INTEGRATION OF NEUROPHYSIOLOGY, ANATOMY, AND BEHAVIOR WITH MATHEMATICS AND STATISTICS IN A WORKSHOP
COURSE
C.M. Stoeppel1, S. OConnell4, A.K. Hensley2, D.M. Bhatt2, S. Logsdon2, G. Richardson3, A. Johnstone2, M. Lancaster5, K. Viele5, S. Kim6, S. Dasari2, R.L. Cooper2*.
Introduction
Experimental
Extracellular experiments:
Overview of course:
The workshop consisted of Dr. Cooper training the students in
dissection and synaptic transmission, processing tissue for
TEM, Dr. Kim teaching data handling for 3D rendering of the
TEM data, and Dr. Viele instructing the students in data
handling for analysis and computational assessments of the
above obtained physiological data. In the final session, the
students wrote a draft manuscript Introduction, Methods,
Results, and Discussion.
Outline of course:
DAY 1: Overview of workshop- solve all minor details
(housing). Overview of campus/Lexington. Laboratory safetywhere to go and what to do. Use of laboratory equipmentgeneral (pH meter, etc.) & detailed use of electrophysiology
equipment.
Day 2: Dissect crayfish abdomen, learn to measure
Membrane Potentials and what effects potentials. Evoked
NMJ potentials note differences in phasic and tonic NMJs.
Examine effects of neuromodulators (5-HT) on synaptic
transmission. Introduce different types of staining of neurons
(methylene blue and CoCl fills).
Day 3: Process dye fills. Clear tissues mount for light
microscopy viewing. Learn how to measure and analyze data
on a computer. Learn new dissection: leg opener muscle.
Learn staining procedures (methylene blue and vital dyes)
Day 4: Learn how to measure extracellular spikes and
quantify responses on the computer. Examine effects of
neuromodulators (5-HT) on sensory function. Photography:
dissecting and compound scopes.
Day 5: Quantal analysis by three or four different approaches.
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Staining:
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Extracellular approach:
Recording extracellular spikes with
a suction electrode.
Methylene blue of motor nerve
terminals for the superficial flexor
muscles of the crayfish
Ventral dissection stained with methylene blue
1. Ventral nerve cord
2. Segmentalganglion
3. Superficial flexorVentral dissection stained with methylene blue
Recordings were made with extracelluar electrodes, the signals from the cut nerve
endings will be small in amplitude. To be able to visualize the signals, one needs to
amplify the electrical response with either a preamplifier and/or the gain on the
oscilloscope. If the preamplifier can be set at a gain of 1,000 times, the oscilloscope
setting should be around 0.1 or 0.2 Volts/division. If audio amplifiers are available, they
can be used to help hear the signals. The suction electrodes consisted of a syringe with
a hypodermic needle attached, a replaceable tip made of polyethylene tubing, and two
insulated silver wires.
Intracellular experiments:
Measuring membrane potentials in crayfish muscle fibers:
The students learned how to properly record the potential across
a membrane, with glass electrodes, in the DEL1 and DEL2
muscles in a crayfish. The students furthered their investigation
of membrane potentials by determining the effects of increased
extracellular K+ levels. Using several solutions of increasing K+
levels, the cells were covered and allowed to soak for 5 min.
The resting membrane potential was then recorded again. The
students graphed their values for the resting potential against
the log potassium concentration and compared these values to
the theoretical
constant
field equation” values determined by using the Nerst equation.
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Em = -RT ln Pk[K+]i + PNa[Na+]i + PCl[Cl-]o +PCa[Ca+2]i
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F
Pk[K+]o + PNa[Na+]o + PCl[Cl-]i + PCa[Ca+2]o
Day 7: Day off.
Day 8: Lecture on synaptic transmission and structure. Learn
to process tissure for TEM. Visit EM facility.
Day 9: Continue to process tissue for data analysis. Teach
students about thin sectioning and post fixation. Drosophila
larva dissection. Stain neuromuscular junctions (NMJs) with
antibodies.
With the confocal microscope students
learned the use of optical sections to reduce
background staining. Here a anti-GABA
antibody was used with a secondary tagged
with TRITC.
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Day 14: Day off.
Day 15: Continue with Computational processing and building
3-D rendering models. Break into work groups for writing up
components of a draft manuscript.
Day 16: Go over all results and student presentations.
“The faculty involved in the KBRIN course were excellent and
made the most beneficial aspect of KBRIN possible – the
inclusion of many activities and aspects of Neuroanatomy in an
intense two-week course. Students covered a wide array of
methods, lectures, readings, and observations in biology,
anatomy, electrophysiology, electron microscopy, and statistics.
The most beneficial aspect of the workshop was the breadth of
material covered by the incredible faculty.”
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How can this workshop be improved? Explain.
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Lucifer yellow staining of a Rz and P cell in the ganglion by
intracellular pressure injection.
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Experimental
Theoretical
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Electron microscopy
To record excitatory and inhibitory junctional potentials (EJP's and IJP's). Record action
potentials extracellularly from the superficial branch of the third root using a fine-tipped
suction electrode applied to the side of the nerve, and match different sized spikes in the
nerve with junctional potentials in the muscle fibers. By penetrating several muscle
fibers, one can see that not all fibers are innervated by all the efferent neurons, and that
the same neuron may elicit different-sized junctional potentials in different fibers. Make a
map to show distribution of the EJP's and IJP's in the muscle. We also induced reflex
firing of some motor neurons by stroking the sides of the abdomen with a fine brush.
Leech Ganglion- identified neuron cell body
•A square impulse was applied to the
Rz cell.
•The initial hyperpolarization and final
depolarization indicates the
capacitance artifact.
•The artifact hyperpolarization and
depolarization also indicate the length
of the square impulse applied to the Rz
cell.
•Length of square impulse stimulus
was approximately 300 ms.
These are traces of individual spontaneous events
that were used for measures. We compared these
types of traces to examine if subsets of events were
present. The purpose was to look for quantal events
that fell into distint groupings.
Mathematical
Day 12: Field studies in crayfish. Caving in KY.
Day 13: Statistical computation with Dr. Viele and Mr. Mark
Lancaster. The quantal recordings obtained from days 4-5
were used for this learning process.
What do you think was the most beneficial aspects of
KBRIN? Explain.
“Having the opportunity to be there was the most beneficial
aspect! It was a challenging, motivating, and inspiring
experience.
This workshop as a whole was extremely beneficial to an
undergraduate student such as myself. Not only did we, the
participants, gain more insight and knowledge of
neurophysiology; but also, we experienced first hand how other
fields of study such as computational mathematics and statistics
are an integral part of biological research.”
Intracellular approach
Day 10: Processing TEM images. Continue to process tissue
for data analysis- tissue in resin blocks. Taking images on the
confocal microscope of stained neuromuscular junctions.
Record synaptic transmission from NMJs of Drosophila (wild
type and mutant lines)
Day 11: Mount fly muscles. Taking images on the confocal
microscope. Behavioral assays in Crayfish and Fruit flies.
In addition to the direct quantal counts, the area of the
evoked and spontaneous events was measured over
time in each preparation for comparison within a
preparation to determine if the area of the quantal units
were altered. The area of the evoked and spontaneous
events was determined by the Simpson's method. The
rich text files were then used in conjunction with
subroutines written in "R basic". This software is
freeware and maintained by CRAN (Comprehensive R
Archive Network) and downloadable from http://cran.rproject.org. The computational assessment involved the
students learning how to write short programs in R for
analysis of the synaptic measures.
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Em = membrane potential
Px = permeability coefficients
Student’s views
Synaptic field potentials were also measured with focal
macropatch electrodes to assess presynaptic vesicular
events. The varicosities on the living terminals were
visualized using the vital fluorescent dye 4-Di-2-ASP
(Molecular Probes). The synaptic potentials were
obtained using the loose patch technique by lightly
placing a 10-20 m fire-polished glass electrode directly
over a spatially isolated varicosity along the nerve
terminal. The evoked field excitatory postsynaptic
potentials (fEPSPs) and field miniature excitatory
postsynaptic potentials (fmEPSPs) were recorded and
analyzed to determine the mean quantal content (m).
Direct counts of the number of evoked quantal events
and failures in evoked release were used as an index of
altering synaptic function.
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Changing Extracellular K+
[K+]o
Day 6: Learn to process tissue for TEM.
One can see individual axons to correlate to the different size
extracellular spikes that were recorded. Also the goal was to correlate
innervation patterns on the muscle for the different terminals.
Computational
Relative amplitude
The goal was to train future students in various science
disciplines to the integrative nature of science so that they can
better prepare themselves with the appropriate training during
the remaining years of undergraduate schooling and help to
direct their efforts and thus competitiveness towards particular
graduate programs.
Anatomical
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Rp (mV)
The undergraduate students were exposed to the marvels of
Neuroscience and were able to experience the future of the
field by learning "Hands-On" neurophysiology, genetics,
electron microscopy, 3-D rendering, computational/statistical
analysis, and neuropharmacology in a workshop laboratory
based atmosphere.
Goal of course:
1. Ag, Univ KY, Lexington, KY, USA
2. Biol, Univ KY, Lexington, KY, USA
3. Biol, Bellarmine Univ, Louisville, KY, USA
4. Psych, N. KY Univ, Highland Heights, KY, USA
5. Stats, Univ. KY, Lexington, KY, USA
6. Math, Univ of KY, Lexington, KY, USA
Mitochondria in Crayfish Opener Muscle
shows a mitochondria in crayfish opener
muscle that is 1236 nm in length, as
measured by the EM. The cristae of the
mitochondria can clearly be seen in this
micrograph. Muscle cells are located around
the mitochondria that, in the tissue would
extend out of the plane of the page.
Sarcoplasmic reticulum is located between
the actin and the myosin filaments. The
magnification of this picture is 164,000X with
80,000 volts applied to the electrons.
Nerve Terminal in Crayfish Opener Muscle
shows the nerve terminal of the crayfish
opener muscle. As can be seen, five
mitochondria exist inside the nerve terminal.
There are many vesicles inside the nerve
terminal; some are docked on the outside edge
of the synapse while others are floating freely
in the middle of the synapse. Four active
zones can be seen as dark bands on the
outside of the synapse. The magnification of
this picture is 81,800 X with 80,000 volts
applied to the electrons.
Here the purpose was for students to learn data
handling for 3-D rendering of the 2-D TEM images. In
order to correctly measure and reconstruct in a 3-D
view the serial sections from 2-D electron
micrographs, errors in measurements and
stereological corrections for section thickness need
consideration. When projections from fragments of
synaptic structures, such as the dense bodies or clear
core synaptic vesicles, are projected in only part of a
section the true dimension or locations are readily
misrepresented. Dr. Kim went over various ways that
these problems could be addressed with mathamatical
treatment of measures while keeping in mind the
errors in the measures and views of the biological
problem.
“I think a more comprehensive research project would have
provided additional insight into various factors that comprise
scientific research, most importantly the statistical analysis of
our data (see question 6 for details). To provide an example:
Day 1 we're divided into two groups with each group being
given a different research project outlining the hypothesis.
Suggestions of what tools, techniques, assays, etc. are
provided. This exercise allows for three things: 1) we learn to
work as a team (most research is not solitary), 2) it necessitates
critical thinking skills, and 3) the results/data will have greater
meaning in the statistical analysis portion of the workshop. This
project should not eliminate other experiments/techniques
covered in the workshop.”
“Although the math and computer programming components of
the workshop were very good in theory, I had difficulty grasping
some of the concepts pertaining to these topics. I think that
future workshops should still contain these subjects; but,
perhaps the material should be prepared in a manner in which
non-math and non-computer minded persons can better
comprehend.”
What other scientific experiments/techniques/other would
you like included in this workshop?
“I would have liked to do a bit more dissection, identification and
physiology work. I also would have liked to deal with drugs or
modulators to examine the effect they had on action potentials
and our observations at large.”
Funding for course:
Funding for this course was provided by NIH-KBRIN
(Kentucky Biomedical Research Infrastructure Network).
National Institutes of Health and the National Center for
Research Resources Grant P20 RR16481, Summer 2004.