The BDNF effects on dendritic spines of mature hippocampal neurons depend on neuronal activity

Abstract

The fine tuning of neural networks during development and learning relies upon both functional and structural plastic processes. Changes in the number as well as in the size and shape of dendritic spines are associated to long-term activity-dependent synaptic plasticity. However, the molecular mechanisms translating functional into structural changes are still largely unknown. In this context, neurotrophins, like Brain-Derived Neurotrophic Factor (BDNF), are among promising candidates. Specifically BDNF-TrkB receptor signaling is crucial for activity-dependent strengthening of synapses in different brain regions. BDNF application has been shown to positively modulate dendritic and spine architecture in cortical and hippocampal neurons as well as structural plasticity in vitro. However, a global BDNF deprivation throughout the central nervous system (CNS) resulted in very mild structural alterations of dendritic spines, questioning the relevance of the endogenous BDNF signaling in modulating the development and the mature structure of neurons in vivo. Here we show that a loss-of-function approach, blocking BDNF results in a significant reduction in dendritic spine density, associated with an increase in spine length and a decrease in head width. These changes are associated with a decrease in F-actin levels within spine heads. On the other hand, a gain-of-function approach, applying exogenous BDNF, could not reproduce the increase in spine density or the changes in spine morphology previously described. Taken together, we show here that the effects exerted by BDNF on the dendritic architecture of hippocampal neurons are dependent on the neuron's maturation stage. Indeed, in mature hippocampal neurons in vitro as shown in vivo BDNF is specifically required for the activity-dependent maintenance of the mature spine phenotype.

Keywords: dendrites; hippocampus; neurotrophins; spines; structural plasticity.

Figure 1

(A) Experimental timeline. DIV23 hippocampal neurons were treated for 24 h before fixation. (B) Representative image of a typical DIV23 fEGFP expressing hippocampal neuron used in the experiments; the inserts on the right show a close up of the cell body labeled with fEGFP (above) or with an immunohistochemistry against CaMKII (middle) merged to show colocalization (below): scale bars are 50 μm. (B′) High-magnification image of typical dendritic stretches from control (left), BDNF (middle) and BDNF antibodies (right) treated neurons. Scale bar, 5 μm. (C) Graphs comparing dendritic spine density between BSA (control), BDNF and BDNF antibodies (BDNF-Abs) treated DIV22 hippocampal neurons. (D) Histogram comparing the spine head width between hippocampal neurons upon BSA (control), BDNF or BDNF antibodies (BDNF-Abs) treatment. (E) Histogram of the dendritic spine length in control, BDNF or BDNF antibodies (BDNF-Abs) treated neurons. (F) Graph showing the binning of spines according to their spine head width and comparing the proportion of spines within each category in response to a BSA (control), BDNF or BDNF antibodies (BDNF-Abs) treatment. (G) Graph showing the binning of spines according to their spine length and comparing the proportion of spines within each category in response to a BSA (control), BDNF or BDNF antibodies (BDNF-Abs) treatment. (H) Graph plotting dendritic complexity in relation to the distance to the cell body of hippocampal neurons under control conditions or treated with BDNF or BDNF antibodies (BDNF-Abs). (H′) Histogram of the total number of intersections for BSA (control), BDNF and BDNF antibodies (BDNF-Abs) treated neurons. The number in the columns represents the number of cells analyzed. Significance is indicated as follows: ^*p < 0.05; ^**p < 0.01; ^***p < 0.001. Error bars indicate s.e.m. ANOVA, post-hoc Tukey's Multiple Comparison Test.

Figure 2

(A) Experimental timeline. DIV16 hippocampal neurons were treated for 24 h before fixation (B) Graphs comparing dendritic spine density between control, BDNF and BDNF-antibodies (BDNF-Abs) treated DIV16 hippocampal neurons. (C) Histogram comparing the spine head width between hippocampal neurons upon BSA (control), BDNF or BDNF antibodies treatment. (D) Histogram of the dendritic spine length in BSA (control), BDNF or BDNF antibodies (BDNF-Abs) treated neurons. (E) Graph showing the binning of spines according to their spine head width and comparing the proportion of spines within each category in response to a BSA (control), BDNF or BDNF antibodies (BDNF-Abs) treatment. (F) Graph showing the binning of spines according to their spine length and comparing the proportion of spines within each category in response to a BSA (control), BDNF or BDNF antibodies (BDNF-Abs) treatment. (G) Graph plotting dendritic complexity in relation to the distance to the cell body of hippocampal neurons under control conditions or treated with BDNF or BDNF antibodies (BDNF-Abs). (G′) Histogram of the total number of intersections for BSA (control), BDNF and BDNF antibodies (BDNF-Abs) treated neurons. The number in the columns represents the number of cells analyzed. Significance is indicated as follows: ^*p < 0.05; ^**p < 0.01; ^***p < 0.001. Error bars indicate s.e.m. ANOVA, post-hoc Tukey's Multiple Comparison Test.

Figure 3

(A) Maximal intensity projection of a representative dendrite of hippocampal neurons expressing fEGFP (above) and RFP-Lifeact (middle) merged to show colocalization (below). Scale bar, 2 μm. (B) High magnification images showing a single dendritic spine expressing fEGFP (above) and RFP-Lifeact (middle) and merged to show their colocalization in the spine head (below) Scale bar, 2 μm. (C) Histogram plotting the normalized mean fluorescent intensity for RFP-Lifeact within the spine head of DIV23 BSA (control), BDNF and BDNF antibodies (BDNF-Abs) treated neurons. (D) Histogram showing the normalized mean intensity for RFP-Lifeact within the spine head of DIV16 hippocampal neurons. The number in the columns represents the number of cells analyzed. Significance is indicated as follows: ^**p < 0.01; ^***p < 0.001. Error bars indicate s.e.m. ANOVA, post-hoc Tukey's Multiple Comparison Test.

Figure 4

(A) Experimental timeline. DIV22 primary hippocampal neurons were treated 15 min with BDNF before fixation. (B) Images of primary hippocampal cultures (22 DIV) treated with either BSA (control) (left) or BDNF (right) and stained with an anti-phospho-TrkB receptor antibodies. Scale bar, 40 μm. (C) Histogram showing the normalized fluorescence intensity for the phospho-TrkB receptor under control conditions or treated with BDNF. (D) Graph showing the number of phospho-TrkB positive hippocampal neurons in control and BDNF treated hippocampal primary cultures. (E) Experimental timeline. DIV22 primary hippocampal neurons were treated 5 min with BDNF during calcium imaging. (F) Pseudo line-scan showing the global calcium transients occurring in hippocampal primary cultures before, during and after BDNF applications. (G) Graph showing the frequency of calcium transients in hippocampal primary neurons before, during and after BDNF application. (H) Experimental timeline. DIV22 primary hippocampal neurons were treated for 3 h with BDNF before fixation. (I) Images of primary hippocampal cultures (22 DIV) treated with either BSA (control) (above) or BDNF (below) and stained with an anti-c-fos antibody. Scale bar, 20 μm. (J) Graph comparing the normalized mean intensity for the immunohistochemistry against c-fos in control or BDNF treated hippocampal primary neurons. (K) Histogram of the number of c-fos positive neurons in control and BDNF treated hippocampal cultures. The number in the columns represents the number of analyzed experiments. Significance is indicated as follows: ^*p < 0.05; ^**p < 0.01; ^***p < 0.001. Error bars indicate s.e.m. Unpaired two-tailed Student Test.

Figure 5

(A) Experimental timeline. BSA, BDNF, BDNF antibodies (BDNF-Abs) or TrkB receptor bodies (TrkB-Fc) were applied on DIV3 hippocampal neurons for 72 h. (B) Micrographs showing MAP2 positive BSA (control) (above), BDNF (middle) or BDNF antibodies (BDNF-Abs; below) treated primary hippocampal neurons. Scale bar, 25 μm. (C) Histogram comparing neurite length of DIV6 hippocampal neurons after application of BSA (control), BDNF, BDNF antibodies (BDNF-Abs), TrkB receptor bodies (TrkB-Fc) or combined BDNF and TrkB receptor bodies (TrkB-Fc). (D) Histogram comparing the number of primary neurites of DIV6 hippocampal neurons after application of BSA (control), BDNF, BDNF antibodies (BDNF-Abs), TrkB receptor bodies (TrkB-Fc) or combined BDNF and TrkB receptor bodies (TrkB-Fc). (E) Histogram comparing the number of branching points for the dendrites of DIV6 hippocampal neurons after application of BSA (control), BDNF, BDNF antibodies (BDNF-Abs), TrkB receptor bodies (TrkB-Fc) or combined BDNF and TrkB receptor bodies (TrkB-Fc). The number in the columns represents the number of cells analyzed. Significance is indicated as follows: ^*p < 0.05; ^**p < 0.01; ^***p < 0.001. Error bars indicate s.e.m. ANOVA, post-hoc Tukey's Multiple Comparison Test.

Figure 6

(A) Experimental timeline. Primary neurons were cultivated for 2 weeks in high (3.5 mM) Mg²⁺ medium and treated with BSA (control), BDNF or TrkB-Fc in low (1.5 mM) Mg²⁺. (B) Graph comparing the frequency of calcium transients in hippocampal primary neurons cultivated in high or low Mg²⁺ containing medium. (C) Graph comparing the amplitude of calcium transients in hippocampal primary neurons cultivated in high or low Mg²⁺ containing medium. (D) Histogram of the total number of intersections in hippocampal primary neurons cultivated in high or low Mg²⁺ containing medium. (E) Graphs comparing dendritic spine density between neurons cultivated in low or high Mg²⁺ and treated with BSA (control), BDNF and TrkB receptor bodies (TrkB-Fc). (F) Histogram comparing the spine head width between primary hippocampal neurons cultivated in high Mg²⁺ and treated with BSA (control), BDNF and TrkB receptor bodies (TrkB-Fc). (G) Histogram of the dendritic spine length between primary hippocampal neurons cultivated in high Mg²⁺ and treated with BSA (control), BDNF and TrkB receptor bodies (TrkB-Fc). (H) Graph showing the binning of spines according to their spine head width and comparing the proportion of spines within each category in response to a BSA (control), BDNF or BDNF antibodies (BDNF-Abs) treatment. (I) Graph showing the binning of spines according to their spine length and comparing the proportion of spines within each category in response to a control, BDNF or TrkB receptor bodies (TrkB-Fc) treatment. The number in the columns represents the number of cells analyzed. Significance is indicated as follows: ^*p < 0.05; ^***p < 0.001. Error bars indicate s.e.m. Unpaired two-tailed Student Test and ANOVA, post-hoc Tukey's Multiple Comparison Test.

Figure 7

(A) Experimental timeline. Primary neurons were cultivated for 2 weeks in high Mg²⁺ medium and treated with BSA (control), BDNF or TrkB-Fc in high Mg²⁺. (B) Graphs comparing dendritic spine density between neurons cultivated in low or high Mg²⁺ and treated with BSA (control), BDNF and TrkB receptor bodies (TrkB-Fc) in high Mg²⁺. (C) Histogram comparing the spine head width between primary hippocampal neurons cultivated and treated in high Mg²⁺ with BSA (control), BDNF and TrkB receptor bodies (TrkB-Fc). (D) Histogram of the dendritic spine length between primary hippocampal neurons cultivated and treated in high Mg²⁺ with BSA (control), BDNF and TrkB receptor bodies (TrkB-Fc). (E) Graph showing the binning of spines according to their spine head width and comparing the proportion of spines within each category in response to a BSA (control), BDNF or BDNF antibodies (BDNF-Abs) treatment in high Mg²⁺. (F) Graph showing the binning of spines according to their spine length and comparing the proportion of spines within each category in response to a BSA (control), BDNF or TrkB receptor bodies (TrkB-Fc) treatment in high Mg²⁺. The number in the columns represents the number of cells analyzed. Significance is indicated as follows: ^*p < 0.05; ^***p < 0.001. Error bars indicate s.e.m. Unpaired two-tailed Student Test and ANOVA, post-hoc Tukey's Multiple Comparison Test.