In this video, Heather Rhodes and her undergraduate students will demonstrate how to set up, conduct, and interpret results from an inquiry-based laboratory exercise using optogenetics in C. elegans. Specifically, Rhodes will:

• Explain how to assemble simple, low-cost equipment to run an inquiry-based optogenetics experiment with undergraduate students.
• Demonstrate optogenetics stimulation to manipulate motor behavior in C. elegans with her students.

After watching this video, you should be able to better understand how to integrate optogenetic approaches into laboratory exercises for undergraduate and high school students.

Visit the Community forum for all eight modules to share your insights and best practices, ask questions, and engage with other training series’ participants.

Speaker

Heather J. Rhodes, PhD

Heather J. Rhodes is an associate professor in the department of biology and director of the neuroscience program at Denison University. Her research focuses on the neural underpinnings of social and reproductive communication in the African clawed frog. She teaches undergraduate courses on multicellular life, comparative physiology, neurophysiology, and neuroscience. Rhodes earned her PhD in neurobiology from Duke University and completed postdoctoral training at Boston University and the Marine Biological Laboratories as a Grass Fellow.

An Accessible Approach to Optogenetics in the Classroom Using Drosophila

By Heather J. Rhodes

Optogenetics is such an important and exciting tool in neuroscience research that I decided to add it to my undergraduate courses at Denison University, the liberal arts college where I teach.

Although I’d never used optogenetics as part of my own research, I’ve now successfully added a Drosophila optogenetics lab to one of my courses with low costs, reasonable preparation, and meaningful impact.

Students benefited from a hands on experience with in vivo optogenetics, and they even designed their own experiments.

In this article, and Implementing Optogenetics in the Classroom Part Two: C. elegans, I share my advice for adopting optogenetics approaches for teaching purposes.

Why Drosophila

Drosophila are an effective model system for optogenetics. You can order a variety of genetic lines and they are relatively easy to raise and cross, allowing you to express channel rhodopsin or other effectors (halorhodopsin or TRPA1, for instance) in specific subsets of neurons.

Because the Drosophila larva are translucent, you can activate channel rhodopsin in vivo with an LED light source while observing behavior. In the lab I describe below, larvae exhibit an instantaneous rigid paralysis when blue light is applied. This is dramatic and easy to measure.

Furthermore, many schools already use Drosophila for genetics or other courses, meaning you may already have most of the supplies you need right down the hall.

Exploring the Literature

My first foray into optogenetics was a two part lab in an upper-level neurophysiology class. I had seen papers describing undergraduate labs (Pulver et al., 2011, Berni et al., 2010, and Titlow et al., 2015), but these involved a level of complexity I wasn’t ready for as a beginner and tools I didn’t have in my teaching lab (such as fluorescent microscopy).

When I found a paper targeted at a high school audience (Titlow et al., 2014), I felt I’d found something I could use. Titlow and colleagues provided supply lists and instructions for constructing a simple and inexpensive blue light source (a blue LED powered by a 9V battery, taped to a standard microscope eyepiece to focus the light).

Setting Up the Experiments

Using this guidance, I knew I could execute the experiment with nothing more than my existing dissecting scopes. A colleague who works with Drosophila showed me how to rear my flies, then let me use his space and equipment to do so.

If you don’t have local help, there are many online resources to get started with Drosophila, including here and here.

Based on Titlow et al., I wrote a two part lab exercise (download the Drosophila lab handout).

In the first lab session, students learned about optogenetics and the particular construct we utilized: a fly line that expressed channel rhodopsin in all glutamatergic neurons, including  all motor neurons.

Students developed a hypothesis about what would happen when they activated all glutamatergic neurons simultaneously. They developed a simple method to quantify larval movement and then activated the channel rhodopsin and observed the results.

This was successful, with obvious results — tetanic paralysis — that the students could easily observe, repeat, share, and discuss.

As soon as the light was removed, locomotion resumed. Fly larvae must be fed all-trans retinol (ATR) in their food to produce functional channel rhodopsin, so a control group of genetically identical larva without ATR fail to show the paralysis in response to blue light.

Challenging Students to Design an Experiment

After completing this inquiry-based exploration, students were then challenged to design their own experiment for the second part of the lab.

They could choose four possible fly crosses, using either a cholinergic or glutamatergic promoter with either channel rhodopsin or a heat sensitive, excitatory ion channel (TRPA1) as the effector gene (see Berni et al., 2010).

Working in groups of 3-4, students proposed an experimental question, developed a hypothesis with a clear rationale, designed a treatment, and specified how they would quantify and collect data.

I prepared the requisite crosses, and students were able to perform their experiments two weeks later.

Students designed interesting experiments and asked questions such as: Will activation of cholinergic neurons with channel rhodopsin disrupt olfactory responses, and will activation of cholinergic neurons with TRPA1 influence photophobic behavior?

Other students tested how different wavelengths of light activated channel rhodopsin or explored how different temperatures induced TRPA1 activity. By letting students design their own experiments, they were given additional experience with the technique, ownership of the work they did, and a sense of the wide range of applications for this technology.

What Students Gained

I found no substantial downsides to this lab. Setting up fly crosses and rearing flies were the only significant efforts, and a strong lab teaching assistant could manage that workload.

The upsides, on the other hand, were tremendous. Students developed an understanding of what optogenetics entails and how it can be used in neuroscience. Given the range of flies available, this lab could also be structured to explore how particular groups of neurons or other neurotransmitter systems contribute to behavior.

I look forward to running this lab again, and encourage others to use it with your own students. All you really need is a microscope, some blue LEDs, and the right flies.

Download the Drosophila lab handout.

Visit the Community forum for all eight modules to share your insights and best practices, ask questions, and engage with other training series’ participants.

Complete this short survey to provide valuable feedback about Module 5 to SfN and the series faculty.

About the Contributor

Heather J. Rhodes, PhD

Heather Rhodes is an associate professor of biology and a program director for neuroscience at Denison University.

An Accessible Approach to Optogenetics in the Classroom Using C. elegans

By Heather J. Rhodes

After the success I had using optogenetics with Drosophila in my undergraduate neurophysiology class last year (read, Implementing Optogenetics in the Classroom Part One: Drosophila), I decided to try working with another species — C. elegans — to conduct an optogenetics activity in my neuroscience senior seminar class this year.

Getting Familiar With a New Species

Like Drosophila larvae, C. elegans are transparent, which makes in vivo optogenetics very easy. By shining light of the wavelength that stimulates channel rhodopsin onto their bodies, you can observe behavioral changes.

Also, like Drosophila, you can order lines of C. elegans worms that express channel rhodopsin or other effectors in specific populations of neurons.

For my course, I used strain ZX460, which expresses channel rhodopsin in cholinergic neurons. Activation of channel rhodopsin in this line induces shortening of body length and a distinct coiling behavior.

Ordering and Maintaining C. elegans

Even as a C. elegans novice, I found this species was even easier to raise and maintain than Drosophila.

There is a huge repository of information on ordering and maintaining worms available online, particularly WormBook and the Caenorhabditis Genetics Center. To maintain worms, you need special agar plates seeded with a particular strain of E. coli. These agar plates can then be stored in an incubator or at room temperature.

Because I only needed to maintain a small number of plates for a couple of weeks, I contacted a C. elegans lab at a neighboring university. They provided me with a couple dozen plates and a small culture of E. coli.

All-trans retinol (ATR, Sigma R2500) is required for functional channel rhodopsin expression. I added ATR to the E. coli culture on some plates, leaving others without ATR to serve as a control group (as described in Goodman et al., 2014).

To deliver the appropriate light stimulus to activate the channel rhodopsin, I used a simple and inexpensive tool described by Titlow et al., 2014. It consists of a blue LED powered by a 9V battery, taped to a standard microscope eyepiece to focus the light.

Conducting the Experiment

Students then observed the C. elegans under a standard student dissecting scope.

I also supplied students with an inexpensive adapter that aligns a cell phone camera to the microscope eyepiece, allowing them to easily take photos and video of worm behavior.

To watch a demonstration of this experiment in progress, including requirement equipment and video footage of results, watch, Using Optogenetics with C. elegans for Inquiry-Based Student Experiments.

Leading an Inquiry-Based Student Activity

I developed an inquiry-based student activity (download the C. elegans lab handout) that could be shortened or expanded to fill a 50 minute class or a three hour lab. The activity was successful because it enabled students to observe changes in behavior driven by the optogenetic stimulation.

Students compared responses to control worms that were genetically identical but lacked the co-factor ATR. They were therefore able to see the contractile and coiling behaviors were driven by optogenetic activation of cholinergic neurons, independent of any photophobic behavior the light exposure may induce.

Students were also prompted to think about the function of these cholinergic neurons in C. elegans.

An additional component that could easily be added to this activity would be an examination of the anatomy of the cholinergic system within the worm. The cholinergic neurons in this line also express eYFP. If an appropriate fluorescent microscope is available, students could examine the location of labeled neurons, further expanding the scope of the laboratory exercise.

Supplies and Budget

• C. elegans strain ZX460: $7 (plus an annual $25 fee to use the CGC)
• NGM plates, E. coli: costs vary depending on quantities and needs
• ATR: approximately $50 for 25 mg (plus another $50 for shipping on dry ice)
• Ethanol (one ml) to dissolve ATR
• LED, battery, and snap connector: less than $10
• Soldering iron to connect LED to snap connector
• Microscope eyepiece with 10x lens: borrow from existing scope
• Dissecting microscope
• Microscope/smart phone adapter (optional): approximately $25

Download the C. elegans lab handout.

Visit the Community forum for all eight modules to share your insights and best practices, ask questions, and engage with other training series’ participants.

Complete this short survey to provide valuable feedback about Module 5 to SfN and the series faculty.

About the Contributor

Heather J. Rhodes, PhD

Heather Rhodes is an associate professor of biology and a program director for neuroscience at Denison University.