Short-term facilitation is a widely observed form of synaptic enhancement that is not well understood. Although presynaptic calcium has long been implicated in this process, its role is unclear, particularly at synapses in the mammalian brain. We tested the role of presynaptic residual free calcium ([Ca]res) in facilitation of synapses between granule cells and Purkinje cells in rat cerebellar slices. Paired-pulse facilitation of synaptic currents resulted in an approximately 2.5-fold enhancement that decayed with a time constant of approximately 200 msec, as assessed by voltage-clamp recordings. Measurements of [Ca]res using fluorescent calcium-sensitive indicators revealed that [Ca]res decayed more rapidly than did facilitation. Manipulation of [Ca]res dynamics by introducing EGTA into presynaptic terminals sped the decays of [Ca]res and facilitation in a dose-dependent manner. When [Ca]res was reduced to a brief impulse lasting several milliseconds, facilitation was still present, although reduced in amplitude and duration. Facilitation decayed with an intrinsic time constant of approximately 40 msec. These results suggest that facilitation at this synapse is produced by a calcium-driven process with a high affinity and a slow effective off-rate. A combination of [Ca]res dynamics and the properties of a calcium-driven reaction determine the time course and amplitude of facilitation.

We used actin tagged with enhanced green fluorescent protein (EGFP-actin) to characterize the distribution and dynamics of actin in living presynaptic terminals in rat CNS neurons. Actin was preferentially concentrated around--and appeared to surround--the presynaptic vesicle cluster. In resting terminals, approximately 30% of actin was found to be in a polymerized but dynamic state, with a remodeling time scale of approximately 20 s. During electrical activity, actin was further polymerized and recruited from nearby axonal regions to the regions surrounding vesicles. Treatment of terminals with the actin monomer-sequestering agent latrunculin-A completely dispersed the actin network and abolished activity-dependent actin dynamics. We used a variety of methods to examine the role of actin in the presynaptic vesicle cycle. These data rule out a propulsive role for actin, either in maintaining the vesicle cluster or in guiding vesicle recycling. Instead, we propose that actin acts as a scaffolding system for regulatory molecules in the nerve terminal.

At fast chemical synapses the rapid release of neurotransmitter that occurs within a few milliseconds of an action potential is followed by a more sustained elevation of release probability, known as delayed release. Here we characterize the role of calcium in delayed release and test the hypothesis that facilitation and delayed release share a common mechanism. Synapses between cerebellar granule cells and their postsynaptic targets, stellate cells and Purkinje cells, were studied in rat brain slices. Presynaptic calcium transients were measured with calcium-sensitive fluorophores, and delayed release was detected with whole-cell recordings. Calcium influx, presynaptic calcium dynamics, and the number of stimulus pulses were altered to assess their effect on delayed release and facilitation. Following single stimuli, delayed release can be separated into two components: one lasting for tens of milliseconds that is steeply calcium-dependent, the other lasting for hundreds of milliseconds that is driven by low levels of calcium with a nearly linear calcium dependence. The amplitude, calcium dependence, and magnitude of delayed release do not correspond to those of facilitation, indicating that these processes are not simple reflections of a shared mechanism. The steep calcium dependence of delayed release, combined with the large calcium transients observed in these presynaptic terminals, suggests that for physiological conditions delayed release provides a way for cells to influence their postsynaptic targets long after their own action potential activity has subsided.

Calcium ions act presynaptically to modulate synaptic strength and to trigger neurotransmitter release. Here we detect stimulus-evoked changes in residual free calcium ([Ca2+]i) in rat cerebellar granule cell presynaptic terminals. Granule cell axons, known as parallel fibers, and their associated boutons, were labeled with several calcium indicators. When parallel fibers were extracellularly activated with stimulus trains, calcium accumulated in the terminals, producing changes in the fluorescence of the indicators. During the stimulus train, the fluorescence change per pulse became progressively smaller with the high affinity indicators Fura-2 and calcium green-2 but remained constant with the low affinity dyes BTC and furaptra. In addition, fluorescence transients of high affinity dyes were slower than those of low affinity indicators, which appear to accurately report the time course of calcium transients. Simulations show that differences in the observed transients can be explained by the different affinities and off rates of the fluorophores. The return of [Ca2+]i to resting levels can be approximated by an exponential decay with a time constant of 150 ms. On the basis of the degree of saturation in the response of high affinity dyes observed during trains, we estimate that each action potential increases [Ca2+]i in the terminal by several hundred nanomolar. These findings indicate that in these terminals [Ca2+]i transients are much larger and faster than those observed in larger boutons, such as those at the neuromuscular junction. Such rapid [Ca2+]i dynamics may be found in many of the terminals in the mammalian brain that are similar in size to parallel fiber boutons.

After exocytosis, synaptic vesicles are recycled locally in the synaptic terminal and are refilled with neurotransmitter via vesicular transporters. The biophysical mechanisms of refilling are poorly understood, but it is clear that the generation of a proton gradient across the vesicle membrane is crucial. To better understand the determinants of vesicle refilling, we developed a novel method to measure unambiguously the kinetics of synaptic vesicle reacidification at individual synaptic terminals. Hippocampal neurons transfected with synapto-pHluorin (SpH), a synaptic vesicle-targeted lumenal GFP (green fluorescent protein), whose fluorescence is quenched when protonated (pK a ϳ 7.1), were rapidly surface-quenched immediately after trains of repetitive electrical stimulation. The recently endocytosed alkaline pool of SpH is protected from such surface quenching, and its fluorescence decay reflects reacidification kinetics. These measurements indicate that, after compensatory endocytosis, synaptic vesicles reacidify with first-order kinetics ( ϳ 4 -5 s) and that their rate of reacidification is subject to slowing by increased external buffer.