Exocytosis neurotransmitters

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. 

Figure 1 General concept representation of synaptic transmission. Vesicles that contain neurotransmitter molecules dock to the cell membranes and release their contents into the synapse by exocytosis. Neurotransmitters can diffuse across the synapse and bind to postsynaptic receptors or can diffuse out of the synapse. This extrasynaptic neurotransmitter can bind to presynaptic receptors, diffuse away and activate receptors on distal neurons, or be cleared from the extracellular space by transporters.

Smooth Muscle Contraction Platelet Aggregation Endothelial Cell Retraction Exocytosis (Neurotransmitters) Lymphocyte Receptor Capping Cell Migration... 

In humans, the hypothalamic-derived protein and the hormone noncovalent complexes are packaged in neurosecretory granules, then migrate along axons at a rate of 1 4 mm/h until they reach the posterior pituitary where they are stored prior to release into the bloodstream by exocytosis (67). Considerable evidence suggests that posterior pituitary hormones function as neurotransmitters (68) vasopressin acts on the anterior pituitary to release adrenocorticotropic hormone [9002-60-2] (ACTH) (69) as well as on traditional target tissues such as kidneys. Both hormones promote other important central nervous system (CNS) effects (9,70). 

Other agents are also used for the treatment of manic-depressive disorders based on preliminary clinical results (177). The antiepileptic carbamazepine [298-46-4] has been reported in some clinical studies to be therapeutically beneficial in mild-to-moderate manic depression. Carbamazepine treatment is used especially in bipolar patients intolerant to lithium or nonresponders. A majority of Hthium-resistant, rapidly cycling manic-depressive patients were reported in one study to improve on carbamazepine (178). Carbamazepine blocks noradrenaline reuptake and inhibits noradrenaline exocytosis. The main adverse events are those found commonly with antiepileptics, ie, vigilance problems, nystagmus, ataxia, and anemia, in addition to nausea, diarrhea, or constipation. Carbamazepine can be used in combination with lithium. Several clinical studies report that the calcium channel blocker verapamil [52-53-9] registered for angina pectoris and supraventricular arrhythmias, may also be effective in the treatment of acute mania. Its use as a mood stabilizer may be unrelated to its calcium-blocking properties. Verapamil also decreases the activity of several neurotransmitters. Severe manic depression is often treated with antipsychotics or benzodiazepine anxiolytics. 

In this lecture we will be concerned by exocytosis of neurotransmitters by chromaffin cells. These cells, located above kidneys, produce the adrenaline burst which induces fast body reactions they are used in neurosciences as standard models for the study of exocytosis by catecholaminergic neurons. Prior to exocytosis, adrenaline is contained at highly concentrated solutions into a polyelectrolyte gel matrix packed into small vesicles present in the cell cytoplasm and brought by the cytoskeleton near the cell outer membrane. Stimulation of the cell by divalent ions induces the fusion of the vesicles membrane with that of the cell and hence the release of the intravesicular content into the outer-cytoplasmic region. 

Neurotransmitter Transporters. Figure 3 Dopamine turnover at a presynaptic nerve terminal, (a) Dopamine is produced by tyrosine hydroxylase (TH). When secretory vesicles are filled, they join the releasable pool of vesicles at the presynaptic membrane. Upon exocytosis, the diffusion of released dopamine is limited by reuptake via DAT. (b) If DAT is inactive, dopamine spreads in the cerebrospinal fluid but cannot accumulate in secretory vesicles. This results in a compensatory increase of dopamine hydroxylase activity and a higher extracellular dopamine level mice with inactive DAT are hyperactive. 

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+... 

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...

Figure 5.1 Mechanism of action at a chemical synapse. The arrival of an action potential at the axon terminal causes voltage-gated Ca++ channels to open. The resulting increase in concentration of Ca++ ions in the intracellular fluid facilitates exocytosis of the neurotransmitter into the synaptic cleft. Binding of the neurotransmitter to its specific receptor on the postsynaptic neuron alters the permeability of the membrane to one or more ions, thus causing a change in the membrane potential and generation of a graded potential in this neuron.

Vesicular proteins and lipids that are destined for the plasma membrane leave the TGN sorting station continuously. Incorporation into the plasma membrane is typically targeted to a particular membrane domain (dendrite, axon, presynaptic, postsynaptic membrane, etc.) but may or may not be triggered by extracellular stimuli. Exocytosis is the eukaryotic cellular process defined as the fusion of the vesicular membrane with the plasma membrane, leading to continuity between the intravesicular space and the extracellular space. Exocytosis carries out two main functions it provides membrane proteins and lipids from the vesicle membrane to the plasma membrane and releases the soluble contents of the lumen (proteins, peptides, etc.) to the extracellular milieu. Historically, exocytosis has been subdivided into constitutive and regulated (Fig. 9-6), where release of classical neurotransmitters at the synaptic terminal is a special case of regulated secretion [54]. 

Secretory cells, including neurons, also possess a specialized regulated secretory pathway. Vesicles in this pathway have soluble proteins, peptides or neurotransmitters stored and concentrated within secretory vesicles. At that point, these vesicles are actively transported to a site for extracellular delivery in response to a specific extracellular signal. Exocytosis through regulated secretion accomplishes different functions, including the... 

One characteristic of regulated exocytosis is the ability to store secretory vesicles in a reserve pool for utilization upon stimulation. In the presynaptic terminal, this principle is expanded to define multiple pools of synaptic vesicles a ready releasable pool, a recycled synaptic vesicle pool and a larger reserve pool. This reserve pool assures that neurotransmitter is available for release in response to even the highest physiological demands. Neurons can fire so many times per minute because synaptic vesicles from the ready releasable pool at a given synapse undergo exocytosis in response to a single action potential. 

Those vesicles have been primed by docking at the active zone and are therefore ready for exocytosis upon arrival of an action potential. However, for the synapse to respond rapidly and repeatedly under heavy physiological demand, these exocytosed vesicles must be rapidly replaced. This is accomplished first from the recycled pool of vesicles and, as the demand increases, from the reserve pool. To be recycled, synaptic vesicles must be reloaded quickly after they release their contents. The sequence of events that is triggered by neurotransmitter exocytosis is known as the synaptic vesicle cycle [73,74] (Fig. 9-8). 

Chemical transmission between nerve cells involves multiple steps 167 Neurotransmitter release is a highly specialized form of the secretory process that occurs in virtually all eukaryotic cells 168 A variety of methods have been developed to study exocytosis 169 The neuromuscular junction is a well defined structure that mediates the presynaptic release and postsynaptic effects of acetylcholine 170 Quantal analysis defines the mechanism of release as exocytosis 172 Ca2+ is necessary for transmission at the neuromuscular junction and other synapses and plays a special role in exocytosis 174 Presynaptic events during synaptic transmission are rapid, dynamic and interconnected 175... 

Ca2+ is necessary for transmission at the neuromuscular junction and other synapses and plays a special role in exocytosis. In most cases in the CNS and PNS, chemical transmission does not occur unless Ca2+ is present in the extracellular fluid. Katz and Miledi [16] elegantly demonstrated the critical role of Ca2+ in neurotransmitter release. The frog NMJ was perfused with salt solution containing Mg2+ but deficient in Ca2+. A twin-barrel micropipet, with each barrel filled with 1.0mmol/l of either CaCl2 or NaCl, was placed immediately adjacent to the terminal. The sodium barrel was used to depolarize the nerve terminal electrically and the calcium barrel to apply Ca2+ ionotophoretically. Depolarization without Ca2+ failed to elicit an EPP (Fig. 10-6A). If Ca2+ was applied just before the depolarization, EPPs were evoked (Fig. 10-6B). In contrast, EPPs could not be elicited if the Ca2+ pulse immediately followed the depolarization (Fig. 10-6C). EPPs occurred when a Ca2+ pulse as short as 1 ms preceded the start of the depolarizing pulse by as little as 50-100 (xs. The experiments demonstrated that Ca2+ must be present when a nerve terminal is depolarized in order for neurotransmitter to be released. 

Release is another area of difference conventional neurotransmitters are secreted from small synaptic vesicles (SSVs) after cytosolic [Ca2+] transiently reaches concentrations of 50-100 pmol/1, while peptides are released from LDCVs at lower concentrations of cytosolic [Ca2+] (Fig. 18-3). Conventional neurotransmitter release is thought to occur very close to the site of Ca2+ entry (see Chs 9,10), while neuropeptides are typically released at a distance from the site of Ca2+ entry (Fig. 18-4). Furthermore the Ca2+ that stimulates exocytosis from LDCVs may come from either internal stores or the extracellular fluid. Thus, the location of LDCVs relative to the site of Ca2+ influx has a very crucial impact on the intensity of stimulation necessary for secretion to occur. 

Subtypes of Rab, particularly Rab3, have been implicated in the regulation of exocytosis and neurotransmitter release at nerve terminals (see also Chs 9 and 10) [30,31]. One possible scheme by which this might occur is shown in Figure 19-5. In its GTP-bound form, Rab associates with synaptic vesicles and interacts with other membrane proteins to create a complex unfavorable for vesicle docking and perhaps fusion. Upon depolarization of the nerve terminal, a Rab GAP is activated, which results in dissociation of the GDP form of Rab from the vesicle membrane. This enables the synaptic vesicle to proceed with... 

Hormonal actions on target neurons are classified in terms of cellular mechanisms of action. Hormones act either via cell-surface or intracellular receptors. Peptide hormones and amino-acid derivatives, such as epinephrine, act on cell-surface receptors that do such things as open ion-channels, cause rapid electrical responses and facilitate exocytosis of hormones or neurotransmitters. Alternatively, they activate second-messenger systems at the cell membrane, such as those involving cAMP, Ca2+/ calmodulin or phosphoinositides (see Chs 20 and 24), which leads to phosphorylation of proteins inside various parts of the target cell (Fig. 52-2A). Steroid hormones and thyroid hormone, on the other hand, act on intracellular receptors in cell nuclei to regulate gene expression and protein synthesis (Fig. 52-2B). Steroid hormones can also affect cell-surface events via receptors at or near the cell surface. 

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