Fig 2: Histochemical staining of the body musculature with phalloidin (red, (a–c)) and immunoreactivity to 5-HT7 serotonin receptor (green, (d–k)) in metacercariae larvae of Opisthorchis felineus. (a,b)—phalloidin staining of body wall musculature in the anterior (a) and posterior (b) body parts of O. felineus, the longitudinal (long arrows), diagonal (short arrows) and circular (arrowheads) muscle fibres, vs—the ventral sucker; (c)—muscle fibres extending from the body wall to the ventral sucker (short thick arrows), vs— ventral sucker; (d)—staining to serotonin receptor (5-HT7-IR) in the body wall muscles of O. felineus, overview, os— the oral sucker (long arrow), vs—the ventral sucker (short arrow); (e)—posterior body region, the pattern of 5-HT7-IR shows the circular (thin arrows) and diagonal (thick arrows) muscle fibres; (f)—5-HT7-IR in the muscle fibres extending from the body wall to the ventral sucker and in the longitudinal muscles of the intestine (arrows); (g)—the body wall musculature of the anterior region of the body, stained with 5-HT7-IR, the longitudinal (long arrows) and circular (short arrows) filaments are indicated, part of the oral sucker musculature (os) is visible; (h,i)—more detailed views on the 5-HT7-IR elements along the body muscles: (h)—the longitudinal (long arrows) and diagonal (short arrows) muscle fibres are indicated; (i)—punctate pattern of 5-HT7-IR along the circular muscle fibres (arrows), confocal and transmission light microscopy; (j)—5-HT7 immunoreactivity in muscle fibres running from the body wall to the ventral sucker (arrows) and in the round structures along the body margin (arrowheads); (k)—5-HT7-IR in the posterior body region, the muscle fibres running from the body wall to the ventral sucker (long arrows) and the longitudinal muscles of the excretory bladder (short arrows), vs—ventral sucker. Scale bars: (a–c,e,f,k)—50 µm; (d)—100 µm; (g)—30 µm; (h), (i)—10 µm; (j)—20 µm.

Fig 3: A phylogenetic tree of serotonin 5-HT7 receptors in cestodes, trematodes and planarian species with the annotated genome sequences.

Fig 4: Immunoreactivity to serotonin (b–f) and 5-HT7 serotonin receptor (g–l) in cysticercoid larvae of Hymenolepis diminuta. (a)—cysticercoid of H. diminuta with suckers (s) in the scolex, transmission light microscopy, whole mount; (b)—5-HT-IR in the scolex of H. diminuta, the lateral ganglia (thick arrow) and the rostellar ganglia (short arrow), the serotonergic neurons (arrowheads), the ventral nerve cords (thin long arrows) and transversal commissures between them (thin short arrows), the serotonergic nerve fibres (the rostellar nerves) extending from the lateral to the rostellar ganglia are visible; (c)—5-HT-IR in the longitudinal nerve cords (long thin arrow), in commissures between them (short arrows), in the nerve cells along the nerve cords (arrowheads), in the nerve plexus and initial part of the longitudinal nerve cords (short thick arrows); (d)—several pairs of 5-HT-IR neurons (arrowheads) situated along the major nerve cords (long thin arrows), the lateral ganglia are visible; (e)—5-HT-IR in the longitudinal nerve cords (thin long arrows) and in the commissures between them (short arrows), in the nerve cells along nervous cords (arrowheads); (f)—5-HT-IR in the lateral ganglion (long thick arrow), in the rostellar ganglion (short thick arrow), in the nerve fibres of the plexus within suckers (thin arrows); (g)—5-HT7-IR along the longitudinal and transversal muscle fibers comprising the worm body wall (thin long arrows) and in the muscles of suckers (thick arrows), 5-HT7-IR is visible in the oval structures, probably the flame cells of the excretory system (arrowheads); (h)—5-HT7-IR along the radial the muscle of the suckers (short arrow) and in the oval structures, the flame cells of the excretory system (arrowheads); (i)—5-HT7-IR in the longitudinal muscle fibres of body (thin arrows), the 5 HT7 -IR is visible in the oval structures (short thick arrows), the flame cells of the excretory system; (j)—5-HT7-IR in the longitudinal and transversal (arrowheads) muscle fibres of the body and in the muscles of the sucker (short thick arrow), 5-HTR7-IR is also present in the oval structures, the flame cells of the excretory system (long arrows); (k)—5-HT7-IR in the oval structures (long arrows), the flame cells, scattered in the cysticercoid body; (l)—phalloidin staining of the oval structures having positive 5-HT7-IR, the larger magnification; (m)—5-HT7-IR in the oval structures (long arrows), presumably, the flame cells of the excretory system, the large magnification. Scale bars: (a,b,d,f,g–j)—20 µm; (c,e,k–m)—10 µm.

Fig 5: Knockdown of the 5-HT7 receptor in the ACC blocks the ameliorative effects of exercise on pain. A, Experimental schematic. The virus was injected 21 d before the behavior test. B, Fluorescence image of AAV-CaMKII-EGFP-shRNA (Htr7) infection in the ACC. Scale bar, 100 µm. C, D, Protein level of the 5-HT7 receptor. n = 3. (E, F) Effect of knockdown of the 5-HT7 receptor in the ACC on exercise-induced alleviation of mechanical allodynia during the time course (E) and at day 18 (F). n = 10. G, H, Effect of knockdown of the 5-HT7 receptor in the ACC on exercise-induced alleviation of thermal hyperalgesia during the time course (G) and at day 18 (H). n = 10. I, J, Typical trace (I) and cartogram (J) from the open field test. Dotted box, center zone of the open field. n = 10. K, L, Typical trace (K) and cartogram (L) from the elevated plus maze test. n = 10. Among the typical traces (I, K), the CFA, shRNA and exercise (CFA & sh-NC & Ex) group trace is shown at the top, and the CFA, shRNA and exercise group (CFA & sh-Htr7 & Ex) trace is shown at the bottom. Unpaired Student’s t tests were used to compare the CFA, sh-NC and exercise group (CFA & sh-NC & Ex) and the CFA, sh-Htr7 and exercise group (CFA & sh-Htr7 & Ex) (D, J, L, **P < 0.01), and repeated one-way ANOVA was used to compare the time points (E–H, *P < 0.05, **P < 0.01). The data are presented as the mean ± SEM.