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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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Inhibitory synapses preferentially form bulk endosomes and are slightly larger as compared to excitatory synapses.(A) Experimental procedure. Functional synapses of rat hippocampal neurons were fluorescently labelled with αSyt1-cypHer5. In parallel, inhibitory synapses were identified with an anti-vGAT (vesicular GABA transporter) antibody, labelled with the green fluorescent dye Oyster488®. The cells were then electrically stimulated with 1200 AP, 40 Hz in the presence of 50 µM dextran-TMR. (B) Representative images of αSyt1-cypHer5 and anti-vGAT-oyster488® antibody-stained hippocampal neurons that were exposed to dextran-TMR during the electrical stimulation (“image”). Quantification was done by a Laplace operator based peak-detection (“mask”). (C) vGAT-positive synapses showed a larger percentage of dextran-TMR-staining than vGAT-negative ones or the total of synapses. The boxplot indicates median (red line), 25th and 75th percentiles (blue box) and extreme values (whiskers). Wilcoxon signed rank test, ***p<0.001. N = 4 experiments with 4 to 7 fields of view each; (D) vGAT-positive synapses were larger than vGAT-negative ones or the total of synapses.
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pone-0038188-g006: Inhibitory synapses preferentially form bulk endosomes and are slightly larger as compared to excitatory synapses.(A) Experimental procedure. Functional synapses of rat hippocampal neurons were fluorescently labelled with αSyt1-cypHer5. In parallel, inhibitory synapses were identified with an anti-vGAT (vesicular GABA transporter) antibody, labelled with the green fluorescent dye Oyster488®. The cells were then electrically stimulated with 1200 AP, 40 Hz in the presence of 50 µM dextran-TMR. (B) Representative images of αSyt1-cypHer5 and anti-vGAT-oyster488® antibody-stained hippocampal neurons that were exposed to dextran-TMR during the electrical stimulation (“image”). Quantification was done by a Laplace operator based peak-detection (“mask”). (C) vGAT-positive synapses showed a larger percentage of dextran-TMR-staining than vGAT-negative ones or the total of synapses. The boxplot indicates median (red line), 25th and 75th percentiles (blue box) and extreme values (whiskers). Wilcoxon signed rank test, ***p<0.001. N = 4 experiments with 4 to 7 fields of view each; (D) vGAT-positive synapses were larger than vGAT-negative ones or the total of synapses.

Mentions: Specific subsets of inhibitory interneurons are characterized by high rates of activity [38], suggesting that they may utilize ADBE to compensate for this elevated activity. Therefore, we next determined whether ADBE was more prevalent in inhibitory synaptic terminals in our hippocampal cultures. To identify inhibitory synapses, we marked GABAergic neurons with a fluorescently-labelled antibody against the luminal domain of the vesicular GABA transporter (αvGAT-oyster488) [39] simultaneously with the αSyt1-cypHer5 incubation. Then we induced ADBE with 1200 AP at 40 Hz in the presence of dextran-TMR as before (Fig. 6A). We identified a subpopulation of the αSyt1-cypHer5-positive boutons to be vGAT-positive within our cultures (34.3±10.7%), which is in line with findings from other studies [40]. ADBE could be visualized by dextran-TMR-uptake in both vGAT-positive and –negative synaptic terminals (Fig. 6B). Interestingly, the vGAT-positive synaptic terminals displayed a significantly higher percentage of dextran-TMR-labeling compared to the total number of synapses as defined by the αSyt1-cypHer5 signal (p<0.001) (Fig. 6C). Conversely, vGAT-negative synaptic terminals showed significantly less dextran-TMR-uptake (p<0.001). Thus GABAergic synaptic terminals are more likely to utilize ADBE as an endocytosis mechanism than non-GABAeric terminals.


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)

Inhibitory synapses preferentially form bulk endosomes and are slightly larger as compared to excitatory synapses.(A) Experimental procedure. Functional synapses of rat hippocampal neurons were fluorescently labelled with αSyt1-cypHer5. In parallel, inhibitory synapses were identified with an anti-vGAT (vesicular GABA transporter) antibody, labelled with the green fluorescent dye Oyster488®. The cells were then electrically stimulated with 1200 AP, 40 Hz in the presence of 50 µM dextran-TMR. (B) Representative images of αSyt1-cypHer5 and anti-vGAT-oyster488® antibody-stained hippocampal neurons that were exposed to dextran-TMR during the electrical stimulation (“image”). Quantification was done by a Laplace operator based peak-detection (“mask”). (C) vGAT-positive synapses showed a larger percentage of dextran-TMR-staining than vGAT-negative ones or the total of synapses. The boxplot indicates median (red line), 25th and 75th percentiles (blue box) and extreme values (whiskers). Wilcoxon signed rank test, ***p<0.001. N = 4 experiments with 4 to 7 fields of view each; (D) vGAT-positive synapses were larger than vGAT-negative ones or the total of synapses.
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Related In: Results  -  Collection

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pone-0038188-g006: Inhibitory synapses preferentially form bulk endosomes and are slightly larger as compared to excitatory synapses.(A) Experimental procedure. Functional synapses of rat hippocampal neurons were fluorescently labelled with αSyt1-cypHer5. In parallel, inhibitory synapses were identified with an anti-vGAT (vesicular GABA transporter) antibody, labelled with the green fluorescent dye Oyster488®. The cells were then electrically stimulated with 1200 AP, 40 Hz in the presence of 50 µM dextran-TMR. (B) Representative images of αSyt1-cypHer5 and anti-vGAT-oyster488® antibody-stained hippocampal neurons that were exposed to dextran-TMR during the electrical stimulation (“image”). Quantification was done by a Laplace operator based peak-detection (“mask”). (C) vGAT-positive synapses showed a larger percentage of dextran-TMR-staining than vGAT-negative ones or the total of synapses. The boxplot indicates median (red line), 25th and 75th percentiles (blue box) and extreme values (whiskers). Wilcoxon signed rank test, ***p<0.001. N = 4 experiments with 4 to 7 fields of view each; (D) vGAT-positive synapses were larger than vGAT-negative ones or the total of synapses.
Mentions: Specific subsets of inhibitory interneurons are characterized by high rates of activity [38], suggesting that they may utilize ADBE to compensate for this elevated activity. Therefore, we next determined whether ADBE was more prevalent in inhibitory synaptic terminals in our hippocampal cultures. To identify inhibitory synapses, we marked GABAergic neurons with a fluorescently-labelled antibody against the luminal domain of the vesicular GABA transporter (αvGAT-oyster488) [39] simultaneously with the αSyt1-cypHer5 incubation. Then we induced ADBE with 1200 AP at 40 Hz in the presence of dextran-TMR as before (Fig. 6A). We identified a subpopulation of the αSyt1-cypHer5-positive boutons to be vGAT-positive within our cultures (34.3±10.7%), which is in line with findings from other studies [40]. ADBE could be visualized by dextran-TMR-uptake in both vGAT-positive and –negative synaptic terminals (Fig. 6B). Interestingly, the vGAT-positive synaptic terminals displayed a significantly higher percentage of dextran-TMR-labeling compared to the total number of synapses as defined by the αSyt1-cypHer5 signal (p<0.001) (Fig. 6C). Conversely, vGAT-negative synaptic terminals showed significantly less dextran-TMR-uptake (p<0.001). Thus GABAergic synaptic terminals are more likely to utilize ADBE as an endocytosis mechanism than non-GABAeric terminals.

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