Prenatal to early postnatal nicotine exposure impairs central chemoreception and modifies breathing pattern in mouse neonates: a probable link to sudden infant death syndrome

Abstract

Nicotine is a neuroteratogen and is the likely link between maternal cigarette smoking during pregnancy and sudden infant death syndrome (SIDS). Osmotic minipumps were implanted in 5-7 d CF1 pregnant mice to deliver nicotine bitartrate (60 mg Kg(-1) day(-1)) or saline (control) solutions for up to 28 d. Prenatal to early postnatal nicotine exposure did not modify the number of newborns per litter or their postnatal growth; however, nicotine-exposed neonates hypoventilated and had reduced responses to hypercarbia (inhalation of air enriched with 10% CO(2) for 20 min) and hypoxia (inhalation of 100% N(2) for 20 s) at postnatal days 0-3 (P0-P3). In contrast, at postnatal day 8, nicotine-exposed neonates were indistinguishable from controls. Isolated brainstem-spinal cord preparations obtained from P0 to P3 nicotine-exposed neonates showed fictive respiration with respiratory cycles longer and more irregular than those of controls, as indicated by high short- and long-term variability in Poincaré plots. In addition, their responses to acidification were reduced, indicating compromise of central chemoreception. Furthermore, the cholinergic contribution to central chemosensory responses switched from muscarinic receptor to nicotinic receptor-based mechanisms. No significant astrogliosis was detectable in the ventral respiratory group of neurons with glial fibrillary acidic protein immunohistochemistry. These results indicate that nicotine exposure affects the respiratory rhythm pattern generator and causes a decline in central chemoreception during early postnatal life. Consequently, breathing would become highly vulnerable, failing to respond to chemosensory demands. Such impairment could be related to the ventilatory abnormalities observed in SIDS.

Figure 1.

Ventilatory response to hypercarbia in control (A) and nicotine-exposed (B) P3 pups. Plethysmographic recordings of control (nonexposed) and nicotine-exposed P3 pups obtained before (arrow 1), during (arrow 2), and after (arrow 3) inhalation of air containing 10% CO₂. Fast sweeps of the recordings indicated by arrows are displayed in the respective traces below. Bar indicates period of hypercarbia. Note that large spikes correspond to oscillations produced by the displacement of trunk and extremities (movement artifacts). Measurements of ventilatory variables were done during periods that recordings were free of movement artifacts (traces 1–3).

Figure 2.

Mice exposed to prenatal-perinatal nicotine hypoventilate during early postnatal life. V_E, f_R, and V_T were evaluated in nicotine-exposed (filled squares, n = 13) and control (open squares, n = 13) pups. Data are expressed as mean ± SEM. ANOVA two-way analysis revealed a significant nicotine effect (df = 1, F ratios and respective p indicated for each variable) and a significant effect of aging upon V_E (F ratio = 14.63, df = 3, p < 0.0001), f_R (F ratio = 23.66, df = 3, p < 0.0001), and V_T (F ratio = 10.13, df = 3, p < 0.0001); ^#p < 0.01 indicates significant differences between nicotine-exposed pups and controls at a specific age as determined with the Newman–Keuls post hoc test. Note that V_E and V_T are normalized using the weights of the respective neonates.

Figure 3.

Prenatal-perinatal nicotine reduces the respiratory response to hypercarbia in early postnatal life in mouse neonates. Time course of changes in V_E, f_R, and V_T induced by hypercapnia (inhalation of air with 10% CO₂ for 20 min) in nicotine-exposed (filled squares, n = 13) and control (open squares, n = 13) pups. Hypercapnic stimulation is indicated by the bar. Data are expressed as means ± SEM. Significant nicotine effects upon V_E, f_R, and V_T were observed at P0, P1, and P3, but not in P8 (df = 1, F ratios and p indicated for each postnatal age). The symbols * and ^# indicate significant differences (p < 0.05 and p < 0.01, respectively) between nicotine-exposed pups and controls at a specific time, as determined with the Newman–Keuls post hoc test.

Figure 4.

Prenatal-perinatal nicotine reduces the respiratory response to hypoxia in early postnatal life in mouse neonates. Time course of changes in V_E, f_R, and V_T induced by hypoxia (inhalation of 100% N2 for 20 s) in nicotine-exposed (filled squares, n = 12) and control (open squares, n = 13) pups; hypoxia administration is indicated by the bar. Data are expressed as means ± SEM. Significant effects of nicotine upon V_E and V_T were observed at P0, P1, and P3, but not P8 (df = 1, F ratios and p indicated for each postnatal age). Note that significant effects of nicotine upon f_R were observed only at P0; * and ^# indicate significant differences (p < 0.05 and p < 0.01, respectively) between nicotine-exposed pups and controls at a specific time as determined by the Newman–Keuls post hoc test.

Figure 5.

Prenatal-perinatal nicotine reduces both the frequency of fictive respiration and the response elicited by central chemoreceptor activation in the brainstem-spinal cord preparation from P0–P3 neonates. A, Integrated inspiratory burst recorded from C4 ventral roots in isolated brainstem spinal cord preparations (see diagram in inset) obtained from control and nicotine-exposed P2 mice during superfusion with aCSF pH 7.4 and pH 7.3. Note the increase in frequency and the reduction in amplitude of the bursts induced by acidification. B, Basal frequency of fictive respiration in nicotine-exposed preparations (filled squares, n = 9) is lower than those in controls (open squares, n = 10); reduction in amplitude and increase in frequency in response to acidification were expressed as percentage of the respective basal values. Changes in amplitude (C) were less pronounced, but no differences in frequency were observed (D) in nicotine-exposed animals compared with controls after changing the pH of the brainstem superfusion from pH 7.4 to 7.3. Data are expressed as mean ± SEM. ANOVA two-way analysis revealed a significant effect of nicotine upon basal frequency and response in amplitude induced by acidification (df = 1, F ratios and respective p indicated in each graph). Symbols * and ^# indicate significant differences (p < 0.05 and p < 0.01, respectively) at a specific age as determined by the Newman–Keuls post hoc test.

Figure 6.

Poincaré plots of cycles T_{n + 1} as function of the previous cycle, T_n, for fictive respiration recorded from brainstem–spinal cord preparations obtained from control (open squares, n = 10) and nicotine-exposed (filled squares, n = 9) P0 and P3 mice. Two-way ANOVA revealed a significant effect of nicotine upon long- and short-term variability in P0 (F ratio = 119.45, p < 0.0001; F ratio = 13.16, p = 0.0003, respectively) and P3 (F ratio = 164.29, p < 0.0001; F ratio = 76.03, p < 0.0001, respectively) pups. ^†p < 0.01, significant difference from pH 7.4. ^#p < 0.01, significant difference between nicotine-exposed and control preparations at specific pH conditions as determined with the Newman–Keuls post hoc test; subscripts L and S indicate long- and short-term variability, respectively.

Figure 7.

Cholinergic contribution to the central chemosensory responses is modified by prenatal to early postnatal nicotine exposure. Changes in amplitude (top graph) and frequency (bottom graph) of fictive respiration induced by acidification of the superfusion medium in isolated brainstem–spinal cord preparations are expressed as percentage of basal values. Control (open bars) and nicotine-exposed (filled bars) preparations from P2–P3 neonates were acidified in the absence or presence of muscarinic receptor blocker (atrop, atropine 100 μm) or nicotinic receptor blocker (hexam, hexamethonium 100 μm). ANOVA revealed significant differences in amplitude and frequency for control preparations (F ratio = 8.31, p = 0.001; F ratio = 5.14, p = 0.01, respectively). Note that muscarinic but not nicotinic blockade abolished the changes in amplitude and frequency (^†p < 0.05 and ^††p < 0.01 with respect to controls in the absence of blockers, Dunnett's test). In nicotine-exposed preparations, significant differences were observed in frequency, but not amplitude (F ratio = 4.14, p = 0.03; F ratio = 1.93, p = 0.16, respectively). In contrast to control preparations, nicotinic but not muscarinic blockade affected the acidification-induced responses (^§p < 0.05 with respect to nicotine-exposed preparations in absence of blockers, Dunnett's test). Significant differences between control and nicotine-exposed preparations are indicated (^#p < 0.01 and *p < 0.05, ANOVA). Bars and vertical lines indicate mean and SEM, respectively, for data points. Numbers of preparations are indicated inside the bars. Dotted lines indicate no change with respect to basal values (100% of basal).

Figure 8.

GFAP immunohistochemistry detected no gliosis in the respiratory ventral group region of P1 and P3 neonates after prenatal nicotine administration. In A, B and E, F, somatostatin-positive cell bodies (in green) and GFAP-positive cells (astrocyte marker, in red) at the pre-Bötzinger complex of control and nicotine-exposed P1 and P3 neonates. In C, D and G, H, somatostatin nerve endings in the ventral respiratory group (in green) and GFAP-positive cells (in red) of control and nicotine-exposed P1 and P3 neonates. Scale bar, 50 μm. Area occupied by GFAP-positive pixels was estimated from an area of 230 × 230 μm positioned on the ventral respiratory group using ImageJ software. GFAP labeling did not differ between controls (n = 5) and nicotine-exposed (n = 6) neonates evaluated in P1 and P3. However, it increased twofold from 1.2 ± 0.2% (n = 10) in P1 to 2.4 ± 0.4% (n = 12) in P3 neonates (p = 0.04, ANOVA).