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.

Retrospective analysis of a STEM outreach event reveals positive influences on student attitudes toward STEM careers but not scientific methodology.

Substantial, involved, and expensive efforts to promote the dissemination of scientific knowledge and career interest in Science, Technology, Engineering, and Mathematics (STEM) are enthusiastically supported by many scientific, federal, and local organizations. The articulated underlying goals for these efforts include an enhanced public understanding of science and science-related policy, an increased diversity in STEM careers, and an increase in the future STEM workforce. This effort is primarily driven by an underperformance of the United States that includes poor test performance and limited number of students pursuing STEM degrees. Despite this investment, attitudes toward STEM have not notably changed. The goal of this project was to determine students' attitudes toward STEM in response to a previously established scientific outreach event. This event was used to address three common goals in STEM outreach: STEM literacy, diversity and inclusion, and career preparedness. We found there was a notable difference in the attitudes toward scientific activities and interest in pursuing a "Science Career" after participation in this event. Strikingly, interest in hypothesis development, the keystone of all STEM disciplines, was the least liked of all the activities offered during the event. Our data suggest that events designed to enhance interest in pursuing a STEM career may benefit from different elements compared with events designed to increase understanding of STEM literacy concepts, such as hypothesis development.

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.

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.

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.

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.

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.

Consequences of Puberty on Efficacy of Intraocular Pressure-Lowering Drugs in Male Dutch-Belted Rabbits.

PURPOSE: To investigate the changes in intraocular pressure (IOP), aqueous flow, and outflow facility, as well as efficacy of IOP-lowering drugs before and after sexual development in rabbits. METHODS: Male Dutch-belted rabbits were studied at night between the ages of 8 and 44 weeks. During these times, body weight, testicular volume, and serum testosterone were measured to monitor sexual maturity. Ocular measurements included anterior chamber depth, central corneal thickness, IOP, aqueous flow, and outflow facility. Systemic acetazolamide or topical timolol, latanoprost, or saline were administered pre- and postpuberty to assess drug effects on these parameters. RESULTS: Body weight, testicular volume, and serum testosterone increased until 28 weeks of age. IOP increased during prepuberty (R2 = 0.49, P = 0.003), dropped significantly during puberty, rising again immediate postpuberty, and changing little thereafter. Postpuberty compared with prepuberty found higher IOP (P < 0.0001), slower aqueous flow (P = 0.008), lower outflow facility (not statistically significant, P = 0.07), increased central cornea thickness, and increased anterior chamber volume. Timolol lowered IOP both pre- and postpuberty, whereas, latanoprost and acetazolamide decreased IOP postpuberty only. CONCLUSIONS: As male rabbits mature, the cornea thickens and the anterior chamber volume increases. At the same time, aqueous flow slows, yet, IOP increases. This suggests that decreased outflow facility and/or increased episcleral venous pressure might contribute to the puberty-related changes in IOP. Underdevelopment of tissues of the outflow pathways may contribute to the differences in drug efficacy in rabbits when young compared with after sexual maturity.

Aqueous Flow Measured by Fluorophotometry in the Mouse.

PURPOSE: A fluorophotometer designed to measure aqueous flow in murine eyes was tested with artificial fluorescein chambers and in live mice with different anesthesia regimens, aqueous flow suppressants, and an anterior chamber cannulation method. METHODS: Two hours following topical fluorescein application, one group of CD-1 mice was anesthetized with ketamine/xylazine, 2,2,2-tribromoethanol, or ketamine alone. Cornea and anterior chamber fluorescein concentrations were measured periodically for 60 to 90 minutes by fluorophotometric scans to calculate aqueous flow. Later, a subgroup of mice underwent aqueous flow measurement by anterior chamber cannulation. A third group was treated with timolol, dorzolamide, and vehicle in a crossover manner 1 hour prior to fluorophotometric scans. RESULTS: Aqueous flow with ketamine/xylazine anesthesia (0.09 ± 0.05 µL/min, mean ± SD, n = 24) was slower than with tribromoethanol or ketamine alone (P < 0.001). Timolol reduced aqueous flow from 0.20 ± 0.07 µL/min to 0.07 ± 0.03 µL/min (P = 0.001) under tribromoethanol anesthesia and from 0.14 ± 0.03 µL/min to 0.10 ± 0.02 µL/min (P = 0.004) under ketamine anesthesia but not under ketamine/xylazine anesthesia. Dorzolamide reduced aqueous flow from 0.09 ± 0.03 to 0.06 ± 0.03 µL/min (P = 0.04) under ketamine/xylazine anesthesia. Aqueous flow by anterior chamber cannulation (0.20 ± 0.13 µL/min) was greater (P = 0.05) than by fluorophotometry (0.09 ± 0.07 µL/min). CONCLUSIONS: A new noninvasive fluorophotometric method detected effects of general anesthesia and known aqueous suppressants on aqueous flow in mice. Aqueous flow measured by fluorophotometry was slower than by cannulation, and was technically easier with less variability. The mouse fluorophotometer is useful for repeated measurements of aqueous flow in the murine eye making crossover and longitudinal studies possible.

Improvement in outflow facility by two novel microinvasive glaucoma surgery implants.

PURPOSE: To determine improvement in outflow facility (C) in human anterior segments implanted with a novel Schlemm's canal scaffold or two trabecular micro-bypasses. METHODS: Human anterior segments were isolated from 12 pairs of eyes from donors with no history of ocular disease and then perfused at 50, 40, 30, 20, and 10 mm Hg pressures for 10 minutes each. Baseline C was calculated from perfusion pressures and flow rates. The scaffold was implanted into Schlemm's canal of one anterior segment, and two micro-bypasses were implanted three clock-hours apart in the contralateral anterior segment. Outflow facility and resistance were compared at various standardized perfusion pressures and between each device. RESULTS: Compared to baseline, C increased by 0.16 ± 0.12 µL/min/mm Hg (74%) with the scaffold, and 0.08 ± 0.12 µL/min/mm Hg (34%) with two micro-bypasses. The scaffold increased C at perfusion pressures of 50, 40, 30, and 20 mm Hg (P < 0.005). Two micro-bypasses increased C at a perfusion pressure of 40 mm Hg (P < 0.05). CONCLUSIONS: Both implants effectively increased C in human eyes ex vivo. The scaffold increased C by a greater percentage (73% vs. 34%) and at a greater range of perfusion pressures (20 to 50 mm Hg vs. 40 mm Hg) than the two micro-bypasses, suggesting that the 8-mm dilation of Schlemm's canal by the scaffold may have additional benefits in lowering the outflow resistance. The Hydrus Microstent scaffold may be an effective therapy for increasing outflow facility and thus reducing the IOP in patients with glaucoma.

A novel 8-mm Schlemm's canal scaffold reduces outflow resistance in a human anterior segment perfusion model.

PURPOSE: To study the effect on outflow facility and outflow resistance of a nitinol microstent implanted into Schlemm's canal. METHODS: Using a constant pressure perfusion method, outflow facility and outflow resistance were measured in 26 pairs of dissected anterior segments from donated human eyes. Measurements were made at perfusion pressures of 10, 20, 30 and 40 mm Hg. The Hydrus Microstent was placed in Schlemm's canal of one eye and the contralateral eye underwent a sham procedure. Outflow facility and outflow resistance were measured again after the microstent implantation or sham procedure. RESULTS: The Hydrus Microstent significantly increased outflow facility from 0.33 ± 0.17 µL/min/mm Hg to 0.52 ± 0.19 µL/min/mm Hg (P < 0.001). Outflow resistance was significantly reduced from 4.38 ± 3.03 mm Hg/µL/min at baseline to 2.34 ± 1.04 mm Hg/µL/min (P < 0.001) with the microstent. There was a linear correlation between outflow resistance at baseline and decrease in outflow resistance with the microstent (R(2) = 0.89, P < 0.0001). CONCLUSIONS: The increase in outflow facility and decrease in resistance supports the potential use of the Hydrus Microstent as a surgical option to reduce intraocular pressure (IOP). The IOP-lowering effect may be higher in eyes with higher outflow resistance (and IOP) as compared with eyes with lower outflow resistance (and IOP).