Fig. 2

Neurotransmitter distribution in larval VNC cell types. A Feature plots showing expression of neurotransmitter marker genes VAChT, Gad1, VGlut, Tbh, SerT, and TH. B Histogram showing the percentage of neurons in the VNC cell atlas classified as cholinergic, GABAergic, glutamatergic, and monoaminergic according to marker gene expression. C Histogram depicting the frequency of different combinations of neurotransmitter co-expression. D-I In vivo expression patterns of neurotransmitters. D Maximum intensity projection image depicting the distribution of GABAergic neurons (labeled by anti-GABA immunoreactivity) in a representative abdominal segment. The yellow dashed line marks the midpoint of the ganglion along the AP axis; GABA+ cells principally accumulate in the posterior portion of each ganglion. E Quantification of the number of GABA+ cells, the proportion of cells in each ganglion that were GABA+, and the percentage of GABA+ cells located in the posterior half of the ganglion. Points represent measurements from an individual ganglion, and measurements were grouped according to segmental identity (thoracic or abdominal). *P < 0.05, unpaired t-test with Welch’s correction. F Maximum intensity projection image depicting the distribution of serotonergic neurons labeled by anti-5-HT immunoreactivity in a representative abdominal segment. G Quantification of 5-HT+ cell distribution in the VNC. T1 typically has 3 pairs of 5-HT+ cells and all other segments typically have 2 pairs of 5-HT+ cells. Yellow arrowheads mark cell bodies. *P < 0.05, Kruskal-Wallis test with a post-hoc Dunn’s test. H Maximum intensity projection image depicting the distribution of dopaminergic neurons labeled by anti-TH immunoreactivity. Cell bodies are indicated as follows: white arrow marks medially located high TH expressing cell; white arrowheads mark laterally located intermediate TH expressing cells; yellow arrowheads indicated laterally located low TH expressing cells. I Quantification of cell distribution in the VNC. *P < 0.05, Kruskal-Wallis test with a post-hoc Dunn’s test. J UMAP plot of isolated, re-clustered TH+ cells shaded according to their cluster of origin. K Feature plots showing expression of GABAergic neuron marker genes Gad1 in TH+ cells. L Maximum intensity projection image depicting double-labeling of dopaminergic neurons with antibodies to TH and GABA. The white arrow indicates the medial high-TH neuron which is GABA-, the white arrowhead marks a TH+, GABA- cell, and yellow arrows indicate TH+, GABA+ double-positive cells. M Histogram depicting the number of TH+, GABA+ double-positive cells in each segment of the larval VNC. TH+, GABA+ double-positive cells are principally located in segments A2 and A3, which have the largest number of TH+ cells overall. *P < 0.05, Kruskal-Wallis test with a post-hoc Dunn’s test. Genotype for all panels: brp-T2A-QF2w / +; QUAS-mcd8GFP / +