About author: Aishwarya Ramakrishnan works as a Ph.D. student under the guidance of Dr. Sheeba Vasu at JNCASR, Bangalore. She has completed her Masters in Biotechnology from Maharaja Sayajirao University of Baroda, Vadodara, and her Bachelor’s in Biotechnology from the University of Mumbai. She is interested in understanding the neuronal mechanisms that govern circadian rhythms using Drosophila as a model system. For her Ph.D. thesis, she investigated the roles played by gap junction genes in regulating circadian rhythm properties. Her research has uncovered a novel mechanism by which a gap junction protein, Innexin2, regulates an important circadian rhythm property, the free-running period of activity rhythms in Drosophila.
Interview Questions
How would you explain your research outcomes to the non-scientific community?
Most organisms from bacteria to humans possess a genetically encoded clock that internally measures time on a 24-hour time scale and regulates various physiological and metabolic processes. E.g. – sleep and wake timings, feeding times, rhythms in core body temperature, etc. These internal clocks are called circadian clocks, and the rhythms that they regulate are called circadian rhythms. Disruption of these rhythms, as occurs with modern 24/7 lifestyles (shift-work, nocturnal light exposure), is a significant cause of mental and physical illnesses like obesity and metabolic disorders. Circadian clocks can synchronize to external, periodic cues in the environment like light and dark cycles. But even in the absence of any such external cues, the clock continues to function with a near 24-hour periodicity. This is called the ‘free-running’ period,which is a fundamental property of circadian clocks. The central clock that controls the sleep-wake cycles or activity-rest rhythms is known to be located in the brain. Over the years, several research groups have performed experiments to understand how neurons in the brain regulate circadian rhythms using various model organisms. We study the neuronal basis of circadian rhythms using the fruitfly, Drosophila melanogaster. Many discoveries about the molecular, genetic, and neuronal mechanisms governing circadian timekeeping have been made using this model system. Drosophila has about 150 clock neurons distributed bilaterally in the brain, and these neurons communicate amongst each other to generate coherent activity-rest rhythms. Previously, several neuropeptides and neurotransmitters (chemical synapses) were shown to be essential to regulate aspects of activity-rest rhythms. Neuronal cells can also communicate via electrical synapses or gap junctions which are proteins that allow for the transfer of ions and small molecules directly from one cell to the other, thus ‘coupling’ the cells electrically.
We examined if gap junctions have any role in regulating circadian activity-rest rhythms in Drosophila by downregulating the expression of each of the eight classes of gap junction genes (Innexins) using genetic tools in circadian neurons. We find that knockdown of a gap junction gene, Innexin2 lengthens the free-running period of activity-rest rhythms by about an hour (these flies now have a 25-hour free-running period instead of 24 hours shown by control flies). This suggests that Innexin2 is necessary to determine a near 24-hour periodicity in Drosophila. Taking advantage of the wide range of molecular-genetic tools in Drosophila, we show that this protein functions in the circadian circuit in the mature, adult stages and not during development. We found that Innexin2 is present and works in a critical subset (about 16 cells) of circadian neurons. Downregulation of its levels causes a delay in the accumulation of a core clock protein in the circadian neurons and increases the levels of a critical neuropeptide, Pigment Dispersing Factor (PDF), in the axonal terminals of a clock neuronal subset. We hypothesize that knockdown of Innexin2 levels could affect the membrane properties of circadian neurons, ultimately translating into molecular and behavioural phenotypes.
How do these findings contribute to your research area?
We believe our findings will be of great interest to Chronobiologists and Neurobiologists. The study uncovers a novel mechanism by which circadian timekeeping is regulated in Drosophila. Communication among neurons or glia in a neuronal network can be brought about by both electrical and chemical synapses. Although chemical synapses are very well-studied in most organisms and behaviours, the mechanisms by which electrical synapses functions are currently not well-understood. We show that apart from chemical synapses, electrical synapses/gap junctions in the circadian network are also important to regulate a very fundamental property of the clock, its free-running period. There are very few reports which have examined the roles of gap junction genes in adult neuronal circuits of Drosophila. We believe our study will help create a better appreciation for electrical synapses in neural circuits for regulating several behaviours across organisms.
our study will help create a better appreciation for electrical synapses in neural circuits for regulating several behaviours across organisms.
What was the exciting moment during your research?
I remember two particularly exciting moments. First, when I saw for the first time that the gap junction protein of my interest, Innexin2 is actually expressed in the circadian neurons. We obtained the antibody for this protein from a lab in Germany. As researchers in India can relate, it is a challenging task to get perishable reagents and fly lines from outside India, and it often takes months of planning. Waiting for about 6-7 months for this antibody finally, when I was able to stain the brains, I saw beautiful fluorescent images showing the presence of these proteins on circadian neurons. That indeed was a very satisfying feeling. Second, when I got the results from a particularly tedious but a crucial time point dissection experiment which took about 4-5 months from planning to dissecting (throughout the day and night) to imaging and analysis. Eventually, it was all worth it when I was finally able to plot the results. The cellular clock proteins had beautifully shifted in the absence of Innexin2, and it was exhilarating to be able to capture that kind of shift in my experiment.
What do you hope to do next?
With respect to this project, many questions yet remain unanswered. My work probably has raised more questions than providing answers about how gap junctions work in the circadian systems. We have exciting ideas and plans to continue this project in multiple directions that will help figure out the mechanisms by which these proteins function to modulate our daily rhythms. As for me, I would love to keep working in the field of circadian biology. I will be joining a postdoc lab soon to work on a different aspect of circadian rhythms in Drosophila, and I’m looking forward to it.
Where do you seek scientific inspiration from?
It’s a bit difficult to pinpoint to a single source of inspiration in my journey. Honestly, I never thought I would pursue research as a career. After my master’s, when I started working in a lab, I realized how much I enjoy thinking about hypotheses on how biological systems work, performing experiments, and interpreting results. Hence,I decided to do a Ph.D. in neuroscience because I was interested in the subject back from my M.Sc. days, thanks to the fantastic coursework at MSU. But once I started working on neural circuits of circadian clocks, I knew there was no turning back. I had found something that I wanted to pursue as a long-term research interest. I wish to acknowledge few people who have played significant roles in helping me make this decision. My Supervisor, Dr. Sheeba Vasu who put her trust in me and gave me the independence and encouragement to pursue a question that interests me, a very comfortable lab environment that helped me flourish as a researcher, and most importantly, some very amazing and like-minded colleagues with whom I share my interests in chronobiology.
How do you intend to help Indian science improve?
It is an exciting time to be in the Indian scientific community. There are so many talented researchers working on many interesting and challenging questions of science. It would be great to facilitate a more collaborative research environment in the Indian science community, as that would result in sharing of expertise and resources, which will significantly benefit the progress of science, in my opinion.
Lab group photo
Reference
Ramakrishnan, A., & Sheeba, V. (2021). Gap junction protein Innexin2 modulates the period of free-running rhythms in Drosophila melanogaster. iScience,Volume 24, Issue 9, 103011. https://www.cell.com/iscience/fulltext/S2589-0042(21)00979-2
Edited by: Nikita Nimbark