neurobiology questions

Dave Rintoul drintoul at ksu.ksu.edu
Wed Oct 12 15:39:34 EST 1994


Greetings,

As promised, here are some questions about basic neurobiology, which
will eventually appear in a companion volume to accompany the next
edition of a major cell biology textbook.  These questions are of the
"middling-hard" category, and are entitled "Putting concepts to work,"
,and "Developing Problem-solving Skills."  I would greatly appreciate
it if interested parties would take some time and review these
questions and answers, and then point out to me any inaccuracies,
omissions, or other errors. I can be reached by e-mail at
drintoul at ksu.ksu.edu.  All assistance will be acknowledged in the
preface to the companion volume, which is due out in early 1995.
Thanks in advance.

Dave Rintoul
Assoc. Professor
Biolgy Division - Kansas State University

----

PART C: Putting Concepts to Work [answers appear in brackets]

43. Neurons in higher vertebrates are located either in the central
nervous system (CNS) or in the peripheral nervous system (PNS). In the
table below, write CNS or PNS in the blanks opposite each neuronal
cell type to indicate the typical location of this neuronal cell.

a. interneuron			___
b. sensory neuron			___
c. motor neuron			___

[a. CNS
b. PNS
c. PNS]


44. Changes in ion permeability of a neuronal membrane will alter the
membrane potential of a cell. In the spaces provided, write H if the
indicated change in ion permeability will hyperpolarize the cell, and
D if it will depolarize the cell.

a. increase in K+ permeability		___
b. decrease in Cl- permeability		___
c. increase in Na+ permeability		___
d. decrease in K+ permeability		___

[a. H b. D c. D d. D]

45. Fibroblasts and most other non-neuronal cells exhibit an
inside-negative electric potential. However, when they are
depolarized, fibroblasts do not produce an action potential even
though the concentrations of Na+ and K+ inside and outside fibroblasts
are identical to those associated with neurons? Why do fibroblasts not
generate an action potential?

[Fibroblasts have no voltage-sensitive channels, a necessary
prerequisite for the generation and maintenance of action potentials.]

46. How do inhibitory synapes differ from excitatory synapses?

[Inhibitory synapses contain ion channels specific for ions (e.g. Cl-,
K+) which hyperpolarize the post-synaptic cell.  Excitatory synapses
contain ion channels which admit ions (Na+) which depolarize the
cell.]

47. Cyanide and carbon monoxide (CO) inhibit the electron transport
chain of the mitochondria and can inhibit neuronal firing. The effect
of these drugs on impulse transmission takes hours to occur, whereas
the effect of Na+ and K+ channel blockers occurs within seconds. Why
does it take so long for cyanide and CO to inhibit impulse
transmission?

[Cyanide and CO inhibit ATP synthesis in mitochondria, leading to a
decrease in cytosolic pools of ATP; this reduction, in turn, decreases
the activity of the Na+-K+ ATPase. However, because very few Na+-K+
ions traverse the plasma membrane during the course of the action
potential, it takes several hours for cyanide or CO to compromise the
Na+-K+ gradient to the extent that neither the resting potential nor
the action potential can be sustained.]

48. Which property of the voltage-gated Na+ channel ensures that
action potentials will only be propagated unidirectionally?

[Sodium channels have a brief refractory period immediately after
channel opening; during this refractory period the channels are
closed, but are also insensitive to voltage changes.  This ensures
that those "upstream" channels which were previously stimulated will
not open again, and those "downstream" channels will be the only ones
that respond to the membrane depolarization.}

49. Myelination of an axon increases the speed of propagation of an
action potential. Why?

Ions can flow into a myelinated axon only at the exposed, myelin-free
sites (the nodes of Ranvier). These sites contain most of the
voltage-gated Na+ channels in the axon.  Thus a region of
depolarization (elevated Na+ levels) can travel along the axon to the
next node, without having to elicit a response from voltage-gated
channels between the nodes.  The action potential "jumps" from node to
node, and the conduction velocity of a myelinated axon is greater
because of this behavior.  In addition, passive spread, and subsequent
diminution, of the depolarization is reduced by the low ion
permeability of the membrane in the myelinated regions.]

50. According to one current model, the nicotinic acetylcholine
receptor molecule has five subunits (2) arranged around a central
channel, or pore, whose diameter is slightly less than 1 nm, which is
much larger than the dimensions of Na+ or K+ ions. Why is the channel
pore so large compared with the size of the ions that pass through it?

[Both Na+ and K+ ions are hydrated; that is, there is a shell of bound
water surrounding these cations. The pore of the Na+ channel must thus
be large enough to permit passage of the hydrated cations, which are
considerably larger than the unhydrated cations.]

51. What is the function of the voltage-gated calcium channels that
are found at axon terminals? What would happen to synaptic
transmission if the pre- and post-synaptic cells were incubated in
Ca++-free medium?

[The voltage-gated Ca2+ channels at axon terminals respond to membrane
depolarization by allowing influx of Ca2+ from the extracellular space
into the presynaptic cell.  The Ca2+ ions bind to proteins that
connect the synaptic vesicles to the plasma membrane, inducing
membrane fusion and exocytosis of vesicle contents into the synapitc
cleft.  These channels are the agents that are responsible for
transducing an electrical signal (membrane depolarization) into a
chemical signal (neurotransmitter release into the synaptic cleft).
If the synapse was incubated in Ca2+-free medium, membrane
depolarization of the pre-synaptic cell would not result in membrane
depolarization of the post-synaptic cell.]

52. Explain why stimulation of the rod photoreceptors of the eye can
be considered the "reverse" of a typical neuron.

[In the dark, the rod is depolarized and secretes neurotransmitter.
When "stimulated" with light, it hyperpolarizes and secretion of
neurotransmitter is inhibited.  A typical sensory neuron is
depolarized when it is stimulated.

53. Synaptic stimulation can be prolonged if an active form of the
neurotransmitter remains in the synaptic cleft. Many pharmacologically
active compounds act by inhibiting re-uptake or hydrolysis of
neurotransmitters in the synaptic cleft. Match the drugs below with
the neurotransmitter whose action they prolong.

Prozac			acetylcholine
Cocaine			serotonin
nerve gas		dopamine

[Prozac inhibits uptake of serotonin.
Cocaine inhibits uptake of dopamine.
Nerve gas inhibits hydrolysis of acetylcholine.]

54. Many voltage-sensitive channel proteins have transmembrane helices
with lysine or argine at approximately every third or fourth residue.
What is the proposed function for such conserved domains, and how are
they thought to work, on a molecular level?

[These positively-charged amino acids are localized on one side of the
helix, and are thought to function as the voltage "sensor." When the
membrane is depolarized, this helix is thought to move toward the
exoplasmic surface, which is now negatively charged (see Figure 21-17
in MCB).  The conformational change associated with this motion is
thought to open the channel.]

PART D: Developing Problem-solving Skills

56. What is the effect on
synaptic activity of a rod cell if it is injected with cGMP? Explain
this effect.

[Injection of cGMP into a rod cell will enhance synaptic activity.
This is because the cGMP keeps the sodium channels open, resulting in
membrane depolarization, and enhanced neurotransmitter release at the
synaptic end of the rod cell.]

57. If the external sodium concentration is decreased, the magnitude
of the action potential in a squid giant axon is decreased. Why? What
would happen if the external sodium concentration were reduced to
zero?

[According to the Nernst equation, the magnitude of membrane
depolarization during an action potential is dependent upon the
permeability of the membrane to sodium, and upon the pre-existing
concentration gradient across the membrane.  Decreasing the magnitude
of the concentration gradient will decrease the magnitude of the
depolarization observed during the action potential.  Decreasing the
external sodium concentration to zero would eliminate the action
potential altogether, since Na+ influx could not occur at all.]

58. During a summer internship at Woods Hole, you explore some of the
aspects of the action potential using giant squid axons. When you
impale an isolated neuron with a microelectrode, you observe two
curious anomalies: (1) the resting potential is 20 mV more negative
than what has been reported in the literature, and (2) no action
potential is elicited when the cell is depolarized. You then examine
the voltage-sensitive Na+ channels using a patch-clamping technique
and find that they are functional. What might be the cause of both the
hyperpolarized resting potential and lack of an action potential in
your first experiment?

[The most probable reason for the hyperpolarized resting potential and
lack of an action potential is that there is too little or no Na+ in
the external medium. The resulting decrease in the Na+ "leak current"
would account for the hyperpolarized resting potential.  Furthermore,
in the absence of a Na+ gradient, no action potential could be
generated even when the Na+ channels open normally. Although an
increased permeability to K+ ions also could account for the
hyperpolarized resting potential, only a defect in ENa could result in
no action potential. The lack of Na+ ions in the form of NaCl would
ultimately cause lysis of the cells due to an osmotic imbalance.]

61. Drosophila shaker mutants have a defective K+ channel that causes
delayed repolarization of the plasma membrane of axons. However, this
type of delay also can result from other membrane-specific defects.
Suggest one other possible defect that would contribute to delayed
repolarization of neurons. Which techniques are most suitable for
demonstrating such a defect?

[A defective Na+ channel that cannot be inactivated in the wild-type
manner also could contribute to delayed repolarization of neurons. The
best techniques for demonstrating such a defect are voltage clamping
and patch clamping.]

62. Assume that you have cloned the normal gene for a novel potassium
channel from a marine invertebrate. You have also isolated the gene
for a charybdotoxin-resistant form of this channel protein. You
suspect that the functional channel contains multiple copies of the
polypeptide encoded by this gene. You can express both the normal and
mutant genes in Xenopus oocytes, and do patch-clamp studies of single
channels in this system. In an oocyte which was injected with a
mixture of mRNAs for these channel proteins (75% normal and 25% toxin
resistant), you find that 42% of the channels are toxin-sensitive. If
the mRNA mixture is 50/50, you find that 12% of the channels are
toxin-sensitive. Assuming that the two types of polypeptides mix
randomly during channel assembly, and assuming that one copy of the
normal polypeptide will render a channel toxin-sensitive, how many
polypeptides make up a functional potassium channel in this marine
invertebrate?

[The fraction of toxin-sensitive channels in a mixture of sensitive
and resistant polypeptides is A raised to the nth power, where A =
fraction of sensitive polypeptides and n = number of polypeptides per
channel.  Thus 0.75n should 0.56 if n =2, 0.42 if n = 3, and 0.32 if n
= 4.  Similarly, 0.5n = 0.25 if n = 2, 0.125 if n = 3, and 0.0625 if n
= 4.  The observed data are consistent with the hypothesis that there
are three polypeptides per channel.]

65. You notice an unusual defect in a mutant cholinergic neuronal cell
line, which can be induced to differentiate in culture. When two
adjacent mutant neurons form a synapse, they are deficient in their
ability to transmit an action potential from the presynaptic neuron to
the postsynaptic neuron, whereas wild-type cells do not exhibit a
similar defect. How could you determine whether the mutant cells are
defective in (a) the amount of neurotransmitter in presynaptic
vesicles, (b) the ability of the vesicles to be released into the
synaptic cleft, and/or (c) the responsiveness of the postsynaptic
receptor to acetylcholine.

[a. You could determine if the mutant cells contain less
neurotransmitter than wild-type cells by quantitative ultrastructural
immunochemistry using an antibody directed against acetylcholine. If
the defect is located here, then mutant cells would contain fewer gold
particles in transmitter vesicles than wild-type cells.

b. Radioimmunoassay techniques could be used to compare the ability of
mutant and wild-type cells to release acetylcholine into the synaptic
cleft. In this case, 50 mM K+ is added to the extracellular medium to
depolarize all the neurons in the culture and then the extracellular
medium is collected and analyzed for acetylcholine.

c. A defect in the postsynaptic receptors could be detected by
measuring the changes in membrane potential in mutant postsynaptic
cells with a microelectrode following addition of a suprathreshold
level of acetylcholine to the extracellular medium. Similar
postsynaptic responses in both mutant and wild-type cells would
indicate that the postsynaptic receptors are functional in both
cases.]

66. Cells in the adrenal medulla are embryologically related to
neurons. When chromaffin cels from the adrenal medulla are removed and
placed in cell culture, they have a typical rounded morphology.
However, when presented with nerve growth factor, they differentiate
into neuronal-like cells. What characteristics would have to be
examined to determine if this cell is physiologically, as well as
morphologically, a neuron?

[Cells which are of neuronal lineage might contain voltage-sensitive
channels, one of the characteristic features of an "excitable" cell.
In addition, they might be able to synthesize, store, and release
neurotransmitters.  Both of these features are found in adrenal
chromaffin cells which have been treated with nerve growth factor.]

67. You have isolated a new psychoactive drug from a South American
lizard, after noting that certain Indian tribes in South America use
extracts of the skin from this lizard in religious ceremonies. You are
able to prepare a radioactive derivative of it. You would like to
determine the mode of action of this compound, and have hypothesized
that it mimics the action of a neurotransmitter. Unfortunately, the
identity of this neurotransmitter is unknown.

a. How would you use this radioactive compound to test your
hypothesis?

b. Assuming that your experiments described in (a) supported the
hypothesis, what other experiments could you do to help confirm this
hypothesis?

b. What other hypotheses could be invoked to explain the mode of
action of this drug on a molecular level?

[a. If the new drug is mimicking a neurotransmitter, it should bind to
the same receptor as that neurotransmitter.  One "shotgun" approach
might be to incubate brain slices, or a membrane preparation from
brain, in the presence of the radioactive drug, both in the presence
and absence of a large excess of unlabeled neurotransmitter.  If any
of the unlabeled neurotransmitters inhibit binding of the radiolabeled
drug, this neurotransmitter is a good candidate for further studies.

b. Further tests of the hypothesis would be to add the drug to neurons
which are known to tbe sensitive to this neurotransmitter, and assess
the ability of the drug to induce electrical changes in the target
neurons.

c. The drug could inhibit uptake or metabolism of a neurotransmitter.]


-- 
Dave Rintoul                 Internet: drintoul at ksu.ksu.edu
Biology Division - KSU     Latitude 39.18, Longitude -96.34
Manhattan KS 66506-4901                Compuserve: 71634,32
(913)-532-6663 or 5832                  FAX: (913)-532-6653



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