Chronic central serotonin depletion attenuates ventilation and body temperature in young but not adult Tph2 knockout rats

Abstract

Genetic deletion of brain serotonin (5-HT) neurons in mice leads to ventilatory deficits and increased neonatal mortality during development. However, it is unclear if the loss of the 5-HT neurons or the loss of the neurochemical 5-HT led to the observed physiologic deficits. Herein, we generated a mutant rat model with constitutive central nervous system (CNS) 5-HT depletion by mutation of the tryptophan hydroxylase 2 (Tph2) gene in dark agouti (DA(Tph2-/-)) rats. DA(Tph2-/-) rats lacked TPH immunoreactivity and brain 5-HT but retain dopa decarboxylase-expressing raphe neurons. Mutant rats were also smaller, had relatively high mortality (∼50%), and compared with controls had reduced room air ventilation and body temperatures at specific postnatal ages. In adult rats, breathing at rest and hypoxic and hypercapnic chemoreflexes were unaltered in adult male and female DA(Tph2-/-) rats. Body temperature was also maintained in adult DA(Tph2-/-) rats exposed to 4°C, indicating unaltered ventilatory and/or thermoregulatory control mechanisms. Finally, DA(Tph2-/-) rats treated with the 5-HT precursor 5-hydroxytryptophan (5-HTP) partially restored CNS 5-HT and showed increased ventilation (P < 0.05) at a developmental age when it was otherwise attenuated in the mutants. We conclude that constitutive CNS production of 5-HT is critically important to fundamental homeostatic control systems for breathing and temperature during postnatal development in the rat.

Keywords: apnea; chemoreflex; control of breathing; neonate; serotonin.

Fig. 1.

Zinc-finger nuclease (ZFN)-targeted mutation of the Tph2 gene in the rat eliminated tryptophan hydroxylase (TPH) immunoreactivity. A: basepair sequence of exon 7 of the native Tph2 allele [Tph2 wild type (WT)] and the 10- and 11-basepair deletions generated in two mutant lines of dark agouti (DA) rats (M2 and M3). B: tissue sections through the raphe obscurus nucleus of adult male rats that were stained to simultaneously detect TPH (red) and dopa decarboxylase (DDC; green). Compared with WT (DA^Tph2+/+; top), DA^Tph2−/− rats (bottom) showed a complete loss of TPH immunoreactivity but retained immunoreactivity for DDC that was similar to WT, indicating the presence of neurons in the DA^Tph2−/− brainstem that would otherwise be serotonergic. The composite images show the coincidence of TPH and DDC staining in the WT rat (yellow) and emphasize the lack of TPH staining in the mutant. The composite images also show cell nuclei marked by DAPI staining (blue).

Fig. 2.

DA^Tph2−/− rats had reduced growth and increased mortality. A: DA^Tph2−/− rats were smaller beginning at P7 and remained smaller thereafter up to 60 days of age (P < 0.001, two-way ANOVA). B: weights of DA^Tph2−/− rats (green) ware expressed as a percentage of the WT weight at each age and plotted across age (days). C: survival (%pups born for each genotype) at each postnatal (P) age showed that after an initial high rate of mortality in DA^Tph2−/− pups there was additional mortality on or after P10 that was rare in control [WT and heterozygous (Het)] pups.

Fig. 3.

Ventilation was disrupted at distinct postnatal ages in DA^Tph2−/− rats. A: representative plethysmographic recordings of basal ventilation (measured as voltage deflections) in 1 WT (WT 125; blue) and a DA^Tph2−/− littermate (DA^Tph2−/− 127; green) rat at P1, P3, and P12 while breathing room air. Note that ventilation in the DA^Tph2−/− rat alternated between regular and highly irregular with apneas and large, atypical breaths compared with the WT, but that there was improvement by P3. However, by P12 ventilation was reduced and irregular in the DA^Tph2−/− rat. B: Poincare plots comparing the inter-breath interval (IBI) among consecutive breaths during room air exposure of WT (n = 9; 5,425 breaths) and DA^Tph2−/− (n = 9; 3,594 breaths) rats from P1-2. C: variability in the IBI was calculated by distance from the line of identity (“width” from LOI) or distance from the mean IBI along the LOI (“length”; see results), and averaged for each genotype and age. Note that variability in the IBI was significantly greater (P < 0.003) in DA^Tph2−/− rats at P1-2 and P11-12 but not at other ages tested (two-way ANOVA).

Fig. 4.

Ventilation and body temperatures were reduced in DA^Tph2−/− rats at distinct time points during development. Minute ventilation (V̇e; ml·min⁻¹·100 g⁻¹; A), breathing frequency (breaths/minute; B), tidal volume (V_T; ml/100 g; C), and rectal temperatures (body temperature; °C; D) were plotted across postnatal age in 2- or 3-day bins. Note that resting V̇e is reduced in DA^Tph2−/− rats (green; n = 7–15) compared with controls (WT and Het; blue; n = 6–15), due to a combination of reductions in frequency and V_T. Note also that body temperatures in DA^Tph2−/− rats were markedly lower compared with controls beginning on P13 despite the ambient temperature being warmed to ∼27°C. *Significant effect of genotype within age (P < 0.05, two-way ANOVA).

Fig. 5.

Resting ventilation is augmented in DA^Tph2−/− rats treated with 5-HTP. A: representative plethysmographic recordings of basal ventilation (measured in voltage deflections) of individual DA^Tph2−/− and WT rat pups from P12-16 breathing room air before (left) and after 3 injections of 5-HTP (10 mg/kg) or saline, respectively, administered over 24 h (right). Mean group data (±SE) for V̇e (B; expressed as ml·min⁻¹·100 g⁻¹) and body temperature (C; °C) in DA^Tph2−/− rats (n = 9) and WT littermates (n = 12) while breathing room air. Saline injections in WT pups had no effect on V̇e, but 5-HTP injections increased V̇e compared with preinjection control and compared with saline-treated WT pups. *Significant effect of genotype in room air (RA); **significant differences from preinjection control and saline-injected WT pups in B and C (P < 0.05, two-tailed t-test). D: HPLC measurements of whole brains from the saline-injected WT and 5-HTP-treated DA^Tph2−/− rats demonstrated the presence of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) after treatment, with little or no effects on norepinephrine (NE) or dopamine (DA) levels [P < 0.05; two-way repeated-measures (RM) ANOVA; *significant difference from saline-treated WT rats].

Fig. 6.

Minimal effects of Tph2 knockout on ventilation at rest or during hypercapnia in adult male rats. Ventilation (V̇e; ml·min⁻¹·100 g⁻¹; A), breathing frequency (breaths/min; B), V̇e [expressed as a percentage of room air breathing (%control); C], and tidal volume (V_T; ml·breath⁻¹·100 g⁻¹; D) were compared among control (WT and Het) and DA^Tph2−/− adult rats. Breathing frequency was significantly lower in room air and 7% CO₂, and V_T slightly greater in 7% CO₂ in DA^Tph2−/− rats compared with controls. *Significant effect of genotype (P < 0.05, two-way RM ANOVA).

Fig. 7.

Minimal effects of Tph2 knockout on ventilation at rest or during hypoxia in adult rats. Ventilation (V̇e; ml·min⁻¹·100 g⁻¹; A), breathing frequency (breaths/min; B), V̇e [expressed as a percentage of room air breathing, (%control); C], and tidal volume (V_T; ml·breath⁻¹·100 g⁻¹; D) were compared among control (WT and Het) and DA^Tph2−/− adult rats. Breathing frequency was significantly lower and V_T higher in DA^Tph2−/− rats compared with controls when exposed to either RA or 12% O₂. *Significant effect of genotype (P < 0.05; 2-way RM ANOVA).

Fig. 8.

Tph2 knockout rats maintain body temperature during a cold challenge. Rectal temperature (T_R; °C) was measured 30 min before (room temperature; 23°C) and at 30-min intervals for up to 4 h while exposed to an ambient temperature of 4°C. Note that control (WT and Het) and DA^Tph2−/− rats both maintain body temperature during environmental cooling (P > 0.05, two-way ANOVA).