Prenatal nicotine exposure alters central cardiorespiratory responses to hypoxia in rats: implications for sudden infant death syndrome

Abstract

Maternal cigarette smoking and prenatal nicotine exposure are the highest risk factors for sudden infant death syndrome (SIDS). During hypoxia, respiratory frequency and heart rate transiently increase and subsequently decrease. These biphasic cardiorespiratory responses normally serve to prolong survival during hypoxia by reducing the metabolic demands of cardiac and respiratory muscles. However, exaggerated responses to hypoxia may be life threatening and have been implicated in SIDS. Heart rate is primarily determined by the activity of brainstem preganglionic cardioinhibitory vagal neurons (CVNs) in the nucleus ambiguus. We developed an in vitro rat brainstem slice preparation that maintains rhythmic inspiratory-related activity and contains fluorescently labeled CVNs. Synaptic inputs to CVNs were examined using patch-clamp electrophysiological techniques. Hypoxia evoked a biphasic change in the frequency of both GABAergic and glycinergic IPSCs in CVNs, comprised of an initial increase followed by a decrease in IPSC frequency. Prenatal exposure to nicotine changed the GABAergic response to hypoxia from a biphasic response to a precipitous decrease in spontaneous GABAergic IPSC frequency. This study establishes a likely neurochemical mechanism for the heart rate response to hypoxia and a link between prenatal nicotine exposure and an exaggerated bradycardia during hypoxia that may contribute to SIDS.

Figure 1.

pH and pO₂ profiles of the thick medullary slice when the perfusate was saturated with a 95% O₂-5% CO₂ gas mixture and when the perfusate was saturated with 75% N₂-20% O₂-5% CO₂ to evoke hypoxia. a, Top trace, pH progressively declined from the upper surface of the tissue to a relative minimum in the slice core. After this point, pH progressively increased as the electrode was advanced to the lower surface of the tissue (n = 7). Bottom trace, pH profile of the thick medullary slice under hypoxic conditions (n = 7). b, Top trace, pO₂ progressively declined from the upper surface of the tissue to a relative minimum in the slice core. After this point, pO₂ progressively increased as the electrode was advanced to the lower surface of the tissue (n = 7). Bottom trace, pO₂ profile of the thick medullary slice under hypoxic conditions (n = 6). c, Time course of pO₂ changes in the thick medullary slice at a depth of 300 μm. Perfusion with hypoxic media caused a rapid drop in pO₂, which dropped below the hypoxic threshold in ∼2 min and reached a steady state shortly thereafter (n = 7). The interval between the start of hypoxic perfusate (at time 0) and pO₂ dropping below the hypoxic threshold was defined as transition (Trans). Error bars represent SEM.

Figure 2.

Hypoxia causes a biphasic change in glycinergic IPSC frequency. a, Inspiratory-related bursting activity was recorded from the hypoglossal rootlet (XII) and electronically integrated (∫XII). Fluorescently identified CVNs were patch clamped in the whole-cell configuration, and glycinergic neurotransmission was isolated by focal application of NMDA, non-NMDA, and GABA_A receptor antagonists. The frequency of glycinergic IPSCs in CVNs was significantly increased during inspiratory bursts (n = 13; p < 0.01 using a paired Student's t test). Hypoxia induced a significant increase in the frequency of spontaneous IPSCs in CVNs during the transition to hypoxia. Spontaneous glycinergic IPSC frequency was also significantly reduced during hypoxia relative to glycinergic IPSC frequency during the transition to hypoxia (p < 0.05; n = 13). Inspiratory glycinergic IPSC frequency also transiently increased during transition (p < 0.05) and subsequently significantly decreased during hypoxia (p < 0.05; n = 13). b, Average spontaneous glycinergic IPSC frequency ± SE. c, Average inspiratory glycinergic IPSC frequency ± SE. ^*p < 0.05; ^**p < 0.01. All statistical comparisons were performed using Fisher's protected F procedure unless otherwise indicated.

Figure 3.

Hypoxia induces a biphasic change in GABAergic IPSC frequency in CVNs. GABAergic synaptic activity was isolated by focal application of the NMDA, non-NMDA, and glycinergic antagonists AP-5 (50 μmol/l), CNQX (50 μmol/l), and strychnine (1 μm). a, The frequency of GABAergic IPSCs in CVNs was significantly increased during inspiratory bursts (n = 13; p < 0.01 using a paired Student's t test). During the transition to hypoxia, spontaneous (p < 0.01; n = 13) GABAergic IPSC frequency was significantly increased. During hypoxia, the frequency of both spontaneous (n = 13; p < 0.05) and inspiratory GABAergic IPSCs in CVNs was significantly decreased (n = 13; p < 0.05). b, Average spontaneous GABAergic IPSC frequency ± SE. c, Average inspiratory GABAergic IPSC frequency ± SE. ^*p < 0.05; ^**p < 0.01. All statistical comparisons were performed using Fisher's protected F procedure unless otherwise indicated.

Figure 4.

Hypoxia induces a rapid decrease in the frequency of both spontaneous and inspiratory GABAergic IPSCs in CVNs from animals exposed to nicotine prenatally. a, In animals prenatally exposed to nicotine, the inspiratory-related increase in GABAergic IPSC frequency was significantly increased compared with control animals (n = 13 Unexposed; n = 10 Prenatal Nicotine; p < 0.05 using an unpaired Student's t test). b, c, Hypoxia induced a rapid decrease in the frequency of both spontaneous and inspiratory GABAergic IPSCs in CVNs. Values are mean GABAergic IPSC frequency ± SE. Double asterisks denote p < 0.01 versus mean control frequency. d, During hypoxia, the frequency of spontaneous GABAergic IPSCs in CVNs in animals exposed to nicotine prenatally was significantly lower than the frequency of spontaneous GABAergic IPSCs in unexposed animals (random regression model testing group main effect, p < 0.05; group × quadratic time trend interaction, p < 0.005; n = 13 unexposed CVNs; n = 10 PN CVNs). e, Under control conditions, the frequency of inspiratory GABAergic IPSCs in CVNs was significantly greater in animals prenatally exposed to nicotine compared with unexposed animals (unpaired Student's t test; p < 0.01). Hypoxia abolished the elevation of inspiratory GABAergic IPSC frequency in animals prenatally exposed to nicotine (random regression model testing group main effect, p > 0.05; group × quadratic time trend interaction, p > 0.05).