RESUMEN
Many physiologic effects of l-glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, are mediated via signaling by ionotropic glutamate receptors (iGluRs). These ligand-gated ion channels are critical to brain function and are centrally implicated in numerous psychiatric and neurologic disorders. There are different classes of iGluRs with a variety of receptor subtypes in each class that play distinct roles in neuronal functions. The diversity in iGluR subtypes, with their unique functional properties and physiologic roles, has motivated a large number of studies. Our understanding of receptor subtypes has advanced considerably since the first iGluR subunit gene was cloned in 1989, and the research focus has expanded to encompass facets of biology that have been recently discovered and to exploit experimental paradigms made possible by technological advances. Here, we review insights from more than 3 decades of iGluR studies with an emphasis on the progress that has occurred in the past decade. We cover structure, function, pharmacology, roles in neurophysiology, and therapeutic implications for all classes of receptors assembled from the subunits encoded by the 18 ionotropic glutamate receptor genes. SIGNIFICANCE STATEMENT: Glutamate receptors play important roles in virtually all aspects of brain function and are either involved in mediating some clinical features of neurological disease or represent a therapeutic target for treatment. Therefore, understanding the structure, function, and pharmacology of this class of receptors will advance our understanding of many aspects of brain function at molecular, cellular, and system levels and provide new opportunities to treat patients.
Asunto(s)
Receptores de Glutamato , Receptores Ionotrópicos de Glutamato , Animales , Sistema Nervioso Central , Ácido Glutámico , Humanos , Neurotransmisores , Receptores Ionotrópicos de Glutamato/genéticaRESUMEN
Amacrine cells are interneurons composing the most diverse cell class in the mammalian retina. They help encode visual features such as edges or directed motion by mediating excitatory and inhibitory interactions between input (i.e. bipolar) and output (i.e. ganglion) neurons in the inner plexiform layer (IPL). Like other brain regions, the retina also contains glial cells that contribute to neurotransmitter uptake, metabolic regulation and neurovascular control. Here, we report that in mouse retina (of either sex), an abundant, though previously unstudied inhibitory amacrine cell is coupled directly to Müller glia. Electron microscopic reconstructions of this amacrine type revealed chemical synapses with known retinal cell types and extensive associations with Müller glia, the processes of which often completely ensheathe the neurites of this amacrine cell. Microinjecting small tracer molecules into the somas of these amacrine cells led to selective labelling of nearby Müller glia, leading us to suggest the name "Müller glia-coupled amacrine cell," or MAC. Our data also indicate that MACs release glycine at conventional chemical synapses, and viral retrograde transsynaptic tracing from the dorsal lateral geniculate nucleus (dLGN) showed selective connections between MACs and a subpopulation of RGC types. Visually-evoked responses revealed a strong preference for light increments; these "ON" responses were primarily mediated by excitatory chemical synaptic input and direct electrical coupling with other cells. This initial characterization of the MAC provides the first evidence for neuron-glia coupling in the mammalian retina and identifies the MAC as a potential link between inhibitory processing and glial function.Significance Statement:Gap junctions between pairs of neurons or glial cells are commonly found throughout the nervous system and play multiple roles, including electrical coupling and metabolic exchange. In contrast, gap junctions between neurons and glia cells have rarely been reported and are poorly understood. Here we report the first evidence for neuron-glia coupling in the mammalian retina, specifically between an abundant (but previously unstudied) inhibitory interneuron and Müller glia. Moreover, viral tracing, optogenetics and serial electron microscopy provide new information about the neuron's synaptic partners and physiological responses.
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Directionally tuned signalling in starburst amacrine cell (SAC) dendrites lies at the heart of the circuit that detects the direction of moving stimuli in the mammalian retina. The relative contributions of intrinsic cellular properties and network connectivity to SAC direction selectivity remain unclear. Here we present a detailed connectomic reconstruction of SAC circuitry in mouse retina and describe two previously unknown features of synapse distributions along SAC dendrites: input and output synapses are segregated, with inputs restricted to proximal dendrites; and the distribution of inhibitory inputs is fundamentally different from that observed in rabbit retina. An anatomically constrained SAC network model suggests that SACSAC wiring differences between mouse and rabbit retina underlie distinct contributions of synaptic inhibition to velocity and contrast tuning and receptive field structure. In particular, the model indicates that mouse connectivity enables SACs to encode lower linear velocities that account for smaller eye diameter, thereby conserving angular velocity tuning. These predictions are confirmed with calcium imaging of mouse SAC dendrites responding to directional stimuli.
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Conectoma , Percepción de Movimiento/fisiología , Vías Nerviosas/fisiología , Retina/citología , Retina/fisiología , Sinapsis/fisiología , Células Amacrinas/fisiología , Animales , Calcio/metabolismo , Simulación por Computador , Dendritas/fisiología , Potenciales Postsinápticos Excitadores , Femenino , Masculino , Ratones , Modelos Neurológicos , Inhibición Neural , Estimulación Luminosa , Terminales Presinápticos/metabolismo , Conejos , Retina/anatomía & histología , Células Bipolares de la Retina/fisiología , Especificidad de la EspecieRESUMEN
Inhibition shapes activity and signal processing in neural networks through numerous mechanisms mediated by many different cell types. Here, we examined how one type of GABAergic interneuron in the retina, the A17 amacrine cell, influences visual information processing. Our results suggest that A17s, which make reciprocal feedback inhibitory synapses onto rod bipolar cell (RBC) synaptic terminals, extend the luminance range over which RBC synapses compute temporal contrast and enhance the reliability of contrast signals over this range. Inhibition from other amacrine cells does not influence these computational features. Although A17-mediated feedback is mediated by both GABAA and GABAC receptors, the latter plays the primary role in extending the range of contrast computation. These results identify specific functions for an inhibitory interneuron subtype, as well as specific synaptic receptors, in a behaviorally relevant neural computation.
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Células Amacrinas/fisiología , Retroalimentación Fisiológica/fisiología , Neuronas GABAérgicas/fisiología , Inhibición Neural/fisiología , Células Bipolares de la Retina/fisiología , Sinapsis/fisiología , Animales , Ratas , Ratas Sprague-DawleyRESUMEN
UNLABELLED: Feedforward (FF) inhibition is a common motif in many neural networks. Typically, excitatory inputs drive both principal neurons and interneurons; the interneurons then inhibit the principal neurons, thereby regulating the strength and timing of the FF signal. The interneurons introduce a likely nonlinear processing step that could distort the excitation/inhibition (E/I) ratio in the principal neuron, potentially degrading the reliability of computation in the circuit. In the retina, FF inhibition is an essential feature of the circuitry underlying direction selectivity (DS): glutamatergic bipolar cells (BCs) provide excitatory input to direction-selective ganglion cells (DSGCs) and GABAergic starburst amacrine cells (SACs), and the SACs then provide FF inhibition onto DSGCs. Robust DS computation requires a consistent synaptic E/I ratio in the DSGC in various visual conditions. Here, we show in mouse retina that the E/I ratio is maintained in DSGCs over a wide stimulus contrast range due to compensatory mechanisms in the diverse population of presynaptic BCs. BC inputs to SACs exhibit higher contrast sensitivity, so that the subsequent nonlinear transformation in SACs reduces the contrast sensitivity of FF inhibition to match the sensitivity of direct excitatory inputs onto DSGCs. Measurements of light-evoked responses from individual BC synaptic terminals suggest that the distinct sensitivity of BC inputs reflects different contrast sensitivity between BC subtypes. Numerical simulations suggest that this network arrangement is crucial for reliable DS computation. SIGNIFICANCE STATEMENT: Properly balanced excitation and inhibition are essential for many neuronal computations across brain regions. Feedforward inhibition circuitry, in which a common excitatory source drives both the principal cell and an interneuron, is a typical mechanism by which neural networks maintain this balance. Feedforward circuits may become imbalanced at low stimulation levels, however, if the excitatory drive is too weak to overcome the activation threshold in the interneuron. Here we reveal how excitation and inhibition remain balanced in direction selective ganglion cells in the mouse retina over a wide visual stimulus range.
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Retroalimentación Fisiológica/fisiología , Percepción de Movimiento/fisiología , Red Nerviosa/fisiología , Inhibición Neural/fisiología , Plasticidad Neuronal/fisiología , Neuronas Retinianas/fisiología , Animales , Femenino , Masculino , Ratones , Estimulación Luminosa/métodos , Campos VisualesRESUMEN
Microglia, the principal resident immune cell of the CNS, exert significant influence on neurons during development and in pathological situations. However, if and how microglia contribute to normal neuronal function in the mature uninjured CNS is not well understood. We used the model of the adult mouse retina, a part of the CNS amenable to structural and functional analysis, to investigate the constitutive role of microglia by depleting microglia from the retina in a sustained manner using genetic methods. We discovered that microglia are not acutely required for the maintenance of adult retinal architecture, the survival of retinal neurons, or the laminar organization of their dendritic and axonal compartments. However, sustained microglial depletion results in the degeneration of photoreceptor synapses in the outer plexiform layer, leading to a progressive functional deterioration in retinal light responses. Our results demonstrate that microglia are constitutively required for the maintenance of synaptic structure in the adult retina and for synaptic transmission underlying normal visual function. Our findings on constitutive microglial function are relevant in understanding microglial contributions to pathology and in the consideration of therapeutic interventions that reduce or perturb constitutive microglial function. SIGNIFICANCE STATEMENT: Microglia, the principal resident immune cell population in the CNS, has been implicated in diseases in the brain and retina. However, how they contribute to the everyday function of the CNS is unclear. Using the model of the adult mouse retina, we examined the constitutive role of microglia by depleting microglia from the retina. We found that in the absence of microglia, retinal neurons did not undergo overt cell death or become structurally disorganized in their processes. However, connections between neurons called synapses begin to break down, leading to a decreased ability of the retina to transmit light responses. Our results indicate that retinal microglia contribute constitutively to the maintenance of synapses underlying healthy vision.
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Microglía/fisiología , Neuronas/fisiología , Retina/citología , Sinapsis/fisiología , Animales , Muerte Celular/genética , Modelos Animales de Enfermedad , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Proteínas del Ojo/metabolismo , Femenino , Expresión Génica/genética , Regulación de la Expresión Génica/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Nistagmo Optoquinético/genética , ARN no Traducido/genética , ARN no Traducido/metabolismo , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8A/metabolismo , Sinapsis/genética , Trastornos de la Visión/genética , Trastornos de la Visión/patología , Trastornos de la Visión/fisiopatología , Vías Visuales/fisiologíaRESUMEN
Synaptic ribbons are presynaptic protein structures found at many synapses that convey graded, "analog" sensory signals in the visual, auditory, and vestibular pathways. Ribbons, typically anchored to the presynaptic membrane and surrounded by tethered synaptic vesicles, are thought to regulate or facilitate vesicle delivery to the presynaptic membrane. No direct evidence exists, however, to indicate how vesicles interact with the ribbon or, once attached, move along the ribbon's surface to reach the presynaptic release sites at its base. To address these questions, we have created, validated, and tested a passive vesicle diffusion model of retinal rod bipolar cell ribbon synapses. We used axial (bright-field) electron tomography in the scanning transmission electron microscopy to obtain 3D structures of rat rod bipolar cell terminals in 1-µm-thick sections of retinal tissue at an isotropic spatial resolution of â¼3 nm. The resulting structures were then incorporated with previously published estimates of vesicle diffusion dynamics into numerical simulations that accurately reproduced electrophysiologically measured vesicle release/replenishment rates and vesicle pool sizes. The simulations suggest that, under physiologically realistic conditions, diffusion of vesicles crowded on the ribbon surface gives rise to a flow field that enhances delivery of vesicles to the presynaptic membrane without requiring an active transport mechanism. Numerical simulations of ribbon-vesicle interactions predict that transient binding and unbinding of multiple tethers to each synaptic vesicle may achieve sufficiently tight association of vesicles to the ribbon while permitting the fast diffusion along the ribbon that is required to sustain high release rates.
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Simulación por Computador , Modelos Neurológicos , Neurotransmisores/metabolismo , Terminales Presinápticos/fisiología , Células Bipolares de la Retina/fisiología , Vesículas Sinápticas/metabolismo , Animales , Difusión , Tomografía con Microscopio Electrónico , Femenino , Masculino , Método de Montecarlo , Terminales Presinápticos/ultraestructura , Ratas , Ratas Sprague-Dawley , Células Bipolares de la Retina/ultraestructuraRESUMEN
Sensory processing in the auditory system requires that synapses, neurons, and circuits encode information with particularly high temporal and spectral precision. In the amphibian papillia, sound frequencies up to 1 kHz are encoded along a tonotopic array of hair cells and transmitted to afferent fibers via fast, repetitive synaptic transmission, thereby promoting phase locking between the presynaptic and postsynaptic cells. Here, we have combined serial section electron microscopy, paired electrophysiological recordings, and Monte Carlo diffusion simulations to examine novel mechanisms that facilitate fast synaptic transmission in the inner ear of frogs (Rana catesbeiana and Rana pipiens). Three-dimensional anatomical reconstructions reveal specialized spine-like contacts between individual afferent fibers and hair cells that are surrounded by large, open regions of extracellular space. Morphologically realistic diffusion simulations suggest that these local enlargements in extracellular space speed transmitter clearance and reduce spillover between neighboring synapses, thereby minimizing postsynaptic receptor desensitization and improving sensitivity during prolonged signal transmission. Additionally, evoked EPSCs in afferent fibers are unaffected by glutamate transporter blockade, suggesting that transmitter diffusion and dilution, and not uptake, play a primary role in speeding neurotransmission and ensuring fidelity at these synapses.
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Células Ciliadas Auditivas/citología , Neurotransmisores/metabolismo , Transducción de Señal/fisiología , Transmisión Sináptica/fisiología , Algoritmos , Animales , Ácido Aspártico/farmacología , Benzotiadiazinas/farmacología , Calcio/metabolismo , Simulación por Computador , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Femenino , Ácido Glutámico/metabolismo , Células Ciliadas Auditivas/ultraestructura , Masculino , Microscopía Electrónica , Modelos Neurológicos , Técnicas de Placa-Clamp , Rana catesbeiana , Transducción de Señal/efectos de los fármacosRESUMEN
Neuronal microcircuits, small, localized signaling motifs involving two or more neurons, underlie signal processing and computation in the brain. Compartmentalized signaling within a neuron may enable it to participate in multiple, independent microcircuits. Each A17 amacrine cell in the mammalian retina contains within its dendrites hundreds of synaptic feedback microcircuits that operate independently to modulate feedforward signaling in the inner retina. Each of these microcircuits comprises a small (<1 µm) synaptic varicosity that typically receives one excitatory synapse from a presynaptic rod bipolar cell (RBC) and returns two reciprocal inhibitory synapses back onto the same RBC terminal. Feedback inhibition from the A17 sculpts the feedforward signal from the RBC to the AII, a critical component of the circuitry mediating night vision. Here, we show that the two inhibitory synapses from the A17 to the RBC express kinetically distinct populations of GABA receptors: rapidly activating GABA(A)Rs are enriched at one synapse while more slowly activating GABA(C)Rs are enriched at the other. Anatomical and electrophysiological data suggest that macromolecular complexes of voltage-gated (Cav) channels and Ca(2+)-activated K(+) channels help to regulate GABA release from A17 varicosities and limit GABA(C)R activation under certain conditions. Finally, we find that selective elimination of A17-mediated feedback inhibition reduces the signal to noise ratio of responses to dim flashes recorded in the feedforward pathway (i.e., the AII amacrine cell). We conclude that A17-mediated feedback inhibition improves the signal to noise ratio of RBC-AII transmission near visual threshold, thereby improving visual sensitivity at night.
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Inhibición Neural/fisiología , Retina/citología , Retina/fisiología , Sinapsis/fisiología , Visión Ocular/fisiología , Animales , Canales de Calcio Tipo L/metabolismo , Oscuridad , Retroalimentación Fisiológica/fisiología , Inmunohistoquímica , Inmunoprecipitación , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp , Estimulación Luminosa , Ratas Sprague-Dawley , Receptores de GABA/metabolismo , Umbral Sensorial/fisiología , Técnicas de Cultivo de TejidosRESUMEN
GLT-1, the major glutamate transporter in the adult brain, is abundantly expressed in astrocytic processes enveloping synapses. By limiting glutamate escape into the surrounding neuropil, GLT-1 preserves the spatial specificity of synaptic signaling. Here we show that the amyloid-ß peptide Aß1-42 markedly prolongs the extracellular lifetime of synaptically released glutamate by reducing GLT-1 surface expression in mouse astrocytes and that this effect is prevented by the vitamin E derivative Trolox. These findings indicate that astrocytic glutamate transporter dysfunction may play an important role in the pathogenesis of Alzheimer's disease and suggest possible mechanisms by which several current treatment strategies could protect against the disease.
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Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Astrocitos/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Ácido Glutámico/metabolismo , Fragmentos de Péptidos/metabolismo , Sinapsis/metabolismo , Péptidos beta-Amiloides/farmacología , Animales , Antioxidantes/farmacología , Cromanos/farmacología , Interacciones Farmacológicas , Espacio Extracelular/metabolismo , Femenino , Hipocampo/citología , Hipocampo/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Neurológicos , Neuronas/metabolismo , Técnicas de Cultivo de Órganos , Fragmentos de Péptidos/farmacología , Sinapsis/efectos de los fármacosRESUMEN
Many inherited retinal diseases target photoreceptors, which transduce light into a neural signal that is processed by the downstream visual system. As photoreceptors degenerate, physiological and morphological changes to retinal synapses and circuitry reduce sensitivity and increase noise, degrading visual signal fidelity. Here, we pharmacologically targeted the first synapse in the retina in an effort to reduce circuit noise without sacrificing visual sensitivity. We tested a strategy to partially replace the neurotransmitter lost when photoreceptors die with an agonist of receptors that ON bipolars cells use to detect glutamate released from photoreceptors. In rd10 mice, which express a photoreceptor mutation that causes retinitis pigmentosa (RP), we found that a low dose of the mGluR6 agonist l-2-amino-4-phosphonobutyric acid (L-AP4) reduced pathological noise induced by photoreceptor degeneration. After making in vivo electroretinogram recordings in rd10 mice to characterize the developmental time course of visual signal degeneration, we examined effects of L-AP4 on sensitivity and circuit noise by recording in vitro light-evoked responses from individual retinal ganglion cells (RGCs). L-AP4 decreased circuit noise evident in RGC recordings without significantly reducing response amplitudes, an effect that persisted over the entire time course of rod photoreceptor degeneration. Subsequent in vitro recordings from rod bipolar cells (RBCs) showed that RBCs are more depolarized in rd10 retinas, likely contributing to downstream circuit noise and reduced synaptic gain, both of which appear to be ameliorated by hyperpolarizing RBCs with L-AP4. These beneficial effects may reduce pathological circuit remodeling and preserve the efficacy of therapies designed to restore vision.
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The neurovascular unit (NVU), comprising vascular, glial and neural elements, supports the energetic demands of neural computation, but this aspect of the retina's trilaminar vessel network is poorly understood. Only the innermost vessel layer - the superficial vascular plexus (SVP) - is ensheathed by astrocytes, like brain capillaries, whereas glial ensheathment in other layers derives from radial Müller glia. Using serial electron microscopy reconstructions from mouse and primate retina, we find that Müller processes cover capillaries in a tessellating pattern, mirroring the tiled astrocytic endfeet wrapping brain capillaries. However, gaps in the Müller sheath, found mainly in the intermediate vascular plexus (IVP), permit different neuron types to contact pericytes and the endothelial cells directly. Pericyte somata are a favored target, often at spine-like structures with a reduced or absent vascular basement lamina. Focal application of adenosine triphosphate (ATP) to the vitreal surface evoked Ca2+ signals in Müller sheaths in all three vascular layers. Pharmacological experiments confirmed that Müller sheaths express purinergic receptors that, when activated, trigger intracellular Ca2+ signals that are amplified by IP3-controlled intracellular Ca2+ stores. When rod photoreceptors die in a mouse model of retinitis pigmentosa (rd10), Müller sheaths dissociate from the deep vascular plexus (DVP) but are largely unchanged within the IVP or SVP. Thus, Müller glia interact with retinal vessels in a laminar, compartmentalized manner: glial sheathes are virtually complete in the SVP but fenestrated in the IVP, permitting direct neural-to-vascular contacts. In the DVP, the glial sheath is only modestly fenestrated and is vulnerable to photoreceptor degeneration.
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Fast synaptic transmission requires tight colocalization of Ca(2+) channels and neurotransmitter vesicles. It is generally thought that Ca(2+) channels are expressed abundantly in presynaptic active zones, that vesicles within the same active zone have similar release properties, and that significant vesicle depletion only occurs at synapses with high release probability. Here we show, at excitatory CA3âCA1 synapses in mouse hippocampus, that release from individual vesicles is generally triggered by only one Ca(2+) channel and that only few functional Ca(2+) channels may be spread in the active zone at variable distances to neighboring neurotransmitter vesicles. Using morphologically realistic Monte Carlo simulations, we show that this arrangement leads to a widely heterogeneous distribution of release probability across the vesicles docked at the active zone, and that depletion of the vesicles closest to Ca(2+) channels can account for the Ca(2+) dependence of short-term plasticity at these synapses. These findings challenge the prevailing view that efficient synaptic transmission requires numerous presynaptic Ca(2+) channels in the active zone, and indicate that the relative arrangement of Ca(2+) channels and vesicles contributes to the heterogeneity of release probability within and across synapses and to vesicle depletion at small central synapses with low average release probability.
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Canales de Calcio/metabolismo , Modelos Neurológicos , Neuronas/metabolismo , Neurotransmisores/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Animales , Región CA1 Hipocampal/metabolismo , Región CA3 Hipocampal/metabolismo , Potenciales Postsinápticos Excitadores , Ratones , Ratones Endogámicos C57BL , Método de Montecarlo , Técnicas de Placa-Clamp , Vesículas Sinápticas/metabolismoRESUMEN
Inward rectifying potassium (Kir) channels participate in regulating potassium concentration (K(+)) in the central nervous system (CNS), including in the retina. We explored the contribution of Kir channels to retinal function by delivering Kir antibodies (Kir-Abs) into the rat eye in vivo to interrupt channel activity. Kir-Abs were coupled to a peptide carrier to reach intracellular epitopes. Functional effects were evaluated by recording the scotopic threshold response (STR) and photopic negative response (PhNR) of the electroretinogram (ERG) noninvasively with an electrode on the cornea to determine activity of the rod and cone pathways, respectively. Intravitreal delivery of Kir2.1-Ab coupled to the peptide carrier diminished these ERG responses equivalent to dimming the stimulus 10- to 100-fold. Immunohistochemistry (IHC) showed Kir2.1 immunostaining of retinal bipolar cells (BCs) matching the labeling pattern obtained with conventional IHC of applying Kir2.1-Ab to fixed retinal sections postmortem. Whole-cell voltage-clamp BC recordings in rat acute retinal slices showed suppression of barium-sensitive Kir2.1 currents upon inclusion of Kir2.1-Ab in the patch pipette. The in vivo functional and structural results implicate a contribution of Kir2.1 channel activity in these electronegative ERG potentials. Studies with Kir4.1-Ab administered in vivo also suppressed the ERG components and showed immunostaining of Müller cells. The strategy of administering Kir antibodies in vivo, coupled to a peptide carrier to facilitate intracellular delivery, identifies roles for Kir2.1 and Kir4.1 in ERG components arising in the proximal retina and suggests this approach could be of further value in research.
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Canales de Potasio/metabolismo , Retina/metabolismo , Animales , Anticuerpos/metabolismo , Anticuerpos/farmacología , Anticuerpos/fisiología , Bario/metabolismo , Bario/farmacología , Bario/fisiología , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/fisiología , Citoplasma/metabolismo , Citoplasma/fisiología , Electrorretinografía , Degeneración Nerviosa/metabolismo , Neuroglía/metabolismo , Neuroglía/fisiología , Técnicas de Placa-Clamp , Potasio/metabolismo , Potasio/farmacología , Potasio/fisiología , Canales de Potasio/farmacología , Canales de Potasio/fisiología , Canales de Potasio de Rectificación Interna , Ratas , Ratas Mutantes , Retina/efectos de los fármacos , Retina/fisiología , Retinaldehído/metabolismo , Retinaldehído/farmacología , Retinaldehído/fisiologíaRESUMEN
While Generative Adversarial Networks (GANs) can now reliably produce realistic images in a multitude of imaging domains, they are ill-equipped to model thin, stochastic textures present in many large 3D fluorescent microscopy (FM) images acquired in biological research. This is especially problematic in neuroscience where the lack of ground truth data impedes the development of automated image analysis algorithms for neurons and neural populations. We therefore propose an unpaired mesh-to-image translation methodology for generating volumetric FM images of neurons from paired ground truths. We start by learning unique FM styles efficiently through a Gramian-based discriminator. Then, we stylize 3D voxelized meshes of previously reconstructed neurons by successively generating slices. As a result, we effectively create a synthetic microscope and can acquire realistic FM images of neurons with control over the image content and imaging configurations. We demonstrate the feasibility of our architecture and its superior performance compared to state-of-the-art image translation architectures through a variety of texture-based metrics, unsupervised segmentation accuracy, and an expert opinion test. In this study, we use 2 synthetic FM datasets and 2 newly acquired FM datasets of retinal neurons.
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Microscopía , Mallas Quirúrgicas , Humanos , Imagenología Tridimensional/métodos , Procesamiento de Imagen Asistido por Computador/métodos , NeuronasRESUMEN
In the retina, rod and cone pathways mediate visual signals over a billion-fold range in luminance. AII ("A-two") amacrine cells (ACs) receive signals from both pathways via different bipolar cells, enabling AIIs to operate at night and during the day. Previous work has examined luminance-dependent changes in AII gap junction connectivity, but less is known about how surrounding circuitry shapes AII receptive fields across light levels. Here, we report that moderate contrast stimuli elicit surround inhibition in AIIs under all but the dimmest visual conditions, due to actions of horizontal cells and at least two ACs that inhibit presynaptic bipolar cells. Under photopic (daylight) conditions, surround inhibition transforms AII response kinetics, which are inherited by downstream ganglion cells. Ablating neuronal nitric oxide synthase type-1 (nNOS-1) ACs removes AII surround inhibition under mesopic (dusk/dawn), but not photopic, conditions. Our findings demonstrate how multiple layers of neural circuitry interact to encode signals across a wide physiological range.
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Células Amacrinas , Retina , Células Amacrinas/fisiología , Retina/fisiología , Células Fotorreceptoras Retinianas Conos , Uniones Comunicantes/fisiologíaRESUMEN
Experience regulates synapse formation and function across sensory circuits. How inhibitory synapses in the mammalian retina are sculpted by visual cues remains unclear. By use of a sensory deprivation paradigm, we find that visual cues regulate maturation of two GABA synapse types (GABAA and GABAC receptor synapses), localized across the axon terminals of rod bipolar cells (RBCs)-second-order retinal neurons integral to the night-vision circuit. Lack of visual cues causes GABAA synapses at RBC terminals to retain an immature receptor configuration with slower response profiles and prevents receptor recruitment at GABAC synapses. Additionally, the organizing protein for both these GABA synapses, LRRTM4, is not clustered at dark-reared RBC synapses. Ultrastructurally, the total number of ribbon-output/inhibitory-input synapses across RBC terminals remains unaltered by sensory deprivation, although ribbon synapse output sites are misarranged when the circuit develops without visual cues. Intrinsic electrophysiological properties of RBCs and expression of chloride transporters across RBC terminals are additionally altered by sensory deprivation. Introduction to normal 12-h light-dark housing conditions facilitates maturation of dark-reared RBC GABA synapses and restoration of intrinsic RBC properties, unveiling a new element of light-dependent retinal cellular and synaptic plasticity.
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Retina , Privación Sensorial , Animales , Retina/fisiología , Células Bipolares de la Retina/fisiología , Terminales Presinápticos/metabolismo , Sinapsis/fisiología , Ácido gamma-Aminobutírico/metabolismo , MamíferosRESUMEN
Synaptic plasticity is a fundamental feature of the CNS that controls the magnitude of signal transmission between communicating cells. Many electrical synapses exhibit substantial plasticity that modulates the degree of coupling within groups of neurons, alters the fidelity of signal transmission, or even reconfigures functional circuits. In several known examples, such plasticity depends on calcium and is associated with neuronal activity. Calcium-driven signaling is known to promote potentiation of electrical synapses in fish Mauthner cells, mammalian retinal AII amacrine cells, and inferior olive neurons, and to promote depression in thalamic reticular neurons. To measure local calcium dynamics in situ, we developed a transgenic mouse expressing a GCaMP calcium biosensor fused to Connexin 36 (Cx36) at electrical synapses. We examined the sources of calcium for activity-dependent plasticity in retina slices using confocal or Super-Resolution Radial Fluctuations imaging. More than half of Cx36-GCaMP gap junctions responded to puffs of glutamate with transient increases in fluorescence. The responses were strongly dependent on NMDA receptors, in keeping with known activity-dependent signaling in some amacrine cells. We also found that some responses depended on the activity of voltage-gated calcium channels, representing a previously unrecognized source of calcium to control retinal electrical synaptic plasticity. The high prevalence of calcium signals at electrical synapses in response to glutamate application indicates that a large fraction of electrical synapses has the potential to be regulated by neuronal activity. This provides a means to tune circuit connectivity dynamically based on local activity.
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Calcio , Uniones Comunicantes , Ratones , Animales , Uniones Comunicantes/fisiología , Retina , Conexinas/genética , Células Amacrinas/fisiología , Mamíferos , Proteína delta-6 de Union ComunicanteRESUMEN
Glutamate uptake by transporters expressed in astrocytes combines with synaptic structure to regulate the time that synaptically released glutamate remains in the extracellular space and, consequently, the duration and location of postsynaptic receptor activation. Both factors change greatly in the rodent hippocampus during the second postnatal week when most synapses become established and begin to mature, processes that are influenced by synaptically released glutamate. Transporter expression increases, potentially speeding removal of synaptically released glutamate, whereas extracellular space decreases, thereby slowing dilution. We investigated whether these competing changes influence the glutamate concentration time course and postsynaptic responses in the CA1 region of the mouse hippocampus during this critical period of synaptic development. Our results suggest that the glutamate concentration time course remains relatively consistent over this period, although the primary mechanisms regulating glutamate clearance change. Before the second postnatal week, clearance of synaptically released glutamate depends primarily on diffusion into large extracellular spaces, whereas later in development it relies more on increased uptake capacity. Thus, increased transporter expression during this period accompanies structural changes in the neuropil, preserving a relatively consistent glutamate concentration time course and ensuring that postsynaptic receptor activation remains brief and primarily localized to receptors close to release sites.
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Ácido Glutámico/metabolismo , Sinapsis/metabolismo , Algoritmos , Animales , Animales Recién Nacidos , Astrocitos/metabolismo , Western Blotting , Difusión , Electroforesis en Gel de Poliacrilamida , Fenómenos Electrofisiológicos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Inmunohistoquímica , Luminiscencia , Masculino , Ratones , Microscopía Electrónica , Neurópilo/metabolismo , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas de Transporte Vesicular de Glutamato/metabolismoRESUMEN
Feedback inhibition at reciprocal synapses between A17 amacrine cells and rod bipolar cells (RBCs) shapes light-evoked responses in the retina. Glutamate-mediated excitation of A17 cells elicits GABA (gamma-aminobutyric acid)-mediated inhibitory feedback onto RBCs, but the mechanisms that underlie GABA release from the dendrites of A17 cells are unknown. If, as observed at all other synapses studied, voltage-gated calcium channels (VGCCs) couple membrane depolarization to neurotransmitter release, feedforward excitatory postsynaptic potentials could spread through A17 dendrites to elicit 'surround' feedback inhibitory transmission at neighbouring synapses. Here we show, however, that GABA release from A17 cells in the rat retina does not depend on VGCCs or membrane depolarization. Instead, calcium-permeable AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (AMPARs), activated by glutamate released from RBCs, provide the calcium influx necessary to trigger GABA release from A17 cells. The AMPAR-mediated calcium signal is amplified by calcium-induced calcium release (CICR) from intracellular calcium stores. These results describe a fast synapse that operates independently of VGCCs and membrane depolarization and reveal a previously unknown form of feedback inhibition within a neural circuit.