Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dendritic Targeting of BDNF and TrkB mRNAs in Hippocampal Neurons

doi: 10.1523/JNEUROSCI.17-24-09492.1997

Figure Lengend Snippet: Maximal distance of dendritic staining

Article Snippet: Fixed and permeabilized cells were preincubated for 30 min at rt in 3% BSA in PBS, incubated 3 hr at rt with an antibody recognizing the TrkB full length isoform (Santa Cruz Ab794, made in rabbit, diluted 1:100 in 3% BSA in PBS), or anti-BDNF [(Promega, Madison, WI) made in chicken, diluted 1:100 in 3% BSA in PBS].

Techniques: Control

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dendritic Targeting of BDNF and TrkB mRNAs in Hippocampal Neurons

doi: 10.1523/JNEUROSCI.17-24-09492.1997

Figure Lengend Snippet: Subcellular distribution of BDNF and TrkB mRNAs in cultured hippocampal neurons. Staining by nonradioactive in situ hybridization with digoxigenin-labeled riboprobes and oligonucleotides. Fields are viewed with Nomarski optics, except immunofluorescence in B and F.A, The BDNF riboprobe labels the cell soma and a process identified as a dendrite in B by a double-labeling with an anti-MAP2 monoclonal antibody. C, No staining is observed with a BDNF sense riboprobe. D, The BDNF antisense oligonucleotide probe shows somatodendritic labeling.E, The TrkB riboprobe stains the cell soma and a dendritic process identified as a dendrite, in F, by the anti-MAP2 antibody. G, No labeling is detected with the TrkB sense riboprobe. H, The TrkB antisense oligonucleotide probe labels the cell soma and a dendrite.Arrowheads indicate labeling at dendritic branchings or varicosities. Scale bar (shown in H): 20 μm forA–H.

Article Snippet: Fixed and permeabilized cells were preincubated for 30 min at rt in 3% BSA in PBS, incubated 3 hr at rt with an antibody recognizing the TrkB full length isoform (Santa Cruz Ab794, made in rabbit, diluted 1:100 in 3% BSA in PBS), or anti-BDNF [(Promega, Madison, WI) made in chicken, diluted 1:100 in 3% BSA in PBS].

Techniques: Cell Culture, Staining, In Situ Hybridization, Labeling, Immunofluorescence

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dendritic Targeting of BDNF and TrkB mRNAs in Hippocampal Neurons

doi: 10.1523/JNEUROSCI.17-24-09492.1997

Figure Lengend Snippet: High potassium increases the dendritic localization of TrkB, but not of β-actin, mRNAs in cultured hippocampal neurons. Nonradioactive in situhybridization on cultured hippocampal neurons viewed with Nomarski optics. A, The TrkB riboprobe stains both cell bodies and dendrites. B, On depolarization with 10 mm KCl for 3 hr, the dendritic labeling is detectable at a much greater distance from the cell body. C, The β-actin probe labels the cell bodies under control conditions and (D) after 3 hr depolarization in 10 mm KCl. Scale bar (shown in D): 20 μm forA–D.

Article Snippet: Fixed and permeabilized cells were preincubated for 30 min at rt in 3% BSA in PBS, incubated 3 hr at rt with an antibody recognizing the TrkB full length isoform (Santa Cruz Ab794, made in rabbit, diluted 1:100 in 3% BSA in PBS), or anti-BDNF [(Promega, Madison, WI) made in chicken, diluted 1:100 in 3% BSA in PBS].

Techniques: Cell Culture, Labeling, Control

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dendritic Targeting of BDNF and TrkB mRNAs in Hippocampal Neurons

doi: 10.1523/JNEUROSCI.17-24-09492.1997

Figure Lengend Snippet: Quantitative analysis of dendritic localization of BDNF and TrkB mRNAs after depolarization of hippocampal neurons in culture. Bars in A and Bindicate the fold increase, with respect to controls, of the mean maximal distance at which the in situ labeling in dendrites was detectable (MDDL). A, BDNF mRNA. Depolarization with either 10 (K) or 20 mm KCl (KK) increases the mean maximal distance of dendritic labeling (MDDL) at 3 hr (3 h K and 3 h KK) but not at 30 min (30′ K and 30′ KK). The average MDDL measured with 10 mm KCl (3 h K) is larger than with 20 mm KCl (3 h KK). B, TrkB mRNA. Both KCl concentrations induce a significant increase in MDDL of TrkB mRNA after 30 min (30′ K, 30′ KK) and a stronger increase at 3 hr (3 h K, 3 h KK). ○, Significantly different with respect to the 3 h K-stimulated; ⋄, significantly different with respect to the 3 h KK stimulated; and *, significantly different with respect to the control (no stimulation). Error bars represent SE. The corresponding numerical values, number of dendrites measured, and significance values are shown in Table ​Table11.C, D, E, F, Correlation plots of the MDDL versus dendritic length. Each point refers to one MDDL determination, together with the length of the corresponding dendrite, under the experimental conditions indicated. The scatter plots were fitted by linear regression lines through the origin. C, Slope = 0.30, correlation coefficient r = 0.57; D, slope = 0.68; r = 0.80; E, slope = 0.26; r = 0.31; F, slope = 0.67; r = 0.72.

Article Snippet: Fixed and permeabilized cells were preincubated for 30 min at rt in 3% BSA in PBS, incubated 3 hr at rt with an antibody recognizing the TrkB full length isoform (Santa Cruz Ab794, made in rabbit, diluted 1:100 in 3% BSA in PBS), or anti-BDNF [(Promega, Madison, WI) made in chicken, diluted 1:100 in 3% BSA in PBS].

Techniques: In Situ, Labeling, Control

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dendritic Targeting of BDNF and TrkB mRNAs in Hippocampal Neurons

doi: 10.1523/JNEUROSCI.17-24-09492.1997

Figure Lengend Snippet: The effects of KCl depolarization do not depend on new mRNA synthesis. Nonradioactive in situ hybridization on cultured hippocampal neurons viewed with Nomarski optics. In control conditions, cells were kept in the presence of actinomycin-d for 3.5 hr and then stained with (A) BDNF or (C) TrkB riboprobes. After a pretreatment of 30 min with actinomycin-d, cultures were depolarized for 3 hr in 10 mm KCl in the continuous presence of actinomycin-d and stained with (B) BDNF or (D) TrkB riboprobes. Scale bar (shown inD): 20 μm for A–D. E, Quantitative analysis of the MDDL for BDNF and TrkB mRNAs with actinomycin-d. Bars indicate the fold increase, with respect to controls, of the mean maximal distance at which the in situ labeling was detectable (MDDL). Results are similar for both BDNF and TrkB mRNAs: treatment of control cultures with actinomycin-d for 3.5 hr (C act.D) reduces the MDDL with respect to untreated controls (C), whereas a strong increase of MDDL induced by 3 hr in 10 mm KCl also occurs in the presence of actinomycin-d (3h K act.D). Error bars represent SE. *, Significantly different from controls. The corresponding numerical values, the number of dendrites measured, and the significance values are shown in Table ​Table11.

Article Snippet: Fixed and permeabilized cells were preincubated for 30 min at rt in 3% BSA in PBS, incubated 3 hr at rt with an antibody recognizing the TrkB full length isoform (Santa Cruz Ab794, made in rabbit, diluted 1:100 in 3% BSA in PBS), or anti-BDNF [(Promega, Madison, WI) made in chicken, diluted 1:100 in 3% BSA in PBS].

Techniques: In Situ Hybridization, Cell Culture, Control, Staining, In Situ, Labeling

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dendritic Targeting of BDNF and TrkB mRNAs in Hippocampal Neurons

doi: 10.1523/JNEUROSCI.17-24-09492.1997

Figure Lengend Snippet: Effects of Ca2+-free medium on the KCl-induced increase in dendritic localization of BDNF and TrkB mRNAs. Quantitative analysis of the MDDL for BDNF and TrkB mRNAs in nominally Ca2+-free medium supplemented with 10 μm EGTA or BAPTA-AM. Bars indicate the fold increase, with respect to controls, of the mean maximal distance at which the in situ labeling was detectable (MDDL) for 80–150 dendrites. A, In nominally Ca2+-free medium the MDDL for BDNF mRNA, induced by depolarization with 10 mm KCl for 3 hr, does not change with respect to controls. The effects are identical in the presence of either EGTA or BAPTA-AM. B, MDDL for TrkB mRNA. At the level of p ≤ 0.05 (Student’st test), no significant variation in MDDL with respect to controls can be observed when cultures are depolarized under Ca2+-free conditions. Identical effects are seen with either EGTA or BAPTA-AM. C, Unstimulated controls in normal medium. CEGTA, Unstimulated controls maintained 3 hr in Ca2+-free medium containing EGTA;CBAPTA-AM, unstimulated controls maintained 3 hr in Ca2+-free medium containing BAPTA-AM; 3h K EGTA, neurons stimulated with 10 mm KCl in Ca2+-free medium containing EGTA; 3h K BAPTA-AM, neurons stimulated with 10 mm KCl in Ca2+-free medium containing BAPTA-AM. Error bars represent SE.

Article Snippet: Fixed and permeabilized cells were preincubated for 30 min at rt in 3% BSA in PBS, incubated 3 hr at rt with an antibody recognizing the TrkB full length isoform (Santa Cruz Ab794, made in rabbit, diluted 1:100 in 3% BSA in PBS), or anti-BDNF [(Promega, Madison, WI) made in chicken, diluted 1:100 in 3% BSA in PBS].

Techniques: In Situ, Labeling

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dendritic Targeting of BDNF and TrkB mRNAs in Hippocampal Neurons

doi: 10.1523/JNEUROSCI.17-24-09492.1997

Figure Lengend Snippet: Effects of TTX, kinurenic acid, and nifedipine on the KCl-induced increase in dendritic localization of BDNF and TrkB mRNAs. Quantitative analysis of the MDDL for BDNF and TrkB mRNAs in the presence of TTX, kinurenic acid, and nifedipine. Barsindicate the fold increase, with respect to controls, of the mean maximal distance at which the in situ labeling was detectable (MDDL). A, Continuous presence of tetrodotoxin (TTX) inhibits the increase in MDDL for BDNF mRNA, induced by depolarization with 10 mmKCl for 3 hr (3h K TTX). The glutamate receptor antagonist kinurenic acid partially counteracts the KCl-induced increase by either 10 (3h K kinu) or 20 mmKCl (3h KK kinu). B, The L-type Ca2+ channel blocker nifedipine has distinct effects at different KCl concentrations, with a partial inhibition at 10 mm KCl (3h K nife) and an almost complete inhibition at 20 mm KCl (3h KK nife).C, TTX strongly inhibits the MDDL increase for TrkB mRNA in 10 mm KCl (3h K TTX). The glutamate receptor antagonist kinurenic acid reduced the 10 mm KCl depolarization effects (3h K kinu) more effectively than at 20 mm KCl (3h KK kinu). D, In contrast, nifedipine almost completely abolishes the effects induced by 20 mm KCl (3h KK nife) and only partially inhibits those induced by 10 mm KCl depolarization (3h K nife). Error bars represent SE. ○, Significantly different with respect to the 3 h K-stimulated; ⋄, significantly different with respect to the 3 h KK stimulated; *, significantly different with respect to the control (no stimulation). Also see Table ​Table11.

Article Snippet: Fixed and permeabilized cells were preincubated for 30 min at rt in 3% BSA in PBS, incubated 3 hr at rt with an antibody recognizing the TrkB full length isoform (Santa Cruz Ab794, made in rabbit, diluted 1:100 in 3% BSA in PBS), or anti-BDNF [(Promega, Madison, WI) made in chicken, diluted 1:100 in 3% BSA in PBS].

Techniques: In Situ, Labeling, Inhibition, Control

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dendritic Targeting of BDNF and TrkB mRNAs in Hippocampal Neurons

doi: 10.1523/JNEUROSCI.17-24-09492.1997

Figure Lengend Snippet: A short incubation in high potassium increases BDNF and TrkB protein levels in the dendritic compartment. Immunohistochemistry on cultured hippocampal neurons. Each picture represents the integration in a single projection of a series of five optical sections obtained with a confocal microscope, as described in Materials and Methods. A, Anti-BDNF immunostaining of control cultures. B, Anti-BDNF immunostaining after 10 mm KCl depolarization for 10 min. C, Anti-BDNF staining after a preincubation with nocodazole for 6 hr followed by 10 min of 10 mm KCl depolarization in the continuous presence of nocodazole. D, Staining with anti-TrkB antibody in control conditions. E, Staining with anti-TrkB antibodies after 10 min depolarization in 10 mm KCl. F, Anti-TrkB staining after a preincubation with nocodazole for 6 hr followed by 10 min of 10 mm KCl depolarization in the continuous presence of nocodazole. Scale bar (shown in F): 20 μm forA–F.

Article Snippet: Fixed and permeabilized cells were preincubated for 30 min at rt in 3% BSA in PBS, incubated 3 hr at rt with an antibody recognizing the TrkB full length isoform (Santa Cruz Ab794, made in rabbit, diluted 1:100 in 3% BSA in PBS), or anti-BDNF [(Promega, Madison, WI) made in chicken, diluted 1:100 in 3% BSA in PBS].

Techniques: Incubation, Immunohistochemistry, Cell Culture, Microscopy, Immunostaining, Control, Staining

Journal: The Journal of Neuroscience

Article Title: Activity-Dependent Dendritic Targeting of BDNF and TrkB mRNAs in Hippocampal Neurons

doi: 10.1523/JNEUROSCI.17-24-09492.1997

Figure Lengend Snippet: Quantitative analysis of BDNF and TrkB protein levels in proximal and distal regions of the dendrites. Immunofluorescence for BDNF or TrkB was acquired by five confocal sections and integrated in a single projection, in control conditions and after 10 min depolarization with 10 mm KCl, in the presence or absence of cycloheximide (A, C) and of nocodazole (B, D). Fluorescent density of BDNF (A, B) and TrkB (C, D) was determined in proximal and distal regions of the projections of labeled dendrites as described in Materials and Methods. Bars represent the mean fold increase of the fluorescence density of 45 dendrites, with respect to the controls (=1.0). Error bars represent SE.A, Incubation of cells in 10 mm KCl for 10 min leads to a strong increase of BDNF fluorescence density in both proximal and distal regions (10′K). Incubation of control cells with the protein synthesis inhibitor cycloheximide does not alter the basal levels of fluorescence density for BDNF in proximal and distal regions (C + Cyclo). Cycloheximide completely inhibits the increase in fluorescence density induced by the 10 mm KCl stimulus (10′K + Cyclo).B, After pretreatment of cells with nocodazole for 6 hr the levels of BDNF fluorescence density in the proximal dendrites depolarized for 10 min with 10 mm KCl in the continuous presence of nocodazole (10′K + Noco) were comparable to control (C + Noco), whereas a significant fluorescence density increase could be detected in the distal dendrites (10′K + Noco). C, Incubation of cells in 10 mm KCl for 10 min led to a strong increase of fluorescence density for TrkB in the distal region (10′K) but not in the proximal region, and this effect was abolished by cycloheximide (10′K + Cyclo). Incubation of control cells with cycloheximide reduces the basal levels of TrkB fluorescence density in the proximal but not in the distal regions (C + Cyclo). D, After pretreatment of cells with nocodazole for 6 hr, followed by a depolarization for 10 min with 10 mm KCl in continuous presence of nocodazole, the TrkB fluorescence density in the proximal dendrites (10′K + Noco) was comparable to the controls (C + Noco), whereas a fluorescence density increase could be detected in the distal dendrites (10′K + Noco). Significance with respect to controls: op ≤ 0.05; *p ≤ 0.01; **p ≤ 0.001.E, Western blot for the soluble and microtubule cellular pools of tubulin in hippocampal neurons in culture. M1, Soluble tubulin fraction; M2, polymerized tubulin fraction. After 6 hr incubation with nocodazole, the soluble, unpolymerized tubulin fraction is doubled.

Article Snippet: Fixed and permeabilized cells were preincubated for 30 min at rt in 3% BSA in PBS, incubated 3 hr at rt with an antibody recognizing the TrkB full length isoform (Santa Cruz Ab794, made in rabbit, diluted 1:100 in 3% BSA in PBS), or anti-BDNF [(Promega, Madison, WI) made in chicken, diluted 1:100 in 3% BSA in PBS].

Techniques: Immunofluorescence, Control, Labeling, Fluorescence, Incubation, Western Blot