In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however it is most commonly performed at E15 as shown in this video.
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"To identify the morphological features and proliferative status of cells in the ventral telencephalon, we performed in utero intraventricular retrovirus injections in embryonic mouse brains at gestational day 12.5 (E12.5) (Walantus et al., 2007), coinciding with peak neurogenesis (Butt et al., 2005; Miyoshi et al., 2007). "
"In utero intraventricular injections of E14 mice embryos were performed as described by Walantus et al. (2007). Pregnant Swiss mice in the 14 gestational day were anesthetized with intraperitoneal injection of 2-2-2 Tribromoethanol (Sigma Aldrich) 1 mg/g of body weight. "
[Show abstract][Hide abstract] ABSTRACT: The major neural stem cell population in the developing cerebral cortex is composed of the radial glial cells, which generate glial cells and neurons. The mechanisms that modulate the maintenance of the radial glia (RG) stem cell phenotype, or its differentiation, are not yet completely understood. We previously demonstrated that the transforming growth factor-β1 (TGF-β1) promotes RG differentiation into astrocytes in vitro (Glia 2007; 55:1023-33) through activation of multiple canonical and non-canonical signaling pathways (Dev Neurosci 2012; 34:68-81). However, it remains unknown if TGF-β1 acts in RG-astrocyte differentiation in vivo. Here, we addressed the astrogliogenesis induced by TGF-β1 by using the intraventricular in utero injection in vivo approach. We show that injection of TGF-β1 in the lateral ventricles of E14,5 mice embryos resulted in RG fibers disorganization and premature gliogenesis, evidenced by appearance of GFAP positive cells in the cortical wall. These events were followed by decreased numbers of neurons in the cortical plate (CP). Together, we also described that TGF-β1 actions are region-dependent, once RG cells from dorsal region of the cerebral cortex demonstrated to be more responsive to this cytokine compared with RG from lateral cortex either in vitro as well as in vivo. Our work demonstrated that TGF-β1 is a critical cytokine that regulates RG fate decision and differentiation into astrocytes in vitro and in vivo. We also suggest that RG cells are heterogeneous population that acts as distinct targets of TGF-β1 during cerebral cortex development.
Full-text · Article · Nov 2014 · Frontiers in Cellular Neuroscience
"Body temperature was kept constant at 37 • C with a heating blanket . A volume of 0.7–3 µl of MAM (several concentrations, see Results) or saline (NaCl 0.9%) was injected in the lateral ventricle of several embryos using a glass pipette (1.2 mm outer diameter, 0.69 mm inner diameter, Harvard Apparatus) coupled to an automatic microinjector (VisualSonics Inc, Canada) using standard approaches (Walantus et al., 2007). In initial attempts, we used ultrasound control for guiding injections (VeVo 770, VisualSonics Inc, Canada), but we found it was not required for embryos older than E14. "
[Show abstract][Hide abstract] ABSTRACT: Developmental cortical malformations comprise a large spectrum of histopathological brain abnormalities and syndromes. Their genetic, developmental and clinical complexity suggests they should be better understood in terms of the complementary action of independently timed perturbations (i.e., the multiple-hit hypothesis). However, understanding the underlying biological processes remains puzzling. Here we induced developmental cortical malformations in offspring, after intraventricular injection of methylazoxymethanol (MAM) in utero in mice. We combined extensive histological and electrophysiological studies to characterize the model. We found that MAM injections at E14 and E15 induced a range of cortical and hippocampal malformations resembling histological alterations of specific genetic mutations and transplacental mitotoxic agent injections. However, in contrast to most of these models, intraventricularly MAM-injected mice remained asymptomatic and showed no clear epilepsy-related phenotype as tested in long-term chronic recordings and with pharmacological manipulations. Instead, they exhibited a non-specific reduction of hippocampal-related brain oscillations (mostly in CA1); including theta, gamma and HFOs; and enhanced thalamocortical spindle activity during non-REM sleep. These data suggest that developmental cortical malformations do not necessarily correlate with epileptiform activity. We propose that the intraventricular in utero MAM approach exhibiting a range of rhythmopathies is a suitable model for multiple-hit studies of associated neurological disorders.
Full-text · Article · Apr 2014 · Frontiers in Systems Neuroscience