Materials and Reagents

1. Animals
   
   1. The study has been performed on adult male crayfish, Procambarus clarkii , fished near Bordeaux (France) in the "Réserve naturelle de Bruges" and stored in an animal house with 12:12 dark light cycle. Crayfish were fed ad libitum with pellets (Novo Prawn). Crayfish used for this study were 8.7 ± 0.8 cm and 22.1 ± 1.1 g. We generally used male crayfish in our experiments (distinguished by their first abdominal appendages), but this protocol is also suitable for experiments on females.
      
   2. Before any experiment, crayfish were isolated in enriched aquariums (75 cm x 40 cm x 50 cm), filled with recycled oxygenated water and enriched with pebbles and plastic tubes (behind or inside which they can hide). Animals were kept isolated at least 3 weeks before any experiment in order to erase past life histories and to avoid social interactions.
      
   3. Water must be chlorine free (agitating fresh tap water during 24 h is generally enough to remove chlorine). Change water after each session or use a conventional filtering system for aquarium.
      
   4. Fishnets are used to manipulate crayfish.
   
   
2. Reagents
   
   1. Ringer: Crayfish were injected with a classical invertebrate ringer containing 195 NaCl, 5 KCl, 13 CaCl₂, 2 MgCl₂ and 3 HEPES (Sigma-Aldrich) with a pH of 7.65
      
   2. Chlordiazepoxide hydrochloride (CDZ) (Sigma-Aldrich) (dissolved in crayfish ringer and was used at a dose 15 µg/g)

Equipment

1. Dark/light (D/L) plus maze
   
   1. Customized apparatus (65 x 65 cm, see Figure 1) is adapted to the size of adult crayfish. Smaller or larger dimensions might be necessary for young crayfish or other species.
      
   2. The illuminated arms were made of transparent Plexiglas and dark arms of opaque Plexiglas (Figure 1).
      
   3. The maze is filled with ca. 7 cm of oxygenated freshwater. It is placed in the center of a larger round tank (Figure 2). The round tank (1.5 m in diameter) is also filled with 7 cm freshwater to equilibrate water pressure on each maze wall and to allow for good oxygenation and recycling of water (Figure 2).
      
      
      Figure 1. Dark/light plus maze. The total length of the maze is 65 cm x 65 cm. Each arm is 25 cm long and 15 cm wide. The middle zone is 15 cm x 15 cm. The light is generated by four sources comprising two LEDs. Each source of light is placed on the outside face of the opaque Plexiglas.
      
      
      Figure 2. The dark/light plus maze is placed in a larger tank (diameter 1.5 m). Right: Room light switch on. Left: Room light switch off under experimental conditions.
      
      
   4. Illumination of the light arms is performed by a series of LEDs (white light; voltage; 3.5 V, light intensity; 880 to 2,530 mcd) disposed outside the plus maze, on the outward side of the dark arms on the outmost extremities (see Figure 3).
      
      
      Figure 3. Two LEDs placed at the end of the dark arm illuminate a light arm. The height of the maze wall is 20 cm.
   
   
2. Other devices
   
   1. A video camera must be placed above the maze. We used a handycam (Sony inc, model: HDR-XR155) but any commercial camera can be used.
      
   2. A pump (NewJet) is placed on a large tank between experimental sessions to agitate and oxygenate water.
      
   3. Light intensity was assessed with a luxmeter (Chauvin Arnoux).

Software

1. We used Ethovision v8 (Noldus) but any tracking software can be used. Measured parameters are listed in Table 1.
   
   Table 1. List of parameters that can be used to analyse the anxiety-like behaviour in the D/L plus maze
   

   Name of variable (and abbreviation used in Figure 2)	Definition	Measurement	Range
   Total distance moved (Distance)	Distance moved in the entire arena.	cm	≥0
   % Time in zone (%T light, dark)	Time spent in a zone (dark or light)/(divided by total time)*100	%	0-100
   Latency to first in light (Lat.Light)	Time from start time to first entry in a light arm	Second (s)	0-600
   Latency to first in dark (Lat.Dark)	Time from start time to first entry in a dark arm	Second (s)	0-600
   Mean number of entry in light arms (Nb. ent. Dark)	Number of entry from middle zone to a light arm	NA	≥0
   Mean number of entry in dark arms (Nb. ent. Light)	Number of entry from middle zone to a dark arm	NA	≥0
   Mean duration per visit in light arms	Time in light arms/mean number of entry	Second (s)	≥0
   Mean duration per visit in dark arms	Time in dark arms/mean number of entry	Second (s)	≥0
   Retreat Ratio (RR)	Number of retreat / total number of attempts	NA	0-1

Procedure