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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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Formation of bulk endosomes correlates with the recycling pool size.(A) Experimental procedure. Hippocampal neurons were simultaneously loaded with 50 µM dextran-TMR and 2.5 µM FM1-43 by a 1200 AP, 40 Hz electrical stimulation. After washing away extracellular dye, images for FM1-43 and dextran-TMR were acquired. Complete destaining of FM1-43 was done by two subsequent electrical stimulations (2×900 AP, 30 Hz). (B) Representative images of dextran-TMR and FM1-43 labelled hippocampal neurons. Scale bar, 10 µm. (C) The number of dextran-TMR-positive spots increases with dF(FM). All synapses in 5 fields of view of each of 3 individual experiments were evaluated by sorting according to their fluorescence intensity and subsequent binning into 8 groups containing equal numbers of terminals. The percentage of dextran-positive terminals in each group was determined. Displayed is the mean of all fields of view. R, spearman’s rho.
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pone-0038188-g004: Formation of bulk endosomes correlates with the recycling pool size.(A) Experimental procedure. Hippocampal neurons were simultaneously loaded with 50 µM dextran-TMR and 2.5 µM FM1-43 by a 1200 AP, 40 Hz electrical stimulation. After washing away extracellular dye, images for FM1-43 and dextran-TMR were acquired. Complete destaining of FM1-43 was done by two subsequent electrical stimulations (2×900 AP, 30 Hz). (B) Representative images of dextran-TMR and FM1-43 labelled hippocampal neurons. Scale bar, 10 µm. (C) The number of dextran-TMR-positive spots increases with dF(FM). All synapses in 5 fields of view of each of 3 individual experiments were evaluated by sorting according to their fluorescence intensity and subsequent binning into 8 groups containing equal numbers of terminals. The percentage of dextran-positive terminals in each group was determined. Displayed is the mean of all fields of view. R, spearman’s rho.

Mentions: ADBE only occurs in a subset of synaptic terminals (Fig. 1). Therefore we next questioned if any physiological characteristics potentially predict which synaptic terminals undergo ADBE. One potential predictor could be the size of the total SV recycling pool. This has been quantified previously by loading synaptic terminals with a styryl dye such as FM1-43 during a saturating stimulus train. This loading is then followed by a similar saturating stimulus to evoke complete dye unloading [24], [28]. To investigate the relation of synapse size and initiation of ADBE, we loaded FM1-43 and dextran-TMR in parallel with 1200 AP, 40 Hz. We observed a positive correlation (R = 0.9762) between SV recycling pool size and initiation of ADBE (Fig. 4). However, FM1-43 also labels ADBE [23] meaning that the quantification of SV pool size by dye unloading could be skewed by retention of dye in bulk endosomes. Therefore, we determined whether the αSyt1-cypHer5 signal was a more suitable method to determine the size of the total SV recycling pool. Cells were incubated for 1 h with αSyt1-cypHer5 and then FM1-43 was loaded with 1200 AP at 30 Hz (Fig. 5A). After washing away extracellular FM1-43, a punctate pattern was observed for both FM1-43 and αSyt1-cypHer5 staining (Fig. 5B). Three subsequent stimulations evoked a complete unloading of the SV recycling pool, enabling the determination of the fluorescence difference dF(FM) for every synaptic terminal (Fig. 5C). We found that αSyt1-cypHer5 fluorescence intensity correlates well (R = 0.7575) with the dF(FM) values from FM1-43-stained boutons (Fig. 5D). Thus, αSyt1-cypHer5 fluorescence intensity is an appropriate measure of total SV pool size.


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)

Formation of bulk endosomes correlates with the recycling pool size.(A) Experimental procedure. Hippocampal neurons were simultaneously loaded with 50 µM dextran-TMR and 2.5 µM FM1-43 by a 1200 AP, 40 Hz electrical stimulation. After washing away extracellular dye, images for FM1-43 and dextran-TMR were acquired. Complete destaining of FM1-43 was done by two subsequent electrical stimulations (2×900 AP, 30 Hz). (B) Representative images of dextran-TMR and FM1-43 labelled hippocampal neurons. Scale bar, 10 µm. (C) The number of dextran-TMR-positive spots increases with dF(FM). All synapses in 5 fields of view of each of 3 individual experiments were evaluated by sorting according to their fluorescence intensity and subsequent binning into 8 groups containing equal numbers of terminals. The percentage of dextran-positive terminals in each group was determined. Displayed is the mean of all fields of view. R, spearman’s rho.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366995&req=5

pone-0038188-g004: Formation of bulk endosomes correlates with the recycling pool size.(A) Experimental procedure. Hippocampal neurons were simultaneously loaded with 50 µM dextran-TMR and 2.5 µM FM1-43 by a 1200 AP, 40 Hz electrical stimulation. After washing away extracellular dye, images for FM1-43 and dextran-TMR were acquired. Complete destaining of FM1-43 was done by two subsequent electrical stimulations (2×900 AP, 30 Hz). (B) Representative images of dextran-TMR and FM1-43 labelled hippocampal neurons. Scale bar, 10 µm. (C) The number of dextran-TMR-positive spots increases with dF(FM). All synapses in 5 fields of view of each of 3 individual experiments were evaluated by sorting according to their fluorescence intensity and subsequent binning into 8 groups containing equal numbers of terminals. The percentage of dextran-positive terminals in each group was determined. Displayed is the mean of all fields of view. R, spearman’s rho.
Mentions: ADBE only occurs in a subset of synaptic terminals (Fig. 1). Therefore we next questioned if any physiological characteristics potentially predict which synaptic terminals undergo ADBE. One potential predictor could be the size of the total SV recycling pool. This has been quantified previously by loading synaptic terminals with a styryl dye such as FM1-43 during a saturating stimulus train. This loading is then followed by a similar saturating stimulus to evoke complete dye unloading [24], [28]. To investigate the relation of synapse size and initiation of ADBE, we loaded FM1-43 and dextran-TMR in parallel with 1200 AP, 40 Hz. We observed a positive correlation (R = 0.9762) between SV recycling pool size and initiation of ADBE (Fig. 4). However, FM1-43 also labels ADBE [23] meaning that the quantification of SV pool size by dye unloading could be skewed by retention of dye in bulk endosomes. Therefore, we determined whether the αSyt1-cypHer5 signal was a more suitable method to determine the size of the total SV recycling pool. Cells were incubated for 1 h with αSyt1-cypHer5 and then FM1-43 was loaded with 1200 AP at 30 Hz (Fig. 5A). After washing away extracellular FM1-43, a punctate pattern was observed for both FM1-43 and αSyt1-cypHer5 staining (Fig. 5B). Three subsequent stimulations evoked a complete unloading of the SV recycling pool, enabling the determination of the fluorescence difference dF(FM) for every synaptic terminal (Fig. 5C). We found that αSyt1-cypHer5 fluorescence intensity correlates well (R = 0.7575) with the dF(FM) values from FM1-43-stained boutons (Fig. 5D). Thus, αSyt1-cypHer5 fluorescence intensity is an appropriate measure of total SV pool size.

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