Quantal Size Research Articles

Differential Sorting of the Vesicular Glutamate Transporter 1 into a Defined Vesicular Pool Is Regulated by Light Signaling Involving the Clock Gene Period2

Synaptic strength depends on the amount of neurotransmitter stored in synaptic vesicles. The vesicular transmitter content has recently been shown to be directly dependent on the expression levels of vesicular neurotransmitter transporters indicating that the transport capacity of synaptic vesicles is a critical determinant for synaptic efficacy. Using synaptic vesicles prepared from whole brain at different times of the day we now show that the amount of vesicular glutamate transporter (VGLUT) 1 undergoes strong diurnal cycling. VGLUT1 protein levels are high before the start of the light period, decline at noon, increase again before start of the dark period, and decline again at midnight. Mice kept in complete darkness showed within a 24-h period only a single peak of VGLUT1 expression in the middle of the rest phase. In contrast, mice lacking the period gene Period 2, a core component of the circadian clock, did not show any light-cycle-dependent changes of VGLUT1 levels. No other of several synaptic vesicle proteins examined underwent circadian cycling. Circadian cycling of VGLUT1 was not seen when analyzing homogenate or synaptosomes, the starting fraction for vesicle preparation. Circadian cycling of VGLUT1 was also not reflected at the mRNA level. We conclude that nerve terminals are endowed with mechanisms that regulate quantal size by changing the copy number of transporters in synaptic vesicles. A reduced amount of VGLUT1 per vesicle is probably achieved by means of selective sorting controlled by clock genes.

Experience-Dependent Formation and Recruitment of Large Vesicles from Reserve Pool

The sizes and contents of transmitter-filled vesicles have been shown to vary depending on experimental manipulations resulting in altered quantal sizes. However, whether such a presynaptic regulation of quantal size can be induced under physiological conditions as a potential alternative mechanism to alter the strength of synaptic transmission is unknown. Here we show that presynaptic vesicles of glutamatergic synapses of Drosophila neuromuscular junctions increase in size as a result of high natural crawling activities of larvae, leading to larger quantal sizes and enhanced evoked synaptic transmission. We further show that these larger vesicles are formed during a period of enhanced replenishment of the reserve pool of vesicles, from which they are recruited via a PKA- and actin-dependent mechanism. Our results demonstrate that natural behavior can induce the formation, recruitment, and release of larger vesicles in an experience-dependent manner and hence provide evidence for an additional mechanism of synaptic potentiation.

Fast Flies Take a Quantum Leap

Presynaptic regulation of quantal size is an appealing mechanism for changing synapse strength. In this issue of Neuron, Steinert et al. describe an activity-dependent increase in synapse strength mediated by the formation and release of large synaptic vesicles at the Drosophila neuromuscular junction.

Activity-dependent regulation of vesicular glutamate and GABA transporters: A means to scale quantal size

The functional balance of glutamatergic and GABAergic signaling in neuronal cortical circuits is under homeostatic control. That is, prolonged alterations of global network activity leads to opposite changes in quantal amplitude at glutamatergic and GABAergic synapses. Such scaling of excitatory and inhibitory transmission within cortical circuits serves to restore and maintain a constant spontaneous firing rate of pyramidal neurons. Our recent work shows that this includes alterations in the levels of expression of vesicular glutamate (VGLUT1 and VGLUT2) and GABA (VIAAT) transporters. Other vesicle markers, such as synaptophysin or synapsin, are not regulated in this way. Endogenous regulation at the level of mRNA and synaptic protein controls the number of transporters per vesicle and hence, the level of vesicle filling with transmitter. Bidirectional and opposite activity-dependent regulation of VGLUT1 and VIAAT expression would serve to adjust the balance of glutamate and GABA release and therefore the level of postsynaptic receptor saturation. In some excitatory neurons and synapses, co-expression of VGLUT1 and VGLUT2 occurs. Bidirectional and opposite changes in the levels of two excitatory vesicular transporters would enable individual neocortical neurons to scale up or scale down the level of vesicular glutamate storage, and thus, the amount available for release at individual synapses. Regulated vesicular transmitter storage and release via selective changes in the level of expression of vesicular glutamate and GABA transporters indicates that homeostatic plasticity of synaptic strength at cortical synapses includes presynaptic elements.

G protein βγ-subunits activated by serotonin mediate presynaptic inhibition by regulating vesicle fusion properties

Neurotransmitters are thought to be released as quanta, where synaptic vesicles deliver packets of neurotransmitter to the synaptic cleft by fusion with the plasma membrane. However, synaptic vesicles may undergo incomplete fusion. We provide evidence that G protein-coupled receptors inhibit release by causing such incomplete fusion. 5-hydroxytryptamine (5-HT) receptor signaling potently inhibits excitatory postsynaptic currents (EPSCs) between lamprey reticulospinal axons and their postsynaptic targets by a direct action on the vesicle fusion machinery. We show that 5-HT receptor-mediated presynaptic inhibition, at this synapse, involves a reduction in EPSC quantal size. Quantal size was measured directly by comparing unitary quantal amplitudes of paired EPSCs before and during 5-HT application and indirectly by determining the effect of 5-HT on the relationship between mean-evoked EPSC amplitude and variance. Results from FM dye-labeling experiments indicate that 5-HT prevents full fusion of vesicles. 5-HT reduces FM1-43 staining of vesicles with a similar efficacy to its effect on the EPSC. However, destaining of FM1-43-labeled vesicles is abolished by lower concentrations of 5-HT that leave a substantial EPSC. The use of a water-soluble membrane impermeant quenching agent in the extracellular space reduced FM1-43 fluorescence during stimulation in 5-HT. Thus vesicles contact the extracellular space during inhibition of synaptic transmission by 5-HT. We conclude that 5-HT, via free Gbetagamma, prevents the collapse of synaptic vesicles into the presynaptic membrane.

Gβγ reduces the number and quantal size of exocytotic events in neurosecretory chromaffin cells

G-protein βγ subunits (Gβγ) inhibit regulated exocytosis by several mechanisms including modulation of voltage gated Ca2+ channels (VGCC). We have shown that Gβγ also binds to the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex. Gβγ interacts with individual SNARE proteins and the ternary SNARE complex in vitro. Furthermore, Gβγ competes with Ca2+-dependent synaptotagmin binding to the SNARE complex. We postulate that Gβγ inhibits exocytosis by parallel mechanisms that target both Ca2+ influx (VGCC) and the exocytotic fusion event. To investigate this possibility, we explored Gβγ function in bovine adrenal chromaffin cells. GFP-fused Gβ was expressed with Gγ in chromaffin cells. Confocal microscopy confirmed plasma membrane targeting, and functional expression was confirmed electrophysiologically using prepulse facilitation (reversal of tonic VGCC inhibition). We used Ca2+ ionophores to bypass VGCC and directly stimulate secretion. Exocytotic events were detected using carbon fiber amperometry. The total number of exocytotic fusion events (amperometric spikes) was significantly reduced (~ 42%) in Gβγ expressing cells compared to GFP expressing control cells. In addition, the mean charge and peak amplitude of the amperometric spikes were significantly reduced in Gβγ-expressing cells. These data support the hypothesis that Gβγ can inhibit secretion downstream from VGCC. We propose that Gβγ attenuate exocytosis by parallel mechanisms, both inhibiting VGCC, and reducing the number and quantal size of exocytotic events by interacting with the SNARE complex. Supported by NIH EY10291

Syntillas Release Ca 2+ at a Site Different from the Microdomain Where Exocytosis Occurs in Mouse Chromaffin Cells

Spontaneous, short-lived, focal cytosolic Ca 2+ transients were found for the first time and characterized in freshly dissociated chromaffin cells from mouse. Produced by release of Ca 2+ from intracellular stores and mediated by type 2 and perhaps type 3 ryanodine receptors (RyRs), these transients are quantitatively similar in magnitude and duration to Ca 2+ syntillas in terminals of hypothalamic neurons, suggesting that Ca 2+ syntillas are found in a variety of excitable, exocytotic cells. However, unlike hypothalamic nerve terminals, chromaffin cells do not display syntilla activation by depolarization of the plasma membrane, nor do they have type 1 RyRs. It is widely thought that focal Ca 2+ transients cause “spontaneous” exocytosis, although there is no direct evidence for this view. Hence, we monitored catecholamine release amperometrically while simultaneously imaging Ca 2+ syntillas, the first such simultaneous measurements. Syntillas failed to produce exocytotic events; and, conversely, spontaneous exocytotic events were not preceded by syntillas. Therefore, we suggest that a spontaneous syntilla, at least in chromaffin cells, releases Ca 2+ into a cytosolic microdomain distinct from the microdomains containing docked, primed vesicles. Ryanodine (100 μM) reduced the frequency of Ca 2+ syntillas by an order of magnitude but did not alter the frequency of spontaneous amperometric events, suggesting that syntillas are not involved in steps preparatory to spontaneous exocytosis. Surprisingly, ryanodine also increased the total charge of individual amperometric events by 27%, indicating that intracellular Ca 2+ stores can regulate quantal size.

Low-Threshold Exocytosis Induced by cAMP-Recruited Ca V3.2 ( α1H) Channels in Rat Chromaffin Cells

We have studied the functional role of Ca V3 channels in triggering fast exocytosis in rat chromaffin cells (RCCs). Ca V3 T-type channels were selectively recruited by chronic exposures to cAMP (3 days) via an exchange protein directly activated by cAMP (Epac)-mediated pathway. Here we show that cAMP-treated cells had increased secretory responses, which could be evoked even at very low depolarizations (−50, −40 mV). Potentiation of exocytosis in cAMP-treated cells did not occur in the presence of 50 μM Ni 2+, which selectively blocks T-type currents in RCCs. This suggests that the “low-threshold exocytosis” induced by cAMP is due to increased Ca 2+ influx through cAMP-recruited T-type channels, rather than to an enhanced secretion downstream of Ca 2+ entry, as previously reported for short-term cAMP treatments (20 min). Newly recruited T-type channels increase the fast secretory response at low voltages without altering the size of the immediately releasable pool. They also preserve the Ca 2+ dependence of exocytosis, the initial speed of vesicle depletion, and the mean quantal size of single secretory events. All this indicates that cAMP-recruited Ca V3 channels enhance the secretory activity of RCCs at low voltages by coupling to the secretory apparatus with a Ca 2+ efficacy similar to that of already existing high-threshold Ca 2+ channels. Finally, using RT-PCRs we found that the fast inactivating low-threshold Ca 2+ current component recruited by cAMP is selectively associated to the α 1H (Ca V3.2) channel isoform.

Cisatracurium: myographical and electrophysiological studies in the isolated rat muscle

Myographical and electrophysiological studies of cisatracurium were performed, in vitro, in the isolated sciatic nerve-extensor digitorum longus muscle preparation of the rat. Indirect twitches were generated at 0.1 Hz and tetanic contractions at 50 Hz. endplate potentials (epps) were generated in trains of 50 Hz. The electrophysiological variables used in the analysis of the epps were: amplitude of the first epp in the train, average amplitude of the 30 degrees to the 59 degrees epp in the train (epps-plateau), tetanic rundown (percent loss in amplitude of epps-plateau relative to the first epp in the train), quantal size and quantal content. The myographical results showed that the inhibitory concentration 50% (IC(50)) of cisatracurium for the blockade of twitches (0.48 microm) is 12 times its IC(50) for the induction of tetanic fade (0.04 microm). The electrophysiological results showed a concentration dependent decrease in the amplitudes of first epps in the trains and of epps-plateau in the two used concentrations (0.13 microm and 0.38 microm). The tetanic rundown was intensified only in the presence of the higher (0.38 microm) concentration of cisatracurium. In cisatracurium 0.13 microm (a concentration which affects only tetanic contractions, inducing their fade, while leaving the twitch unaffected) there was a decrease in the quantal content of the first epp and of epps-plateau in the train. In cisatracurium (0.38 microm), a concentration, which affects the twitch, there was a decrease of the quantal size and of quantal content of epps-plateau, but not of the quantal content of the first epp in the train. The results indicate that the fade of the tetanic contraction induced by cisatracurium at the concentration of 0.13 microm is entirely because of a pre-synaptic blocking effect while the decrease in the twitch induced by cisatracurium at the concentration of 0.38 microm is due to a post-synaptic blocking effect.

Protein Kinase B/Akt Is a Novel Cysteine String Protein Kinase That Regulates Exocytosis Release Kinetics and Quantal Size

Protein kinase B/Akt has been implicated in the insulin-dependent exocytosis of GLUT4-containing vesicles, and, more recently, insulin secretion. To determine if Akt also regulates insulin-independent exocytosis, we used adrenal chromaffin cells, a popular neuronal model. Akt1 was the predominant isoform expressed in chromaffin cells, although lower levels of Akt2 and Akt3 were also found. Secretory stimuli in both intact and permeabilized cells induced Akt phosphorylation on serine 473, and the time course of Ca2+-induced Akt phosphorylation was similar to that of exocytosis in permeabilized cells. To determine if Akt modulated exocytosis, we transfected chromaffin cells with Akt constructs and monitored catecholamine release by amperometry. Wild-type Akt had no effect on the overall number of exocytotic events, but slowed the kinetics of catecholamine release from individual vesicles, resulting in an increased quantal size. This effect was due to phosphorylation by Akt, because it was not seen in cells transfected with kinase-dead mutant Akt. As overexpression of cysteine string protein (CSP) results in a similar alteration in release kinetics and quantal size, we determined if CSP was an Akt substrate. In vitro 32P-phosphorylation studies revealed that Akt phosphorylates CSP on serine 10. Using phospho-Ser10-specific antisera, we found that both transfected and endogenous cellular CSP is phosphorylated by Akt on this residue. Taken together, these findings reveal a novel role for Akt phosphorylation in regulating the late stages of exocytosis and suggest that this is achieved via the phosphorylation of CSP on serine 10.

A Single Vesicular Glutamate Transporter Is Sufficient to Fill a Synaptic Vesicle

Quantal size is the postsynaptic response to the release of a single synaptic vesicle and is determined in part by the amount of transmitter within that vesicle. At glutamatergic synapses, the vesicular glutamate transporter (VGLUT) fills vesicles with glutamate. While elevated VGLUT expression increases quantal size, the minimum number of transporters required to fill a vesicle is unknown. In Drosophila DVGLUT mutants, reduced transporter levels lead to a dose-dependent reduction in the frequency of spontaneous quantal release with no change in quantal size. Quantal frequency is not limited by vesicle number or impaired exocytosis. This suggests that a single functional unit of transporter is both necessary and sufficient to fill a vesicle to completion and that vesicles without DVGLUT are empty. Consistent with the presence of empty vesicles, at dvglut mutant synapses synaptic vesicles are smaller, suggesting that vesicle filling and/or transporter level is an important determinant of vesicle size.

Intragranular pH rapidly modulates exocytosis in adrenal chromaffin cells

Several drugs produce rapid changes in the kinetics of exocytosis of catecholamines, as measured at the single event level with amperometry. This study is intended to unveil whether the mechanism(s) responsible for these effects involve changes in the intravesicular pH. Cell incubation with bafilomycin A1, a blocker of the vesicular proton pump, caused both a deceleration in the kinetics of exocytosis and a reduction in the catecholamine content of vesicle. These effects were also observed upon reduction of proton gradient by nigericin or NH4Cl. pH measurements using fluorescent probes (acridine orange, quinacrine or enhanced green fluorescent protein-synaptobrevin) showed a strong correlation between vesicular pH and the kinetics of exocytosis. Hence, all maneuvers tested that decelerated exocytosis also alkalinized secretory vesicles and vice versa. On the other hand, calcium entry caused a transient acidification of granules. We therefore propose that the regulation of vesicular pH is, at least partially, a necessary step in the modulation of the kinetics of exocytosis and quantal size operated by some cell signals.

The complex regulation of dopamine output: A review of current themes

Most studies of neurotransmission examine ‘private’ synapses where input from a presynaptic terminal is tightly coupled to a postsynaptic site. In contrast, dopamine release occurs via ‘social’ synapses where release of a quantum of neurotransmitter diffuses to tens to hundreds of postsynaptic sites. This overflow is controlled in part by the neuronal firing pattern and effects on vesicular mobilization and saturation of the dopamine uptake transporter (DAT). Presynaptic effects that mediate diffusion thus modulate social synapses, and dopaminergic terminals have evolved means to express a rich repertoire of presynaptic states. Addictive drugs enhance dopamine diffusion by a variety of mechanisms: cocaine blocks DAT, nicotine activates burst neuronal firing while depressing release at low firing rates, while amphetamine reduces quantal size, induces stimulation-independent DAT reverse transport, and depresses neuronal firing. Even at the level of a single terminal, social synapses seem adapted for complex modulation of neural circuits, and means to comprehend these interactions are only now beginning to be explored.

Regulation of synaptic vesicles pools within motor nerve terminals during short-term facilitation and neuromodulation

The reserve pool (RP) and readily releasable pool (RRP) of synaptic vesicles within presynaptic nerve terminals were physiologically differentiated into distinctly separate functional groups. This was accomplished in glutamatergic nerve terminals by blocking the glutamate transporter with dl-threo-beta-benzyloxyaspartate (TBOA; 10 microM) during electrical stimulation with either 40 Hz of 10 pulses within a train or 20- or 50-Hz continuous stimulation. The 50-Hz continuous stimulation decreased the excitatory postsynaptic potential amplitude 60 min faster than for the 20-Hz continuous stimulation in the presence of TBOA (P < 0.05). There was no significant difference between the train stimulation and 20-Hz continuous stimulation in the run-down time in the presence of TBOA. After TBOA-induced synaptic depression, the excitatory postsynaptic potentials were rapidly (<1 min) revitalized by exposure to serotonin (5-HT, 1 microM) in every preparation tested (P < 0.05). At this glutamatergic nerve terminal, 5-HT promotes an increase probability of vesicular docking and fusion. Quantal recordings made directly at nerve terminals revealed smaller quantal sizes with TBOA exposure with a marked increase in quantal size as well as a continual appearance of smaller quanta upon 5-HT treatment after TBOA-induced depression. Thus 5-HT was able to recruit vesicles from the RP that were not rapidly depleted by acute TBOA treatment and electrical stimulation. The results support the notion that the RRP is selectively activated during rapid electrical stimulation sparing the RP; however, the RP can be recruited by the neuromodulator 5-HT. This suggests at least two separate kinetic and distinct regulatory paths for vesicle recycling within the presynaptic nerve terminal.

Release kinetics, quantal parameters and their modulation during short‐term depression at a developing synapse in the rat CNS

We have characterized developmental changes in the kinetics and quantal parameters of action potential (AP)-evoked neurotransmitter release during maturation of the calyx of Held synapse. Quantal size (q) and peak amplitudes of evoked EPSCs increased moderately, whereas the fraction of vesicles released by single APs decreased. During synaptic depression induced in postnatal day (P) 5-7 synapses by 10-100 Hz stimulation, q declined rapidly to 40-12% of its initial value. The decrease in q was generally smaller in more mature synapses (P12-14), but quite severe for frequencies > or = 300 Hz. The stronger decline of q in immature synapses resulted from a slower recovery from desensitization, presumably due to delayed glutamate clearance. Recovery from this desensitization followed an exponential time course with a time constant of approximately 480 ms in P5-7 synapses, and sped up > 20-fold during maturation. Deconvolution analysis of EPSCs revealed a significant acceleration of the release time course during development, which was accompanied by a 2-fold increase of the peak release rate. During long 100 Hz trains, more mature synapses were able to sustain average rates of 8-10 quanta s(-1) per active zone for phasic release. The rates of asynchronous vesicle release increased transiently > 35-fold immediately after such stimuli and decayed rapidly with an exponential time constant of approximately 50 ms to low resting levels of spontaneous release. However, even following extended periods of 100 Hz stimulation, the amount of asynchronous release was relatively minor with peak rates of less than 5% of the average rate of synchronous release measured at steady state during the tetani. Therefore, a multitude of mechanisms seems to converge on the generation of fast, temporally precise and reliable high-frequency transmission at the mature calyx of Held synapse.

Transmission Between Type II Hair Cells and Bouton Afferents in the Turtle Posterior Crista

Synaptic activity was recorded with sharp microelectrodes during rest and during 0.3-Hz sinusoidal stimulation from bouton afferents identified by their efferent-mediated inhibitory responses. A glutamate antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) decreased quantal size (qsize) while lowering external Ca(2+) decreased quantal rate (qrate). Miniature excitatory postsynaptic potentials (mEPSPs) had effective durations (qdur) of 3.5-5 ms. Their timing was consistent with Poisson statistics. Mean qsizes ranged in different units from 0.25 to 0.73 mV and mean qrates from 200 to 1,500/s; there was an inverse relation across the afferent population between qrate and qsize. qsize distributions were consistent with the independent release of variable-sized quanta. Channel noise, measured during AMPA-induced depolarizations, was small compared with quantal noise. Excitatory responses were larger than inhibitory responses. Peak qrates, which could approach 3,000/s, led peak excitatory mechanical stimulation by 40 degrees . Quantal parameters varied with stimulation phase with qdur and qsize being maximal during inhibitory stimulation. Voltage modulation (vmod) was in phase with qrate and had a peak depolarization of 1.5-3 mV. On average, 80% of vmod was accounted for by quantal activity; the remaining 20% was a nonquantal component that persisted in the absence of quantal activity. The extracellular accumulation of glutamate and K(+) are potential sources of nonquantal transmission and may provide a basis for the inverse relation between qrate and qsize. Comparison of the phases of synaptic and spike activity suggests that both presynaptic and postsynaptic mechanisms contribute to variations across afferents in the timing of spikes during sinusoidal stimulation.

Rapid Vesicular Release, Quantal Variability, and Spillover Contribute to the Precision and Reliability of Transmission at a Glomerular Synapse

The amplitude and shape of EPSC waveforms are thought to be important determinants of information processing and storage in the brain, yet relatively little is known about the origins of EPSC variability or how it affects synaptic signaling. We investigated the stochastic determinants of AMPA receptor-mediated EPSC variability at cerebellar mossy fiber-granule cell (MF-GC) connections by combining multiple-probability fluctuation analysis (MPFA) and deconvolution methods. The properties of MF connections with a single release site and the effects of the rapidly equilibrating competitive antagonist kynurenic acid on EPSCs suggest that receptors are not saturated by glutamate during a quantal event and that quanta sum linearly over a wide range of release probabilities. MPFA revealed an average of five vesicular release sites per MF-GC connection. Our results show that the time course of vesicular release is rapid (decay, tau = 75 micros) and independent of release probability, introducing little jitter in the shape or timing of the quantal component of the EPSC at physiological temperature. Moreover, the peak vesicular release rate per release site after an action potential (AP) (approximately 3 ms(-1)) is substantially higher than previously reported for central synapses. Interaction of amplitude fluctuations arising from quantal release and quantal size with the slower, low variability spillover-mediated current produce substantial variability in EPSC shape. Our simulations of MF-GC transmission suggest that quantal variability and transmitter spillover extend the voltage from which AP threshold can be crossed, improving reliability, and that fast vesicular release allows precise signaling across MF connections with heterogeneous weights.

Gβγ Decreases Quantal Size

In a study of rat adrenal chromaffin cells, which have dense-core secretory granules, Chen et al . reported that release of Gβγ in response to activation of G i/o -coupled receptors inhibited secretion by reducing quantal size. The reduction in quantal size was measured by amperometry in response to application of adenosine triphosphate (ATP), opioids, or somatostatin. Although each agonist partially inhibited calcium signaling, each also inhibited secretion induced by caffeine, which bypasses plasma membrane calcium channels to promote release of calcium directly from intracellular stores. Thus, application of ATP, for example, which can inhibit plasma membrane calcium channels, does not affect calcium transients induced by caffeine, yet still inhibits secretion. The agonists did not inhibit secretion stimulated by activation of muscarinic acetylcholine receptors (mAChR), but pharmacological inhibition of protein kinase C (PKC), which is downstream of the G q -coupled mAChR, restored ATP-, opioid,-, and somatostatin-mediated inhibition of secretion. ATP was confirmed to act through the G i/o -coupled receptor P2Y. Somatostatin and the opioid agonist tested also act through a G i/o -coupled receptor; however, the effects of these agonists appeared to be mediated through the βγ and not the α subunits. The mechanism by which βγ inhibited secretion appeared not to involve the known kinases downstream of βγ, because pharmacological inhibition of phosphoinositide 3-kinase (PI3K), phospholipase β2, serine-threonine kinases, or tyrosine kinases did not affect caffeine-induced secretion. Sequestration of βγ with a membrane-permeable peptide blocked ATP-induced inhibition of caffeine-induced secretion, and whole-cell dialysis of Gβ 1 γ 2 into the cells inhibited depolarization-induced secretion. The authors propose that Gβγ limits the open time of the fusion pore, but the exact target of βγ for this effect remains to be determined. X.-K. Chen, L.-C. Wang, Y. Zhou, Q. Cai, M. Prakriya, L.-K. Duan, Z.-H. Sheng, C. Lingle, Z. Zhou, Activation of GPCRs modulates quantal size in chromaffin cells through G βγ and PKC. Nat. Neurosci. 8 , 1160-1168 (2005). [PubMed]

Paired Motor Neuron–Muscle Recordings in Zebrafish Test the Receptor Blockade Model for Shaping Synaptic Current

The transparent spinal cord and electrically compact fast muscle of zebrafish offer the first opportunity to perform simultaneous whole-cell patch-clamp recordings from both motor neuron and target skeletal muscle in situ. Our paired recordings reveal the fastest reported kinetics for both spontaneous and evoked synaptic currents at any synapse and a large quantal size that facilitates the resolution of spontaneous synaptic currents. We used this preparation to test the recent proposal that open channel block of the acetylcholine receptor by acetylcholine modulates the kinetics and timing of transmission between nerve and muscle in larval zebrafish (Legendre et al., 2000). Contrary to the predictions of this model, we find similar delay and onset kinetics of synaptic current at positive and negative muscle membrane potentials, even after inhibition of acetylcholinesterase. In contrast, blockade of motor neuron K channels by 4-aminopyridine prolonged the action potential and introduced a significant delay and slowing of evoked synaptic currents, demonstrating our ability to measured altered transmitter release with this system. We conclude that the kinetics of neuromuscular synaptic currents in zebrafish is not governed by receptor block.

Activation of GPCRs modulates quantal size in chromaffin cells through Gβγ and PKC

Exocytosis proceeds by either full fusion or 'kiss-and-run' between vesicle and plasma membrane. Switching between these two modes permits the cell to regulate the kinetics and amount of secretion. Here we show that ATP receptor activation reduces secretion downstream from cytosolic Ca2+ elevation in rat adrenal chromaffin cells. This reduction is mediated by activation of a pertussis toxin-sensitive G(i/o) protein, leading to activation of G(betagamma) subunits, which promote the 'kiss-and-run' mode by reducing the total open time of the fusion pore during a vesicle fusion event. Furthermore, parallel activation of the muscarinic acetylcholine receptor removes the inhibitory effects of ATP on secretion. This is mediated by a G(q) pathway through protein kinase C activation. The inhibitory effects of ATP and its reversal by protein kinase C activation are also shared by opioids and somatostatin. Thus, a variety of G protein pathways exist to modulate Ca2+-evoked secretion at specific steps in fusion pore formation.