Biology154
Molecular and Cellular Neurobiology
FINAL EXAM
Fall 2004-2005
December 8, 2004
Name:
Student ID number:
Student email:
Section TA Name:
Section Day & Time:
DO NOT OPEN YOUR EXAM UNTIL YOU ARE TOLD TO DO SO!
You have 3 hours to complete this exam.
This is an open book exam. All notes, lecture slides, textbooks are allowed.
Computers are not allowed.
Please make sure that your answers are brief and specific. If incorrect information
is included in your answer, you can lose points even if the correct information is also
there.
If you write legibly the graders will look more kindly on your answer.
NOTE: Some questions may have multiple correct answers; as long as yours is
reasonable, logical, and consistent, you can get full credit. Be aware of point values
for each question and budget time accordingly. Some questions may be challenging
but don’t panic; grading will be on a curve. Good luck!
Please limit your answers to the space provided.
At the conclusion of this exam, please sign the honor code statement on the next page.
1
Honor code statement:
In signing, I acknowledge my responsibility in the maintenance of the honor code.
In preparing for and in completing this exam, I have adhered fully to the
requirements of the honor code of Stanford University
__________________
Signature
Point allocation (for graders only)
Q 1 ______________/20
Q 5 ______________/20
Q 2 ______________/20
Q 6 ______________/12
Q 3 ______________/20
Q 7 ______________/12
Q 4 ______________/12
Q 8 ______________/12
Total ______________/128
2
QUESTION 1 (20 pts)
1. Comparing the brain representation of the senses of vision, olfaction and taste,
what is a common principle? (1 pt) Which two senses are more similar to each other
and why? (3 pts)
2. Charles likes to knock out genes in mice and test the effects of their taste
preference (as measured by relative licking rate in two bottle choice assay as shown
in the figure below). When he knocked out the gene encoding a signal transduction
component expressed in taste cells, phospholipase C (PLC2), he found the
following results.
(wild-type (white) vs. knockout (dark gray). Hint: AceK/Sac have the similar taste
as glucose; PROP/Den/SOA tastes bitter).
What are the two ways of explaining the result of this experiment? (6 pts)
3
3. To further understand the organizational principle, Charles introduced into the
PLC2 knockout mice a transgene expressing PLC2 under the control of a bitter
receptor promoter and tested the taste choices (rescue: light grey bars in the figure
above). What conclusions can you make from this rescue experiment? (3 pts)
4. In a separate experiment (unrelated to the above figure), Charles identified a
human taste receptor for a novel ligand X that does not taste anything to mice (i.e.
the mice drink equally from pure water and X-containing water). He generated a
first strain of transgenic mice that express this human receptor using a bitter
receptor promoter. Mice strongly prefer pure water over X-containing water. He
then generated a second strain of transgenic mice that express receptor X using a
sweet receptor promoter. Mice prefer X-containing water than pure water. What
can you learn from these two experiments? (4 pts). For fun, Charles then created
mice that have one copy each of the above two transgenes. Which water do you
think the mice will choose -- pure water or X-containing water? Explain your
prediction. (3 pts)
4
QUESTION 2 (20 pts)
Inspired by Roger Sperry’s eye rotation experiments, you decided to search for the
“chemoaffinity tags” that might mediate cell-cell recognition during synapse
formation. You decided to use a simple organism, C. elegans to explore this
question.
In wild type animals, you found there was one neuron A, which forms synapses on
muscle B. The axon of A also contacts muscles C and D, but does not form synaptic
connections onto either C or D. (Refer to Figure I.) You hypothesize that there are
molecules on A that can distinguish B, C and D in forming synaptic connections. In
preliminary experiments, you found defects in synaptic patterning in two types of
animals. The results are described and shown below:
(Figure II.) In animals II, cell B is displaced so that B does not contact A and A
forms synapses on D.
(Figure III.) In animal III, cell B is eliminated and A forms synapses on D.
Stars represent synapses.
1. What does this tell you about B’s role in forming a presynaptic compartment in
cell A? (1 pt) What type of cue does cell B most likely possess (diffusible or
membrane attached)? Why? (2 pt)
5
2. In a genetic screen, you uncovered a number of mutants in which A forms
synapses on D instead of B. What neuronal developmental processes could have
been affected by these mutations? List three. (6 pts)
3. You went ahead and cloned two of these mutants, x and y. You found that X and
Y encode two membrane molecules that have characteristics of adhesion molecules.
What are the key experiments to further understand the function of X and Y in A-B
synapse formation? List three. (6 pts)
4. Assume that X functions in neuron A and Y functions in muscle B, how can you
show that the potential X-Y interaction is sufficient to initiate synapse formation? (3
pts)
5. In the absence of B, A forms synapses with D but not C. What did we learn from
this result about how specific synapses are formed? (2 pts)
6
QUESTION 3 (20 pts)
You are investigating the cellular mechanisms of temperature sensation in mammals.
Based on previous studies, you hypothesize that TRP channels may mediate temperature
sensitivity. To test this hypothesis you clone many of the rat TRP channels and express
each one individually in HEK293T cells, which lack TRP channels. You can then
characterize each channel’s response to changes in temperature.
You record from a TRP-expressing cell using a whole-cell patch clamp configuration (the
electrode is continuous with the inside of the cell). Below are shown the results of an
experiment where you recorded the current (I) at a range of different temperatures over
time. You conducted this experiment while holding the membrane potential at +100mV or
–80mV. The x-axis represents time.
(a) Is this heat sensitive or cold sensitive channel? (2 pts)
(b) Briefly explain why the TRP current is positive when the cell is held at +100mV, but
negative at –80mV. (5 pts)
(c) How would an increase in extracellular K+ affect the membrane potential under
normal conditions? (3 pts) How would an increase in extracellular Cl- affect
membrane potential? (3 pts)
(d) Lastly, you want to determine which cations pass through this TRP channel. Describe
an experiment to differentiate between the permeability of the channel to Ca2+, Na+ and K+.
(6 pts)
7
QUESTION 4 (12 pts)
(a) Among primates, humans, apes, old world monkeys (OWM), and the Howler
monkey (a species of new world monkeys) have full trichromatic vision. This means
that they have three distinct loci for opsin genes. On the other hand, all new world
monkeys (NWM) except Howler do not possess full trichromatic vision:
heterozygous females can be fully trichromatic, but all males are dichromatic. Full
trichromatic vision is believed to have evolved through a gene duplication event of
opsin genes. Based on this, label (with an X) in the phylogenetic tree shown below
where you think the gene duplication event(s) took place that led to full trichromatic
vision during evolution. (4 pts)
8
Olfactory receptor (OR) genes are encoded by the largest gene family in the
mammalian genomes. As shown in the figure below, a large proportion of OR genes
are pseudogenes. For example, ~51% of human OR genes are pseudogenes.
(b) What is a pseudogene? (3 pts)
(c) Explain briefly the relationship between the proportion of OR pseudogenes and
full trichromatic vision. (3 pts)
(d) During evolution, which do you think happened first, change in the proportion
of OR pseudogenes, or emergence of full trichromatic vision? Why? (2 pts)
9
QUESTION 5 (20 pts)
1. Ciliary and rhabdom photoreceptors represent two evolutionarily distinct cell
types involved in light detection. As result of their independent origins, they employ
a number of molecularly distinct mechanisms to convert the photoisomerization of
11 cis-retinal into a change in membrane potential. Outline three such differences
(4 pts).
2. As the transduction cascades in these two photoreceptor types are so
different, you wonder how the electrophysiological properties of the two
photoreceptor types might differ. You begin by recording the dark noise
signal in Drosophila photoreceptors (just as Denis Baylor did with
vertebrate rods), and compare it to the single photon response. You
observe the following:
What does this result tell you about the discrete noise component seen in
normal (WT) flies? (2 pts) How does this differ from what Baylor observed
in vertebrate rods? (1 pt)
10
3. Gaq1 is a mutation in the promoter region of the alpha subunit of a
particular G protein, Gq, which only causes the amount Gq protein to be
reduced by approximately 100-fold compared to the normal level. All other
transduction components are unaffected. The lowest trace in this figure
records the dark noise signal in Gaq1 mutant photoreceptors. What does
this observation tell you about the source of the discrete dark noise? (2
pts).
Encouraged by this work, you go on examine the light-induced responses
of normal and Gaq1 photoreceptors, administering brief light flashes to
each, and recording the responses of a large population of photoreceptors
(panels A and B), as well as the responses of single photoreceptors (panels
C and D, displaying only single photon responses). You observe the
following:
What are the two key differences between the light-induced responses of
normal (WT) and Gaq1 mutant photoreceptors? (2 pts) Suggest a molecular
explanation for these differences that takes into account the nature of the
Gaq1 mutation? (6 pts) Suggest an experimental test of one aspect of your
model. (2 pts)
11
QUESTION 6 (12 pts)
You are a graduate student in Eric Kandel’s lab studying the molecular basis of
learning in Aplysia. You use a reduced cell culture preparation in which a sensory
neuron (SN) synapses onto two different motor neurons (MN).
You find a peptide, FMRFamide (FMRFa), which modulates the strength of the SNMN synapse. Shown below is data from an experiment in which you puff FMRFa
or serotonin (5HT) locally onto the individual synapse depicted. 24 hours later you
record the change in EPSP amplitude in motor neurons (Y axis).
1. Focus on panel A. What is the effect of 5HT on synaptic strength? What is the
effect of FMRFa? (2 pts) Briefly describe four general mechanisms by which
FMRFa may be affecting synaptic strength. (4 pts)
12
2. You also puff 5HT and FMRFa simultaneously onto different synapses (Panel B).
EPSP amplitude is measured as before. You note that FMRFa application at one
SN-MN synapse changes the response of the other synapse to 5HT. You guess that
FMRFa binding to its receptor on SNs initiates these synaptic changes. You have at
your disposal antibodies to the FMRFa receptor, which can bind to and inactivate
the receptor. You inject them into the SN. But when you repeat the experiments
depicted in Panel A and B, the results are unchanged. Propose a model for FMRFa
function consistent with these results. (4 pts) Design an experiment to test your
model. (2 pts)
13
QUESTION 7 (12 pts)
1. List 2 lines of evidence that supports that synaptic transmission requires calcium.
(4 pts)
2. While searching the mouse genome, you identify this very interesting protein which has
homology to a C. elegans protein, abc-1. Abc-1 has been previously shown to be involved in
synapse function. You wish to determine if this putative mouse homolog, mabc-1, has any
effect on synapses. You decide to go ahead and make the loss-of-function mouse mutant.
The mouse does not exhibit gross morphological defects of motor neurons or muscles. To
characterize this mouse mutant, you decide to inject aldicarb, an acetylcholinesterase
inhibitor, into the mouse muscle. This causes hypercontraction of the muscle in wild type
animals, but in mabc-1 knockout mice, you don’t observe hypercontraction of the muscle.
Interpret this result. List 5 possible explanations where mabc-1 could be acting in synaptic
transmission. (5 pts)
3. You decide to do further characterization by injecting levimasole, an acetylcholine
receptor agonist, into the mouse muscle. This also normally causes hypercontraction of the
muscle in wild type animals. However, you also don’t observe hypercontraction of the
muscle in your mabc-1 knockout mouse. Where is mabc-1 most likely acting in the process
of synaptic transmission? Design one experiment, which could test your model. (4 pts)
14
QUESTION 8 (12 pts)
1. We learned that the mammalian SCN (suprachiasmatic nucleus) has an intrinsic
rhythm that is responsible for mediating the circadian rhythm in mice. Explain
how the SCN is entrained to natural light/dark cycles. (2 sentence max) (2 pts)
2. Organisms as diverse as mammals, insects, plants, and yeast have circadian
rhythms that can be entrained to natural light/dark cycles; however, pulses of light
during the dark half of a cycle can phase shift circadian rhythms. When Drosophila
is entrained to natural light/dark cycles, it is most active at dawn (morning) and
dusk (evening). Below is a histogram of locomotor activity for Drosophila. If wildtype or mutant flies are exposed to a pulse of light at the given point (asterisk),
answer the following questions about how behavior will change.
For each of the following flies, at what point (A, B, or C) would you expect the peak
of the next period of activity to occur? Why? (2 pts each)
wild-type:
cry (cryptochrome mutant):
tim (timeless null mutant):
15
3. A group of scientists interested in circadian rhythms showed that subsets of
neurons in Drosophila control morning and evening activity. Below is a figure that
shows locomotor behavior of light-dark (LD) -adapted flies over a 24 hour period in
DD (constant darkness) conditions. White represents subjective day and grey
represents subjective night.
Explain how ablation of both C & P subsets of neurons affects the circadian rhythm.
(1 pt)
4. When only the P subset of neurons is ablated, the evening part of the circadian
rhythm is restored (histogram on the far right). Further experiments showed that
ablation of the C neurons resulted in restoration of the morning rhythm. Draw a
diagram of the expected locomotor activity of flies in the C ablation experiment.
What does this result imply about the role of C and P neurons in establishing the
circadian rhythm in Drosophila? (2 pts)
5. Ablation of C and P neurons is accomplished by inducing cell death of these
neurons. You can imagine that the loss of these neurons may have unexpected
effects on development of the brain or brain function. In the course of making these
genetic cell ablation constructs, it was found that many genes involved in the
circadian clock are expressed in C and P neurons. Describe a more elegant set of
genetic experiments that would directly test the importance of a circadian rhythm in
these neurons. (3 pts)
16