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C R Slater

Publications and source records attributed to C R Slater.

At least 19 recordsLinked to original sources
PubMed2001

Sodium channel mRNAs at the neuromuscular junction: distinct patterns of accumulation and effects of muscle activity.

Voltage-gated sodium channels (VGSCs) are highly concentrated at the neuromuscular junction (NMJ) in mammalian skeletal muscle. Here we test the hypothesis that local upregulation of mRNA contributes to this accumulation. We designed radiolabeled antisense RNA probes, specific for the "adult" Na(V)1.4 and "fetal" Na(V)1.5 isoforms of VGSC in mammalian skeletal muscle, and used them in in situ hybridization studies of rat soleus muscles. Na(V)1.4 mRNA is present throughout normal adult muscles but is highly concentrated at the NMJ, in which the amount per myonucleus is more than eightfold greater than away from the NMJ. Na(V)1.5 mRNA is undetectable in innervated muscles but is dramatically upregulated by denervation. In muscles denervated for 1 week, both Na(V)1.4 and Na(V)1.5 mRNAs are present throughout the muscle, and both are concentrated at the NMJ. No Na(V)1.5 mRNA was detectable in denervated muscles stimulated electrically for 1 week in vivo. Neither denervation nor stimulation had any significant effect on the level or distribution of Na(V)1.4 mRNA. We conclude that factors, probably derived from the nerve, lead to the increased concentration of VGSC mRNAs at the NMJ. In addition, the expression of Na(V)1.5 mRNA is downregulated by muscle activity, both at the NMJ and away from it.

Animals↗
PubMed2001

Safety factor at the neuromuscular junction.

Reliable transmission of activity from nerve to muscle is necessary for the normal function of the body. The term 'safety factor' refers to the ability of neuromuscular transmission to remain effective under various physiological conditions and stresses. This is a result of the amount of transmitter released per nerve impulse being greater than that required to trigger an action potential in the muscle fibre. The safety factor is a measure of this excess of released transmitter. In this review we discuss the practical difficulties involved in estimating the safety factor in vitro. We then consider the factors that influence the safety factor in vivo. While presynaptic transmitter release may be modulated on a moment to moment basis, the postsynaptic features that determine the effect of released transmitter are not so readily altered to meet changing demands. Different strategies are used by different species to ensure reliable neuromuscular transmission. Some, like frogs, rely on releasing a large amount of transmitter while others, like man, rely on elaborate postsynaptic specialisations to enhance the response to transmitter. In normal adult mammals, the safety factor is generally 3-5. Both pre- and postsynaptic components change during development and may show plasticity in response to injury or disease. Thus, both acquired autoimmune and inherited congenital diseases of the neuromuscular junction (NMJ) can significantly reduce, or even transiently increase, safety factor.

Action Potentials↗
PubMed2000

Ectopic expression of NCAM in skeletal muscle of transgenic mice results in terminal sprouting at the neuromuscular junction and altered structure but not function.

The neuromuscular system provides an excellent model for the analysis of molecular interactions involved in the development and plasticity of synaptic contacts. The neural cell adhesion molecule (NCAM) is believed to be involved in the development and plasticity of the neuromuscular junction, in particular the axonal sprouting response observed in paralyzed and denervated muscle. In order to explore the role of myofiber NCAM in modulating the differentiation of motor neurons, we generated transgenic mice expressing a GPI-anchored NCAM isoform that is normally found in developing and denervated muscle, under the control of a skeletal muscle-specific promoter. This results in the constitutive expression of NCAM at postnatal ages, a time when the endogenous mouse NCAM is absent from the myofiber. We found that a significant number of neuromuscular junctions in adult transgenic animals displayed terminal sprouting (>20%) reminiscent of that elicited in response to cessation of neuromuscular activity. Additionally, a significant increase in the size and complexity of neuromuscular synapses as a result of extensive intraterminal sprouting was detected. Electrophysiological studies, however, revealed no significant alterations of neuromuscular transmission at this highly efficient synapse. Sprouting in response to paralysis or following nerve crush was also significantly enhanced in transgenic animals. These results suggest that in this ectopic expression model NCAM can directly modulate synaptic structure and motor neuron-muscle interactions. The results contrast with knockout experiments of the NCAM gene, where very limited changes in the neuromuscular system were observed.

Acetylcholinesterase↗
PubMed1999

Synaptic vesicle dynamics in rat fast and slow motor nerve terminals.

We have investigated whether rat motor nerve terminals with different in vivo activity patterns also have different vesicle trafficking characteristics. To do this, we monitored, using combined optical and electrical techniques, the rate of exocytosis (during different frequencies and patterns of activity), the releasable pool size, and the recycle time of synaptic vesicles in terminals on soleus (slow-twitch) and extensor digitorum longus [(EDL); fast-twitch] muscle fibers. EDL terminals had a higher initial quantal content (QC) than soleus, but during tonic or phasic stimulation at 20-80 Hz, EDL QC ran down to a greater extent than soleus QC. By recording loss of fluorescence from exocytosing vesicles labeled with the dye FM1-43, EDL terminals were found to destain faster than those in soleus. Simultaneous intracellular recording of end plate potentials, to count the number of vesicles released, permitted estimation of the total vesicle pool (VP) size and the recycle time by combining the optical and electrophysiological data. Soleus vesicle pool was larger than EDL, but recycle time was not significantly different. These terminals, therefore, are adapted to their in vivo activity patterns by alterations in QC and VP size but not recycle time.

Adaptation, Physiological↗
PubMed1998

An early stage in sodium channel clustering at developing rat neuromuscular junctions.

Voltage-gated sodium channels (VGSCs) are concentrated in the postsynaptic membrane at the adult rat neuromuscular junction (NMJ). We have used immunolabelling to determine the pattern of initial VGSC accumulation during development. At birth, but not 3 days before, VGSC labelling is detectable at the NMJ and in the perijunctional (periJ) membrane but not elsewhere. A much higher density cluster of VGSCs forms at the NMJ itself 1-2 weeks later. If the nerve is cut 2 days after birth, VGSC labelling persists in the periJ region for at least 4 weeks but the clustering of VGSCs at the NMJ fails to develop. Thus an early, stable accumulation of VGSCs develops near the NMJ at least a week before high density postsynaptic VGSC clusters form.

Animals↗
PubMed1998

beta-Spectrin is colocalized with both voltage-gated sodium channels and ankyrinG at the adult rat neuromuscular junction.

Voltage-gated sodium channels (VGSCs) are concentrated in the depths of the postsynaptic folds at mammalian neuromuscular junctions (NMJs) where they facilitate action potential generation during neuromuscular transmission. At the nodes of Ranvier and the axon hillocks of central neurons, VGSCs are associated with the cytoskeletal proteins, beta-spectrin and ankyrin, which may help to maintain the high local density of VGSCs. Here we show in skeletal muscle, using immunofluorescence, that beta-spectrin is precisely colocalized with both VGSCs and ankyrinG, the nodal isoform of ankyrin. In en face views of rat NMJs, acetylcholine receptors (AChRs), and utrophin immunolabeling are organized in distinctive linear arrays corresponding to the crests of the postsynaptic folds. In contrast, beta-spectrin, VGSCs, and ankyrinG have a punctate distribution that extends laterally beyond the AChRs, consistent with a localization in the depths of the folds. Double antibody labeling shows that beta-spectrin is precisely colocalized with both VGSCs and ankyrinG at the NMJ. Furthermore, quantification of immunofluorescence in labeled transverse sections reveals that beta-spectrin is also concentrated in perijunctional regions, in parallel with an increase in labeling of VGSCs and ankyrinG, but not of dystrophin. These observations suggest that interactions with beta-spectrin and ankyrinG help to maintain the concentration of VGSCs at the NMJ and that a common mechanism exists throughout the nervous system for clustering VGSCs at a high density.

Animals↗
PubMed1998

Utrophin mRNA expression in muscle is not restricted to the neuromuscular junction.

Utrophin is normally present exclusively in synaptic regions of skeletal muscle fibers, although it is expressed extrasynaptically in certain pathological situations, where it has been proposed to compensate for the absence of dystrophin in Duchenne muscular dystrophy patients and mdx mice. Recently there have been conflicting reports regarding the preferential expression of utrophin mRNA at the neuromuscular junction. Using in situ hybridization with RNA probes, we show a clear accumulation of autoradiographic labeling at more than 90% of neuromuscular junctions (identified by histochemical demonstration of cholinesterase activity). The intensity of this labeling is proportional to the number of junctional myonuclei in the section. Some clusters of labeling were found associated with nonmuscle nuclei (e.g., blood vessels, nerves), where utrophin is present. In addition, labeling for utrophin mRNA was associated with about 25% of extrajunctional myonuclei, where the protein is not present. The mean labeling per nucleus at junctional myonuclei was at least 10 times greater than at extrajunctional myonuclei. We discuss the possible regulatory mechanisms involved in the heterogeneous expression of utrophin mRNA in skeletal muscle.

Amino Acid Sequence↗
PubMed1998

An improved method for the simultaneous demonstration of mRNA and esterase activity at the human neuromuscular junction.

The aim of this study was to develop a simple means of studying the distribution of mRNA coding for post-synaptic proteins at the human neuromuscular junction. A reliable method by which to identify the junctions in tissue sections after in situ hybridization was essential. A method is described for combining the histochemical demonstration of esterase activity at the neuromuscular junction with autoradiographic localization of mRNA by in situ hybridization in the same cryostat section of skeletal muscle. The indigogenic esterase method of Strum and Hall-Craggs (1982) was modified in such a way that it is able to survive the multiple steps involved in in situ hybridization and autoradiography. The protocol is simple and reproducible and has been used successfully on sections of both rat and human skeletal muscle. To demonstrate the method, sections were reacted to reveal esterase activity and were then processed for in situ hybridization using a 35S-labelled probe specific for the epsilon-subunit of the acetylcholine receptor. The reaction product was retained after the lengthy in situ hybridization and autoradiographic procedures. To our knowledge, this is the first demonstration of acetylcholine receptor mRNA by in situ hybridization at human neuromuscular junctions.

Animals↗
PubMed1997

Differential localization of voltage-dependent calcium channel alpha1 subunits at the human and rat neuromuscular junction.

Neurotransmitter release is regulated by voltage-dependent calcium channels (VDCCs) at synapses throughout the nervous system. At the neuromuscular junction (NMJ) electrophysiological and pharmacological studies have identified a major role for P- and/or Q-type VDCCs in controlling acetylcholine release from the nerve terminal. Additional studies have suggested that N-type channels may be involved in neuromuscular transmission. VDCCs consist of pore-forming alpha1 and regulatory beta subunits. In this report, using fluorescence immunocytochemistry, we provide evidence that immunoreactivity to alpha1A, alpha1B, and alpha1E subunits is present at both rat and human adult NMJs. Using control and denervated rat preparations, we have been able to establish that the subunit thought to correspond to P/Q-type channels, alpha1A, is localized presynaptically in discrete puncta that may represent motor nerve terminals. We also demonstrate for the first time that alpha1A and alpha1B (which corresponds to N-type channels) may be localized in axon-associated Schwann cells and, further, that the alpha1B subunit may be present in perisynaptic Schwann cells. In addition, the alpha1E subunit (which may correspond to R/T-type channels) seems to be localized postsynaptically in the muscle fiber membrane and concentrated at the NMJ. The possibility that all three VDCCs at the NMJ are potential targets for circulating autoantibodies in amyotrophic lateral sclerosis is discussed.

Amyotrophic Lateral Sclerosis↗
PubMed1997

The contribution of postsynaptic folds to the safety factor for neuromuscular transmission in rat fast- and slow-twitch muscles.

1. At the rat neuromuscular junction, the postsynaptic folds and the voltage-gated sodium channels (VGSCs) within them are thought to amplify the effects of postsynaptic currents. In this study, the contribution of this effect to the safety factor for neuromuscular transmission, the ratio of the normal quantal content to the number of quanta required to reach threshold, has been estimated. 2. Normal quantal content was determined in isolated nerve-muscle preparations of rat soleus and extensor digitorum longus (EDL) muscles in which muscle action potentials were blocked by mu-conotoxin. The quantal content estimated from voltage recordings was 61.8 and 79.4 in soleus and EDL, respectively, and from charge measurements derived from current recordings was 46.3 (soleus) and 65.1 (EDL). 3. The threshold for action potential generation in response to nerve stimulation was determined from endplate potentials (EPPs) and endplate currents (EPCs) in preparations partially blocked with d-tubocurarine. The number of quanta required to reach threshold was estimated from voltage recordings to be 19.7 (soleus) and 23.2 (EDL) and from charge measurements derived from current recordings to be 13.3 (soleus) and 13.0 (EDL). 4. When intracellular electrodes were used to inject current into the muscle fibre, the total charge required to reach threshold was approximately twice that of the nerve-evoked threshold EPC. 5. The safety factor for nerve-evoked responses at the junction was 3.5 (soleus) and 5.0 (EDL). In the extrajunctional region the safety factor estimated from injected currents was 1.7 (soleus) and 2.5 (EDL). 6. It is concluded that the effect of the postsynaptic folds and the VGSCs within them is to double the safety factor. At normal frequencies of nerve impulse activity in vivo, this effect is likely to be crucial for ensuring effective neuromuscular transmission.

Action Potentials↗
PubMed1997

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. Utrophin-deficient mice are healthy and show no signs of weakness. However, their NMJs have reduced numbers of AChRs (alpha-bungarotoxin [alpha-BgTx] binding reduced to approximately 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.

Animals↗
PubMed1997

Utrophin abundance is reduced at neuromuscular junctions of patients with both inherited and acquired acetylcholine receptor deficiencies.

Congenital myasthenic syndromes are a heterogeneous group of conditions in which muscle weakness resulting from impaired neuromuscular transmission is often present from infancy. One form of congenital myasthenic syndrome is due to a reduction of the number of acetylcholine receptors (AChRs) at the neuromuscular junction. We describe four new cases of AChR deficiency, characterized by a reduction in both miniature endplate potential amplitude and AChR abundance accompanied by elongation of the neuromuscular junction and some decrease in postsynaptic folding. A number of cytoplasmic proteins are normally associated with the postsynaptic membrane and may contribute to the clustering of AChRs at the neuromuscular junction. We therefore investigated the expression of several of these proteins in these AChR-deficiency patients. In each patient, immunolabelling of the neuromuscular junction for rapsyn, dystrophin, beta-dystroglycan and a form of beta-spectrin was strong but that for utrophin was markedly reduced or absent. This suggested that a defect in utrophin expression might underlie the congenital AChR deficiency. However, a reduction in utrophin labelling was also seen in three patients with adult acquired autoimmune myasthenia gravis in whom AChR loss results directly from the extracellular binding of autoantibodies. We conclude that the loss of AChRs in AChR deficiency does not result from the absence of rapsyn or beta-dystroglycan and that reduction of utrophin is probably secondary to the loss of AChRs. The possible role of AChRs and/or utrophin in determining the extent of postsynaptic folding is discussed.

Action Potentials↗
PubMed1997

Microdosimetry spectra of the Loma Linda proton beam and relative biological effectiveness comparisons.

Protons have long been recognized as low LET radiation in radiotherapy. However, a detailed account of LET (linear energy transfer) and RBE (relative biological effectiveness) changes with incident beam energy and depth in tissue is still unresolved. This issue is particularly important for treatment planning, where the physical dose prescription is calculated from a RBE using cobalt as the reference radiation. Any significant RBE changes with energy or depth will be important to incorporate in treatment planning. In this paper we present microdosimetry spectra for the proton beam at various energies and depths and compare the results to cell survival studies performed at Loma Linda. An empirically determined biological weighting function that depends on lineal energy is used to correlate the microdosimetry spectra with cell survival data. We conclude that the variations in measured RBE with beam energy and depth are small until the distal edge of the beam is reached. On the distal edge, protons achieve stopping powers as high as 100 keV/micron, which is reflected in the lineal energy spectra taken there. Lineal energy spectra 5 cm beyond the distal edge of the Bragg peak also show a high LET component but at a dose rate 600 times smaller than observed inside the proton field.

Animals↗
PubMed1996

Spatial relationships of utrophin, dystrophin, beta-dystroglycan and beta-spectrin to acetylcholine receptor clusters during postnatal maturation of the rat neuromuscular junction.

At the adult mammalian neuromuscular junction, acetylcholine receptors are concentrated at the tops of the postsynaptic folds and voltage-gated sodium channels are concentrated in their depths. It is likely that this arrangement involves linkage of the ion channels to components of the underlying membrane cytoskeleton. In rats, the mature distribution of acetylcholine receptors arises as part of the developmental remodelling of the junctional region during the first few weeks after birth. We have followed the changes during this period in the distribution of four proteins associated with the postsynaptic cytoskeleton at mature neuromuscular junctions (utrophin, dystrophin, beta-dystroglycan and beta-spectrin) to see if any of them co-localizes with acetylcholine receptors during the remodelling process, as would be required if it serves to link acetylcholine receptors to the cytoskeleton. Each protein was visualized with specific monoclonal antibodies and its distribution at various stages was compared with that of the acetylcholine receptors, labelled with alpha-bungarotoxin. We also related the changes in distribution of these postsynaptic proteins to the main stages in fold formation and, in the Discussion, to reported observations of the accumulation of voltage gated sodium channels during development. Our results show that utrophin labelling is closely co-localized with that of acetylcholine receptors throughout postnatal maturation. beta-dystroglycan labelling is present at most sites of high acetylcholine receptors density throughout maturation although it often extends beyond the region of highest acetylcholine receptors labelling density. By contrast, dystrophin and beta-spectrin labelling is not consistently concentrated at most neuromuscular junctions until after P7 and P14 respectively.

Age Factors↗
PubMed1995

Action potential generation in rat slow- and fast-twitch muscles.

1. In skeletal muscle fibres, voltage-gated sodium channels are concentrated at the neuromuscular junction. The effect of this accumulation of sodium channels on action potential generation was investigated in rat slow- and fast-twitch muscle fibres. 2. Intracellular microelectrodes were used to generate and record action potentials, from an imposed membrane potential of -75 and -90 mV, in junctional and extrajunctional regions of the muscle fibre. To identify junctional regions, preparations were incubated with 5 x 10(-7) M d-tubocurarine (dTC) to block muscle contraction in response to nerve stimulation whilst allowing endplate potentials (EPPs) to be recorded. Injection of rectangular depolarizing current pulses initiated action potentials at the endplate with threshold values several millivolts lower than those generated elsewhere in the fibre. In addition, the maximum rate of rise of the action potential was greater at the endplate than in extrajunctional regions. 3. In other muscles, neuromuscular transmission was partially blocked with dTC (2 x 10(-7) M), such that repetitive nerve stimulation evoked action potentials and EPPs in the same fibre. The threshold of these nerve-evoked action potentials was approximately 50% lower than values derived from action potentials generated by current injection. 4. It is concluded that the threshold for action potential generation is significantly lower at the neuromuscular junction than in extrajunctional regions of skeletal muscle fibres. Furthermore, nerve-evoked current is more effective at generating an action potential than is injected current.

Action Potentials↗
PubMed1994

Release and synthesis of acetylcholine at ectopic neuromuscular junctions in the rat.

1. The ability of axons in the superficial fibular nerve to synthesize and release acetylcholine (ACh) has been studied before and after the formation of ectopic neuromuscular junctions (NMJs) with denervated soleus muscles of adult rats. 2. The central end of the severed fibular nerve was transplanted to the surface of the soleus muscle. After 3.5-5 weeks the soleus muscle was denervated in one group of rats by cutting the tibial nerve, allowing the formation of functional ectopic NMJs within a few days. In other rats the tibial nerve remained intact, preventing the formation of functional ectopic NMJs. 3. A month later the content of ACh, the levels of activity of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), and the amount of ACh released by depolarization by exposure to 50 mM KCl were measured in segments of isolated muscles that (i) contained normal or ectopic NMJs, (ii) were free of nerve or (iii) contained nerve that had not made NMJs. 4. Regions of muscles with ectopic nerve growth in which new NMJs had not formed contained substantial amounts of ACh and ChAT but no AChE. No detectable release of ACh could be evoked from these regions. 5. In muscles in which ectopic NMJs had formed after cutting the tibial nerve, the amounts of ACh and ChAT were about one-fifth of those in the regions of innervation of control muscles. ACh release could be evoked from the region of ectopic nerve growth in amounts nearly as great as those released from NMJs in normal and contralateral control muscles. 6. We conclude that the ability of the terminal parts of mature motor axons to synthesize and store ACh is largely independent of functional contact with muscle fibres. By contrast, the ability to release ACh in substantial amounts only develops when NMJs are formed. The possible significance of this situation for the development of synapses is discussed.

Acetylcholine↗
PubMed1992

Different distributions of dystrophin and related proteins at nerve-muscle junctions.

The distributions of dystrophin, 'dystrophin-related protein' (DRP) and beta-spectrin were compared with that of acetylcholine receptors (AChRs) at rat nerve-muscle junctions (NMJs) using immunofluorescence techniques. In sections, monoclonal antibodies (MAbs) to dystrophin and beta-spectrin labelled the entire sarcolemma but were concentrated at the NMJs while those to DRP labelled only NMJs. In permeabilized muscle fibres, DRP was precisely co-localized with the AChRs, whereas the zone of high density labelling of dystrophin and beta-spectrin extended 0.3-0.4 microns beyond the AChRs. Within the NMJ, the labelling of DRP appeared as a series of interconnecting lines similar to that of AChRs. However, labelling of dystrophin and beta-spectrin was consistently more punctate. These data suggest DRP is more closely associated with AChRs than are dystrophin or beta-spectrin.

Animals↗