uperficial layers of the dorsal column as a sensory division of the spinal cord. In addition, microglia in SOD1G93A mice show substantial differences in inflammatory activity within the affected lateral column of spinal cord when compared to control mice, namely highly reactive microglia in pre-clinical stages and ameboid transformed and activated microglia with reduced injury-directed response in clinical stages. MB, a phenothiazine compound, has been used to treat malaria, is the first line treatment of methemoglobinemia and is frequently used in the treatment of ifosfamide-induced encephalopathy. Recently, beneficial effects in Alzheimer’s disease have been reported. Here, we used transgenic mouse combined with time-lapse 2-photon laser-scanning microscopy to investigate the effect of MB on microglial behavior in the SOD1-G93A mouse model for ALS. MB-treated animals were subjected to in vivo imaging to study MB effects on microglial behavior at a cellular level in an intact environment within lateral tracts of the spinal cord, which largely convey efferent signals, i. e. for motor functions. To confirm local anti-inflammatory effects, we analyzed the influence of MB on cytokine and chemokine release from microglia of control and advanced clinical SOD1G93A mice in vitro. Furthermore, we investigated the effect of MB on disease onset, progression and survival as well as on the motor behavior in the ALS model. Additionally, we performed immunohistochemical studies with regard to microglia and motor neurons. Materials and Methods Ethics statement The experiments were performed according to the ethical Vorapaxar guidelines of the national animal protection law and were authorized by the ethical committee of the State of Lower Saxony. Furthermore, the ARRIVE Guidelines have been followed. Mouse strains and motor tests Mice transgenic for the mutated human SOD1G93A were originally obtained from the Jackson Laboratory, USA 1Gur/ J) and bred at the Max Planck Institute for Experimental Medicine Gottingen. Transgenic mice were maintained in the hemizygous state by mating G93A males with B6SJL hybrid females for more than 10 generations. Animals were fed standard AIN93G diets. Drug administration was orally in drinking water or by intraperitoneal injection, started at the age of 8 or 7 weeks. Neuropathology is first detected at this time in SOD1G93A mice but without clinical expression of 
disease. The oral study used 10 mice per group that were treated with MB 3, 10, 30 and 100 mg per kg body weight per day. The control group consisted of 16 mice on a standard diet. The distribution of sexes was similar in each group. Water consumption was monitored in 12 mice from each group throughout the oral experiment in the different clinical stages. The i. p. study used 16 mice per group and received either a daily injection of 0.9% NaCl solution alone as control or 10 mg MB per kg per day dissolved in 0.9% NaCl. The distribution between the sexes was also similar in both groups. For 2P-LSM experiments, hemizygote male TgN mice were crossbred with female TgHxTgN mice to obtain SOD1G93A mice with fluorescently labeled microglia and projection neurons,. Doubletransgenic TgHxTgN mice were previously obtained by crossbreeding homozygous CX3CR1EGFP mice, in which the expression of EGFP in microglia is achieved by inserting the EGFP reporter gene into the Cx3cr1 locus encoding the chemokine receptor CX3CR1, with transgenic THY1-EYFP mice expressing the yellow fluorescen