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...

Rationale and objectivesThymic stromal lymphopoietin (TSLP), an epithelial upstream cytokine, initiates production of type-2 (T2) cytokines with eosinophilia and possibly airway hyperresponsiveness (AHR) in asthma.This study aimed to determine whether tezepelumab (a human monoclonal antibody targeting TSLP) decreases AHR and airway inflammation in patients with symptomatic asthma on maintenance treatment with inhaled corticosteroids.Methods and measurementsIn this double-blind, placebo-controlled randomised trial adult patients with asthma and AHR to mannitol received either 700 mg tezepelumab or placebo intravenously at 4-week intervals for 12 weeks. AHR to mannitol was assessed, and a bronchoscopy was performed at baseline and after 12 weeks. The primary outcome was the change in AHR from baseline to week-12 and secondary outcomes were changes in airway inflammation.ResultsForty patients were randomised to receive either tezepelumab (n=20) or placebo (n=20). The mean change in PD15 with tezepelumab was 1.9 DD (95% CI 1.2 to 2.5) versus 1·0 (95% CI 0.3 to 1.6) with placebo; p=0.06. Nine (45%) tezepelumab and three (16%) placebo patients had a negative PD15 test at week-12, p=0.04. Airway tissue and BAL eosinophils decreased by 74% (95% CI −53 to −86) and 75% (95% CI −53 to −86) respectively with tezepelumab compared with an increase of 28% (95% CI −39 to 270) and a decrease of 7% (95% CI −49 to 72) respectively with placebo, p=0.004 and p=0.01.ConclusionsInhibiting TSLP-signalling with tezepelumab reduced the proportion of patients with AHR and decreased eosinophilic inflammation in BAL and airway tissue.

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. Furthermore, SD may contribute differently to the pathogenesis of widely heterogeneous clinical conditions. Indeed, clinical disorders related to SD vary in their presentation and severity, ranging from benign headache conditions (migraine syndromes) to severely disabling events, such as cerebral ischemia, or even death in people with epilepsy. Although the characteristics and mechanisms of SD have been dissected using a variety of approaches, ranging from cells to human models, this phenomenon remains only partially understood because of its complexity and the difficulty of obtaining direct experimental data. Currently, clinical monitoring of SD is limited to patients who require neurosurgical interventions and the placement of subdural electrode strips. Significantly, SD events recorded in humans display electrophysiological features that are essentially the same as those observed in animal models. Further research using existing and new experimental models of SD may allow a better understanding of its core mechanisms, and of their differences in different clinical conditions, fostering opportunities to identify and develop targeted therapies for SD-related disorders and their worst consequences.

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 2+ imaging. We developed a method to extract and quantify electrophysiological transients coupled with Ca 2+ events and we applied this tool to analyze two different epilepsy models and to assess the efficacy of the anti-epileptic drug valproate. Finally, by cross correlating the imaging data with the LFP, we demonstrated that the cerebellum is the main source of epileptiform transients. We have also shown that each transient was preceded by the activation of a sparse subset of neurons mostly located in the optic tectum. of human pathologies, including behavioral aspects of the seizure phenotype, such as locomotor patterns and loss of posture [11,13]. Up to now, locomotion represents the main readout of the epileptic phenotype and the pattern and speed of swimming behavior of larvae can indeed be measured using automated locomotion-tracking [14] to provide information on seizure severity and on the outcome of administered drugs. Electrophysiological recordings [9,[15][16][17][18] permit activity monitoring in intact larvae, but the analysis of the electrophysiology data relies mostly on their visual inspection. Moreover, it is often difficult to tell true epileptiform activity apart from physiological events, such as eye and tail movements [18] on account of the high sensibility of the electric signal to muscle activity due to the small dimension of the entire organism. Therefore, electrophysiology requires additional information for the unequivocal identification of the mutant phenotypes and for the evaluation of anti-epileptic compounds.Recently, deep learning classifiers have been employed to identify electrophysiological events [19] and, although these methods are very fast and effective, they are unavoidably affected by the need of an a priori classification of epilepsy features and by the degree of completeness of the training dataset. A high-throughput local field potential (LFP) recording platform has also recently been used to analyze high-order statistical moments for an unsupervised detection of seizures [20]. However, these electrophysiological methods for seizure identification and classification are still prone to be affected by motion artefacts. Moreover, a complete physio-pathological interpretation of LFP recordings is hindered by the impossibility of identifying the sources of the electrophysiological signals and the spatial dynamics of epileptic activity of the underlying neuronal populations.A complementary window on brain function is provided by imaging the fluorescence of ...