Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific <em>In Utero</em> Electroporation in the C57BL/6 Mouse

Abstract

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable. Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

<em>In Utero</em> Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

“…With respect to existing methods, it visualizes discriminating aspects of connectivity, such as the number of axonal branches per neuron, their precise topography, and their anatomical location. By altering the position of the electrodes, it is possible to target other neuron populations, such as the cingulate cortex (keep the same angle between the electrodes and the brain, but change the orientation of the poles) or the hippocampus 5 , and perform similar experiments labeling individual neurons or broader populations, depending on the desired strategy. However, there are limitations to this, as not all populations are equally accessible or equally selectively labeled.…”

“…NOTE: This protocol for IUE is adapted from others that have been previously published 5,6,7 . This manuscript describes a protocol for the IUE of E15.5 embryos, with modifications in the reporter strategy that allow for the study of the morphology of single neurons 8 and their electrophysiological properties in a separate experiment using standard GFP reporter plasmids.…”

Diversity in striatal synaptic circuits arises from distinct embryonic progenitor pools in the ventral telencephalon

“…Tα1-Cre and CβA-FLEx constructs (and other combinations of constructs) were thoroughly mixed and injected at equal ratios (stock concentration of all plasmids was ∼3.0 μg/μl, so final concentration of two plasmid constructs in 1:1 ratio was 1.5 μg/μl) and exhibited robust and faithful expression at these concentrations ( Ellender et al., 2019 ). The negative pole of the Tweezertrode (Sonidel) was placed just above the primordial ear outside the uterine muscle and the positive pole was placed slightly lower at the contralateral cheek region ( Baumgart and Baumgart, 2016 ). Five pulses (50 ms duration separated by 200 - 950 ms) at 42-55V were given with a BTX ECM 830 pulse generator (Genetronics).…”

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

“…Third, the expression of the tAKARα sensor in the mouse cortex by in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). Excellent protocols for surgeries for IUE 24,25 and stereotaxic injection of viral particles 26 have been previously published. The key parameters we used are described below.…”