Expression of the <i>Helix</i> Pedal Peptide (HPep) gene in statocysts of snails exposed to microgravity and under different ground conditions.

<p>Expression of the <i>Helix</i> Pedal Peptide (HPep) gene in statocysts of snails exposed to microgravity and under different ground conditions.</p

Averaged Cumulative Neural Responses to Tilt.

<p>The total number of spikes was collected over a 2 s interval after the onset of tilt in control and postflight snails for both M-2 and M-3 experiments. The tilt duration was comparable for the M-2 (1100 ms; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017710#pone-0017710-g003" target="_blank">Fig. 3A</a>) and M-3 (1075 ms; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017710#pone-0017710-g003" target="_blank">Fig. 3B</a>) experiments. The control and flight snails had highly significant differences in magnitude of tilt response in the M-3 experiments (*p<0.01, Student's t- test for difference between the means).</p

Negative gravitaxis response in control and postflight snails.

<p><b>A.</b> Phases of the stereotypic response to sudden shift of the snail with platform from horizontal to “head down” position. <b>B.</b> Latency of gravitaxis reaction phases acquired during Foton M-2 experiments. The plot shows averaged (±SEM) time of the behavioral responses at 4 phases of the negative gravitaxis response in 14 flight and 8 control snails. Flight snails were faster in their response to pitch stimulation at each phase, and the difference reach level of significance p<0.05 at the later phases T3 and T4. <b>C.</b> Changes in latency of gravitaxis reaction of T2 phase acquired during Foton M-3 experiments. The plot shows averaged (±SEM) time of the behavioral responses at the T2 phase in 5 flight and 6 control snails tested before (black columns) and after (open columns) flight. Flight snails were faster than control snails as a group in their response to pitch stimulation, insignificant at T1 (not shown) but significant (p<0.02) at T2 phase. Post-flight gravitaxis responses were significantly faster (shorter latency of T2; p<0.04) than pre-flight responses recorded in the same snail.</p

Postflight changes in directional sensitivity of statocyst response to tilt.

<p><b>A, B:</b> Electrophysiological responses to tilt in statocyst nerve of 4 control and 5 postflight snails (<b>A</b>; Foton M-2) and 8 control and 8 postflight snails (<b>B</b>; Foton M-3) are shown at head down and head up (or tail down) orientations. Scales are expanded in each plot for illustrative purposes. <b>C, D</b>: averaged difference between statocyst nerve responses to tilt corresponding to “head-up” and “head-down” positions are plotted for M-2 (<b>C</b>) and M-3 (<b>D</b>) experiments. A response near zero indicates no directional preference. In both M-2 and M-3 series control and postflight snails the statocyst response demonstrated the opposite directional selectivity. A significant difference between postflight and control snails was observed in the middle portion of tilt in both M-2 and M-3 experiments (p<0.02, RM-ANOVA with Tukey post-hoc analysis).</p

Experimental paradigm of behavioral and physiology tests.

<p><b>A.</b> Cartoon of tilt platform used in neural recordings. Petri dish is fixed on a platform that can be mechanically tilted to a maximum displacement angle of 19°. In Foton M-2 experiments the tilt duration was fixed to 1.1 s (17.3°/s peak tilt velocity) and in Foton M-3 experiments the tilt duration was varied in 4 steps from 550–3020 ms (6.3–34.5°/s peak tilt velocity). Activity of the statocyst nerve was recorded using electrically separate chambers for statocyst and cerebral ganglion with the nerve passing over a Vaseline bridge. <b>B.</b> Head-down or head-up tilt of snail correspond to tilting platform with the preparation oriented at 0° (middle panel) and 180° (lower panel), respectively. <b>C.</b> Example of whole nerve response to tilt and cell sorting technique. Traces from bottom to top: whole nerve statocyst discharge (bar = 5 µV), platform position during tilt stimulus was recorded using a potentiometer (bar = 10°), and six identified cells in this preparation labeled Cell 1–6 (bar = 2 spikes/s).</p

Postflight increase of statocyst response to vestibular stimulation.

<p><b>A.</b> Averaged statocyst nerve responses (mean spike rate ± SEM sampled at 0.2 s bin width) of 5 postflight (open circles) and 4 control (filled circles) snails (Foton M-2) to platform tilt. The increased response of the postflight snails to tilt was insignificant. The stimulation and recording protocols were improved for the Foton M-3 experiments. Averaged statocyst nerve responses (mean spike rate ± SEM sampled at 0.3 s bin width) of 8 postflight (open circles) and 8 control (filled circles) snails to platform tilt of 1075 ms ramp time or 17.7°/s (<b>B</b>; close to M-2 ramp time), and at a faster (<b>D</b>; 550 ms or 34.5°/s) and a slower (<b>E</b>; 3020 ms or 6.3°/s) ramp times. At all tilt speeds the magnitude of the statocyst response was significantly increased (indicated by * in each plot) in postflight snails. <b>C.</b> Cumulative number of spikes over 2 s period following the onset of tilt for M-2 and M-3 experiments. Spike numbers were taken from time 0–2 s in the plots shown in panels A and B for control and postflight snails to allow a more direct comparison between the two missions. Control data were comparable in both missions, but the postflight results were significantly different in M-3 experiments (p<0.01, Student's t-test). <b>F</b>. The significant hypersensitivity of the statocyst to tilt following µG exposure is shown by plotting the total number of spikes (mean ± SEM) over a 4 s period following tilt onset at 4 peak velocities in the 8 control and 8 postflight snails (p<0.01**; p<0.02*).</p

Localization of neurons expressing preproHPep gene in snail CNS and statocyst using <i>in situ</i> hybridization.

<p>Left panels (<b>A, C, E, G</b>) are images taken from control snails; right panels (<b>B, D, F, I</b>) are those taken from postflight snails. The staining in control and postflight snails was qualitatively similar in the CNS structures, but consistently different in the statocyst. <b>A, B</b>: cerebral ganglia; <b>C, D</b>: suboesophageal ganglia complex; <b>E, F</b>: pedal ganglia; <b>G, I</b>: statocysts. Note the labelled statocyst receptor cells in postflight snails in I (indicated by arrows) and lack of staining in control snails in G. <b>H</b>: for illustrative purposes the immunohistochemistry of HPep in a preflight snail shows the location of 3 receptors with respect to the statocyst nerve. Expression of this gene was observed only in these cells in all preparations. Calibration: A–F, 500 µm; G–I, 50 µm.</p