Intracellular chloride ([Cl−] i ) and pH (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 twophoton excitation protocol to simultaneously determine the changes of pH i and [Cl − ] i in response to hypercapnia and seizure activity.I ntracellular ion concentrations are controlled by plasmalemmal transporters and channels, which generate and dissipate ionic electrochemical gradients, respectively (1). In recent years, regulation of the intracellular Cl − concentration ([Cl − ] i ) in neurons has attracted lots of attention, because it is the main ion that carries current across GABA A (and also, glycine) receptors. Changes in [Cl − ] i exert an immediate effect on the reversal potential of GABAergic currents (E GABA ) and, thereby, on the properties of GABA A receptor-mediated transmission (2-4). The "ionic plasticity" of GABAergic signaling involves not only the passive flux of Cl − ions through membrane channels but also, a number of ion transporters that regulate [Cl − ] i . Furthermore, this mechanism is under the control of intracellular signaling cascades that regulate the expression patterns as well as functional properties of ion transporters and channels (5, 6). With regard to long-term ionic modulation of GABAergic transmission, a case in point is the decrease in [Cl − ] i that is generally thought to take place during maturation of most central neurons. According to this widely accepted scenario, the Na-K-2Cl cotransporter NKCC1 accumulates Cl − in immature neurons, thereby promoting depolarizing GABA responses (3, 7-9), which is followed by developmental upregulation of the neuron-specific K-Cl cotransporter KCC2 that is required for the generation of classical hyperpolarizing inhibitory postsynaptic potentials (IPSPs) (10).A wealth of electrophysiological evidence dating back to the work in vivo by Eccles and coworkers (11) has provided evidence for active regulation of [Cl − ] i in mammalian central neurons and its crucial effect on the driving force of Cl − in inhibitory synapses (1). However, thus far, there are no direct data on neuronal [Cl − ] i measured in vivo at the single-cell level in the living brain, and for in...

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.

See Contreras and Hippenmeyer (doi:) for a scientific commentary on this article.Autism spectrum disorders (ASDs) are complex conditions with diverse aetiologies. Szczurkowska et al. demonstrate that two ASD-related molecules – FGFR2 and Negr1 – physically interact to act on the same downstream pathway, and regulate cortical development and ASD-relevant behaviours in mice. Identifying common mechanisms in ASDs may reveal targets for pharmacological intervention.

g-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in adults, acting through ionotropic chloride-permeable GABA A receptors (GABA A Rs), and metabotropic GABA B Rs coupled to calcium or potassium channels, and cAMP signalling. During early development, GABA is the main neurotransmitter and is not hyperpolarizing, as GABA A R activation is depolarizing while GABA B Rs lack coupling to potassium channels. Despite extensive knowledge on GABA A Rs as key factors in neuronal development, the role of GABA B Rs remains unclear. Here we address GABA B R function during rat cortical development by in utero knockdown (short interfering RNA) of GABA B R in pyramidal neuron progenitors. GABA B R knockdown impairs neuronal migration and axon/dendrite morphological maturation by disrupting cAMP signalling. Furthermore, GABA B R activation reduces cAMP-dependent phosphorylation of LKB1, a kinase involved in neuronal polarization, and rescues LKB1 overexpression-induced defects in cortical development. Thus, non-hyperpolarizing activation of GABA B Rs during development promotes neuronal migration and morphological maturation by cAMP/LKB1 signalling.

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. Genetic analysis indicates that 20% to 30% of the total open reading frame encodes for integral membrane proteins (1). Although less abundant than cytosolic proteins, membrane-passing proteins contribute to the regulation of all major cell processes and signaling pathways. In particular, membrane proteins play an important role in the establishment of functional neuronal circuitries during development. This process initially entails the growth, guidance, and stabilization of neuronal processes (axons and dendrites) in a timely, ordered manner involving cell surface molecules that sense the extracellular surroundings and activate signaling cascades (2).Then, specialized cell-to-cell connections, the synapses, are formed. These connections allow information to flow from one neuron to another and relay the precise juxtaposition and interactions between the pre-and postsynaptic membrane proteins to support their final functional establishment. Several families of synaptic transmembrane or membrane proteins, such as semaphorin, neuroligin, neurexin, and the immunoglobulin superfamily (IgSF), 1 are implicated in neurite formation and synapse establishment (3). However, the picture of membrane proteins expressed in neurons is still far from being completely resolved, and it is expected that many other key molecules are awaiting identification (4). Thus, uncovering the nature of the dynamic multiprotein complexes expressed at the plasma membrane will possibly strongly support the understanding of the mechanism controlling structural and functional neuron development. Here, we describe a biochemical approach to isolate and quantify proteins exposed at the extracellular side of the plasma membrane. Our assay utilized affinity purification on streptavidin resin of biotinylated membrane proteins extracted from a crude synaptosomal preparation. We combined this cell surface biotinylation assay with MS/MS analysis and label-free quantification to investigate protein patterns characterizing immature and m...

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.

New dentate granule cells (DGCs) are continuously generated, and integrate into the preexisting hippocampal network in the adult brain. How an adult-born neuron with initially simple spindle-like morphology develops into a DGC, consisting of a single apical dendrite with further branches, remains largely unknown. Here, using retroviruses to birth date and manipulate newborn neurons, we examined initial dendritic formation and possible underlying mechanisms. We found that GFP-expressing newborn cells began to establish a DGC-like morphology at ∼7 d after birth, with a primary dendrite pointing to the molecular layer, but at this stage, with several neurites in the neurogenic zone. Interestingly, the Golgi apparatus, an essential organelle for neurite growth and maintenance, was dynamically repositioning in the soma of newborn cells during this initial integration stage. Two weeks after birth, by which time most neurites in the neurogenic zone were eliminated, a compact Golgi apparatus was positioned exclusively at the base of the primary dendrite. We analyzed the presence of Golgi-associated genes using single-cell transcriptomes of newborn DGCs, and among Golgi-related genes, found the presence of and, regulators of embryonic neuronal development. When we knocked down either of these two proteins, we found Golgi mislocalization and extensive aberrant dendrite formation. Furthermore, overexpression of a mutated form of STRAD, underlying the disorder polyhydramnios, megalencephaly, and symptomatic epilepsy, characterized by abnormal brain development and intractable epilepsy, caused similar defects in Golgi localization and dendrite formation in adult-born neurons. Together, our findings reveal a role for Golgi repositioning in regulating the initial integration of adult-born DGCs. Since the discovery of the continuous generation of new neurons in the adult hippocampus, extensive effort was directed toward understanding the functional contribution of these newborn neurons to the existing hippocampal circuit and associated behaviors, while the molecular mechanisms controlling their early morphological integration are less well understood. Dentate granule cells (DGCs) have a single, complex, apical dendrite. The events leading adult-born DGCs' to transition from simple spindle-like morphology to mature dendrite morphology are largely unknown. We studied establishment of newborn DGCs dendritic pattern and found it was mediated by a signaling pathway regulating precise localization of the Golgi apparatus. Furthermore, this Golgi-associated mechanism for dendrite establishment might be impaired in a human genetic epilepsy syndrome, polyhydramnios, megalencephaly, and symptomatic epilepsy.

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.