Glial Ca2+ signaling in the spinal cord and the myelin-protective effect of the GABAb receptor in oligodendrocyte precursor cells

The highly complex network of glial cells in the central nervous system relies intracellularly and intercellularly on multilayered communication through the secondary messenger Ca2+ and is fundamentally different in its characteristics from the communication system of neurons, which is more rapidly timed. The Ca2+-based communication system is inherent in all glial cells, whether astroglia, microglia, or the cells of oligodendroglial lineage. Eech cell receives, processes, and sends signals through Ca2+ concentration changes and, embedded in a syncytium of cells, provides information about its activity. If a cellular stress response occurs, Ca2+ concentration is an important indicator of the switch in cellular function, from gene activity to protein turnover and integrity to targeted cell death. Such steps are cell type-specific and task-related, as well as controllable by surrounding cells or disease. This cellular Ca2+ activity can be visualized under cell type-specific promotors using the genetically encoded Ca2+ sensor GCaMP3 and has been comparatively analyzed in this study in spinal glial cells using two-photon microscopy in vivo and ex vivo. In the development and maintenance of myelin by oligodendrocytes, finely tuned Ca2+ signals are crucial for functional myelin quality. These mature oligodendrocytes arise throughout life from their precursors, NG2 glia, also a sensitive Ca2+ dependent process. The inhibitory neurotransmitter γ-aminobutyric acid (GABA) has a depolarizing effect on these cells via its ionotropic receptor (GABAA) on one hand and leads to an increase of intracellular Ca2+ via the metabotropic receptor (GABAB), presumably involving G proteins and phospholipase C, on the other hand. Already in clinical therapy of spasticity in multiple sclerosis, receptor agonists against GABA receptors are used to provide relief to patients. Conditional receptor loss in oligodendroglial precursor cells (OPCs) and its effect on Ca2+ homeostasis, as well as its combination with a toxic demyelination model by a cuprizone diet, were investigated in mouse spinal cord in this study. Because the peripheral arrangement of lipid-rich white matter in the spinal cord is readily accessible, two-photon microscopy in vivo was used. This study revealed cell type-specific and regionally dependent Ca2+ activity for microglia, astroglia as well as oligodendroglial precursor cells. Furthermore, loss of the metabotropic GABAB receptor in oligodendroglial precursor cells showed reduced Ca2+ activity and altered myelin structure and, at a later time point, a myelin protective effect with hardly altered Ca2+ signaling activity after toxic demyelination.Das hoch komplexe Netzwerk aus Gliazellen im Zentralnervensystem stützt sich intra- und interzellulär auf die vielschichtige Kommunikation durch den sekundären Botenstoff Ca2+ und ist grundlegend anders in seiner Charakteristik als das Kommunikationssystem der Neurone, das zeitlich schneller getaktet ist. Die auf Ca2+ basierende Kommunikation ist allen Gliazellen eigen, ob Astroglia, Mikroglia oder Zellen der oligodendroglialen Linie. Jede dieser Zellen erhält, verarbeitet und sendet durch Ca2+ Konzentrationsänderungen Signale aus und gibt, eingebettet in einem Synzytium aus Zellen, Aufschluss über seine Aktivität. Kommt es zur zellulären Stressantwort ist die Ca2+ Konzentration ein wichtiger Indikator für die Umstellung der Zellfunktionen, von der Genaktivität über den Proteinumsatz und -integrität bis hin zum gezielten Zelltod. Solche Schritte sind zelltypspezifisch und aufgabenbezogen sowie durch umliegende Zellen oder Krankheiten manipulierbar. Diese zelleigene Ca2+ Aktivität kann unter zelltypspezifischen Promotoren mittels dem genetisch kodierten Kalziumsensor GCaMP3 visualisiert werden und ist in dieser Studie in spinalen Gliazellen mittels Zwei-Photonen Mikroskopie in vivo und ex vivo vergleichend analysiert worden. Bei der Entwicklung und Aufrechterhaltung des Myelins durch Oligodendrozyten sind fein abgestimmte Ca2+ Signale ausschlaggebend für funktionales Myelin. Reife Oligodendrozyten entstehen zeitlebens aus ihren Vorläufern, den NG2-Glia unter anderem durch einen sensiblen, Ca2+ abhängigen Prozess. Der inhibitorische Neurotransmitter γ-Aminobuttersäure (GABA) wirkt an diesen Zellen zum einen depolarisierend über seinen ionotropen Rezeptor (GABAA) und zum anderen positiv auf die Erhöhung des intrazellulären Ca2+ über den metabotropen Rezeptor (GABAB), vermutlich G-Proteine und Phospholipase C involvierend. Bereits in der klinischen Therapie von Spastiken bei Multipler Sklerose werden Agonisten gegen GABA Rezeptoren eingesetzt um den Patienten Linderung zu verschaffen. Der konditionelle Rezeptorverlust in oligodendroglialen Vorläuferzellen und dessen Auswirkung auf die Ca2+ Homöostase, sowie dessen Kombination mit einem toxischen Demyelinisierungsmodell durch eine Cuprizon Diät wurden in dieser Studie im Rückenmark von Mäusen untersucht. Da die periphere Anordnung der lipidreichen weißen Substanz im Rückenmark leicht zugänglich ist, konnte hier ebenfalls die Zwei-Photonen Mikroskopie in vivo eingesetzt werden. In dieser Studie zeigten sich zelltypspezifische und für Mikro- und Astroglia sowie oligodendrogliale Vorläuferzellen regional abhängige Ca2+ Aktivitäten. Des Weiteren führte der Verlust des metabotropen GABAB Rezeptors in oligodendroglialen Vorläuferzellen zu einer reduzierten Ca2+ Aktivität und veränderter Myelin Struktur sowie zu einem späteren Untersuchungszeitpunkt zu einem Myelin protektiven Effekt mit kaum veränderter Ca2+ Signalaktivität nach toxischer Demyelinisierung

Astrocytes and Microglia Exhibit Cell-Specific Ca2+ Signaling Dynamics in the Murine Spinal Cord

The spinal cord is the main pathway connecting brain and peripheral nervous system. Its functionality relies on the orchestrated activity of both neurons and glial cells. To date, most advancement in understanding the spinal cord inner mechanisms has been made either by in vivo exposure of its dorsal surface through laminectomy or by acute ex vivo slice preparation, likely affecting spinal cord physiology in virtue of the necessary extensive manipulation of the spinal cord tissue. This is especially true of cells immediately responding to alterations of the surrounding environment, such as microglia and astrocytes, reacting within seconds or minutes and for up to several days after the original insult. Ca2+ signaling is considered one of the most immediate, versatile, and yet elusive cellular responses of glia. Here, we induced the cell-specific expression of the genetically encoded Ca2+ indicator GCaMP3 to evaluate spontaneous intracellular Ca2+ signaling in astrocytes and microglia. Ca2+ signals were then characterized in acute ex vivo (both gray and white matter) as well as in chronic in vivo (white matter) preparations using MSparkles, a MATLAB-based software for automatic detection and analysis of fluorescence events. As a result, we were able to segregate distinct astroglial and microglial Ca2+ signaling patterns along with method-specific Ca2+ signaling alterations, which must be taken into consideration in the reliable evaluation of any result obtained in physiological as well as pathological conditions. Our study revealed a high degree of Ca2+ signaling diversity in glial cells of the murine spinal cord, thus adding to the current knowledge of the astonishing glial heterogeneity and cell-specific Ca2+ dynamics in non-neuronal networks

Cannabidiol Exerts a Neuroprotective and Glia-Balancing Effect in the Subacute Phase of Stroke

Pharmacological agents limiting secondary tissue loss and improving functional outcomes after stroke are still limited. Cannabidiol (CBD), the major non-psychoactive component of Cannabis sativa, has been proposed as a neuroprotective agent against experimental cerebral ischemia. The effects of CBD mostly relate to the modulation of neuroinflammation, including glial activation. To investigate the effects of CBD on glial cells after focal ischemia in vivo, we performed time-lapse imaging of microglia and astroglial Ca2+ signaling in the somatosensory cortex in the subacute phase of stroke by in vivo two-photon laser-scanning microscopy using transgenic mice with microglial EGFP expression and astrocyte-specific expression of the genetically encoded Ca2+ sensor GCaMP3. CBD (10 mg/kg, intraperitoneally) prevented ischemia-induced neurological impairment, reducing the neurological deficit score from 2.0 ± 1.2 to 0.8 ± 0.8, and protected against neurodegeneration, as shown by the reduction (more than 70%) in Fluoro-Jade C staining (18.8 ± 7.5 to 5.3 ± 0.3). CBD reduced ischemia-induced microglial activation assessed by changes in soma area and total branch length, and exerted a balancing effect on astroglial Ca2+ signals. Our findings indicate that the neuroprotective effects of CBD may occur in the subacute phase of ischemia, and reinforce its strong anti-inflammatory property. Nevertheless, its mechanism of action on glial cells still requires further studies

Novel algorithms for improved detection and analysis of fluorescent signal fluctuations

Fluorescent dyes and genetically encoded fuorescence indicators (GEFI) are common tools for visualizing concentration changes of specifc ions and messenger molecules during intra- as well as intercellular communication. Using advanced imaging technologies, fuorescence indicators are a prerequisite for the analysis of physiological molecular signaling. Automated detection and analysis of fuorescence signals require to overcome several challenges, including correct estimation of fuorescence fuctuations at basal concentrations of messenger molecules, detection, and extraction of events themselves as well as proper segmentation of neighboring events. Moreover, event detection algorithms need to be sensitive enough to accurately capture localized and low amplitude events exhibiting a limited spatial extent. Here, we present two algorithms (PBasE and CoRoDe) for accurate baseline estimation and automated detection and segmentation of fuorescence fuctuations

Defining the causes of sporadic Parkinson's disease in the global Parkinson's genetics program (GP2)

The Global Parkinson’s Genetics Program (GP2) will genotype over 150,000 participants from around the world, and integrate genetic and clinical data for use in large-scale analyses to dramatically expand our understanding of the genetic architecture of PD. This report details the workflow for cohort integration into the complex arm of GP2, and together with our outline of the monogenic hub in a companion paper, provides a generalizable blueprint for establishing large scale collaborative research consortia

Multi-ancestry genome-wide association meta-analysis of Parkinson?s disease

Although over 90 independent risk variants have been identified for Parkinson’s disease using genome-wide association studies, most studies have been performed in just one population at a time. Here we performed a large-scale multi-ancestry meta-analysis of Parkinson’s disease with 49,049 cases, 18,785 proxy cases and 2,458,063 controls including individuals of European, East Asian, Latin American and African ancestry. In a meta-analysis, we identified 78 independent genome-wide significant loci, including 12 potentially novel loci (MTF2, PIK3CA, ADD1, SYBU, IRS2, USP8, PIGL, FASN, MYLK2, USP25, EP300 and PPP6R2) and fine-mapped 6 putative causal variants at 6 known PD loci. By combining our results with publicly available eQTL data, we identified 25 putative risk genes in these novel loci whose expression is associated with PD risk. This work lays the groundwork for future efforts aimed at identifying PD loci in non-European populations

Cannabidiol Exerts a Neuroprotective and Glia-Balancing Effect in the Subacute Phase of Stroke

Pharmacological agents limiting secondary tissue loss and improving functional outcomes after stroke are still limited. Cannabidiol (CBD), the major non-psychoactive component of Cannabis sativa, has been proposed as a neuroprotective agent against experimental cerebral ischemia. The effects of CBD mostly relate to the modulation of neuroinflammation, including glial activation. To investigate the effects of CBD on glial cells after focal ischemia in vivo, we performed time-lapse imaging of microglia and astroglial Ca2+ signaling in the somatosensory cortex in the subacute phase of stroke by in vivo two-photon laser-scanning microscopy using transgenic mice with microglial EGFP expression and astrocyte-specific expression of the genetically encoded Ca2+ sensor GCaMP3. CBD (10 mg/kg, intraperitoneally) prevented ischemia-induced neurological impairment, reducing the neurological deficit score from 2.0 ± 1.2 to 0.8 ± 0.8, and protected against neurodegeneration, as shown by the reduction (more than 70%) in Fluoro-Jade C staining (18.8 ± 7.5 to 5.3 ± 0.3). CBD reduced ischemia-induced microglial activation assessed by changes in soma area and total branch length, and exerted a balancing effect on astroglial Ca2+ signals. Our findings indicate that the neuroprotective effects of CBD may occur in the subacute phase of ischemia, and reinforce its strong anti-inflammatory property. Nevertheless, its mechanism of action on glial cells still requires further studies

Drugs used to treat pediatric emergencies

This clinical report is a revision of “Preparing for Pediatric Emergencies: Drugs to Consider.” It updates the list, indications, and dosages of medications used to treat pediatric emergencies in the prehospital, pediatric clinic, and emergency department settings. Although it is not an all-inclusive list of medications that may be used in all emergencies, this resource will be helpful when treating a vast majority of pediatric medical emergencies. Dosage recommendations are consistent with current emergency references such as the Advanced Pediatric Life Support and Pediatric Advanced Life Support textbooks and American Heart Association resuscitation guidelines

Molecular allelokaryotyping of pediatric acute lymphoblastic leukemias by high-resolution single nucleotide polymorphism oligonucleotide genomic microarray

Pediatric acute lymphoblastic leukemia (ALL) is a malignant disease resulting from accumulation of genetic alterations. A robust technology, single nucleotide polymorphism oligonucleotide genomic microarray (SNP-chip) in concert with bioinformatics offers the opportunity to discover the genetic lesions associated with ALL. We examined 399 pediatric ALL samples and their matched remission marrows at 50 000/250 000 SNP sites using an SNP-chip platform. Correlations between genetic abnormalities and clinical features were examined. Three common genetic alterations were found: deletion of ETV6, deletion of p16INK4A, and hyperdiploidy, as well as a number of novel recurrent genetic alterations. Uniparental disomy (UPD) was a frequent event, especially affecting chromosome 9. A cohort of children with hyperdiploid ALL without gain of chromosomes 17 and 18 had a poor prognosis. Molecular allelokaryotyping is a robust tool to define small genetic abnormalities including UPD, which is usually overlooked by standard methods. This technique was able to detect subgroups with a poor prognosis based on their genetic status