Activity of Tachykinin1-Expressing Pet1 Raphe Neurons Modulates the Respiratory Chemoreflex.

Homeostatic control of breathing, heart rate, and body temperature relies on circuits within the brainstem modulated by the neurotransmitter serotonin (5-HT). Mounting evidence points to specialized neuronal subtypes within the serotonergic neuronal system, borne out in functional studies, for the modulation of distinct facets of homeostasis. Such functional differences, read out at the organismal level, are likely subserved by differences among 5-HT neuron subtypes at the cellular and molecular levels, including differences in the capacity to coexpress other neurotransmitters such as glutamate, GABA, thyrotropin releasing hormone, and substance P encoded by the Tachykinin-1 (Tac1) gene. Here, we characterize in mice a 5-HT neuron subtype identified by expression of Tac1 and the serotonergic transcription factor gene Pet1, referred to as the Tac1-Pet1 neuron subtype. Transgenic cell labeling showed Tac1-Pet1 soma resident largely in the caudal medulla. Chemogenetic [clozapine-N-oxide (CNO)-hM4Di] perturbation of Tac1-Pet1 neuron activity blunted the ventilatory response of the respiratory CO2 chemoreflex, which normally augments ventilation in response to hypercapnic acidosis to restore normal pH and PCO2Tac1-Pet1 axonal boutons were found localized to brainstem areas implicated in respiratory modulation, with highest density in motor regions. These findings demonstrate that the activity of a Pet1 neuron subtype with the potential to release both 5-HT and substance P is necessary for normal respiratory dynamics, perhaps via motor outputs that engage muscles of respiration and maintain airway patency. These Tac1-Pet1 neurons may act downstream of Egr2-Pet1 serotonergic neurons, which were previously established in respiratory chemoreception, but do not innervate respiratory motor nuclei.SIGNIFICANCE STATEMENT Serotonin (5-HT) neurons modulate physiological processes and behaviors as diverse as body temperature, respiration, aggression, and mood. Using genetic tools, we characterize a 5-HT neuron subtype defined by expression of Tachykinin1 and Pet1 (Tac1-Pet1 neurons), mapping soma localization to the caudal medulla primarily and axonal projections to brainstem motor nuclei most prominently, and, when silenced, observed blunting of the ventilatory response to inhaled CO2Tac1-Pet1 neurons thus appear distinct from and contrast previously described Egr2-Pet1 neurons, which project primarily to chemosensory integration centers and are themselves chemosensitive.

Partial Raphe Dysfunction in Neurotransmission Is Sufficient to Increase Mortality after Anoxic Exposures in Mice at a Critical Period in Postnatal Development.

Sudden infant death syndrome (SIDS) cases often have abnormalities of the brainstem raphe serotonergic (5-HT) system. We hypothesize that raphe dysfunction contributes to a failure to autoresuscitate from multiple hypoxic events, leading to SIDS. We studied autoresuscitation in two transgenic mouse models in which exocytic neurotransmitter release was impaired via conditional expression of the light chain from tetanus toxin (tox) in raphe neurons expressing serotonergic bacterial artificial chromosome drivers Pet1 or Slc6a4. These used recombinase drivers targeted different portions of medullary raphe serotonergic, tryptophan hydroxylase 2 (Tph2)(+) neurons by postnatal day (P) 5 through P12: approximately one-third in triple transgenic Pet1::Flpe, hßactin::cre, RC::PFtox mice; approximately three-fourths inSlc6a4::cre, RC::Ptox mice; with the first model capturing a near equal number of Pet1(+),Tph2(+) versus Pet1(+),Tph2(low or negative) raphe cells. At P5, P8, and P12, "silenced" mice and controls were exposed to five, ∼37 s bouts of anoxia. Mortality was 5-10 times greater in "silenced" pups compared with controls at P5 and P8 (p = 0.001) but not P12, with cumulative survival not differing between experimental transgenic models. "Silenced" pups that eventually died took longer to initiate gasping (p = 0.0001), recover heart rate (p = 0.0001), and recover eupneic breathing (p = 0.011) during the initial anoxic challenges. Variability indices for baseline breathing distinguished "silenced" from controls but did not predict mortality. We conclude that dysfunction of even a portion of the raphe, as observed in many SIDS cases, can impair ability to autoresuscitate at critical periods in postnatal development and that baseline indices of breathing variability can identify mice at risk. SIGNIFICANCE STATEMENT: Many sudden infant death syndrome (SIDS) cases exhibit a partial (∼26%) brainstem serotonin deficiency. Using recombinase drivers, we targeted different fractions of serotonergic and raphe neurons in mice for tetanus toxin light chain expression, which prevented vesicular neurotransmitter release. In one model, approximately one-third of medullary Tph2(+) neurons are silenced by postnatal (P) days 5 and 12, along with some Pet1(+),Tph2(low or negative) raphe cells; in the other, approximately three-fourths of medullary Tph2(+) neurons, also with some Tph2(low or negative) cells. Both models demonstrated excessive mortality to anoxia (a postulated SIDS stressor) at P5 and P8. We demonstrated fatal vulnerability to anoxic stress at a specific time in postnatal life induced by a partial defect in raphe function. This models features of SIDS.

An augmented CO2 chemoreflex and overactive orexin system are linked with hypertension in young and adult spontaneously hypertensive rats.

KEY POINTS: Activation of central chemoreceptors by CO2 increases sympathetic nerve activity (SNA), arterial blood pressure (ABP) and breathing. These effects are exaggerated in spontaneously hypertensive rats (SHRs), resulting in an augmented CO2 chemoreflex that affects both breathing and ABP. The augmented CO2 chemoreflex and the high ABP are measureable in young SHRs (postnatal day 30-58) and become greater in adult SHRs. Blockade of orexin receptors can normalize the augmented CO2 chemoreflex and the high ABP in young SHRs and normalize the augmented CO2 chemoreflex and significantly lower the high ABP in adult SHRs. In the hypothalamus, SHRs have more orexin neurons, and a greater proportion of them increase their activity with CO2 . The orexin system is overactive in SHRs and contributes to the augmented CO2 chemoreflex and hypertension. Modulation of the orexin system may be beneficial in the treatment of neurogenic hypertension. ABSTRACT: Activation of central chemoreceptors by CO2 increases arterial blood pressure (ABP), sympathetic nerve activity and breathing. In spontaneously hypertensive rats (SHRs), high ABP is associated with enhanced sympathetic nerve activity and peripheral chemoreflexes. We hypothesized that an augmented CO2 chemoreflex and overactive orexin system are linked with high ABP in both young (postnatal day 30-58) and adult SHRs (4-6 months). Our main findings are as follows. (i) An augmented CO2 chemoreflex and higher ABP in SHRs are measureable at a young age and increase in adulthood. In wakefulness, the ventilatory response to normoxic hypercapnia is higher in young SHRs (mean ± SEM: 179 ± 11% increase) than in age-matched normotensive Wistar-Kyoto rats (114 ± 9% increase), but lower than in adult SHRs (226 ± 10% increase; P < 0.05). The resting ABP is higher in young SHRs (122 ± 5 mmHg) than in age-matched Wistar-Kyoto rats (99 ± 5 mmHg), but lower than in adult SHRs (152 ± 4 mmHg; P < 0.05). (ii) Spontaneously hypertensive rats have more orexin neurons and more CO2 -activated orexin neurons in the hypothalamus. (iii) Antagonism of orexin receptors with a dual orexin receptor antagonist, almorexant, normalizes the augmented CO2 chemoreflex in young and adult SHRs and the high ABP in young SHRs and significantly lowers ABP in adult SHRs. (iv) Attenuation of peripheral chemoreflexes by hyperoxia does not abolish the augmented CO2 chemoreflex (breathing and ABP) in SHRs, which indicates an important role for the central chemoreflex. We suggest that an overactive orexin system may play an important role in the augmented central CO2 chemoreflex and in the development of hypertension in SHRs.

Altered 5-HT2A/C receptor binding in the medulla oblongata in the sudden infant death syndrome (SIDS): Part II. Age-associated alterations in serotonin receptor binding profiles within medullary nuclei supporting cardiorespiratory homeostasis.

The failure of chemoreflexes, arousal, and/or autoresuscitation to asphyxia may underlie some sudden infant death syndrome (SIDS) cases. In Part I, we showed that some SIDS infants had altered 5-hydroxytryptamine (5-HT)2A/C receptor binding in medullary nuclei supporting chemoreflexes, arousal, and autoresuscitation. Here, using the same dataset, we tested the hypotheses that the prevalence of low 5-HT1A and/or 5-HT2A/C receptor binding (defined as levels below the 95% confidence interval of controls-a new approach), and the percentages of nuclei affected are greater in SIDS versus controls, and that the distribution of low binding varied with age of death. The prevalence and percentage of nuclei with low 5-HT1A and 5-HT2A/C binding in SIDS were twice that of controls. The percentage of nuclei with low 5-HT2A/C binding was greater in older SIDS infants. In >80% of older SIDS infants, low 5-HT2A/C binding characterized the hypoglossal nucleus, vagal dorsal nucleus, nucleus of solitary tract, and nuclei of the olivocerebellar subnetwork (important for blood pressure regulation). Together, our findings from SIDS infants and from animal models of serotonergic dysfunction suggest that some SIDS cases represent a serotonopathy. We present new hypotheses, yet to be tested, about how defects within serotonergic subnetworks may lead to SIDS.

Antagonism of orexin receptors significantly lowers blood pressure in spontaneously hypertensive rats.

In normal rats, central administration of orexin or exposure to certain forms of stress can induce significant increases in blood pressure and sympathetic nerve activity, which can be blocked by orexin receptor antagonists. The resting blood pressure is, however, unaffected by such antagonists, but is significantly lower in rodents with total loss of orexin, such as prepro-orexin knockout mice and orexin neuron-ablated orexin/ataxin-3 transgenic rats. We hypothesize that orexin is involved in the pathophysiology and maintenance of high blood pressure in the spontaneously hypertensive rat (SHR), a model of primary hypertension. To test this hypothesis, we measured orexin-A mRNA expression in the rostral ventrolateral medulla and antagonized both orexin receptors using an orally administered potent dual orexin receptor antagonist, almorexant, in SHRs and normotensive Wistar-Kyoto rats. In SHRs, there was a strong trend towards an increased orexin-A mRNA expression in the rostral ventrolateral medulla, and blocking orexin receptors markedly lowered blood pressure (from 182/152 ± 5/6 to 149/119 ± 9/8 mmHg; P < 0.001), heart rate (P < 0.001), sympathetic vasomotor tone (P < 0.001) and the noradrenaline levels in cerebrospinal fluid and plasma (P < 0.002). The significant antihypertensive effects of almorexant were observed in wakefulness and non-rapid eye movement sleep during both dark and light phases of the diurnal cycle only in SHRs. Blocking orexin receptors had no effect on blood pressure and sympathetic tone in normotensive Wistar-Kyoto rats. Our study links the orexin system to the pathogenesis of high blood pressure in SHRs and suggests that modulation of the orexin system could be a potential target in treating some forms of hypertension.

Development of brainstem 5-HT1A receptor-binding sites in serotonin-deficient mice.

The sudden infant death syndrome is associated with a reduction in brainstem serotonin 5-hydroxytryptamine (5-HT) and 5-HT(1A) receptor binding, yet it is unknown if and how these findings are linked. In this study, we used quantitative tissue autoradiography to determine if post-natal development of brainstem 5-HT(1A) receptors is altered in two mouse models where the development of 5-HT neurons is defective, the Lmx1b(f/f/p) , and the Pet-1⁻/⁻ mouse. 5-HT(1A) receptor agonist-binding sites were examined in both 5-HT-source nuclei (autoreceptors) and in sites that receive 5-HT innervation (heteroreceptors). In control mice between post-natal day (P) 3 and 10, 5-HT(1A) receptor binding increased in several brainstem sites; by P25, there were region-specific increases and decreases, refining the overall binding pattern. In the Lmx1b(f/f/p) and Pet-1⁻/⁻ mice, 5-HT(1A)-autoreceptor binding was significantly lower than in control mice at P3, and remained low at P10 and P25. In contrast, 5-HT(1A) heteroreceptor levels were comparable between control and 5-HT-deficient mice. These data define the post-natal development of 5-HT(1A)-receptor binding in the mouse brainstem. Furthermore, the data suggest that 5-HT(1A)-heteroreceptor deficits detected in sudden infant death syndrome are not a direct consequence of a 5-HT neuron dysfunction nor reduced brain 5-HT levels. To elucidate the developmental relationship between serotonin (5-HT) levels and 5-HT(1A) receptors in the brainstem, we examined 5-HT(1A) binding in two 5-HT-deficient mouse models. In nuclei containing 5-HT neurons, 5-HT(1A) binding was decreased (autoreceptors), while binding was maintained in projection sites (heteroreceptors). Thus, brainstem 5-HT(1A)-heteroreceptor-binding sites do not appear developmentally sensitive to reduced brain 5-HT levels.

Caffeine improves the ability of serotonin-deficient (Pet-1-/-) mice to survive episodic asphyxia.

BACKGROUND: In neonatal rodents, serotonin (5-HT) neurons are critical for successful autoresuscitation. We hypothesized that caffeine, a respiratory stimulant, would hasten the onset of gasping and improve autoresuscitation in 5-HT-deficient, Pet-1(-/-) mice. METHODS: Using a head-out system and electrocardiogram, we measured respiratory and heart rate (HR) responses of Pet-1(-/-) rodents and their littermates during episodic asphyxia at postnatal days 8-9 (P8-9). After a baseline recording, we injected either vehicle or caffeine (i.p.) at doses of 1, 5, or 10 mg/kg. We then induced 10 brief (~30 s) episodes of asphyxia, each interspersed with 5 min of room air to allow autoresuscitation. In addition to measuring survival, we measured the duration of hypoxic apnea (time to initiate gasping) and time to recover eupnea and HR. RESULTS: Caffeine had a dose-dependent effect of hastening the onset of gasping, recovery of breathing, and restoration of HR in Pet-1(-/-) (but not in wild-type) rodents, thereby improving survival across asphyxic episodes. Increased survival was strongly correlated with hastened onset of gasping. CONCLUSION: Our data suggest that caffeine reduces mortality relating to asphyxia and 5-HT deficiency. These findings may be relevant for efforts to reduce the incidence of sudden infant death syndrome (SIDS), given that SIDS is associated with failed autoresuscitation and reduced brainstem 5-HT.

Sudden and unexpected death in early life: proceedings of a symposium in honor of Dr. Henry F. Krous.

Reported here are the proceedings of a symposium given in honor of Dr. Henry F. Krous upon his retirement as Clinical Professor of Pathology and Pediatrics at the University of California Schools of Medicine, and as Director of the San Diego SIDS/SUDC Research Project. Dr. Krous' distinguished 37-year-career was dedicated to research into sudden unexpected death in infancy and childhood, notably the sudden infant death syndrome (SIDS) and sudden unexplained death in childhood (SUDC). The presentations were given at the International Conference on Stillbirth, SIDS, and infant survival on October 5, 2012, in Baltimore, MD, USA. Eight colleagues of Dr. Krous whose own professional careers were touched by his efforts discussed forensic issues related to SIDS, tissue banking, animal models in SIDS, brainstem studies in SIDS, genetic studies in SIDS, establishment of a SUDC registry, neuropathologic research in SUDC, and potential shared mechanisms underlying sudden and unexpected death in early life. The wide scope of the presentations crossed the disciplines of forensic pathology, pediatric pathology, neuropathology, neuroscience, physiology, genetics, and bereavement, and attest to Dr. Krous' far-reaching influence upon SIDS and SUDC research.

Essential role of Phox2b-expressing ventrolateral brainstem neurons in the chemosensory control of inspiration and expiration.

Phox2b-expressing neurons of the retrotrapezoid nucleus (RTN), located in the ventrolateral brainstem, are sensitive to changes in PCO(2)/pH, have excitatory projections to the central respiratory rhythm/pattern generator, and their activation enhances central respiratory drive. Using in vivo (conscious and anesthetized rats) and in situ (arterially perfused rat brainstem-spinal cord preparations) models, we evaluated the functional significance of this neuronal population for both resting respiratory activity and the CO(2)-evoked respiratory responses by reversibly inhibiting these neurons using the insect peptide allatostatin following transduction with a lentiviral construct to express the G-protein-coupled Drosophila allatostatin receptor. Selective inhibition of the Phox2b-expressing neurons in the ventrolateral brainstem, including the RTN, using allatostatin was without effect on resting respiratory activity in conscious rats, but decreased the amplitude of the phrenic nerve discharge in anesthetized rats and the in situ rat preparations. Postinspiratory activity was also reduced in situ. In the absence or presence of the peripheral chemoreceptor input, inhibiting the Phox2b-expressing neurons during hypercapnia abolished the CO(2)-evoked abdominal expiratory activity in anesthetized rats and in situ preparations. Inspiratory responses evoked by rising levels of CO(2) in the breathing air were also reduced in anesthetized rats with denervated carotid bodies and conscious rats with peripheral chemoreceptors intact (by 28% and 60%, respectively). These data indicate a crucial dependence of central expiratory drive upon Phox2b-expressing neurons of the ventrolateral brainstem and support the hypothesis that these neurons contribute in a significant manner to CO(2)-evoked increases of inspiratory activity.

Brainstem serotonin deficiency in the neonatal period: autonomic dysregulation during mild cold stress.

Based on previous studies in adult animals, devoid of 5-HT neurones, showing altered thermoregulation in cold stress (4°C) and a reduced ventilatory response to CO2, we hypothesized that neonatal mice lacking 60-70% of their 5-HT neurones (Pet-1(-/-)) would have: (1) a reduced thermogenic response to a mild drop in ambient temperature (TA), (2) reduced V(E) and heart rate (HR) responses to mild cooling that reflect this reduced thermogenic response, and (3) a reduced ventilatory response to CO2 after postnatal day 12 (P12), when 5-HT neurones become chemosensitive in vitro. We first determined that a 60-70% loss of 5-HT-positive neurones results in a ~90% loss of 5-HT from the brainstems of Pet-1(-/-) animals. We then subjected Pet-1(-/-) and wild-type (WT) mice (N = 5) to mild environmental cooling (T(A) = 29°C) at ~P12. T(A) was initially held at 34°C for ~20 min, reduced to 29°C over 15 min and held for an additional 10 min at steady state, and then returned to 34°C. From 34°C to 29°C, there was a robust increase in V(O2) in P12WT, but not Pet-1(-/-) animals (68±19.9% versus -16±8%, respectively; P = 0.002). On average, body temperature (T(B)) dropped 1.1°C more in Pet-1(-/-) compared to WT animals (P = 0.03). HR remained unchanged in WT but dropped 22±2.3% in Pet-1(-/-) animals (P = 0.01). Genotype had no effect on tail temperature (T(T)), either at 34°C or 29°C. After cooling, values for V(O2) and HR of Pet-1(-/-) animals were no different to values predicted by Q10 effects alone, while values of WT animals were greater than predicted. V(E) increased in WT with cooling, while it decreased in Pet-1(-/-) animals (P = 0.002). Still, Pet-1(-/-) animals hyperventilated relative to WT (increased V(E)/V(O2)) irrespective of T(A) (P = 0.002). As tested in a separate group of pups, there was no difference in the ventilatory response to CO2 between WT and Pet-1(-/-) animals, either at P5 or P15. We conclude that during neonatal life in mouse pups: (1) brainstem 5-HT is critical for the thermogenic response to a mild drop in environmental temperature probably via a sympathetically-mediated increase in brown fat metabolism; (2) reduced thermogenesis probably contributes to the reduced HR and V(O2) observed with 5-HT deficiency; and (3) the presence of some brainstem 5-HT is sufficient for an appropriate ventilatory response to hypercapnia up until P15. Infants with reduced brainstem 5-HT could be prone to cardiovascular and respiratory abnormalities resulting from compromised thermogenesis.

Postnatal loss of brainstem serotonin neurones compromises the ability of neonatal rats to survive episodic severe hypoxia.

Pet-1(-/-) mice with a prenatal, genetically induced loss of 5-hydroxytryptamine (5-HT, serotonin) neurones are compromised in their ability to withstand episodic environmental anoxia via autoresuscitation. Given the prenatal role of 5-HT neurones in the development of neural networks, here we ask if a postnatal loss of 5-HT neurones also compromises autoresuscitation. We treated neonatal rat pups at postnatal day (P)2-3 with an intra-cisternal injection of 5,7-dihydroxytryptamine (5,7-DHT; ~40 µg; n = 8) to pharmacologically lesion the 5-HT system, or vehicle (control; n = 14). At P7-10 we exposed unanaesthetized treated and control pups to 15 episodes of environmental anoxia (97% N(2), 3% CO(2)). Medullary 5-HT content was reduced 80% by 5,7-DHT treatment (P < 0.001). Baseline ventilation (V(E)), metabolic rate (V(O(2))), ventilatory equivalent (V(E)/V(O(2))), heart rate (HR), heart rate variability (HRV) and arterial haemoglobin saturation (S(aO(2))) were no different in 5-HT-deficient pups compared to controls. However, only 25% of 5-HT-deficient pups survived all 15 episodes of environmental anoxia, compared to 79% of control littermates (P = 0.007). High mortality of 5,7-DHT-treated pups was associated with delayed onset of gasping (P < 0.001), delayed recovery of HR from hypoxic-induced bradycardia (P < 0.001), and delayed recovery of eupnoea from hypoxic-induced apnoea (P < 0.001). Treatment with 5,7-DHT affected neither the gasping pattern once initiated, nor HR, V(E)/V(O(2)) or S(aO(2)) during the intervening episodes of room air. A significant increase in HRV occurred in all animals with repeated exposure, and in 5-HT-deficient pups this increase occurred immediately prior to death. We conclude that a postnatal loss of brainstem 5-HT content compromises autoresuscitation in response to environmental anoxia. This report provides new evidence in rat pups that 5-HT neurones serve a physiological role in autoresuscitation. Our data may be relevant to understanding the aetiology of the sudden infant death syndrome (SIDS), in which there is medullary 5-HT deficiency and in some cases evidence of severe hypoxia and failed autoresuscitation.

Bradycardia in serotonin-deficient Pet-1-/- mice: influence of respiratory dysfunction and hyperthermia over the first 2 postnatal weeks.

Neonatal rodents deficient in medullary serotonin neurons have respiratory instability and enhanced spontaneous bradycardias. This study asks if, in Pet-1(-/-) mice over development: 1) the respiratory instability leads to hypoxia; 2) greater bradycardia is related to the degree of hypoxia or concomitant hypopnea; and 3) hyperthermia exacerbates bradycardias. Pet-1(+/+), Pet-1(+/-), and Pet-1(-/-) mice [postnatal days (P) 4-5, P11-12, P14-15] were held at normal body temperature (T(b)) and were then made 2 degrees C hypo- and hyperthermic. Using a pneumotach-mask system with ECG, we measured heart rate, metabolic rate (Vo(2)), and ventilation. We also calculated indexes for apnea-induced hypoxia (total hypoxia: apnea incidence x O(2) consumed during apnea = microl.g(-1).min(-1)) and bradycardia (total bradycardia: bradycardia incidence x magnitude = beats missed/min). Resting heart rate was significantly lower in all Pet-1(-/-) animals, irrespective of T(b). At P4-5, Pet-1(-/-) animals had approximately four- to eightfold greater total bradycardia (P < 0.001), owing to an approximately two- to threefold increase in bradycardia magnitude and a near doubling in bradycardia incidence. Pet-1(-/-) animals had a significantly reduced Vo(2) at all T(b); thus there was no genotype effect on total hypoxia. At P11-12, total bradycardia was nearly threefold greater in hyperthermic Pet-1(-/-) animals compared with controls (P < 0.01). In both genotypes, bradycardia magnitude was positively related to the degree of hypopnea (P = 0.02), but there was no genotype effect on degree of hypopnea or total hypoxia. At P14-15, genotype had no effect on total bradycardia, but Pet-1(-/-) animals had up to seven times more total hypoxia (P < 0.001), owing to longer and more frequent apneas and a normalized Vo(2). We infer from these data that 1) Pet-1(-/-) neonates are probably not hypoxic from respiratory dysfunction until P14-15; 2) neither apnea-related hypoxia nor greater hypopnea contribute to the enhanced bradycardias of Pet-1(-/-) neonates from approximately P4 to approximately P12; and 3) an enhancement of a temperature-sensitive reflex may contribute to the greater bradycardia in hyperthermic Pet-1(-/-) animals at approximately P12.

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.

Antagonism of orexin receptor-1 in the retrotrapezoid nucleus inhibits the ventilatory response to hypercapnia predominantly in wakefulness.

Recent data from transgenic mice suggest that orexin plays an important role in the ventilatory response to CO(2) during wakefulness. We hypothesized that orexin receptor-1 (OX(1)R) in the retrotrapezoid nucleus (RTN) contributes to chemoreception. In unanaesthetized rats, we measured ventilation using a whole-body plethysmograph, together with EEG and EMG. We dialysed the vehicle and then SB-334867 (OX(1)R antagonist) into the RTN to focally inhibit OX(1)R and studied the effects of both treatments on breathing in air and in 7% CO(2). During wakefulness, SB-334867 caused a 30% reduction of the hyperventilation induced by 7% CO(2) (mean +/- S.E.M., 135 +/- 10 ml (100 g)(-1) min(-1)) compared with vehicle (182 +/- 10 ml (100 g)(-1) min(-1)) (P < 0.01). This effect was due to both decreased tidal volume and breathing frequency. There was a much smaller, though significant, effect in sleep (9% reduction). Neither basal ventilation nor oxygen consumption was affected. The number and duration of apnoeas were similar between control and treatment periods. No effect was observed in a separate group of animals who had the microdialysis probe misplaced (peri-RTN). We conclude that projections of orexin-containing neurons to the RTN contribute, via OX(1)Rs in the region, to the hypercapnic chemoreflex control during wakefulness and to a lesser extent, non-rapid eye movement sleep.

Acoustic plethysmography measures breathing in unrestrained neonatal mice.

Measurement of breathing volumes in neonatal mice is of growing importance in order to characterize the influence of development and genetic modifications on respiratory control to evaluate hypotheses concerned with human infant deficits that may affect sudden infant death syndrome, for example. Current techniques require undesirable physical constraints or incur possible artifacts specific to very small animals. We have examined the utility of a recently proposed approach using an acoustic resonance procedure that does not require undue physical constraint beyond placement in the acoustic plethysmograph. We show here that this approach can be applied to baby mice 5 days after birth and that it can be accurately calibrated. In addition, this approach should be useful to study unrestrained neonatal mice under conditions where body temperature approaches environmental temperature and barometric plethysmography cannot be used.

Ventilatory effects of muscimol microdialysis into the rostral medullary raphé region of conscious rats.

We hypothesized that inhibition of the rostral medullary raphe region (MRR), a putative central chemoreceptor location, with the GABA(A) receptor agonist muscimol would decrease ventilatory responses to hypercapnia and hypoxia in conscious rats, and that its known effect at this site on body temperature might alter its effect upon these ventilatory responses. At ambient temperatures of 24.5-26.5 degrees C (Cool), microdialysis of 1mM muscimol into the MRR significantly decreased body temperature by approximately 0.5 degrees C, increased the ventilatory response to 7% CO(2) and decreased the response to 10% O(2). At ambient temperatures of 29.5-30.5 degrees C (Warm), 1 mM muscimol microdialysis no longer decreased body temperature and increased the ventilatory response to hypercapnia and to hypoxia. Muscimol did not significantly affect the VE/VO2 ratio at either temperature. Muscimol significantly increased the hypercapnic ventilatory responses in Cool and Warm conditions and the hypoxic response in Warm conditions, which indicates the presence of an inhibitory effect of rostral MRR neurons sensitive to muscimol. In the Cool condition the ventilatory response to hypoxia is inhibited but appropriately so for the lower VO2 .

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 x 100 nl) (a) substance P-saporin (SP-SAP; 1 microm) to kill NK1R-expressing neurones, (b) a novel conjugate of a monoclonal antibody to the serotonin transporter (SERT) and saporin (anti-SERT-SAP; 1 microm) to kill serotonergic neurones, or (c) SP-SAP and anti-SERT-SAP together to kill both types. Controls received IgG-SAP injections (1 microm). 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, Tukey's 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 CO(2) in wakefulness and sleep by a similar amount. SP-SAP treatment decreased the averaged CO(2) 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, Tukey's 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.

CO2 dialysis in nucleus tractus solitarius region of rat increases ventilation in sleep and wakefulness.

To evaluate the function of widely distributed central chemoreceptors during sleep and wakefulness in the rat, we focally stimulate single chemoreceptor sites during naturally occurring sleep-wake cycles by microdialysis of artificial cerebrospinal fluid equilibrated with 25% CO2. In retrotrapezoid nucleus, this increased ventilation (tidal volume) by 24% only in wakefulness (Li A, Randall M, and Nattie E. J Appl Physiol 87: 910-919, 1999). In caudal medullary raphé, it increased ventilation (frequency) by 15-20% only in sleep (Nattie EE and Li A. J Appl Physiol 90: 1247-1257, 2001). Here, in nucleus tractus solitarius (NTS), focal acidification significantly increased ventilation by 11% in sleep and 7% in wakefulness rostrally (n = 5) and by 16% in sleep and 28% in wakefulness caudally (n = 5). The sleep-wake cycle was unaltered. Dialysis with 5% CO2 had no effect. Dialysis with 50% CO2 caudally did not further stimulate ventilation but did disrupt sleep. Central chemoreceptors in the NTS affect breathing in both sleep and wakefulness. The threshold for arousal in caudal NTS is greater than that for the stimulation of breathing.

Inhibition of medullary raphe serotonergic neurons has age-dependent effects on the CO2 response in newborn piglets.

Medullary raphé serotonergic neurons are chemosensitive in culture and are situated adjacent to blood vessels in the brain stem. Selective lesioning of serotonergic raphé neurons decreases the ventilatory response to systemic CO2 in awake and sleeping adult rats. Abnormalities in the medullary serotonergic system, including the raphé, have been implicated in the sudden infant death syndrome (48). In this study, we ask whether serotonergic neurons in the medullary raphé and extra-raphé regions are involved in the CO2 response in unanesthetized newborn piglets, 3-16 days old. Whole body plethysmography was used to examine the ventilatory response to 5% CO2 before and during focal inhibition of serotonergic neurons by 8-hydroxy-2-di-n-propylaminotetralin (8-OH-DPAT), a 5-HT1A receptor agonist. 8-OH-DPAT (10 or 30 mM in artificial cerebrospinal fluid) decreased the CO2 response in wakefulness in an age-dependent manner, as revealed by a linear regression analysis that showed a significant negative correlation (P < 0.001) between the percent change in the CO2 response and piglet age. Younger piglets showed an exaggerated CO2 response. Control dialysis with artificial cerebrospinal fluid had no significant effect on the CO2 response. Additionally, 8-OH-DPAT increased blood pressure and decreased heart rate independent of age (P < 0.05). Finally, sleep cycling was disrupted by 8-OH-DPAT, such that piglets were awake more and asleep less (P < 0.05). Because of the fragmentary sleep data, it was not possible to examine the CO2 response in sleep. Inhibition of serotonergic medullary raphé and extra-raphé neurons decreases ventilatory CO2 sensitivity and alters cardiovascular variables and sleep cycling, which may contribute to the sudden infant death syndrome.