Fig. 4. Elevation of cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) by exogenous serotonin microinjected to the blastocoel. (A) Ventral view of a larva hooked at the tip of a glass needle (black arrow) near stomach (white arrow). (B) Propagation of [Ca²⁺]_i wave in a same pluteus larva as in A. Microinjection of serotonin into the blastocoel near stomach (B, 0'', yellow arrow) triggered elevation of [Ca²⁺]_i on the larval surface. The micropipette was kept inserted to the blastocoel without releasing serotonin for 15 s to monitor [Ca²⁺]_i level before serotonin application. Initial elevation of [Ca²⁺]_i occurred near serotonin injection site immediately after onset of serotonin release (B,16'', arrow 1), that transiently propagated posteriorly for about 350 µm in 2 s (C, double-headed arrow) on the left side (B, 16'', post), then traveled to the right side of larva by 17 s (B, 17'', arrow 2 and curved arrow), and the other one anteriorly (B, 16'', ant) for 50 µm before diminishing earlier than the posterior propagation at 20 s (B, 20''). The numbers shown on upper left corner in (B) show the time in second from when micropipette was inserted (time zero, 0') and thereafter. Transient [Ca²⁺]_i elevation returned close to visual time zero level in 30 s (B, 30''), and to background level in 60 s as shown by a time-course of the wave intensity (C). [Ca²⁺]_i elevation occurred soon after the completion of serotonin releasing (C, green arrow). A second fluorescence wave toward the anterior direction diminished in 2 s after the initial [Ca²⁺]_i elevation (B, from 16'' to 18'', 18'' arrowhead). Rainbow-colored bar shows relative intensity of fluorescence, as described in Materials and methods. (C) Entire time-course of [Ca²⁺]_i elevation from 15 s before onset of serotonin release (time zero, green arrow) to 60 s. (D) Double stained immunohistochemistry of serotonin (red) and serotonin receptor cell network (green). The serotonin receptor cell network is a major structure in the blastocoelar space. (E) Double stained immunohistochemistry of serotonin (negative immunoreaction) and serotonin receptor cell network (green) of p-chlorophenylalanine (pCPA)-treated larva. pCPA inhibited serotonin synthesis at the apical ganglion and perturbed the formation of serotonin receptor cell network. (F) Same pCPA-treated larvae as that used to examine [Ca²⁺]_i elevation. Red arrow shows an oil droplet from the glass micropipette, and is used as a marker of injection. The stomach is smaller than that of normal larvae (black arrow). (G) pCPA-treated larva lacked intrinsic high level of [Ca²⁺]_i at stomach (0', arrow). (H) 18 s after serotonin injection, no elevation of [Ca²⁺]_i occurred. (I) Time course of [Ca²⁺]_i elevation in pCPA-treated larva. No [Ca²⁺]_i elevation occurred. Bars, 100 µm.