http://repub.eur.nl/res/aut/15569/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/27596/ 2010-03-26T00:00:00Z Synaptic vesicle fusion in brain synapses occurs in phases that are either tightly coupled to action potentials (synchronous), immediately following action potentials (asynchronous), or as stochastic events in the absence of action potentials (spontaneous). Synaptotagmin-1, -2, and -9 are vesicleassociated Ca2+sensors for synchronous release. Here we found that double C2 domain (Doc2) proteins act as Ca2+sensors to trigger spontaneous release. Although Doc2 proteins are cytosolic, they function analogously to synaptotagmin-1 but with a higher Ca2+sensitivity. Doc2 proteins bound to N-ethylmaleimidesensitive factor attachment receptor (SNARE) complexes in competition with synaptotagmin-1. Thus, different classes of multiple C2 domain-containing molecules trigger synchronous versus spontaneous fusion, which suggests a general mechanism for synaptic vesicle fusion triggered by the combined actions of SNAREs and multiple C2 domain-containing proteins. http://repub.eur.nl/res/pub/15038/ 2009-03-04T00:00:00Z In this thesis, we first examined how the calyx of Held forms during the first postnatal days and what the function of calyceal filopodia is (Chapter 2). Furthermore, we investigated whether forms of synaptic plasticity longer than facilitation, could be induced at the calyx of Held synapse. In contrast to a study that showed post-tetanic depression (Forsythe et al., 1998), we found that EPSCs were much larger after prolonged stimulation (Chapter 3). We set out to examine what made this synapse double its output after a period of high activity. We used several experimental approaches to try and answer this question. In particular we started by looking at spontaneous release, trains of action potentials and levels of residual calcium. We combined pre- and postsynaptic recordings of electrical activity and investigated calcium influx and calcium buffering during a single action potential (Chapter 4). Lastly, we compared changes in Pr and RRP more closely duri! ng the decay of PTP and extended our analysis to PTP at physiological temperature (Chapter 5). http://repub.eur.nl/res/pub/29094/ 2008-04-08T00:00:00Z The calyx of Held is probably the largest synaptic terminal in the brain, forming a unique one-to-one connection in the auditory ventral brainstem. During early development, calyces have many collaterals, whose function is unknown. Using electrophysiological recordings and fast-calcium imaging in brain slices, we demonstrate that these collaterals are involved in synaptic transmission. We show evidence that the collaterals are pruned and that the pruning already begins 1 week before the onset of hearing. Using two-photon microscopy to image the calyx of Held in neonate rats, we report evidence that both axons and nascent calyces are structurally dynamic, showing the formation, elimination, extension, or retraction of up to 65% of their collaterals within 1 hour. The observed dynamic behavior of axons may add flexibility in the choice of postsynaptic partners and thereby contribute to ensuring that each principal cell eventually is contacted by a single calyx of Held. http://repub.eur.nl/res/pub/35404/ 2007-06-01T00:00:00Z The size of the readily releasable pool (RRP) of vesicles was measured in control conditions and during post-tetanic potentiation (PTP) in a large glutamatergic terminal called the calyx of Held. We measured excitatory postsynaptic currents evoked by a high frequency train of action potentials in slices of 4-11-day-old rats. After a tetanus the cumulative release during such a train was enlarged by approximately 50%, indicating that the size of the RRP was increased. The amount of enhancement depended on the duration and frequency of the tetanus and on the age of the rat. After the tetanus, the size of the RRP decayed more slowly (t1/2= 10 versus 3 min) back to control values than the release probability. This difference was mainly due to a very fast initial decay of the release probability, which had a time constant compatible with an augmentation phase (τ ≈ 30 s). The overall decay of PTP at physiological temperature was not different from room temperature, but the increase in release probability (Pr) was restricted to the first minute after the tetanus. Thereafter PTP was dominated by an increase in the size of the RRP. We conclude that due to the short lifetime of the increase in release probability, the contribution of the increase in RRP size during post-tetanic potentiation is more significant at physiological temperature. © 2007 The Author. Journal compilation http://repub.eur.nl/res/pub/13673/ 2005-04-01T00:00:00Z We studied synaptic plasticity in the calyx of Held synapse, an axosomatic synapse in the auditory brainstem, by making whole-cell patch clamp recordings of the principal cells innervated by the calyces in a slice preparation of 7- to 10-day-old rats. A 5 min 20 Hz stimulus train increased the amplitude of excitatory postsynaptic currents (EPSCs) on average more than twofold. The amplitude of the synaptic currents took several minutes to return to control values. The post-tetanic potentiation (PTP) was accompanied by a clear increase in the frequency, but not the amplitude, of spontaneous EPSCs, which returned to baseline more rapidly than the potentiation of evoked release. The size of the readily releasable pool of vesicles was increased by about 30%. In experiments in which presynaptic measurements of the intracellular calcium concentration were combined with postsynaptic voltage clamp recordings, PTP was accompanied by an increase in the presynaptic calcium concentration to about 210 nM. The decay of the PTP matched the decay of this increase. When the decay of the calcium transient was shortened by dialysing the terminal with EGTA, the PTP decay sped up in parallel. Our experiments suggest that PTP at the calyx of Held synapse is due to a long-lasting increase in the presynaptic calcium concentration following a tetanus, which results in an increase in the release probability of the vesicles of the readily releasable pool. Although part of the PTP can be explained by a direct activation of the calcium sensor for phasic release, other mechanisms are likely to contribute as well.