BIPN 140 Problem Set 6

Transcription

1 BIPN 140 Problem Set 6 1) Hippocampus is a cortical structure in the medial portion of the temporal lobe (medial temporal lobe in primates. a) What is the main function of the hippocampus? The hippocampus is critical for memory formation, spatial navigation & spatial memory b) Draw the tri-synaptic circuit of the hippocampus. c) How can an LTP be induced and measured in the hippocampus? i) Administer multiple tetani (high-frequency electrical stimulus, e.g. 100 Hz) at the Schaffer collaterals and measure EPSP in the CA1 pyramidal neurons. Alternatively, administer tetani at the mossy fiber and measure EPSP in CA3 neurons or administer tetani at the performant path and measure EPSP at the granule cells. d) In hippocampal slice recording, what are the two key requirements (neurotransmitter receptor and ion) for LTP to occur? Briefly describe the experiments that demonstrate the necessity for these two components. i) NMDA Receptors are Required for LTP (1) Bath application of a NMDA-R blocker, APV, blocks LTP without affecting basal EPSP amplitudes. ii) Ca2+ influx is Required for LTP (1) Infusion of Ca2+ chelators (e.g. BAPTA) via patch pipet blocks LTP. e) How does LTP involve coincidence detection? i) LTP occurs when both presynaptic and postsynaptic neurons are active around the same time. In other words, LTP occurs when the presynaptic neurons fire action potentials to release glutamate and the postsynaptic membrane is sufficiently depolarized. Therefore, these two events need to be tightly linked in time (coincidence) in order for LTP to occur.

2 2) Synaptic facilitation and depression can occur at the same synapse. Although both occur due to repeated firing of the presynaptic neuron, synaptic facilitation leads to a larger amplitude PSP, whereas synaptic depression decreases the amplitude of the PSP. The nature of the presynaptic stimulus determines which form of short term plasticity will likely occur. a) What are the mechanisms for synaptic facilitation and depression? Synaptic facilitation occurs when elevated calcium (remaining after a depolarization opens voltage gated Ca2+ channels) persists in the presynaptic terminal. When a subsequent AP occurs, Ca2+ levels are elevated above the concentration caused by a single AP alone, which results in a higher probability of vesicle release and a larger PSP. Synaptic depression occurs when the readily releasable pool of synaptic vesicles is depleted due to previous AP firing. The result is fewer vesicles available for release, resulting in a smaller PSP. b) Presynaptic terminal A has a higher probability of release upon single AP firing than presynaptic terminal B. Both neurons have a similarly sized pool of readily releasable SVs. Relative to each other, would you expect A or B to favor synaptic facilitation over depression? Why? I would expect B to favor synaptic facilitation over depression. A higher initial probability of release means that the terminal is more likely to deplete its readily releasable pool of synaptic vesicles. This would result in depression, rather than facilitation. c) Presynaptic terminal A has a larger readily releasable pool of SVs and fewer voltage-gated calcium channels than terminal B. Relative to each other, would you expect A or B to favor synaptic facilitation over depression? Why? I would expect that terminal A would favor facilitation over depression. A larger pool of readily releasable vesicles means that terminal A is less likely to deplete its readily releasable SV pool upon firing the first action potential. d) A compound called DM-Nitrophen releases bound Ca 2+ when exposed to UV light. An experiment is performed in which DM-Nitrophen is injected into the presynaptic terminal in the presence of low extracellular Ca 2+. A UV light is flashed at the same time as a depolarizing pulse is delivered to the presynaptic neuron while recording from the postsynaptic neuron. In comparison to a PSP measured in a control experiment without flashing UV light, would you expect the PSP to be larger or smaller when the UV light is paired with stimulation? Does this experiment provide mechanistic evidence for synaptic facilitation or for synaptic depression? I would expect a larger PSP when depolarization of the presynaptic neuron and UV light are used in combination (as compared to depolarization alone). This experiment provides evidence for synaptic facilitation because it mimics persistent elevated Ca2+ remaining from previous AP firing. Because in this experiment there is not an AP close in time to the one triggering the AP, there is no chance for depletion, and so this experiment does not mimic depression.

3 3. You are performing electrophysiology recording on a rat brain slice focusing on the Schaffer Collateral (SC) pathway. a) What are the pre- and postsynaptic cells that are involved in this pathway? Where can they be found? Presynaptic CA3 pyramidal cells and postsynaptic CA1 pyramidal cells. b) When you applied tetanic stimulations (100Hz x 5) in the presynaptic neuron, what would be the postsynaptic events that occur assuming late-ltp is achieved? Please describe the change in EPSP amplitude compared to baseline, time-span of this response, as well as the key molecules that are involved for initiating and stabilizing late LTP. Glutamate binding to the postsynaptic AMDA-receptors would allow these receptors open, which in turn depolarizes the membrane. The increase in membrane potential prompted the removal of the Mg 2+ plugs from NMDA-receptors, which would allow influx of Ca 2+ ions into the postsynaptic cell. The increase in intracellular [Ca 2+ ] (binding to calmodulin, thus activating CAMKII), as well as the binding of glutamate to mglur, would then activate the enzyme adenylyl cyclase, which hydrolyzes ATP into camp. As the concentration of camp level elevates, this second messenger would then activate protein kinase A (PKA). PKA would be translocated to the nucleus (soma) and phosphorylate camp response element-binding protein (CREB), which acts on CRE to mediate transcription and translation of plasticity related proteins. These proteins would be trafficked back to the spine via active transport at where the initial LTP occurred and stablize the LTP. The amplitude of EPSPs in this case will be maintained at much higher levels as compared to baseline for a long period of time (can be from hours to years). d) A 100-Hz stimulation is applied to another SC pathway (pathway 2), where no LTP on the same postsynaptic neuron is observed. What is this property unique to the long-term potentiation (LTP) called? What would you do, besides changing the frequency of the stimulation for pathway 2, in order to induce a LTP at this pathway? LTP is input-specific. When the stimulation of pathway 2 is administered nearly at the same time when the first LTP is initiated, then the likelihood for it to achieve LTP is much higher. This property of LTP is called associativity, an important factor for associative learning to take place during which two stimuli are required. 4. From lecture 12, you learned that LTD can also promote learning. a) What would be one example of a learning-based LTD? The NMDA-receptor-independent, cerebellar LTD for motor learning in the eye puff experiment. b) What are the main players (types of cells) for this type of LTD mentioned in a)? The parallel fibers (granule cells) with weak excitatory input, the climbing fibers with strong excitatory input, as well as a Purkinje cell, which receives both of these inputs. c) What is the coincidence detector for this LTD?

4 Protein kinase C (PKC). When intracellular Ca 2+ is low, it stays inactive. As soon as inputs from climbing fibers stimulates the opening of the voltage-gated calcium channels at the Purkinje cell, the now sufficient amount of calcium ions would allow PKC to activate kinases, which would then phosphorylate the AMPA receptors and drive internalization of these receptors. d) What would happen in the initiation of LTD, if a drug is introduced to the extracellular space, which may potentially block voltage-gated sodium channels? 6) Short and long term plasticity can occur in the same synapse. Likewise, long term potentiation and depression can also be observed in the same synapse depending on the nature of the stimulation. a) How is the mechanism of long term synaptic plasticity different from short term synaptic plasticity? Short term synaptic plasticity operates through presynaptic mechanisms, whereas long term plasticity operates through postsynaptic mechanisms (although there may be some presynaptic contributions as well). b) Hippocampal LTP and LTD are both induced by stimulating the presynaptic neuron (but at different frequencies), which results in glutamate release and activation of postsynaptic NMDA receptors. How does AMPA receptor phosphorylation contribute to the establishment of LTP versus LTD? What is the role of a calcium inducible phosphatase, particularly with respect to calcium concentrations necessary to effectively activate it? AMPA-Rs are highly mobile, and AMPA-R phosphorylation influences whether AMPA-Rs will be trafficked to synaptic or extrasynaptic sites. When AMPA receptors are phosphorylated following multiple high frequency pulses and large and fast Ca2+ influx, more AMPA-Rs are recruited to the synaptic site, resulting in LTP. Phosphatases can dephosphorylate synaptic AMPA-Rs, leading to internalization of synaptic AMPA-Rs and smaller EPSP, as occurs with LTD. The amount of phosphorylation is tied to the activity of both kinases (which add phosphate groups) and phosphatases. The amount of calcium flowing into the postsynaptic terminal can shift the balance to favor kinases or phosphatases. Phosphatases are activated with lower calcium concentrations. In conditions where Ca2+ concentrations remain low, calcium-dependent phosphatases would be activated more effectively than calcium-dependent kinases would be. This occurs with a low frequency stimulation (1 Hz for min) and favors LTD.

5 c) Calcineurin is a calcium-dependent phosphatase. Assuming that you have a drug that specifically inactivates calcineurin, design an experiment to test its role in LTD. Predict how LTD would be affected in the presence of the inhibitor. Compare EPSP amplitude before and after a 1 Hz pulse injected into the presynaptic neuron for min between neurons in the presence and absence of a calcineurin inhibitor. This stimulus should induce LTD at synapses between the two neurons in control conditions (no calcineurin inhibitor). If the magnitude of LTD (the change in EPSP amplitude before and after stimulation) is smaller in the presence of calcineurin inhibitor, this suggests that calcineurin is involved in the production of LTD. d) Would you expect calciunerin inhibition to also affect LTP? Would overexpression of calcineurin influence LTP? Why or why not? I would expect calcineurin inhibition to affect LTP. Because calcineurin only requires low levels of Ca2+ to be activated, it would also be activated when Ca2+ levels rise higher, as they would in conditions that favor LTP. This activation counteracts the activity of kinases, which phosphorylate AMPA receptors and produce LTP. Because of this, a reduction in calcineurin activity would facilitate the induction of LTP, and an overexpression of calcineurin would make LTP induction more difficult. 7) You are studying a cultured rat hippocampal neuron with three unknown receptors: A, B, and C (as shown in the figure below), activation of which all lead to the phosphorylation of CREB. You apply drugs X, Y, and Z to determine the identity of these receptors. Each drug is an agonist for its receptor and can only bind to one receptor. A MAP Kinase inhibitor was also applied in order to study the mechanism of action of each drug. Use the figure and results from the table below to determine which receptor each drug binds to.

6 Drug (+) Inhibitor/ (-) Drug (+) Inhibitor/ (+) Drug (-) Inhibitor/ (+) Drug X Y Z Table 1: Percentages of CREB phosphorylation in this neuron under different conditions. The inhibitor binds and inactivated MAP kinase completely. Experiments: A separate drug was used that inhibited MAP kinase completely in this neuron. Drug Z was A separate drug was used that inhibited MAP kinase completely in this neuron. Drug Y was A separate drug was used that inhibited MAP kinase completely in this neuron. Drug X was A control was used in which MAP kinase molecules were left intact. Drug Z was A control was used in which MAP kinase molecules were left intact. Drug Y was A control was used in which MAP kinase molecules were left intact. Drug X was Results: No change in phosphorylated levels of CREB relative to baseline (Inhibitor was applied but no drug Z was applied) A 30% increase in phosphorylated levels of (Inhibitor was applied but no drug Y was applied) A 50% increase in phosphorylated levels of (Inhibitor was applied but no drug x was applied) A 15% increase in phosphorylated levels of (neither inhibitor nor drug Z was applied) An 45% increase in phosphorylated level of (neither inhibitor nor drug Y was applied) A 50% increase in phosphorylation level of

7 (neither inhibitor nor drug X was applied) Based on the results, identify which receptors each of these drugs bind to. Drug X binds to and activates G protein-coupled receptor -Both in the case when MAP kinases was removed and left intact the phosphorylation levels of CREB increased by the same amount. Therefore, MAP kinase in not involved in the pathway Drug Z binds to and activates Receptor Tyrosine Kinase -In control we have CREB phosphorylation while in experimental we do not, because MAP kinase was removed. Drug Y binds to and opens the ionotropic Ca channels -Compared to the control, phosphorylation levels of CREB was decreased in experimental because MAP kinase is one component of pathway. If MAP kinase is unblocked you have two pathways that phosphorylate CREB. When blocked you only have one pathway that phosphorylates and that is why we see diminished levels of phosphorylation in the experimental vs the control.