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Unformatted text preview: Transmitter Release Page 7-1 Neurons communicate with each other at synapses. For chemical synapses, the first step in this interneuronal communication is the release of neurotransmitter from the presynaptic terminal. Characteristics of transmitter release (1) The amount of neurotransmitter released by a presynaptic terminal depends on the amount of depolarization of that terminal. Experiments: (a) The stellate ganglion of the squid was used to study the relationship between the membrane potential of the presynaptic terminals and the amount of transmitter release. The presynaptic fiber and postsynaptic fiber were impaled with microelectrodes to simultaneously record the presynaptic depolarization and the postsynaptic response. (b)When tetrodotoxin (TTX) was added, the presynaptic action potential gradually decreased. The amplitude of the postsynpatic action potential also decreased. (c) (b) Page 7-2 (c) In another set of experiments, the presynaptic action potential was first completely eliminated by adding TTX. The experimenters then applied brief current pulses to the presynaptic terminal creating depolarizations of known amplitude. They then indirectly measured how much neurotransmitter was released by measuring the amplitude of the postsynaptic potential. (d) The results of experiments (b) and (c) are similar, indicating that the normal fluxes of sodium and potassium ions responsible for the action potential are not necessary for transmitter release; deplolarization is the trigger. In these experiments, the transmitter release was not seen until around 45mV of presynaptic depolarization. After that, release increased rapidly with increasing depolarization. (2) Calcium must also be present in the extracellular fluid at the synapse. (b) (c) If calcium is removed from the fluid or if large divalent cations that can block calcium channels (such as magnesium or cobalt) are added, then no transmitter is released. It turns out that the main effect of depolarization on the presynaptic terminal is to open voltage gated calcium channels, causing calcium to enter the presynaptic terminal. Experiments: Page 7-3 Action potential was blocked by TTX and TEA. The equilibrium potential for calcium in a typical neuron is about +60mV. (a) If the presynaptic terminal is depolarized to a level below +60mV (left), voltage gated calcium channels open, an inward calcium current is seen, and transmitter is released causing a postsynaptic potential. (b) If the terminal is depolarized to exactly the calcium equilibrium potential, the voltage gated calcium channels open, but no calcium current is seen (no driving force). When no calcium enters the terminal, no transmitter is released, as is shown by the lack of a postsynaptic potential. (c) When the +60mV pulse is turned off, there is a small calcium current because the voltage gated calcium channels were opened by the pulse and cannot close instantaneously. These experiments showed that depolarization alone does not cause transmitter release, but that calcium entry without depolarization does cause release. Quantal Release - Physiological evidence Both physiological and anatomical evidence has shown that neurotransmitter is released from the presynaptic terminal from vesicles. Page 7-4 Quantal release of neurotransmitter means that transmitter is always released in multimolecular packets containing the same number of neurotransmitter molecules. Evidence (1) Miniature end plate potentials (MEPPs) The first evidence came from the observation of MEPPs, which are spontaneous postsynaptic potentials of about 1mV amplitude at the neuromuscular junction: (a) MEPPs are due to acetylcholine release, because they are abolished by botulinum toxin, which is know to block ACh release. (b) Each MEPP cannot be the response to a single molecule of transmitter. This conclusion comes from two pharmacological experiments: Curare which is an AChR antagonist decreased the size of MEPPs. If each MEPP represented the response to one transmitter molecule, curare could decrease the frequency of MEPPs or abolish them completely, but would not affect their amplitude. Prostigmine which inhibits acetylcholinesterase (an enzyme that destroys ACh) increases the size of MEPPs. If each MEPP were the response to one molecule, prostigmine could increase the frequency of MEPPs, but cold not affect the amplitude. (2) The postsynaptic potential amplitude was always an integer multiple of the MEPP amplitude. (a) Further evidence for quantal release came from experiments looking at the postsynaptic response to a presynaptic stimulus of constant size. For these experiments, the amount of transmitter release was kept very low by reducing extracellular calcium and adding extracellular magnesium to block some voltage gated calcium channels. (b) Under these conditions, sometimes the stimulus produced no postsynaptic potential at all. Sometimes it produced a postsynaptic potential with a 1mV amplitude, looking just like a MEPP. Sometimes it produced a potential two or three or four or five times as big. (c) Looking at the statistics of how many times each of these sizes of postsynaptic potentials were seen provided yet more evidence for the quantal theory. The experimental data (at a mammalian NMJ in high magnesium solution) were an extremely good fit for the statistical model of what would be seen if quanta are released independently.
1 mV Page 7-5 Vesicle Hypothesis - Anatomical evidence Page 7-6 Soon after the experimental demonstration that transmitter release is quantal, the first anatomical evidence for vesicular release was obtained. Evidence: Vesicles were seen in electron micrographs of nerve terminals. If a presynaptic stimulus was applied (causing transmitter release), and then the tissue was immediately frozen, the vesicles could be seen fusing with the cell membrane, suggesting that transmitter is released by exocytosis. Each vesicle contains one quantum of transmitter Further experiments tied together physiological evidence of quantal release and anatomical evidence of vesicular release. (a) For these experiments, the neuron at the neuromuscular junction was stimulated and then the tissue was immediately frozen and prepared for freeze-fracture electron microscopy. In this technique, the tissue is fractured along the surfaces of membranes. This allows direct observation and counting of fused vesicles. This experiment was done in different concentrations of 4aminopyridine (4-AP) which increases the number of vesicles that fuse. (b) In separate electrophysiological experiments, the amplitude of the postsynaptic potential in response to the same stimulus was measured again at different concentrations of 4-AP. Then the anatomical count of the number of fused vesicle was compared with the physiological measurement of the number of quanta released for each concentration of 4-AP. There was a perfect match between the number of vesicles fusing and the number of quanta released. Release of vesicle contents by exocytosis Page 7-7 (1) Voltage gated calcium channels are found extremely close to vesicles lined up at the active site of a presynaptc terminal. This allows for transient large increases of calcium concentration at the active site, allowing for rapid fusing of vesicles with the membrane. (2) Calcium induced exocytosis can occur only when vesicles are first filled with neurotransmitter, then tethered to the membrane, and then docked to docking proteins at the active site. The docking proteins are located extremely close to the voltage gated calcium channels. (3) After docking, vesicles are primed by the binding of syaptotagmin ("Calcium sensor"). Synaptotagmin acts to stop the vesicle from fusing with the membrane in the absence of calcium, and to facilitate fusion when calcium is present. (4) After transmitter is released from vesicles, the vesicles are incorporated into the cell membrane. This can be seen as an increase in the capacitance of the neuron (Membrane size is increased. More space for ions) Page 7-8 (5) New vesicles are made by the formation of clathrin coated pits which pinch off the membrane to become coated vesicles which are then recycled into new synaptic vesicles. These vesicles are then filled with transmitter, and the cycle begins again. http://mcdb.colorado.edu/courses/3280/lectures/class10.html ...
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- Summer '08