Procedures

Procedure 1: Animal housing and husbandry

Procedure PDF:

 

Procedure 2: Open Field

Procedure PDF:

 

Equipment, software, and supplies

• Open field arena, square shaped, clear polycarbonate arena (44.5 x 44.5 x 25.4 cm) with removable clear acrylic aerated lids. Infrared photobeam sensors at floor level and 3 inches from arena floor (16 x 16). (Med Associates, #MED-OFAS-515U)
• Environmental control chamber, each arena placed within a sound attenuated, ventilated cabinet containing 2 incandescent lights (18.5 inches from the center of arena floor), providing illumination of 60 +/- 10 lux (Med Associates, #MED-OFA-017)
• Activity monitor software (Med Associates, v. 7.0.5.10 SOF-812)
• Forceps, for all mouse transfers

Reagents and solutions

• 70% ethanol, to sanitize arena between subjects
• Virkon wipes, 1% Virkon working solution used to sanitize arena between test cohorts
• Paper towels
• Spor-Klenz, used to sterilize forceps between animals (1:32 in water) (Steris Life Science)

Environmental Conditions

Holding Area and/or Sample Collection Area

Facility: Subjects transported from housing room to anteroom adjacent to procedure room

Acclimation Period: minimum 30 min

Description of Holding Enclosure: Home cages

Max # Animals Per Enclosure: 1

Acoustic Background: Ambient

Testing Area

Facility: Procedure room

Acclimation Period: Mice immediately placed in test chambers

Room Space (dimensions): 20'9" x 10'2" with 24 arenas, each within its own environmental chamber; double stacked along each side of the room

Light Source: Overhead incandescent lights at ~500 lux. Lighting within chambers is provided by 2 28-V lamps, providing 14 lux

Light Intensity: ~500 lux in room; 14 lux at testing floor of arena

Temperature (°C): 21.7 +/- 3

Humidity: 50 +/- 20%

Acoustic Background: Ambient

Acoustic Deprivation: Sound attenuating chamber

Handling With: Forceps

Collection Method: Whole Mouse

Technician Protection: Gloves, Mask, Lab Protective Clothing

# Mice Tested at the Same Time: 24

Female and Male Mice Tested in Same Test Group: Yes

Sanitation of Equipment: 70% ethanol between animals; Virkon solution between cohorts

Notes: Testing conducted during light phase, beginning at least 60 min after lights on and concluding at least 30 min prior to lights off

Additional ARRIVE Guidelines

Allocation of Animals to Test Group: Subjects pre-assigned to test chamber and counterbalanced based on strain and sex.

Data Collection: Automated

Data QC: Data reviewed as generated for technical issues, verification of subject IDs, etc. Observed aberrations are noted.

Procedure 3: Light Dark

Procedure PDF:

 

Equipment, software, and supplies

• Open field arena, see Open Field procedure
• Environmental control chamber, see Open Field procedure
• Activity monitor software, see Open Field procedure
• Forceps, to handle all animal transfers
• Light-dark box, insert (divides arena into light zone and dark zone) constructed of black polycarbonate material that does not interrupt infrared beams; placed in back half of open field arena with entry door opening positioned toward front of arena thereby reducing light levels in dark side of the box

Reagents and solutions

• 70% ethanol, used to sanitize between subjects
• Virkon wipes, used to sanitize arena at end of testing day
• Paper towels
• Spor-Klenz, used to sterilize forceps between animals (1:32 in water) (Steris Life Science)

Environmental Conditions

Holding Area and/or Sample Collection Area

Facility: Anteroom to procedure room; subjects transported from housing room to anteroom on wheeled rack

Acclimation Period: 30 min

Description of Holding Enclosure: Home cages

Max # Animals Per Enclosure: 1

Acoustic Background: Ambient

Testing Area

Facility: Procedure room

Acclimation Period: Mice are wheeled into procedure room and immediately placed in chambers

Room Space (dimensions): 10'9" x 10'2"; 24 arenas placed within environmental chambers and double stacked on both sides of room

Light Source: Overhead fluorescent light at ~500 lux; lighting within chambers provided by 2 28-V lamps giving 14 lux

Light Intensity: ~500 lux in room; 14 lux at testing floor

Temperature (°C): 21.7 +/- 3

Humidity: 50 +/- 20%

Acoustic Background: Ambient

Acoustic Deprivation: Sound attenuating chamber

Handling With: Forceps

Collection Method: Whole Mouse

Technician Protection: Gloves, Mask, Lab Protective Clothing

# Mice Tested at the Same Time: 24

Female and Male Mice Tested in Same Test Group: Yes

Sanitation of Equipment: 70% ethanol between subjects; Virkon between cohorts or at end of day

Notes: Test conducted during light phase, beginning at least 60 min after lights on and concluding at least 30 min prior to lights off. Subjects placed in center of arena on light side, facing dark side.

Additional ARRIVE Guidelines

Allocation of Animals to Test Group: Subjects pre-assigned a test chamber; counterbalanced by strain and sex

Data Collection: Automated

Data QC: Data are reviewed as generated for technical issues, verification of subject IDs, etc. Observed aberrations are noted.

Procedure 4: Hole Board

Procedure PDF:

 

Equipment, software, and supplies

• Open field arena, see Open Field procedure
• Environmental control chamber, see Open Field procedure
• Activity monitor software, see Open Field procedure
• Forceps, used to handle and transfer mice by tail
• Holeboard, consists of two components: metal floor with 16 evenly spaced holes and an underplate containing 16 holes matching those in the floor with wire mesh for baiting the holes

Reagents and solutions

• 70% ethanol, used to sanitize arena between subjects
• Virkon wipes, used to sanitize arena at end of testing day (1:32 in water)
• Paper towels
• Spor-Klenz, used to sterilize forceps between animals (Steris Life Science)

Environmental Conditions

Holding Area and/or Sample Collection Area

Facility: Anteroom to procedure room

Acclimation Period: 30 min

Description of Holding Enclosure: Home cages

Max # Animals Per Enclosure: 1

Acoustic Background: Ambient

Testing Area

Facility: Procedure room

Acclimation Period: Subjects are transported from the anteroom to procedure room where they are immediately placed in chambers

Room Space (dimensions): 20'9" x 10'2". 24 arenas within their own environmental chambers; double stacked and located around perimeter

Light Source: Overhead fluorescent lights providing ~500 lux; lighting within chambers provided by 2 28-V lamps yielding 14 lux

Light Intensity: ~500 lux overhead; 14 lux at testing floor

Temperature (°C): 21.7 +/- 3

Humidity: 50 +/- 20%

Acoustic Background: Ambient

Acoustic Deprivation: Sound attenuating chamber

Handling With: Forceps

Collection Method: Whole Mouse

Technician Protection: Gloves, Mask, Lab Protective Clothing

# Mice Tested at the Same Time: 24

Female and Male Mice Tested in Same Test Group: Yes

Sanitation of Equipment: 70% ethanol between subjects; Virkon at end of testing day

Notes: Testing conducted during light phase, beginning at least 60 min after lights on and concluding at least 30 min prior to lights off.

Additional ARRIVE Guidelines

Allocation of Animals to Test Group: Subjects pre-assigned test chamber; counterbalanced based on strain and sex

Data Collection: Automated

Data QC: Data are reviewed as generated for technical issues, verification of subject IDs, etc. Observed aberrations noted.

Procedure 5: Novelty Place Preference

Procedure PDF:

 

Equipment, software, and supplies

• Novelty Place Preference apparatus, rectangular shaped, 3 chambered arena (acrylic or polycarbonate): white/black chambers are 5" x 6.5" x 5"; gray center chamber is 5" x 3.5" x 5". Automatic doors between chambers and clear aerated lid; bottom of arena contains pairs of horizontal infrared photobeam sensors at the level of the floor (Med Associates, St. Albans, VT, USA)
• Environmental control chamber, each arena placed within a sound attenuated, ventilated cabinet with dimensions 25" x 26" x 21" (Med Associates, St.Albans, VT, USA)
• MED-PC IV software (Med Associates, St. Albans, VT, USA)
• Forceps, used to handle all mouse transfers

Reagents and solutions

• 70% ethanol, used to sanitize arena between subjects
• Virkon wipes, 1% solution used to sanitize arena between cohorts of mice and at end of day (Lanxess Corporation, Pittsburgh, PA)
• Paper towels
• Spor-Klenz, used to sterilize forceps between animals (1:32 in water) (Steris Life Science)

Environmental Conditions

Holding Area and/or Sample Collection Area

Facility: Anteroom to procedure room

Acclimation Period: Subjects transported from housing room where they are left undisturbed for 30 min

Description of Holding Enclosure: Home cage

Max # Animals Per Enclosure: 1

Acoustic Background: Ambient

Testing Area

Facility: Procedure room

Acclimation Period: Subjects wheeled in from the anteroom and immediately placed in the test chamber

Room Space (dimensions): 10' x 11'; 16 arenas, each within its own environmental chamber; double stacked, located along 2 walls of the room

Light Source: Overhead fluorescent light providing ~500 lux; 28-V lamps in each chamber producing ~12 lux each

Light Intensity: ~500 lux overhead; ~12 lux within each chamber

Temperature (°C): 21.7 +/- 3

Humidity: 50% +/- 20%

Acoustic Background: Ambient

Acoustic Deprivation: Sound attenuating chamber

Handling With: Forceps

Collection Method: Whole Mouse

Technician Protection: Gloves, Mask, Lab Protective Clothing

# Mice Tested at the Same Time: 16

Female and Male Mice Tested in Same Test Group: Yes

Sanitation of Equipment: 70% ethanol between subjects; 1% Virkon between cohorts

Notes: Visual cues: each compartment is a different color and has a different floor type. Test conducted during light phase, beginning at least 60 min after lights on and concluding at least 30 min prior to lights off.

Additional ARRIVE Guidelines

Allocation of Animals to Test Group: Subjects pre-assigned a test chamber; counterbalanced across test chambers by strain and sex

Data Collection: Automated

Data QC: Data are reviewed as generated for technical issues, verification of subject ID, etc. Observed aberrations are noted.

Procedure 6: Reversal Learning

Procedure PDF:

 

Equipment, software, and supplies

• Operant Conditioning Chamber, each chamber fitted with horizontal array of 5 nose-poke apertures on one side of box and photocell-equipped food delivery magazine on the opposite side; each nose-poke aperture equipped with infrared photocell beams (Med Associates, ENV-307W-CT, St. Albans, VT, USA)
• Environmental Control Chamber, each chamber is placed within a ventilated, sound attenuated cabinet (Med Associates, ENV-022MD, St. Albans, VT, USA)
• White noise producer (Med Associates, ENV-303HDW, St. Albans, VT, USA)
• House light (Med Associates, ENV-227M, Test 1217, St. Albans, VT, USA)
• Variable speed pump (Med Associates, PHM-122-18, St. Albans, VT, USA)
• MedPC IV software (Med Associates, v. 4.2, St. Albans, VT, USA)
• Scale
• Paper towels
• Cotton swabs
• Luer lock syringes, 5 mL

Reagents and solutions

• Boost, Strawberry Creamy (Nestle HealthCare Nutrition, Inc., Florham Park, NJ, USA)
• 70% ethanol, used to sanitize chambers between subjects
• Virkon sanitization agent (Lanxess Corp., Pittsburgh, PA, USA)

Environmental Conditions

Testing Area

Facility: Procedure room

Acclimation Period: Subjects moved from housing room to procedure room on wheeled cart and immediately placed in chambers.

Room Space (dimensions): 10' x 10' with 12 chambers in the room

Light Source: Room lighting (550 lux) turned off when chamber doors are open; house light in chamber provides 7 lux

Light Intensity: Overhead light 550 lux; chamber light 7 lux

Temperature (°C): 21.7 +/- 3

Humidity: 50% +/- 20%

Acoustic Background: White noise

Acoustic Deprivation: Sound attenuating chamber

Handling With: Forceps

Collection Method: Whole Mouse

Technician Protection: Gloves, Mask, Lab Protective Clothing

# Mice Tested at the Same Time: 12

Female and Male Mice Tested in Same Test Group: Yes

Sanitation of Equipment: 70% ethanol between subjects; 1% Virkon between cohorts or at end of day

Notes: Testing conducted during light phase, beginning at least 60 min after lights on and concluding at least 60 min before lights off. There is a daily pre-testing welfare assessment (see procedure pdf).

Additional ARRIVE Guidelines

Allocation of Animals to Test Group: Randomization and counterbalancing based on strain and sex

Data Collection: Automated

Data QC: Data are reviewed for accuracy and technical errors

Procedure 7: Cocaine Locomotor Sensitization

Procedure PDF:

 

Equipment, software, and supplies

• Open Field Arena, square, clear polycarbonate arena (44.5 x 44.5 x 25.4 cm) with removable acrylic aerated lids (custom manufactured); external to the perimeter of arena at floor level are horizontal infrared photobeam sensors (16 x 16); additional pair of sensors is located 3 inches above floor (Med Associates, MED-OFAS-515U, St. Albans, VT, USA)
• Environmental Control Chamber, each arena placed in sound attenuated, ventilated cabinet (26" x 20" x 22"), with incandescent lights affixed in upper rear two corners of cabinet, 18.5" from center of arena floor (Med Associates, MED-OFA-017, St. Albans, VT, USA)
• Activity Monitor Software (Med Associates, 7.0.5.10 SOF-812, St. Albans, VT, USA)
• Forceps, used to handle all mouse transfers
• Paper towels
• Syringes, 1.0 mL
• Needles, 28G

Reagents and solutions

• Cocaine, dose = 10 mg/kg in saline
• Saline
• 70% ethanol, used to sanitize arena between subjects
• Virkon wipes, 1% solution used to sanitize arena between test cohorts (Lanxess)
• Spor-Klenz, used to sterilize forceps between subjects (1:32 in water) (Steris Life Science)

Environmental Conditions

Holding Area and/or Sample Collection Area

Facility: Anteroom to procedure room

Acclimation Period: Subjects are wheeled from housing room to anteroom where they are acclimated for 30 min while mice are being weighed

Description of Holding Enclosure: Home cages

Max # Animals Per Enclosure: 1

Acoustic Background: Ambient

Testing Area

Facility: Procedure room

Acclimation Period: Subjects are wheeled into procedure room from anteroom and placed in chambers immediately

Room Space (dimensions): 20'9" x 10'2" with 24 arenas, double stacked and located on both sides of room

Light Source: Overhead fluorescent light producing ~500 lux; chamber lighted by 2 28-V lamps giving 14 lux at testing floor

Light Intensity: Overhead ~500 lux; chamber 14 lux

Temperature (°C): 21.7 +/- 3

Humidity: 50% +/- 20%

Acoustic Background: Ambient

Acoustic Deprivation: Sound attenuating chamber

Handling With: Forceps

Restraint: Mice restrained for cocaine or saline injections

Collection Method: Whole Mouse

Technician Protection: Gloves, Mask, Lab Protective Clothing

# Mice Tested at the Same Time: 24

Female and Male Mice Tested in Same Test Group: Yes

Sanitation of Equipment: 70% ethanol between subjects; 1% Virkon between cohorts or at end of day

Notes: Mice are tested on 3 consecutive days and then every other day until day 11, with additional tests on day 12 and 19. Mice receive IP cocaine injections on days 3, 5, 7, 9, 11, 19. Mice receive IP saline on days 1, 2, 12.

Additional ARRIVE Guidelines

Allocation of Animals to Test Group: Randomization and counterbalancing based on strain and sex

Data Collection: Automated

Data QC: Data are reviewed as generated for technical issues, verification of subject ID, etc. Observed aberrations are noted

Procedure 8: Cocaine Intravenous Self-administration v1.0

Procedure PDF:

 

Equipment, software, and supplies

• Operant Conditioning Chamber, fitted on front wall with two retractable levers on right and left sides and food hopper in center panel; directly over each lever (2-3 inches above) are red stimulus lights modified with Plexiglas floor (custom manufactured), fitted to cover floor railing (Med Associates, ENV-307W-CT, St. Albans, VT, USA)
• Counterbalance (Med Associates, PHM-124MW, St. Albans, VT, USA)
• Tether (Med Associates, PHM-KVAH62T/MED, St. Albans, VT, USA)
• Swivel (Med Associates, PHM-115IP-25, St. Albans, VT, USA)
• PE tubing
• Catheter, permits quick aseptic connection and disconnection of catheterized mouse and infusion tether (Instech, VAB)
• Single speed syringe pump, 120 V (Med Associates, PHM-100, St. Albans, VT, USA)
• Expanded PVC sound attenuating cubicle (Med Associates, ENV-022V, St. Albans, VT, USA)
• Protective aluminum cap (Instech, VAB62CAP)
• Scale
• Restrainer (Braintree Scientific, Inc., TV-150 STD and TV-150 SM, Braintree, MA, USA)
• Syringes and needles, 1 cc syringe with 25G 1 cc BD U-100 insulin syringe with slip tip; 60 mL BD syringes with Luer-Lok tips (Becton-Dickinson)
• Paper towels

Reagents and solutions

• Heparin sodium injection, USP 10,000 units/10 mL (NDC Code: 63739-931-28)
• Baytril (enrofloxacin) injectable solution (2.27%), broad-spectrum antibiotic (Bayer)
• Brevital (methohexital), ultrashort acting barbiturate used to confirm catheter patency
• Wavicide 01 disinfectant solution (Performance Health, 081545508, Akron, OH, USA)
• Cocaine HCl, supplied by National Institute on Drug Abuse Supply Program, Division of Therapeutics and Medical Consequences

Environmental Conditions

Testing Area

Facility: Procedure room

Acclimation Period: Subjects transported in home cages from housing room to procedure room on wheeled rack.

Room Space (dimensions): 21' x 10' with 32 chambers, each placed on separate shelves located next to each other along each side of room.

Light Source: Overhead fluorescent light producing ~500 lux

Light Intensity: ~500 lux

Temperature (°C): 21.7 +/- 3

Humidity: 50% +/- 20%

Acoustic Background: Ambient

Handling With: Forceps

Restraint: Yes, during injections.

Anesthesia: Yes, during jugular vein catheterization.

Technician Protection: Gloves, Mask, Lab Protective Clothing

# Mice Tested at the Same Time: 32

Female and Male Mice Tested in Same Test Group: Yes

Notes: Testing conducted during light phase, beginning at least 30 min after lights on and concluded at least 30 min prior to lights off. Subjects undergo jugular vein catheterization prior to testing (see Procedure pdf for details).

Investigator Notes: Mice underwent one of two versions of the Intravenous Self-Administration procedure. Versions can be found per-animal in the download file for expanded data.

Procedure 9: Cocaine Intravenous Self-administration v2.0

Procedure PDF:

 

Equipment, software, and supplies

• Operant Conditioning Chamber, fitted on front wall with two retractable levers on right and left sides and food hopper in center panel; directly over each lever (2-3 inches above) are red stimulus lights modified with Plexiglas floor (custom manufactured), fitted to cover floor railing (Med Associates, ENV-307W-CT, St. Albans, VT, USA)
• Counterbalance (Med Associates, PHM-124MW, St. Albans, VT, USA)
• Tether (Med Associates, PHM-KVAH62T/MED, St. Albans, VT, USA)
• Swivel (Med Associates, PHM-115IP-25, St. Albans, VT, USA)
• PE tubing
• Catheter, permits quick aseptic connection and disconnection of catheterized mouse and infusion tether (Instech, VAB)
• Single speed syringe pump, 120 V (Med Associates, PHM-100, St. Albans, VT, USA)
• Expanded PVC sound attenuating cubicle (Med Associates, ENV-022V, St. Albans, VT, USA)
• Protective aluminum cap (Instech, VAB62CAP)
• Scale
• Restrainer (Braintree Scientific, Inc., TV-150 STD and TV-150 SM, Braintree, MA, USA)
• Syringes and needles, 1 cc syringe with 25G 1 cc BD U-100 insulin syringe with slip tip; 60 mL BD syringes with Luer-Lok tips (Becton-Dickinson)
• Paper towels

Reagents and solutions

• Heparin sodium injection, USP 10,000 units/10 mL (NDC Code: 63739-931-28)
• Baytril (enrofloxacin) injectable solution (2.27%), broad-spectrum antibiotic (Bayer)
• Brevital (methohexital), ultrashort acting barbiturate used to confirm catheter patency
• Wavicide 01 disinfectant solution (Performance Health, 081545508, Akron, OH, USA)
• Cocaine HCl, supplied by National Institute on Drug Abuse Supply Program, Division of Therapeutics and Medical Consequences

Environmental Conditions

Testing Area

Facility: Procedure room

Acclimation Period: Subjects transported in home cages from housing room to procedure room on wheeled rack.

Room Space (dimensions): 21' x 10' with 32 chambers, each placed on separate shelves located next to each other along each side of room.

Light Source: Overhead fluorescent light producing ~500 lux

Light Intensity: ~500 lux

Temperature (°C): 21.7 +/- 3

Humidity: 50% +/- 20%

Acoustic Background: Ambient

Handling With: Forceps

Restraint: Yes, during injections.

Anesthesia: Yes, during jugular vein catheterization.

Collection Method: Whole Mouse

Technician Protection: Gloves, Mask, Lab Protective Clothing

# Mice Tested at the Same Time: 32

Female and Male Mice Tested in Same Test Group: Yes

Notes: Testing conducted during light phase, beginning at least 30 min after lights on and concluded at least 30 min prior to lights off. Subjects undergo jugular vein catheterization prior to testing (see Procedure pdf for details).

Investigator Notes: Mice underwent one of two versions of the Intravenous Self-Administration procedure. Versions can be found per-animal in the download file for expanded data.

Procedure 10: Molecular Rhythms in Primary Fibroblasts

Procedure PDF:

 

Equipment, software, and supplies

• Tissue culture incubator
• Syringe filter (0.2µm)
• Tubes (1.5 mL)
• Tweezers
• Paper towels
• Scissors
• Tube shaker
• Square cover glass (22 mm) (VWR, 48368-062)
• Sterile coverslip (40 mm) (Thermo Scientific, 22-038-999)
• TC-treated tissue culture dishes (60 mm) (Corning, 08-772-21)
• TC-treated tissue culture dishes (35 mm)
• Lumicycle luminometer

Reagents and solutions

• Dulbecco's Modified Eagles Medium (DMEM) (HyClone, SH3024302)
• Penicillin Streptomycin Glutamine solution (HyClone, SV3008201)
• HEPES (1M) (Gibco, 15-630-080)
• Beetle Luciferin, Potassium Salts (Promega, PR-E1602)
• Bmal1-dLuc Lentivirus (VectorBuilder)
• Forskolin (Sigma, F6886)
• TrypLE Express Enzyme (1X), no phenol red (Gibco, 12-604-021)
• DPBS without calcium and magnesium (Gibco, 14-190-144)
• Collagenase, Type D, powder (Gibco, 17-104-019)
• Pronase Protease, Streptomyces griseus (Millipore, 53-702-25KU)
• High-vacuum grease (Corning, 14-635-5D)
• Cell culture grade water (HyClone, SH3052901)
• Amphotericin B solution (Sigma, A2942)
• Gentamycin Sulfate (Millipore, 345815)

Environmental Conditions

Holding Area and/or Sample Collection Area

Facility: Procedure room

Description of Holding Enclosure: Home cage

Anesthesia: Yes

Collection Method: Whole ear