Vesicle pools and short-term synaptic depression: lessons from a large synapse

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Vesicle pools and short-term synaptic depression: lessons from a large synapse Ralf Schneggenburger, Takeshi Sakaba and Erwin Neher Depletion of a pool of readily releasable vesicles during repetitive presynaptic activity is a candidate mechanism for the induction of short-term synaptic depression. The large, calyx-type synaptic terminals in the brainstem auditory pathway, and especially the calyx of Held, offer unique possibilities for studying the cellular mechanisms leading to synaptic depression. Recent work at these synapses using presynaptic whole-cell patch-clamp recordings has revealed a large pool of readily releasable vesicles. During prolonged presynaptic depolarization, vesicles are released in kinetically distinct phases, indicating heterogeneity of release probability between vesicles. Heterogeneity might endow synapses with a rapid phase of depression at the onset of activity, followed by sustained and surprisingly large synaptic strength during the steady-state phase of depression. By influencing the synaptic output during repetitive activity, vesicle pool dynamics are expected to modulate information processing in neuronal networks of the CNS.

Ralf Schneggenburger* Takeshi Sakaba Erwin Neher Max-Planck Institut für Biophysikalische Chemie, Abteilung Membranbiophysik, Am Fassberg, D-37077 Göttingen, Germany. *e-mail: rschneg@ gwdg.de

Short-term plasticity of synaptic transmission is recognized as an important element of information processing in neuronal networks [1–3]. Upon repetitive presynaptic action-potential (AP) activity, the postsynaptic response will not follow with uniform strength, but will be modified in a time- and activity-dependent manner, leading either to facilitation, or to depression, or to a mixture of both forms of short-term plasticity. In recent years, synaptic depression has been studied at large, excitatory synapses with calyceal (cup-shaped) morphology that are located in the auditory pathway (Fig. 1). This review focuses on recent progress that has been made in studying the role of vesicle pool dynamics in synaptic depression. The conceptual basis for understanding the strength of a given synaptic connection has been established by the quantal hypothesis, originating from work on the nerve–muscle synapse [4]. This concept, combined with the binomial model of synaptic transmission [5–7], states that the amplitude of a postsynaptic current (PSC) is determined by the postsynaptic quantal size q, the number N of release sites, and the probability p that release of a quantum of transmitter occurs at a site, according to the equation PSC = Npq

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This concept is still a useful framework, separating the possible pre- and postsynaptic parameters that are subject to modification during short-term plasticity. It also serves to identify the site http://tins.trends.com

of action of molecular targets that mediate signaling steps in synaptic transmission. Depending on the definition of N, two meanings of release probability p have emerged. If N is regarded as the number of vesicles (Nves) in the readily releasable pool, then p refers to the probability that a vesicle is released from the pool (pves). By contrast, if N is regarded as the number of morphologically defined active zones (Naz) then p will deal with the probability that vesicle fusion occurs at a given active zone (paz). Knowledge of release probability p is of crucial importance for understanding the contribution of vesicle pool depletion to synaptic depression. This is because a term (1−p) is proportional to the number of vesicles (or, alternatively, to the number of active zones) remaining a short time after a presynaptic stimulus, when recovery is still negligible [8–10]. The number of readily releasable vesicles, Nves, and the number of active zones, Naz, can be used to derive the average release probabilities pves and paz, given that the number of vesicles released during a presynaptic AP (the ‘quantal content’) is known. Thus, determining the quantal content, and Nves and Naz, is an important prerequisite for understanding the role of vesicle pool dynamics in short-term plasticity at a given synaptic connection. Calyceal synapses in the auditory pathway as models for transmission

Synaptic transmission has been studied at the ‘endbulb of Held’ synapses in the ventral cochlear nucleus (VCN; Fig. 1a), or at the homologous synapses in the nucleus magnocellularis of the avian auditory system [11], as well as at the ‘calyx of Held’ synapses in the medial nucleus of the trapezoid body (MNTB). The calyx of Held is a projection of the globular bushy cells in the VCN onto principal cells in the MNTB. Each principal neuron in the MNTB receives input from only one calyx-type axon terminal [12,13] (Fig. 1). Owing to their unusually large size of ~10–15 µm, these nerve terminals are accessible to whole-cell patch-clamp recording [14,15], offering unique advantages for studying the cellular mechanisms of short-term depression. Thus, the presynaptic membrane potential can be measured and controlled by voltage-clamp, and Ca2+ currents

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can be recorded directly from the presynaptic compartment [15–22]. Furthermore, whole-cell patch-clamp has been used to modify the biochemical state of the presynaptic cytosol [23], and to introduce Ca2+ indicators [24] and caged-Ca2+ compounds [25–27], an experimental approach that has led to the quantification of the intracellular Ca2+ sensitivity of synaptic vesicle fusion [26,27]. At the calyceal synapses in the auditory system, release of glutamate activates postsynaptic AMPA- as well as NMDA-receptors [11,12,28]. The amplitude of the NMDA component of the EPSCs is, however, downregulated during postnatal development [29–31]. Calyceal synapses are specialized for rapid information transfer [32]. The release of many quanta in a short period of time rapidly depolarizes the postsynaptic neuron and guarantees that a presynaptic AP is reliably, and with minimal time delay, converted into a postsynaptic spike. One prerequisite for this function is the large number of active zones within calyceal nerve terminals. The number of active zones, Naz, at calyces of Held from 8–10-day-old rats has been estimated by two approaches. First, an ultrastructural analysis with electron microscopy and serial reconstruction of an entire calyx of Held has shown 600 morphologically defined active zones [33]. Second, a non-stationary EPSC variance analysis has given an estimate of the binomial parameter N with 637 ± 117 (mean ± SEM), interpreted to reflect an upper limit of the number of functional active zones [34]. The two estimates are in good agreement, and show that calyces of Held from postnatal day 8–10 rats – used for many of the functional studies reviewed here – already have a large number of active zones. On the ultrastructural level, single active zones of calyceal synapses are similar to those of small, bouton-like central synapses [33,35–37], although differences have also been observed [33,37]. Calyceal synapses can thus be regarded as parallel arrangements of several hundred conventional active zones, which are activated by a presynaptic AP in a single, large axon terminal. Quantal content and number of releasable vesicles with AP stimulation

When the calyx of Held was stimulated with single presynaptic APs (Fig. 2a), EPSCs in the range of 1–15 nA were observed at negative holding potentials of −70 to –80 mV [15,16,25,27,30,38–40]. The variability in EPSC amplitude was largely caused by cell-to-cell differences [25,30,38]. In the example shown in Fig. 2a, an EPSC of ~5 nA was observed. When this value is divided by the quantal size estimated from mEPSC amplitude distributions of spontaneous, miniature EPSCs (mEPSCs; 30–40 pA on average at –70 to –80 mV [16,25,34,39–41]), a quantal content of ~150 can be calculated, assuming that quanta add up linearly to form an evoked EPSC. Quantal contents at the calyx of Held have been http://tins.trends.com

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Fig. 1. Calyceal synapses in the mammalian auditory pathway. (a) Calyx-like synaptic connections of the auditory brainstem pathway of mammals (modified from Ref. [13]). Several auditory fibers arising from the spiral ganglion in the inner ear make synapses onto a given bushy cell in the anterior part of the ventral cochlear nucleus (VCN), forming ‘endbulb of Held’ synapses. The ‘calyx of Held’ synapses arise from a projection of globular bushy cells onto inhibitory principal cells of the contralateral medial nucleus of the trapezoid body (MNTB). The balance of synaptic excitation (from the VCN) and inhibition (from the MNTB) in the lateral superior olive (LSO) is thought to play a role in sound source localization [32,76]. (b) Image of a calyx of Held nerve terminal filled with a fluorescent Ca2+ indicator dye via a whole-cell patch-clamp pipette (Pre). Scale bar, 10 µm. Reproduced, with permission, from Ref. [27].

estimated at 210 ± 22 [16], 140 [25], 178 ± 86 [27] and at an average of 157 [34]. In two studies [34,41], EPSC variance analysis was used to estimate quantal sizes during evoked transmission. As these were similar to the quantal size from mEPSC amplitude distributions [34,41], the assumption of linear summation of quanta was verified for AP-evoked EPSCs under conditions of normal release probability. Relating the range of average quantal contents found in the various studies (150–210) to the estimated number of active zones at the calyx of Held (~600; see Refs [33,34]), it can be concluded that a given active zone is used with fairly low probability (paz ∼0.25–0.4) during a single AP stimulus (Fig. 2a, right panel). When stimulated repetitively with presynaptic APs, the calyx of Held [15,19,23,25,26,30,34,38,40–42], as well as calyceal synapses in the VCN [43,44] and chick nucleus magnocellularis [11,45,46], showed strong synaptic depression. The number of readily releasable vesicles, Nves, has been estimated at the calyx of Held by summing up peak EPSC amplitudes during depressing trains at high stimulation frequencies (Fig. 2b; see Refs [25,26,30,40]), in an analysis similar to the one first applied at the nerve–muscle synapse

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Fig. 2. Estimates of quantal content, and the size of a readily releasable vesicle pool at the calyx of Held synapse. (a) Simultaneous whole-cell recording of a presynaptic action potential (AP) after afferent fiber stimulation (upper trace), and the AP-evoked EPSC (lower trace) (reproduced, with permission, from Ref. [15]). It is thought that stimulation with a single AP leads to vesicle fusion at one out of roughly three active zones at the calyx of Held, as illustrated in the scheme on the right-hand side. (b) Pool size estimate after afferent fiber stimulation at 100 Hz with 4 mM extracellular Ca2+. The cumulative plot of peak EPSC amplitudes (inset) was fitted with a line for times larger than 100 ms (red line), and back-extrapolated to time 0 (reproduced, with permission, from Ref. [25]). During short trains of APs, it is thought that vesicle fusion occurs once or twice at a given active zone. (c) Estimates of the time course of quantal release and vesicle fusion after stimulating the calyx of Held with prolonged (>10 ms) presynaptic Ca2+ currents (top). The EPSC was recorded with cyclothiazide and kynurenic acid (middle) to avoid use-dependent decreases in postsynaptic quantal size, q. Cumulative transmitter release was estimated by EPSC deconvolution (bottom; reproduced, with permission, from Ref. [54]). The inset shows the increase in presynaptic membrane capacitance (∆Cm) after step depolarizations with various durations (reproduced, with permission, from Ref. [57]). The estimated time-courses of pool depletion are similar for capacitance measurements and EPSC deconvolution. During such prolonged presynaptic Ca2+ currents, vesicle fusion probably occurs several times within the first few milliseconds at a given active zone [57].

[47]. This analysis assumes that transmission during the steady-state phase of depression is limited by a constant re-supply of vesicles. The contribution of re-supply was eliminated by back-extrapolating a linear fit to the late phase (>100 ms) in the cumulative EPSC amplitude plot to time zero (Fig. 2b, inset). A pool size was then estimated by dividing the cumulative EPSC amplitude by the quantal size (~30–40 pA). The resulting value of 600–700 was http://tins.trends.com

considered to represent the pool of vesicles immediately available during short trains of high frequency stimulation [25]. This pool size estimate has been shown to increase with postnatal maturation [30,40]. One of the assumptions underlying the analysis in Fig. 2b is that synaptic depression is mediated exclusively by presynaptic mechanisms. However, postsynaptic factors, such as desensitization [11,45,46,48] or saturation of AMPA receptors [48] have been shown to contribute to synaptic depression at calyceal synapses. Therefore, a reduction in postsynaptic quantal size has to be taken into account. This has been done recently in non-stationary EPSC fluctuation analysis [49], in which the analysis of means, variances and covariances of successive EPSCs was used to calculate the average quantal size q for each EPSC in the train. The quantal size was reduced to ~40% of its initial value for the 3rd to the 5th EPSC during 100 Hz trains at 4 mM Ca2+ [41]. This reduction was prevented by cyclothiazide and kynurenic acid, indicating that AMPA-receptor desensitization and saturation contributed to synaptic depression, in agreement with earlier results [11,45,46,48]. When the corrected quantal sizes were used for an estimate of cumulative release, an average of ~900 quanta was obtained [41]. Comparing this value with the estimated number of active zones (~600; [33,34]), it can be concluded that a given active zone is used, on average, once or twice during short trains of presynaptic APs (Fig. 2b, right panel). Pool size estimates using prolonged presynaptic Ca2++ stimuli

In the studies reviewed so far, EPSCs were evoked by presynaptic APs (Fig. 2a). During APs, the voltage-gated Ca2+ currents are brief, with a width at half amplitude of 360 ± 10 µs at room temperature [18]. This brief Ca2+ influx can be regarded as a ‘delta-pulse’-like stimulus, during which the release probability rises and falls rapidly, with an estimated half-width of <0.5 ms at calyceal synapses [16,27,50]. Therefore, the amount of transmitter release is approximately proportional to the EPSC amplitude, if the time constant of decay of the underlying postsynaptic quantal event is sufficiently long. Because the calyx of Held is accessible to wholecell patch-clamp recordings, presynaptic Ca2+ currents can be activated by prolonged voltage-clamp steps (Fig. 2c). The depolarization-evoked transmitter release will then continue for times that are longer than the decay of the underlying quantal events (which, for AMPA-receptor-mediated EPSCs, is <1 ms because the auditory neurons express AMPA-receptor subunits that confer rapid gating properties) [51,52]. Thus, the EPSC amplitude no longer represents a useful measure of the amount of transmitter release.

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To estimate transmitter release during prolonged stimuli faithfully, two alternative methods have been used at the calyx of Held. First, transmitter release rates were extracted by deconvolving the evoked EPSCs with the mEPSC waveform [48,53,54], after correcting for the effects of glutamate spill-over [11,55,56]. These experiments were done in the presence of cyclothiazide and kynurenic acid to minimize use-dependent decreases in q caused by desensitization and/or saturation of AMPA-receptors (see previous discussion). Second, presynaptic wholecell capacitance was used as a measure of synaptic vesicle fusion at the calyx of Held [57]. Assuming an average vesicle diameter and a specific membrane capacitance, the observed capacitance changes were converted to the number of vesicles. In both types of study [53,54,57], significantly larger pool sizes were obtained than from high frequency AP stimulation. With a presynaptic voltage step to −10 mV, cumulative transmitter release estimated from deconvolution analysis slowed down after a few milliseconds despite a constant presynaptic Ca2+ current (Fig. 2c), indicating the depletion of a pool of readily releasable vesicles. Estimates of Nves were 2409 ± 415 (ranging from 1500 to 4000 between cells) [54] and 2246 ± 303 [53]. Similarly, when the calyx of Held was stimulated by uncaging of Ca2+, EPSC deconvolution showed that 1785 ± 870 vesicles (ranging 800–3500) were released within 10 ms, provided the presynaptic Ca2+ concentration was higher than ~10 µM [27]. When transmitter release was measured with capacitance following Ca2+ currents of varying duration [57], the time-course of pool depletion was found to be 2.9 ms (Fig. 2c, inset). This is similar to the time-course of cumulative release from EPSC deconvolution (Fig. 2c, lower panel). The estimate for Nves from capacitance measurements was 3300–5200, somewhat larger than the ones from EPSC deconvolution analysis. However, considering the limitations of each method, the agreement in pool size estimate between EPSC deconvolution and capacitance measurements is reasonable. These studies show that, upon strong stimulation with prolonged Ca2+ currents, each active zone must be used several times within <5 ms (Fig. 2c, right panel). Therefore, multivesicular release [58–60] probably occurs at individual active zones under such stimulus conditions. The pool size estimates based on prolonged Ca2+ stimuli, in which release was estimated by EPSC deconvolution or capacitance measurements (~1800 [27], 2400 [54] and 3300–5200 [57]) are significantly larger than the ones based on summing peak EPSC amplitudes during trains of afferent fiber stimulations (~700 [25], ~810 [26], ~380–940 at various stages of development [30], and ~900 after correction for the reduction in q during depression [41]). This might indicate that only a subset of the pool is released during short high frequency trains, http://tins.trends.com

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owing to heterogeneity of release probability [53,54]. However, differences between the two methods must also be considered. Thus, the analysis of peak EPSC amplitudes (Fig. 2b) does not account for asynchronously released vesicles; by contrast, such vesicles will be detected by EPSC deconvolution [48] and capacitance measurements [57]. Indications of heterogeneous release probability

Comparing the quantal content of EPSCs evoked by presynaptic APs (~150–210; see Fig. 2a and Refs [16,25,27,34]) to the number of readily releasable vesicles Nves estimated by strong, pool-depleting stimuli (2000–5000; [53,54,57]), an average pves of 0.03–0.10 is calculated. This value is too small to induce significant vesicle pool depletion in a simple model that assumes the release of a constant fraction of a homogenous pool of available resources by each AP [8]. Heterogeneous release probability between vesicles in the readily releasable pool might point to a solution of this paradox. Non-uniform release probabilities have previously been reported at central excitatory synapses [61–64], and probably reflect heterogeneity in p between different active zones [64]. At the calyx of Held, the first indications of heterogeneity in p came from the study of Wu and Borst [65]. They stimulated transmitter release during recovery from synaptic depression by AP-like depolarization, followed by a prolonged, 10 ms step depolarization [65]. The EPSC amplitude in response to prolonged steps recovered faster than the EPSC triggered by AP-like depolarizations, and the release kinetics in response to prolonged steps was slowed during the recovery phase. This indicated that release probability was not constant during recovery from depression, showing the importance of separating changes in p and N during use-dependent changes of synaptic transmission (see also Ref. [66]). Evidence for heterogeneity of p was also obtained for prolonged step depolarizations under resting conditions of the synapse [53,54], by dissecting the time-course of transmitter release by EPSC deconvolution [48]. Two phases of cumulative transmitter release, with time constants of 3.1 ± 0.2 ms and 30.5 ± 2.0 ms [54], could be distinguished with 0.5 mM EGTA in the presynaptic terminal, indicating the presence of both a rapidly and a slowly releasing pool of vesicles. The slowly releasing pool had an intrinsically fast recovery from depletion (~200 ms), whereas the rapidly releasing pool recovered on a much slower time scale, taking seconds [54]. This difference in recovery time can explain the change in p during recovery from depression [65], if it is assumed that vesicles in the slowly releasing pool have low p upon AP-stimulation. Furthermore, Sakaba and Neher [54] showed that the intrinsically slow recovery of the rapidly releasing pool can be speeded up in a Ca2+– calmodulin dependent manner. This confirms,

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Fig. 3. Cellular mechanisms leading to short-term synaptic depression. Three classes of mechanisms, their sites of action, and the quantal parameter that is affected in each case, are shown. (a) Decreased presynaptic Ca2+ influx might occur after changes in the presynaptic action potential (AP) waveform during depression, or because of other mechanisms that cause a decreased Ca2+ current (ICa) such as Ca2+ current inactivation [19], feedback inhibition via metabotropic autoreceptors [38,40], or depletion of extracellular Ca2+ from the synaptic cleft [79]. These mechanisms will lead to a reduction of release probability, p. (b) A reduction in the number of release-competent vesicles can occur because of depletion of a pool of vesicles with initially high p, or otherwise because of an a posteriori reduction of release probability of those vesicles remaining after a first round of exocytosis. This class of mechanisms will manifest itself as a change in p, or alternatively N. (c) The size of the postsynaptic quantal size, q might be reduced by desensitization and/or saturation of postsynaptic receptor channels.

and extends, previous observations of a Ca2+-dependent step during recovery from synaptic depression [42,67,68]. The consequences of slowly and rapidly releasing vesicle pools [54] for transmitter release during trains of presynaptic APs are not understood in detail, but it can be assumed that the two pools will cause heterogeneity in p during AP stimulation. Because the intracellular Ca2+ signal associated with presynaptic APs is significantly shorter (~0.5 ms; [26,27]) than the estimated release time constant of the slow pool (~10 ms at physiological Ca2+ buffering [54]), it is likely that the slow pool is hardly tapped by AP-evoked release early during stimulus trains, whereas the rapidly releasing pool might undergo (partial) depletion. During the sustained phase of depression, the initially reluctantly releasable pool of slow vesicles [53,54] might be released more effectively, either because of Ca2+ build-up and subsequent facilitation of transmitter release [65], and/or because of mobilization via another, as yet unknown, intracellular second messenger postulated to act on the synaptic vesicle priming factor munc13-1 [69]. Several mechanisms might account for heterogeneity of p between vesicles. First, intrinsic, molecular differences between rapidly and slowly releasing vesicles could cause different release rates following an otherwise similar presynaptic Ca2+ signal. In terms of kinetic models for Ca2+ binding and vesicle fusion [26,27,70,71], this would imply that vesicles differ in Ca2+-sensitivity, or in the maximal fusion rate upon full binding of Ca2+ to the Ca2+ sensor http://tins.trends.com

for vesicle fusion. Differences in the Ca2+ binding kinetics and maximal fusion rates have been shown for a fast and a slow vesicle pool in adrenal chromaffin cells [70,71]. Additionally, or alternatively, vesicles could differ in their location relative to presynaptic Ca2+ channels. This implies that different vesicles ‘see’ different intracellular Ca2+ signals, causing vesicle fusion to occur at different rates [72]. Yet another possibility is that heterogeneity of p is induced only after a preceding stimulus, such that vesicles remaining after a first round of exocytosis would experience a decrease in p (Fig. 3b). Such a posteriori heterogeneity mechanisms would include Ca2+ adaptation of the vesicle fusion machinery [73] and ‘lateral inhibition’ between vesicles in the readily releasable pool [74,75]. Cellular mechanisms involved in synaptic depression

Further cellular mechanisms upstream (Fig. 3a) or downstream (Fig. 3c) of vesicle fusion have been shown to participate in generating short-term synaptic depression, as reviewed recently [76]. Briefly, a decrease in presynaptic Ca2+ influx during trains of presynaptic APs leading to reduced release probability p (Fig. 3a) might be caused by changes in AP waveform [77] (see also Ref. [78]), by Ca2+ current inactivation [19], by feedback activation [38,40] of metabotropic glutamate receptors known to downregulate presynaptic Ca2+ currents via G-protein activation [17,20], or by depletion of Ca2+ from the synaptic cleft [79]. However, these mechanisms are probably not major contributors to depression at the calyx of Held of postnatal-day 8–10 rats, because the first and the last AP in a train of 20 stimuli induce the same integral Ca2+ influx, despite changes in AP waveform [77]. Apart from presynaptic mechanisms, postsynaptic factors contribute to synaptic depression (Fig. 3c). Thus, a reduction in quantal size has been demonstrated in chick calyceal synapses [11,45,46] as a consequence of AMPA-receptor desensitization, as well as in calyceal synapses in the VCN [44] and at the calyx of Held [41,48]. Conclusion

Studies using presynaptic whole-cell voltage-clamp experiments, combined with EPSC deconvolution [27,48,53,54] or presynaptic capacitance measurements [57], have revealed a large number of releasable vesicles at the calyx of Held synapse. For a realistic prediction of synaptic output during repetitive activity, it is thus necessary to consider heterogeneity in release probability [53,54,65] and further cellular mechanisms that participate in generating short-term synaptic depression (Fig. 3). Heterogeneity of p is just emerging as a factor in shaping the synaptic output during trains of presynaptic activity. Questions to be answered in the

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near future are: (1) Is heterogeneity of p caused by intrinsic differences between vesicles, or by differential localization of vesicles with respect to Ca2+ channels? (2) Which molecular factors determine the differences in p, and is there differential modulation of rapidly and slowly releasable vesicle pools by second messengers [80] or during postnatal development? (3) How does References 1 Abbott, L.F. et al. (1997) Synaptic depression and cortical gain control. Science 275, 220–224 2 Tsodyks, M.V. and Markram, H. (1997) The neural code between neocortical pyramidal neurons depends on neurotransmitter release probability. Proc. Natl. Acad. Sci. U. S. A. 94, 719–723 3 Nadim, F. et al. (1999) Synaptic depression creates a switch that controls the frequency of an oscillatory circuit. Proc. Natl. Acad. Sci. U. S. A. 96, 8206–8211 4 Katz, B. (1969) The Release of Neural Transmitter Substances, Liverpool University Press 5 Johnson, E.W. and Wernig, A. (1971) The binomial nature of transmitter release at the crayfish neuromuscular junction. J. Physiol. 218, 757–767 6 Zucker, R. (1973) Changes in the statistics of transmitter release during facilitation. J. Physiol. 229, 787–810 7 Quastel, D.M.J. (1997) The binomial model in fluctuation analysis of quantal neurotransmitter release. Biophys. J. 72, 728–753 8 Liley, A.W. and North, K.A.K. (1953) An electrical investigation of effects of repetitive stimulation on mammalian neuromuscular junction. J. Neurophysiol. 16, 509–527 9 Weis, S. et al. (1999) Properties of a model of Ca++-dependent vesicle pool dynamics and short term synaptic depression. Biophys. J. 77, 2418–2429 10 Dittman, J.S. et al. (2000) Interplay between facilitation, depression, and residual calcium at three presynaptic terminals. J. Neurosci. 20, 1374–1385 11 Trussell, L.O. et al. (1993) Desensitization of AMPA receptors upon multiquantal neurotransmitter release. Neuron 10, 1185–1196 12 Forsythe, I.D. and Barnes-Davies, M. (1993) The binaural auditory pathway: excitatory amino acid receptors mediate dual timecourse excitatory postsynaptic currents in the rat medial nucleus of the trapezoid body. Proc. R. Soc. London Ser. B 251, 151–157 13 Forsythe, I.D. et al. (1995) The calyx of Held: a model for transmission at mammalian glutamatergic synapses. In Excitatory Amino Acids and Synaptic Transmission (Wheal, H. and Thomson, A., eds), pp. 133–144, Academic Press 14 Forsythe, I.D. (1994) Direct patch recording from identified presynaptic terminals mediating glutamatergic EPSCs in the rat CNS, in vitro. J. Physiol. 479, 381–387 15 Borst, J.G.G. et al. (1995) Pre- and postsynaptic whole-cell recordings in the medial nucleus of the trapezoid body of the rat. J. Physiol. 489, 825–840 16 Borst, J.G.G. and Sakmann, B. (1996) Calcium influx and transmitter release in a fast CNS synapse. Nature 383, 431–434 17 Takahashi, T. et al. (1996) Presynaptic calcium current modulation by metabotropic glutamate receptor. Science 274, 594–597

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heterogeneity of p between vesicles in the readily releasable pool contribute to the synaptic transfer function at different frequencies of presynaptic activity? Resolving some of these issues will allow us to gain deeper insights into the mechanisms of short-term plasticity, and their consequences for information processing in neuronal assemblies.

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Life span: getting the signal from the nervous system Catherine A. Wolkow Life span is determined by both environmental and genetic influences. The importance of genes is illustrated by the fact that single gene mutations extend life span in nematodes, fruit flies and mice. Recent reports reveal that the life span of Caenorhabditis elegans is controlled by insulin-like signals from the nervous system. Hormones that control life span have recently been identified in fruit flies and mice. These findings suggest that neuroendocrine pathways could constitute an important determinant of life span across phylogeny. This review examines the evidence for nervous system control of longevity and discusses the implications for popular models for aging.

Death is an inevitable event for all organisms. In the absence of accidents and illness, most species have a characteristic life span that is under genetic control. Life span can vary greatly, from the two weeks of Caenorhabditis elegans to the average 80 years of humans. Single gene mutations that can result in a http://tins.trends.com

significantly lengthened life span have been identified in yeast, nematodes, fruit flies and mice (see Ref. [1] for a recent review). Genetic studies of human longevity suggest that genes also influence human life span, albeit in a complicated fashion [2,3]. The free-radical theory of aging

The free-radical theory of aging hypothesizes that life span is limited by the accumulation of cellular damage from reactions with free radicals (see Ref. [4] for a review). Free radicals are produced primarily as by-products of metabolism but are also derived from environmental sources. A major source of free radicals is superoxide (−O•), produced in mitochondria. The cell clears superoxide by the action of superoxide dismutase (SOD), which converts superoxide into

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