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Crawdad: An Online Lab Manual for Neurophysiology

Robert A. Wyttenbach, Bruce R. Johnson, Ronald R. Hoy

Instructors should email Bob Wyttenbach for access to the instructor's manual.

Crawdad Labs

The manual has several appendices that should be used along with the lab descriptions. Appendix A is the overall introduction to the crayfish neuromuscular preparation used in labs 2-6. Appendix B contains suggestions for successful dissecting. Appendix C is a reference for techniques and troubleshooting of electrophysiological recording. Appendix D lists all the neuromodulators, neurotransmitters, and drugs that you might apply to your preparations. Appendix E provides equation calculators and analysis tools.

The references included at the end of each lab exercise are intended as supplemental material. You do not need to look these up unless you want to cite them in a lab report or pursue a topic further. Most journal references are followed by links labeled [doi] or [pdf]. Doi (digital object identifier) links are supposed to be stable over time and redirect your browser to the article on a journal’s site. Pdf links are provided where no doi exists. They may go directly to a publisher’s site or to the NCBI site at the National Institutes of Health, which gives access to full text or an abstract. Many journals give access only to subscribers; you may not be able to get to articles using an off-campus network.

Figures with the magnify icon in the upper-left corner can be magnified. Enlarge the window to make the image larger or place the cursor over the image and roll the mouse wheel to zoom in or out (click and drag to pan across a zoomed image).

Compatibility

This site has been tested with 2016 versions of Chrome, Firefox, Internet Explorer, Edge, Safari, and Opera. In Windows, we recommend using Chrome, Opera, Firefox, or Edge because Explorer does not expand figures when windows are enlarged. Crawdad is also compatible with iOS devices (iPhone and iPad). The iOS version has all functions of the desktop site, including videos and scalable images. We have not tested with Android, Kindle, or Windows Mobile devices. They may not show the videos, but other aspects of the site should be functional.

Instructor’s Supplements

Introduction

This series of neurophysiology exercises comes from a variety of sources: some are based on classic exercises and some are original. All have been adapted to fit the framework of a semester-long neurophysiology lab course at Cornell University. See The Crawdad Project (below) for more detailed credits.

These exercises have both directed and investigative components. Dissection and recording procedures are outlined step-by-step, as are the basic experiments. However, the experiments and questions are designed so that students think creatively about the material and design their own experiments.

Lab Sequence

Most of these labs can stand on their own and could be inserted singly or in groups into an existing lab course. If the entire set is used, the following sequence fits well with a standard organization of lecture material:

This sequence starts with technically easy exercises (membrane properties, motor nerve) and works up to more difficult ones (synapse, snail brain). The Crayfish Neuromuscular labs (2-6) logically go together in sequence. They might be followed by the snail labs, which are technically more difficult and require the intracellular recording experience gained in labs 4-6. We often give students two periods for the first snail lab, for a total of three sessions with the snail brain. The Plant Action Potential lab, although technically easier than intracellular recording of muscle and neurons, fits most logically after coverage of animal action potentials. The Stretch Receptor lab, being technically easy, can be used whenever sensory systems are covered in the course. We may do it at the end of the semester, so that students can end with a satisfying lab that generates data readily, or perhaps just before or after spring break, when students may need something easy. It could also fit after the Motor Nerve Recording lab, since it shows one of the sensory inputs to reflex activity seen in the motor nerve.

The two exercises that most easily stand on their own are the Motor Nerve Recording and Stretch Receptor labs. Both are technically easy and succeed reliably, while requiring only a minimal extracellular recording setup.

Even if you do not plan to use all of these exercises, we suggest that you look over the write-ups and graphics for each of them. Much of this material, particularly from the two snail brain labs and the two synapse labs, could be used to illustrate lecture concepts even if they are not used as part of the laboratory sequence. Figures and videos can be downloaded directly from Appendix E, Resources.

Lab Reports

The student manual contains questions and suggested data analyses (answers appear in the corresponding sections of the instructor’s supplements) that can form the basis of a lab report. You may wish to add your own questions or change the emphasis, depending on the lecture content of your course. In general, we prefer to have students write their reports in the form of brief scientific papers, with introduction, methods, results, and discussion sections, where the discussion would incorporate answers to the questions posed in the manual. If you use this approach, some pairs of labs can be combined into single reports, such as the two motor nerve labs, the two synapse labs, and the two snail brain labs.

Many of these labs lend themselves to quantitative analysis of the results, especially given access to current computer software. The questions in the student manual suggest such analysis where appropriate; the supplements contain examples of typical results and their analysis.

Instructor Support

The supplement to each lab contains a statement of the objectives of the lab, the equipment and supplies required, troubleshooting tips, example results, and answers to the questions posed in the student manual. Appendix E, Resources, has a table of all figures and videos, including links with which you can download items for use in your own class presentations.

We have created a web site (crawdad.cornell.edu) and mailing list to support instructors who are using this material. The web site contains suggestions for suppliers of lab materials and equipment and links to other neuroscience sites. The mailing list allows ongoing discussion among users of the Crawdad material. We monitor this list and will offer advice as needed. We encourage you to subscribe to this list so that those who teach this material can become a mutually supporting community.

Please direct any questions or suggestions about the content of the labs to Crawdad authors. Technical issues about the CD or web site should be directed to Sinauer support.

Appendices

To avoid repetition and lengthy asides in the labs, much of the background material for the lab exercises is given in appendices. The crayfish neuromuscular sequence (Labs 2-6) has a single introduction in Appendix A, Crayfish Neuromuscular Preparation, that students should read. General information about dissection and recording procedures, including basic troubleshooting, is given in the student versions of Appendix B, Dissection Tips, and Appendix C, Recording Tips. Students should read these early in the semester and to refer to them as needed during the labs.

References

The references included at the end of each student lab exercise are intended primarily as supplemental material. They are not necessarily cited in the lab text. It is not expected that students would read these unless they wish to pursue a topic further, but we find it helpful to have copies available in the teaching lab. The references we list are not always the most recent ones, but are the ones we have used to teach this material. Literature suggestions are welcome and will be added to the lab updates on our web site.

The Crawdad Project

Origins and Credits

The lab exercises presented here form the core of Cornell’s Principles of Neurophysiology course, taught by Bruce Johnson since 1987. Many of these exercises are derived from standard research or teaching preparations. We present them with our own slant and more contemporary references. We hope that this presentation and the addition of videos will make this manual useful even for faculty who already teach these or similar labs.

The crayfish neuromuscular sequence is derived from Ron Hoy’s thesis work and experiences in the lab of Don Kennedy at Stanford. Bruce Johnson first taught these exercises with Ron Hoy and Ron Harris-Warrick in the Neural Systems and Behavior course at the Marine Biological Laboratory. The recording simulation and electronics exercise is adapted from one used by Bruce Land and Mike May in an earlier version of Cornell’s Neurophysiology course; the electric fish component was inspired by lab exercises used by Carl Hopkins of Cornell. The snail preparation was introduced to us by Ron Harris-Warrick of Cornell. Plant action potential recording was introduced to us by Lin Davidson and further developed with help from Randy Wayne, both of Cornell. Steve Zottoli of Williams College contributed to the development of some of the crayfish exercises. Previous coinstructors in the Principles of Neurophysiology course contributed to the development of all of the exercises. In particular, we thank Mike May, Peter Brodfuehrer, Satoshi Amagai, Bing Zhang, Rob Levini, Michael Oshinsky, Bruce Carlson, Matt Weeg, and Pete Lovell.

We routinely use some of these preparations as demonstrations in workshops for high school students and teachers, summer science camps, Chautauqua science workshops, and at colleges and universities in upstate New York. These exercises have also formed the core of other neurobiology courses taught by Bruce Johnson and Bob Wyttenbach, including the undergraduate Hughes Scholars techniques courses at Cornell, the Neurobiology of Animal Behavior course at the Shoals Marine Laboratory, and graduate neuroscience courses at the Panum Institute of the University of Copenhagen. We thank the student and faculty participants in all of these workshops and courses for their constructive feedback. Special thanks goes to the faculty participants in our three National Science Foundation-funded Crawdad workshops, on whom we tested early versions of the lab write-ups and videos in 1996-8. Their feedback and enthusiasm have been especially helpful in shaping this project.

Figure Credits

Figures 8.2, 7.1, and B.1 are by Margaret Nelson; Figures 7.2 and A.1 are based on drawings by Margaret Nelson. Figures 8.1 and 8.3 are based on a drawing by Satoshi Amagai. Dissecting tool images in Figures B.2 and B.3 are courtesy of Fine Science Tools. Figure C.1 is redrawn from Loeb and Gans (1986). Figure 8.6 is redrawn from Kerkut et al. (1975). Figure 2.5i is based on sections cut by Elke Buschbeck. The Crawdad logo is based on Figure 1 of T.H. Huxley’s classic book, The Crayfish (1880, reprinted 1977 by MIT Press, Cambridge, MA).

Funding

Development of the Crawdad CD-ROM was funded by National Science Foundation grant 955095. Opinions expressed are those of the authors and not necessarily those of the Foundation. Development of these labs prior to this grant was made possible by funding from the Howard Hughes Medical Institute, the National Institute of Mental Health, the Grass Foundation, the Division of Biological Sciences at Cornell University, and the Section of Neurobiology and Behavior at Cornell University.

The Authors

Robert A. Wyttenbach is a Senior Lecturer in the Emory University Neuroscience and Behavioral Biology program. He was formerly a Senior Research Associate in Neurobiology and Behavior at Cornell University. He earned a B.S. in Organismal Biology and a B.A. in Computer Science from the University of Kansas. He then spent two years at Oxford University, earning a B.A. in Philosophy and Psychology, before going to Cornell University, where he earned his Ph.D. As a graduate student, post-doc, and research associate, he has been active in developing and refining invertebrate neurophysiology labs and in producing software for use in the teaching lab. He has taught neurophysiology labs at Emory, Cornell, to Hughes Scholars, at the Shoals Marine Lab, and to college faculty at Cornell’s Crawdad workshop, the Cornell/ADInstruments Crawdad and CrawFly workshops, and Faculty for Undergraduate Neuroscience workshops. His thesis research applied a psychophysical approach to hearing in crickets, including habituation, categorical perception, and sound localization. His subsequent research investigated hearing in mosquitoes.

Bruce Johnson is a Senior Research Associate in the Department of Neurobiology and Behavior at Cornell University. He earned biology degrees at Florida State University (B.A.), Florida Atlantic University (M.S.), and at the Marine Biological Laboratory in Woods Hole (Ph.D.) through the Boston University Marine Program. At Cornell he teaches a laboratory course entitled Principles of Neurophysiology and continues development of model preparations for student neuroscience laboratories. Bruce has directed and taught in neuroscience faculty workshops sponsored by NSF (Crawdad), ADInstruments (Crawdad and CrawFly), and the Faculty for Undergraduate Neuroscience (FUN), and in neuroscience courses at the Universities of Copenhagen and Cologne, the Marine Biological Laboratory, and the Shoals Marine Laboratory. He has received outstanding educator and distinguished teaching awards at Cornell University, and the FUN Educator of the Year Award. He is a past president of FUN, and presently the Associate Editor for the Journal of Undergraduate Neuroscience Education. Bruce's research addresses the cellular and synaptic mechanisms of motor network plasticity.??

Ronald R. Hoy is a Professor of Neurobiology and Behavior at Cornell University. He earned his Ph.D. in Biology at Stanford University in 1969 with Donald Kennedy, who pioneered the use of crayfish as a model system for studying neuronal integration. Hoy’s experience with crayfish in the Kennedy lab convinced him that he had found an ideal animal for teaching neurophysiology, which he has utilized at every opportunity for the past three decades. He used crayfish labs in his undergraduate teaching at Cornell and at the Marine Biological Laboratory in Woods Hole, where he was codirector of the Neural Systems and Behavior course from 1979 to 1985. Hoy has been a chairman of the Section of Neurobiology and Behavior and served a term as associate dean of the College of Arts and Science. He serves on the Trustee Boards of the Cornell Laboratory of Ornithology and of the Grass Foundation. Hoy wrote the National Science Foundation grant that supported development of this CD-ROM and, along with Wyttenbach and Johnson, taught in the three Crawdad workshops. His research interests are bioacoustics, animal communication, neuroethology, and evolutionary neurobiology.

The authors wish to acknowledge Dan Harris-Warrick, an undergraduate at Carleton College at the time, for his invaluable assistance as a video editor for the 1999 edition of Crawdad.