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. 2025 Jun;92:None. doi: 10.1016/j.conb.2025.103027

Mapping and decoding neuropeptide signaling networks in nervous system function

Isabel Beets 1,⁎, Jan Watteyne 1
PMCID: PMC12178255  PMID: 40262384

Abstract

Neuropeptides are widespread signaling molecules that are central to brain function in all animals. Recent advances in profiling their expression across neural circuits, in conjunction with detailed biochemical characterization of their interactions with receptors, have made it feasible to build brain-wide maps of neuropeptide signaling. Here, we discuss how recent reconstructions of neuropeptide signaling networks, from mammalian brain regions to nervous system-wide maps in C. elegans, reveal conserved organizational features of neuropeptidergic networks. Furthermore, we review recent technical breakthroughs in in vivo sensors for peptide release, receptor binding, and intracellular signaling that bring a mechanistic understanding of neuropeptide networks within experimental reach. Finally, we describe how the architecture of neuropeptide signaling networks can change throughout evolution or even the lifetime of individuals, which highlights the complexities that must be considered to understand how these molecules modulate circuit activity and behavior across different contexts.

Highlights

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    The mapping of neuropeptide signaling networks reveals conserved organizing features.

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    New in vivo sensors allow spatiotemporal decoding of dense neuropeptide networks.

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    Neuropeptide network architecture changes throughout an animal's lifetime and evolution.

Introduction

Nervous system function relies on two main modes of neuronal communication mediated by synaptic– or wired connections, and by “wireless” extrasynaptic signaling between neurons. The structure of synaptic connectomes has been comprehensively mapped by huge efforts in the field of connectomics that have generated complete wiring diagrams of nervous systems through anatomical reconstruction of synaptic contacts [1, 2, 3, 4]. However, the organization of wireless signaling networks by diffusible signaling molecules, such as monoamines and neuropeptides, has been a long-standing challenge because these communication routes cannot be inferred merely from neuroanatomy. Understanding the organizing principles of extrasynaptic signaling is particularly challenging for neuropeptides, the largest and most diverse group of neural messengers [5,6]. Neuropeptides, which mainly bind to G protein-coupled receptors (GPCRs), are ubiquitously expressed in animal nervous systems and have been implicated in nearly all brain functions [5, 6, 7∗∗, 8∗∗]. They play conserved roles in the regulation of many biological processes, including metabolism, development, reproduction, and the control of behavior, learning, and motivational states [9, 10, 11]. Despite their abundance, little is known about how networks of neuropeptide pathways are organized and interact in animal brains, in part because of technical challenges to map neuropeptide transmission between neurons. A mechanistic understanding of these networks, however, has recently come within experimental reach thanks to technological advances that enable charting neuropeptidergic interactions and visualizing neuropeptide activity in vivo.

Here we review recent insights into the structure and the conserved features of neuropeptide signaling networks, gleaned from comprehensive mapping of neuropeptide-receptor interactions and single-neuron gene expression in vertebrate and invertebrate nervous systems. We then cover in vivo sensors that have been recently developed for visualizing neuropeptide activity, and that allow measuring which neuropeptidergic pathways are active and when, focusing on developments over the past few years. Finally, we discuss examples of context-dependent plasticity that illustrate how the architecture of neuropeptide signaling networks changes over broad timescales. Together, these recent developments provide exciting new possibilities to delineate adaptive features and conserved principles of neuropeptide networks that are fundamental to nervous system function.

Mapping neuropeptide signaling networks – conserved organizing principles

Since the discovery of the first brain opioid receptor in the 1970's [12,13], many peptide-activated receptors have been found to regulate brain function. Animal genomes usually encode around 100–150 different peptide GPCRs, and a similar diversity is encountered in peptide-encoding genes [14,15]. Most peptides are released by neurons, referred to as neuropeptides, although they can also be expressed outside of the nervous system. In recent years, our understanding of neuropeptide and GPCR expression expanded enormously through single-cell RNA sequencing and systematic fluorescent reporter studies that have enabled cellular-resolution mapping of gene expression in animal brains [7,16, 17, 18, 19∗, 20]. A common finding of these studies has been the ubiquitous expression of neuropeptide and receptor genes in nervous systems; most neurons express multiple neuropeptides and peptide-activated GPCRs, often in combinations that are unique to a neuronal cell type [7,8,16,19,20]. Complex combinatorial expression patterns of neuropeptides and receptors have been found across animal phyla, including in sea anemones [21], C. elegans [7,22], zebrafish [19], and mammals [8,16], which suggests that states and information flow in the peptide network are specified through the concerted actions of multiple neuropeptides. Indeed, seminal studies of small invertebrate circuits revealed that neuromodulator interactions produce emergent effects, which are not easily predicted from the actions of individual modulators [23], and the mapping of neuropeptide networks has revealed dozens of putative interacting pathways (see below).

The broad neuronal expression of peptides and receptors suggests that they form an extensive network. Neuropeptide networks have been mapped by combining data from single-neuron transcriptomics with knowledge on peptide-receptor interactions, to predict neuropeptidergic signaling paths between neurons (Figure 1a) [6,24]. These network maps, referred to as neuropeptidergic connectomes, consist of nodes – representing individual neurons or cell types – that are connected by edges when cells express matching receptor-ligand pairs. The mapping of neuropeptide networks has long been hampered by a gap in knowledge on peptide-receptor interactions, but this knowledge expanded enormously with large-scale reverse pharmacology studies that have systematically screened peptide-GPCR pairs in heterologous expression systems [21,25, 26∗∗, 27]. Combined with gene expression studies, these datasets enabled reconstructing neuropeptidergic connectomes for the nervous system of C. elegans [24], the human brain [28], and brain regions such as the mouse cortex [8], the zebrafish telencephalon [19], the Drosophila clock circuit [29], the neuroglandular cells in Nematostella vectensis [21], and a neurosecretory center in Platynereis dumerilii [30]. Each network map highlights the vast potential for neuropeptidergic communication in animal brains, including many new neuron interactions.

Figure 1.

Figure 1

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The construction of neuropeptide signaling maps and in vivo sensors for peptide transmission reveal organizational features of neuropeptidergic networks.

a) The integration of single-cell gene expression resources with biochemical peptide-GPCR interaction datasets allows mapping the dense network of neuron-to-neuron neuropeptide signaling pathways, or ‘neuropeptide connectome’. Graphical representations show individual neurons as nodes connected by gray edges, depicting neuropeptidergic interactions. Individual neuropeptide-GPCR signaling networks typically differ in their topology, ranging from local networks in which both peptide and receptor are expressed in a few cell types, broadcasting networks formed by a small number of sender cells that transmit peptide signals to many receivers, to pervasive networks with a many-to-many signaling structure.

b) Neuropeptidergic connectomes across species show conserved organizing features of interacting peptidergic pathways. Peptidergic crosstalk arises from promiscuity in receptor-ligand pairing. In addition, cell-type specific combinatorial expression of neuropeptides and their receptors establishes interactions through i) convergent signaling in which different peptide-GPCR signals are integrated within the same target cell, ii) divergent signaling in which a cell releases neuropeptides that act on different receptor and cell targets, iii) neuropeptide cascades in which a peptide receptor controls the release of a non-agonist peptide with which it is co-expressed, and iv) autocrine signaling in which peptides signal back onto their sending cell.

c) The development of different in vivo sensors provides spatiotemporal access to different aspects of peptide transmission in neuropeptide networks: i) the timing and location of their release within circuits, ii) physicochemical diffusion and degradation in the extracellular space, iii) the spatial range and temporal dynamics of their interaction with receptors, and iv) intracellular signaling responses and integration into the biochemical state of target cells.

d) Plasticity in neuropeptide and receptor expression, during development, experience, or evolution, shapes the architecture of neuropeptide signaling networks across broad timescales.

C. elegans was the first organism for which a neuropeptidergic connectome of its entire nervous system has been constructed (Figure 2) [24]. This was achieved by mapping comprehensive resources for gene expression and receptor-ligand interactions onto the worm's defined neuroanatomy. A transcriptomic atlas, generated by single-cell RNA sequencing, revealed the pattern of neuropeptide and receptor expression in each neuron class of the C. elegans nervous system [7]. In addition, a recent deorphanization study identified 461 peptide-receptor pairs in C. elegans by systematically screening >55,000 possible interactions in heterologous cells [26]. Many receptor-ligand interactions and gene expression patterns were also validated in vivo [7,24,26]. The neuropeptidergic connectome generated from these datasets reveals a dense network that is potentially more connected than the known synaptic connectome, with >10-fold more neuron interactions [24]. Overall, the topology of the neuropeptide network differs from that of synaptic and monoamine networks in C. elegans (Figure 2a); for example, only 5 % of peptidergic connections overlap with synaptic connections between neurons, indicating that most neuropeptidergic pathways mediate extrasynaptic interactions [24]. As several C. elegans GPCRs remain orphan and more peptide receptors have been characterized [26,31,32], new extrasynaptic pathways may also be identified in the future. While highly connected, the C. elegans neuropeptidergic connectome displays several organizational features, such as neuron clusters with similar peptidergic connectivity [24]. Some of the hub neurons in the network overlap with those in the synaptic connectome, while others are unique to the neuropeptidergic connectome (Figure 2b) [24]. Interestingly, networks of individual neuropeptide-receptor pairs also exhibit diverse topologies. Some neuropeptides and receptors are expressed in only one or a few neuron classes, forming local networks, whereas others are widely expressed and may constitute broadcasting or pervasive networks [24]. A similar spectrum of topologies has been identified in vertebrate peptide networks [8,16,19], although the functional implications of distinct network architectures remain to be uncovered.

Figure 2.

Figure 2

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Neuropeptide network map of the C. elegans nervous system.

a) Juxtaposition of synaptic (chemical synapses and gap junctions), monoamine, and neuropeptide signaling maps of the C. elegans nervous system, derived from anatomical and gene expression analyses of different individuals. Data from Refs. [3,24,78,80]. Neuronal nodes are colored based on their anatomical classification, and their size is defined by their relative degree (total sum of number of incoming and outgoing connections). Node positions are not the same in all layers.

b) Position of the ten highest-degree hub neurons in the synaptic (blue) and neuropeptide (green) network, represented on the map of synaptic contacts in the C. elegans nervous system. Some neurons are among the highest-degree hubs in both synaptic and neuropeptide networks (orange). Data from Ref. [3,24].

c) Example of different neuropeptide signaling cascades found in the neuropeptidergic connectome of C. elegans, superimposed on the synaptic network of the C. elegans nervous system. Data from Ref. [3,24].

d) Diversity in autocrine peptide connections across the C. elegans nervous system. Cell body size indicates the number of autocrine neuropeptide-GPCR pairs co-expressed in that neuron. Circuits in which autocrine connections are especially prominent (those of the oxygen-sensing and locomotor circuit) are highlighted. Data from Ref. [24].

Comparing expression-based neuropeptide networks across species reveals several conserved features. Peptide networks commonly display a high density, which suggests that most neuronal cell types in animal brains are interconnected via neuropeptide signaling [8,19,24,33]. Another emerging theme is the abundant co-expression of neuropeptide pathways, indicative of combinatorial interactions (Figure 1b). For example, network analysis delineated dozens of peptidergic cascades in the C. elegans nervous system (Figure 2c), and more than half of C. elegans and mouse cortical neurons exhibit autocrine peptide connections (Figure 2d) [8,24]. Evidence from in vivo studies supports these types of interactions, as the activation of peptide GPCRs has been shown to regulate the release of their peptide ligands and/or other neuropeptides in the same neurons [34, 35, 36]. Fine-tuned modulation can also be achieved by co-expression of peptide GPCRs with similar or opposing actions on downstream signaling events, such as second messenger production [37∗∗, 38, 39]. A recent study in mice, for example, showed that satiation during feeding is gradually determined by competing peptidergic signals from hunger-promoting and satiety-promoting neurons, which have opposing effects on cAMP signaling in common target neurons [37]. Finally, peptidergic crosstalk can also arise from promiscuity in receptor-ligand pairing, as receptor deorphanization studies have identified many cross-interactions between neuropeptide ligands and receptors [25,26]. While most neurons express multiple neuropeptides and receptors, some of which are promiscuous, it remains unclear if neuropeptides are redundant or have cumulative actions. A recent study in C. elegans delineated functional redundancy between peptide receptors by phenotypic profiling of a combinatorial mutant library [40]. They used a CRISPR/Cas9-based approach to disrupt 1654 GPCR-encoding genes in 284 strains, by mutating a set of closely related receptors in each strain. Combined with the C. elegans neuropeptidergic connectome, similar strategies could be used to probe for interactions of co-expressed neuropeptides and GPCRs [40]. One of the main challenges in neuroscience will be to delineate how the interplay of neuropeptidergic signals, along with other communication routes, controls neural dynamics and behavior.

Measuring neuropeptidergic activity – spatiotemporally resolved peptide networks

The complex properties of neuropeptide signaling networks have driven the need for development of specialized tools that provide spatiotemporal access to neuropeptide transmission. Understanding which expression-based neuropeptidergic interactions are functional and when has been challenging due to a lack of specific in vivo sensors. In recent years, diverse tools have been developed for directly monitoring neuropeptide transmission. These methods are rapidly advancing our understanding of the temporal dynamics [41, 42∗, 43], compartmental release [44,45], and cellular integration of neuropeptide signals [37]. Here, we discuss examples of tools that can be used to probe the spatiotemporal organization of peptidergic networks, by measuring neuropeptide release, receptor binding, and downstream signaling (Figure 1c). As this is a fast-moving field, we mainly focus on developments over the past few years.

Diverse optical methods have been developed that directly measure the release of peptide-containing dense-core vesicles (DCVs). For example, peptides have been genetically labeled with pH-sensitive fluorescent indicators that report pH changes in the DCV lumen during exocytosis [46]. These reporters can be used to spatiotemporally resolve individual release events, especially when using techniques like total internal reflection fluorescence (TIRF) microscopy that only visualize DCVs just below the plasma membrane [47,48]. Similarly, neuropeptides have been tagged with fluorogen-activating proteins (FAPs) that fluorescence upon contact with extracellularly applied membrane-impermeant fluorogens, for example, used to visualize DCV exocytosis in the Drosophila brain [45,49]. Fluorescently labeled neuropeptides may show altered trafficking, which led to the development of a DCV release sensor that does not interfere with endogenous peptidergic signaling. This universal sensor consists of a pH-sensitive GFP variant attached to the luminal side of the DCV-specific membrane protein CYB561 [50]. Altogether, these tools are particularly useful for resolving the temporal dynamics and compartmentalization of neuropeptide release in peptide networks. Drosophila clock neurons, for example, were shown to use distinct mechanisms for the release of neuropeptides from either the nerve terminals or the soma, which in turn control different aspects of rhythmic behavior [45].

In addition, the development of genetically modified GPCRs that report peptide binding has provided a suite of versatile tools for studying neuropeptide transmission in vivo [51]. Fluorescent GPCR-based sensors allow monitoring where, how far, and how quickly neuropeptides travel once released and thus provide valuable tools for testing whether expression-based neuropeptidergic interactions are functional. These genetically encoded sensors are designed by inserting a fluorescent protein into the intracellular loop of a GPCR selectively binding the neuropeptide of interest. The receptor essentially acts as a ligand-recognition module, coupling ligand-induced conformational changes to an increase in fluorescence emission. Several GPCR-based peptide sensors have been developed for neuropeptides such as mammalian orexin [52], oxytocin [44,53], somatostatin [51], opioids [42,43], and Drosophila short neuropeptide F [41]. Their high sensitivity and rapid temporal binding kinetics have made them suited for real-time monitoring of endogenous neuropeptide signaling, even in freely behaving animals. Fluorescent GPCR sensors for oxytocin have for instance revealed ultradian oscillations in oxytocin levels, and compartmental release of oxytocin peptides during mating, in freely behaving mice [44,53]. In addition, real-time GPCR sensors are exquisitely suited to measure the spatiotemporal spread of neuropeptides through extracellular space. Various reports have documented extrasynaptic dispersion; for example, neuropeptides can reach receptors over 100 μm away from their release sites within seconds in the mouse brain [37,42,54].

Real-time fluorescent GPCR sensors report the binding of a receptor to its peptide ligand but not its intracellular signaling; because the insertion of a fluorescent reporter blocks coupling to effectors such as G proteins, these sensors typically do not induce downstream signaling cascades [42,51]. Other approaches are therefore needed to understand how and in which cells neuropeptides modulate cellular physiology. Transcriptional GPCR activity integrators mark cells in which GPCRs are activated by coupling the receptor's intracellular signaling cascade to the production of a transcriptional reporter [55]. Here, the C-terminus of a GPCR is fused to a non-native transcription factor through a tobacco etch virus (TEV)–protease cleavage site. Upon ligand activation, a TEV-protease fused to β-arrestin - a protein recruited to the GPCR following receptor activation [56] - is brought in close proximity of its target sequence, which releases the transcription factor and subsequently induces reporter-gene expression. The incorporation of a light-sensitive LOV protein that cages the TEV-protease cleavage site in the dark further increases temporal resolution by only allowing transcriptional activity to be induced during defined time windows [57,58]. While these transcriptional GPCR activity integrators are commonly used to monitor GPCR activation in cultured cells, they are only scarcely applied to study neuromodulator signaling in vivo [57,59]. They have complementary strengths to real-time GPCR sensors, such as their ability to stably report receptor activation over large populations of cells, and will hopefully be used more frequently, with further improvements, for in vivo studies in the future.

Monitoring changes in the levels of second messengers such as calcium, cAMP, and diacylglycerol downstream of G protein activation provides a straightforward way of measuring functional responses of peptide-GPCR signaling, especially since GPCR intracellular signaling usually involves at least one of these second messengers [60]. By far the most commonly recorded second messenger is cytoplasmic free calcium, largely owing to its levels serving as valuable proxy for neural activity [61]. A large body of calcium imaging studies has uncovered the multiplicity of ways in which neuropeptides modulate brain function by affecting neural activity. Recent advances have made it possible to analyze these effects at scale through large-volume calcium recordings in freely behaving animals and in combination with optical neuron stimulation. These studies revealed broad effects of neuropeptide signaling on neuron population dynamics, as expected from their ubiquitous expression. Neuropeptides modulate persistent neural activity patterns observed during behavioral states, such as aggression in mice and persistent foraging states in C. elegans [62,63]. In addition, neuropeptide signaling influences large-scale population dynamics on relatively short (<second) timescales, as recently shown in C. elegans [64]. This work combined whole-brain calcium imaging with optogenetic stimulation of each head neuron to delineate signal propagation across the C. elegans nervous system [64]. Many instances of fast, acute calcium transients were found to directly depend on DCV signaling, often in neurons that express receptors for neuropeptides released from the optically stimulated neuron [64]. While these calcium recordings already underscore the broad impact of the neuropeptide network on neural function, this is likely an underestimation since sensors to measure other second messengers, such as cAMP and diacylglycerol, are yet to be systematically applied at brain scale.

One particularly promising direction is the combination of these tools to connect different physiological aspects of neuropeptide action, or even to track the actions of multiple neuropeptides simultaneously. One elegant study used both real-time GPCR and cAMP sensors to show that stochastic neuropeptide Y and αMSH signals are integrated in hypothalamic target neurons and compete, through opposing effects on cAMP signaling, to calibrate the rate of satiation [37]. Similarly, simultaneously visualizing the effects of multiple neuropeptides and other neuroactive molecules, as was recently done in Drosophila [41] and the mouse brain [65], offers powerful new ways for dissecting information processing in brain circuits. Altogether, these new tools to measure neuropeptidergic dynamics and activity in vivo will bring a mechanistic understanding of neuropeptide networks and their functional principles.

Remodeling of neuropeptide signaling networks – peptidergic plasticity

While current in silico and functional analyses of expression-based neuropeptide networks are largely considering neuropeptide connectomes as static structures superimposed on neuroanatomy, transient or persistent changes in neuropeptide and receptor expression can widely impact the structure and function of these networks (Figure 1d). For example, neuropeptide networks are likely remodeled during development. In vertebrates, the extensive functional and structural changes that the brain undergoes from birth to adulthood are accompanied by changes in the expression of neuropeptides, such as galanin, that contribute to neurocircuit maturation [66,67]. Similarly, in C. elegans temporally regulated neuropeptide expression during post-embryonic nervous system development underpins temporal transitions in exploratory behavior [68]. Besides developmental programs, neuropeptide expression is shaped by experience and sex. Recent atlases of sex-specific gene expression in C. elegans unveil the sexually dimorphic expression of numerous neuropeptides and receptors, which in some cases drives sex-specific roles in stress responses or behaviors such as associative learning [69,70]. Neuronal transcriptomes also reveal broad peptide expression changes in response to sensory experience [71] or disruption of homeostasis, like sleep deprivation [72]. Network analysis of these types of peptidergic plasticity has been little-explored but may provide insight into the adaptive features or core conserved elements of peptide networks.

Such insights can also be gleaned from comparisons of neuropeptide networks across species. Neuropeptide gene expression can evolve rapidly; for example, regulatory changes in the neuropeptide gene encoding pigment dispersing factor (PDF) have been identified as a hotspot for circadian plasticity evolution in closely related Drosophila species [73]. Comparison of gene expression throughout the nervous system of three Caenorhabditis species also revealed striking patterns of evolutionary changes in neuropeptide and receptor expression [74]. Yet despite these widespread differences at the individual gene level, the overall topology of the neuropeptide network is conserved, with many neuron-to-neuron connections being mediated by distinct sets of neuropeptide-receptor pairs in different species [74]. This suggests that neuropeptide network structure is intimately linked to function, and that some aspects of its core functionality are maintained even when neuropeptide signaling adapts to distinct ecological demands. With the emergence of new transcriptomic datasets for different species and contexts, comparative analyses will shed further light on the common principles of peptide network architecture.

Concluding thoughts

The recent developments reviewed here are bringing forth an exciting new phase in our understanding of the structure and the functioning of nervous systems. Neuropeptidergic circuits are increasingly recognized as fundamental building blocks of animal brains [75], sparked by the extensive peptide networks that have been mapped in both invertebrates and vertebrates. While neuropeptides are well-known regulators of physiology and behavior, the extent to which they interconnect virtually all neurons has inspired an appreciation of their prominent roles in nervous system function, as modulatory signals– regulating the actions of fast neurotransmitters, and as primary information transmitters in complex behaviors [6,50,75,76]. Indeed, neuropeptidergic communication has a broad impact on neural circuit activity, as is evident from its effects on neuron population dynamics in studies from C. elegans to mammals [28,62∗, 63∗∗, 64∗∗]. The high density of peptide networks suggests a major role for local neuropeptide signaling in the control of circuit activity and behavior, which is supported by a large body of work on small invertebrate circuits [10,23,77,78]. While fewer local peptidergic circuits have been explored in vertebrates, accumulating evidence shows that they are important for complex behaviors such as learning [50,79]. Other aspects of neuropeptide pathway organization also exhibit striking parallels across animal species, such as cell type-specific combinatorial neuropeptide and receptor expression and receptor-ligand promiscuity. While peptidergic systems in vertebrates were initially considered to be largely different from invertebrates, comparative analyses in the past decade have revealed deep molecular and functional conservation of neuropeptide systems [9,14,15,26]. Given these conserved organizing features, we anticipate that further comparative studies of invertebrate and vertebrate neuropeptide networks will reveal principles that are fundamental to the function of nervous systems.

One of the main challenges ahead is to uncover the operating principles of dense neuropeptide networks. This will require functional studies, which are now primarily focusing on one pathway at a time, to be upscaled to the analysis of network activity. The recently developed suite of in vivo sensors for different peptides and receptor signaling cascades, combined with neuropeptide signaling maps, will be instrumental to understand how neurons process simultaneous neuropeptide signals with similar or opposing effects. This will be aided by future work that systematically explores the effects of neuropeptide-receptor pathways on cellular physiology. Another key aspect to investigate is which neuropeptidergic interactions are accessible and when, considering that peptidergic plasticity can alter network structure. The development of neuropeptide release reporters and GPCR-based sensors also provides access to little-explored processes that determine the spatiotemporal scope of neuropeptide signaling, like compartmental release, diffusion, and degradation of peptides. Ultimately, integrative approaches and computational models that relate spatiotemporally resolved patterns of neuropeptide transmission with anatomy, circuits, and behavior will provide exciting new possibilities to uncover how neuropeptides control internal brain states and behavior.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We acknowledge grant support from the European Research Council (ERC 950328), the Research Foundation Flanders (FWO G036524N and 12AKR24N), and the Baillet Latour Fund.

This review comes from a themed issue on Molecular Neuroscience 2025

Edited by Peter Scheiffele and Yulong Li

Data availability

No data was used for the research described in the article.

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