Increased nicotinic receptor desensitization in hypoglossal motor neurons following chronic developmental nicotine exposure

Abstract

Neuronal nicotinic acetylcholine receptors (nAChRs) are expressed on hypoglossal motor neurons (XII MNs) that innervate muscles of the tongue. Activation of XII MN nAChRs evokes depolarizing currents, which are important for regulating the size and stiffness of the upper airway. Although data show that chronic developmental nicotine exposure (DNE) blunts cholinergic neurotransmission in the XII motor nucleus, it is unclear how nAChRs are involved. Therefore, XII MN nAChR desensitization and recovery were examined in tissues from DNE or control pups using a medullary slice preparation and tight-seal whole cell patch-clamp recordings. nAChR-mediated inward currents were evoked by brief pressure pulses of nicotine or the α4β2 nAChR agonist RJR-2403. We found that, regardless of treatment, activatable nAChRs underwent desensitization, but, following DNE, nAChRs exhibited increased desensitization and delayed recovery. Similar results were produced using RJR-2403, showing that DNE influences primarily the α4β2 nAChR subtype. These results show that while some nAChRs preserve their responsiveness to acute nicotine following DNE, they more readily desensitize and recover more slowly from the desensitized state. These data provide new evidence that chronic DNE modulates XII MN nAChR function, and suggests an explanation for the association between DNE and the incidence of central and obstructive apneas.

Fig. 1.

A: cartoon of the medullary slice preparation and experimental configuration showing the rostral surface, including the XII motor nucleus, the spinal tract of the trigeminal nerve (5SP), preBotzinger complex (preBotC), nucleus ambiguous (NA), inferior olivary nucleus (IO), and the intermediate reticular formation (IRt). B: high resolution image (×40) of an isolated XII MN along with a recording pipette and a pressure ejection “puffing” pipette. C: nicotine-activated inward current showing nAChR desensitization current fade during a 1-s pressure pulse. D: example of slow nicotinic acetylcholine receptor (nAChR) desensitization. Continuous bath application of nicotine (0.5 μM) for 15 min reduces the response to pressure pulses (20 psi) of nicotine (2 mM), documenting desensitization of the nAChRs. Note that the nAChR-mediated inward current amplitude slowly recovers during the washout period. Arrows indicate pulse timing. Horizontal white bar indicates normal aCSF and the black bar indicates aCSF + nicotine (0.5 μM). The pressure pipette was positioned 10 μm from the XII MN. psi, pounds per square inch; V-clamp, voltage clamp.

Fig. 2.

Developmental nicotine exposure (DNE) affects the desensitization and recovery of nAChR currents. A: examples of inward currents induced by nicotine pressure pulses (2 mM; 50 ms) in control (CON) and DNE animals during a 4-min pressure-pulse protocol. Pressure pulses occurred at successively longer intervals following a control pulse at time 0. B: summary data showing DNE-induced differences in the desensitization and recovery of nAChR-mediated peak currents. All data are means ± SE, *P < 0.05. C: quantification of time-dependent nAChR recovery using a single exponential fit to the average data shown in B. DNE slows nAChR recovery (control; n = 17 cells; DNE, nicotine-exposed, n = 16 cells).

Fig. 3.

DNE affects desensitization and recovery of α4β2 nAChR currents. A: examples of inward currents induced by pressure pulses of the α4β2 agonist RJR-2403 (300 μM; 20 ms) in CON and DNE animals during a 4-min pressure-pulse protocol. Pressure pulses occurred at 5 s, 30 s, 60 s, 120 s, and 240 s following a control pulse at time 0. B: RJR-2403-induced currents are almost abolished by the α4β2 antagonist DH-β-E (0.1 μM) delivered to the bath superfusate (n = 3). C: summary data showing DNE-induced differences in the desensitization and recovery of α4β2 nAChR-mediated peak currents. All data are means ± SE, *P < 0.05. D: quantification of time-dependent nAChR recovery using a single exponential fit to the average data shown in C. DNE greatly slows α4β2 nAChR recovery (n = 5, CON; n = 7, DNE).