Cellular Neurobiology BIPN 140 Fall 2016 Problem Set #1

Transcription

1 Cellular Neurobiology BIPN 140 Fall 2016 Problem Set #1 1. (Antonia) You are studying a neuron with an arterial cuff under a microscope. However, you knock over a bunch of chemicals onto the plate because of your coffee jitters (you ve been caffeinating all night to keep up with BIPN 140 reading). Anxiously, you look at the neuron again and see a bulge forming on one side of the cuff, as shown in the figure below: a. Would you normally expect the bulge forming on only one side of the arterial cuff? Why? i. No, there should be build-up forming on both sides of the cuff in a normal neuron. The formation of the build-up on both sides is due to orthograde and retrograde axoplasmic transport. b. What type of axoplasmic transport can still be observed? i. Orthograde transport can still be observed, since there is build-up forming on the side of the cell body. Orthograde transport moves materials away from the cell body (towards to synapse). c. At what rate can the functioning axoplasmic transport propagate? i. Orthograde transport can propagate at either a fast or a slow rate. Slow rate moves materials 1 mm/day. Fast rate moves materials 400 mm/day. d. Which motor protein has most likely been damaged by the chemicals? i. Dynein has most likely been damaged, since retrograde transport cannot be observed. There is no build-up of materials on the side of the synapse. 2. (Stephanie) For each of the following questions, decide which technique would be more suitable and provide a brief explanation. a. To compare the density of hippocampal neurons between a wild-type and Alzheimer s disease mouse model (Nissl vs. Golgi staining). i. Nissl staining is more appropriate for this application because it binds to nucleic acids, labeling (ideally) the cell body of all cells. As neuronal processes are not labeled with this technique, the density of staining correlates with cell density. Golgi staining sparsely labels both cell bodies and neuronal processes, so not only would not all neurons be labeled, but it would be difficult to distinguish changes in cell density from morphological changes. b. To identify brain regions which project their axons to the substantia nigra (anterograde versus retrograde tracer). 1

2 . Because the target structure (i.e. substantia nigra) is known, the easiest method would be a retrograde tracer, which are taken up by axons and transported to the cell body. An anterograde tracer would be appropriate if the question were instead to identify the targets to which the substantia nigra projects. c. To study dopaminergic signaling during a reward task in humans (PET vs. fmri).. Although fmri is generally preferable for many measurements due to its safety compared to PET, it is not ideal for probing signaling associated with a specific neurotransmitter because it measures activity through the proxy of blood flow (BOLD signal). With injection of a radiolabeled dopamine precursor, PET can specifically measure dopamine signaling. d. To confirm that a knockout mouse model does not express the targeted protein (immunohistochemistry [antibody labeling] versus a reporter gene [GFP expression regulated by the promoter of the targeted protein]).. Immunohistochemistry is preferable. Because expression of the reporter gene requires only that transcriptional regulation of the target protein is not impaired, it is possible that GFP labeling would be identical regardless of whether a functional protein is being expressed. Immunohistochemistry labels the protein itself, so assuming the antibody is specific to only the targeted protein, a loss of signal after labeling would confirm that the protein of interest was knocked out successfully. 3) (Milad) Below are three figures showing an action potential profile measured using the recording methods discussed in class. Based on the depolarization/repolarization schemes determine which recording method was used: intracellular, extracellular, or neither. The +/- on the y-axis describe the voltage measured by the recording electrode relative to the reference electrode, while the x-axis is a measure of time. The reference electrode is placed at a reasonable distance away from the recording electrode. (For practice, justify each answer to see if the recording methods actually make sense to you). A-This figure represents an intracellular recording of an action potential B,C- Both of these figures represent an extracellular recording of an action potential With intracellular recordings a recording electrode is poked into the cell so that it is bathed in the intracellular fluid (ICF), while the reference electrode is placed along the axon in the extracellular fluid (ECF). Relative to the reference electrode, the recording electrode measures a negative voltage because the cell is inside-negative compared to the ECF. For this reason, 2

3 the recording should start at a negative voltage. When the cell is stimulated to fire an action potential it becomes depolarized due to the influx of Na, causing the recording electrode to measure a more positive membrane voltage relative to the reference electrode. This is why we get a positive deflection. When the cell repolarizes due to efflux of K + the recording electrode will measure a more negative membrane voltage relative to the reference electrode. That is why we get a negative deflection. With extracellular recordings both the recording and reference electrodes are placed in the ECF, separated by some distance so that the reference electrode will not measure the AP. Due to the fact the both of the electrodes are in the same environment, i.e. both in ECF, they measure the same voltage. Therefore the difference in voltage between the two is 0 and our recording should start at zero. When the cell is stimulated to fire an action potential there will be Na + influx and the solution around the recording electrode will become less positive relative to the reference electrode. That is why the recording will initially be negative. As K + begins to efflux from the cell, the solution around the recording electrode becomes more positive relative to the reference electrode. That is why the recording becomes positive. Figures B and C are both showing extracellular recordings, just with the y-axis flipped. 4) (Antonia) Fill out the missing blanks: Name Location (CNS/PNS) Function Functional fact Schwann cells PNS Production of myelin around the axon of a neuron. Oligodendrocytes CNS Involved in the production of myelin around axons. Radial glial cells CNS They produce the substrate for neuronal migration. Microglia CNS Remove debris and waste in the brain. Astrocytes CNS They are involved in: buffering extracellular potassium concentrations, inactivation of neurotransmitters (help in the take up), release of neurotransmitters, formation of the blood brain Myelin helps propagate the action potentials at a higher speed. Multiple sclerosis is an immune disease that attacks the myelin sheets. Neuronal migration is important for the formation of connections and differentiation of cells into either neurons or astrocytes. These cells act as immune responders to brain injuries and trauma. Very dynamic. The most numerous cells in the brain. 3

4 barrier by forming a sheath around capillaries Ependymal cells CNS Involved in the production of the cerebrospinal fluid. Cerebrospinal fluid provides proteins and nutrients to the brain and the spinal cord. 5) (Milad) You are studying the concept of frequency coding in the lab by injecting a strong stimulus into the axon of a neuron. You take three measurements: the control, a case when a drug (Drug X) is applied, and the case when the drug is washed off. Provided below are action potential profiles showing how the Vm is influenced in the three scenarios. Furthermore, the frequencies of action potentials were also provided for these three scenarios. You are using an intracellular recording to make your measurements, and Drug X completely blocks the pore of the type(s) of channel(s) it binds to. Also, you are using a 500 Hz frequency train of suprathreshold stimuli (threshold of neuron is always reached with these stimuli). The absolute refractory period is 2 msec while the relative refractory period is 4 msec for the untreated neuron. a. Why is Drug X causing this effect on the action potential and how does this effect decrease the frequency of action potentials? b. Why don't the data from the wash match those of the control? c. You are given a list of 4 drugs that could rectify the effects of drug X. Which of these drugs will ameliorate the effects of Drug X and which will not? Drug A Effect Increases the translation and transport of voltage-gated Na+ channels to the membrane. 4

5 B C D Increases the translation and transport of voltage-gated K+ channels to the membrane. Increases the conductance of the unaffected channels the drug X normally binds to. Decreases the number of K+ leak channels. A) Drug X is a voltage-gated K+ channel inhibitor that acts in a very similar manner to Tetramethyl ammonium ions (TEA+; it comes as the chloride salt). These data show that Drug X blocks some of the channels and decreases conductance of the neuron to K+; as a result less K+ is able to flow out of the cell and it takes the cell longer to repolarize, as the dashed line indicates in the figure above. The cell does not repolarize as much as the control simply due to the fact that K+ conductance is decreased. If the drug has blocked all of the voltage-gated K+ channels then repolarization would depend on the Na/K ATPase and that would take a much longer time. The frequency of action potentials decreases because the relative refractory period for each action potential is increased. B) This question has a pretty simple answer. The wash just isn t removing all of Drug X as we would like. Therefore, some of the channels will remain closed and still cause the cell to repolarize more slowly. However, in this case many fewer channels are blocked compared to the drug trial. C) To fix the problem that this drug causes we want to do one underlying thing: increase the conductance of K+. As stated above, drug X decreases conductance of K+ so we need to counter this by increasing K+ conductance! A- Increasing the number of voltage-gated Na+ channels will do nothing to increase K+ conductance so this drug doesn t work B- Increasing the number of voltage-gated K+ channels in the membrane increases the K+ conductance so this drug works, C- This drug increases the permeability of the voltage-gated K+ channels that are already on the membrane and unaffected, so this drug works. D- This drug would actually decrease the permeability of K+ so it does not work. 6) (Stephanie) Recording and stimulating at the cell body of a neuron (approximated as a sphere), you would like to calculate values for the passive membrane properties of your neuron. You inject a rectangular pulse of current of magnitude I during interval x (see below). a) On the graph of voltage and time (below), which equation describes time interval y? We consider the neuron as a sphere, so the decrease in Vm will be given by Vt=Vo e(-t/τ). Note that here, our Vo (initial voltage) is V on this graph because this is the initial value of voltage as we enter the interval in which the voltage is decreasing. b) Based on this equation and using your graph, how could you estimate the value of τ? From the beginning of the decreasing interval, when t=τ, Vt=V (1/e)=0.37V. So τ is equal to the amount of time elapsed from the beginning of y to when V=0.37V. 5

6 c) When t is large relative to τ (i.e. when V is approximately V ), the capacitive current of the circuit will be negligible. Using this interval, how would you calculate the resistance of the cell in terms of Vo, V, and I (ignoring any resistance from the electrode itself)? How would you calculate the capacitance using your knowledge from part b? From Ohms law, V=IR. So V=V - Vo, and you are injecting a known current I, then R=(V - Vo)/I. Since τ=rc, then C=τ/R. d) If you were determining these properties in a living, functional neuron, why might it be preferable to inject a hyperpolarizing current, as opposed to a depolarizing current? If your depolarizing current is too strong, you might evoke an action potential, which uses active membrane properties and will obscure your data. 7) (PeiXi) You are carrying out an experiment using an isolated piece of a sciatic nerve of a Hungarian frog, immersed in a solution resembling its native environment. a) By placing a cold block along this axon at a distance away from where battery-driven, abovethreshold electrical stimulations of the axon take place, what would the propagation of the action potentials look like before and after they reach the block? What about the electrical current (what would the extracellular recordings look like)? (Hint: what kind of effect is the cold block eliciting?) This experiment is based on Hodgkin s experiment carried out with a crab axon. The idea of the cold block is to prevent ion channels from opening and generating an action potential. When supra-threshold electrical stimulation occurs, local inward currents generated by action potentials flow passively along the axons, depolarizing neighboring membrane, opening voltagegated Na + channels and initiating the next action potential until they reach the cold block where ion channels don t open. The action potential fails to propagate while the inward current from previous APs continues to travel down the axon. However, recordings measuring the passive current without further inward current flow near the cold block indicate a decaying current flow as it passes through the block, hinting that the inward current leaks out of the membrane over the distance it travels. Action potentials may or may not (most likely not) travel past the block depending on whether the current is large enough (suprathrehold) to allow further propagation. b) Draw the action potentials before they reach the cold block. What happens when the block is removed? 6

7 The action potentials generated right before they reach the cold block would be reduced in amplitude compared to APs further away from the cold block. The reason is that the low temperature of the block projects to a distance away from itself, thus lowering the temperature of the neighboring axonal region. As the cold temperature prevents most ion channels from opening, the ion flow across the membrane is less prominent than at warmer temperatures when more ion channels are opened, making the action potential smaller in amplitude. Only when the block is no longer in place would the action potential be likely (depending on the size of the current) to travel further (concluded by experiments following Hodgkin s) along the axon. c) What is this experiment suggesting? It demonstrates that passive membrane properties such as the membrane s resistance to current flow through resting ion channels determines whether an action potential propagates along the axon 8) (PeiXi) a) An intracellular recording is used to measure the voltage across the membrane of a neuron. The membrane potential measured is 0 mv. What would be the concentration of extracellular K+ ions when the intracellular concentration of K+ ions is 50 mm, considering that the membrane is only permeable to K+ + ions, and the neuron is bathed at 20 o C? b) In lecture, the following graph was presented. Why is there a deviation of the curve from the line? What do the line and curve each represent? When measuring the resting potential of a neuron, which depends mainly on the concentration of K + ions intra/extracellularly because its permeability to K + ions is much higher than for other 7

8 ions, the deviation indicates that other ions such as sodium and chloride ions are actually slightly permeable and thus influence the resting potential. The straight line in this case is the theoretical prediction that the change in membrane potential (equilibrium potential of K + ions) is proportional to the logarithm of the extracellular K + concentration while intracellular [K + ] remains unchanged, usually with a slope of 58 mv per tenfold change at room temperature without taking permeability of the membrane to other ions into consideration. The curve, in contrast, represents the actual change in membrane potential when other ions are included. c) Determine the membrane potential of this neuron with the given data, assuming that it is bathed at a 20 o C environment. What would the membrane potential be when extracellular [Na] is lowered to 70 mm? Ion Intracellular (mm) Extracellular (mm) Permeability K Na Cl Ca Another calculation problem. According to the GHK equation, Vm = 58 log!"!!!"#!!"#!"!!"#!!"#[!"! ]!" => Vm = mV!"!!!"!!"#!"!!"!!"#!"!!"# When [Na + ] out = 70mM, Vm = 58 log!"!!!"#!!"#!"!!"#!!"#[!"! ]!" => Vm = mV!"!!!"!!"#!"!!"!!"#!"!!"# 8