Neuromuscular Junction Preparations

Isolated Nerve Terminals as a Model System for the Study of Botulinum and Tetanus Toxins 197 

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In 1972 Ginsborg and Hirst (1972) described for the first time that the nucleoside adenosine inhibited acetylcholine release at the rat-diaphragm neuromuscular junction and that this effect was abolished by theophylline. This observation was soon supported by Ribeiro and Walker (1975) using the frog neuromuscular junction preparation. 

With these thoughts in mind, we examined the effects of various drugs on GABA-mediated synapses in the crayfish. While there are a number of GABAergic neuromuscular junction preparations in invertebrates which are accessible for physiological research, the crayfish stretch receptor is one of very few invertebrate preparations which can be used for studies of synapses between nerves. The anatomy and physiology of these receptor organs from the abdomen of the crayfish has been extensively studied (6) ... 

Tetanus immunoglobulin is an example of an antibody preparation used to induee passive immunization against a mierobial toxin. Tetanus (lockjaw) is an infectious disease caused by the bacterium, Clostridium tetani. Bacterial spores can commonly contaminate surface wounds and the resulting bacterial cells produce a toxin as they multiply. The toxin interferes with normal neurological function, particularly at neuromuscular junctions. The result is spasmodic contraction of muscles and, if untreated, mortality rates are high. Treatment with antibiotics and anti-toxin, however, is highly effective if administered promptly. 

A group of esterases hydrolyze simple oxygen esters. Some of these are designed to hydrolyze a particular ester or small group of esters, while others have a more nonspecific action. Acetylcholinesterase609 611a is specific for acetylcholine (Eq. 12-25), a neurotransmitter that is released at many nerve synapses and neuromuscular junctions (Chapter 30). The acetylcholine, which is very toxic in excess, must be destroyed rapidly to prepare the synapse for transmission of another impulse ... 

Neuromuscular junctions can be visualized by light microscopy following labeling of the presynaptic nerve terminal and the postsynaptic acetylcholine receptors. For best results, muscles should be prepared for longitudinal sections, and NMJs in an en face orientation can be imaged. In almost all muscles, the end plate band is near the middle of the muscle and this represents the region of interest. 

ATP is a well-established co-transmitter to noradrenaline in the central (Poelchen et al. 2001) as well as peripheral (von Kugelgen and Starke 1991) nervous system. Therefore, the functions of presynaptic P2X receptors have been investigated in various preparations containing noradrenergic nerve terminals (Cunha and Ribeiro 2000). Early evidence for a stimulation and positive feedback modulation of noradrenaline via presynaptic P2X receptors has been obtained in sympathetic neurons (Boehm 1999). At the neuromuscular junction, ATP stimulated the release of acetylcholine (1994), and this effect was suggested to be mediated by presynaptic P2X7 receptors (Moores et al. 2005). 

Calcium performs a variety of cellular functions in muscle and nerve that ultimately result in muscular contraction. Excellent descriptions of calcium s function in muscle and nerve are to be found in the reviews by Hoyle (37), Cohen (38), and Robertson (39). At the neuromuscular junction, the excitable cells are very sensitive to changes in extracellular concentrations of calcium. Curtis (40) and Luttgau (41) described a fall in the resting action potential and electrical resistance when the extracellular calcium concentration fell below 10 M. The action potential and electrical resistance returned to normal following addition of calcium to this vitro preparation. The magnitude of the Initial muscle membrane action potential, that which regulates the propagation of further muscle contraction, is also mediated by the extracellular calcium concentration. While the inward flow of sodium ions from the extracellular space remains the dominant factor in the mechanism of muscle membrane depolarization, calcium ion flux appears to mediate the cell s permeability to sodium ions. This effect is particularly true in cardiac tissue (W). 

White and Stedman (39) suggested that. In addition to Inhibiting AChE, OP compounds have an effect on the site where the ACh molecule reacts at the neuromuscular Junction. Rlker and Wescoe (40) showed a direct agonist action of neostigmine at the neuromuscular junction, and many others have found that neostigmine and some other antl-ChE agents have anticurare effects not apparently related to Inhibition of ChE (41). Additional observations Indicated that preparations chat had been denervated for over 20 d responded with a contracture when exposed to sarin. 

In general, sensitivity of a given preparation to clostridial neurotoxins depends on the type of neurotoxin. For instance, BoNT/A is particularly potent in poisoning peripheral cholinergic nerve terminals (i.e. neuromuscular junctions), whereas TeTx is most potent for CNS preparations. However, all eight neurotoxins are able to inhibit exocy-... 

A number of invertebrate polyamine toxins have recently been shown to be open channel blockers of glutamate receptors initially at the neuromuscular junction of locusts [54] and recently on central mammalian neurones [54-57], These toxins include argiotoxin 636, Joro spider toxin and philanthotoxin. Although these polyamine toxins block NMDA receptors at least in some preparations, in others they also block responses mediated by AMPA receptors more selectively [58-60]. 

The basis for the lack of response to 3,4-DAP by the other serotypes is not well understood. At a functional level, serotype A-intoxicated neuromuscular junctions undergo an attenuated but synchronous release of ACh following stimulation preparations intoxicated by serotypes B, D, and F produce asynchronous release where the ACh quanta are dispersed and cannot summate to produce suprathreshold EPPs (Lundh et al., 1977 Molgo et al., 1980 Thesleff, 1989). It is readily apparent that the lack of synchrony would prevent 3,4-DAP from restoring transmitter release however, the factors that lead to asynchronous release are not currently understood. 

The first neurotransmitter to be discovered was acetylcholine, discovered by Loewi in the early 1920s. These studies were conducted with isolated preparations of heart and indicated that a substance inhibitory to the chro-notrophy of the heart was released by inhibitory nerves. Since then, numerous chemicals have been proposed as putative neurotransmitters, and reasonable estimates suggest that there are at least 50 candidates for the role of neurotransmitters. Like acetylcholine, many of the other neurotransmitters that were discovered early were found in peripheral tissues such as the heart or the vertebrate neuromuscular junction. At such sites, there is the release of a neurotransmitter at discrete sites where the pre- and postsynaptic membranes are in intimate contact. However, as research progressed over the past two decades, it became apparent that there were many situations where a nerve terminal was not closely associated with a postsynaptic membrane containing a unique receptor for the transmitter in question, but in fact was so situated that the released chemical could either diffuse into the circulation (i.e., neurohypophysis or adrenal medulla) or into the extracellular space to act on a distant receptor. Chemicals so released are usually not considered neurotransmitters so much as neurohormones or neuromodulators. 

The ensuing discussion will deal with that major category of receptors that are essentially components of cellular membranes. For example, the acetylcholine receptor involving skeletal muscles exerts its effect at the end of the motor nerve and its junction with the muscle (neuromuscular junction, see Chapter 7) by a depolarizing action. The fact that receptors are embedded in muscle cell membranes can be surmised by the fact that the contractile effect can be initiated by simply applying acetylcholine to the surface of the muscle preparation intracellular injection of the agonist produces no effect. A more interesting... 

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