Timothy A. Ryan

The kinetics of synaptic vesicle recycling measured at single presynaptic boutons

We used the fluorescent membrane probe FM 1-43 to label recycling synaptic vesicles within the presynaptic boutons of dissociated hippocampal neurons in culture. Quantitative time-lapse fluorescence imaging was employed in combination with rapid superfusion techniques to study the dynamics of synaptic vesicles within single boutons. This approach enabled us to measure exocytosis and to analyze the kinetics of endocytosis and the preparation of endocytosed vesicles for re-release (repriming). Our measurements indicate that under sustained membrane depolarization, endocytosis persists much longer than exocytosis, with a t1/2 approximately 60 s (approximately 24 degrees C); once internalized, vesicles become reavailable for exocytosis in approximately 30 s. Furthermore, we have shown that endocytosis is not dependent on membrane potential and, unlike exocytosis, that it is independent of extracellular Ca2+.

Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system.

Following the fusion of synaptic vesicles with the presynaptic plasma membrane of nerve terminals by the process of exocytosis, synaptic-vesicle components are recycled to replenish the vesicle pool. Here we use a pH-sensitive green fluorescent protein to measure the residence time of VAMP, a vesicle-associated SNARE protein important for membrane fusion, on the surfaces of synaptic terminals of hippocampal neurons following exocytosis. The time course of VAMP retrieval depends linearly on the amount of VAMP that is added to the plasma membrane, with retrieval occurring between about 4 seconds and 90 seconds after exocytosis, and newly internalized vesicles are rapidly acidified. These data are well described by a model in which endocytosis appears to be saturable, but proceeds with an initial maximum velocity of about one vesicle per second. We also find that, following exocytosis, a portion of the newly inserted VAMP appears on the surface of the axon.

The Use of pHluorins for Optical Measurements of Presynaptic Activity

Genetically encoded reporters for optical measurements of presynaptic activity hold significant promise for measurements of neurotransmission within intact or semi-intact neuronal networks. We have characterized pH-sensitive green fluorescent protein-based sensors (pHluorins) of synaptic vesicle cycling at nerve terminals. pHluorins have a pK approximately 7.1, which make them ideal for tracking synaptic vesicle lumen pH upon cycling through the plasma membrane during action potentials. A theoretical analysis of the expected signals using this approach and guidelines for future reporter development are provided.

Impaired PtdIns(4,5)P2 synthesis in nerve terminals produces defects in synaptic vesicle trafficking.

Phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) has an important function in cell regulation both as a precursor of second messenger molecules and by means of its direct interactions with cytosolic and membrane proteins. Biochemical studies have suggested a role for PtdIns(4,5)P2 in clathrin coat dynamics, and defects in its dephosphorylation at the synapse produce an accumulation of coated endocytic intermediates. However, the involvement of PtdIns(4,5)P2 in synaptic vesicle exocytosis remains unclear. Here, we show that decreased levels of PtdIns(4,5)P2 in the brain and an impairment of its depolarization-dependent synthesis in nerve terminals lead to early postnatal lethality and synaptic defects in mice. These include decreased frequency of miniature currents, enhanced synaptic depression, a smaller readily releasable pool of vesicles, delayed endocytosis and slower recycling kinetics. Our results demonstrate a critical role for PtdIns(4,5)P2 synthesis in the regulation of multiple steps of the synaptic vesicle cycle.

Synapsin dispersion and reclustering during synaptic activity.

Presynaptic modulation of synaptic transmission provides an important basis for control of synaptic function. The synapsins, a family of highly conserved proteins associated with synaptic vesicles, have long been implicated in the regulation of neurotransmitter release. However, direct physiological measurements of the molecular mechanisms have been lacking. Here we show that in living hippocampal terminals, green fluorescent protein (GFP)-labeled synapsin Ia dissociates from synaptic vesicles, disperses into axons during action potential (AP) firing, and reclusters to synapses after the cessation of synaptic activity. Using various mutated forms of synapsin Ia that prevent phosphorylation at specific sites, we performed simultaneous FM 4-64 measurements of vesicle pool mobilization along with synapsin dispersion kinetics. These studies indicate that the rate of synapsin dispersion is controlled by phosphorylation, which in turn controls the kinetics of vesicle pool turnover. Thus synapsin acts as a phosphorylation-state-dependent regulator of synaptic vesicle mobilization, and hence, neurotransmitter release.

Actin has a molecular scaffolding, not propulsive, role in presynaptic function

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, ∼30% of actin was found to be in a polymerized but dynamic state, with a remodeling time scale of ∼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.

Vesicle pool mobilization during action potential firing at hippocampal synapses

Using the fluorescent membrane label FM 1-43, we have measured the release, reuptake, and repriming of synaptic vesicles in response to action potential stimulation of cultured hippocampal neurons. We find that approximately 90% of a recycling vesicle pool is released during 60 s of 10 Hz action potential firing, and that a single action potential releases approximately 0.5% of that pool. Our data also indicate that endocytic reuptake of vesicle membrane externalized by 10 Hz action potentials lags exocytosis, with a half-time on the order of 20 s, and that the minimum time for repriming of an endocytosed vesicle is on the order of 15 s. Finally, we find that once vesicles have undergone this repriming period, they become functionally mixed in the vesicle pool within a few minutes; the probability of release for recently recycled vesicles is indistinguishable from that of vesicles that have resided within the bouton for much longer periods.

Synaptic Vesicle Mobilization Is Regulated by Distinct Synapsin I Phosphorylation Pathways at Different Frequencies

During action potential firing, the rate of synapsin dissociation from synaptic vesicles and dispersion into axons controls the rate of vesicle availability for exocytosis at the plasma membrane. Here we show that synapsin Ias dispersion rate tracks the synaptic vesicle pool turnover rate linearly over the range 5-20 Hz and that the molecular basis for this lies in regulation at both the calcium-calmodulin-dependent kinase (CaM kinase) and the mitogen-activated protein (MAP) kinase/calcineurin sites. Our results show that CaM kinase sites control vesicle mobilization at low stimulus frequency, while MAP kinase/calcineurin sites are critical at both lower and higher stimulus frequencies. Thus, multiple signaling pathways serve to allow synapsins control of vesicle mobilization over different stimulus frequencies.

Calcium accelerates endocytosis of vSNAREs at hippocampal synapses

A pH-sensitive form of green-fluorescent protein (GFP) fused to the lumenal domain of VAMP (synapto-pHluorin) provides a sensitive optical probe to track the net balance between exocytosis and endocytosis of this protein at small synaptic terminals of the central nervous system. Here we used a reversible proton-pump blocker that prevents vesicle re-acidification upon endocytosis to trap vesicles in the alkaline state during recycling. In combination with optical measurements of synapto-pHluorin, we used alkaline trapping to examine the kinetic components of exocytosis and endocytosis separately at synaptic terminals. Using this approach, we show that, in addition to controlling exocytosis, intracellular calcium levels tightly regulate the speed of endocytosis, increasing it to a maximal speed of approximately one vesicle per second.

Decreased synaptic vesicle recycling efficiency and cognitive deficits in amphiphysin 1 knockout mice.

The function of the clathrin coat in synaptic vesicle endocytosis is assisted by a variety of accessory factors, among which amphiphysin (amphiphysin 1 and 2) is one of the best characterized. A putative endocytic function of amphiphysin was supported by dominant-negative interference studies. We have now generated amphiphysin 1 knockout mice and found that lack of amphiphysin 1 causes a parallel dramatic reduction of amphiphysin 2 selectively in brain. Cell-free assembly of endocytic protein scaffolds is defective in mutant brain extracts. Knockout mice exhibit defects in synaptic vesicle recycling that are unmasked by stimulation and suggest impairments at multiple stages of the cycle. These defects correlate with increased mortality due to rare irreversible seizures and with major learning deficits, suggesting a critical role of amphiphysin for higher brain functions.