Synapse vesicles

The short delays between Ca2+ influx and exocytosis have important implications for the mechanism of fusion of synaptic vesicles (see Ch. 9). In this short time, a synaptic vesicle cannot move significant distances and must be already at the release site. From the diffusion constant of Ca2+ in squid axoplasm, one can calculate that Ca2+ could diffuse a distance of only 850 A, somewhat greater than the diameter of a synaptic vesicle. Therefore, in fast synapses, vesicle exocytosis sites must be close to the triggering Ca2+ channels.. Vesicles are exposed to [Ca2+] of a few hundred micromoles near the cytoplasmic mouth of the channels. 

Synaptic Transmission. Figure 1 Synaptic transmission. The presynaptic terminal contains voltage-dependent Na Superscript and Ca2+ channels, vesicles with a vesicular neurotransmitter transporter VNT, a plasmalemmal neurotransmitter transporter PNT, and a presynaptic G protein-coupled receptor GPCR with its G protein and its effector E the inset also shows the vesicular H+ pump. The postsynaptic cell contains two ligand-gated ion channels LGIC, one for Na+ and K+ and one for Cl-, a postsynaptic GPRC, and a PNT. In this synapse, released transmitter is inactivated by uptake into cells. 

Synaptic vesicles are the organelles in axon terminals that store neurotransmitters and release them by exocytosis. There are two types, the large dense-core vesicles, diameter about 90 nm, that contain neuropeptides, and the small synaptic vesicles, diameter about 50nm, that contain non-peptide transmitters. About ten vesicles per synapse are docked to the plasma membrane and ready for release, the readily releasable pool . Many more vesicles per synapse are stored farther away from the plasma membrane, the resting pool . When needed, the latter vesicles may be recruited into the readily releasable pool. Neuronal depolarization and activation of voltage-sensitive Ca2+... 

Due to their physicochemical properties trace amines can pass the cell membrane to a limited extent by passive diffusion, with the more lipophilic PEA and TRP crossing membranes more readily than the more polar amines TYR. and OCT. In spite of these features, trace amines show a heterogeneous tissue distribution in the vertebrate brain, and for TYR. and OCT storage in synaptic vesicles as well as activity-dependent release have been demonstrated. So far, trace amines have always been found co-localized with monoamine neurotransmitters, and there is no evidence for neurons or synapses exclusively containing trace amines. 

Figure 6. Transport of material along the nerve axon. Materials such as neurotransmitter peptides are synthesized in the cell body and sequestered in vesicles at the Golgi. Vesicles are then transported down the axon towards the synapse by kinesin motors. Other materials are transported from the synapse to the cell body by dynein motors.

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. 

Figure 3.1 Schematic representation of a generic excitatory synapse in the brain. The presynaptic terminal releases the transmitter glutamate by fusion of transmitter vesicles with the nerve terminal membrane. Glutamate diffuses rapidly across the synaptic cleft to bind to and activate AMPA and NMDA receptors. In addition, glutamate may bind to metabotropic G-protein-coupled glutamate receptors located perisynaptically to cause initiation of intracellular signalling via the G-protein, Gq, to activate the enzyme phospholipase and hence produce inositol triphosphate (IP3) which can release Ca from intracellular calcium stores...

At most synapses a conventional NT is synthesised from an appropriate precursor in the nerve terminal, stored in vesicles, released, acts on postsynaptic receptors and is... 

ACh and considered to be the vesicles in the labile releasable pool. The evidence for and the actual mechanism of the vesicular release of ACh, mostly gained from studies at peripheral synapses, has been covered in Chapter 4. 

However, as early as the 1970s, it was obvious that end-product inhibition of TH could not be the main factor regulating the rate of noradrenaline synthesis. Clearly, the hydroxylation of tyrosine takes place in the cytoplasm and so it must be cytoplasmic noradrenaline that governs enzyme activity. Yet, it is vesicle-bound transmitter that undergoes impulse-evoked release from the neuron. Also, when neurons are releasing noradrenaline, its reuptake from the synapse is increased and, even though some of this transmitter ends up in the vesicles, or is metabolised by MAO, there should be a transient increase in the concentration of cytoplasmic noradrenaline which would increase end-product inhibition of TH. 

As with the other monoamines, 5-HT is found primarily in storage vesicles (30-35 nm diameter) where serotonin-binding proteins (SBPs) have also been identified. These seem to form a macromolecular complex with 5-HT. In fact, three such proteins have now been characterised, but only one of them, 45kDa SBP, appears to be secreted into the synapse along with 5-HT. Whether they serve any role other than forming an osmotically inert storage matrix for 5-HT is unknown. 

The release of some peptides may differ from that of other transmitters, depending on the firing rate of the neurons. The large vesicles needed to store a peptide may need a greater rate of depolarisation for membrane fusion and release of the contents. In the salivary gland the release of vasoactive intestinal polypeptide requires high-frequency stimulation whereas acetylcholine is released by all stimuli. Due to the complexities and problems of access to CNS synapses it is not known if the same occurs here but there is no reason why this should not. In sensory C-fibres a prolonged stimulus appears to be a prerequisite for the release of substance P. 

Iproniazid also prevents the reserpine syndrome in rats. Reserpine blocks vesicular uptake of monoamines which, as a consequence, leak from the storage vesicles into the cytosol. Although these monoamines would normally be metabolised by MAO, they are conserved when a MAO inhibitor (MAOI) is present, and so co-administration of reserpine and a MAOI leads to accumulation of monoamines in the neuronal cytosol. It is now known that, when the concentration of cytoplasmic monoamines is increased in this way, they are exported to the synapse on membrane-bound monoamine transporters. The ensuing increase in monoamine transmission, despite the depletion of the vesicular pool, presumably accounts for the effects of iproniazid on the behaviour of reserpine-pretreated rats. 

Figure 20.1 Schematic diagram illustrating how antidepressants increase the concentration of extraneuronal neurotransmitter (noradrenaline and/or 5-HT). In the absence of drug (b), monoamine oxidase on the outer membrane of mitochondria metabolises cytoplasmic neurotransmitter and limits its concentration. Also, transmitter released by exocytosis is sequestered from the extracellular space by the membrane-bound transporters which limit the concentration of extraneuronal transmitter. In the presence of a MAO inhibitor (a), the concentration of cytoplasmic transmitter increases, causing a secondary increase in the vesicular pool of transmitter (illustrated by the increase in the size of the vesicle core). As a consequence, exocytotic release of transmitter is increased. Blocking the inhibitory presynaptic autoreceptors would also increase transmitter release, as shown by the absence of this receptor in the figure. In the presence of a neuronal reuptake inhibitor (c), the membrane-bound transporter is inactivated and the clearance of transmitter from the synapse is diminished...

As described above, because MAO is bound to mitochondrial outer membranes, MAOIs first increase the concentration of monoamines in the neuronal cytosol, followed by a secondary increase in the vesicle-bound transmitter. The enlarged vesicular pool will increase exocytotic release of transmitter, while an increase in cytoplasmic monoamines will both reduce carrier-mediated removal of transmitter from the synapse (because the favourable concentration gradient is reduced) and could even lead to net export of transmitter by the membrane transporter. That MAOIs increase the concentration of extracellular monoamines has been confirmed using intracranial microdialysis (Ferrer and Artigas 1994). 

Once returned to the presynaptic terminal, dopamine is repackaged into synaptic vesicles via the vesicular monoamine transporter (VMAT) or metabolized to dihydroxyphenylacetic acid (DOPAC) by monoamine oxidase (MAO). Two alternative pathways are available for dopamine catabolism in the synapse, depending on whether the first step is catalyzed by MAO or catechol-O-methyltransferase (COMT). Thus, dopamine can be either deaminated to 3,4-dihydroxyphenylacetic acid (DOPAC) or methylated to 3-methoxytyramine (3-MT). In turn, deamination of 3-MT and methylation of DOPAC leads to homovanillic acid (HVA). In humans, cerebrospinal fluid levels of HVA have been used as a proxy for levels of dopaminergic activity within the brain (Stanley et al. 1985). 

In neurochemical terms, amphetamine and cocaine boost monoamine activity. Amphetamine has a threefold mode of action first, it causes dopamine and noradrenaline to leak into the synaptic cleft second, it boosts the amount of transmitter released during an action potential and third, it inhibits the reuptake of neurotransmitter back into presynaptic vesicles. These three modes all result in more neurotransmitter being available at the synapse, thus generating an increase in postsynaptic stimulation. Cocaine exerts a similar overall effect, but mainly by reuptake inhibition. The main neurotransmitters affected are dopamine and noradrenaline, although serotonin is boosted to a lesser extent. These modes of action are outlined in Chapter 3, and the neurochemical rationale for drug tolerance is covered more fully in Chapter 10. The main differences between amphetamine and cocaine are their administration routes (summarised above) and the more rapid onset and shorter duration of action for cocaine. 

Frederickson, C.J., Suh, S.W., Silva, D. and Thompson, R.B. (2000). Importance of zinc in the central nervous system the zinc-containing neuron. J. Nutr., 130, 1471S-1483S Holt, M. and Jahn, R. (2004). Synaptic vesicles in the fast lane. Science, 303, 1986-1987 Kandel, E.R. (2001). The molecular biology of memory storage a dialogue between genes and synapses. Science, 294, 1030-1038... 

In addition to their transporter function, perisynaptic transporters function as buffers to confine the extracellular free glutamate to receptors nearest the postsynaptic release sites. The total concentration of glial transporters in some CNS synaptic regions is estimated to be sufficient to bind the glutamate content of three to five vesicles per synapse [45]. This buffering , which occurs mostly in end-feet of astrocytes, is essential for reliable synaptic transmission of high-frequency signals [46]. 

At least two classes of regulated secretion can be defined [54]. The standard regulated secretion pathway is common to all secretory cells (i.e. adrenal chromaffin cells, pancreatic beta cells, etc.) and works on a time scale of minutes or even longer in terms of both secretory response to a stimulus and reuptake of membranes after secretion. The second, much faster, neuron-specific form of regulated secretion is release of neurotransmitters at the synapse. Release of neurotransmitters may occur within fractions of a second after a stimulus and reuptake is on the order of seconds. Indeed, synaptic vesicles may be recycled and ready for another round of neurotransmitter release within 1-2 minutes [64]. These two classes of regulated secretion will be discussed separately after a consideration of secretory vesicle biogenesis. 

Some neurons can fire several hundred times per second, secreting neurotransmitter each time. Synapses are remarkable secretion machines destined to undergo millions of repeated exocytic cycles in their lifetime. So how does this process work so fast and so efficiently Answers have gradually emerged from the work of many different laboratories and model systems [73, 74]. An exhaustive description is beyond the scope of this chapter, but a summary of key events and specializations of the synaptic vesicle cycle is useful. This will show how synaptic transmission is optimized spatially and biochemically (see also Chs 6,10 and 22). 

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