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Regulation of early embryonic behavior by nitric oxide in the pond snail Helisoma trivolvis

Alison G. Cole, Aniseh Mashkournia, Shawn C. Parries and Jeffrey I. Goldberg^*

Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9

View larger version (12K): [in a new window]   Fig. 2. Effect of NO donors on the embryonic rotation behavior. Application of SNAP (A) or SNP (B) at various concentrations resulted in a significant increase in the rate of embryonic rotation. In both cases, the most effective dose produced a twofold increase in rotation rate, in contrast to the fourfold increase produced by 100 µm serotonin (5-HT). (A) N=252 embryos; (B) N=147 embryos. (C) SNAP (10 µmol l-1) and SNP (25 µmol l-1) solutions that were pre-exposed to light for 24 h at room temperature (x-SNAP; x-SNP) had no effect on rotation rate. The NO-donating capability of SNAP and SNP are exhausted by this treatment, leaving only stable NO by-products. N=68 embryos. Asterisks represent statistically significant differences compared to the control (artificial pondwater, APW) treatment (P<0.05).

View larger version (11K): [in a new window]   Fig. 3. Effect of NOS inhibitors on the embryonic rotation behavior. (A) Application of 10 mmol l-1 L-NAME resulted in a significant decrease in the rate of embryonic rotation (P<0.01, N=100 embryos), whereas 10 mmol l-1 D-NAME had no effect. (B) Application of 100 µmol l-1 7-NI also produced a significant inhibition of rotation rate (P<0.001, N=63 embryos). In A+B, 100 µmol l-1 serotonin (5-HT) produced the expected three- to fivefold increase in rotation rate. (C) NO donors bypass the inhibitory effect of NOS inhibitors. Coapplication of 10 mmol l-1 L-NAME with either 1 µmol l-1 (1) or 10 µmol l-1 (10) SNAP resulted in a significant increase in embryonic rotation rate, rather than the decrease produced by L-NAME alone (P<0.001, N=137 embryos). Asterisks represent statistically significant differences compared to the control (artificial pondwater, APW) treatment.

View larger version (15K): [in a new window]   Fig. 5. Effect of NO donors and NOS inhibitors on the inter-surge rate of rotation. Both 10 µmol l-1 SNAP (A) and 25 µmol l-1 SNP (B) caused a significant increase in the mean rate of slow tonic rotation. In contrast, 10 mmol l-1 L-NAME (C) and 100 µmol l-1 7-NI (D) caused a significant reduction in the mean rate of slow tonic rotation. Asterisks represent statistically significant differences compared to the control (artificial pondwater, APW) treatment. (A) P<0.001, N=32 embryos; (B) P<0.001, N=26 embryos; (C) P<0.001, N=14 embryos; (D) P<0.0001, N=22 embryos.

View larger version (14K): [in a new window]   Fig. 6. Effect of NO donors and NOS inhibitors on the frequency of rotational surges. Both 10 µmol l-1 SNAP (A) and 25 µmol l-1 SNP (B) had no effect on the frequency of rotational surges. In contrast, 10 mmol l-1 L-NAME (C) and 100 µmol l-1 7-NI (D) caused a significant reduction in the frequency of rotational surges. Asterisks represent statistically significant differences (P<0.001) compared to the control (artificial pondwater, APW) treatment. (A) N=34 embryos; (B) N=30 embryos; (C) N=17 embryos; (D) N=23 embryos.

View larger version (17K): [in a new window]   Fig. 7. Effect of NO donors and NOS inhibitors on the amplitude of rotational surges. Both 10 µmol l-1 SNAP (A) and 25 µm SNP (B) caused a significant increase in the mean amplitude of rotational surges, expressed in rotations per minute (r.p.m.). In contrast, 10 mmol l-1 L-NAME (C) and 100 µmol1-1 7-NI (D) had no effect on the mean amplitude of rotational surges. Asterisks represent statistically significant differences compared to the control (artificial pondwater, APW) treatment. (A) P<0.05, N=44 surges from 34 embryos; (B) P<0.05, N=39 surges from 30 embryos; (C) N=15 surges from 17 embryos; (D) N=13 surges from 23 embryos.

View larger version (81K): [in a new window]   Fig. 1. Localization of NOS in H. trivolvis embryos as revealed by NADPH-d staining. (A) Dorsal-anterior view of stage E25 embryo. NOS was expressed bilaterally in the dendritic knobs of ENC1 (arrows), the ciliary cells of the dorsolateral ciliary bands (arrowheads) and the protonephridia (n). The entire buccal mass (bu) was diffusely stained throughout, with greatest intensity occurring in the ciliated dorsal region. Staining also occurred in several unidentified cells near the embryonic surface (asterisks) that were widely spaced throughout the dorsal half of the embryo, including the region of the primordial mantle. The box outlines a deeper focal plane of the same embryo that was spliced in to reveal staining in dorsolateral ciliary cells (arrowheads) and the protonephridium on the right side. Scale bar, 40 µm. (B) Longitudinal profile of stage E35 embryo at the level of the left ENC1 (anterior at left). The dendritic knob (black arrow), apical dendrite (small black arrowhead), soma (white arrow) and proximal descending axon (white arrowhead) of ENC1 were all stained at this stage. In addition, intense staining occurred in a dorsolateral ciliary cell (upper black arrowhead), the dorsal buccal region (bu) and the protonephridium (lower black arrowhead). The diffusely stained radular sac (r) is also shown. Scale bar, 30 µm. (C,D) Summary diagrams of NAPHD-d staining in H. trivolvis embryos corresponding to the stages and orientations presented in A (C) and B (D). Only structures depicted in blue were stained. At stage E25 (C), only ENC1's dendritic knob was reliably stained (arrow), whereas by stage E35 (D), its apical dendrite, soma and proximal axon were also stained. At both stages, the dorsolateral cilia (dl), buccal region (bu), radular sac (r), protonephridia (n) and unidentified dorsal superficial cells (asterisks) were stained. Surprisingly, the ciliary cells comprising the pedal ciliary band (p) were not reliably stained at either stage.

View larger version (19K): [in a new window]   Fig. 4. Effect of L-NAME on subcomponents of embryonic rotation. Changes in rotation rate were measured over 5 s intervals. (A) In the presence of APW, two representative embryos displayed low basal rotation rates (0.3-1.2 r.p.m.) that were interrupted by periodic surges of faster rotation. (B) In the presence of 10 mmol l-1 L-NAME, two representative embryos displayed lower basal rotation rates (0-0.05 r.p.m.) and fewer surges. Arrows indicate the peak rotation rate during each surge in A and B.