Cellular source and circuit context organize functional specificity in the Drosophila NPF system
Madison N. Endres, Tarandeep Singh Dadyala, Kevin William ChristieiD, I.G. SinakevitchiD, Lisha ShaoiD
Abstract
Neuropeptides regulate diverse and sometimes opposing functions, yet how a single peptide achieves functional specificity remains unclear. Receptor diversity can provide specificity, but not in systems with only one receptor. Drosophila neuropeptide F (NPF) acts through a single receptor, providing a model to test whether specificity is organized by the peptide's cellular source. Using the whole-brain connectome, we defined four adult NPF cell types with no shared first-order synaptic partners and separate networks. These cell types made distinct and partly opposing contributions to reinforcement, feeding, mating, and energy storage. Most peptide-dependent effects were undetectable after population-level NPF reduction, showing that global perturbation can obscure source-specific functions. Changing neuronal activity and reducing NPF in the same cells produced different, sometimes opposing, phenotypes, so neuronal activation cannot be equated with peptide action. Thus, cellular source and circuit context generate functional specificity that population-level perturbation conceals.
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In-depth summary
Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (17 Sept 2026) · CC BY 4.0
Four connectome-defined Drosophila NPF cell types sit in separate circuits with distinct, sometimes opposing functions; population-wide NPF knockdown hides these, and activity manipulation can differ from peptide loss.
A neuropeptide acting through a single receptor cannot rely on receptor diversity for functional specificity, so the paper tests whether the cellular source of the peptide organizes its functions. It says earlier work had not mapped the function of adult NPF neurons cell type by cell type using the connectome.
Key results
ShownThe four adult NPF cell types (DM, L1, P1, P2) sit in largely separate input-output networks in the FlyWire/FAFB connectome.
No synapses between different NPF cell types; only one first-order presynaptic partner shared (DM and L1); no shared first-order postsynaptic partners; second-order partner overlap modest, with cosine similarity highest between DM and L1. · NPF cell types possess distinct connectomic…
ShownActivating or silencing each cell type gave different behavioral effects. Only DM changed positional preference, DM, L1 and P2 activation each produced appetitive memory, and DM and P2 activation drove calcium responses in PAM dopaminergic axons.
P1 activation decreased food intake; silencing DM or L1 increased it; activating DM or P1 reduced copulation. PAM responses were in the γ4 and β'2 compartments for DM and P2 only. N = 15-25 (positional preference), 11-35 (learning), 22-24 (feeding), 29-59 (mating). · Distinct NPF cell types differentially recruit…; Neural activity manipulations reveal…; Figure legends
ShownCell-type-specific npf RNAi produced distinct peptide-dependent phenotypes, but pan-NPF knockdown retained only the glycogen reduction.
DM knockdown reduced feeding, copulation and glycogen; L1 reduced feeding, triglyceride and glycogen; P1 increased feeding; P2 reduced glycogen. NPF-GAL4 knockdown showed no feeding, mating or triglyceride change. N = 20-23 (feeding), 29-36 (mating), 20 (metabolites). · Cell-type-specific NPF knockdown reveals…; Figure legends
ShownIn DM and L1, neuronal silencing and npf knockdown gave opposite feeding phenotypes, whereas in P1 activation and knockdown agreed, so manipulating neuronal activity does not always report NPF action.
In DM and L1, neuronal silencing increased feeding while NPF knockdown decreased it, consistent with DM/L1-derived NPF promoting intake while the neurons' aggregate activity-dependent output suppresses it. In P1, activation and knockdown agreed, both pointing to P1-derived NPF suppressing feeding. · Neural activity manipulations reveal…; Cell-type-specific NPF knockdown reveals…; Neuronal activity is not equivalent to peptide…
ShownNPF contributes conditionally to the effects of activating the whole NPF population, depending on stimulation intensity.
npf RNAi attenuated activation-induced feeding suppression at 6 mW/cm² but not 12 mW/cm², and positional preference at 0.6 mW/cm² but not 1 mW/cm². Appetitive memory at 1 mW/cm² was not reduced in fed or starved flies. · NPF peptide contributes conditionally to…; Neuronal activity is not equivalent to peptide…; Figure legends
Methods, data and reproducibility
The authors defined four NPF cell types in the FlyWire/FAFB connectome, generated stable split-GAL4 drivers for DM, P1 and P2, and used an existing driver for L1. They compared optogenetic activation or silencing with cell-type-restricted npf RNAi across calcium imaging, behavioral and metabolic assays, and also tested pan-NPF activation combined with RNAi.
- FlyWire/Codex whole-brain connectome of FAFB, snapshot 783 · Drosophila melanogaster
- Cell-type-specific and pan-NPF activation/silencing: positional preference, olfactory learning, feeding and mating · Drosophila melanogaster · n = N = 15-25 (position), 11-35 (learning), 22-24 (feeding), 29-59 (mating)
- Cell-type-specific and pan-NPF npf RNAi: feeding, mating, triglyceride and glycogen · Drosophila melanogaster · n = N = 20-23 (feeding), 29-36 (mating), 20 (metabolites)
- Ex vivo two-photon calcium imaging of PAM dopaminergic axons during NPF cell-type stimulation · Drosophila melanogaster
- Methods & tools
- FlyWire/Codex, NeuronBridge, split-GAL4 drivers, CsChrimson, A1ACR, GCaMP7s, flyPad/optoPad, Fly Flirt, MATLAB, GraphPad Prism, Jaccard similarity and hypergeometric tests, cosine similarity of synapse-weighted partner vectors
- Software & environment
- Connectome analysis used FlyWire/Codex snapshot 783 with custom Python code; analysis used GraphPad Prism and custom MATLAB scripts. No version pins or containers stated.
- Reproducibility gaps
- Raw imaging and behavioral data and newly generated fly lines are available only on request from the lead contact. No public data repository or accession is given.
Limitations (5)
- Only four sexually monomorphic central cell types were studied, not the whole NPF system; synaptic connectomics cannot identify the full set of extrasynaptic peptide targets. · stated by the authors
- RNAi reduced but did not eliminate NPF immunoreactivity, and knockdown was not adult-restricted, so developmental compensation and residual peptide could shape phenotypes. · stated by the authors
- Positional preference and conditioning were tested only with activity manipulation, and metabolic readouts only with knockdown, so the two perturbation maps are not fully symmetric. Obscuring in pan-NPF knockdown is not shown to be cancellation. · stated by the authors
- The pan-NPF driver also labels gut enteroendocrine cells, so the pan versus cell-type comparison mixes central and peripheral sources. · noted in this summary
- Group assays count each cohort as N = 1, and the sample sizes (for example 11-35 for learning) are modest for the many comparisons across cell types and assays. · noted in this summary
Who should read:Read this if you study neuropeptide circuits or want connectome-guided cell-type drivers and a worked comparison of neuronal activation versus peptide knockdown in Drosophila.
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