RESUMEN
From mouse to primate, there is a striking discontinuity in our current understanding of the neural coding of motion direction. In non-primate mammals, directionally selective cell types and circuits are a signature feature of the retina, situated at the earliest stage of the visual process. In primates, by contrast, direction selectivity is a hallmark of motion processing areas in visual cortex, but has not been found in the retina, despite significant effort. Here we combined functional recordings of light-evoked responses and connectomic reconstruction to identify diverse direction-selective cell types in the macaque monkey retina with distinctive physiological properties and synaptic motifs. This circuitry includes an ON-OFF ganglion cell type, a spiking, ON-OFF polyaxonal amacrine cell and the starburst amacrine cell, all of which show direction selectivity. Moreover, we discovered that macaque starburst cells possess a strong, non-GABAergic, antagonistic surround mediated by input from excitatory bipolar cells that is critical for the generation of radial motion sensitivity in these cells. Our findings open a door to investigation of a precortical circuitry that computes motion direction in the primate visual system.
Asunto(s)
Conectoma , Macaca , Retina , Células Amacrinas/fisiología , Animales , Potenciales Evocados Visuales/fisiología , Macaca/fisiología , Mamíferos , Ratones , Primates/fisiología , Retina/fisiología , Células Ganglionares de la Retina/fisiología , Sinapsis/fisiologíaRESUMEN
The mammalian retina contains a small number of retinal ganglion cells that express melanopsin, a retinal based visual pigment, and generate a depolarizing response to light in the absence of rod and cone driven synaptic input; hence they are referred to as intrinsically photosensitive retinal ganglion cells (ipRGCs). They have been shown to be comprised of a number of sub-types and to provide luminance information that participates primarily in a variety of non-imaging forming visual functions. Here I review what is currently known about the cascade of events that couple the photoisomerization of melanopsin to the opening of a non-selective cation channel. While these events conform in a general sense to the prevailing model for invertebrate phototransduction, in which visual pigment signals through a G protein of the Gq class and a phospholipase C cascade to open a TRPC type ion channel, none of the molecular elements in the melanopsin transduction process have been unequivocally identified. This has given rise to the possibility that the underlying mechanism responsible for intrinsic photosensitivity is not same in all ipRGC sub-types and to the recognition that signal transduction in ipRGCs is more complex than originally thought.
Asunto(s)
Fototransducción , Células Ganglionares de la Retina/fisiología , Animales , Humanos , Luz , Fototransducción/efectos de la radiación , Células Ganglionares de la Retina/efectos de la radiaciónRESUMEN
Following photoreceptor degeneration, ON and OFF retinal ganglion cells (RGCs) in the rd-1/rd-1 mouse receive rhythmic synaptic input that elicits bursts of action potentials at â¼ 10 Hz. To characterize the properties of this activity, RGCs were targeted for paired recording and morphological classification as either ON alpha, OFF alpha or non-alpha RGCs using two-photon imaging. Identified cell types exhibited rhythmic spike activity. Cross-correlation of spike trains recorded simultaneously from pairs of RGCs revealed that activity was correlated more strongly between alpha RGCs than between alpha and non-alpha cell pairs. Bursts of action potentials in alpha RGC pairs of the same type, i.e. two ON or two OFF cells, were in phase, while bursts in dissimilar alpha cell types, i.e. an ON and an OFF RGC, were 180 degrees out of phase. This result is consistent with RGC activity being driven by an input that provides correlated excitation to ON cells and inhibition to OFF cells. A2 amacrine cells were investigated as a candidate cellular mechanism and found to display 10 Hz oscillations in membrane voltage and current that persisted in the presence of antagonists of fast synaptic transmission and were eliminated by tetrodotoxin. Results support the conclusion that the rhythmic RGC activity originates in a presynaptic network of electrically coupled cells including A2s via a Na(+)-channel dependent mechanism. Network activity drives out of phase oscillations in ON and OFF cone bipolar cells, entraining similar frequency fluctuations in RGC spike activity over an area of retina that migrates with changes in the spatial locus of the cellular oscillator.
Asunto(s)
Degeneración Retiniana/fisiopatología , Células Ganglionares de la Retina/fisiología , Potenciales de Acción/fisiología , Animales , Electrofisiología , RatonesRESUMEN
The amplitude and time course of stimulus-evoked second messenger signals carried by intracellular changes in free calcium ([Ca](free)) depend on the total influx of Ca(2+), the fraction bound to endogenous buffer and the rate of extrusion. Estimates of the values of these three parameters in proximal dendrites of 15 mouse α retinal ganglion cells were made using the "added buffer" method and found to vary greatly from one experiment to the next. The variations in the measured parameters were strongly correlated across the sample of cells. This reduced the variability in the amplitude and time course of the dendritic Ca(2+) signal and suggests that the expression of Ca(2+) channels, binding proteins and extrusion mechanisms is homeostatically coordinated to maintain the amplitude and kinetics of the Ca(2+) signal within a physiologically appropriate range.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Dendritas/metabolismo , Células Ganglionares de la Retina/metabolismo , Potenciales de Acción/fisiología , Animales , Hipocampo/metabolismo , Masculino , RatonesRESUMEN
Here we review evidence that loss of photoreceptors due to degenerative retinal disease causes an increase in the rate of spontaneous ganglion spike discharge. Information about persistent spike activity is important since it is expected to add noise to the communication between the eye and the brain and thus impact the design and effective use of retinal prosthetics for restoring visual function in patients blinded by disease. Patch-clamp recordings from identified types of ON and OFF retinal ganglion cells in the adult (36-210 d old) rd1 mouse show that the ongoing oscillatory spike activity in both cell types is driven by strong rhythmic synaptic input from presynaptic neurons that is blocked by CNQX. The recurrent synaptic activity may arise in a negative feedback loop between a bipolar cell and an amacrine cell that exhibits resonant behavior and oscillations in membrane potential when the normal balance between excitation and inhibition is disrupted by the absence of photoreceptor input.
RESUMEN
Second-harmonic generation (SHG) by membrane-incorporated probes is a nonlinear optical signal that is voltage-sensitive and the basis of a sensitive method for imaging membrane potential. The voltage dependence of SHG by four different probes, three retinoids (all-trans retinal), and two new retinal analogs, 3-methyl-7-(4'-dimethylamino-phenyl)-2,4,6-heptatrienal (AR-3) and 3,7-dimethyl-9-(4'-dimethylamino-phenyl)-2,4,6,8-nonatetraenal (AR-4), and a styryl dye (FM4-64), were compared in HEK-293 cells. Results were analyzed by fitting data with an expression based on an electrooptic mechanism for SHG, which depends on the complex-valued first- and second-order nonlinear electric susceptibilities (χ² and χ³) of the probe. This gave values for the voltage sensitivity at the cell's resting potential, the voltage where the SHG is minimal, and the amplitude of the signal at that voltage for each of the four compounds. These measures show that χ² and χ³ are complex numbers for all compounds except all-trans retinal, consistent with the proximities of excitation and/or emission wavelengths to molecular resonances. Estimates of probe orientation and location in the membrane electric field show that, for the far-from-resonance case, the shot noise-limited signal/noise ratio depends on the location of the probe in the membrane, and on χ³ but not on χ².
Asunto(s)
Imagenología Tridimensional/métodos , Potenciales de la Membrana/fisiología , Retinaldehído/análogos & derivados , Electricidad , Células HEK293 , Humanos , Compuestos de Piridinio/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Retinaldehído/metabolismo , Factores de TiempoRESUMEN
Retinal ganglion cells (RGCs) are the output cells of the retina; they convert synaptic input into spike output that carries visual information to the brain. Synaptic inputs are received, integrated and communicated to the spike initiation zone of the axon by dendrites whose properties are poorly understood. Here simultaneous patch-clamp recording and 2-photon Ca(2+) imaging are used to study voltage- and light-evoked Ca(2+) signals in the dendrites of identified types of mouse RGCs from parallel ON and OFF pathways, which encode the onset and offset of light, respectively. The results show pathway-specific differences in voltage-dependent Ca(2+) signaling. While both ON and OFF cells express high-voltage-activated (HVA) Ca(2+) channels, only OFF RGCs also express low-voltage-activated (LVA) Ca(2+) channels. LVA Ca(2+) channels in OFF cells are deinactivated by hyperpolarization from the resting potential and give rise to rebound excited Ca(2+) spikes at the termination of a step of either hyperpolarizing current or light. This suggests that the differential expression of voltage-gated Ca(2+) channels in ON and OFF RGC dendrites contributes to differences in the way the two cell types process visual stimuli.
Asunto(s)
Señalización del Calcio/fisiología , Dendritas/metabolismo , Retina/citología , Células Ganglionares de la Retina/clasificación , Células Ganglionares de la Retina/citología , Animales , Fenómenos Biofísicos/efectos de los fármacos , Fenómenos Biofísicos/fisiología , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Dendritas/efectos de los fármacos , Estimulación Eléctrica/métodos , Femenino , Técnicas In Vitro , Activación del Canal Iónico/efectos de los fármacos , Proteínas Luminiscentes/genética , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Níquel/farmacología , Técnicas de Placa-Clamp/métodos , Estimulación Luminosa/métodos , Células Ganglionares de la Retina/efectos de los fármacos , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacología , Antígenos Thy-1/genéticaRESUMEN
In the primate retina the small bistratified, "blue-yellow" color-opponent ganglion cell receives parallel ON-depolarizing and OFF-hyperpolarizing inputs from short (S)-wavelength sensitive and combined long (L)- and middle (M)-wavelength sensitive cone photoreceptors, respectively. However, the synaptic pathways that create S versus LM cone-opponent receptive field structure remain controversial. Here, we show in the macaque monkey retina in vitro that at photopic light levels, when an identified rod input is excluded, the small bistratified cell displays a spatially coextensive receptive field in which the S-ON-input is in spatial, temporal, and chromatic balance with the LM-OFF-input. ON pathway block with l-AP-4, the mGluR6 receptor agonist, abolished the S-ON response but spared the LM-OFF response. The isolated LM component showed a center-surround receptive field structure consistent with an input from OFF-center, ON-surround "diffuse" cone bipolar cells. Increasing retinal buffering capacity with HEPES attenuated the LM-ON surround component, consistent with a non-GABAergic outer retina feedback mechanism for the bipolar surround. The GABAa/c receptor antagonist picrotoxin and the glycine receptor antagonist strychnine did not affect chromatic balance or the basic coextensive receptive field structure, suggesting that the LM-OFF field is not generated by an inner retinal inhibitory pathway. We conclude that the opponent S-ON and LM-OFF responses originate from the excitatory receptive field centers of S-ON and LM-OFF cone bipolar cells, and that the LM-OFF- and ON-surrounds of these parallel bipolar inputs largely cancel, explaining the small, spatially coextensive but spectrally antagonistic receptive field structure of the blue-ON ganglion cell.
Asunto(s)
Percepción de Color/fisiología , Visión de Colores/fisiología , Retina/citología , Células Ganglionares de la Retina/fisiología , Campos Visuales/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Fenómenos Biofísicos , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas del GABA/farmacología , Glicinérgicos/farmacología , Técnicas In Vitro , Macaca , Modelos Neurológicos , Estimulación Luminosa/métodos , Picrotoxina/farmacología , Propionatos/farmacología , Células Fotorreceptoras Retinianas Conos/fisiología , Células Ganglionares de la Retina/clasificación , Células Ganglionares de la Retina/efectos de los fármacos , Estricnina/farmacología , Vías Visuales/efectos de los fármacosRESUMEN
Dendritic signals play an essential role in processing visual information in the retina. To study them in neurites too small for electrical recording, we developed an instrument that combines a multi-photon (MP) microscope with a through-the-objective high-resolution visual stimulator. An upright microscope was designed that uses the objective lens for both MP imaging and delivery of visual stimuli to functionally intact retinal explants or eyecup preparations. The stimulator consists of a miniature liquid-crystal-on-silicon display coupled into the optical path of an infrared-excitation laser-scanning microscope. A pair of custom-made dichroic filters allows light from the excitation laser and three spectral bands ('colors') from the stimulator to reach the retina, leaving two intermediate bands for fluorescence imaging. Special optics allow displacement of the stimulator focus relative to the imaging focus. Spatially resolved changes in calcium-indicator fluorescence in response to visual stimuli were recorded in dendrites of different types of mammalian retinal neurons.
Asunto(s)
Microscopía de Fluorescencia por Excitación Multifotónica/instrumentación , Retina/fisiología , Animales , Calcio/metabolismo , Dendritas/fisiología , Dendritas/efectos de la radiación , Rayos Láser , Matemática , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Neuronas/fisiología , Óptica y Fotónica , Estimulación Luminosa , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Conejos , Retina/efectos de la radiación , Programas InformáticosRESUMEN
Glutamate released from photoreceptors controls the activity and output of parallel pathways in the retina. When photoreceptors die because of degenerative diseases, surviving retinal networks are left without their major source of input, but little is known about how photoreceptor loss affects ongoing synaptic activity and retinal output. Here, we use patch-clamp recording and two-photon microscopy to investigate morphological and physiological properties of identified types of ON and OFF retinal ganglion cells (RGCs) in the adult (36-210 d old) retinal degeneration rd-1/rd-1 mouse. We find that strong rhythmic synaptic input drives ongoing oscillatory spike activity in both ON and OFF RGCs at a fundamental "beating" frequency of approximately 10 Hz. Despite this aberrant activity, ON and OFF cells maintain their characteristic dendritic stratification, intrinsic firing properties, including rebound firing in OFF cells, balance of synaptic excitation and inhibition, and dendritic calcium signaling. Thus, RGCs are inherently stable during degeneration-induced retinal activity.
Asunto(s)
Degeneración Retiniana/fisiopatología , Células Ganglionares de la Retina/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Animales , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BLRESUMEN
The responses of individual salamander L-cones to light steps of moderate intensity (bleaching 0.3-3% of the total photopigment) and duration (between 5 and 90 s) were recorded using suction electrodes. Light initially suppressed the circulating current, which partially recovered or "sagged" over several seconds. The sensitivity of the cone to dim flashes decreased rapidly after light onset and approached a minimum within 500 ms. Background light did not affect the rising phase of the dim flash response, a measure of the initial gain of phototransduction. When the light was extinguished, the circulating current transiently exceeded or "overshot" its level in darkness. During the overshoot, the sensitivity of the cone required several seconds to recover. The sag and overshoot remained in voltage-clamped cones. Comparison with theory suggests that three mechanisms cause the sag, overshoot, and slow recovery of sensitivity after the light step: a gradual increase in the rate of inactivation of the phototransduction cascade during the light step, residual activity of the transduction cascade after the step is extinguished, and an increase in guanylate cyclase activity during the light step that persists after the light is extinguished.
Asunto(s)
Adaptación Ocular/fisiología , Ambystoma/fisiología , Estimulación Luminosa/métodos , Células Fotorreceptoras de Vertebrados/fisiología , AnimalesRESUMEN
Negative feedback from horizontal cells to cone photoreceptors is regarded as the critical pathway for the formation of the antagonistic surround of retinal neurons, yet the mechanism by which horizontal cells accomplish negative feedback has been difficult to determine. Recent evidence suggests that feedback uses a novel, non-GABAergic pathway that directly modulates the calcium current in cones. In non-mammalian vertebrates, enrichment of retinal pH buffering capacity attenuates horizontal cell feedback, supporting one model in which feedback occurs by horizontal cell modulation of the extracellular pH in the cone synaptic cleft. Here we test the effect of exogenous pH buffering on the response dynamics of H1 horizontal cells and the center-surround receptive field structure of parasol ganglion cells in the macaque monkey retina. Enrichment of the extracellular buffering capacity with HEPES selectively attenuates surround antagonism in parasol ganglion cells. The H1 horizontal cell light response includes a slow, depolarizing component that is attributed to negative feedback to cones. This part of the response is attenuated by HEPES and other pH buffers in a dose-dependent manner that is correlated with predicted buffering capacity. The selective effects of pH buffering on the parasol cell surround and H1 cell light response suggests that, in primate retina, horizontal cell feedback to cones is mediated via a pH-dependent mechanism and is a major determinant of the ganglion cell receptive field surround.
Asunto(s)
Luz , Protones , Retina/citología , Células Ganglionares de la Retina , Células Horizontales de la Retina , Potenciales de Acción/fisiología , Potenciales de Acción/efectos de la radiación , Animales , Tampones (Química) , Relación Dosis-Respuesta a Droga , HEPES/farmacología , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Macaca fascicularis , Macaca nemestrina , Estimulación Luminosa/métodos , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/fisiología , Células Ganglionares de la Retina/efectos de la radiación , Células Horizontales de la Retina/efectos de los fármacos , Células Horizontales de la Retina/fisiología , Células Horizontales de la Retina/efectos de la radiación , Visión OcularRESUMEN
Detection of image motion direction begins in the retina, with starburst amacrine cells (SACs) playing a major role. SACs generate larger dendritic Ca(2+) signals when motion is from their somata towards their dendritic tips than for motion in the opposite direction. To study the mechanisms underlying the computation of direction selectivity (DS) in SAC dendrites, electrical responses to expanding and contracting circular wave visual stimuli were measured via somatic whole-cell recordings and quantified using Fourier analysis. Fundamental and, especially, harmonic frequency components were larger for expanding stimuli. This DS persists in the presence of GABA and glycine receptor antagonists, suggesting that inhibitory network interactions are not essential. The presence of harmonics indicates nonlinearity, which, as the relationship between harmonic amplitudes and holding potential indicates, is likely due to the activation of voltage-gated channels. [Ca(2+)] changes in SAC dendrites evoked by voltage steps and monitored by two-photon microscopy suggest that the distal dendrite is tonically depolarized relative to the soma, due in part to resting currents mediated by tonic glutamatergic synaptic input, and that high-voltage-activated Ca(2+) channels are active at rest. Supported by compartmental modeling, we conclude that dendritic DS in SACs can be computed by the dendrites themselves, relying on voltage-gated channels and a dendritic voltage gradient, which provides the spatial asymmetry necessary for direction discrimination.
Asunto(s)
Células Amacrinas/fisiología , Dendritas/fisiología , Células Amacrinas/citología , Células Amacrinas/efectos de los fármacos , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Señalización del Calcio , Potenciales Evocados Visuales/efectos de los fármacos , Antagonistas del GABA/farmacología , Técnicas In Vitro , Activación del Canal Iónico , Potenciales de la Membrana , Modelos Neurológicos , Movimiento (Física) , Estimulación Luminosa , Conejos , Visión Ocular/fisiologíaRESUMEN
The A1 cell is an axon-bearing amacrine cell of the primate retina with a diffusely stratified, moderately branched dendritic tree (approximately 400 microm diameter). Axons arise from proximal dendrites forming a second concentric, larger arborization (>4 mm diameter) of thin processes with bouton-like swellings along their length. A1 cells are ON-OFF transient cells that fire a brief high frequency burst of action potentials in response to light (Stafford & Dacey, 1997). It has been hypothesized that A1 cells receive local input to their dendrites, with action potentials propagating output via the axons across the retina, serving a global inhibitory function. To explore this hypothesis we recorded intracellularly from A1 cells in an in vitro macaque monkey retina preparation. A1 cells have an antagonistic center-surround receptive field structure for the ON and OFF components of the light response. Blocking the ON pathway with L-AP4 eliminated ON center responses but not OFF center responses or ON or OFF surround responses. Blocking GABAergic inhibition with picrotoxin increased response amplitudes without affecting receptive field structure. TTX abolished action potentials, with little effect on the sub-threshold light response or basic receptive field structure. We also used multi-photon laser scanning microscopy to record light-induced calcium transients in morphologically identified dendrites and axons of A1 cells. TTX completely abolished such calcium transients in the axons but not in the dendrites. Together these results support the current model of A1 function, whereby the dendritic tree receives synaptic input that determines the center-surround receptive field; and action potentials arise in the axons, which propagate away from the dendritic field across the retina.
Asunto(s)
Células Amacrinas/fisiología , Axones/fisiología , Polaridad Celular/fisiología , Dendritas/fisiología , Potenciales de Acción/efectos de los fármacos , Células Amacrinas/efectos de los fármacos , Aminobutiratos/farmacología , Animales , Axones/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Dendritas/efectos de los fármacos , Electrofisiología , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas del GABA/farmacología , Macaca fascicularis , Macaca nemestrina , Microelectrodos , Estimulación Luminosa , Picrotoxina/farmacología , Retina/citología , Retina/fisiología , Células Bipolares de la Retina/fisiología , Células Fotorreceptoras Retinianas Conos/fisiología , Tetrodotoxina/farmacología , Campos VisualesRESUMEN
Neuronal discharge is driven by either synaptic input or cell-autonomous intrinsic pacemaker activity. It is commonly assumed that the resting spike activity of retinal ganglion cells (RGCs), the output cells of the retina, is driven synaptically, because retinal photoreceptors and second-order cells tonically release neurotransmitter. Here we show that ON and OFF RGCs generate maintained activity through different mechanisms: ON cells depend on tonic excitatory input to drive resting activity, whereas OFF cells continue to fire in the absence of synaptic input. In addition to spontaneous activity, OFF cells exhibit other properties of pacemaker neurons, including subthreshold oscillations, burst firing, and rebound excitation. Thus, variable weighting of synaptic mechanisms and intrinsic properties underlies differences in the generation of maintained activity in these parallel retinal pathways.
Asunto(s)
Potenciales de Acción/fisiología , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/fisiología , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Estimulación Luminosa/métodos , Retina/citología , Retina/fisiologíaRESUMEN
Although signals controlled by single molecules are expected to be inherently variable, rod photoreceptors generate reproducible responses to single absorbed photons. We show that this unexpected reproducibility-the consistency of amplitude and duration of rhodopsin activity-varies in a graded and systematic manner with the number but not the identity of phosphorylation sites on rhodopsin's C terminus. These results indicate that each phosphorylation site provides an independent step in rhodopsin deactivation and that collectively these steps tightly control rhodopsin's active lifetime. Other G protein cascades may exploit a similar mechanism to encode accurately the timing and number of receptor activation.
Asunto(s)
Fotones , Células Fotorreceptoras Retinianas Bastones/metabolismo , Rodopsina/metabolismo , Animales , Arrestina/metabolismo , Electrofisiología , Ratones , Ratones Transgénicos , Modelos Biológicos , Mutación , Técnicas de Placa-Clamp , Fosforilación , Rodopsina/genética , Visión OcularRESUMEN
G-protein-coupled enzyme cascades are used by eukaryotic cells to detect external signals and transduce them into intracellular messages that contain biological information relevant to the cell's function. Since G-protein-coupled receptors that are designed to detect different kinds of external signals can generate the same kind of intracellular response, effective signaling requires that there are mechanisms to increase signal specificity and fidelity. Here we examine the kinetic equations for the initial three stages in a generic G-protein-coupled cascade and show that the physical properties of the transduction pathway result in two intrinsic features that benefit signaling. 1), The response to a single activated receptor is naturally confined to a localized spatial domain, which could improve signal specificity by reducing cross talk. 2), The peak of the response generated by such a signaling domain is limited. This saturation effect reduces trial-to-trial variability and increases signaling fidelity by limiting the response to receptors that remain active for longer than average. We suggest that this mechanism for reducing response fluctuations may be a contributing factor in making the single photon responses of vertebrate retinal rods so remarkably reproducible.
Asunto(s)
Proteínas de Unión al GTP/química , Transducción de Señal , Animales , Biofisica/métodos , Catálisis , Regulación de la Expresión Génica , Cinética , Modelos Biológicos , Modelos Estadísticos , Fotones , Estructura Terciaria de Proteína , Receptores de Superficie Celular/metabolismo , Células Fotorreceptoras Retinianas Bastones , Factores de TiempoRESUMEN
The detection of image motion is fundamental to vision. In many species, unique classes of retinal ganglion cells selectively respond to visual stimuli that move in specific directions. It is not known which retinal cell first performs the neural computations that give rise to directional selectivity in the ganglion cell. A prominent candidate has been an interneuron called the 'starburst amacrine cell'. Using two-photon optical recordings of intracellular calcium concentration, here we find that individual dendritic branches of starburst cells act as independent computation modules. Dendritic calcium signals, but not somatic membrane voltage, are directionally selective for stimuli that move centrifugally from the cell soma. This demonstrates that direction selectivity is computed locally in dendritic branches at a stage before ganglion cells.
Asunto(s)
Células Amacrinas/metabolismo , Señalización del Calcio , Dendritas/metabolismo , Percepción de Movimiento/fisiología , Células Ganglionares de la Retina/metabolismo , Percepción Visual/fisiología , Células Amacrinas/citología , Células Amacrinas/efectos de los fármacos , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Tamaño de la Célula , Oscuridad , Dendritas/efectos de los fármacos , Antagonistas del GABA/farmacología , Luz , Percepción de Movimiento/efectos de los fármacos , Estimulación Luminosa , Fotones , Piridazinas/farmacología , Conejos , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/efectos de los fármacos , Percepción Visual/efectos de los fármacosRESUMEN
Visual transduction in retinal photoreceptors operates through a dynamic interplay of two second messengers, Ca(2+) and cGMP. Ca(2+) regulates the activity of guanylate cyclase (GC) and the synthesis of cGMP by acting on a GC-activating protein (GCAP). While this action is critical for rapid termination of the light response, the GCAP responsible has not been identified. To test if GCAP1, one of two GCAPs present in mouse rods, supports the generation of normal flash responses, transgenic mice were generated that express only GCAP1 under the control of the endogenous promoter. Paired flash responses revealed a correlation between the degree of recovery of the rod a-wave and expression levels of GCAP1. In single cell recordings, the majority of the rods generated flash responses that were indistinguishable from wild type. These results demonstrate that GCAP1 at near normal levels supports the generation of wild-type flash responses in the absence of GCAP2.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Guanilato Ciclasa/metabolismo , Células Fotorreceptoras Retinianas Bastones/fisiología , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Expresión Génica , Proteínas Activadoras de la Guanilato-Ciclasa , Ratones , Ratones Noqueados , Ratones Transgénicos , Células Fotorreceptoras Retinianas Bastones/citología , Células Fotorreceptoras Retinianas Bastones/metabolismoRESUMEN
The visual process is initiated by the photoisomerization of 11-cis-retinal to all-trans-retinal. For sustained vision the 11-cis-chromophore must be regenerated from all-trans-retinal. This requires RPE65, a dominant retinal pigment epithelium protein. Disruption of the RPE65 gene results in massive accumulation of all-trans-retinyl esters in the retinal pigment epithelium, lack of 11-cis-retinal and therefore rhodopsin, and ultimately blindness. We reported previously (Van Hooser, J. P., Aleman, T. S., He, Y. G., Cideciyan, A. V., Kuksa, V., Pittler, S. J., Stone, E. M., Jacobson, S. G., and Palczewski, K. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8623-8628) that in Rpe65-/- mice, oral administration of 9-cis-retinal generated isorhodopsin, a rod photopigment, and restored light sensitivity to the electroretinogram. Here, we provide evidence that early intervention by 9-cis-retinal administration significantly attenuated retinal ester accumulation and supported rod retinal function for more than 6 months post-treatment. In single cell recordings rod light sensitivity was shown to be a function of the amount of regenerated isorhodopsin; high doses restored rod responses with normal sensitivity and kinetics. Highly attenuated residual rod function was observed in untreated Rpe65-/- mice. This rod function is likely a consequence of low efficiency production of 11-cis-retinal by photo-conversion of all-trans-retinal in the retina as demonstrated by retinoid analysis. These studies show that pharmacological intervention produces long lasting preservation of visual function in dark-reared Rpe65-/- mice and may be a useful therapeutic strategy in recovering vision in humans diagnosed with Leber congenital amaurosis caused by mutations in the RPE65 gene, an inherited group of early onset blinding and retinal degenerations.