Search

The escape response and rhythmic swimming in zebrafish are distinct behaviors mediated by two functionally distinct motoneuron (Mn) types. The primary (1°Mn) type depresses, has a large quantal content (Qc), and a high release probability (Pr). Conversely, the secondary (2°Mn) type facilitates and has low and variable Qc and Pr. This functional duality matches well the distinct associated behaviors, with the 1°Mn providing the strong, singular C-bend initiating escape and the 2°Mn confers weaker, rhythmic contractions. Contributing to these functional distinctions is our identification of P/Q type calcium channels mediating transmitter release in 1°Mns and N type channels in 2°Mns. Remarkably, despite these functional and behavioral distinctions, all ~15 individual synapses on each muscle cell are shared by a 1°Mn bouton and at least one 2°Mn bouton. This novel blueprint of synaptic sharing provides an efficient way of controlling two different behaviors at the level of a single postsynaptic cell.

The CaV2.2 (N-type) and CaV2.1 (P/Q-type) voltage-dependent calcium channels are prevalent throughout the nervous system where they mediate synaptic transmission, but the basis for the selective presence at individual synapses still remains an open question. The CaV2.1 channels have been proposed to respond more effectively to brief action potentials (APs), an idea supported by computational modeling. However, the side-by-side comparison of CaV2.1 and CaV2.2 kinetics in intact neurons failed to reveal differences. As an alternative means for direct functional comparison we expressed zebrafish CaV2.1 and CaV2.2 α-subunits, along with their accessory subunits, in HEK293 cells. HEK cells lack calcium currents, thereby circumventing the need for pharmacological inhibition of mixed calcium channel isoforms present in neurons. HEK cells also have a simplified morphology compared to neurons, which improves voltage control. Our measurements revealed faster kinetics and shallower voltage-dependence of activation and deactivation for CaV2.1. Additionally, recordings of calcium current in response to a command waveform based on the motorneuron AP show, directly, more effective activation of CaV2.1. Analysis of calcium currents associated with the AP waveform indicate an approximately fourfold greater open probability (PO) for CaV2.1. The efficient activation of CaV2.1 channels during APs may contribute to the highly reliable transmission at zebrafish neuromuscular junctions.

In the early 1950s, Katz and his colleagues capitalized on the newly developed intracellular microelectrode recording technique to investigate synaptic transmission. For study they chose frog neuromuscular junction (NMJ), which was ideally suited due to the accessibility and large size of the muscle cells. Paradoxically, the large size precluded the use of next generation patch clamp technology. Consequently, electrophysiological study of synaptic function shifted to small central synapses made amenable by patch clamp. Recently, however, the unique features offered by zebrafish have rekindled interest in the NMJ as a model for electrophysiological study of synaptic transmission. The small muscle size and synaptic simplicity provide the singular opportunity to perform in vivo spinal motoneuron-target muscle patch clamp recordings. Additional incentive is provided by zebrafish lines harboring mutations in key synaptic proteins, many of which are embryonic lethal in mammals, but all of which are able to survive well past synapse maturation in zebrafish. This mini-review will highlight features that set zebrafish NMJs apart from traditional NMJs. We also draw into focus findings that offer the promise of identifying features that define release sites, which serve to set the upper limit of transmitter release. Since its conception several candidates representing release sites have been proposed, most of which are based on distinctions among vesicle pools in their state of readiness for release. However, models based on distinctions among vesicles have become enormously complicated and none adequately account for setting an upper limit for exocytosis in response to an action potential (AP). Specifically, findings from zebrafish NMJ point to an alternative model, positing that elements other than vesicles per se set the upper limits of release.

Summary The recruitment of motoneurons during force generation follows a general pattern that has been confirmed across diverse species [1–3]. Motoneurons are recruited systematically according to synaptic inputs and intrinsic cellular properties and corresponding to movements of different intensities. However, much less is known about the output properties of individual motoneurons and how they affect the translation of motoneuron recruitment to the strength of muscle contractions. In larval zebrafish, spinal motoneurons are recruited in a topographic gradient according to their input resistance (Rin) at different swimming strengths and speeds. Whereas dorsal, lower-Rin primary motoneurons (PMns) are only activated during behaviors that involve strong and fast body bends, more ventral, higher-Rin secondary motoneurons (SMns) are recruited during weaker and slower movements [4–6]. Here we perform in vivo paired recordings between identified spinal motoneurons and skeletal muscle cells in larval zebrafish. We characterize individual motoneuron outputs to single muscle cells and show that the strength and reliability of motoneuron outputs are inversely correlated with motoneuron Rin. During repetitive high-frequency motoneuron drive, PMn synapses undergo depression, whereas SMn synapses potentiate. We monitor muscle cell contractions elicited by single motoneurons and show that the pattern of motoneuron output strength and plasticity observed in electrophysiological recordings is reflected in muscle shortening. Our findings indicate a link between the recruitment pattern and output properties of spinal motoneurons that can together generate appropriate intensities for muscle contractions. We demonstrate that motoneuron output properties provide an additional peripheral mechanism for graded locomotor control at the neuromuscular junction.

Rapsyn-deficient myasthenic syndrome is characterized by a weakness in voluntary muscle contraction, a direct consequence of greatly reduced synaptic responses that result from poorly clustered acetylcholine receptors. As with other myasthenic syndromes, the general muscle weakness is also accompanied by use-dependent fatigue. Here, we used paired motor neuron target muscle patch-clamp recordings from a rapsyn-deficient mutant line of zebrafish to explore for the first time the mechanisms causal to fatigue. We find that synaptic responses in mutant fish can follow faithfully low-frequency stimuli despite the reduced amplitude. This is in part helped by a compensatory increase in the number of presynaptic release sites in the mutant fish. In response to high-frequency stimulation, both wild-type and mutant neuromuscular junctions depress to steady-state response levels, but the latter shows exaggerated depression. Analysis of the steady-state transmission revealed that vesicle reloading and release at individual release sites is significantly slower in mutant fish during high-frequency activities. Therefore, reductions in postsynaptic receptor density and compromised presynaptic release collectively serve to reduce synaptic strength to levels that fall below the threshold for muscle action potential generation, thus accounting for use-dependent fatigue. Our findings raise the possibility that defects in motor neuron function may also be at play in other myasthenic syndromes that have been mapped to mutations in muscle-specific proteins.SIGNIFICANCE STATEMENTUse-dependent fatigue accompanies many neuromuscular myasthenic syndromes, including muscle rapsyn deficiency. Here, using a rapsyn-deficient line of zebrafish, we performed paired motor neuron target muscle patch-clamp recordings to investigate the mechanisms causal to this phenomenon. Our findings indicate that the reduced postsynaptic receptor density resulting from defective rapsyn contributes to weakness, but is not solely responsible for use-dependent fatigue. Instead, we find unexpected involvement of altered transmitter release from the motor neuron. Specifically, slowed reloading of vesicle release sites leads to augmented synaptic depression during repeated action potentials. Even at moderate stimulus frequencies, the depression levels for evoked synaptic responses fall below the threshold for the generation of muscle action potentials. The associated contraction failures are manifest as use-dependent fatigue.

Mutations in muscle ACh receptors cause slow-channel syndrome (SCS) and Escobar syndrome, two forms of congenital myasthenia. SCS is a dominant disorder with mutations reported for all receptor subunits except γ. Escobar syndrome is distinct, with mutations located exclusively in γ, and characterized by developmental improvement of muscle function. The zebrafish mutant line, twister , models SCS in terms of a dominant mutation in the α subunit (α twi ) but shows the behavioral improvement associated with Escobar syndrome. Here, we present a unique electrophysiological study into developmental improvement for a myasthenic syndrome. The embryonic α twi βδγ receptor isoform produces slowly decaying synaptic currents typical of SCS that transit to a much faster decay upon the appearance of adult ε, despite the α twi mutation. Thus, the continued expression of α twi into adulthood is tolerated because of the ε expression and associated recovery, raising the likelihood of unappreciated myasthenic cases that benefit from the γ−ε switch.

Slow channel syndrome (SCS) is an autosomal dominant disease resulting from mutations in muscle acetylcholine receptor (AChR) subunits. The associated fatigue and muscle degeneration are proposed to result from prolonged synaptic responses that overload intracellular calcium. Single channel studies on reconstituted receptors bearing human mutations indicate that the prolonged responses result from an increase in receptor open duration and, in some cases, increased sensitivity to acetylcholine (ACh). We show that both of these aberrant receptor properties are recapitulated in heterozygotic zebrafish bearing an L258P mutation in the α subunit, thus affording the unique opportunity to compare the single channel properties of mutant receptors to the synaptic currents in vivo. Whole cell recordings revealed synaptic currents that decayed along a multi-exponential time course, reflecting receptors containing mixtures of wild type and mutant α subunits. Treatment with quinidine, an open channel blocker used to treat the human disorder, restored fast synaptic current kinetics and the ability to swim. Quinidine block also revealed that mutant receptors generate a large steady state current in the absence of ACh. The spontaneous openings reflected a destabilization of the closed state, leading to an apparent increase in the sensitivity of these receptors to ACh. The effective block by quinidine on synaptic currents as well as non-liganded openings points to dual sources for the calcium-dependent myopathy in certain forms of SCS.

Synaptic depression is prominent among synapses, but the underlying mechanisms remain uncertain. Here, we use paired patch clamp recording to study neuromuscular transmission between the caudal primary motor neuron and target skeletal muscle in zebrafish. This synapse has an unusually low number of release sites, all with high probabilities of release in response to low-frequency stimulation. During high-frequency stimulation, the synapse undergoes short-term depression and reaches steady-state levels of transmission that sustain the swimming behavior. To determine the release parameters underlying this steady state, we applied variance analysis. Our analysis revealed two functionally distinct subclasses of release sites differing by over 60-fold in rates of vesicle reloading. A slow reloading class requires seconds to recover and contributes to depression onset but not the steady-state transmission. By contrast, a fast reloading class recovers within tens of milliseconds and is solely responsible for steady-state transmission. Thus, in contrast to most current models that assign levels of steady-state depression to vesicle availability, our findings instead assign this function to nonuniform release site kinetics. The duality of active-site properties accounts for the highly nonlinear dependence of steady-state depression levels on frequency.

Fast and slow skeletal muscle types in larval zebrafish can be distinguished by a fivefold difference in the time course of their synaptic decay. Single-channel recordings indicate that this difference is conferred through kinetically distinct nicotinic acetylcholine receptor (AChR) isoforms. The underlying basis for this distinction was explored by cloning zebrafish muscle AChR subunit cDNAs and expressing them in Xenopus laevis oocytes. Measurements of single-channel conductance and mean open burst duration assigned α(2)βδε to fast muscle synaptic current. Contrary to expectations, receptors composed of only αβδ subunits (presumed to be α(2)βδ(2) receptors) recapitulated the kinetics and conductance of slow muscle single-channel currents. Additional evidence in support of γ/ε-less receptors as mediators of slow muscle synapses was reflected in the inward current rectification of heterologously expressed α(2)βδ(2) receptors, a property normally associated with neuronal-type nicotinic receptors. Similar rectification was reflected in both single-channel and synaptic currents in slow muscle, distinguishing them from fast muscle. The final evidence for α(2)βδ(2) receptors in slow muscle was provided by our ability to convert fast muscle synaptic currents to those of slow muscle by knocking down ε subunit expression in vivo. Thus, for the first time, muscle synaptic function can be ascribed to a receptor isoform that is composed of only three different subunits. The unique functional features offered by the α(2)βδ(2) receptor likely play a central role in mediating the persistent contractions characteristic to this muscle type.

We have identified a zebrafish mutant line, bajan, in which compromised motility and fatigue result from a point mutation in the gene coding choline acetyltransferase (ChAT), the enzyme responsible for acetylcholine (ACh) synthesis. Although the mutation predicts loss of ChAT function, bajan inexplicably retains low levels of neuromuscular transmission. We exploited this residual activity and determined the consequences for synaptic function. The attenuated synaptic responses were a direct consequence of a decrease in both resting mean quantal size and quantal content. To replicate behavioral fatigue in swimming, motorneurons were stimulated at high frequencies. A prominent reduction in quantal content, reflecting vesicle depletion, was coincident with a small additional reduction in quantal size. In humans, defective ChAT leads to episodic apnea, a form of congenital myasthenic syndrome characterized by use-dependent fatigue. In contrast to bajan, however, afflicted individuals exhibit a normal resting quantal size and quantal content. The fatigue in humans results from a pronounced long-lasting drop in quantal size with little or no change in quantal content. These differences have important implications for interpreting fatigue as well as on understanding the impact of ACh availability on vesicle filling and recycling.

Fast and slow skeletal muscle types are readily distinguished in larval zebrafish on the basis of differences in location and orientation. Additionally, both muscle types are compact, rendering them amenable to in vivo patch clamp study of synaptic function. Slow muscle mediates rhythmic swimming, but it does so purely through synaptic drive, as these cells are unable to generate action potentials. Our patch clamp recordings from muscle pairs of zebrafish reveal a network of electrical coupling in slow muscle that allows sharing of synaptic current within and between segmental boundaries of the tail. The synaptic current exhibits slow kinetics (τdecay ∼4 ms), which further facilitates passage through the low pass filter, a consequence of the electrically coupled network. In contrast to slow muscle, fast skeletal muscle generates action potentials to mediate the initial rapid component of the escape response. The combination of very weak electrical coupling and synaptic kinetics (τdecay

On initial formation of neuromuscular junctions, slow synaptic signals interact through an electrically coupled network of muscle cells. After the developmental onset of muscle excitability and the transition to fast synaptic responses, electrical coupling diminishes. No studies have revealed the functional importance of the electrical coupling or its precisely timed loss during development. In the mutant zebrafish shocked ( sho) electrical coupling between fast muscle cells persists beyond the time that it would normally disappear in wild-type fish. Recordings from sho indicate that muscle depolarization in response to motor neuron stimulation remains slow due to the low-pass filter characteristics of the coupled network of muscle cells. Our findings suggest that the resultant prolonged muscle depolarizations contribute to the premature termination of swimming in sho and the delayed acquisition of the normally rapid touch-triggered movements. Thus the benefits of gap junctions during early synapse development likely become a liability if not inactivated by the time that muscle would normally achieve fast autonomous function.

The physiologies of cells depend on electrochemical signals carried by ion channels and receptors. Venomous animals produce an enormous variety of peptide toxins with high affinity for specific ion channels and receptors. The mammalian prototoxin lynx1 shares with α-bungarotoxin the ability to bind and modulate nicotinic receptors (nAChRs); however, lynx1 is tethered to the membrane via a GPI anchor. We show here that several classes of neurotoxins, including bungarotoxins and cobratoxins, retain their selective antagonistic properties when tethered to the membrane. Targeted elimination of nAChR function in zebrafish can be achieved with tethered α-bungarotoxin, silencing synaptic transmission without perturbing synapse formation. These studies harness the pharmacological properties of peptide toxins for use in genetic experiments. When combined with specific methods of cell and temporal expression, the extension of this approach to hundreds of naturally occurring peptide toxins opens a new landscape for cell-autonomous regulation of cellular physiology in vivo.

Upon touch, twitch once zebrafish respond with one or two swimming strokes instead of typical full-blown escapes. This use-dependent fatigue is shown to be a consequence of a mutation in the tetratricopeptide domain of muscle rapsyn, inhibiting formation of subsynaptic acetylcholine receptor clusters. Physiological analysis indicates that reduced synaptic strength, attributable to loss of receptors, is augmented by a potent postsynaptic depression not seen at normal neuromuscular junctions. The synergism between these two physiological processes is causal to the use-dependent muscle fatigue. These findings offer insights into the physiological basis of human myasthenic syndrome and reveal the first demonstration of a role for rapsyn in regulating synaptic function.

Physiological analysis of two lines of paralytic mutant zebrafish, relaxed and sofa potato , reveals defects in distinct types of receptors in skeletal muscle. In sofa potato the paralysis results from failed synaptic transmission because of the absence of acetylcholine receptors, whereas relaxed mutants lack dihydropyridine receptor-mediated release of internal calcium in response to the muscle action potential. Synaptic structure and function appear normal in relaxed , showing that muscle paralysis per se does not impede proper synapse development. However, sofa potato mutants show incomplete development of the postsynaptic complex. Specifically, in the absence of ACh receptors, clusters of the receptor-aggregating protein rapsyn form in the extrasynaptic membrane but generally fail to localize to the subsynaptic region. Our results indicate that, although rapsyn molecules are capable of self-aggregation, interaction with ACh receptors is required for proper subsynaptic localization.

The transcriptional repressor, REST, helps restrict neuronal traits to neurons by blocking their expression in nonneuronal cells. To examine the repercussions of REST expression in neurons, we generated a neuronal cell line that expresses REST conditionally. REST expression inhibited differentiation by nerve growth factor, suppressing both sodium current and neurite growth. A novel corepressor complex, CoREST/HDAC2, was shown to be required for REST repression. In the presence of REST, the CoREST/HDAC2 complex occupied the native Nav1.2 sodium channel gene in chromatin. In neuronal cells that lack REST and express sodium channels, the corepressor complex was not present on the gene. Collectively, these studies define a novel HDAC complex that is recruited by the C-terminal repressor domain of REST to actively repress genes essential to the neuronal phenotype.

Fully grown oocytes of Xenopus laevis undergo resumption of the meiotic cycle when treated with the steroid hormone progesterone. Previous studies have shown that meiotic maturation results in profound downregulation of specific endogenous membrane proteins in oocytes. To determine whether the maturation impacts the functional properties of exogenously expressed membrane proteins, we used cut-open recordings from Xenopus oocytes expressing several types of Na+and K+channels. Treatment of oocytes with progesterone resulted in a downregulation of heterologously expressed Na+and K+channels without a change in the kinetics of the currents. The time course of progesterone-induced ion channel inhibition was concentration dependent. Complete elimination of Na+currents temporally coincided with development of germinal vesicle breakdown, while elimination of K+currents was delayed by ∼2 h. Coexpression of human β1-subunit with rat skeletal muscle α-subunit in Xenopus oocytes did not prevent progesterone-induced downregulation of Na+channels. Addition of 8-bromo-cAMP to oocytes or injection of heparin before progesterone treatment prevented the loss of expressed currents. Pharmacological studies suggest that the inhibitory effects of progesterone on expressed Na+and K+channels occur downstream of the activation of cdc2 kinase. The loss of channels is correlated with a reduction in Na+channel immunofluorescence, pointing to a disappearance of the ion channel-forming proteins from the surface membrane.

Studies on the amphibian neuromuscular junction have indicated that N-type calcium channels are the sole mediators of stimulus-evoked neurotransmitter release. We show, via both presynaptic and postsynaptic voltage-clamp measurements, that dihydropyridine (DHP)-sensitive calcium channels also contribute to stimulus-evoked release at developingXenopusneuromuscular junctions. Whereas inhibition of postsynaptic responses by ω-conotoxin (ω-Ctx) GVIA has been taken previously as evidence that only N-type channels mediate transmitter release, we find that both N-type and DHP-sensitive calcium currents are sensitive to this toxin. The unusual sensitivity of DHP-sensitive calcium channels to ω-Ctx GVIA in presynaptic terminals raises the possibility that this channel type may have escaped detection in previous physiological studies on adult frog neuromuscular junctions. Alternatively, the additional channel isoforms may be present only during early development, when they may serve to strengthen collectively presynaptic release during critical periods of synaptogenesis.

Asynchronous transmission plays a prominent role at certain synapses but lacks the mechanistic insights of its synchronous counterpart. The current view posits that triggering of asynchronous release during repetitive stimulation involves expansion of the same calcium domains underlying synchronous transmission. In this study, live imaging and paired patch clamp recording at the zebrafish neuromuscular synapse reveal contributions by spatially distinct calcium sources. Synchronous release is tied to calcium entry into synaptic boutons via P/Q type calcium channels, whereas asynchronous release is boosted by a propagating intracellular calcium source initiated at off-synaptic locations in the axon and axonal branch points. This secondary calcium source fully accounts for the persistence following termination of the stimulus and sensitivity to slow calcium buffers reported for asynchronous release. The neuromuscular junction and CNS neurons share these features, raising the possibility that secondary calcium sources are common among synapses with prominent asynchronous release. DOI: http://dx.doi.org/10.7554/eLife.01206.001, eLife digest Neurons communicate with one another at junctions called synapses. The arrival of an electrical signal known as an action potential at the first (presynaptic) neuron causes calcium ions to flood into the cell. This in turn causes the neuron to release packages of chemicals called neurotransmitters into the synapse. These activate receptors on the second (postsynaptic) neuron, triggering a new action potential that travels down the axon to the next synapse. The ions that trigger the release of the neurotransmitters are thought to enter the neuron through special calcium channels on or near the synapse. A sudden discrete influx of calcium ions causes the neuron to release many packages of transmitter simultaneously. This is called synchronous release. By contrast, when successive action potentials occur in the same neuron, the ions entering through the calcium channels accumulate inside the cell. This is thought to account for a sustained release of the neurotransmitter that continues even in the absence of nerve action potentials. This is called asynchronous release. Wen et al. have now obtained evidence that these two forms of release might be triggered by calcium from different sources. The work was performed using a synapse between nerve and muscle cells in zebrafish: it has been shown that channels called P/Q calcium channels control the release of neurotransmitters at this synapse in zebrafish. Mutant zebrafish with greatly reduced numbers of P/Q channels showed reduced synchronous release, but normal asynchronous release. Blocking the P/Q channels with a specific toxin in normal zebrafish eliminated synchronous release but left asynchronous release intact. Imaging experiments on these toxin-treated zebrafish revealed that a wave of calcium ions that propagated from a distant source coincided with the onset of asynchronous release. This wave of calcium fully accounted for the delayed onset and the persistence of asynchronous release following termination of the action potentials. This study further demonstrates that asynchronous release can be triggered by calcium ions that do not enter through the P/Q calcium channels. Waves of calcium have been described in the nervous system before, but their significance has always been unclear. The work of Wen et al. offers the first possible explanation for the role of these waves, and further experiments are now needed to determine whether this process happens at other types of synapses. DOI: http://dx.doi.org/10.7554/eLife.01206.002

Shepherd, Dawn and Paul Brehm. Two types of ACh receptors contribute to fast channel gating on mouse skeletal muscle. J. Neurophysiol. 78: 2966–2974, 1997. Single-channel recordings from mouse C2 myotubes indicate that maturation of skeletal muscle is accompanied by the appearance of two types of fast acetylcholine (ACh) receptor channels that are each functionally distinct from the embryonic receptor type present at early stages of differentiation. The embryonic receptor type has a low conductance (45 pS) and long channel open time, rendering slowly decaying synaptic currents. One fast channel type that appears during muscle maturation is distinguished from the embryonic receptor type on the basis of both higher conductance (65 pS) and shorter open time. However, single-channel recordings from differentiated mouse skeletal muscle cell line (C2) point to the existence of a second fast receptor type, which has a conductance similar to the embryonic receptor type (45 pS), yet significantly reduced mean channel open time. Analyses of individual channel function at high ACh concentrations directly demonstrate the coexistence of two kinetically distinct types of 45 pS ACh receptors. Openings by fast type and slow embryonic type of 45 pS receptors occurred in bursts, allowing distinction on the basis of both mean open time and open probability for individual receptors. The embryonic type of 45 pS receptor has an open time approximately twofold longer than the fast-receptor counterpart. Additional differences were reflected in the open probability distributions for fast and slow 45 pS receptor types. Both types of 45 pS receptor were kinetically distinguishable from the 65 pS receptor. We found no support for the idea that the slow and fast 45 pS receptor types result from the interconversion of dual gating modes involving the same receptor protein. Our results are consistent with the idea that the acquisition of fast synaptic current decay, required at mature neuromuscular synapses, is the result of the up-regulation of two distinct fast types of nicotinic ACh receptors during skeletal muscle development.

A long-held tenet of neuromuscular transmission is that calcium-dependent neurotransmitter release is mediated by N-type calcium channels in frog but P/Q-type channels in mammals. The N-type assignment in frog is based principally on pharmacological sensitivity to ω-conotoxin GVIA. Our studies show that zebrafish neuromuscular transmission is also sensitive to ω-conotoxin GVIA. However, positional cloning of a mutant line with compromised neuromuscular function identified a mutation in a P/Q- rather than N-type channel. Cloning and heterologous expression of this P/Q-type channel confirmed a block by ω-conotoxin GVIA raising the likelihood that all vertebrates, including frog, use the P/Q-type calcium channel for neuromuscular transmission. In addition, our P/Q defective mutant line offered a means of testing the ability of roscovitine, known to potentiate frog neuromuscular transmission, to mediate behavioral and functional rescue. Acute treatment led to rapid improvement of both, pointing to potential therapeutic benefit for myasthenic disorders involving calcium channel dysfunction.

The continuous presence of nerve growth factor (NGF) is thought to be required for the elaboration of neuronal-like traits in PC12 cells. Surprisingly, we find that a 1 min exposure to NGF is sufficient to engage a longer-term genetic program leading to the acquisition of membrane excitability. Whereas continuous exposure to NGF causes the induction of a family of sodium channels, the effect of a brief exposure is to induce selectively expression of the peripheral nerve-type sodium channel gene PN1, through a distinct signaling pathway requiring immediate-early genes. A 1 min exposure of PC12 cells to interferon-gamma also causes PN1 gene induction, suggesting that the "triggered" NGF and interferon-gamma signaling pathways share common molecular intermediates.

Epithelial calcium action potentials in Obelia geniculata trigger brief light flashes from specialized cells by direct activation of cytoplasmic calcium-activated photoprotein obelin. During a series of action potentials, sequential flashes undergo characteristic facilitation and decrement with no change in associated spike waveform. Analysis of the subcellular light distribution shows that facilitation results from two processes: recruitment of calcium entry sites and increased light from previously responding localized sites. We propose a model that accounts for the localized flash facilitation and decrement observed in vivo and is based upon the kinetics of calcium binding and emission of obelin. In this model, obelin emits light only when three calcium ions are bound. Changes in flash intensity during successive action potentials result from calcium bound persistently to unexpended obelin, effectively lowering the number of calcium ions required for subsequent activation. Accordingly, facilitation or decrement results from the time-dependent availability of singly and doubly bound obelin.

During vertebrate skeletal muscle development, multiple forms of long- open-time (slow-type) ACh receptor channels are replaced by at least two different types of short-open-time (fast-type) ACh receptors. Expression of ACh receptors in Xenopus oocytes indicates that the substitution of an epsilon subunit for a gamma subunit may account for both types of fast-gated channel types in adult muscle. Unlike the various forms of the embryonic receptor, in which functional diversity is achieved through alterations in subunit composition, the two major fast-gated forms expressed in oocytes have identical subunit composition. These findings provide a structural basis for both types of short-open-time ACh receptor types found in adult muscle.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Paired spinal motorneuron and target muscle recordings were used to examine transmitter depletion and subsequent recovery during high frequency stimulation. The skeletal muscle is sufficiently compact that the unitary synaptic events (∼400pA average) were fully resolved through whole cell voltage clamp. Also, the evoked endplate current reflected the sum of 20Hz led to drops in quantal content and eventual failure, with no associated change in unitary quantal size. The time required to deplete 80% of the transmitter corresponded to 25 sec at 20Hz, 10 sec at 50Hz and 5 sec at 100 Hz. Recovery occurred abruptly after a 40 sec rest and the rate of recovery was calcium-dependent. Analysis of the depletion profile during 100 Hz stimulation revealed two partially overlapping processes. Initially, all release was synchronous upon repolarization of the motorneuron action potential. With continued stimulation the release was delayed and asynchronous with respect to the action potential. The quantal size underlying both synchronous and asynchronous modes was unchanged during the depletion and following recovery. At 100 Hz the synchronous endplate current converted to asynchronous unitary openings in a manner that was qualitatively reciprocal. Moreover, traces composed principally of synchronous events had few asynchronous events and conversely traces with the largest numbers of asynchronous events lacked synchronous release. This reciprocity suggested that the two modes share common release sites. However, measurements of total synchronous and asynchronous events during the time of maximal overlap revealed a period of facilitation, suggesting that the synchronous and asynchronous modes were capable of additivity and might represent different release sites. Experiments are ongoing to distinguish between these alternative possibilities.

Electrophysiological studies indicate that ion channels are present throughout the animal kingdom. Between distantly-related phyla, similarities in ion selectivity, gating, pharmacology and kinetics have obscured insights into how these proteins might have evolved. Moreover, deduced structures of a limited number of chordate and arthropod ion channels have shown that functionally disparate channel types often share a common structure. At least six different superfamilies of structurally-related ion channels have been described thus far, providing some clues for ‘ancestral’ proteins of a specific family. By contrast, evolutionary links (should they exist) among these superfamilies are less clear, requiring further structural analysis of ion channels in lower invertebrates, single-celled organisms and plants.

Asset–liability management for nonlife insurers is more complicated than quotidian duration-matching analysis of changing interest rates. When stochastic liabilities are correlated with a number of financial variables, enterprise-wide models are needed to quantify the impacts of possible investment and hedging strategies. Keywords: assets; liabilities; ALM ; duration; ERM ; investments; portfolio; risk return; efficient frontier; reinsurance; hedging

Truncated escape responses characteristic of the zebrafish shocked mutant result from a defective glial glycine transporter (GlyT1). In homozygous GlyT1 mutants, irrigating brain ventricles with glycine-free solution rescues normal swimming. Conversely, elevating brain glycine levels restores motility defects. These experiments are consistent with previous studies that demonstrate regulation of global glycine levels in the CNS as a primary function of GlyT1. As GlyT1 mutants mature, their ability to mount an escape response naturally recovers. To understand the basis of this recovery, we assay synaptic transmission in primary spinal motor neurons by measuring stimulus-evoked postsynaptic potentials. At the peak of the motility defect, inhibitory synaptic potentials are both significantly larger and more prolonged indicating a prominent role for GlyT1 in shaping fast synaptic transmission. However, as GlyT1 mutants naturally regain their ability to swim, the amplitude of inhibitory potentials decreases to below wild-type levels. In parallel with diminishing synaptic potentials, the glycine concentration required to evoke the mutant motility defect increases 61-fold during behavioral recovery. Behavioral recovery is also mirrored by a reduction in the levels of both glycine receptor protein and transcript. These results suggest that increased CNS glycine tolerance and reduced glycine receptor expression in GlyT1 mutants reflect compensatory mechanisms for functional recovery from excess nervous system inhibition.

Acetylcholine receptor (AcChoR) subunit mRNAs transcribed from mouse BC3H-1 cDNAs were injected into Xenopus oocytes and the expressed AcChoR channels were examined by single channel recording. Injection of alpha-, beta-, gamma-, and delta-subunit mRNAs produced two predominant channel classes with conductances of approximately 50 and approximately 12 pS, while infrequent openings of approximately 25-pS channels were also observed. Injection of alpha-, beta-, and gamma-subunit mRNAs produced a single class of approximately 12-pS AcChoR channels, which resembled the smallest conductance channels present in alpha beta gamma omega-injected oocytes. Assembly of delta-less channels may thus explain the lowest conductance AcChoR channels in alpha beta gamma delta-injected oocytes and might also account for similar channels that have been observed in vertebrate skeletal muscle.

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 musclein 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.

Zebrafish acquire the ability for fast swimming early in development. The motility mutant accordion (acc) undergoes exaggerated and prolonged contractions on both sides of the body, interfering with the acquisition of patterned swimming responses. Our whole cell recordings from muscle indicate that the defect is not manifested in neuromuscular transmission. However, imaging of skeletal muscle of larval acc reveals greatly prolonged calcium transients and associated contractions in response to depolarization. Positional cloning of acc identified a serca mutation as the cause of the acc phenotype. SERCA is a sarcoplasmic reticulum transmembrane protein in skeletal muscle that mediates calcium re-uptake from the myoplasm. The mutation in SERCA, a serine to phenylalanine substitution, is likely to result in compromised protein function that accounts for the observed phenotype. Indeed, direct evidence that mutant SERCA causes the motility dysfunction was provided by the finding that wild type fish injected with an antisense morpholino directed against serca, exhibited accordion-like contractions and impaired swimming. We conclude that the motility dysfunction in embryonic and larval accordion zebrafish stems directly from defective calcium transport in skeletal muscle rather than defective CNS drive.

Clustering of nicotinic muscle acetylcholine receptors (AChRs) requires association with intracellular rapsyn, a protein with an intrinsic ability to self-cluster. Previous studies onsofa potato(sop), an AChR null line of zebrafish, have suggested that AChRs may play an active role in subsynaptic localization of rapsyn clusters. To test this proposal directly, we identified and cloned the gene responsible for thesopphenotype and then attempted to rescue subsynaptic localization of the receptor-rapsyn complex in mutant fish.sopcontains a leucine to proline mutation at position 28, near the N terminus of the zebrafish AChR δ subunit. Transient expression of mutant δ subunit insopfish was unable to restore surface expression of muscle AChRs. In contrast, expression of wild-type δ subunit restored the ability of muscle to assemble surface receptors along with the ability of fish to swim. Most importantly, the ability of rapsyn clusters to localize effectively to subsynaptic sites also was rescued in large part. Our results point to direct involvement of the AChR molecule in restricting receptor-rapsyn clusters to the synapse.

Before establishing terminal synapses with their final muscle targets,migrating motor axons form en passant synaptic contacts with myotomal muscle. Whereas signaling through terminal synapses has been shown to play important roles in pre- and postsynaptic development, little is known about the function of these early en passant synaptic contacts. Here, we show that increased neuromuscular activity through en passant synaptic contacts affects pre- and postsynaptic development. We demonstrate that in zebrafish twistermutants, prolonged neuromuscular transmission causes motor axonal extension and muscular degeneration in a dose-dependent manner. Cloning of twister reveals a novel, dominant gain-of-function mutation in the muscle-specific nicotinic acetylcholine receptor α-subunit, CHRNA1. Moreover, electrophysiological analysis demonstrates that the mutant subunit increases synaptic decay times, thereby prolonging postsynaptic activity. We show that as the first en passant synaptic contacts form, excessive postsynaptic activity in homozygous embryos severely impedes pre- and postsynaptic development, leading to degenerative defects characteristic of the human slow-channel congenital myasthenic syndrome. By contrast, in heterozygous embryos, transient and mild increase in postsynaptic activity does not overtly affect postsynaptic morphology but causes transient axonal defects, suggesting bi-directional communication between motor axons and myotomal muscle. Together, our results provide compelling evidence that during pathfinding, myotomal muscle cells communicate extensively with extending motor axons through en passant synaptic contacts.

Slow channel syndrome (SCS), a congenital form of myasthenia, results from a gain of function mutation in the muscle nicotinic acetylcholine receptor (AChR). The mutant line twister represents a zebrafish equivalent of SCS as a result of a L258P mutation in the M2 region of the α-subunit. Zebrafish heterozygous for the twister mutation lack the ability to perform coordinated swimming functions during early development, presumably due to the greatly prolonged end-plate currents (EPC) resulting from altered AChR kinetics. EPCs recorded from fast skeletal muscle of twister decay along a triple exponential time course (1.1, 8.6 and 70.2 ms) compared to a single exponential decay typical of wild-type muscle. Paradoxically, swimming behavior improves during development. The improvement coincides with the selective disappearance of the slowest component of synaptic decay. We recorded single channel properties of heterologously expressed zebrafish subunits αtwi along with β,δ,e or γ subunits to identify the basis of this slowest component. αtwiβδe receptors exhibit a mean open time that corresponds to the intermediate time constant of twister decay. By contrast, the embryonic receptor isoform αtwiβδγ exhibited a greatly prolonged open time that corresponded to the slowest component of synaptic decay in twister. This result is unexpected given the similarity in mean open times for wild type αβδe (0.5 ms) and αβδγ (1.2 ms) receptors. We propose that behavioral and functional recovery results from the observed developmental decrease in γ subunit mRNA expression, facilitating a switch from αtwiβδγ to αtwiβδe receptors. In support of this idea, morpholino RNA knockdown of γ subunit expression improved swimming performance and reduced the contribution of the slow component to synaptic current decay, presumably through an increase in αtwiβδe AChRs.

Calcium channels play dual roles in cell signaling by promoting membrane depolarization and allowing entry of calcium ions. Patch-clamp recordings of calcium and calcium-dependent currents from the soma of Xenopus spinal neurons indicate key functional differences from those of presynaptic terminals. Both terminals and somas exhibit prominent high-voltage-activated (HVA) calcium current, but only the soma expresses additional low-voltage-activated (LVA) T-type current. Further differences are reflected in the HVA current; N- and R-type channels are predominant in the soma while the terminal calcium current is composed principally of N type with smaller contribution by L- and R-type channels. Potential physiological significance for these different distributions of channel types may lie in the differential channel kinetics. Activation of somatic HVA calcium current occurs more slowly than HVA currents in terminals. Additionally, somatic LVA calcium current activates and deactivates much more slowly than any HVA calcium current. Fast-activating and -deactivating calcium current may be critical to processing the rapid exocytotic response in terminals, whereas slow LVA and HVA calcium currents may play a central role in shaping the somatic firing pattern. In support of different kinetic behavior between these two compartments, we find that somatic calcium current activates a prominent slow chloride current not observed in terminal recordings. This current activates in response to calcium entering through either LVA or HVA channels and likely functions as a modulator of excitability or synaptic input. The restriction of this channel type to the soma lends further support to the idea that differential expression of fast and slow channel types in these neurons is dictated by differences in signaling requirements for somatic and terminal compartments.

The functional properties of most sodium channels are too similar to permit identification of specific sodium channel types underlying macroscopic current. Such discrimination would be particularly advantageous in the nervous system in which different sodium channel family isoforms are coexpressed in the same cell. To test whether members of the μ-conotoxin family can discriminate among known neuronal sodium channel types, we examined six toxins for their ability to block different types of heterologously expressed sodium channels. PIIIA μ-conotoxin blocked rat brain type II/IIA (rBII/IIA) and skeletal muscle sodium current at concentrations that resulted in only slight inhibition of rat peripheral nerve (rPN1) sodium current. Recordings from variant lines of PC12 cells, which selectively express either rBII/IIA or rPN1 channel subtypes, verified that the differential block by PIIIA also applied to native sodium current. The sensitivity to block by PIIIA toxin was then used to discriminate between rBII/IIA and rPN1 sodium currents in NGF-treated PC12 cells in which both mRNAs are induced. During the first 24 hr of NGF-treatment, PN1 sodium channels accounted for over 90% of the sodium current. However, over the ensuing 48 hr period, a sharp rise in the proportion of rBII/IIA sodium current occurred, confirming the idea, based on previous mRNA measurements, that two distinct sodium channel types appear sequentially during neuronal differentiation of PC12 cells.

Cut-open recordings from Xenopus oocytes expressing either nerve (PN1) or skeletal muscle (SkM1) Na(+) channel alpha subunits revealed slow inactivation onset and recovery kinetics of inward current. In contrast, recordings using the macropatch configuration resulted in an immediate negative shift in the voltage-dependence of inactivation and activation, as well as time-dependent shifts in kinetics when compared to cut-open recordings. Specifically, a slow transition from predominantly slow onset and recovery to exclusively fast onset and fast recovery from inactivation occurred. The shift to fast inactivation was accelerated by patch excision and by agents that disrupted microtubule formation. Application of positive pressure to cell-attached macropatch electrodes prevented the shift in kinetics, while negative pressure led to an abrupt shift to fast inactivation. Simultaneous electrophysiological recording and video imaging of the cell-attached patch membrane revealed that the pressure-induced shift to fast inactivation coincided with rupture of sites of membrane attachment to cytoskeleton. These findings raise the possibility that the negative shift in voltage-dependence and the fast kinetics observed normally for endogenous Na(+) channels involve mechanical destabilization. Our observation that the beta1 subunit causes similar changes in function of the Na(+) channel alpha subunit suggests that beta1 may act through interaction with cytoskeleton.

During prolonged application of transmitter, ligand-gated ion channels enter a nonconducting desensitized state. Studies onTorpedoelectroplax nicotinic acetylcholine (ACh) receptors have shown that entry into the desensitized state is accelerated by protein kinase A-dependent (PKA) receptor phosphorylation. To examine the effects of phosphorylation on desensitization of muscle-type ACh receptors, we expressed the frog embryonic receptor type inXenopusoocytes. Treatment of embryonic muscle ACh receptors with 8-Br cAMP had no measurable effect on the rate of entry into a desensitized state, but it greatly accelerated the recovery from desensitization. Three complementary approaches to reduce the levels of receptor phosphorylation provided additional evidence for a role of PKA-dependent phosphorylation in rescuing receptors from long-term desensitization. Inactivation of the endogenous PKA activity by coexpression of an inhibitor protein, treatment of receptors with phosphatase, and removal of phosphorylation sites by site-specific subunit mutation all resulted in slowed recovery. Our findings point to the existence of two distinct desensitized states: one requiring several seconds for full recovery and a second state from which recovery requires minutes. Receptors lacking PKA phosphorylation sites exhibit a pronounced increase in the slowly recovering component of desensitization, suggesting that receptor phosphorylation speeds overall recovery by reducing the entry into a deep desensitized state. This newly described effect of phosphorylation on ACh receptor function may serve as an important modulator of postsynaptic receptor sensitivity.

Changes in the functional properties of acetylcholine receptor (AchR) channels were followed in the C2 muscle cell line over the period of 1 to 17 days following myotube formation. Up to 1 week after myotube formation, the predominant class of channel exhibited low (45 pS) conductance and long mean channel open time (14 msec), characteristic of the major type of AchR in embryonic skeletal muscle. Three additional Ach-activated currents with conductances lower than 45 pS and long channel open times were also observed. Seven to 10 days following myotube formation, channels of 45 pS and 65 pS and short (2-6 msec) mean open duration were observed, characteristic of receptor channels in adult muscle. Increases in ϵ subunit mRNA levels preceded the functional expression of channels with brief open durations. Our results show that C2 muscle cells maintained in culture express a full range of functionally distinct AchR types in the absence of nerve.

1. Nicotinic acetylcholine (ACh) receptors in developing vertebrate skeletal muscle exhibit functional heterogeneity in both conductance and kinetics. To assess the contributions of receptors differing in subunit composition to the heterogeneity, various combinations of mouse alpha beta delta gamma epsilon subunit RNAs were tested for the ability to express functional receptors in Xenopus oocytes. 2. Two combinations of dual-subunit RNAs (alpha delta and alpha gamma) resulted in detectable ACh-activated currents and six different combinations of three or more subunit RNAs produced significant numbers of functional channels. The order of combinations yielding the greatest amount of current was alpha beta gamma > alpha beta delta = alpha delta epsilon > alpha delta gamma > alpha delta > alpha gamma. 3. The extent to which a channel type with three different subunits was expressed was highly dependent upon the ratios of RNAs coding for the different subunits. For alpha beta delta receptors the efficiency of expression was alpha: beta: delta (1/5:1/5:1) >> (1:1:1) >> (1/5:1:1/5) > (1:1/5:1/5). 4. The level of expression of three-subunit combinations was also critically dependent upon the order of RNAs injected. When alpha delta or alpha gamma RNA combinations were co-injected 2 days prior to the injection of beta RNA, the expression was 2-5 times greater than when alpha beta injection was followed by injection of delta or gamma RNA. 5. Single-channel measurements revealed that alpha beta delta channels were not expressed in the presence of alpha beta delta epsilon RNAs, even under conditions when the amount of delta RNA injected was 5-fold higher than the amount of epsilon RNA. 6. These data indicate that the functional expression of subunit-omitted receptors depends critically upon the relative amounts of the five different subunit RNAs. Receptors composed of three different subunits express in the presence of the subunit RNAs characteristic of embryonic muscle (alpha beta delta gamma), but are not observed with the combination of RNAs characteristic of adult muscle (alpha beta delta epsilon).

During the transition from embryonic to adult skeletal muscle, a decreased mean channel open time and accelerated desensitization of nicotinic acetylcholine (ACh) receptors result from the substitution of an epsilon subunit for gamma. A single ACh receptor channel of the embryonic type, expressed in Xenopus oocytes, interconverts between gating modes of short and long open time, whereas the adult receptor channel resides almost exclusively in the gating mode with short open time. Differences in the fraction of time spent in either gating mode account for the subunit dependence of both receptor open time and desensitization. Therefore, developmental changes in the kinetics of muscle ACh receptors may be imparted through subunit-dependent stabilization of intrinsic gating modes.