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Key physiological parameters dictate triggering of activity-dependent bulk endocytosis in hippocampal synapses.

Wenzel EM, Morton A, Ebert K, Welzel O, Kornhuber J, Cousin MA, Groemer TW - PLoS ONE (2012)

Bottom Line: Furthermore we observed a strong correlation between SV pool size and ability to perform ADBE.We also identified that inhibitory nerve terminals were more likely to utilize ADBE and had a larger SV recycling pool.These results implicate ADBE as a key modulator of both hippocampal neurotransmission and plasticity.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry and Psychotherapy, University of Erlangen-Nürnberg, Erlangen, Germany. eva.wenzel@rr-research.no

ABSTRACT
To maintain neurotransmission in central neurons, several mechanisms are employed to retrieve synaptically exocytosed membrane. The two major modes of synaptic vesicle (SV) retrieval are clathrin-mediated endocytosis and activity-dependent bulk endocytosis (ADBE). ADBE is the dominant SV retrieval mode during intense stimulation, however the precise physiological conditions that trigger this mode are not resolved. To determine these parameters we manipulated rat hippocampal neurons using a wide spectrum of stimuli by varying both the pattern and duration of stimulation. Using live-cell fluorescence imaging and electron microscopy approaches, we established that stimulation frequency, rather than the stimulation load, was critical in the triggering of ADBE. Thus two hundred action potentials, when delivered at high frequency, were sufficient to induce near maximal bulk formation. Furthermore we observed a strong correlation between SV pool size and ability to perform ADBE. We also identified that inhibitory nerve terminals were more likely to utilize ADBE and had a larger SV recycling pool. Thus ADBE in hippocampal synaptic terminals is tightly coupled to stimulation frequency and is more likely to occur in terminals with large SV pools. These results implicate ADBE as a key modulator of both hippocampal neurotransmission and plasticity.

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The initiation of ADBE depends on the frequency rather than the number of AP.(A) Experimental procedure. Functional synapses of rat hippocampal neurons were fluorescently labelled with anti-synaptotagmin1-cypHer5 (αSyt1-cypHer5) which fluoresces due to the acidic pH in the acidic vesicular lumen after endocytosis. The cells were then electrically stimulated with varying intensities (see panel (D)) in the presence of 50 µM dextran-TMR, which serves as a marker for bulk membrane invaginations. (B) Representative images of αSyt1-cypHer5 antibody-stained hippocampal neurons that were stimulated with 1200 AP, 80 Hz in the presence of dextran-TMR (“image”). Quantification of dextran-positive terminals was done by a Laplace operator based peak-detection (“mask”) followed by colocalization analysis. (C) Representative images of αSyt1-cypHer5 antibody-stained hippocampal neurons that were exposed to dextran-TMR without electrical stimulation. Automatically detected regions of interest are indicated in the lower panel. (D) Dextran-TMR positive boutons indicating terminals with bulk endosomes were quantified for varying numbers of action potentials (50, 200, 400, 800, 1200) and varying frequencies (10, 40, 80 Hz) of electrical stimulation. N = 3 to 4 experiments with 3 to 6 fields of view each; error bars represent SEM; One-way ANOVA for groups of constant numbers of AP and varying Hz: 200 AP: p<0.05; 400 AP: p>0.05; 800 AP: p<0.05; 1200 AP: p<0.01; Post-hoc test: Dunnett’s test. Asterisks indicate significant differences to unstimulated control at the 5% probability level for a 15 group comparison with a group size of 12 (critical value 2.87).
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pone-0038188-g001: The initiation of ADBE depends on the frequency rather than the number of AP.(A) Experimental procedure. Functional synapses of rat hippocampal neurons were fluorescently labelled with anti-synaptotagmin1-cypHer5 (αSyt1-cypHer5) which fluoresces due to the acidic pH in the acidic vesicular lumen after endocytosis. The cells were then electrically stimulated with varying intensities (see panel (D)) in the presence of 50 µM dextran-TMR, which serves as a marker for bulk membrane invaginations. (B) Representative images of αSyt1-cypHer5 antibody-stained hippocampal neurons that were stimulated with 1200 AP, 80 Hz in the presence of dextran-TMR (“image”). Quantification of dextran-positive terminals was done by a Laplace operator based peak-detection (“mask”) followed by colocalization analysis. (C) Representative images of αSyt1-cypHer5 antibody-stained hippocampal neurons that were exposed to dextran-TMR without electrical stimulation. Automatically detected regions of interest are indicated in the lower panel. (D) Dextran-TMR positive boutons indicating terminals with bulk endosomes were quantified for varying numbers of action potentials (50, 200, 400, 800, 1200) and varying frequencies (10, 40, 80 Hz) of electrical stimulation. N = 3 to 4 experiments with 3 to 6 fields of view each; error bars represent SEM; One-way ANOVA for groups of constant numbers of AP and varying Hz: 200 AP: p<0.05; 400 AP: p>0.05; 800 AP: p<0.05; 1200 AP: p<0.01; Post-hoc test: Dunnett’s test. Asterisks indicate significant differences to unstimulated control at the 5% probability level for a 15 group comparison with a group size of 12 (critical value 2.87).

Mentions: Statistical analysis was performed by MATLAB (The MathWorks Inc., Natick) or Microsoft Excel. Error bars indicate SEM unless otherwise indicated. For single group comparisons unpaired t-tests were applied (6C, D). For multiple group comparisons Tukey’s Multiple Comparison Test (Fig. 3) or a Dunnett’s test against unstimulated control (Fig. 1D) was performed. The critical value was determined for a 15 group comparison with a group size of 12 each according to [30]. In Fig. 5E the αSyt1-cypHer5 fluorescence intensity was correlated to the percentage of dextran-positive boutons. Synapses of several fields of view from 3 individual experiments (1200 AP, 40 Hz stimulations) were sorted according to their fluorescence intensities in 8 equally sized bins as described [29] and the percentage of dextran-positive terminals evaluated. Depicted are mean percentages +/− SEM. R: coefficient of determination (Spearman’s rho).


Key physiological parameters dictate triggering of activity-dependent bulk endocytosis in hippocampal synapses.

Wenzel EM, Morton A, Ebert K, Welzel O, Kornhuber J, Cousin MA, Groemer TW - PLoS ONE (2012)

The initiation of ADBE depends on the frequency rather than the number of AP.(A) Experimental procedure. Functional synapses of rat hippocampal neurons were fluorescently labelled with anti-synaptotagmin1-cypHer5 (αSyt1-cypHer5) which fluoresces due to the acidic pH in the acidic vesicular lumen after endocytosis. The cells were then electrically stimulated with varying intensities (see panel (D)) in the presence of 50 µM dextran-TMR, which serves as a marker for bulk membrane invaginations. (B) Representative images of αSyt1-cypHer5 antibody-stained hippocampal neurons that were stimulated with 1200 AP, 80 Hz in the presence of dextran-TMR (“image”). Quantification of dextran-positive terminals was done by a Laplace operator based peak-detection (“mask”) followed by colocalization analysis. (C) Representative images of αSyt1-cypHer5 antibody-stained hippocampal neurons that were exposed to dextran-TMR without electrical stimulation. Automatically detected regions of interest are indicated in the lower panel. (D) Dextran-TMR positive boutons indicating terminals with bulk endosomes were quantified for varying numbers of action potentials (50, 200, 400, 800, 1200) and varying frequencies (10, 40, 80 Hz) of electrical stimulation. N = 3 to 4 experiments with 3 to 6 fields of view each; error bars represent SEM; One-way ANOVA for groups of constant numbers of AP and varying Hz: 200 AP: p<0.05; 400 AP: p>0.05; 800 AP: p<0.05; 1200 AP: p<0.01; Post-hoc test: Dunnett’s test. Asterisks indicate significant differences to unstimulated control at the 5% probability level for a 15 group comparison with a group size of 12 (critical value 2.87).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366995&req=5

pone-0038188-g001: The initiation of ADBE depends on the frequency rather than the number of AP.(A) Experimental procedure. Functional synapses of rat hippocampal neurons were fluorescently labelled with anti-synaptotagmin1-cypHer5 (αSyt1-cypHer5) which fluoresces due to the acidic pH in the acidic vesicular lumen after endocytosis. The cells were then electrically stimulated with varying intensities (see panel (D)) in the presence of 50 µM dextran-TMR, which serves as a marker for bulk membrane invaginations. (B) Representative images of αSyt1-cypHer5 antibody-stained hippocampal neurons that were stimulated with 1200 AP, 80 Hz in the presence of dextran-TMR (“image”). Quantification of dextran-positive terminals was done by a Laplace operator based peak-detection (“mask”) followed by colocalization analysis. (C) Representative images of αSyt1-cypHer5 antibody-stained hippocampal neurons that were exposed to dextran-TMR without electrical stimulation. Automatically detected regions of interest are indicated in the lower panel. (D) Dextran-TMR positive boutons indicating terminals with bulk endosomes were quantified for varying numbers of action potentials (50, 200, 400, 800, 1200) and varying frequencies (10, 40, 80 Hz) of electrical stimulation. N = 3 to 4 experiments with 3 to 6 fields of view each; error bars represent SEM; One-way ANOVA for groups of constant numbers of AP and varying Hz: 200 AP: p<0.05; 400 AP: p>0.05; 800 AP: p<0.05; 1200 AP: p<0.01; Post-hoc test: Dunnett’s test. Asterisks indicate significant differences to unstimulated control at the 5% probability level for a 15 group comparison with a group size of 12 (critical value 2.87).
Mentions: Statistical analysis was performed by MATLAB (The MathWorks Inc., Natick) or Microsoft Excel. Error bars indicate SEM unless otherwise indicated. For single group comparisons unpaired t-tests were applied (6C, D). For multiple group comparisons Tukey’s Multiple Comparison Test (Fig. 3) or a Dunnett’s test against unstimulated control (Fig. 1D) was performed. The critical value was determined for a 15 group comparison with a group size of 12 each according to [30]. In Fig. 5E the αSyt1-cypHer5 fluorescence intensity was correlated to the percentage of dextran-positive boutons. Synapses of several fields of view from 3 individual experiments (1200 AP, 40 Hz stimulations) were sorted according to their fluorescence intensities in 8 equally sized bins as described [29] and the percentage of dextran-positive terminals evaluated. Depicted are mean percentages +/− SEM. R: coefficient of determination (Spearman’s rho).

Bottom Line: Furthermore we observed a strong correlation between SV pool size and ability to perform ADBE.We also identified that inhibitory nerve terminals were more likely to utilize ADBE and had a larger SV recycling pool.These results implicate ADBE as a key modulator of both hippocampal neurotransmission and plasticity.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry and Psychotherapy, University of Erlangen-Nürnberg, Erlangen, Germany. eva.wenzel@rr-research.no

ABSTRACT
To maintain neurotransmission in central neurons, several mechanisms are employed to retrieve synaptically exocytosed membrane. The two major modes of synaptic vesicle (SV) retrieval are clathrin-mediated endocytosis and activity-dependent bulk endocytosis (ADBE). ADBE is the dominant SV retrieval mode during intense stimulation, however the precise physiological conditions that trigger this mode are not resolved. To determine these parameters we manipulated rat hippocampal neurons using a wide spectrum of stimuli by varying both the pattern and duration of stimulation. Using live-cell fluorescence imaging and electron microscopy approaches, we established that stimulation frequency, rather than the stimulation load, was critical in the triggering of ADBE. Thus two hundred action potentials, when delivered at high frequency, were sufficient to induce near maximal bulk formation. Furthermore we observed a strong correlation between SV pool size and ability to perform ADBE. We also identified that inhibitory nerve terminals were more likely to utilize ADBE and had a larger SV recycling pool. Thus ADBE in hippocampal synaptic terminals is tightly coupled to stimulation frequency and is more likely to occur in terminals with large SV pools. These results implicate ADBE as a key modulator of both hippocampal neurotransmission and plasticity.

Show MeSH
Related in: MedlinePlus