Visualization and genetic manipulation of dendrites and spines in the mouse cerebral cortex and hippocampus using in utero electroporation.
JoVE. 2012-07-26; (65):
DOI: 10.3791/4163
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1. J Vis Exp. 2012 Jul 26;(65). pii: 4163. doi: 10.3791/4163.
Visualization and genetic manipulation of dendrites and spines in the mouse
cerebral cortex and hippocampus using in utero electroporation.
Pacary E(1), Haas MA, Wildner H, Azzarelli R, Bell DM, Abrous DN, Guillemot F.
Author information:
(1)Division of Molecular Neurobiology, MRC National Institute for Medical
Research.
In utero electroporation (IUE) has become a powerful technique to study the
development of different regions of the embryonic nervous system (1-5). 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 (6-8). 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.
DOI: 10.3791/4163
PMCID: PMC3476406
PMID: 22872172 [Indexed for MEDLINE]