Fig. 2: Courtship and agonistic calling differentially activates midbrain periaqueductal gray (PAG).

a–c Male midshipman (Porichthys notatus) produce three, acoustically distinct vocalisations during the breeding season. Scale bars = 500 ms for a–c. d Midshipman vocal motor network, sagittal view. Abbreviations: PAG rostral (r) and caudal (c) zones; preoptic area, POA; vocal motor (VMN), vocal pacemaker (VPN) and vocal prepacemaker (VPP) nuclei ventral tuberal hypothalamus, vT. ei, eii Nissl-stained, coronal sections illustrating PAG zones (rostral and caudal superficial: rs, cs; caudal deep, cd; levels indicated in d); fi, fii are higher magnification views of ei and eii, respectively. Scale bars = 250 µm for fi, fii. LL lateral lemniscus, MLF medial longitudinal fasciculus, nLV nucleus lateralis valvulae, PAGm medial PAG, TeM midbrain tectum, Pe periventricular layer of auditory division of torus semicircularis, PTT paratoral tegmentum, PTT. g Male inside an artificial nest; photograph taken during lights-on to maximise clarity of the housing conditions. h Nesting male presented with 3-D printed model of midshipman male, picture taken at “night” under red light conditions. hi Photograph of male in the nest taken with a front facing camera. i Bar plots (mean ± SEM) illustrating number of c-fos mRNA expressing cells in PAGrs (ANOVA: p = 0.24; see table S2), PAGcs (ANOVA: p = 1.579e-05) and PAGcd (ANOVA: p = 0.0001) in silent (n = 5 animals), silent and hearing (n = 3), courtship calling (n = 8) and agonistic calling (n = 6) males. *denotes significant BH corrected post-hoc differences for Analysis of Variance (ANOVA) tests, p < 0.05. j–m Relationship of call number to c-fos expressing cell number in PAGcs and PAGcd. Solid trend lines denote significant relationships p < 0.05 (see table S2 for statistics ). n–p Representative staining of c-fos mRNA (green) in PAGcs and PAGcd (see Fig. S3 for PAGrs). Scale bars = 250 µm. ni, ii, oi, ii, pi, ii Scale bars = 50 µm.