RESUMEN
In retinitis pigmentosa (RP), rod and cone photoreceptors degenerate, depriving downstream neurons of light-sensitive input, leading to vision impairment or blindness. Although downstream neurons survive, some undergo morphological and physiological remodeling. Bipolar cells (BCs) link photoreceptors, which sense light, to retinal ganglion cells (RGCs), which send information to the brain. While photoreceptor loss disrupts input synapses to BCs, whether BC output synapses remodel has remained unknown. Here we report that synaptic output from BCs plummets in RP mouse models of both sexes owing to loss of voltage-gated Ca2+ channels. Remodeling reduces the reliability of synaptic output to repeated optogenetic stimuli, causing RGC firing to fail at high-stimulus frequencies. Fortunately, functional remodeling of BCs can be reversed by inhibiting the retinoic acid receptor (RAR). RAR inhibitors targeted to BCs present a new therapeutic opportunity for mitigating detrimental effects of remodeling on signals initiated either by surviving photoreceptors or by vision-restoring tools.
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Células Bipolares de la Retina , Sinapsis , Tretinoina , Animales , Células Bipolares de la Retina/efectos de los fármacos , Células Bipolares de la Retina/fisiología , Ratones , Tretinoina/farmacología , Masculino , Femenino , Sinapsis/efectos de los fármacos , Sinapsis/fisiología , Retinitis Pigmentosa/fisiopatología , Retinitis Pigmentosa/genética , Degeneración Retiniana/fisiopatología , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Ganglionares de la Retina/fisiología , Células Ganglionares de la Retina/efectos de los fármacosRESUMEN
Magnetogenetics was developed to remotely control genetically targeted neurons. A variant of magnetogenetics uses magnetic fields to activate transient receptor potential vanilloid (TRPV) channels when coupled with ferritin. Stimulation with static or RF magnetic fields of neurons expressing these channels induces Ca2+ transients and modulates behavior. However, the validity of ferritin-based magnetogenetics has been questioned due to controversies surrounding the underlying mechanisms and deficits in reproducibility. Here, we validated the magnetogenetic approach Ferritin-iron Redistribution to Ion Channels (FeRIC) using electrophysiological (Ephys) and imaging techniques. Previously, interference from RF stimulation rendered patch-clamp recordings inaccessible for magnetogenetics. We solved this limitation for FeRIC, and we studied the bioelectrical properties of neurons expressing TRPV4 (nonselective cation channel) and transmembrane member 16A (TMEM16A; chloride-permeable channel) coupled to ferritin (FeRIC channels) under RF stimulation. We used cultured neurons obtained from the rat hippocampus of either sex. We show that RF decreases the membrane resistance (Rm) and depolarizes the membrane potential in neurons expressing TRPV4FeRIC RF does not directly trigger action potential firing but increases the neuronal basal spiking frequency. In neurons expressing TMEM16AFeRIC, RF decreases the Rm, hyperpolarizes the membrane potential, and decreases the spiking frequency. Additionally, we corroborated the previously described biochemical mechanism responsible for RF-induced activation of ferritin-coupled ion channels. We solved an enduring problem for ferritin-based magnetogenetics, obtaining direct Ephys evidence of RF-induced activation of ferritin-coupled ion channels. We found that RF does not yield instantaneous changes in neuronal membrane potentials. Instead, RF produces responses that are long-lasting and moderate, but effective in controlling the bioelectrical properties of neurons.
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Ferritinas , Neuronas , Animales , Ferritinas/metabolismo , Ratas , Neuronas/fisiología , Masculino , Femenino , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/genética , Células Cultivadas , Campos Magnéticos , Ratas Sprague-Dawley , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Hipocampo/fisiología , Hipocampo/citologíaRESUMEN
The development of fluorescent dyes that emit and absorb light at wavelengths greater than 700 nm and that respond to biochemical and biophysical events in living systems remains an outstanding challenge for noninvasive optical imaging. Here, we report the design, synthesis, and application of near-infrared (NIR)-absorbing and -emitting optical voltmeter based on a sulfonated, phosphine-oxide (po) rhodamine for voltage imaging in intact retinas. We find that po-rhodamine based voltage reporters, or poRhoVRs, display NIR excitation and emission profiles at greater than 700 nm, show a range of voltage sensitivities (13 to 43% ΔF/F per 100 mV in HEK cells), and can be combined with existing optical sensors, like Ca2+-sensitive fluorescent proteins (GCaMP), and actuators, like light-activated opsins ChannelRhodopsin-2 (ChR2). Simultaneous voltage and Ca2+ imaging reveals differences in activity dynamics in rat hippocampal neurons, and pairing poRhoVR with blue-light based ChR2 affords all-optical electrophysiology. In ex vivo retinas isolated from a mouse model of retinal degeneration, poRhoVR, together with GCaMP-based Ca2+ imaging and traditional multielectrode array (MEA) recording, can provide a comprehensive physiological activity profile of neuronal activity, revealing differences in voltage and Ca2+ dynamics within hyperactive networks of the mouse retina. Taken together, these experiments establish that poRhoVR will open new horizons in optical interrogation of cellular and neuronal physiology in intact systems.
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Rayos Infrarrojos , Imagen Óptica , Óxidos/química , Fosfinas/química , Rodaminas/química , Animales , Calcio/metabolismo , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Ratones , Neuronas/citología , Neuronas/metabolismo , Retina/citología , Retina/diagnóstico por imagen , Retina/metabolismoRESUMEN
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.
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Retroalimentación Fisiológica/fisiología , Células Fotorreceptoras Retinianas Conos/fisiología , Células Horizontales de la Retina/fisiología , Sinapsis/fisiología , Animales , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/fisiología , Conexinas/metabolismo , Femenino , Glutamatos/fisiología , Concentración de Iones de Hidrógeno , Masculino , Protones , Receptores AMPA/metabolismo , Células Fotorreceptoras Retinianas Conos/ultraestructura , Células Horizontales de la Retina/ultraestructura , Sinapsis/ultraestructura , Transmisión Sináptica/fisiología , Proteína 2 de Membrana Asociada a Vesículas/genética , Proteína 2 de Membrana Asociada a Vesículas/fisiología , Pez CebraRESUMEN
Degenerative retinal diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD) affect millions of people around the world and lead to irreversible vision loss if left untreated. A number of therapeutic strategies have been developed over the years to treat these diseases or restore vision to already blind patients. In this Review, we describe the development and translational application of light-sensitive chemical photoswitches to restore visual function to the blind retina and compare the translational potential of photoswitches with other vision-restoring therapies. This therapeutic strategy is enabled by an efficient fusion of chemical synthesis, chemical biology, and molecular biology and is broadly applicable to other biological systems. We hope this Review will be of interest to chemists as well as neuroscientists and clinicians.
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Compuestos Azo/uso terapéutico , Ceguera/terapia , Degeneración Retiniana/terapia , Animales , Ceguera/patología , Regulación de la Expresión Génica , Humanos , Luz , Optogenética , Procesos Fotoquímicos , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Neuronas Retinianas/efectos de los fármacos , Neuronas Retinianas/patología , Neuronas Retinianas/efectos de la radiación , Transducción de SeñalRESUMEN
Photoswitchable neurotransmitter receptors are powerful tools for precise manipulation of neural signaling. However, their applications for slow or long-lasting biological events are constrained by fast thermal relaxation of cis-azobenzene. We address this issue by modifying the ortho positions of azobenzene used in the tethered ligand. In cultured cells and intact brain tissue, conjugating inhibitory neurotransmitter receptors with one of the derivatives, dMPC1, allows bidirectional receptor control with 380 and 500 nm light. Moreover, the receptors can be locked in either an active or an inactive state in darkness after a brief pulse of light. This strategy thus enables both rapid and sustained manipulation of neurotransmission, allowing optogenetic interrogation of neural functions over a broad range of time scales.
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Compuestos Azo/metabolismo , Antagonistas de Receptores de GABA-A/metabolismo , Receptores de GABA-A/metabolismo , Transmisión Sináptica/efectos de los fármacos , Animales , Compuestos Azo/síntesis química , Compuestos Azo/química , Compuestos Azo/efectos de la radiación , Células Cultivadas , Femenino , Antagonistas de Receptores de GABA-A/síntesis química , Antagonistas de Receptores de GABA-A/química , Antagonistas de Receptores de GABA-A/efectos de la radiación , Humanos , Ligandos , Masculino , Ratones , Optogenética/métodos , Embarazo , Estereoisomerismo , Rayos UltravioletaRESUMEN
Optogenetics is an emerging technique that enables precise and specific control of biological activities in defined space and time. This technique employs naturally occurring or engineered light-responsive proteins to manipulate the physiological processes of the target cells. To better elucidate the molecular bases of neural functions, substantial efforts have been made to confer light sensitivity onto ion channels and neurotransmitter receptors that mediate signaling events within and between neurons. The chemical strategies for engineering light-switchable channels/receptors and the neuronal implementation of these tools are discussed.
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Canales Iónicos/metabolismo , Neuronas/metabolismo , Optogenética/métodos , Receptores de Neurotransmisores/metabolismo , Transducción de Señal , Animales , Cisteína/genética , Cisteína/metabolismo , Humanos , Canales Iónicos/genética , Luz , Procesos Fotoquímicos , Ingeniería de Proteínas/métodos , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Receptores de Neurotransmisores/genéticaRESUMEN
Lateral inhibition at the first synapse in the retina is important for visual perception, enhancing image contrast, color discrimination, and light adaptation. Despite decades of research, the feedback signal from horizontal cells to photoreceptors that generates lateral inhibition remains uncertain. GABA, protons, or an ephaptic mechanism have all been suggested as the primary mediator of feedback. However, the complexity of the reciprocal cone to horizontal cell synapse has left the identity of the feedback signal an unsolved mystery.
Asunto(s)
Lateralidad Funcional , Modelos Neurológicos , Inhibición Neural , Retina/fisiología , Células Fotorreceptoras Retinianas Conos/fisiología , Células Horizontales de la Retina/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Animales , Canales de Calcio Tipo N/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Proteínas del Tejido Nervioso/agonistas , Proteínas del Tejido Nervioso/metabolismo , Receptores de GABA/metabolismo , Retina/citología , Células Fotorreceptoras Retinianas Conos/citología , Células Horizontales de la Retina/citología , Células Fotorreceptoras Retinianas Bastones/citología , Transmisión Sináptica , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Local anesthetics effectively suppress pain sensation, but most of these compounds act nonselectively, inhibiting activity of all neurons. Moreover, their actions abate slowly, preventing precise spatial and temporal control of nociception. We developed a photoisomerizable molecule, quaternary ammonium-azobenzene-quaternary ammonium (QAQ), that enables rapid and selective optical control of nociception. QAQ is membrane-impermeant and has no effect on most cells, but it infiltrates pain-sensing neurons through endogenous ion channels that are activated by noxious stimuli, primarily TRPV1. After QAQ accumulates intracellularly, it blocks voltage-gated ion channels in the trans form but not the cis form. QAQ enables reversible optical silencing of mouse nociceptive neuron firing without exogenous gene expression and can serve as a light-sensitive analgesic in rats in vivo. Because intracellular QAQ accumulation is a consequence of nociceptive ion-channel activity, QAQ-mediated photosensitization is a platform for understanding signaling mechanisms in acute and chronic pain.
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Canales Iónicos/metabolismo , Nocicepción/efectos de los fármacos , Nocicepción/efectos de la radiación , Animales , Compuestos Azo/química , Compuestos Azo/farmacología , Línea Celular , Células HEK293 , Humanos , Canales Iónicos/antagonistas & inhibidores , Ratones , Terminaciones Nerviosas/efectos de los fármacos , Terminaciones Nerviosas/efectos de la radiación , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/efectos de la radiación , Estimulación Luminosa , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Compuestos de Amonio Cuaternario/química , Compuestos de Amonio Cuaternario/farmacología , Ratas , Receptores Purinérgicos P2X7/metabolismo , Médula Espinal/citología , Médula Espinal/efectos de los fármacos , Médula Espinal/efectos de la radiación , Canales Catiónicos TRPV/metabolismo , Factores de TiempoRESUMEN
Cone photoreceptors and horizontal cells (HCs) have a reciprocal synapse that underlies lateral inhibition and establishes the antagonistic center-surround organization of the visual system. Cones transmit to HCs through an excitatory synapse and HCs feed back to cones through an inhibitory synapse. Here we report that HCs also transmit to cone terminals a positive feedback signal that elevates intracellular Ca(2+) and accelerates neurotransmitter release. Positive and negative feedback are both initiated by AMPA receptors on HCs, but positive feedback appears to be mediated by a change in HC Ca(2+), whereas negative feedback is mediated by a change in HC membrane potential. Local uncaging of AMPA receptor agonists suggests that positive feedback is spatially constrained to active HC-cone synapses, whereas the negative feedback signal spreads through HCs to affect release from surrounding cones. By locally offsetting the effects of negative feedback, positive feedback may amplify photoreceptor synaptic release without sacrificing HC-mediated contrast enhancement.
Asunto(s)
Retroalimentación Fisiológica , Células Fotorreceptoras Retinianas Conos/fisiología , Células Horizontales de la Retina/fisiología , Sinapsis/fisiología , Ambystoma , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Ácido Glutámico/metabolismo , Técnicas In Vitro , Lagartos , Potenciales de la Membrana/efectos de los fármacos , Quinoxalinas/farmacología , Conejos , Receptores AMPA/antagonistas & inhibidores , Receptores AMPA/metabolismo , Receptores de Glutamato/metabolismo , Retina/citología , Retina/efectos de los fármacos , Retina/fisiología , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Horizontales de la Retina/efectos de los fármacos , Células Horizontales de la Retina/metabolismo , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Pez Cebra , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
Bipolar cells of the retina carry visual information from photoreceptors in the outer retina to retinal ganglion cells (RGCs) in the inner retina. Bipolar cells express L-type voltage-gated Ca2+ channels at the synaptic terminal, but generally lack other types of channels capable of regenerative activity. As a result, the flow of information from outer to inner retina along bipolar cell processes is generally passive in nature, with no opportunity for signal boost or amplification along the way. Here we report the surprising discovery that blocking voltage-gated Na+ channels profoundly reduces the synaptic output of one class of bipolar cell, the type 6 ON bipolar cell (CBC6), despite the fact that the CBC6 itself does not express voltage-gated Na+ channels. Instead, CBC6 borrows voltage-gated Na+ channels from its neighbor, the inhibitory AII amacrine cell, with whom it is connected via an electrical synapse. Thus, an inhibitory neuron aids in amplification of an excitatory signal as it moves through the retina, ensuring that small changes in the membrane potential of bipolar cells are reliably passed onto downstream RGCs.
RESUMEN
Rab3A is a synaptic vesicle-associated protein found throughout the nervous system, but its precise function is unknown. Genetic knock-out studies show that Rab3A is not necessary for vesicular release or replenishment at conventional synapses in the brain. Here we explore the function of Rab3A at ribbon synapses in the retina of the tiger salamander (Ambystoma tigrinum). Fluorescently labeled Rab3A, delivered into rods and cones through a patch pipette, binds to and dissociates from synaptic ribbons. Experiments using nonphosphorylatable GDP analogs and a GTPase-deficient Rab3A mutant indicate that ribbon binding and dissociation are governed by a GTP hydrolysis cycle. Paired recordings from presynaptic photoreceptors and postsynaptic OFF-bipolar cells show that the Rab3A mutant blocks synaptic release in an activity-dependent manner, with more frequent stimulation leading to more rapid block. The frequency dependence of block by exogenous Rab3A suggests that it acts competitively with synaptic vesicles to interfere with their resupply to release sites. Together, these findings suggest a crucial role of Rab3A in delivering vesicles to Ca²âº-dependent release sites at ribbon synapses.
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Células Fotorreceptoras de Vertebrados/fisiología , Sinapsis/metabolismo , Vesículas Sinápticas/metabolismo , Proteína de Unión al GTP rab3A/metabolismo , Ambystoma , Animales , Femenino , GTP Fosfohidrolasas/genética , Masculino , Mutación , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/efectos de los fármacos , Retina/metabolismo , Células Bipolares de la Retina/fisiología , Proteína de Unión al GTP rab3A/genética , Proteína de Unión al GTP rab3A/farmacologíaRESUMEN
Rod and cone photoreceptors degenerate in retinitis pigmentosa and age-related macular degeneration, robbing the visual system of light-triggered signals necessary for sight. However, changes in the retina do not stop with the photoreceptors. A stereotypical set of morphological and physiological changes, known as remodeling, occur in downstream retinal neurons. Some aspects of remodeling are homeostatic, with structural or functional changes compensating for partial loss of visual inputs. However, other aspects are nonhomeostatic, corrupting retinal information processing to obscure vision mediated naturally by surviving photoreceptors or artificially by vision-restoration technologies. In this review, I consider the mechanism of remodeling and its consequences for residual and restored visual function; discuss the role of retinoic acid, a critical molecular trigger of detrimental remodeling; and discuss strategies for suppressing retinoic acid biosynthesis or signaling as therapeutic possibilities for mitigating vision loss.
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Neuronas Retinianas , Trastornos de la Visión , Humanos , Retina , Células Fotorreceptoras Retinianas Conos , TretinoinaRESUMEN
The retina, behind the transparent optics of the eye, is the only neural tissue whose physiology and pathology can be non-invasively probed by optical microscopy. The aberrations intrinsic to the mouse eye, however, prevent high-resolution investigation of retinal structure and function in vivo. Optimizing the design of a two-photon fluorescence microscope (2PFM) and sample preparation procedure, we found that adaptive optics (AO), by measuring and correcting ocular aberrations, is essential for resolving putative synaptic structures and achieving three-dimensional cellular resolution in the mouse retina in vivo. Applying AO-2PFM to longitudinal retinal imaging in transgenic models of retinal pathology, we characterized microvascular lesions with sub-capillary details in a proliferative vascular retinopathy model, and found Lidocaine to effectively suppress retinal ganglion cell hyperactivity in a retinal degeneration model. Tracking structural and functional changes at high-resolution longitudinally, AO-2PFM enables microscopic investigations of retinal pathology and pharmacology for disease diagnosis and treatment in vivo.
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Retina , Degeneración Retiniana , Ratones , Animales , Retina/patología , Células Ganglionares de la Retina , Degeneración Retiniana/patología , Microscopía Fluorescente , Óptica y FotónicaRESUMEN
To better understand neural circuits and behavior, microbial opsins have been developed as optogenetic tools for stimulating or inhibiting action potentials with high temporal and spatial precision. However, if we seek a more reductionist understanding of how neuronal circuits operate, we also need high-resolution tools for perturbing the function of synapses. By combining photochemical tools and molecular biology, a wide variety of light-regulated neurotransmitter receptors have been developed, enabling photo-control of excitatory, inhibitory, and modulatory synaptic transmission. Here we focus on photo-control of GABAA receptors, ligand-gated Cl- channels that underlie almost all synaptic inhibition in the mammalian brain. By conjugating a photoswitchable tethered ligand onto a genetically-modified subunit of the GABAA receptor, light-sensitivity can be conferred onto specific isoforms of the receptor. Through gene editing, this attachment site can be knocked into the genome, enabling photocontrol of endogenous GABAA receptors. This strategy can be employed to explore the cell biology and neurophysiology of GABAA receptors. This includes investigating how specific isoforms contribute to synaptic and tonic inhibition and understanding the roles they play in brain development, long-term synaptic plasticity, and learning and memory.
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Optogenética , Receptores de GABA-A , Animales , Encéfalo/metabolismo , Humanos , Ligandos , Mamíferos/genética , Mamíferos/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Sinapsis/metabolismo , Ácido gamma-Aminobutírico/genética , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Rod and cone photoreceptors degenerate in retinitis pigmentosa (RP). While downstream neurons survive, they undergo physiological changes, including accelerated spontaneous firing in retinal ganglion cells (RGCs). Retinoic acid (RA) is the molecular trigger of RGC hyperactivity, but whether this interferes with visual perception is unknown. Here, we show that inhibiting RA synthesis with disulfiram, a deterrent of human alcohol abuse, improves behavioral image detection in vision-impaired mice. In vivo Ca2+ imaging shows that disulfiram sharpens orientation tuning of visual cortical neurons and strengthens fidelity of responses to natural scenes. An RA receptor inhibitor also reduces RGC hyperactivity, sharpens cortical representations, and improves image detection. These findings suggest that photoreceptor degeneration is not the only cause of vision loss in RP. RA-induced corruption of retinal information processing also degrades vision, pointing to RA synthesis and signaling inhibitors as potential therapeutic tools for improving sight in RP and other retinal degenerative disorders.
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Degeneración Retiniana , Retinitis Pigmentosa , Animales , Modelos Animales de Enfermedad , Ratones , Degeneración Retiniana/tratamiento farmacológico , Degeneración Retiniana/etiología , Degeneración Retiniana/metabolismo , Células Ganglionares de la Retina/metabolismo , Retinitis Pigmentosa/tratamiento farmacológico , Retinitis Pigmentosa/metabolismo , Tretinoina/metabolismo , Tretinoina/farmacologíaRESUMEN
Photopharmacological compounds such as azobenzene-based photoswitches have been shown to control the conductivity of ionic channels in a light-dependent manner and are considered a potential strategy to restore vision in patients with end-stage photoreceptor degeneration. Here, we report the effects of DENAQ, a second-generation azobenzene-based photoswitch on retinal ganglion cells (RGC) in canine retinas using multi-electrode array (MEA) recordings (from nine degenerated and six WT retinas). DENAQ treatment conferred increased light sensitivity to RGCs in degenerated canine retinas. RGC light responses were observed in degenerated retinas following ex vivo application of 1 mM DENAQ (n = 6) or after in vivo DENAQ injection (n = 3, 150 µL, 3-10 mM) using 455 nm light at intensities as low as 0.2 mW/cm2. The number of light-sensitive cells and the per cell response amplitude increased with light intensity up to the maximum tested intensity of 85 mW/cm2. Application of DENAQ to degenerated retinas with partially preserved cone function caused appearance of DENAQ-driven responses both in cone-driven and previously non-responsive RGCs, and disappearance of cone-driven responses. Repeated stimulation slowed activation and accelerated recovery of the DENAQ-driven responses. The latter is likely responsible for the delayed appearance of a response to 4 Hz flicker stimulation. Limited aqueous solubility of DENAQ results in focal drug aggregates associated with ocular toxicity. While this limits the therapeutic potential of DENAQ, more potent third-generation photoswitches may be more promising, especially when delivered in a slow-release formulation that prevents drug aggregation.
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The ability to stimulate select neurons in isolated tissue and in living animals is important for investigating their role in circuits and behavior. We show that the engineered light-gated ionotropic glutamate receptor (LiGluR), when introduced into neurons, enables remote control of their activity. Trains of action potentials are optimally evoked and extinguished by 380 nm and 500 nm light, respectively, while intermediate wavelengths provide graded control over the amplitude of depolarization. Light pulses of 1-5 ms in duration at approximately 380 nm trigger precisely timed action potentials and EPSP-like responses or can evoke sustained depolarizations that persist for minutes in the dark until extinguished by a short pulse of approximately 500 nm light. When introduced into sensory neurons in zebrafish larvae, activation of LiGluR reversibly blocks the escape response to touch. Our studies show that LiGluR provides robust control over neuronal activity, enabling the dissection and manipulation of neural circuitry in vivo.
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Conducta Animal/fisiología , Iluminación/métodos , Neuronas/fisiología , Receptores de Ácido Kaínico/fisiología , Potenciales de Acción/fisiología , Potenciales de Acción/efectos de la radiación , Animales , Animales Modificados Genéticamente , Animales Recién Nacidos , Conducta Animal/efectos de la radiación , Células Cultivadas , Cisteína/genética , Relación Dosis-Respuesta en la Radiación , Estimulación Eléctrica/métodos , Potenciales Postsinápticos Excitadores , Hipocampo/citología , Larva , Leucina/genética , Mutación , Neuronas/efectos de los fármacos , Neuronas/efectos de la radiación , Técnicas de Placa-Clamp/métodos , Estimulación Física/métodos , Ratas , Receptores de Ácido Kaínico/genética , Transfección/métodos , Pez Cebra , Receptor de Ácido Kaínico GluK2RESUMEN
Light hyperpolarizes cone photoreceptors, causing synaptic voltage-gated Ca(2+) channels to open infrequently. To understand neurotransmission under these conditions, we determined the number of L-type Ca(2+) channel openings necessary for vesicle fusion at the cone ribbon synapse. Ca(2+) currents (I(Ca)) were activated in voltage-clamped cones, and excitatory postsynaptic currents (EPSCs) were recorded from horizontal cells in the salamander retina slice preparation. Ca(2+) channel number and single-channel current amplitude were calculated by mean-variance analysis of I(Ca). Two different comparisons-one comparing average numbers of release events to average I(Ca) amplitude and the other involving deconvolution of both EPSCs and simultaneously recorded cone I(Ca)-suggested that fewer than three Ca(2+) channel openings accompanied fusion of each vesicle at the peak of release during the first few milliseconds of stimulation. Opening fewer Ca(2+) channels did not enhance fusion efficiency, suggesting that few unnecessary channel openings occurred during strong depolarization. We simulated release at the cone synapse, using empirically determined synaptic dimensions, vesicle pool size, Ca(2+) dependence of release, Ca(2+) channel number, and Ca(2+) channel properties. The model replicated observations when a barrier was added to slow Ca(2+) diffusion. Consistent with the presence of a diffusion barrier, dialyzing cones with diffusible Ca(2+) buffers did not affect release efficiency. The tight clustering of Ca(2+) channels, along with a high-Ca(2+) affinity release mechanism and diffusion barrier, promotes a linear coupling between Ca(2+) influx and vesicle fusion. This may improve detection of small light decrements when cones are hyperpolarized by bright light.
Asunto(s)
Canales de Calcio Tipo L/fisiología , Calcio/metabolismo , Activación del Canal Iónico/fisiología , Luz , Retina/citología , Células Fotorreceptoras Retinianas Conos/fisiología , Sinapsis/fisiología , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Animales , Bario/farmacología , Fenómenos Biofísicos/efectos de los fármacos , Calcio/farmacología , Agonistas de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Quelantes/farmacología , Simulación por Computador , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Técnicas In Vitro , Activación del Canal Iónico/efectos de los fármacos , Masculino , Modelos Biológicos , Nifedipino/farmacología , Técnicas de Placa-Clamp , Probabilidad , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Células Horizontales de la Retina/efectos de los fármacos , Células Horizontales de la Retina/fisiología , Sinapsis/efectos de los fármacos , UrodelosRESUMEN
Currently available optogenetic tools, including microbial light-activated ion channels and transporters, are transforming systems neuroscience by enabling precise remote control of neuronal firing, but they tell us little about the role of indigenous ion channels in controlling neuronal function. Here, we employ a chemical-genetic strategy to engineer light sensitivity into several mammalian K(+) channels that have different gating and modulation properties. These channels provide the means for photoregulating diverse electrophysiological functions. Photosensitivity is conferred on a channel by a tethered ligand photoswitch that contains a cysteine-reactive maleimide (M), a photoisomerizable azobenzene (A), and a quaternary ammonium (Q), a K(+) channel pore blocker. Using mutagenesis, we identify the optimal extracellular cysteine attachment site where MAQ conjugation results in pore blockade when the azobenzene moiety is in the trans but not cis configuration. With this strategy, we have conferred photosensitivity on channels containing Kv1.3 subunits (which control axonal action potential repolarization), Kv3.1 subunits (which contribute to rapid-firing properties of brain neurons), Kv7.2 subunits (which underlie "M-current"), and SK2 subunits (which are Ca(2+)-activated K(+) channels that contribute to synaptic responses). These light-regulated channels may be overexpressed in genetically targeted neurons or substituted for native channels with gene knockin technology to enable precise optopharmacological manipulation of channel function.