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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.Significance Statement Photoreceptor degenerative disorders such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD) lead to vision impairment or blindness. Vision mediated by surviving photoreceptors or artificial vision restoration technologies, rely on bipolar cells retaining normal function despite photoreceptor death. We find that in two animal models of RP, synaptic transmission from both rod and cone bipolar cells is severely impaired owing to diminished voltage-gated calcium current, preventing postsynaptic amacrine cells and retinal ganglion cells from properly receiving and encoding visual information. We find that an inhibitor of the retinoic acid receptor restores both the calcium current and synaptic release from bipolar cells. These discoveries about bipolar cells reveal a new functional deficit in blindness and a potential therapeutically important solution.
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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 , Neurônios , Animais , Ferritinas/metabolismo , Ratos , Neurônios/fisiologia , Masculino , Feminino , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/genética , Células Cultivadas , Campos Magnéticos , Ratos Sprague-Dawley , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Hipocampo/fisiologia , Hipocampo/citologiaRESUMO
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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Raios Infravermelhos , Imagem Óptica , Óxidos/química , Fosfinas/química , Rodaminas/química , Animais , Cálcio/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Retina/citologia , Retina/diagnóstico por imagem , Retina/metabolismoRESUMO
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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Retroalimentação Fisiológica/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Horizontais da Retina/fisiologia , Sinapses/fisiologia , Animais , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/fisiologia , Conexinas/metabolismo , Feminino , Glutamatos/fisiologia , Concentração de Íons de Hidrogênio , Masculino , Prótons , Receptores de AMPA/metabolismo , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Células Horizontais da Retina/ultraestrutura , Sinapses/ultraestrutura , Transmissão Sináptica/fisiologia , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/fisiologia , Peixe-ZebraRESUMO
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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Compostos Azo/uso terapêutico , Cegueira/terapia , Degeneração Retiniana/terapia , Animais , Cegueira/patologia , Regulação da Expressão Gênica , Humanos , Luz , Optogenética , Processos Fotoquímicos , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Neurônios Retinianos/efeitos dos fármacos , Neurônios Retinianos/patologia , Neurônios Retinianos/efeitos da radiação , Transdução de SinaisRESUMO
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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Compostos Azo/metabolismo , Antagonistas de Receptores de GABA-A/metabolismo , Receptores de GABA-A/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Animais , Compostos Azo/síntese química , Compostos Azo/química , Compostos Azo/efeitos da radiação , Células Cultivadas , Feminino , Antagonistas de Receptores de GABA-A/síntese química , Antagonistas de Receptores de GABA-A/química , Antagonistas de Receptores de GABA-A/efeitos da radiação , Humanos , Ligantes , Masculino , Camundongos , Optogenética/métodos , Gravidez , Estereoisomerismo , Raios UltravioletaRESUMO
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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Canais Iônicos/metabolismo , Neurônios/metabolismo , Optogenética/métodos , Receptores de Neurotransmissores/metabolismo , Transdução de Sinais , Animais , Cisteína/genética , Cisteína/metabolismo , Humanos , Canais Iônicos/genética , Luz , Processos Fotoquímicos , Engenharia de Proteínas/métodos , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Receptores de Neurotransmissores/genéticaRESUMO
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.
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Lateralidade Funcional , Modelos Neurológicos , Inibição Neural , Retina/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Horizontais da Retina/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Animais , Canais de Cálcio Tipo N/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/metabolismo , Receptores de GABA/metabolismo , Retina/citologia , Células Fotorreceptoras Retinianas Cones/citologia , Células Horizontais da Retina/citologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Transmissão Sináptica , Ácido gama-Aminobutírico/metabolismoRESUMO
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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Canais Iônicos/metabolismo , Nociceptividade/efeitos dos fármacos , Nociceptividade/efeitos da radiação , Animais , Compostos Azo/química , Compostos Azo/farmacologia , Linhagem Celular , Células HEK293 , Humanos , Canais Iônicos/antagonistas & inibidores , Camundongos , Terminações Nervosas/efeitos dos fármacos , Terminações Nervosas/efeitos da radiação , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/efeitos da radiação , Estimulação Luminosa , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/farmacologia , Ratos , Receptores Purinérgicos P2X7/metabolismo , Medula Espinal/citologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/efeitos da radiação , Canais de Cátion TRPV/metabolismo , Fatores de TempoRESUMO
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.
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Retroalimentação Fisiológica , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Horizontais da Retina/fisiologia , Sinapses/fisiologia , Ambystoma , Animais , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Técnicas In Vitro , Lagartos , Potenciais da Membrana/efeitos dos fármacos , Quinoxalinas/farmacologia , Coelhos , Receptores de AMPA/antagonistas & inibidores , Receptores de AMPA/metabolismo , Receptores de Glutamato/metabolismo , Retina/citologia , Retina/efeitos dos fármacos , Retina/fisiologia , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Horizontais da Retina/efeitos dos fármacos , Células Horizontais da Retina/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Peixe-Zebra , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
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.
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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/fisiologia , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Proteína rab3A de Ligação ao GTP/metabolismo , Ambystoma , Animais , Feminino , GTP Fosfo-Hidrolases/genética , Masculino , Mutação , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismo , Células Bipolares da Retina/fisiologia , Proteína rab3A de Ligação ao GTP/genética , Proteína rab3A de Ligação ao GTP/farmacologiaRESUMO
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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Neurônios Retinianos , Transtornos da Visão , Humanos , Retina , Células Fotorreceptoras Retinianas Cones , TretinoínaRESUMO
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 , Degeneração Retiniana , Camundongos , Animais , Retina/patologia , Células Ganglionares da Retina , Degeneração Retiniana/patologia , Microscopia de Fluorescência , Óptica e FotônicaRESUMO
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 , Animais , Encéfalo/metabolismo , Humanos , Ligantes , Mamíferos/genética , Mamíferos/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Sinapses/metabolismo , Ácido gama-Aminobutírico/genética , Ácido gama-Aminobutírico/farmacologiaRESUMO
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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Degeneração Retiniana , Retinose Pigmentar , Animais , Modelos Animais de Doenças , Camundongos , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/etiologia , Degeneração Retiniana/metabolismo , Células Ganglionares da Retina/metabolismo , Retinose Pigmentar/tratamento farmacológico , Retinose Pigmentar/metabolismo , Tretinoína/metabolismo , Tretinoína/farmacologiaRESUMO
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.
Assuntos
Comportamento Animal/fisiologia , Iluminação/métodos , Neurônios/fisiologia , Receptores de Ácido Caínico/fisiologia , Potenciais de Ação/fisiologia , Potenciais de Ação/efeitos da radiação , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Comportamento Animal/efeitos da radiação , Células Cultivadas , Cisteína/genética , Relação Dose-Resposta à Radiação , Estimulação Elétrica/métodos , Potenciais Pós-Sinápticos Excitadores , Hipocampo/citologia , Larva , Leucina/genética , Mutação , Neurônios/efeitos dos fármacos , Neurônios/efeitos da radiação , Técnicas de Patch-Clamp/métodos , Estimulação Física/métodos , Ratos , Receptores de Ácido Caínico/genética , Transfecção/métodos , Peixe-Zebra , Receptor de GluK2 CainatoRESUMO
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.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Luz , Retina/citologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Sinapses/fisiologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Bário/farmacologia , Fenômenos Biofísicos/efeitos dos fármacos , Cálcio/farmacologia , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Quelantes/farmacologia , Simulação por Computador , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Modelos Biológicos , Nifedipino/farmacologia , Técnicas de Patch-Clamp , Probabilidade , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Células Horizontais da Retina/efeitos dos fármacos , Células Horizontais da Retina/fisiologia , Sinapses/efeitos dos fármacos , UrodelosRESUMO
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.