Research News

Zebrafish goes CARTing!

A comprehensive study, the first of its kind in its in depth analysis, of CART (a neuropeptide with roles in appetite regulation) expression during different developmental stages of zebrafish has been recently reported by biologists at IISER Pune.

A peptide that determines your appetite and a model system where neuronal sensing of glucose, the critical energy currency, can be easily monitored—together, they make a good team to study development of feeding behavior. Dr Aurnab Ghose and Prof Nishikant Subhedar’s groups at IISER Pune have exploited this combination to monitor expression of the CART neuropeptide during different stages of zebrafish development.

Zebrafish as a useful system to study glucose sensing

With well-characterized developmental stages, translucent embryos and a fully sequenced genome, zebrafish is a useful vertebrate model organism for neuronal development and gene function studies. “Zebrafish has a simpler version of the vertebrate brain with lower number of neurons and simple neuronal circuits,” says Subhedar, who has a long-standing experience in studying neuropeptide action in other vertebrate models such as rodents. He adds, “Aurnab Ghose’s laboratory has already established zebrafish colonies at different developmental stages and is involved in understanding neuronal circuits. So, this fish seemed ideal to track CART peptide during development and to probe its role in glucose sensing.”

The different developmental stages that he is referring to are the embryonic, larval, juvenile and adult phases in the lifetime of zebrafish. Eggs released by female zebrafish are fertilized externally to form an embryo. During the next few days, the larvae are dependent on yolk sac for their nutrition. As the larvae develop, they become nutritionally independent of the yolk sac and learn to swim and feed actively.

What triggers the fish to realize hunger and seek food and what molecular features define satiety? Such questions on glucose sensing in the brain can be addressed using zebrafish: the first step that the authors took in this direction is to monitor CART expression in the brain of zebrafish in different stages of development. This information was not previously available for zebrafish, or for any other vertebrate.

CART in zebrafish: The whereabouts and possible new roles

CART (cocaine and amphetamine regulated transcript) codes for a neuropeptide involved in energy metabolism and several other biological functions. It is widely distributed in the brain, spinal cord and some peripheral tissues. CART peptide has earlier been found to produce satiety i.e., to inhibit hunger.

Along with project student Arghya Mukherjee (presently pursuing Ph.D. at FMI, Basel, Switzerland), Ghose and Subhedar looked for the presence of CART in various parts of brain during different stages of zebrafish development. This, in itself, was an extensive study and has generated an atlas of CART distribution in brain across different developmental stages.

In this study, CART peptide was found to appear as early as 24 hours post fertilization and was widely distributed by 30 days post fertilization. CART appears to be enriched in a number of sensory areas suggesting a general function in processing sensory information. Intriguingly, CART expression in the major neuronal tracts appear to peak precisely at the time of laying out of the brain communication highways. This may suggest a completely novel role for CART as a developmental cue.

Shown here is the entopeduncular nucleus (EN) region, which the authors propose is the primary site of glucose sensing in the zebrafish brain. Most neurons in the EN express the neurotransmitter/neuromodulator NPY (green), while a subset also expresses CART (red). Blue is DAPI staining of the nuclei 

 “There were a few interesting outcomes from this study,” says Ghose, “when we exposed 15-day old larvae to glucose, we observed an increase in CART containing neurons in a certain region of the brain. This suggests that the development of the glucose sensing system is timed to coincide with initiation of active free-feeding. Secondly, presence of CART in the pineal gland has been observed for the first time. This may give an insight into the workings of the pineal, a photosensory organ, in regulating day-night rhythms.”

“It would be really interesting to look for electrical activity in these glucose responsive neurons. Since zebrafish is great for genetic and pharmacological manipulations as well as imaging, this system should provide a unique advantage in teasing out the neuronal circuitry in glucose sensing,” says Ghose who is excited about the new angles one can now explore using the zebrafish model.

The study titled “Ontogeny of the cocaine- and amphetamine-regulated transcript (CART) neuropeptide system in the brain of zebrafish, Danio rerio” has been accepted for publication in the Journal of Comparative Neurology and is authored by Arghya Mukherjee, Nishikant Subhedar and Aurnab Ghose.

This research was supported by grants from the Indian Institute of Science Education and Research (IISER) Pune and the Department of Science and Technology (DST), Government of India.

Reported by Shanti Kalipatnapu

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