Except panel b, which shows a representative photomicrograph of 5-HT immunostaining of the HSN neurons, all the other panels show photomicrographs of 5-HT immunostaining of the head region. All the animals shown were adults with anterior to the left.
a. In WT animals, seven neurons in four classes (NSM, ADF, RIH and AIM) showed 5-HT immunoreactivity. c. In mod-5/SERT mutants, only NSM and ADF showed 5-HT immunoreactivity. d. In cat-1/VMAT mutants, 5-HT immunoreactivity in NSM and ADF was significantly reduced and undetectable in AIM and RIH. e. mod-5/SERT mutants bearing the AN::mod-5 transgene restored AIM 5-HT immunoreactivity, and f. showed 5-HT immunoreactivity in ectopic neurons. The percentage of each neuronal type stained in these strains is summarized in Table 1.

tph-1 mutants, and transgenic tph-1 and tph-1;mod-5 mutants expressing GFP-tagged TPH-1 in ADF, Ex[ADF::tph-1]), or in NSM, Ex[NSM::tph-1], were analyzed by anti-5-HT antibody staining. Photos show merge of green fluorescence of the transgenes and red fluorescence of 5-HT immunoreactivity to discern the neurons that express TPH-1 and the neurons that accumulate 5-HT.
a – b. tph-1 mutants showed no discernable 5-HT immunoreactivity in the head neurons (a) or in HSN (b).
c. tph-1 mutants bearing the Ex[ADF::tph-1] transgene showed 5-HT immunoreactivity in ADF, as well as in NSM, RIH and AIM.
d. tph-1;mod-5 double mutants bearing Ex[ADF::tph-1] showed 5-HT immunoreactivity in ADF only.
e. tph-1 mutants expressing the Ex[NSM::tph-1] transgene showed 5-HT immunoreactivity in NSM, as well as in RIH and AIM.
f. tph-1;mod-5 double mutants bearing Ex[NSM::tph-1] showed 5-HT immunoreactivity in NSM only.
All the animals shown were adults with anterior to the left. The arrows indicate GFP-tagged TPH-1 in ADF dendrites (d) and NSM processes (e). Quantification of the percentage of each neuronal type stained in the strains is presented in Table 1.

Representative photomicrographs of 5-HT immunostaining of WT and indicated mutants untreated and treated with 2 mg/ml of 5-HT or 0.1 mg/ml of 5-HTP for 5.5 hr. All the animals shown have anterior to the left.
a - b. tph-1 and tph-1;mod-5 mutants treated with exogenous 5-HT. Applying 5-HT restored 5-HT immunoreactivity in tph-1 mutants but not in tph-1;mod-5 double mutants.
c – e. bas-1/decarboxylase mutants untreated and treated with 5-HT or 5-HTP. Untreated bas-1 mutants showed no discernable 5-HT immunoreactivity. bas-1 mutants treated with 5-HT restored 5-HT immunoreactivity. 5-HTP treatment did not improve 5-HT immunoreactivity in bas-1 mutants.
f – k. 5-HTP treatment of WT, tph-1 mutants and tph-1;mod-5 double mutants. f – h show the head region, and i –k showed the middle to posterior portion of the body. 5-HT immunoreactivity was observed in all of the 5-HT-producing neurons in these genetic backgrounds. In addition, 5-HT immunoreactivity was detected in the dopaminergic neurons (CEP, ADE, and PDE), consistent with the expression pattern of bas-1/decarboxylase (Hare and Loer, 2004). Quantification of the percentage of each neuronal types stained in the strains is presented in Table 2.

a. The structure of mod-5 GFP reporters. Intron (line)/exon (bar) structure of the mod-5 gene is adapted from (Ranganathan et al., 2001). The structure of mod-5 GFP reporters is schematically depicted.
b. Pmod-5::gfp(A) expression in WT animals. Photo shows GFP expression in ADF, RIH and AIM, as well as in non-serotonergic cell g2, in a merged image of Nomarski microscopy and fluorescence microscopy.
c. Pmod-5::gfp(B) expression in WT animals. Photo shows merge of green fluorescence of the transgene and red fluorescence of DiI staining of the amphid chemosensory neurons indicating GFP expression in NSM, as well as in ADF, RIH and AIM.
d – f. MOD-5 immunostaining of WT and mod-5 mutants. d. In WT animals, MOD-5/SERT immunoreactivity was evenly distributed in AIM soma and axon, but it appeared to be enriched in axons in NSM. e. No staining was detected in any neuron in mod-5(n822) opal mutant (not shown), although rare and very faint staining of NSM axons (indicated by a thin arrow) was observed in mod-5(n3114) mutant. f. The AN::mod-5 transgene restored MOD-5 immunoreactivity in AIM, as well as in several ectopic cells (indicated by a thick arrow), but not in NSM in mod-5 mutants. NR, nerve ring.
g – h. Distribution of GFP-tagged CAT-1/VMAT in WT animals. CAT-1::GFP showed punctate pattern in the soma and processes in NSM, ADF and HSN, as well as in dopaminergic neurons including PDE. Notice that the pattern of CAT-1::GFP overlaps with MOD-5 immunoreactivity in NSM, but not in ADF. All the animals shown were adults with anterior to the left.

a – f. Photomicrographs of 5-HT immunostaining of mutants affecting SV secretion (snt-1 and snb-1), DCV secretion (unc-31), and both forms of secretion (unc-13, unc-18, unc-64 and unc-104). All the animals shown were adults with anterior to the left. Arrows point to bulged 5-HT immunoreactivity at the end of NSM axons, which could reflect the accumulation of unreleased 5-HT vesicles in the mutant neurons.
g. Quantification of 5-HT immunoreactivity in AIM. Data represent the average of 15 animals per strain ± SEM, and the value of the mutants is normalized to that of WT stained in parallel. The experiments have been performed multiple times and the results from the other trials were similar. ***, p < 0.001 Student’s t-test.

a – c. Locomotion rates of WT, mod-5 mutants, and transgenic mod-5 worms following brief food deprivation. PN denotes transgenes expressed in the 5-HT-producing neurons NSM and ADF. AN denotes transgenes under the control of the mbr-1 promoter, which is expressed in the 5-HT-absorbing neurons AIM and several non-serotonergic neurons (Kage et al., 2005). Each bar represents the average of at least three trials ± SEM, with five animals per strain per trial.
a. mod-5 mutants bearing the Ex[AN::mod-5] transgene fully corrected exaggerated slowing response.
b. Expressing the tetanus toxin light chain in WT animals by the Ex[PN::TeTx] transgene reduced slowing response, as seen in 5-HT deficient mutant tph-1. However, the Ex[AN::TeTx] transgene did not enhance slowing response as compared with WT animals.
c. Differential rescue of exaggerated slowing response of mod-5 mutants by transgenic expression of MOD-5 in 5-HT-producing neurons (PN::mod-5) and in non-serotonergic neurons (Pgcy-27::mod-5 and Pgcy-36::mod-5). The difference between mod-5 mutants and the transgenic animals is indicated (*, p<0.05, ***, p<0.001). mod-5; Ex[PN::mod-5] and mod-5; Ex[Pgcy-27::mod-5] differed from WT, p<0.001, p<0.01, respectively.
d. Quantification of 5-HT immunoreactivity in AIM. Data represent the average of 15 animals per strain ± SEM, and the value of the transgenic animals were normalized to that of non-transgenic WT stained in parallel. ***, p<0.001.
e - f. MOD-5 immunostaining of transgenic mod-5 mutants. e. The Ex[PN::mod-5] tansgene restored MOD-5 immunoreactivity in NSM and ADF, showing the specificity of the promoter for 5-HT-absorbing neurons. f. The Ex[Pgcy-36::mod-5] transgene produced ectopic MOD-5 expression in the URX and AQR neurons, consistent with the expression pattern of gcy-36 (Ortiz et al., 2006).
g - h. 5-HT immunostaining of transgenic mod-5 mutants. Noticing the 5-HT immunoreactivity in the ectopic neurons URX and AQR.

The ADF chemosensory neurons sense external cues; they may couple environmental food signals and 5-HT neurotransmission. The NSM secretory neurons detect food passing through the pharynx; they may couple food ingestion and 5-HT neurotransmission. Following food deprivation, ADF and NSM release 5-HT at the synapses, as well as into extrasynaptic space to induce slowing response. MOD-5/SERT in RIH and AIM absorb 5-HT from extrasynaptic space, preventing exaggerated response.