Hello everyone! It’s a been over a month since my project began on studying the diet and attempting taste manipulation of the Drosophila melanogaster.
Before my experiments could begin I faced many software and hardware issues. The flyPAD itself is an extremely thin 0.6mm PCB board so every slight bend of it can result in a breaking of the soldered connections with each channel’s circuits.
I transfer flies by sucking them up…don’t worry there’s a cotton stopper. Human Sips.
In order to change my flies’ taste perception, the light-sensitive proteins which were inserted into their ‘sweet’ tasting neurons had to be stimulated by an intense red light. For a proper response, this light stimulation has to happen almost exactly when the flies take sips of the target food. To make this happen, a code was programmed into Bonsai so that an LED is turned on nearly instantaneously to when food is touched by a fly, triggered by a change in capacitance between the electrodes. This is how the flies’ taste neurons are activated at the same instant they sip certain foods to influence their food choice preference.
The instant the fly’s proboscis (mouth) touches food, intense red light shines
To get to the finalized rig I use today, I experienced firsthand just how much debugging and problem-solving is involved in research. Below is a pictorial formula of how I got to my final experimental setup:
Got my flyPAD
Integrate circuit and solder under a microscope… this is what was breaking every time the .6mm boar bent… whch was all the time
Creating circuit and code for LEDs to sink up with each channel
I soldered a shield for easy LED plugin
LEDs set up with flyPAD
soldered LEDs to their drivers, which increase the light intensity
Another arduino programmed to pulse 100 Hz light to give flies recovery time of 900 ms
Painted acrylic to avoid unwanted light penetration of neighboring LEDs
The bonsai workflow for LED stimulation
Over many hours of adjusting my setup and learning how to insert the food, I finally got everything working so that experiments could get underway.
To see if the flies’ taste can be changed, I had to determine what their natural food preference was. I chose banana and avocado to compare. The cumulative number of sips showed banana was preferred over avocado, as avocados have almost no sugar in them.
To see if food choice preference can be altered, LEDs were activated upon contact with avocado. This fired the gr5a sweet neurons in the flies.
As predicted, the light stimulation successfully activated the flies’ sweet neurons to alter what they perceive as sweet tasting.
To verify these results, I ran a positive and negative control. The positive, showing the desired effect which is expected from the independent variable, was the proboscis extension (seen with the green arrow) from a gr5a fly upon stimulation from the 625nm red LED light.
As for the negative control, which does not produce the desired outcome of the experiment, I put gr5a flies that had not been feeding on all trans-retinal, under the same LED treatment targeted on avocado. All trans-retinal must be ingested to activate the proteins in the flies’ neurons, otherwise light stimulation will not fire the neurons. As expected, these flies still preferred the more sugary banana over the bland avocado.
Now that I know that the alteration of Drosophila taste preference is possible, I plan to study the nutrients within the flies’ diet.
It’s easy to eat sugar, but when offered your favourite sweet snack and a healthy food, it’s hard to eat what you know is better for you. I will create this scenario for my flies. Specifically, I am interested to see if the flies will eat protein after being deprived of it, even when a tasty, sugary apple is also up for grabs.
When deprived of protein, it has been found in the past that females will eat more protein when offered it again as opposed to males since they have to produce eggs. I want to see if this is still the case with natural foods rich in protein. I hypothesize that the flies will still prefer to eat a more sugary food as opposed to the nutrient they are deprived of. If this is the case, I will use optogenetics once again to see if I can make them eat the food they require the most. Are flies’ instincts to eat the nutrients they require stronger than their pickiness of taste? This is an interesting comparison to humans – as so many of us are more than willing to indulge in decadent desserts rather than eating our veggies any day of the week. Will optogenetics one day make its way into the human realm to revolutionize eating and health forever?
Thank you for taking a look at my post! I can’t wait to see what response I get out of my flies in the near future.
Introduction to the project
Hi, everyone! Last week marked the halfway point of my time as a fellow here at Backyard Brains! Recently, I’ve succeeded in building a rig and recording video footage of my Squid Hatchlings! I’m excited because it means I can start gathering quantifiable data! The squiddos have kept me pretty busy during these weeks, and they have been so fascinating to study (and photograph obsessively, like the proud parent that I am).
The main goal of my summer project is to document and begin to understand the behavior of squid hatchlings. Mostly I have been investigating how they complete the complex task of navigating away from where they’re born to seek prey and space to grow up. From previous studies, we suspect that they use their vision to help them navigate because they tend to swim towards sources of light, especially when they are very young. Since we already know a lot about what kind of light exists at the different depths, being able to specifically quantify the strength of their preference for different colors and intensities of light would help us understand where they like to be in the ocean at different ages.
In order to study how their light preferences change over time, I first had to figure out how to separate the eggs into individual containers that could still be aerated from a single pump. The containers needed to be able to let in oxygenated water, but keep hatchlings inside once they were born. The solution was a series of seven water bottle covered in tiny pinholes which rest in a bucket of water. As long as I check them regularly, I always know the age of a hatchling in any given bottle.
Home sweet home.
Next, I needed to write the computer software that could track the squid when I filmed them reacting to different environmental conditions. Since my camera and the background of the tank are stationary when I film, the solution was pretty simple. I break down the video into frames and find the average image over time to figure out what the background looks like. Then, I remove the background from each frame, apply some filters and am left with an image of just the squid! This method is called foreground detection and it is a commonly used method in image processing and computer vision. Once I have this simplified image, I can calculate the population of squid that are in each segment of their tank and see how they move over time. (Click the Gif’s to see the sweet action!)
Next, I needed to build an experimental setup in order to actually test the squid with variable conditions. After several failed attempts, I ended up with an acrylic box covered in black paper that has a hole for the iPhone to take a video through. This way, the only light that the squid are exposed to while I record them is the light that I produce and control. At first, I wrongly assumed that I should be testing squid with light from the top (since the sun is above them in their natural environment), and was getting very… boring results. Besides their sense of light, the squid also have a sense of gravity and are negatively geotactic, meaning that they like to stay at the surface of the water. So the squid in the dark would be at the surface of the water and I’d turn on a light and they’d just… stay at the surface. I wasn’t sure how to quantify this and I knew that these weren’t the results I wanted, since there was no differentiation of their reactions. I felt exactly like this:
My exact face after four hours of attempting to get results… I brought them outside the lab to see if the fresh air would help them? I just got soaked in the rain and still saw nothing interesting…
Finally, I had the idea to put the LEDs on the sides of the tank to test their reactions. I wouldn’t be forcing them to swim away from the surface, but I could hopefully still see them track the light. It worked! I could finally see the squid making an active effort to follow the light and can quantify their reactions to it.
Now I am in the process of designing the exact experiment that I want to study consistently across all groups of squid as they age. While I have been exploring and contemplating, I’ve seen some pretty cool effects. It seems that young squid (within 24 hours of hatching) react extremely strongly to low light levels (around 100 lux) and much less strongly to very high light levels (around 600 lux).
Two recordings from samples at <12 hours since hatching. The x-axis here represents the region of the tank where section 1 is the far left (in this case, closest to the ligt) and section 7 is the far right. The y-axis shows the average percentage of the population that can be found in the section over the course of a 2-minute video.
In testing some older squid, I’ve also observed that they don’t seem to react to red light. Perhaps they don’t see it at all, at the least they don’t care about it. It’s not conclusive yet, and I’d like to test more groups, but you can see the graph of their reaction below.
I’ve also seen that the older squid seem to react strongly to very strong intensities of light, especially compared to the younger squid. This might provide some interesting insight into the life of the squid and where they live in the ocean at different ages.
I think that that is all for now! After recording some more data I hope to have some awesome graphs to share with you that show how the squid react to low light, high light, and colored light as they age! With that information, hopefully we will be able to piece together the narrative of the early life of the loligo pealeii and improve our understanding of their abilities and behaviors.
It’s ET again with some updates to the BYB World Neuro Tour. Arriving in Rio de Janeiro, my friend Geo and I travelled down the Atlantic coast to spread the word of Neuroscience (for details see: onneurotour.blogspot.com or here). In Montevideo we decided to buy a VW Kombi, named her ‘Brunhilde’, and wanted to find our way quickly to Patagonia. Unfortunately, the long buying process and a friend’s visit from Germany forced us to separate in Montevideo. Waiting for ‘Brunhilde’ I used the time to visited a team of doctors at the Hospital de Clinicas where we were able to detect uterine contractions with the EMG Spiker shield in a pregnant woman.Moreover, I was invited to set up a stand at the Brain Week of Montevideo where kids enthusiastically used the BYB equipment. Special thanks to Sofia Letaief, Alejandra Mondino, and Prof. Jose Diaz.
Once allowed to leave Uruguay we arrived quickly at the Peninsula Valdes, where Brunhilde decided to take off one of her wheels. Stuck in the middle of nowhere I was adopted by the local park rangers as heavy rains blocked the routes. After many days in the Peninsula without signal we were able to repair the Kombi and a cascade of happy coincidences directed me to Mirta Anton. Together we organized the first of three ‘conferencias’ in Trelew. Thanks to her help the ‘conferencia’ was advertised and hosted by the local newspaper El Chubut (see here). Additionally, she made it possible that the NeuroTour will appear in a TV documentary of the Channel 12 called ‘Nueva Mirada’ in July. After this first stop in Patagonia we headed in direction Cordillera with two more ‘conferencias’ in Esquel and El Bolson. Special thanks to Andres Barcena and Alumine Honik who organized these events!
The three Patagonian conferencias with their spiritual minded attendants highlighted interesting aspects and applications of the BYB equipment. First, the necessity and power of the intention revealed by the Human-Human-Interface, where the controller has to perform the action willingly. Second, the possibility to control your heart rate in the ECG experiment via respiratory exercises. Third, the built-in alpha-wave amplitude to sound conversion of the EEG experiment can be used to increase your meditation performance. This is also called biofeedback therapy and is often used to help patients with neurological disorders such as schizophrenia and depression.
Finally, I would like to thank all Patagonians that supported and helped me during my stay in that incredibly beautiful region. Now, I’m heading up north along the Panamericana to visit BYB co-founder Tim in Santiago.