Main job responsibilities#

The main responsibilities of this job are the dissection of Drosophila central nervous system, screening hemidriver combinations for neurons of interest for different lab members with confocal microscopy, other immunohistochemistry experiments, stock maintenance, making of various fly foods, running behavioral experiments for lab members, and computational neuroanatomy.

Fly food#

We have various protocols for different types of fly foods. The main foods you will make are ATR molasses and German food. Depending on what’s going on with Genesee, you might have to make this food from scratch or from a packet. This page goes over the different types of food and their protocols.

Dissections#

We conduct dissections of the Drosophila brain to image using confocal microscopy. The best way to learn this skill is through hands-on training but there is a guide on how to dissect that can be helpful. Dissections can be challenging when you first start, so it can be a good idea to get tips on technique from the current RA, particularly for removal of trachea.

Screening hemidriver combinations#

Another main component of this job involves screening different hemidriver combinations for neurons of interest. Generally, the process is to identify a neuron (or neurons) that you want to either image or conduct experiments with and then find various AD and GDBDs on NeuronBridge and Bloomington. The next step is to combine the AD, GDBD and a GFP line together, and then image these crosses on the confocal microscope. Here is a guide on how to find lines on NeuronBridge and a guide on the crosses necessary to these screens and how to make a stable split-gal4 line.

Immunohistochemistry Experiments

After dissection and fixation, the next step is to stain the brains using antibodies (usually against GFP) in order to visualize any neurons labeled in a line. The guide I have linked is the same one for dissections but scroll down to read the section on immnunohistochemistry.

Behavioral Experiments#

We conduct behavioral experiments through optogenetic activation or inhibition of neurons of interest, as well as with other silencing or activating techniques to determine how neurons of interest impact behavior. The behaviors of interest most often studied include start stop transitions, steering, forward and rotational speed, pursuit, and exploratory behaviors. The key part of this skill is learning how to mount experimental flies in cone holders and techniques to elicit good walking behavior. This page goes over best practices for good behavior. Mounting flies is very challenging at first, so don’t be discouraged! The current RA will help you with this skill.

Confocal Microscopy#

We use the NIF (Neurobiology Imaging Facility Core) here at Harvard to do all of our confocal imaging. Currently, Michelle Ocana (michelle_ocana@hms.harvard.edu) is the director of the core and she is the one who you should email for questions about the confocal scopes. When you get started, you should email Michelle to get trained on the different scopes (currently there are three - SPE, SP8 and Stellaris).

How to get good images

Below are tips for getting good-quality confocal images:

1. Sometimes confocal images will not look as good if the reagents you are using are getting old such as antibodies, fix or NGS. Be sure to replace your PFA every few months as that can effect how your antibodies penetrate the brain.
2. Getting a good stain is also dependent on your dissection technique and washing technique as both can damage the brain, so if your brains are looking worse for wear once you get to the scope, check that you are being careful with your washes and dissections.
3. When imaging, it is important that for your neuropil stain you can see distinctly see different brain structures, this can involve fiddling with the gain, laser power, averaging or contrast. The same goes for your GFP or labeling channel; you want high signal to noise so adjusting the contrast and gain can be a good way to get good visualization of neurons - this will also be beneficial for later computational neuroanatomy too.
4. Depending on what scope you are using, minimizing cross talk between channels should be a priority over the speed of the image.

Other immunohistochemistry experiments#

In addition to staining against GFP, there are other immunohistochemistry techniques we use in the lab to answer different scientific questions. For example, we can use MCFO to stochastically label neurons in a split-gal4 line. Here is a guide that outlines the different genetic tools we have to answer identify and answer different questions about neurons.

MCFO Tips

1. It is good practice to continue flipping your MCFO crosses every 2-3 days after the first vial starts eclosing. If the flies are getting old, making sure you have enough virgins to make a new cross can also be useful. Because this type of experiment involves a large number of samples and has high variability, you will need a continuous stream of flies. Even if you end up not needing some of the later flips of your MCFO cross, its better to not need it than to be set back 2-3 weeks if you don’t have any more flies.
2. There has been an issue with MCFO specifically in the past where the brains will disintegrate after starting the staining process which can cause you lose all your brains. Below are possible issues and their fixes.
3. Change out your PBS, PBST and paraformaldehyde every 2-3 months. If your paraformaldehyde gets too old, this can decrease the ability of antibodies to penetrate your tissue.
4. Change out the NGS, primary antibodies (if you can) and secondary antibodies. Sometimes older antibodies can cause a stain not to work or may be the cause of your brains disintegrating.
5. Older flies tend to be harder to dissect as there will be more fat build up. The brain integrity from the get go may also be lower than a younger fly as well so you have to be careful when washing your brains.
6. Brains that are kept in the fridge for longer than a week can also lose their integrity over time, so it is wise to stain as quickly after dissection as possible.

Computational neuroanatomy#

Finally, after you have screened lines possible hemidriver combinations, you will need to determine if this split-gal4 line actually contains the neuron that you want. In some cases, it can be very clear, and in others it can be more confusing. There are few ways to address this issue. The first way can be to conduct MCFO to get single neuron morphology which can then be compared to databases such as neuprint. You can find the protocol for MCFO in Tettra and also linked it in the “Other immunohistochemistry experiments” section above. The second way is to register your light level images and then co-plot these registered images with neurons from neuprint. Finally, you can trace and register the neuron from your light level image and co-plot it with a neuron from neuprint as well. Here is a guide that walks you through all of the computational options.

How to learn#

While reading each section of the lab’s training manual, you should write down questions and then ask those questions of a person in lab. If you don’t have any questions, you probably haven’t read that section carefully enough. Then, when you start to do these things with your own hands, go back to the manual are re-check the instructions. If you realize you don’t understand something, it’s never too late to ask a question. If you notice something that needs updating, go ahead and revise it! Learning comes from reading, asking, doing, checking, and revising.

Things to note#

• Ask questions, early and often!
• When possible, make a point of going to lunch with other lab members. It’s a good way to get to know the other lab members as well as get introduced to the science.
• Ask questions of many different people whenever possible. That way, you get to know many people, you tap into many sources of expertise, and you spread out the burden of your questions.
• Write down detailed notes about your projects so you can remember what you did and can refer back to inform others. Example: full genotype, genotypes crossed, age of the flies, antibodies used, etc.
• Please update this guide as needed!

Quick guide to reagents#

• ATR: 1 vial (1 gram) + 20 ml of Ethyl Alcohol
  • ATR lives in the freezer
  • Ethyl alcohol - glass bottle in the orange cabinet
  • Measure 20 mL of ethyl alcohol into a 50 mL falcon tube. Crack open the ATR powder and dump it into the ethyl alcohol. Make sure to get all the powder out. Then vortex the tube until the powder has dissolved. Wrap the tube in foil so that no light gets in- VERY IMPORTANT. ATR is light sensitive. Label the tube “ATR” and then the date. Store it in the freezer at the front of lab.
  • IMPORTANT: There should always be a 1 gram vial of ATR powder in the freezer. That means every time you make a new tube of ATR solution, immediately slack Stephen and ask him to order another gram.
• PFA: is found in the hood - we use it as it comes
  • Crack open a vial and pour the contents into a scintillation vial. Wrap the vial in foil so that no light gets in. PFA is light sensitive. Label the vial with a piece of tape that says “32% PFA” and then the date.
• PBS: found in the back shelves. It comes as 10X, you have to make it 1X.
  • To make this, fill a 500 mL round media bottle with 300 mL millipore water from the water station at the front of the lab via a graduated cylinder. Then add 70 mL of 10X PBS found above the microwave in the back of lab also via a graduated cylinder. Label the bottle “PBS” and the date.
• PBST: 1X PBS solution with a 0.44% concentration of TritonX (found in the back shelf in the “T” section, comes as 1.0 concentration)
  • To make this, follow the exact steps above to create 300 mL of your 1X PBS solution. Then locate the TritonX bottle in the T section on the chemical shelf and add 1.32 mL of TritonX to your 300 mL of PBS 1X. Give it a good stir. Label the bottle “PBST” and the date.
• Tegosept solution for German food:
  • 5g Tegosept (p-Hydroxy-benzoic acid): in a large white bucket on the back shelf in the main lab
  • 47.5 mL pure ethanol: inside a flammable cabinet (be careful of the concentration as we have multiple ethanol bottles of different concentration)
  • 2.5 mL millipore water
  • When it is ready, stored in the fridge.
• Propionic acid: found in the orange cabinet, in the “P” section