Increased central cholinergic drive contributes to the apneas of serotonin-deficient rat pups during active sleep

Abstract

Infant rat pups lacking central nervous system (CNS) serotonin (5-hydroxytryptamine; 5-HT) have unstable breathing during prolonged periods of active sleep. Given that cholinergic neurons are drivers of active sleep and project to respiratory patterning regions in the brainstem, we hypothesized that 5-HT preserves respiratory stability in active sleep by dampening central cholinergic drive. We used whole-body plethysmography coupled with nuchal electromyography to monitor the breathing pattern of 2-wk-old tryptophan hydroxylase 2 (TPH2)+/+ and TPH2-deficient (TPH2−/−) pups in active sleep, before and after muscarinic blockade. For the group 1 experiment we injected methylatropine (Ap-M), a CNS-impermeant form of atropine, followed ~30 min later by an injection of atropine sulfate (Ap-S), the CNS-permeant form (both 1 mg/kg, 10 μl bolus iv); both injections occurred within an active sleep episode. We analyzed the effect of each drug on the coefficient of variation of the respiratory period (CV-P) during active sleep. For the group 2 experiment rats were cycled through several episodes of active and quiet sleep before administration of Ap-S (1 mg/kg, 200 μl ip) or vehicle. We assessed the effect of Ap-S on the apnea indices of both genotypes during quiet and active sleep. In group 1 Ap-S significantly reduced the CV-P of TPH2−/− pups (P = 0.03), an effect not observed in TPH2+/+ pups or following Ap-M. In group 2 the apnea index of TPH2−/− pups was significantly reduced following Ap-S injection (P = 0.04), whereas the apnea index of TPH2+/+ littermates was unaffected (P = 0.58). These findings suggest that central 5-HT reduces apnea and stabilizes breathing by reducing cholinergic signaling through muscarinic receptors.

NEW & NOTEWORTHY Serotonin in the central nervous system (CNS) is necessary for maintaining the stability of breathing in the early postnatal period, particularly during active sleep. Here we show that the administration of atropine to the CNS selectively stabilizes the respiratory pattern of tryptophan hydroxylase 2-deficient rat pups and reduces their apneas. This suggests that CNS serotonin stabilizes breathing at least in part by reducing central cholinergic drive.

INTRODUCTION

An array of neuromodulators within respiratory neuronal networks have the potential to modulate the frequency, amplitude, and pattern of the respiratory motor output to maintain blood gas and pH homeostasis in the face of intrinsic or extrinsic stressors. Serotonin (5-hydroxytryptamine; 5-HT), which within the central nervous system (CNS) is synthesized mainly in the raphe nuclei of the brain stem and midbrain, has complex effects within the respiratory network—some neurons are excited whereas others are inhibited, depending on the subtype of 5-HT receptor expressed (e.g., 5-HT₂ vs. 5-HT₁) (5, 31). In addition to increasing respiratory frequency (12, 19), Pena and Ramirez showed that 5-HT stabilizes the respiratory rhythm through the activation of 5-HT_2A receptors (31). Data obtained from knockout mice and rats deficient in 5-HT neurons, or specifically in 5-HT, also support a stabilizing role for 5-HT on the respiratory rhythm. For example, adult mice lacking 5-HT within the CNS (tryptophan hydroxylase 2 knockout; TPH2−/−) have increased apnea frequency during non-rapid eye movement (NREM) sleep (or “quiet sleep” in infancy) (26). Infant TPH2−/− rats also display a highly unstable respiratory pattern punctuated with frequent apneas and tachypneas (22, 35). We discovered that although TPH2−/− rats have decreased respiratory frequency and hypoventilate in both quiet and active sleep, their apnea and general respiratory instability emerge almost exclusively in active sleep [equivalent to rapid eye movement sleep (REM) in adults] (35).

5-HT neurons within the midbrain, pons, and medulla are one of several types of neurons whose activity depends on state of vigilance. Others include catecholaminergic neurons in the locus coeruleus, orexin neurons in the perifornical region and lateral hypothalamus and histaminergic neurons in the posterior hypothalamus. Most 5-HT neurons have relatively high activity during wakefulness, reduced activity during NREM, and virtually no activity in REM sleep (27). 5-HT neurons in the dorsal raphe play a significant role in the regulation of sleep, a function that is associated with, and is partially due to, their state-dependent firing pattern (27). In adult mice, a deficiency of 5-HT is associated with prolonged but fewer episodes of both REM (8, 21) and NREM sleep (33). We have shown similar findings in infant TPH2−/− rats (35); the prolonged active sleep episodes displayed by young TPH2−/− rats suggest that there is a facilitation or disinhibition of populations of neurons that maintain active sleep. In keeping with this idea, 5-HT in the laterodorsal tegmentum (LDT) reduces REM episode duration with little effect on REM incidence (20), an effect that is likely due to activation of inhibitory 5-HT_1A receptors on cholinergic neurons in this region (34). That said, the overall role of 5-HT on REM sleep architecture is not clear cut: 5-HT_2A receptor activation within the LDT has been shown to decrease the incidence of REM sleep with little effect on duration (3), and 5-HT_1A activation in the pedunculopontine tegmentum, another cholinergic REM-promoting nucleus, decreases the incidence of REM sleep (18).

Given the evidence that 5-HT, through 5-HT_1A receptors, is able to inhibit a major group of cholinergic, REM/active sleep-driving neurons in the pons (34), it may be that increased cholinergic drive contributes to the unstable breathing pattern of infant tryptophan hydroxylase 2-deficient (TPH2−/−) rats during prolonged active sleep. Indeed, cholinergic neurons originating in the pontine tegmentum project to respiratory neurons replete with muscarinic receptors, including regions in the dorsolateral pons (6, 25). To evaluate the role of central muscarinic drive in the respiratory phenotype of infant TPH2−/− rats, we treated them and their TPH2+/+ littermates with two forms of atropine, one that can permeate the CNS and one that cannot. Our findings suggest that enhanced muscarinic signaling in the CNS contributes to the apneas and destabilized breathing displayed by 5-HT-deficient rat pups during active sleep.

METHODS

Ethical Approval

All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Missouri at Columbia, MO, in accordance with national guidelines.

Animals

For this study we used TPH2−/− and wild-type (TPH2+/+) rat pups at postnatal days 14–16. Heterozygous (TPH2+/−) females were bred with TPH2+/− males to generate TPH2−/− and +/+ pups. Dams were fed ad libitum on standard rat chow and kept on a 12-h light-dark cycle. The generation and genotyping of the TPH2−/− rat line on Dark Agouti background has been previously described (22). Briefly, Kaplan and colleagues utilized custom-made zinc-finger nucleases to induce a 10-bp deletion within exon 7 of the TPH2 gene (22). Heterozygous (TPH2+/−) males and females were identified by PCR using the forward primer 5′-GGCCTTTAGGTCCTGAGGTT-3′ and the reverse primer 5′-CCCTTCTCCACAGAAGTGCT-3′ over 35 cycles at an annealing temperature of 65°C. PCR products were run on 15% gels to discern the wild-type and knockout alleles. For both experiments we used a mixture of male and female animals and as we did not observe any effects of sex, data from both were combined before statistical analyses. Initial experiments indicated a relatively high degree of variability from animal to animal with respect to apnea incidence and the effect of atropine. Animals numbers used are based on a sample-size analysis performed on pilot experiments.

Determining Vigilance State

Wakefulness, quiet sleep, and active sleep were determined using a combination of nuchal electromyography (EMG) along with established behavioral criteria that we and others have previously described (14, 35). In quiet sleep pups were completely immobile, with their head resting on the bottom of the chamber or on the dorsal surface of the forelimbs. Associated with the decrease in EMG amplitude that occurred after the transition from quiet sleep to active sleep (Fig. 1) was the appearance of myoclonic twitching of the whiskers, ears, and limbs, which also generated bursts of activity on the EMG record. A highly stable respiratory pattern and low heart-rate variability also differentiated quiet sleep from active sleep (Fig. 1). Associated with the abrupt increase in EMG amplitude during arousal was lifting of the head and flexion of the forepaws. In group 1 experiments, we initially observed that the presence of both an intravenous catheter and nuchal EMG electrodes reduced the appearance of active sleep. Because of this, we instrumented only a subset of animals (n = 3 TPH2+/+ and n = 4 TPH2−/−) to confirm the specific behaviors associated with active sleep and arousal from sleep. We could also determine changes in heart rate in response to drug application in these animals, as the nuchal electrodes also detected cardiac ECG activity, which was superimposed on the EMG record (35). The remainder of group 1 animals (n = 6 TPH2+/+ and n = 6 TPH2−/− pups) were performed without EMG electrodes, and active sleep and arousal were determined solely by the behavioral criteria that characterize each state, as described above. All group 2 animals were instrumented with nuchal EMG electrodes as they did not have venous catheters and thus displayed sufficient amounts of active sleep throughout their protocol. Digital video records, made using the Video Capture Module of LabChart V8 (ADInstruments, Colorado Springs CO), were used to confirm episodes of quiet and active sleep as well as arousals.

Fig. 1.

Identification of the transition from quiet sleep (QS) to active sleep (AS). Period (P) of individual breaths, heart rate (HR), raw nuchal electromyography (EMG) signals, and the raw respiratory volume (V_T) of a rat pup just before and after the transition to AS from QS (top). Magnified EMG and respiratory trace shown in the inset (bottom). Note that AS is characterized by an abrupt decrease in respiratory stability, reflected in greater variability in P and reduced EMG amplitude (indicated in the margins of the magnified trace on the bottom by the vertical arrows and dashed lines that highlight the amplitude of the signal). bpm, beats/min.

Surgery

EMG electrodes.

Pups were removed from the dam and anesthetized with 2–3% isoflurane. Paw pinch was used to assess whether anesthesia was sufficient. An insulated stainless steel wire electrode with a terminal suture pad was sutured under the neck muscle, and another was sutured into the flank muscle to serve as a ground (cat. no. E-363/76, PlasticsOne, Roanoke, VA); pad dimension: thickness, 0.66 mm; width, 1 mm; length, 3.18 mm; wire dimensions: diameter, 0.25 mm; length, 37.5 mm). Lidocaine (~100–200 µl of 20 mg/ml) was applied to each of the electrode sites before experimentation.

Femoral venous catheters.

Femoral venous catheters were implanted into group 1 pups immediately before testing, as described previously (24). Briefly, catheters consisting of polyethylene-10 tubing were heated, stretched, and filled with sterile heparinized 0.9% saline (100 μl/ml) before surgery. Although the pups were under ~2% isoflurane, a skin incision was made in the left groin for visualization and dissection of the left femoral vein under a ×20 dissecting microscope. Using 5-0 surgical suture, the vein was tied just distal to the insertion site, and an incision was then made in the vein for insertion of the PE-10 catheter. The tip of the catheter was advanced into the common iliac vein. Liquid lidocaine was used as an analgesic at the incision site, which was sutured with 5-0 surgical silk.

Experimental Setup

Data were recorded from unrestrained animals kept within a water-perfused glass plethysmographic chamber (volume: 100 ml), attached to a programmable water bath/pump to maintain chamber temperature at 31°C, within the thermoneutral range for pups this age (28, 35). Air (21% O₂, balance N₂) from a gas cylinder or wall air was passed through a flowmeter before entering the chamber via a 20-G needle pushed through one of the rubber stoppers that seal the chamber. Chamber pressure was kept near atmospheric by pulling the gas from the opposite end of the chamber with a pump, also through a 20-G needle. Air flow through the chamber was held constant at ~250 ml/min. Analog signals from the respiratory pressure transducer were fed into a Powerlab data acquisition system (ADInstruments) and analyzed in LabChart V8 (ADInstruments).

Experimental Groups and Protocols

Group 1: Assessing the effects of acute muscarinic blockade on respiratory stability during active sleep.

All experiments were performed during the daytime (8 AM–5 PM), with one TPH2−/− and one TPH2+/+ pup tested each day. To control for any effect of time of day, we balanced the number of TPH2+/+ and TPH2−/− pups tested in the morning and afternoon. We used a total of 9 TPH2+/+ and 10 TPH2−/− pups for group 1 experiments. Animals were introduced to the plethysmographic chamber and allowed a settling period of ~30 min to establish periods of quiet sleep. After 60 min, animals would typically display periods of active sleep. At this time, we continually observed the animal and the EMG record to identify the transition from quiet to active sleep (Fig. 1). Approximately 60 s after the initiation of active sleep we administered an intravenous bolus of methylatropine (Ap-M), a form of atropine that is incapable of crossing the blood-brain barrier (17, 23). After ~1 h, and again ~60 s after the identification of a period of active sleep, pups were given an intravenous bolus of atropine sulfate (Ap-S), a form of atropine that has far more access to the CNS compared with Ap-M. Both drugs were administered as 10 μl bolus in saline; final dose: 1 mg/kg. We chose to delay each injection ~60 s after the appearance of active sleep to collect sufficient preinjection data for respiratory pattern analysis while at the same time minimizing the chances of the animal waking up (each episode is ~1.5–2.5 min long, on average). We chose to inject Ap-M first to control for and quantify any potential effect of altered heart rate on respiratory stability and because Ap-M has, at the very least, far less access to the CNS compared with Ap-S. Thus, any change in respiratory stability in response to Ap-S is very likely to be mediated centrally.

Group 2: Assessing the effects of muscarinic blockade on apnea index and sleep architecture.

This group of animals allowed us to analyze larger sections of data to determine effects of central muscarinic blockade on apnea indices accurately, as well as the incidence and duration of quiet and active sleep episodes. We treated 14 TPH2+/+ and 17 TPH2−/− with Ap-S, with another 13 TPH2+/+ and 16 TPH2−/− given vehicle alone (saline). Pups were introduced to the chamber and allowed to behave freely for the first hour to establish normal sleep cycling. Approximately 2 h after the start of the protocol, after arousal from an episode of active sleep, the pup was removed from the chamber and given an intraperitoneal injection of Ap-S (200-µl volume in saline; final dose 1 mg/kg) or vehicle alone. Pups were returned to the chamber and allowed to cycle for an additional 2 h through wakefulness, quiet sleep, and active sleep.

Data and Statistical Analysis

Group 1.

We measured the effect of Ap-M and Ap-S on the coefficient of variation of the respiratory period [CV-P (expressed as a % of the average period)] as an index of respiratory stability. Some pups immediately woke up during drug injection. Rather than completely exclude these animals from analysis, we decided to do two separate analyses. First, in both TPH2+/+ and TPH2−/− pups that did not arouse in response to the injection, we measured CV-P immediately before and after drug injection (i.e., within the same period of active sleep; TPH2+/+: n = 5 Ap-M and n = 4 Ap-S; TPH2−/−: n = 4 Ap-S and n = 7 Ap-M). For animals that woke up during or within a few seconds of the injection, we measured CV-P in the next active sleep period following arousal (usually only a few minutes later). For both TPH2+/+ and TPH2−/− animals, we used two-factor, repeated-measures analysis of variance within IBM SPSS software (Armonk, NY) to determine whether there were significant effects of Ap-M and Ap-S on CV-P [factor 1: drug; factor 2: time (i.e., pre- vs. postinjection)]. The data for TPH2+/+ pups were not normally distributed so data were rank-transformed before testing. When significant main effects were resolved, pairwise multiple comparisons were performed using Holm-Sidak post hoc analyses within IBM SPSS.

Group 2.

This group allowed us to assess the effects of Ap-S on apnea and severe apnea indices across several minutes of quiet and active sleep. We first measured the average respiratory period in the last two quiet and active sleep episodes before Ap-S injection and then measured them in the first two quiet and active sleep episodes after injection (~5 min of data before and after injection). Apnea was defined as any period greater than 150% of the average period that was not preceded by an augmented breath (invariably associated with arousal). Severe apnea was defined as any period greater than 200% of the average period. Periods were determined using the Cyclic Measurements function within LabChart (ADInstruments), with apneas identified by setting guidelines to the threshold for apnea described above. We determined apnea indices, respiratory frequency (f), tidal volume, heart rate (using the ECG record that was superimposed on the EMG record), and CV-P in the quiet and active sleep periods immediately before and after the injection of Ap-S. To determine whether Ap-S had any significant effects on the variables described above, we performed both a three-factor repeated-measures ANOVA within IBM SPSS, with genotype, drug (Ap-S or vehicle), and time (pre- and postinjection) as factors, as well as two-factor repeated-measures ANOVAs within each genotype (factor 1: drug; factor 2: time). When significant main effects were identified, we used Holm-Sidak post hoc analyses for pairwise comparisons.

The longer duration records obtained in group 2 animals also allowed us to determine quantitatively how muscarinic blockade affects sleep architecture and whether there were any interactive effects between muscarinic blockade and 5-HT deficiency. To this end, we performed a between-group analysis of the number and duration of quiet and active sleep episodes, as well as the proportion of time (%) spent in each state, during the full 2-h postinjection period. We chose this approach, rather than assessing the within-animal effect of the drug, because we observed that sleep architecture itself changed markedly from the first 2-h preinjection period to the last 2-h postinjection period (i.e., there was a large effect of time alone on sleep the frequency and duration of active sleep episodes).

RESULTS

Central, but Not Peripheral, Muscarinic Blockade Stabilizes the Respiratory Pattern of TPH2−/− Rats

Group 1 experiments involved the intravenous injection of Ap-M, which is mostly inaccessible to the CNS, followed by the injection of Ap-S, which has substantially more access. Both injections were performed while the animal was in an episode of active sleep. In TPH2−/− pups, the influence of Ap-S on the respiratory period variability was qualitatively distinct from that of Ap-M [compare Fig. 2A (Ap-M) with Fig. 2B (Ap-S)]. Ap-M had essentially no significant effect on the period variability displayed by TPH2−/− pups during active sleep; if anything, Ap-M destabilized the respiratory pattern of these animals (Fig. 2A). In contrast, the respiratory period of TPH2−/− pups were immediately stabilized following intravenous injection of Ap-S (Fig. 2B). The effects of Ap-M and Ap-S on period variability are summarized in Fig. 3, A and B. Data from animals that did not arouse in response to the injections are shown in Fig. 3A. Neither the injection of Ap-M nor Ap-S had any significant influence on the CV-P of TPH2+/+ pups (drug effect: P = 0.99; Fig. 3A). Similar to TPH2+/+, Ap-M had no significant effect on the CV-P of TPH2−/− pups (post hoc: P = 0.35). Unlike TPH2+/+, Ap-S significantly reduced the CV-P of TPH2−/− animals (P = 0.039) (genotype × drug interaction for TPH2−/−: P = 0.05).

Fig. 2.

Atropine sulfate (Ap-S) but not methylatropine (Ap-M) stabilizes the respiratory pattern of TPH2−/− pups during active sleep. A: heart rate (HR), respiratory period (P), and the raw respiratory trace [tidal volume (V_T)] of a tryptophan hydroxylase-deficient (TPH2−/−) rat pup before and after intravenous injection of Ap-M (injection at arrowhead and dashed line). Note the small increase in HR and reduced HR variability following Ap-M injection, with no qualitative effect on variability of P. B: P and V_T of a TPH2−/− rat pup before and after intravenous injection of Ap-S (injection at arrowhead and dashed line). Insets show close-ups of the respiratory pattern before and after injection. Note the stabilization of the respiratory pattern, reflected in reduced variability of P, following Ap-S injection. bpm, beats/min.

Fig. 3.

Central blockade of muscarinic receptors stabilizes the breathing pattern of tryptophan hydroxylase-deficient (TPH2−/−) pups during active sleep. A: coefficient of respiratory period variability [CV-P (%)] of wild-type (+/+) and TPH2-deficient (−/−) pups before and after injection of methylatropine (Ap-M) and atropine sulfate (Ap-S). These animals did not arouse in response to the injection (i.e., data are analyzed within the same period of active sleep); TPH2+/+: n = 5 Ap-M and n = 4 Ap-S; TPH2−/−: n = 4 Ap-S and n = 7 Ap-M. B: CV-P of TPH2+/+ (n = 10) and TPH2−/− (n = 9) pups before and after injection of Ap-M and Ap-S. Data are from all animals tested, irrespective of whether or not they aroused in response to the injection. For animals that aroused, data were obtained from the next active sleep episode following injection. C: Both Ap-M and Ap-S led to small changes in heart rate (ΔHR; beats/min) but there was no influence of genotype (n = 3 TPH2+/+ and n = 4 TPH2−/−) on the response. D: Neither Ap-M nor Ap-S had any significant influence on respiratory frequency (Δf; breaths/min) for TPH2+/+ (n = 3) or TPH2−/− (n = 4). *Significant difference between pre- and postinjection CV-P (P < 0.05).

Given the relatively small numbers of animals that did not arouse in response to injection, we also analyzed the effects of the drugs on stability in pups that did wake up and combined all data for analysis. For pups that woke up, we measured CV-P in the episode of active sleep before injection, comparing it with CV-P in the subsequent active sleep episode (which typically occurred within a few minutes of the injection). We combined data from animals that woke up with those that remained asleep and compared the pre- and postdrug CV-P. Again, in TPH2+/+ pups neither Ap-M nor Ap-S had any significant effects on the variability of the respiratory pattern (drug effect: P = 0.31; Fig. 3B, left). In contrast, in TPH2−/− pups the change in CV-P depended strongly on which drug was injected (drug × time: P = 0.002; Fig. 3B, right). Ap-M actually had a destabilizing effect on TPH2−/− breathing, increasing CV-P by 43% (post hoc: P = 0.048; Fig. 3B, right). On the other hand, Ap-S had a significant stabilizing effect on their breathing, decreasing CV-P by 25% (post hoc: P = 0.006; Fig. 3B, right). A few animals (n = 3 TPH2+/+ and n = 4 TPH2−/−) were instrumented with EMG electrodes, which also detected cardiac electrical activity thereby allowing us to determine heart rate responses to Ap-M and Ap-S. As we have found previously (24), heart rate changed minimally in response to either Ap-M or Ap-S—there was an ~30 beat/min (5–10% of resting heart rate) increase following Ap-M and ~10 beat/min increase following the subsequent injection of Ap-S (Fig. 3C). Finally, the stabilizing effect of Ap-S was unrelated to a change in respiratory rate, given that neither drug when injected intravenously in this time frame had any immediate effect on rate (Fig. 3D).

Muscarinic Blockade Reduces the Apnea Index of TPH2−/− Rat Pups

Group 2 experiments involved the intraperitoneal injection of Ap-S after multiple episodes of active and quiet sleep and then monitoring breathing and sleep architecture following the injection. We determined the apnea indices (no. of events/h) within the last two active and quiet sleep episodes immediately before injection and compared them with apnea indices during the first two active and quiet sleep episodes immediately after injection (usually within ~30 min of the injection). Compared with group 1 experiments, this approach generated more data that allowed us to determine more accurately the effects of Ap-S on apnea indices. We analyzed data from two episodes of active and quiet sleep (~5 min of each total) before and after Ap-S treatment because we were concerned about the influence of time-dependent factors on breathing, as well as the uncertainty with respect to the biological half-life of the drug within the CNS. As we found in a previous study, 2-wk-old TPH2−/− rats had a large number of apneas during active sleep (35), significantly more than TPH2+/+ littermates (P = 0.002; Fig. 4A, left). TPH2−/− tended to have more severe apneic episodes as well (P = 0.06; Fig. 4A). There were very few apneas during quiet sleep, with no significant effect of 5-HT deficiency on their incidence (Fig. 4A, right).

Fig. 4.

Tryptophan hydroxylase-deficient (TPH2−/−) pups have more apneas than TPH2+/+ littermates. A: boxplots show apnea and severe apnea indices (no. events/hour) for TPH2+/+ (open boxes; n = 28 from drug and vehicle experiments) and TPH2−/− pups (gray boxes; n = 33 total) in active (left) and quiet sleep (right). For these and box plots in other figures, plots show the median (horizontal line within the box), as well as the 25th and 75th percentiles (top and bottom of the box) and the 5th and 95th percentiles (closed circles above and below the box). Note that in active sleep, TPH2−/− pups have significantly more apneas than TPH2+/+. Very few apneas occur for either genotype during quiet sleep. *Significant difference between TPH2+/+ and TPH2−/− (P < 0.05). B: raw data from a TPH2−/− pup showing respiratory period (P) and raw respiratory volume (V_T) before (left) and after (right) atropine sulfate (Ap-S) treatment. Note the decrease in apneas (indicated by the arrowheads) following Ap-S treatment. C: box plots showing apnea index of TPH2−/− and TPH2+/+ pups in active sleep (left) and quiet sleep (right), before and after Ap-S treatment (n = 17 TPH2−/−; n = 15 TPH2+/+) or vehicle alone (n = 16 TPH2−/−; n = 13 TPH2+/+). †Note that Ap-S has a significant effect on the apnea index of TPH2−/− pups, with no significant effect on TPH2+/+. *Significantly increased apnea index of TPH2−/− compared with TPH2+/+ pups was eliminated following Ap-S treatment but not saline.

Mirroring its effect in group 1 experiments, Ap-S had a stabilizing effect on the respiratory pattern of TPH2−/− pups (Fig. 4B). Overall, considering data from both genotypes, Ap-S treatment significantly reduced apnea indices compared with the effect of vehicle alone (3FRMA, drug × time: P = 0.036; Fig. 4C, left). Ap-S (but not saline) significantly reduced the apnea index of TPH2−/− pups (from ~126 to 70; 2FRMA, drug × time: P = 0.04; post hoc comparing pre- and post-Ap-S: P = 0.01; Fig. 4C, left). In contrast, the effect of Ap-S on the apnea index of TPH2+/+ pups was smaller and not statistically different than the effect of vehicle alone (2FRMA, drug × time: P = 0.58; Fig. 4C, left). Thus, Ap-S eliminated the difference in apnea index that existed between the genotypes before treatment (Fig. 4C, left; note absence of genotype effect post-Ap-S). This suggests that Ap-S had a stronger effect on the apnea index of TPH2−/− pups compared with TPH2+/+. Muscarinic blockade has no influence on the apnea indices of either TPH2+/+ or TPH2−/− during quiet sleep, which for both genotypes was very low (Fig. 4C, right). There were no significant effects of Ap-S on the severe apnea indices of either TPH2+/+ or TPH2−/− pups (not shown).

In both active (Fig. 5A) and quiet sleep (Fig. 5B), TPH2−/− had a lower respiratory f compared with TPH2+/+. Ap-S significantly increased the f of TPH2+/+ and TPH2−/− pups, to a greater degree than vehicle alone (drug × time: P = 0.001). Ap-S increased f in both active (~20 breaths/min for both genotypes; P = 0.001 compared with saline; Fig. 5, A and C) and quiet sleep (~32 breaths/min for TPH2−/− and 18 breaths/min for TPH2+/+; P = 0.002 compared with saline; Fig. 5B). It was plausible that the stimulatory effect of Ap-S on f was a major factor in the decrease in apnea index experienced by TPH2−/− pups during active sleep. However, regression analysis revealed that for TPH2−/− pups, there was only a weak association between the decrease in apnea index and the increase in f (R² = 0.28; P = 0.02) and absolutely no association between the two variables in TPH2+/+ pups (Fig. 5D). Thus, it appears that the decrease in apnea index displayed by TPH2−/− pups following Ap-S was not solely because of an increase in f but also an independent mechanism. Finally, Ap-S had no influence on tidal volume or overall ventilation (not shown).

Fig. 5.

Atropine sulfate (Ap-S) increases respiratory frequency in both genotypes. Shown are respiratory frequency (f) in active (A) and quiet sleep (B) for tryptophan hydroxylase 2 (TPH2)+/+ (open boxes) and TPH2−/− littermates (dark gray boxes), before and after intraperitoneal injection of Ap-S. Note the higher f in TPH2+/+ pups compared with TPH2−/− (geno: P < 0.001). In both states, Ap-S injection increased f in both genotypes, to a greater degree than vehicle alone; drug × time: †P < 0.01. Summary of the change in f during active sleep for both genotypes following Ap-S or saline (Sal) alone; treatment: †P < 0.05 (C). In TPH2−/− pups there was small, yet statistically significant relationship between the change in apnea index (AI) and the change in f (D).

As expected, in both active and quiet sleep TPH2−/− had a significantly lower heart rate than TPH2+/+. Similar to group 1 animals, both TPH2+/+ and TPH2−/− pups experienced a small increase in heart rate following Ap-S injection (~25–30 beats/min; Fig. 6, A–C). However, statistically this increase was no different than that induced by saline alone. For both genotypes there was no significant relationship between the increase in heart rate and the change in apnea index (Fig. 6D).

Fig. 6.

Atropine sulfate (Ap-S) has no significant effect on heart rate. Shown are heart rate (HR; beats/min) in active sleep (A) and quiet sleep (B) for tryptophan hydroxylase (TPH2)+/+ (open boxes) and TPH2−/− littermates (dark gray boxes), before and after intraperitoneal injection of Ap-S. HR is higher in TPH2+/+ pups compared with TPH2−/− (geno: P < 0.001). Note that although the HR of both genotypes increased following Ap-S, the increase was not significantly different from the rise in HR following saline alone; time: ^P < 0.001. Summary of the change in HR following Ap-S and saline (Sal) during active sleep (C). For both genotypes the Ap-S-induced change in apnea index during active sleep was not significantly related to the change in HR (D).

Muscarinic Blockade Reduces the Amount of Active Sleep in Both TPH2+/+ and TPH2−/− Pups

We previously showed that TPH2−/− pups had fewer episodes of active sleep but because each episode was prolonged compared with TPH2+/+, the overall proportion of time spent in active sleep was no different than TPH2+/+ (35). With the present study we have partially confirmed this finding. TPH2−/− pups given saline alone had ~33% fewer episodes of active sleep compared with TPH2+/+, but each episode was ~73% longer (Fig. 7). For both genotypes, Ap-S treatment significantly reduced the number of episodes of active sleep and increased the number of episodes of quiet sleep (drug × state: P < 0.001). Ap-S had a stronger influence on TPH2+/+ compared with TPH2−/− pups; Ap-S eliminated the difference between the genotypes with regard to the number of active sleep episodes they experience (genotype × drug × state: P = 0.037; Fig. 7A, left). Ap-S had no effect on the duration of active sleep episodes; TPH2−/− pups still displayed longer active sleep episodes even after treatment (genotype × state: P < 0.001; Fig. 7B). Considering both quiet and active sleep combined, TPH2−/− pups spent more time asleep than TPH2+/+ (overall genotype: P < 0.001; Fig. 7C). For both genotypes, Ap-S treatment led to an increase in the proportion of the time spent in quiet sleep, reducing the amount of active sleep (drug × state: P < 0.001; Fig. 7C). To summarize, TPH2−/− pups had fewer but longer episodes of active sleep compared with TPH2+/+ littermates and spent more time asleep compared with TPH2+/+. For both genotypes, muscarinic blockade reduced the number of active sleep episodes—this eliminated the effect of 5-HT deficiency on the frequency of active sleep and reduced the overall amount of active sleep. However, the longer active sleep episodes displayed by TPH2−/− animals persisted even after muscarinic blockade.

Fig. 7.

Atropine sulfate (Ap-S) normalizes the frequency of active sleep (AS) episodes displayed by TPH2−/− pups but not their duration. Shown are the number of sleep episodes (A), duration (B), and % total time for AS (left) and quiet sleep (QS; right) experienced by tryptophan hydroxylase (TPH2)+/+ (open bars) and TPH2−/− pups (dark gray bars), in the 2 h following Ap-S or saline (Sal). Note that for both genotypes, Ap-S reduced both the number of active sleep episodes and the proportion of time spent in active sleep. The effect on episode frequency was stronger in TPH2+/+ animals compared with TPH2−/−. Ap-S had no influence on the duration of active sleep episodes. *Genotype effect: P < 0.05; †drug × state interaction: P < 0.05; ††genotype × drug × state: P = 0.037.

DISCUSSION

Based on the observations that 5-HT-deficient TPH2−/− pups have increased apnea exclusively in active sleep as well as prolonged episodes of active sleep (35), the purpose of this study was to address the hypothesis that enhanced cholinergic drive contributes to their increased apnea and respiratory instability. Our data support this hypothesis in that the apneas and unstable breathing of 2-wk-old TPH2−/− rat pups were eliminated following central muscarinic blockade (i.e., via Ap-S), whereas peripheral blockade alone (i.e., via Ap-M) was without effect. We showed that Ap-S (which can permeate the CNS) selectively reduced the variability of the respiratory rhythm as well as the apnea index of pups deficient in central 5-HT; following Ap-S the apnea index of TPH2−/− pups was statistically the same as TPH2+/+ controls. These data suggest that CNS 5-HT functions as a negative regulator of the cholinergic system; a loss of 5-HT leads to abnormally high cholinergic drive within respiratory circuits, leading to destabilized breathing.

Differential Effects of Central and Peripheral Cholinergic Drive on the Respiratory Pattern of 5-HT-Deficient Rat Pups

For group 1 experiments, our strategy involved injecting TPH2+/+ and TPH2−/− animals first with Ap-M and then Ap-S to assess, and to control for, any potential effects of peripheral (i.e., cardiac) muscarinic blockade on the respiratory pattern, before central muscarinic blockade. We could not apply the blockers in the reverse order as Ap-S does not discriminate between central and peripheral muscarinic receptors, because of its ability to cross the blood-brain barrier and because atropine has a relatively long biological half-life (1). Although Ap-M had no significant effects on the CV-P of TPH2+/+ pups, it led to a marked destabilization of the breathing of TPH2−/− pups (CV-P increased by 43%). Thus, the subsequent stabilizing influence of Ap-S on the breathing pattern of TPH2−/− occurred despite a destabilizing influence of peripheral muscarinic blockade. In other words, our data likely underestimate the stabilizing effect of central 5-HT on the respiratory pattern through a dampening of central cholinergic drive. The effect of Ap-M and Ap-S on heart rate was small (<10%), and as we found no correlation between it and the change in apnea index, it is unlikely that the stabilizing effect of Ap-S (or the destabilizing effect of Ap-M) has anything to do with the change in heart rate. We do note that heart rate variability was decreased following Ap-M (see Fig. 1A and Fig. 2A), which could influence respiratory stability through an interaction between cardiovascular or autonomic nuclei and respiratory patterning neurons.

Regulation of Cholinergic Drive to Respiratory Neurons by 5-HT: Potential Sites and Mechanisms of Action

Given that Ap-S normalized the unstable breathing of TPH2−/− pups, our interpretation is that central 5-HT stabilizes the respiratory pattern during active sleep by decreasing cholinergic drive through central muscarinic receptors, potentially those expressed within patterning nuclei. At which site(s) is 5-HT acting to regulate cholinergic drive negatively? There are numerous possibilities, given the widespread nature of serotonergic projections within the brainstem. The inhibitory serotonergic drive to the cholinergic pontine LDT may function not only to terminate active/REM sleep episodes but also to reduce cholinergic drive to respiratory patterning regions. The basis for this idea was not only the prolonged episodes of active sleep displayed by TPH2−/− rats but also previous research showing an inhibitory effect of 5-HT_1A agonists on cholinergic LDT neurons (34). However, other cholinergic respiratory neurons also modulate the respiratory rhythm, including the recently described preinspiratory complex, a cholinergic nucleus that projects to multiple regions involved in respiratory patterning (4).

With respect to the target of enhanced muscarinic drive in TPH2−/− pups, a region of particular interest is the parabrachial/Kolliker Fuse nucleus (PB/KF). It has been known for some time that the PB/KF region (otherwise known as the pontine respiratory group or historically the “pneumotaxic center”) has powerful effects on the timing of respiratory phase switching. Decades ago it was shown that electrical stimulation of this region halted inspiration and promoted exhalation (11). But the PB/KF does not function exclusively in this manner, as both electrical stimulation and the application of glutamate to other regions of the PB/KF can actually terminate expiration and promote inspiration (9, 11). That said, there is now ample evidence that either increased excitatory neurotransmission (9, 15) or decreased inhibitory neurotransmission (2) specifically to the KF can result in increased apneas and an unstable breathing pattern. With regard to the role of 5-HT, it was recently demonstrated that signaling through inhibitory 5-HT_1A receptors in the KF regulates respiratory rhythm variability and reduces apnea frequency (15). It is possible that a lack of 5-HT_1A signaling in the PB/KF could contribute to the apneas displayed by TPH2−/− rat pups. But given that their respiratory phenotype emerges exclusively in active sleep, it seems likely that other factors associated with active sleep also make an important contribution. Our data suggest that aberrant cholinergic drive is one such factor.

Acetylcholine has variable and sometimes conflicting effects on breathing because of the array of nicotinic and muscarinic receptors and intracellular signaling pathways subsequently activated (7, 32). Even when considering muscarinic signaling alone, the ultimate effect of ACh on respiratory neurons depends on the specific receptor subtype involved. There are five known muscarinic receptor (mAChR) subtypes expressed within the brainstem (M1–M5). M1, M3, and M5 receptors are coupled to G_q/11 and increase neuronal excitability, whereas M2 and M4 mAChRs are coupled to G_i/o and reduce neuronal excitability. As atropine is a nonselective mAChR antagonist, our experimental approach is unable to provide any resolution with respect to which muscarinic pathway is responsible for the apnea and respiratory instability displayed by TPH2−/− pups. It is possible that the stabilizing effect of Ap-S on the breathing of TPH2−/− pups was due to muscarinic blockade within the pre-Bӧtzinger Complex. Indeed, all five muscarinic receptors are expressed in the pre-Bӧtzinger Complex. Our data indicate that blockade of all muscarinic receptors leads to an increase in respiratory frequency, an effect that is not altered by a loss of serotonergic signaling. An increase in frequency was also observed in goats following the direct application of atropine to the pre-Bӧtzinger Complex, in both wakefulness and NREM sleep (29). Taken together these data suggest that, in sum, cholinergic drive through muscarinic receptors at the level of the pre-Bӧtzinger Complex is actually inhibitory to respiratory rhythm generation. Although it may be that the reduced apnea following muscarinic blockade was due simply to increased respiratory frequency, we think this is unlikely. First, in group 1 animals there was an immediate stabilizing effect of Ap-S, without a concurrent increase in frequency. Second, in group 2 animals, we found only a weak correlation between the increase in frequency and the reduction in apnea index. Thus, it appears acetylcholine has effects on respiratory rhythm that are independent from its effects on respiratory pattern and which are not influenced by a loss of central 5-HT.

Along with apneas, 5-HT-deficient rat pups also display increased tachypneic episodes compared with wild-type littermates (35). However, although we did not systematically analyze their appearance in relation to apneas, broadly there was not strong association between the two events; i.e., apneas did not consistently follow tachypneas, as occurs in Cheyne-Stokes breathing, for example. Thus, we do not attribute the apneas to altered chemoreceptor and/or loop gain. We note that in the early postnatal period, 5-HT has little influence on the hypoxic and hypercapnic ventilatory responses (13, 35), suggesting 5-HT has little influence on chemoreceptor gain.

Here, and in our previous study, we show that despite having longer episodes of active sleep, TPH2−/− pups actually have fewer of them. Cholinergic mechanisms are not only responsible for the maintenance of active/REM sleep episodes but also their initiation. With this in mind, given that they have fewer active sleep episodes, it may be that TPH2−/− pups have reduced, not increased, acetylcholine release within the pontine tegmentum compared with wild-type littermates. Indeed, here we show that in TPH2+/+ animals there is a larger decrease in the number of active sleep episodes following Ap-S treatment compared with TPH2−/− pups; i.e., Ap-S treatment eliminates the effect of genotype on the frequency of active sleep. Moreover, atropine had no significant effect on the duration of active sleep episodes in TPH2−/− pups, suggesting that increased cholinergic drive probably does not contribute to this phenotype. With these observations and the variable influence of the PB/KF region on respiratory patterning in mind (9), it may be possible that the apnea and respiratory instability displayed by TPH2−/− pups is due to reduced, rather than increased, cholinergic drive.

Potential Role of Interdependence of Neuromodulators in the Effect of Atropine

Our interpretation of these data is also based on the assumption that the blockade of muscarinic receptors was without secondary effects on other neuromodulatory systems. However, there is accumulating evidence that this assumption may be flawed. Blockade of muscarinic receptors not only increases the release of 5-HT but also Substance P, an excitatory neuromodulator with potent effects on the control of breathing, as well as glycine and γ-aminobutyric acid (GABA) (16, 29). Thus, the decrease in the apnea index of TPH2−/− pups may be a secondary effect of one or more of these factors. Substance P, for example, is a coreleased neuromodulator synthesized by serotonergic neurons whose receptor (neurokinin-1 receptor) behaves like 5-HT_2A receptors in that it activates G_q/11 and typically has stimulatory effects on breathing (30, 31). This could also explain the increase in respiratory frequency that was experienced by both genotypes following Ap-S treatment.

Conclusions

Given that animals lacking central 5-HT have increased apnea during prolonged episodes of active sleep, we designed this study to test the hypothesis that serotonin acts to stabilize breathing in active sleep by inhibiting central cholinergic drive. Our findings support this hypothesis in that only Ap-S, a form of atropine that can cross the blood-brain barrier, stabilized the respiratory pattern of TPH2−/− pups, whereas Ap-M, a form of atropine that does not have access to the CNS, actually destabilized their breathing. Ap-S treatment also led to a marked reduction in the apnea frequency of TPH2−/− pups. Our data bring to light a previously unknown interaction between the 5-HT and cholinergic systems in the control of breathing in early life. These findings are relevant to Sudden Infant Death Syndrome, which is associated with apnea and sleep abnormalities, as well as evidence of 5-HT and cholinergic system dysfunction.

GRANTS

Funding for this work was provided by an American Heart Association Scientist Development Grant (grant no. 14SDG18560022, to K. Cummings), an NIH R01 Grant (grant no. 1R01-HL136710-01A1, to K. Cummings), and an NIH F31 Predoctoral Fellowship (grant no. 1F31-HL136067-01, to J. Magnusson).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

K.J.C. conceived and designed research; M.R.D., J.L.M., and K.J.C. performed experiments; M.R.D. and K.J.C. analyzed data; M.R.D. and K.J.C. interpreted results of experiments; K.J.C. prepared figures; K.J.C. drafted manuscript; M.R.D., J.L.M., and K.J.C. edited and revised manuscript; M.R.D., J.L.M., and K.J.C. approved final version of manuscript.