Journal: Brain Sciences

Article Title: Neonatal Handling Positively Modulates Anxiety, Sensorimotor Gating, Working Memory, and Cortico-Hippocampal Neuroplastic Adaptations in Two Genetically Selected Rat Strains Differing in Emotional and Cognitive Traits

doi: 10.3390/brainsci15080776

Figure Lengend Snippet: Western blot analysis of the BDNF ( A , B ), trkB ( C , D ), and the PSA-NCAM ( E , F ), in the prefrontal (prelimbic/infralimbic) cortex of the RHA and the RLA rats, either untreated controls (CTRL) or treated with neonatal handling (NH). The values represent the densitometric analysis of the BDNF/GAPDH ( B ), trkB/GAPDH ( D ), and the PSA-NCAM/GAPDH ( F ) band grey optical density (O.D.) ratios. The bars denote the mean ± S.E.M. of 9–10 rats, in each experimental group. ***: p < 0.001 (post hoc Duncan’s multiple range test).

Article Snippet: Molecular weight (mw) standards (Precision Plus Protein Western C Standards, Cat# 161–0376, Bio-Rad, Hercules, CA, USA) and recombinant human BDNF protein (rhBDNF) (Cat# B-257, Alomone Labs, Jerusalem, Israel) were run in parallel.

Techniques: Western Blot

Journal: Brain Sciences

Article Title: Neonatal Handling Positively Modulates Anxiety, Sensorimotor Gating, Working Memory, and Cortico-Hippocampal Neuroplastic Adaptations in Two Genetically Selected Rat Strains Differing in Emotional and Cognitive Traits

doi: 10.3390/brainsci15080776

Figure Lengend Snippet: Western blot analysis of the BDNF ( A , B ), trkB ( C , D ), and the PSA-NCAM ( E , F ), in the anterior cingulate cortex of the RHA and the RLA rats, either untreated controls (CTRL) or treated with neonatal handling (NH). The values represent the densitometric analysis of the BDNF/GAPDH ( B ), trkB/GAPDH ( D ), and the PSA-NCAM/GAPDH ( F ) band grey optical density (O.D.) ratios. The bars denote the mean ± S.E.M. of 9–10 rats, in each experimental group. **: p < 0.01 (post hoc Duncan’s multiple range test).

Article Snippet: Molecular weight (mw) standards (Precision Plus Protein Western C Standards, Cat# 161–0376, Bio-Rad, Hercules, CA, USA) and recombinant human BDNF protein (rhBDNF) (Cat# B-257, Alomone Labs, Jerusalem, Israel) were run in parallel.

Techniques: Western Blot

Journal: Brain Sciences

Article Title: Neonatal Handling Positively Modulates Anxiety, Sensorimotor Gating, Working Memory, and Cortico-Hippocampal Neuroplastic Adaptations in Two Genetically Selected Rat Strains Differing in Emotional and Cognitive Traits

doi: 10.3390/brainsci15080776

Figure Lengend Snippet: Western blot analysis of the BDNF ( A , B ), trkB ( C , D ), and the PSA-NCAM ( E , F ), in the ventral hippocampus of the RHA and the RLA rats, either untreated controls (CTRL) or treated with neonatal handling (NH). The values represent the densitometric analysis of the BDNF/GAPDH ( B ), trkB/GAPDH ( D ), and the PSA-NCAM/GAPDH ( F ) band grey optical density (O.D.) ratios. The bars denote the mean ± S.E.M. of 9–10 rats, in each experimental group. *: p < 0.05; **: p < 0.02 (post hoc Duncan’s multiple range test).

Article Snippet: Molecular weight (mw) standards (Precision Plus Protein Western C Standards, Cat# 161–0376, Bio-Rad, Hercules, CA, USA) and recombinant human BDNF protein (rhBDNF) (Cat# B-257, Alomone Labs, Jerusalem, Israel) were run in parallel.

Techniques: Western Blot

Journal: Brain Sciences

Article Title: Neonatal Handling Positively Modulates Anxiety, Sensorimotor Gating, Working Memory, and Cortico-Hippocampal Neuroplastic Adaptations in Two Genetically Selected Rat Strains Differing in Emotional and Cognitive Traits

doi: 10.3390/brainsci15080776

Figure Lengend Snippet: Western blot analysis of the BDNF ( A , B ), trkB ( C , D ), and the PSA-NCAM ( E , F ), in the dorsal hippocampus of the RHA and the RLA rats, either untreated controls (CTRL) or treated with neonatal handling (NH). The values represent the densitometric analysis of the BDNF/GAPDH ( B ), trkB/GAPDH ( D ), and the PSA-NCAM/GAPDH ( F ) band grey optical density (O.D.) ratios. The bars denote the mean ± S.E.M. of 9–10 rats, in each experimental group.

Article Snippet: Molecular weight (mw) standards (Precision Plus Protein Western C Standards, Cat# 161–0376, Bio-Rad, Hercules, CA, USA) and recombinant human BDNF protein (rhBDNF) (Cat# B-257, Alomone Labs, Jerusalem, Israel) were run in parallel.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Axonal PLC-γ activity is required for BDNF long-distance signaling

doi: 10.1101/2023.11.10.566602

Figure Lengend Snippet: (A) DIV 7 compartmentalized mouse cortical neurons were retrograde labeled with Ctb555 (red) overnight. At DIV 8, the cell body compartment was treated with TrkB-Fc, and the AC was treated with DyLight 488-conjugated streptavidin alone (Control) or with biotinylated BDNF coupled to DyLight 488-conjugated streptavidin (f-BDNF, 150 ng/mL) for 6 hours in the absence (BDNF, green) or presence of 5 μM U-73122 (BDNF + U-173122) or 20 μM BAPTA, AM (BDNF + BAPTA). Then, the cells were fixed, mounted in Mowiol containing Hoechst (blue) and prepared for visualization. The circles in the BDNF panel indicate the smallest BDNF endosome considered for the analysis shown in B. Scale bar, 5 μm (B) Quantification of BDNF-positive vesicles in cell bodies of neurons containing Ctb555. Only vesicles larger than 20*10 -2 μm 2 as shown in the BDNF treatment group by the circles in panel A were considered for the analysis. Forty-five neurons from three independent compartmentalized cultures were considered. Statistical analysis was performed by one-way ANOVA followed by the Bonferroni correction for multiple comparisons. **** P<0.0001. (C) Left panels, representative images of DyLight 488-conjugated streptavidin (green) associated fluorescence. White lines are selecting the region of the microgroove label with Ctb555 (red) shown in the right panels. White arrows indicate DyLight 488-conjugated streptavidin positive vesicles (control) or f-BDNF positive vesicles in the lower panels. Right panels, Representative images of axons (labeled with Ctb555, red) in microgrooves of neurons treated as described in A. Scale bar, 5 μm. (D) Quantification of BDNF-positive vesicles in microgrooves of axons containing Ctb555. Forty-five microgrooves from three independent compartmentalized cultures were considered for the analysis. Statistical analysis was performed by one-way ANOVA followed by the Bonferroni correction for multiple comparisons. **** P<0.0001.

Article Snippet: To prepare fluorescent BDNF (f-BDNF), commercially available biotinylated BDNF (Alomone Labs, cat# B-250-B) was coupled to DyLight 488-conjugated streptavidin (Invitrogen, cat# 21832) by incubation at 37°C for 20 minutes; the reaction consisted of 6 µL of a 6 µM solution of DyLight 488-conjugated streptavidin and 2 µL of a 6 µM solution of biotinylated BDNF diluted in 72 µL of neurobasal medium supplemented with 0.1% BSA (molar range 1:1).

Techniques: Labeling, Control, Fluorescence

Journal: Frontiers in Molecular Neuroscience

Article Title: BDNF-TrkB Signaling Coupled to nPKCε and cPKCβI Modulate the Phosphorylation of the Exocytotic Protein Munc18-1 During Synaptic Activity at the Neuromuscular Junction

doi: 10.3389/fnmol.2018.00207

Figure Lengend Snippet: BDNF/TrkB signaling pathway modulates Munc18-1 phosphorylation under muscle contraction conditions. Representative Western blot bands and quantification show that both exogenous BDNF and 47/TrkB significantly decrease pMunc18-1 levels after stimulation resulting in contraction. Moreover, exogenous BDNF decreases Munc18-1 levels under these conditions. Data are mean percentage ± SEM, * p < 0.05 ( n = 5).

Article Snippet: For BDNF exogenous incubations we used h-BDNF (Alomone Labs; Cat# B-250) in a working solution of 10 mM.

Techniques: Western Blot

Journal: eLife

Article Title: Rbfox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels

doi: 10.7554/eLife.49673

Figure Lengend Snippet: ( A ) TrkB activation by BDNF is decreased by increased levels of TrkB.T1. HEK293 cells stably expressing TrkB.FL were transfected with increasing amounts of a TrkB.T1 expressing plasmid (0.3 µg, 0.5 µg and 1 µg) before BDNF treatment (5 ng/ml for 5 min). Immunoblots were probed with antibodies against TrkB extracellular domain to detect both TrkB.FL and TrkB.T1, phospho-TrkB.FL at tyrosine 515 (p-TrkB.FL), phospho-ERK (p-ERK), ERK and Gapdh as controls. ( B ) TrkB.T1- /- neurons have increased BDNF signaling. Immunoblot analysis of E18.5 WT and TrkB.T1- /- primary hippocampal neurons cultured for 6 days in vitro before BDNF treatment (1 ng/ml for 5 min; black bar). Antibodies are as in ( A ) except for the β-III-tubulin antibody used as neuronal marker. ( C, D ) Immunoblot quantification analysis from ( B ) of p-TrkB.FL ( C ) and p-ERK ( D ) expressed respectively, as relative percentage of p-TrkB.FL over total TrkB.FL and phospho-ERK over total ERK. n = 3 ± SEM. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001 (Student’s t-test).

Article Snippet: Primary neurons were starved in Neurobasal media without B27 supplement (Gibco) for 5 hr before BDNF treatment for 5 min (1 ng/ml; Alomone Labs, Cat# B-250).

Techniques: Activation Assay, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Western Blot, Cell Culture, In Vitro, Marker

Journal: eLife

Article Title: Rbfox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels

doi: 10.7554/eLife.49673

Figure Lengend Snippet: T1 receptor allele. ( A ) Averaged time-course of the field excitatory postsynaptic potential (fEPSP) slope in hippocampal slices from control (Ctrl = Nes Cre, n = 10), N-cre;Fox1( Nes-Cre;R26-Rbfox1 +/flox ; n = 10) and N-cre;Fox1;T1+/- ( Nes-Cre;R26-Rbfox1 +/flox ;TrkB.T1 +/- ; n = 13) mice after extracellular application of BDNF (20 ng/ml). Values are expressed as fEPSP percentage of the baseline values (average of 5 min before BDNF application, mean ± SEM). BDNF application (20 min) is indicated by the black horizontal bar. ( B ) Histogram showing mean ± SEM of the fEPSP slope average at 50 min after BDNF infusion; *** = p ≤ 0.001; ** = p ≤ 0.01; * = p ≤ 0.05 (Student’s t-test). ( C ) Representative recording traces of slices before (baseline = black line) and 50 min after BDNF application (red line). ( D ) Western blot analysis of hippocampus expression of TrkB, Rbfox1 and Gapdh of animals analyzed in ( A–C ).

Article Snippet: Primary neurons were starved in Neurobasal media without B27 supplement (Gibco) for 5 hr before BDNF treatment for 5 min (1 ng/ml; Alomone Labs, Cat# B-250).

Techniques: Western Blot, Expressing

Journal: eLife

Article Title: Rbfox1 up-regulation impairs BDNF-dependent hippocampal LTP by dysregulating TrkB isoform expression levels

doi: 10.7554/eLife.49673

Figure Lengend Snippet: T1 heterozygous animals. ( A ) Averaged time-course of changes in field excitatory postsynaptic potential (fEPSP) slope after extracellular application of BDNF (20 ng/ml) in mouse hippocampal slices from control (Ctrl; Nes -cre, n = 10) and TrkB.T1 heterozygous animals (T1+/-; n = 8). Values are expressed as fEPSP percentage of baseline values (average of 5 min before BDNF application, mean ± SEM). BDNF application (20 min) is indicated by the black horizontal bar. ( B ) Histogram showing the average mean ± SEM of the fEPSP slope at 50 min after BDNF infusion (n.s.: Student’s t-test). ( C ) Western blot analysis of hippocampal expression of TrkB, Rbfox1 and Gapdh of animals as in ( A ).

Article Snippet: Primary neurons were starved in Neurobasal media without B27 supplement (Gibco) for 5 hr before BDNF treatment for 5 min (1 ng/ml; Alomone Labs, Cat# B-250).

Techniques: Western Blot, Expressing