Rapid synaptic vesicle endocytosis in cone photoreceptors of salamander retina.

Following synaptic vesicle exocytosis, neurons retrieve the fused membrane by a process of endocytosis to provide a supply of vesicles for subsequent release and maintain the presynaptic active zone. Rod and cone photoreceptors use a specialized structure called the synaptic ribbon that enables them to sustain high rates of neurotransmitter release. They must also employ mechanisms of synaptic vesicle endocytosis capable of keeping up with release. While much is known about endocytosis at another retinal ribbon synapse, that of the goldfish Mb1 bipolar cell, less is known about endocytosis in photoreceptors. We used capacitance recording techniques to measure vesicle membrane fusion and retrieval in photoreceptors from salamander retinal slices. We found that application of brief depolarizing steps (\u3c100 \u3ems) to cones evoked exocytosis followed by rapid endocytosis with a time constant ∼250 ms. In some cases, the capacitance trace overshot the baseline, indicating excess endocytosis. Calcium had no effect on the time constant, but enhanced excess endocytosis resulting in a faster rate of membrane retrieval. Surprisingly, endocytosis was unaffected by blockers of dynamin, suggesting that cone endocytosis is dynamin independent. This contrasts with synaptic vesicle endocytosis in rods, which was inhibited by the dynamin inhibitor dynasore and GTPγS introduced through the patch pipette, suggesting that the two photoreceptor types employ distinct pathways for vesicle retrieval. The fast kinetics of synaptic vesicle endocytosis in photoreceptors likely enables these cells to maintain a high rate of transmitter release, allowing them to faithfully signal changes in illumination to second-order neurons

Ca2+ Diffusion through Endoplasmic Reticulum Supports Elevated Intraterminal Ca2+ Levels Needed to Sustain Synaptic Release from Rods in Darkness.

UNLABELLED: In addition to vesicle release at synaptic ribbons, rod photoreceptors are capable of substantial slow release at non-ribbon release sites triggered by Ca(2+)-induced Ca(2+) release (CICR) from intracellular stores. To maintain CICR as rods remain depolarized in darkness, we hypothesized that Ca(2+) released into the cytoplasm from terminal endoplasmic reticulum (ER) can be replenished continuously by ions diffusing within the ER from the soma. We measured [Ca(2+)] changes in cytoplasm and ER of rods from Ambystoma tigrinum retina using various dyes. ER [Ca(2+)] changes were measured by loading ER with fluo-5N and then washing dye from the cytoplasm with a dye-free patch pipette solution. Small dye molecules diffused within ER between soma and terminal showing a single continuous ER compartment. Depolarization of rods to -40 mV depleted Ca(2+) from terminal ER, followed by a decline in somatic ER [Ca(2+)]. Local activation of ryanodine receptors in terminals with a spatially confined puff of ryanodine caused a decline in terminal ER [Ca(2+)], followed by a secondary decrease in somatic ER. Localized photolytic uncaging of Ca(2+) from o-nitrophenyl-EGTA in somatic ER caused an abrupt Ca(2+) increase in somatic ER, followed by a slower Ca(2+) increase in terminal ER. These data suggest that, during maintained depolarization, a soma-to-terminal [Ca(2+)] gradient develops within the ER that promotes diffusion of Ca(2+) ions to resupply intraterminal ER Ca(2+) stores and thus sustain CICR-mediated synaptic release. The ability of Ca(2+) to move freely through the ER may also promote bidirectional communication of Ca(2+) changes between soma and terminal. SIGNIFICANCE STATEMENT: Vertebrate rod and cone photoreceptors both release vesicles at synaptic ribbons, but rods also exhibit substantial slow release at non-ribbon sites triggered by Ca(2+)-induced Ca(2+) release (CICR). Blocking CICR inhibits \u3e50% of release from rods in darkness. How do rods maintain sufficiently high [Ca(2+)] in terminal endoplasmic reticulum (ER) to support sustained CICR-driven synaptic transmission? We show that maintained depolarization creates a [Ca(2+)] gradient within the rod ER lumen that promotes soma-to-terminal diffusion of Ca(2+) to replenish intraterminal ER stores. This mechanism allows CICR-triggered synaptic release to be sustained indefinitely while rods remain depolarized in darkness. Free diffusion of Ca(2+) within the ER may also communicate synaptic Ca(2+) changes back to the soma to influence other critical cell processes

Properties of ribbon and non-ribbon release from rod photoreceptors revealed by visualizing individual synaptic vesicles.

Vesicle release from rod photoreceptors is regulated by Ca(2+) entry through L-type channels located near synaptic ribbons. We characterized sites and kinetics of vesicle release in salamander rods by using total internal reflection fluorescence microscopy to visualize fusion of individual synaptic vesicles. A small number of vesicles were loaded by brief incubation with FM1-43 or a dextran-conjugated, pH-sensitive form of rhodamine, pHrodo. Labeled organelles matched the diffraction-limited size of fluorescent microspheres and disappeared rapidly during stimulation. Consistent with fusion, depolarization-evoked vesicle disappearance paralleled electrophysiological release kinetics and was blocked by inhibiting Ca(2+) influx. Rods maintained tonic release at resting membrane potentials near those in darkness, causing depletion of membrane-associated vesicles unless Ca(2+) entry was inhibited. This depletion of release sites implies that sustained release may be rate limited by vesicle delivery. During depolarizing stimulation, newly appearing vesicles approached the membrane at ∼800 nm/s, where they paused for ∼60 ms before fusion. With fusion, vesicles advanced ∼18 nm closer to the membrane. Release events were concentrated near ribbons, but lengthy depolarization also triggered release from more distant non-ribbon sites. Consistent with greater contributions from non-ribbon sites during lengthier depolarization, damaging the ribbon by fluorophore-assisted laser inactivation (FALI) of Ribeye caused only weak inhibition of exocytotic capacitance increases evoked by 200-ms depolarizing test steps, whereas FALI more strongly inhibited capacitance increases evoked by 25 ms steps. Amplifying release by use of non-ribbon sites when rods are depolarized in darkness may improve detection of decrements in release when they hyperpolarize to light

Calmodulin enhances ribbon replenishment and shapes filtering of synaptic transmission by cone photoreceptors.

At the first synapse in the vertebrate visual pathway, light-evoked changes in photoreceptor membrane potential alter the rate of glutamate release onto second-order retinal neurons. This process depends on the synaptic ribbon, a specialized structure found at various sensory synapses, to provide a supply of primed vesicles for release. Calcium (Ca(2+)) accelerates the replenishment of vesicles at cone ribbon synapses, but the mechanisms underlying this acceleration and its functional implications for vision are unknown. We studied vesicle replenishment using paired whole-cell recordings of cones and postsynaptic neurons in tiger salamander retinas and found that it involves two kinetic mechanisms, the faster of which was diminished by calmodulin (CaM) inhibitors. We developed an analytical model that can be applied to both conventional and ribbon synapses and showed that vesicle resupply is limited by a simple time constant, τ = 1/(Dρδs), where D is the vesicle diffusion coefficient, δ is the vesicle diameter, ρ is the vesicle density, and s is the probability of vesicle attachment. The combination of electrophysiological measurements, modeling, and total internal reflection fluorescence microscopy of single synaptic vesicles suggested that CaM speeds replenishment by enhancing vesicle attachment to the ribbon. Using electroretinogram and whole-cell recordings of light responses, we found that enhanced replenishment improves the ability of cone synapses to signal darkness after brief flashes of light and enhances the amplitude of responses to higher-frequency stimuli. By accelerating the resupply of vesicles to the ribbon, CaM extends the temporal range of synaptic transmission, allowing cones to transmit higher-frequency visual information to downstream neurons. Thus, the ability of the visual system to encode time-varying stimuli is shaped by the dynamics of vesicle replenishment at photoreceptor synaptic ribbons

Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype.

The human CLCA4 (chloride channel regulator, calcium-activated) modulates the intestinal phenotype of cystic fibrosis (CF) patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring null variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype

A Holistic Systems Approach to Characterize the Impact of Pre- and Post-natal Oxycodone Exposure on Neurodevelopment and Behavior

Background: Increased risk of oxycodone (oxy) dependency during pregnancy has been associated with altered behaviors and cognitive deficits in exposed offspring. However, a significant knowledge gap remains regarding the effect of in utero and postnatal exposure on neurodevelopment and subsequent behavioral outcomes. Methods: Using a preclinical rodent model that mimics oxy exposure in utero (IUO) and postnatally (PNO), we employed an integrative holistic systems biology approach encompassing proton magnetic resonance spectroscopy (1H-MRS), electrophysiology, RNA-sequencing, and Von Frey pain testing to elucidate molecular and behavioral changes in the exposed offspring during early neurodevelopment as well as adulthood. Results: 1H-MRS studies revealed significant changes in key brain metabolites in the exposed offspring that were corroborated with changes in synaptic currents. Transcriptomic analysis employing RNA-sequencing identified alterations in the expression of pivotal genes associated with synaptic transmission, neurodevelopment, mood disorders, and addiction in the treatment groups. Furthermore, Von Frey analysis revealed lower pain thresholds in both exposed groups. Conclusions: Given the increased use of opiates, understanding the persistent developmental effects of these drugs on children will delineate potential risks associated with opiate use beyond the direct effects in pregnant women

Single cell derived mRNA signals across human kidney tumors.

Funder: Department of HealthTumor cells may share some patterns of gene expression with their cell of origin, providing clues into the differentiation state and origin of cancer. Here, we study the differentiation state and cellular origin of 1300 childhood and adult kidney tumors. Using single cell mRNA reference maps of normal tissues, we quantify reference "cellular signals" in each tumor. Quantifying global differentiation, we find that childhood tumors exhibit fetal cellular signals, replacing the presumption of "fetalness" with a quantitative measure of immaturity. By contrast, in adult cancers our assessment refutes the suggestion of dedifferentiation towards a fetal state in most cases. We find an intimate connection between developmental mesenchymal populations and childhood renal tumors. We demonstrate the diagnostic potential of our approach with a case study of a cryptic renal tumor. Our findings provide a cellular definition of human renal tumors through an approach that is broadly applicable to human cancer

Hyperpolarization-Activated Current (Ih) in Ganglion-Cell Photoreceptors

Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and serve as the primary retinal drivers of non-image-forming visual functions such as circadian photoentrainment, the pupillary light reflex, and suppression of melatonin production in the pineal. Past electrophysiological studies of these cells have focused on their intrinsic photosensitivity and synaptic inputs. Much less is known about their voltage-gated channels and how these might shape their output to non-image-forming visual centers. Here, we show that rat ipRGCs retrolabeled from the suprachiasmatic nucleus (SCN) express a hyperpolarization-activated inwardly-rectifying current (Ih). This current is blocked by the known Ih blockers ZD7288 and extracellular cesium. As in other systems, including other retinal ganglion cells, Ih in ipRGCs is characterized by slow kinetics and a slightly greater permeability for K+ than for Na+. Unlike in other systems, however, Ih in ipRGCs apparently does not actively contribute to resting membrane potential. We also explore non-specific effects of the common Ih blocker ZD7288 on rebound depolarization and evoked spiking and discuss possible functional roles of Ih in non-image-forming vision. This study is the first to characterize Ih in a well-defined population of retinal ganglion cells, namely SCN-projecting ipRGCs

Euclid preparation: XXXI. The effect of the variations in photometric passbands on photometric-redshift accuracy

The technique of photometric redshifts has become essential for the exploitation of multi-band extragalactic surveys. While the requirements on photometric redshifts for the study of galaxy evolution mostly pertain to the precision and to the fraction of outliers, the most stringent requirement in their use in cosmology is on the accuracy, with a level of bias at the sub-percent level for the Euclid cosmology mission. A separate, and challenging, calibration process is needed to control the bias at this level of accuracy. The bias in photometric redshifts has several distinct origins that may not always be easily overcome. We identify here one source of bias linked to the spatial or time variability of the passbands used to determine the photometric colours of galaxies. We first quantified the effect as observed on several well-known photometric cameras, and found in particular that, due to the properties of optical filters, the redshifts of off-axis sources are usually overestimated. We show using simple simulations that the detailed and complex changes in the shape can be mostly ignored and that it is sufficient to know the mean wavelength of the passbands of each photometric observation to correct almost exactly for this bias; the key point is that this mean wavelength is independent of the spectral energy distribution of the source. We use this property to propose a correction that can be computationally efficiently implemented in some photometric-redshift algorithms, in particular template-fitting. We verified that our algorithm, implemented in the new photometric-redshift code Phosphoros, can effectively reduce the bias in photometric redshifts on real data using the CFHTLS T007 survey, with an average measured bias Δz over the redshift range 0.4 ≤ z ≤ 0.7 decreasing by about 0.02, specifically from Δz ≃ 0.04 to Δz ≃ 0.02 around z = 0.5. Our algorithm is also able to produce corrected photometry for other applications

Euclid preparation. XXXI. The effect of the variations in photometric passbands on photometric-redshift accuracy

The technique of photometric redshifts has become essential for the exploitation of multi-band extragalactic surveys. While the requirements on photo-zs for the study of galaxy evolution mostly pertain to the precision and to the fraction of outliers, the most stringent requirement in their use in cosmology is on the accuracy, with a level of bias at the sub-percent level for the Euclid cosmology mission. A separate, and challenging, calibration process is needed to control the bias at this level of accuracy. The bias in photo-zs has several distinct origins that may not always be easily overcome. We identify here one source of bias linked to the spatial or time variability of the passbands used to determine the photometric colours of galaxies. We first quantified the effect as observed on several well-known photometric cameras, and found in particular that, due to the properties of optical filters, the redshifts of off-axis sources are usually overestimated. We show using simple simulations that the detailed and complex changes in the shape can be mostly ignored and that it is sufficient to know the mean wavelength of the passbands of each photometric observation to correct almost exactly for this bias; the key point is that this mean wavelength is independent of the spectral energy distribution of the source}. We use this property to propose a correction that can be computationally efficiently implemented in some photo-z algorithms, in particular template-fitting. We verified that our algorithm, implemented in the new photo-z code Phosphoros, can effectively reduce the bias in photo-zs on real data using the CFHTLS T007 survey, with an average measured bias Delta z over the redshift range 0.4<z<0.7 decreasing by about 0.02, specifically from Delta z~0.04 to Delta z~0.02 around z=0.5. Our algorithm is also able to produce corrected photometry for other applications.Comment: 19 pages, 13 figures; Accepted for publication in A&