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

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

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

Green fluorescent genetically encoded calcium indicator based on calmodulin/M13-peptide from fungi

<div><p>Currently available genetically encoded calcium indicators (GECIs) utilize calmodulins (CaMs) or troponin C from metazoa such as mammals, birds, and teleosts, as calcium-binding domains. The amino acid sequences of the metazoan calcium-binding domains are highly conserved, which may limit the range of the GECI key parameters and cause undesired interactions with the intracellular environment in mammalian cells. Here we have used fungi, evolutionary distinct organisms, to derive CaM and its binding partner domains and design new GECI with improved properties. We applied iterative rounds of molecular evolution to develop FGCaMP, a novel green calcium indicator. It includes the circularly permuted version of the enhanced green fluorescent protein (EGFP) sandwiched between the fungal CaM and a fragment of CaM-dependent kinase. FGCaMP is an excitation-ratiometric indicator that has a positive and an inverted fluorescence response to calcium ions when excited at 488 and 405 nm, respectively. Compared with the GCaMP6s indicator <i>in vitro</i>, FGCaMP has a similar brightness at 488 nm excitation, 7-fold higher brightness at 405 nm excitation, and 1.3-fold faster calcium ion dissociation kinetics. Using site-directed mutagenesis, we generated variants of FGCaMP with improved binding affinity to calcium ions and increased the magnitude of FGCaMP fluorescence response to low calcium ion concentrations. Using FGCaMP, we have successfully visualized calcium transients in cultured mammalian cells. In contrast to the limited mobility of GCaMP6s and G-GECO1.2 indicators, FGCaMP exhibits practically 100% molecular mobility at physiological concentrations of calcium ion in mammalian cells, as determined by photobleaching experiments with fluorescence recovery. We have successfully monitored the calcium dynamics during spontaneous activity of neuronal cultures using FGCaMP and utilized whole-cell patch clamp recordings to further characterize its behavior in neurons. Finally, we used FGCaMP <i>in vivo</i> to perform structural and functional imaging of zebrafish using wide-field, confocal, and light-sheet microscopy.</p></div

<i>In vitro</i> properties of the purified FGCaMP indicator.

<p>(A, B) Absorbance (A), excitation and emission spectra (B) for FGCaMP in Ca²⁺-free and Ca²⁺-bound states. (C, D) Intensity and dynamic range for FGCaMP as a function of pH at 402 (C) and 490 nm excitation (D), respectively. The dynamic range (fold) at each pH value was measured as the ratio of FGCaMP fluorescence intensity in the absence of Ca²⁺ to that in the presence of Ca²⁺ at 402 nm excitation (C) and vice versa at 490 nm excitation (D). Error represents the standard deviation for the average of three records. (E) Maturation curves for mEGFP and FGCaMP in Ca²⁺-bound state at 402 nm excitation. (F) Photobleaching curves for FGCaMP in Ca²⁺-free state (at 355 nm excitation), in Ca²⁺-bound state (at 470 nm excitation), mEGFP, and mTagBFP2.</p

Response of FGCaMP to Ca²⁺ concentration changes in HeLa Kyoto cells and FRAP of FGCaMP and control GECIs at different Ca²⁺ concentrations in HeLa Kyoto cells.

<p>(A) Confocal image of HeLa Kyoto cells expressing FGCaMP calcium sensor. (B) The graph illustrates changes in the green fluorescence of FGCaMP in HeLa Kyoto cells excited at 405 (cyan line) or 488 nm (green line) in response to the addition of 2 mM CaCl₂ and 5 μM ionomycin. The changes correspond to the area indicated with white circles on the panel A. (C) Example of HeLa Kyoto cells expressing FGCaMP calcium sensor used for FRAP experiment. An example of FRAP area having a size of around 1 μm² is indicated with a white asterisk. (D)-(F) The graphs illustrate FRAP induced changes in green fluorescence of FGCaMP and control GECIs at physiological Ca²⁺ concentrations and in response to the addition of 2 mM CaCl₂ and 5 μM ionomycin. Error bars are standard deviations shown for each 20^th dot on plots.</p