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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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Dextran-TMR cannot be released from synaptic terminals by exocytosis.(A) Experimental procedure. Functional synapses of rat hippocampal neurons were fluorescently labelled with αSyt1-cypHer5. Dextran-TMR was subsequently loaded with 1200 AP, 80 Hz (Stim. 1). After removing extracellular dextran-TMR (wash), images in the Cy5- and TRITC channels were acquired during a second electrical stimulation with 1200 AP, 80 Hz (Stim 2). (B) αSyt1-cypHer5 fluorescence intensity over time for a representative experiment (>900 synapses). A.u., arbitrary units. (C) The corresponding dextran-TMR fluorescence intensity over time during the second stimulation was unaltered by the electrical stimulation. A.u., arbitrary units.
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pone-0038188-g002: Dextran-TMR cannot be released from synaptic terminals by exocytosis.(A) Experimental procedure. Functional synapses of rat hippocampal neurons were fluorescently labelled with αSyt1-cypHer5. Dextran-TMR was subsequently loaded with 1200 AP, 80 Hz (Stim. 1). After removing extracellular dextran-TMR (wash), images in the Cy5- and TRITC channels were acquired during a second electrical stimulation with 1200 AP, 80 Hz (Stim 2). (B) αSyt1-cypHer5 fluorescence intensity over time for a representative experiment (>900 synapses). A.u., arbitrary units. (C) The corresponding dextran-TMR fluorescence intensity over time during the second stimulation was unaltered by the electrical stimulation. A.u., arbitrary units.

Mentions: We next determined whether accumulated dextran-TMR could be subsequently released from synaptic terminals by high frequency stimulation. No release of the reporter was observed under these conditions (Fig. 2). Thus dextran-TMR specifically reports the generation of bulk endosomes, without dilution of the fluorescent signal from either 1) direct fusion of endosomes with the plasma membrane or 2) generation of dextran-TMR-loaded SVs from these endosomes in agreement with previous studies [18], [31]. Further evidence that large dextrans accurately report ADBE comes from studies, where the number of dextran puncta visualized using fluorescence microscopy closely correlate with the number of HRP-labelled endosomes (but not HRP-SVs) in nerve terminals across a range of different stimulation trains [32]. Manipulations that specifically inhibit ADBE cause a parallel reduction in both dextran uptake and HRP endosome number, but show no effect on HRP-labelled SVs [33], [34]. Moreover, large dextrans have been found to be associated with endosomal markers by fluorescence microscopy of fibroblasts [35] and finally ultrastructural analysis of photoconverted FITC-dextran confirmed the localization of dextrans in endosomal structures [36].


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)

Dextran-TMR cannot be released from synaptic terminals by exocytosis.(A) Experimental procedure. Functional synapses of rat hippocampal neurons were fluorescently labelled with αSyt1-cypHer5. Dextran-TMR was subsequently loaded with 1200 AP, 80 Hz (Stim. 1). After removing extracellular dextran-TMR (wash), images in the Cy5- and TRITC channels were acquired during a second electrical stimulation with 1200 AP, 80 Hz (Stim 2). (B) αSyt1-cypHer5 fluorescence intensity over time for a representative experiment (>900 synapses). A.u., arbitrary units. (C) The corresponding dextran-TMR fluorescence intensity over time during the second stimulation was unaltered by the electrical stimulation. A.u., arbitrary units.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366995&req=5

pone-0038188-g002: Dextran-TMR cannot be released from synaptic terminals by exocytosis.(A) Experimental procedure. Functional synapses of rat hippocampal neurons were fluorescently labelled with αSyt1-cypHer5. Dextran-TMR was subsequently loaded with 1200 AP, 80 Hz (Stim. 1). After removing extracellular dextran-TMR (wash), images in the Cy5- and TRITC channels were acquired during a second electrical stimulation with 1200 AP, 80 Hz (Stim 2). (B) αSyt1-cypHer5 fluorescence intensity over time for a representative experiment (>900 synapses). A.u., arbitrary units. (C) The corresponding dextran-TMR fluorescence intensity over time during the second stimulation was unaltered by the electrical stimulation. A.u., arbitrary units.
Mentions: We next determined whether accumulated dextran-TMR could be subsequently released from synaptic terminals by high frequency stimulation. No release of the reporter was observed under these conditions (Fig. 2). Thus dextran-TMR specifically reports the generation of bulk endosomes, without dilution of the fluorescent signal from either 1) direct fusion of endosomes with the plasma membrane or 2) generation of dextran-TMR-loaded SVs from these endosomes in agreement with previous studies [18], [31]. Further evidence that large dextrans accurately report ADBE comes from studies, where the number of dextran puncta visualized using fluorescence microscopy closely correlate with the number of HRP-labelled endosomes (but not HRP-SVs) in nerve terminals across a range of different stimulation trains [32]. Manipulations that specifically inhibit ADBE cause a parallel reduction in both dextran uptake and HRP endosome number, but show no effect on HRP-labelled SVs [33], [34]. Moreover, large dextrans have been found to be associated with endosomal markers by fluorescence microscopy of fibroblasts [35] and finally ultrastructural analysis of photoconverted FITC-dextran confirmed the localization of dextrans in endosomal structures [36].

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