Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology^1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) ^4,5,6 in Drosophila motor neurons using the GAL4-UAS system ⁷, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.     

Developmental Changes in Calmodulin-Kinase II Activity at Brain Synaptic Junctions: Alterations in Holoenzyme Composition

Synaptic junctions (SJs) from rat forebrain were isolated at increasing postnatal ages and examined for endogenous protein kinase activities. Our studies focused on the postnatal maturation of the multifunctional protein kinase designated Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II). This kinase is comprised of a major 50-kilodalton (kDa) and a minor 60-kDa subunit. Experiments examined the developmental properties of CaM-kinase II associated with synaptic plasma membranes (SPMs) and synaptic junctions (SJs), as well as the holoenzyme purified from cytosolic extracts. Large developmental increases in CaM-kinase II activity of SJ fractions were observed between postnatal days 6 and 20; developmental changes were examined for a number of properties including (a) autophosphorylation, (b) endogenous substrate phosphorylation, (c) exogenous substrate phosphorylation, and (d) immunoreactivity. Results demonstrated that forebrain CaM-kinase II undergoes a striking age-dependent change in subunit composition. In early postnatal forebrain the 60-kDa subunit constitutes the major catalytic and immunoreactive subunit of the holoenzyme. The major peak of CaM-kinase II activity in SJ fractions occurred at approximately postnatal day 20, a time near the end of the most active period of in vivo synapse formation. Following this developmental age, CaM-kinase II continued to accumulate at SJs; however, its activity was not as highly activated by Ca2+ plus calmodulin.     

Calmodulin-Dependent Protein Phosphorylation in Synaptic Junctions

Synaptic junctions (SJs) from rat forebrain were examined for Ca2+/calmodulin (CaM)-dependent kinase activity and compared to synaptic plasma membrane (SPM) and postsynaptic density (PSD) fractions. The kinase activity in synaptic fractions was examined for its capacity to phosphorylate endogenous proteins or exogenous synapsin I, in the presence or absence of Ca2+ plus CaM. When assayed for endogenous protein phosphorylation, SJs contained approximately 25-fold greater amounts of Ca2+/CAM-dependent kinase activity than SPMs, and fivefold more activity than PSDs. When kinase activities were measured by phosphorylation of exogenous synapsin I, SJs contained fourfold more activity than SPMs, and 10-fold more than PSDs. The phosphorylation of SJ proteins of 60- and 50-kilodalton (major PSD protein) polypeptides were greatly stimulated by Ca2+/CaM; levels of phosphorylation for these proteins were 23- and 17-fold greater than basal levels, respectively. Six additional proteins whose phosphorylation was stimulated 6-15-fold by Ca2+/CAM were identified in SJs. These proteins include synapsin I, and proteins of 240, 207, 170, 140, and 54 kilodaltons. The 54-kilodalton protein is a highly phosphorylated form of the major PSD protein and the 170-kilodalton component is a cell-surface glycoprotein of the postsynaptic membrane that binds concanavalin A. The CaM-dependent kinase in SJ fractions phosphorylated endogenous phosphoproteins at serine and/or threonine residues. Ca2+-dependent phosphorylation in SJ fractions was strictly dependent on exogenous CaM, even though SJs contained substantial amounts of endogenous CaM (15 micrograms CaM/mg SJ protein). Exogenous CaM, after being functionally incorporated into SJs, was rapidly removed by sequential washings. These observations suggest that the SJ-associated CaM involved in regulating Ca2+-dependent protein phosphorylation may be in dynamic equilibrium with the cytoplasm. These findings indicate that a brain CaM-dependent kinase(s) and substrate proteins are concentrated at SJs and that CaM-dependent protein phosphorylation may play an important role in mechanisms that underlie synaptic communication.     

Neuromuscular Junctions in Flight and Tymbal Muscles of the Cicada

The tymbal muscle fiber in the cicada closely resembles the indirect flight muscle fiber in its structural detail. We agree with other authors that the tymbal muscle is a modified indirect flight muscle. The peripheral nerve branches to the tymbal and flight muscle fibers are similar to those in the wasp leg. The axon is loosely mantled by irregular turns of the mesaxon, enclosing cytoplasm. The nerve is therefore a tunicated nerve. The neuromuscular junction in the high frequency muscle fibers shows direct apposition of plasma membranes of axon and muscle fiber, large numbers of mitochondria and synaptic vesicles in the axon, and concentrations of mitochondria, aposynaptic granules, and endoplasmic reticulum in the postsynaptic area of the muscle fiber. Of special interest is the multitude of intracellular, opposing membranes in the postsynaptic area. They form laminated stacks and whorls, vesicles, cysternae, and tubules. They occasionally show continuity with the plasma membrane, the outer nuclear envelope, and the circumfibrillar endoplasmic reticulum. The membrane system in this area is designated "rete synapticum." It is believed to add to the electrical capacity of the neuromuscular junction, to serve in transmission of potentials, and possibly is the site of the oscillating mechanism in high-frequency muscle fibers.     

Time Lapse in Vivo Visualization of Developmental Stabilization of Synaptic Receptors at Neuromuscular Junctions

The lifetime of nicotinic acetylcholine receptors (AChRs) in neuromuscular junctions (NMJs) is increased from <1 day to >1 week during early postnatal development. However, the exact timing of AChR stabilization is not known, and its correlation to the concurrent embryonic to adult AChR channel conversion, NMJ remodeling, and neuromuscular diseases is unclear. Using a novel time lapse in vivo imaging technology we show that replacement of the entire receptor population of an individual NMJ occurs end plate-specifically within hours. This makes it possible to follow directly in live animals changing stabilities of end plate receptors. In three different, genetically modified mouse models we demonstrate that the metabolic half-life values of synaptic AChRs increase from a few hours to several days after postnatal day 6. Developmental stabilization is independent of receptor subtype and apparently regulated by an intrinsic muscle-specific maturation program. Myosin Va, an F-actin-dependent motor protein, is also accumulated synaptically during postnatal development and thus could mediate the stabilization of end plate AChR.     

Analysis of Neurotrophic Factors in Limb and Extraocular Muscles of Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is currently an incurable fatal motor neuron syndrome characterized by progressive weakness, muscle wasting and death ensuing 3–5 years after diagnosis. Neurotrophic factors (NTFs) are known to be important in both nervous system development and maintenance. However, the attempt to translate the potential of NTFs into the therapeutic options remains limited despite substantial number of approaches, which have been tested clinically. Using quantitative RT-PCR (qRT-PCR) technique, the present study investigated mRNA expression of four different NTFs: brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4) and glial cell line-derived neurotrophic factor (GDNF) in limb muscles and extraocular muscles (EOMs) from SOD1^G93A transgenic mice at early and terminal stages of ALS. General morphological examination revealed that muscle fibres were well preserved in both limb muscles and EOMs in early stage ALS mice. However, in terminal ALS mice, most muscle fibres were either atrophied or hypertrophied in limb muscles but unaffected in EOMs. qRT-PCR analysis showed that in early stage ALS mice, NT-4 was significantly down-regulated in limb muscles whereas NT-3 and GDNF were markedly up-regulated in EOMs. In terminal ALS mice, only GDNF was significantly up-regulated in limb muscles. We concluded that the early down-regulation of NT-4 in limb muscles is closely associated with muscle dystrophy and dysfunction at late stage, whereas the early up-regulations of GDNF and NT-3 in EOMs are closely associated with the relatively well-preserved muscle morphology at late stage. Collectively, the data suggested that comparing NTFs expression between limb muscles and EOMs from different stages of ALS animal models is a useful method in revealing the patho-physiology and progression of ALS, and eventually rescuing motor neuron in ALS patients.     

Postsynaptic Abnormalities at the Neuromuscular Junctions of Utrophin-deficient Mice

Utrophin is a dystrophin-related cytoskeletal protein expressed in many tissues. It is thought to link F-actin in the internal cytoskeleton to a transmembrane protein complex similar to the dystrophin protein complex (DPC). At the adult neuromuscular junction (NMJ), utrophin is precisely colocalized with acetylcholine receptors (AChRs) and recent studies have suggested a role for utrophin in AChR cluster formation or maintenance during NMJ differentiation. We have disrupted utrophin expression by gene targeting in the mouse. Such mice have no utrophin detectable by Western blotting or immunocytochemistry. Utrophindeficient mice are healthy and show no signs of weakness. However, their NMJs have reduced numbers of AChRs (α-bungarotoxin [α-BgTx] binding reduced to ~60% normal) and decreased postsynaptic folding, though only minimal electrophysiological changes. Utrophin is thus not essential for AChR clustering at the NMJ but may act as a component of the postsynaptic cytoskeleton, contributing to the development or maintenance of the postsynaptic folds. Defects of utrophin could underlie some forms of congenital myasthenic syndrome in which a reduction of postsynaptic folds is observed.     

Molecular Mechanism of Active Zone Organization at Vertebrate Neuromuscular Junctions

Organization of presynaptic active zones is essential for development, plasticity, and pathology of the nervous system. Recent studies indicate a trans-synaptic molecular mechanism that organizes the active zones by connecting the pre- and the postsynaptic specialization. The presynaptic component of this trans-synaptic mechanism is comprised of cytosolic active zone proteins bound to the cytosolic domains of voltage-dependent calcium channels (P/Q-, N-, and L-type) on the presynaptic membrane. The postsynaptic component of this mechanism is the synapse organizer (laminin β2) that is expressed by the postsynaptic cell and accumulates specifically on top of the postsynaptic specialization. The pre- and the postsynaptic components interact directly between the extracellular domains of calcium channels and laminin β2 to anchor the presynaptic protein complex in front of the postsynaptic specialization. Hence, the presynaptic calcium channel functions as a scaffolding protein for active zone organization and as an ion-conducting channel for synaptic transmission. In contrast to the requirement of calcium influx for synaptic transmission, the formation of the active zone does not require the calcium influx through the calcium channels. Importantly, the active zones of adult synapses are not stable structures and require maintenance for their integrity. Furthermore, aging or diseases of the central and peripheral nervous system impair the active zones. This review will focus on the molecular mechanisms that organize the presynaptic active zones and summarize recent findings at the neuromuscular junctions and other synapses.     

The Neuromuscular Junction: Measuring Synapse Size,Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.     

The Distribution of Pre- and Postsynaptic Inhibition at Crustacean Neuromuscular Junctions

The relative contribution of pre- and postsynaptic mechanisms to peripheral inhibition has been analyzed in the abdominal slow flexor muscles of crayfish and lobsters. The conductance of the muscle fiber membrane may be increased to five or more times its resting value by repetitive stimulation of the peripheral inhibitory axon, and this effect accounts for all of the attenuation exerted by the inhibitor against excitatory junctional potentials. No "critical interval" has been found at which an inhibitory nerve impulse produces anomalously large reduction of a following depolarizing junctional potential; electrotonic depolarizations and junctional potentials are identically affected under all phase conditions. The presynaptic inhibitory mechanism is, therefore, absent in this system. In the dactyl opener muscle, on the contrary, most of the attenuation of excitatory junctional potentials is achieved presynaptically, though equally large postjunctional conductance changes are also seen (Dudel and Kuffler, 1961). The difference is correlated with a difference in the reflex operation of the two muscles. Reflex inhibition in the abdominal slow flexors is primarily central, whereas in the dactyl opener, inhibition is brought about by an increase in inhibitory nerve discharge frequency without central suppression of the single excitatory axon. The function of peripheral inhibition in the abdominal flexors is presumably to terminate residual depolarization by reducing the long time-constant of the muscle fibers.     

Developmental Changes in the Oligosaccharide Composition of Synaptic Junctional Glycoproteins

Synaptic junctions (SJs) were isolated from the forebrains of rats ranging in postnatal age from 10 days to greater than 1 year. SJ glycoproteins that react with Concanavalin A (Con A) were isolated by chromatography on Con A-agarose and separated by gel electrophoresis. The concentrations of the major SJ Con A binding (Con A+) glycoproteins (apparent Mr 180,000, 130,000, and 110,000) increased between 10 and 28 days, with GP180 and GP110 showing greater relative increases than GP130. Con A binding oligosaccharides associated with 10-day SJs were sensitive to digestion with endoglycosidase C11 and alpha-mannosidase, indicating that they were of the high-mannose type, as previously shown for 28-day SJs. Con A+ oligosaccharides from rats of increasing postnatal age were analyzed by chromatography on Biogel P-4. Two major oligosaccharides, containing five and eight mannose residues, were present in SJs of all ages examined. During development the ratio of man5 to man8 oligosaccharides increased, so that man5 constituted the predominant species in 28-day and adult SJs. Peptide mapping experiments showed that GP180, GP130, and GP110 were each associated with a unique polypeptide composition. Little or no change in peptide composition of the major SJ glycoproteins occurred during development.     

Changes in the Parameters of Quantal Acetylcholine Release after Activation of PAR1-Type Thrombin Receptors at the Mouse Neuromuscular Junctions

In mature and newly formed neuromuscular synapses of mouse skeletal muscles, miniature endplate potentials (MEPPs) and multiquantal endplate potentials (EPPs) evoked by a single stimulation of the nerve were recorded using intracellular microelectrode technique. The mechanisms underlying the changes in spontaneous and evoked acetylcholine (ACh) release caused by the activation of PAR1-type muscle receptors induced by their peptide agonist TRAP6-NH₂ were studied. It has been shown for the first time that, in either mature or newly formed motor synapses, the activation of PAR1 that lack presynaptic localization causes a sustained increase in the MEPP amplitude due to the increase in the ACh quantal size at the presynaptic level. It was found that phospholipase C (PLC) participates in the signaling mechanism triggered by the PAR1 activation. Exogenously applied brain-derived neurotrophic factor (BDNF) mimics the effect of activation of PAR1 by TRAP6-NH₂. Moreover, an increase in the MEPP amplitude caused by the peptide PAR1 agonist was fully prevented by blocking the BDNF receptors–tropomyosin receptor kinases B (TrkB). Thus, it has been shown for the first time that the increase in ACh quantal size due to the activation of PAR1 in motor synapses is mediated by a complex signaling cascade that starts at the postsynaptic level of the motor synapse and ends at the presynaptic level. It is expected that the activation of PAR1 at the muscle fiber membrane followed by the PLC upregulation results in the release of neurotrophin BDNF as a retrograde signal. Its effect on the presynaptic TrkB receptors triggers the cascade leading to an increase in the quantal size of ACh.     

Caenorhabditis elegans wsp-1 Regulation of Synaptic Function at the Neuromuscular Junction

The Rho GTPase members and their effector proteins, such as the Wiskott-Aldrich syndrome protein (WASP), play critical roles in regulating actin dynamics that affect cell motility, endocytosis, cell division, and transport. It is well established that Caenorhabditis elegans wsp-1 plays an essential role in embryonic development. We were interested in the role of the C. elegans protein WSP-1 in the adult nematode. In this report, we show that a deletion mutant of wsp-1 exhibits a strong sensitivity to the neuromuscular inhibitor aldicarb. Transgenic rescue experiments demonstrated that neuronal expression of WSP-1 rescued this phenotype and that it required a functional WSP-1 Cdc42/Rac interactive binding domain. WSP-1-GFP fusion protein was found localized presynaptically, immediately adjacent to the synaptic protein RAB-3. Strong genetic interactions with wsp-1 and other genes involved in different stages of synaptic transmission were observed as the wsp-1(gm324) mutation suppresses the aldicarb resistance seen in unc-13(e51), unc-11(e47), and snt-1 (md290) mutants. These results provide genetic and pharmacological evidence that WSP-1 plays an essential role to stabilize the actin cytoskeleton at the neuronal active zone of the neuromuscular junction to restrain synaptic vesicle release.     

Mutational Analysis of Rab3 Function for Controlling Active Zone Protein Composition at the Drosophila Neuromuscular Junction

At synapses, the release of neurotransmitter is regulated by molecular machinery that aggregates at specialized presynaptic release sites termed active zones. The complement of active zone proteins at each site is a determinant of release efficacy and can be remodeled to alter synapse function. The small GTPase Rab3 was previously identified as playing a novel role that controls the distribution of active zone proteins to individual release sites at the Drosophila neuromuscular junction. Rab3 has been extensively studied for its role in the synaptic vesicle cycle; however, the mechanism by which Rab3 controls active zone development remains unknown. To explore this mechanism, we conducted a mutational analysis to determine the molecular and structural requirements of Rab3 function at Drosophila synapses. We find that GTP-binding is required for Rab3 to traffick to synapses and distribute active zone components across release sites. Conversely, the hydrolytic activity of Rab3 is unnecessary for this function. Through a structure-function analysis we identify specific residues within the effector-binding switch regions that are required for Rab3 function and determine that membrane attachment is essential. Our findings suggest that Rab3 controls the distribution of active zone components via a vesicle docking mechanism that is consistent with standard Rab protein function.     

Recycling of Synaptic Vesicles at the Frog Neuromuscular Junction in the Presence of Strontium

Abstract: In these experiments, we followed the exocytosis and endocytosis of synaptic vesicles with the vital dye FM1-43 and asked whether calcium is important for membrane retrieval at the frog neuromuscular junction. We replaced calcium with equimolar amounts of strontium and monitored the staining of recycling vesicles by inducing exocytosis with electrical stimulation. Trains of 2,400 (2 or 20 Hz) or 4,200 (20 Hz) pulses failed to induce FM1-43 internalization in the presence of strontium, but they did in the presence of calcium. This effect of strontium was not due to a decrease in exocytosis, because FM1-43 release was similar in the presence of calcium or strontium. The impairment in endocytosis, observed as inhibition of FM1-43 internalization, could be overcome by longer periods of stimulation (6,000 pulses at 2 or 20 Hz) in the presence of strontium (1.8 m M ) or by increasing the extracellular concentration of strontium to 10 m M (2,400 action potentials at 20 Hz). It is suggested that endocytosis is dependent on calcium influx and that strontium is much less effective in replacing calcium for endocytosis than it is for exocytosis.     

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.     

The Quantal Nature of Synaptic Transmission at the Neuromuscular Junction of a Spider

Spontaneous and evoked release of transmitter at neuromuscular junctions in three different leg muscles of a tarantula (Dugesiella hentzi) was investigated. In most cases the spontaneous miniature potentials were released independently, although bursts from single synaptic junctions occasionally occurred. In contrast to recent findings in other arthropod muscles, focal extracellular recording from junctional areas revealed that the evoked release of transmitter quanta followed Poisson's theorem at low quantal content synaptic junctions in arachnid muscles.     

低温对茶树叶片膜脂脂肪酸和蛋白质的影响

本文研究低温胁迫过程中龙井43和大叶云峰叶片膜脂脂肪酸的变化。结果发现,低温下不同品种茶树叶片膜脂脂肪酸配比变化趋势不同,较抗寒的龙井43,不饱和脂肪酸指数(IUFA)和亚麻酸(183)比例随低温期间不同阶段呈现出"低-高-低"的变化趋势;而不抗寒的大叶云峰的变化趋势无明显规律性。此外,还研究了越冬过程中龙井43叶片可溶性蛋白和膜蛋白的变化,发现低温期间龙井43叶片可溶性蛋白含量和组分基本稳定,而膜蛋白含量在低温胁迫时大幅度上升,且经低温诱导出现了46KD、38KD两种新的蛋白组分,并在温度升高后消失。     

Spontaneous Activity of Murine Neuromuscular Junctions in the Presence of Dantrolene

By recording miniature end-plate potentials (mEPP), the effects of dantrolene (10-100 M), a blocker of ryanodine receptors, were studied on the isolated diaphragm of mice. The effects to be studied were as follows: on spontaneous secretion of acetylcholine quanta and on the pattern of interaction with ryanodine effects. Two-hour-long application of dantrolene to the muscle caused no significant changes in the amplitude and dispersion (²) of mEPP, nor on its time course. In the presence of 100 M dantrolene, the mean frequency of mEPP increased, on average, by 58.3 ± 5.9% (P < 0.05). Dantrolene suppressed in a dose-dependent manner a number of ryanodine effects (this agent was used in a concentration of 0.5 M as an intensifier of intracellular Ca²⁺ mobilization): it completely prevented the appearance of the population of high-amplitude (the so-called giant) mEPP, reduced by 50-80% the increment of the mEPP amplitude dispersion, and increased by 25-45% the mEPP mean amplitude; the above effects were induced by ryanodine application for 120 min. After preliminary application of dantrolene (10-100 M), ryanodine caused an effect not observable in the absence of dantrolene: mEPP became more frequent (140-210%). Thus, when acting on motor synapses, dantrolene behaves as a nontoxic agent, inducing only a presynaptic effect – a moderate increase in the mEPP frequency. The dual character of interaction between dantrolene and ryanodine in motor synapses was observed: on the one hand, dantrolene acts as a physiological antagonist of ryanodine by reducing the ryanodine-induced increase of dispersion and mEPP amplitude; on the other hand, dantrolene unmasks the ability of ryanodine to increase the mEPP frequency.