CSB332 Exam Review Notes

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Cell and Systems Biology
Francis Bambico

CSB332 Exam Review Notes Slide 3 - Neurotransmitter is released into the synaptic cleft. Neurotransmitters can activate three subtypes of receptors. o These receptors may be located on the postsynaptic cell (called postsynaptic receptors). o Neurotransmitters can stray from the synaptic region and activate extra-synaptic receptors located outside of the synaptic region, especially when there is a spill over or when there is an uncontrollable release of neurotransmitters. o Neurotransmitters can stay in the synaptic cleft and activate receptors that are located on the presynaptic neuron (called presynaptic autoreceptors). Autoreceptors meaning receptors for the self (e.g., self-receptors). Autoreceptors can be located on the presynaptic axon or somatic dendritic sites of the neuron (e.g., cell bodies, dendrites). Slide 4 - This is another mechanism that regulates neurotransmitter release. - This is a sympathetic neuron releasing NE. NE binds to the α2-adrenergic receptor on its own presynaptic axon. Autoreceptors are typically coupled to an inhibitory G protein (Go or Gi). α2- adrenergic receptor is coupled to a Gi protein. When activated by NE, the beta and gamma subunits of the G protein will dissociate and will decrease the conductance of the Ca2+ channel. It will prevent the further influx of Ca2+ into presynaptic axon. The increase in the levels of presynaptic Ca2+ is responsible for exocytosis of neurotransmitters. The decrease in Ca2+ concentrations would lead to the prevention of further release of NE in synaptic cleft. Slide 5 - This is a third mechanism regulating neurotransmitter release. - This is different from presynaptic autoinhibition because it is caused by the release of neurotransmitter from presynaptic axon. - This mechanism is via retrograde messengers, which are molecules that are released by the postsynaptic back into synaptic cleft. When released, retrograde messengers activate presynaptic receptors that are inhibitory. An example of retrograde inhibitory molecules is endogenous cannabinoids, which are the brain’s cannabis-like ligand that activates cannabinoid CB1 and CB2 receptors in the CNS. - When the metabotropic and ionotropic receptors on the postsynaptic neuron are activated, it leads to the increase in Ca2+ concentration, leads to the synthesis of endogenous cannabinoids, leads to the enhanced release of endogenous cannabinoids into the synaptic cleft as retrograde messengers. Two examples of endocannabinoids are AEA and 2-AG. Once in the synaptic cleft, they activate cannabinoid CB1 or CB2 receptors. CB1 receptors are mainly located on the presynaptic axons of glutamatergic and GABAergic interneurons. These receptors are negatively coupled to adenylyl cyclase or Gi protein, which results in inhibitory action on the presynaptic axon. This results in decrease in Ca2+ influx and increase in Ca2+ efflux. This prevents further neurotransmitter release into synaptic cleft, thus preventing further activation of the postsynaptic neuron. This is what THC does. Slide 6 - Endocannabinoids mediate two types of short-term synaptic plasticity. - DSI occurs in type 2 or inhibitory synapses. DSE occurs in type 1 or excitatory synapses. DSI and DSE refers to the process in which endocannabinoid release as retrograde messengers prevent further neurotransmitter release from the presynaptic axon, thereby inhibiting excitation or inhibition of the postsynaptic cell. - The CB1R is expressed on the presynaptic axon. The presynaptic axon is a GABAergic (e.g., inhibitory or type 2 synapse) interneuron. When the postsynaptic cell is activated by neighbouring excitatory inputs, the depolarization would result in the activation of enzymes (DAGL and PLCβ1) that would result in an increased synthesis of endocannabinoids (2-AG). 2-AG is synthesized in the postsynaptic cell on demand (e.g., in an activity dependent manner). The postsynaptic cell should be activated and depolarized before the levels of endocannabinoids concentration are increased in the postsynaptic cell. This will result in the diffusion of endocannabinoids as retrograde messengers back into the synaptic cleft. This will activate CB1R in the presynaptic axon, resulting in the blockade of Ca2+ conductance, preventing the further release of neurotransmitters (GABA) into the synaptic cleft. The prevention of GABA release will result in the excitation or disinhibition of the postsynaptic cell. - This process is called DSI because the inhibition of the GABA neuron on the postsynaptic cell is suppressed. This only occurs when the postsynaptic cell is depolarized by neighboring cells. Slide 7 - We tested the effects of exogenous cannabinoids and URB597. - The problem with using direct CB1 receptor agonists (e.g., THC) is that injecting it into the patient will activate all CB1 receptors in the brain. CB1 receptor is the most abundant G protein coupled receptor in the brain, so it will affect many structures in the brain. It will decrease the activity of many regions in the brain. Hence, it will produce unwanted side effects. o Munchies (hypothalamus) o Psychosis at high doses (cerebral cortex) o Problems in coordination of movement (highest density of CB1 receptors in the cerebellum) - There are very low CB1 receptor densities in the vital regions of the brain (e.g., medulla oblongata) that controls vital processes, which is why no one has died from overdose of cannabis. Slide 10 - Depending on the type of information being received, it relays this information into different parts of the brain for LTM storage or encoding. There are two general types of memory information. - Explicit memory o Memory information that you can readily formulate a proposition or declaration or narration. This is information that you can transmit verbally to another person. o Semantic memory  Memory about factual information. o Episodic memory  Memory storage for personal events or experiences. - Implicit memory o Memory information that you can’t efficiently narrate to a person, but you can demonstrate. Unconscious form of memory that you can’t transmit to another person, but you can demonstrate it. o Procedural memory  Memories of skills and habits. o Non-associative learning  Ways of obtaining information from the environment via non-associative ways. This is information that is reflexive or easily accessed without being conscious about it or associating it with other things. o Associative learning  Ways of obtaining or storing information by associating the information with other information in the brain.  Skeletal muscles  Stored information via associations that modify how you behave or move.  Emotional response  Stored information via associative learning for emotional responses. Slide 12 - Learning induces structural changes in the brain. - If there is a modification in the structure of the synapse, then there will also be modifications in the function of the synapse. If a synapse becomes larger, then the signal that you can get from neurons in the synapse will also get larger. - Hebbian synaptic plasticity occurs when the presynaptic axon and the postsynaptic neuron are activated almost simultaneously. The postsynaptic cell should also be activated at the same time as the presynaptic axon. Slide 13 - Habituation and sensitization are very simple types of learned information (e.g., non-associative implicit memory). - Sensitization is different and somewhat opposite from habituation. Slide 14 - Aplysia is a marine snail. Researchers observed of reflexive behaviours in the Aplysia such as gill- withdrawal reflex or siphon-withdrawal reflex. Researchers have determined the mechanism that mediates sensitization in the Aplysia. How can the Aplysia demonstrate sensitization? If the defensive gill-withdrawal behaviour gets stronger than initially displayed. One mechanism used to explain sensitization in the Aplysia gill-withdrawal reflex is a mechanism called presynaptic facilitation. - Presynaptic facilitation involves the activity of two neurons: a serotonergic neuron and the main motor neuron that mediates the gill-withdrawal reflex. It is mediated by two biochemical pathways that occur within the motor neuron. There are two parts of the body: the siphon and the gill. - Under normal conditions, when you apply a tactile (weak) stimulus on the siphon, it will activate sensory neurons, leading to subtle activation of the motor neuron that induces gill-withdrawal but the response is not strong. There is a weak synapse between the sensory neuron of the siphon and the motor neuron of the gill. o You can demonstrate the weak intersynaptic connection. If you apply a tactile stimulus and record the action potential generated in the sensory neuron and record the EPSP exhibited by the motor neuron, then you will see that there is a strong sensory neuron action potential but the EPSP recorded in the motor neuron is weak. This is correlated with a very weak gill-withdrawal reflex. - If you apply a sensitizing (strong) stimulus (e.g., electric shock) to the tail, it will activate so many neurons. It will activate the sensory neurons in the tail, many interneurons in many other parts of the brain, the sensory neuron in the siphon, and the motor neuron in the gill. After applying the sensitizing stimulus, you examine the changes that occur in the initially very weak pathway. - After the application of the sensitizing stimulus, you apply a tactile stimulus on the siphon. The gill-withdrawal reflex will be sensitized. The response of the gill-withdrawal reflex will be dramatically amplified. o If you record the action potential generated by the sensory neuron that mediates siphon stimulation and the EPSP from the motor neuron cell body, you will see that after the application of the sensitizing stimulus, you will find that there will be no change in the generation of the sensory neuron action potential, but the response to the tactile stimulation will be much greater. This is suggested by an increased in the amplitude of the EPSP recorded from the motor neuron. This correlates with the strength of the gill- withdrawal reflex. Sensitization has taken place. - The activation of the interneuron that is very close to the area of the sensitizing stimulus does something to the presynaptic axon that was initially weak. It primed the presynaptic axon. It increased the excitability of the presynaptic sensory axon. - The strength of the presynaptic axon is enhanced or increased by the prior exposure to the sensitizing stimulus. Slide 15 - Before sensitization, the synaptic connectivity or the amount of neurotransmitters released is very low. This is the baseline. - When you apply the sensitizing stimulus on the tail, it will activate the facilitating interneuron and release massive amounts of serotonin. Serotonin will bind serotonin receptors along the presynaptic axon plasma membrane. This is an axoaxonic synapse. o Serotonin receptors are metabotropic receptors (GPCRs). There is only one type of ionotropic serotonin receptors, which is the 5-HT3 receptor. Activating the serotonin receptors will mobilize G proteins and activate some enzymes inside the cytosol. The two enzymes associated with G proteins coupled to the serotonin receptors are AC and PLC.  AC is associated with a Gs protein. When Gs protein is activated, it will activate the cAMP system.  When AC is activated, it will lead to an increase in the intracellular levels of cAMP, which is the second intracellular messenger in the Gs cAMP system. cAMP will travel, phosphorylate, and activate another enzyme called PKA. PKA is cAMP-dependent. o When PKA is activated, it does two things.  PKA phosphorylates potassium channels. When potassium channels are phosphorylated, it will lead to an increase in the inward conductance of potassium channels. This will eventually lead to depolarization.  PKA interacts with synaptic vesicles in the free or reserved pool of synaptic vesicles around the core of the presynaptic terminal axon. PKA will mobilize the synaptic vesicles and move them towards the active zone. There are a lot of macromolecules in the presynaptic zone that interacts with the synaptic vesicles and tethers them to the plasma membrane. There will be more synaptic vesicles and more readily releasable pool of neurotransmitters in the active zone.  PLC is associated with a Gq protein. When a Gq protein is activated, it will activate the phosphoinositol system.  The activation of PLC leads to the activation of PKC. PKC will mobilize synaptic vesicles to the active zone. - When you apply another tactile stimulus in the siphon, the sensory neuron will be activated, the action potential will travel down the presynaptic axon, and the amount of neurotransmitters would get dramatically increased. More neurotransmitters are released. More glutamate being released will activate more glutamate receptors on the motor neuron, a larger EPSP will be generated in the motor neuron, and a greater or longer gill-withdrawal reflex will be observed. The synapse has been sensitized after a single exposure to a sensitizing stimulus. Slide 16 - LTP and LTD are associated with consolidation or depositing information from STM to LTM. - If new information has been retained in the brain, something has to change in the synaptic connectivities that mediate this information. One way to figure that out is whether there are changes in the synaptic function of that synapse. One way to examine synaptic function is to examine the EPSP or IPSP in response to a stimulus. Slide 17 - There are basic, fundamental forms of learning that is different in terms of mechanism with higher forms of learning. These types of learning have psychological realities. - The mechanisms of habituation have been demonstrated in a vertebrate model. Habituation results in an increase in the frequent of the stimulation of the presynaptic axon. This means that the organism is repeatedly exposed to a stimulus, which results in a decrease of the specific response. The response can be studied in the Aplysia californica as the gill withdrawal reflex or the siphon withdrawal reflex. - The mechanism of habituation has to do with the fact that if you apply high frequency stimulation in the presynaptic axon, then it will eventually result in the inactivation of all voltage-gated calcium channels. If you expose an organism to a specific stimulus several times, then it will result in high frequency stimulation of a presynaptic axon, and repeated stimulation of the presynaptic axon results in the inactivation of voltage-gated calcium channels. o If an action potential arrives at the presynaptic axon, then it will result in the opening of voltage-gated calcium channels because they are required for exocytosis or transmitter release. o If you do this several times (e.g., high frequency stimulation, high frequency action potential generation), then all of the activated voltage-gated calcium channels will eventually switch into an inactivated mode. You won’t be able to give the presynaptic axon calcium channels to recover from its inactivated mode. This results in a decrease in exocytosis. o After a while, the inactivated calcium channels revert back to its activatable state. This will recover the habituation response. This type of learning is called dishabituation. - Habituation is a form of non-Hebbian learning because the mechanism only occurs in the presynaptic axon. It does not involve the activity of the postsynaptic neuron. Slide 18 - Hippocampal formation is the medial most part of the temporal lobe. When the temporal lobe forms during development, the inferior part of the temporal lobe folds onto itself in the medial afferent of the brain and it forms the hippocampal formation. The hippocampal formation is composed of several structures: dentate gyrus and hippocampus. o The hippocampus is composed of several sub fields: CA3, CA2, CA1. CA stands for Cornu Ammonis (e.g., ram’s horn). CA3 is the input area of the hippocampus. CA1 is the output area of the hippocampus. o The dentate gyrus receives axons that are formed by the perforant fiber pathway. These axons come from many parts brain. This is the input region of the hippocampal formation. It receives axons from many parts of the brain coming from the subiculum and entorhinal cortex. It can receive information from the amydgala, prefrontal cortex, basal ganglia, etc. Many parts of the brain can input into the hippocampus because the hippocampus is involved in memory. The perforant fiber pathway synapses onto granule cells of the dentate gyrus. The dentate gyrus contains granule cells. They are called granule cells because they have tiny cell bodies. o The granule cells send axons to the input region of the hippocampus proper. These neurons are pyramidal neurons. All of the synapses are glutamatergic or excitatory synapses. pyramidal neurons in the CA3 region receives afferents from the dentate gyrus granule cells. This is called the mossy fiber. o The pyramidal neurons in the CA3 region send many axonal collaterals. One set of collateral synapse with the pyramidal neurons in the CA1 region. There are also axons that extend laterally to the other hemisphere of the brain called the hippocampal commissure. Therefore, the bundles of axons are collectively called the Schaffer collateral commissural pathway. The CA1 pyramidal cells then send axons to many other parts of the brain outside of the hippocampus. Slide 19 - It looks like is an interlocking C shaped structure. - To examine whether synaptic changes occur in any synapse, you can lower a recording electrode to any cell body (e.g., dentate gyrus granule cells) and examine changes in postsynaptic potentials. Since these are mainly glutamatergic synapses, so the type postsynaptic potential that you record from the cell bodies of the dentate gyrus granule cells will be EPSPs resulting from activation of glutamatergic receptors in the cell bodies. - The dentate gyrus receives axons from the perforant fiber pathway. The perforant fiber pathway cell bodies normally generate action potentials that are in low frequencies. - The researchers wanted to the manipulate the action potentials generated in the perforant pathway arriving in the cell bodies of the dentate gyrus and see if this will influence the strength/magnitude of the EPSPs recorded from the dentate gyrus cell bodies. o The researchers lowered a stimulating electrode to manipulate the frequency of presynaptic action potential. First, they mimicked normal firing frequency of the axons of the perforant fiber pathway. There will be no change from normal conditions. o Then they apply high frequency stimulation. They will increase the number of action potentials at a given time. This type of high frequency stimulation is called tetanic stimulation or the conditioning training of stimulations.  Apply stimulation of 100 Hz for 5 seconds, for example  Examine the EPSP generated in the cell body in response to a test stimulus (e.g., one action potential)  Compare the magnitude of the EPSP in response to the test stimulus before and after the application of the tetanic stimulation Slide 20 - The mechanism of non-associative LTP has something to do with the number of action potentials arriving at the presynaptic axon. Many action potentials arriving at the presynaptic terminal results in the opening of presynaptic voltage-gated calcium channels. The increase in the opening of presynaptic calcium channels results an increase of calcium concentration in the presynaptic axon cytosolic environment. This increase in calcium concentration results in the activation of enzymes (e.g., AC) and activation of more downstream enzymes (e.g., PKA, PKC). The activation of PKA and PKC results in the mobilization of synaptic vesicles down to the presynaptic membrane active zone. When a test pulse is applied to the presynaptic axon, then it will release more neurotransmitters, which will activate more postsynaptic receptors and increase EPSP. - Non-associative LTP can be observed all of these three pathways. All of these pathways can exhibit non-associative LTP. Non-associative LTP will be dependent upon PKA, NMDA receptors, etc. - The mossy fiber CA3 pathway does not produce associative LTP and it only produces non- associative LTP. If you apply an inhibitor of PKA, you will abolish the increase in EPSP, and thus abolish LTP. - The Schaffer collateral CA1 pathway and direct perforant CA1 pathway produces both associative and non-associative LTP, and can be blocked by NMDA receptors and other calcium channel blockers (e.g., nitrendipine). Nitrendipine is a blocker for L-type voltage-gated calcium channels. Slide 21 - The blue dots are the magnitude of EPSPs without the application of the tetanic stimulation. - The red dots are the magnitude of the EPSPs after one bout of stimulation, after a second bout of stimulation, after a third bout of stimulation, and a fourth bout of tetanic stimulation. - They found that the presynaptic tetanic stimulation progressively increases the magnitude of EPSPs recorded from the granule cell bodies. - This illustrates LTP. The experience (e.g., high frequency stimulation) of the presynaptic axon results in a greater EPSP that is recorded from the postsynaptic cell. This is a function of how many times you apply the tetanic stimulation onto the presynaptic axon. - This suggests that the synaptic strength of the synapse is dramatically increased in response to an increase in the frequency of stimulation of the presynaptic axon. Slide 22 - Another way of examining the synaptic strength of the synapse in response to the presynaptic stimulation is to examine how long it takes for the enhanced EPSP to return to normal amplitude without further stimulating the presynaptic axon. - The temporal duration or decay of LTP induced by the tetanic stimulation is correlated with the number of times you tetanically stimulate the presynaptic axon. - This graph represents how long it takes for the EPSP to return back to normal. After tetanic stimulation, in all cases, the EPSP is increased 2.5 times the normal EPSP amplitude. They examined the changes in the strength of the EPSP. After one bout of tetatnic stimulation, the EPSP decays much faster than 4 or 8 bouts. After 8 bouts of tetanic stimulation, the EPSP amplitude initially drops, but then it will be retained for long periods of time, even for months. - What explains the difference in temporal decay of EPSP? o There are several phases of LTP. o The longer lasting form of LTP is explained by more stable changes. o The increase in EPSP amplitude suggests that there are changes going on in the synapse. The change is more stable if you apply greater number of tetanic stimulations to the presynaptic axon. o The shorter lasting LTP in response to one tetanic stimulation is called the early phase of LTP. The longer lasting LTP in response to four or eight tetanic stimulation is called the late phase of LTP. There are differences in terms of the amount of change that occurs in the synapse. The changes in the synapse are greater and more stable in the late phase of LTP in comparison to the early phase of LTP. Slide 23 - LTP1 is not long lasting, so the changes are non-genomic mechanisms. - LTP2 and LTP3 results in more stable, longer lasting LTP because it recruits genomic mechanisms in addition to non-genomic mechanisms. Slide 24 - How does calcium increase in the postsynaptic cell as a result of high frequency presynaptic axonal stimulation? - The glutamate receptor in the postsynaptic neuron that is involved in LTP is mainly composed of two types: AMPA receptor and NMDA receptor. AMPA and NMDA receptors are both ionotropic receptors that mediate the generation of EPSPs in the postsynaptic neuron. Only the NMDA receptor is permeable to calcium. AMPA receptors are not permeable to calcium. Embryonic forms of AMPA receptors do allow calcium to enter, but the adult form does not allow calcium to enter the cell. AMPA receptors are only permeable to sodium. - NMDA receptors are important for LTP and learning. NMDA receptors are permeable to both calcium and sodium. Under normal conditions, calcium is not able to enter the cell. You need an increase in calcium concentration or calcium entry before LTP can be generated. However, magnesium is plugged into the pore of the NMDA receptor, and does not allow calcium or any ions to enter into the neuron. How can you increase the entry of calcium into the neuron? o One has to depolarize the postsynaptic neuron to dislodge the magnesium. If you depolarize the interior of the plasma membrane, then the buildup of positive ions inside will dislodge magnesium and free the pore of the NMDA receptor to allow calcium to get into the glutamatergic postsynaptic neuron. o Further depolarization of the postsynaptic neuron will activate another channel, including voltage-gated calcium channels. This is another way by which calcium can enter into the neuron and increase the cytosolic concentration of calcium. For example, the L-type calcium channels. Slide 25 - Presynaptic axons are releasing glutamate in response to stimulation. AMPA receptors are not permeable to calcium, but only permeable to sodium. NMDA receptors are permeable to both sodium and calcium, but there is a magnesium plug under normal conditions or low level of stimulation. Many axons should activate many AMPA receptors to be able to depolarize the postsynaptic neuron. The buildup of positive concentration inside the neuron will result in depolarization and dislodge the magnesium within the pore of the NMDA receptor and allow calcium to enter the postsynaptic neuron. - The other way for calcium to get into the postsynaptic neuron is via voltage-gated calcium channels. Slide 26 - You don’t have to apply the tetanic stimulation one presynaptic input to generate an increase in the EPSP. You can apply the tetanic stimulation on another presynaptic axon, which will possibly increase the EPSP in the other synapse in response to a test pulse. - You can apply the tetanic stimulation on the presynaptic axon of synapse I/B, and you can generate LTP on the postsynaptic neuron in response to a test pulse applied on the presynaptic axon of synapse II/A. - The EPSPs are measured in CA1 pyramidal neurons. The recording electrode is lowered into the cell body of the CA1 pyramidal neuron. Then two stimulating electrodes are lowered onto two different set of presynaptic axon fibers. We want to determine whether activity of these presynaptic axons will result in changes in EPSP evoked by a test pulse. - If you stimulate the middle segment of an axon, then the action potentials can flow bidirectionally or back to the dendritic spine of the postsynaptic neuron. - The researchers were able to induce LTP in an initially weak synapse (e.g., synapse II). - Control o This is the baseline EPSP response. We are interested synapse formed by axon II on the postsynaptic dendritic spine. This is the synapse that we are interested in analyzing and examining the changes in the strength of. o You apply a test pulse on axon II corresponding to one bout of stimulation (e.g., one action potential). You record the EPSP. - 4 min after tetanus at II o This is control A. o You apply the tetan
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