Restoring and sustaining human postmortem retinal light responses with scalable methods for testing degenerative disease therapies.

Neuro- and retinal degenerative diseases rob millions of aging individuals of their independence. Researching these diseases in human tissue has been hindered by the immediate loss of electric activity in neurons after the circulation ceases. Recent studies indicate that limited neuronal activity can be revived postmortem, even in the retina.

Closed-perfusion transretinal ERG setup for preclinical drug and nanostructure testing.

Objective: The isolated mammalian retina may serve as a sensitive biosensor for preclinical drug testing, including eye drugs and a broader range of pharmaceuticals. To facilitate testing with minimal amounts of drug molecules or nanostructures, we developed a closed-perfusion transretinal electroretinography (tERG) setup.

Methods: The major challenge with small amounts of circulating perfusate was maintaining retinal viability and stability during long experiments.

Acyl-CoA synthetase 6 controls rod photoreceptor function and survival by shaping the phospholipid composition of retinal membranes.

The retina is light-sensitive neuronal tissue in the back of the eye. The phospholipid composition of the retina is unique and highly enriched in polyunsaturated fatty acids, including docosahexaenoic fatty acid (DHA). While it is generally accepted that a high DHA content is important for vision, surprisingly little is known about the mechanisms of DHA enrichment in the retina.

Modeling complex age-related eye disease.

Modeling complex eye diseases like age-related macular degeneration (AMD) and glaucoma poses significant challenges, since these conditions depend highly on age-related changes that occur over several decades, with many contributing factors remaining unknown. Although both diseases exhibit a relatively high heritability of >50%, a large proportion of individuals carrying AMD- or glaucoma-associated genetic risk variants will never develop these diseases. Furthermore, several environmental and lifestyle factors contribute to and modulate the pathogenesis and progression of AMD and glaucoma.

Optimizing the Setup and Conditions for Ex Vivo 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 retinal diseases, and testing therapeutic interventions. The ex vivo electroretinogram (ERG) allows the quantification of contributions from individual cell types in the isolated retina by addition of specific pharmacological agents and evaluation of tissue-intrinsic changes independently of systemic influences. Retinal light responses can be measured using a specialized ex vivo ERG specimen holder and recording setup, modified from existing patch clamp or microelectrode array equipment.

Revival of light signalling in the postmortem mouse and human retina.

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.

Identification of small-molecule allosteric modulators that act as enhancers/disrupters of rhodopsin oligomerization.

The elongated cilia of the outer segment of rod and cone photoreceptor cells can contain concentrations of visual pigments of up to 5 mM. The rod visual pigments, G protein-coupled receptors called rhodopsins, have a propensity to self-aggregate, a property conserved among many G protein-coupled receptors. However, the effect of rhodopsin oligomerization on G protein signaling in native cells is less clear.

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

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.

Diabetic photoreceptors: Mechanisms underlying changes in structure and function.

Based on clinical findings, diabetic retinopathy (DR) has traditionally been defined as a retinal microvasculopathy. Retinal neuronal dysfunction is now recognized as an early event in the diabetic retina before development of overt DR. While detrimental effects of diabetes on the survival and function of inner retinal cells, such as retinal ganglion cells and amacrine cells, are widely recognized, evidence that photoreceptors in the outer retina undergo early alterations in diabetes has emerged more recently.

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

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.

Homeostatic plasticity in the retina is associated with maintenance of night vision during retinal degenerative disease.

Neuronal plasticity of the inner retina has been observed in response to photoreceptor degeneration. Typically, this phenomenon has been considered maladaptive and may preclude vision restoration in the blind. However, several recent studies utilizing triggered photoreceptor ablation have shown adaptive responses in bipolar cells expected to support normal vision.

Rod phototransduction and light signal transmission during type 2 diabetes.

Introduction: Diabetic retinopathy is a major complication of diabetes recently associated with compromised photoreceptor function. Multiple stressors in diabetes, such as hyperglycemia, oxidative stress and inflammatory factors, have been identified, but systemic effects of diabetes on outer retina function are incompletely understood. We assessed photoreceptor physiology in vivo and in isolated retinas to better understand how alterations in the cellular environment compared with intrinsic cellular/molecular properties of the photoreceptors, affect light signal transduction and transmission in the retina in chronic type 2 diabetes.

Functional diversity of human intrinsically photosensitive retinal ganglion cells.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are a subset of cells that participate in image-forming and non-image-forming visual responses. Although both functional and morphological subtypes of ipRGCs have been described in rodents, parallel functional subtypes have not been identified in primate or human retinas. In this study, we used a human organ donor preparation method to measure human ipRGCs' photoresponses.

Sensitivity of Mammalian Cone Photoreceptors to Infrared Light.

Two-photon vision arises from the perception of pulsed infrared (IR) laser light as color corresponding to approximately half of the laser wavelength. The physical process responsible for two-photon vision in rods has been delineated and verified experimentally only recently. Here, we sought to determine whether IR light can also be perceived by mammalian cone photoreceptors via a similar activation mechanism.

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

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.

Stereospecific modulation of dimeric rhodopsin.

The classic concept that GPCRs function as monomers has been challenged by the emerging evidence of GPCR dimerization and oligomerization. Rhodopsin (Rh) is the only GPCR whose native oligomeric arrangement was revealed by atomic force microscopy demonstrating that Rh exists as a dimer. However, the role of Rh dimerization in retinal physiology is currently unknown.

Investigating the Ca-dependent and Ca-independent mechanisms for mammalian cone light adaptation.

Vision is mediated by two types of photoreceptors: rods, enabling vision in dim light; and cones, which function in bright light. Despite many similarities in the components of their respective phototransduction cascades, rods and cones have distinct sensitivity, response kinetics, and adaptation capacity. Cones are less sensitive and have faster responses than rods.

Regulation of calcium homeostasis in the outer segments of rod and cone photoreceptors.

Calcium plays important roles in the function and survival of rod and cone photoreceptor cells. Rapid regulation of calcium in the outer segments of photoreceptors is required for the modulation of phototransduction that drives the termination of the flash response as well as light adaptation in rods and cones. On a slower time scale, maintaining proper calcium homeostasis is critical for the health and survival of photoreceptors.

Guanylate cyclase-activating protein 2 contributes to phototransduction and light adaptation in mouse cone photoreceptors.

Light adaptation of photoreceptor cells is mediated by Ca-dependent mechanisms. In darkness, Ca influx through cGMP-gated channels into the outer segment of photoreceptors is balanced by Ca extrusion via Na/Ca, K exchangers (NCKXs). Light activates a G protein signaling cascade, which closes cGMP-gated channels and decreases Ca levels in photoreceptor outer segment because of continuing Ca extrusion by NCKXs.

The Na/Ca, K exchanger NCKX4 is required for efficient cone-mediated vision.

Calcium (Ca) plays an important role in the function and health of neurons. In vertebrate cone photoreceptors, Ca controls photoresponse sensitivity, kinetics, and light adaptation. Despite the critical role of Ca in supporting the function and survival of cones, the mechanism for its extrusion from cone outer segments is not well understood.

The Na(+)/Ca(2+), K(+) exchanger 2 modulates mammalian cone phototransduction.

Calcium ions (Ca(2+)) modulate the phototransduction cascade of vertebrate cone photoreceptors to tune gain, inactivation, and light adaptation. In darkness, the continuous current entering the cone outer segment through cGMP-gated (CNG) channels is carried in part by Ca(2+), which is then extruded back to the extracellular space. The mechanism of Ca(2+) extrusion from mammalian cones is not understood.

A novel Ca2+-feedback mechanism extends the operating range of mammalian rods to brighter light.

Sensory cells adjust their sensitivity to incoming signals, such as odor or light, in response to changes in background stimulation, thereby extending the range over which they operate. For instance, rod photoreceptors are extremely sensitive in darkness, so that they are able to detect individual photons, but remain responsive to visual stimuli under conditions of bright ambient light, which would be expected to saturate their response given the high gain of the rod transduction cascade in darkness. These photoreceptors regulate their sensitivity to light rapidly and reversibly in response to changes in ambient illumination, thereby avoiding saturation.

Autophagy supports color vision.

Cones comprise only a small portion of the photoreceptors in mammalian retinas. However, cones are vital for color vision and visual perception, and their loss severely diminishes the quality of life for patients with retinal degenerative diseases. Cones function in bright light and have higher demand for energy than rods; yet, the mechanisms that support the energy requirements of cones are poorly understood.

A new mouse model for stationary night blindness with mutant Slc24a1 explains the pathophysiology of the associated human disease.

Mutations that affect calcium homeostasis (Ca(2+)) in rod photoreceptors are linked to retinal degeneration and visual disorders such as retinitis pigmentosa and congenital stationary night blindness (CSNB). It is thought that the concentration of Ca(2+) in rod outer segments is controlled by a dynamic balance between influx via cGMP-gated (CNG) channels and extrusion via Na(+)/Ca(2+), K(+) exchangers (NCKX1). The extrusion-driven lowering of rod [Ca(2+)]i following light exposure controls their light adaptation and response termination.

Simultaneous ex vivo functional testing of two retinas by in vivo electroretinogram system.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans.