Abstract
Evoked neuropeptide secretion in the central nervous system occurs slowly, but the basis for slow release is not fully understood. Whereas exocytosis of single synaptic vesicles in neurons and of dense-core vesicles (DCVs) in endocrine cells have been directly visualized, single DCV exocytic events in neurons of the central nervous system have not been previously studied. We imaged DCV exocytosis in primary cultured hippocampal neurons using fluorescent propeptide cargo and total internal reflectance fluorescence microscopy. The majority of Ca(2+)-triggered exocytic events occurred from immobile plasma-membrane-proximal DCVs in the cell soma, whereas there were few events in the neurites. Strikingly, DCVs in the cell soma exhibited 50-fold greater release probabilities than those in neurites. Latencies to depolarization-evoked fusion for DCVs were surprisingly long, occurring with an average time constant (tau) of 16 seconds for DCVs in the soma and even longer for DCVs in neurites. All of the single DCV release events exhibited rapid fusion-pore openings and closures, the kinetics of which were highly dependent upon Ca(2+) levels. These ;kiss-and-run' events were associated with limited cargo secretion. Thus, the slow evoked release of neuropeptides could be attributed to very prolonged latencies from stimulation to fusion and transient fusion-pore openings that might limit cargo secretion.
Highlights
A detailed understanding of how a nervous system functions requires analysis of its chemical signaling, which is largely mediated by regulated vesicle exocytosis
Visualization of single neuropeptide-containing dense-core vesicles (DCVs) To directly image stimulus-secretion coupling of DCVs in neurons, primary cultured hippocampal neurons were transfected with plasmids encoding atrial-natriuretic-factor–EGFP (ANF-EGFP), which is targeted to DCVs (Burke et al, 1997; 76 Journal of Cell Science 122 (1)
All of the ANF-EGFP-containing puncta in the evanescent field were positive for secretogranin II (SgII) (Fig. 1B, first row), a DCV-content marker (Huttner et al, 1991), which confirmed their identity as DCVs
Summary
A detailed understanding of how a nervous system functions requires analysis of its chemical signaling, which is largely mediated by regulated vesicle exocytosis. Regulated exocytosis in neurons involves at least two types of secretory vesicles: synaptic vesicles (SVs) and dense-core vesicles (DCVs) (De Camilli and Jahn, 1990). DCVs store and release a diverse array of modulators, including neuropeptides, monoamines and neurotrophins (Bean et al, 1994; Lessmann et al, 2003; Li et al, 2005), that regulate many crucial processes, such as neuronal survival, synaptic plasticity and learning (Chen and Strickland, 1997; Poo, 2001; Strand et al, 1991). Whereas exocytic and endocytic events have been extensively studied for SVs in neurons and DCVs in endocrine cells (Neher, 1998; Penner and Neher, 1989; Ryan, 2001), the vesicle cycle for DCVs in neurons is still poorly understood. Little is known about the cellular location of DCV exocytosis, the latency between stimulation and DCV fusion, release probabilities for DCVs, the kinetics of individual DCV exocytic events or the termination of exocytosis and DCV-membrane retrieval
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