Search

Vertebrates need oxygen to survive. The central nervous system has an especially high energy demand, so brain and retinal neurons quickly die in anoxia. But fish of the genus Carassius are exceptionally anoxia-tolerant: the crucian carp (C. carassius) can survive months without oxygen in ice-covered ponds, and the common goldfish (C. auratus) can withstand hours of anoxia at room temperature. These fish previously offered insights into anoxia tolerance in the brain, heart, and liver. Here, we advance Carassius spp. as models to study anoxia tolerance in the retina. Electroretinogram and evoked potential recordings show that crucian carp reversibly downregulate their visual systems in anoxia, probably to save ATP. Notably, Carassius suppress their visual systems nearly twice as much as anoxia-tolerant turtles, Trachemys and Chrysemys spp., which are often promoted as the champions of anoxia tolerance. We summarize what is known about anoxia tolerance in the goldfish and crucian carp retinas, including cellular pathways which may protect retinal neurons from excitotoxic cell death. We compare the Carassius retina with two relevant models: natural anoxia tolerance in the turtle brain, and ischemic preconditioning in the rat retina. All three models include mitochondria as oxygen sensors: mitochondria depolarize due to mitochondrial ATP-dependent K + channels, possibly to trigger neuroprotective second messenger cascades. The Carassius retina is an accessible and inexpensive model, with over 70 fruitful years of history in vision research. As a model for anoxia tolerance, it may provide new insights into diseases of the eye (like diabetes, macular degeneration, and eye stroke)., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Seasonal modifications in the structure of cellular membranes occur as an adaptive measure to withstand exposure to prolonged environmental change. Little is known about whether such changes occur independently of external cues, such as photoperiod or temperature, or how they may impact the central nervous system. We compared membrane properties of neurons isolated from the retina of goldfish (Carassius auratus), an organism well adapted to extreme environmental change, during the summer and winter months. Goldfish were maintained in a facility under constant environmental conditions throughout the year. Analysis of whole-retina phospholipid composition using mass spectrometry-based lipidomics revealed a twofold increase in phosphatidylethanolamine species during the winter, suggesting an increase in cell membrane fluidity. Atomic force microscopy was used to produce localized, nanoscale-force deformation of neuronal membranes. Measurement of Young's modulus indicated increased membrane-cortical stiffness (or decreased elasticity) in neurons isolated during the winter. Voltage-clamp electrophysiology was used to assess physiological changes in neurons between seasons. Winter neurons displayed a hyperpolarized reversal potential (Vrev) and a significantly lower input resistance (Rin) compared with summer neurons. This was indicative of a decrease in membrane excitability during the winter. Subsequent measurement of intracellular Ca2+ activity using Fura-2 microspectrofluorometry confirmed a reduction in action potential activity, including duration and action potential profile, in neurons isolated during the winter. These studies demonstrate chemical and biophysical changes that occur in retinal neurons of goldfish throughout the year without exposure to seasonal cues, and suggest a novel mechanism of seasonal regulation of retinal activity., (© 2022. Published by The Company of Biologists Ltd.)

The light-absorbing retina has an exceptionally high oxygen demand, which imposes two conflicting needs: high rates of blood perfusion and an unobstructed light path devoid of blood vessels. This review discusses mechanisms and physiological trade-offs underlying retinal oxygen supply in vertebrates and examines how these physiological systems supported the evolution of vision.

Neurons of the retina require oxygen to survive. In hypoxia, neuronal ATP production is impaired, ATP-dependent ion pumping is reduced, transmembrane ion gradients are dysregulated, and intracellular Ca2+ concentration ([Ca2+]i) increases enough to trigger excitotoxic cell death. Central neurons of the common goldfish (Carassius auratus) are hypoxia tolerant, but little is known about how goldfish retinas withstand hypoxia. To study the cellular mechanisms of hypoxia tolerance, we isolated retinal interneurons (horizontal cells; HCs), and measured [Ca2+]i with Fura-2. Goldfish HCs maintained [Ca2+]i throughout 1 h of hypoxia, whereas [Ca2+]i increased irreversibly in HCs of the hypoxia-sensitive rainbow trout (Oncorhynchus mykiss) with just 20 min of hypoxia. Our results suggest mitochondrial ATP-dependent K+ channels (mKATP) are necessary to stabilize [Ca2+]i throughout hypoxia. In goldfish HCs, [Ca2+]i increased when mKATP channels were blocked with glibenclamide or 5-hydroxydecanoic acid, whereas the mKATP channel agonist diazoxide prevented [Ca2+]i from increasing in hypoxia in trout HCs. We found that hypoxia protects against increases in [Ca2+]i in goldfish HCs via mKATP channels. Glycolytic inhibition with 2-deoxyglucose increased [Ca2+]i, which was rescued by hypoxia in a mKATP channel-dependent manner. We found no evidence of plasmalemmal KATP channels in patch-clamp experiments. Instead, we confirmed the involvement of KATP in mitochondria with TMRE imaging, as hypoxia rapidly (<5 min) depolarized mitochondria in a mKATP channel-sensitive manner. We conclude that mKATP channels initiate a neuroprotective pathway in goldfish HCs to maintain [Ca2+]i and avoid excitotoxicity in hypoxia. This model provides novel insight into the cellular mechanisms of hypoxia tolerance in the retina., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Horizontal cells (HCs) are neurons of the outer retina, which provide inhibitory feedback onto photoreceptors and contribute to image processing. HCs in teleosts are classified into four subtypes (H1-H4), each having different roles: H1-H3 feed back onto different sets of cones, H4 feed back onto rods, and only H1 store and release the inhibitory neurotransmitter, γ-aminobutyric acid (GABA). Dissociated HCs exhibit spontaneous Ca 2+ -based action potentials (APs), yet it is unclear if APs occur in situ, or if all subtypes exhibit APs. We measured intracellular Ca 2+ and report APs in slice preparations of the goldfish retina. In HCs furthest from photoreceptors (i.e., H3/H4), APs were less frequent, with greater duration and area under the curve (a measure of Ca 2+ flux). Next, we classified acutely dissociated HCs into subtypes by integrating the ratio of dendritic field size vs. soma size (r d/s ). H1 and H2 subtypes had low r d/s values (<8); H3/H4 had high r d/s (>12). To verify this model, H1s were identified by immunoreactivity for GABA and 95% of these cells had an r d/s < 4. In Ca 2+ imaging experiments, as r d/s increased, AP duration and area under the curve increased, while frequency decreased. Our results demonstrate the presence of Ca 2+ -based APs in the goldfish retina in situ and show that HC subtypes H1 through H4 exhibit progressively longer and less frequent spontaneous APs. These results suggest that APs may play an important role in inhibitory feedback, and may have implications for understanding the relative contributions of HC subtypes in the outer retina., (© 2020 Wiley Periodicals LLC.)

Horizontal cells (HCs) are interneurons of the outer retina that undergo graded changes in membrane potential during the light response and provide feedback to photoreceptors. We characterized spontaneous Ca 2+ -based action potentials (APs) in isolated goldfish ( Carassius auratus ) HCs with electrophysiological and intracellular imaging techniques. Transient changes in intracellular Ca 2+ concentration ([Ca 2+ ] i ) were observed with fura-2 and were abolished by removal of extracellular Ca 2+ or by inhibition of Ca 2+ channels by 50 µM Cd 2+ or 100 µM nifedipine. Inhibition of Ca 2+ release from stores with 20 µM ryanodine or 50 µM dantrolene abolished Ca 2+ transients and increased baseline [Ca 2+ ] i . This increased baseline was prevented by blocking L-type Ca 2+ channels with nifedipine, suggesting that Ca 2+ -induced Ca 2+ release from stores may be needed to inactivate membrane Ca 2+ channels. Caffeine (3 mM) increased the frequency of Ca 2+ transients, and the store-operated channel antagonist 2-aminoethyldiphenylborinate (100 μM) counteracted this effect. APs were detected with voltage-sensitive dye imaging (FluoVolt) and current-clamp electrophysiology. In current-clamp recordings, regenerative APs were abolished by removal of extracellular Ca 2+ or in the presence of 5 mM Co 2+ or 100 µM nifedipine, and APs were amplified with 15 mM Ba 2+ . Collectively, our data suggest that during APs Ca 2+ enters through L-type Ca 2+ channels and that Ca 2+ stores (gated by ryanodine receptors) contribute to the rise in [Ca 2+ ] i . This work may lead to further understanding of the possible role APs have in vision, such as transitioning from light to darkness or modulating feedback from HCs to photoreceptors. NEW & NOTEWORTHY Horizontal cells (HCs) are interneurons of the outer retina that provide inhibitory feedback onto photoreceptors. HCs respond to light via graded changes in membrane potential. We characterized spontaneous action potentials in HCs from goldfish and linked action potential generation to a rise in intracellular Ca 2+ via plasma membrane channels and ryanodine receptors. Action potentials may play a role in vision, such as transitioning from light to darkness, or in modulating feedback from HCs to photoreceptors.

The retina is part of the central nervous system, and shares the characteristically high metabolism of the brain. The high energy demand of the retina is normally matched with a large supply of metabolites. When supply does not equal demand (e.g. if retinal blood flow is impaired), retinal neurons are at risk of excitotoxic cell death and vision is impaired or lost. Understanding the energetic budget of the retina is therefore crucial for understanding the pathology and treatment of retinal disease. In this minireview I give an overview of the energetics of the retina, with a focus on lessons learnt from comparative physiology. Retinas of all species studied thus far receive blood flow from choroidal capillaries. Additionally, fish, reptiles, and birds each have unique structures to increase metabolite supply. Primates and some mammals also have intra- and supraretinal vasculature to supply the retina, while other mammals rely solely on the choroid at the cost of retinal thickness. Neuroglobin, an oxygen-binding protein, may assist in oxygen delivery to counteract large diffusion distances from capillaries to mitochondria. Energy demand differs among models, as does mitochondrial location. More ATP is consumed in the dark due to Na + /K + ATPase activity to counteract the dark current, whereas phototransduction dominates ATP demand in the light. Photoreceptor metabolism is therefore especially high, and may be sustained with phosphocreatine and lactate shuttles. This comparative physiology approach raises new research questions, and suggests caution in comparing animal models of retinal disease, as they differ greatly in vasculature and energetics., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Horizontal cells (HCs) are inhibitory interneurons of the vertebrate retina. Unlike typical neurons, HCs are chronically depolarized in the dark, leading to a constant influx of Ca 2+ Therefore, mechanisms of Ca 2+ homeostasis in HCs must differ from neurons elsewhere in the central nervous system, which undergo excitotoxicity when they are chronically depolarized or stressed with Ca 2+ HCs are especially well characterized in teleost fish and have been used to unlock mysteries of the vertebrate retina for over one century. More recently, mammalian models of the retina have been increasingly informative for HC physiology. We draw from both teleost and mammalian models in this review, using a comparative approach to examine what is known about Ca 2+ pathways in vertebrate HCs. We begin with a survey of Ca 2+ -permeable ion channels, exchangers, and pumps and summarize Ca 2+ influx and efflux pathways, buffering, and intracellular stores. This includes evidence for Ca 2+ -permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and N-methyl-d-aspartate receptors and for voltage-gated Ca 2+ channels. Special attention is given to interactions between ion channels, to differences among species, and in which subtypes of HCs these channels have been found. We then discuss a number of unresolved issues pertaining to Ca 2+ dynamics in HCs, including a potential role for Ca 2+ in feedback to photoreceptors, the role for Ca 2+ -induced Ca 2+ release, and the properties and functions of Ca 2+ -based action potentials. This review aims to highlight the unique Ca 2+ dynamics in HCs, as these are inextricably tied to retinal function., (Copyright © 2017 the American Physiological Society.)