Key Physiological Parameters Dictate Triggering of Activity-Dependent Bulk Endocytosis in Hippocampal Synapses

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.

Exocytosis and Endocytosis: Modes, Functions, and Coupling Mechanisms

“…EM examination after intense nerve stimulation reveals endosome-like structures or cisternae much larger than regular vesicles (Figure 3 b i ) (6, 44, 56–58, 78, 79). Some of these structures take up extracellular horseradish peroxidase or FM dyes, suggesting their endocytic origin (Figure 3 b i ).…”

“…Some of these structures take up extracellular horseradish peroxidase or FM dyes, suggesting their endocytic origin (Figure 3 b i ). Imaging reveals large fluorescent spots that may correspond to endosome-like structures taking up extracellular dyes (Figure 3 b ii ) (79–83). Endosome-like structures may be generated by bulk endocytosis or by vesicle-vesicle fusion.…”

“…This finding may account for the previous observation that calcium upregulates rapid and slow endocytosis at hippocampal synapses (99, 127), retinal nerve terminals (94), the calyx of Held (98), and hair cells (104). It explains why more intense stimulation, which generates higher calcium influx, induces more endosome-like structures that may be caused by bulk endocytosis at various synapses (6, 44, 56–58, 78, 79, 131) and induces endocytosis overshoot at chromaffin cells and pituitary cells (95, 105). Accordingly, calcium influx is suggested as the universal trigger for all forms of vesicle endocytosis in secretory cells in which calcium triggers exocytosis (49).…”

Bulk endocytosis at neuronal synapses

“…Interestingly, when preparations were rested after 15 min stimulation, the cisternae disappeared and SVs reappeared, suggesting the SVs could have derived from the cisternae [1]. Since this initial seminal study, a considerable number of studies have demonstrated that ADBE also occurs in rat hippocampal [6,17] and cerebellar granule [18] neurons, lamprey reticulospinal synapses [5], mouse calyx of Held [19] and auditory inner hair cells [20], snake [21] and Caenorhabditis elegans motor terminals [22], and goldfish bipolar neurons [8].…”

Advances in chemical labeling of proteins in living cells

“…With a pKa of 6.1, Cypher 5 is only fluorescent under acidic conditions. Traditionally, Cypher5 has been conjugated to antibodies to study receptor internalization and the pH change in the endocytic compartments (Adie et al, 2002; Nordberg et al, 2007; Wenzel et al, 2012). However, antibody conjugation is not practical and readily available.…”