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Lecture 2

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Rutsuko Ito

Lecture 2 Notes Neurotransmission Introduction: Chemical neurotransmission Chemical neurotransmission recap: Dominant form of neuron to neuron transmission of information. Arrive of an action potential at the presynaptic terminal triggers a cascade of cellular events that result in the release of a neurochemical substance (neurotransmitter) across the synaptic cleft. Neurotransmitter release: The neurotransmitter binds to receptors embedded in the postsynaptic membrane to bring about a change in the electrical property of the postsynaptic cell (in the form of graded potentials). Mechanism of neurotransmitter release: Opening of voltage gated Ca2+ channels in the presynaptic terminal. Allows the influx of Ca2+ ions to cause fusion of neurotransmitter filled vesicles with the presynaptic membrane. The vesicles then release the neurotransmitter into the synaptic cleft, which in turn diffuses across to bind to postsynaptic receptors. Two types of postsynaptic receptors: 1) Ionotropic, 2) metabotropic. Ionotropic postsynaptic receptors: Transmembrane molecules that can 'open' or 'close' a channel that would allow smaller particles (ions) to travel in and out of the cell. These receptors are usually closed until a neurotransmitter binds to the receptor, causing a conformational change in the channel protein. Ionotropic receptors are also known as 'ligand-gated transmembrane ion channels' Metabotropic postsynaptic receptors: Do not have a 'channel' that opens or closes. Instead, they are linked to a g-protein. When the neurotransmitter binds the metabotropic receptor, the receptor activates the g-protein. The g-protein is then released and goes on to activate other molecules (secondary messengers) Secondary mesengers: A chemical whose function is to go and activate other particles. 1) The secondary messenger can binds to and opens ion channels located elsewhere on the membrane, or 2) activate other intermediate molecules inside the cell Presynaptic mechanisms Fatt and Katz: Discovery of spontaneous miniature end plate potentials (MEPPs) in the neuromuscular junction of a frog. End plates: The postsynaptic terminals of muscle fibers Mechanisms of neurotransmitter release: When an action potential reaches the axon terminal of a motor neuron, vesicles carrying neurotransmitters (mainly acetylcholine) are exocytosed and the contents are released into the neuromuscular junction. These neurotransmitters bind to receptors on the postsynaptic membrane and lead to its depolarization. MEPPs: Miniature end plate potentials. In the absence of an action potential, acetylcholine vesicles spontaneously leak into the neuromuscular junction and cause very small depolarizations in the postsynaptic membrane. This small response of around ~0.5 mV in amplitude is called an MEPP and is generated by one acetylcholine containing vesicle (quantum). It represents the smallest possible depolarization which can be induced in a muscle. These small depolarizations are not enough to reach threshold and so an action potential in the postsynaptic membrane does not occur. Each acetylcholine vesicle contains approximately 5000 acetylcholine molecules which reflects the opening of many individual ACh receptors. Addition of receptor agonists would increase MEPP amplitude, while addition of receptor antagonists would decrease MEPP amplitude Threshold potential: When action potential causes the release of many acetylcholine vesicles, acetylcholine diffuses across the neuromuscular junction and binds to ligand gated nicotinic receptors (non-selective cation channels) on the muscle fiber. This allows for increased conductance of Na+ and K+ which results in depolarization of the sarcolemma (muscle cell membrane). The small depolarization associated with the release of acetylcholine from one synaptic vesicle is called a MEPP. MEPPs are additive and lead to greater depolarization of the postsynaptic membrane and become EPP (end plate potentials). When EPPs cause the membrane to reach threshold, the voltage gated ion channels in the postsynaptic membrane open. This causes an influx of sodium ions and a sharp spike in depolarization. This spike causes and action potential to occur and propagate down the postsynaptic membrane leading to muscle contraction. It is important to note that EPPs are NOT action potentials, but instead trigger them. Delcastillo and Katz: Discovered that when motor neuron is stimulated under conditions that are unfavorable to neurotransmitter release (such as low Ca2+), the magnitude of the recorded end plate potentials (EPPs) fluctuated in a stepwise manner (what does this mean?). They observed that the smallest evoked EPPs were approximately the size of an MEPP and other EPPs were of sizes at integral multiples of MEPPS. These findings led to the quantal hypothesis. Quantal hypothesis: neurotransmitters are released from the presynaptic terminal in discrete units (quanta). An MEPP is a response to a spontaneous release of a single quantum, while an EPP is a response to some 100s of quanta being released. Ca2+ levels control the release probability of the quanta, such that low calcium levels often result in failures in releasing quanta. Anatomical evidence of vesicular / quantal release Neuroanatomical basis for quantal release: 1) A single ACh channel opening generates ~0.5 uV. For a quantal size of 0.5 mV MEPP, 1000 channels must open. Each ACh channel requires 2 ACh molecules, but some will be destroyed by AChE or lost in the synaptic cleft before reaching the target. An estimate of ~5000 ACh molecules is needed, while a synaptic vesicle is estimated to contain about 4000-10000 molecules -- so theres a good correspondence between the number of ACh molecules estimated to be in a quantum and the amount of ACh contained within a vesicle. 2) Free fracture technique allowed researchers observe vesicles in the act of exocytosis. This was achieved by blocking K+ channels with AP- 4 (to allow for a longer AP propagation) and then quickly freezing the presynaptic axon terminals at precise time points following the stimulation. Numerous 'omega' profiles (look like the greek letter omega) were observed at the active zone, close to the location of presynaptic Ca2+ channels and postsynaptic receptors. Two accounts of mechanisms of vesicular release Heuser and Reese: Found that the number of 'fused vesicles' matched the number of quanta measured from the response. Identified 'pits' outside the active zone which could be recycled vesicles. Proposed the full fusion account of vesicular neurotransmitter release Full fusion model: Vesicles fuse fully with membrane at active zone. Membrane is then taken from a region different from the active zone to form new vesicles. New vesicles are filled with synaptic transmitter molecules. Evidence for the full fusion view: Involves the incorporation of vesicle membrane into the cell membrane. This leads to an increase in the total cell surface area of the presynaptic membrane. Given that capacitance measure is a function of the membrane surface area and membrane capacitance (thickness of membrane - usually constant), any increase in capacitance measure is likely to reflect the full fusion (incorporation) of the synaptic vesicle with the cell membrane (increase in surface area). Ceccarelli, Hurlbut and Mauro: Proposed kiss and run account of vesicular neurotransmitter release. Kiss and run model: Partial fusion of active vesicle forms a pore through active zones of presynaptic membrane. Discharges transmitter molecules (but not entire contents) into synaptic cleft. Intact vesicle separates from membrane and replenishes its store of transmitter molecules towards the interior of the presynaptic neuron. Evidence for kiss-and-run view: Amperometric recording from an adrenal chromaffin cell generates a signal that shows the current produced when secreted neurotransmitters (e.g. serotonin) strike a carbon fiber electrode (CFE) positioned near the cell. A low level signal (foot) represents the leakage of neurotransmitters through a small flickering fusion pore (for about 100 m/s). This precedes a spike that results from the rapid dilation of the fusion pore. Hence the release of the remaining contents of the granule (spike). During the foot, the signal may fluctuate, as if the fusion pore size were rapidly opening and closing. Which mechanism?: Recording of membrane capacitance and amperometric current provides evidence for both models of vesicular release. Limitations of amperometric current measurement: Provides powerful insight into exocytosis at the single vesicle level, but does not capture exocytotic events from the entire cell surface. Molecular evidence for vesicular (kiss-and-run) fusion Molecular basis of kiss-and-run fusion: Vesicular fusion requires SNARE proteins which are located on the vesicle and cell (target) membranes. SNARE proteins: Synaptobrevin (vSNARE), syntaxin and SNAP-25 (tSNARE). SNARE fusion mechanism: 1) Docking of the vesicle with the cell (target) membrane to form a loose trans-SNARE complex. 2) Complex forms a tight trans-SNARE complex. 3) Formation of pore through the fused vesicle and cell membrane. 4) Opening of the pore is a Ca2+ dependent process that changes the SNARE complex from trans to cis conformation. 5) The pore fully opens to allow the vesicle to release its contents under the cis-SNARE complex. 6) Vesicle disengages from cell (target) membrane for another cycle. Importance of Ca2+ in vesicular fusion: Probability of vesicle fusion increases drastically within less than 0.2 m/s after Ca2+ influx and returns to much lower levels within 1 m/s. It is critical that change in intracellular Ca2+ is sensed by the cell. Ca2+ sensor: Synaptotagmin has two Ca2+ binding sites as well as phospholipid binding properties. It is the Ca2+ sensor on the cell vesicle membrane that results in the fusion signal with the presynaptic membrane. Importance of Ca2+ sensor / Geppert study: Generated a strain of mice with functional disruption of the synaptotagmin gene (knockout) and studied synaptic responses of hippocampal embryonic neurons. Evoked responses are greatly diminished in mutant cells and display a large degree of fluctuation between each stimulus. Two pools of vesicles in the terminal bouton: 1) readily releasable pool of vesicles located at or close to the active zone of the presynaptic membrane (kiss-and-run vesicles). These reform rapidly after electrical stimulation of the neuron. 2) reserve pool of vesicles located away from the activate zone of the presynaptic membrane. These vesicles are reformed relatively slowly from the infolding of the cell membrane. During intense electrical stimulation of the neuron, vesicles of the reserve pool are mobilized over 10-15 s and migrate to the active zone. Relative number of vesicles in each pool: Differ in different parts of the nervous system and are modulated by extracellular events. Why should we care which mechanism is used?: One of the unknowns of the kiss-and-run model is whether or not all of the neurotransmitter substances are released from the vesicle upon fusion. If this is not the case, then it may have a huge impact on our understanding of transmitter release (i.e. is it quantal?) Postsynaptic mechanism of neurotransmission Variation in the actions of neurotransmitter substances: 1) Many neurotransmitters can cause more than one postsynaptic response, dictated mostly by pharmacologically distinct post-synaptic receptors. 2) The same neurotransmitter can have different effects in different regions of the brain or even in neighboring cells with the same local circuit (divergence). 3) Different neurotransmitters can modify the same ionic currents. Postsynaptic excitation Postsynaptic excitation: Increases the probability of action potential discharge by depolarization. Usually achieved by the opening of non-selective cation channels. Degree of excitation in postsynaptic neuron: Depends on many factors: 1) Amount and frequency of neurotransmitter release. 2) Lifetime of neurotransmitter release in synaptic cleft (long lasting?). 3) Responsiveness, number and location of postsynaptic receptors. 4) Excitation state of the postsynaptic cell Type of receptor: 1) Ionotropic, characterized by fast postsynaptic currents. 2) Metabotropic, characterized by slow postsynaptic current
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