Neurotransmitters single-vesicle release

Release of the neurotransmitter dopamine from a single active zone on a cell surface can be monitored by placing a nanoelectrode next to the cell, as shown at the opening of this chapter. When stimulated by ion injected near the cell, vesicles release dopamine by exocytosis. Each dopamine molecule that diffuses to the nanoelectrode gives up two electrons. Panel c at the beginning of the chapter shows four pulses measured over a period of 1 minute near one active zone. Each pulse lasts 10 milliseconds and has a peak current of — 10-50 picoamperes. The number of electrons in a pulse tells us how many dopamine molecules were released from one vesicle. 

The earliest experiments of this type employed voltammetry as a detection scheme. Fast-scan cyclic voltammetry can be used to discriminate between released substances with different oxidation/reduction properties. Voltammetric measurements of absolute concentrations released from single vesicles undergoing exocytosis have proven to be difficult however, because the amount of transmitter released is only at zeptomole levels and because the events occur on the millisecond time scale [13]. Furthermore, although elicited by chemical stimulation, these events do not occur at precise times. Although several neurotransmitters have been identified on the basis of characteristic voltammograms, it is difficult to distinguish catecholamines with this technique. However, fast-scan cyclic voltammetry has been used to identify the seaeted catecholamines norepinephrine and epinephrine [14]. 

In contrast, constant potential amperometry has allowed the quantitative aspects of single exocytotic release events to be studied in detail. This technique provides specific information on the amplitude, kinetics, and location of individual release events from single cells. Secretion is resolved as a series of current spikes that represent the electrooxidation of released substances. Wightman et al. have shown that each amperometric current spike detected represents the oxidation of neurotransmitter from a single exocytotic event [8]. In addition, the technique holds the potential to provide clues about the fusion pore complex, which manifests itself as a pre-spike foot that is observed directly prior to some release events. A drawback of this technique, however, is that chemical identification must be sacrificed for temporal resolution. This is a concern when one considers the complex biological matrix present in synaptic vesicles. 

Yakushenko, A., Katelhon, E., Wolfrum, B. 2013. Parallel on-chip analysis of single vesicle neurotransmitter release. Anal. Chem. 85 5483-5490. 

Neurons constitute the most striking example of membrane polarization. A single neuron typically maintains thousands of discrete, functional microdomains, each with a distinctive protein complement, location and lifetime. Synaptic terminals are highly specialized for the vesicle cycling that underlies neurotransmitter release and neurotrophin uptake. The intracellular trafficking of a specialized type of transport vesicles in the presynaptic terminal, known as synaptic vesicles, underlies the ability of neurons to receive, process and transmit information. The axonal plasma membrane is specialized for transmission of the action potential, whereas the plasma... 

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. 

Sensitive electrochemical techniques have also been developed to directly measure the release of oxidizable neurotransmitters such as catecholamines (CAs) and serotonin (5-hydroxytryptamine, 5-HT). Current flows in the circuit when the potential of the electrode is positive enough to withdraw electrons from, i.e. oxidize, the released neurotransmitter. The technique is very sensitive and readily detects the release of individual quanta of neuro transmitter resulting from the fusion of single secretory vesicles to the plasmalemma (Fig. 10-2). 

Specific membrane components must be delivered to their sites of utilization and not left at inappropriate sites [3]. Synaptic vesicles and other materials needed for neurotransmitter release should go to presynaptic terminals because they serve no function in an axon or cell body. The problem is compounded because many presynaptic terminals are not at the end of an axon. Often, numerous terminals occur sequentially along a single axon, making en passant contacts with multiple targets. Thus, synaptic vesicles cannot merely move to the end of axonal MTs. Targeting of synaptic vesicles thus becomes a more complex problem. Similar complexities arise with membrane proteins destined for the axolemma or a nodal membrane. 

Nitric oxide (NO) and carbon monoxide are atypical neurotransmitters. They are not stored in synaptic vesicles, are not released in by exocytosis, and do not act at postsynaptic membrane receptor proteins. NO is generated in a single step from the amino acid arginine through the action of the NO synthase (NOS). The form of NOS initially purified was designated nNOS (neuronal NOS), the macrophage form is termed inducible NOS (iNOS), and the endothelial from is called eNOS. 

A synaptic vesicle cycle. The number of synaptic vesicles in a single synapse in the brain varies from fewer than 100 to several hundred. In specialized synapses there may be thousands. However, at any moment only a fraction of the total are in the "active zone," often aligned along the presynaptic membrane (Fig. 30-20A) or in specialized ribbons such as those in Fig. 30-10B. The vesicles are normally reused repeatedly, undergoing a cycle of filling with neurotransmitter, translocation to the active zone, ATP-dependent priming, exocytosis with release of the neurotransmitter into the synaptic cleft, coating with clathrin, endocytosis, and acidification as outlined in Fig. 30-20B.554-557 The entire cycle may be completed within 40-60 s to avoid depletion of active vesicles.558 559 A key event in the cycle is the arrival of an action potential at the presynaptic neuron end. 

Careful monitoring of the membrane potential of the muscle membrane at a synapse with a cholinergic motor neuron has demonstrated spontaneous, intermittent, and random 2-ms depolarizations of about 0.5-1.0 mV in the absence of stimulation of the motor neuron. Each of these depolarizations is caused by the spontaneous release of acetylcholine from a single synaptic vesicle. Indeed, demonstration of such spontaneous small depolarizations led to the notion of the quanta release of acetylcholine (later applied to other neurotransmitters) and thereby led to the hypothesis of vesicle exocytosis at synapses. The release of one acetyl-choline-containing synaptic vesicle results In the opening of about 3000 ion channels In the postsynaptic membrane, far short of the number needed to reach the threshold depolarization that induces an action potential. Clearly, stimulation of muscle contraction by a motor neuron requires the nearly simultaneous release of acetylcholine from numerous synaptic vesicles. 

Release of acetylcholine from the storage vesicles is initiated by an action potential that has traveled dovirn the axon to the presynaptic nerve membrane. This action potential leads to opening of voltage-dependent calcium channels, affording an influx of Ca and exocytotic release of acetylcholine into the synapse. The increase in intracellular Ca may induce fusion of acetylcholine storage vesicles virith the presynaptic membrane before release of the neurotransmitter. Each synaptic vesicle contains a quantum of acetylcholine one quantum represents betvireen 12,000 and 60,000 molecules of acetylcholine. A single action potential causes the release of several hundred quanta of acetylcholine into the synapse. 

The release of neurotransmitters from neuronal terminals in the brain can be viewed as a quantal event. When an action potential arrives at a nerve terminal, a finite number of synaptic vesicles fuse with the terminal membrane and release their contents into the extracellular space. This release event occurs very rapidly studies of exocytotic events in single isolated cells show that they occur on a sub-miUisecond timescale [34, 35]. Furthermore, the event is spatially discrete, since the nerve terminals themselves have dimensions of just a few hundred nanometers [36, 37). With this description in mind, it is reasonable to regard each nerve terminal as... 

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