Eugene E. Nattie

The Brainstem and Serotonin in the Sudden Infant Death Syndrome

The sudden infant death syndrome (SIDS) is the sudden death of an infant under one year of age that is typically associated with sleep and that remains unexplained after a complete autopsy and death scene investigation. A leading hypothesis about its pathogenesis is that many cases result from defects in brainstem-mediated protective responses to homeostatic stressors occurring during sleep in a critical developmental period. Here we review the evidence for the brainstem hypothesis in SIDS with a focus upon abnormalities related to the neurotransmitter serotonin in the medulla oblongata, as these are the most robust pathologic findings to date. In this context, we synthesize the human autopsy data with genetic, whole-animal, and cellular data concerning the function and development of the medullary serotonergic system. These emerging data suggest an important underlying mechanism in SIDS that may help lead to identification of infants at risk and specific interventions to prevent death.

Brainstem serotonergic deficiency in sudden infant death syndrome.

CONTEXT Sudden infant death syndrome (SIDS) is postulated to result from abnormalities in brainstem control of autonomic function and breathing during a critical developmental period. Abnormalities of serotonin (5-hydroxytryptamine [5-HT]) receptor binding in regions of the medulla oblongata involved in this control have been reported in infants dying from SIDS. OBJECTIVE To test the hypothesis that 5-HT receptor abnormalities in infants dying from SIDS are associated with decreased tissue levels of 5-HT, its key biosynthetic enzyme (tryptophan hydroxylase [TPH2]), or both. DESIGN, SETTING, AND PARTICIPANTS Autopsy study conducted to analyze levels of 5-HT and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA); levels of TPH2; and 5-HT(1A) receptor binding. The data set was accrued between 2004 and 2008 and consisted of 41 infants dying from SIDS (cases), 7 infants with acute death from known causes (controls), and 5 hospitalized infants with chronic hypoxia-ischemia. MAIN OUTCOME MEASURES Serotonin and metabolite tissue levels in the raphé obscurus and paragigantocellularis lateralis (PGCL); TPH2 levels in the raphé obscurus; and 5-HT(1A) binding density in 5 medullary nuclei that contain 5-HT neurons and 5 medullary nuclei that receive 5-HT projections. RESULTS Serotonin levels were 26% lower in SIDS cases (n = 35) compared with age-adjusted controls (n = 5) in the raphé obscurus (55.4 [95% confidence interval {CI}, 47.2-63.6] vs 75.5 [95% CI, 54.2-96.8] pmol/mg protein, P = .05) and the PGCL (31.4 [95% CI, 23.7-39.0] vs 40.0 [95% CI, 20.1-60.0] pmol/mg protein, P = .04). There was no evidence of excessive 5-HT degradation assessed by 5-HIAA levels, 5-HIAA:5-HT ratio, or both. In the raphé obscurus, TPH2 levels were 22% lower in the SIDS cases (n = 34) compared with controls (n = 5) (151.2% of standard [95% CI, 137.5%-165.0%] vs 193.9% [95% CI, 158.6%-229.2%], P = .03). 5-HT(1A) receptor binding was 29% to 55% lower in 3 medullary nuclei that receive 5-HT projections. In 4 nuclei, 3 of which contain 5-HT neurons, there was a decrease with age in 5-HT(1A) receptor binding in the SIDS cases but no change in the controls (age x diagnosis interaction). The profile of 5-HT and TPH2 abnormalities differed significantly between the SIDS and hospitalized groups (5-HT in the raphé obscurus: 55.4 [95% CI, 47.2-63.6] vs 85.6 [95% CI, 61.8-109.4] pmol/mg protein, P = .02; 5-HT in the PGCL: 31.4 [95% CI, 23.7-39.0] vs 71.1 [95% CI, 49.0-93.2] pmol/mg protein, P = .002; TPH2 in the raphé obscurus: 151.2% [95% CI, 137.5%-165.0%] vs 102.6% [95% CI, 58.7%-146.4%], P = .04). CONCLUSION Compared with controls, SIDS was associated with lower 5-HT and TPH2 levels, consistent with a disorder of medullary 5-HT deficiency.

Impaired Respiratory and Body Temperature Control Upon Acute Serotonergic Neuron Inhibition

Inducible neuron inhibition reveals essential roles for serotonergic neurons in respiratory and body temperature homeostasis. Physiological homeostasis is essential for organism survival. Highly responsive neuronal networks are involved, but their constituent neurons are just beginning to be resolved. To query brain serotonergic neurons in homeostasis, we used a neuronal silencing tool, mouse RC::FPDi (based on the synthetic G protein–coupled receptor Di), designed for cell type–specific, ligand-inducible, and reversible suppression of action potential firing. In mice harboring Di-expressing serotonergic neurons, administration of the ligand clozapine-N-oxide (CNO) by systemic injection attenuated the chemoreflex that normally increases respiration in response to tissue carbon dioxide (CO2) elevation and acidosis. At the cellular level, CNO suppressed firing rate increases evoked by CO2 acidosis. Body thermoregulation at room temperature was also disrupted after CNO triggering of Di; core temperatures plummeted, then recovered. This work establishes that serotonergic neurons regulate life-sustaining respiratory and thermoregulatory networks, and demonstrates a noninvasive tool for mapping neuron function.

Central chemoreception is a complex system function that involves multiple brain stem sites

central chemoreception refers to detection of CO2/pH within the brain and the subsequent reflex effects on breathing. It involves multiple sites within the hindbrain ([9][1], [12][2], [19][3]) as focal acidification in vivo uniquely at these sites stimulates breathing, indicating detection and

Central chemosensitivity, sleep, and wakefulness.

Abstract Neurons in many regions of the lower brain are chemosensitive in vitro. Focal acidification of these same and other regions in vivo can stimulate breathing indicating the presence of chemoreception. Why are there so many sites for central chemoreception? This review evaluates data obtained from unanesthetized rats at three central chemoreceptor sites, the retrotrapezoid nucleus (RTN), the medullary raphe, and the nucleus tractus solitarius (NTS) and extends ideas concerning two hypotheses, which were recently formulated (Nattie, E., 2000. Respir. Physiol. 122, 223–235). (1) The high overall sensitivity of the respiratory control system in the unanesthetized state to small increases in arterial CO 2 relies on an additive or greater effect of these multiple chemoreceptor sites. (2) Chemoreceptor sites can vary in effectiveness dependent on the state of arousal. These ideas fit into a more speculative and general hypothesis that central chemoreceptors are organized in a hierarchical manner as proposed for temperature sensing and thermoregulation (Satinoff, E., 1978. Science 201, 16–22). The presence of a number of chemosensitive sites with varying thresholds, sensitivity, and arousal dependence provides finely tuned control and stability for breathing.

Medullary serotonergic neurones and adjacent neurones that express neurokinin-1 receptors are both involved in chemoreception in vivo

Neurokinin‐1 receptor (NK1R)‐expressing neurones that are involved in chemoreception at the retrotrapezoid nucleus ( Nattie & Li, 2002b ) are also prominent at locations that contain medullary serotonergic neurones, which are chemosensitive in vitro. In medullary regions containing both types, we evaluated their role in central chemoreception by specific cell killing. We injected (2×100 nl) (a) substance P–saporin (SP‐SAP; 1μm) to kill NK1R‐expressing neurones, (b) a novel conjugate of a monoclonal antibody to the serotonin transporter (SERT) and saporin (anti‐SERT‐SAP; 1μm) to kill serotonergic neurones, or (c) SP‐SAP and anti‐SERT‐SAP together to kill both types. Controls received IgG‐SAP injections (1μm). There was no double‐labelling of NK1R‐immunoreactive (ir) and tryptophan‐hydroxylase (TPOH)‐ir neurones. Cell (somatic profile) counts showed that NK1R‐ir neurones in the SP‐SAP group were reduced by 31%; TPOH‐ir neurones in the anti‐SERT‐SAP group by 28%; and NK1R‐ir and TPOH‐ir neurones, respectively, in the combined lesion group by 55% and 31% (P < 0.001; two‐way ANOVA; P < 0.05, Tukeys post hoc test). The treatments had no significant effect on sleep/wake time, body temperature, or oxygen consumption but all three reduced the ventilatory response to 7% inspired CO2 in wakefulness and sleep by a similar amount. SP‐SAP treatment decreased the averaged CO2 responses (3, 7 and 14 days after lesions) in wakefulness and sleep by 21% and 16%, anti‐SERT‐SAP decreased the responses by 15% and 18%, and the combined treatment decreased the responses by 12% and 12% (P < 0.001; two‐way ANOVA; P < 0.05, Tukeys post hoc test). We conclude that separate populations of serotonergic and adjacent NK1R‐expressing neurones in the medulla are both involved in central chemoreception in vivo.

Substance P-saporin lesion of neurons with NK1 receptors in one chemoreceptor site in rats decreases ventilation and chemosensitivity

All medullary central chemoreceptor sites contain neurokinin‐1 receptor immunoreactivity (NK1R‐ir). We ask if NK1R‐ir neurons and processes are involved in chemoreception. At one site, the retrotrapezoid nucleus/parapyramidal region (RTN/Ppy), we injected a substance P‐saporin conjugate (SP‐SAP; 0.1 pmol in 100 nl) to kill NK1R‐ir neurons specifically, or SAP alone as a control. We made measurements for 15 days after the injections in two groups of rats. In group 1, with unilateral injections made in the awake state via a pre‐implanted guide cannula, we compared responses within rats using initial baseline data. In group 2, with bilateral injections made under anaesthesia at surgery, we compared responses between SP‐SAP‐ and SAP‐treated rats. SP‐SAP treatment reduced the volume of the RTN/Ppy region that contained NK1R‐ir neuronal somata and processes by 44 % (group 1) and by 47 and 40 % on each side, respectively (group 2). Ventilation (V̇E) and tidal volume (VT) were decreased during air breathing in sleep and wakefulness (group 2; P < 0.001; two‐way ANOVA) and Pa,CO2 was increased (group 2; P < 0.05; Students t test). When rats breathed an air mixture containing 7 % CO2 during sleep and wakefulness, V̇E and VT were lower (groups 1 and 2; P < 0.001; ANOVA) and the ΔV̇E in air containing 7 % CO2 compared to air was decreased by 28‐30 % (group 1) and 17‐22 % (group 2). SP‐SAP‐treated rats also slept less during air breathing. We conclude that neurons with NK1R‐ir somata or processes in the RTN/Ppy region are either chemosensitive or they modulate chemosensitivity. They also provide a tonic drive to breathe and may affect arousal.

Multiple sites for central chemoreception: their roles in response sensitivity and in sleep and wakefulness.

Central chemoreceptors appear to be widely distributed in the brainstem. Why are there so many central chemoreceptor sites? This review focuses on two hypotheses. (1) The high sensitivity of the respiratory control system as a whole to small changes in systemic P(CO(2)) results from an additive, or greater, effect of the multiple central chemoreceptor sites. Each site provides a fraction of the total response and, importantly, provides tonic excitatory input in eucapnia as well. (2) Individual central chemoreceptor sites vary in effectiveness depending on the arousal or vigilance state of the animal. For example, some sites are more important in wakefulness; others in sleep. Proof for these hypotheses depends critically on obtaining accurate measures of stimulus intensity at each chemoreceptor site in vivo.

Medullary serotonergic neurones modulate the ventilatory response to hypercapnia, but not hypoxia in conscious rats.

Serotonergic neurones in the mammalian medullary raphe region (MRR) have been implicated in central chemoreception and the modulation of the ventilatory response to hypercapnia, and may also be involved in the ventilatory response to hypoxia. In this study, we ask whether ventilatory responses across arousal states are affected when the 5‐hydroxytryptamine 1A receptor (5‐HT1A) agonist (R)‐(+)‐8‐hydroxy‐2(di‐n‐propylamino)tetralin (DPAT) is microdialysed into the MRR of the unanaesthetized adult rat. Microdialysis of 1, 10 and 30 mm DPAT into the MRR significantly decreased absolute ventilation values during 7% CO2 breathing by 21%, 19% and 30%, respectively, in wakefulness compared to artificial cerebrospinal fluid (aCSF) microdialysis, due to decreases in tidal volume (VT) and not in frequency (f), similar to what occurred during non‐rapid eye movement (NREM) sleep. The concentration‐dependence of the hypercapnic ventilatory effect might be due to differences in tissue distribution of DPAT. DPAT (30 mm) changed room air breathing pattern by increasing f and decreasing VT. As evidenced by a sham control group, repeated experimentation and microdialysis of aCSF alone had no effect on the ventilatory response to 7% CO2 during wakefulness or sleep. Unlike during hypercapnia, microdialysis of 30 mm DPAT into the MRR did not change the ventilatory response to 10% O2. Additionally, 10 and 30 mm DPAT MRR microdialysis decreased body temperature, and 30 mm DPAT increased the percentage of experimental time in wakefulness. We conclude that serotonergic activity in the MRR plays a role in the ventilatory response to hypercapnia, but not to hypoxia, and that MRR 5‐HT1A receptors are also involved in thermoregulation and arousal.

Catecholamine neurones in rats modulate sleep, breathing, central chemoreception and breathing variability

Brainstem catecholamine (CA) neurones have wide projections and an arousal‐state‐dependent activity pattern. They are thought to modulate the processing of sensory information and also participate in the control of breathing. Mice with lethal genetic defects that include CA neurones have abnormal respiratory control at birth. Also the A6 region (locus coeruleus), which contains CA neurones sensitive to CO2in vitro, is one of many putative central chemoreceptor sites. We studied the role of CA neurones in the control of breathing during sleep and wakefulness by specifically lesioning them with antidopamine β‐hydroxylase–saporin (DBH‐SAP) injected via the 4th ventricle. After 3 weeks there was a 73–84% loss of A5, A6 and A7 tyrosine hydroxylase (TH) immunoreactive (ir) neurones along with 56–60% loss of C1 and C2 phenyl ethanolamine‐N‐methyltransferase (PNMT)‐ir neurones. Over the 3 weeks, breathing frequency decreased significantly during air and 3 or 7% CO2 breathing in both wakefulness and non‐REM (NREM) sleep. The rats spent significantly less time awake and more time in NREM sleep. REM sleep time was unaffected. The ventilatory response to 7% CO2 was reduced significantly in wakefulness at 7, 14 and 21 days (−28%) and in NREM sleep at 14 and 21 days (−26%). Breathing variability increased in REM sleep but not in wakefulness or NREM sleep. We conclude that CA neurones (1) promote wakefulness, (2) participate in central respiratory chemoreception, (3) stimulate breathing frequency, and (4) minimize breathing variability in REM sleep.