Subtype-selective contribution of muscarinic acetylcholine receptors for filial imprinting in newly-hatched domestic chicks

Muscarinic acetylcholine receptors (mAChRs) play an important role in many brain functions. Our previous study revealed that the injection of mAChRs antagonist scopolamine into the intermediate medial mesopallium (IMM) region, which is critical for filial imprinting, impairs memory formation. In avian brains, four mAChR subtypes have been identified (M₂, M₃, M₄ and M₅). M₃ and M₅ receptors increase the excitability of neurons, whereas M₂ and M₄ receptors reduce the excitability. Because the scopolamine blocks all subtypes, the previous study did not identify which subtype contributes to the memory formation. By injecting several types of mAChR antagonists into the IMM, in this study we determined which mAChR subtype plays a critical role in imprinting. First, the effects of antagonists on the excitatory receptor subtypes M₃ and M₅ were examined. Injection of the M₃ antagonist (DAU5884) at 20 mM or the M₅ antagonist (ML381) at 2 mM impaired imprinting. Considering the pKi value of DAU5884, the impairment seems to be caused by DAU5884 binding to M₃ and/or M₄ receptors. Second, the effect of antagonists on the inhibitory receptor subtype M₂ was examined. The results showed that the M₂ antagonist (AQ-RA741) impaired imprinting at a concentration of 20 mM. Considering the pKi value of AQ-RA741, the impairment seems to be caused by AQ-RA741 binding to M₂ and/or M_4. The findings of this study suggests that the excitatory receptor subtypes M₃ and M₅ and the inhibitory receptor subtype M₂ and/or M₄ cooperate to achieve the appropriate balance of acetylcholine signaling to execute imprinting.