Jentsch2 project protocol

Drug study: Cocaine self-administration behavior and capacities for inhibitory control in 4 BXD recombinant inbred strains of mice   (2013)

Jentsch JD
With: Cervantes MC, Laughlin RE

Jentsch2 Protocol

Project protocol - Contents

Workflow and sampling

Data collected *
Mice anethetized, indwelling jugular catheters implanted
Daily cocaine self-administration sessions (2h per day, 7 d per wk): progressive schedules (fixed ratio 1 (FR1), FR2, FR5)
Acquisition performance, number of infusions earned, discrimination index, time-out response
3 Second cohort of mice assessed for locomotor responses to non-contingent cocaine administration Horizontal beam breaks

*Supplementary data are available for this project. See Jentsch2

Equipment and supplies

  • Ventilation system for anesthesia
  • Circulating water heating pad
  • Catheter (Camcaths, Ely, Cambridgeshire UK)
  • Sutures (SOFSILK 4-0)
  • Operant conditioning chambers with computer running Med-PC (Med Associates, St. Albans VT)
  • Pump-driven syringe
  • Activity chambers (Columbus Instruments, Columbus OH)

Reagents and solutions

  • Cocaine base (NIDA); dissolved in concentrated HCl before being brought to volume in 0.9% sterile saline (pH ~6.5)
  • Saline
  • Oxygen/isoflurane
  • Propofol
  • Triple antibiotic ointment
  • Ticarcillin clavulanate (Glaxo Smith Klein, Research Triangle Park NC)
  • Heparin

Strain selection and acclimation

    Strains were selected based on reversal learning, assessed in Jentsch1. Out of 51 RI strains tested, BXD31 and BXD38 exhibited performance that placed them in the top 20% of all strains and are referred to as "good reversal learning (GRL) strains"; BXD42 and BXD68 exhibited reversal learning performance in the lowest 5% of the sample and are referred to as "poor reversal learning (PRL) strains".

    Mice were handled for 5 days prior to catheter implantation.

Procedure: Catheter implantation, maintenance, and patency

    1. Mice are anesthetized under an oxygen/isoflurane (1.5-2.0%) ventilation system and supported on a circulating-water heating pad.
    2. A catheter is inserted through a ventral supra-jugular incision (placed 1.2 cm dorsal to the heart, anchored with suture knots above and below a silicon bead) and is tunneled subcutaneously over the shoulder and externalized through a midscapular dorsal incision.
    3. Incisions are sutured and treated with triple antibiotic ointment.
    4. Mice are allowed to recover 6 days prior to testing.
    5. A 20 µL solution containing the antibiotic ticarcillin clavulanate is dissolved in heparinized (30 units/mL) 0.9% saline and flushed through the catheter immediately after implantation, daily throughout recovery, and before and after each self-administration session.
    6. Catheter patency is evaluated with a 20 µL infusion of 1% propofol (10 mg/mL) before starting self-administration and between each experimental phase (if prominent sedation is not apparent within 3 s of infusion, the mouse is removed from the study).

Procedure: Cocaine self-administration

Operant conditioning chambers are enclosed in sound-attenuating cabinets and computer controlled. Chambers are fitted with two levers, assigned as active and inactive (in a counterbalanced fashion). A single-channel fluid swivel connects a pump-driven syringe to the indwelling catheter (infusion speed = 10 µL/s). Completion of a response schedule on the active lever triggers a 20 µL infusion containing a unit dose of 0.5 mg/kg cocaine and initiates flashing of the house light for 20 s (post-infusion period). Daily self-administration sessions last 2h and are conducted 7 days a week. A maximum of 65 cocaine infusions can be obtained in each session.

    1. Mice train for acquisition of cocaine self-administration under a fixed ratio 1 (FR1) schedule of reinforcement (5 days).
    2. If mice acquire and show stable behavior for FR1 (see criteria below), they are evaluated under a FR2 schedule (4 days).
    3. If mice acquire and show stable behavior for FR2 (see criteria below), they are evaluated under a FR5 schedule (5 days)

    Criteria for stable acquisition and cocaine-maintained responding for FR1 and FR2 (FR5 tests are administered without completion criteria):

    • a) 20% or less variation in number of infusions earned and greater than 70% discrimination index (active responses divided by total responses) across three consecutive days
    • b) 10 infusions minimum earned per session
    • c) 5 and 4 days minimum per schedule, respectively

    Mice with patent catheters are considered to have failed to acquire self-administration if they earned no infusions over 7 consecutive days or if they earned fewer than 10 infusions per session over 3 consecutive days.

Procedure: Locomotor activity

Activity chambers consist of large acrylic cages placed within an infrared grid; clean cages are used for each session and each subject.

    1. Mice are habituated to the experimental setup for 30 min across three consecutive days.
    2. Mice are injected with cocaine (1 mL/100g body weight) at four doses: 0, 5, 10, 20 mg/kg using a repeated measures, cyclic Latin square design, with 2-3 days between tests.
    3. Number of photobeam breaks is recorded, and activity measured as the average activity counts, excluding the first 15 min to allow for drug onset.

Data collected by investigator

  • total number of days to reach criteria
  • number of infusions earned per session
  • number of post-infusion responses (active responses during time-out period)
  • discrimination index (active responses divided by total responses)
  • number of horizontal beam breaks

*Supplementary data are available for this project. See Jentsch2