Differential control of central cardiorespiratory interactions by hypercapnia and the effect of prenatal nicotine

Abstract

Hypercapnia evokes a strong cardiorespiratory response including gasping and a pronounced bradycardia; however, the mechanism responsible for these survival responses initiated in the brainstem is unknown. To examine the effects of hypercapnia on the central cardiorespiratory network, we used an in vitro medullary slice that allows simultaneous examination of rhythmic respiratory-related activity and inhibitory synaptic neurotransmission to cardioinhibitory vagal neurons (CVNs). Hypercapnia differentially modulated inhibitory neurotransmission to CVNs; whereas hypercapnia selectively depressed spontaneous glycinergic IPSCs in CVNs without altering respiratory-related increases in glycinergic neurotransmission, it decreased both spontaneous and inspiratory-associated GABAergic IPSCs. Because maternal smoking is the highest risk factor for sudden infant death syndrome (SIDS) and prenatal nicotine exposure is proposed to be the link between maternal smoking and SIDS, we examined the cardiorespiratory responses to hypercapnia in animals exposed to nicotine in the prenatal and perinatal period. In animals exposed to prenatal nicotine, hypercapnia evoked an exaggerated depression of GABAergic IPSCs in CVNs with no significant change in glycinergic neurotransmission. Hypercapnia altered inhibitory neurotransmission to CVNs at both presynaptic and postsynaptic sites. Although the results obtained in this study in vitro cannot be extrapolated with certainty to in vivo responses, the results of this study provide a likely neurochemical mechanism for hypercapnia-evoked bradycardia and the dysregulation of this response with exposure to prenatal nicotine, creating a higher risk for SIDS.

Figure 1.

Hypercapnia evokes biphasic changes in central respiratory activity. A, Changing the perfusate from aCSF equilibrated with 95% O₂/5% CO₂ to aCSF equilibrated with 92% O₂/8% CO₂ elicited a biphasic change in respiratory activity as burst frequency initially significantly increased and subsequently returned to control levels, as shown in are presentative experiment. B, Respiratory burst frequency (Freq.) significantly increased during hypercapnia in both unexposed (□; p < 0.01; n = 15) and prenatal nicotine-exposed (○; n = 9) animals. C, Hypercapnia significantly depressed burst duration in both unexposed and prenatal nicotine-exposed animals. D, Hypercapnia induced a small, but not statistically significant, rise in burst amplitude (Amp.) in both unexposed and prenatal nicotine-exposed animals. There were no statistically significant differences in the respiratory responses between unexposed and prenatal nicotine-exposed animals. In this and all subsequent figures, *^,+p < 0.05 and **^,++p < 0.01 in unexposed and prenatal nicotine-treated animals, respectively.

Figure 2.

Hypercapnia selectively depresses spontaneous 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 significantly increased during inspiratory bursts. Hypercapnia elicited a significant decrease in spontaneous glycinergic IPSCs but did not alter inspiratory-evoked IPSCs. IPSC amplitude was not modulated by inspiratory bursts; however, hypercapnia significantly inhibited glycinergic IPSC amplitude. A typical experiment from an unexposed animal is shown in A, and the average data from 11 cells are shown in B. C, In animals exposed to nicotine prenatally, hypercapnia significantly inhibited the frequency of spontaneous but not inspiratory glycinergic IPSCs. IPSC amplitude was not modulated by inspiratory bursts, although hypercapnia significantly decreased IPSC amplitude. There were no significant differences in unexposed and prenatal nicotine-exposed animals. *p < 0.05; ^{**, ++} p < 0.01.

Figure 3.

Hypercapnia diminishes GABAergic IPSC frequency. GABAergic synaptic activity was isolated by focal application of NMDA, non-NMDA, and glycinergic receptor antagonists. A, As shown for a typical experiment, GABAergic IPSC frequency significantly increased during respiratory bursts. B, After exposure to hypercapnia, both spontaneous and inspiratory GABAergic IPSC frequency was depressed. C, GABAergic IPSC amplitude was not modulated by respiratory bursts; however, hypercapnia diminished IPSC amplitude. *p < 0.05; **p < 0.01.

Figure 4.

Prenatal nicotine exposure exaggerates GABAergic IPSC frequency depression during hypercapnia. A, In animals exposed to nicotine prenatally, the inspiratory-evoked increase in GABAergic frequency was significantly increased compared with control animals, as shown in a typical experiment. B, Hypercapnia evoked a significant decrease in both spontaneous and inspiratory GABAergic IPSC amplitude. C, GABAergic IPSC amplitude was not modulated by respiratory bursts; however, hypercapnia diminished IPSC amplitude in animals exposed to nicotine prenatally. ⁺p < 0.05; ⁺⁺p < 0.01.

Figure 5.

Hypercapnia inhibits GABAergic and glycinergic mIPSCs in CVNs. A, Representative traces showing the effect of hypercapnia on inhibitory mIPSCs. Application of TTX (1 μm), NMDA, non-NMDA, and GABAergic or glycinergic receptor antagonists induces TTX-insensitive, glycinergic, or GABAergic mIPSCs, respectively. Both the frequency and amplitude of GABAergic and glycinergic mIPSCs were significantly reduced after exposure to hypercapnia. B, Average data for seven cells. *p < 0.05; **p < 0.01.

Figure 6.

Hypercapnia diminishes CVN postsynaptic responses to inhibitory neurotransmitters. Focal application of GABA (100 μm) or glycine (100 μm) was applied under conditions of synaptic blockade with TTX (1 μm), CNQX (50 μm), AP-5 (50 μm), and strychnine (1 μm) or gabazine (25 μm). A, Representative traces showing the effect of hypercapnia on GABA- and glycine-evoked inward currents in CVNs. As shown in the average data, hypercapnia significantly reduced postsynaptic responses in CVNs to both GABA (n = 8) and glycine (n = 9). *p < 0.05; **p < 0.01.