Additional file1: Figure S1. of Effects of FTY720 on brain neurogenic niches in vitro and after kainic acid-induced injury

Neurosphere culture differentiation into neurons and oligodendrocytes. Dose-response for FTY720-P treatment and immunofluorescence on fixed cells was performed using anti-β-III tubulin or CNPase as markers of mature neurons or oligodendrocytes, respectively, (representative photomicrographs of the staining are shown in the upper panel of the figure). Total nuclei were stained with DAPI. Fluorescence intensity both from neurons (β-III tubulin) or oligodendrocytes (CNPase), and total nuclei (DAPI) were measured using a fluorescence plate reader with appropriate excitation and emission filters. Differentiation was evaluated as a ratio of β-III tubulin or CNPase fluorescence intensity over total nuclei fluorescence intensity (graphs in the lower panel of the figure). The data are expressed as mean ± SEM of four independent experiments. Statistical analysis: two tailed Student t test; *p < 0.05 and **p < 0.01 vs control. (TIFF 4858 kb

Additional file 2: Figure S2. of Effects of FTY720 on brain neurogenic niches in vitro and after kainic acid-induced injury

Immunostaining with Ki67 showed no relevant difference with BrdU staining. (a) Representative images of immunoperoxidase staining of Ki67 in ipsilateral and contralateral DG in all experimental groups. Scale bar: 200 μm. DG is surrounded in white dotted line. GCL and SGZ are indicated with arrows. Arrowheads indicate the magnified view in the box. (b) Quantitative analysis of the immunoperoxidase staining for Ki67 in ipsilateral and contralateral SGZ of the dorsal hippocampus in control, FTY720, KA and KA + FTY720-treated animals. Positive cells were considered to be within the SGZ if they were within two cell body diameters of the border between the GCL and the hilus. Data were plotted as the mean of Ki67+ cells per slice ± SEM. Statistical analysis: Mann Whitney test; *p < 0.05 vs control. Two-way ANOVA showed no significant difference between ipsilateral and contralateral Ki67+ cells in SGZ. N = 4 for each experimental group. (TIFF 3666 kb

Additional file 3: Figure S3. of Effects of FTY720 on brain neurogenic niches in vitro and after kainic acid-induced injury

Intraperitoneal treatment with FTY720 increases the number of new DCX-positive cells in the SGZ. Animals were sacrificed 8 days after surgery, and 40-μm-thick coronal slices of the dorsal hippocampus were obtained. Double immufluorescence staining for BrdU and DCX was then performed in ipsilateral DG in vehicle (control), FTY720 (ip), KA (icv) and KA (icv) + FTY720 (ip) injected animals. BrdU and DCX-positive cells were counted using confocal acquired images over the total SGZ of the ipsi DG in two sections per animal, corresponding to two levels of the dorsal hippocampus. DCX and BrdU colocalization was determined examining three-dimensional orthogonal reconstructions of confocal layers (ImageJ software). Data are presented as (a, b) mean of positive cells per slice ± SEM and (c) percentage of DXC+ cells over BrdU+ total cells. Statistical analysis: one-way ANOVA followed by Bonferroni post hoc test; *p < 0.05, **p < 0.01. The number of animals per group: control = 3, FTY720 = 3, KA = 2, KA + FTY720 = 2. (TIFF 410 kb

Additional file 1: Figure S1. of Synaptic plasticity and spatial working memory are impaired in the CD mouse model of Williams-Beuren syndrome

PPF of EPSCs was unchanged in CD mice. (DOCX 54 kb

Functional glutamate transport in rodent optic nerve axons and glia

Recent findings suggest that synaptic-type glutamate signaling operates between axons and their supporting glial cells. Glutamate reuptake will be a necessary component of such a system. Evidence for glutamate-mediated damage of oligodendroglia somata and processes in white matter suggests that glutamate regulation in white matter structures is also of clinical importance. The expression of glutamate transporters was examined in postnatal Day 14-17 (P14-17) mouse and in mature mouse and rat optic nerve using immuno-histochemistry and immuno-electron microscopy. EAAC1 was the major glutamate transporter detected in oligodendroglia cell membranes in both developing and mature optic nerve, while GLT-1 was the most heavily expressed transporter in the membranes of astrocytes. Both EAAC1 and GLAST were also seen in adult astrocytes, but there was little membrane expression of either at P14-17. GLAST, EAAC1, and GLT-1 were expressed in P14-17 axons with marked GLT-1 expression in the axolemma, while in mature axons EAAC1 was abundant at the node of Ranvier. Functional glutamate transport was probed in P14-17 mouse optic nerve revealing Na+-dependent, TBOA-blockable uptake of D-aspartate in astrocytes, axons, and oligodendrocytes. The data show that in addition to oligodendroglia and astrocytes, axons represent a potential source for extracellular glutamate in white matter during ischaemic conditions, and have the capacity for Na+-dependent glutamate uptake. The findings support the possibility of functional synaptic-type glutamate release from central axons, an event that will require axonal glutamate reuptake

Severity of Cannabis Use.

<p>^aFourth edition of the Diagnostic and Statistical Manual of Mental Disorders, revised edition (DSM-IV-TR).²³</p><p>^bEuropean Addiction Severity Index (Europ-ASI).²⁵</p><p>Severity of Cannabis Use.</p

Increased Panx1 channel activity in EAE.

<p>(<b>A</b>) Bar histograms of the mean ± s.e.m. values of the relative intensity of YoPro uptake (test/control) recorded from Panx1 wild type (WT) and Panx1 knockout (KO) spinal cord slices of control and EAE mice. **P<0.005, ***P<0.001 (ANOVA followed by Newman-Keuls multiple comparison test). Nine Panx1 WT non-EAE, 9 Panx1 WT EAE, 8 Panx1 KO non-EAE, and 13 Panx1 KO EAE mice were used, with a minimum of 167 slices examined per group. Samples from EAE mice were then separated into mildly affected (flaccid tail, hind-limb weakness, clinical score range 0–3) and severely affected (hind-limb paralysis with possible trunk and forelimb involvement, clinical score range 4–7). (<b>B</b>) Bar histograms of the mean ± s.e.m. values of the relative intensity of propidium iodide (P.I.) uptake (test/control) recorded from Panx1 wild type (WT) and Panx1 knockout (KO) spinal cord slices of control and EAE mice. ***P<0.001, (ANOVA followed by Newman-Keuls post hoc comparison). Seven Panx1 WT non-EAE, 5 Panx1 WT EAE, 5 Panx1 KO non-EAE, and 7 Panx1 KO EAE mice were used, with a minimum of 71 slices per group examined. (<b>C</b>) Bar histograms of the mean ± s.e.m. values of ATP (pmol/mg protein) recorded from ACSF bathing Panx1 wild-type (WT) and Panx1 knockout (KO) spinal cord slices of control and EAE mice. In parentheses are the numbers of animals used. (<b>D</b>) Bar histograms of the mean ± s.e.m. values of Panx1 transcript (normalized to ribosomal 18S) measured from RNA samples extracted from cerebellum of naïve and EAE Panx1 WT mice. Samples are from 3 naïve and 3 EAE mice. ***P<0.001 and *P<0.05 (ANOVA followed by Newman-Keuls multiple comparison test); P value in part <b>D</b> was obtained using unpaired t-test analysis.</p

Blockade of Panx1 channels ameliorates signs of EAE.

<p>Time course of the mean ± s.e.m. values of neurological scores recorded from rats (<b>A, B</b>) and mice (<b>C, D</b>) with EAE. (<b>A</b>) Daily intraperitoneal injections of 5 mg/kg mefloquine (MFQ) but not 1 mg/kg MFQ improved EAE outcome in rats. (<b>B–D</b>) Daily injections of MFQ (5 mg/kg) administered to (<b>B</b>) rats and (<b>C</b>) mice at 7 days post immunization (dpi) till, respectively 14 and 20 dpi, and to (<b>D</b>) mice starting at 14 dpi till 38 dpi is shown to ameliorate the EAE symptoms. P values were calculated from all data points using Mann-Whitney test. (<b>E</b>) Bar histograms showing the mean ± s.e.m. values of conduction latency in the corticospinal pathway of mice with EAE untreated (black bar) and treated (gray bar) with MFQ. In parentheses are the numbers of animals used. P values were obtained from unpaired t-test.</p

Deficient IL-1β release from activated Panx1 KO splenic macrophages.

<p>Bar histograms showing the mean ± s.e.m. values of interleukin-1β (IL-1β) measured from media bathing Panx1 wild type (WT) and Panx1 knockout (KO) macrophage cultures that were untreated, treated with lipopolysaccharide (LPS) and treated with LPS and stimulated with 5 mM ATP. Samples were obtained from 9 mice pooled into three groups and ELISA run in triplicates. ***P<0.001 (ANOVA followed by Newman-Keuls multiple comparison test).</p

Delayed onset of EAE in Panx1 KO mice.

<p>(<b>A - right</b>) Time course of clinical signs recorded from Panx1 wild type (WT) and Panx1 knockout (KO) mice immunized for MOG. (<b>A-left</b>) The graph depicts the initial acute phase of the disease (blue rectangle in <b>A</b>) showing that up to day 12 post-immunization, a significant difference in clinical scores was detected between the two genotypes. Symbols represent mean ± s.e.m. Twenty two Panx1 WT and 22 Panx1 KO female mice were immunized. Thirteen days post-immunization (dpi), 5 animals of each genotype were used for histopathology; 6 Panx1 WT and 2 Panx1 KO died or were euthanized due to the severity of EAE symptoms. *P<0.01, unpaired t-test. (<b>B</b>) Hematoxylin & eosin stained sections of spinal cord from Panx1 WT and Panx1 KO mice obtained at the acute phase of EAE (12–13 dpi). Ventral funiculus is outlined in white box and inflammatory lesions are outlined in cyan. Bar histograms on the right represent the mean ± s.e.m values of the percent lesion area in each genotype. Note that Panx1 KO EAE spinal cords exhibit significantly less infiltrating cells than spinal cords from WT mice. Five Panx1 WT and 5 Panx1 KO mice were used for histology; three spinal cord sections from sacral to thoracic areas were analyzed from each mouse. (<b>C</b>) Hematoxylin & eosin stained sections of spinal cord from Panx1 WT and Panx1 KO mice obtained at the chronic phase of EAE (35 dpi). Bar histograms on the right represent the mean ± s.e.m values of the percent lesion area in each genotype. At this stage of disease, Panx1 WT and Panx1 KO spinal cords exhibit similar extent of lesion areas. Five Panx1 WT and 5 Panx1 KO mice were used for histology. Three sections from sacral to thoracic spinal cords were analyzed from each mouse. Quantification of lesions is presented as percent area of ventral funiculus white matter occupied by inflammatory cells. P values were obtained using unpaired t-test.</p