RESUMO
A major stumbling block to understanding neural circuits is the extreme anatomical and functional diversity of interneurons. Subsets of interneurons can be targeted for manipulation using Cre mouse lines, but Cre expression is rarely confined to a single interneuron type. It is essential to have a strategy that further restricts labeling in Cre driver lines. We now describe an approach that combines Cre driver mice, recombinant adeno-associated virus, and rabies virus to produce sparse but binary labeling of select interneurons--frequently only a single cell in a large region. We used this approach to characterize the retinal amacrine and ganglion cell types in five GABAergic Cre mouse (Mus musculus) lines, and identified two new amacrine cell types: an asymmetric medium-field type and a wide-field type. We also labeled several wide-field amacrine cell types that have been previously identified based on morphology but whose connectivity and function had not been systematically studied due to lack of genetic markers. All Cre-expressing amacrine cells labeled with an antibody to GABA. Cre-expressing RGCs lacked GABA labeling and included classically defined as well as recently identified types. In addition to the retina, our technique leads to sparse labeling of neurons in the cortex, lateral geniculate nucleus, and superior colliculus, and can be used to express optogenetic tools such as channelrhodopsin and protein sensors such as GCaMP. The Cre drivers identified in this study provide genetic access to otherwise hard to access cell types for systematic analysis including anatomical characterization, physiological recording, optogenetic and/or chemical manipulation, and circuit mapping.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Retina/citologia , Adenoviridae/genética , Animais , Cálcio/metabolismo , Channelrhodopsins , Feminino , Regulação da Expressão Gênica/genética , Técnicas In Vitro , Integrases/genética , Integrases/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Vias Neurais/metabolismo , Técnicas de Patch-Clamp , Ácido gama-Aminobutírico/genéticaRESUMO
Postsynaptic kainate receptors mediate excitatory synaptic transmission over a broad range of temporal frequencies. In heterologous systems, the temporal responses of kainate receptors vary when different channel-forming and auxiliary subunits are co-expressed but how this variability relates to the temporal differences at central synapses is incompletely understood. The mammalian cone photoreceptor synapse provides advantages for comparing the different temporal signalling roles of kainate receptors, as cones release glutamate over a range of temporal frequencies, and three functionally distinct Off bipolar cell types receive cone signals at synapses that contain either AMPA or kainate receptors, all with different temporal properties. A disadvantage is that the different receptor subunits are not identified. We used in situ hybridization, immunocytochemistry, and pharmacology to identify the kainate receptor and auxiliary subunits in ground squirrel (Ictidomys tridecimlineatus) cb1a/b, cb2, and cb3a/b Off bipolar cell types. As expected, the types showed distinct subunit expression patterns. Kainate receptors mediated â¼80% of the synaptic response in cb3a/b cells and were heteromers of GluK1 and GluK5. Cb3a/b cells contained message for GluK1 and GluK5, and also GluK3 and the auxiliary subunit Neto1. The synaptic responses in cb1a/b cells were mediated by GluK1-containing kainate receptors that behaved differently from the receptors expressed by cb3a/b cells. AMPA receptors mediated the entire synaptic response in cb2 cells and the remaining synaptic response in cb3a/b cells. We conclude that GluK1 is the predominant kainate receptor subunit in cb1 and cb3 Off bipolar cells. Different temporal response properties may result from selective association with GluK3, GluK5, or Neto1.
Assuntos
Receptores de Ácido Caínico/metabolismo , Células Bipolares da Retina/metabolismo , Transmissão Sináptica , Animais , Potenciais Pós-Sinápticos Excitadores , Ácido Glutâmico/metabolismo , Subunidades Proteicas , Receptores de Ácido Caínico/efeitos dos fármacos , Receptores de Ácido Caínico/genética , Células Bipolares da Retina/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/metabolismo , Sciuridae , Fatores de Tempo , Receptor de GluK3 CainatoRESUMO
In congenital stationary night blindness type 2 (CSNB2)-a disorder involving the Cav1.4 (L-type) Ca2+ channel-visual impairment is mild considering that Cav1.4 mediates synaptic release from rod and cone photoreceptors. Here, we addressed this conundrum using a Cav1.4 knockout (KO) mouse and a knock-in (G369i KI) mouse expressing a non-conducting Cav1.4. Surprisingly, Cav3 (T-type) Ca2+ currents were detected in cones of G369i KI mice and Cav1.4 KO mice but not in cones of wild-type mouse, ground squirrel, and macaque retina. Whereas Cav1.4 KO mice are blind, G369i KI mice exhibit normal photopic (i.e., cone-mediated) visual behavior. Cone synapses, which fail to form in Cav1.4 KO mice, are present, albeit enlarged, and with some errors in postsynaptic wiring in G369i KI mice. While Cav1.4 KO mice lack evidence of cone synaptic responses, electrophysiological recordings in G369i KI mice revealed nominal transmission from cones to horizontal cells and bipolar cells. In CSNB2, we propose that Cav3 channels maintain cone synaptic output provided that the nonconducting role of Cav1.4 in cone synaptogenesis remains intact. Our findings reveal an unexpected form of homeostatic plasticity that relies on a non-canonical role of an ion channel.
RESUMO
Barlow (1953) studied summation in ganglion cell receptive fields and observed a fine discrimination of spatial information from which he inferred that retinal interneurons use analog signals to process images. Subsequent intracellular recordings confirmed that the interneurons of the outer retina, including photoreceptors, horizontal cells, and bipolar cells, respond to light with slow, graded changes in membrane potential. Analog processing may enable interneurons to discriminate fine gradations in light intensity and spatiotemporal pattern, but at the expense of the speed, temporal precision, and threshold discrimination that are characteristic of all-or-nothing Na(+) spikes. We show that one type of mammalian On bipolar cell, the ground squirrel cb5b, has a large tetrodotoxin (TTX)-sensitive Na(+) current. When recorded from in the perforated patch configuration, cb5b cells can signal the onset of a light step with 1-3 all-or-nothing action potentials that attain a peak amplitude of -10 to -20 mV (peak width at half-height equals 2-3 ms). When exposed to a continuous, temporally fluctuating stimulus, cb5b cells generate both graded and spiking responses. Cb5b cells spike with millisecond precision, selecting for stimulus sequences in which transitions to light are preceded by a period of darkness. The axon terminals of cb5b bipolar cells costratify with the dendrites of amacrine and ganglion cells that encode light onset with a short latency burst of spikes. The results support the idea that a spiking On bipolar cell is part of a dedicated retinal pathway for rapidly and reliably signaling dark to light transitions.
Assuntos
Potenciais de Ação/fisiologia , Membrana Celular/fisiologia , Células Bipolares da Retina/fisiologia , Canais de Sódio/fisiologia , Visão Ocular/fisiologia , Potenciais de Ação/efeitos da radiação , Animais , Membrana Celular/efeitos da radiação , Feminino , Luz , Masculino , Mamíferos , Técnicas de Cultura de Órgãos , Estimulação Luminosa/métodos , Células Bipolares da Retina/efeitos da radiação , Sciuridae , Canais de Sódio/efeitos da radiação , Visão Ocular/efeitos da radiaçãoRESUMO
A fundamental organizing plan of the retina is that visual information is divided into ON and OFF streams that are processed in separate layers. This functional dichotomy originates in the ON and OFF bipolar cells, which then make excitatory glutamatergic synapses onto amacrine and ganglion cells in the inner plexiform layer. We have identified an amacrine cell (AC), the sign-inverting (SI) AC, that challenges this fundamental plan. The glycinergic, ON-stratifying SI-AC has OFF light responses. In opposition to the classical wiring diagrams, it receives inhibitory inputs from glutamatergic ON bipolar cells at mGluR8 synapses, and excitatory inputs from an OFF wide-field AC at electrical synapses. This "inhibitory ON center - excitatory OFF surround" receptive-field of the SI-AC allows it to use monostratified dendrites to conduct crossover inhibition and push-pull activation to enhance light detection by ACs and RGCs in the dark and feature discrimination in the light.
Assuntos
Células Amácrinas , Retina , Interneurônios , Dissidências e Disputas , Sinapses ElétricasRESUMO
Neural computations arise from highly precise connections between specific types of neurons. Retinal ganglion cells (RGCs) with similar stratification patterns are positioned to receive similar inputs but often display different response properties. In this study, we used intersectional mouse genetics to achieve single-cell type labeling and identified an object motion sensitive (OMS) AC type, COMS-AC(counter-OMS AC). Optogenetic stimulation revealed that COMS-AC makes glycinergic synapses with the OMS-insensitive HD2p-RGC, while chemogenetic inactivation showed that COMS-AC provides inhibitory control to HD2p-RGC during local motion. This local inhibition, combined with the inhibitory drive from TH2-AC during global motion, explains the OMS-insensitive feature of HD2p-RGC. In contrast, COMS-AC fails to make synapses with W3(UHD)-RGC, allowing it to exhibit OMS under the control of VGlut3-AC and TH2-AC. These findings reveal modular interneuron circuits that endow structurally similar RGC types with different responses and present a mechanism for redundancy-reduction in the retina to expand coding capacity.
Assuntos
Retina , Células Ganglionares da Retina , Camundongos , Animais , Retina/fisiologia , Células Ganglionares da Retina/fisiologia , Transmissão Sináptica , Interneurônios , Sinapses/fisiologiaRESUMO
Neurons enhance their computational power by combining linear and nonlinear transformations in extended dendritic trees. Rich, spatially distributed processing is rarely associated with individual synapses, but the cone photoreceptor synapse may be an exception. Graded voltages temporally modulate vesicle fusion at a cone's ~20 ribbon active zones. Transmitter then flows into a common, glia-free volume where bipolar cell dendrites are organized by type in successive tiers. Using super-resolution microscopy and tracking vesicle fusion and postsynaptic responses at the quantal level in the thirteen-lined ground squirrel, Ictidomys tridecemlineatus, we show that certain bipolar cell types respond to individual fusion events in the vesicle stream while other types respond to degrees of locally coincident events, creating a gradient across tiers that are increasingly nonlinear. Nonlinearities emerge from a combination of factors specific to each bipolar cell type including diffusion distance, contact number, receptor affinity, and proximity to glutamate transporters. Complex computations related to feature detection begin within the first visual synapse.
Assuntos
Células Fotorreceptoras Retinianas Cones , Sinapses , Animais , Células Fotorreceptoras Retinianas Cones/fisiologia , Sinapses/fisiologia , Mamíferos , Retina/fisiologiaRESUMO
Cone photoreceptors transmit signals at high temporal frequencies and mediate fine spatial vision. High-frequency transmission requires a high rate of glutamate release, which could promote spillover to neighboring cells, whereas spatial vision requires that cones within a tightly packed array signal light to postsynaptic bipolar cells with minimal crosstalk. Glutamate spread from the cone terminal is thought to be limited by presynaptic transporters and nearby glial processes. In addition, there is no ultrastructural evidence for chemical synapses between mammalian cones, although such synapses have been described in lower vertebrate retinas. We tested for cone-cone glutamate diffusion by recording from adjacent cone pairs in the ground squirrel retina, and instead found that the glutamate released by one cone during electrical stimulation activates glutamate transporter Cl(-) conductances on neighboring cones. Unlike in other systems, where crosstalk is diminished by increasing the temperature and by moving to a more intact preparation, glutamate spread persisted at physiological temperatures (37°C) and in retinal flat mounts. The glutamate-gated anion conductance in cones has a reversal potential of â¼-30 mV compared with a cone resting potential of â¼-50 mV; thus, crosstalk should have a depolarizing effect on the cone network. Cone-cone glutamate spread is regulated by the physiological stimulus, light, and under physiological conditions can produce a response of â¼2 mV, equivalent to 13-20% of a cone's light response. We conclude that in the absence of discrete chemical synapses, glutamate flows between cones during a light response and may mediate a spatially distributed positive feedback.
Assuntos
Ácido Glutâmico/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Transmissão Sináptica/fisiologia , Visão Ocular/fisiologia , Sistema X-AG de Transporte de Aminoácidos/fisiologia , Animais , Canais de Cloreto/fisiologia , Estimulação Elétrica/métodos , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp/métodos , Estimulação Luminosa/métodos , Receptor Cross-Talk/fisiologia , Retina/metabolismo , Retina/fisiologia , SciuridaeRESUMO
A cone photoreceptor releases glutamate at ribbons located atop narrow membrane invaginations that empty onto a terminal base. The unique shape of the cone terminal suggests that there are two transmitter microenvironments: within invaginations, where concentrations are high and exposures are brief; and at the base, where concentrations are low and exposure is smoothed by diffusion. Using multicell voltage-clamp recording, we show that different subtypes of Off bipolar cells sample transmitter in two microenvironments. The dendrites of an AMPA receptor-containing cell insert into invaginations and sense rapid fluctuations in glutamate concentration that can lead to transient responses. The dendrites of kainate receptor-containing cells make basal contacts and respond to a smoothed flow of glutamate that produces sustained responses. Signaling at the cone to Off bipolar cell synapse illustrates how transmitter spillover and synapse architecture can combine to produce distinct signals in postsynaptic neurons.
Assuntos
Ácido Glutâmico/metabolismo , Células Bipolares da Retina/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Animais , Dendritos/metabolismo , Dendritos/ultraestrutura , Potenciais Evocados Visuais/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Cinética , Microscopia Confocal , Modelos Neurológicos , Receptores de AMPA/fisiologia , Receptores de Ácido Caínico/fisiologia , Células Bipolares da Retina/ultraestrutura , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Sciuridae , Sinapses/ultraestrutura , Proteínas Vesiculares de Transporte de Glutamato/metabolismoRESUMO
The mammalian retina is fundamentally dichromatic, with trichromacy only recently emerging in some primates. In dichromats, an array of short wavelength-sensitive (S, blue) and middle wavelength-sensitive (M, green) cones is sampled by approximately ten bipolar cell types, and the sampling pattern determines how retinal ganglion cells and ultimately higher visual centers encode color and luminance. By recording from cone-bipolar cell pairs in the retina of the ground squirrel, we show that the bipolar cell types sample cone signals in three ways: one type receives input exclusively from S-cones, two types receive mixed S/M-cone input and the remaining types receive an almost pure M-cone signal. Bipolar cells that carry S- or M-cone signals can have a role in color discrimination and may contact color-opponent ganglion cells. Bipolar cells that sum signals from S- and M-cones may signal to ganglion cells that encode luminance.
Assuntos
Percepção de Cores/fisiologia , Sensibilidades de Contraste/fisiologia , Retina , Células Bipolares da Retina/fisiologia , Vias Visuais/fisiologia , Animais , Biotina/análogos & derivados , Biotina/farmacocinética , Cálcio/metabolismo , Cor , Imageamento Tridimensional/métodos , Imuno-Histoquímica/métodos , Técnicas In Vitro , Luz , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Modelos Neurológicos , Técnicas de Patch-Clamp/métodos , Estimulação Luminosa/métodos , Receptores de Glutamato Metabotrópico/metabolismo , Retina/anatomia & histologia , Retina/fisiologia , Células Bipolares da Retina/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/fisiologia , Opsinas de Bastonetes/metabolismo , Sciuridae , Vias Visuais/anatomia & histologia , Vias Visuais/efeitos dos fármacosRESUMO
Adeno-associated virus (AAV) is the most commonly used viral vector for both biological and gene therapeutic applications. Although many methods have been developed to measure quantity attributes of AAV, they are often technically challenging and time-consuming. Here, we report a method to titer AAV with GelGreen® dye, a safe green fluorescence nucleic acid dye recently engineered by Biotium company (Fremont, CA). This method, hereinafter referred to as GelGreen method, provides a fast (â¼30 min) and reliable strategy for AAV titration. To validate GelGreen method, we measured genome titer of an AAV reference material AAV8RSM and compared our titration results with those determined by Reference Material Working Group (ARMWG). We showed that GelGreen results and capsid enzyme-linked immunosorbent assay results are comparable with each other. We also showed that GelRed® dye, a red fluorescence dye from Biotium, can be used to directly "visualize" AAV genome titer on a conventional gel imager, presenting an especially direct approach to estimate viral quantity. Finally, we showed that GelGreen and GelRed dyes can also be used to quantify self-complementary AAV (scAAV) and crudely purified AAV samples. In summary, we described a technique to titer AAV by using new generation of safe DNA dyes. This technique is simple, safe, reliable, and cost efficient. It has potential to be broadly applied for quantifying and normalizing AAV viral vectors.
Assuntos
DNA Viral/análise , Dependovirus/genética , Corantes Fluorescentes/química , Vetores Genéticos/análise , DNA Viral/genética , Vetores Genéticos/genética , HumanosRESUMO
The distinct absorbance spectra of the cone photopigments form the basis of color vision, but ultrastructural and physiological evidence shows that mammalian cones are electrically coupled. Coupling between cones of the same spectral type should average voltage noise in adjacent photoreceptors and improve the ability to resolve low-contrast spatial patterns. However, indiscriminate coupling between spectral types could compromise color vision by smearing chromatic information across channels. Here we show, by measuring the junctional conductance between green-green and blue-green cone pairs in slices from the dichromatic ground-squirrel retina, that green-green cone pairs are routinely coupled with an average conductance of 220 pS, whereas coupling is undetectable in blue-green cone pairs. Together with a lack of tracer coupling and the selective localization of connexin proteins, our results show that signals in blue and green cones are processed separately in the photoreceptor layer.
Assuntos
Percepção de Cores/fisiologia , Junções Comunicantes/fisiologia , Rede Nervosa/fisiologia , Retina/citologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Animais , Conexinas/metabolismo , Adaptação à Escuridão/fisiologia , Condutividade Elétrica , Corantes Fluorescentes/metabolismo , Imuno-Histoquímica/métodos , Técnicas In Vitro , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Microscopia Confocal/métodos , Modelos Neurológicos , Técnicas de Patch-Clamp/métodos , Estimulação Luminosa , Células Fotorreceptoras Retinianas Cones/citologia , Sciuridae , Proteína delta-2 de Junções ComunicantesRESUMO
The mammalian retina harbors over 100 different cell types. To understand how retinal circuits work, it is essential to systematically access each type. A widely used approach for achieving targeted transgene expression exploits promoter-driven Cre lines. However, Cre expression in a given transgenic line in the retina and elsewhere in the brain is rarely confined to a single cell type, contributing ambiguity to the interpretation of results from broadly applied manipulations. To obtain unambiguous information about retinal processing, it is desirable to have strategies for further restricting transgene expression to a few or even to a single cell type. We employed an intersectional strategy based on a Cre/Flp double recombinase system to target amacrine and ganglion cell types in the inner retina. We analyzed expression patterns in seven Flp drivers and then created combinational mouse lines by selective cross breeding with Cre drivers. Breeding with Flp drivers can routinely remove labeling from more than 90% of the cells in Cre drivers, leading to only a handful cell types, typically 2-3, remaining in the intersection. Cre/Flp combinatorial mouse lines enabled us to identify and anatomically characterize retinal cell types with greater ease and demonstrated the feasibility of intersectional strategies in retinal research. In addition to the retina, we examined Flp expression in the lateral geniculate nucleus and superior colliculus. Our results establish a foundation for future application of intersectional strategies in the retina and retino-recipient regions.
Assuntos
Células Amácrinas/fisiologia , DNA Nucleotidiltransferases , Corpos Geniculados/fisiologia , Integrases , Células Ganglionares da Retina/fisiologia , Colículos Superiores/fisiologia , Células Amácrinas/metabolismo , Animais , DNA Nucleotidiltransferases/metabolismo , Feminino , Corpos Geniculados/metabolismo , Integrases/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Modelos Animais , Células Ganglionares da Retina/metabolismo , Colículos Superiores/metabolismoRESUMO
BACKGROUND: Cone photoreceptors are noisy because of random fluctuations of photon absorption, signaling molecules, and ion channels. However, each cone's noise is independent of the others, whereas their signals are partially shared. Therefore, electrically coupling the synaptic terminals prior to forward transmission and subsequent nonlinear processing can appreciably reduce noise relative to the signal. This signal-processing strategy has been demonstrated in lower vertebrates with rather coarse vision, but its occurrence in mammals with fine acuity has been doubted (even though gap junctions are present) because coupling would blur the neural image. RESULTS: In ground squirrel retina, whose triangular cone lattice resembles the human fovea, paired electrical recordings from adjacent cones demonstrated electrical coupling with an average conductance of approximately 320 pS. Blur caused by this degree of coupling had a space constant of approximately 0.5 cone diameters. Psychophysical measurements employing laser interferometry to bypass the eye's optics suggest that human foveal cones experience a similar degree of neural blur and that it is invariant with light intensity. This neural blur is narrower than the eye's optical blur, and we calculate that it should improve the signal-to-noise ratio at the cone terminal by about 77%. CONCLUSIONS: We conclude that the gap junctions observed between mammalian cones, including those in the human fovea, represent genuine electrical coupling. Because the space constant of the resulting neural blur is less than that of the optical blur, the signal-to-noise ratio can be markedly improved before the nonlinear stages with little compromise to visual acuity.
Assuntos
Comunicação Celular/fisiologia , Junções Intercelulares/fisiologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Animais , Condutividade Elétrica , Eletrofisiologia , Mamíferos , Técnicas de Patch-Clamp , SciuridaeRESUMO
Ribbon synapses mediate continuous release in neurons that have graded voltage responses. While mammalian retinas can signal visual flicker at 80-100 Hz, the time constant, τ, for the refilling of a depleted vesicle release pool at cone photoreceptor ribbons is 0.7-1.1 s. Due to this prolonged depression, the mechanism for encoding high temporal frequencies is unclear. To determine the mechanism of high-frequency signaling, we focused on an "Off" cone bipolar cell type in the ground squirrel, the cb2, whose transient postsynaptic responses recovered following presynaptic depletion with a τ of â¼0.1 s, or 7- to 10-fold faster than the τ for presynaptic pool refilling. The difference in recovery time course is caused by AMPA receptor saturation, where partial refilling of the presynaptic pool is sufficient for a full postsynaptic response. By limiting the dynamic range of the synapse, receptor saturation counteracts ribbon depression to produce rapid recovery and facilitate high-frequency signaling.
Assuntos
Potenciais Pós-Sinápticos Excitadores/fisiologia , Retina/fisiologia , Células Bipolares da Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Transdução de Sinais , Sinapses/fisiologia , Animais , Cálcio/metabolismo , Estimulação Elétrica/métodos , Exocitose/fisiologia , Transdução de Sinais/fisiologia , Transmissão Sináptica/fisiologiaRESUMO
Decades of research have focused on the circuit connectivity between retinal neurons, but only a handful of amacrine cells have been described functionally and placed in the context of a specific retinal circuit. Here, we identify a circuit where inhibition from a specific amacrine cell plays a vital role in shaping the feature selectivity of a postsynaptic ganglion cell. We record from transgenically labeled CRH-1 amacrine cells and identify a postsynaptic target for CRH-1 amacrine cell inhibition in an atypical retinal ganglion cell (RGC) in mouse retina, the Suppressed-by-Contrast (SbC) RGC. Unlike other RGC types, SbC RGCs spike tonically in steady illumination and are suppressed by both increases and decreases in illumination. Inhibition from GABAergic CRH-1 amacrine cells shapes this unique contrast response profile to positive contrast. We show the existence and impact of this circuit, with both paired recordings and cell-type-specific ablation.
Assuntos
Células Amácrinas/fisiologia , Retina/fisiologia , Células Ganglionares da Retina/fisiologia , Células Amácrinas/citologia , Células Amácrinas/metabolismo , Animais , Camundongos , Camundongos Transgênicos , Estimulação Luminosa , Retina/citologia , Retina/metabolismo , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Transdução de Sinais , Transmissão SinápticaRESUMO
We developed two-photon scanning patterned illumination microscopy (2P-SPIM) for super-resolution two-photon imaging. Our approach used a traditional two-photon microscopy setup with temporally modulated excitation to create patterned illumination fields. Combing nine different illuminations and structured illumination reconstruction, super-resolution imaging was achieved in two-photon microscopy. Using 2P-SPIM we achieved a lateral resolution of 141 nm, which represents an improvement by a factor of 1.9 over the corresponding diffraction limit. We further demonstrated super-resolution cellular imaging by 2P-SPIM to image actin cytoskeleton in mammalian cells and three-dimensional imaging in highly scattering retinal tissue.
Assuntos
Microscopia de Fluorescência/métodos , Citoesqueleto de Actina/ultraestrutura , Meios de Contraste , Desenho de Equipamento , Fluorescência , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Imageamento Tridimensional/instrumentação , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/instrumentação , Modelos Teóricos , Nanosferas/ultraestrutura , Imagens de Fantasmas , Compostos de Quinolínio , Retina/ultraestrutura , Técnicas de Cultura de TecidosRESUMO
The dynamic range of visual coding is extended by having separate ganglion cell types that respond to light increments and decrements. Although the primordial color vision system in mammals contains a well-characterized ganglion cell that responds to blue light increments (a blue On center cell), less is known about ganglion cells that respond to blue light decrements (blue Off center cells). We identified a regular mosaic of blue Off center ganglion cells in the ground squirrel. Contrary to the standard scheme, blue Off responses came from a blue On bipolar and inverting amacrine cell.
Assuntos
Visão de Cores/fisiologia , Estimulação Luminosa/métodos , Neurônios Retinianos/fisiologia , Vias Visuais/fisiologia , Potenciais de Ação/fisiologia , Animais , Percepção de Cores/fisiologia , Vias Neurais/fisiologia , Células Ganglionares da Retina/fisiologia , Neurônios Retinianos/citologia , SciuridaeRESUMO
In daylight vision, parallel processing starts at the cone synapse. Cone signals flow to On and Off bipolar cells, which are further divided into types according to morphology, immunocytochemistry, and function. The axons of the bipolar cell types stratify at different levels in the inner plexiform layer (IPL) and can interact with costratifying amacrine and ganglion cells. These interactions endow the ganglion cell types with unique functional properties. The wiring that underlies the interactions among bipolar, amacrine, and ganglion cells is poorly understood. It may be easier to elucidate this wiring if organizational rules can be established. We identify 13 types of cone bipolar cells in the ground squirrel, 11 of which contact contiguous cones, with the possible exception of short-wavelength-sensitive cones. Cells were identified by antibody labeling, tracer filling, and Golgi-like filling following transduction with an adeno-associated virus encoding for green fluorescent protein. The 11 bipolar cell types displayed two organizational patterns. In the first pattern, eight to 10 of the 11 types came in pairs with partially overlapping axonal stratification. Pairs shared morphological, immunocytochemical, and functional properties. The existence of similar pairs is a new motif that might have implications for how signals first diverge from a cone to bipolar cells and then reconverge onto a costratifying ganglion cell. The second pattern is a mirror symmetric organization about the middle of the IPL involving at least seven bipolar cell types. This anatomical symmetry may be associated with a functional symmetry in On and Off ganglion cell responses.