ABSTRACT
Techniques enabling DNA delivery into mouse retinal cells using in utero electroporation are available. However, these techniques target the central retina and do not enable the electroporation of the ventro-temporal retina where ipsilateral retinal ganglion cells are located. Here, we describe a protocol to specifically electroporate the ventro-temporal retina, a critical approach to manipulate ipsilaterally projecting retinal ganglion cells and contralaterally projecting neurons located in the same region of the retina. The procedure is adaptable to target other retinal quadrants. For complete details on the use and execution of this protocol, please refer to Louail et al. (2020).
Subject(s)
Electroporation , Retinal Ganglion Cells , Animals , Female , PregnancyABSTRACT
Axonal arbors in many neuronal networks are exuberant early during development and become refined by activity-dependent competitive mechanisms. Theoretical work proposed non-competitive interactions between co-active axons to co-stabilize their connections, but the demonstration of such interactions is lacking. Here, we provide experimental evidence that reducing cyclic AMP (cAMP) signaling in a subset of retinal ganglion cells favors the elimination of thalamic projections from neighboring neurons, pointing to a cAMP-dependent interaction that promotes axon stabilization.
Subject(s)
Axons/metabolism , Cyclic AMP/metabolism , Neurons/metabolism , Humans , Signal TransductionABSTRACT
Calcium is a second messenger crucial to a myriad of cellular processes ranging from regulation of metabolism and cell survival to vesicle release and motility. Current strategies to directly manipulate endogenous calcium signals lack cellular and subcellular specificity. We introduce SpiCee, a versatile and genetically encoded chelator combining low- and high-affinity sites for calcium. This scavenger enables altering endogenous calcium signaling and functions in single cells in vitro and in vivo with biochemically controlled subcellular resolution. SpiCee paves the way to investigate local calcium signaling in vivo and directly manipulate this second messenger for therapeutic use.
Subject(s)
Calcium/metabolism , Genetic Techniques , Adenosine Triphosphate/metabolism , Animals , Calcium Signaling/drug effects , Cell Death/drug effects , Cell Movement/drug effects , Cell Survival/drug effects , Chelating Agents/pharmacology , HEK293 Cells , Humans , Mice, Inbred C57BL , Neurons/cytology , Neurons/drug effects , Signal Transduction/drug effects , Subcellular Fractions/metabolism , Thapsigargin/pharmacologyABSTRACT
cGMP is critical to a variety of cellular processes, but the available tools to interfere with endogenous cGMP lack cellular and subcellular specificity. We introduce SponGee, a genetically encoded chelator of this cyclic nucleotide that enables in vitro and in vivo manipulations in single cells and in biochemically defined subcellular compartments. SponGee buffers physiological changes in cGMP concentration in various model systems while not affecting cAMP signals. We provide proof-of-concept strategies by using this tool to highlight the role of cGMP signaling in vivo and in discrete subcellular domains. SponGee enables the investigation of local cGMP signals in vivo and paves the way for therapeutic strategies that prevent downstream signaling activation.