Convention says that both sides of the brain should be symmetrical. However, contradicting evidence of asymmetric structure can be found even in the common fruit fly (Drosophila melanogaster). This study investigates possible causes of this asymmetrical body (AB) and explores the mechanisms behind communication within the neural circuit. In particular, we examine the asymmetrical morphology of major AB input neurons. We also develop an image analysis system accurately calculate the change of volume in the AB. Our work aims to enhance understanding of the brain using Drosophila as a model organism for studying neuronal networks, specifically with regards to how the coordinated activity of connected neurons gives rise to brain function in an asymmetrical manner.

Author: Arsalan Hashmi
University of California, Santa Barbara
Mentors: Professor Carlos Lois, Aubrie De La Cruz, and Ting-Hao Huang
California Institute of Technology
Editor: Hannah Chen

Introduction

Previously, it was believed that both sides of the brain in Drosophila were completely identical, but recent studies have shown that one side of the brain has an asymmetrical body (AB). This AB is a neuropil — a network of dendrites, axons, and synapses connecting neuronal cell bodies — on the left side of the brain which is only a fourth the size of the corresponding right neuropil (Figure 1). We aim to investigate how different brain functions such as memory and movement may be attributed to these different structures in the left and right hemispheres.

The asymmetrical circuit in question involves two neurons, SA1 and SA2, which innervate into the right AB. Our goal is to investigate its mechanism and function from both a developmental and a behavioral basis. We first use an inward rectified potassium channel that prevents neuronal firing and then analyze the resulting changes in development. Then, I study Drosophila courtship behavior, which could be a gateway to understanding their short- and long-term memory. Previous results have shown that while the asymmetric body does not play a role in short-term memory, it does in long-term memory, which we intend to test [1].

Development

To explore the morphology of the AB on a molecular level, we performed genetic crosses. In immature SA1/SA2, we introduced Kir2.1, an inward rectifying potassium channel that prevents the neurons from firing action potentials. The flies carrying Kir2.1-eGFP were collected, dissected, and immunostained. We captured images under a confocal microscope, which were then merged into a 3D stacked image (Figure 2).

Using the image processing package Fiji, I implemented an image analysis system to accurately calculate the volume and intensity of the RFP/GFP signal within the brains (Figure 3). RFP/GFP allows us to visualize neural activity and confirm that there is a change occurring. The procedure involved mapping out specific structures in the stacked confocal image and calculating the area of each slice.

Initial analysis of the results (Figure 4) showed conformational changes in the samples containing Kir2.1 in comparison to control. As shown below, the sample containing Kir2.1 has more visible branches, indicating added neurite projections. The asymmetric body also became more symmetrical. It was concluded that Kir2.1 does in fact impact the neurons in the region and has some role to play in the development of Drosophila brain. In future work, we aim to excite the neurons to test whether calcium influx is a factor in altering the SA1/SA2 morphology.

Behavior

We examined courtship rejection of males from pre-mated females to understand how their retention and long-term memory work when separated and placed back together. Our control experiment was designed around protocols used in a previous study (Figure 5) [2].

We first set up an assay that allows observation of mating patterns of wildtype Drosophila in real-time, dividing the virgin males and pre-mated females into separate vials. During the training period, individual males and females were placed into the same wells together, left for a nine-hour period, and then separated for 24 hours. We tested whether the males have memory of the courtship rejection from the training period by comparing their actions (Figure 6) with typical mating behaviors (Figure 7).

Results did indicate that females reject the courtship behavior as expected, but the sample size was too small to make any firm conclusions. Additionally, we ran into difficulty gathering the right tools to repeat the experiment. We did not have access to an aspirator for fly transfer, so we used anesthesia instead. We also had to use flies with “dirty” drivers, meaning they had expressions of non-interest neurons.

This first trial served as a valuable learning experience on how to conduct the experiment efficiently. In the future, we plan to repeat the experiment with consideration of the issues we faced. We also aim to perform the courtship behavioral assay on flies with silenced SA1/SA2 from Kir2.1 to compare to the wildtypes.

Acknowledgments

Special thanks to Sophia, Javier, the Read de Alaniz Group and the McNair staff and cohort for their support back at my home institution, UCSB. Thank you to my Caltech mentors Carlos, Aubrey, Ting-Hao and the rest of the Lois lab. Thank you to the coordinators and fellow WAVE/SURF/Amgen Fellows. Lastly, thank you to the Chen Institute for funding and providing me with a platform to research.

References

[1] Pascual, A., Huang, K.-L., Neveu, J., & Préat, T. (2004). Brain asymmetry and long-term memory. Nature, 427(6975), 605–606. https://doi.org/10.1038/427605a

[2] Koemans, T. S., Oppitz, C., Donders, R. A. T., van Bokhoven, H., Schenck, A., Keleman, K., & Kramer, J. M. (2017). Drosophila courtship conditioning as a measure of learning and memory. Journal of Visualized Experiments : JoVE, 124, 55808. https://doi.org/10.3791/55808