Readily Releasable Pool Size Research Articles

A maximum of two readily releasable vesicles per docking site at a cerebellar single active zone synapse.

Recent research suggests that in central mammalian synapses, active zones contain several docking sites acting in parallel. Before release, one or several synaptic vesicles (SVs) are thought to bind to each docking site, forming the readily releasable pool (RRP). Determining the RRP size per docking site has important implications for short-term synaptic plasticity. Here, using mouse cerebellar slices, we take advantage of recently developed methods to count the number of released SVs at single glutamatergic synapses in response to trains of action potentials (APs). In each recording, the number of docking sites was determined by fitting with a binomial model the number of released SVs in response to individual APs. After normalization with respect to the number of docking sites, the summed number of released SVs following a train of APs was used to estimate of the RRP size per docking site. To improve this estimate, various steps were taken to maximize the release probability of docked SVs, the occupancy of docking sites, as well as the extent of synaptic depression. Under these conditions, the RRP size reached a maximum value close to two SVs per docking site. The results indicate that each docking site contains two distinct SV-binding sites that can simultaneously accommodate up to one SV each. They further suggest that under special experimental conditions, as both sites are close to full occupancy, a maximal RRP size of two SVs per docking site can be reached. More generally, the results validate a sequential two-step docking model previously proposed at this preparation.

A maximum of two readily releasable vesicles per docking site at a cerebellar single active zone synapse

Recent research suggests that in central mammalian synapses, active zones contain several docking sites acting in parallel. Before release, one or several synaptic vesicles (SVs) are thought to bind to each docking site, forming the readily releasable pool (RRP). Determining the RRP size per docking site has important implications for short-term synaptic plasticity. Here, using mouse cerebellar slices, we take advantage of recently developed methods to count the number of released SVs at single glutamatergic synapses in response to trains of action potentials (APs). In each recording, the number of docking sites was determined by fitting with a binomial model the number of released SVs in response to individual APs. After normalization with respect to the number of docking sites, the summed number of released SVs following a train of APs was used to estimate of the RRP size per docking site. To improve this estimate, various steps were taken to maximize the release probability of docked SVs, the occupancy of docking sites, as well as the extent of synaptic depression. Under these conditions, the RRP size reached a maximum value close to two SVs per docking site. The results indicate that each docking site contains two distinct SV-binding sites that can simultaneously accommodate up to one SV each. They further suggest that under special experimental conditions, as both sites are close to full occupancy, a maximal RRP size of two SVs per docking site can be reached. More generally, the results validate a sequential two-step docking model previously proposed at this preparation.

PLC regulates spontaneous glutamate release triggered by extracellular calcium and readily releasable pool size in neocortical neurons.

Dynamic physiological changes in brain extracellular calcium ([Ca2+]o) occur when high levels of neuronal activity lead to substantial Ca2+ entry via ion channels reducing local [Ca2+]o. Perturbations of the extracellular microenvironment that increase [Ca2+]o are commonly used to study how [Ca2+] regulates neuronal activity. At excitatory synapses, the Ca2+-sensing receptor (CaSR) and other G-protein coupled receptors link [Ca2+]o and spontaneous glutamate release. Phospholipase C (PLC) is activated by G-proteins and is hypothesized to mediate this process. Patch-clamping cultured neocortical neurons, we tested how spontaneous glutamate release was affected by [Ca2+]o and inhibition of PLC activity. We used hypertonic sucrose (HS) to evaluate the readily releasable pool (RRP) and test if it was affected by inhibition of PLC activity. Spontaneous glutamate release substantially increased with [Ca2+]o, and inhibition of PLC activity, with U73122, abolished this effect. PLC-β1 is an abundant isoform in the neocortex, however, [Ca2+]o-dependent spontaneous release was unchanged in PLC-β1 null mutants (PLC-β1-/-). U73122 completely suppressed this response in PLC-β1-/- neurons, indicating that this residual [Ca2+]o-sensitivity may be mediated by other PLC isoforms. The RRP size was substantially reduced after incubation in U73122, but not U73343. Phorbol esters increased RRP size after PLC inhibition. Together these data point to a strong role for PLC in mediating changes in spontaneous release elicited by [Ca2+]o and other extracellular cues, possibly by modifying the size of the RRP.

The activation of mGluR4 rescues parallel fiber synaptic transmission and LTP, motor learning and social behavior in a mouse model of Fragile X Syndrome

BackgroundFragile X syndrome (FXS), the most common inherited intellectual disability, is caused by the loss of expression of the Fragile X Messenger Ribonucleoprotein (FMRP). FMRP is an RNA-binding protein that negatively regulates the expression of many postsynaptic as well as presynaptic proteins involved in action potential properties, calcium homeostasis and neurotransmitter release. FXS patients and mice lacking FMRP suffer from multiple behavioral alterations, including deficits in motor learning for which there is currently no specific treatment.MethodsWe performed electron microscopy, whole-cell patch-clamp electrophysiology and behavioral experiments to characterise the synaptic mechanisms underlying the motor learning deficits observed in Fmr1KO mice and the therapeutic potential of positive allosteric modulator of mGluR4.ResultsWe found that enhanced synaptic vesicle docking of cerebellar parallel fiber to Purkinje cell Fmr1KO synapses was associated with enhanced asynchronous release, which not only prevents further potentiation, but it also compromises presynaptic parallel fiber long-term potentiation (PF-LTP) mediated by β adrenergic receptors. A reduction in extracellular Ca2+ concentration restored the readily releasable pool (RRP) size, basal synaptic transmission, β adrenergic receptor-mediated potentiation, and PF-LTP. Interestingly, VU 0155041, a selective positive allosteric modulator of mGluR4, also restored both the RRP size and PF-LTP in mice of either sex. Moreover, when injected into Fmr1KO male mice, VU 0155041 improved motor learning in skilled reaching, classical eyeblink conditioning and vestibuloocular reflex (VOR) tests, as well as the social behavior alterations of these mice.LimitationsWe cannot rule out that the activation of mGluR4s via systemic administration of VU0155041 can also affect other brain regions. Further studies are needed to stablish the effect of a specific activation of mGluR4 in cerebellar granule cells.ConclusionsOur study shows that an increase in synaptic vesicles, SV, docking may cause the loss of PF-LTP and motor learning and social deficits of Fmr1KO mice and that the reversal of these changes by pharmacological activation of mGluR4 may offer therapeutic relief for motor learning and social deficits in FXS.

Impaired Replenishment of Cortico-Striatal Synaptic Glutamate in Huntington's Disease Mouse Model.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by expansion of CAG repeats in the Huntingtin gene (HTT). Studies suggest cortical to striatal (C-S) projections, which regulate movement and provide cell survival signals to SPNs, are altered in the pre-manifest and early symptomatic stages of HD. But whether and how presynaptic cortical terminals are affected in HD is not well explored. Test size and replenishment of readily releasable pool (RRP), and assess glutamate refill of C-S synapses in HD models. Immunocytochemistry was applied in C-S co-cultures generated from FVB/N (WT: wildtype) mice and YAC128, an HD mouse model expressing human HTT with 128 CAG repeats on the FVB/N background; Whole-cell patch clamp recordings from striatal neurons were performed both in cultures, with or without osmotic stimuli, and in acute brain slices from 6-month-old early symptomatic YAC128 mice and WT following prolonged trains of electrical stimuli in corpus callosum. We found no change in the average size or vesicle replenishment rate of RRP in C-S synapses of YAC128, compared with WT, cultures at day in vitro 21, a time when immunocytochemistry showed comparable neuronal survival between the two genotypes. However, YAC128 C-S synapses showed a slowed rate of recovery of glutamate release in co-cultures as well as in acute brain slices. Mutant HTT expression impairs glutamate refill but not RRP size or replenishment in C-S synapses. This work provides a foundation for examining the contribution of deficits in presynaptic cortical terminals on HD progression.

Outside-in regulation of the readily releasable pool of synaptic vesicles by α2δ-1.

The readily releasable pool (RRP) of synaptic vesicles is a key determinant of phasic neurotransmission. Although the size of the RRP is tightly regulated by intracellular factors, there is little evidence for its modification by extracellular signals. By studying the homogeneous population of synapses present in autaptic microcultures, we show that pregabalin, a prototypical gabapentinoid, decreases the effective RRP size. Simultaneous imaging of presynaptic calcium influx and recording of postsynaptic responses shows that the effect is not related to a reduction of calcium entry. The main cause is the impairment of the functional coupling among N-type calcium channels and the RRP, resembling an increase of intracellular mobile calcium buffers. The ectodomain of neurexin-1α shows a similar action to pregabalin, acting as an endogenous ligand of α2δ-1 that reduces the RRP size without affecting presynaptic calcium influx. The regulatory actions described for pregabalin and the ectodomain of neurexin-1α are mutually exclusive. The overexpression of α2δ-1 enhances the effect of pregabalin and the ectodomain of neurexin-1α on neurotransmission by decreasing their effective concentration. In contrast, knockdown of α2δ-1 causes a profound inhibition of synaptic transmission. These observations prompt to consider α2δ-1 as an outside-in signaling platform that binds exogenous and endogenous cues for regulating the coupling of voltage-gated calcium channels to synaptic vesicles.

SNAP-25 phosphorylation at Ser187 is not involved in Ca2+ or phorbolester-dependent potentiation of synaptic release

SNAP-25, one of the three SNARE-proteins responsible for synaptic release, can be phosphorylated by Protein Kinase C on Ser-187, close to the fusion pore. In neuroendocrine cells, this phosphorylation event potentiates vesicle recruitment into releasable pools, whereas the consequences of phosphorylation for synaptic release remain unclear. We mutated Ser-187 and expressed two mutants (S187C and S187E) in the context of the SNAP-25B-isoform in SNAP-25 knockout glutamatergic autaptic neurons. Whole-cell patch clamp recordings were performed to assess the effect of Ser-187 phosphorylation on synaptic transmission. Blocking phosphorylation by expressing the S187C mutant did not affect synapse density, basic evoked or spontaneous neurotransmission, the readily-releasable pool size or its Ca2+-independent or Ca2+-dependent replenishment. Furthermore, it did not affect the response to phorbol esters, which activate PKC. Expressing S187C in the context of the SNAP-25A isoform also did not affect synaptic transmission. Strikingly, the – potentially phosphomimetic – mutant S187E reduced spontaneous release and release probability, with the largest effect seen in the SNAP-25B isoform, showing that a negative charge in this position is detrimental for neurotransmission, in agreement with electrostatic fusion triggering. During the course of our experiments, we found that higher SNAP-25B expression levels led to decreased paired pulse potentiation, probably due to higher release probabilities. Under these conditions, the potentiation of evoked EPSCs by phorbol esters was followed by a persistent down-regulation, probably due to a ceiling effect. In conclusion, our results indicate that phosphorylation of Ser-187 in SNAP-25 is not involved in modulation of synaptic release by Ca2+ or phorbol esters.

A Tripartite Interaction Among the Calcium Channel α1- and β-Subunits and F-Actin Increases the Readily Releasable Pool of Vesicles and Its Recovery After Depletion.

Neurotransmitter release is initiated by the influx of Ca2+ via voltage-gated calcium channels. The accessory β-subunit (CaVβ) of these channels shapes synaptic transmission by associating with the pore-forming subunit (CaVα1) and up-regulating presynaptic calcium currents. Besides CaVα1, CaVβ interacts with several partners including actin filaments (F-actin). These filaments are known to associate with synaptic vesicles (SVs) at the presynaptic terminals and support their translocation within different pools, but the role of CaVβ/F-actin association on synaptic transmission has not yet been explored. We here study how CaVβ4, the major calcium channel β isoform in mamalian brain, modifies synaptic transmission in concert with F-actin in cultured hippocampal neurons. We analyzed the effect of exogenous CaVβ4 before and after pharmacological disruption of the actin cytoskeleton and dissected calcium channel-dependent and -independent functions by comparing the effects of the wild-type subunit with the one bearing a double mutation that impairs binding to CaVα1. We found that exogenously expressed wild-type CaVβ4 enhances spontaneous and depolarization-evoked excitatory postsynaptic currents (EPSCs) without altering synaptogenesis. CaVβ4 increases the size of the readily releasable pool (RRP) of SVs at resting conditions and accelerates their recovery after depletion. The enhanced neurotransmitter release induced by CaVβ4 is abolished upon disruption of the actin cytoskeleton. The CaVα1 association-deficient CaVβ4 mutant associates with actin filaments, but neither alters postsynaptic responses nor the time course of the RRP recovery. Furthermore, this mutant protein preserves the ability to increase the RRP size. These results indicate that the interplay between CaVβ4 and F-actin also support the recruitment of SVs to the RRP in a CaVα1-independent manner. Our studies show an emerging role of CaVβ in determining SV maturation toward the priming state and its replenishment after release. We envision that this subunit plays a role in coupling exocytosis to endocytosis during the vesicle cycle.

Chronic morphine reduces the readily releasable pool of GABA, a presynaptic mechanism of opioid tolerance.

Chronic treatment with opioids, such as morphine, leads to analgesic tolerance. While postsynaptic opioid tolerance is well documented, the involvement of presynaptic mechanisms remains unclear. We show that chronic morphine reduces the ability of periaqueductal grey (PAG) neurons to maintain GABAergic transmission. This depression of GABAergic transmission was due to a reduction in the effective size of the readily releasable pool. This also led to a reduction in opioid presynaptic inhibition; these presynaptic adaptations need to be considered in the development of strategies to reduce opioid tolerance. The midbrain periaqueductal grey (PAG) plays a critical role in tolerance to the analgesic actions of opioids such as morphine. While numerous studies have identified the postsynaptic adaptations induced by chronic morphine treatment in this and other brain regions, the presence of presynaptic adaptations remains uncertain. We examined GABAergic synaptic transmission within rat PAG brain slices from animals which underwent a low dose morphine treatment protocol which produces tolerance, but not withdrawal. Evoked GABAergic IPSCs (inhibitory postsynaptic currents) were less in morphine compared to control saline treated animals. Postsynaptic GABAA receptor mediated currents and desensitization, presynaptic release probability (Pr ), and inhibition by endogenous neurotransmitters were similar in morphine and saline treated animals. By contrast, the effective size of the readily releasable pool (RRP) was smaller in morphine treated animals. While the μ-opioid agonist DAMGO produced a reduction in Pr and RRP size in saline treated animals, it only reduced Pr in morphine treated animals. Consequently, DAMGO-induced inhibition of evoked IPSCs during short burst stimulation was less in morphine, compared to saline treated animals. These results indicate that low dose chronic morphine treatment reduces presynaptic μ-opioid inhibition by reducing the size of the pool of vesicles available for action potential dependent release. This novel presynaptic adaptation may provide important insights into the development of efficacious pain therapies that can circumvent the development of opioid tolerance.

SAD-B Phosphorylation of CAST Controls Active Zone Vesicle Recycling for Synaptic Depression

Short-term synaptic depression (STD) is a commonform of activity-dependent plasticity observed widely in the nervous system. Few molecular pathways that control STD have been described, but the active zone (AZ) release apparatus provides a possible link between neuronal activity and plasticity. Here, we show that an AZ cytomatrix protein CAST and an AZ-associated protein kinase SAD-B coordinately regulate STD by controlling reloading of the AZ with release-ready synaptic vesicles. SAD-B phosphorylates the N-terminal serine (S45) of CAST, and S45 phosphorylation increases with higher firing rate. A phosphomimetic CAST (S45D) mimics CAST deletion, which enhances STD by delaying reloading of the readily releasable pool (RRP), resulting in a pool size decrease. A phosphonegative CAST (S45A) inhibits STD and accelerates RRP reloading. Our results suggest that the CAST/SAD-B reaction serves as a brake on synaptic transmission by temporal calibration of activity and synaptic depression via RRP size regulation.

Actin- and Myosin-Dependent Vesicle Loading of Presynaptic Docking Sites Prior to Exocytosis

Variance analysis of postsynaptic current amplitudes suggests the presence of distinct docking sites (also called release sites) where vesicles pause before exocytosis. Docked vesicles participate in the readily releasable pool (RRP), but the relation between docking site number and RRP size remains unclear. It is also unclear whether all vesicles of the RRP are equally release competent, and what cellular mechanisms underlie RRP renewal. We address here these questions at single glutamatergic synapses, counting released vesicles using deconvolution. We find a remarkably low variance of cumulative vesicle counts during action potential trains. This, combined with Monte Carlo simulations, indicates that vesicles transit through two successive states before exocytosis, so that the RRP is up to 2-fold higher than the docking site number. The transition to the second state has a very rapid rate constant, and is specifically inhibited by latrunculin B and blebbistatin, suggesting the involvement of actin and myosin.

Dynamin-1 deletion enhances post-tetanic potentiation and quantal size after tetanic stimulation at the calyx of Held.

Post-tetanic potentiation (PTP) is attributed mainly to an increase in release probability (Pr ) and/or readily-releasable pool (RRP) in many synapses, but the role of endocytosis in PTP is unknown. Using the calyx of Held synapse from tissue-specific dynamin-1 knockout (cKO) mice (P16-20), we report that cKO synapses show enhanced PTP compared to control. We found significant increases in both spontaneous excitatory postsynaptic current (spEPSC) amplitude and RRP size (estimated by a train of 30 APs at 100Hz) in cKO over control during PTP. Actin depolymerization blocks the increase in spEPSC amplitude in both control and cKO, and it abolishes the enhancement of PTP in cKO. PTP is sensitive to the PKC inhibitor GF109203X in both control and cKO. We conclude that an activity-dependent quantal size increase contributes to the enhancement of PTP in cKO over control and an altered endocytosis affects short-term plasticity through quantal size changes. High-frequency stimulation leads to post-tetanic potentiation (PTP) at many types of synapses. Previous studies suggest that PTP results primarily from a protein kinase C (PKC)-dependent increase in release probability (Pr ) and/or readily-releasable pool (RRP) of synaptic vesicles (SVs), but the role of SV endocytosis in PTP is unknown. Using the mature calyx of Held (P16-20), we report that tissue-specific ablation of dynamin-1 (cKO), an endocytic protein crucial for SV regeneration, enhances PTP in cKO over control. To explore the mechanism of this enhancement, we estimated the changes in paired-pulse ratios (PPRs) and RRP size during PTP. RRP was estimated by the back-extrapolation of cumulative EPSC amplitudes during a train of 30 action potentials at 100Hz (termed RRPtrain ). We found an increase in RRPtrain during PTP in both control and cKO, but no significant changes in the PPR. Moreover, the amplitude and frequency of spontaneous excitatory postsynaptic currents (spEPSCs) increased during PTP in both control and cKO; however, the spEPSC amplitude in cKO during PTP was significantly larger than in control. Actin depolymerization reagent latrunculin-B (Lat-B) abolished the activity-dependent increase in spEPSC amplitude in both control and cKO, but selectively blocked the enhancement of PTP in cKO, without affecting PTP in control. PKC inhibitor GF109203X nearly abolished PTP in both control and cKO. These data suggest that the quantal size increase contributes to the enhancement of PTP in dynamin-1 cKO, and this change depends on strong synaptic activity and actin polymerization.

Synaptotagmin-1 and -7 Are Redundantly Essential for Maintaining the Capacity of the Readily-Releasable Pool of Synaptic Vesicles.

In forebrain neurons, Ca2+ triggers exocytosis of readily releasable vesicles by binding to synaptotagmin-1 and -7, thereby inducing fast and slow vesicle exocytosis, respectively. Loss-of-function of synaptotagmin-1 or -7 selectively impairs the fast and slow phase of release, respectively, but does not change the size of the readily-releasable pool (RRP) of vesicles as measured by stimulation of release with hypertonic sucrose, or alter the rate of vesicle priming into the RRP. Here we show, however, that simultaneous loss-of-function of both synaptotagmin-1 and -7 dramatically decreased the capacity of the RRP, again without altering the rate of vesicle priming into the RRP. Either synaptotagmin-1 or -7 was sufficient to rescue the RRP size in neurons lacking both synaptotagmin-1 and -7. Although maintenance of RRP size was Ca2+-independent, mutations in Ca2+-binding sequences of synaptotagmin-1 or synaptotagmin-7—which are contained in flexible top-loop sequences of their C2 domains—blocked the ability of these synaptotagmins to maintain the RRP size. Both synaptotagmins bound to SNARE complexes; SNARE complex binding was reduced by the top-loop mutations that impaired RRP maintenance. Thus, synaptotagmin-1 and -7 perform redundant functions in maintaining the capacity of the RRP in addition to nonredundant functions in the Ca2+ triggering of different phases of release.

A three-pool model dissecting readily releasable pool replenishment at the calyx of held.

Although vesicle replenishment is critical in maintaining exo-endocytosis recycling, the underlying mechanisms are not well understood. Previous studies have shown that both rapid and slow endocytosis recycle into a very large recycling pool instead of within the readily releasable pool (RRP), and the time course of RRP replenishment is slowed down by more intense stimulation. This finding contradicts the calcium/calmodulin-dependence of RRP replenishment. Here we address this issue and report a three-pool model for RRP replenishment at a central synapse. Both rapid and slow endocytosis provide vesicles to a large reserve pool (RP) ~42.3 times the RRP size. When moving from the RP to the RRP, vesicles entered an intermediate pool (IP) ~2.7 times the RRP size with slow RP-IP kinetics and fast IP-RRP kinetics, which was responsible for the well-established slow and rapid components of RRP replenishment. Depletion of the IP caused the slower RRP replenishment observed after intense stimulation. These results establish, for the first time, a realistic cycling model with all parameters measured, revealing the contribution of each cycling step in synaptic transmission. The results call for modification of the current view of the vesicle recycling steps and their roles.

Synaptic vesicle glycoprotein 2A modulates vesicular release and calcium channel function at peripheral sympathetic synapses.

Synaptic vesicle glycoprotein (SV)2A is a transmembrane protein found in secretory vesicles and is critical for Ca(2+) -dependent exocytosis in central neurons, although its mechanism of action remains uncertain. Previous studies have proposed, variously, a role of SV2 in the maintenance and formation of the readily releasable pool (RRP) or in the regulation of Ca(2+) responsiveness of primed vesicles. Such previous studies have typically used genetic approaches to ablate SV2 levels; here, we used a strategy involving small interference RNA (siRNA) injection to knockdown solely presynaptic SV2A levels in rat superior cervical ganglion (SCG) neuron synapses. Moreover, we investigated the effects of SV2A knockdown on voltage-dependent Ca(2+) channel (VDCC) function in SCG neurons. Thus, we extended the studies of SV2A mechanisms by investigating the effects on vesicular transmitter release and VDCC function in peripheral sympathetic neurons. We first demonstrated an siRNA-mediated SV2A knockdown. We showed that this SV2A knockdown markedly affected presynaptic function, causing an attenuated RRP size, increased paired-pulse depression and delayed RRP recovery after stimulus-dependent depletion. We further demonstrated that the SV2A-siRNA-mediated effects on vesicular release were accompanied by a reduction in VDCC current density in isolated SCG neurons. Together, our data showed that SV2A is required for correct transmitter release at sympathetic neurons. Mechanistically, we demonstrated that presynaptic SV2A: (i) acted to direct normal synaptic transmission by maintaining RRP size, (ii) had a facilitatory role in recovery from synaptic depression, and that (iii) SV2A deficits were associated with aberrant Ca(2+) current density, which may contribute to the secretory phenotype in sympathetic peripheral neurons.

Impaired synaptic vesicle recycling contributes to presynaptic dysfunction in lipoprotein lipase-deficient mice

Lipoprotein lipase (LPL) is expressed at high levels in hippocampal neurons, although its function is unclear. We previously reported that LPL-deficient mice have learning and memory impairment and fewer synaptic vesicles in hippocampal neurons, but properties of synaptic activity in LPL-deficient neurons remain unexplored. In this study, we found reduced frequency of miniature excitatory postsynaptic currents (mEPSCs) and readily releasable pool (RRP) size in LPL-deficient neurons, which led to presynaptic dysfunction and plasticity impairment without altering postsynaptic activity. We demonstrated that synaptic vesicle recycling, which is known to play an important role in maintaining the RRP size in active synapses, is impaired in LPL-deficient neurons. Moreover, lipid assay revealed deficient docosahexaenoic acid (DHA) and arachidonic acid (AA) in the hippocampus of LPL-deficient mice; exogenous DHA or AA supplement partially restored synaptic vesicle recycling capability. These results suggest that impaired synaptic vesicle recycling results from deficient DHA and AA and contributes to the presynaptic dysfunction and plasticity impairment in LPL-deficient neurons.

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.

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.

Intrinsic variability in Pv, RRP size, Ca2+ channel repertoire, and presynaptic potentiation in individual synaptic boutons

The strength of individual synaptic contacts is considered a key modulator of information flow across circuits. Presynaptically the strength can be parsed into two key parameters: the size of the readily releasable pool (RRP) and the probability that a vesicle in that pool will undergo exocytosis when an action potential fires (Pv). How these variables are controlled and the degree to which they vary across individual nerve terminals is crucial to understand synaptic plasticity within neural circuits. Here we report robust measurements of these parameters in rat hippocampal neurons and their variability across populations of individual synapses. We explore the diversity of presynaptic Ca2+ channel repertoires and evaluate their effect on synaptic strength at single boutons. Finally, we study the degree to which synapses can be differentially modified by a known potentiator of presynaptic function, forskolin. Our experiments revealed that both Pv and RRP spanned a large range, even for synapses made by the same axon, demonstrating that presynaptic efficacy is governed locally at the single synapse level. Synapses varied greatly in their dependence on N or P/Q type Ca2+ channels for neurotransmission, but there was no association between specific channel repertoires and synaptic efficacy. Increasing cAMP concentration using forskolin enhanced synaptic transmission in a Ca2+-independent manner that was inversely related with a synapse's initial Pv, and independent of its RRP size. We propose a simple model based on the relationship between Pv and calcium entry that can account for the variable potentiation of synapses based on initial probability of vesicle fusion.