User:Kinkreet/MCBII/Neuronal Signalling

Most of the synapses are chemical synapses; electrical synapses are few in adults, and primarily act in development. The speed for information to travel in the mind is not very fast, the fastest neuron can process at ~300Hz. An action potential takes about half a millisecond to establish. So in reality, there is a delay of ~0.5 seconds for your brain to process it, but your mind tries to make sense of the world. An example of this is our perception of our sight - there is a small part of our retina that can see accurately and crispy, our mind creates a construct for us to deal with.

Calyx of Held
Many of the studies on synaptic junctions are done on calyx of Held, a particularly large (maybe the largest) synapse in the mammalian auditory central nervous system. It is used because it has a large nerve terminal (∼15 μm diameter) that envelops the soma of the post-synaptic neuron, making 500-600 synapses, of which ~200 fire each time upon the presynaptic potential signal. Due to its large size, it allows for electrophysiological recordings of the membrane potential and thus able to record pre- and post-synaptic events. The synapses of calyx of Held contain less vesicles (125 ± 82 per active zone) than others such as cerebellar, cortical, or hippocampal synapses (>200 synapses per active zone), and also less docked and free vesicles as well.

Generation and Propagation of Action Potential
Naturally, due to cells have different ionic concentrations on the inner side of the membrane compared to the other, this allows all cells to have a potential difference across the membrane. Inside the membrane is about ~(-)70mV and outside is ~(+)40mV, mostly due to the presence of Na+ ions. This ionic gradient is maintained passively by the plasma membrane, and actively using ATPase antiport pump which pumps 3 Na+ ions out of the cell while taking in 2 K+ simultaneously. There are also potassium channels which allows potassium to leave the cell down its own electrochemical gradient, although it will never reach its own equilibrium point, because the presence of Na+. The voltage at which dynamic equilibrium occurs is determined by the concentration of ions on either side of the membrane, and this can be estimated using the Nernst Equation.


 * $$E = \frac{R T}{z F} \ln\frac{[\text{ion outside cell}]}{[\text{ion inside cell}]} = 2.303\frac{R T}{z F} \log_{10}\frac{[\text{ion outside cell}]}{[\text{ion inside cell}]}.$$

When a neuron receives a synaptic signal which when integrated (if at all) is above the action potential, it will depolarize. First Na+ channels are opened to allow for an influx of Na+ ions into the cell until the inside reaches ~30mV, where Na+ has nearly reached its equilibrium potential, but it does not reach it because the Na+ channel shuts quickly. This depolarisation causes neighbouring channels (at Nodes of Ranvier) to open, propagating the signal in both directions from the point of depolarization. The depolarization also increases the permeability of K+ ions to leave the cell, repolarizing the cell. The K+ remains open after repolarization, causing hyperpolarisation, where the voltage inside becomes even more negative than the resting membrane potential; this ensures that the propagating signal do not travels backwards when the set of channels on the next Node of Ranvier opens. Each Node of Ranvier is like an amplifier, maintaining the signal strength until it reaches the axon terminal.

Calcium influx
Almost all neurons have only one axon, but each one can branch near its end to form many presynaptic terminals; most neurons have >500 presynaptic terminals. At the presynaptic terminal, the depolarization activates voltage-gated Ca2+ calcium channels (VGCCs) at ~(-)30mV. Within VGCC, there is a positive S4 region which is usually negatively charged, and hide inside the channel to block ion pass through; when depolarization occurs, S4 becomes positively charged and change conformation, allowing the entry of Ca2+ into the cell. This occurs within 0.3 ms of the action potential signal. The calcium channel opens for a very short period of time because thermodynamically, it favours the close position. The VGCCs opens at the peak of the action potential and remains open until full repolarization, when the potential inside the cell is positive, and thus it is not very favourable for Ca2+ ions to enter into the cell; as the potential becomes more negative, the influx of Ca2+ increases. Most of the current comes at the end of depolarization, where the cell is hyperpolarized, because it is at this point where the driving force is the highest. Therefore, the signal occurs at the end of the action potential, which takes about 0.5 ms.

Calcium ions binds to Ca2+ sensors synaptotagmins 1 and 2, each having at least 5 Ca2+ binding sites. Synaptotagmins are membrane-bound vesicular proteins; they have a short intravesicular domain, a transmembrane region, a short linker sequence and two cytoplasmic domains - C2A and C2B. C2A binds to 3 calcium ions and C2B binds to two; both domains bind with very low affinity intrinsically, but increases a 1000-fold when it associates with phospholipid membranes. Synaptotagmins form homomultimers using its N-terminal domain in the absence of Ca2+ and via its C2B domain in the presence of calcium.

Synaptic Vesicles
First, the vesicles (radius 17–22 nm) are filled with neurotransmitters (typically 5,000 - 20,000 acetylcholine, ACh, molecules per vesicle); once filled, it moves and cluster at the active zone (∼0.05–0.10 μm2). They are then docked and primed for Ca2+-triggered fusion-pore opening.

In a study by Gandhi and Stevens in 2003 GFP18, a modified GFP pH sensor which fluoresce when there are no pH gradient across the membrane, was tagged to vesicular proteins. At rest, proton pumps acidify the vesicles and quenches GFP18 so it does not fluoresce. When the vesicle docks and release the contents, the pH gradient is lost and the GFP18 fluoresces. From this study, it confirms the facts that the probability of vesicular release following Ca2+ influx is not 1, and that the vesicles do not fully fuse, but only a pore is formed.

There are thought to be at least two components of release: a fast and synchronous component which is activated from 50-500μs (dependent on temperature and individual synapse) after Ca2+ influx, and a slower asynchronous component which last for over a second and increases the rate of spontaneous release. In most synapses, release is stimulated by Ca2+ influx through P/Q- (CaV2.1) or N-type Ca2+ channels (CaV2.2), whereas the related R- (CaV2.3) or the more distant l-type Ca2+ channels (CaV1 series) are involved only rarely.

The magnitude of the post-synaptic response is thought to be proportional to the power of 4 of the calcium ion concentration.

Fusion
Fusion is usually mediated by SNARE proteins. SNARE proteins are characterised by homologous 70-residue sequence called the SNARE motif; SNARES are present on both the fusing membranes. There are four types of SNARE motifs - R, Qa, Qb and Qc; to forma a core complex, all four motifs must be present and assembled together to form a four-helical bundle. Each SNARE protein has 1-2 SNARE motifs, and so 2-4 SNARE proteins are required for each core complex. The core complex formation forces the fusing membranes close to each other, encouraging fusion. But it does not cause the fusion itself.

There are three SNARE proteins involved in synaptic vesicular fusion: synaptobrevin (a.k.a. vesicle-associated membrane protein) on synaptic vesicles provides the R motif, syntaxin 1 on the plasma membrane provides the Qa motif, and SNAP-25 on the presynaptic plasma membrane provides the Qb and Qc motif. Synaptophysin is the most abundant vesicle membrane protein; it binds to synaptobrevin. When bound, it prevents synaptobrevin from associating with other SNARE proteins, and thus is thought to negatively regulate neurotransmitter release. This is supported by evidence that chronic blockade of glutamate receptors caused an increase in neurotransmitter release but a decrease in the synaptobrevin/synaptophysin complex.

Complexins bind to the core complex and promotes the action of synaptotagmin 1. Synaptotagmins 1 associates with the complexin/SNARE core complex in the absence of Ca2+. Upon binding of Ca2+, the C2 domains associates with the phospholipid membrane and destabilizes the intermediate and enable pore formation. This is the mechanism in which calcium influx triggers vesicle fusion.

After fusion, vesicles can be recycled in three ways:
 * Kiss-and-stay - where the vesicle do not undock and remains at the active zone. It is reacidified and neurotransmitter are added to it, and will release as soon as it has been primed and calcium ions are present.
 * Kiss-and-run - where the vesicles undock and is refilled and reacidified locally, it can then dock again as usual
 * Endosomal recycling - clathrin is used to coat the vesicle and endocytose them back into the cell, where they are reacidified, maybe through the endosome, and then refilled

The kiss-and-stay and kiss-and-run models is rapid (the fast component) and used preferentially at low stimulation; endosomal recycling (slow component) starts to have an effect at higher stimulation (stimulation at higher frequencies). The rate of endocytosis is thought to be related to the number of unretrieved vesicles, and may be related to membrane tension. Synapsin are thought to regulate the number of synaptic vesicles available for release via exocytosis at any one time.

The conversion from action potential to synaptic release is consistently low, only 10-20% of all action potentials that reaches the terminal result in release. The success depends on the intracellular messengers, extracellular modulators and the number of times the synapse has been used. Even at rest, there is a chance that the vesicles will release, causing small postsynaptic currents. The neurotransmitters diffuse across the synapse rapidly. The delay between release and a post-synaptic response is ~0.1 ms. The time for the neurotransmitter to diffuse across is even shorter, thought to be only ~1.6μs.

Binding of Receptor
After release of neurotransmitters, the concentration of neurotransmitters reaches to ~1mM; they diffuse across the synaptic cleft and bind to specific receptors and initiate a reaction cascade, which usually lead to one of these:
 * A rapid opening of ligand-gated ion channels that lead to an influx of current, which depolarizes the post-synaptic neuron.
 * The release of secondary messengers that modulate ion channels
 * Activation of GTP-binding proteins which associates with ion channels

The nicotinic ACh receptor is a member of the pentameric “Cys-loop” superfamily of transmittergated ion channels, which includes neuronal ACh receptors, GABAA receptors, 5-HT3 receptors and glycine receptors. They are ligand-gated ion channels that when ACh is bound, opens and allow sodium ions to flow into the cell, depolarising the cell and generate another action potential, or end-plate potential if it is a muscle cell. The potential change will decay as it moves away from the ion channel, because the membrane may be leaky or have channels which lets out ions. AChR (or the other nicotinic receptors) closes rapidly and ACh dissociates from the receptor within 1-5 ms and are degraded.

The structure of the nicotinic acetlycholine receptor is elucidated in 2005 at 4Å. It is found to be a large (290kDa) pentameric glycoprotein made up of 5 homologous subunits (α, γ, α, β, δ). Each subunit has a large extracellular N-terminal domain, and transmembrane domain containing four segments, and a small cytosolic C-terminal tail. The structure of the cytosolic domain is not determined because they are not stable enough for a sharp diffraction pattern. They assemble to form a pore using the TM2 segment of the transmembrane domain, with two binding sites for ACh. The pore will open up when both of these sites are bound. Nicotinic ACh receptors are neural modulators, but not at synapses in the brain. In the brain, glutamine (for stimulatory) and GABA (for inhibitory) are used.

Patch-clamping
Patch-clamping is a way to record the action of a single or a few ion channels. The neuron to be studied is placed on glass and excised. A micropipette with a little bit of suction is then used to form a closed seal on the cell surface with the cell membrane. The opening at the end of the pipette is typically 1 μm2, ensuring only a few channels are covered.

The pipette is then filled with a bath solution and a chlorided silver wire inserted to record the potential at the bath solution. Different molecules, such as a neurotransmitter, can be added to the bath solution and the potential change recorded. As each time a channel opens up, the potential changes by a defined amount, by looking at how many steps the potential changes from being fully closed to fully opened, one can see the effect of different concentrations of neurotransmitters on the proportion of channels that opens (open probability), the conductance of each channel, and also the thermodynamics of binding. Many channels exhibits a fast association and slow dissociation, suggesting thermodynamical stability when ligand binds.