Transgenic mice lacking serotonin neurons have severe apnea and high mortality during development

Abstract

Central serotonin (5-HT) neurons modulate many vital brain functions, including respiratory control. Whether breathing depends critically on 5-HT neurons, or whether their influence is excitatory or inhibitory, remains controversial. Here we show that neonatal Lmx1b(flox/flox;ePet-Cre/+) mice (also called Lmx1b(f/f/p) mice), which selectively lack serotonin neurons, display frequent and severe apnea lasting as long as 55 s. This was associated with a marked decrease in ventilation to less than one-half of normal. These respiratory abnormalities were most severe during the postnatal period, markedly improving by the time the pups were 2-4 weeks old. Despite the severe breathing dysfunction, many of these mice survived, but there was a high perinatal mortality, and those that survived had a decrease in growth rate until the age at which the respiratory defects resolved. Consistent with these in vivo observations, respiratory output was markedly reduced in isolated brainstem-spinal cord preparations from neonatal Lmx1b(f/f/p) mice and completely blocked in perfused brain preparations from neonatal rats treated with selective antagonists of 5-HT(2A) and neurokinin 1 (NK-1) receptors. The ventilatory deficits in neonatal Lmx1b(f/f/p) mice were reversed in vitro and in vivo with agonists of 5-HT(2A) and/or NK-1 receptors. These results demonstrate that ventilatory output in the neonatal period is critically dependent on serotonin neurons, which provide excitatory drive to the respiratory network via 5-HT(2A) and NK-1 receptor activation. These findings provide insight into the mechanisms of sudden infant death syndrome, which has been associated with abnormalities of 5-HT neurons and of cardiorespiratory control.

Figure 1.

Mice lacking 5-HT neurons have severe and frequent apnea during early development, which resolves with maturation. A, Plethysmography recording from a P4 Lmx1b^f/f/p mouse with a prolonged (35 s) apnea and erratic low-amplitude breaths (raw trace; inspiration is an upward deflection, and expiration is downward). B, Recordings from two P4 mice (upper traces). The WT mouse (black trace) had continuous, rhythmic breathing, whereas the Lmx1b^f/f/p mouse (blue trace) had short periods of low-amplitude and low-frequency rhythmic breathing separated by prolonged apnea. The WT and Lmx1b^f/f/p mice both had episodes of vocalization (arrows) that were equivalent in amplitude (after normalization to weight; see Results), demonstrating that Lmx1b^f/f/p mice were capable of large-amplitude inspiratory efforts and that these could be accurately measured. Recordings from the same two mice at P12 (lower traces) show marked improvement of the Lmx1b^f/f/p mouse, with regular breathing and no apnea. C, Changes in apnea with age in WT (black; n = 13) and Lmx1b^f/f/p (blue; n = 14) mice. Lmx1b^f/f/p mice spent more time apneic (total apnea duration as a percentage of total study time, measured at ambient temperatures of 24 and 30°C; **p ≤ 0.013), and had a greater apnea frequency [inset (24°C)], up to the age of P9 (*p ≤ 0.001). D, The percentage of Lmx1b^f/f/p (blue) mice with prolonged (>5 s) apnea was greater (*p < 0.006; χ² with Yates correction) relative to WT mice (black) at the ages of P2 and P4, and this declined with postnatal development. E, The frequency of prolonged apneas was also greater (*p < 0.001) in Lmx1b^f/f/p mice at these same ages.

Figure 2.

Ventilation is reduced in Lmx1b^f/f/p mice during postnatal development. A–E, Data are presented from two groups of mice: one studied using flow-through plethysmography at 27°C (A, B) and another studied using stop-flow plethysmography at 24 and 30°C (C–E). A, Ventilation, which was normalized to weight and expressed relative to WT mice (black; n = 8–14 animals per data point), was lower in Lmx1b^f/f/p mice (blue; n = 5–13) up to the age of P28 (*p ≤ 0.02), but was normal in adults. B, Similarly, the ratio of ventilation to oxygen consumption [calculated in ml/min in this case (see Materials and Methods)] was also reduced in Lmx1b^f/f/p mice (n = 5–6) relative to WT mice (n = 8–9) from P20–P28 mice but was normal in adults. *p ≤ 0.045. C, Ventilation was also reduced (p ≤ 0.015) in Lmx1b^f/f/p mice (n = 6–12) compared with WT mice (n = 7–10) up to P6 at a warm ambient temperature (30°C; open symbols), and up to P12 at 24°C (p ≤ 0.014; solid symbols). D, Breath amplitude (normalized to weight and expressed as a percentage of WT value) was lower in Lmx1b^f/f/p mice up to P4 at both temperatures (p ≤ 0.006) and up to P9 at 24°C (p < 0.001). E, Breathing frequency was also lower in Lmx1b^f/f/p mice up to P9 at both temperatures (p ≤ 0.004) and up to P12 at 24°C (p < 0.001). *Difference between genotypes at 24°C; **difference between genotypes at both temperatures. Data points were shifted along the x-axis by ±0.2 d of age in C and D for clarity. Adult data shown here are reproduced from an earlier publication for comparison (Hodges et al., 2008). F, Breathing was more irregular in Lmx1b^f/f/p mice relative to WT mice (**p < 0.05) from P2 to P6, as quantified by the CV IBI when measured at ambient temperatures of 24°C (solid symbols) and 30°C (open symbols). G, Animal temperature was slightly lower in Lmx1b^f/f/p mice relative to WT mice at an ambient temperature of 27°C at the ages of P12, P20, and P28 (*p ≤ 0.049) but was not significantly different at other ages, including adults.

Figure 3.

The growth of Lmx1b^f/f/p mice was reduced during development in parallel with the presence of frequent apnea. A, Body weight was equal at birth (P0; inset) but then was lower (*p < 0.05) in Lmx1b^f/f/p mice (blue; n = 24) relative to WT mice (black; n = 24) until P55. B, Growth rates (calculated as weight gained per day as a percentage of weight on the previous day) in Lmx1b^f/f/p mice was less than that in WT mice up to ∼P12, but then growth accelerated at P15, coincident with the age at which apnea resolved in Lmx1b^f/f/p mice.

Figure 4.

Abnormal breathing in Lmx1b^f/f/p mice is because of loss of excitatory drive to the respiratory network from central 5-HT neurons. A, Raw (first and third traces) and integrated (second and fourth traces) signals from the hypoglossal (XII) and cervical (C1–C4) nerve roots of isolated brainstem–spinal cord (en bloc) preparations from P2 mice showing synchronized inspiratory bursts. B, Recordings from XII nerve roots show lower respiratory frequency in Lmx1b^f/f/p (blue) relative to WT (black) preparations under control conditions (left traces). Respiratory frequency was increased by bath application of DOI (10 μm) (right traces). C, Respiratory frequency was markedly lower in Lmx1b^f/f/p (n = 8) compared with WT (n = 12) brainstem–spinal cord preparations under control conditions (*p < 0.001). Respiratory burst frequency was significantly increased by DOI in Lmx1b^f/f/p mice (p < 0.05) to a level that was equal to WT preparations during the final 10 min of application (p = 0.51). Addition of substance P further increased respiratory output in WT and Lmx1b^f/f/p preparations, with no difference between genotypes (p = 1.0). D, E, Hypoglossal (XII) and cervical (C1–C4) nerve burst durations (D) and amplitudes (E) were not different between genotypes during the control period or during bath application of DOI (p > 0.05). Cervical nerve burst duration increased (p = 0.012) in Lmx1b^f/f/p preparations, and XII nerve burst amplitude decreased with DOI compared with control in the WT preparations (p < 0.001). *Significant effect of DOI relative to control. F–I, Traces of raw XII nerve activity in Lmx1b^f/f/p preparations during control conditions (F), bath application of substance P (1 μm) (G), and exposure to 3.0 mm K⁺ (H) or 6.0 mm K⁺ (I).

Figure 5.

Endogenous 5-HT and substance P receptor activation are critical for generation of respiratory output in the perfused neonatal rat nervous system in situ. A, Recordings of integrated XII nerve activity from the neonatal rat in situ preparation. The 5-HT_2A receptor antagonist MDL 11,939 caused a large reduction of inspiratory burst frequency and amplitude when applied via the perfusate. B–D, MDL 11,939 (n = 4) and the NK-1 receptor antagonist SR 140333 (n = 4) caused a progressive decrease both in frequency (B) (p < 0.001; ANOVA) and amplitude (shown as percentage of control values; p < 0.001; ANOVA) of integrated PN (C) and hypoglossal nerve (XII) (D) inspiratory bursts, with eventual complete elimination of both motor outputs as the concentrations of drugs were increased. ^#Significant decrease from control values for MDL 11,939; *significant decrease from control values for both antagonists.

Figure 6.

5-HT_2A receptor stimulation reverses the respiratory defect in neonatal Lmx1b^f/f/p mice in vivo. A, Plethysmography traces in a P2 Lmx1b^f/f/p mouse before (top) and 60 min after (bottom) subcutaneous injection of DOI (0.1 μg/gm). B, The percentage of time spent apneic decreased (p ≤ 0.029) and relative ventilation increased (p ≤ 0.015) in Lmx1b^f/f/p mice (n = 6) for >4 h after DOI. *Significant difference between control and DOI treatment in Lmx1b^f/f/p mice.