<em>Ex Utero</em> Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Abstract

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Reversible and Irreversible Effects of Electroporation on Contractility and Calcium Homeostasis in Isolated Cardiac Ventricular Myocytes

“…Cells following exposure to electric fields below the IRE threshold may however regain homeostasis and survive. 1 Technologies leveraging pulsed electric field (PEF) to achieve reversible and IRE have been developed in a broad range of medical specialties including oncology, 2–6 neurology, 7,8,9,10 dermatology 11,12 and most recently, cardiology. 13,14,15,16 Specifically, the use of PEF to ablate aberrant cardiac tissues via IRE is rapidly emerging as a promising energy modality in the treatment of cardiac arrhythmias.…”

Both GEF domains of the autism and epilepsy-associated Trio protein are required for proper tangential migration of GABAergic interneurons

“…Timed-pregnant females were sacrificed by cervical dislocation and embryos were collected at e13.5, e14.5, e15.5 or e18.5 as previously described 15, 112 . Animals at P21 were deeply anesthetized with Pentobarbital (100 mg/kg, i.p.).…”

“…Electroporation of the MGE was conducted at e13.5 as previously described 112 , with minor modifications. Briefly, endo-free plasmid DNA solutions, mixed with 0.01% Fast Green (Cat.…”

“…Trio WT and Trio cKO embryonic brains were processed for organotypic slice cultures as previously described 112 . Briefly, e13.5 brains were dissected out of the skull in cold L-15 medium (Invitrogen), embedded in 4% low-melting point agarose and cut with a vibratome into 250 µ m-thick slices collected in sterile oxygenated artificial cerebrospinal fluid (ACSF; see 112 for composition).…”

Both GEF domains of the autism and epilepsy-associated Trio protein are required for proper tangential migration of GABAergic interneurons

“…Electroporation of the MGE was conducted at e13.5 as previously described 112 , with minor modi cations. Brie y, endo-free plasmid DNA solutions, mixed with 0.01% Fast Green (Cat.…”