Cellular Neurobiology / BIPN 140

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1 SECOND MIDTERM EXAMINATION Fall, 2015 GENERAL INSTRUCTIONS 1. Please write your name on ALL 6 pages. 2. Please answer each question IN THE SPACE ALLOTTED. 1) /10 pts 2) /10 pts 3) /15 pts 4) /15 pts 5) /15 pts 6) /15 pts 7) /20 pts TOTAL /100 pts 8) /10 pts EXTRA CREDIT WAIVER: By signing this waiver I give permission for this exam to be left for me to pick up in the vestibule by the elevator on the 3rd floor of Pacific Hall. I understand that I may only pick up my own exam. I realize that the Division of Biological Sciences and its staff cannot take responsibility for exams, which may be stolen or lost, from this area. If I choose not to sign this waiver, I acknowledge that my exam will only be available for pickup 10:00 am-4:00 pm, Monday-Friday from the Exam Depot Window outside the north entrance to Pacific Hall. 1. (10 points) You have successfully isolated a pair of neurons and placed them on a culture dish filled with artificial cerebrospinal fluid. One of the neurons synapses onto the other one through an excitatory chemical synapse. Describe and explain what would happen to the amplitudes of A) excitatory postsynaptic potential (EPSP) and B) miniepsp if you significantly lower the calcium concentration in the artificial cerebrospinal fluid surrounding the presynaptic terminal. (A) The amplitude of EPSP would decrease. That is because lowering the extracellular calcium concentration will reduce the calcium current entering the presynaptic terminal upon opening of the 1

2 voltage-gated calcium channels. That would reduce the number of synaptic vesicles released, leading to a smaller EPSP. (4 points) (B) The amplitude of miniepsp would not change. That is because the density of postsynaptic neurotransmitter receptors is likely unchanged. (2 points) C) Later on, you found that the presynaptic neurons you isolated express a mutated form of voltage-gated calcium channel with a much reduced calcium conductance. What kind of short-term plasticity would most likely be induced by a paired-pulse stimulus? Please explain your reasoning. Short-term potentiation is most likely to occur. This is due to less calcium entering the cell during the stimulation, thus allowing calcium to accumulate over stimuli to favor paired pulse facilitation. (4 points) 2. (10 points) A group of scientists are testing the effects of four different drugs on different inotropic receptors that are present within the membrane of ventral tegmental neurons. They hope to find the right drug that targets an ionotropic receptor to yield inhibition (IPSP) in order to treat epileptic seizures originating from those neurons. They obtained the following data: Receptor Type A B C D Drug-evoked postsynaptic potential (PSP) + 2 mv - 1 mv + 4 mv - 4 mv A) Of the drugs that target the four receptor types, which ones are certain to be effective in treating epileptic seizures based on this data alone? Activation of B and D receptors would definitely lead to inhibition, because a hyperpolarizing PSP is always inhibitory. Conclusions cannot be made on whether the depolarizing currents are inhibitory or excitatory based on this data alone. (5 points) B) Upon doing further experiments, they discovered that the conductance for K + is relatively high for receptor A, and very low for receptor C. Based on this information, activation of which receptor type would be more likely neural protective in the event of an epileptic seizure? Explain your reasoning. Activation of receptor A would more likely be protective, because a relatively high conductance to K+ means that the reversal potential is more likely below AP threshold, thus the activation of this receptor would most likely lead to IPSCs (despite the depolarizing current). (5 points) 2

3 3. (15 points) You have been given a brand new project to study motor neurons and the neuromuscular junction: A) Which of the antibodies can be used to identify and differentiate cholinergic neurons from other classes of neurons? Choose all options that apply and explain your reasoning. Anti-VGLUT (vesicular glutamate transporter) Anti-ChAT (choline acetyltransferase) Anti-GAD (glutamic acid decarboxylase) Anti-VAChT (vesicular acetylcholine transporter) Anti-acetylcholine esterase Anti-glutaminase Anti-ChAT and anti-vacht. Choline acetyltransferase is an enzyme responsible for the synthesis of acetylcholine by catalyzing the transfer of acetyl group from acetyl-coa to choline. VAChT is a transporter protein responsible for loading acetylcholine into synaptic vesicles. Therefore, either ChAT or VAChT can be used as a selective marker to define a cholinergic neuron. (5 pts) B) By reading some recent literatures, you find that cholinergic neurons may also release GABA together with acetylcholine. Which other antibodies from the list should also label the neurons? Explain your reasoning. Anti-glutaminase and anti-gad. Glutamic acid decarboxylase is an enzyme responsible for the synthesis of GABA from glutamate, which is synthesized by glutaminase from glutamine. Both antibodies will give positive staining of GABAergic neurons. (5 pts) C) You apply curare, a nicotinic acetylcholine receptor (nachr) antagonist to your prep. What would you observe when recording postsynaptic potential regarding to evoked EPP amplitude? Explain your reasoning. Curare is a competitive inhibitor of acetylcholine, preventing the binding of acetylcholine to the nicotinic acetylcholine receptor, thus reducing the number of nachr s channels that can be opened when acetylcholine is released from presynaptic neuron. As a result, the amplitude of postsynaptic potentials is decreased. (5 pts) 4. (15 points) You manage to isolate a pair of synaptically connected neurons from a mouse brain, one being a presynaptic glutamatergic neuron and the other is its postsynaptic partner. A) Describe how you would determine which type of ionotropic glutamate receptors is present in the postsynaptic neuron by performing intracellular recording, assuming that kainate receptor is not present in the postsynaptic neuron. The best way is to observe EPSCs at different command voltages in the presence of each receptor s antagonist. If the AMPAR is present, clamping the postsynaptic neuron at its resting potential while stimulation the presynaptic neuron would give you a sharp transient EPSC solely mediated by AMPAR because NMDAR passes cations only when the postsynaptic membrane is depolarized. At resting potential, AMPAR antagonist (e.g. CNQX) will completely abolish EPSC whereas NMDAR antagonist (e.g. APV) will not have any effect. (5 points) If NMDAR is also present, clamping the postsynaptic neuron at a more depolarized membrane potential would give you an EPSC mediated by both AMPAR and NMDAR. Applying AMPAR antagonist 3

4 will block the AMPAR-mediated current, allowing you to observe only NMDAR-mediated current which is characterized by a slower decay. Correct drawings of currents at different voltage commends are also accepted as answers. (5 points) B) What is a silent synapse? A silent synapse is one which does not yield a detectable postsynaptic response when stimulated under resting membrane potential. These are glutamatergic and result from having NMDA receptors but no AMPA receptors. Therefore, at resting membrane potential, glutamate release from the presynaptic terminal cannot elicit a PSP: the NMDA receptors remain blocked Mg2+ at negative potentials, and no AMPA receptors are present to generate a postsynaptic current or depolarization. (5 points) 5. (15 points) On a neuronal dendrite at the CA1 region of the hippocampus, three spines are located spatially close to each other, as shown in the graph. Here is your previous experimental result: when you give a strong tetanus to path 2 along with a weak tetanus to path 3, both synapses undergo LTP. A) Which LTP property does it demonstrate? Why would the synapse receiving weak tetanus from path 3 be strengthened? This is called associativity, which is based on the temporal and spatial summation of the EPSPs elicited from both path 2 and path 3. As a result, the summed EPSP is large enough to depolarize the postsynaptic neuron and remove the Mg 2+ block of NMDA receptor at synapse 3, allowing LTP to occur. (5 points) B) After several trains of strong tetanus on path 2, you could no longer observe further increase in EPSP magnitude. What LTP property does it demonstrate? LTP has reached saturation. (2 points) C) Despite the fact that further LTP cannot be induced at synapse 2, a temporary increase in the magnitude of EPSP can still be observed when a strong tetanus is given to Schaffer Collateral axon 2 that could last for up to several minutes. What is most likely causing the increase in the magnitude of EPSP here? Explain your reasoning. Post-tetanic potentiation, which is due to the increase in the size of readily releasable SV pool and release probability. (3 points) 4

5 D) Afterwards, you decided to give a 15-min long, low-frequency (e.g. 1 Hz) stimulus on path 2 and found that the magnitude of EPSP returned to baseline. What property of long-term plasticity does it demonstrate? What could be the most likely mechanism underlying the reduction in EPSP amplitude? LTP/LTD can be bidirectionally regulated. (2 points) LTD is triggered by the prolonged low frequency stimulus. NMDA-Rs allows a slow rise in intracellular Ca2+ and activation of phosphatases to promote internalization of AMPA-Rs. (3 points) 6. (15 points) Scientists have shown that late-phase LTP requires protein synthesis. A) Summarize the molecular pathway for late-phase LTP. Repetitive strong tetani => Ca2+ influx through NMDA receptors => Ca2+ and calmodulin activates adenylyl cyclase => increase in camp => activation of PKA => CREB phosphorylation (activation) => transcription/translation of new genes. (10 points) B) Describe the mechanism underlying specificity of late-phase LTP. That is, how does the neuron know which synapse is the one to deliver its newly synthesized protein products? Local tag is produced at the specific synapses that were activated during tetanus. Newly synthesized proteins are then only captured by tagged synapses. (2 points) C) By looking at the experimental results given in the graph, do you think the process of making local synaptic tags requires protein synthesis? (Red line on the top indicates application of protein synthesis inhibitor. S1 and S2 are two synapses on the same neuron. The three arrows on the bottom left indicate strong tetanus stimulation applied to S1, and the three arrows on the bottom right indicate strong tetanus stimulation applied to S2). Making local synaptic tags does not require protein synthesis, as S2 is able to hijack the new protein products (shown by the LTP time course, which is more than 3 hours, indicative of a latephase LTP) even though it is activated when protein synthesis is blocked in the neuron. (3 points) 5

6 7. (20 points) There are two major types of kinases in neurons: Tyr kinases and Ser/Thr kinases. A) What is the general function of kinases? Kinases phosphorylate proteins (or phosphorylation). (2 points) B) Give two examples of Ser/Thr kinase commonly found in neurons. PKA, CaMKII, PKC, PKG, MAPK, MAPKK etc. (4 points) C) Give an example of a receptor tyrosine kinase commonly found in neurons TrkA, TrkB, ErbB, EphB (Eph receptors) etc. (2 points) D) Compare and contrast the general mechanisms and immediate cellular events of activating receptor tyrosine kinases vs Ser/Thr kinases. Upon ligand binding, receptor tyrosine kinases would dimerize and phosphorylate its partner (auto-phosphorylation). Activated receptor tyrosine kinases will recruit other signaling proteins or scaffolding proteins to activate other intracellular signaling pathways. (6 points) In contrast, Ser/Thr kinases can be activated by second messengers (Ca2+, camp, cgmp etc) through disinhibition (ligand binds to the inhibitory subunit, thereby exposing the catalytic subunits) or by phosphorylation by other kinases. Activated S/T kinases will phosphorylate other substrates to propagate the signaling cascade leading to a cellular response. (6 points) EXTRA CREDIT BONUS QUESTION (10 points). 8. In the paper, Engineering a memory with LTD and LTP, Nabavi et al. provided invaluable experimental evidence supporting a causal link between these forms of long-term synaptic plasticity and memory. A) Instead of using a tone, what was the technique that they used to stimulate the auditory cortex? Optogenetics. They expressed channelrhodopsin2 (or a light-activated cation channel) in the neurons of the auditory cortex so that they can be activated by light. (3 points) B) How did they verify that their experimental protocol can trigger LTD? They used in vivo extracellular field recording in the rats. They performed the experimental optogenetic protocol intended to trigger LTD, then observed that EPSP responses remained significantly reduced for a long time afterwards (LTD). (5 points) C) In order to trigger fear in the rats, they stimulated neurons in the auditory cortex that project to which region of the brain? Amygdala or lateral amygdala. (2 points) -THE END- 6

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