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-]i in adult mouse neocortex, using in vivo two-photon imaging. We found a two-fold increase in baseline [Cl-]i in layer 2/3 pyramidal neurons, from day to night, with marked effects upon both physiological cortical processing and seizure susceptibility. Importantly, the night-time activity can be converted to the day-time pattern by local inhibition of NKCC1, while inhibition of KCC2 converts day-time [Cl-]i towards night-time levels. Changes in the surface expression and phosphorylation of the cation-chloride cotransporters, NKCC1 and KCC2, matched these pharmacological effects. When we extended the dark period by 4 h, mice remained active, but [Cl-]i was modulated as for animals in normal light cycles. Our data thus demonstrate a daily [Cl-]i modulation with complex effects on cortical excitability.

Diffuse alveolar damage patterns reflect the immunological and molecular heterogeneity in fatal COVID-19.

BACKGROUND: Severe COVID-19 lung disease exhibits a high degree of spatial and temporal heterogeneity, with different histological features coexisting within a single individual. It is important to capture the disease complexity to support patient management and treatment strategies. We provide spatially decoded analyses on the immunopathology of diffuse alveolar damage (DAD) patterns and factors that modulate immune and structural changes in fatal COVID-19. METHODS: We spatially quantified the immune and structural cells in exudative, intermediate, and advanced DAD through multiplex immunohistochemistry in autopsy lung tissue of 18 COVID-19 patients. Cytokine profiling, viral, bacteria, and fungi detection, and transcriptome analyses were performed. FINDINGS: Spatial DAD progression was associated with expansion of immune cells, macrophages, CD8+ T cells, fibroblasts, and (lymph)angiogenesis. Viral load correlated positively with exudative DAD and negatively with disease/hospital length. In all cases, enteric bacteria were isolated, and Candida parapsilosis in eight cases. Cytokines correlated mainly with macrophages and CD8+T cells. Pro-coagulation and acute repair were enriched pathways in exudative DAD whereas intermediate/advanced DAD had a molecular profile of elevated humoral and innate immune responses and extracellular matrix production. INTERPRETATION: Unraveling the spatial and molecular immunopathology of COVID-19 cases exposes the responses to SARS-CoV-2-induced exudative DAD and subsequent immune-modulatory and remodeling changes in proliferative/advanced DAD that occur side-by-side together with secondary infections in the lungs. These complex features have important implications for disease management and the development of novel treatments. FUNDING: CNPq, Bill and Melinda Gates Foundation, HC-Convida, FAPESP, Regeneron Pharmaceuticals, and the Swedish Heart & Lung Foundation.

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-/y mice). We found that the speed and directionality of migrating neuroblasts were deeply perturbed in OPHN1-/y mice. The significant reduction in speed was due to altered chloride (Cl-) homeostasis, while the overactivation of the OPHN1 downstream signaling pathway, RhoA kinase (ROCK), caused abnormalities in the directionality of the neuroblast progression in mutants. Blocking the cation-Cl- cotransporter KCC2 almost completely rescued the migration speed while proper directionality was restored upon ROCK inhibition. Our data unveil a strong impact of OPHN1 on GABAergic inhibitory interneurons and identify putative targets for successful therapeutic approaches.

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. We demonstrate the generation of a knockout mosaic of the autism/epilepsy related gene PTEN in which the genotype of each neuron is reliably identified, and the neuronal phenotype is accurately characterized by two-photon microscopy.

Evolution of Epileptiform Activity in Zebrafish by Statistical-Based Integration of Electrophysiology and 2-Photon Ca2+ 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 Ca2+ imaging. We developed a method to extract and quantify electrophysiological transients coupled with Ca2+ 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.

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. 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.

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

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.