Delayed non-match to sample (DNMTS) training protocol

DNMTS was performed as previously described²⁰. Specifically, the rats were initially habituated to the operant conditioning chambers with the three levers extended. The animals were trained for 2 days to lever press for food reward on a continuous reinforcement schedule (i.e. pressing of any lever would result in the delivery of a sucrose pellet to the hopper). On the subsequent 2 days the levers were programmed to retract once pressed, delivering a pellet and then extending again. This was also on a continuous reinforcement schedule aimed to habituate the animals to the retraction and extension of the levers. On day 5, the same program was used with the exception that one specific lever could not be reinforced more than 3 consecutive times. This modification was aimed to force the animals to perform alternate lever pressing, thereby suppressing lever preferences to obtain reward.

The next phase of training involved randomised presentation of the front lever (left or right) and once pressed the extension of the back-lever was triggered. The reward was delivered only after the back lever was pressed. These lever combinations were repeated 60 times (30 left/center and 30 right/center) at 10 s intervals; this procedure was repeated for 2 days.

Training in the non-match-to-sample task was comprised of 90 trials in a maximum 90 min session daily. At the start of each session the house light is on with the levers in the retracted position. The animals were initially trained on the task contingencies with no enforced delay between the sample and the choice component (0-delay condition). At the start of each trial one response lever was randomly selected and inserted into the chamber (the “sample”). As soon as the lever press response was registered the lever was retracted and the rear lever on the opposite wall extended. Once the response on the back lever was registered the two front levers were extended into the chamber together (the “choice”). If a correct response was registered (i.e. a response on the non-matching to sample lever) the levers retract and a pellet delivered to the hopper, the house light remained on and an inter-trial interval of 10 s was initiated before the next trial began. If an incorrect response was registered (i.e. a response on the initial sample lever) no pellet was delivered, the house light extinguished and the 10 s interval initiated before the next trial started. Rats were required to meet a criterion of 85% for 3 consecutive days on this program before introduction of the delay. In the next stage of training a randomised 1 to 5 s delay was introduced between the response on the sample lever and the extension of the rear lever. This phase lasted for 3 days.

In the final stage of training, a random delay of 1–30 s was introduced requiring the rat to wait for the extension of the rear lever before moving to the choice phase. Training continued on this phase of the task until the animals’ performance reached a plateau (40 sessions).

Testing Following surgery, animals were allowed to recover for 48 h before resuming testing in the DNMTS task. Animals were treated with Vehicle or Analgesia 23 h before test in the DNMTS task to avoid potential acute drug effects interfering with task performance. Animals were tested each day from 48 h post-surgery/control procedure up to and including day 8 post-surgery. Each daily session was composed of 90 trials of different delay lengths. Completion of the 90 trials each day was used as a control for any potential drug-induced interference in task performance. As such any animal which did not complete the 90 trials each day was not included in the analysis of DNMTS data. MA Control and MA + Anesthesia groups each had one animal removed leaving n = 10 for each group. The MA + Surgery Model (Bup-0.05 mg/kg) had 7 animals excluded, leaving n = 5. The MA + Surgery Model (Bup-0.1 mg/kg) had 6 animals excluded, leaving n = 6. The MA + Surgery Model (Par-75 mg/kg) had 3 animals excluded, leaving n = 9. The MA + Surgery Model (Par-150 mg/kg) had 1 animal excluded, leaving n = 11. Bup treatment may have effects the animal’s ability to perform the task.