RESUMO
Learning has been associated with modifications of synaptic and circuit properties, but the precise changes storing information in mammals have remained largely unclear. We combined genetically targeted voltage imaging with targeted optogenetic activation and silencing of pre- and post-synaptic neurons to study the mechanisms underlying hippocampal behavioral timescale plasticity. In mice navigating a virtual-reality environment, targeted optogenetic activation of individual CA1 cells at specific places induced stable representations of these places in the targeted cells. Optical elicitation, recording, and modulation of synaptic transmission in behaving mice revealed that activity in presynaptic CA2/3 cells was required for the induction of plasticity in CA1 and, furthermore, that during induction of these place fields in single CA1 cells, synaptic input from CA2/3 onto these same cells was potentiated. These results reveal synaptic implementation of hippocampal behavioral timescale plasticity and define a methodology to resolve synaptic plasticity during learning and memory in behaving mammals.
Assuntos
Região CA1 Hipocampal , Hipocampo , Camundongos , Animais , Região CA1 Hipocampal/fisiologia , Hipocampo/fisiologia , Plasticidade Neuronal/fisiologia , Aprendizagem/fisiologia , Neurônios , Transmissão Sináptica/fisiologia , MamíferosRESUMO
ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology.
Assuntos
Channelrhodopsins/química , Channelrhodopsins/metabolismo , Ativação do Canal Iônico , Animais , Channelrhodopsins/ultraestrutura , Microscopia Crioeletrônica , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Modelos Moleculares , Optogenética , Filogenia , Ratos Sprague-Dawley , Bases de Schiff/química , Células Sf9 , Relação Estrutura-AtividadeRESUMO
Calcium imaging is a powerful tool for recording from large populations of neurons in vivo. Imaging in rhesus macaque motor cortex can enable the discovery of fundamental principles of motor cortical function and can inform the design of next generation brain-computer interfaces (BCIs). Surface two-photon imaging, however, cannot presently access somatic calcium signals of neurons from all layers of macaque motor cortex due to photon scattering. Here, we demonstrate an implant and imaging system capable of chronic, motion-stabilized two-photon imaging of neuronal calcium signals from macaques engaged in a motor task. By imaging apical dendrites, we achieved optical access to large populations of deep and superficial cortical neurons across dorsal premotor (PMd) and gyral primary motor (M1) cortices. Dendritic signals from individual neurons displayed tuning for different directions of arm movement. Combining several technical advances, we developed an optical BCI (oBCI) driven by these dendritic signalswhich successfully decoded movement direction online. By fusing two-photon functional imaging with CLARITY volumetric imaging, we verified that many imaged dendrites which contributed to oBCI decoding originated from layer 5 output neurons, including a putative Betz cell. This approach establishes new opportunities for studying motor control and designing BCIs via two photon imaging.
Assuntos
Interfaces Cérebro-Computador , Cálcio/metabolismo , Dendritos/fisiologia , Microscopia Intravital/instrumentação , Microscopia Intravital/métodos , Córtex Motor/diagnóstico por imagem , Imagem Multimodal/métodos , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Dendritos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Implantes Experimentais , Macaca mulatta , Masculino , Modelos Neurológicos , Atividade Motora/fisiologia , Córtex Motor/fisiologia , Neurônios/fisiologia , FótonsRESUMO
Perceptual experiences may arise from neuronal activity patterns in mammalian neocortex. We probed mouse neocortex during visual discrimination using a red-shifted channelrhodopsin (ChRmine, discovered through structure-guided genome mining) alongside multiplexed multiphoton-holography (MultiSLM), achieving control of individually specified neurons spanning large cortical volumes with millisecond precision. Stimulating a critical number of stimulus-orientation-selective neurons drove widespread recruitment of functionally related neurons, a process enhanced by (but not requiring) orientation-discrimination task learning. Optogenetic targeting of orientation-selective ensembles elicited correct behavioral discrimination. Cortical layer-specific dynamics were apparent, as emergent neuronal activity asymmetrically propagated from layer 2/3 to layer 5, and smaller layer 5 ensembles were as effective as larger layer 2/3 ensembles in eliciting orientation discrimination behavior. Population dynamics emerging after optogenetic stimulation both correctly predicted behavior and resembled natural internal representations of visual stimuli at cellular resolution over volumes of cortex.
Assuntos
Neocórtex/fisiologia , Neocórtex/ultraestrutura , Neurônios/fisiologia , Percepção Visual/fisiologia , Animais , Organismos Aquáticos/genética , Células Cultivadas , Channelrhodopsins/genética , Holografia/métodos , Camundongos , Imagem Molecular , Opsinas/genética , Optogenética , Orientação/fisiologia , Estimulação Luminosa , Percepção Visual/genéticaRESUMO
To decipher dynamic brain information processing, current genetically encoded calcium indicators (GECIs) are limited in single action potential (AP) detection speed, combinatorial spectral compatibility, and two-photon imaging depth. To address this, here, we rationally engineered a next-generation quadricolor GECI suite, XCaMPs. Single AP detection was achieved within 3-10 ms of spike onset, enabling measurements of fast-spike trains in parvalbumin (PV)-positive interneurons in the barrel cortex in vivo and recording three distinct (two inhibitory and one excitatory) ensembles during pre-motion activity in freely moving mice. In vivo paired recording of pre- and postsynaptic firing revealed spatiotemporal constraints of dendritic inhibition in layer 1 in vivo, between axons of somatostatin (SST)-positive interneurons and apical tufts dendrites of excitatory pyramidal neurons. Finally, non-invasive, subcortical imaging using red XCaMP-R uncovered somatosensation-evoked persistent activity in hippocampal CA1 neurons. Thus, the XCaMPs offer a critical enhancement of solution space in studies of complex neuronal circuit dynamics. VIDEO ABSTRACT.
Assuntos
Potenciais de Ação/fisiologia , Axônios/metabolismo , Córtex Cerebral/metabolismo , Hipocampo/metabolismo , Interneurônios/metabolismo , Células Piramidais/metabolismo , Animais , Córtex Cerebral/citologia , Feminino , Hipocampo/citologia , Interneurônios/citologia , Camundongos , Camundongos Transgênicos , Células Piramidais/citologia , Ratos , Ratos Sprague-DawleyRESUMO
Categorically distinct basic drives (for example, for social versus feeding behaviour1-3) can exert potent influences on each other; such interactions are likely to have important adaptive consequences (such as appropriate regulation of feeding in the context of social hierarchies) and can become maladaptive (such as in clinical settings involving anorexia). It is known that neural systems regulating natural and adaptive caloric intake, and those regulating social behaviours, involve related circuitry4-7, but the causal circuit mechanisms of these drive adjudications are not clear. Here we investigate the causal role in behaviour of cellular-resolution experience-specific neuronal populations in the orbitofrontal cortex, a major reward-processing hub that contains diverse activity-specific neuronal populations that respond differentially to various aspects of caloric intake8-13 and social stimuli14,15. We coupled genetically encoded activity imaging with the development and application of methods for optogenetic control of multiple individually defined cells, to both optically monitor and manipulate the activity of many orbitofrontal cortex neurons at the single-cell level in real time during rewarding experiences (caloric consumption and social interaction). We identified distinct populations within the orbitofrontal cortex that selectively responded to either caloric rewards or social stimuli, and found that activity of individually specified naturally feeding-responsive neurons was causally linked to increased feeding behaviour; this effect was selective as, by contrast, single-cell resolution activation of naturally social-responsive neurons inhibited feeding, and activation of neurons responsive to neither feeding nor social stimuli did not alter feeding behaviour. These results reveal the presence of potent cellular-level subnetworks within the orbitofrontal cortex that can be precisely engaged to bidirectionally control feeding behaviours subject to, for example, social influences.
Assuntos
Comportamento Alimentar/fisiologia , Vias Neurais/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/fisiologia , Comportamento Social , Animais , Condicionamento Operante/fisiologia , Ingestão de Energia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Optogenética , Recompensa , Análise de Célula ÚnicaRESUMO
Increasing the volumetric imaging speed of light-sheet microscopy will improve its ability to detect fast changes in neural activity. Here, a system is introduced for brain-wide imaging of neural activity in the larval zebrafish by coupling structured illumination with cubic phase extended depth-of-field (EDoF) pupil encoding. This microscope enables faster light-sheet imaging and facilitates arbitrary plane scanning-removing constraints on acquisition speed, alignment tolerances, and physical motion near the sample. The usefulness of this method is demonstrated by performing multi-plane calcium imaging in the fish brain with a 416×832×160 µm field of view at 33 Hz. The optomotor response behavior of the zebrafish is monitored at high speeds, and time-locked correlations of neuronal activity are resolved across its brain.
Assuntos
Cálcio/metabolismo , Luz , Microscopia/métodos , Neurônios/metabolismo , Animais , Encéfalo/citologia , Caenorhabditis elegans , Imagem MolecularRESUMO
We introduce a scanless optical method to image neuronal activity in three dimensions simultaneously. Using a spatial light modulator and a custom-designed phase mask, we illuminate and collect light simultaneously from different focal planes and perform calcium imaging of neuronal activity in vitro and in vivo. This method, combining structured illumination with volume projection imaging, could be used as a technological platform for brain activity mapping.
Assuntos
Imageamento Tridimensional/métodos , Neurônios/fisiologia , Animais , Hipocampo/fisiologia , Processamento de Imagem Assistida por Computador , Camundongos , Camundongos Endogâmicos C57BL , Peixe-ZebraRESUMO
Imaging three-dimensional structures represents a major challenge for conventional microscopies. Here we describe a Spatial Light Modulator (SLM) microscope that can simultaneously address and image multiple targets in three dimensions. A wavefront coding element and computational image processing enables extended depth-of-field imaging. High-resolution, multi-site three-dimensional targeting and sensing is demonstrated in both transparent and scattering media over a depth range of 300-1,000 microns.
RESUMO
We demonstrate quantitative multicolor three-dimensional (3D) subdiffraction imaging of the structural arrangement of fluorescent protein fusions in living Caulobacter crescentus bacteria. Given single-molecule localization precisions of 20-40 nm, a flexible locally weighted image registration algorithm is critical to accurately combine the super-resolution data with <10 nm error. Surface-relief dielectric phase masks implement a double-helix response at two wavelengths to distinguish two different fluorescent labels and to quantitatively and precisely localize them relative to each other in 3D.
Assuntos
Proteínas de Bactérias/ultraestrutura , Caulobacter crescentus/química , CorRESUMO
The use of complementary engineered point spread functions is proposed for the joint tasks of depth estimation and image recovery over an extended depth of field. A digital imaging system with a dynamically adjustable pupil is demonstrated experimentally. The implementation of a broadband, passive camera is demonstrated with a fractional ranging error of 4/10(4) at a working distance of 1 m. Once the depth and brightness information of a scene are obtained, a synthetic camera is defined and images rendered computationally to emphasize particular features such as image focusing at different depths.
Assuntos
Algoritmos , Interpretação Estatística de Dados , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Iluminação/métodos , Modelos Estatísticos , Simulação por ComputadorRESUMO
The 3D orientation and location of individual molecules is an important marker for the local environment and the state of a molecule. Therefore dipole localization and orientation estimation is important for biological sensing and imaging. Precise dipole localization is also critical for superresolution imaging. We propose and analyze wide field microscope configurations to simultaneously measure these parameters for multiple fixed dipole emitters. Examination of the images of radiating dipoles reveals how information transfer and precise detection can be improved. We use an information theoretic analysis to quantify the performance limits of position and orientation estimation through comparison of the Cramer-Rao lower bounds in a photon limited environment. We show that bi-focal and double-helix polarization-sensitive systems are attractive candidates for simultaneously estimating the 3D dipole location and orientation.
Assuntos
Algoritmos , Simulação por Computador , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia/métodos , Fótons , HumanosRESUMO
Super-resolution imaging with photo-activatable or photo-switchable probes is a promising tool in biological applications to reveal previously unresolved intra-cellular details with visible light. This field benefits from developments in the areas of molecular probes, optical systems, and computational post-processing of the data. The joint design of optics and reconstruction processes using double-helix point spread functions (DH-PSF) provides high resolution three-dimensional (3D) imaging over a long depth-of-field. We demonstrate for the first time a method integrating a Fisher information efficient DH-PSF design, a surface relief optical phase mask, and an optimal 3D localization estimator. 3D super-resolution imaging using photo-switchable dyes reveals the 3D microtubule network in mammalian cells with localization precision approaching the information theoretical limit over a depth of 1.2 µm.
Assuntos
Processamento de Imagem Assistida por Computador , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Óptica e Fotônica , Animais , Desenho de Equipamento , Humanos , FótonsRESUMO
Photo-activation localization microscopy is a far-field superresolution imaging technique based on the localization of single molecules with subdiffraction limit precision. Known under acronyms such as PALM (photo-activated localization microscopy) or STORM (stochastic optical reconstruction microscopy), these techniques achieve superresolution by allowing only a sparse, random set of molecules to emit light at any given time and subsequently localizing each molecule with great precision. Recently, such techniques have been extended to three dimensions, opening up unprecedented possibilities to explore the structure and function of cells. Interestingly, proper engineering of the three-dimensional (3D) point spread function (PSF) through additional optics has been demonstrated to theoretically improve 3D position estimation and ultimately resolution. In this paper, an optimal 3D single-molecule localization estimator is presented in a general framework for noisy, aberrated and/or engineered PSF imaging. To find the position of each molecule, a phase-retrieval enabled maximum-likelihood estimator is implemented. This estimator is shown to be efficient, meaning it reaches the fundamental Cramer-Rao lower bound of x, y, and z localization precision. Experimental application of the phase-retrieval enabled maximum-likelihood estimator using a particular engineered PSF microscope demonstrates unmatched low-photon-count 3D wide-field single-molecule localization performance.
RESUMO
We present a double-helix point spread function (DH-PSF) based three-dimensional (3D) microscope with efficient photon collection using a phase mask fabricated by gray-level lithography. The system using the phase mask more than doubles the efficiency of current liquid crystal spatial light modulator implementations. We demonstrate the phase mask DH-PSF microscope for 3D photo-activation localization microscopy (PM-DH-PALM) over an extended axial range.