Defects in breathing and thermoregulation in mice with near-complete absence of central serotonin neurons

Abstract

Serotonergic neurons project widely throughout the CNS and modulate many different brain functions. Particularly important, but controversial, are the contributions of serotonin (5-HT) neurons to respiratory and thermoregulatory control. To better define the roles of 5-HT neurons in breathing and thermoregulation, we took advantage of a unique conditional knock-out mouse in which Lmx1b is genetically deleted in Pet1-expressing cells (Lmx1b(f/f/p)), resulting in near-complete absence of central 5-HT neurons. Here, we show that the hypercapnic ventilatory response in adult Lmx1b(f/f/p) mice was decreased by 50% compared with wild-type mice, whereas baseline ventilation and the hypoxic ventilatory response were normal. In addition, Lmx1b(f/f/p) mice rapidly became hypothermic when exposed to an ambient temperature of 4 degrees C, decreasing core temperature to 30 degrees C within 120 min. This failure of thermoregulation was caused by impaired shivering and nonshivering thermogenesis, whereas thermosensory perception and heat conservation were normal. Finally, intracerebroventricular infusion of 5-HT stimulated baseline ventilation, and rescued the blunted hypercapnic ventilatory response. These data identify a previously unrecognized role of 5-HT neurons in the CO(2) chemoreflex, whereby they enhance the response of the rest of the respiratory network to CO(2). We conclude that the proper function of the 5-HT system is particularly important under conditions of environmental stress and contributes significantly to the hypercapnic ventilatory response and thermoregulatory cold defense.

Figure 1.

Breath-to-breath variability in WT and Lmx1b^f/f/p mice. A, B, Poincaré plot of the IBI of sequential breaths during baseline normoxic ventilation in WT (A; n = 7) and Lmx1b^f/f/p (B; n = 7) mice. C, Mean width (short-term variation) and length (long-term variation) in room air (RA), 5% and 7% CO₂. D, Relationship of scatter plot width and breathing frequency in WT mice under varying conditions. Note that Lmx1b^f/f/p data fall along the line fit to the WT data (variability = 14,660 × frequency^−2.4754), indicating the variation in IBI was caused by differences in breathing frequency rather than breathing instability. Unpaired t test was used; *p < 0.05 for WT versus Lmx1b^f/f/p; ^#p < 0.05 for RA versus 5% or 7% CO₂. Data are mean ± SEM.

Figure 2.

Lmx1b^f/f/p mice have decreased ventilatory drive and their hypercapnic ventilatory response is severely blunted. A, Plethysmography recordings from WT (blue) and Lmx1b^f/f/p (red) mice at rest, and while breathing 5% and 7% CO₂ in normoxia (21% O₂). Dashed lines highlight decreased slope of the inspiratory phase (ventilatory drive), and vertical lines mark the IBI. B, C, Ventilation (B) and breathing frequency (C) during hypercapnia under normoxic (NO) and hyperoxic (HO; 50% O₂) conditions (n = 10 WT NO, 8 Lmx1b^f/f/p NO, 11 WT HO, 8 Lmx1b^f/f/p HO). D, E, Ventilation (D) and convection requirement (E; V_E/V_O2; expressed as a percentage of control) during exposure to room air (RA) and hypoxia (n = 8 WT, 8 Lmx1b^f/f/p). For two-way ANOVA (genotype and condition as factors), **p < 0.05. For unpaired t test, *p < 0.05 (WT versus Lmx1b^f/f/p) for both normoxia and hyperoxia. For paired t test, ^#p < 0.05 for normoxia versus hyperoxia in Lmx1b^f/f/p. Data are mean ± SEM.

Figure 3.

Lmx1b^f/f/p mice fail to maintain their body temperature during cold exposure. A, mean core body temperature (T_Core; top) and activity [arbitrary units (A.U.); bottom] over 24 h in WT (blue) and Lmx1b^f/f/p (red) mice at 24°C. B, Mean T_Core and activity by phase [light (L); dark (D)] at 24°C (n = 6 WT, 5 Lmx1b^f/f/p) and 30°C (n = 6 WT, 6 Lmx1b^f/f/p). C–F, Core temperatures (T_Core) in individual WT (n = 5; blue) and Lmx1b^f/f/p (n = 7; red) mice before, during and after exposure to 4°C (C, D) or 12°C (E, F). Lines in D and F are discontinuous to align recovery period, and arrows in F indicate removal of animals from the cold caused by marked hypothermia. Unpaired t test was used; *p < 0.05 for WT versus Lmx1b^f/f/p; ^#p < 0.05 for light versus dark; ^†p < 0.05 for 24°C versus 30°C. Data are mean ± SEM.

Figure 4.

Lmx1b^f/f/p mice have normal thermosensory perception and activate heat conservation mechanisms normally in response to cold. A, Visible light (top) and infrared (bottom) images of thermocline. B, Summary data from thermocline experiments (n = 5 WT, 6 Lmx1b^f/f/p). Note that the mean temperature that mice selected over time (top inset) and overall (bottom inset) were not different between genotypes. C, Thermographic images of tail (arrows) surface temperatures in WT (left) and Lmx1b^f/f/p (right) mice. D, Summary data of tail surface temperature versus ambient temperature when mice were exposed to cold (n = 10 WT, 8 Lmx1b^f/f/p). Data are mean ± SEM.

Figure 5.

Shivering and brown adipose tissue activation are both impaired in Lmx1b^f/f/p mice. A, Recordings of nuchal muscle activity (top two traces, raw data; bottom trace, RMS) in warm (top trace; 28°C) and cold (bottom two traces; 4°C) T_Amb. Note that regular shivering was induced by cold exposure in the WT mouse (left; blue), but bursts were less frequent in the Lmx1b^f/f/p mouse (right; red). B, Group means (n = 4 WT, blue; 4 Lmx1b^f/f/p, red) of burst frequency before, during and after cold exposure. Insets, Total nuchal muscle activity (top; expressed as absolute and Δ total EMG), and burst duration (bottom) 1 h before [warm (W)] and during the last 30 min of cold exposure [cold (C)]. C, Thermographic images of the shaved back in WT (right) and Lmx1b^f/f/p (left) mice during cold exposure. D, Summary data of surface temperature differences between the IBAT region (solid circles in C) and the lower back (dashed circles in C) at T_Amb of 35, 10, and 7°C. Note that there was significant IBAT activation in both WT (n = 8) and Lmx1b^f/f/p (n = 8) mice at 10 and 7°C, but the activation was less in Lmx1b^f/f/p mice at 7°C. E, Thermographic image of neonatal (P5) WT and Lmx1b^f/f/p mice at T_Amb of 20°C. Arrows denote the head. F, Line profile summary data of back surface temperatures at three ages (P2–P3, P4, and P5–P6). Note the visible yellow (warm) band in the P5 WT mouse, indicating IBAT activation. Two-way ANOVA (genotype and time, or ambient temperature, and tail surface temperature as factors) or unpaired t test was used; ^†p < 0.05 for warm versus cold; *p < 0.05 for WT versus Lmx1b^f/f/p. Data are mean ± SEM.

Figure 6.

Exogenous 5-HT rescues the hypercapnic ventilatory response. A, Plethysmography recordings from an Lmx1b^f/f/p mouse breathing 0% CO₂ and 7% CO₂ (both in 50% O₂) before (red), and on the second day of intracerebroventricular infusion of 5 mm 5-HT (black). B, Effect of CO₂ on ventilation in WT mice (blue; n = 7), Lmx1b^f/f/p mice before intracerebroventricular infusion (red; n = 9), and Lmx1b^f/f/p mice on the second day of intracerebroventricular infusion of 5-HT at concentrations (within the pump) of 0.5 (brown; n = 5), 1.0 (purple; n = 7), and 5.0 mm (black; n = 6). Data were obtained in 50% O₂. Inset, Baseline ventilation in 21% O₂. C, Mean T_Core in Lmx1b^f/f/p mice with (black) or without (red) intracerebroventricular 5-HT infusion. Data were combined from all animals at 5-HT concentrations of 1, 5, and 25 mm. Two-way ANOVA (p < 0.001 for 5-HT level and condition), and unpaired t test were used; ^$p < 0.05 for intracerebroventricular 5-HT versus control; *p < 0.05 for 1.0 mm 5-HT versus control; ^†p < 0.05 for 5.0 mm 5-HT versus control; ^#p < 0.05 for Lmx1b^f/f/p control and 0.5 mm 5-HT versus WT. Data are mean ± SEM.