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1.
Cell ; 184(15): 4048-4063.e32, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34233165

RESUMO

Microglia, the resident immune cells of the brain, have emerged as crucial regulators of synaptic refinement and brain wiring. However, whether the remodeling of distinct synapse types during development is mediated by specialized microglia is unknown. Here, we show that GABA-receptive microglia selectively interact with inhibitory cortical synapses during a critical window of mouse postnatal development. GABA initiates a transcriptional synapse remodeling program within these specialized microglia, which in turn sculpt inhibitory connectivity without impacting excitatory synapses. Ablation of GABAB receptors within microglia impairs this process and leads to behavioral abnormalities. These findings demonstrate that brain wiring relies on the selective communication between matched neuronal and glial cell types.


Assuntos
Microglia/metabolismo , Inibição Neural/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Animais Recém-Nascidos , Comportamento Animal , Regulação da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Parvalbuminas/metabolismo , Fenótipo , Receptores de GABA-B/metabolismo , Sinapses/fisiologia , Transcrição Gênica
3.
Nature ; 608(7924): 750-756, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35948630

RESUMO

Microglia are specialized macrophages in the brain parenchyma that exist in multiple transcriptional states and reside within a wide range of neuronal environments1-4. However, how and where these states are generated remains poorly understood. Here, using the mouse somatosensory cortex, we demonstrate that microglia density and molecular state acquisition are determined by the local composition of pyramidal neuron classes. Using single-cell and spatial transcriptomic profiling, we unveil the molecular signatures and spatial distributions of diverse microglia populations and show that certain states are enriched in specific cortical layers, whereas others are broadly distributed throughout the cortex. Notably, conversion of deep-layer pyramidal neurons to an alternate class identity reconfigures the distribution of local, layer-enriched homeostatic microglia to match the new neuronal niche. Leveraging the transcriptional diversity of pyramidal neurons in the neocortex, we construct a ligand-receptor atlas describing interactions between individual pyramidal neuron subtypes and microglia states, revealing rules of neuron-microglia communication. Our findings uncover a fundamental role for neuronal diversity in instructing the acquisition of microglia states as a potential mechanism for fine-tuning neuroimmune interactions within the cortical local circuitry.


Assuntos
Microglia , Neocórtex , Células Piramidais , Córtex Somatossensorial , Animais , Contagem de Células , Camundongos , Microglia/classificação , Microglia/fisiologia , Neocórtex/citologia , Neocórtex/fisiologia , Células Piramidais/classificação , Células Piramidais/fisiologia , Análise de Célula Única , Córtex Somatossensorial/citologia , Córtex Somatossensorial/fisiologia , Transcriptoma
4.
Nat Methods ; 19(3): 311-315, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34824477

RESUMO

Highly multiplexed tissue imaging makes detailed molecular analysis of single cells possible in a preserved spatial context. However, reproducible analysis of large multichannel images poses a substantial computational challenge. Here, we describe a modular and open-source computational pipeline, MCMICRO, for performing the sequential steps needed to transform whole-slide images into single-cell data. We demonstrate the use of MCMICRO on tissue and tumor images acquired using multiple imaging platforms, thereby providing a solid foundation for the continued development of tissue imaging software.


Assuntos
Processamento de Imagem Assistida por Computador , Neoplasias , Diagnóstico por Imagem , Humanos , Processamento de Imagem Assistida por Computador/métodos , Neoplasias/diagnóstico por imagem , Neoplasias/patologia , Software
5.
Nature ; 569(7756): 413-417, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31043747

RESUMO

A technology that simultaneously records membrane potential from multiple neurons in behaving animals will have a transformative effect on neuroscience research1,2. Genetically encoded voltage indicators are a promising tool for these purposes; however, these have so far been limited to single-cell recordings with a marginal signal-to-noise ratio in vivo3-5. Here we developed improved near-infrared voltage indicators, high-speed microscopes and targeted gene expression schemes that enabled simultaneous in vivo recordings of supra- and subthreshold voltage dynamics in multiple neurons in the hippocampus of behaving mice. The reporters revealed subcellular details of back-propagating action potentials and correlations in subthreshold voltage between multiple cells. In combination with stimulation using optogenetics, the reporters revealed changes in neuronal excitability that were dependent on the behavioural state, reflecting the interplay of excitatory and inhibitory synaptic inputs. These tools open the possibility for detailed explorations of network dynamics in the context of behaviour. Fig. 1 PHOTOACTIVATED QUASAR3 (PAQUASAR3) REPORTS NEURONAL ACTIVITY IN VIVO.: a, Schematic of the paQuasAr3 construct. b, Photoactivation by blue light enhanced voltage signals excited by red light in cultured neurons that expressed paQuasAr3 (representative example of n = 4 cells). c, Model of the photocycle of paQuasAr3. d, Confocal images of sparsely expressed paQuasAr3 in brain slices. Scale bars, 50 µm. Representative images, experiments were repeated in n = 3 mice. e, Simultaneous fluorescence and patch-clamp recordings from a neuron expressing paQuasAr3 in acute brain slice. Top, magnification of boxed regions. Schematic shows brain slice, patch pipette and microscope objective. f, Simultaneous fluorescence and patch-clamp recordings of inhibitory post synaptic potentials in an L2-3 neuron induced by electrical stimulation of L5-6 in acute slice. g, Normalized change in fluorescence (ΔF/F) and SNR of optically recorded post-synaptic potentials (PSPs) as a function of the amplitude of the post-synaptic potentials. The voltage sensitivity was ΔF/F = 40 ± 1.7% per 100 mV. The SNR was 0.93 ± 0.07 per 1 mV in a 1-kHz bandwidth (n = 42 post-synaptic potentials from 5 cells, data are mean ± s.d.). Schematic shows brain slice, patch pipette, field stimulation electrodes and microscope objective. h, Optical measurements of paQuasAr3 fluorescence in the CA1 region of the hippocampus (top) and glomerular layer of the olfactory bulb (bottom) of anaesthetized mice (representative traces from n = 7 CA1 cells and n = 13 olfactory bulb cells, n = 3 mice). Schematics show microscope objective and the imaged brain region. i, STA fluorescence from 88 spikes in a CA1 oriens neuron. j, Frames from the STA video showing the delay in the back-propagating action potential in the dendrites relative to the soma. k, Sub-Nyquist fitting of the action potential delay and width shows electrical compartmentalization in the dendrites. Experiments in k-m were repeated in n = 2 cells from n = 2 mice.


Assuntos
Potenciais de Ação , Hipocampo/citologia , Hipocampo/fisiologia , Optogenética/métodos , Algoritmos , Animais , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Bacteriorodopsinas/genética , Bacteriorodopsinas/metabolismo , Células Cultivadas , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Neurônios/metabolismo , Caminhada
6.
Bioinformatics ; 39(6)2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37267161

RESUMO

MOTIVATION: Imaging Spatial Transcriptomics techniques characterize gene expression in cells in their native context by imaging barcoded probes for mRNA with single molecule resolution. However, the need to acquire many rounds of high-magnification imaging data limits the throughput and impact of existing methods. RESULTS: We describe the Joint Sparse method for Imaging Transcriptomics, an algorithm for decoding lower magnification Imaging Spatial Transcriptomics data than that used in standard experimental workflows. Joint Sparse method for Imaging Transcriptomics incorporates codebook knowledge and sparsity assumptions into an optimization problem, which is less reliant on well separated optical signals than current pipelines. Using experimental data obtained by performing Multiplexed Error-Robust Fluorescence in situ Hybridization on tissue from mouse brain, we demonstrate that Joint Sparse method for Imaging Transcriptomics enables improved throughput and recovery performance over standard decoding methods. AVAILABILITY AND IMPLEMENTATION: Software implementation of JSIT, together with example files, is available at https://github.com/jpbryan13/JSIT.


Assuntos
Perfilação da Expressão Gênica , Transcriptoma , Animais , Camundongos , Hibridização in Situ Fluorescente/métodos , Perfilação da Expressão Gênica/métodos , Software , Algoritmos
7.
J Neurosci ; 39(25): 4889-4908, 2019 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-30952812

RESUMO

Optical tools for simultaneous perturbation and measurement of neural activity open the possibility of mapping neural function over wide areas of brain tissue. However, spectral overlap of actuators and reporters presents a challenge for their simultaneous use, and optical scattering and out-of-focus fluorescence in tissue degrade resolution. To minimize optical crosstalk, we combined an optimized variant (eTsChR) of the most blue-shifted channelrhodopsin reported to-date with a nuclear-localized red-shifted Ca2+ indicator, H2B-jRGECO1a. To perform wide-area optically sectioned imaging in tissue, we designed a structured illumination technique that uses Hadamard matrices to encode spatial information. By combining these molecular and optical approaches we made wide-area functional maps in acute brain slices from mice of both sexes. The maps spanned cortex and striatum and probed the effects of antiepileptic drugs on neural excitability and the effects of AMPA and NMDA receptor blockers on functional connectivity. Together, these tools provide a powerful capability for wide-area mapping of neuronal excitability and functional connectivity in acute brain slices.SIGNIFICANCE STATEMENT A new technique for simultaneous optogenetic stimulation and calcium imaging across wide areas of brain slice enables high-throughput mapping of neuronal excitability and synaptic transmission.


Assuntos
Anticonvulsivantes/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Imagem Óptica/métodos , Transmissão Sináptica/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Células HEK293 , Humanos , Camundongos , Rede Nervosa/efeitos dos fármacos , Optogenética , Estimulação Luminosa , Ratos
9.
Nat Chem Biol ; 12(8): 586-92, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27272565

RESUMO

Cell signaling relies extensively on dynamic pools of redox-inactive metal ions such as sodium, potassium, calcium and zinc, but their redox-active transition metal counterparts such as copper and iron have been studied primarily as static enzyme cofactors. Here we report that copper is an endogenous regulator of lipolysis, the breakdown of fat, which is an essential process in maintaining body weight and energy stores. Using a mouse model of genetic copper misregulation, in combination with pharmacological alterations in copper status and imaging studies in a 3T3-L1 white adipocyte model, we found that copper regulates lipolysis at the level of the second messenger, cyclic AMP (cAMP), by altering the activity of the cAMP-degrading phosphodiesterase PDE3B. Biochemical studies of the copper-PDE3B interaction establish copper-dependent inhibition of enzyme activity and identify a key conserved cysteine residue in a PDE3-specific loop that is essential for the observed copper-dependent lipolytic phenotype.


Assuntos
Cobre/farmacologia , AMP Cíclico/metabolismo , Lipólise/efeitos dos fármacos , Inibidores da Fosfodiesterase 3/farmacologia , Células 3T3-L1 , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Relação Dose-Resposta a Droga , Camundongos , Estrutura Molecular , Relação Estrutura-Atividade
10.
J Neurosci ; 36(8): 2458-72, 2016 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-26911693

RESUMO

Optical imaging of voltage indicators based on green fluorescent proteins (FPs) or archaerhodopsin has emerged as a powerful approach for detecting the activity of many individual neurons with high spatial and temporal resolution. Relative to green FP-based voltage indicators, a bright red-shifted FP-based voltage indicator has the intrinsic advantages of lower phototoxicity, lower autofluorescent background, and compatibility with blue-light-excitable channelrhodopsins. Here, we report a bright red fluorescent voltage indicator (fluorescent indicator for voltage imaging red; FlicR1) with properties that are comparable to the best available green indicators. To develop FlicR1, we used directed protein evolution and rational engineering to screen libraries of thousands of variants. FlicR1 faithfully reports single action potentials (∼3% ΔF/F) and tracks electrically driven voltage oscillations at 100 Hz in dissociated Sprague Dawley rat hippocampal neurons in single trial recordings. Furthermore, FlicR1 can be easily imaged with wide-field fluorescence microscopy. We demonstrate that FlicR1 can be used in conjunction with a blue-shifted channelrhodopsin for all-optical electrophysiology, although blue light photoactivation of the FlicR1 chromophore presents a challenge for applications that require spatially overlapping yellow and blue excitation.


Assuntos
Corantes Fluorescentes/análise , Hipocampo/química , Hipocampo/fisiologia , Proteínas Luminescentes/análise , Neurônios/química , Neurônios/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Células HEK293 , Células HeLa , Humanos , Masculino , Microscopia de Fluorescência/métodos , Técnicas de Cultura de Órgãos/métodos , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteína Vermelha Fluorescente
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