α4* Nicotinic Receptors in preBötzinger Complex Mediate Cholinergic/Nicotinic Modulation of Respiratory Rhythm

Acetylcholine and nicotine can modulate respiratory patterns by acting on nicotinic acetylcholine receptors (nAChRs) in the preBotzinger complex (preBotC). To further explore the molecular composition of these nAChRs, we studied a knock-in mouse strain with a leucine-to-alanine mutation in the M2 pore-lining region (L9′A) of the nAChR α4 subunit; this mutation renders α4-containing receptors hypersensitive to agonists. We recorded respiratory-related rhythmic motor activity from hypoglossal nerve (XIIn) and patch-clamped preBotC inspiratory neurons in an in vitro medullary slice preparation from neonatal mice. Nicotine affected respiratory rhythm at concentrations ∼100-fold lower in the homozygous L9′A knock-in mice compared with wild-type mice. Bath application of 5 nm nicotine increased the excitability of preBotC inspiratory neurons, increased respiratory frequency, and induced tonic/seizure-like activities in XIIn in L9′A mice, effects similar to those induced by 1 μm nicotine in wild-type mice. In L9′A mice, microinjection of low nanomolar concentrations of nicotine into the preBotC increased respiratory frequency, whereas injection into the ipsilateral hypoglossal (XII) nucleus induced tonic/seizure-like activity. The α4*-selective nAChR antagonist dihydro-β-erythroidine produced opposite effects and blocked the nicotinic responses. These data, showing that nAChRs in the preBotC and XII nucleus in L9'A mice are hypersensitive to nicotine and endogenous ACh, suggest that functional α4* nAChRs are present in the preBotC. They mediate cholinergic/nicotinic modulation of the excitability of preBotC inspiratory neurons and of respiratory rhythm. Furthermore, functional α4* nAChRs are present in XII nucleus and mediate cholinergic/nicotinic modulation of tonic activity in XIIn.