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2.
Neuron ; 110(12): 2024-2040.e10, 2022 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-35452606

RESUMO

General anesthetics induce loss of consciousness, a global change in behavior. However, a corresponding global change in activity in the context of defined cortical cell types has not been identified. Here, we show that spontaneous activity of mouse layer 5 pyramidal neurons, but of no other cortical cell type, becomes consistently synchronized in vivo by different general anesthetics. This heightened neuronal synchrony is aperiodic, present across large distances, and absent in cortical neurons presynaptic to layer 5 pyramidal neurons. During the transition to and from anesthesia, changes in synchrony in layer 5 coincide with the loss and recovery of consciousness. Activity within both apical and basal dendrites is synchronous, but only basal dendrites' activity is temporally locked to somatic activity. Given that layer 5 is a major cortical output, our results suggest that brain-wide synchrony in layer 5 pyramidal neurons may contribute to the loss of consciousness during general anesthesia.


Assuntos
Anestésicos Gerais , Células Piramidais , Anestesia Geral , Anestésicos Gerais/farmacologia , Animais , Dendritos/fisiologia , Camundongos , Células Piramidais/fisiologia , Inconsciência
3.
Science ; 368(6495): 1108-1113, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32499439

RESUMO

Enabling near-infrared light sensitivity in a blind human retina may supplement or restore visual function in patients with regional retinal degeneration. We induced near-infrared light sensitivity using gold nanorods bound to temperature-sensitive engineered transient receptor potential (TRP) channels. We expressed mammalian or snake TRP channels in light-insensitive retinal cones in a mouse model of retinal degeneration. Near-infrared stimulation increased activity in cones, ganglion cell layer neurons, and cortical neurons, and enabled mice to perform a learned light-driven behavior. We tuned responses to different wavelengths, by using nanorods of different lengths, and to different radiant powers, by using engineered channels with different temperature thresholds. We targeted TRP channels to human retinas, which allowed the postmortem activation of different cell types by near-infrared light.


Assuntos
Cegueira/terapia , Ouro , Raios Infravermelhos , Nanotubos , Degeneração Retiniana/terapia , Limiar Sensorial/efeitos da radiação , Canais de Cátion TRPC/fisiologia , Visão Ocular/efeitos da radiação , Animais , Cegueira/fisiopatologia , Modelos Animais de Doenças , Potenciais Evocados Visuais/fisiologia , Potenciais Evocados Visuais/efeitos da radiação , Engenharia Genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Estimulação Luminosa , Ratos , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Fotorreceptoras Retinianas Cones/efeitos da radiação , Degeneração Retiniana/fisiopatologia , Células Ganglionares da Retina/fisiologia , Células Ganglionares da Retina/efeitos da radiação , Limiar Sensorial/fisiologia , Serpentes , Canais de Cátion TRPC/genética , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/fisiologia , Visão Ocular/fisiologia , Córtex Visual/fisiopatologia , Córtex Visual/efeitos da radiação
4.
Nat Neurosci ; 23(5): 625-637, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32284608

RESUMO

Decades of research support the idea that associations between a conditioned stimulus (CS) and an unconditioned stimulus (US) are encoded in the lateral amygdala (LA) during fear learning. However, direct proof for the sources of CS and US information is lacking. Definitive evidence of the LA as the primary site for cue association is also missing. Here, we show that calretinin (Calr)-expressing neurons of the lateral thalamus (Calr+LT neurons) convey the association of fast CS (tone) and US (foot shock) signals upstream from the LA in mice. Calr+LT input shapes a short-latency sensory-evoked activation pattern of the amygdala via both feedforward excitation and inhibition. Optogenetic silencing of Calr+LT input to the LA prevents auditory fear conditioning. Notably, fear conditioning drives plasticity in Calr+LT neurons, which is required for appropriate cue and contextual fear memory retrieval. Collectively, our results demonstrate that Calr+LT neurons provide integrated CS-US representations to the LA that support the formation of aversive memories.


Assuntos
Condicionamento Clássico/fisiologia , Medo/fisiologia , Vias Neurais/fisiologia , Plasticidade Neuronal/fisiologia , Animais , Complexo Nuclear Basolateral da Amígdala/fisiologia , Calreticulina/metabolismo , Sinais (Psicologia) , Memória/fisiologia , Camundongos , Neurônios/fisiologia , Transdução de Sinais/fisiologia , Tálamo/fisiologia
5.
Nat Methods ; 16(11): 1105-1108, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31527839

RESUMO

Light-sheet microscopy is an ideal technique for imaging large cleared samples; however, the community is still lacking instruments capable of producing volumetric images of centimeter-sized cleared samples with near-isotropic resolution within minutes. Here, we introduce the mesoscale selective plane-illumination microscopy initiative, an open-hardware project for building and operating a light-sheet microscope that addresses these challenges and is compatible with any type of cleared or expanded sample ( www.mesospim.org ).


Assuntos
Microscopia de Fluorescência/instrumentação , Animais , Embrião de Galinha , Microscopia de Fluorescência/métodos , Software
6.
Neuron ; 99(1): 117-134.e11, 2018 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-29937281

RESUMO

Many brain regions contain local interneurons of distinct types. How does an interneuron type contribute to the input-output transformations of a given brain region? We addressed this question in the mouse retina by chemogenetically perturbing horizontal cells, an interneuron type providing feedback at the first visual synapse, while monitoring the light-driven spiking activity in thousands of ganglion cells, the retinal output neurons. We uncovered six reversible perturbation-induced effects in the response dynamics and response range of ganglion cells. The effects were enhancing or suppressive, occurred in different response epochs, and depended on the ganglion cell type. A computational model of the retinal circuitry reproduced all perturbation-induced effects and led us to assign specific functions to horizontal cells with respect to different ganglion cell types. Our combined experimental and theoretical work reveals how a single interneuron type can differentially shape the dynamical properties of distinct output channels of a brain region.


Assuntos
Retroalimentação , Interneurônios/fisiologia , Células Ganglionares da Retina/fisiologia , Células Horizontais da Retina/fisiologia , Visão Ocular/fisiologia , Animais , Cálcio/metabolismo , Camundongos , Modelos Neurológicos , Células Fotorreceptoras de Vertebrados , Células Bipolares da Retina , Sinapses
7.
Nat Biotechnol ; 36(1): 81-88, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29251729

RESUMO

Genetic engineering by viral infection of single cells is useful to study complex systems such as the brain. However, available methods for infecting single cells have drawbacks that limit their applications. Here we describe 'virus stamping', in which viruses are reversibly bound to a delivery vehicle-a functionalized glass pipette tip or magnetic nanoparticles in a pipette-that is brought into physical contact with the target cell on a surface or in tissue, using mechanical or magnetic forces. Different single cells in the same tissue can be infected with different viruses and an individual cell can be simultaneously infected with different viruses. We use rabies, lenti, herpes simplex, and adeno-associated viruses to drive expression of fluorescent markers or a calcium indicator in target cells in cell culture, mouse retina, human brain organoid, and the brains of live mice. Virus stamping provides a versatile solution for targeted single-cell infection of diverse cell types, both in vitro and in vivo.


Assuntos
Encéfalo/virologia , Nanopartículas de Magnetita/administração & dosagem , Análise de Célula Única/métodos , Vírus/genética , Animais , Engenharia Genética/tendências , Humanos , Nanopartículas de Magnetita/química , Camundongos , Organoides/metabolismo , Organoides/virologia , Retina/metabolismo , Retina/virologia , Distribuição Tecidual , Viroses/genética , Viroses/metabolismo , Replicação Viral/genética
8.
Nat Neurosci ; 20(7): 960-968, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28530661

RESUMO

How neuronal computations in the sensory periphery contribute to computations in the cortex is not well understood. We examined this question in the context of visual-motion processing in the retina and primary visual cortex (V1) of mice. We disrupted retinal direction selectivity, either exclusively along the horizontal axis using FRMD7 mutants or along all directions by ablating starburst amacrine cells, and monitored neuronal activity in layer 2/3 of V1 during stimulation with visual motion. In control mice, we found an over-representation of cortical cells preferring posterior visual motion, the dominant motion direction an animal experiences when it moves forward. In mice with disrupted retinal direction selectivity, the over-representation of posterior-motion-preferring cortical cells disappeared, and their responses at higher stimulus speeds were reduced. This work reveals the existence of two functionally distinct, sensory-periphery-dependent and -independent computations of visual motion in the cortex.


Assuntos
Células Amácrinas/fisiologia , Percepção de Movimento/fisiologia , Retina/fisiologia , Córtex Visual/fisiologia , Animais , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Toxina Diftérica/farmacologia , Feminino , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Estimulação Luminosa , Retina/efeitos dos fármacos , Retina/metabolismo , Córtex Visual/metabolismo , Vias Visuais/fisiologia
9.
Science ; 349(6243): 70-4, 2015 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-26138975

RESUMO

Individual cortical neurons can selectively respond to specific environmental features, such as visual motion or faces. How this relates to the selectivity of the presynaptic network across cortical layers remains unclear. We used single-cell-initiated, monosynaptically restricted retrograde transsynaptic tracing with rabies viruses expressing GCaMP6s to image, in vivo, the visual motion-evoked activity of individual layer 2/3 pyramidal neurons and their presynaptic networks across layers in mouse primary visual cortex. Neurons within each layer exhibited similar motion direction preferences, forming layer-specific functional modules. In one-third of the networks, the layer modules were locked to the direction preference of the postsynaptic neuron, whereas for other networks the direction preference varied by layer. Thus, there exist feature-locked and feature-variant cortical networks.


Assuntos
Terminações Pré-Sinápticas/fisiologia , Células Piramidais/fisiologia , Córtex Visual/fisiologia , Animais , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Potenciais Evocados Visuais , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Camundongos , Movimento (Física) , Rede Nervosa/citologia , Rede Nervosa/fisiologia , Neuroimagem , Vírus da Raiva , Análise de Célula Única
10.
Neuron ; 79(6): 1078-85, 2013 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-23973208

RESUMO

Inferring the direction of image motion is a fundamental component of visual computation and essential for visually guided behavior. In the retina, the direction of image motion is computed in four cardinal directions, but it is not known at which circuit location along the flow of visual information the cardinal direction selectivity first appears. We recorded the concerted activity of the neuronal circuit elements of single direction-selective (DS) retinal ganglion cells at subcellular resolution by combining GCaMP3-functionalized transsynaptic viral tracing and two-photon imaging. While the visually evoked activity of the dendritic segments of the DS cells were direction selective, direction-selective activity was absent in the axon terminals of bipolar cells. Furthermore, the glutamate input to DS cells, recorded using a genetically encoded glutamate sensor, also lacked direction selectivity. Therefore, the first stage in which extraction of a cardinal motion direction occurs is the dendrites of DS cells.


Assuntos
Dendritos/fisiologia , Percepção de Movimento/fisiologia , Orientação/fisiologia , Células Ganglionares da Retina/citologia , Sinapses/fisiologia , Vias Visuais/fisiologia , Potenciais de Ação , Animais , Animais Recém-Nascidos , Colina O-Acetiltransferase/metabolismo , Estimulação Elétrica , Proteínas Relacionadas à Folistatina/genética , Proteínas Relacionadas à Folistatina/metabolismo , Ácido Glutâmico/metabolismo , Proteínas de Fluorescência Verde/genética , Imageamento Tridimensional , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Confocal , Rede Nervosa/citologia , Rede Nervosa/fisiologia , Optogenética , Técnicas de Patch-Clamp , Vírus da Raiva/fisiologia , Retina/citologia , Células Bipolares da Retina/classificação , Células Bipolares da Retina/fisiologia , Células Ganglionares da Retina/fisiologia , Transdução Genética
11.
Nat Methods ; 9(2): 201-8, 2012 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-22231641

RESUMO

The understanding of brain computations requires methods that read out neural activity on different spatial and temporal scales. Following signal propagation and integration across a neuron and recording the concerted activity of hundreds of neurons pose distinct challenges, and the design of imaging systems has been mostly focused on tackling one of the two operations. We developed a high-resolution, acousto-optic two-photon microscope with continuous three-dimensional (3D) trajectory and random-access scanning modes that reaches near-cubic-millimeter scan range and can be adapted to imaging different spatial scales. We performed 3D calcium imaging of action potential backpropagation and dendritic spike forward propagation at sub-millisecond temporal resolution in mouse brain slices. We also performed volumetric random-access scanning calcium imaging of spontaneous and visual stimulation-evoked activity in hundreds of neurons of the mouse visual cortex in vivo. These experiments demonstrate the subcellular and network-scale imaging capabilities of our system.


Assuntos
Encéfalo/fisiologia , Fótons , Potenciais de Ação , Animais , Camundongos , Neurônios/fisiologia , Córtex Visual/citologia , Córtex Visual/fisiologia
12.
IEEE Trans Biomed Eng ; 57(2): 384-96, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19535317

RESUMO

A computational method providing online, automated 3-D reconstruction of the right atrium of the human heart is presented in this paper. Endocardial boundaries were extracted from transesophageal ultrasound data by a topographic cellular contour extraction (TCCE) algorithm. The TCCE method was implemented on a continuous-time, analog, massively parallel processor, and on a digital serial processor. Processing speeds were 500 or 40 frames per second, depending on the hardware used. Extracted boundary point sets were rendered into a 3-D mesh and the volume of the cavity was quantified. Accuracy of volume quantification was validated on 60 in vitro static phantoms and 12 clinical subjects. For the clinical recordings, reference volumes were estimated from manually delineated endocardial boundaries. The average error of volume quantification by the TCCE method was 8% +/-5% ( n = 12), the average of the interobserver variability between two independent human experts was 5% +/-2% ( n = 6). Interactive planning of atrial septal defect closure in pediatric cardiology is presented as an example that demonstrates a potential clinical application of the method.


Assuntos
Algoritmos , Ecocardiografia Transesofagiana/métodos , Átrios do Coração/anatomia & histologia , Processamento de Imagem Assistida por Computador/métodos , Criança , Humanos , Variações Dependentes do Observador , Imagens de Fantasmas , Reprodutibilidade dos Testes
13.
Curr Biol ; 17(11): 981-8, 2007 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-17524644

RESUMO

Intrinsically photosensitive melanopsin-containing retinal ganglion cells (ipRGCs) control important physiological processes, including the circadian rhythm, the pupillary reflex, and the suppression of locomotor behavior (reviewed in [1]). ipRGCs are also activated by classical photoreceptors, the rods and cones, through local retinal circuits [2, 3]. ipRGCs can be transsynaptically labeled through the pupillary-reflex circuit with the derivatives of the Bartha strain of the alphaherpesvirus pseudorabies virus(PRV) [4, 5] that express GFP [6-12]. Bartha-strain derivatives spread only in the retrograde direction [13]. There is evidence that infected cells function normally for a while during GFP expression [7]. Here we combine transsynaptic PRV labeling, two-photon laser microscopy, and electrophysiological techniques to trace the local circuit of different ipRGC subtypes in the mouse retina and record light-evoked activity from the transsynaptically labeled ganglion cells. First, we show that ipRGCs are connected by monostratified amacrine cells that provide strong inhibition from classical-photoreceptor-driven circuits. Second, we show evidence that dopaminergic interplexiform cells are synaptically connected to ipRGCs. The latter finding provides a circuitry link between light-dark adaptation and ipRGC function.


Assuntos
Células Ganglionares da Retina/fisiologia , Opsinas de Bastonetes/metabolismo , Vias Visuais/fisiologia , Células Amácrinas/fisiologia , Células Amácrinas/virologia , Animais , Proteínas de Fluorescência Verde/análise , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/metabolismo , Camundongos , Células Ganglionares da Retina/efeitos da radiação , Células Ganglionares da Retina/virologia , Transmissão Sináptica
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