Unlocking Neural Sexual Dimorphism: Neurotransmitter Remodeling and Multi-Transmitter Co-transmission

2026-08-17 11:37

Keywords: neurotransmitter; neuronal atlas; sexual dimorphism; nervous system; Caenorhabditis elegans

Introduction

Neurotransmitters mediate neuronal signal transmission and serve as key molecules for deciphering behavioral regulation and sexually dimorphic neural circuits in animals. Caenorhabditis elegans (C. elegans) exhibits two sexes, males and hermaphrodites, with distinct sexually dimorphic neural circuits. However, a whole-body neurotransmitter expression atlas at single-cell resolution has long been lacking, and conventional reporter systems suffer from positional deviation and aberrant expression. A study published in eLife adopted CRISPR knock-in combined with the NeuroPAL multicolor labeling system to systematically map neurotransmitter distribution across neurons, glial cells and non-neural cells.

Construction of SunyBiotech

SunyBiotech generated some strains that served as core experimental tools for systematic mapping of neurotransmitters and the discovery of novel neurotransmitter expression sites:

PHX7566 unc-47(syb7566[unc-47::sl2::gfp::h2b]) III

PHX6486 cat-1(syb6486[cat-1::sl2::gfp::h2b]) X

PHX6451 tph-1(syb6451[tph-1::sl2::gfp::h2b]) II

PHX7786 tbh-1(syb7786[tbh-1::sl2::gfp::h2b]) X

PHX7768 tdc-1(syb7768[gfp::linker::h2b::t2a::tdc-1]) II

PHX8255 cat-2(syb8255[cat-2::sl2::gfp::h2b]) II

PHX7290 snf-3(syb7290[snf-3::TagRFP::sl2::gfp::h2b]) II

PHX8870 oct-1(syb8870[oct-1::sl2::gfp::h2b]) I

PHX1048 hdl-1(syb1048[hdl-1::gfp]) IV

PHX4208 hdl-1(syb4208[hdl-1::t2a::3xnls::cre]) IV

PHX4491 unc-17(syb4491[unc-17::t2a::gfp:h2b]) IV

PHX4257 eat-4(syb4257[eat-4::t2a::gfp::h2b]) III

PHX5923 bas-1(syb5923[bas-1::sl2::gfp::h2b]) III

1.Neuronal Transmitter Patterns and Comprehensive Neurotransmitter Classification

This study first analyzed shared neural circuits between the two sexes. Acetylcholine, glutamate and GABA are the three major primary neurotransmitters in shared neurons, accounting for over 70% of all neurons. Their stable transmitter phenotypes sustain fundamental physiological functions such as feeding and locomotion (Fig. 1A). Two distinct neuronal signaling modes were identified: one group possesses complete neurotransmitter synthesis pathways to autonomously synthesize and vesicularly release 5-HT (Fig. 1B). The other lacks synthetic enzymes and uptakes exogenous transmitters via transporters including mod-5, oct-1 and snf-3 (Fig. 1C-1E). Pure peptidergic neurons such as PVQ were also identified, which lack transporters for classical small-molecule transmitters and rely solely on neuropeptides for signaling (Fig. 1F). A comprehensive classification framework for neuronal transmitters was thereby established.  

Figure 1. Neuronal Transmitter Patterns and Comprehensive Neurotransmitter Classification

2. Novel Neurotransmitters and Systemic Intercellular Signaling Networks

This work also expanded the understanding of classical neurotransmitters and uncovered unprecedented neurotransmission mechanisms. Betaine was validated as a novel synaptic messenger; specific neurons co-express vesicular transporters and betaine uptake proteins, enabling betaine release (Fig. 1E). Numerous unc-47-positive neurons lack GABA synthetase, indicating UNC-47 may mediate transport of inhibitory transmitters including glycine. Widespread bas-1 expression independent of known pathways suggests multiple uncharacterized monoamine signaling routes (Fig. 2A-2B). This study further demonstrates that chemical signaling is not restricted to nervous tissues, and a pervasive systemic intercellular communication network exists throughout C. elegans.

Figure 2. Novel Neurotransmitters and Systemic Intercellular Signaling Networks

3. Male-Specific Co-transmission and Sexually Dimorphic Neurotransmitter Remodeling

The research finally explored sex differences in neural circuits and revealed the molecular basis of male-specific neural regulation. Male-specific neurons in the tail show far higher levels of neurotransmitter co-expression than shared neurons, with some neurons releasing multiple signaling molecules simultaneously (Fig. 3A). Ray neurons and CP neurons associated with mating display prominent transmitter co-expression. Several male-specific neurons express only a single synthetic enzyme, implying the presence of novel trace amines (Fig. 3B-3C). Comparative analysis of homologous PVN and AIM neurons between sexes revealed upregulated transporter expression in male PVN neurons and complete transmitter phenotype switching in adult male AIM neurons (Fig. 1F and 3D). These findings confirm that sexual differentiation sculpts male-exclusive mating circuits via two mechanisms: quantitative modulation of neurotransmitter abundance and reprogramming of neuronal identity.

Figure 3. Male-Specific Co-transmission and Sexually Dimorphic Neurotransmitter Remodeling  

Conclusion

This study constructs the first single-cell-resolution whole-body neurotransmitter expression atlas of C. elegans and reveals the core mechanisms of neural circuit sexual dimorphism. Male-specific neurons function through multi-transmitter co-transmission, whereas neurons shared by both sexes achieve sex-specific differentiation via neurotransmitter abundance adjustment and cellular identity remodeling. This study validates betaine as a novel synaptic messenger and identifies multiple potential new neurotransmitter pathways. The findings provide a crucial reference model and dataset for investigations into neural sexual differentiation and neurotransmitter evolution.

Reference

Wang C, Vidal B, Sural S, Loer C, Aguilar GR, Merritt DM, Toker IA, Vogt MC, Cros CC, Hobert O. A neurotransmitter atlas of C. elegans males and hermaphrodites. Elife. 2024 Oct 18;13:RP95402.

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