Name KEY - University of California, San Diegoclasses.biology.ucsd.edu/bipn140.FA16/2015 BIPN140 Final Exam Ke… · Yes. 2. (10 points) During development of the nervous system,

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Cellular Neurobiology Biology 140 Fall 2015

FINAL EXAMINATION

GENERAL INSTRUCTIONS: 1. Please write your name on ALL pages. 2. Please answer the question in the SPACE ALLOTTED. 3. For full credit, state assumptions and show calculations. 1) __________/ 10 pts Rodolfo 2) __________/ 10 pts Rodolfo 3) __________/ 30 pts Eric 4) __________/ 15 pts Rodolfo 5) __________/ 20 pts Eric 6) __________/ 15 pts Huanqiu 7)__________/ 14 pts Huanqiu 8)__________/ 16 pts Varoth 9) __________/ 20 pts Varoth 10) _________/ 20 pts Huanqiu 11) _________/ 10 pts Varoth 12) _________/ 20 pts Varoth BONUS: 1) __________/ 10 pts Varoth BONUS: 2) __________/ 10 pts Rodolfo TOTAL ___________/200 pts Bonus Total __________/20 pts

COURSE GRADE ____________ 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 that 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. Name Date  Histograms of grades for the final exam and for the entire course are at the end, with grade cutoffs for the course.  

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1. (10 points) A. In recent years, a new form of brain plasticity has been recognized: activity-dependent neurotransmitter switching. When the presynaptic neuron switches the identity of the neurotransmitter that it expresses, what happens to the transmitter receptors that the postsynaptic neurons express? Why is this important? (4 points)

When the presynaptic transmitter switches, the postsynaptic receptors switch to match the transmitter. If the receptor population did not change to match the newly expressed transmitter the synapse would not function. B. What are Reserve Pool Neurons? (4 points)

Reserve Pool neurons are neurons that are already wired into circuits, express one or more transmitters and have functions, but can acquire a different transmitter and acquire another function. C. Can activity-dependent neurotransmitter switching take place in both the developing and the mature nervous system? (2 points)

Yes. 2. (10 points) During development of the nervous system, it is crucial for the commissural axons to cross and reach the other side of the midline. Below are images showing the tissue around the midline of a normal Drosophila larva (animal A) and 2 mutants (animals B and C). Axons are stained in black and the midline is labeled with a dotted line. In the normal animal, axons can cross the midline properly. The two mutants exhibit defects caused by mutations in receptors involved in guiding axons to cross the midline. Choose the most likely receptor mutation that would cause the defects shown in animals B and C. Then explain why that mutation would give rise to that defect in the mutant. In your explanation, remember to mention the ligand that binds to that receptor. (modified from Kidd, Bland and Goodman, Cell, 1999) A. Animal B has a mutation in (5 points): The DCC receptor

Animal A (Normal, wild-type)

Animal B Animal C

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Netrin binds to DCC receptor and attracts commissural axons to the midline. So if DCC receptors are mutated, then the axons will no longer be drawn to the midline. The explains why there is no axon crossing the midline in animal B. B. Animal C has a mutation in (5 points): The Robo receptor Slit binds to Robo receptor and repels the commissural axon from the midline. So if Robo receptors are mutated but the netrin signaling is still intact, then axons will remain trapped in the midline, as shown in animal C. 3. (30 points) Neurulation in the mammalian embryo. In the paragraph below, fill in the blanks with proper terms selected from the following table. Each blank in the paragraph corresponds to the number in the neurulation diagram provided below.

 BONE  

MORPHOGENETIC  PROTEIN  (BMP)  

EMBRYONIC  STEM  CELLS   GASTRULATION   NEUREGULIN  

NEURAL  INDUCTION   NEURAL  TUBE   NEUROBLASTS   NEUROECTODERM  

SONIC  HEDGEHOG  (SHH)   BLASTULA   NOGGIN/CHORDIN   SOMITES  

MUSCLE  FIBERS  

NEURAL  GROOVE   NOTOCHORD   NEURAL  CREST  

The germ layer that lies immediately above the …..[1]….., called ectoderm, gives rise to the entire nervous system. The signal from …..[1]….. causes the overlying ectoderm to become .....[2]…... This process is called …..[3]….. . When the ectodermal cells are exposed to …..[4]….., they assume an epidermal fate, forming structures associated with the skin. The binding of …..[4]….. to receptor serine kinase is blocked in the presence of endogenous antagonists, …..[5]....., causing the ectodermal cells to assume a neural fate. As neurulation proceeds, the neural plate

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begins to fold inward creating …..[6]….. . Once the edges of the neural plate meet in the midline, the formation of …..[7]….. is complete. On the dorsal side of the …..[7]….., another population of precursor cells forms a region called …..[8]….. . The mesoderm adjacent to the …..[7]..... thickens and subdivides into structures called …..[9]….. . The cells in …..[7]….. are capable of self-renewal and possess the capacity to give rise to the full range of cell classes found in mature nervous system. Asymmetrical division of neural stem cells gives rise to post-mitotic cells, called …..[10]….. which will develop into neurons. (2 points each) 1. Notochord 2. Neuroectoderm 3. Neural induction 4. BMPs 5. Noggin/Chordin 6. Neural groove 7. Neural tube 8. Neural crest 9. Somites 10. Neuroblasts 4. (15 points) Synaptogenesis involves multiple sequential steps and intracellular events. A. What is the function of Agrin and what is its receptor during neuromuscular junction development? (6 points) Agrin is released from the motor neuron axon terminal and binds to MuSK receptor tyrosine kinase on the muscle fiber to recruit Rapsyn, an AChR interacting protein. This interaction ultimately leads to AChR clustering in the muscle fiber around the innervating motor neuron axon terminal. B. During early development, each muscle fiber is innervated by multiple motor neurons. However, individual mature muscle fiber is innervated by only one motor neuron. Please provide a brief description of the fate of the motor neurons that retract from their original innervation sites during development. (3 points) The motor neurons that retract from their original innervation sites do not die. Instead, they maintain innervation to other target muscle fibers at which they are the dominant motor neuron. C. Multiple signaling molecules are involved in the development of central synapses. Let’s say that HEK293 cells (a cell line derived from human embryonic kidney cells) are transfected with ephrinB so that ephrinB is abundantly expressed on their cell surfaces. What effects will be seen on the dissociated developing glutamatergic neurons that are co-cultured with the transfected HEK293 cells? (6 points) Upon binding to ephrin B, EphB receptors expressed in the glutamatergic neurons will be activated, leading to EphB aggregation, NMDARs clustering, and formation of postsynaptic density. As a result, you will be able to observe more mature dendritic spines in the neuronal culture.

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5. (20 points) Please answer the following questions about human memory and Alzheimer’s disease. A. Group the following examples of memory into either declarative or non-declarative memory. (6 points) i. Remembering how to swim even though you haven’t swum for many years ii. Remembering that the school shuttle arrives at your bus stop at 9am iii. Writing a post on Facebook about your past Thanksgiving dinner iv. Remembering the meaning of the word “neurobiology” v. Remembering how to solve the rubik’s cube vi. Remembering the function of kinases Declarative: ii, iii, iv, vi Non-declarative: i, v B. Studies on patients with traumatic brain injuries in the medial temporal lobe brought critical insights into the formation and storage of memory. In particular, studies on the amnesic patient H.M. revealed that different categories of memory (e.g. working memory, declarative memory, non-declarative memory) are likely mediated by different brain regions. Please provide a brief explanation of how this conclusion is reached. (5 points) H.M. had bilateral lobotomy of the medial temporal lobe and could no longer form new declarative memory. However, he could still learn to perform a new motor skill (mirror tracing), indicating that his working memory (remember the rule of the task) and non-declarative memory (or procedural memory) were not affected by the loss of medial temporal lobe. These observations support the conclusion that the medial temporal lobe is important for the formation of declarative memory but dispensable for working and procedural memory. C. One of the initial problems in the brains of Alzheimer patients is the decline in cholinergic receptors. Which of the following drugs is commonly used to treat this problem? Explain why that drug would be effective. (5 points) i. Acetylcholinesterase inhibitors ii. Glutaminase inhibitors iii. Choline acetyltransferase (ChAT) inhibitors iv. Serine hydroxymethyltransferase inhibitors Inhibiting acetylcholinesterase would slow down the breakdown of acetylcholine in the synaptic cleft. This can compensate for the reduction in the number of cholinergic receptors in the brain of Alzheimer patients. D. What is an intracellular pathological evidence of the Alzheimer’s disease? (2 points) Neurofibrillary tangles. E. What is an extracellular pathological evidence of the Alzheimer’s disease? (2 points) Deposits of amyloid / Aβ-amyloid peptide/ forming Aβ-amyloid plague.

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6. (15 points) Please answer the following questions about olfaction. A. Describe the molecular mechanism of olfactory transduction in mammals, starting from the activation of the odorant receptor. (10 points) Activation of an odorant receptor by odorant Activation Golf Activation of adenyl cyclase III (ACIII) causes increase of cAMP level cAMP opens cAMP-gated nonselctive cation channel (CNG channel) Na+ and Ca2+ influx through the nonselctive cation channel causes depolarization Ca2+ opens a calcium-gated chloride channel, Anoctamin 2 (Ano2) Cl- efflux to further depolarize olfactory receptor neuron B. Opening of a chloride channel usually hyperpolarizes a neuron. Why does the opening of chloride channels cause depolarization in mammalian olfactory receptor neurons? Please explain your reasoning. (5 points) Mammalian olfactory receptor neurons express a sodium potassium chloride exchanger/ Na+ K+ 2Cl- (NKCC1) that elevates the intracellular chloride concentration. This makes the reversal potential of the chloride channel higher than the resting potential of the neuron. Therefore, opening the chloride channel will cause depolarization instead of hyperpolarization. 7. (14 points) G-protein signaling is responsible for diverse cellular events in response to neurotransmitter release. A. As opposed to activation of an ionotropic receptor, please describe two major functional differences when a metabotropic receptor is activated. (4 points) Signal amplification Allowing longer-lasting responses and integration of multiple signals B. GTPγS is a non-hydrolyzable GTP analog that prevents activated G protein from being inactivated. Among the key molecules (GEFs and GAPs) that regulate G protein signaling, which type of molecule will no longer be functionally meaningful when GTPγS is applied intracellularly? Please explain your reasoning. (5 points) GAPs (GTPase Activating Proteins) will become functionally meaningless. Even though they could still activate the GTPase activity of a G protein, the GTPase can no longer hydrolyze GTPγS associated with the G protein to GDP to terminate the response. C. If an activated metabotropic receptor couples to Gi to regulate protein kinase A (PKA) activity, please describe the most likely effect of intracellular GTPγS application on PKA activity. Please provide a brief description of the signaling pathway and explain your reasoning. (5 points) PKA activity will likely decline. Gi inhibits adenylyl cyclase => lowering intracellular cAMP level => reducing PKA activity because PKA activation (or disinhibition) requires cAMP.

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8. (16 points) You find that the following ion channels are present in the neurons you study. Channel A: a chloride-selective channel Channel B: a non-selective cation channel that is permeable to Na+ and K+

Voltage clamping while activating each type of channel reveals properties of each channel in the following I-V plot. (Vrest – resting potential; EK, ECl, ENa – reversal potential of K+, Cl-, Na+ respectively.

Fill in the blanks below to describe the effects that are generated by activating each of these channels. The action potential threshold of this neuron is -40 mV.

Channel Reversal Potential

(mV)

Excitatory or inhibitory

postsynaptic potential?

Inward or outward current?

Depolarizing or hyperpolarizing

postsynaptic potential?

A -58

Inhibitory PSP

Inward current

Depolarizing

PSP

B 0

Excitatory PSP

Inward current

Depolarizing

PSP 9. (20 points) Please answer the following questions about long-term synaptic plasticity. A. When you apply a tetanic stimulus to the Schaffer collateral, which type of hippocampal neurons should you record from in order to measure the postsynaptic response? (4 points) CA1 pyramidal neurons. B. What is the glutamate receptor that is critical for the induction of LTP? Based on its conductance and activation requirement, what are the key functional features that

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allow this type of glutamate receptor to mediate LTP? Please explain your reasoning based on these two functional features. (16 points) NMDA receptor; it is permeable to Ca2+ and activation of NMDA receptor requires sufficient depolarization to remove Mg2+ block. Transient and substantial Ca2+ influx leads to activation of various kinases that eventually leads to more AMPA-R trafficking to the postsynaptic sites to strengthen the synapse (LTP). The requirement of depolarization explains why LTP will only occur when high-frequency stimuli or coincidental stimuli are delivered presynaptically.

10. (20 points) Passive membrane properties are always a challenge for students in BIPN140. A. Explain why the actual curve deviates from the theoretical straight line when the [K+]o concentration is reduced in the following graph. (4 points)

Neuroscience, 4th Edition, Fig 2.7 At low [K+]o, other ions proportionally make a larger contribution to the overall Vm (according to the GHK equation, whereas the theoretical curve is derived from the Nernest equation), which is the reason why the Vm deviates towards the inside-positive direction.

B. How would you manipulate the ionic environment of the cell culture medium to correct this deviation? (2 points) Remove Na+ ions from the cell culture medium (and any other ions besides K+). C. A squid axon membrane has the following properties: radius = 200 µm, Vrest = ‐65 mV, Vthreshold = -30 mV, rm = 5*104 ohm*cm, Cm=10‐6 F/cm2, Ri = 100 ohm*cm, and the AP amplitude is 50 mV. In order to make an AP propagating along the axonal membrane unable to regenerate, Hodgkin decided to use a cold block over a certain length of the membrane, which inactivated the voltage-gated ion channels along this length. Assuming that the cold block does not alter other passive membrane properties, how long should the cold block be in order to make the AP unable to regenerate on the opposite side of the cold block? (14 points)

Vx = V0*e‐x/λ

Vx = -30-(-65) = 35 mV V0 = 50 mV-(-65) = 115 mV ri = Ri/πa2 = 100 ohm*cm /π/(0.2cm)2 = 795.8 ohm/cm λ =(rm/ri)0.5=(5*104ohm*cm/795.8 ohm/cm)0.5 = 7.93 cm 35 = 115 *e‐x/7.93cm = 9.43 cm.

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Therefore the cold block should be at least 9.43cm long. 11. (10 points) A. In the vertebrate i) central nervous system and ii) peripheral nervous system, which type of glial cell is responsible for the myelination of axons? (2 points) CNS: Oligodendrocytes. PNS: Schwann cells. B. How does myelination affect the membrane resistance, the membrane capacitance, and the length constant of a neuron? (2 points) Myelination increases membrane resistance, decreases membrane capacitance, and increases the length constant. C. The dendrite of neuron A has a longer length constant than the dendrite of neuron B. Will passive current flow along the dendrite diminish more in neuron A or B? (2 points) Neuron B. D. The following three axons are of the same length. Axons A and C are of the same thickness. Rank the length constant of these axons in descending order of magnitude, assuming the properties of the axonal membrane are the same in all three cases. (2 points)

Axon A> Axon C > Axon B

E. A depolarizing current is injected into the left end of each of the above three axons and current is propagated passively to the right end of the axons. Rank the magnitude of the current detected at the right end of the axons in ascending order. (2 points)

Axon B < Axon C < Axon A 12. (20 points) You are studying an isolated neuron in a dish, outside of a living organism. In this setting, you have a complete control over the local ionic environment because you can control the makeup of the solution in the dish. Normally, the neurons would be bathed in artificial cerebrospinal fluid (ACSF), a bicarbonate buffered salt solution mimicking the composition of the cerebrospinal fluid but you are wondering what would happen if you alter some of the ionic concentrations.

The table below lists the ionic concentrations of ACSF. The ‘resting permeability’ column lists the permeability of each ion at resting potential when the neuron is bathed in ACSF. The ‘intracellular’ column lists the ionic concentrations inside the neuron. All concentrations are in millimolar (mM).

Axon Myelin

Axon A

Axon

Axon C

Axon

Axon B

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ION RESTING PERMEABILITY

INTRACELLULAR (MM)

ACSF (MM)

K+ 100 140 5 NA+ 4 10 150 CL- 10 12 120

A. Based on this chart, what are the Nernst potentials for K+, Na+, Cl- and Ca2+ if the neuron is bathed in ACSF at 20°C? (9 points) According to the Nernst equation, Eion = , therefore EK = 58log(5/140) = -84 mV ENa = 58log(150/10)= +68 mV ECl = -58log(120/12)= -58 mV B. What would be the resting potential of this neuron when the cell is bathed in ACSF at 20°C? (Assume that Ca2+ permeability is negligible.) (7 points) According to the GHK equation, Vm = Therefore, Vm = -64 mV C. When the neuron is bathed in ACSF at 20°C, which ion experiences the smallest driving force if we apply a voltage of -80 mV across the membrane? What direction would the net flux be for that ion? (2 points) K+; intracellular => extracellular (or K+ outward current) D. When the neuron is bathed in ACSF at 20°C, which ion experiences the largest driving force if we apply a voltage of -80 mV across the membrane? What direction would the net flux be for that ion? (2 points) Na+; extracellular => intracellular (or Na+ inward current) BONUS 1. (10 points EXTRA CREDIT) From the paper on non-synaptic inhibition between compartmentalized olfactory receptor neurons: A. (4 points) Please compare and contrast the immediate anatomical environments in which insect and mammalian olfactory receptor neurons (ORNs) reside. Insect ORNs are compartmentalized in sensory hairs (sensilla), whereas mammalian ORNs are distributed in the olfactory epithelium. B. (6 points) The authors discovered that transient activation of one ORN inhibits the chronic activity of its neighbor. Please describe the experiments that allowed the authors to conclude that activation of one ORN is both necessary and sufficient to inhibit the chronic response of its neighbor. The authors first presented a background odorant to chronically activate one ORN. Subsequently, a pulse of second odorant was delivered to transiently activate the second ORN in the same sensory hair. The authors observed that transient activation of the second ORN inhibits the chronic response of the first ORN. Necessity: genetic ablation of the second ORN abolished the inhibition of the first ORN.

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Sufficiency: Instead of using odorant to activate the second ORN, the authors activated the second ORN expressing ChR2 directly with blue light. This activation also leads to inhibition of the first, chronically activated ORN. BONUS 2. (10 points EXTRA CREDIT) The paper by Yacubova and Komuro investigated the role of ligands and calcium signaling in neuronal migration. A. (2 points) What cells did the authors study, and in what experimental preparation? The authors studied migration of cerebellar granule neurons in slices and in culture B. (4 points) What ligand regulates cell migration, and what were its effects? Endogenous somatostatin regulates this migration. It accelerates migration at the onset of migration and slows migration at the termination of migration. C. (4 points) What was the effect of this ligand on calcium signaling, and what experiment did they perform to link calcium signaling to migration? Somatostatin increases the frequency of calcium transients at the onset of migration and decreases the frequency of calcium transients at the termination of migration. When calcium transients were eliminated by addition of a calcium chelator (BAPTA), the effects of somatostatin on migration were abolished.

The End Grade cutoffs >350 A+; >334 A; >325 A-; >304 B+; >282 B; >260 B-; >239 C+; >217 C; >195 C-; >130 D; <130 F

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