Journal: PLoS ONE

Article Title: Sigma-1 Receptor Enhances Neurite Elongation of Cerebellar Granule Neurons via TrkB Signaling

doi: 10.1371/journal.pone.0075760

Figure Lengend Snippet: (A) CGNs immunostained for Sig-1R (red) and TrkB (green). Scale Bar: 10 µm. (B and C) CGNs were cultured for 24 h with or without PRE-084. As for cultures incubated with K252a, a pan-tropomyosin receptor kinase (trk) K252a was added at the same time as PRE was added to the culture. Representative images of CGNs are shown in (B). The neurite lengths were quantified from three independent experiments. The mean lengths of neurite are shown in the graph (C). Scale bar: 20 µm. (D) The same effects were observed when the cells were cultured in the presence of BDNF; n = 3, ** P <0.01, Scheffe’s test.

Article Snippet: The following reagents were used in this study: 2-(4-Morpholinethyl)1-phenylcyclohexanecarboxylate (PRE-084), a specific Sig-1R agonist; 1-[3,4-dichlorophenyl]ethyl]-4-methylpiprazine (BD1063), a Sig-1R-specific antagonist (Tocris Bioscience, Minneapolis, MN, USA), brain-derived neurotrophic factor (BDNF; Peprotech, Rocky Hill, NJ, USA), and K252a (Alomone Labs, Jerusalem, Israel) were used.

Techniques: Cell Culture, Incubation

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Tissue-Type Plasminogen Activator Regulates the Neuronal Uptake of Glucose in the Ischemic Brain

doi: 10.1523/JNEUROSCI.1241-12.2012

Figure Lengend Snippet: A. Experimental design used to study the effect of tPA on neuronal survival. Letters denote time of treatment with tPA after exposure to oxygen-glucose deprivation (OGD) conditions. B – D. Mean cell survival (panels B & C) and release of LDH into the culture media (panel D) in Wt cerebral cortical neurons treated with 5 nM of proteolytically active (panels B & D) or inactive tPA (itPA; panels C & D), or 10 nM of plasmin (panel C), 5, or 30, or 60, or 120, or 180, or 360 minutes after exposure to 55 minutes of OGD conditions. n = 20 in B and 15 in C and D. * in B: p < 0.05 compared to neurons exposed to OGD conditions without subsequent treatment with tPA. * in C: p < 0.05 compared to neurons left untreated after exposure to OGD conditions. Ns: non-significant. * in D: p < 0.05 compared to cells exposed to OGD conditions without subsequent treatment. Lines denote SD. E. Mean cell survival in Wt cerebral cortical neurons exposed to 55 minutes of OGD conditions and treated 1 hour later with 5 nM of tPA, alone or in combination with either 10 µM of MK-801 or 60 nM of the receptor associated protein (RAP), or 100 nM of the tyrosine kinase receptor B (TrkB) inhibitor K-252a. n = 10. * p < 0.05 compared to neurons treated with tPA alone, or with a combination of tPA and K-252a. Lines denote SD. F. Mean volume of the ischemic lesion in Wt and Plg−/− mice treated one hour after tMCAO with saline solution (white bars) or rtPA 1 – 9 mg/Kg/IV (gray bars). n = 10 per group. * p < 0.05 compared to Wt mice treated with saline solution. ** p < 0.05 compared to mice treated with 1 mg/Kg/IV of rtPA. *** p < 0.05 compared to Plg−/− mice treated with saline solution. Bars depict mean volume of the ischemic lesion in mm3. Lines denote SD.

Article Snippet: Other reagents were human recombinant tissue-type plasminogen activator (Genentech Inc.), the phosphoinositide (PI) 3-kinase/Akt inhibitor Wortmannin, methanol, methyl salicylate and triphenyltetrazolium chloride (Sigma Aldrich), the 3-(4,5- Dimethylthiazol -2-yl)-2,5-di phenyl tetrazolium bromide (MTT) assay (ATCC), the LDH release assay (Roche), the Receptor-Associated Protein (RAP; kindly provided by Dr. Dudley K. Strickland, University of Maryland), the NMDAR antagonist MK-801 (Tocris Bioscience), rapamycin and the TrkB inhibitor K-252a (Calbiochem), HIF-1α shRNA, scramble shRNA lentiviral particles, anti-GLUT3 antibodies and TRITC-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology), anti-HIF-1α antibodies (Abcam), antibodies against the p70S6 kinase (p70 S6K ) phosphorylated at Thr389 (Cell Signaling), heparin sodium (Abraxis Pharmaceutical Products), blue latex (Connecticut Valley Biological Supply), ApopTag Plus Fluorescein in Situ Apoptosis Detection Kit (Chemicon International), 4'-6-Diamidino-2-phenylindole (DAPI; Invitrogen), triphenyltetrazolium chloride (TTC; Sigma-Aldrich), 2- N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG; Molecular Probes), and 18-Fluorodeoxyglucose (PETNET).

Techniques: Saline

Journal: Neural Plasticity

Article Title: Divergent Roles of p75 NTR and Trk Receptors in BDNF's Effects on Dendritic Spine Density and Morphology

doi: 10.1155/2012/578057

Figure Lengend Snippet: Role of Trk and p75 NTR in BDNF's effects on dendritic spine density and morphology. (a) Representative examples of dendritic segments of CA1 pyramidal neurons maintained in serum-containing media (SM) and treated with the function-blocking antibody of p75 NTR , REX (50 μ g/mL), and BDNF (250 ng/mL) for 48 hrs (scale bar represents 2 μ m). (b) Dendritic spine density expressed per 10 μ m of apical dendrite. (c) Proportion of each morphological type of dendritic spine, expressed as a fraction of the total spine population. (d) Representative examples of dendritic segments of CA1 pyramidal neurons maintained in SM and treated with k-252a (200 nM) and BDNF (250 ng/mL) for 48 hrs. (e) Dendritic spine density expressed per 10 μ m of apical dendrite. (f) Proportion of each morphological type of dendritic spines, expressed as a fraction of the total spine population. * P < 0.05, ** P < 0.01, and *** P < 0.001, after a one-way ANOVA.

Article Snippet: Slices were kept in a serum containing media throughout the course of the experiments and were randomly assigned to the following groups: (1) control, serum-containing media; (2) BDNF (250 ng/mL); (3) anti-p75 NTR antibody REX (50 μ g/mL), known to block p75 NTR function [ ] (provided by L. Reichardt, UCSF); (4) BDNF in the presence of REX antibody; (5) k-252a (200 nM, in DMSO; Calbiochem; San Diego, CA) to block autophosphorylation and activation of tyrosine kinase domains of plasma membrane neurotrophin receptors [ ]; (6) BDNF in the presence of k-252a; (7) TTX (1 μ M; Alomone Labs; Jerusalem, Israel); (8) TTX in the presence of k-252a.

Techniques: Blocking Assay

Journal: Neural Plasticity

Article Title: Divergent Roles of p75 NTR and Trk Receptors in BDNF's Effects on Dendritic Spine Density and Morphology

doi: 10.1155/2012/578057

Figure Lengend Snippet: Role of neuronal activity in k-252a's effects on dendritic spine density and morphology. (a) Representative examples of dendritic segments of CA1 pyramidal neurons maintained in serum-containing media (SM) and treated with k-252a (200 nM), TTX (1 μ M), or both k-252a and TTX for 48 hrs (scale bar represents 2 μ m). (b) Dendritic spine density expressed per 10 μ m of apical dendrite. (c) Proportion of each morphological type of dendritic spines, expressed as a fraction of the total spine population. * P < 0.05, ** P < 0.01, and *** P < 0.001, after a one-way ANOVA.

Article Snippet: Slices were kept in a serum containing media throughout the course of the experiments and were randomly assigned to the following groups: (1) control, serum-containing media; (2) BDNF (250 ng/mL); (3) anti-p75 NTR antibody REX (50 μ g/mL), known to block p75 NTR function [ ] (provided by L. Reichardt, UCSF); (4) BDNF in the presence of REX antibody; (5) k-252a (200 nM, in DMSO; Calbiochem; San Diego, CA) to block autophosphorylation and activation of tyrosine kinase domains of plasma membrane neurotrophin receptors [ ]; (6) BDNF in the presence of k-252a; (7) TTX (1 μ M; Alomone Labs; Jerusalem, Israel); (8) TTX in the presence of k-252a.

Techniques: Activity Assay

Journal: Neural Plasticity

Article Title: Divergent Roles of p75 NTR and Trk Receptors in BDNF's Effects on Dendritic Spine Density and Morphology

doi: 10.1155/2012/578057

Figure Lengend Snippet: Quantitative results of dendritic spine analyses .

Article Snippet: Slices were kept in a serum containing media throughout the course of the experiments and were randomly assigned to the following groups: (1) control, serum-containing media; (2) BDNF (250 ng/mL); (3) anti-p75 NTR antibody REX (50 μ g/mL), known to block p75 NTR function [ ] (provided by L. Reichardt, UCSF); (4) BDNF in the presence of REX antibody; (5) k-252a (200 nM, in DMSO; Calbiochem; San Diego, CA) to block autophosphorylation and activation of tyrosine kinase domains of plasma membrane neurotrophin receptors [ ]; (6) BDNF in the presence of k-252a; (7) TTX (1 μ M; Alomone Labs; Jerusalem, Israel); (8) TTX in the presence of k-252a.

Techniques:

Journal: Neural Plasticity

Article Title: Divergent Roles of p75 NTR and Trk Receptors in BDNF's Effects on Dendritic Spine Density and Morphology

doi: 10.1155/2012/578057

Figure Lengend Snippet: Summary of total dendritic length, cells, slices, and individual spines sampled for the quantitative dendritic spine analyses.

Article Snippet: Slices were kept in a serum containing media throughout the course of the experiments and were randomly assigned to the following groups: (1) control, serum-containing media; (2) BDNF (250 ng/mL); (3) anti-p75 NTR antibody REX (50 μ g/mL), known to block p75 NTR function [ ] (provided by L. Reichardt, UCSF); (4) BDNF in the presence of REX antibody; (5) k-252a (200 nM, in DMSO; Calbiochem; San Diego, CA) to block autophosphorylation and activation of tyrosine kinase domains of plasma membrane neurotrophin receptors [ ]; (6) BDNF in the presence of k-252a; (7) TTX (1 μ M; Alomone Labs; Jerusalem, Israel); (8) TTX in the presence of k-252a.

Techniques: