DOP-2 went to a party, got high on alcohol/dopamine and danced like a peacock

Dr. Pratima Pandey and Anuradha Singh’s joint interview with Bio Patrika hosting “Vigyan Patrika”, a series of author interviews.

Pratima Pandey obtained her Ph.D. from CSIR, Institute of Microbial Technology, where she studied the halotolerant mechanism in a highly halotolerant yeast Debaryomyces hansenii. Post Ph. D., she joined Dr. Lynn Boyd’s lab at the University of Alabama Huntsville, USA, where she utilized an invertebrate, Caenorhabditis elegans, to study the neurological disorder Huntington’s disease. It initiated her long-lasting love for this model organism and neuronal studies. Before joining Dr. Kavita Babu’s lab at IISER Mohali, she worked with Dr. Harbinder Singh Dhillon in Delaware State University, USA, and started working on dopamine signalling pathways in C. elegans. In Dr. Kavita Babu’s lab, Pratima has investigated the molecular mechanism of neurotransmitters and their receptors at the C. elegans synapses and Neuromuscular junction. Her long-term goal is to decipher the cross-modulatory effects of neurotransmitters during interaction of the nervous system with the environment and the coordination of internal homeostasis.

Anuradha Singh is currently pursuing Ph.D. in Neuroscience in the lab of Dr. Kavita Babu at the Indian Institute of Science Education and Research. Mohali, India. Her research focus in on understanding the role of DOP-2, a dopamine autoreceptor, in ethanol dependent locomotion of Caenorhabditis elegans. She obtained her Masters of Science in Molecular and Human Genetics from Banaras Hindu University, Varanasi, Uttar Pradesh, India.

Here, first authors talk about their work published in Plos Genetics.

How would you explain your paper’s key results to the non-scientific community?

This is a story of alcohol-sensitive worms who were physically affected when exposed to an increased dose of alcohol. They could not walk their natural sinusoidal path but just kept dragging themselves in circular flat tracks. Alcohol is not an isolated abusive problem but leads to various other diseases such as obesity, fatty liver, oral cancer, and immense mental and emotional devastation that slowly plague society. With this complexity, there are daunting questions faced by researchers. Studies across various species showed that alcohol intake increases neurotransmitter dopamine release that induces the reward pathway, and we feel elated. This also results in loss of control over behaviours such as movement, sex, and speech. We studied how alcohol affected movement through dopamine in a nematode, Caenorhabditis elegans. It is fascinating to look at this miniscule worm and be in awe at its vast organ system, the genetic diversity that shows the high level of conservation with numerous aspects of mammalian systems. Our studies in this organism showed that if the gateman monitoring dopamine balance known as dopamine autoreceptor (DOP-2) is absent, dopamine will escape uncontrolled and affect the muscle activity, showing hyper contraction. Drunk animals have to drag themselves instead of their wavy movement where opposite muscles contract and relax. It could explain why drunk people cannot pass the sobriety walk and turn test.

What are the possible consequences of these findings for your research area?

The dopamine system in C. elegans is highly compact, with just 8 pairs of neurons compared to ~ 4 lakhs of neurons in mammals. Thus, it was easier to study the dopamine ethanol connection in this organism. Recently it has emerged as an invaluable tool to investigate various diseases and addiction to psychoactive drugs, including cocaine, methamphetamine, caffeine, and alcohol. Dopamine is involved in worm’s feeding, movement, learning, and memory. DOP-2 was found to have a minimal role, if any, when studied for dopamine-dependent behaviours, although its expression in dopamine neurons indicated it to be an autoreceptor or regulator of dopamine. C. elegans lives in soil and rotten fruits, so it is expected to possess an evolutionarily developed neuronal circuit beneficial for our alcohol-dependent investigations. In C. elegans, deletion mutants are available for most of the protein-encoding genes; hence we planned to test DOP-2 deletion worms for movement defect/s in the presence of alcohol (ethanol). We used increasing doses of ethanol and spread that on well-dried assay plates. Ethanol functions in a concentration-dependent manner, acting as a stimulant at lower concentrations and a depressant at higher concentrations. DOP-2 worms, when exposed to 400 mM ethanol, showed a fascinating behaviour called EIS (Ethanol Induced Sedative) behaviour. We utilized it to delineate synaptic dopamine signalling through dopamine receptor DOP-2, and showed that EIS behaviour is a direct outcome of increased dopamine levels. We showed that PDE neuron synapses onto DVA interneuron where NLP-12 (a neuropeptide) and DOP-1 (Dopamine receptor) are the two molecules important for movement regulation through motor neurons. Interestingly, it is quite distinct from the anterior dopamine circuitry that functions extra-synaptically to control various behaviours. Moreover, EIS behaviour can be used to screen for an array of chemicals/conditions that can enhance dopamine signalling and could serve to treat various dopamine-associated diseases.

What was the exciting moment (eureka moment) during your research?

The eureka movement in this project was when I found the drunk worms were moving in circles. I was exhausted from doing all kinds of assays to solve the mystery behind the function of DOP-2 in C. elegans. This molecule was expressed on all the Dopamine (DA) synthesizing neurons and few other neurons. However, its role in any dopamine-dependent behaviour could not be resolved. Thus, I thought that maybe if we escalate external conditions, we will get to know its function. So we exposed these animals to bit higher levels of alcohol. In spite of our efforts, there was no visible outcome during the time assays were performed. I Left the worms on ethanol plates overnight and checked the plates the next morning. I was surprised to find unusual circular tracks on the plates without any waves in movement as usually found in Wild type worms which showed normal tracks and had left the plates. I repeated this overnight exposure of worms to alcohol again and, after consistent results, finally discussed with Kavita, who was equally excited. Call it serendipity, luck, or our observation, we finally had phenotype and the assay to investigate the function of dopamine autoreceptor, DOP-2 in C. elegans. During that time, Anuradha joined our lab and started working with me on this project. Together we delineated the step-by-step signalling pathway that linked the loss of DOP-2 finally to another neurotransmitter, Acetylcholine. In this study, we showed that in the presence of alcohol, there is an increase in dopamine levels. Normally, DOP-2 takes care of that extra dopamine, but in its absence, it travels to the next cell and is welcomed by its receptor DOP-1. Here, it forces the cell to release a chemical neuropeptide, NLP-12. This molecule over activates motor neurons to hyper contract muscles via Acetylcholine. This was clearly affecting the posterior region of the worm as the worm was dragging its tail. In the completion of this fascinating story, Dr. Anidya Ghosh’s lab at NBRC Manesar helped to laser ablate targeted neurons in the transparent worm. It strengthened our results, and we confidently reported that PDE dopaminergic neuron activates DVA interneuron that communicates with motor neurons, and increased signalling through these neurons in DOP-2 mutants showed EIS behaviour.

What do you hope to do next?

Our manuscript creates a paradigm for future studies where post-withdrawal effects of this chronic ethanol exposure will be understood. It is quite relevant for human alcoholic studies as the alcohol concentration used is quite comparable to the drinking limit in humans. The behavioural assay designed by us is so much easier to perform and score that it can be utilized for drug screening to identify molecules with the ability to increase dopamine levels in the organism relevant for DA-associated diseases. Furthermore, alcoholism is hereditary, so transgenerational effects of chronic ethanol exposure will be studied on the progeny, and the knowledge generated can be utilized to overcome Alcohol use disorders (AUDs) in humans.

“Our manuscript creates a paradigm for future studies where post-withdrawal effects of this chronic ethanol exposure will be understood.”

Where do you seek scientific inspiration?

My scientific inspiration comes from the desire to seek answers to problems. I am always fascinated by people’s behaviours and their personalities. Thus I chose to study behaviours and their underlying reasons. I have been in research for a long time now. Still, every small successful experiment gives me immense joy, and I am ridiculed by failing experiments, but they drive me to seek many more answers. Being in Babu’s lab was sheer joy as I got to work with so many young, brilliant students, and scientific discussions ignited many new project ideas. In this project, graduate student Anuradha Singh worked tirelessly with me. It would not have been possible for me to accomplish this research outcome without her hard work and perseverance. It was our teamwork and Dr. Kavita Babu’s genuine support and guidance that sailed us through. It is such a treat to have worked with Dr. Kavita Babu; she is a very considerate, helpful, approachable, and genuine mentor. I learned various life and research lessons during the years spent in her lab. I loved the freedom to pursue my own research questions. Her ever-enthusiastic attitude towards science and your progress helps you move forward.

How do you intend to help Indian science improve?

In my view, it is imperative to encourage young minds at the early stages of their education to inculcate the desire for questioning and research. There are brilliant students who do not get into research either due to a lack of awareness about how to pursue research careers or feel that it is not rewarding. The government has already laid many efforts to popularise science in schools through tinker labs, but still much needs to be done. Students pursuing biology-related degrees in colleges and universities need to have good facilities so that they can pursue research questions, or there should be opportunities for them to undertake research activities in established labs. There is a need for an increase in investment towards the development of scientific ecosystems at large levels, leveraged with the timely release of funds, extending help to the upcoming researchers by providing mentoring options and incorporating feedbacks from scientists regarding their experience with respect to institutes and the funding agencies.

Reference

Pandey P, Singh A, Kaur H, Ghosh-Roy A, Babu K (2021). Increased dopaminergic neurotransmission results in ethanol dependent sedative behaviors in Caenorhabditis elegans. PLOS Genetics 17(2): e1009346. https://doi.org/10.1371/journal.pgen.1009346

Email: pratima.sharma@babulab.org; anuradha.singh@babulab.org

Dr. Kavita Babu lab: https://www.babulab.org/home

Edited by: Manveen K Sethi (Volunteer, Bio Patrika)

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