# A New Method for Imaging Neuropeptide Release in the Brain

> **NIH NIH R21** · UNIVERSITY OF PITTSBURGH AT PITTSBURGH · 2020 · $196,735

## Abstract

Neuropeptides modulate synapses and circuits to control mood and a wide variety of behaviors
including appetite, pain perception and circadian rhythms. Despite the importance of neuropeptides, it
is not possible to detect synaptic neuropeptide release in the intact living brain with current methods.
However, we recently developed a new optical approach for imaging exocytosis of neuropeptide-containing dense-core vesicles (DCVs) at intact living Drosophila synapses. This approach is based
on inserting a fluorogen activating protein (FAP), which confers fluorescence on the normally
nonfluorescent dye malachite green (MG), into a proneuropeptide, thus targeting the FAP to the DCV
lumen. Following extracellular application of membrane impermeant MG derivatives that are small
enough to pass through fusion pores, activity-evoked fusions of individual DCVs can readily be
resolved at the Drosophila neuromuscular junction. Furthermore, we detected a novel mode of DCV
spontaneous exocytosis that is distinguished by its sensitivity to perturbations of the secretory
apparatus (e.g. resistance to tetanus toxin). Finally, preliminary studies show that the neuropeptide-FAP approach is applicable to studying circadian peptidergic neurons in the intact adult Drosophila
brain. Therefore, to demonstrate the utility of FAP imaging, this approach will be used to answer
fundamental questions regarding the function of the circadian circuit in the Drosophila. For example,
we will determine the timing of neuropeptide release by multiple neurons and address whether a
newly discovered mode of spontaneous release accounts for tetanus toxin resistant behavioral effects
of a fly neuropeptide. Furthermore, we will use new dyes, a second spectrally distinct FAP variant and
genomic engineering to enable simultaneous imaging of synaptic release of two neuropeptides under
native transcriptional control. In addition to testing specific hypotheses about peptidergic transmission
in the circadian circuit of the adult fly brain, these studies will serve as proof of principle examples for
applying real time neuropeptide-FAP imaging to other systems including the mammalian central
nervous system.

## Key facts

- **NIH application ID:** 10016856
- **Project number:** 5R21NS115023-02
- **Recipient organization:** UNIVERSITY OF PITTSBURGH AT PITTSBURGH
- **Principal Investigator:** EDWIN S LEVITAN
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $196,735
- **Award type:** 5
- **Project period:** 2019-09-15 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10016856

## Citation

> US National Institutes of Health, RePORTER application 10016856, A New Method for Imaging Neuropeptide Release in the Brain (5R21NS115023-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10016856. Licensed CC0.

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