Linalool acts as a fast and reversible anesthetic in Hydra

Abstract

The ability to make transgenic Hydra lines has allowed for quantitative in vivo studies of Hydra regeneration and physiology. These studies commonly include excision, grafting and transplantation experiments along with high-resolution imaging of live animals, which can be challenging due to the animal's response to touch and light stimuli. While various anesthetics have been used in Hydra studies, they tend to be toxic over the course of a few hours or their long-term effects on animal health are unknown. Here, we show that the monoterpenoid alcohol linalool is a useful anesthetic for Hydra. Linalool is easy to use, non-toxic, fast acting, and reversible. It has no detectable long-term effects on cell viability or cell proliferation. We demonstrate that the same animal can be immobilized in linalool multiple times at intervals of several hours for repeated imaging over 2-3 days. This uniquely allows for in vivo imaging of dynamic processes such as head regeneration. We directly compare linalool to currently used anesthetics and show its superior performance. Linalool will be a useful tool for tissue manipulation and imaging in Hydra research in both research and teaching contexts.

Fig 1. Linalool as an anesthetic.

A. Representative images of Hydra polyps before (i. extended, ii. contracted) and after (iii) incubation in 1 mM linalool (abbreviated to LL). Scale bar: 200 μm. B. 3 hr incubation in linalool concentrations exceeding 3 mM causes lethality. Each point represents a single technical replicate containing 8–10 animals. C. Box plot showing time of full extension after last observed contraction burst during 65 min incubation in linalool concentrations up to 1 mM. 1mM linalool takes 7.53 min (5.44, 9.03) (median (25^th percentile, 75^th percentile)) to anesthetize the animals. (*), (**) and (***) indicate statistically significant difference from 0 mM linalool at p<0.05, p<0.01 and p<0.001 respectively (Mann-Whitney U test). Data from 3 technical replicates containing 3–4 animals each for every concentration. Each data point corresponds to one animal. D. Box plot showing time of first observed contraction burst during 120 min recovery in HM following 65 min of anesthesia in linalool. Animals recover in 12.77 min (7.72, 15.13) (median (25^th percentile, 75^th percentile)) after incubation in 1mM linalool. (*), (**) and (***) indicate statistically significant difference from 0 mM linalool at p<0.05, p<0.01 and p<0.001 respectively (Mann-Whitney U test). Data from 3 technical replicates containing 4 animals each for every concentration. Each data point corresponds to one animal. E. Pinch response. i. Hydra polyp in HM. ii. Polyp in HM shows a global body column contraction in response to pinching. iii. Hydra polyp incubated in 1 mM linalool for 10 min. iv. Anesthetized polyp shows only local swelling after pinch, indicated by black arrowhead. Images representative of n = 5 animals per replicate in 2 technical replicates. F. 30 min feeding response in 4-day starved polyp. i. Hydra polyps in HM readily capture and ingest Artemia (brine shrimp), with multiple Artemia clearly visible within the body column of each animal. ii. Hydra polyps incubated in linalool for 10 min prior to introduction of Artemia have a strongly reduced reaction, and only rarely ingest Artemia. White arrowheads indicate Artemia inside polyps. Several animals have not ingested prey at all, and those that have contain a maximum of one Artemia each. Scale bars for E, F: 1 mm.

Fig 2. Linalool improves outcomes of surgical manipulations in Hydra.

A. Sectioning of body column. i. Experimental schematic. ii. Sections cut in HM. iii. Sections cut in linalool. Scale bar: 400 μm iv. Time required to section a polyp in Hydra medium (HM) (90 ± 45 s (mean ± SD), n = 13, across 3 technical replicates) and in 1mM linalool (40 ± 9 s, n = 16, across 5 technical replicates). v. Thickness of body column sections cut in HM (0.20 ±0.07 mm (mean± SD), n = 66 sections, 15 polyps across 4 technical replicates) and in 1mM linalool (0.18 ± 0.05mm, n = 99 sections, 19 polyps across 6 technical replicates). Error bars represent SDs. (*), (**) and (***) indicate statistical significance at p < 0.05, p < 0.01 and p < 0.001 respectively, calculated using a 2 tailed t-test. B. “Zebra grafting”. i. Experimental schematic. ii. Representative animal grafted and healed in HM. iii. Representative animal grafted and healed in linalool. Scale bars: 400 μm. All grafts are shown in S3 Fig. iv. Time taken to assemble grafts in HM (506 ± 141 s (mean ± SD)) and in 1 mM linalool (524 ± 142) (n = 8, across 2 technical replicates each for HM and 1 mM linalool). v. Number of segments in completed graft in HM (5 ± 2 (mean ± SD)) and in 1 mM linalool (8 ± 1) (n = 8, across 2 technical replicates each for HM and 1 mM linalool). Error bars represent SDs. (*) indicates statistically significant difference from grafts in HM at p < 0.05 (2-tailed t-test). C. Head transplantation into gastric region. i. Experimental schematic. ii. Representative animal grafted and healed in HM. iii. Representative animal grafted and healed in 1mM linalool. Scale bars: 400 μm. D. Head organizer transplantation into gastric region. i. Experimental schematic. ii. Animal grafted in HM imaged daily over 5 days. iii. Animal grafted in 1 mM linalool imaged daily over 5 days. Scale bars: 200 μm. Linalool did not improve hypostome cutting times, which were 60 s (50, 69) (median, (25^th quartile, 75^th quartile), measured for n = 17 grafts) in HM and 50 s, (38, 66) (n = 17) in linalool, but slightly improved success of the induction of ectopic axes (6/25 in HM versus 11/25 in linalool) and significantly shortened grafting time to 134 s (104, 209) (n = 17) compared to 196 s (147, 258) (n = 17) in HM.

Fig 3. Single channel live imaging in linalool.

A. Unconstrained GCaMP6s Hydra imaged at low magnification. i. single image in HM. ii. Maximum intensity t-projection of a 10 s video in HM. iii. Rigid body correction of HM video projection. iv. Single image in 1 mM linalool. v. Maximum intensity t-projection of 10 s video in linalool. vi. Rigid body correction of linalool video projection. Scale bars: 200 μm. B. Single slice from a 7.5 μm thick z-stack of a GCaMP6s animal imaged at 60x magnification with a resolution of 0.25 μm along the z-axis at a 500 ms exposure per slice using blue excitation in (i) HM and (ii) 1 mM linalool. C. Maximum intensity projection of high magnification z-stacks in (i) HM and (ii) 1 mM linalool. Scale bars: 10 μm. D. Coefficient of variation for (i) low magnification imaging in HM (0.175 ± 0.025 (mean ± SD)) and linalool (0.143 ± 0.008) calculated from n = 10 polyps across 2 technical replicates (ii) high magnification imaging in HM (0.188 ± 0.042 (mean ± SD)) and linalool (0.121 ± 0.026) calculated from n = 6 polyps across 2 technical replicates. Error bars represent SDs. (**) indicates statistically significant difference at p < 0.01 as determined by a 2 tailed t-test.

Fig 4. Linalool enables high resolution imaging in multiple channels.

A. Low magnification maximum intensity projection of a z-stack acquired of an open Hydra mouth in 1mM linalool using i. Hoechst 33342, ii. Ectoderm—GFP, iii. Endoderm–DsRed2, iv. overlay. 5 μm slice thickness, 6 slices total. Scale bar: 100 μm. B. High magnification maximum intensity projection of a z-stack of the body column tissue acquired in 1mM linalool using i. Hoechst 33342, ii. Ectoderm–GFP, iii. Nematocysts–SYTO 60, iv. overlay. 0.25 μm z-step, 17 slices total. Scale bar: 10 μm. The reduced animal motion allows for acquisition of multiple z-slices in 3 channels.

Fig 5. Linalool enables repeated high-resolution imaging.

A. Head regeneration in a transgenic HyBra2 promoter::GFP polyp imaged at high resolution every 4 h from 12 h to 48 h. Subset of images shown. Scale bar: 0.5 mm B. Repeated anesthesia and recovery do not impact regeneration speed or outcome (n = 10 animals HM, n = 16 animals linalool, 3 technical replicates). Differences between conditions not statistically significant at p = 0.05 level (Fisher’s Exact test).

Fig 6. Effect of long-term continuous linalool exposure.

A. 3-day incubation in 1 mM linalool does not impact rate of cell division. Slices stained with DRAQ5 (nuclei) and anti-PH3 (phospho-histone H3, dividing cells). i. Representative image of body column sections from polyps incubated 3 days in HM. ii. Representative slice from polyps incubated 3 days in 1 mM linalool. iii. Percentage of dividing cells in animals incubated 3 d in HM or 1mM linalool. Mean ± SD: HM = 1.3 ± 0.6, linalool = 1.6 ± 0.8. n = 18 across 5 technical replicates. Difference not statistically significant at p < 0.05 (2-tailed t-test). Error bars represent SD. Scale bar: 100 μm. B. 3-day incubation in 1 mM linalool does not damage or kill cells. Representative images of polyps stained with propidium iodide after incubating for i. 3 days in HM, ii. 24 h in 0.04% colchicine, and iii. 3 days in 1 mM linalool. iv. Mean number of dead cells per animal after incubation in HM (5 ± 4, n = 38), in colchicine (31 ± 21, n = 28) and linalool (4 ± 3, n = 39). Error bars represent SD. (***) indicates statistically significant difference from linalool at p < 0.001 (2-tailed t-test). Scale bar: 100 μm. C. Long term incubation in linalool does not impact budding. Representative images of a budding polyp continuously incubated and imaged in 1 mM linalool. Scale bar: 500 μm. D. Long term incubation in linalool prevents head regeneration. Error bars represent SD (0 mM n = 17, 0.1 mM n = 20, 0.5 mM n = 40, 0.75 mM n = 19, 1 mM n = 19; 3 technical replicates). (*), (**) and (***) indicate statistically significant difference from 0 mM at p<0.05, p<0.01 and p<0.001 respectively (Fisher’s Exact Test). E. Recovery in HM rescues the head regeneration defect. i. Polyp incubated in HM for 68 h after decapitation. ii. Polyp incubated in 1 mM linalool for 68 h after decapitation. iii. Decapitated polyp recovered for 28 h after 3 d in 1mM linalool, iv. Polyps recovered for 3 d after 3 d in 1mM linalool. Scale bar: 1 mm. v. Head regeneration is suppressed by incubation for 3 d in 1 mM linalool. Only 14/42 polyps incubated in 1 mM linalool regenerated at least one tentacle at the end of 3 d incubation compared to 40/42 polyps in HM (across 4 technical replicates). (***) denotes that the difference is statistically significant at p < 0.001 (Fisher’s Exact test) when comparing overall numbers. vi. Head regeneration is rescued in linalool- incubated animals after 3 d recovery in HM. 35/36 polyps incubated in 1 mM linalool regenerated heads at the end of 3 d recovery compared to 36/36 polyps in HM, across 4 technical replicates. The difference is not statistically significant at p < 0.05 (Fisher’s Exact test) when comparing overall numbers.

Fig 7. Comparison of various Hydra anesthetics.

A. Comparisons of the same animal after 15 min and 60 min of anesthetic exposure. i. 1 mM linalool, ii. 0.04% heptanol, iii. 2% urethane, iv. 0.1% chloretone. Scale bars: 1 mm. B. Maximum intensity projections of GCaMP6s animals at 60x magnification in each anesthetic. Scale bars: 10 μm. C. Maximum intensity projections of two-channel images of watermelon animals stained with Hoechst nuclear dye at 60x magnification. GFP channel, DAPI channel, and merge (overlay) shown for each anesthetic. Scale bars: 10 μm.

Fig 8. Hydra response to 1mM linalool (L), 0.04% heptanol (H), 2% urethane (U), and 0.1% chloretone (C).

A. Percent length of anesthetized Hydra polyps compared to their natural state at 10min incubation. n = 10 animals per condition across 2 technical replicates. Anesthetized lengths similar to the average lengths in HM were recorded in linalool at 103% (87, 112; median (25^th percentile, 75^th percentile)), heptanol at 83% (71, 93) and urethane at 96% (88, 118), while chloretone-treated animals hyperextended at 133% (125, 153). B. Induction times across 2 technical replicates. Linalool n = 13, heptanol n = 14, urethane n = 13, chloretone n = 10. Linalool’s median induction time was 9 min (6, 9) (median (25^th percentile, 75^th percentile)) and thus significantly longer than that of heptanol at 6 min (4, 9), urethane at 5 min (4, 6) and chloretone at 5 min (3, 7). C. Recovery times across 2 technical replicates. Linalool n = 10, heptanol n = 8, urethane n = 12, chloretone n = 10. Median recovery time was 8 min (7, 17) for linalool, 11 min (7,15) for heptanol, 14 min (12, 26) for urethane and 13 min (7,15) for chloretone. (D-F) Pairwise statistical comparisons of data shown in A-C. Pink, red and dark red indicate a statistically significant difference at p<0.05, p<0.01 and p<0.001 respectively, determined using the Mann-Whitney U test between pairs of anesthetics. D. Comparison between percent length distributions. E. Comparison between induction time distributions. F. Comparison between recovery time distributions. G. Overview of the four anesthetics tested, scored on degree of immobilization, animal health following anesthesia, time to induce anesthesia, time to recover from anesthesia, morphology, and ease of use (see Methods).