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: daniel.cattaert@u-bordeaux.fr

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

Materials and methods 1

2

Animals: 3

A total of 267 male crayfish (Procambarus clarkii) averaging 8.2±0.1 cm in length 4

and 15.6±1.5 g in weight were used in our experiments. The crayfish were obtained 5

from swamps near Bordeaux (“Réserve naturelle de Bruges”), reared inside a 6

specific animal house at 20°C with a 12h/12h light/dark cycle and fed pellets ad 7

libitum. To limit the effects of previous life experiences under natural conditions, each 8

animal was isolated at least three weeks before the experiments in a tank 9

(50x30x30cm) equipped with recirculating water. All experiments were performed in 10

accordance with the CNRS guidelines for animal care. 11

12

Induction of stress: 13

Electric fields: Stressed crayfish were placed in a specific tank (30x20x10 cm, light 14

intensity: 15 lux, Fig.2A). Two electrodes placed on opposite sides of the tank were 15

connected to a stimulator (AMPI, Isr.) that delivered repetitive trains (0.1 s duration) 16

consisting of ten 5-ms pulses at 100 Hz. The current intensity was progressively 17

increased until the appearance of an aversive response by the crayfish (i.e., tail flip). 18

Electric trains were then produced at 5-s intervals during the total time (30 min, 19

unless otherwise stated). Unstressed crayfish were also placed in the same tank for 20

the same duration (30 min) but did not receive electric stimulations. 21

22

Drug treatments: 23

Serotonin (5HT) and chlordiazepoxide hydrochloride (CDZ) (Sigma Aldrich, St Louis, 24

MO, USA) were dissolved in crayfish saline (195 mM NaCl, 5 mM KCl, 13 mM CaCl2, 25

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2 mM MgCl2 and 3 mM HEPES, pH 7.65). The crayfish were injected between two 26

abdominal segments directly into the hemolymph. 5HT was used at a final dose of 27

5µg/g fresh weight. We verified (by HPLC, see below) that this treatment resulted in a 28

significant increase in brain and ventral nerve cord 5HT concentrations after 5min. 29

CDZ at 15 µg/g, chosen after performing dose-response curves (Fig.S5). To prevent 30

serotonin effects, a mix of mianserin hydrochloride (5HT2 blocker, Sigma Aldrich) 31

and methysergide maleate salt (5HT1 and 2 blocker, Sigma Aldrich) were applied at 32

1 nmol/g immediately prior to the electric field stress protocol. 33

The timing of single injections was determined after preliminary experiments: 5HT 34

antagonists were effective when applied before stress exposure, while CDZ was 35

efficient immediately after. 36

37

Animal groups: 38

As a consequence of the stress and injection protocols, the animals used in our 39

experiments were separated into 9 different groups before being submitted to the 40

D/L+maze test. All animals had only one visit in the maze: (1) unstressed: animals 41

were placed in the stress tank as described above without receiving electric 42

stimulation; (2) unstressed+saline: unstressed animals were injected with saline 10-43

15 min before the test; (3) unstressed+CDZ: unstressed animals received a CDZ 44

injection 10-15 min before the test; (4) unstressed+5HT: unstressed animals were 45

injected with 5HT 10-15 min before the test; (5) unstressed+5HT and CDZ: 46

unstressed animals were injected with 5HT immediately followed by CDZ and tested 47

10-15 min later; (6) stressed: animals were placed in the stress tank and received 48

electric stimulations for 30 min (unless otherwise specified) 10-15 min before the test; 49

(7) stressed+saline: animals were placed as above and received a saline injection 50

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after the stress procedure 10-15 min prior to the test; (8) stressed+CDZ: animals 51

were placed as above and received a CDZ injection at variable doses (1.5, 7.5 or 15 52

µg /g) after the stress procedure and 10-15 min prior to the D/L+maze test; (9) 53

stressed+5HT antagonists: animals received a mixture of 5HT antagonists before 54

the stress procedure. 55

56

Bioamine level measurements: 57

Bioamines were measured by high performance liquid chromatography with 58

electrochemical detection (RP-HPLC-ECD) as previously described (15). Briefly, the 59

crayfish brain and nerve chain were rapidly extirpated and weighed, then separately 60

homogenized in 200 µL of 0.1N HClO4 by sonication and centrifuged at 13000 rpm 61

for 30 min at 4°C. Aliquots of the supernatants were injected into a reverse-phase 62

HPLC column (Chromasyl Stability C8, 150 x 4.6 mm). The mobile phase (70 mM 63

NaH2PO4, 0.1 mM disodium EDTA, 2 mM sodium octane-1-sulfonate monohydrate, 64

7% methanol, pH 3.9) was delivered at 1 mL/min by a Beckman 128 pump. A 65

coulometric detector (Coulochem II, ESA) equipped with a dual-electrode analytical 66

cell (potentials set at +350 and -270 mV) enabled detection of dopamine and 67

serotonin. Only 5HT displayed significant changes after stress and was thus further 68

analyzed. 69

70

Glucose level measurements: 71

In order to avoid interference of our measurements with circadian variations of 72

glucose titer, all hemolymph sampling were made at the same time of the day 73

(between 10am and 1pm). A 25 µl aliquot of hemolymph was carefully removed with 74

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an Hamilton syringe inserted in soft cuticle of the thoraco-coxal joint of first or second 75

pereiopod, and placed in 100 µl of 3 mM trichloracetic acid (C2HCl302). The extract 76

was centrifuged at 5000 rpm, and 50 µl of the supernatant was placed in 500 µl of 77

reactive solution (Glucose RTU, BioMérieux SA, France) and incubated at 37°C for 78

10 min. The optical density was then measured with a spectrophotometer at 505 nm 79

(Helios Epsilon, ThermoSpectronic, Fisher Scientific Inc.). 80

81

Behavior analysis: the dark/light plus maze protocol 82

Behavior paradigm: The objective of this experiment was to analyze the spontaneous 83

exploration behavior of crayfish confronted with a novel environment. The dark/light 84

(D/L) plus maze is a sub-aquatic arena (total dimensions, 60x60 cm) comprising two 85

dark arms (light intensity, 10 lux) and two illuminated arms (light intensity, 50 lux). 86

Each arm was 25 cm in length and 10 cm in width. This arena was developed to take 87

advantage of the relative aversion of crayfish to light and the spontaneous 88

expression of its exploratory behavior in a new environment. 89

Trial: Each tested animal was first placed in the center of the arena and confined for 90

one minute under a small opaque chamber. After this delay, the crayfish was 91

released, and exploratory behavior was recorded with a video camera (Sony Inc.) 92

placed above the arena. We did not control the start position (head-body direction) of 93

the animal in the maze: its random distribution did not bias subsequent statistical 94

analyses. Animals with no or very low locomotion (distance moved <40cm) were not 95

analyzed. 96

Tracking: We used Ethovision software XT8 (Noldus, NL) to detect and track crayfish 97

in the arena. The different zones of the arena are indicated in Fig.1A. The software 98

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then automatically calculated the different variables described in TableS1, except RR 99

that was calculated by the experimenter. The time spent in the middle area was not 100

considered relevant for comparisons. 101

Statistical analysis: Comparisons of the mean and SEM were obtained and plotted 102

using Prism software (GraphPad, US) and paired or unpaired non-parametric tests 103

were applied; n represents the number of individuals in each group. To compare 104

dark/light differences in a crayfish group, as in Fig.1 or Fig.S7, a Wilcoxon matched-105

pair signed-rank test was performed. A Mann-Whitney test was used to compare 2 106

groups, and 3 or more groups were compared with a Kruskal-Wallis test followed by 107

a Dunn's multiple comparison test. A value of P<0.05 was considered significant (*, 108

P<0.05, **, P<0.01 and ***, P<0.001). 109

PCA: Principal component analysis was performed using R software (Ade4 package) 110

based on the behavioral variables described in Table 1. In a first step, the 111

contribution of each variable to the variance in the first and second components of 112

the PCA was calculated (Fig.4A). Only the variables whose contribution to the 113

variance of a given component was larger than the mean contribution of all variables 114

to that component (i.e., 1428 for component 1 and 1429 for component 2) were 115

considered (see *). This method revealed that three variables were mostly 116

responsible for animal variance in the first component (%time in light: 2393; latency 117

to light: 2188; and retreat ratio: 2062). Similarly, three variables were mostly 118

responsible for animal variance in the second component (walked distance: 3659; 119

mean time per visit in light arms: 2444; and latency to first entry in dark arms: 2232). 120

This analysis indicates that the first component of the PCA (horizontal axis) mostly 121

represented avoidance of light arms. On the left side are animals that spent 122

substantial time in the light arms; on the right are animals that hesitated to enter the 123

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light arms (large latency and high level of retreat, a behavior characteristic of anxious 124

animals). Therefore, the first component of the PCA seems mostly indicative of the 125

anxiety level. Using a similar method, the second component was interpreted as 126

representing crayfish locomotion capacities: positive values represent high capacities 127

(i.e., large distance walked), whereas negative values represent walking impairment 128

(slow walking velocity responsible for longer visit time and longer latency to enter the 129

dark compartment). 130

In the second step, the location of each animal group was represented on the plane 131

defined by the first and second components of the PCA (Fig.4B). The separation 132

between pairs of groups was evaluated by calculating the inertia, which was defined 133

as the ratio of the between-group variance to the global variance. The statistical 134

significance of inertia for group separation was estimated using a Monte Carlo 135

permutation test (1000 runs) and fixed to P<0.01. In each run, the simulated inertia 136

was calculated. The distribution of simulated inertia values was then compared to the 137

real inertia. A P-value was then calculated as the ratio of the number of simulations in 138

which the simulated inertia was larger than the real inertia to the total number of runs. 139

Table S1: Definitions of the variables used for behavioral analysis in the 140

D/L+maze. 141

Table S2: Means ± SEM of all behavioral variables. 142

The PCA column indicates ensembles of significantly different groups resulting from 143

PCA analysis. 144

145

Supplementary Figure 1: Stress significantly increases glucose titers. 146

Glucose titers (means±SEM) were measured in the hemolymph of crayfish (n=6) 147

before the stress protocol (0.040±0.003 µg/µl), after 30 min of stress (post stress, 148

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0.15±0.03 µg/µl) and 30 min later (0.21±0.06 µg/µl, **: P<0.002, Kruskal-Wallis). By 149

contrast, in unstressed animals, glucose titers at t=0 (0,05±0,01 µg/µl, n=8), t=30 150

(0,06±0,01 µg/µl, n=7) and t=60 min (0,076±0,007 µg/µl, n=3) were non significantly 151

different (P=0.37, Kruskal-Wallis). 152

153

Supplementary Figure 2: Effect of stress duration on the behavioral variables 154

measured in the light arms of the D/L+maze. 155

(A-C) Increasing the duration of the stressful stimulation gradually decreased (A) the 156

number of entries in light arms (P<0.001, Kruskal-Wallis), and increased both (B) the 157

latency to first entry in light arms (P<0.001, Kruskal-Wallis) and (C) the retreat ratio 158

(P<0.001, Kruskal-Wallis). For each of these variables, a plateau was reached after 159

30 min of exposure to stress (*, **, ***: Dunn's multiple comparison test). U: values 160

from unstressed group for comparison. The number of animals (n) is in parentheses. 161

162

Supplementary Figure 3: Stability of avoidance behavior. 163

(A-C) Time course of stress-induced changes: (A) number of entries in light arms, (B) 164

latency to first entry in light arms and (C) RR. These three behavioral variables were 165

significantly different from unstressed crayfish (P<0,0001; Kruskal Wallis) and 166

remained stable during the first 30 min after stress (*, **, ***,: Dunn's multiple 167

comparison test). After 60 min, a recovery to unstressed values progressively 168

occurred, and total recovery was reached after 90 min. The bars represent 169

means±SEM. The number of animals (n) is in parentheses. 170

171

Supplementary Figure 4: 5HT induces an increase in glucose titers. 172

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Glucose titers were measured at t=0 (immediately before 5HT injection) and t=15min. 173

In the sham group, the same procedure was applied without 5HT injection. 174

Glucose titers significantly increased from 0.034±0.004 µg/µl at t=0 to 0.09±0.02 175

µg/µl at t=15min after 5HT injection (n=7, P<0.01, Dunn's multiple comparison test). 176

By contrast, in sham group, glucose titer remained unchanged (0,036±0,009 µg/µl, 177

n=5, P>0.05, Dunn's multiple comparison test). The number of animals (n) is in 178

parentheses. 179

180

Supplementary Figure 5: Dose-dependent effect of CDZ on the recovery of 181

behavior after a 30-min stress exposure 182

Three groups of animals received different doses of CDZ (1.5, 7.5 or 15 µg/g) 183

immediately after a 30-min stress exposure and 10-15 min before behavioral test. 184

Behavioral variables were then measured in the D/L+maze (A-D). Increasing the 185

dose of injected CDZ progressively restored the behavior of stressed animals toward 186

control values for: (A) %time spent in light arms; (B) retreat ratios; (C) number of 187

entries in the light arms; and (D) latency to first entry in the light arms 188

(U=unstressed). For %time spent in the light arms, a dose of 7.5 µg/g was efficient to 189

induce a partial recovery of behavior (P<0.05, compared to saline injection at CDZ 190

dose=0, Dunn's multiple comparison test). A significant effect on the %time in light 191

arms, RR and mean number of entries was observed at a dose of 15 µg/g (P<0.05, 192

P<0.01, compared to saline injection at CDZ dose=0, Dunn's multiple comparison 193

test). The number of animals (n) is in parentheses (B-D, same animals as in A). 194

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Supplementary Figure 6: CDZ injection does not prevent increased glucose 196

titer in stressed crayfish. 197

The increase in glucose titers (0.03±0.006 µg/µl before stress to 0.12±0.02 µg/µl after 198

stress) persisted 30 min after the stress protocol and CDZ injection (0.21±0.05 µg/µl, 199

n=5, P=0.004, Kruskal-Wallis; compare with Fig.S1). 200

201

Supplementary Figure 7: Schematic diagram summarizing the response to 202

stress in crayfish and the assumed site of drug action. 203

A stressor or 5HT injection elicit a rise in brain 5HT followed by a response to stress 204

consisting in at least 2 components: a metabolic mobilization that prepares acute 205

response to stress (e.g. fight, escape etc.) and the anxiety-like behavior that 206

anticipates future threatening situations. This scheme is based on experiments using 207

5HT antagonists and CDZ. When administered immediately before stress exposure 208

5HT antagonists blocked the emergence of the stress response caused by 5HT 209

increase (14), whereas CDZ prevented anxiety-like behavior when administrated 210

after stress exposure, without blocking the metabolic response. 211

212

Supplementary movie 1: Spontaneous behavior of unstressed crayfish in the 213

D/L+maze 214

Supplementary movie 2: Example of a retreat. 215

Supplementary movie 3: Spontaneous behavior of stressed crayfish in the 216

D/L+maze. 217

Supplementary movie 4: Spontaneous behavior of stressed crayfish after CDZ 218

injection. 219

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Name of variables Definition Measurement Range

Total distance walked

(Distance)*

Distance walked in the entire arena.

cm ≥0

%Time in dark

(Time spent in dark/ total time)*100

% 0-100

%Time in light *

(Time spent in light/ total time)*100

% 0-100

Latency to first in light

*

Time from start time to first entry in a light

arm

Second (s) 0-600

Latency to first in dark

*

Time from start time to first entry in a dark

arm

Second (s) 0-600

Mean number of entries into light

arms

Number of entries from the middle zone

to a light arm

NA ≥0

Mean number of entries into dark

arms

Number of entries from the middle zone

to a dark arm

NA ≥0

Mean duration per visit in light arms

*

Time in light arms/mean number

of entries

Second (s) ≥0

Mean duration per visit in dark arms

*

Time in dark arms/mean number

of entries

Second (s) ≥0

Retreat Ratio (RR)

*

Number of retreats/total

number of attempts

NA 0-1

Table S1 223

224

Table S2225

Distance walked

(cm)

%Time in Light arms

%Time in dark arms

Latency to first entry in

light (sec)

Latency to first entry in

dark (sec)

Mean number

of entries in light

Mean number

of entries in dark

Mean duration per visit in light (sec)

Mean duration per visit in dark (sec)

Retreat ratio

PCA

Unstressed (N=55)

468±26

35.7±1.7

43±2 92±15

49±14 4.7±0.3 5.5±0.4 61±8 63±8 0.26±0.03 a

Stressed (N=36)

379±41

11.6±1.9

66±4 311±41

42±17 2±0.3 5.1±0.7 26.5±4.7 169±32 0.65±0.05 b

Unstressed+Saline (N=19)

542±53

34±4

48.5±5 92±22

18.7±10 5.3±0.7 6.3±0.6 42.8±6.6 59±13 0.25±0.05 a

Unstressed+CDZ (N=12)

430±60

37.8±2.6

42±3 114±31

46±18 4.6±0.8 5.3±0.7 59.2±7.2 56±7 0.08±0.03 a

Stressed+Saline (N=14)

340±83

6±2.5

80±5 383±69

19±12 1.6±0.53

5.4±1.5 10.2±3.8 264±66 0.8±0.07 b

Stressed+CDZ (N=22)

468±43

23.7±3

48.4±2.6 147±25

40±12 3.7±0.4 6.3±0.6 43.7±6.4 56.4±7.3 0.38±0.06 a

Stressed + 5HT antagonists

(N=12)

332±65

22.7±3.2

59±3.8 101±38

87±28 3.5±0.7 3.3±0.6 57±16.7 147±25 0.22±0.07 a

Unstressed + 5HT (N=15)

138±30

12±4.6

65±8 395±69

15±6 0.7±0.3 2±0.3 47.4±17 272±55 0.68±0.11 b

5HT+CDZ (N=12)

171±31

46±8.6

35±7 171±53

129±66 1.9±0.3 1.7±0.4 157±32 133±38 0.08±0.06 a

226 Supp Fig 1 227

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Supp Fig 2 229

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236 Supp Fig 5 237

238

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Supp Fig 6 240

241

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242 Supp Fig 7 243

Group Distance percentlight meanTpervisitD meanTperVisitL latdark latlight Retreat uncdz1 uncdz 639.2940696 35.5 26.76 21.3 31.6 2.8 0.166666667 uncdz2 uncdz 306.8625448 41.63333333 96.53333333 83.26666667 0.6 133.8 0 uncdz3 uncdz 955.6140085 43.63333333 25.08888889 23.8 41 0.6 0 uncdz4 uncdz 287.0651426 42.33333333 47.85 84.66666667 0 197 0 uncdz5 uncdz 368.969036 40.4 64.8 60.6 97.6 9.2 0 uncdz6 uncdz 385.926389 30.36666667 39.35 45.55 110.2 223 0.333333333 uncdz7 uncdz 473.5581576 21.86666667 62.3 43.73333333 3.6 58 0 uncdz8 uncdz 293.1375279 39.87999983 59.88 79.75999967 2.16 72.48 0 uncdz9 uncdz 257.5701526 24.23999983 91.20000067 48.47999967 61.199999 346.8 0.25 uncdz10 uncdz 594.0833556 49.03999983 26.55 49.03999983 7.679999 135.36 0.25 uncdz11 uncdz 348.1 33.31666667 63.56 66.63333333 0 174.2 0 uncdz12 uncdz 254 51.68333333 71.03333333 103.3666667 194.6 18.72 0 saline1 saline 340.3613442 55.23333333 66.2 110.4666667 2 139.8 0.4 saline2 saline 288.781882 1.533333333 93.28 9.2 8.4 412 0.8 saline3 saline 595.5826614 34.9 32.5 29.91428571 0 81.4 0.125 saline4 saline 632.6728202 24.56666667 44.625 29.48 0.8 144.8 0.285714286 saline5 saline 439.0815646 2 60.025 12 0.4 26.4 0.666666667 saline6 saline 524.0092461 31.36666667 40.25 20.91111111 20.6 1.2 0 saline7 saline 755.8660169 46.13333333 23.84444444 27.68 0 15.6 0 saline8 saline 525.7711662 25.23333333 38.35 50.46666667 0.8 145.6 0.5 saline9 saline 345.6094344 41.1 71 82.2 5.4 61.6 0.25 saline10 saline 214.864382 54 76.5 108 168.6 2.2 0 saline11 saline 589.2085969 43.32000017 24.95999975 43.32000017 0.239999 74.16 0.142857143 saline12 saline 349.9285 48.75999983 7.36 32.50666656 113.519999 8.4 0 saline13 saline 698.4727135 37.2 38.60999975 31.88571429 0 94.08 0.125 saline14 saline 547.0530159 23.46666667 52.28571429 35.2 0 118.4 0.2 saline15 saline 530.5332219 23.93333333 57.6 35.9 0 119.8 0.428571429 saline16 saline 286.797816 4.166666667 284.8 25 0 100.8 0 saline17 saline 834.6785249 61.66666667 25.26666667 46.25 34.6 0 0.111111111 saline18 saline 1205.381799 49.53333333 19.50909091 24.76666667 0 39 0.2 saline19 saline 593.5733558 39.1 60.8 58.65 0 163 0.5 Shockscdz1 Shockscdz 402.7828819 10.4 44.62857143 31.2 1.4 89.6 0.666666667 Shockscdz2 Shockscdz 674.6009035 24.26666667 37.46666667 20.8 0 28 0.222222222 Shockscdz3 Shockscdz 262.4950962 2.266666667 57.56 6.8 160.4 270.8 0.714285714 Shockscdz4 Shockscdz 394.2750668 15.13333333 47.66666667 30.26666667 4.8 105.4 0.5 Shockscdz5 Shockscdz 793.8186308 28.3 30.48 33.96 0.4 87 0.375 Shockscdz6 Shockscdz 319.303261 10.53333333 82.92 63.2 3.4 200.8 0.75 Shockscdz7 Shockscdz 354.0151303 38.26666667 64.95 57.4 3 130.8 0 Shockscdz8 Shockscdz 434.0101632 29.8 48.35 29.8 171.4 108.8 0.25 Shockscdz9 Shockscdz 451.2550502 55.86666667 25.44 67.04 68 16.2 0 Shockscdz10 Shockscdz 748.8868518 13.16666667 33.3 26.33333333 0.8 183.8 0.666666667 Shockscdz11 Shockscdz 458.2332622 23.33333333 59.83333333 35 1 186 0.428571429 Shockscdz12 Shockscdz 520.9515922 43.46666667 26.34285714 37.25714286 5.6 36.6 0 Shockscdz13 Shockscdz 213.6260832 35.33333333 70.93333333 106 26.4 183.4 0 Shockscdz14 Shockscdz 958.488 23.63333333 27.32307692 23.63333333 12.2 70.4 0.25 Shockscdz15 Shockscdz 676.1408345 32.5 32.45 39 10.8 57.2 0.375 Shockscdz16 Shockscdz 564.8101201 19.16666667 42.82857143 23 43 0 0.444444444 Shockscdz17 Shockscdz 500.9987564 11.2 45.875 16.8 0 233.6 0.5 Shockscdz18 Shockscdz 540.1067595 20.23333333 34.08571429 30.35 107.6 229.8 0.2 Shockscdz19 Shockscdz 234.8980817 18.9 171.5 56.7 94.2 393.2 0 Shockscdz20 Shockscdz 248.9773395 46.1 121.1 138.3 109.2 4.2 0.6

Shockscdz21 Shockscdz 308.7403466 8.533333333 71.44 51.2 12 450.4 0.833333333 Shockscdz22 Shockscdz 244.3432035 12.56666667 63.85 37.7 46 177.4 0.6 shock1 shock 360.7876854 31.26666667 47.24 62.53333334 9.8 57 0.625 shock2 shock 271.1281908 11.13333333 52.55 33.39999999 5.6 115.8 0.75 shock3 shock 276.897618 23.4 76.55 46.8 1.8 40 0.5 shock4 shock 434.7771743 17.36666667 47.8 34.73333334 12.4 141.8 0.571428571 shock5 shock 184.0235141 4.533333333 225.2 27.2 2.4 479.2 0.666666667 shock6 shock 576.8268386 3.933333333 40.52 23.6 11.8 573.6 0.9 shock7 shock 336.1705527 0 98.8 0 2 600 1 shock8 shock 400.1250814 0 48.47272727 0 14 600 1 shock9 shock 1005.880399 0.933333333 23.56666667 5.599999998 1.2 13.2 0.944444444 shock10 shock 811.3807497 11.93333333 26.22857143 17.9 1.2 36.2 0.6 shock11 shock 229.1875714 19.33333333 127.5333333 38.66666666 72.4 25.6 0.25 shock12 shock 66.09465616 0 598.4 0 1.4 600 1 shock13 shock 693.9897708 18.13333333 30.21818182 18.13333333 1 39.2 0.454545455 shock14 shock 165.8809183 0 284.5 0 22.4 600 1 shock15 shock 105.0491732 0 600 0 0 600 1 shock16 shock 433.3812738 26.93333333 46.96666667 40.4 2.2 138.6 0.555555556 shock17 shock 317.9367058 6.133333333 131.8 12.26666667 1.6 346.8 0.5 shock18 shock 342.3932297 0 569.8 0 30.4 600 1 shock19 shock 631.6346459 12.16666667 49.96666667 36.5 15.6 116.6 0.5 shock20 shock 906.4415526 14.56666667 64.86666667 29.13333333 5.4 48.4 0.571428571 shock21 shock 432.6887651 0.479999833 102.6240002 2.879999 0.479999 597.36 0.75 shock22 shock 173.0215002 0 430 0 170 600 1 shock23 shock 72.5770153 0 595.199999 0 5.04 600 1 shock24 shock 793.7144205 28.39999967 30.8399999 28.39999967 0.72 68.88 0.571429 shock25 shock 81.24024092 0 581.519999 0 18.72 600 1.000000 shock26 shock 612.3389906 20 45.805714 30 0.479999 136.08 0.300000 shock27 shock 358.718665 7.359999833 76.01999975 22.0799995 10.8 312 0.666000 shock28 shock 201.1277 28.24 130.96 84.72 2.64 345.6 0 shock29 shock 270.4307 7.4 78.92 44.4 0 498 0.75 shock30 shock 189.6703 19.08 206.52 57.24 19.2 394.32 0 shock31 shock 102.3194 0.24 379.92 1.44 220.32 27.12 0.5 shock32 shock 438.0144 44.12 41.24 52.944 1.679999 38.16 0.2857143 shock33 shock 125.8295 12.84 18.72 38.52 534.24 468.24 0 shock34 shock 646.7 25.83333 47.65714286 30.999996 30 87.36 0.375 shock35 shock 236.8 22.2 84.06666 133.2 205 63.12 0.8 shock36 shock 361.6 0 60.76667 0 78 600 1 C1 control 485.8376644 44.53333333 27.88 53.44 16.6 59.6 0.375 C2 control 284.3527409 51.4 48.6 61.68 298.8 8.2 0.166666667 C3 control 291.3686063 35.93333333 73.8 71.86666666 13 82.4 0.25 C4 control 465.3244163 24.96666667 69.4 37.45000001 1.4 96 0.333333333 C5 control 545.0826018 30.9 55.23333333 37.08 0 35.4 0.166666667 C6 control 75.17618558 39.9 296.6 239.4 29 360.8 0 C7 control 209.7302638 44.93333333 121.8 89.86666666 80.8 16.6 0.25 C8 control 441.3340386 23 53.86666667 46 0.4 189.8 0.571428571 C9 control 343.7384969 50.23333333 65 75.35 2.4 49.4 0.2 C10 control 504.3654857 53.63333333 30.1 53.63333333 1.6 47.2 0 C11 control 331.2081994 38.93333333 66.8 77.86666666 0.8 85 0 C12 control 376.4830552 29.1 70.28 58.2 2.6 205 0.4 C13 control 203.0314598 50.76666667 74.2 152.3 0.4 121.4 0 C14 control 466.5598529 28.53333333 44.05714286 42.8 2.4 40.4 0.333333333 C15 control 361.6686349 41.9 66.85000001 62.85 6.4 122.8 0.2 C16 control 84.46449781 67.5 121 405 479.2 54.6 0 C17 control 639.6 43.93333333 64.35 87.86666666 0 84.6 0.333333333 C18 control 511.9410958 42.56666667 58.44 42.56666667 69 17.2 0.5 C19 control 640.6204798 41.06666667 40.84 49.28 13.8 178.6 0.454545455 C20 control 450.3 37.46666667 26.38 32.11428572 5.6 87.6 0 C21 control 883.1 21.33333333 36.875 18.28571428 16.4 8.4 0.533333333

C22 control 642.6632038 23.23333333 41.175 23.23333333 0 47.4 0.454545455 C23 control 689.2744388 38.96666667 34.45714286 38.96666667 11.6 245.8 0.25 C24 control 568.2733103 31.73333333 67.4 47.6 104.2 0.8 0.2 C25 control 626.7254641 27.63333333 44.17142857 27.63333333 0 153.4 0 C26 control 316.0482326 27.2 53.1 40.8 99.6 252.8 0.2 C27 control 373.0136026 12.9 117.45 25.8 2.4 152 0.25 C28 control 474.0429454 30.43333333 29.83333333 45.65 2.4 29.4 0 C29 control 506.3338623 31.83333333 59.64 38.2 32.6 0 0.2 C30 control 1123.722919 40.53333333 15.38571429 17.37142857 23.6 0 0.285714286 C31 control 484.5271017 28.03333333 28.475 33.64 0.8 44.6 0.375 C32 control 502.4 14.43333333 227.8 21.65 75.4 1 0.666666667 C33 control 483.9515851 67.46666667 9.914285713 57.82857143 0 166.8 0 C34 control 333.6737083 30.13333333 27.85 36.16 6.8 79.8 0 C35 control 595.2 30.83333333 128.3 185 2.2 568.2 0.444444444 C36 control 211.9 5.366666667 262.3 4.6 277.4 0 0.5 C37 control 541.9966776 34.7 35.83333333 34.7 30 0 0.25 C38 control 337.8 35.96666667 82.90000001 71.93333334 1.6 143.6 0 C39 control 849.6716716 49.9 16.54 29.94 0.6 30.6 0.166666667 C40 control 453.9928645 30.93333333 29.06666667 46.4 1.2 28.8 0.333333333 C41 control 726.1814586 38 29.13333333 38 21.6 121.4 0.142857143 C42 control 466.2283147 50.53333333 30.46666667 60.64 4.2 31.8 0.4 C43 control 466.2113293 13.6 57.94285714 27.2 5.4 180.6 0.8 C44 control 543.719804 36 31.91428571 36 143 24.2 0.125 C45 control 717.6215181 27.4 30.02 27.4 0 11.6 0.285714286 C46 control 200.3453754 43.36666667 61.00000002 86.73333334 442.6 3.6 0.666666667 C47 control 368.1716798 34.83333333 42.84 69.66666666 46.2 0.8 0 C48 control 527.4733093 28.53333333 43.74285714 34.24 50 0 0 C49 control 315.1849215 5.733333333 66.28 17.2 3.2 417.8 1 C50 control 525.7711662 25.23333333 38.35 50.46666666 0.8 145.6 0.5 C51 control 345.6094344 41.1 71 82.2 5.4 61.6 0.25 C52 control 214.864382 54 76.5 108 168.6 2.2 0 C53 control 589.2085969 43.32000017 24.95999975 43.32000017 0.239999 74.16 0.142857143 C54 control 349.9285 48.75999983 7.36 32.50666655 113.519999 8.4 0 C55 control 698.4727135 37.2 38.60999975 31.88571429 0 94.08 0.125 Shocksaline1 shockssaline 1005.880399 0.933333333 23.56666667 5.6 1.2 13.2 0.944444444 Shocksaline2 shockssaline 811.3807497 11.93333333 26.22857143 17.9 1.2 95 0.6 Shocksaline3 shockssaline 229.1875714 19.33333333 127.5333333 38.66666667 72.4 25.6 0.25 Shocksaline4 shockssaline 66.09465616 0 598.4 0 1.4 600 1 Shocksaline5 shockssaline 693.9897708 18.13333333 30.21818182 18.13333333 1 39.2 0.454545455 Shocksaline6 shockssaline 165.8809183 0 600 0 22.4 600 1 Shocksaline7 shockssaline 105.0491732 0 284.5 0 0 600 1 Shocksaline8 shockssaline 433.3812738 26.93333333 600 40.4 2.2 138.6 0.555555556 Shocksaline9 shockssaline 317.9367058 6.133333333 46.96666667 12.26666667 1.6 346.8 0.5 Shocksaline10 shockssaline 54.27248825 0 131.8 0 0 600 1 Shocksaline11 shockssaline 545.8 0.166666667 49.2 1 0.8 596 1 Shocksaline12 shockssaline 55.7 1.466666667 469.8 8.8 154 504 1 Shocksaline13 shockssaline 170 0 137.5 0 2 600 1 Shocksaline14 shockssaline 100 0 570 0 5 600 1 5HTCDZ1 5HTCDZ 142.9096659 21.9 52 131.4 0.6 340 0.666666667 5HTCDZ2 5HTCDZ 460.5330954 46.2 46.44 69.3 0 100.2 0 5HTCDZ3 5HTCDZ 107.6838964 70.63333333 99.4 423.8 0.8 111.2 0 5HTCDZ4 5HTCDZ 169.2521881 30.8 117.1 92.4 133.2 9.6 0 5HTCDZ5 5HTCDZ 88.54163508 88.96666667 0 266.9 600 18.8 0 5HTCDZ6 5HTCDZ 111.2721365 95.86666667 0 287.6 600 0 0

5HTCDZ7 5HTCDZ 254.7852897 35.53333333 109.8 106.6 7.8 129.2 0 5HTCDZ8 5HTCDZ 144.3470776 14.86666667 197.2 89.2 0 190.8 0 5HTCDZ9 5HTCDZ 96.86 18.43333333 422.2 110.6 2.8 489.6 0 5HTCDZ10 5HTCDZ 211.3 77.96666667 77 155.9333333 0 82 0 5HTCDZ11 5HTCDZ 57.7 12.3 361.6 73.8 23.6 524.8 0 5HTCDZ12 5HTCDZ 211.3 39.96666667 108.7 79.93333333 179.6 54.2 0.25 anta5HT-chocs1 anta5HT-chocs 210.5783474 14.06666667 181.6 42.2 7.6 217.2 0.333333333 anta5HT-chocs2 anta5HT-chocs 130.0792677 19.3 216.6 115.8 11 133.2 0 anta5HT-chocs3 anta5HT-chocs 114.7175446 36.23333333 309.2 217.4 291 49 0.5 anta5HT-chocs4 anta5HT-chocs 734.4453731 27.73333333 38.225 20.8 0.4 68.2 0.2 anta5HT-chocs5 anta5HT-chocs 512.6423465 23.56666667 72.86666667 20.2 234 11.8 0.222222222 anta5HT-chocs6 anta5HT-chocs 389.4128248 38.63333333 103.7333333 57.95 50 0 0 anta5HT-chocs7 anta5HT-chocs 198.6840884 15.9 229.3 47.7 68.6 11 0 anta5HT-chocs8 anta5HT-chocs 734.7167757 25.53333333 49.93333333 25.53333333 26.2 0.8 0.333333333 anta5HT-chocs9 anta5HT-chocs 142.1908805 13.23333333 230.8 39.7 48 385.2 0.333333333 anta5HT-chocs10 anta5HT-chocs 188.5059893 0.5 128.6 3 8.4 310.8 0.75 anta5HT-chocs11 anta5HT-chocs 407.6 36.96666667 65.55 36.96666667 124.6 14.2 0 anta5HT-chocs12 anta5HT-chocs 221.8 20.63333333 140.3333333 61.9 177.4 10.4 0 5HT1 5HT 175.4343047 33.9 44 203.4 4 48.4 0 5HT2 5HT 431.137218 42.03333333 79.75 84.06666667 0 55.4 0.33 5HT3 5HT 59.9854731 0 558.4 0 41.8 600 1 5HT4 5HT 60.20733746 0 566 0 34.2 600 1 5HT5 5HT 103.4825277 0 299.5 0 0 600 1 5HT6 5HT 114.1087434 0 600.2 0 0 600 1 5HT7 5HT 40.82 0 580.2 0 20 600 1 5HT8 5HT 280.1365253 51.8 98.7 103.6 65.4 0 0 5HT9 5HT 211.5769245 21.93333333 85.2 131.6 3 430.2 0.5 5HT10 5HT 295.1846026 0.833333333 89 5 0 3.8 1 5HT11 5HT 55.60977954 0 235.1 0 48.4 600 1 5HT12 5HT 54.0847328 12.13333333 354 72.8 5 527.4 0.5 5HT13 5HT 49.63724908 0 147.8 0 0 600 1 5HT14 5HT 40.01 18.53333333 60.5 111.2 3.8 64.8 0 5HT15 5HT 101.9 0 280 0 2 600 1