Sex- and age-based differences in the effect of central serotonin on arterial blood pressure regulation

Abstract

Medullary serotonin (5-hydroxytryptamine; 5-HT) neurons project to multiple autonomic nuclei in the central nervous system (CNS). Infant rats lacking 5-HT have low arterial blood pressure (ABP) in quiet sleep, but the role of 5-HT in ABP regulation across vigilance states in adults has not been studied. We hypothesized that in adults, CNS 5-HT deficiency leads to hypotension mainly in quiet wakefulness (QW) and non-rapid eye movement (NREM) sleep, when 5-HT neurons are active. We tested male and female tryptophan hydroxylase 2 knockout rats (TPH2−/−), specifically deficient in CNS 5-HT, and wild-type (TPH2+/+) controls at 2–3, 5–8, and 12–13 mo of age. Compared with TPH2+/+, mean arterial pressure of 5–8- and 12–13-mo-old (middle-aged) male TPH2−/− rats was significantly elevated (∼10 mmHg) in QW and rapid eye movement (REM) sleep. Middle-aged male TPH2−/− rats also had more frequent extreme hypertensive events during prolonged episodes of REM sleep. Female TPH2−/− had normal ABP. The low- and very-low-frequency components of systolic ABP variability were significantly higher in middle-aged male, but not female, TPH2−/− rats compared with in TPH2+/+ rats, suggesting elevated sympathetic vascular tone in male TPH2−/− rats. However, the hypertension of male TPH2−/− rats was not ameliorated by ganglionic blockade. Hearts and lungs of middle-aged male TPH2−/− rats were significantly heavier than those of TPH2+/+ rats. We show that a loss of CNS 5-HT leads to high ABP only in middle-aged males during wakefulness and REM sleep, possibly due to increased vascular tone. It should be investigated whether elevated ventricular afterload associated with CNS 5-HT deficiency initiates cardiac remodeling or alters pulmonary hemodynamics.

NEW & NOTEWORTHY The role of serotonin in arterial blood pressure (ABP) regulation across states of vigilance is unknown. We hypothesized that adult rats devoid of CNS serotonin (TPH2−/−) have low ABP in wakefulness and NREM sleep, when serotonin neurons are active. However, TPH2−/− rats experience higher ABP than TPH2+/+ rats in wakefulness and REM only, a phenotype present only in older males and not females. CNS serotonin may be critical for preventing high ABP in males with aging.

INTRODUCTION

Neurons within the caudal raphe nuclei that release serotonin (5-hydroxytryptamine; 5-HT) project to autonomic regions within the brainstem and spinal cord and can modulate sympathetic and parasympathetic nerve activity in response to a variety of physiological stressors (34). Although studies using 5-HT receptor agonists and antagonists have attempted to clarify the role of specific 5-HT signaling pathways in arterial blood pressure (ABP) regulation, the picture they paint is somewhat muddled. This is in no small part due to the wide array of 5-HT receptors expressed by presympathetic nuclei, some of which have opposing actions within intracellular signaling pathways (34). For example, the rostral ventrolateral medulla (RVLM), a nucleus key to the generation of sympathetic nerve activity, expresses both excitatory (e.g., 5-HT₂) and inhibitory (e.g., 5-HT_1A) 5-HT receptors. Predictably, microinjection of the 5-HT_1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin, into the RVLM reduces ABP (23). Conversely, microinjection of 2,5-dimethoxy-4-iodoamphetamine (DOI), a specific 5-HT_2A receptor agonist, increases ABP (21). 5-HT_2A receptors are also expressed within the nucleus of the solitary tract (NTS), and DOI microinjection into this region elicits bradycardia and hypotension, typical responses of baroreceptor activation (5, 28).

Although these earlier studies provide insight into how the activation of specific 5-HT receptors in discrete regions of the central nervous system (CNS) affects ABP regulation, they cannot resolve how a more global change in serotonergic activity within the CNS alters ABP. This is a relevant question because 5-HT concentrations in the CNS fluctuate in a diurnal and sleep-state-dependent manner and can be altered by diet and common pharmaceutical treatments (e.g., selective 5-HT reuptake inhibitors). Findings from studies using freely behaving animals are conflicting; although pharmacological depletion of 5-HT increases the ABP of adult rats (15, 17), adult tryptophan-hydroxylase 2 knockout mice (TPH2−/−), specifically lacking 5-HT in the CNS (with no loss in the periphery), have reduced ABP (1). The reduced ABP of TPH2−/− mice is most evident in the evening (1), suggesting an interaction between 5-HT and factors related to state of vigilance or circadian pattern. Indeed, the activity of 5-HT neurons themselves depends on vigilance state, with maximal firing during wakefulness, minimal or absent activity in rapid eye movement (REM) sleep, and an intermediate level of activity in non-rapid eye movement (NREM) sleep (22). We have recently shown that compared with wild-type (TPH2+/+) controls, infant TPH2−/− rats have reduced ABP during quiet sleep (similar to NREM sleep in adults) but not during active sleep (or REM sleep in adults) (20). The lower ABP of infant TPH2−/− rats was ameliorated by systemic phentolamine treatment (20). Taken together, the aforementioned observations strongly suggest that in infant animals, 5-HT facilitates an increase in ABP by increasing sympathetic vascular tone.

The precise role of CNS 5-HT in ABP regulation across states of vigilance in adult animals has not been studied and is our focus here. Based on our findings from neonatal TPH2−/− rats (20) and others’ findings from adult TPH2−/− mice (1), we hypothesized that adult TPH2−/− rats would have reduced ABP compared with TPH2+/+ controls due to reduced sympathetic vascular tone. We further hypothesized that these phenotypes would be most evident in wakefulness and NREM sleep when 5-HT neurons are active and less evident during REM sleep when they are quiescent. Our results do not support this hypothesis in that middle-aged (5–8 and 12–13 mo old) male TPH2−/− rats have increased ABP compared with TPH2+/+ rats. Moreover, the high ABP of middle-aged male TPH2−/− rats was evident in wakefulness and REM sleep, but not in NREM sleep, and was associated with significantly more frequent and intense ABP surges (i.e., extreme hypertensive events, EHEs) in REM sleep. Middle-aged male TPH2−/− rats had increased low- and very-low-frequency components of systolic blood pressure variability. Interestingly, significant sex-based differences were also observed, as the ABP of female TPH2−/− rats was not different from that of TPH2+/+ rats. The increase in ABP observed in male TPH2−/− rats was also associated with an increase in heart weight compared with controls, suggesting that chronic increases in left ventricular afterload in the absence of CNS 5-HT could initiate cardiac remodeling, potentially setting the stage for the subsequent development of more complicated cardiovascular disease.

METHODS

Ethical approval.

All animal experiments were approved by the University of Missouri Institutional Animal Care and Use Committee and performed in accordance with guidelines set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th edition, 2011).

Animals and treatments.

We used adult rats deficient in tryptophan hydroxylase 2 (TPH2−/−); TPH2 catalyzes the rate-limiting step in 5-HT biosynthesis specifically in the CNS; hence, TPH2−/− animals are nearly devoid of 5-HT within the CNS, with no loss in the peripheral tissues (16). Compared with TPH2+/+ littermates, TPH2−/− rats have the same levels of noradrenaline and dopamine in the CNS (16). Male and female TPH2−/− and wild-type (TPH2+/+) controls were tested at three ages: 2–3 mo, 5–8 mo, and 12–13 mo. There were no differences in body weight between male or female TPH2−/− and TPH2+/+ at 2–3 or 12–13 mo. At 5–8 mo of age, male TPH2−/− rats weighed slightly (∼10%) more than controls (male TPH2+/+: 331 g ± 6 g; male TPH2−/−: 360 g ± 7 g; P < 0.05), whereas there was no difference in the body weight of females. Animals had unrestricted access to water and standard rat chow and were kept on a 12-h light/dark cycle. Unless otherwise indicated, all rodents received the same surgical procedures, described as follows.

EEG and EMG surgery.

Rats were surgically implanted with electroencephalographic (EEG) and electromyographic (EMG) electrodes (Plastics One Inc., Roanoke, VA) to assess vigilance state. Sterile surgery was performed under isoflurane anesthesia; depth of anesthesia was assessed using hindlimb withdrawal. Rats were secured in a stereotaxic frame for implantation of EEG and EMG electrodes. Two EMG electrodes were implanted into the dorsal neck muscles and three EEG electrodes were screwed into the skull, located as follows: the first electrode at 2 mm rostral to bregma and 2 mm lateral to the midline, the second electrode at 3 mm caudal to bregma and 2 mm lateral to the midline ipsilateral to the first screw, and a ground electrode was placed contralateral, and at a rostral-caudal position equidistant to the other two screws. Following implantation, the EEG/EMG electrode wires were connected to a six-prong plastic pedestal (Plastics One Inc., Roanoke, VA). Dental acrylic (Lang Dental Manufacturing Co. Inc., Wheeling, IL) was used to secure the cap and electrodes to the skull. Following the surgery, animals were given Banamine (2.5 mg/kg; Zoetis Inc., Kalamazoo, MI) to prevent postoperative pain and Baytril (5–10 mg/kg; Bayer Healthcare, LLC; Shawnee Mission, KS) to help prevent bacterial infection. Animals were allowed a minimum of a 7-day recovery from surgery before testing. At least once during the week before testing, animals were acclimated to the recording chamber for 1–2 h.

Assessment of sleep-wake states.

Vigilance states were determined by EEG/EMG signal as well as visual inspection of behavior during the study. Vigilance states were classified as active wakefulness (not included in analysis), quiet wakefulness (QW), NREM sleep, and REM sleep, based on previously described criteria (3). QW was identified by low-voltage EEG with relatively high-voltage EMG. NREM sleep was identified by appearance of high-voltage spikes in the EEG signal suggestive of high delta activity, along with relatively reduced voltage in the EMG signal compared with QW. REM sleep was identified by low-voltage EEG accompanied by little to no EMG activity (Fig. 1). Behavioral scoring was performed continuously over the course of the 4-h recording using both EEG and EMG records as well as behavioral observation. REM sleep was confirmed visually with characteristic myoclonic twitching of the distal muscles. Cardiorespiratory variables were recorded during each state.

Fig. 1.

Measuring cardiorespiratory variables across different states of vigilance. Shown are records of the electromyograph (EMG), the electroencephalograph (EEG), mean arterial pressure (MAP), heart rate (HR), and respiration (tidal volume, V_T) for an adult rat during a transition from non-rapid eye movement (NREM) to rapid eye movement (REM) sleep. NREM is characterized by relatively high EMG amplitude compared with REM, as well as high-amplitude spikes in the EEG signal suggesting high delta power. Transitions to REM were identified by a decrease in both EMG and EEG amplitude. Episodes of REM sleep were confirmed by observing characteristic behavior features of REM, including myoclonic twitching of the face and whiskers.

Femoral artery/venous catheterization.

Two days before testing, all adult animals were implanted with a femoral arterial catheter for the measurement of ABP, as we have described in a previous publication (20). Group 2 animals (see Experimental groups and protocols for cardiorespiratory measurements in unanesthetized animals for details) were also implanted with a femoral venous catheter for injection of drugs. Briefly, rats were anesthetized under 2% isoflurane and a ∼1-cm skin incision was made in the left groin. A ×20 dissecting microscope was used to dissect out the left femoral artery. The artery was then tied with 5-0 surgical suture just distal to the epigastric branch. An incision was made on the left femoral artery for insertion of the PE10 portion of the custom-made catheter of PE10 fused to PE50 tubing (BD, Sparks, MD). Similar procedures were used for insertion of the femoral venous catheter. Catheters were secured in place and tunneled through the back with a small portion exteriorized between the shoulder blades to allow easy access for flushing daily with sterile heparinized 0.9% saline (100 μL/mL) until testing and euthanasia. Lidocaine was applied to the surgical sites postsurgery, and both incision sites were sutured with sterile 5-0 surgical silk.

Whole body plethysmography.

We used whole body plethysmography to assess respiratory variables of interest during wakefulness and sleep, similar to previous studies in our laboratory (20, 37). Animals were provided air from the wall, passed through a flowmeter before entering the chamber (flow = 1 L/min). Chamber pressure was maintained close to atmospheric by pulling air from the opposite end of the chamber by a wall vacuum. Changes in chamber pressure related to breathing were measured using a differential pressure transducer (Validyne), connected to the animal chamber on one side and an empty reference chamber on the other. The reference chamber was used to account for thermal drift and was connected to the test chamber via a ∼10 cm length of small-diameter tubing. Analog signals from transducers for both arterial pressure and respiration were fed into a Powerlab data acquisition system (ADInstruments) and analyzed in LabChart 7.3.7 (ADInstruments).

Experimental groups and protocols for cardiorespiratory measurements in unanesthetized animals.

Experiments were performed during the “light period” (8 AM to 5 PM) on two groups of animals. For both groups, animals were allowed 4 h to recover from light isoflurane anesthesia, administered to facilitate the setup of a catheter tube extension and an EEG/EMG guidewire. Group 1 animals were used to assess resting cardiorespiratory variables across QW, NREM, and REM sleep: 2–3 mo TPH2−/− rats (n = 8 males, 6 females) and TPH2+/+ controls (n = 9 males, 9 females); 5–8 mo TPH2−/− rats (n = 9 males, 15 females) and TPH2+/+ controls (n = 12 males, 12 females); and 12–13 mo TPH2−/− rats (n = 6 males, 4 females) and TPH2+/+ controls (n = 9 males, 7 females). After a 4-h recovery and acclimatization period, variables were collected for an additional 4 h (total time in chamber: ∼8 h). Animals had access to a small dish of moistened rat chow throughout the course of the experiment.

We also assessed the effects of central 5-HT deficiency on baseline parasympathetic and sympathetic tone to the heart and vasculature. For males, this required a second set of 5–8-mo-old TPH2−/− and TPH2+/+ rats (group 2; n = 11 of each). Resting variables were obtained from female rats following experiments on males, and we were able to assess parasympathetic and sympathetic in these same animals following the recording of resting cardiorespiratory variables. Females and group 2 males were injected with atropine methyl nitrate (1 mg/kg, iv, Sigma Chemical), followed by the ganglionic blocker hexamethonium (30 mg/kg, iv, Sigma Chemical). The acute heart rate (HR) response to atropine and hexamethonium was used to quantify the parasympathetic and sympathetic drive to the heart, respectively. The ABP response to hexamethonium was used to quantify the sympathetic drive to the vasculature. For analyses of baseline variables in males, the baseline data from groups 1 and 2 were combined (total n = 23 male TPH2+/+ and 20 male TPH2−/−).

Transthoracic echocardiography and cardiopulmonary morphological analyses.

Transthoracic echocardiography was performed with a 12-MHz pediatric transducer using a GE Vivid I Ultrasound system to assess in vivo heart rate, stroke volume, and cardiac output under 0.5%–1.0% inhaled isoflurane anesthesia, as previously described (9, 10). We assessed cardiac variables in 8 male TPH2+/+ and 8 male TPH2−/− rats in the 5–8 mo cohort and 8 TPH2+/+ and 12 TPH2−/− rats in the 12–13 mo cohort. Cardiac (5–8- and 12–13-mo-old rats) morphology and lung (12–13-mo-old rats) morphology were determined using postmortem weight. Tibia length was measured to assess phenotypical differences in normal age-related growth between TPH2+/+ and TPH2−/− animals.

Data and statistical analysis.

For all animals, we determined mean arterial pressure (MAP, mmHg), systolic arterial blood pressure (sBP, mmHg), diastolic arterial blood pressure (dBP, mmHg), heart rate (HR, beats/min), respiratory frequency (f; breaths/min), tidal volume (V_T; mL/kg), and ventilation [V̇e (f × V_T); mL/min/kg]. In a subset of animals, we measured metabolic rate (V̇o₂, mL/min/kg) and the metabolic equivalent (V̇e/V̇o₂). Raw respiratory and ABP data were collected at a sampling rate of 1 kHz.

Using the Spectrum function within LabChart 7 (Hahn window with a Fast Fourier Transfer size of 512), we conducted spectral analysis of the low-frequency (LF) and very-low-frequency (vLF) components of sBP variability. The vLF component of sBP variability likely originates from sympathetic nervous system activity or changes in vascular myogenic activity related to humoral factors or local regulation; for example, epinephrine, renin-angiotensin system, or endothelial-derived nitric oxide. The LF component originates mostly from sympathetic nerve activity (18, 26, 27). We chose not to analyze the high-frequency component of sBP variability because its origin is unclear. The power of the very low (vLF, 0–0.2 Hz, mmHg²) and low (LF, 0.2–0.75 Hz, mmHg²) components of systolic blood pressure variability was quantified continually within LabChart 7 across the course of the 4-h experiment, spanning all episodes of QW, NREM sleep, and REM sleep (see Table 3 for total time in each state). In each animal, an average power for vLF and LF was calculated for each state, and for each genotype, we present averages of these individual averages. We acknowledge that the analysis of these components of sBP variability provides an indirect measure of sympathetic vascular tone; regardless, it at least partially overcomes the challenges related to recording sympathetic nerve activity destined solely for the vasculature.

Table 3.

Sleep architecture in male and female TPH2+/+ and TPH2−/− rats

			Male			Female
		Geno	QW	NREM	REM	QW	NREM	REM
2–3 mo	No. of episodes	+/+	42 ± 6	38 ± 5	13 ± 2	40 ± 3	37 ± 2	19 ± 1
		−/−	38 ± 2	28 ± 3	9 ± 1	40 ± 3	33 ± 3	9 ± 1*
	Duration, s	+/+	46 ± 6	170 ± 18	109 ± 16	43 ± 4	152 ± 8	99 ± 4
		−/−	55 ± 5	212 ± 22	218 ± 28*	62 ± 10	191 ± 27	234 ± 26*
	Total time, s	+/+	1,878 ± 285	5,759 ± 307	1,557 ± 236	1,667 ± 173	5,495 ± 188	1,856 ± 108
		−/−	2,106 ± 352	5,687 ± 397	1,877 ± 341	2,402 ± 269	5,992 ± 643	2,058 ± 197
	% time	+/+	20 ± 3	63 ± 2	17 ± 3	18 ± 2	62 ± 2	20 ± 1
		−/−	22 ± 4	59 ± 3	19 ± 3	23 ± 3	57 ± 4	20 ± 2
5–8 mo	No. of episodes	+/+	20 ± 2	31 ± 3	19 ± 2	22 ± 3	32 ± 4	20 ± 3
		−/−	19 ± 2	17 ± 2*	7 ± 3*	28 ± 3	24 ± 2	7 ± 1*
	Duration, s	+/+	86 ± 8	184 ± 11	94 ± 5	76 ± 7	177 ± 11	91 ± 7
		−/−	95 ± 8	405 ± 44*	268 ± 18*	85 ± 6	250 ± 19*	201 ± 11*
	Total time, s	+/+	1,635 ± 163	5,420 ± 332	1,707 ± 134	1,608 ± 229	5,297 ± 435	1,640 ± 181
		−/−	1,672 ± 137	5,923 ± 358	1,817 ± 146	2,210 ± 186	5,636 ± 377	1,380 ± 139
	% time	+/+	20 ± 2	61 ± 2	19 ± 1	20 ± 4	61 ± 3	19 ± 1
		−/−	19 ± 2	62 ± 2	19 ± 1	25 ± 3	60 ± 2	15 ± 1
12–13 mo	No. of episodes	+/+	27 ± 4	39 ± 4	26 ± 4	32 ± 9	51 ± 4	19 ± 5
		−/−	29 ± 11	23 ± 2*	8 ± 2*	34 ± 8	34 ± 3*	7 ± 2*
	Duration, s	+/+	52 ± 5	179 ± 17	102 ± 22	52 ± 4	113 ± 9	73 ± 3
		−/−	94 ± 15*	284 ± 42*	233 ± 39*	93 ± 12*	162 ± 19*	191 ± 38*
	Total time, s	+/+	1,430 ± 362	6,705 ± 355	2,447 ± 177	1,687 ± 409	5,565 ± 382	1,341 ± 160
		−/−	3,236 ± 1,051	6,255 ± 1,054	1,558 ± 263	3,317 ± 936	5,455 ± 362	1,445 ± 569
	% time	+/+	15 ± 3	63 ± 3	22 ± 2	20 ± 7	64 ± 4	16 ± 4
		−/−	27 ± 9	59 ± 7	15 ± 2	31 ± 8	54 ± 4	14 ± 5

Shown are number of episodes of non-rapid eye movement (NREM) sleep, quiet wakefulness (QW), and rapid eye movement (REM) sleep; sleep episode duration; total time; and % time in each state in male and female rats at the three ages. Animal numbers are the same as in Table 1. Data shown are averages ± SE

^*Significant differences (denoted by symbol and bold font; genotype: P < 0.05).

For each genotype, we also quantified the number and magnitude of ABP surges occurring during REM sleep (i.e., “extreme hypertensive events,” EHEs). EHEs were defined as any elevation in ABP that was at least 20 mmHg above the average ABP during NREM sleep for that animal. For both sexes and genotypes, we also analyzed the number of EHEs occurring over time within episodes of REM sleep.

Baseline cardiorespiratory variables for unanesthetized, 5–8-mo-old male rats from groups 1 and 2 were combined for analyses (TPH2+/+: n = 23; TPH2−/−: n = 20). Cardiorespiratory data from episodes of QW, NREM sleep, and REM sleep were analyzed if the episodes were at least 10 s in duration. For each animal, we present average data (±95% confidence intervals) across each of the three states. For males and females, significant effects of genotype and vigilance state on ABP, HR, sBP variability, respiratory, and sleep variables were determined using two-factor, repeated-measures ANOVA (2FRMA) in GraphPad Prism (GraphPad Software, San Diego, CA). We analyzed the number of EHEs occurring over time across episodes of REM in 5–8-mo-old male and female TPH2−/− and TPH2+/+ controls, dividing each episode into 30-s bins and counting the number of EHEs per bin. Significant effects of genotype and bin number on the number of EHEs were assessed using a negative binomial regression in IBM SPSS Statistics. Significant effects of genotype on heart weight, lung weight, stroke volume, heart rate, and cardiac output in anesthetized animals were determined using two-factor ANOVA in GraphPad Prism. Differences were considered statistically significant at P values ≤ 0.05.

RESULTS

CNS 5-HT deficiency leads to elevated ABP during wakefulness and REM sleep.

Here, we hypothesized that adult male and female TPH2−/− rats would have low ABP, similar to neonatal TPH2−/− rats and adult TPH2−/− mice (1, 20). We also hypothesized that the ABP of TPH2−/− rats would be affected mostly in QW and NREM sleep, while in TPH2+/+ animals, 5-HT neurons are firing and releasing 5-HT (22). As aging and sex have well-characterized effects on ABP, we tested both male and female TPH2−/− animals at three ages: 2–3, 5–8, and 12–13 mo.

At 2–3 mo of age, the MAP of male TPH2−/− rats was the same as that of TPH2+/+ littermates (Fig. 2A, left). At 5–8 mo of age, the MAP of male TPH2−/− rats was elevated compared with TPH2+/+ rats during QW and REM sleep but was not different during NREM sleep (genotype × state: P < 0.0001; post hoc QW: P = 0.006; REM sleep: P = 0.0001; NREM sleep: P = 0.19; Fig. 2B, left). Increased sBP (genotype: P = 0.0006) and dBP (genotype: P = 0.02) contributed to the elevated ABP of 5–8-mo-old male TPH2−/− rats. Male TPH2−/− rats also displayed higher MAP than TPH2+/+ littermates at 12–13 mo of age (genotype: P = 0.026; Fig. 2C, left). There was a tendency for this phenotype to be more prominent during QW and REM sleep (state × genotype: P = 0.10), similar to what we observed in the 5–8-mo-old cohort. Elevated dBP contributed significantly to the high MAP of 12–13-mo-old male TPH2−/− rats, and there was also a strong tendency for elevated sBP compared with that seen in TPH2+/+ rats (Table 1). Unlike male TPH2−/− rats, at all ages, the MAP of female TPH2−/− rats was not significantly different from that of TPH2+/+ rats (Fig. 2, A–C, right). In males, at all three ages, and irrespective of genotype, MAP was highest in REM sleep compared with the other states (P < 0.01 at 2–3 mo and P < 0.001 in middle-aged rats; Fig. 2). The MAP of female rats also showed a state dependency; at 2–3 mo of age, MAP of both TPH2+/+ and TPH2−/− rats was highest in REM sleep (P < 0.01, Fig. 2). In middle-aged females, the MAP of TPH2−/− rats was highest in REM sleep, whereas in TPH2+/+ rats, there was no influence of state. At 5–8 mo, the MAP of both male and female TPH2−/− rats was lowest in NREM sleep, a phenotype absent in TPH2+/+ rats (P < 0.001 in males, and P < 0.05 in females; Fig. 2B).

Fig. 2.

Arterial blood pressure is elevated in middle-aged male, but not female, TPH2−/− rats. Shown are for mean arterial pressure (MAP) in male (left) and female (right) TPH2−/− rats (filled circles) and TPH2+/+ littermates (open circles) at 2–3 mo (A, male: n = 9 +/+ and 8 −/−; female: n = 9 +/+ and 6 −/−), 5–8 mo (B, male: n = 23 +/+ and 20 −/−; female: n = 12 +/+ and 15 −/−), and 12–13-mo-old (C, male: n = 9 +/+ and 6 −/−; female: n = 7 +/+ and 4 −/−). Two factor, repeated-measures ANOVA with post hoc analyses revealed significant effects of genotype (*P < 0.05; ***P < 0.001; ****P < 0.0001) and significant effects of state (#P < 0.05; ##P < 0.01; ###P < 0.001; ###P < 0.0001). For this and subsequent figures, data are expressed as means ± 95% confidence interval.

Table 1.

Systolic and diastolic arterial blood pressure data

			Male			Female
		Geno	QW	NREM	REM	QW	NREM	REM
2–3 mo	sBP	+/+	148 ± 5	148 ± 6	150 ± 6	142 ± 7	141 ± 7	143 ± 7
		−/−	154 ± 6	152 ± 7	158 ± 7	142 ± 9	139 ± 7	143 ± 7
	dBP	+/+	100 ± 7	101 ± 7	100 ± 7	92 ± 7	92 ± 7	95 ± 7
		−/−	104 ± 8	102 ± 9	108 ± 8	89 ± 6	86 ± 6	93 ± 5
5–8 mo	sBP	+/+	139 ± 2	138 ± 2	142 ± 1	136 ± 3	136 ± 3	138 ± 4
		−/−	149 ± 2*	146 ± 2*	153 ± 2*	140 ± 3	138 ± 3	144 ± 3
	dBP	+/+	94 ± 1	94 ± 1	96 ± 1	92 ± 3	92 ± 3	94 ± 4
		−/−	99 ± 2*	96 ± 2	104 ± 2*	99 ± 3	97 ± 3	103 ± 3
12–13 mo	sBP	+/+	147 ± 3	148 ± 3	153 ± 4	132 ± 5	133 ± 6	135 ± 7
		−/−	158 ± 5	156 ± 5	164 ± 5	130 ± 3	128 ± 3	133 ± 1
	dBP	+/+	95 ± 2	96 ± 2	100 ± 2	81 ± 7	82 ± 7	84 ± 8
		−/−	104 ± 2**	103 ± 2	110 ± 2**	78 ± 3	77 ± 3	79 ± 3

Data represent 2–3-mo-old TPH2−/− rats (n = 8 males, 6 females) and TPH2+/+ controls (n = 9 males, 9 females); 5–8-mo-old TPH2−/− rats (n = 20 males, 15 females) and TPH2+/+ controls (n = 23 males, 12 females); and 12–13-mo-old TPH2−/− rats (n = 6 males, 4 females) and TPH2+/+ controls (n = 9 males, 7 females) during non-rapid eye movement (NREM) sleep, quiet wakefulness (QW), and rapid eye movement sleep (REM). Data are means ± SE. dBP, diastolic arterial blood pressure; sBP, systolic arterial blood pressure. Genotype effect:

^*P < 0.05,

^**P < 0.01.

At all ages and in both sexes, heart rate was unaffected by a loss of CNS 5-HT (Table 2).

Table 2.

Heart rate of male and female TPH2+/+ and TPH2−/− rats during quiet wake, non-rapid eye movement sleep, and rapid eye movement sleep

		Male			Female
	Geno	QW	NREM	REM	QW	NREM	REM
2–3 mo	+/+	438 ± 18	433 ± 18	411 ± 8	459 ± 14	454 ± 15	443 ± 17
	−/−	433 ± 10	419 ± 11	431 ± 7	440 ± 7	422 ± 9	431 ± 9
5–8 mo	+/+	369 ± 4	350 ± 4	337 ± 4	428 ± 12	413 ± 12	394 ± 12
	−/−	367 ± 4	342 ± 5	351 ± 5	410 ± 6	392 ± 7	397 ± 9
12–13 mo	+/+	362 ± 11	350 ± 13	350 ± 13	450 ± 14	437 ± 17	422 ± 18
	−/−	373 ± 14	369 ± 13	392 ± 22	412 ± 15	399 ± 16	424 ± 11

Animal numbers are the same as in Table 1. NREM, non-rapid eye movement sleep, REM, rapid eye movement sleep; QW, quiet wakefulness. Data are means ± SE.

NS 5-HT deficiency leads to extreme hypertensive events during prolonged periods of REM sleep.

Blood pressure is normally erratic in REM sleep compared with QW and NREM sleep, displaying periodic surges that do not occur in the other states (29). In addition to having generally higher ABP in REM compared with TPH2+/+ rats, we wondered whether middle-aged male and female TPH2−/− rats might have more frequent or intense ABP surges during REM. We, therefore, analyzed both the number and overall impact of ABP surges (extreme hypertensive events, EHEs) in 5–8- and 12–13-mo-old male and female TPH2+/+ and TPH2−/− rats during REM sleep. We defined EHEs as events where MAP increased at least 20 mmHg more than the average MAP of each animal during NREM sleep (Fig. 3A). Indeed, both TPH2+/+ and TPH2−/− rats experienced EHEs throughout REM sleep. These episodes did not appear to be associated with arousals from sleep (Fig. 3B); in contrast, arousals from REM sleep were often associated with an abrupt decrease in MAP (Fig. 3C). We found that 5–8-mo-old male TPH2−/− rats had ∼23 more EHEs per hour of REM sleep (P = 0.03; Fig. 4A, left). By 12–13 mo of age, there was no longer any effect of genotype on EHE frequency; in TPH2+/+ rats, EHE frequency increased with age (post hoc: P = 0.025), whereas age had no effect in TPH2−/− rats (P = 0.99). In this way, EHE frequency in younger TPH2−/− rats was as high as in older TPH2+/+ rats. The magnitude of the EHEs was also greater with a loss of CNS 5-HT; at both 5–8 and 12–13 mo of age, the maximum MAP experienced by male TPH2−/− rats during EHEs was considerably elevated compared with the maximum experienced by TPH2+/+ rats (5–8 mo: P < 0.0001; 12–13 mo: P = 0.02; Fig. 4A, middle). We calculated the cumulative area under the MAP curve during all EHEs, i.e., total area under the curve (total AUC), to determine the effect of CNS 5-HT deficiency on the overall impact of EHEs on MAP in REM sleep. Total AUC was significantly higher in 5–8-mo-old male TPH2−/− rats compared with age-matched male TPH2+/+ rats (P = 0.038; Fig. 4A, right). In fact, the total AUC of younger TPH2−/− rats was the same as older TPH2+/+ rats. There was no effect of genotype at 12–13 mo of age (Fig. 3B, right).

Fig. 3.

Severe surges in arterial blood pressure occur during rapid eye movement (REM) sleep (extreme hypertensive events, EHEs). A: raw records of mean arterial pressure (MAP) and heart rate (HR) of a male TPH2+/+ rat and male TPH2−/− littermate during an episode or REM sleep. Note the presence of EHEs is indicated by the arrows. B: raw record of an EHE occurring at the beginning of a REM sleep episode following a non-rapid eye movement (NREM) episode. C: records from a different animal than in (B) show that there is no increase in EMG amplitude (i.e., no arousal) when the EHE occurs (indicated by arrowheads) and that MAP typically falls following arousal from REM (indicated by arrowhead and “a”).

Fig. 4.

Middle-aged male TPH2−/− rats have more frequent and severe surges in arterial blood pressure during rapid eye movement (REM) sleep. A, left: 5–8-mo-old male TPH2−/− rats (filled circles) had more frequent extreme hypertensive events (EHE) than TPH2+/+ rats (open circles). EHE frequency of 12–13-mo-old male TPH2+/+ rats was increased compared with 5–8-mo-old rats and was the same as 12–13-mo-old male TPH2−/− rats. A, middle: maximum MAP reached during EHEs was significantly greater in male TPH2−/− rats at both ages. A, right: sum of the area under the MAP curve (total AUC) was significantly greater in 5–8-mo-old male TPH2−/− rats compared with in 5–8-mo-old male TPH2+/+ rats. B, left: at both ages, female TPH2−/− rats had the same frequency of EHEs as female TPH2+/+ rats. B, middle: 5–8-mo-old female TPH2−/− rats experienced a significantly elevated maximum MAP during the events, but at both ages had the same total AUC as TPH2+/+ rats. B, right: 2-factor, repeated-measures ANOVA revealed significant effects of genotype or age. *P < 0.05; **P < 0.01; ****P < 0.0001. Animal numbers are the same as in Fig. 2.

Unlike male TPH2−/− rats, at both ages, female TPH2−/− rats had the same number of EHEs as female TPH2+/+ rats (5–8 mo: P = 0.53; 12–13 mo: P = 0.94; Fig. 4B, left). Like males, 5–8-mo-old female TPH2−/− rats experienced a significantly higher maximum MAP than female TPH2+/+ rats during the EHEs (P = 0.014; Fig. 4B, middle). Females were also somewhat protected in that their total AUC was not affected by a loss of CNS 5-HT (Fig. 4B, right). Taken together, our data suggest that CNS 5-HT mitigates the number and severity of EHEs occurring in REM sleep, an effect especially evident in males. It is also interesting that with a loss of CNS 5-HT, males prematurely age with regard to the frequency of the hypertensive events and their total impact on ABP in REM sleep.

The ABP of middle-aged male TPH2−/− rats was highest during REM sleep, in part because of the appearance of more frequent and intense EHEs in this state. To address the possibility that mechanisms involved in the initiation or maintenance of REM sleep also contribute to the increased EHEs of middle-aged male TPH2−/− rats, we first analyzed the number and duration of REM sleep episodes in both genotypes at all three ages. At all ages, and in both sexes, REM sleep episodes were approximately twice as long in 5-HT-deficient rats (Fig. 5, A and B; Table 3). Middle-aged TPH2−/− rats also had prolonged episodes of NREM sleep, but to a lesser degree compared with REM sleep (Fig. 5, A and B, middle and right; Table 3). Despite prolonged NREM episodes, the total time and overall proportion of time spent in NREM sleep were the same in middle-aged male and female TPH2−/− and TPH2+/+ rats, because TPH2−/− rats experienced significantly fewer sleep episodes (Table 3). There was a strong tendency for 12–13-mo-old TPH2−/− rats to spend less total time (state × genotype: P = 0.076) and a smaller proportion of time (state × genotype: P = 0.056) in REM sleep (Table 3).

Fig. 5.

Middle-aged male TPH2−/− rats have extreme hypertensive events (EHEs) during longer episodes of rapid eye movement (REM) sleep. Shown is the average duration of episodes or quiet wakefulness (QW), non-rapid eye movement (NREM) sleep, and REM sleep for (A) male and (B) female TPH2+/+ rats (open circles) and TPH2−/− littermates (closed circles) at 2–3 (left), 5–8 (middle), and 12–13 mo of age (right). C: number of EHEs over time (30-s time bins) in REM sleep episodes in 5–8-mo-old male and female TPH2−/− rats and TPH2+/+ littermates. Note the male TPH2−/− rats have more EHEs than TPH2+/+ rats as the REM episodes progressed with time. Two-factor, repeated-measures ANOVA revealed a significant interaction between genotype and time (†P < 0.05), and a post hoc analysis revealed significant effects of genotype: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Animal numbers are the same as in Fig. 2.

We then addressed the possibility that the increased overall EHE frequency in middle-aged male TPH2−/− rats was related to their prolonged REM episodes. To this end, for both genotypes and sexes, we analyzed the number of EHEs occurring over time across all episodes of REM. The number of EHEs occurring over time was highly dependent on time and on sex. Although middle-aged TPH2−/− males had the same number of EHEs as TPH2+/+ rats at the start of REM sleep, they experienced more than double the number of EHEs as TPH2+/+ rats as REM progressed (genotype × time: P < 0.0001; Fig. 5C, left). In contrast, the occurrence of EHEs in middle-aged female TPH2−/− rats was the same as in TPH2+/+ rats (genotype: P = 0.15; Fig. 5C, right). In fact, female TPH2−/− rats actually had fewer events in the early part of REM compared with TPH2+/+ rats (genotype × time: P < 0.001).

Taken together, these analyses suggest that with a loss of CNS 5-HT, middle-aged males and females alike experience prolonged periods of REM sleep. However, CNS 5-HT is more important in males for mitigating the frequency of ABP surges as REM progresses. Females, on the other hand, are protected from these surges in ABP.

CNS 5-HT deficiency alters systolic blood pressure variability in males but not females.

We performed fast Fourier transform function for power spectral analysis on sBP, another indicator of sympathetic vascular tone. We analyzed the proportion of very low (vLF%) and low (LF%) frequency components of the total sBP variability (19, 26). The vLF component reflects changes in sympathetic outflow related to hormonal activity, thermoregulation, or blood flow to meet local metabolic demands, whereas the LF component reflects mostly sympathetic drive to the vasculature (19, 26). In 2–3-mo-old male rats, there was no effect of genotype on vLF% or LF%. At 5–8 mo of age, male TPH2−/− rats displayed elevated vLF% (P = 0.04; Fig. 6A, top left) and LF% (P = 0.03; Fig. 6A, top middle) compared with TPH2+/+ controls. At 12–13 mo of age, male TPH2−/− rats also had significantly elevated vLF% (P = 0.03, Fig. 6A, bottom left) and LF% (P = 0.03; Fig. 6A, bottom middle). Unlike males, there was no effect of 5-HT deficiency on any of the components of sBP variability in female TPH2−/− rats of either age (Fig. 6, A and B, bottom). These LF and vLF data suggest that a loss of CNS 5-HT increases the sympathetic component of vascular tone in middle-aged males but not females.

Fig. 6.

Middle-aged male, but not female, TPH2−/− rats have higher proportion of low-frequency (LF) and very low-frequency (vLF) components of systolic blood pressure variability. Proportion of vLF% and LF% components of systolic blood pressure variability in (A) 5–8-mo-old male and female TPH2+/+ (open circles) and TPH2−/− rats (closed circles) and (B) 12–13-mo-old male and female TPH2+/+ and TPH2−/− rats. Two-factor, repeated-measures ANOVA revealed a significant genotype effect: *P < 0.05. Animal numbers are the same as in Fig. 2.

To further address the possibility that the high ABP of male TPH2−/− rats was due to altered autonomic control, we treated 5–8-mo-old male TPH2−/− and TPH2+/+ controls with atropine methyl nitrate (iv, 1 mg/kg) and hexamethonium (iv, 30 mg/kg) to induce complete autonomic blockade to the heart and vasculature. We measured the subsequent atropine-induced increase in HR and MAP and hexamethonium-induced decrease in MAP as indicators of parasympathetic drive to the heart and the sympathetic component of vascular tone respectively (see raw records in Fig. 7A). The rise in MAP following atropine and the fall in MAP following hexamethonium were not influenced by genotype (Fig. 7B). Compared with male TPH2+/+ rats, MAP was still elevated in 5–8-mo-old male TPH2−/− rats following full ganglionic blockade (P = 0.025; Fig. 7A). Likewise, in 5–8-mo-old females, there was no effect of genotype on the magnitude of the fall in MAP induced by hexamethonium (Fig. 7C). MAP was the same in female TPH2−/− and TPH2+/+ rats at baseline, after atropine, and following hexamethonium (P = 0.14; Fig. 7C).

Fig. 7.

Ganglionic blockade does not normalize the elevated blood pressure of TPH2−/− rats. A: raw records of mean arterial pressure (MAP) and heart rate (HR) in a male TPH2+/+ rat (left) and a TPH2−/− rat (right) following administration of atropine and hexamethonium (Hex) to establish complete autonomic blockade to the heart and vasculature. B: MAP at baseline (base) and following intravenous (iv) administration of atropine and Hex in male TPH2+/+ (open circles, n = 11) and TPH2−/− rats (filled circles, n = 11). C: responses of female TPH2+/+ (n = 8) and TPH2−/− (n = 9) rats to atropine and Hex. Note that the rise in MAP following atropine and the fall in MAP following Hex were not influenced by genotype in either sex. Two-factor, repeated-measures ANOVA revealed a significant effect of genotype on MAP in males only (*P < 0.05).

CNS 5-HT deficiency leads to subtle, vigilance state-dependent changes in the control of breathing.

As pups, TPH2−/− rats hypoventilate due to reductions in both respiratory frequency (f) and tidal volume (V_T) (16), effects of 5-HT deficiency that do not depend on vigilance state (36). Kaplan et al. (16) also reported that, as adults, male TPH2−/− rats have reduced f, but normal ventilation (V̇e), due to slightly increased V_T. We wondered if, as adults, the effects of central 5-HT deficiency on breathing pattern might depend on state of vigilance. Indeed, we found that at 2–3 mo of age, TPH2−/− rats had reduced f in REM sleep (by ∼11 breaths/min), an effect not observed in NREM or QW (genotype × state: P < 0.001; post hoc, genotype effect in REM sleep: P = 0.04; Table 4). As was found previously (16), the V_T of 2–3-mo-old TPH2−/− rats tended to be elevated compared with TPH2+/+, such that there was no effect of genotype on either V̇e or V̇e^/V̇o₂. At 5–8 mo of age, the f of TPH2−/− rats was again slightly but significantly lower in REM sleep (by ∼8 breaths/min; post hoc, genotype effect in REM sleep: P < 0.0001), while being slightly greater than that of TPH2+/+ rats in NREM and QW (post hoc, genotype effect in NREM and QW: P < 0.001 for both). The decreased f of 5–8-mo-old TPH2−/− rats in REM sleep and increased f in NREM sleep and QW led to a significant interactive effect of CNS 5-HT deficiency on V̇e when examining across all vigilance states (genotype × state: P < 0.0001); yet there was no significant effect of genotype in any one state (post hoc, genotype effect in each state: P > 0.05; Table 4). Further, there was no effect of genotype on V̇e^/V̇o₂ at this age, suggesting little to no influence of central 5-HT deficiency on arterial blood gases. As in male TPH2−/− rats, the f of female TPH2−/− rats was slightly but significantly reduced compared with female TPH2+/+ rats in REM sleep (∼10 breaths/min lower; post hoc: P = 0.001). As in males, there was no effect of genotype on the V̇e of females in any state (post hoc genotype effect in each state: P > 0.05). Finally, the subtle respiratory phenotypes that were evident in male TPH2−/− rats at 2–3 and 5–8 mo of age were absent in 12–13-mo-old animals (Table 4). Taken together, in both sexes, a chronic loss of central 5-HT decreases respiratory frequency in REM sleep in animals less than 1 yr of age but has little influence over ventilation or arterial blood gases at rest, as has been reported previously (16).

Table 4.

Resting respiratory variables of male TPH2+/+ and TPH2−/− rats

			f			VT			V̇e			V̇e/V̇o2
	Geno	Mass	QW	NREM	REM	QW	NREM	REM	QW	NREM	REM	QW	NREM	REM
2–3 mo	+/+ n = 9	227 ± 9	84 ± 3	80 ± 3	92 ± 2	12.3 ± 0.9	11.9 ± 1.0	11.5 ± 0.8	1058 ± 121	964 ± 113	1012 ± 100	15.8 ± 1.3	14.6 ± 1.3	15.3 ± 1.7
	−/− n = 8	221 ± 16	87 ± 4	84 ± 4	81 ± 4†	13.6 ± 1.0	12.8 ± 1.0	12.9 ± 1.0	1,190 ± 149	1,087 ± 133	1,039 ± 122	17.2 ± 1.3	16.0 ± 1.3	15.2 ± 1.1
5–8 mo	+/+ n = 23	330 ± 5	65 ± 1	62 ± 1	78 ± 1	10.9 ± 0.3	10.0 ± 0.3	8.7 ± 0.4	707 ± 24	624 ± 21	672 ± 27	25.6 ± 1.0	24.3 ± 1.0	23.8 ± 0.9
	−/− n = 20	359 ± 6*	73 ± 1†	68 ± 1†	70 ± 1†	10.6 ± 0.3	9.6 ± 0.4	8.4 ± 0.4	786 ± 29†	660 ± 32†	596 ± 32†	26.2 ± 0.9	24.8 ± 1.2	23.6 ± 1.2
12–13 mo	+/+ n = 9	382 ± 8	65 ± 1	65 ± 1	79 ± 2	9.7 ± 0.8	9.3 ± 0.8	8.7 ± 0.7	627 ± 48	600 ± 49	682 ± 54	25.3 ± 2.1	24.8 ± 2.1	28.5 ± 2.2
	−/− n = 6	390 ± 11	69 ± 3	70 ± 2	76 ± 2†	9.9 ± 1.3	9.5 ± 1.3	9.2 ± 1.3	681 ± 90	657 ± 90	687 ± 92	24.4 ± 3.1	24.2 ± 3.0	25.7 ± 3.2

Shown are mass (g), respiratory frequency (f, breaths/min), tidal volume (V_T, mL·breath⁻¹·kg⁻¹), ventilation (V̇e, mL·min⁻¹·kg⁻¹), and the ventilatory equivalent (V̇e/V̇o₂) of male TPH2−/− rats and TPH2+/+ littermates in quiet wakefulness (QW), non-rapid eye movement sleep (NREM) and rapid eye movement sleep (REM). Animal numbers are indicated.

^*Significant genotype effect: P < 0.05.

^†Significant genotype-state interaction: P < 0.05.

High blood pressure of male TPH2−/− rats is associated with increased heart and lung weight.

Given their high blood pressure, the hearts and lungs of 5–8- and 12–13-mo-old male TPH2−/− and TPH2+/+ rats were examined for evidence of cardiac remodeling potentially related to chronic increases in left ventricular afterload. At both 5–8 and 12–13 mo of age, whole heart weight was significantly increased in TPH2−/− animals compared with in their TPH2+/+ counterparts (genotype effect: P = 0.0002; Fig. 8A). Wet lung weight was also significantly increased in TPH2−/− rats (genotype effect: P = 0.01; Fig. 8B). Tibia length was the same between genotypes, indicating that postmortem increases in heart and lung weight in TPH2−/− rats were not due to differences in normal age-related growth (Fig. 8C). As there were no significant differences in tibia length between groups, absolute heart and lung weights were used for group morphological analyses as previously reported (35). Despite evidence of cardiac remodeling, cardiac function was normal in TPH2−/− rats compared with in their TPH2+/+ littermates, with no significant differences in HR (Fig. 8D), stroke volume (Fig. 8E), or cardiac output (Fig. 8F). Cardiac morphology was not assessed in female animals, given that no chronic increases in left ventricular afterload were observed in female TPH2−/− rats.

Fig. 8.

Postmortem heart and lung weight is increased in male TPH2−/− rats. A: heart weight (HW) was significantly increased in both 5–8-mo-old (n = 10) and 12–13-mo-old (n = 11) male TPH2−/− rats compared with in TPH2+/+ rats (n = 8 of both 5–8 and 12–13 mo old). B: lung weight (LW) was significantly greater in 12–13-mo-old male TPH2−/− rats (closed circles, n = 11) compared with in TPH2+/+ rats (open circles, n = 7) [*P < 0.05; 2-factor, repeated-measures ANOVA]. C: tibia length (TL) was not different between the genotypes, suggesting no difference in age-related growth. There were no effects of genotype on heart rate (HR; D), stroke volume (SV; E), or cardiac output (CO; F), comparing 5–8- and 12–13-mo-old animals (data from both 5–8- and 12–13-mo-old animals; n = 12 TPH2+/+ and n = 16 TPH2−/−).

DISCUSSION

The major hypothesis of this study was that central 5-HT helps maintain blood pressure during wakefulness and NREM sleep, when serotonergic neurons are active and releasing synaptic 5-HT. Unexpectedly, we found that the ABP of 5–8- and 12–13-mo-old male TPH2−/− rats was elevated compared with that of male TPH2+/+ littermates during REM sleep and wakefulness but was normal during NREM sleep. In REM, middle-aged male TPH2−/− rats had more frequent and intense EHEs compared with TPH2+/+ rats, with MAP increasing to nearly 20 mmHg more than in TPH2+/+ rats during these events. TPH2−/− rats also had prolonged periods of REM sleep, and the more frequent EHEs of male TPH2−/− rats only became apparent as REM episodes progressed. Females were in general protected from the ABP phenotypes associated with a loss of CNS 5-HT. Our analyses of sBP variability suggest that the vascular component of sympathetic activity is higher in middle-aged male TPH2−/− rats compared with in TPH2+/+ rats. Finally, increased heart and lung weights in male TPH2−/− animals compared with in TPH2+/+ rats are potentially indicative of early stages of compensated cardiopulmonary disease as demonstrated by cardiac hypertrophy observed in parallel with normal systolic function.

A loss of CNS 5-HT leads to more extreme hypertensive events in prolonged periods of REM sleep.

We found that, compared with that of TPH2+/+ rats, the MAP of middle-aged (5–8 and 12–13 mo old) male TPH2−/− rats was elevated in wakefulness and, in particular, during REM sleep (Fig. 2B). It is well known that cardiovascular function varies according to vigilance state. ABP and HR are normally highest in wakefulness and lowest in NREM sleep. In REM sleep, there is a withdrawal of parasympathetic activity and a redistribution of sympathetic activity that together increase ABP and HR back to values observed in wakefulness (30). Efferent activity to the heart and vasculature is also more variable in REM sleep compared with in NREM sleep and QW, leading to marked fluctuations in ABP and HR that do not occur in the other states. Our data indicate that the ABP of middle-aged male TPH2−/− rats deficient in CNS 5-HT was particularly volatile during REM sleep, with considerably more frequent and extreme hypertensive events (EHEs) compared with that seen in male TPH2+/+ rats, events that do not appear to be associated with an increase in EMG amplitude that would indicate a cortical or subcortical arousal. Thus, CNS 5-HT is necessary in middle-age males for mitigating these extreme events during REM sleep. Females, on the other hand, are generally protected against the deleterious effects of CNS 5-HT depletion on ABP regulation, including the frequency and overall impact of EHEs in REM sleep (see Females are protected from the deleterious effects of CNS 5-HT deficiency on ABP regulation).

We previously showed that as infants, TPH2−/− rats have longer episodes of active sleep (i.e., REM), while the average duration of quiet sleep (i.e., NREM) episodes was the same as for TPH2+/+ rats (8, 36). Here we found that as middle-aged adults, male and female TPH2−/− rats have prolonged episodes of REM and NREM sleep, with REM sleep duration being especially affected. However, the overall proportion of time spent in sleep is no different than that seen in their TPH2+/+ counterparts because they have fewer episodes of sleep. The prolonged sleep episodes of rats deficient in CNS 5-HT may be due to a generalized increase in the threshold for arousal, an idea recently posited by Solarewicz et al. (31), who described a similar phenotype in TPH2−/− mice. We do not know if there is a common mechanism underlying the prolonged REM sleep episodes and increased EHEs of male TPH2−/− rats during REM sleep. That said, our data suggest that the frequency of EHEs becomes higher than that seen in TPH2+/+ rats toward the end of REM sleep episodes. Neurohumoral factors that are known to be involved in prolonging REM sleep may therefore be involved in the EHE phenotype of male TPH2−/− rats. One such factor is acetylcholine; the activity of REM-generating cholinergic neurons in the pontine tegmentum (e.g., the laterodorsal tegmentum) may be increased in TPH2−/− rats, especially because 5-HT has known inhibitory effects on these regions (33). We previously showed that central cholinergic blockade reduced the apnea and stabilized the breathing of infant TPH2−/− rats (8), and it may be that enhanced cholinergic drive also contributes to the high ABP of middle-aged male TPH2−/− rats. Moreover, increased cholinergic activity may underlie the high ABP of male TPH2−/− rats in wakefulness as well, given that acetylcholine is not only a REM sleep driver but also a major component of the ascending arousal system that helps maintain wakefulness.

Evidence for and against increased sympathetic drive contributing to the high ABP of male TPH2−/− rats.

The elevated LF in 5–8- and 12–13-mo-old TPH2−/− rats compared with in TPH2+/+ rats suggested that the sympathetic component of vascular tone is elevated in the absence of central 5-HT; this could result from lack of 5-HT_1A-mediated inhibition in the RVLM, for example (23, 25). However, the results from our ganglionic blockade experiment did not support this conclusion. We hypothesized that, following hexamethonium administration, male TPH2−/− rats would have a greater drop in MAP compared with controls, suggesting greater sympathetic vascular tone in the absence of CNS 5-HT. However, the MAP of TPH2−/− rats was still elevated compared with that of TPH2+/+ rats following treatment (i.e., the fall in MAP was the same in each genotype; Fig. 4). On the surface, this finding suggests that elevated sympathetic nerve activity probably does not significantly contribute to the high ABP of male TPH2−/− rats. vLF is also elevated in male TPH2−/− rats, suggesting a humoral or local origin for their hypertension. Possibilities include elevated epinephrine, angiotensin II, or reduced endothelial nitric oxide production. Future experiments should also examine whether a loss of CNS 5-HT leads, over time, to adaptive changes in the vasculature (e.g., altered expression or signaling from α-adrenergic receptors) that normalize the low MAP of male TPH2−/− rats as neonates (20) but that eventually lead to hypertension in adulthood, as we show here.

Females are protected from the deleterious effects of CNS 5-HT deficiency on ABP regulation.

Our data show that ABP of middle-aged females was, for the most part, not influenced by a lack of CNS 5-HT. Despite experiencing slightly higher peaks in MAP during EHEs, in all states, the MAP of female TPH2−/− rats was not significantly different from that of female TPH2+/+ rats. Although middle-aged female TPH2−/− rats also had prolonged episodes of REM sleep, unlike males, they did not exhibit increased EHEs as REM sleep progressed. The proportion of vLF and LF components of systolic blood pressure variability was also not increased in female TPH2−/− rats. Regardless of the mechanism underlying the high ABP of middle-aged male TPH2−/− rats, be it increased sympathetic vascular tone or a factor associated with prolonged REM sleep, it would be of interest to investigate a potential interaction between 5-HT and estrogen, or at least the possibility that estrogens can compensate for 5-HT in ABP regulation. Estrogens have well-described, inhibitory effects on sympathetic nerve activity and ABP (11). It may be that estrogens act through similar pathways as 5-HT within the CNS and can compensate for a lack of 5-HT to maintain normal ABP regulation. There is some evidence that estrogen can enhance the inhibitory effects of 5-HT_1A receptor activation on cardiovascular function, for example (2). It is important to also note that, in our hands, the MAP of female rats was more variable than that of males, irrespective of genotype. Although yet to be tested, this variability could very well be due to the influence of the estrous cycle and associated fluctuations in female sex hormones (32). For example, females with lower MAP may be in estrus or proestrus when estrogens are high. We also acknowledge that, due to high mortality, we could test only a small number of 12–13-mo-old females. Thus, although there is no trend indicating high blood pressure in this cohort of TPH2−/− rats, our finding of lack of any difference in blood pressure should be considered with some degree of caution.

Upon initial inspection, our findings appear to be in conflict with previous studies that showed reduced ABP regulation in TPH2−/− mice (1). However, Alenina et al. (1) found reduced blood pressure in TPH2−/− mice only in the evening. We conducted all of our experiments during the daytime (sleep period for rodents). It would be of interest to monitor the ABP of middle-aged TPH2−/− rats in the evening, as there may be a diurnal influence of CNS 5-HT neurons on the control of ABP. In addition, Alenina et al. (1) used only relatively young TPH2−/− mice (∼4 mo of age). TPH2−/− rats have low ABP in early life (20), and our data suggest that they only become hypertensive with aging. Whether the regulation of ABP in TPH2−/− mice is also age dependent should be investigated.

Evidence of cardiopulmonary involvement in middle-aged adults deficient in central 5-HT.

Evidence of increased postmortem heart and lung weight in male TPH2−/− rats suggests that the loss of 5-HT in the CNS can lead to the beginning stages of cardiovascular disease. Although further investigation is required, the combination of cardiac hypertrophy and normal resting systolic function in male TPH2−/− animals suggests a compensated functional phenotype with myocardial remodeling occurring likely secondary to sustained increases in afterload (i.e., increased ABP). These changes, when considered together with a parallel increase in lung weight, imply that over time male TPH2−/− rats may be more susceptible to the development of chronic diseases such as heart failure. Considering the potential impact of the estrous cycle and associated fluctuations in female sex hormones on the variability of not only ABP but also heart and lung structure and function, we decided to not examine cardiopulmonary involvement in the female groups. That said, given well-established increases in cardiovascular risk for women postmenopause, clearly a more detailed interrogation of how central 5-HT influences the development of cardiovascular disease with both aging and/or the loss of sex hormones is warranted.

Chronic loss of CNS 5-HT has minimal influence over the control of breathing in adulthood.

Infant animals deficient in central 5-HT have severe defects in the control of breathing; the most consistent observation is reduced respiratory frequency, increased incidence and severity of apnea, and hypoventilation (6, 7, 14, 16, 37). The current data suggest that as adults, TPH2−/− rats have only subtle deficits in breathing, including slightly reduced respiratory frequency in younger adults during REM sleep; however, given that there is no effect on V̇e^/V̇o₂, this hypopnea probably is of no consequence in terms of blood gas homeostasis during REM sleep. Our findings largely corroborate the findings by Kaplan et al. (16) in adult TPH2−/− rats and Hickner et al. (12) in TPH2−/− mice and suggest that there are compensatory mechanisms in the neural respiratory networks that maintain ventilation in the face of a chronic loss of central 5-HT. The most parsimonious explanation is that compensation is provided by substance P and/or thyrotropin releasing factor, neuropeptides that are also released from serotonergic neurons. However, adult mice lacking nearly all of their 5-HT neurons (lmx1b−/−) also have normal ventilation at rest (13), suggesting that other forms of plasticity within the respiratory control network can maintain ventilation in the absence of 5-HT neurons and their array of coreleased factors.

Significance.

Our findings suggest that central 5-HT plays a critical role in preventing an age-related increase in blood pressure in male rats. Given that TPH2−/− rats are not obese and have no other identifiable metabolic phenotypes, our data suggest that defects within the serotonergic system of the brainstem could underpin some cases of essential hypertension in middle-aged males. This new knowledge is important because essential hypertension accounts for more than 90% of all hypertensive cases and brings with it comorbidities including stroke, myocardial infarction, and heart failure (4, 24). In this way, a loss of central 5-HT, or defects in central 5-HT signaling, may put males at higher risk for these cardiovascular diseases with aging. 5-HT-deficient rats, like the spontaneously hypertensive rat, are also prone to extreme hypertensive events in REM sleep. TPH2−/− rats experience surges in blood pressure that approach 160 mmHg. This also suggests that defects in the CNS 5-HT system could put males at risk for severe, life-threatening cardiovascular events during REM sleep.

GRANTS

This work was funded by National Heart, Lung, and Blood Institute Grant project R01 HL136710 02 (to K. J. Cummings) and project R01 HL112998 (to C. A. Emter).

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

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