Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using <em>In utero</em> Electroporation

Abstract

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system [1][2][3][4][5] . To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex [6][7][8] . Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/ or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9 . However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 ( Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures ( Figure 1B) but also at a specific time point of development ( Figure 1C).Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells.In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines. Video LinkThe video component of this article can be found at https://www.jove.com/video/4163/ ProtocolIn the United Kingdom, mice are housed, bred, and treated according to the guidelines approved by the Home Office under the Animal (Scientific Procedures) Act 1986. Preparation: DNA Solution and Needles1. Purify plasmid DNA with an Endotoxin free Maxi-prep kit. Prepare plasmid DNA solution for injection to desired concentrations in water...

Plasticity Related Gene 3 (PRG3) overcomes myelin-associated growth inhibition and promotes functional recovery after spinal cord injury

“…( N ) Immunohistochemical staining identifies endogenous PRG3 expression (green) is located primarily in the hippocampus in the adult mouse brain (Red = TuJ1). ( O ) Scheme of the experimental protocol for cortical in utero electroporation [55]. ( P ) Representative example of electroporated brain section showing pyramidal neurons positive for GFP expressing pyramidal neurons (left) and PRG3-positive pyramidal cells (right).…”

Neuronal LRP4 regulates synapse formation in the developing CNS

“…In utero electroporation of E14.5 timed pregnant mice (C57/Bl6; commercially obtained by Janvier Labs, Le Genest-Saint-Isle, France) was performed as previously described (Pacary et al, 2012). Specifically, pregnant mice were anesthetized with isoflurane (induction, 3%; surgery, 1.5%).…”

Genesis of Heterotopia in BCNU Model of Cortical Dysplasia, Detected by Means of in utero Electroporation

“…This technique enables the introduction, locally and temporally, of a marker gene into cortical or other neural progenitor cells in developing embryos in the uterus in order to visualize the process of migration and layer formation [12,13,14]. In utero electroporation has been successfully applied to the study of neuronal generation and migration during the development of different brain areas [15,16,17].…”