optimizing-setup-conditions-for-ex-vivo-electroretinogram-to-study

Optimizing the Setup and Conditions for <em>Ex Vivo</em> Electroretinogram to Study Retina Function in Small and Large Eyes

Measurements of retinal neuronal light responses are critical to investigating the physiology of the healthy retina, determining pathological changes in ...

University of Utah

Photoreceptors are specialized neurons that rely on Ca to regulate phototransduction and neurotransmission. Photoreceptor dysfunction and degeneration occur when intracellular Ca homeostasis is disrupted. Ca homeostasis is maintained partly by mitochondrial Ca uptake through the mitochondrial Ca uniporter (MCU), which can influence cytosolic Ca signals, stimulate energy production, and trigger apoptosis. Here we discovered that zebrafish cone photoreceptors express unusually low levels of MCU. We expected that this would be important to prevent mitochondrial Ca overload and consequent cone degeneration. To test this hypothesis, we generated a cone-specific model of MCU overexpression. Surprisingly, we found that cones tolerate MCU overexpression, surviving elevated mitochondrial Ca and disruptions to mitochondrial ultrastructure until late adulthood. We exploited the survival of MCU overexpressing cones to additionally demonstrate that mitochondrial Ca uptake alters the distributions of citric acid cycle intermediates and accelerates recovery kinetics of the cone response to light. Cones adapt to mitochondrial Ca stress by decreasing MICU3, an enhancer of MCU-mediated Ca uptake, and selectively transporting damaged mitochondria away from the ellipsoid toward the synapse. Our findings demonstrate how mitochondrial Ca can influence physiological and metabolic processes in cones and highlight the remarkable ability of cone photoreceptors to adapt to mitochondrial stress.

Increasing Ca in photoreceptor mitochondria alters metabolites, accelerates photoresponse recovery, and reveals adaptations to mitochondrial stress.

Rods and cones use intracellular Ca to regulate many functions, including phototransduction and neurotransmission. The Mitochondrial Calcium Uniporter (MCU) complex is thought to be the primary pathway for Ca entry into mitochondria in eukaryotes. We investigate the hypothesis that mitochondrial Ca uptake via MCU influences phototransduction and energy metabolism in photoreceptors using a mcu zebrafish and a rod photoreceptor-specific Mcu mouse. Using genetically encoded Ca sensors to directly examine Ca uptake in zebrafish cone mitochondria, we found that loss of MCU reduces but does not eliminate mitochondrial Ca uptake. Loss of MCU does not lead to photoreceptor degeneration, mildly affects mitochondrial metabolism, and does not alter physiological responses to light, even in the absence of the Na/Ca, K exchanger. Our results reveal that MCU is dispensable for vertebrate photoreceptor function, consistent with its low expression and the presence of an alternative pathway for Ca uptake into photoreceptor mitochondria.

Mitochondrial Calcium Uniporter (MCU) deficiency reveals an alternate path for Ca uptake in photoreceptor mitochondria.

Sensing changes in the environment is crucial for survival. Animals from invertebrates to vertebrates use both visual and olfactory stimuli to direct survival behaviors including identification of food sources, finding mates, and predator avoidance. In primary sensory neurons there are signal transduction mechanisms that convert chemical or light signals into an electrical response through ligand binding or photoactivation of a receptor, that can be propagated to the olfactory and visual centers of the brain to create a perception of the odor and visual landscapes surrounding us. The fundamental principles of olfactory and phototransduction pathways within vertebrates are somewhat analogous. Signal transduction in both systems takes place in the ciliary sub-compartments of the sensory cells and relies upon the activation of G protein-coupled receptors (GPCRs) to close cyclic nucleotide-gated (CNG) cation channels in photoreceptors to produce a hyperpolarization of the cell, or in olfactory sensory neurons open CNG channels to produce a depolarization. However, while invertebrate phototransduction also involves GPCRs, invertebrate photoreceptors can be either ciliary and/or microvillar with hyperpolarizing and depolarizing responses to light, respectively. Moreover, olfactory transduction in invertebrates may be a mixture of metabotropic G protein and ionotropic signaling pathways. This review will highlight differences of the visual and olfactory transduction mechanisms between vertebrates and invertebrates, focusing on the implications to the gain of the transduction processes, and how they are modulated to allow detection of small changes in odor concentration and light intensity over a wide range of background stimulus levels.

Transduction and Adaptation Mechanisms in the Cilium or Microvilli of Photoreceptors and Olfactory Receptors From Insects to Humans.

Death is defined as the irreversible cessation of circulatory, respiratory or brain activity. Many peripheral human organs can be transplanted from deceased donors using protocols to optimize viability. However, tissues from the central nervous system rapidly lose viability after circulation ceases, impeding their potential for transplantation. The time course and mechanisms causing neuronal death and the potential for revival remain poorly defined. Here, using the retina as a model of the central nervous system, we systemically examine the kinetics of death and neuronal revival. We demonstrate the swift decline of neuronal signalling and identify conditions for reviving synchronous in vivo-like trans-synaptic transmission in postmortem mouse and human retina. We measure light-evoked responses in human macular photoreceptors in eyes removed up to 5 h after death and identify modifiable factors that drive reversible and irreversible loss of light signalling after death. Finally, we quantify the rate-limiting deactivation reaction of phototransduction, a model G protein signalling cascade, in peripheral and macular human and macaque retina. Our approach will have broad applications and impact by enabling transformative studies in the human central nervous system, raising questions about the irreversibility of neuronal cell death, and providing new avenues for visual rehabilitation.

Article	Year
Increasing Ca in photoreceptor mitochondria alters metabolites, accelerates photoresponse recovery, and reveals adaptations to mitochondrial stress. Cell death and differentiation\| PubMed ID: 31371786	2020
Mitochondrial Calcium Uniporter (MCU) deficiency reveals an alternate path for Ca uptake in photoreceptor mitochondria. Scientific reports\| PubMed ID: 32994451	2020
Transduction and Adaptation Mechanisms in the Cilium or Microvilli of Photoreceptors and Olfactory Receptors From Insects to Humans. Frontiers in cellular neuroscience\| PubMed ID: 33867944	2021
Revival of light signalling in the postmortem mouse and human retina. Nature\| PubMed ID: 35545677	2022

Fatima Abbas

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