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. 2022 Dec 13;16:1006594. doi: 10.3389/fnins.2022.1006594

FIGURE 13.

FIGURE 13

Crinotocin precursor expression in Antedon mediterranea and in vitro effects of crinotocin on arm preparations. (A–C) Arm sections of A. mediterranea labeled with antisense probes for crinotocin precursor transcripts using mRNA in situ hybridization. bn, brachial nerve; l, ligament; m, muscle; p, pinnule; o, ossicle. s, syzygy; Scale bars: 120 μm for (A,B); 210 μm for (C). (A) Transverse section of the arm, with the square showing the region containing the brachial nerve, which is shown at higher magnification in panel (B). (B) High magnification image of the brachial nerve, showing the presence of crinotocin precursor-expressing cells in the core and margins of the nerve. (C) Sagittal section of an arm, showing labeled cells in the brachial nerve along its length and in branches of the brachial nerve that innervate ligaments and muscles (arrow). (D) Effects of crinotocin (10–5 M) on arm preparations, with five tests on different preparations from two representative animals shown. After addition of crinotocin (arrow), recording was continued for several minutes. Variability in responses both within and between animals is observed. For example, with animal 11 crinotocin caused a gradual and small flexion in some arm preparations, whereas in most animals (e.g., animal 15) crinotocin caused an abrupt and large flexion in most arm preparations. (E) Concentration-dependent effect of crinotocin on mean arm flexion rate. Five different concentrations were tested (10–4 M, 10–5 M, 10–6 M, 10–7 M, 10–8 M), and an example test per concentration for a representative animal is shown. Responses are greatest at the highest concentrations tested (10–4 M and 10–5 M), and visibly decrease at intermediate concentrations until little or no visible effect is observed at the lowest concentrations tested (10–7 M and 10–8 M). (F) Concentration-response curve for the effect of crinotocin on arm preparations. Data from 143 arm preparations from four animals were used to generate this concentration-response curve, but one data point for the 10–4 M concentration was excluded as an outlier. See Supplementary File 5. Error bars represent standard error of the mean (SEM). The graph was generated using GraphPad Prism. (G) Anesthetization of arm preparations blocks the response of arms to crinotocin. ASW: pretreated in ASW alone then treated with ASW alone; CRIN: pretreated in ASW alone then treated with 10–5 M crinotocin; CRIN+PRO: anesthetized in 10–3 M procaine hydrochloride then treated with 10–5 M crinotocin in anesthetic solution. Numbers of preparations are shown in parentheses. Error bars represent SEM. The graph was generated using GraphPad Prism.