Rossi Et Al. Delayed Alternation Prefrontal Cortical Mechanisms Underlying Delayed Alternation in Mice Abbreviated Title: Delayed Alternation 2

34 The prefrontal cortex (PFC) has been implicated in the maintenance of task-relevant information 35 during goal-directed behavior. Using a combination of lesions, local inactivation, and 36 optogenetics, we investigated the functional role of the medial prefrontal cortex (mPFC) in mice 37 using a novel operant delayed alternation task. Task difficulty was manipulated by changing the 38 duration of the delay between two sequential actions. In Experiment 1, we showed that 39 excitotoxic lesions of the mPFC impaired acquisition of delayed alternation with long delays (16 40 seconds), whereas lesions of the dorsal hippocampus and ventral striatum, areas connected with 41 the PFC, did not produce any deficits. Lesions of dorsal hippocampus, however, significantly 42 impaired reversal learning, when the rule was changed from alternation to repetition. In 43 Experiment 2, we showed that local infusions of muscimol (an agonist of the GABA-A receptor) 44 into mPFC impaired performance even when the animal is well trained, suggesting that the 45 mPFC is not only critical for acquisition but also for successful performance. In Experiment 3, 46 to examine the mechanisms underlying the role of GABAergic inhibition, we used Cre-inducible 47 Channelrhodopsin-2 to activate Parvalmumin (PV)-expressing GABAergic interneurons in the 48 mPFC of PV-Cre transgenic mice as they performed the task. Using whole-cell patch clamp 49 recording, we demonstrated that activation of PV-expressing interneurons in vitro with blue light 50 in brain slices reliably produced spiking and inhibited nearby pyramidal projection neurons. 51 With similar stimulation parameters, in vivo stimulation significantly impaired delayed 52 alternation performance. Together these results demonstrate a critical role for the mPFC in the 53 acquisition and performance of the delayed alternation task. 54 55