problems with research data

Dave Rintoul drintoul at ksu.ksu.edu
Thu Oct 6 16:15:27 EST 1994


Greetings, 

As previously announced on several of these newsgroups, I am preparing
a revision of a companion volume or study guide for a major
cell/molecular biology textbook.  The questions in this study guide
range from easy (vocabulary building) through conceptual to harder
questions dealing with analysis of research data.  I am responsible
for chapters on various topics for which I have limited (indeed, zero)
expertise, but I am willing to learn.  One of these chapters is on
neurons, and their properties.  I am interested in soliciting
suggestions for questions of the "analysis of research data" category.
If you could suggest research papers that might be appropriate for this 
exercise, or types (areas?) of questions that may be appropriate,
I would be very grateful.  A good candidate would be a paper that
deals in a straighforward manner with properties of neuronal cells, or
channels, and that does NOT require a tremendous amount of background
explanation (i.e. jargon, history) in order to be understood by an
advanced undergraduate or beginning graduate student.  A representative
example from the last edition is reproduced below to give you an idea of
what sort of paper and what level of questions I would be utilizing.  Thanks
for any input you can provide.

Dave

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

---representative question follows------

One of the three glutamate receptor subtypes, the N-methyl-d-aspartate
(NMDA) receptor, may be involved in the death of hippocampal cells in
the central nervous system during episodes of cerebral ischemia (lack
of O2 to the brain). According to this hypothesis, the large increase
in extracellular glutamate in the hippocampus that occurs during
cerebral ischemia overstimulates the NMDA receptors, leading to a
large influx of Ca2+ ions into hippocampal neurons and their
subsequent death. This mechanism is thought to be the primary reason
why hippocampal neurons are among the first to die during cerebral
ischemia.

In order to investigate NMDA-elicited cell death, Carl Cotman of the
University of California has studied the effect of NMDA on the
survival of embryonic hippocampal cells in vitro. In these studies,
hippocampal neurons were isolated from 18-day-old rat embryos and
plated in culture; the number of neurons surviving was determined
periodically over a 2-week period using a trypan blue exclusion test.
Among other things Cotman analyzed the ability of MK801, a
noncompetitive NMDA blocker, to inhibit NMDA-elicited cell death. Data
representative of Cotman's results are shown in Figure 20-7.

[fig. 20-7 shows a 14 day time course with three curves.  Cells
surviving is graphed on the y axis.  Curve A = cells cultured in
the presence of MK801, there is little or no decline in cell survival
from 100% on day 0 to 95% on day 14.  Curve B = cells cultured in MK801
and NMDA; there is a slight decline in cell survival to about 80% on day
14.  This coincides with the control curve (no additions).  Curve C =
cells cultured in NMDA, this coincides with curve B until d 7, then
cell survival declines to a nadir of 0 at day 14.]

a. Which data presented in Figure 20-7 supports the hypothesis that
activation of NMDA receptors by NMDA causes cell death?

b. Did the embryonic hippocampal cells used in these experiments have
NMDA receptors present throughout the 2-week study period?

c. Do the data suggest that MK801 might affect the survival of
hippocampal neurons by more than one mechanism?

d. As shown in Figure 20-7, NMDA did not elicit death of hippocampal
cells during the first 7 days of the study period. How could you
distinguish between the two following explanations for this
observation: (1) no NMDA receptors are present during this period and
(2) receptors are present but they do not permit entry of Ca2+ ions
into the cells, which subsequently leads to cell death.

e. From day 7 to day 14 of the study period, NMDA caused a substantial
increase in cell death. How could you determine if this effect was
caused, directly or indirectly, by the influx of Ca2+ ions from the
extracellular medium into the hippocampal neurons?

f. Assume that Ca2+ is the major contributor to cell death in this
system and that NMDA receptors are present at all times during the
study period. When the Ca2+ ionophore A23187 is added at day 2 and day
10, the cells exhibit quite different sensitivities to this drug,
which increases the membrane permeability to Ca2+ ions, as shown in
Figure 20-8. Based on these data and the stated assumptions, how might
the two-phase +NMDA curve in Figure 20-7 be explained?

g. Isolated hippocampal cells are incubated with [3H] glutamate for
two different time periods, after culturing -- day 7 - 14 and day 14 -
21; the cells are then prepared for quantitative autoradiography. When
the fixed cells are examined, the same number of autoradiographic
grains are found in the preparations from each culture time period.
Are these results consistent with the data presented in Figures 20-7
and 20-8?

[Figure 20-8 shows the effect of A23187 on survival of 2-day and 10-day
cells. The 2-day cells have half maximal killing at about 0.1 micromolar
A23187; the 10 day cells have half-maximal killing at about 5 micromolar
A23187.]

ANSWERS

a. The hypothesis is supported by the observation that cell death is
much greater in the presence than in the absence of NMDA (+NMDA curve
versus control curve). In addition, the specificity of this effect is
indicated by the ability of MK801, a blocker of NMDA, to inhibit it;
that is, the survival curve in the presence of MK801 + NMDA is similar
to the control curve.

b. The absence of any effect of NMDA on cell survival during the first
7 days in culture suggests that either no NMDA receptors or no
functional receptors were present in the embryonic hippocampal cells
during this period. Additional studies would be needed to confirm this
conclusion, however (see part d).

c. MK801 alone increased the survival rate from day 1 in culture
compared with control cultures, whereas MK801 + NMDA blocked the
NMDA-elicited rapid cell death beginning at day 7. These findings
suggest that MK801 can inhibit cell death by two different mechanisms,
one of which prevents activation of NMDA receptors by NMDA and the
other of which does not involve these receptors.

d. The best way to distinguish these explanations would be binding
studies with [3H] glutamate, using NMDA to displace the radioactive
glutamate. If receptors are present, then binding should be observed;
in this case, presumably, the receptors, though present, do not allow
an influx of Ca2+ ions early in the culture period.

e. One way to demonstrate whether NMDA-elicited cell death is related
to the influx of Ca2+ ions would be to compare the NMDA effect at high
and low external Ca2+ concentrations. One problem with this
experimental approach is that cells need some extracellular Ca2+ to
remain attached to the substratum; thus depleting external Ca2+ might
cause some effects unrelated to the NMDA effect.

f. The data in Figure 20-8 indicate that 2-day cells are less
sensitive to the toxic effects of Ca2+ than 10-day cells, presumably
because they are less permeable to Ca2+ ions. That is, an
approximately 100-fold higher concentration of Ca2+ ionophore is
required to cause 50-percent cell death in 2-day cells than in 10-day
cells. These findings suggest that the inability of NMDA elicit cell
death during the first 7 days of culture, as shown in Figure 20-7,
results from the inability of the stimulated receptor to permit an
influx of Ca2+ ions from the extracellular medium.

g. This experiment provides no useful information because the
glutamate binding observed may reflect nonspecific binding to various
cell structures and/or binding to the other two glutamate receptor
subtypes. In order to demonstrate the presence of the NMDA receptor,
competition experiments using NMDA to displace the radioactive
glutamate are necessary.

-- 
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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