RESUMO
Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 µm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine spatially and molecularly defined subregions. EASI-FISH also facilitates iterative reanalysis of scRNA-seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.
Assuntos
Região Hipotalâmica Lateral/metabolismo , Hibridização in Situ Fluorescente , Animais , Biomarcadores/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Região Hipotalâmica Lateral/citologia , Imageamento Tridimensional , Masculino , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA/metabolismo , RNA-Seq , Análise de Célula Única , Transcrição GênicaRESUMO
Chemogenetics enables noninvasive chemical control over cell populations in behaving animals. However, existing small-molecule agonists show insufficient potency or selectivity. There is also a need for chemogenetic systems compatible with both research and human therapeutic applications. We developed a new ion channel-based platform for cell activation and silencing that is controlled by low doses of the smoking cessation drug varenicline. We then synthesized subnanomolar-potency agonists, called uPSEMs, with high selectivity for the chemogenetic receptors. uPSEMs and their receptors were characterized in brains of mice and a rhesus monkey by in vivo electrophysiology, calcium imaging, positron emission tomography, behavioral efficacy testing, and receptor counterscreening. This platform of receptors and selective ultrapotent agonists enables potential research and clinical applications of chemogenetics.
Assuntos
Células Quimiorreceptoras/efeitos dos fármacos , Antagonistas Nicotínicos/farmacologia , Agentes de Cessação do Hábito de Fumar/farmacologia , Vareniclina/análogos & derivados , Vareniclina/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Animais , Células Quimiorreceptoras/fisiologia , Engenharia Genética , Haplorrinos , Humanos , Ligantes , Camundongos , Mutação , Domínios Proteicos , Receptores de Glicina/agonistas , Receptores de Glicina/genética , Receptores 5-HT3 de Serotonina/genética , Tropizetrona/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/genéticaRESUMO
Transforming synaptic input into action potential output is a fundamental function of neurons. The pattern of action potential output from principal cells of the mammalian hippocampus encodes spatial and nonspatial information, but the cellular and circuit mechanisms by which neurons transform their synaptic input into a given output are unknown. Using a combination of optical activation and cell type-specific pharmacogenetic silencing in vitro, we found that dendritic inhibition is the primary regulator of input-output transformations in mouse hippocampal CA1 pyramidal cells, and acts by gating the dendritic electrogenesis driving burst spiking. Dendrite-targeting interneurons are themselves modulated by interneurons targeting pyramidal cell somata, providing a synaptic substrate for tuning pyramidal cell output through interactions in the local inhibitory network. These results provide evidence for a division of labor in cortical circuits, where distinct computational functions are implemented by subtypes of local inhibitory neurons.
Assuntos
Interneurônios/citologia , Inibição Neural/fisiologia , Sinapses/fisiologia , Animais , Biofísica , Córtex Cerebral/citologia , Channelrhodopsins , Proteínas de Ligação a DNA/genética , Dependovirus/genética , Estimulação Elétrica , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas GABAérgicos/farmacologia , Glutamato Descarboxilase/genética , Ácido Glutâmico/farmacologia , Técnicas In Vitro , Interneurônios/fisiologia , Canais Iônicos de Abertura Ativada por Ligante/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Neurológicos , Mutação/genética , Inibição Neural/efeitos dos fármacos , Inibição Neural/genética , Parvalbuminas/genética , Técnicas de Patch-Clamp , Estimulação Luminosa , Células Piramidais/citologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia , Piridazinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Glicina/genética , Sinapses/genética , Transmissão Sináptica , Fatores de Transcrição/genética , Transdução Genética , Valina/análogos & derivados , Valina/farmacologia , Ácido gama-Aminobutírico/farmacologiaRESUMO
Understanding the structure and function of neural circuits is central is neuroscience research. To address the associated questions, new genetically encoded tools have been developed for mapping, monitoring, and manipulating neurons. Essential to implementation of these tools is their selective delivery to defined neuronal populations in the brain. This has been facilitated by recent improvements in cell type-specific transgene expression using recombinant adeno-associated viral vectors. Here, we highlight these developments and discuss areas for improvement that could further expand capabilities for neural circuit analysis.