Vesicles, neuromuscular junctions

To achieve their different effects NTs are not only released from different neurons to act on different receptors but their biochemistry is different. While the mechanism of their release may be similar (Chapter 4) their turnover varies. Most NTs are synthesised from precursors in the axon terminals, stored in vesicles and released by arriving action potentials. Some are subsequently broken down extracellularly, e.g. acetylcholine by cholinesterase, but many, like the amino acids, are taken back into the nerve where they are incorporated into biochemical pathways that may modify their structure initially but ultimately ensure a maintained NT level. Such processes are ideally suited to the fast transmission effected by the amino acids and acetylcholine in some cases (nicotinic), and complements the anatomical features of their neurons and the recepter mechanisms they activate. Further, to ensure the maintenance of function in vital pathways, glutamate and GABA are stored in very high concentrations (10 pmol/mg) just as ACh is at the neuromuscular junction. 

Ceccarelli, B., Hurlbut, W. P. and Mauro, A. Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction. /. Cell Biol. 57 499-524,1973. 

A variety of methods have been developed to study exocytosis. Neurotransmitter and hormone release can be measured by the electrical effects of released neurotransmitter or hormone on postsynaptic membrane receptors, such as the neuromuscular junction (NMJ see below), and directly by biochemical assay. Another direct measure of exocytosis is the increase in membrane area due to the incorporation of the secretory granule or vesicle membrane into the plasma membrane. This can be measured by increases in membrane capacitance (Cm). Cm is directly proportional to membrane area and is defined as Cm = QAJV, where Cm is the membrane capacitance in farads (F), Q is the charge across the membrane in coulombs (C), V is voltage (V) and Am is the area of the plasma membrane (cm2). The specific capacitance, Q/V, is the amount of charge that must be deposited across 1 cm2 of membrane to change the potential by IV. The specific capacitance, mainly determined by the thickness and dielectric constant of the phospholipid bilayer membrane, is approximately 1 pF/cm2 for intracellular organelles and the plasma membrane. Therefore, the increase in plasma membrane area due to exocytosis is proportional to the increase in Cm. 

This technique has permitted the dynamics of the exo-cytic/endocytic cycle to be investigated. At the neuromuscular junction, a readily releasable pool (RRP) and a reserve pool of vesicles coexist, the latter being released... 

Van der Kloot, W. Loading and recycling of synaptic vesicles in the Torpedo electric organ and the vertebrate neuromuscular junction. Prog. Neurobiol. 71, 269-303, 2003. 

FIGURE18-4 Neuropeptides and conventional neurotransmitters are released from different parts of the nerve terminal. A neuromuscular junction containing both large dense-core vesicles (containing the neuropeptide SCP) and also small synaptic vesicles (containing acetylcholine) was stimulated for 30 min at 12 Hz (3.5 s every 7 s). Depletion of the small clear vesicles at the muscle face and of the peptide granules at the nonmuscle face of the nerve terminal was observed. After stimulation, there was an increase in the number of large dense-core vesicles within one vesicle diameter of the membrane. (Adapted from reference [37].)... 

Measuring muscle-evoked responses to repetitive motor nerve electrical stimulation permits detection of presyn-aptic neuromuscular junction dysfunction. In botulism and the Lambert-Eaton syndrome, repetitive stimulation elicits a smaller than normal skeletal muscle response at the beginning of the stimulus train, due to impaired initial release of acetylcholine-containing vesicles from presyn-aptic terminals of motor neurons followed by a normal or accentuated incremental muscle response during repeated stimulation. This incremental response to repetitive stimulation in presynaptic neuromuscular disorders can be distinguished from the decremental response that characterizes autoimmune myasthenia gravis, which affects the postsynaptic component of neuromuscular junctions. 

Botulinum exotoxin impedes release of neurotransmitter vesicles from cholinergic terminals at neuromuscular junctions. Botulinum exotoxin is ingested with food or, in infants, synthesized in situ by anaerobic bacteria that colonize the gut. A characteristic feature of botulinum paralysis is that the maximal force of muscle contraction increases when motor nerve electrical stimulation is repeated at low frequency, a phenomenon attributable to the recruitment of additional cholinergic vesicles with repetitive depolarization of neuromuscular presynaptic terminals. Local administration of Clostridium botulinum exotoxin is now in vogue for its cosmetic effects and is used for relief of spasticity in dystonia and cerebral palsy [21]. 

Lambert-Eaton syndrome is an antibody-mediated neuromuscular junction disorder. This disorder, which is most frequently encountered in patients with small-cell lung carcinoma, is characterized clinically by weakness and hyporeflexia. The impaired release of acetylcholine vesicles from presynaptic terminals at neuromuscular junctions that causes the weakness is a consequence of autoantibodies against small cell carcinoma epitopes that cross-react with and downregulate the expression of motor nerve terminal Ca2+ channels [39]. (See in Ch. 43.)... 

All botulin neurotoxins act in a similar way. They only differ in the amino-acid sequence of some protein parts (Prabakaran et al., 2001). Botulism symptoms are provoked both by oral ingestion and parenteral injection. Botulin toxin is not inactivated by enzymes present in the gastrointestinal tracts. Foodborne BoNT penetrates the intestinal barrier, presumably due to transcytosis. It is then transported to neuromuscular junctions within the bloodstream and blocks the secretion of the neurotransmitter acetylcholine. This results in muscle limpness and palsy caused by selective hydrolysis of soluble A-ethylmalemide-sensitive factor activating (SNARE) proteins which participate in fusion of synaptic vesicles with presynaptic plasma membrane. SNARE proteins include vesicle-associated membrane protein (VAMP), synaptobrevin, syntaxin, and synaptosomal associated protein of 25 kDa (SNAP-25). Their degradation is responsible for neuromuscular palsy due to blocks in acetylcholine transmission from synaptic terminals. In humans, palsy caused by BoNT/A lasts four to six months. 

Schematic representation of the neuromuscular junction. ACh, acetylcholine AChE, acetylcholinesterase JF, junctional folds M, mitochondrion V, transmitter vesicle.

Mechanism of action. The cellular actions of bot-ulinum toxin at the neuromuscular junction have recently been clarified.84 This toxin is attracted to glycoproteins located on the surface of the presynaptic terminal at the skeletal neuromuscular junction.33 Once attached to the membrane, the toxin enters the presynaptic terminal and inhibits proteins that are needed for acetylcholine release (Figure 13-4).84 Normally, certain proteins help fuse presynaptic vesicles with the inner surface of the presynaptic terminal, thereby allowing the vesicles to release acetylcholine via exocytosis. Botulinum toxin cleaves and destroys these fusion proteins, thus making it impossible for the neuron to release acetylcholine into the synaptic cleft.32,84 Local injection of botulinum toxin into specific muscles will therefore decrease muscle excitation by disrupting synaptic transmission at the neuromuscular junction. The affected muscle will invariably undergo some degree of paresis and subsequent... 

FIGURE 13-4 Mechanism of action of botulinum toxin at the skeletal neuromuscular junction. At a normal synapse (shown on left], fusion proteins connect acetylcholine (ACh] vesicles with the presynaptic membrane, and ACh is released via exocytosis. Botulinum toxin (represented by BTX on the right] binds to the presynaptic terminal, and enters the terminal where it destroys the fusion proteins so that ACh cannot be released. See text for details. 

Schematic representation of the neuromuscular junction. (V, transmitter vesicle M, mitochondrion ACh, acetylcholine AChE, acetylcholinesterase JF, junctional folds.) (Reproduced, with permission, from Drachman DB Myasthenia gravis. N Engl J Med 1978 298 135.)...

Fig. 20.8. Neuromuscular junctions analyzed by transmission electron microscopy. (A) In wild-type mice, the motor nerve terminal (MN) is depressed into the muscle fiber surface. The terminal is polarized, with small clear vesicles near the presynaptic membrane and mitochondria in the more proximal portion of the terminal. The postsynaptic membrane has deep convolutions (junctional folds, JF) and the membrane near the tops of these folds is very electron dense because of the high density of acetylcholine receptors (arrowheads). (B) In some myasthenias where the nerve sprouts but remains in contact with the muscle, terminals with mitochondria and vesicles are observed in the absence of any postsynaptic specialization. Presumably these are sprouting terminals that have not established a functional connection. (C) Partial innervation of postsynaptic sites is evident as elaborate junctional folds in the muscle membrane with no overlying nerve terminal. In these examples, the interpretations were aided by light microscopy examination of other samples as described in Fig. 20.8 in parallel with electron microscopy. The mutation shown in (B, C) is an unpublished ENU-induced allele of agrin.

Betz WJ, Bewick GS (1992) Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction. Science 255 200-3... 

Betz WJ, Bewick GS (1993) Optical monitoring of transmitter release and synaptic vesicle recycling at the frog neuromuscular junction. J Physiol 460 287-309 Betz WJ, Mao F, Smith CB (1996) Imaging exocytosis and endocytosis. Curr Opin Neurobiol 6 365-71... 

Wu LG, Betz WJ (1996) Nerve activity but not intracellular calcium determines the time course of endocytosis at the frog neuromuscular junction. Neuron 17 769-79 Zenisek D, Steyer JA, Feldman ME, Aimers W (2002) A membrane marker leaves synaptic vesicles in milliseconds after exocytosis in retinal bipolar cells. Neuron 35 1085-97 Zhou FM, Liang Y, Salas R, Zhang L, De Biasi M, Dani JA (2005) Corelease of dopamine and serotonin from striatal dopamine terminals. Neuron 46 65-74 Zucker RS, Regehr WG (2002) Short-term synaptic plasticity. Annu Rev Physiol 64 355—405... 

Couteaux R, Pdcot-Dechavassine M (1970) Synaptic vesicles and pouches at the level of active zones of the neuromuscular junction C R Acad Sci Hebd Seances Acad Sci D 271 2346-9... 

Sellin LC, Kauffman JA, Dasgupta BR (1983) Comparison of the effects of botulinum neurotoxin types A and E at the rat neuromuscular junction. Med Biol 61 120-5 Sheridan RE (1998) Gating and permeability of ion channels produced by botulinum toxin types A and E in PC12 cell membranes. Toxicon 36 703-17 Shone CC, Hambleton P, Melling J (1987) A 50-kDa fragment from the NH2-terminus of the heavy subunit of Clostridium botulinum type A neurotoxin forms channels in lipid vesicles. Eur J Biochem 167 175-80... 

Ceccarelli B, Grohovaz F, Hurlbut WP (1979) Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. I. Effects of black widow spider venom and Ca2+-free solutions on the structure of the active zone. J Cell Biol 81 163-77 Ceccarelli B, Hurlbut WP (1980) Ca2+-dependent recycling of synaptic vesicles at the frog neuromuscular junction. J Cell Biol 87 297-303... 

Frontali N, Ceccarelli B, Gorio A et al (1976) Purification from black widow spider venom of a protein factor causing the depletion of synaptic vesicles at neuromuscular junctions. J Cell Biol... 

Heuser, J. E., and Reese, T. S. (1973). Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. Cell Biol 57, 315-344. 

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