Journal: International Journal of Molecular Sciences

Article Title: Fingolimod Modulates Dendritic Architecture in a BDNF-Dependent Manner

doi: 10.3390/ijms21093079

Figure Lengend Snippet: Fingolimod-phosphate (FTY720-P) regulates neuronal architecture in a BDNF-dependent manner. ( A ) Representative Neurolucida tracings, used to perform the Sholl analysis of dendritic complexity for feGFP expressing neurons treated for 24h with: DMSO (black), 2nM FTY720-P (gray), DMSO + TrkBFc (dark blue) and 2nM FTY720-P + TrkBFc (light blue). Scale bar: 100 μm. ( B ) Sholl analysis displayed as number of dendritic intersections against distance from the cell body for DMSO (black), 2nM FTY720-P (gray), DMSO + TrkBFc (dark blue) and 2nM FTY720-P + TrkBFc (light blue) and ( C ) total dendritic complexity of all treatment groups. F value in ( B ) refers to comparison between all the 4 treatment groups. ( D ) Dendritic spine densities for DMSO (black), 2nM FTY720-P (gray), DMSO + TrkBFc (dark blue) and 2nM FTY720-P + TrkBFc (light blue) calculated using segments of secondary dendritic branches as shown in ( E ) feGFP panel. The Syn panel displays the corresponding staining of the pre-synaptic marker SynapsinI/II and the merge panel shows the images with overlapping SynapsinI/II puncta (red, pre-synapse) to its matching feGFP dendrite segment (green, post-synapse). The arrows point to coinciding puncta, indicative of mature synapse between the post and pre-synaptic compartments. Scale bar: 5μm. ( F ) The graph compares the fraction of SynapsinI/II positive feGFP labelled spines for DMSO (black), 2nM FTY720-P (gray), DMSO + TrkBFc (dark blue) and 2nM FTY720-P + TrkBFc (light blue). ( G ) Representative fields of view (FOV) of the DMSO, 2nM FTY720-P, DMSO + TrkBFc and 2nM FTY720-P + TrkBFc treated hippocampal cultures stained for c-fo s . Scale bar: 100μm. ( H ) Quantification of the proportion of c-Fos expressing neurons represented as normalized fraction for DMSO (black), 2nM FTY720-P (gray), DMSO + TrkBFc (dark blue) and 2nM FTY720-P + TrkBFc (light blue) treated cultures. All graphs represent data as mean + SEM. Numbers in the bars show either total number of neurons or of FOV analyzed, obtained from ≥3 sets of independent experiments. Two-way ANOVA followed by Bonferroni post-hoc test was used in ( B ). For ( C , D , F ) and ( H ) one-way ANOVA with Bonferroni post-hoc was used. Denotations for significance are * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Recombinant Human BDNF protein (R&D systems, Minneapolis, MN, USA) was dissolved in sterile PBS with 0.1% BSA at a concentration of 50 ng/μL.

Techniques: Expressing, Comparison, Staining, Marker

Journal: International Journal of Molecular Sciences

Article Title: Fingolimod Modulates Dendritic Architecture in a BDNF-Dependent Manner

doi: 10.3390/ijms21093079

Figure Lengend Snippet: Treatment with the non-phosphorylated Fingolimod (FTY720) modulates neuronal architecture. ( A ) | Representative Neurolucida tracings from feGFP positive hippocampal neurons used for the Sholl analysis from cultures treated either with DMSO or 10nM FTY20 for 24 h. Scale bar: 100μm. ( B ) | Dendritic complexity shown by the number of dendritic intersections plotted against the distance from the soma for DMSO (black) and FTY720 (blue) treated neurons. The F value shows the statistical comparison between the two groups. The inset graph represents total dendritic complexity upon treatment with DMSO (black) and FTY720 (blue). ( C ) | Total dendritic length for DMSO (black) and FTY720 (blue) treated neurons. ( D ) | Representative stretches from dendrites of eGFP transfected hippocampal neurons showing dendritic spine protrusions, treated either with DMSO or FTY720 for 24h. Scale bar: 5μm. ( E ) | The graph shows dendritic spine density for DMSO (black) and FTY720 (blue) treated neurons . ( F ) | Representative images of fields of view (FOV) from primary hippocampal cultures stained with anti phospho-ERK1/2 antibody, 30 min post-application of one of the following: DMSO, 10nM FTY720, DMSO_100, 100nM FTY720, DMSO_100 + TrkB-Fc, 100nM FTY720 + TrkB-Fc or 40ng recombinant BDNF protein as a positive control. Scale bar: 100μm. ( G ) | The graph displays the fraction of pERK1/2 expressing neurons relative to the total number of MAP2 + neurons. The data is normalized to the respective controls and compared between the different treatment groups: DMSO (black), 10nM FTY720 (light blue solid), DMSO100 (gray), 100nM FTY720 (dark blue solid), 40ng recombinant BDNF (magenta), DMSO100 + TrkB-Fc (gray open), 100nM FTY720 + TrkB-Fc (dark blue open). All data is plotted as mean + SEM. Numbers in the bars show total number of neurons or FOV analyzed, obtained from ≥3 sets of independent experiments. Two-way ANOVA followed by Bonferroni post-hoc test was used in ( B ). For ( B ) total intersections, ( C ) and ( E ) unpaired Student’s t-test and for ( G ) one-way ANOVA with Bonferroni post-hoc was used. Denotations for significance are * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: Recombinant Human BDNF protein (R&D systems, Minneapolis, MN, USA) was dissolved in sterile PBS with 0.1% BSA at a concentration of 50 ng/μL.

Techniques: Comparison, Transfection, Staining, Recombinant, Positive Control, Expressing