(a) Schematic representation of neuronal types with markers used for validation. Genes between brackets were predicted from RNAseq but not validated. (b) Sympathetic neuronal types verified in vivo by a combinatorial immunohistochemical approach. NA1-5 neurons all express TH and TRKA; ACh1-2 express VACHT and GFRα2; Glut neurons express VGLUT2. NA1 express RARRES1; NA2 and NA3 express NPY; NA2 but not NA3 are RET+ and Htr3aEGFP+, therefore NA2 are NPY+ RET+Htr3aEGFP+ and NA3 are NPY+RET-Htr3aEGFP-. NA2 also express GFRα3. NA4-5 was separated from NA1-3 based on their expression of GFRα2. NA4 and NA5 neurons were separated based on expression of ENC1. Hence, NA4 are GFRα2+ENC1+, whereas NA5 are GFRα2+ENC1-. ACh1 were separated from ACh2 neurons based on expression of SST. Ret expression was present at low levels (Retlow) in NA2 and NA4-5 but not in NA3. NA1 neurons were mostly RET- (see also Supplementary Fig. 1d). ACh1 and ACh2 express Ret at high levels (Rethigh). (c) Violin plots showing the mRNA expression of the selection of genes used to validate the existence of predicted neuronal groups. (d-t) Extended validation of neuronal types. (d) Triple immunohistochemistry for NPY, TOMATO (recapitulating TrkA expression) and RARRES1 (upper panel) and RET (lower panel) on adult SGs from TrkACre/+;R26RTOM/+ animals. Sections were first incubated and imaged with the first set of primary antibodies (rabbit anti-NPY, chicken anti-TOMATO and goat anti-RARRES1), then eluted (Glycine+SDS pH2, see Methods), then re-incubated with the second set of primary antibodies (rabbit anti-NPY, chicken anti-TOMATO and goat anti-RET). Inset and arrowheads show that NA1 RARRES1+NPY- neurons are generally negative for RET expression. NA1 neurons occasionally expressed NPY (20% of all RARRES1+ cells) and RET (30% of all RARRES1+ cells) but never together. (e) Triple immunohistochemistry for FST, NPY and GFP (recapitulating Htr3a expression) on P35 SGs from Htr3a-EGFP mice. Upper panel: FST+NPY- NA1 neurons express Htr3a (inset) whereas NA3 neurons are FST+NPY+Htr3aEGFP- (arrowheads); lower panel: NA2 neurons are FST+NPY+Htr3aEGFP+(inset). (f) Triple immunohistochemistry for GFRα3, GFP (recapitulating Ret expression) and NPY on adult SGs from RetCFP/+ mice shows NA2 cells are GFRα3+Retlow+NPY+ (inset). (g) Triple immunohistochemistry for NPY, RET and GFP (recapitulating Htr3a expression) on P35 SGs from Htr3a-EGFP mice. NA2 cells (inset NA2) are NPY+RET+Htr3aEGFP+, whereas NA3 cells (inset NA3) are NPY+ only. (h) Triple immunohistochemistry for GAL, NPY and GFP (recapitulating Htr3a expression) on on P35 SGs from Htr3a-EGFP mice. NA2 (inset) and NA3 cells (arrowheads) express GAL. (i) Triple immunohistochemistry for ESR1, NPY and GFP (recapitulating Htr3a expression) on P35 SGs from Htr3a-EGFP mice shows ESR1 is expressed in NA2 cells (inset). (j) Triple immunohistochemistry for CDH8, NPY and FST (both in green) and TH on SGs from adult wild-type mice. NA1-3 neurons do not express CDH8, which is expressed uniquely by NA4-5 groups (inset). (k) Triple immunohistochemistry for CDH8, GFP (recapitulating Ret expression) and TRKA on P11 SGs from RetCFP mice shows NA4-5 CDH8+ cells express Retlow (inset). (l) Triple immunohistochemistry for TYRO3, GFP (recapitulating Ret expression) and TH on adult SGs from RetCFP mice shows NA4-5 Retlow cells express TYRO3 (inset). TYRO3 is also expressed by ACh1-2 (TH-Rethigh neurons, arrowheads). (m) Triple immunohistochemistry for GFRα2, GFP (recapitulating Ret expression) and NPY on adult SGs from RetCFP mice shows NA4-5 Retlow cells express GFRA2. GFRA2 is expressed also by ACh1-2 (NPY- Rethigh neurons, arrowheads). (n) Triple immunohistochemistry for ENC1, TOMATO (recapitulating TrkA expression) and GFRΑ2 on adult SGs from TrkACre/+;R26RTOM/+ animals shows that NA5 GFRα2+ ENC1-negative cells (arrowheads) could be distinguished from ENC1-expressing NA4 cells (inset). (o) Triple immunohistochemistry for CNTN6, CDH8, and TH on P11 SGs from wild-type animals shows that NA5 GFRα2+ CNTN6-negative cells (arrowheads) cells can be distinguished from CNTN6-expressing NA4 cells (inset). (p) Triple immunohistochemistry for ENC1, GFP (recapitulating Ret expression) and TH on adult SGs from RetCFP/+ mice confirms that both NA4 ENC1+ (inset) and NA5 ENC1- (arrowheads) cells express Retlow. (q) Triple immunohistochemistry for SST, VACHT (Slc18a3) and DAPI on adult SGs from Wnt1-Cre;R26RTOM/+ animals shows that cholinergic neurons are divided into VACHT+SST- (ACh1 group) and VACHT+SST+ (ACh2 group) neurons (inset). (r) Triple immunohistochemistry for VACHT, GFP (recapitulating Ret expression) and VIP on adult SGs from RetCFP mice shows ACh1-2 VACHT+ neurons express Rethigh and VIP (inset). (s) Triple immunohistochemistry for VACHT, TH and ISL1 on P8 SGs from wild-type animals shows ISL1+ neurons that are associated to the sympathetic ganglion but do not express either TH or VACHT (arrowheads), thus they do not belong to NA1-5 or ACh1-2. (t) Double immunohistochemistry for VGLUT2 (Slc17a6) and TH on P8 SGs from wild-type animals shows that TH- neurons (arrowheads) express VGLUT2 and are therefore glutamatergic. The staining was carried out on a section consecutive of the one shown in (r). (u) Proportion of identified sympathetic cell types in the superior cervical ganglion (SCG), stellate ganglion and thoracic ganglia 1-4 and 5-12. In the SCG, only 55.8% of all neurons could be identified to belong to either NA1-5 or ACh1-2 neuronal types. Thus, about half of these neurons belong to classes of neurons yet to be discovered. (v) Soma size of sympathetic types. NA2, NA4 and NA5 show a significantly larger soma size compared to NA1, NA3, ACh1 and ACh2 (F(6, 14) = 30.94, P < 0.0001, one-way analysis of variance, n=3. Post-test: BonferroniÕs multiple comparison test. All data are presented as mean ± s.e.m.). (w) Double immunohistochemistry for NPY and RET on adult stellate sections from DbhCre;R26Tomato mice. Note NA2 neurons clustered together within the ganglion (dashed line). (b), (d-t) and (w) are representative images from experiments carried out on at least two animals. Scale bars: 5 μm (b), 20 μm (d-t), 50 μm (w).