Readily Releasable Pool Research Articles

Enhancing the fidelity of neurotransmission by activity-dependent facilitation of presynaptic potassium currents.

Neurons convey information in bursts of spikes across chemical synapses where the fidelity of information transfer critically depends on synaptic input-output relationship. With a limited number of synaptic vesicles (SVs) in the readily-releasable pool (RRP), how nerve terminals sustain transmitter release during intense activity remains poorly understood. Here we report that presynaptic K+ currents evoked by spikes facilitate in a Ca2+-independent but frequency- and voltage-dependent manner. Experimental evidence and computer simulations demonstrate this facilitation originates from dynamic transition of intermediate gating states of voltage-gated K+ channels (Kvs), and specifically attenuates spike amplitude and inter-spike potential during high-frequency firing. Single or paired recordings from a mammalian central synapse further reveal that facilitation of Kvs constrains presynaptic Ca2+ influx, thereby efficiently allocating SVs in the RRP to drive postsynaptic spiking at high rates. We conclude that presynaptic Kv facilitation imparts neurons with a powerful control of transmitter release to dynamically support high-fidelity neurotransmission.

Exposure to mission relevant doses of 1 GeV/Nucleon (56)Fe particles leads to impairment of attentional set-shifting performance in socially mature rats.

Previous ground-based experiments have shown that cranial irradiation with mission relevant (20 cGy) doses of 1 GeV/nucleon (56)Fe particles leads to a significant impairment in Attentional Set Shifting (ATSET) performance, a measure of executive function, in juvenile Wistar rats. However, the use of head only radiation exposure and the biological age of the rats used in that study may not be pertinent to determine the likelihood that ATSET will be impaired in Astronauts on deep space flights. In this study we have determined the impact that whole-body exposure to 10, 15 and 20 cGy of 1 GeV/nucleon (56)Fe particles had on the ability (at three months post exposure) of socially mature (retired breeder) Wistar rats to conduct the attentional set-shifting paradigm. The current study has established that whole-body exposures to 15 and 20 (but not 10) cGy of 1 GeV/nucleon (56)Fe particles results in the impairment of ATSET in both juvenile and socially mature rats. However, the exact nature of the impaired ATSET performance varied depending upon the age of the rats, whether whole-body versus cranial irradiation was used and the dose of 1 GeV/u (56)Fe received. Exposure of juvenile rats to 20 cGy of 1 GeV/nucleon (56)Fe particles led to a decreased ability to perform intra-dimensional shifting (IDS) irrespective of whether the rats received head only or whole-body exposures. Juvenile rats that received whole-body exposure also had a reduced ability to habituate to the assay and to complete intra-dimensional shifting reversal (IDR), whereas juvenile rats that received head only exposure had a reduced ability to complete compound discrimination reversal (CDR). Socially mature rats that received whole-body exposures to 10 cGy of 1 GeV/nucleon (56)Fe particles exhibited no obvious decline in set-shifting performance; however those exposed to 15 and 20 cGy had a reduced ability to perform simple discrimination (SD) and compound discrimination (CD). Exposure to 20 cGy of 1 GeV/nucleon (56)Fe particles also led to a decreased performance in IDR and to ∼25% of rats failing to habituate to the task. Most of these rats started to dig for the food reward but rapidly (within 15 s) gave up digging, suggesting that they had developed appropriate procedural memories about food retrieval, but had an inability to maintain attention on the task. Our preliminary data suggests that whole-body exposure to 20 cGy of 1 GeV/nucleon (56)Fe particles reduced the cholinergic (but not the GABAergic) readily releasable pool (RRP) in nerve terminals of the basal forebrain from socially-mature rats. This perturbation of the cholinergic RRP could directly lead to the loss of CDR and IDR performance, and indirectly [through the metabolic changes in the medial prefrontal cortex (mPFC)] to the loss of SD and CD performance. These findings provide the first evidence that attentional set-shifting performance in socially mature rats is impaired after whole-body exposure to mission relevant doses (15 and 20 cGy) of 1 GeV/nucleon (56)Fe particles, and importantly that a dose reduction down to 10 cGy prevents that impairment. The ability to conduct Discrimination tasks (SD and CD) and reversal learning (CDR) is reduced after exposure to 15 and 20 cGy of 1 GeV/nucleon (56)Fe particles, but at 20 cGy there is an additional decrement, ∼ 25% of rats are unable to maintain attention to task. These behavioral decrements are associated with a reduction in the cholinergic RRP within basal forebrain, which has been shown to play a major role in regulating the activity of the PFC.

Distinct actions of Rab3 and Rab27 GTPases on late stages of exocytosis of insulin.

Rab GTPases associated with insulin-containing secretory granules (SGs) are key in targeting, docking and assembly of molecular complexes governing pancreatic β-cell exocytosis. Four Rab3 isoforms along with Rab27A are associated with insulin granules, yet elucidation of the distinct roles of these Rab families on exocytosis remains unclear. To define specific actions of these Rab families we employ Rab3GAP and/or EPI64A GTPase-activating protein overexpression in β-cells from wild-type or Ashen mice to selectively transit the entire Rab3 family or Rab27A to a GDP-bound state. Ashen mice carry a spontaneous mutation that eliminates Rab27A expression. Using membrane capacitance measurements we find that GTP/GDP nucleotide cycling of Rab27A is essential for generation of the functionally defined immediately releasable pool (IRP) and central to regulating the size of the readily releasable pool (RRP). By comparison, nucleotide cycling of Rab3 GTPases, but not of Rab27A, is essential for a kinetically rapid filling of the RRP with SGs. Aside from these distinct functions, Rab3 and Rab27A GTPases demonstrate considerable functional overlap in building the readily releasable granule pool. Hence, while Rab3 and Rab27A cooperate to generate release-ready SGs in β-cells, they also direct unique kinetic and functional properties of the exocytotic pathway.

Syntaxin 1B, but Not Syntaxin 1A, Is Necessary for the Regulation of Synaptic Vesicle Exocytosis and of the Readily Releasable Pool at Central Synapses

Two syntaxin 1 (STX1) isoforms, HPC-1/STX1A and STX1B, are coexpressed in neurons and function as neuronal target membrane (t)-SNAREs. However, little is known about their functional differences in synaptic transmission. STX1A null mutant mice develop normally and do not show abnormalities in fast synaptic transmission, but monoaminergic transmissions are impaired. In the present study, we found that STX1B null mutant mice died within 2 weeks of birth. To examine functional differences between STX1A and 1B, we analyzed the presynaptic properties of glutamatergic and GABAergic synapses in STX1B null mutant and STX1A/1B double null mutant mice. We found that the frequency of spontaneous quantal release was lower and the paired-pulse ratio of evoked postsynaptic currents was significantly greater in glutamatergic and GABAergic synapses of STX1B null neurons. Deletion of STX1B also accelerated synaptic vesicle turnover in glutamatergic synapses and decreased the size of the readily releasable pool in glutamatergic and GABAergic synapses. Moreover, STX1A/1B double null neurons showed reduced and asynchronous evoked synaptic vesicle release in glutamatergic and GABAergic synapses. Our results suggest that although STX1A and 1B share a basic function as neuronal t-SNAREs, STX1B but not STX1A is necessary for the regulation of spontaneous and evoked synaptic vesicle exocytosis in fast transmission.

Temperature dependence of vesicular dynamics at excitatory synapses of rat hippocampus.

How vesicular dynamics parameters depend on temperature and how temperature affects the parameter change during prolonged high frequency stimulation was determined by fitting a model of vesicular storage and release to the amplitudes of the excitatory post-synaptic currents (EPSC) recorded from CA1 neurons in rat hippocampal slices. The temperature ranged from low (13°C) to higher and more physiological temperature (34°C). Fitting the model of vesicular storage and release to the EPSC amplitudes during a single pair of brief high-low frequency stimulation trains yields the estimates of all parameters of the vesicular dynamics, and with good precision. Both fractional release and replenishment rate decrease as the temperature rises. Change of the underlying 'basic' parameters (release coupling, replenishment coupling and readily releasable pool size), which the model-fitting also yields is complex. The replenishment coupling between the readily releasable pool (RRP) and resting pool increases with temperature (which renders the replenishment rate higher), but this is more than counterbalanced by greater RRP size (which renders the replenishment rate lower). Finally, during long, high frequency patterned stimulation that leads to significant synaptic depression, the replenishment rate decreases markedly and rapidly at low temperatures (<22°C), but at high temperatures (>28°C) the replenishment rate rises with stimulation, making synapses better able to maintain synaptic efficacy.

Stress and corticosterone increase the readily releasable pool of glutamate vesicles in synaptic terminals of prefrontal and frontal cortex

Stress and glucocorticoids alter glutamatergic transmission, and the outcome of stress may range from plasticity enhancing effects to noxious, maladaptive changes. We have previously demonstrated that acute stress rapidly increases glutamate release in prefrontal and frontal cortex via glucocorticoid receptor and accumulation of presynaptic SNARE complex. Here we compared the ex vivo effects of acute stress on glutamate release with those of in vitro application of corticosterone, to analyze whether acute effect of stress on glutamatergic transmission is mediated by local synaptic action of corticosterone. We found that acute stress increases both the readily releasable pool (RRP) of vesicles and depolarization-evoked glutamate release, while application in vitro of corticosterone rapidly increases the RRP, an effect dependent on synaptic receptors for the hormone, but does not induce glutamate release for up to 20 min. These findings indicate that corticosterone mediates the enhancement of glutamate release induced by acute stress, and the rapid non-genomic action of the hormone is necessary but not sufficient for this effect.

Modulation of Synaptic Vesicle Exocytosis in Muscle-Dependent Long-Term Depression at the Amphibian Neuromuscular Junction

We have labeled recycling synaptic vesicles at the somatic Bufo marinus neuromuscular junction with the styryl dye FM2-10 and provide direct evidence for refractoriness of exocytosis associated with a muscle activity-dependent form of long-term depression (LTD) at this synapse. FM2-10 dye unloading experiments demonstrated that the rate of vesicle exocytosis from the release ready pool (RRP) of vesicles was more than halved in the LTD (induced by 20 min of low frequency stimulation). Recovery from LTD, observed as a partial recovery of nerve-evoked muscle twitch amplitude, was accompanied by partial recovery of the refractoriness of RRP exocytosis. Unexpectedly, paired pulse plasticity, another routinely used indicator of presynaptic forms of synaptic plasticity, was unchanged in the LTD. We conclude that the LTD induces refractoriness of the neuromuscular vesicle release machinery downstream of presynaptic calcium entry.

Short-term synaptic depression is topographically distributed in the cochlear nucleus of the chicken.

In the auditory system, sounds are processed in parallel frequency-tuned circuits, beginning in the cochlea. Activity of auditory nerve fibers reflects this frequency-specific topographic pattern, known as tonotopy, and imparts frequency tuning onto their postsynaptic target neurons in the cochlear nucleus. In birds, cochlear nucleus magnocellularis (NM) neurons encode the temporal properties of acoustic stimuli by "locking" discharges to a particular phase of the input signal. Physiological specializations exist in gradients corresponding to the tonotopic axis in NM that reflect the characteristic frequency (CF) of their auditory nerve fiber inputs. One feature of NM neurons that has not been investigated across the tonotopic axis is short-term synaptic plasticity. NM offers a rather homogeneous population of neurons with a distinct topographical distribution of synaptic properties that is ideal for the investigation of specialized synaptic plasticity. Here we demonstrate for the first time that short-term synaptic depression (STD) is expressed topographically, where unitary high CF synapses are more robust with repeated stimulation. Correspondingly, high CF synapses drive spiking more reliably than their low CF counterparts. We show that postsynaptic AMPA receptor desensitization does not contribute to the observed difference in STD. Further, rate of recovery from depression, a presynaptic property, does not differ tonotopically. Rather, we show that another presynaptic feature, readily releasable pool (RRP) size, is tonotopically distributed and inversely correlated with vesicle release probability. Mathematical model results demonstrate that these properties of vesicle dynamics are sufficient to explain the observed tonotopic distribution of STD.

Modes and Regulation of Endocytic Membrane Retrieval in Mouse Auditory Hair Cells

Synaptic vesicle recycling sustains high rates of neurotransmission at the ribbon-type active zones (AZs) of mouse auditory inner hair cells (IHCs), but its modes and molecular regulation are poorly understood. Electron microscopy indicated the presence of clathrin-mediated endocytosis (CME) and bulk endocytosis. The endocytic proteins dynamin, clathrin, and amphiphysin are expressed and broadly distributed in IHCs. We used confocal vglut1-pHluorin imaging and membrane capacitance (Cm) measurements to study the spatial organization and dynamics of IHC exocytosis and endocytosis. Viral gene transfer expressed vglut1-pHluorin in IHCs and targeted it to synaptic vesicles. The intravesicular pH was ∼6.5, supporting only a modest increase of vglut1-pHluorin fluorescence during exocytosis and pH neutralization. Ca(2+) influx triggered an exocytic increase of vglut1-pHluorin fluorescence at the AZs, around which it remained for several seconds. The endocytic Cm decline proceeded with constant rate (linear component) after exocytosis of the readily releasable pool (RRP). When exocytosis exceeded three to four RRP equivalents, IHCs additionally recruited a faster Cm decline (exponential component) that increased with the amount of preceding exocytosis and likely reflects bulk endocytosis. The dynamin inhibitor Dyngo-4a and the clathrin blocker pitstop 2 selectively impaired the linear component of endocytic Cm decline. A missense mutation of dynamin 1 (fitful) inhibited endocytosis to a similar extent as Dyngo-4a. We propose that IHCs use dynamin-dependent endocytosis via CME to support vesicle cycling during mild stimulation but recruit bulk endocytosis to balance massive exocytosis.

A Sequential Vesicle Pool Model with a Single Release Sensor and a Ca2+-Dependent Priming Catalyst Effectively Explains Ca2+-Dependent Properties of Neurosecretion

Neurotransmitter release depends on the fusion of secretory vesicles with the plasma membrane and the release of their contents. The final fusion step displays higher-order Ca2+ dependence, but also upstream steps depend on Ca2+. After deletion of the Ca2+ sensor for fast release – synaptotagmin-1 – slower Ca2+-dependent release components persist. These findings have provoked working models involving parallel releasable vesicle pools (Parallel Pool Models, PPM) driven by alternative Ca2+ sensors for release, but no slow release sensor acting on a parallel vesicle pool has been identified. We here propose a Sequential Pool Model (SPM), assuming a novel Ca2+-dependent action: a Ca2+-dependent catalyst that accelerates both forward and reverse priming reactions. While both models account for fast fusion from the Readily-Releasable Pool (RRP) under control of synaptotagmin-1, the origins of slow release differ. In the SPM the slow release component is attributed to the Ca2+-dependent refilling of the RRP from a Non-Releasable upstream Pool (NRP), whereas the PPM attributes slow release to a separate slowly-releasable vesicle pool. Using numerical integration we compared model predictions to data from mouse chromaffin cells. Like the PPM, the SPM explains biphasic release, Ca2+-dependence and pool sizes in mouse chromaffin cells. In addition, the SPM accounts for the rapid recovery of the fast component after strong stimulation, where the PPM fails. The SPM also predicts the simultaneous changes in release rate and amplitude seen when mutating the SNARE-complex. Finally, it can account for the loss of fast- and the persistence of slow release in the synaptotagmin-1 knockout by assuming that the RRP is depleted, leading to slow and Ca2+-dependent fusion from the NRP. We conclude that the elusive ‘alternative Ca2+ sensor’ for slow release might be the upstream priming catalyst, and that a sequential model effectively explains Ca2+-dependent properties of secretion without assuming parallel pools or sensors.

Function suggests nano-structure: towards a unified theory for secretion rate, a statistical mechanics approach

The inventory of secretory granules along the plasma membrane can be viewed as maintained in two restricted compartments. The release-ready pool represents docked granules available for an initial stage of fast, immediate secretion, followed by a second stage of granule set-aside secretion pool, with significantly slower rate. Transmission electron microscopy ultra-structural investigations correlated with electrophysiological techniques and mathematical modelling have allowed the categorization of these secretory vesicle compartments, in which vesicles can be in various states of secretory competence. Using the above-mentioned approaches, the kinetics of single vesicle exocytosis can be worked out. The ultra-fast kinetics, explored in this study, represents the immediately available release-ready pool, in which granules bound to the plasma membrane are exocytosed upon Ca(2+) influx at the SNARE rosette at the base of porosomes. Formalizing Dodge and Rahamimoff findings on the effect of calcium concentration and incorporating the effect of SNARE transient rosette size, we postulate that secretion rate (rate), the number (X) of intracellular calcium ions available for fusion, calcium capacity (0 ≤ M ≤ 5) and the fusion nano-machine size (as measured by the SNARE rosette size K) satisfy the parsimonious M-K relation rate ≈ C × [Ca(2+)](min(X,M))e(-K/2).

Point Mutation in Syntaxin-1A Causes Abnormal Vesicle Recycling, Behaviors, and Short Term Plasticity

Syntaxin-1A is a t-SNARE that is involved in vesicle docking and vesicle fusion; it is important in presynaptic exocytosis in neurons because it interacts with many regulatory proteins. Previously, we found the following: 1) that autophosphorylated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), an important modulator of neural plasticity, interacts with syntaxin-1A to regulate exocytosis, and 2) that a syntaxin missense mutation (R151G) attenuated this interaction. To determine more precisely the physiological importance of this interaction between CaMKII and syntaxin, we generated mice with a knock-in (KI) syntaxin-1A (R151G) mutation. Complexin is a molecular clamp involved in exocytosis, and in the KI mice, recruitment of complexin to the SNARE complex was reduced because of an abnormal CaMKII/syntaxin interaction. Nevertheless, SNARE complex formation was not inhibited, and consequently, basal neurotransmission was normal. However, the KI mice did exhibit more enhanced presynaptic plasticity than wild-type littermates; this enhanced plasticity could be associated with synaptic response than did wild-type littermates; this pronounced response included several behavioral abnormalities. Notably, the R151G phenotypes were generally similar to previously reported CaMKII mutant phenotypes. Additionally, synaptic recycling in these KI mice was delayed, and the density of synaptic vesicles was reduced. Taken together, our results indicated that this single point mutation in syntaxin-1A causes abnormal regulation of neuronal plasticity and vesicle recycling and that the affected syntaxin-1A/CaMKII interaction is essential for normal brain and synaptic functions in vivo.

Inhibition of catecholamine secretion by iron-rich and iron-deprived multiwalled carbon nanotubes in chromaffin cells

The assay of the toxic effects of carbon nanotubes (CNTs) on human health is a stringent need in view of their expected increasing exploitation in industrial and biomedical applications. Most studies so far have been focused on lung toxicity, as the respiratory tract is the main entry of airborne particulate, but there is also recent evidence on the existence of toxic effects of multiwalled carbon nanotubes (MWCNTs) on neuronal and neuroendocrine cells (Belyanskaya et al., 2009; Xu et al., 2009; Gavello et al., 2012). Commercial MWCNTs often contain large amounts of metals deriving from the catalyst used during their synthesis. Since metals, particularly iron, may contribute to the toxicity of MWCNTs, we compared here the effects of two short MWCNTs samples (<5μm length), differing only in their iron content (0.5 versus 0.05% w/w) on the secretory responses of neurotransmitters in mouse chromaffin cells.We found that both iron-rich (MWCNT+Fe) and iron-deprived (MWCNT−Fe) samples enter chromaffin cells after 24h exposure, even though incorporation was attenuated in the latter case (40% versus 78% of cells). As a consequence of MWCNT+Fe or MWCNT−Fe exposure (50–263μg/ml, 24h), catecholamine secretion of chromaffin cells is drastically impaired because of the decreased Ca2+-dependence of exocytosis, reduced size of ready-releasable pool and lowered rate of vesicle release. On the contrary, both MWCNTs were ineffective in changing the kinetics of neurotransmitter release of single chromaffin granules and their quantal content. Overall, our data indicate that both MWCNT samples dramatically impair secretion in chromaffin cells, thus uncovering a true depressive action of CNTs mainly associated to their structure and degree of aggregation. This cellular “loss-of-function” is only partially attenuated in iron-deprived samples, suggesting a minor role of iron impurities on MWCNTs toxicity in chromaffin cells exocytosis.

The Bruchpilot cytomatrix determines the size of the readily releasable pool of synaptic vesicles

Synaptic vesicles (SVs) fuse at a specialized membrane domain called the active zone (AZ), covered by a conserved cytomatrix. How exactly cytomatrix components intersect with SV release remains insufficiently understood. We showed previously that loss of the Drosophila melanogaster ELKS family protein Bruchpilot (BRP) eliminates the cytomatrix (T bar) and declusters Ca(2+) channels. In this paper, we explored additional functions of the cytomatrix, starting with the biochemical identification of two BRP isoforms. Both isoforms alternated in a circular array and were important for proper T-bar formation. Basal transmission was decreased in isoform-specific mutants, which we attributed to a reduction in the size of the readily releasable pool (RRP) of SVs. We also found a corresponding reduction in the number of SVs docked close to the remaining cytomatrix. We propose that the macromolecular architecture created by the alternating pattern of the BRP isoforms determines the number of Ca(2+) channel-coupled SV release slots available per AZ and thereby sets the size of the RRP.

Is replenishment of the readily releasable pool associated with vesicular movement?

At the excitatory synapse of rat hippocampus the short-term synaptic depression observed during long high-frequency stimulation is associated with slower replenishment of the readily-releasable pool. Given that the replenishment rate is also not [Ca(++)]o sensitive this puts into question a widely held notion that the vesicles-constrained by the cytoskeleton and rendered free from such constraints by Ca(++) entry that renders them more mobile-are important in the replenishment of the readily-releasable pool. This raises a question-Is vesicular replenishment of the readily releasable pool associated with significant movement? To answer this question we evaluated how okadaic acid and staurosporine (compounds known to affect vesicular mobility) influence the replenishment rate. We used patterned stimulation on the Schaffer collateral fiber pathway and recorded the excitatory post-synaptic currents (EPSCs) from rat CA1 neurons, in the absence and presence of these drugs. The parameters of a circuit model with two vesicular pools were estimated by minimizing the squared difference between the ESPC amplitudes and simulated model output. [Ca(2+)]o did not influence the progressive decrease of the replenishment rate during long, high frequency stimulation. Okadaic acid did not significantly affect any parameters of the vesicular storage and release system, including the replenishment rate. Staurosporine reduced the replenishment coupling, but not the replenishment rate, and this is owing to the fact that it also reduces the ability of the readily releasable pool to contain quanta. Moreover, these compounds were ineffective in influencing how the replenishment rate decreases during long, high frequency stimulation. In conclusion at the excitatory synapses of rat hippocampus the replenishment of the readily releasable pool does not appear to be associated with a significant vesicular movement, and during long high frequency stimulation [Ca(++)]o does not influence the progressive decrease of vesicular replenishment.

Exocyst Sec5 Regulates Exocytosis of Newcomer Insulin Granules Underlying Biphasic Insulin Secretion

The exocyst complex subunit Sec5 is a downstream effector of RalA-GTPase which promotes RalA-exocyst interactions and exocyst assembly, serving to tether secretory granules to docking sites on the plasma membrane. We recently reported that RalA regulates biphasic insulin secretion in pancreatic islet β cells in part by tethering insulin secretory granules to Ca2+ channels to assist excitosome assembly. Here, we assessed β cell exocytosis by patch clamp membrane capacitance measurement and total internal reflection fluorescence microscopy to investigate the role of Sec5 in regulating insulin secretion. Sec5 is present in human and rodent islet β cells, localized to insulin granules. Sec5 protein depletion in rat INS-1 cells inhibited depolarization-induced release of primed insulin granules from both readily-releasable pool and mobilization from the reserve pool. This reduction in insulin exocytosis was attributed mainly to reduction in recruitment and exocytosis of newcomer insulin granules that undergo minimal docking time at the plasma membrane, but which encompassed a larger portion of biphasic glucose stimulated insulin secretion. Sec5 protein knockdown had little effect on predocked granules, unless vigorously stimulated by KCl depolarization. Taken together, newcomer insulin granules in β cells are more sensitive than predocked granules to Sec5 regulation.

Deleterious effects of soluble amyloid-β oligomers on multiple steps of synaptic vesicle trafficking

Growing evidence supports a role for soluble amyloid-β oligomer intermediates in the synaptic dysfunction associated with Alzheimer's disease (AD), but the molecular mechanisms underlying this effect remain unclear. We found that acute treatment of cultured rat hippocampal neurons with nanomolar concentrations of Aβ oligomers reduced the recycling pool and increased the resting pool of synaptic vesicles. Endocytosis of synaptic vesicles and the regeneration of fusion-competent vesicles were also severely impaired. Furthermore, the release probability of the readily-releasable pool (RRP) was increased, and recovery of the RRP was delayed. All these effects were prevented by antibody against Aβ. Moreover reduction of the pool size was prevented by inhibiting calpain or CDK5, while the defects in endocytosis were averted by overexpressing phosphatidylinositol-4-phosphate-5-kinase type I-γ, indicating that these two downstream pathways are involved in Aβ oligomers-induced presynaptic dysfunction.

Complexin facilitates exocytosis and synchronizes vesicle release in two secretory model systems

Complexins (Cplxs) are small, SNARE-associated proteins believed to regulate fast, calcium-triggered exocytosis. However, studies have pointed to either an inhibitory and/or facilitatory role in exocytosis, and the role of Cplxs in synchronizing exocytosis is relatively unexplored. Here, we compare the function of two types of complexin, Cplx 1 and 2, in two model systems of calcium-dependent exocytosis. In mouse neuromuscular junctions (NMJs), we find that lack of Cplx 1 significantly reduces and desynchronizes calcium-triggered synaptic transmission; furthermore, high-frequency stimulation elicits synaptic facilitation, instead of normal synaptic depression, and the degree of facilitation is highly sensitive to the amount of cytoplasmic calcium buffering. In Cplx 2-null adrenal chromaffin cells, we also find decreased and desynchronized evoked release, and identify a significant reduction in the vesicle pool close to the calcium channels (immediately releasable pool, IRP). Viral transduction with either Cplx 1 or 2 rescues both the size of the evoked response and the synchronicity of release, and it restores the IRP size. Our findings in two model systems are mutually compatible and indicate a role of Cplx 1 and 2 in facilitating vesicle priming, and also lead to the new hypothesis that Cplxs may synchronize vesicle release by promoting coupling between secretory vesicles and calcium channels.

Presynaptic Calcium Influx Controls Neurotransmitter Release in Part by Regulating the Effective Size of the Readily Releasable Pool

The steep calcium dependence of synaptic strength that has been observed at many synapses is thought to reflect a calcium dependence of the probability of vesicular exocytosis (p), with the cooperativity of three to six corresponding to the multiple calcium ion binding sites on the calcium sensor responsible for exocytosis. Here we test the hypothesis that the calcium dependence of the effective size of the readily releasable pool (RRP) also contributes to the calcium dependence of release at the calyx of Held synapse in mice. Using two established methods of quantifying neurotransmitter release evoked by action potentials (effective RRP), we find that when calcium influx is changed by altering the external calcium concentration, the calcium cooperativity of p is insufficient to account for the full calcium dependence of EPSC size; the calcium dependence of the RRP size also contributes. Reducing calcium influx by blocking R-type voltage-gated calcium channels (VGCCs) with Ni(2+), or by blocking P/Q-type VGCCs with ω-agatoxin IVA also changes EPSC amplitude by reducing both p and the effective RRP size. This suggests that the effective RRP size is dependent on calcium influx through VGCCs. Furthermore, activation of GABAB receptors, which reduces presynaptic calcium through VGCCs without other significant effects on release, also reduces the effective RRP size in addition to reducing p. These findings indicate that calcium influx regulates the RRP size along with p, which contributes to the calcium dependence of synaptic strength, and it influences the manner in which presynaptic modulation of presynaptic calcium channels affects neurotransmitter release.

Potential molecular mechanisms for decreased synaptic glutamate release in dysbindin-1 mutant mice

Behavioral genetic studies of humans have associated variation in the DTNBP1 gene with schizophrenia and its cognitive deficit phenotypes. The protein encoded by DTNBP1, dysbindin-1, is expressed in forebrain neurons where it interacts with proteins mediating vesicular trafficking and exocytosis. It has been shown that loss of dysbindin-1 results in a decrease in glutamate release in the prefrontal cortex; however the mechanisms underlying this decrease are not fully understood. In order to investigate this question, we evaluated dysbindin-1 null mutant mice, using electrophysiological recordings of prefrontal cortical neurons, imaging studies of vesicles, calcium dynamics and Western blot measures of synaptic proteins and Ca2+ channels.Dysbindin-1 null mice showed a decrease in the ready releasable pool of synaptic vesicles, decreases in quantal size, decreases in the probability of release and deficits in the rate of endo- and exocytosis compared with wild-type controls. Moreover, the dysbindin-1 null mice show decreases in the [Ca2+]i,expression of L- and N-type Ca2+channels and several proteins involved in synaptic vesicle trafficking and priming. Our results provide new insights into the mechanisms of action of dysbindin-1.