Fig 2: Features of two main classes of calcitonin gene-related peptide (CGRP)-positive axons in the major pelvic ganglion (MPG). (A) CGRP-positive axons surrounding several MPG neurons. Some neurons are associated with dense CGRP-positive, substance P (SP)-negative varicosities, but one neuron is surrounded by an axonal plexus that is both CGRP- and SP-positive. (B) Cholera toxin, subunit B (CTB)-positive neurons retrogradely labelled from the bladder body. Some of these CTB neurons are associated with varicose axons that are SP- or CGRP-positive, or labelled for both peptides. (C) Varicose CGRP axon encircling an MPG neuron, showing CGRP in the varicosities and the inter-varicose segments of the axon. This varicose plexus is vesicular acetycholine transporter (VAChT)-negative, but the neuron is also supplied by VAChT-positive, CGRP-negative terminals. Another CGRP-positive, VAChT-negative plexus of axons is at the bottom right of the image, and on the left, a field of neurons with VAChT-positive but weak or absent CGRP labelling. (D) A region of MPG where many of the neurons are supplied by boutons immunoreactive for VAChT-positive boutons, with or without CGRP immunoreactivity. (E) Higher magnification of noradrenergic neuron supplied by boutons that show both CGRP- and VAChT-immunoreactivity, amongst other noradrenergic neurons with no nearby CGRP-positive boutons. Calibration in panel A represents (µm): A (40), B (30), C (20), D (40), E (20). [Colour figure can be viewed at wileyonlinelibrary.com]

Fig 3: Peptidergic nociceptor markers are reduced in DRG from Nav-Tsc2 mice. Immunohistochemisty of adult L4 DRG transverse sections stained for SP and TuJ1 (A, B) or NF200 and CGRP (D, E), NF200 and TrkA (G, H), or NF200 and IB4 (J, K). Scale bar: 100 µm. Arrows point to neurons that colabeled with NF200. C, F, G, L, Total number of L4 DRG neurons labeling with markers as indicated. Individual animals plotted with mean ± SEM; N = 4-6; N.S. p > 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 4: Visualisation of sensory and autonomic neural projections to the lower urinary tract labelled following cholera toxin, subunit B (CTB) injection into the bladder body (C, H, I), bladder trigone (A, D) or proximal urethra (B, E, F, G). (A) L6 dorsal root ganglion, showing numerous CTB‐labelled neurons. (B) In B1, the pelvic nerve is shown at low magnification, with part of the major pelvic ganglion (MPG) at the top of the panel. Many MPG neurons are labelled with CTB. The pelvic nerve is shown at higher magnification in B2. The five fascicles of the pelvic nerve are numbered, with the smaller of fascicles 1 and 2 obscured by the larger fascicle. The majority of CTB‐positive axons are located in fascicles 3 to 5. Peptidergic calcitonin gene‐related peptide (CGRP) axons are located in all fascicles. (C, D) CTB‐positive axons aggregated in pelvic nerve fascicles 3–5 (fascicle 5 not shown in C). (E) CTB‐positive autonomic ganglion neurons in the MPG, showing bright immunoreactivity in cell bodies and faint labelling of proximal axons (arrows). (F) Pelvic nerve showing aggregation of CTB‐positive axons in fascicles 3–5 and their site of entry to the MPG (top); co‐staining for tyrosine hydroxylase (TH) shows the location of fascicles 1 and 2. (G) and (H) provide higher magnification views of fascicles 3–5 close to the MPG (G) and ~ 1 mm along the pelvic nerve (H); sensory and sympathetic axons are present in each fascicle. (I) Transverse section through a pelvic nerve fascicle containing many CTB‐positive axons, showing that many are CGRP‐positive but none are TH‐positive. A–E, G, H: wide‐field fluorescence microscopy; F, I, confocal microscopy. Calibration bar in panel a represents (µm): A (500), B1 (500); B2 (160); C (400), D (200), E (60), F (180), G and H (60), I (10). [Colour figure can be viewed at wileyonlinelibrary.com]

Fig 5: Tsc2 deletion in Nav1.8-positive neurons activates mTORC1 signaling and increases soma size. A, Western blotting of L4 DRG from adult Nav-Tsc2 mice showed decreased expression of Tsc2 and increased phosphorylation of the direct mTORC1 target S6 kinase T389 relative to control mice; N = 8–10. B, C, TMP histochemistry of adult control and Nav-Tsc2 L4 DRG. Scale bar: 50 µm; N = 6. D, E, Immunohistochemistry of adult control and Nav-Tsc2 L4 DRG for CGRP and NF200. Arrows point to CGRP-positive, NF200-negative neurons; N = 5. F, Average cell area of labeled neurons. Individual animals plotted with mean ± SEM; N.S, p > 0.05, ****p < 0.0001.