RESUMEN
We describe a method for light-inducible and tissue-selective cell ablation using a genetically encoded photosensitizer, miniSOG (mini singlet oxygen generator). miniSOG is a newly engineered fluorescent protein of 106 amino acids that generates singlet oxygen in quantum yield upon blue-light illumination. We transgenically expressed mitochondrially targeted miniSOG (mito-miniSOG) in Caenorhabditis elegans neurons. Upon blue-light illumination, mito-miniSOG causes rapid and effective death of neurons in a cell-autonomous manner without detectable damages to surrounding tissues. Neuronal death induced by mito-miniSOG appears to be independent of the caspase CED-3, but the clearance of the damaged cells partially depends on the phagocytic receptor CED-1, a homolog of human CD91. We show that neurons can be killed at different developmental stages. We further use this method to investigate the role of the premotor interneurons in regulating the convulsive behavior caused by a gain-of-function mutation in the neuronal acetylcholine receptor acr-2. Our findings support an instructive role for the interneuron AVB in controlling motor neuron activity and reveal an inhibitory effect of the backward premotor interneurons on the forward interneurons. In summary, the simple inducible cell ablation method reported here allows temporal and spatial control and will prove to be a useful tool in studying the function of specific cells within complex cellular contexts.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Flavoproteínas/metabolismo , Proteínas Luminiscentes/metabolismo , Neuronas/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Caspasas/genética , Caspasas/metabolismo , Muerte Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Flavoproteínas/genética , Interneuronas/citología , Interneuronas/metabolismo , Interneuronas/efectos de la radiación , Luz , Proteínas Luminiscentes/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Mitocondrias/metabolismo , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Neuronas Motoras/efectos de la radiación , Neuronas/citología , Neuronas/efectos de la radiación , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Oxígeno Singlete/metabolismo , Factores de TiempoRESUMEN
Single-cell characterization and perturbation of neurons provides knowledge critical to addressing fundamental neuroscience questions including the structure-function relationship and neuronal cell-type classification. Here we report a robot for efficiently performing in vivo single-cell experiments in deep brain tissues optically difficult to access. This robot automates blind (non-visually guided) single-cell electroporation (SCE) and extracellular electrophysiology, and can be used to characterize neuronal morphological and physiological properties of, and/or manipulate genetic/chemical contents via delivering extraneous materials (for example, genes) into single neurons in vivo. Tested in the mouse brain, our robot successfully reveals the full morphology of single-infragranular neurons recorded in multiple neocortical regions, as well as deep brain structures such as hippocampal CA3, with high efficiency. Our robot thus can greatly facilitate the study of in vivo full morphology and electrophysiology of single neurons in the brain.