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Preferences between black-center and white-center patterns were tested before the first conditioning stage and at 30 min or 1 day after completing the second conditioning stage.
The group injected with epinastine before the first conditioning stage exhibited a significantly increased preference for the CS2, thus indicating that appetitive second-order conditioning is achieved.
The group injected with fluphenazine before the first conditioning stage exhibited a significantly increased preference for the control pattern (and thus significantly decreased preference for the CS2), thus indicating that aversive second-order conditioning is achieved.
For the study using second-order conditioning, other groups of animals were each injected with 3 μl of saline or saline containing 1 μM epinastine or 500 μM fluphenazine at 30 min before the first conditioning stage, before the second conditioning stage or before the final test in second-order conditioning.
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In contrast, epinastine impaired appetitive second-order conditioning when it was injected before the second conditioning stage.
In contrast, fluphenazine impaired aversive second-order conditioning when it was injected before the second conditioning stage.
In contrast, the group injected with epinastine before the second conditioning stage exhibited no significantly increased preference for the CS2, thus indicating complete impairment of second-order conditioning.
In contrast, the group injected with fluphenazine before the second conditioning stage exhibited no significantly increased preference for the control pattern, thus indicating complete impairment of second-order conditioning.
The group injected with saline before the second conditioning stage exhibited a significantly increased preference for the CS2, the level being as high as that in a group of intact animals.
The first conditioning trial was given 24 hours later.
Therefore, additional conditioning trials act as confirmations of the information gained during the first conditioning trial.
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