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Department of Biology, Ursinus College, Collegeville, Pennsylvania 19426
Submitted August 13, 2001; Revised March 1, 2002; Accepted March 25, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: vesicle transport • vesicle fusion • synaptic vesicles • green fluorescent protein • fluorescence microscopy • Caenorhabditis elegans • undergraduate laboratory exercise
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Neurons send signals to other neurons or to muscle cells by releasing neurotransmitters. Neurotransmitters are chemicals that diffuse through the space between the cells and stimulate a response in the adjacent cell. In this manner, a neuron can stimulate a muscle to contract. Before they are released, the neurotransmitters are packaged into vesicles in the cell bodies of the neurons and transported down the length of the axon. The vesicles accumulate at the end of a neuron until a signal stimulates the vesicles to fuse with the cell membrane and release their contents into the synapse (Figure 1).
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Students are first asked to design an experiment to determine which worms have a defect in nervous system function and which worms have a normal nervous system. If students make the connection that a defect in the nervous system would affect movement of the worm, then they can design a simple experiment observing movement of the different strains of worms using a dissecting microscope. Worms with a normally functioning nervous system would move quickly on a plate, while worms with a defect in nervous system function would move slowly or not at all.
After distinguishing between normal and mutant worms based on movement of the organisms, students are asked to design an experiment to determine what type of nervous system defect is affecting the worm's behavior. The nervous system in the worms has been labeled with the fluorescent tag GFP, which causes bioluminescence in the Pacific Northwest jellyfish, Aequorea victoria. After GFP absorbs blue light, it emits green light. GFP can be incorporated into other living organisms and used as a fluorescent tag (Chalfie et al., 1994). In this study, GFP is attached to a protein on synaptic vesicle membranes (Nonet, 1999). Using this knowledge, students can design an experiment in which they compare the appearance of the nervous system in normal worms to the appearance of the nervous system in mutant worms. Because the location of synaptic vesicles in neurons can be identified by a green glow visualized with a fluorescent microscope (Figure 2), students may choose to observe the worms under the fluorescent microscope and distinguish between vesicle transport and vesicle fusion mutants based on the localization of GFP in the nervous system. As they have already determined which worms have a normal nervous system, they have established a control for their study of the subcellular localization of synaptic vesicles in the mutant worms.
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Caenorhabditis elegans is an ideal organism for studying the nervous system in an undergraduate laboratory. The worms are simple, nonparasitic, and develop quickly. Because they are only 1 mm long, the plates they grow on take up little space. Populations can be frozen long term, so that a stock can be thawed shortly before the laboratory exercise. Moreover, the nervous system of C. elegans has been studied and characterized extensively at the cellular level (White et al., 1986; Hall and Russell, 1991). Surprisingly, the nervous system of this small worm functions in many ways like our own nervous system (Bargmann, 1998). Genes that are involved in human neurological disorders, such as Alzheimer's disease, have counterparts in C. elegans (Levitan and Greenwald, 1995; McDermott et al., 1996). Using C. elegans in a laboratory exercise presents an opportunity to emphasize the importance of model organisms in basic research.
The following methods were designed to make this experiment feasible for a large introductory cell biology course for undergraduates. During a semester, we found it possible to run six laboratory sections, each with approximately 20 students. The students worked in groups of four to enhance cooperative learning in the laboratory (Johnson et al., 1991; Advisory Committee to the National Science Foundation Directorate for Education and Human Resources, 1996; Strum-Kenny, 1998). The necessary equipment for this laboratory exercise should be found in most colleges or universities.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Materials to Gather a Week Before the Laboratory Exercise
Techniques Students May Choose to Use During the Laboratory Exercise
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Determining Types of Nervous System Defects
Students next designed experiments to determine what types of defects were
present in the nervous systems of C. elegans strains RK001 and NM440.
The students were aware from discussions and readings in their cell biology
course that synaptic vesicles must travel to the appropriate location in a
neuron and fuse with the plasma membrane to release neurotransmitter. They
also knew that the synaptic vesicles of all the worms they were studying were
tagged with GFP and that they had access to a fluorescence microscope. The
second experiment asked them to analyze a complex phenomenon: how nervous
system function is affected by different defects in neurons. Because the
concepts were complex, group discussions were critical for experimental
design. By working in a group, students pooled their basic knowledge of
nervous system function and their awareness of the tools available, and chose
to design experiments to observe the pattern of GFP in the worms using the
fluorescent microscope. Because they had already deduced that the nervous
system of strain NM1233 was normal, they used the appearance of its nervous
system as a control for normal distribution of synaptic vesicles.
Using the fluorescent microscope to observe cellular structure was somewhat challenging for the students. The microscope was larger and more complex than ones with which the students were familiar. Therefore, a longer time was needed to find worms for observation than with the stereomicroscope. One suggestion to make the microscope observation run efficiently would be to have three lab groups each bring a slide to the fluorescent microscope at one time. The laboratory instructor can arrange that slides of NM1233, NM440, and RK001 worms are each brought to the fluorescent microscope by a different group. If one person from each lab group finds a worm on their slide with bright field microscopy and then switches the microscope to fluorescence microscopy, each student in the room can then take a turn observing the green glow in the nervous system. In this manner, three worm strains can be observed using one fluorescent microscope and the lab groups can collect data for all the strains in a short period of time.
Students observed that the pattern of GFP in the nervous system, which represented the location of synaptic vesicles, varied for different strains. In the control for a normal nervous system, strain NM1233, they visualized the nerve ring, and ventral and dorsal nerve cords (Figure 3). Although the vesicles are found at synapses, the synapses are close enough together that the nerve ring and nerve cords appear as lines under a low magnification, rather than as punctate spots. When students observed C. elegans strain RK001, which they had already determined had a defect in nervous system function, they saw the same pattern of GFP as in NM1233 (Figure 3). Students discussed the idea that a worm with a defect in nervous system function could still have synaptic vesicles located at synapses. They deduced that the vesicles must be transported properly along axons but were unable to release neurotransmitter. Therefore, they determined that strain RK001 represents a mutation in the gene unc-13, which regulates vesicle fusion. When students observed the GFP pattern in strain NM440, they visualized a different pattern. In these worms, nerve cells bodies were visible in the head as small ovals. Some cell bodies were visible along the ventral nerve cord, but the thin line representing synapses along the ventral nerve cord was no longer visible. The dorsal nerve cord was no longer visible (Figure 3). In young larvae, the cell bodies in the ventral nerve cord may be close enough together to look like a line (data not shown). Students knew that the strain had a defect in nervous system function and that the vesicles must not be arriving at the appropriate location in neurons. Through group discussion, they concluded that the vesicles must not be transported properly, as would be consistent with a mutation in unc-104, which produces a motor protein needed for vesicle trafficking along axons. By combining data from two experiments that they designed, students determined which worms had defects in their nervous systems, and what type of defect at the subcellular level prevented the organism from moving properly.
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Assessment of the Exercise
Student response to the exercise was evaluated for six laboratory sections.
Each section was part of a first-year cell biology course. For quantitative
analysis, a Likert-scale questionnaire
(Table 1) was adapted from the
"Student Assessment of Learning Gains" website
(Seymour, 1997). The website
is a free site that is designed to help instructors gain feedback about how
elements of their courses help students to learn. For qualitative analysis,
students were asked directed questions to assess how their knowledge of cell
biological topics improved (Table
2 and Figure 4).
Both types of assessment indicated an increase in understanding of cell
biological topics.
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Responses to Likert-Style Questionnaire
Students were asked to respond to several questions addressing how well
they learned cell biological concepts. On the numerical scale provided with
the questions, a score of "one" represented the best possible
answer, "three" represented an average response, and
"five" represented the worst possible answer. Students compared
their level of learning as a result of this exercise to the other exercises in
the course. The questions they addressed were divided into three categories:
1) How much did each of the following aspects of the laboratory exercise help
your learning? 2) As a result of your work in this exercise, how well do you
think that you now understand each of the following? 3) To what extent did you
make gains in any of the following as a result of what you did in this
exercise?
Student responses to the Likert-scale questionnaire are separated into two groups. One group represents students in laboratory sections with instructors who were leading the exercise for the first time. The second group represents students in a laboratory section with an instructor who has led the exercise previously. Responses were divided in this manner, because one section was taught by an instructor who ran the laboratory exercise previously, while all other sections were taught by instructors running the laboratory exercise for the first time. Student response to the exercise varied depending on whether the laboratory instructor had taught the exercise previously.
In response to the three categories of questions, students indicated that they improved their knowledge of cell biology topics better than from an average laboratory exercise. Data collected from sections for which instructors taught the exercise for the first time indicated better than average scores for all questions in the three categories. When error was taken into account, however, all scores from these groups of students ranged poorer than average (Figure 4). Data collected from sections with an experienced instructor indicated that this exercise was better than average for all questions when compared with other exercises in the course. All questions in the three categories were also scored better than or equal to average when error was taken into account (Figure 4). Responses to questions specifically addressing learning of the concepts vesicle transport, neurotransmitter release, affect of nervous system on behavior, and GFP were equal to or better than average. According to the results from a Student's t test, p < 0.05 for all questions asked, indicating that there was a significant difference in responses from students in sections taught by inexperienced versus experienced instructors.
Written Responses to Specific Questions About Learning
Students were asked a series of directed questions for which they provided
written answers rather than numerical scores. Fifty-four students were asked
six questions (Table 2). These
questions are divided into three categories: 1) knowledge of cell biology
topics, 2) experiential learning and teamwork, and 3) comparing this exercise
with other cell biology exercises. Students wrote detailed responses to
questions and indicated that the laboratory exercise played a positive role in
learning cell biology concepts.
Students indicated that they increased their knowledge of specific cell biology concepts. These concepts included vesicle transport and fusion, the affect of cell function on behavior, and the use of model organisms to understand cell biology processes. Students identified advances they made in understanding vesicle transport and fusion, writing that they "Better understood function/importance of motor proteins and vesicle fusion in neurons" and that "We were able to directly correlate the movement with what was happening inside the cell. We learned about vesicles, their transport, and their binding." In response to a question about cell function and behavior, students wrote, "It helped us understand the functionality of the vesicle transport system as well as how mutations in such a system affect the organism" and "After this lab exercise, I was able to understand that mutations occurring in the nervous system can drastically affect the muscle function and movement of an organism." In response to a question designed to assess connections students made between the use of model organisms and an increase in understanding of cell biological topics, a student wrote, "The lab was helpful, because I could apply what I saw in the C. elegans to humans, and understand how some human motor disorders work."
The second category of questions was designed to assess how an inquiry-based team exercise affected the manner in which students thought about cell biology topics. In response to a question asking how designing experiments helped to increase their ability to think about cell biology problems, a student indicated a direct connection between designing experiments and understanding vesicle transport. This student wrote, "Thinking of ways to observe and identify such problems really made us try to go through the whole process of vesicle transport and understand the significance of all aspects of this process." Another student considered how designing an experiment allowed connections to be made between movement of a whole organism and the processes inside of its cells: "The designing of this type of experiment allows you to relate concepts of cell biology like vesicle transport to physical problems, you can learn that the vesicles help carry messages, but in an experiment you can observe how that relates to your physical movement and behavior." Students indicated that working in a team aided the learning process, writing, "The team members combined their knowledge of the process of vesicle fusion at synapses and helped to point out what was being observed. We also collaborated on making an experiment to test why the organisms were damaged. We all had different theories and hypotheses and this helped us think more critically about the mutant worms."
We next asked how the format of this inquiry-based exercise helped students to increase their knowledge as compared with other laboratory exercises in the course. In response, a student commented on the research aspect of the exercise as well as the use of specific tools. This student wrote, "The experiment related to real scientific research problems, showed the uses of model organisms and also combined work with a new microscope." Another student discussed a very specific benefit of this laboratory exercise that was provided by the use of GFP, writing, "In other labs, we inferred what was going on within cells or organisms, but this lab actually enabled us to see everything first hand."
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Connecting Organismal Behavior with Synaptic Vesicle Transport and Fusion
Students successfully designed two experiments to help them to make
connections between function of the nervous system and behavior. Through the
use of team discussions, they surmised that nervous system defects would have
an adverse effect on movement. They designed simple experiments to analyze
movement and categorized strains of worms as having either normal nervous
systems or defective nervous systems. This simple analysis, however, was not
sufficient to determine the type of nervous system defect in the worms.
Through further team discussion, they developed more complex experiments to
deduce whether the worms had defects in vesicle transport or fusion in
neurons. These experiments were made possible through the use of strains with
GFP-tagged synaptic vesicles. Students determined the normal pattern of
GFP-tagged vesicles in worms and compared it with the patterns in worms with
nervous system defects. Through observations of these patterns, they
determined which type of defect was associated with each worm strain. The
simple structure of the nervous system in C. elegans and the fact
that vesicles were tagged with GFP enabled students in an introductory cell
biology course to design and carry out the experiments as a team.
Instructors' Observations of Student Activities
Students were observed to respond well to this laboratory experience. They
made connections between the behavior of a whole organism and the cellular
processes that control this behavior. The lab was also successful technically;
the worms were easy to observe with the stereomicroscope and the method for
preparing slides was straightforward. For some students, it took several
minutes to find the worms with the fluorescent microscope. The students were
less familiar with this larger, more complex microscope than with the compound
microscopes they use frequently in the laboratory. Provided there were plenty
of worms on the slides, students were able to locate worms easily. Students
were very excited to use the fluorescent microscope and to see GFP. During
lecture, we discussed both microscopy and fluorescent tags, and the students
enjoyed seeing them in action.
Assessment of Exercise
The course assessment showed that the exercise was a successful learning
tool. Results from both a quantitative Likert-style questionnaire and
qualitative questions indicated that students increased their knowledge of
cell biology topics. Specifically, students better understood the concepts of
vesicle transport and fusion, the affect of the nervous system on behavior,
and the effectiveness of using GFP in their studies. Both types of assessment
also indicated that students learned better as a result of the inquiry-based
exercise compared with the other exercises in the course. Students indicated
that teamwork and experimental design helped them to learn more effectively.
They also indicated that GFP was a useful tool in this exercise because direct
observations of GFP helped them visualize defects in the nervous system and
correlate them with organismal function.
Results from student assessment with a Likert-style questionnaire indicated that students learned better from an experienced instructor. It is not surprising that an experienced instructor guided students though a complex thought exercise more successfully than inexperienced instructors. We addressed the issue in this article so that a person running the exercise for the first time is not discouraged if they do not initially obtain the desired learning results.
Additional Activity
Because some lab groups must wait to work with the fluorescent microscope,
an additional activity can be included. The waiting lab groups can observe and
draw cells using standard compound microscopes and prepared slides of tissue
sections. This activity familiarizes students with the actual appearance of
the cells that are often drawn schematically during class. In our laboratory
sections, we gave students three different slides and asked them to draw the
appearance of individual cells. From a section of skeletal muscle, they were
asked to draw a muscle cell, including myosin and the boundaries of a
sarcomere. On a slide with a section of gallbladder, students drew epithelial
cells, showing microvilli, if visible. The third slide was a spinal cord
section and students were asked to draw the structure of a neuron. The
exercise helped students to understand that cells with different functions can
have very different shapes. It also showed the students the cell bodies and
axons of neurons, which are relevant to this study.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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* Corresponding author. E-mail address: rkohn{at}ursinus.edu.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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