10 research outputs found
Non-hyperpolarizing GABA B receptor activation regulates neuronal migration and neurite growth and specification by cAMP/LKB1
Îł-Aminobutyric acid is the principal inhibitory neurotransmitter in adults, acting through ionotropic chloride-permeable GABAA receptors (GABAARs), and metabotropic GABABRs coupled to calcium or potassium channels, and cyclic AMP signalling. During early development, Îł-aminobutyric acid is the main neurotransmitter and is not hyperpolarizing, as GABAAR activation is depolarizing while GABABRs lack coupling to potassium channels. Despite extensive knowledge on GABAARs as key factors in neuronal development, the role of GABABRs remains unclear. Here we address GABABR function during rat cortical development by in utero knockdown (short interfering RNA) of GABABR in pyramidal-neuron progenitors. GABABR short interfering RNA impairs neuronal migration and axon/dendrite morphological maturation by disrupting cyclic AMP signalling. Furthermore, GABABR activation reduces cyclic AMP-dependent phosphorylation of LKB1, a kinase involved in neuronal polarization, and rescues LKB1 overexpression-induced defects in cortical development. Thus, non-hyperpolarizing activation of GABABRs during development promotes neuronal migration and morphological maturation by cyclic AMP/LKB1 signalling
Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo
Intracellular chloride ([Cl-](i)) andpH(pH(i)) are fundamental regulators of neuronal excitability. They exert wide-ranging effects on synaptic signaling and plasticity and on development and disorders of the brain. The ideal technique to elucidate the underlying ionic mechanisms is quantitative and combined two-photon imaging of [Cl-](i) and pH(i), but this has never been performed at the cellular level in vivo. Here, by using a genetically encoded fluorescent sensor that includes a spectroscopic reference (an element insensitive to Cl-and pH), we show that ratiometric imaging is strongly affected by the optical properties of the brain. We have designed a method that fully corrects for this source of error. Parallel measurements of [Cl-](i) and pH(i) at the single-cell level in the mouse cortex showed the in vivo presence of the widely discussed developmental fall in [Cl-](i) and the role of the K-Cl cotransporter KCC2 in this process. Then, we introduce a dynamic two-photon excitation protocol to simultaneously determine the changes of pHi and [Cl-](i) in response to hypercapnia and seizure activity.Peer reviewe
Role of mGluR5 in induction of long-term potentiation in the hippocampal CA1 region in mouse.
High-performance and reliable site-directed in vivo genetic manipulation of mouse and rat brain by in utero electroporation with a triple-electrode probe
One of the challenges for modern neuroscience is to understand the role of specific genes in the determination of cellular fate, and in the formation and physiology of neuronal-circuits. Techniques for genetic manipulation in vivo such as in utero electroporation are fundamental tools to address these issues. Here, we describe an established protocol for in utero electroporation in mouse and rat for reliable targeting of the hippocampus, the motor, prefrontal, and visual cortices, and the Purkinje cells of the cerebellum. The method is based on an electroporation configuration entailing commonly used forceps-type electrodes plus an additional third electrode. This configuration allows highly consistent direction of the electric field to the different neurogenic areas by simple and reliable adjustment of relative positions, polarities and/or dimensions of the electrodes. More than 70% of electroporated embryos survive to postnatal ages and around 60-90% express the electroporated vector, depending on the targeted area. By a single electroporation episode, the protocol enables for symmetric transfection in both brain hemispheres. The procedure requires 4 hours of preparation on the first day and it lasts 1 hour, including a surgery time of 30 mins, on the second day
Targeted in vivo genetic manipulation of the mouse or rat brain by in utero electroporation with a triple-electrode probe
This article describes how to reliably electroporate with DNA plasmids rodent neuronal progenitors of the hippocampus; the motor, prefrontal and visual cortices; and the cerebellum in utero. As a Protocol Extension article, this article describes an adaptation of an existing Protocol and offers additional applications. The earlier protocol describes how to electroporate mouse embryos using two standard forceps-type electrodes. In the present protocol, additional electroporation configurations are possible because of the addition of a third electrode alongside the two standard forceps-type electrodes. By adjusting the position and polarity of the three electrodes, the electric field can be directed with great accuracy to different neurogenic areas. Bilateral transfection of brain hemispheres can be achieved after a single electroporation episode. Approximately 75% of electroporated embryos survive to postnatal ages, and depending on the target area, 50–90% express the electroporated vector. The electroporation procedure takes 1 h 35 min. The protocol is suitable for the preparation of animals for various applications, including histochemistry, behavioral studies, electrophysiology and in vivo imaging.LN
Synapsin III Acts Downstream of Semaphorin 3A/CDK5 Signaling to Regulate Radial Migration and Orientation of Pyramidal Neurons In Vivo
Synapsin III (SynIII) is a phosphoprotein that is highly expressed at early stages of neuronal development. Whereas in vitro evidence suggests a role for SynIII in neuronal differentiation, in vivo evidence is lacking. Here, we demonstrate that in vivo downregulation of SynIII expression affects neuronal migration and orientation. By contrast, SynIII overexpression affects neuronal migration, but not orientation. We identify a cyclin-dependent kinase-5 (CDK5) phosphorylation site on SynIII and use phosphomutant rescue experiments to demonstrate its role in SynIII function. Finally, we show that SynIII phosphorylation at the CDK5 site is induced by activation of the semaphorin-3A (Sema3A) pathway, which is implicated in migration and orientation of cortical pyramidal neurons (PNs) and is known to activate CDK5. Thus, fine-tuning of SynIII expression and phosphorylation by CDK5 activation through Sema3A activity is essential for proper neuronal migration and orientation
Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo
Synchronous Bioimaging of Intracellular pH and Chloride Based on LSS Fluorescent Protein
Ion homeostasis regulates
critical physiological processes in the
living cell. Intracellular chloride concentration not only contributes
in setting the membrane potential of quiescent cells but it also plays
a role in modulating the dynamic voltage changes during network activity.
Dynamic chloride imaging demands new tools, allowing faster acquisition
rates and correct accounting of concomitant pH changes. Joining a
long-Stokes-shift red-fluorescent protein to a GFP variant with high
sensitivity to pH and chloride, we obtained LSSmClopHensor, a genetically
encoded fluorescent biosensor optimized for the simultaneous chloride
and pH imaging and requiring only two excitation wavelengths (458
and 488 nm). LSSmClopHensor allowed us to monitor the dynamic changes
of intracellular pH and chloride concentration during seizure like
discharges in neocortical brain slices. Only cells with tightly controlled
resting potential revealed a narrow distribution of chloride concentration
peaking at about 5 and 8 mM, in neocortical neurons and SK-N-SH cells,
respectively. We thus showed that LSSmClopHensor represents a new
versatile tool for studying the dynamics of chloride and proton concentration
in living systems