Liu, Qiang et al. (2011) International Worm Meeting "Short-term synaptic depression and vesicle pool refilling at the C. elegans neuromuscular junction."

Jorgensen, Erik, Liu, Qiang, Watanabe, Shigeki

[International Worm Meeting, 2011]

Short-term plasticity of synaptic transmission is recognized as an important element of information processing in neuronal networks. Acetylcholine release at the C. elegans neuromuscular junction is rapidly depressed upon repetitive stimulation and gradually recovers after a short rest. A simple model is that the readily releasable pool is depleted by extensive release and refilled from either newly endocytosed vesicles or from a large reserve pool. To determine the contributions of different pool refilling mechanisms and identify key molecular players, I use electrophysiological assays to screen candidate genes that function in endocytosis or other vesicle traffic pathways for altered synaptic depression and recovery. From this screen I expect to identify essential proteins that regulate synaptic strength and reveal mechanisms underlying synaptic depression. To target the screen specifically on the presynaptic components of synaptic depression, I built a reference strain to eliminate contributions from the postsynaptic acetylcholine receptors desensitization and the inactivation of channelrhodopsin-2. I used ChIEF, a channelrhodopsin-2 variant with less inactivation, in acr-16(-) background which has little receptor desensitization. Almost all the depression measured from this strain (acr-16;Punc-17::ChIEF::mCherry) is by presynaptic mechanisms. The time course of the depression and recovery measured from this control had two time constants indicating multiple underlying mechanisms. Candidate genes were cherry-picked based on their reported functions in endocytosis and vesicle trafficking steps including docking, priming, clustering and so on. Mutant strains of these candidate genes were crossed into the reference strain. Synaptic depression and recovery at the acetylcholine neuromuscular junction under 10Hz 30 sec stimulations were recorded. Preliminary recordings have been obtained for clathrin (chc-1 ts), dynamin-1 (dyn-1 ts), synaptojanin (unc-26), synapsin (snn-1), RIM (unc-10), liprin-alpha (syd-2), CDK5 (cdk-5), synaptotagmin-1 (snt-1), etc.. One interesting result emerged from the preliminary results is that chc-1 ts did not alter the depression nor the recovery rates at the restrictive temperature. This result calls into doubt endocytosis being the rate-limiting step for the synaptic depression and recovery. Alternatively, clathrin-mediated endocytosis may not be the essential endocytic pathway for synaptic vesicles in C. elegans. Clathrin and other mutants with either augmented or diminished depression or recovery rate will be further characterized using electron microscopy in the absence or presence of neuronal stimulation at different time points.

Qiang Liu et al. (2008) "Channelrhodopsion-2 as a Tool to Study Synaptic Depression at C. elegans Neuromuscular Junctions"

Gunther Hollopeter, Qiang Liu, Erik Jorgensen

[2008]

" Electrical stimulation of the ventral nerve cord has been used to evoke post-synaptic currents (PSCs) at C. elegans neural-muscular junctions (NMJs). However, the technical difficulty and the invasive nature of the electrode have limited its usefulness in delivery of high frequency stimulus trains to study synaptic depression at NMJs. A recently developed optogenetic technique enables the targeted expression of a light-activated ion channel, Channelrhodopsin-2 (ChR2), in order to depolarize cells in dissected electrophysiology preps in a non-invasive, reversible manner. We expressed ChR2 in body-wall muscles, cholinergic and GABAergic motor neurons under the control of specific promoters: Pmyo-3, Punc-17 and Punc-47, respectively. All three lines of worms respond to blue light behaviorally in expected manners: body contraction in Pmyo-3::ChR2 and Punc-17::ChR2 worms, and relaxation in Punc-47::ChR2 worms. Light activation of neuronal ChR2 (Punc-17::ChR2 and Punc-47::ChR2) evoked robust cholinergic or GABAergic post-synaptic currents (PSCs), and repetitive light stimulus at various frequencies up to 20Hz can elicit PSC trains with exponentially decreased amplitudes in these worms. This depression is partially due to the inactivation of ChR2 itself since the same stimulus train produced depression of photocurrents mediated by ChR2 in muscles as well. Pair-pulse analysis with various intervals revealed remarkable differences in the degree of depression and the recovery rate between synaptic currents mediated by ChR2 in neurons and photocurrents mediated by ChR2 in muscles, suggesting strong synaptic depression at NMJs. The observed synaptic depression could presumably result from the depletion of synaptic vesicle pool or the inaction of voltage gated ion channels, or both. ChR2 under Punc-17 is crossed into endocytosis defective mutants as well as voltage gated Ca channel loss of function mutants. Fatigue analysis on these transgenic worms is underway to determine the mechanism of synaptic depression at C. elegans NMJs."

Qiang Liu et al. (2005) International Worm Meeting "Novel Synaptic Connectivity at the C. elegans Neuromuscular Junction"

Bojun Chen, Qiang Liu, Zhao-Wen Wang

[International Worm Meeting, 2005]

In C. elegans, a single presynaptic specialization is usually apposed to several postsynaptic partners at the neuromuscular junction (NMJ). Similarly, a single presynaptic specialization is often associated with two or three postsynaptic elements in the nervous system(White et al, 1986). Because postsynaptic densities are generally indiscernible at chemical synapses in C. elegans, it is unknown whether all the postsynaptic elements form functional synapses with the presynaptic specialization. We addressed this problem with the C. elegans NMJ where axons of motor neurons form en passant synapses with muscle arms of body-wall muscle cells. In the absence of nerve stimulation, synaptic vesicles are released spontaneously, resulting in miniature postsynaptic currents (mPSCs). We recorded mPSCs from pairs of adjacent muscle cells using the dual whole-cell voltage-clamp technique, and determined the frequencies of mPSCs occurring in each cell, and those occurring concurrently. The term Concurrent Index (CI) was used for comparisons. A CI value of 1 equals to the frequency of concurrent mPSCs predicted from pure coincidence. As expected, mPSCs of unrelated cell pairs showed CIs of ~1. Interestingly, some cell pairs showed very high CIs invariably. There were two possible causes for the high CIs: (1) exocytosis of a single synaptic vesicle activated both muscle cells, and (2) electrical coupling allowed mPSCs to spread between the two cells. The second possibility did not appear to be true because the rise times of concurrent and non-concurrent mPSCs were identical, and the CI value was not related to the degree of electrical coupling. To obtain more direct evidence that electrical coupling did not contribute to concurrent mPSCs, we compared CIs between the wild-type and mutants of unc-9, an innexin required for inter-quadrant electrical coupling of body-wall muscle cells (see abstract by Liu, Chen, and Wang). In unc-9(fc16), electrical coupling was abolished in pairs of cells from the two different ventral quadrants (one cell per quadrant), whereas the CI remained high, suggesting that the high CI value was not due to electrical coupling. These observations provide compelling evidence that one presynaptic specialization may form functional synapses with multiple postsynaptic partners at the C. elegans NMJ. They also suggest that the high frequency of mPSCs at the C. elegans NMJ (~50 Hz) are not due to electrical coupling. This type of synaptic connectivity must have a high fidelity since exocytosis of a single synaptic vesicle causes responses in multiple postsynaptic cells. Experiments are under way to record concurrent evoked postsynaptic currents at the C. elegans NMJ.

Qiang Liu et al. (2005) International Worm Meeting "CaMKII Regulates Neurotransmitter Release at the C. elegans Neuromuscular Junction"

Qiang Liu, Bojun Chen, Zhao-Wen Wang

[International Worm Meeting, 2005]

Ca2+/calmodulin-dependent kinase II (CaMKII) plays an important role in synaptic plasticity and development of the nervous system. Malfunction of CaMKII is associated with a variety of diseases including epilepsy. In the nervous system, CaMKII is expressed at both pre- and post-synaptic sites. However, little is known about the potential role of CaMKII in neurotransmitter release. We embarked on a project to study the function of CaMKII in neurotransmitter release using the C. elegans neuromuscular junction (NMJ) as a model synapse. C. elegans has a single CaMKII gene, unc-43. Both gain-of-function and loss-of-function mutants of unc-43 are available for analysis. Miniature and evoked postsynaptic currents (mPSCs and ePSCs) at the NMJ were recorded with the postsynaptic body-wall muscle cell clamped at -60 mV. In the gain-of-function mutant unc-43(n498) (Reiner et al., 1999), both mPSC frequency and ePSC amplitude were significantly reduced compared with those of the wild-type. Postsynaptic receptor sensitivity appeared normal because the amplitude of mPSCs was similar to that of the wild-type. These results suggest that a normal function of presynaptic CaMKII is likely to down-regulate neurotransmitter release. In the null mutant unc-43(js125) (Hawasli et al., 2004), mPSC frequency, mPSC amplitude, and ePSC amplitude were all reduced compared with the wild-type. The reduction of ePSC amplitude is likely due to decreased postsynaptic sensitivity to the neurotransmitter because the mean amplitude of mPSCs was also reduced. In unc-43(js125) mutant that was rescued by expressing the wild-type UNC-43 specifically in neurons using the rab-3 promoter, the mPSC amplitude remained small, consistent with the notion that postsynaptic sensitivity rather than presynaptic quantal size was reduced in the js125 mutant. By contrast, the amplitude of ePSCs was completely restored in spite of the small mPSC amplitude, which is perhaps due to overexpression of the wild-type UNC-43. Experiments are under way to (1) further clarify the functions of pre- and post-synaptic CaMKII in synaptic transmission, and (2) to identify the presynaptic phosphorylation targets of CaMKII.

Qiang Liu et al. (2005) International Worm Meeting "Role of Ryanodine Receptors in Synaptic Release at the C. elegans Neuromuscular Junction"

Michael L Nonet, Ed Maryon, D Kent Morest, Bojun Chen, Arthur R Hand, Zhao-Wen Wang, Qiang Liu, Maya Yankova

[International Worm Meeting, 2005]

Quantal size (amount of neurotransmitter released from exocytosis of a single synaptic vesicle) is highly variable at many synapses. However, little is known about the regulation of quantal size. At the C. elegans neuromuscular junction, amplitudes of miniature postsynaptic currents (mPSCs) varied greatly. Mutations of the ryanodine receptor (RYR) gene unc-68 essentially abolished large-amplitude mPSCs. The amplitude of evoked postsynaptic currents (ePSCs) was also suppressed. These defects were completely rescued by expressing wild-type UNC-68 specifically in neurons but not in muscle cells. A combination of UNC-68 dysfunction and zero extracellular Ca2+ eliminated mPSCs, suggesting that synaptic exocytosis may have an obligatory requirement for Ca2+, and that Ca2+ influx and ryanodine receptor-mediated release are likely the exclusive sources of Ca2+, for synaptic exocytosis. The fraction of large-amplitude mPSCs remained constant following removal of extracellular Ca2+, suggesting that they were not endogenous ePSCs. Large-amplitude mPSCs at vertebrate central synapses are thought to result from synchronized multivesicular release ((Llano et al., 2000; Sharma and Vijayaraghavan, 2003). In contrast, large-amplitude mPSCs at the C. elegans NMJ appear to be monoquantal because (1) large-amplitude events remained after removal of extracellular Ca2+, (2) there was no correlation between the rise time and the amplitude of mPSCs, (3) the rise time of ePSCs, which represent synchronized multivesicular release, was much longer than that of mPSCs, (4) the fraction of large-amplitude mPSCs did not decrease in mutants (unc-64 and ric-4) that are defective in synchronized synaptic exocytosis. Electron microscopy showed that neither the synaptic vesicle size nor the numbers of total and morphologically docked vesicles per synapse were different between the wild-type and the unc-68 mutant, suggesting that the inhibition of mPSCs and ePSCs by RYR mutation was not due to decreased vesicle size or number. Thus we conclude that (1) all mPSCs at the C. elegans NMJ are monoquantal; (2) RYRs are required for the normal quantal size, and are potential regulators of quantal size; (3) synaptic exocytosis, including spontaneous events, appears to have an obligatory requirement for Ca2+. Influx of extracellular Ca2+ and RYR-mediated release from the ER are likely the exclusive sources of Ca2+ for synaptic exocytosis.

Qiang Liu et al. (2004) East Coast Worm Meeting "Ryanodine Receptors Regulate Neurotransmitter Release at the C. elegans Neuromuscular Junction"

Michael Nonet, Qiang Liu, Lawrence Salkoff, Zhao-Wen Wang

[East Coast Worm Meeting, 2004]

Traditionally, calcium influx through voltage-gated calcium channels was thought as the sole source of calcium for synaptic release. Emerging evidence suggests that calcium release from intracellular stores may also play a role. We investigated a potential role of presynaptic ryanodine receptors (RYRs), which are calcium release channels in the endoplasmic reticulum membrane, in spontaneous and evoked synaptic exocytosis using C. elegans as a model system. The existence of a single RYR gene ( ryr-1 ) and the availability of ryr-1 mutants in C. elegans make the analysis convenient. The potential role of RYRs was investigated by analyzing miniature and evoked postsynaptic currents (mPSCs and ePSCs) at the neuromuscular junction. In ryr-1 loss-of-function mutants, both the frequency and mean amplitude of mPSCs were significantly reduced compared with the wild-type. These changes appeared due to presynaptic RYR-1 defect since the sensitivity of body-wall muscle cells to exogenously applied neurotransmitters was similar to that of the wild-type. Acute pharmacological blockade of RYRs produced similar changes in mPSCs as the ryr-1 mutations, suggesting that a secondary developmental defect was not involved. In wild-type preparations, elimination of extracellular calcium reduced mPSC frequency by ~70%. By contrast, similar treatment essentially abolished mPSCs in the ryr-1 mutant. Thus, calcium influx and RYR-mediated calcium release are likely the exclusive sources of calcium for spontaneous synaptic release. The ryr-1 mutant also showed a significant reduction in ePSC amplitude. However, the quantal content (ePSC current integral divided by the mean mPSC current integral) did not change, suggesting that RYRs regulate ePSCs by controlling quantal size but not quantal number. Quantal number appeared to be solely determined by depolarization-mediated calcium influx. Given their large effects on mPSCs and ePSCs, RYRs may play an important role in synaptic plasticity.

Qiang Liu et al. (2004) East Coast Worm Meeting "CaM KII Regulates Neurotransmitter Release at the C. elegans Neuromuscular Junction"

Zhao-Wen Wang, Qiang Liu

[East Coast Worm Meeting, 2004]

Ca 2+ /calmodulin-dependent kinase II (CaM KII) plays an important role in synaptic plasticity and development of the nervous system. Malfunction of CaM KII is associated with a variety of diseases including epilepsy. In the nervous system, CaM KII is expressed at both pre- and post-synaptic sites. Little is known about the potential role of CaM KII in neurotransmitter release. We have embarked on a project to study the function of CaM KII in neurotransmitter release using C. elegans as model system. C. elegans has a single CaM KII gene, unc-43 . Both gain-of-function and loss-of-function mutants are available for analysis. To evaluate the function of CaM KII in neurotransmitter release, miniature and evoked postsynaptic currents (mPSCs and ePSCs) were recorded at the neuromuscular junction with the postsynaptic body-wall muscle cell clamped at -60 mV. We initially analyzed mPSCs and ePSCs in unc-43(n498) , a gain-of-function mutant. Compared with the wild-type, the mutant showed a significant reduction in the amplitude of ePSCs (wild-type 1579 +/- 129 pA, n = 8; n-498 721 +/- 79 pA, n = 12) and in the frequency of mPSCs (wild-type 50.6 +/- 3.4 Hz, n = 22; n-498 22.6 +/- 3.4 Hz, n = 6). Postsynaptic receptor sensitivity appeared normal in the mutant since the amplitude of mPSCs did not change (wild-type 27.4 +/- 1.6 pA, n = 22; n-498 28.4 +/- 4.3 pA, n = 6). These results suggest that a normal function of CaM KII is to regulate neurotransmitter release. Experiments are under way to determine 1) whether unc-43 loss-of-function mutants show opposite changes of mPSCs and ePSCs compared with the gain-of-function mutant; 2) whether the apparent effect on neurotransmitter release is due to an action of presynaptic CaM KII, or a retrograde signal generated by postsynaptic CaM KII; and 3) what are the phosphorylation targets of CaM KII in regulating neurotransmitter release.

Qiang Liu et al. (2005) International Worm Meeting "Specific Electrical Coupling of C. elegans Body-wall Muscle Cells and UNC-9 function"

Zhao-Wen Wang, Qiang Liu, Bojun Chen

[International Worm Meeting, 2005]

Body-wall muscle cells of C. elegans are thought to be electrically coupled. However, evidence of functional gap junctions is lacking. By analyzing transjunctional currents (Ij) using the dual whole-cell voltage-clamp technique, we found that body-wall muscle cells were coupled in a highly organized manner. Within the same quadrant, two adjoining cells were coupled if they were from two different rows, but not if they were from the same row, resulting in a wave-like coupling pattern. Pairs of cells at the same longitudinal level but from separate ventral quadrants were also coupled in spite of the lack of cell body contact. To identify the innexin(s) responsible for the coupling, we analyzed Ij in wild-type worms subjected to RNAi against innexins and in innexin mutants. unc-9 mutants (alleles fc16 and e101) showed an absence of inter-quadrant coupling and a decrease of intra-quadrant coupling, suggesting that UNC-9 is a key component of gap junctions mediating inter-quadrant coupling, and at least one additional innexin contributes to intra-quadrant coupling. Although UNC-7 has been speculated to coassemble with UNC-9 to form gap junctions based on similar mutant phenotypes, both intra- and inter-quadrant couplings were normal in the unc-7(e5) mutant. Expression of unc-9 promoter and gfp transcriptional fusions showed that UNC-9 was expressed in many neurons and various muscles, including body-wall muscle. Consistent with its role in electrical coupling, when a full-length UNC-9::GFP fusion protein was expressed specifically in muscle cells under the control of the myo-3 promoter, fluorescent puncta were observed at muscle cell body junctions, and near the ventral and dorsal nerve cords where muscle arms interdigitate, but were conspicuously absent at membrane regions where there were neither cell body nor muscle arm contacts. Wild-type animals treated with unc-9 RNAi showed a significant reduction in locomotion velocity. Since RNAi did not silence unc-9 in neurons, the observation suggests that UNC-9 in body-wall muscle cells plays an important role in locomotion. The specific coupling pattern suggests that gap junctions may contribute to locomotion by synchronizing ventral or dorsal muscle activites at the same longitudinal level, and by coordinating muscle activities along the longitudinal axis of the animal.

Qiang Liu et al. (2007) International Worm Meeting "Presynaptic CaMKII Modulates Neurotransmitter Release by Activating BK Channels at C. elegans Neuromuscular Junction."

Bojun Chen, Qiang Liu, Qian Ge, Zhao-Wen Wang

[International Worm Meeting, 2007]

CaMKII plays key roles in controlling synaptic strength and plasticity. Although the function of postsynaptic CaMKII is well-known, that of presynaptic CaMKII is poorly defined. To understand the function of presynaptic CaMKII, it is important to identify its molecular targets. We assessed the function of presynaptic CaMKII in neurotransmitter release and the possibility of the BK channel as a mediator of presynaptic CaMKII function by analyzing miniature and evoked postsynaptic currents at the C. elegans neuromuscular junction. The BK channel was chosen as a candidate because (1) it plays a major role in regulating neurotransmitter release, (2) it may be activated by CaMKII, and (3) loss-of-function mutants of both unc-43 CaMKII and slo-1 BK channel were isolated in the same genetic screen as suppressors of the lethargic phenotype of a hypomorphic unc-64 syntaxin mutant (Wang et al., 2001). We found that either loss-of-function (lf) or gain-of-function (gf) of unc-43 inhibited neurotransmitter release. The inhibitory effect of unc-43(gf) was reversed by mutation or blockade of SLO-1. SLO-1 expressed in Xenopus oocytes could be activated by recombinant rat <font face=symbol>a</font>-CaMKII, and this effect of CaMKII was abolished by mutating a threonine residue (T425) at a consensus CaMKII phosphorylation site in the first RCK domain (regulator of conductance for K+) of the channel. The inhibitory effect of unc-43(gf) was not reversed by unc-103(lf), dgk-1(lf), or eat-16(lf), which may suppress behavioral phenotypes of unc-43(gf) (Robatzek and Thomas, 2000). These observations suggest that presynaptic CaMKII is a bidirectional modulator of neurotransmitter release, presumably by phosphorylating different molecular targets, and that its negative modulatory effect on the release is mainly mediated by SLO-1 activation.

Watanabe, Shigeki et al. (2011) International Worm Meeting "Two endocytic pathways revealed by capturing channelrhodopsin induced synaptic transmission."

Jorgensen, Erik, Hollopeter, Gunther, Gu, Mingyu, Davis, Wayne, Watanabe, Shigeki, Liu, Qiang

[International Worm Meeting, 2011]

To sustain release of neurotransmitters at high rates of stimulation, synaptic vesicles need to be recycled locally at a synapse. Two major pathways for endocytosis were identified nearly 40 years ago: clathrin-scaffolds[1] and kiss-and-run[2]. Clathrin-mediated endocytosis is a slow process, which seems to take place distant from release sites. On the other hand, kiss-and-run occurs at release sites immediately after neurotransmission. The existence of either pathway at synapses has not been satisfactorily demonstrated[3]. Moreover, molecular mechanisms behind these pathways are poorly understood[3]. To address these issues, we need to observe synaptic vesicles as well as their associated proteins in action at nanoscale resolution. We developed two techniques that allow such an observation. First, we combined optogenetics with electron microscopy to induce synaptic transmission and capture rapid events at nanoscale resolution. Specifically, we expressed a variant of channelrhodopsin, ChIEF, in the C. elegans acetylcholine neurons, and stimulated them at various times before the high-pressure freezing. Using this technique, we identified two endocytic pathways that act at different times and in different parts of the synapse: a slow process distant from the dense projection and a fast process near the dense projection. Second, we developed a correlative super-resolution fluorescence microscopy and electron microscopy (fEM) imaging technique[4] to identify molecules that act in the processes. We found that the adaptor protein AP2 is localized to both identified endocytic sites, suggesting that AP2 may be functioning in both processes. By combining these two techniques, we are beginning to understand how endocytosis takes place at synapses. References: [1]Heuser, J.E. & Reese, T.S. Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol 57, 315-344 (1973). [2]Ceccarelli, B., Hurlbut, W.P. & Mauro, A. Depletion of vesicles from frog neuromuscular junctions by prolonged tetanic stimulation. J. Cell Biol 54, 30-38 (1972). [3]Royle, S.J. & Lagnado, L. Clathrin-mediated endocytosis at the synaptic terminal: bridging the gap between physiology and molecules. Traffic 11, 1489-1497 (2010). [4]Watanabe, S. et al. Protein localization in electron micrographs using fluorescence nanoscopy. Nat. Methods 8, 80-84 (2011).