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*. 1. Ag, Univ KY, Lexington, KY, USA2. Biol, Univ KY, Lexington, KY, USA

3. Biol, Bellarmine Univ, Louisville, KY, USA4. Psych, N. KY Univ, Highland Heights, KY, USA

5. Stats, Univ. KY, Lexington, KY, USA6. Math, Univ of KY, Lexington, KY, USA

Introduction

 

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.

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.

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.

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.

Anatomical

Experimental Computational Student’s viewsExtracellular experiments:

Ventral dissection stained with methylene blue1. Ventral nerve cord2. Segmentalganglion3. 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.

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.

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-project.org. The computational assessment involved the students learning how to write short programs in R for analysis of the synaptic measures.

Staining:

Extracellular approach:

Lucifer yellow staining of a Rz and P cell in the ganglion by intracellular pressure injection.

Intracellular approach

Electron microscopy

Methylene blue of motor nerveterminals for the superficial flexormuscles of the crayfish

One can see individual axons to correlate to the different size extracellular spikes that were recorded. Also the goal was to correlateinnervation patterns on the muscle for the different terminals.

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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Recording extracellular spikes with

a suction electrode.

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.

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.

•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.

Leech Ganglion- identified neuron cell body

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These are traces of individual spontaneous eventsthat 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

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.

[K+]o

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ExperimentalTheoretical

Changing Extracellular K+

constant field equation”

Em = -RT ln Pk[K+]i + PNa[Na+]i + PCl[Cl-]o +PCa[Ca+2]i

F Pk[K+]o + PNa[Na+]o + PCl[Cl-]i + PCa[Ca+2]o

Em = membrane potentialPx = permeability coefficients

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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.”