Controlling Horizontal Cell-Mediated Lateral Inhibition in Transgenic Zebrafish Retina with Chemogenetic Tools.

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

Localizing Proton-Mediated Inhibitory Feedback at the Retinal Horizontal Cell–Cone Synapse with Genetically-Encoded pH Probes

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

Synaptojanin 1 Is Required for Endolysosomal Trafficking of Synaptic Proteins in Cone Photoreceptor Inner Segments

<div><p>Highly polarized cells such as photoreceptors require precise and efficient strategies for establishing and maintaining the proper subcellular distribution of proteins. The signals and molecular machinery that regulate trafficking and sorting of synaptic proteins within cone inner segments is mostly unknown. In this study, we show that the polyphosphoinositide phosphatase Synaptojanin 1 (SynJ1) is critical for this process. We used transgenic markers for trafficking pathways, electron microscopy, and immunocytochemistry to characterize trafficking defects in cones of the zebrafish mutant, <i>nrc^a14</i>, which is deficient in phosphoinositide phosphatase, SynJ1. The outer segments and connecting cilia of <i>nrc^a14</i> cone photoreceptors are normal, but RibeyeB and VAMP2/synaptobrevin, which normally localize to the synapse, accumulate in the <i>nrc^a14</i> inner segment. The structure of the Endoplasmic Reticulum in <i>nrc^a14</i> mutant cones is normal. Golgi develop normally, but later become disordered. Large vesicular structures accumulate within <i>nrc^a14</i> cone photoreceptor inner segments, particularly after prolonged incubation in darkness. Cone inner segments of <i>nrc^a14</i> mutants also have enlarged acidic vesicles, abnormal late endosomes, and a disruption in autophagy. This last pathway also appears exacerbated by darkness. Taken altogether, these findings show that SynJ1 is required in cones for normal endolysosomal trafficking of synaptic proteins.</p></div

Air pollution exposure may impact the composition of human milk oligosaccharides

Abstract Human milk oligosaccharides (HMOs) impact neonate immunity and health outcomes. However, the environmental factors influencing HMO composition remain understudied. This study examined the associations between ambient air pollutant (AAP) exposure and HMOs at 1-month postpartum. Human milk samples were collected at 1-month postpartum (n = 185). AAP (PM2.5, PM10, NO2) exposure included the 9-month pregnancy period through 1-month postpartum. Associations between AAP with (1) HMO diversity, (2) the sum of sialylated and fucosylated HMOs, (3) 6 a priori HMOs linked with infant health, and (4) all HMOs were examined using multivariable linear regression and principal component analysis (PCA). Exposure to AAP was associated with lower HMO diversity. PM2.5 and PM10 exposure was positively associated with the HMO 3-fucosyllactose (3FL); PM2.5 exposure was positively associated with the sum of total HMOs, sum of fucosylated HMOs, and the HMO 2′-fucosyllactose (2′FL). PCA indicated the PM2.5, PM10, and NO2 exposures were associated with HMO profiles. Individual models indicated that AAP exposure was associated with five additional HMOs (LNFP I, LNFP II, DFLNT, LNH). This is the first study to demonstrate associations between AAP and breast milk HMOs. Future longitudinal studies will help determine the long-term impact of AAP on human milk composition

Dark adaptation affects autophagosomes in <i>nrc^a14</i> cone photoreceptors.

<p>Confocal images of cone photoreceptors in light (A, B) or dark-adapted (C, D) WT and <i>nrc^a14 Tg(TαCP:GFP-LC3)</i> retinas. At 4 dpf, larvae were phenotyped by OKR and placed at 28°C either on a normal light/dark cycle, or in complete darkness. 24 hours later, at 5 dpf, larvae were fixed and retinal slices were generated. After dark incubation, the GFP-LC3 positive puncta in <i>nrc^a14</i> cone photoreceptors appeared enlarged (D, arrowhead). Dark incubation did not significantly affect the number of GFP-LC3 puncta in WT or <i>nrc^a14</i> cone photoreceptors (E). Syn = photoreceptor synapses, IS = inner segment. Scale bar = 2 µm in all images. Graph shows average LC3 puncta in the IS, synapse or entire cell (Total) per cell, error bars are STDEV, from two independent light/dark experiments. For each experiment, 3–5 larvae were used per condition, and 60–150 cells were analyzed per larvae.</p

Some synaptic proteins are mislocalized in <i>nrc^a14</i> cone photoreceptors.

<p><i>Tg(TαCP:spH)</i> WT and <i>nrc^a14</i> 5 dpf retinal slices were stained with an antibody against the ribbon synapse protein RibeyeB (A–F). In WT photoreceptors, RibeyeB was found only at synaptic terminals (A). In <i>nrc^a14</i> cone photoreceptors, RibeyeB (D, arrowhead) and VAMP2 (E, arrowhead) were detected in both synaptic terminals and ISs. Mislocalized signals for RibeyeB (magenta) and VAMP2 (green) were coincident in the ISs of <i>nrc^a14</i> cone photoreceptors (F, arrowhead). In contrast, the synaptic vesicle protein Synaptophysin (Syp-CFP) had a primarily synaptic distribution in both WT (G) and <i>nrc^a14</i> (H) cone photoreceptors. Live confocal images were taken of 5 dpf <i>Tg(TαCP:Syp-CFP)</i> WT and <i>nrc^a14</i> larvae. Syn = photoreceptor synapses, IS = inner segment. Scale bar = 2 µm.</p

<i>nrc^a14</i> cone photoreceptors have normal outer segments and connecting cilia.

<p>(A–B) TEM images of 5 dpf WT (A) and <i>nrc^a14</i> (B) cone photoreceptor OSs. There was no difference in OS appearance or length (G) between <i>nrc^a14</i> and WT cone photoreceptors at 5 dpf (p = 0.7). (C–F) Confocal images of 5 dpf zebrafish larval retinas immunostained using antibodies against the CC proteins acetylated tubulin (C, D) or IFT88 (E, F). The number (I) and length (H) of acetylated tubulin stained CC was not significantly different between WT and <i>nrc^a14</i> photoreceptors (p = 0.9 and 0.4 respectively). There was no significant difference (p = 0.7) in the number of IFT88 stained cilia between WT and <i>nrc^a14</i> (J). Antibody staining is shown in yellow; nuclei were stained with Sytox Green (D, E) or Hoechst (G, H) and are shown in blue. Syn = photoreceptor synapses, IS = inner segment. Scale bar = 1 µm in A and B and 2 µm in C–F. Graphs show mean values, error bars are STDEV for multiple larvae.</p

Figure 4. Dark adaptation increases the number of vesicular structures in <i>nrc^a14</i> photoreceptor inner segments.

<p>TEM images of cone photoreceptors in light (A, B) or dark-adapted (C, D) WT and <i>nrc^a14</i> retinas. At 4 dpf, larvae were phenotyped by OKR and placed at 28°C either on a normal light/dark cycle, or in complete darkness. 24 hours later, at 5 dpf, larvae were fixed for TEM. Dark adaptation exaggerated the vesicular structure phenotype in <i>nrc^a14</i> cone photoreceptor inner segments (B vs. D). Cells were scored as “normal”, “vesiculated” or “severely vesiculated” and the quantification is shown in E. Cells with at least 3 vesicular structures >125 nm were scored as “vesiculated” and examples are shown by arrow heads in B and D, cells that contained at least 20 vesicular structures were scored as “severely vesiculated” and an example is shown by an arrow in D. OS = outer segment, N = nuclei. Scale bar = 2 µm in A–D. Graph shows mean, error bars are STDEV for three independent light/dark experiments. In total at least 400–500 cells from 5 larvae were counted per light condition and genotype.</p

Loss of SynJ1 disrupts endolysosomal structures.

<p>Confocal images of larvae incubated with Lysotracker Red and the transgenic fish lines <i>Tg(TαCP:GFP-Rab7)</i> and <i>Tg(TαCP:GFP-LC3)</i> mark the endolysosomal system. 5 dpf larvae were incubated in Lysotracker and imaged live. In WT retinas (A), Lysotracker Red accumulated primarily in the synapse and in small punctate structures in the IS of cone photoreceptors. In <i>nrc^a14</i> cone photoreceptors (B), Lysotracker Red accumulated primarily in larger, abnormal structures in the IS. Retinal slices from WT (C) and <i>nrc^a14</i> (D) larvae show that abnormal Rab7-positive structures including large perinuclear structures (arrowheads), and doughnut shaped structures (arrows) accumulated in the IS and synapse in the absence of SynJ1. The lack of SynJ1 also caused an increase in the number of LC3-GFP-positive structures, indicating a disruption in autophagy in <i>nrc^a14</i> cones (F). The number of LC3 puncta is increased in both the IS and synapse of <i>nrc^a14</i> cones (G). Syn = photoreceptor synapses, IS = inner segment. Scale bar = 2 µm in all images. Graph shows average LC3 puncta in the IS, synapse or entire cell (Total) per cell, error bars are STDEV for 11 WT larvae and 12 <i>nrc^a14</i> larvae. The number of LC3 puncta per cell for each subcellular compartment or the entire cell was significantly different between WT and <i>nrc^a14</i> larvae (p-value<0.001, donated by ***).</p

Large vesicular structures accumulate in <i>nrc^a14</i> photoreceptor inner segments.

<p>TEM images of cone photoreceptors in WT (A, B) and <i>nrc^a14</i> (C–E) 5 dpf retinas. Large vesicular structures were visible in approximately 80% of <i>nrc^a14</i> cone photoreceptor ISs. Some vesicular structures contained electron dense material (arrow heads, E). Boxes in A and C show areas enlarged in B and D respectively. Box in D shows area enlarged in E. OS = outer segment, N = nuclei. Scale bar = 2 µm in A–D, 1 µm in E.</p