Measuring Behavioral Individuality in the Acoustic Startle Behavior in Zebrafish

Materials and Reagents

1. Petri dishes
   
2. Pipette tip 
   
3. 48-well multi-well plate
   
4. Zebrafish embryos
   
5. Instant ocean sea salt mix (Spectrum Brands, catalog number: SS15-10 )
   
6. DI H₂O
   
7. E3 medium (see Recipes)
   

Equipment

1. P1000 PIPETMAN pipettor
   
2. Stir plate
   
3. zBox parts list (Table 1)
   
   Table 1. ZBox parts list
   

   Manufacturer/Item description	Part number	Quantity
   Newegg - Computer
   Minimum requirements: PC, 8 GB RAM, 2 GHz processor		1
   PCIe Firewire 800 card	9SIA24G28M5974	1
   National Instruments (DAQ)
   NI PCI-6229	779068-01	1
   RC68-68 Cable (1 m)	192061-01	2
   CB-68LP - Unshielded	777145-01	2
   McMaster Carr (Structural external)
   80/20 1”x1” framing 96”	47065T123	4
   80/20 corners	47065T177	20
   80/20 nuts 80x	47065T147	12
   Black PVC, 48”x48”x.	84775K942	1
   236” (back)Black PVC 36”x48”x.236	84775K742	3
   sides and top Super Adhesive Hook	94985K614	10ft
   Loop	9489K874	10ft
   Loctite Black Silicone sealant	74945A81	1
   Black gaffer’s tape	7612A94	1
   Right angle 80/20	47065T223	2
   80/20 fastener	47065T139	2
   Acrylic cement	7528A13	1
   PTFE tape	4591K11	1
   General Purpose Epoxy	Permatex 84101	1
   Nylon 6/6 Thumb Screw	96295A336	1
   Thorlabs (Structural internal)
   Breadboard 24”x36”	MB2436	1
   XT66 Rail	XT66-500	1
   XT66 Dovetail	XT66DP-500	1
   XT66 Dovetail clamp 40 mm	XT66C2	2
   XT66 right angle cross piece	XT66CB	1
   XT66 base plate	XT66P1	1
   XT66 pivot platform	XT66RC	2
   Plate holder	FP01	2
   10” mounting post	P10	1
   Pedestal Base adapter	PB4	1
   Clamping fork	PF175	1
   Post mounting clamp	C1501	1
   Blackout fabric	BK5	1
   Right angle plate	AP90	1
   Edmund Optics
   101 x 127 mm, 45 Degree AOI, Hot Mirror	43-958	2
   Pike F-245 2/3” CCD camera	59-221	1
   Pike Tripod adapter	59-227
   Plastic IR filter	43-949	1
   Navitar
   50MM F/2.8 TELECENTRIC LENS	TC-5028	1
   Unibrain (1394store.com)
   1m (15 ft) IEEE-1394b 9p to 9p screw lock cable		1
   Digi-Key Electronics
   100Kohm resistor	CMF100KHFCT-ND	4
   2-pin recipient	A99613-ND	10
   2-pin socket	A99620-ND	20
   2-pin header	A30770-ND	10
   Yellow LEDs	C503B-AAN-CY0B0251-ND	48
   IR LEDs	475-2919-ND	48
   White LEDs	C503C-WAS-CBADA151-ND	48
   Blue LEDs	C503B-BAN-CY0C0461-ND	48
   1ohm resistor	P1.0W-2BK-ND	3
   270 ohm resistor	PPC270W-1CT-ND	16
   390 ohm resistor	PPC390W-1CT-ND	6
   Diode	1N914B-ND	2
   12 V power supply	EPS377-ND	1
   24 V power supply	EPS357-ND	1
   LEDsupply
   BuckPuck DC LED Drivers	0302x-D-x-xxxx	1
   Machine shop (plastics)
   Custom plastic fabrication		1
   Whatman (GE) plates
   Multi-well plate of desired well number
   Visaton (speakers)	8006	2
   Pyle Audio (amplifier)	PyleHome PCA1	1
   60 Watt incandescent bulb		1
   Simple designs home (lamp)	LD1003-WHT	1

   
   Notes: Assembly of zBox (see Figures 1-7):
   1. Use epoxy to adhere the two side walls into the grooves on the back panel (Figure 2).
      
   2. The screw holes in the LED platforms need to be drilled for 4-40 threads (Figure 3).
      
   3. Use the plastic thumbscrews to mount the 2 LED platforms (one for the imaging IR LEDs and one for LEDs for use in optogenetic experiments) to the slots in the side walls (Figure 4).
      
   4. LED arrays can be wired in 8 x 6 or 12 x 10 arrays depending on the desired amount of light. Due to perspective, the IR imaging LEDs are best arranged so that the arrays extend to the sides of the LED platform. Thus, given the distance from the plate, the IR LEDs can evenly light the extent of the multiwell plate.
      
   5. The BuckPack LED driver can be wired to power the LED arrays and be switched using TTL pulses from the DAQ
      
   6. The multiwell plate platform should be able to slide into the grooves on the side walls.
      
   7. Screw speakers to side of multiwell platform (Figures 4-6).
      
   8. Use diffuser paper to assure that IR light is evenly distributed throughout wells.
      
   9. Mount camera to P10 posts. XT66 rails should be constructed to hold IR mirrors (Figure 7).
      
      
      Figure 1. zBox plans
      
      
      Figure 2. Slide side walls into back panel grooves and epoxy in place
      
      
      Figure 3. Use thumb screws to mount LED platforms into side wall slots
      
      
      Figure 4. Insert multi-well plate platform into top grooves of side walls
      
      
      Figure 5. Detailed view of eaker mounting
      
      
      Figure 6. Completed zBox
      
      
      Figure 7. Camera mounting

Software

1. Matlab2014a or later with Imaging Processing and Data Acquisition toolboxes installed

Procedure