INTEGRATION OF NEUROPHYSIOLOGY, ANATOMY, AND BEHAVIOR WITH MATHEMATICS AND STATISTICS IN A WORKSHOP COURSE

1

• 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.
• 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

Introduction
Experimental
Computational
Students views
Extracellular experiments Ventral
dissection stained with methylene blue 1. Ventral
nerve cord 2. Segmentalganglion 3. Superficial
flexorVentral dissection stained with methylene
blue 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
values determined by using the Nerst equation.
Overview of course 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.
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. 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.r-proje
ct.org. The computational assessment involved the
students learning how to write short programs in
R for analysis of the synaptic measures.
Anatomical
1
3
Staining Extracellular approach
2
Recording extracellular spikes with a suction
electrode.
Goal of course 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. 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.
Methylene blue of motor nerve terminals for the
superficial flexor muscles of the crayfish
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.
What do you think was the most beneficial aspects
of KBRIN? Explain. 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. 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. How can
this workshop be improved? Explain. 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.
One can see individual axons to correlate to the
different size extracellular spikes that were
recorded. Also the goal was to correlate innervati
on patterns on the muscle for the different
terminals.
Outline of course DAY 1 Overview of workshop-
solve all minor details (housing). Overview of
campus/Lexington. Laboratory safety- where to go
and what to do. Use of laboratory equipment-
general (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. Day 6 Learn to process tissue for
TEM. 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.
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. 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. 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.
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.
Intracellular approach
constant field equation Em -RT ln PkKi
PNaNai PClCl-o PCaCa2i F
PkKo PNaNao PClCl-i PCaCa2o
2
2
Em membrane potential Px permeability
coefficients
Lucifer yellow staining of a Rz and P cell in the
ganglion by intracellular pressure injection.
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.
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.
 
Mathematical
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.
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.

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.
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.
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.