In vivo two-photon calcium imaging of cortical neurons in neonatal mice.

In vivo calcium imaging is essential to elucidate unique synchronous activities observed in the developing brain. Here, we present a protocol to image and analyze activity patterns in neonatal mouse neocortex in a single-cell level. We describe steps for in utero electroporation, cranial window surgery, two-photon imaging, and activity correlation analysis. This protocol facilitates the understanding of neuronal activities and activity-dependent circuit formation during development. For complete details on the use and execution of this protocol, please refer to Mizuno et al. (2014),1 Mizuno et al. (2018a),2 and Mizuno et al. (2018b).3.

Plant Extract of Limonium gmelinii Attenuates Oxidative Responses in Neurons, Astrocytes, and Cerebral Endothelial Cells In Vitro and Improves Motor Functions of Rats after Middle Cerebral Artery Occlusion.

There are numerous publications demonstrating that plant polyphenols can reduce oxidative stress and inflammatory processes in the brain. In the present study we have investigated the neuroprotective effect of plant extract isolated from the roots of L. gmelinii since it contains a rich source of polyphenols and other biologically active compounds. We have applied an oxidative and inflammatory model induced by NMDA, H2O2, and TNF-α in human primary neurons and astrocytes, and mouse cerebral endothelial cell (CECs) line in vitro. The levels of ROS generation, NADPH oxidase activation, P-selectin expression, and activity of ERK1/2 were evaluated by quantitative immunofluorescence analysis, confocal microscopy, and MAPK assay. In vivo, sensorimotor functions in rats with middle cerebral artery occlusion (MCAO) were assessed. In neurons NMDA induced overproduction of ROS, in astrocytes TNF-α initiated ROS generation, NADPH oxidase activation, and phosphorylation of ERK1/2. In CECs, the exposure by TNF-α induced oxidative stress and triggered the accumulation of P-selectin on the surface of the cells. In turn, pre-treatment of the cells with the extract of L. gmelinii suppressed oxidative stress in all cell types and pro-inflammatory responses in astrocytes and CECs. In vivo, the treatment with L. gmelinii extract improved motor activity in rats with MCAO.

The Effects of Mobile Phone Radiofrequency Electromagnetic Fields on ß-Amyloid-Induced Oxidative Stress in Human and Rat Primary Astrocytes.

Amyloid beta peptide (Aß) is implicated in the development of pathological reactions associated with Alzheimer's disease (AD), such as oxidative stress, neuro-inflammation and death of brain cells. Current pharmacological approaches to treat AD are not able to control the deposition of Aß and suppression of Aß-induced cellular response. There is a growing body of evidence that exposure to radiofrequency electromagnetic field (RF-EMF) causes a decrease of beta-amyloid deposition in the brains and provides cognitive benefits to Alzheimer's Tg mice. Herein, we investigated the effects of mobile phone radiofrequency EMF of 918 MHz on reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP), activity of NADPH-oxidase, and phosphorylation of p38MAPK and ERK1/2 kinases in human and rat primary astrocytes in the presence of Aß42 and H2O2. Our data demonstrate that EMF is able to reduce Aß42- and H2O2-induced cellular ROS, abrogate Aß42-induced production of mitochondrial ROS and the co-localization between the cytosolic (p47-phox) and membrane (gp91-phox) subunits of NADPH oxidase, while increasing MMP, and inhibiting H2O2-induced phosphorylation of p38MAPK and ERK1/2 in primary astrocytes. Yet, EMF was not able to modulate alterations in the phosphorylation state of the MAPKs triggered by Aß42. Our findings provide an insight into the mechanisms of cellular and molecular responses of astrocytes on RF-EMF exposure and indicate the therapeutic potential of RF-EMF for the treatment of Alzheimer's disease.