www.sciencemag.org/content/344/6188/1293/suppl/DC1
Supplementary Material for Anxiety-like behavior in crayfish is controlled by serotonin Pascal Fossat, Julien Bacqué-Cazenave, Philippe De Deurwaerdère, Jean-Paul Delbecque, Daniel Cattaert*
*Corresponding author. E-mail:
[email protected] Published 13 June 2014, Science 344, 1293 (2014) DOI: 10.1126/science.1248811 This PDF file includes:
Materials and Methods Figs. S1 to S7 Tables S1 and S2 PCA Table Other Supplementary Material for this manuscript includes the following: (available at www.sciencemag.org/content/344/6188/1293/suppl/DC1) Movies S1 to S4
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Materials and methods
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Animals:
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A total of 267 male crayfish (Procambarus clarkii) averaging 8.2±0.1 cm in length
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and 15.6±1.5 g in weight were used in our experiments. The crayfish were obtained
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from swamps near Bordeaux (“Réserve naturelle de Bruges”), reared inside a
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specific animal house at 20°C with a 12h/12h light/dark cycle and fed pellets ad
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libitum. To limit the effects of previous life experiences under natural conditions, each
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animal was isolated at least three weeks before the experiments in a tank
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(50x30x30cm) equipped with recirculating water. All experiments were performed in
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accordance with the CNRS guidelines for animal care.
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Induction of stress:
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Electric fields: Stressed crayfish were placed in a specific tank (30x20x10 cm, light
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intensity: 15 lux, Fig.2A). Two electrodes placed on opposite sides of the tank were
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connected to a stimulator (AMPI, Isr.) that delivered repetitive trains (0.1 s duration)
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consisting of ten 5-ms pulses at 100 Hz. The current intensity was progressively
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increased until the appearance of an aversive response by the crayfish (i.e., tail flip).
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Electric trains were then produced at 5-s intervals during the total time (30 min,
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unless otherwise stated). Unstressed crayfish were also placed in the same tank for
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the same duration (30 min) but did not receive electric stimulations.
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Drug treatments:
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Serotonin (5HT) and chlordiazepoxide hydrochloride (CDZ) (Sigma Aldrich, St Louis,
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MO, USA) were dissolved in crayfish saline (195 mM NaCl, 5 mM KCl, 13 mM CaCl2,
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2 mM MgCl2 and 3 mM HEPES, pH 7.65). The crayfish were injected between two
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abdominal segments directly into the hemolymph. 5HT was used at a final dose of
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5µg/g fresh weight. We verified (by HPLC, see below) that this treatment resulted in a
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significant increase in brain and ventral nerve cord 5HT concentrations after 5min.
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CDZ at 15 µg/g, chosen after performing dose-response curves (Fig.S5). To prevent
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serotonin effects, a mix of mianserin hydrochloride (5HT2 blocker, Sigma Aldrich)
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and methysergide maleate salt (5HT1 and 2 blocker, Sigma Aldrich) were applied at
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1 nmol/g immediately prior to the electric field stress protocol.
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The timing of single injections was determined after preliminary experiments: 5HT
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antagonists were effective when applied before stress exposure, while CDZ was
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efficient immediately after.
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Animal groups:
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As a consequence of the stress and injection protocols, the animals used in our
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experiments were separated into 9 different groups before being submitted to the
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D/L+maze test. All animals had only one visit in the maze: (1) unstressed: animals
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were placed in the stress tank as described above without receiving electric
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stimulation; (2) unstressed+saline: unstressed animals were injected with saline 10-
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15 min before the test; (3) unstressed+CDZ: unstressed animals received a CDZ
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injection 10-15 min before the test; (4) unstressed+5HT: unstressed animals were
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injected with 5HT 10-15 min before the test; (5) unstressed+5HT and CDZ:
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unstressed animals were injected with 5HT immediately followed by CDZ and tested
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10-15 min later; (6) stressed: animals were placed in the stress tank and received
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electric stimulations for 30 min (unless otherwise specified) 10-15 min before the test;
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(7) stressed+saline: animals were placed as above and received a saline injection
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after the stress procedure 10-15 min prior to the test; (8) stressed+CDZ: animals
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were placed as above and received a CDZ injection at variable doses (1.5, 7.5 or 15
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µg /g) after the stress procedure and 10-15 min prior to the D/L+maze test; (9)
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stressed+5HT antagonists: animals received a mixture of 5HT antagonists before
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the stress procedure.
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Bioamine level measurements:
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Bioamines were measured by high performance liquid chromatography with
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electrochemical detection (RP-HPLC-ECD) as previously described (15). Briefly, the
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crayfish brain and nerve chain were rapidly extirpated and weighed, then separately
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homogenized in 200 µL of 0.1N HClO4 by sonication and centrifuged at 13000 rpm
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for 30 min at 4°C. Aliquots of the supernatants were injected into a reverse-phase
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HPLC column (Chromasyl Stability C8, 150 x 4.6 mm). The mobile phase (70 mM
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NaH2PO4, 0.1 mM disodium EDTA, 2 mM sodium octane-1-sulfonate monohydrate,
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7% methanol, pH 3.9) was delivered at 1 mL/min by a Beckman 128 pump. A
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coulometric detector (Coulochem II, ESA) equipped with a dual-electrode analytical
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cell (potentials set at +350 and -270 mV) enabled detection of dopamine and
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serotonin. Only 5HT displayed significant changes after stress and was thus further
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analyzed.
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Glucose level measurements:
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In order to avoid interference of our measurements with circadian variations of
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glucose titer, all hemolymph sampling were made at the same time of the day
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(between 10am and 1pm). A 25 µl aliquot of hemolymph was carefully removed with
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an Hamilton syringe inserted in soft cuticle of the thoraco-coxal joint of first or second
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pereiopod, and placed in 100 µl of 3 mM trichloracetic acid (C2HCl302). The extract
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was centrifuged at 5000 rpm, and 50 µl of the supernatant was placed in 500 µl of
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reactive solution (Glucose RTU, BioMérieux SA, France) and incubated at 37°C for
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10 min. The optical density was then measured with a spectrophotometer at 505 nm
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(Helios Epsilon, ThermoSpectronic, Fisher Scientific Inc.).
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Behavior analysis: the dark/light plus maze protocol
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Behavior paradigm: The objective of this experiment was to analyze the spontaneous
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exploration behavior of crayfish confronted with a novel environment. The dark/light
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(D/L) plus maze is a sub-aquatic arena (total dimensions, 60x60 cm) comprising two
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dark arms (light intensity, 10 lux) and two illuminated arms (light intensity, 50 lux).
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Each arm was 25 cm in length and 10 cm in width. This arena was developed to take
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advantage of the relative aversion of crayfish to light and the spontaneous
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expression of its exploratory behavior in a new environment.
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Trial: Each tested animal was first placed in the center of the arena and confined for
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one minute under a small opaque chamber. After this delay, the crayfish was
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released, and exploratory behavior was recorded with a video camera (Sony Inc.)
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placed above the arena. We did not control the start position (head-body direction) of
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the animal in the maze: its random distribution did not bias subsequent statistical
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analyses. Animals with no or very low locomotion (distance moved 0.05, Dunn's multiple comparison test). The number of animals (n) is in
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parentheses.
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Supplementary Figure 5: Dose-dependent effect of CDZ on the recovery of
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behavior after a 30-min stress exposure
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Three groups of animals received different doses of CDZ (1.5, 7.5 or 15 µg/g)
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immediately after a 30-min stress exposure and 10-15 min before behavioral test.
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Behavioral variables were then measured in the D/L+maze (A-D). Increasing the
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dose of injected CDZ progressively restored the behavior of stressed animals toward
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control values for: (A) %time spent in light arms; (B) retreat ratios; (C) number of
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entries in the light arms; and (D) latency to first entry in the light arms
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(U=unstressed). For %time spent in the light arms, a dose of 7.5 µg/g was efficient to
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induce a partial recovery of behavior (P
