TTX-sensitive and TTX-insensitive control of spontaneous gut motility in the developing zebrafish (Danio rerio) larvae

Abstract

Spontaneous regular gut motility in zebrafish begins around 4 days post fertilisation (d.p.f.) and is modulated by release of acetylcholine and nitric oxide. The role of intrinsic or extrinsic innervation for initiating and propagating the spontaneous contractions, however, is not well understood. By creating spatiotemporal maps, we could examine spontaneous motility patterns in zebrafish larvae in vivo at 4 and 7 d.p.f. in more detail. Tetrodotoxin (TTX) was added to elucidate the importance of nervous control. Anterograde and retrograde contraction waves originated in the same region, just posterior to the intestinal bulb. This area correlates well with the distribution of Hu (human neuronal protein C/D)-immunoreactive nerve cell bodies. Whereas numerous immunoreactive nerve cells were present in the mid and distal intestine at both 4 and 7 d.p.f., fewer cells were seen anterior to the origin of contractions. The overall frequency of contractions (1.16+/-0.15 cycles min(-1), N=14 at 4 d.p.f.; 1.05+/-0.09 cycles min(-1), N=13 at 7 d.p.f.) and the interval between individual anterograde contraction waves (54.8+/-7.9 s at 4 d.p.f., N=14; 56.9+/-4.4 s, N=13 at 7 d.p.f.) did not differ between the two stages but the properties of the contractions were altered. The distance travelled by each wave increased from 591.0+/-43.8 microm at 4 d.p.f. (N=14) to 719.9+/-33.2 microm at 7 d.p.f. (N=13). By contrast, the velocity decreased from 4 d.p.f. (49.5+/-5.5 microm s(-1), N=12) to 7 d.p.f. (27.8+/-3.6 microm s(-1), N=13). At 4 d.p.f., TTX did not affect any of the parameters whereas at 7 d.p.f. anterograde frequency (control 1.07+/-0.12 cycles min(-1), N=8; TTX 0.55+/-0.13 cycles min(-1), N=8) and distance travelled (control 685.1+/-45.9 microm, N=8; TTX 318.7+/-88.7 microm, N=6) were decreased. In conclusion, enteric or extrinsic innervation does not seem to be necessary to initiate spontaneous contractions of the gut in zebrafish larvae. However, later in development, nerves have an increasingly important role as modulators of intestinal activity.