Fig 2: Immunohistochemistry of nerves at and along the posterolateral corner of the prostate (posteroinferior group of the pelvic plexus branches). Near-sagittal sections of specimens from a 77-year-old man. Panel a (HE staining; scale bar, 10 mm) displays the topographical anatomy of nerves within the prostate (PR) and rectum (REC). After rotation, a square in panel a is shown in panel b. Panel b displays nNOS immunostaining in a part of the periprostatic nerves: at this lower magnification (scale bar, 1 mm), strong expression of nNOS can be identified. Panels c, d, and e (adjacent sections), corresponding to a nerve indicated by a circle in panel b, show nNOS, VIP, and TH immunostaining, respectively (scale bar in panel c, 0.1 mm). The periprostatic nerve contains abundant nNOS- and TH-positive fibers, but VIP-positive fibers are rare

Fig 3: Nicotine selectively depolarized Pyr and VIP neurons. (a) Example recordings showing effect of nicotine (1 µM in bath) on membrane potential for each cell type. (b) Group data demonstrating nicotine-induced depolarization in Pyr and VIP cells and no effect on SOM or PV cells. Data points represent individual cells. (c) Nicotinic depolarization by layer for Pyr (left) and VIP (right) neurons. Depolarization was stronger in deeper layers for Pyr neurons. (d) Nicotine increased action potential firing rate in VIP cells (left) and firing appears to peak after several minutes (right, firing rate examined in 30 s bins). For statistical comparisons in this and subsequent figures, asterisks indicate: *p < 0.05, **p < 0.01, ***p < 0.001

Fig 4: Immunohistochemistry of nerves in an area between the rectum and levator ani muscle (posterosuperior group of the pelvic plexus branches). This area is characterized by the existence of VIP-positive nerves. The topographical relationship of the four nerves is shown in Fig. 1d. Panels a, d, g, and j display nNOS immunostaining; panels b, e, h, and k exhibit VIP immunostaining; panels c, f, i, and l exhibit TH immunostaining. One of the four nerves (panel e) does not express VIP. In a single nerve, panel d does not correspond to the site shown in panels e and f, as nNOS- (or TH-) positive nerves are restricted to the upper (or lower) side of the panel. VIP-positive fibers appear to be intermingled with nNOS-positive fibers. In panel i, TH-positive fibers are restricted to the left-hand side of the nerve. All panels were prepared at the same magnification (scale bar in panel a, 0.1 mm)

Fig 5: Effect of local VIP application on SC lumen size. (A) Protocol of VIP administration and schematic diagram of VIP distribution via the cornea. (B) Representative distribution of VIP–FITC (green signal) in the anterior segment (iris, trabecular meshwork, and SC), but not in the posterior segment (retina and optic nerve head). (C) Representative HE staining of the SC lumen and (D) immunohistochemical (CD31) staining of SC lumen. The SC lumen size increased significantly 6 hours and 12 hours after the start of local VIP application and returned to baseline values 24 hours after the start of local VIP application (each group n = 4). All groups were compared with the control. ***P < 0.001, ****P < 0.0001. Scale bars: 50 µm.