A neurotransmitter atlas of C. elegans males and hermaphrodites

Abstract

Mapping neurotransmitter identities to neurons is key to understanding information flow in a nervous system. It also provides valuable entry points for studying the development and plasticity of neuronal identity features. In the C. elegans nervous system, neurotransmitter identities have been largely assigned by expression pattern analysis of neurotransmitter pathway genes that encode neurotransmitter biosynthetic enzymes or transporters. However, many of these assignments have relied on multicopy reporter transgenes that may lack relevant cis-regulatory information and therefore may not provide an accurate picture of neurotransmitter usage. We analyzed the expression patterns of 16 CRISPR/Cas9-engineered knock-in reporter strains for all main types of neurotransmitters in C. elegans (glutamate, acetylcholine, GABA, serotonin, dopamine, tyramine, and octopamine) in both the hermaphrodite and the male. Our analysis reveals novel sites of expression of these neurotransmitter systems within both neurons and glia, as well as non-neural cells. The resulting expression atlas defines neurons that may be exclusively neuropeptidergic, substantially expands the repertoire of neurons capable of co-transmitting multiple neurotransmitters, and identifies novel neurons that uptake monoaminergic neurotransmitters. Furthermore, we also observed unusual co-expression patterns of monoaminergic synthesis pathway genes, suggesting the existence of novel monoaminergic transmitters. Our analysis results in what constitutes the most extensive whole-animal-wide map of neurotransmitter usage to date, paving the way for a better understanding of neuronal communication and neuronal identity specification in C. elegans.

Figure 1.. Background on genes examined in this paper.

(A) Neurotransmitter synthesis and transport pathways. TH = tyrosine hydroxylase; TDC = tyrosine decarboxylase; TBH = tyramine beta hydroxylase; TPH = tryptophan hydroxylase; GAD = glutamic acid decarboxylase; AAAD = aromatic amino acid decarboxylase; VMAT = vesicular monoamine transporter; VAChT = vesicular acetylcholine transporter; VGAT = vesicular GABA transporter. Taken and modified from (Hobert 2013). (B) Graphic comparison of scRNA expression data and previously reported reporter expression data. See Table S1 for a more comprehensive version that includes expression of reporter genes in cells that show no scRNA transcripts. Note that scRNA expression values for eat-4 and unc-47 can be unreliable because they were overexpressed to isolate individual neurons for scRNA analysis (Taylor et al. 2021).

Figure 2.. Schematics of reporter knock-in alleles.

Reporter alleles were generated by CRISPR/Cas9 genome engineering. The SL2 or T2A-based separation of the reporter from the coding sequence of the respective loci enables targeting of the reporter to the nucleus (via the H2B tag), which in turn facilitates identification of the cell expressing a given reporter. Genome schematics are from WormBase (Davis et al. 2022).

Figure 3.. Summary of neurotransmitter usage and atlases.

Refer to Table 1, Table 2, and Supplementary Tables S2 and S3 for individual gene expression, rationale for neurotransmitter assignments, and more detailed notes. (A) ACh = acetylcholine; Glu = glutamate; DA = dopamine; 5-HT = 5-hydroxytryptamine, or serotonin; 5-HTP = 5-hydroxytryptophan; PEOH? = the neuron has the potential to use β-hydroxyphenethylamine, or phenylethanolamine; bas-1-depen-unknown MA? = the neuron has the potential to use bas-1-dependent unknown monoamines (histamine, tryptamine, PEA; also see Fig. S1); unknown MA? = the neuron has the potential to use non-canonical monoamines; (up) = neurotransmitter uptake; (syn) = neurotransmitter synthesis; * = dim and variable expression of respective identity gene(s) is detected. Variability could be due to one of the following reasons: (1) the gene is expressed in some but not all animals; (2) the gene is expressed in every animal but the level of expression is below detection threshold in some. Variability is detected only at low fluorescent intensity; at higher intensities, expression remains consistent. Results for anti-GABA staining in SMD and anti-5-HT staining in VC4, VC5, I5, URX, and PVW (male) are variable based on previous reports (see text for citations). (B) Information from A shown in the context of neuron positions in worm schematics. Note “unknown monoamine” here includes both “bas-1-dependent-unknown MA” and “unknown MA” in A. Neurons marked with “u” can uptake given neurotransmitters but not exclusively; some may also synthesize them, e.g., ADF can both synthesize and uptake 5-HT.

Figure 4.. Expression of eat-4/VGLUT and unc-17/VAChT reporter alleles in the adult hermaphrodite.

Neuronal expression of eat-4(syb4257) and unc-17(syb4491) was characterized with landmark strain NeuroPAL (otIs696 and otIs669, respectively). Only selected neurons are shown for illustrating updates from previous reports. See Table S2 for a complete list of neurons. (A) Dim expression of eat-4(syb4257) in head neurons ASK and ADL is consistent with previous fosmid-based reporter expression. RIC expression is consistent with previous observation using the same reporter allele (Reilly et al. 2022). In addition, dim expression is detected in pharyngeal neuron M5 (also in grayscale inset), previously not detected with eat-4 GFP fosmid-based reporter (otIs388) but visible with eat-4 mCherry fosmid-based reporter (otIs518). (B) Previously uncharacterized eat-4 expression in PDE and DVA neurons is detected with the eat-4(syb4257) reporter allele. Variable expression in PHA is also occasionally detected. No expression is detected in PVQ. Expression in PVN is detected in both sexes but at a much higher level in the male. (C) In the head, prominent expression of unc-17(syb4491) in RIP and dim expression in AWA and AFD neurons are detected. There is no visible expression in RIB, FLP, or AVJ. Consistent with previous reports, AIM expresses unc-17 only in males and not hermaphrodites. In addition, very dim expression of AVG can be detected occasionally in hermaphrodites (representative image showing an animal with no visible expression) and slightly stronger in males (representative image showing an animal with visible expression). Inset, grayscale image showing dim expression for AWA and AFD and no expression for RIB. (D) In the tail, PVN expresses unc-17(syb4491) in both sexes, consistent with previous reports. Scale bars, 10 μm in color images in A, C, and D; 5 μm in B and all grayscale images. Quantification in B is done by normalizing fluorescent intensity of eat-4 GFP to that of the blue channel in the NeuroPAL background. Statistics, Mann-Whitney test.

Figure 5.. Expression of GABAergic reporter alleles in the adult hermaphrodite.

(A) Expression of the unc-25/GAD reporter allele ot1372 is detected in the head, ventral nerve cord, and tail neurons. The expression pattern of this new T2A-based reporter allele is similar to that of a previously described SL2-based reporter allele, unc-25(ot867)(Gendrel et al. 2016). (B) Expression of unc-47/VGAT reporter allele syb7566. Left, the expression pattern of the reporter allele largely matches that of a previously described unc-47 mCherry fosmid-based reporter (otIs564) in the head. Right, a close-up view for the characterization of the reporter allele expression with landmark strain NeuroPAL (otIs669). In the head, consistent with previous reports of the unc-47 fosmid-based reporter (otIs564), dim expression of unc-47(syb7566) in SMD, ALA, and very dim and variable expression in IL1 is detected in both sexes, and unc-47(syb7566) is expressed in ADF only in the male and not hermaphrodite. In addition, the reporter allele is also expressed at a very dim level in the pharyngeal neuron I1 (also in inset) whereas no expression is detected in M1. In the tail, consistent with previous reports of the fosmid, sexually dimorphic expression of the unc-47(syb7566) reporter allele is also detected in PDB, AS11, PVN, and PHC only in the male and not the hermaphrodite. In addition, we also detected very dim expression of PLM in both sexes, confirming potential dim expression of the unc-47 mCherry fosmid-based reporter that was only readily visible after anti-mCherry staining in the past (Serrano-Saiz et al. 2017b). Scale bars, 5 μm for insets and 10 μm for all other images. (C) Expression of unc-46/LAMP reporter allele syb7278 is largely similar to that of the previously described unc-46 mCherry fosmid-based reporter (otIs568). We also observed expression of both the reporter allele and fosmid-based reporter in PVW, PVN, and very dimly in PDA. Scale bars, 10 μm.

Figure 6.. Expression of tph-1/TPH, bas-1/AAAD, and cat-1/VMAT reporter alleles in the adult hermaphrodite.

(A) Dorsoventral view of a hermaphrodite head and midbody expressing tph-1(syb6451). tph-1 expression is detected robustly in the MI neuron and dimly and variably in VC4 & VC5. Neuron identities for MI and VC4 & VC5 were validated using otIs518[eat-4(fosmid)::sl2::mCherry::h2b] and vsls269[ida-1::mCherry], respectively, as landmarks. Inset, grayscale image highlighting dim expression in VC4. (B) Neuronal expression of bas-1(syb5923) characterized with the landmark NeuroPAL (otIs669) strain in the head and midbody regions of young adult hermaphrodites. Dorsoventral view of the adult head shows bas-1/AAAD expression in left-right neuron pairs, including previously reported expression in NSM, CEP, ADF, and ADE (Hare and Loer 2004). Additionally, we observed previously unreported expression in the URB neurons. Non-neuronal bas-1/AAAD expression is detected in other non-neuronal cell types as reported previously (Yu et al. 2023) (also see Fig. 14, S3). (C) Lateral views of young adult hermaphrodite head and midbody expressing cat-1/VMAT (syb6486). Previously unreported cat-1/VMAT expression is seen in RIR, CAN, AUA, ASI (also in inset), and variably, ALA. Non-neuronal expression of cat-1/VMAT is detected in a single midbody cell in the gonad (also see Fig. S3), marked with an asterisk. Scale bars, 10 μm for all color images; 5 μm for the inset in grayscale.

Figure 7.. Expression of cat-2/TH, tdc-1/TDC, and tbh-1/TBH reporter alleles in the adult hermaphrodite.

Neuronal expression was characterized with landmark strain NeuroPAL (otIs669). Lateral views of young adult hermaphrodites expressing reporter alleles for (A) cat-2(syb8255), (B) tbh-1(syb7786), and (C) tdc-1(syb7768). (A) cat-2/TH expression in CEPs, ADE, and PDE match previously reported dopamine straining expression (Sulston et al. 1975). (B) and (C) Head areas are shown, no neuronal expression was detected in other areas. tdc-1 expression matches previous analysis (Alkema et al. 2005). We detected previously unreported expression of tbh-1 in all six IL2 neurons at low levels. Scale bars, 10 μm.

Figure 8.. Expression of mod-5/SERT and oct-1/OCT reporter alleles in adult animals.

Neuronal expression was characterized with landmark strain NeuroPAL (otIs669) and DiD-filling. (A,C) In adult hermaphrodites, mod-5(vlc47) is expressed in sex-shared neurons NSM, ADF, RIH, AIM, consistent with previous reports(Jafari et al. 2011; Maicas et al. 2021). In addition, we also observed expression in the phasmid neuron PHA and dim and variable expression in URX. There is no visible expression in the ventral nerve cord (VNC). (B,D) In adult males, mod-5(vlc47) is visibly expressed in NSM, RIH, AIM, as well as the male-specific neurons CEM, PGA, R3B, R9B, and CP1 to CP6. Expression in ADF is often not detected (see F). (E) DiD-filling confirms mod-5(vlc47) expression in phasmid neuron class PHA, and not PHB, in young adults in both sexes (L4 male image is to facilitate neuron ID in adults, because the positions of the two neuron classes can change in males during the L4 to adult transition). (F) Expression of mod-5(vlc47) in ADF is stronger in hermaphrodites than in males. In comparison, expression is not sexually dimorphic for the reporter alleles of either the 5-HT-synthsizing enzyme tph-1 or the vesicular acetylcholine transporter unc-17. (G) In the tail region of wild type males, male-specific neurons PGA, R1B, R3B, and R9B are stained positive for 5-HT. In a mod-5(n3314) mutant background, staining is completely lost in PGA (41/41 stained animals) and significantly affected for R9B (completely lost in 31/41 animals and much dimmer in the rest), while it remains in all 41 stained animals for R1B and R3B. The staining for CP1 to CP6 are also not affected in mod-5 mutant animals (remaining in 41/41 stained animals; image showing CP5 and CP6). (H,I) In adult animals, oct-1(syb8870) is expressed in the tyraminergic neuron class RIM in both sexes. Expression is not observed in any other neurons. (J,K) Outside the nervous system, oct-1(syb8870) is expressed in body wall muscle (BWM) throughout the worm (J) as well as hypodermal cells and selected head glia (K). Expression is also observed in gonadal cells in the male vas deferens (K). A pan-glial reporter otIs870[mir-228p::3xnls::TagRFP] and a dpy-7p::mCherry reporter stIs10166 [dpy-7p::his-24::mCherry + unc-119(+)] were used for glial and hypodermal identification, respectively. Scale bars, 10 μm.

Figure 9.. Expression of snf-3/BGT1/SLC6A12 in adult animals.

Neuronal expression was characterized with landmark strain NeuroPAL (otIs669) and DiD-filling. (A,B) In the adult hermaphrodite, neuronal expression of snf-3(syb7290) is detected in cat-1/VMAT-positive neurons AUA, CAN, and dimly and variably, RIR and ASI (confirmed with DiD-filling). In addition, it is also expressed in cat-1/VMAT-negative neurons AIB, RIM, RMH, SMD, VA12, DA9, PDA, PHC, PVN as labeled, as well as more neurons listed in Table S1. In the midbody, expression is not in PDE (dopaminergic, cat-1-positive) but in its associated glial cells. It is also detected in multiple vulval support cells (B) and some epithelial cells near the somatic gonad. (C) In the adult male, in addition to its expression in sex-shared neurons as in hermaphrodites, snf-3(syb7290) is also expressed in male-specific neuron class PDC, as well as in PHD and variably in PVV. (D) Similarly to its expression in hermaphrodites, snf-3(syb7290) is detected in CAN and PDE-associated glial cells, but not PDE neurons, in males. (E) In the male tail, snf-3(syb7290) is expressed in a number of glial cells including the spicule sockets and/or sheath cells (dorsal view). It is also detected in the somatic gonad (ventral view). (F) snf-3(syb7290) is broadly expressed in most if not all glia in both sexes. Glial cell type is determined by cell location and the appearance of their nuclei in Normarski. To confirm they are not neurons, a pan-neuronal marker (UPN, or “uber pan-neuronal”, a component in NeuroPAL) is used to determine non-overlapping signals between the two reporters. Head expression in the male is very similar to that in the hermaphrodite and thus not shown. (G) snf-3(syb7290) is broadly expressed in hypodermal and seam cells in both sexes. Scale bars, 10 μm. Asterisks, non-neuronal expression.

Figure 10.. Expression of eat-4/VGLUT and unc-17/VAChT reporter alleles in the adult male.

Neuronal expression of eat-4(syb4257) and unc-17(syb4491) was characterized with landmark strain NeuroPAL (otIs696 and otIs669, respectively). Only selected neurons are shown to illustrate updates from previous studies. See Table S3 for a complete list of neurons. (A) eat-4(syb4257) expression. Top, long panels: CA1, CA2, and CA3 show visible, albeit very dim, novel expression of eat-4 (also expressed in CA4). Bottom panels: CA7 strongly expresses eat-4(syb4257), whereas CP7 does not. Neuron IDs for these two neurons were previously switched (Serrano-Saiz et al. 2017b). (B) unc-17(syb4491) expression. Top, long panels: ventral view of a male ventral nerve cord showing high levels of expression in CA1, CA2, and CA3 and previously unreported low levels of expression in CP1, CP2, and CP3. Middle panels: low levels of expression in CA7 and CP7. There is no visible expression in CP9. Bottom panels: lateral view of a male tail showing previously unreported dim expression in R1B, R4B, R5B, R7B, and R9B; ventral view of the pre-anal ganglion showing expression in DX3/4. Scale bars, 10 μm. (C) The updated neurotransmitter atlas underscores the molecular diversity of the male-specific ventral cord neuron class CA and CP. Based on their expression patterns for neurotransmitter genes, these neurons can be potentially grouped into the following 4 CA and 5 CP sub-classes. See Table S3 and Figs. 10–12 for all genes mentioned in the following. CAs: (1) CA1 to CA4: express unc-17, unc-47, very weak eat-4; (2) CA5, 6, 8: express unc-17 and unc-47; (3) CA7: expresses unc-17, strong eat-4, and no unc-47; (4) CA9: expresses weak unc-17. CPs: (1) CP0: only expresses weak eat-4; (2) CP1 to CP4: express unc-17, unc-47, cat-1, tph-1, bas-1, and stain for 5-HT; (3) CP5, 6: express eat-4, unc-47, cat-1, tph-1, bas-1, and stain for 5-HT; (4) CP7, 8: express very weak unc-17 and unc-47; (5) CP9: expresses strong unc-25 and unc-47, and stain for GABA. The proposed sub-classification for these neuron classes indicates different functions of individual CA and CP neurons; it also provides useful resources for ongoing single-cell sequencing efforts for male-specific neurons.

Figure 11.. Expression of GABAergic reporter alleles in the adult male.

Neuronal expression of unc-25(ot1372) and unc-47(syb7566) reporter alleles was characterized with landmark strain NeuroPAL (otIs669). Only selected neurons are shown to illustrate updates from previous reports. See Table S3 for a complete list of neurons. (A) unc-25(ot1372) is expressed in male-specific CP9 and EF neurons as well as a few sex-shared neurons, all consistent with previous reports (Gendrel et al. 2016; Serrano-Saiz et al. 2017b). (B) unc-47(syb7566) shows expression in male head neuron classes MCM and CEM, the former previously undetected and the latter consistent with fosmid-based reporter otIs564. (C) unc-47(syb7566) shows expression in a number of ventral cord CA and CP neurons, largely consistent with reported otIs564 fosmid-based reporter expression except for no visible expression of syb7566 in CA7 (due to its initial confusion with CP7, described in Fig. 10) and presence of very dim expression in CP7. The syb7566 reporter allele is also not visible in CA9. Scale bars, 10 μm.

Figure 12.. Expression of the cat-1/VMAT, tph-1/TPH, and bas-1/AAAD reporter alleles in the adult male.

Neuronal expression was characterized with landmark strain NeuroPAL (otIs669). (A) Novel cat-1(syb6486) expression is seen in male-specific neurons PDC, PVY, PVX, R2A, and R4B. Consistent with previous reports, it is also expressed in HOA, PGA, R5A, R7A, R9A, R1B, and R8B. Its expression in ventral cord neurons CP1 to CP6 is consistent with earlier studies. (B) tph-1(syb6451) is expressed in male-specific head neuron class CEM and sex-shared neurons ADF, NSM, and MI. Similar to its expression in hermaphrodites, tph-1 in MI was previously undetected. In the tail, in addition to previously determined expression in R1B, R3B, and R9B, tph-1(syb6451) is also expressed at very low levels in R4B and R7B. Ventral cord expression of tph-1(syb6451) in CP1 to CP6 is consistent with previous reports and thus not shown here. (C) bas-1(syb5923) is expressed in previously identified NSM, ADE, PDE, and CEP neurons. In addition, we detected weak expression in URB as in the hermaphrodite. We also updated bas-1/AAAD expression in 39 male-specific neurons and 1 more with variable expression (see Table S3 for complete list). Neurons are also shown in grayscale for clearer visualization in some cases. Scale bars, 10 μm. Asterisks, non-neuronal expression, also see Fig. 14 and Fig. S3.

Figure 13.. Expression of cat-2/TH, tdc-1/TDC and tbh-1/TBH reporter alleles in the adult male.

Neuronal expression was characterized with landmark strain NeuroPAL (otIs669). (A) cat-2(syb8255) is expressed in male-specific neurons R4A, R7A, and R9B. This expression, as well as its expression in sex-shared neurons PDE, CEP, and ADE, is consistent with previous reports (Sulston et al. 1975; Sulston et al. 1980; Lints and Emmons 1999). (B) tdc-1(syb7768) is expressed in sex-shared neurons RIM and RIC and male-specific neurons HOA, R8A, and R8B, all consistent with previous studies (Serrano-Saiz et al. 2017b). We also detected weak expression in R7A. (C) tbh-1(syb7786) is expressed in RIC, consistent with its previously reported expression in hermaphrodites. As in hermaphrodites, we also detected tbh-1(syb7786) in IL2 neurons of the male. In male-specific neurons, previously unreported expression is detected in CEM, HOB, and all type B ray neurons except for R6B. Intriguingly, this expression pattern resembles that of pkd-2 and lov-1, both genes essential for male mating functions (Barr and Sternberg 1999; Barr et al. 2001). Inset, grayscale image showing dim expression for IL2 neurons. Scale bars, 10 μm. Asterisks, non-neuronal expression, also see Fig. 14 and Fig. S3.

Figure 14.. Expression of neurotransmitter pathway genes in non-neuronal cell types.

Multiple neurotransmitter pathway genes show expression in glial cells (A,B) and other non-neuronal cell types (C-E). Also see Fig. S3 for whole-worm views that capture more non-neuronal expression. (A) bas-1(syb5923), cat-2(syb8255), and cat-1(syb6486) reporter alleles exhibit expression in the male spicule glial cell types, largely consistent with previous reports.(Lints and Emmons 1999; Hare and Loer 2004; Leboeuf et al. 2014) (B) top 6 panels: bas-1(syb5923) is expressed in additional, multiple glial cell types in the male tail. Left 3 panels: bas-1(syb5923) crossed into a pan-glial reporter otIs870[mir-228p::3xnls::TagRFP], confirming its expression in glial cells; right 3 panels: bas-1(syb5923) shows no overlap with the pan-neuronal marker component in NeuroPAL (otIs669). Bottom 2 panels: bas-1(syb5923) also shows expression in at least two glial cells in the head. A hermaphrodite head is shown here. Expression is similar in the male. (C) In the hermaphrodite vulval region, tdc-1(syb7768) is expressed in uv1, consistent with previous reports (Alkema et al. 2005). This expression in uv1 is not observed for either cat-1(syb6486) or oct-1(syb8870). An ida-1p::mCherry integrant vsls269[ida-1::mCherry] was used for identifying uv1. (D) Detection of eat-4(syb4257) expression in muscle cells in both sexes, most prominently in the head. (E) cat-1(syb6486), tdc-1(syb7768), and tbh-1(syb7786) are expressed in the male somatic gonad. All three have expression in the vas deferens; additionally, cat-1 and tbh-1 are also expressed in the seminal vesicle.