Joanna Szczurkowska

Non-hyperpolarizing GABAB receptor activation regulates neuronal migration and neurite growth and specification by cAMP/LKB1

γ-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, γ-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.

A cell surface biotinylation assay to reveal membrane associated neuronal cues: Negr1 regulates dendritic arborization.

A complex and still not comprehensively resolved panel of transmembrane proteins regulates the outgrowth and the subsequent morphological and functional development of neuronal processes. In order to gain a more detailed description of these events at the molecular level, we have developed a cell surface biotinylation assay to isolate, detect, and quantify neuronal membrane proteins. When we applied our assay to investigate neuron maturation in vitro, we identified 439 differentially expressed proteins, including 20 members of the immunoglobulin superfamily. Among these candidates, we focused on Negr1, a poorly described cell adhesion molecule. We demonstrated that Negr1 controls the development of neurite arborization in vitro and in vivo. Given the tight correlation existing among synaptic cell adhesion molecules, neuron maturation, and a number of neurological disorders, our assay results are a useful tool that can be used to support the understanding of the molecular bases of physiological and pathological brain function.

Targeted in vivo genetic manipulation of the mouse or rat brain by in utero electroporation with a triple-electrode probe

This protocol is an extension to:Nat. Protoc. 1, 1552–1558 (2006); doi:10.1038/nprot.2006.276; published online 9 November 2006This 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.

Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo

Significance The control of intracellular Cl− and pH plays a crucial role in several neuronal functions, and the study of these processes would be helped by tools for their noninvasive optical measurement in vivo. In this study, we have performed combined measurements of Cl− and pH of individual pyramidal neurons by means of in vivo two-photon imaging, and we provide direct experimental demonstration for the presence of the postnatal developmental shift to lower intraneuronal Cl−. Moreover, we introduce an approach for dynamic and simultaneous monitoring of intraneuronal Cl− and pH in vivo. These methods will open a window for the study of the roles of intraneuronal pH and Cl− in neuronal signaling, plasticity, and disease. Intracellular chloride ([Cl−]i) and pH (pHi) 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 pHi, 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 pHi 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.

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.

Synapsin III Acts Downstream of Semaphorin 3A/CDK5 Signaling to Regulate Radial Migration and Orientation of Pyramidal Neurons In Vivo

Summary 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.

Increased performance in genetic manipulation by modeling the dielectric properties of the rodent brain

Genetic approaches to control DNA expression in different brain areas have provided an excellent system to characterize gene function in health and disease of animal models. With respect to others, in utero electroporation of exogenous DNA into progenitor cells committed to specific brain areas is the optimal solution in terms of simplicity and velocity. Indeed, this method entails one quick and easy surgical procedure aimed at DNA injection in the embryonic brain followed by brief exposure to a strong electric field by a bipolar electrode. Nevertheless, the technique is still lacking the necessary control and reliability in addressing the field. Moving from a theoretical model that accounts for the morphology and the dielectric properties of the embryonic brain, we developed here a set of novel and reliable experimental configurations based on the use of three electrodes for electroporation in mouse. Indeed, by means of a full 3D model of the embryonic brain and the surrounding environment, we showed that the distribution of the electric field can be finely tuned in order to target specific brain regions at a desired temporal window by proper placement of the three electrodes. In the light of this theoretical background, we manufactured a three-electrode device and performed model-guided experimental sessions. The result was an increased spatial control, extended time frames and unprecedented reliability of the genetic manipulation, with respect to the current state of the art. In particular, the outcomes of this method applied into the mouse model are reported here for the first time.

The role of Negr1 in cortical development via NCAM-FGFR2 signaling

Autism spectrum disorder (ASD) affects 0.9% of children and it is recognized as the most genetic of all developmental neuropsychiatric syndromes. Mutations in NEGR1 and FGFR2 genes have been recently identified as ASD candidates. Negr1 is a member of IgLON adhesion protein family but its functions are largely unknown. Our original approach has identified Negr1 as a developmentally regulated synaptic protein. Thus we examined the consequences of Negr1 acute downregulation. Strikingly, we found that Negr1 ablation impairs neuronal maturation in vitro1. A combination of biochemical and imaging investigation has demonstrated that Negr1 influences neurites outgrowth via MAPK signaling organizing trans-synaptic heterodimer. In detail, we demonstrated that ectopic Negr1 is sufficient to improve neurite arborization and rescue the morphological phenotype observed in Negr1 KD cells. This function is dependent on the activation of MAPK pathway through tyrosine kinase receptors. In fact, we found that Negr1 physically and functionally interacts with NCAM and FGFR2, modulates FGFR2 response to FGF and consequently influences MAPK pathway. FGFR2/NCAM pathway plays an important role during brain development. Not surprisingly, our investigation of the radial migration of newly generated cortical neurons, revealed that Negr1-FGFR2-NCAM cross-talk controls cortical organization in vivo. Connectivity dysfunctions have been suggested as causative alterations in ASD. Given the functional, physical and genetic correlation among Negr1 and FGFR2 and NCAM, the study of Negr1/FGFR2/NCAM molecular cross talk may offer new therapeutic opportunities.

KCC2 Regulates Dendritic Spine Formation in a Brain-Region Specific and BDNF Dependent Manner

Abstract KCC2 is the major chloride extruder in neurons. The spatiotemporal regulation of KCC2 expression orchestrates the developmental shift towards inhibitory GABAergic drive and the formation of glutamatergic synapses. Whether KCC2’s role in synapse formation is similar in different brain regions is unknown. First, we found that KCC2 subcellular localization, but not overall KCC2 expression levels, differed between cortex and hippocampus during the first postnatal week. We performed site-specific in utero electroporation of KCC2 cDNA to target either hippocampal CA1 or somatosensory cortical pyramidal neurons. We found that a premature expression of KCC2 significantly decreased spine density in CA1 neurons, while it had the opposite effect in cortical neurons. These effects were cell autonomous, because single-cell biolistic overexpression of KCC2 in hippocampal and cortical organotypic cultures also induced a reduction and an increase of dendritic spine density, respectively. In addition, we found that the effects of its premature expression on spine density were dependent on BDNF levels. Finally, we showed that the effects of KCC2 on dendritic spine were dependent on its chloride transporter function in the hippocampus, contrary to what was observed in cortex. Altogether, these results demonstrate that KCC2 regulation of dendritic spine development, and its underlying mechanisms, are brain-region specific.

Negr1 is required for transition of migrating pyramidal neurons from layer V to layer II/III of the mouse cerebral cortex.

The mammalian cerebral cortex is a remarkably complex structure, and establishment of cortical neural circuitries requires its unique laminar organization. During perinatal development, newborn pyramidal neurons migrate along radial glia fibers, to create the six-layered structure of the neocortex. Disruption in neural migration can lead to brain malformations with functional consequences on proper wiring of the neuronal network, as already described in neurodevelopmental disorders such as Autism Spectrum Disorders (ASD). Common knowledge indicates cell-adhesion molecules (CAMs) as essential for proper neural migration. Neuronal growth regulator 1 (Negr1) is a CAM, and NEGR1 gene mutations have been recently associated to ASD. By in utero electroporation coupled with RNA interference (siRNA), we downregulated Negr1 levels in late-born pyramidal neurons migrating to the superficial layers of the neocortex. We found that Negr1 siRNA caused ectopic positioning of neurons concentrated at the border between layer 5 and layer 4 of the somatosensory cortex. Downregulation of Negr1 did not cause migration defects in the motor or prefrontal cortices. We found that FGFR2 (also associated to autism) and its partner NCAM physically and functionally interact with Negr1. Interestingly, downregulation of NCAM and FGFR2 in utero resulted in a strikingly similar phenotype on neuronal migration as found for Negr1, suggesting that all three molecules belong to the same signaling pathway in vivo. Moreover, we found that, downregulation of Negr1 in the embryonic somatosensory cortex resulted in decreased number of ultrasound vocalizations in pups. These data suggest that Negr1/FGFR2/NCAM complex is necessary for proper neuronal migration of pyramidal neurons in the somatosensory cortex, indicating a possible role for this complex in autism.