Daily rhythm in cortical chloride homeostasis underpins functional changes in visual cortex excitability.

Cortical activity patterns are strongly modulated by fast synaptic inhibition mediated through ionotropic, chloride-conducting receptors. Consequently, chloride homeostasis is ideally placed to regulate activity. We therefore investigated the stability of baseline [Cl] in adult mouse neocortex, using in vivo two-photon imaging.

Nuclear ERK1/2 signaling potentiation enhances neuroprotection and cognition via Importinα1/KPNA2.

Cell signaling is central to neuronal activity and its dysregulation may lead to neurodegeneration and cognitive decline. Here, we show that selective genetic potentiation of neuronal ERK signaling prevents cell death in vitro and in vivo in the mouse brain, while attenuation of ERK signaling does the opposite. This neuroprotective effect mediated by an enhanced nuclear ERK activity can also be induced by the novel cell penetrating peptide RB5.

Stretch-activated ion channel TMEM63B associates with developmental and epileptic encephalopathies and progressive neurodegeneration.

By converting physical forces into electrical signals or triggering intracellular cascades, stretch-activated ion channels allow the cell to respond to osmotic and mechanical stress. Knowledge of the pathophysiological mechanisms underlying associations of stretch-activated ion channels with human disease is limited. Here, we describe 17 unrelated individuals with severe early-onset developmental and epileptic encephalopathy (DEE), intellectual disability, and severe motor and cortical visual impairment associated with progressive neurodegenerative brain changes carrying ten distinct heterozygous variants of TMEM63B, encoding for a highly conserved stretch-activated ion channel.

Microglial extracellular vesicles induce Alzheimer's disease-related cortico-hippocampal network dysfunction.

β-Amyloid is one of the main pathological hallmarks of Alzheimer's disease and plays a major role in synaptic dysfunction. It has been demonstrated that β-amyloid can elicit aberrant excitatory activity in cortical-hippocampal networks, which is associated with behavioural abnormalities. However, the mechanism of the spreading of β-amyloid action within a specific circuitry has not been elucidated yet.

Control of circadian rhythm on cortical excitability and synaptic plasticity.

Living organisms navigate through a cyclic world: activity, feeding, social interactions are all organized along the periodic succession of night and day. At the cellular level, periodic activity is controlled by the molecular machinery driving the circadian regulation of cellular homeostasis. This mechanism adapts cell function to the external environment and its crucial importance is underlined by its robustness and redundancy.

Acute stress impairs sensorimotor gating via the neurosteroid allopregnanolone in the prefrontal cortex.

Ample evidence indicates that environmental stress impairs information processing, yet the underlying mechanisms remain partially elusive. We showed that, in several rodent models of psychopathology, the neurosteroid allopregnanolone (AP) reduces the prepulse inhibition (PPI) of the startle, a well-validated index of sensorimotor gating. Since this GABA receptor activator is synthesized in response to acute stress, we hypothesized its participation in stress-induced PPI deficits.

A Superfolder Green Fluorescent Protein-Based Biosensor Allows Monitoring of Chloride in the Endoplasmic Reticulum.

Though the concentration of chloride has been measured in the cytoplasm and in secretory granules of live cells, it cannot be measured within the endoplasmic reticulum (ER) due to poor fluorescence of existing biosensors. We developed a fluorescent biosensor composed of a chloride-sensitive superfolder GFP and long Stokes-shifted mKate2 for simultaneous chloride and pH measurements that retained fluorescence in the ER lumen. Using this sensor, we showed that the chloride concentration in the ER is significantly lower than that in the cytosol.

Trehalose Treatment in Zebrafish Model of Lafora Disease.

Mutations in the gene encoding laforin cause Lafora disease (LD), a progressive myoclonic epilepsy characterized by drug-resistant seizures and progressive neurological impairment. To date, rodents are the only available models for studying LD; however, their use for drug screening is limited by regulatory restrictions and high breeding costs. To investigate the role of laforin loss of function in early neurodevelopment, and to screen for possible new compounds for treating the disorder, we developed a zebrafish model of LD.

Perturbation of Cortical Excitability in a Conditional Model of PCDH19 Disorder.

PCDH19 epilepsy (DEE9) is an X-linked syndrome associated with cognitive and behavioral disturbances. Since heterozygous females are affected, while mutant males are spared, it is likely that DEE9 pathogenesis is related to disturbed cell-to-cell communication associated with mosaicism. However, the effects of mosaic PCDH19 expression on cortical networks are unknown.

Genetically encoded sensors for Chloride concentration.

Insights into chloride regulation in neurons have come slowly, but they are likely to be critical for our understanding of how the brain works. The reason is that the intracellular Cl level ([Cl]) is the key determinant of synaptic inhibitory function, and this in turn dictates all manner of neuronal network function. The true impact on the network will only be apparent, however, if Cl is measured at many locations at once (multiple neurons, and also across the subcellular compartments of single neurons), which realistically, can only be achieved using imaging.

Altered Cl homeostasis hinders forebrain GABAergic interneuron migration in a mouse model of intellectual disability.

Impairments of inhibitory circuits are at the basis of most, if not all, cognitive deficits. The impact of OPHN1, a gene associate with intellectual disability (ID), on inhibitory neurons remains elusive. We addressed this issue by analyzing the postnatal migration of inhibitory interneurons derived from the subventricular zone in a validated mouse model of ID (OPHN1 mice).

Modelling genetic mosaicism of neurodevelopmental disorders in vivo by a Cre-amplifying fluorescent reporter.

Genetic mosaicism, a condition in which an organ includes cells with different genotypes, is frequently present in monogenic diseases of the central nervous system caused by the random inactivation of the X-chromosome, in the case of X-linked pathologies, or by somatic mutations affecting a subset of neurons. The comprehension of the mechanisms of these diseases and of the cell-autonomous effects of specific mutations requires the generation of sparse mosaic models, in which the genotype of each neuron is univocally identified by the expression of a fluorescent protein in vivo. Here, we show a dual-color reporter system that, when expressed in a floxed mouse line for a target gene, leads to the creation of mosaics with tunable degree.

Evolution of Epileptiform Activity in Zebrafish by Statistical-Based Integration of Electrophysiology and 2-Photon Ca Imaging.

The study of sources and spatiotemporal evolution of ictal bursts is critical for the mechanistic understanding of epilepsy and for the validation of anti-epileptic drugs. Zebrafish is a powerful vertebrate model representing an excellent compromise between system complexity and experimental accessibility. We performed the quantitative evaluation of the spatial recruitment of neuronal populations during physiological and pathological activity by combining local field potential (LFP) recordings with simultaneous 2-photon Ca imaging.

Transient Cognitive Impairment in Epilepsy.

Impairments of the dialog between excitation and inhibition (E/I) is commonly associated to neuropsychiatric disorders like autism, bipolar disorders and epilepsy. Moderate levels of hyperexcitability can lead to mild alterations of the EEG and are often associated with cognitive deficits even in the absence of overt seizures. Indeed, various testing paradigms have shown degraded performances in presence of acute or chronic non-ictal epileptiform activity.

Perineuronal nets control visual input via thalamic recruitment of cortical PV interneurons.

In the neocortex, critical periods (CPs) of plasticity are closed following the accumulation of perineuronal nets (PNNs) around parvalbumin (PV)-positive inhibitory interneurons. However, how PNNs tune cortical function and plasticity is unknown. We found that PNNs modulated the gain of visual responses and γ-oscillations in the adult mouse visual cortex in vivo, consistent with increased interneuron function.

Understanding Spreading Depression from Headache to Sudden Unexpected Death.

Spreading depression (SD) is a neurophysiological phenomenon characterized by abrupt changes in intracellular ion gradients and sustained depolarization of neurons. It leads to loss of electrical activity, changes in the synaptic architecture, and an altered vascular response. Although SD is often described as a unique phenomenon with homogeneous characteristics, it may be strongly affected by the particular triggering event and by genetic background.

Brain-wide Mapping of Endogenous Serotonergic Transmission via Chemogenetic fMRI.

Serotonin-producing neurons profusely innervate brain regions via long-range projections. However, it remains unclear whether and how endogenous serotonergic transmission specifically influences regional or global functional activity. We combined designed receptors exclusively activated by designed drugs (DREADD)-based chemogenetics and functional magnetic resonance imaging (fMRI), an approach we term "chemo-fMRI," to causally probe the brain-wide substrates modulated by endogenous serotonergic activity.

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

Intracellular chloride ([Cl]) and pH (pH) 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] and pH, but this has never been performed at the cellular level in vivo.

Arduino Due based tool to facilitate in vivo two-photon excitation microscopy.

Two-photon excitation spectroscopy is a powerful technique for the characterization of the optical properties of genetically encoded and synthetic fluorescent molecules. Excitation spectroscopy requires tuning the wavelength of the Ti:sapphire laser while carefully monitoring the delivered power. To assist laser tuning and the control of delivered power, we developed an Arduino Due based tool for the automatic acquisition of high quality spectra.

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.

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.

Dendritic Spine Instability in a Mouse Model of CDKL5 Disorder Is Rescued by Insulin-like Growth Factor 1.

Background: CDKL5 (cyclin-dependent kinase-like 5) is mutated in many severe neurodevelopmental disorders, including atypical Rett syndrome. CDKL5 was shown to interact with synaptic proteins, but an in vivo analysis of the role of CDKL5 in dendritic spine dynamics and synaptic molecular organization is still lacking.

Methods: In vivo two-photon microscopy of the somatosensory cortex of Cdkl5(-/y) mice was applied to monitor structural dynamics of dendritic spines.

Parvalbumin-Positive Inhibitory Interneurons Oppose Propagation But Favor Generation of Focal Epileptiform Activity.

Parvalbumin (Pv)-positive inhibitory interneurons effectively control network excitability, and their optogenetic activation has been reported to block epileptic seizures. An intense activity in GABAergic interneurons, including Pv interneurons, before seizures has been described in different experimental models of epilepsy, raising the hypothesis that an increased GABAergic inhibitory signal may, under certain conditions, initiate seizures. It is therefore unclear whether the activity of Pv interneurons enhances or opposes epileptiform activities.