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Nature ; 624(7991): 333-342, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-38092915


The function of the mammalian brain relies upon the specification and spatial positioning of diversely specialized cell types. Yet, the molecular identities of the cell types and their positions within individual anatomical structures remain incompletely known. To construct a comprehensive atlas of cell types in each brain structure, we paired high-throughput single-nucleus RNA sequencing with Slide-seq1,2-a recently developed spatial transcriptomics method with near-cellular resolution-across the entire mouse brain. Integration of these datasets revealed the cell type composition of each neuroanatomical structure. Cell type diversity was found to be remarkably high in the midbrain, hindbrain and hypothalamus, with most clusters requiring a combination of at least three discrete gene expression markers to uniquely define them. Using these data, we developed a framework for genetically accessing each cell type, comprehensively characterized neuropeptide and neurotransmitter signalling, elucidated region-specific specializations in activity-regulated gene expression and ascertained the heritability enrichment of neurological and psychiatric phenotypes. These data, available as an online resource ( ), should find diverse applications across neuroscience, including the construction of new genetic tools and the prioritization of specific cell types and circuits in the study of brain diseases.

Encéfalo , Perfilación de la Expresión Génica , Animales , Ratones , Encéfalo/anatomía & histología , Encéfalo/citología , Encéfalo/metabolismo , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Hipotálamo/citología , Hipotálamo/metabolismo , Mesencéfalo/citología , Mesencéfalo/metabolismo , Neuropéptidos/metabolismo , Neurotransmisores/metabolismo , Fenotipo , Rombencéfalo/citología , Rombencéfalo/metabolismo , Análisis de Expresión Génica de una Sola Célula , Transcriptoma/genética
Methods ; 53(3): 194-200, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21187150


Understanding how specific proteins are degraded by neurons in living animals is a fundamental question with relevance to many neurodegenerative diseases. Dysfunction in the ubiquitin-proteasome system (UPS) specifically has been implicated in several important neurodegenerative diseases including Alzheimer's Disease, Parkinson's Disease, and amyotrophic lateral sclerosis. Research in this area has been limited by the fact that many inhibitors of the UPS given systemically do not cross the blood-brain barrier (BBB) in appreciable levels. This limits the ability to easily test in vivo specific hypotheses generated in reduced systems, like brain slice or dissociated cell culture, about whether the UPS may degrade a particular protein of interest. Although several techniques including intracerebral application via direct syringe injection, catheter-pump systems and drug-eluting beads are available to introduce BBB-impermeant drugs into brain they each have certain limitations and new approaches could provide further insights into this problem. In order to test the role of the UPS in protein degradation in vivo we have developed a strategy to treat mouse cortex with the UPS inhibitor clasto-lactacystin beta-lactone (CLBL) via a "cranial window" and recover the treated tissue for immunoblot analysis. This approach can be used in several different cranial window configurations including single window and double hemi-window arrangements that are tailored for different applications. We have also developed two different strategies for recovering treated cortical tissue including a vibratome/laser capture microscopy (LCM)-based and a vibratome only-based approach, each with its own specific advantages. We have documented UPS inhibition >600µm deep into the cortex with this strategy. This set of techniques in the living mammalian brain is complementary to previously developed approaches and extends the repertoire of tools that can be used to the study protein degradation pathways relevant to neurodegenerative disease.

Enfermedades Neurodegenerativas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Animales , Western Blotting , Craneotomía , Inhibidores de Cisteína Proteinasa/farmacología , Modelos Animales de Enfermedad , Humanos , Ratones , Microscopía Confocal , Microtomía