This CURE has been identified as exemplary based on CUREnet's review criteria.
HideNeurogenetics Laboratory: Mapping a functional circuit for cold nociception in Drosophila
Sarah Clark, Georgia State University
Location: Georgia
Abstract
Drosophila larvae exhibit a characteristic behavioral response to noxious cold: a bilateral full-body contraction along the anterior-posterior axis. Class III multi-dendritic (md) sensory neurons have been shown to be the primary sensory neurons driving this behavior, yet the remainder of the neural circuit is not defined. In this CURE, students work in small groups to identify neural populations that may be involved in the Drosophila larval response to noxious cold. They use the GAL4/UAS expression system to excite or inhibit neural populations using GAL4 lines from the Janelia Farms FlyLight project and assess the impact of their manipulation on the larvae's behavioral response to cold.
Student Goals
- Demonstrate understanding of and ability to appropriately apply principles of Drosophila genetics
- Design a well-controlled study that builds on prior knowledge and makes progress towards a research goal
- Identify and employ the characteristics of research integrity in experimental design, data collection, analysis, and communication
Research Goals
- Identify neural populations that are components of the functional circuit for response to noxious cold in Drosophila larvae
Context
The course was originally designed as an advanced (4000 level) elective for Neuroscience majors, with a plan to develop it into a new option for the required laboratory section for Neuroscience majors after testing it as an elective. The elective offering was as successful as could be expected in the Spring 2020 semester, and this CURE is now a regularly offered course that fulfills the laboratory requirement for Neuroscience majors. It is a one-semester course, though research questions and some data/results carry over to future sections. The class is capped at 26 students, but can work well with as few as 8-10. About 4-5 hours per week of lab time is required, preferably on two separate days. Scheduling on M/W, Tu/Th, or W/F is ideal due to larval generation times; alternate schedules will require students or instructors to set up crosses outside of class time. Students need to have some familiarity experimental design and should be able to read and interpret primary literature. It is also helpful but not required for students to have previous lab course experience or background in cellular or molecular biology and/or genetics.
Target Audience:Major, Upper Division
CURE Duration:A full term
CURE Design
Drosophila larvae have a characteristic behavioral response to noxious cold: when they experience it, they contract bilaterally and remain immobile and contracted for at least several seconds. This response has been shown to be dependent upon Class III multi-dendritic sensory neurons, but the rest of the neural circuit that leads to the behavior is currently undefined. Students in this CURE first acquire basic skills in Drosophila genetics along with knowledge of current methods of investigating and defining neural circuits. Students then design and perform a set of experiments using these methods to assess neural populations that may be involved in the cold nociception circuit. Students work in small groups (2 to 4, depending on class size). Groups are constructed (when possible) such that students with prior experience in lab work, cell/molecular biology, or genetics are evenly distributed among the groups. Throughout the course, emphasis is placed on the idea that there is no such thing as a "bad" result - that ruling out a population of neurons is as meaningful as identifying one that is involved. In this way, all students are able to achieve the goals of acquiring skills and experience with research design. Additionally, the course is structured to be flexible enough that, if a group of students identifies a neural population that appears to be involved in some behavior other than cold nociception, they will be able to pursue characterization of that neural population instead, if desired. The project is developed in coordination with a research professor in the Neuroscience Institute and the research goals align with and contribute to a project he coordinates. The students present their work in a poster session at the end of each semester, and particularly promising lines of inquiry may be followed up by subsequent CURE students or in a research laboratory.
Core Competencies:Analyzing and interpreting data, Planning and carrying out investigations
Nature of Research: Basic Research, Wet Lab/Bench Research
Tasks that Align Student and Research Goals
Student Goals ↓
- Describe the basic principles of genetics in relation to Drosophila (independent assortment, recombination)
- Explain the use of markers and balancer chromosomes (why + how)
- Complete a plan for a genetic cross to generate progeny with a specific genotype
- Execute the planned cross and report the results, then troubleshoot if the resulting genotype does not match the goal
- Explain how the GAL4/UAS system works and why it is an important tool for researchers
- Plan and execute a cross that will demonstrate effective use of the GAL4/UAS system (e.g. "express GFP in the Class III neurons")
- Plan and execute crosses that will express ChETA (optogenetic activation) or tetanus toxin (inhibits neurotransmitter release) in target neural populations
- Describe the Drosophila larval behavioral response to noxious vs. innocuous cold stimuli
- Summarize current knowledge of the neural basis of cold nociception in Drosophila based on provided primary literature
- Define what is meant by "control," "positive control," and "negative control" in an experiment
- Explain the distinction between a circuit component that is necessary vs. one that is sufficient
- Write a research proposal for a study designed to test GAL4 lines for involvement in the cold nociceptive circuit
- Identify examples that do not maintain research integrity for: experimental design, data collection, data analysis, and communication of results
- Propose and discuss circumstances that might have led to the lack of integrity in the examples
- Identify potential "problem areas" in their research design and outline the steps they will take to ensure integrity in their research
- Create and present a poster that summarizes the research they performed, with emphasis on strong research design and ethical practices
Instructional Materials
1.1 Research design.pptx (PowerPoint 2007 (.pptx) 122kB Sep30 21)
1.2 Lab Notebooks.docx (Microsoft Word 2007 (.docx) 17kB Sep30 21)
1.2 Writing A Replicable Procedure.docx (Microsoft Word 2007 (.docx) 18kB Sep30 21)
3.2 Intro to Bipartite Expression Systems.pptx (PowerPoint 2007 (.pptx) 1.7MB Sep30 21)
Assessment
Instructions and scoring rubric for genetic cross exercise (Microsoft Word 2007 (.docx) 13kB Sep30 21)
Genetic notation and crosses.docx (Microsoft Word 2007 (.docx) 14kB Sep30 21)
Fly sorting assignment.docx (Microsoft Word 2007 (.docx) 13kB Sep30 21)
Methods summary assignment and rubric.docx (Microsoft Word 2007 (.docx) 13kB Sep30 21)
Instructional Staffing
In addition to the instructor, there are usually two to three undergraduate assistants/peer mentors and one graduate student assistant. Outstanding students from prior offerings are recruited to serve as peer mentors (there is a course they can enroll in to get class credit for this) and graduate assistants are selected from the PhD students already working in the Drosophila lab in the department.
Author Experience
Sarah Clark, Georgia State University
I believe that increasing opportunities for students from all backgrounds to be involved in discovery-driven research is essential to the process of increasing diversity in the sciences. I have designed this CURE to spark students' interest in research and help them to see themselves as scientists, while giving them the opportunity to learn skills that will prepare them to obtain positions as undergraduate research assistants in campus labs.
Advice for Implementation
Most students will not have had extensive exposure to the concept of using genetics in experiments prior to this course, so I have found that it is important to do plenty of scaffolded, skill-building activities to help them work up to understanding the use of bipartite expression systems in research. Fly stocks for all exercises and experiments need to be fully expanded and producing virgins on the day you will begin an exercise or experiment, so the instructor and/or TAs should plan to begin expanding them at least 2 weeks beforehand, depending on how many productive bottles will be needed.
This course has been taught three times - once in Spring 2020 (half in person, half unexpectedly online), once in Fall 2020 (planned online), and is currently being taught in person in Fall 2021. As an experiential lab, the in-person version has been more successful however the course was effectively modified to be entirely online. This involved video recording of experimental procedures for students to watch, using images of flies instead of real flies (e.g., students chose the pictures of flies with the correct markers to "set up a cross" for their experiments), and the instructor and TAs performing experiments so that students could process and analyze novel data.
Iteration
Students in this course learn to build on published literature through the methods summary assignment, in which they identify and discuss methods from primary research papers that they could use in their own research. They also build on published research through their formal research proposal, which requires them to identify and discuss the foundational literature upon which their work will be based.
Within each semester, students have multiple opportunities improve upon techniques in this course using iterative approaches. For example, while learning to collect virgin flies, the students will sort flies into three piles - definitely virgins, maybe virgins, and not virgins. The "maybe virgin" flies are then kept in a dated vial separate from the "definitely virgin" flies. After 5 days, the students are able to check these "maybe" vials for larvae to see if the flies were in fact virgins, thereby refining their own understanding of what does and does not qualify as a virgin fly.
Most exercises using Drosophila naturally allow for students to check their own work. For instance, students can check whether progeny have appropriate markers to make sure they set up a cross correctly. Students are always allowed to go back and repeat steps of exercises where they may have made errors in order to get full credit for the completed exercise. This reinforces the concept of research as a learning experience.
From one semester to another, students may continue research on particularly promising neural populations identified in previous semesters. Students also write procedures as part of their final project that future students will use.
Using CURE Data
All students present their results in a poster session at the end of each semester. Particularly promising results are followed up by future CURE students in subsequent semesters or by interested students who enroll in independent study. If any publication-worthy results are generated, all students who were involved in generating them are invited to participate in the manuscript-writing process if they want to be listed as authors. Involved students who do not contribute to the writing of the manuscript are listed in acknowledgements.
Resources
"A rough guide to Drosophila mating schemes" by Andreas Prokop (faculty and students)