Properties of Synaptic Vesicle Pools in Mature Central Nerve Terminals

Abstract

Readily releasable and reserve pools of synaptic vesicles play different roles in neurotransmission, and it is important to understand their recycling and interchange in mature central synapses. Using adult rat cerebrocortical synaptosomes, we have shown that 100 mosM hypertonic sucrose caused complete exocytosis of only the readily releasable pool (RRP) of synaptic vesicles containing glutamate or ␥-aminobutyric acid. Repetitive hypertonic stimulations revealed that this pool recycled (and reloaded the neurotransmitter from the cytosol) fully in <30 s and did so independently of the reserve pool. Multiple rounds of exocytosis could occur in the constant absence of extracellular Ca 2؉ . However, although each vesicle cycle includes a Ca 2؉ -independent exocytotic step, some other stage(s) critically require an elevation of cytosolic [Ca 2؉ ], and this is supplied by intracellular stores. Repetitive recycling also requires energy, but not the activity of phosphatidylinositol 4-kinase, which maintains the normal level of phosphoinositides. By varying the length of hypertonic stimulations, we found that ϳ70% of the RRP vesicles fused completely with the plasmalemma during exocytosis and could then enter silent pools, probably outside active zones. The rest of the RRP vesicles underwent very fast local recycling (possibly by kiss-and-run) and did not leave active zones. Forcing the fully fused RRP vesicles into the silent pool enabled us to measure the transfer of reserve vesicles to the RRP and to show that this process requires intact phosphatidylinositol 4-kinase and actin microfilaments. Our findings also demonstrate that respective vesicle pools have similar characteristics and requirements in excitatory and inhibitory nerve terminals.Synaptic vesicles in a nerve terminal that undergo release/recycling consist of the readily releasable pool (RRP) 2 and the reserve pool (1, 2). These vesicle populations play distinct roles in neurotransmission, and their relative sizes change with synapse maturation (3, 4). Therefore, to understand the physiologically relevant properties of the vesicle pools, it is important to study them in mature central nerve terminals. When conducted in acute slices of adult brain, such studies are usually limited to specific regions of the hippocampus and thus do not necessarily reflect the behavior of vesicle pools in the rest of the brain. An alternative, well established system, cerebrocortical synaptosomes, is more universal and has several important advantages: (i) synchronized stimulation of all nerve terminals even by viscous solutions such as sucrose, (ii) direct comparison of terminals containing different transmitters, (iii) high-throughput pharmacological analysis, (iv) evaluation of multiple samples, and (v) uniform neuronal sampling. Finally, synaptosomal studies reveal pathways that are common to most nerve terminals, providing a level of generalization that is difficult to achieve by studying individual synapses.This simplified model, which lacks axons and cell bodies, is a...

Loss of phosphatidylinositol 4-kinase 2α activity causes late onset degeneration of spinal cord axons

“…AP-3B is also required to generate synaptic vesicles in presynaptic endosomes (22). PI4K2␣ is the only PI4K isozyme found in synaptic vesicles, which also contain PtdIns (17,24,25), and both PI4K2␣ and AP-3 have been implicated in formation of the reserve pool (26,27). However, studies based on inhibition of synaptosome function have produced conflicting results concerning a role for PI4K2␣ in neurotransmitter release (24,26,28).…”

“…PI4K2␣ is the only PI4K isozyme found in synaptic vesicles, which also contain PtdIns (17,24,25), and both PI4K2␣ and AP-3 have been implicated in formation of the reserve pool (26,27). However, studies based on inhibition of synaptosome function have produced conflicting results concerning a role for PI4K2␣ in neurotransmitter release (24,26,28). Although detailed studies are needed to comprehensively assess synaptic vesicle function, our preliminary studies on synaptosomes from age-matched Pi4k2a GT/GT and WT mice revealed attenuated glutamate uptake but no difference in glutamate release.…”

Fast Synaptic Vesicle Reuse Slows the Rate of Synaptic Depression in the CA1 Region of Hippocampus

“…This previous finding is in line with our observation that reserve pool vesicles do not significantly contribute to neurotransmission at high frequencies. Preferential recycling of RRP vesicles back to their pool of origin have been observed in dissociated hippocampal cultures (Pyle et al, 2000;Sara et al, 2002) as well as in cortical synaptosomes obtained from adult rats brains (Ashton and Ushkaryov, 2005). In addition, endocytosis gets accelerated with Ca 2ϩ influx in the calyx of Held terminal in the auditory brainstem as well as in dissociated hippocampal cultures (Sankaranarayanan and Ryan, 2001), presumably to support rapid reuse of at least some of the RRP vesicles (but see Li et al, 2005;Zenisek, 2005).…”

Dynamics of Intracellular Oxygen in PC12 Cells upon Stimulation of Neurotransmission

“…3, A and D). This does not happen in response to sucrose, a nonphysiological hypertonic treatment known to produce repeatable NT release in a Ca 2ϩ -dependent manner (41). We found that sustained osmotic shock enhanced the respiration of d PC12 cells in a two-phase mode: the first, which is regulated by i Ca 2ϩ and can be inhibited by BAPTA-AM, and the second, which starts 10 min after sucrose application and can be abolished by chelation of i Ca 2ϩ or e Ca 2ϩ (Fig.…”