Functional Changes in the Snail Statocyst System Elicited by Microgravity

BACKGROUND: The mollusk statocyst is a mechanosensing organ detecting the animal's orientation with respect to gravity. This system has clear similarities to its vertebrate counterparts: a weight-lending mass, an epithelial layer containing small supporting cells and the large sensory hair cells, and an output eliciting compensatory body reflexes to perturbations. METHODOLOGY/PRINCIPAL FINDINGS: In terrestrial gastropod snail we studied the impact of 16- (Foton M-2) and 12-day (Foton M-3) exposure to microgravity in unmanned orbital missions on: (i) the whole animal behavior (Helix lucorum L.), (ii) the statoreceptor responses to tilt in an isolated neural preparation (Helix lucorum L.), and (iii) the differential expression of the Helix pedal peptide (HPep) and the tetrapeptide FMRFamide genes in neural structures (Helix aspersa L.). Experiments were performed 13-42 hours after return to Earth. Latency of body re-orientation to sudden 90° head-down pitch was significantly reduced in postflight snails indicating an enhanced negative gravitaxis response. Statoreceptor responses to tilt in postflight snails were independent of motion direction, in contrast to a directional preference observed in control animals. Positive relation between tilt velocity and firing rate was observed in both control and postflight snails, but the response magnitude was significantly larger in postflight snails indicating an enhanced sensitivity to acceleration. A significant increase in mRNA expression of the gene encoding HPep, a peptide linked to ciliary beating, in statoreceptors was observed in postflight snails; no differential expression of the gene encoding FMRFamide, a possible neurotransmission modulator, was observed. CONCLUSIONS/SIGNIFICANCE: Upregulation of statocyst function in snails following microgravity exposure parallels that observed in vertebrates suggesting fundamental principles underlie gravi-sensing and the organism's ability to adapt to gravity changes. This simple animal model offers the possibility to describe general subcellular mechanisms of nervous system's response to conditions on Earth and in space

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

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

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

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

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