Cholinergic vesicles

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]. 

Botulinum toxin Cholinergic vesicles Prevents release... 

Fig. 1. Isolation of cholinergic vesicles from Torpedo nobiliana by zonal centrifugation in a sucrose-NaCl gradient. The large protein peak corresponds to soluble protein, the small one to membrane bound protein. A few particles corresponding to nerve ending particles may have been formed, as...

The experiments described show that phospholipase A, isolated from the venom of Naja Naja siamensis and able to form lysolecithin from biosynthetically formed labelled lecithin, gradually weakens and breaks down synaptic vesicles from rat brain cortex and cholinergic vesicles from the electric organ of Torpedo nobiliana, thereby... 

Synaptic vesicles, isolated from rat brain cortex, and cholinergic vesicles, isolated from the electric organ of Torpedo nobiliana, were broken down by a phospholipase A from cobra venom. The breakdown was accompanied by a release of acetylcholine. Morphological analysis revealed membrane fragments about one-third the size of the vesicle circumference. Subcellular fractions enriched in nerve ending membranes showed some phospholipase A activity. On the basis of these findings models of transmitter release, involving specific alterations of lipid components in the vesicular membrane, are discussed. 

The neurotransmitter must be present in presynaptic nerve terminals and the precursors and enzymes necessary for its synthesis must be present in the neuron. For example, ACh is stored in vesicles specifically in cholinergic nerve terminals. It is synthesized from choline and acetyl-coenzyme A (acetyl-CoA) by the enzyme, choline acetyltransferase. Choline is taken up by a high affinity transporter specific to cholinergic nerve terminals. Choline uptake appears to be the rate-limiting step in ACh synthesis, and is regulated to keep pace with demands for the neurotransmitter. Dopamine [51 -61-6] (2) is synthesized from tyrosine by tyrosine hydroxylase, which converts tyrosine to L-dopa (3,4-dihydroxy-L-phenylalanine) (3), and dopa decarboxylase, which converts L-dopa to dopamine. 

Unfortunately in routine EM (electron microscope) preparations one cannot identify the NT at individual synapses although structural features (shape of vesicle, asymmetric or symmetric specialisations) may provide a clue. At cholinergic synapses the terminals have clear vesicles (200-400 A) while monoamine terminals (especially NA) have distinct large (500-900 A) dense vesicles. Even larger vesicles are found in the terminals of some neuro-secretory cells (e.g. the neurohypophysis). One terminal can contain more than one type of vesicle and although all of them probably store NTs it is by no means certain that all are involved in their release. 

Whittaker, VP (1987) Cholinergic synaptic vesicles from the electromotor nerve terminals of Torpedo composition and life cycle. Ann. NY Acad. Sci. 493 77-91. 

Choline is supplied to the neuron either from plasma or by metabolism of choline-containing compounds 193 A slow release of acetylcholine from neurons at rest probably occurs at all cholinergic synapses 194 The relationship between acetylcholine content in a vesicle and the quanta of acetylcholine released can only be estimated 194 Depolarization of the nerve terminal by an action potential increases the number of quanta released per unit time 194 All the acetylcholine contained within the cholinergic neuron does not behave as if in a single compartment 194... 

Akert K, Sandri C. An electron-microscopic study of zinc iodide-osmium impregnation of neurons. I. Staining of synaptic vesicles at cholinergic junctions. Brain Res 1968 7 286-295. 

Hicks, B.W. and Parsons, S.M., 1992, Characterization of the P-type and V-type ATPases of cholinergic synaptic vesicles and coupling of nucleotide hydrolysis to acetylchohne transport. J. Neurochem., 58 1211-1220. 

The processes involved in neurochemical transmission in a cholinergic neuron are shown in Figure 9.2. The initial substrates for the synthesis of acetylcholine are glucose and choline. Glucose enters the neuron by means of facilitated transport. There is some disagreement as to whether choline enters cells by active or facilitated transport. Pyruvate derived from glucose is transported into mitochondria and converted to acetylcoenzyme A (acetyl-CoA). The acetyl-CoA is transported back into the cytosol. With the aid of the enzyme choline acetyl-transferase, acetylcholine is synthesized from acetyl-CoA and choline. The acetylcholine is then transported into and stored within the storage vesicles by as yet unknown mechanisms. 

Like the cholinergic transmitter, the noradrenergic transmitter is released by action potentials through ex-ocytosis, the contents of entire vesicles being emptied into the biophase (synaptic or junctional region). Similarly, the formation of transmitter-receptor complexes is a direct function of the concentration of transmitter in the biophase and is readily reversible. In this instance, the receptors are adrenoceptors. 

Natochin M, Gasimov KG, Artemyev NO (2001) Inhibition of GDP/GTP exchange on G alpha subunits by proteins containing G-protein regulatory motifs. Biochemistry 40 5322-5328 Ngsee JK, Miller K, Wendland B, Scheller RH (1990) Multiple GTP-binding proteins from cholinergic synaptic vesicles. J Neurosci 10 317-322... 

As ACh is synthesized, it is stored in the neuron or ganglion in at least three different locations. Eighty-five percent of all ACh is stored in a depot and can be released by neuronal stimulation it is always the newly synthesized neurotransmitter that is released preferentially. The surplus ACh can be released by K depolarization only. Finally, there is stationary ACh, which cannot be released at all. It has been assumed that the neurotransmitter in cholinergic and some other neurons is released through the exocytosis of small transmitter-filled synaptic vesicles. 

The transmitter is present throughout the cholinergic neurones and exists within the axon terminals in vesicles. About 1% of the vesicles are the readily releasable store that maintains transmitter release but more than 80% is in motor nerve endings in the releasable store, which is released in response to a nerve impulse. The remainder of ACh is in the so-called stationary store. The release of ACh may be spontaneous or in response to nerve impulses. Spontaneous release of ACh results in the production of random miniature endplate potentials. It is, however, in response to a nerve impulse that we see a large release of ACh provided there is adequate calcium present in the extracellular fluid. Evoked release of ACh usually results in the production of an endplate potential due to depolarisation of the motor endplate. 

Schematic illustration of a generalized cholinergic junction (not to scale). Choline is transported into the presynaptic nerve terminal by a sodium-dependent choline transporter (CHT). This transporter can be inhibited by hemicholinium drugs. In the cytoplasm, acetylcholine is synthesized from choline and acetyl -A (AcCoA) by the enzyme choline acetyltransferase (ChAT). Acetylcholine is then transported into the storage vesicle by a second carrier, the vesicle-associated transporter (VAT), which can be inhibited by vesamicol. Peptides (P), adenosine triphosphate (ATP), and proteoglycan are also stored in the vesicle. Release of transmitter occurs when voltage-sensitive calcium channels in the terminal membrane are opened, allowing an influx of calcium. The resulting increase in intracellular calcium causes fusion of vesicles with the surface membrane and exocytotic expulsion of acetylcholine and cotransmitters into the junctional cleft (see text). This step can he blocked by botulinum toxin. Acetylcholine s action is terminated by metabolism by the enzyme acetylcholinesterase. Receptors on the presynaptic nerve ending modulate transmitter release. SNAPs, synaptosome-associated proteins VAMPs, vesicle-associated membrane proteins.

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