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Application Note: Upd2/insulin modulate energy sensing neural circuits

Researchers from Fred Hutch have elucidated opposing roles for leptin/Upd2 and insulin in energy sensing neural circuits by using Aivia software to analyze fat-sensing neurons in Drosophila.

Accurate and continuous monitoring of energy stores maintains neural tone and drives behavioral decisions critical for survival in both invertebrates and vertebrates. Until recently, the precise mechanisms by which fat-sensing neurons received and reacted to fat-store information was unknown. Researchers Ava Brent and Akhila Rajan have demonstrated that the adipokine Upd2 induces structural changes in neurons that permit insulin release under nutrient surplus, and that insulin itself restores neural tone via negative feedback on the same neural circuit.

Analyzing fat-store related changes in synaptic structure

The ability to image and segment neurons was critical for Brent and Rajan's research because they needed to analyze subtle alterations to neuron morphology and structure. They focused on the presynaptic enlargements on the ends of axons called boutons. Using Aivia software, they developed an image segmentation protocol for analyzing boutons, thus enabling them to systematically monitor neuron structure.

"What set Aivia apart was the accessibility of the team to help users like us who are trying to do something different. With their help, we were able to develop an Aivia recipe to take our complex imaging data generated using basal morphology reporters and code them into objects. These objects were then described using properties such as number, volume, surface area, and intensity," said Rajan.

Figure 1. Segmentation analysis of Syt-GFP labeled boutons in STAT-expression neurons in the pars intercerebralis (PI) region of the Drosophila brain (reproduced with permission from [1]).

A homeostatic circuit

The resulting Aivia image analysis recipe, was designed to be applied to a large batch of imaging datasets, enabling Brent and Rajan to both count the number of boutons and to analyze structural changes. With their Aivia recipe in hand, they set out to examine to different signaling cues governing the fat-sensing neural circuit in Drosophila.

Brent and Rajan fed flies a high sugar diet to mimic nutrient surplus, monitoring Upd2 and insulin levels as well as bouton numbers. Their Aivia image segmentation recipe revealed a Upd2-dependent decrease in bouton number, and, therefore, synaptic contacts. This decrease in synaptic contacts released a "clamp" on insulin secretion, allowing the hormone to be released in response to nutrient surplus.

Further analyzing their images, Brent and Rajan also identified altered expression of several genes involved in cytoskeleton remodeling, including Aru and Bsg, suggesting that Upd2-dependent alterations to synaptic contacts result from actin cytoskeleton reorganization.

Surprisingly, bouton number returned to baseline after 5 days on the high sugar diet, suggesting the presence of a negative-feedback mechanism. Unexpectedly, the researchers found that insulin itself exerted the negative feedback on the neuronal circuit.

Figure 2. Inhibitory feedback promoting increase of Syt-GFP labeled boutons in response to insulin signaling (reproduced with permission from [1])

Now that the researchers know the how of neural circuit regulation, they want to determine how the critical fat-sensing hormones reach their target neurons in the first place.

"We are asking how these fat hormones transit the blood brain barrier," says Rajan. "To do this, we [will] again rely on imaging adipokine transit and hope to apply the techniques and recipes from our prior experience with Aivia."


  1. Brent, AE, and A Rajan (2020). Insulin and Leptin/Upd2 exert opposing influences on synapse number in fat-sensing neurons. Cell Metabolism, 32:5, 786-800.E7

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