifurcations, followed by the posterior pole of the retina. Stress values were similar throughout all 3 layers of the retina (preretinal, intraretinal, and subretinal layers). CONCLUSIONS: Ocular manifestations from AHT revealed unique retinal characteristics. The model predicted stress patterns consistent with the diffuse retinal hemorrhages (RH) typically found in the posterior pole and around the peripheral retina in AHT. This computer model demonstrated that similar stress forces were produced in different layers of the retina, consistent with the finding that retinal hemorrhages are often found in multiple layers of the retina. These data can help explain the RH patterns commonly found in AHT.

Nr2e3 is a genetic modifier that rescues retinal degeneration and promotes homeostasis in multiple models of retinitis pigmentosa.

Recent advances in viral vector engineering, as well as an increased understanding of the cellular and molecular mechanism of retinal diseases, have led to the development of novel gene therapy approaches. Furthermore, ease of accessibility and ocular immune privilege makes the retina an ideal target for gene therapies. In this study, the nuclear hormone receptor gene Nr2e3 was evaluated for efficacy as broad-spectrum therapy to attenuate early to intermediate stages of retinal degeneration in five unique mouse models of retinitis pigmentosa (RP). RP is a group of heterogenic inherited retinal diseases associated with over 150 gene mutations, affecting over 1.5 million individuals worldwide. RP varies in age of onset, severity, and rate of progression. In addition, ~40% of RP patients cannot be genetically diagnosed, confounding the ability to develop personalized RP therapies. Remarkably, Nr2e3 administered therapy resulted in reduced retinal degeneration as observed by increase in photoreceptor cells, improved electroretinogram, and a dramatic molecular reset of key transcription factors and associated gene networks. These therapeutic effects improved retinal homeostasis in diseased tissue. Results of this study provide evidence that Nr2e3 can serve as a broad-spectrum therapy to treat multiple forms of RP.

Resting and stimulated mouse rod photoreceptors show distinct patterns of vesicle release at ribbon synapses.

The vertebrate visual system can detect and transmit signals from single photons. To understand how single-photon responses are transmitted, we characterized voltage-dependent properties of glutamate release in mouse rods. We measured presynaptic glutamate transporter anion current and found that rates of synaptic vesicle release increased with voltage-dependent Ca2+ current. Ca2+ influx and release rate also rose with temperature, attaining a rate of ∼11 vesicles/s/ribbon at -40 mV (35°C). By contrast, spontaneous release events at hyperpolarized potentials (-60 to -70 mV) were univesicular and occurred at random intervals. However, when rods were voltage clamped at -40 mV for many seconds to simulate maintained darkness, release occurred in coordinated bursts of 17 ± 7 quanta (mean ± SD; n = 22). Like fast release evoked by brief depolarizing stimuli, these bursts involved vesicles in the readily releasable pool of vesicles and were triggered by the opening of nearby ribbon-associated Ca2+ channels. Spontaneous release rates were elevated and bursts were absent after genetic elimination of the Ca2+ sensor synaptotagmin 1 (Syt1). This study shows that at the resting potential in darkness, rods release glutamate-filled vesicles from a pool at the base of synaptic ribbons at low rates but in Syt1-dependent bursts. The absence of bursting in cones suggests that this behavior may have a role in transmitting scotopic responses.

Simultaneous Release of Multiple Vesicles from Rods Involves Synaptic Ribbons and Syntaxin 3B.

First proposed as a specialized mode of release at sensory neurons possessing ribbon synapses, multivesicular release has since been described throughout the central nervous system. Many aspects of multivesicular release remain poorly understood. We explored mechanisms underlying simultaneous multivesicular release at ribbon synapses in salamander retinal rod photoreceptors. We assessed spontaneous release presynaptically by recording glutamate transporter anion currents (IA(glu)) in rods. Spontaneous IA(glu) events were correlated in amplitude and kinetics with simultaneously measured miniature excitatory postsynaptic currents in horizontal cells. Both measures indicated that a significant fraction of events is multiquantal, with an analysis of IA(glu) revealing that multivesicular release constitutes ∼30% of spontaneous release events. IA(glu) charge transfer increased linearly with event amplitude showing that larger events involve greater glutamate release. The kinetics of large and small IA(glu) events were identical as were rise times of large and small miniature excitatory postsynaptic currents, indicating that the release of multiple vesicles during large events is highly synchronized. Effects of exogenous Ca2+ buffers suggested that multiquantal, but not uniquantal, release occurs preferentially near Ca2+ channels clustered beneath synaptic ribbons. Photoinactivation of ribbons reduced the frequency of spontaneous multiquantal events without affecting uniquantal release frequency, showing that spontaneous multiquantal release requires functional ribbons. Although both occur at ribbon-style active zones, the absence of cross-depletion indicates that evoked and spontaneous multiquantal release from ribbons involve different vesicle pools. Introducing an inhibitory peptide into rods to interfere with the SNARE protein, syntaxin 3B, selectively reduced multiquantal event frequency. These results support the hypothesis that simultaneous multiquantal release from rods arises from homotypic fusion among neighboring vesicles on ribbons and involves syntaxin 3B.

Contributions of glutamate transporters and Ca2+-activated Cl- currents to feedback from horizontal cells to cone photoreceptors.

Lateral inhibitory feedback from horizontal cells (HCs) to cones establishes center-surround receptive fields and color opponency in the retina. When HCs hyperpolarize to light, inhibitory feedback to cones increases activation of cone Ca2+ currents (ICa) that can in turn activate additional currents. We recorded simultaneously from cones and HCs to analyze cone currents activated by HC feedback in salamander retina. Depolarization-activated inward tail currents in cones were inhibited by CaCCinh-A01 that inhibits both Ano1 and Ano2 Ca2+-activated Cl- currents (ICl(Ca)). An Ano1-selective inhibitor Ani9 was less effective suggesting that Ano2 is the predominant ICl(Ca) subtype in cones. CaCCinh-A01 inhibited feedback currents more strongly when intracellular Ca2+ in cones was buffered with 0.05 mM EGTA compared to stronger buffering with 5 mM EGTA. By contrast, blocking glutamate transporter anion currents (ICl(Glu)) with TBOA had stronger inhibitory effects on cone feedback currents when Ca2+ buffering was strong. Inward feedback currents ran down at rates intermediate between rundown of glutamate release and ICl(Ca), consistent with contributions to feedback from both ICl(Ca) and ICl(Glu). These results suggest that Cl- channels coupled to glutamate transporters help to speed inward feedback currents initiated by local changes in intracellular [Ca2+] close to synaptic ribbons of cones whereas Ano2 Ca2+-activated Cl- channels contribute to slower components of feedback regulated by spatially extensive changes in intracellular [Ca2+].

Ca2+ sensor synaptotagmin-1 mediates exocytosis in mammalian photoreceptors.

To encode light-dependent changes in membrane potential, rod and cone photoreceptors utilize synaptic ribbons to sustain continuous exocytosis while making rapid, fine adjustments to release rate. Release kinetics are shaped by vesicle delivery down ribbons and by properties of exocytotic Ca2+ sensors. We tested the role for synaptotagmin-1 (Syt1) in photoreceptor exocytosis by using novel mouse lines in which Syt1 was conditionally removed from rods or cones. Photoreceptors lacking Syt1 exhibited marked reductions in exocytosis as measured by electroretinography and single-cell recordings. Syt1 mediated all evoked release in cones, whereas rods appeared capable of some slow Syt1-independent release. Spontaneous release frequency was unchanged in cones but increased in rods lacking Syt1. Loss of Syt1 did not alter synaptic anatomy or reduce Ca2+ currents. These results suggest that Syt1 mediates both phasic and tonic release at photoreceptor synapses, revealing unexpected flexibility in the ability of Syt1 to regulate Ca2+-dependent synaptic transmission.