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.

Acute perturbation of Pet1-neuron activity in neonatal mice impairs cardiorespiratory homeostatic recovery.

Cardiorespiratory recovery from apneas requires dynamic responses of brainstem circuitry. One implicated component is the raphe system of Pet1-expressing (largely serotonergic) neurons, however their precise requirement neonatally for homeostasis is unclear, yet central toward understanding newborn cardiorespiratory control and dysfunction. Here we show that acute in vivo perturbation of Pet1-neuron activity, via triggering cell-autonomously the synthetic inhibitory receptor hM4Di, resulted in altered baseline cardiorespiratory properties and diminished apnea survival. Respiratory more than heart rate recovery was impaired, uncoupling their normal linear relationship. Disordered gasp recovery from the initial apnea distinguished mice that would go on to die during subsequent apneas. Further, the risk likelihood of apnea-related mortality associated with suppression of Pet1 neurons was higher for animals with baseline elevated ventilatory equivalents for oxygen. These findings establish that Pet1 neurons play an active role in neonatal cardiorespiratory homeostasis and provide mechanistic plausibility for the serotonergic abnormalities associated with SIDS.

Pre- and early postnatal nicotine exposure exacerbates autoresuscitation failure in serotonin-deficient rat neonates.

KEY POINTS: Sudden infant death syndrome (SIDS) is one of the leading causes of death during the first year of life and abnormalities linked to serotonin (5-HT) have been identified in many SIDS cases. Cigarette smoking and associated exogenous stressors, e.g. developmental nicotine exposure, may compound these serotonergic defects and any associated defects in cardiorespiratory function. Using neonatal rodent pups subjected to medullary 5-HT deficiency and perinatal nicotine exposure, we examined the impact of this interplay of factors on the neonates' ability to autoresuscitate at specific ages. In perinatal nicotine-exposed 5-HT deficient pups, impaired autoresuscitation along with significantly delayed post-anoxic recovery of normal breathing and heart rate was observed at postnatal day 10 (P10). We found that the interaction between 5-HT deficiency and perinatal nicotine exposure can significantly increase pups' vulnerability to environmental stressors and exacerbate defects in cardiorespiratory protective reflexes to repetitive anoxia during the development period. ABSTRACT: Cigarette smoking during pregnancy increases the risk of sudden infant death syndrome (SIDS), and nicotine replacements, a key ingredient of cigarettes, have been recently prescribed to women who wish to quit smoking during their pregnancy. Serotonin (5-HT) abnormalities have been consistently identified in many SIDS cases. Here we investigated the effects of perinatal nicotine exposure in mild 5-HT deficiency rat neonates on autoresuscitation, a protective cardiorespiratory reflex. The mild 5-HT deficiency was induced by a maternal tryptophan-deficient diet, and nicotine was delivered from embryonic day (E) 4 to postnatal day (P) 10 at 6 mg kg-1  day-1 through an osmotic pump. In P10 rats, nicotine exposure exacerbates autoresuscitation failure (mortality) in mildly 5-HT-deficient rats to a greater extent than in controls (P = 0.029). The recovery of eupnoea and heart rate to baseline values following repetitive anoxic events (which elicit an apnoea accompanied by a bradycardia) is significantly delayed in 5-HT-deficient rats treated with nicotine, making them more susceptible to failure of autoresuscitation (eupnoea recovery: P = 0.0053; heart rate recovery: P = < 0.0001). Neither 5-HT deficiency nor nicotine exposure alone appears to affect the ability to autoresuscitate significantly when compared among the four treatments. The increased vulnerability to environmental stressors, e.g. severe hypoxia, asphyxia, or anoxia, in these nicotine-exposed 5-HT-deficient neonates during postnatal developmental period is evident.

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.

Functional and developmental identification of a molecular subtype of brain serotonergic neuron specialized to regulate breathing dynamics.

Serotonergic neurons modulate behavioral and physiological responses from aggression and anxiety to breathing and thermoregulation. Disorders involving serotonin (5HT) dysregulation are commensurately heterogeneous and numerous. We hypothesized that this breadth in functionality derives in part from a developmentally determined substructure of distinct subtypes of 5HT neurons each specialized to modulate specific behaviors. By manipulating developmentally defined subgroups one by one chemogenetically, we find that the Egr2-Pet1 subgroup is specialized to drive increased ventilation in response to carbon dioxide elevation and acidosis. Furthermore, this subtype exhibits intrinsic chemosensitivity and modality-specific projections-increasing firing during hypercapnic acidosis and selectively projecting to respiratory chemosensory but not motor centers, respectively. These findings show that serotonergic regulation of the respiratory chemoreflex is mediated by a specialized molecular subtype of 5HT neuron harboring unique physiological, biophysical, and hodological properties specified developmentally and demonstrate that the serotonergic system contains specialized modules contributing to its collective functional breadth.

The role of melanin concentrating hormone (MCH) in the central chemoreflex: a knockdown study by siRNA in the lateral hypothalamus in rats.

Melanin concentrating hormone (MCH), a neuropeptide produced mainly in neurons localized to the lateral hypothalamic area (LHA), has been implicated in the regulation of food intake, energy balance, sleep state, and the cardiovascular system. Hypothalamic MCH neurons also have multisynaptic connections with diaphragmatic motoneurons and project to many central chemoreceptor sites. However, there are few studies of MCH involvement in central respiratory control. To test the hypothesis that MCH plays a role in the central chemoreflex, we induced a down regulation of MCH in the central nervous system by knocking down the MCH precursor (pMCH) mRNA in the LHA using a pool of small interfering RNA (siRNA), and measured the resultant changes in breathing, metabolic rate, body weight, and blood glucose levels in conscious rats. The injections of pMCH-siRNA into the LHA successfully produced a ∼ 62% reduction of pMCH mRNA expression in the LHA and a ∼ 43% decrease of MCH levels in the cerebrospinal fluid relative to scrambled-siRNA treatment (P = 0.006 and P = 0.02 respectively). Compared to the pretreatment baseline and the scrambled-siRNA treated control rats, knockdown of MCH resulted in: 1) an enhanced hypercapnic chemoreflex (∼ 42 & 47% respectively; P < 0.05) only in wakefulness; 2) a decrease in body weight and basal glucose levels; and 3) an unchanged metabolic rate. Our results indicate that MCH participates not only in the regulation of glucose and sleep-wake homeostasis but also the vigilance-state dependent regulation of the central hypercapnic chemoreflex and respiratory control.

Mice lacking G0S2 are lean and cold-tolerant.

G 0/G 1 switch gene 2 (G0S2) is a protein that was first identified in a search for lymphocyte G 0/G 1 switch genes. A direct role for G0S2 in cell cycle regulation has proven elusive. Yet, there is prior evidence for G0S2 functioning in tumor suppression, immune regulation and lipolysis. To explore definitively G0S2 functions, mice lacking G0S2 were generated and characterized. G0S2(-/-) mice were born at a Mendelian ratio and were phenotypically normal, with the exception of a possible lactation defect. G0S2(-/-) female mice carried viable pups to term, but could not typically sustain them beyond 48 h. G0S2 is shown here to be most highly expressed in adipose tissue. It is also expressed in liver, skeletal muscle, lung, ventricles of the heart, and components of the kidney. G0S2 loss significantly decreased relative body weight gain as compared with wild-type (WT) (G0S2(+/+)) mice, with a significant decrease in gonadal fat pad weight and a significant increase in serum glycerol levels. This decreased relative body weight gain is not associated with a significant decrease in food intake or increase in activity of G0S2(-/-) mice. In fact, G0S2(-/-) mice were significantly less active at night than G0S2(+/+) mice. When fed with a high fat diet (45% fat diet), G0S2 loss did not prevent diet-induced obesity in mice. Intriguingly, G0S2 loss improved acute cold tolerance, augmenting expression of genes involved in thermogenesis. In summary, in vivo roles for G0S2 were found in lactation, energy balance, and thermogenesis. This study provides a basis for tumor suppressive effects of G0S2 by regulating lipolysis.

Orexin, cardio-respiratory function, and hypertension.

In this review we focus on the role of orexin in cardio-respiratory functions and its potential link to hypertension. (1) Orexin, cardiovascular function, and hypertension. In normal rats, central administration of orexin can induce significant increases in arterial blood pressure (ABP) and sympathetic nerve activity (SNA), which can be blocked by orexin receptor antagonists. In spontaneously hypertensive rats (SHRs), antagonizing orexin receptors can significantly lower blood pressure under anesthetized or conscious conditions. (2) Orexin, respiratory function, and central chemoreception. The prepro-orexin knockout mouse has a significantly attenuated ventilatory CO2 chemoreflex, and in normal rats, central application of orexin stimulates breathing while blocking orexin receptors decreases the ventilatory CO2 chemoreflex. Interestingly, SHRs have a significantly increased ventilatory CO2 chemoreflex relative to normotensive WKY rats and blocking both orexin receptors can normalize this exaggerated response. (3) Orexin, central chemoreception, and hypertension. SHRs have higher ABP and SNA along with an enhanced ventilatory CO2 chemoreflex. Treating SHRs by blocking both orexin receptors with oral administration of an antagonist, almorexant (Almxt), can normalize the CO2 chemoreflex and significantly lower ABP and SNA. We interpret these results to suggest that the orexin system participates in the pathogenesis and maintenance of high blood pressure in SHRs, and the central chemoreflex may be a causal link to the increased SNA and ABP in SHRs. Modulation of the orexin system could be a potential target in treating some forms of hypertension.

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.

Focal microdialysis of CO2 in the perifornical-hypothalamic area increases ventilation during wakefulness but not NREM sleep.

We investigated whether the perifornical-lateral hypothalamic area (PF-LHA), where the orexin neurons reside, is a central chemoreceptor site by microdialysis of artificial cerebrospinal fluid (aCSF) equilibrated with 25% CO(2) into PF-LHA in conscious rats. This treatment is known to produce a focal tissue acidification like that associated with a 6-7 mm Hg increase in arterial [Formula: see text] . Such focal acidification in the PF-LHA significantly increased ventilation up to 15% compared with microdialysis of normal aCSF equilibrated with 5% CO(2) only in wakefulness but not in sleep in both the dark (P=0.004) and light (P<0.001) phases of the diurnal cycle. This response was predominantly due to a significant increase in respiratory frequency (11%, P<0.001). There were no significant effects on ventilation in the group with probes misplaced outside the PF-LHA. These results suggest that PF-LHA functions as a central chemoreceptor site in the central nervous system in a vigilant state dependent manner with predominant effects in wakefulness.

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.

Egr2-neurons control the adult respiratory response to hypercapnia.

`The early growth response 2 transcription factor, Egr2, establishes a population of brainstem neurons essential for normal breathing at birth. Egr2-null mice die perinatally of respiratory insufficiency characterized by subnormal respiratory rate and severe apneas. Here we bypass this lethality using a noninvasive pharmacogenetic approach to inducibly perturb neuron activity postnatally, and ask if Egr2-neurons control respiration in adult mice. We found that the normal ventilatory increase in response to elevated tissue CO2 was impaired, blunted by 63.1 ± 8.7% after neuron perturbation due to deficits in both respiratory amplitude and frequency. By contrast, room-air breathing was unaffected, suggesting that the drive for baseline breathing may not require those Egr2-neurons manipulated here. Of the multiple brainstem sites proposed to affect ventilation in response to hypercapnia, only the retrotrapezoid nucleus, a portion of the serotonergic raphé, and a portion of the A5 nucleus have a history of Egr2 expression. We recently showed that acute inhibition of serotonergic neurons en masse blunts the CO2 chemoreflex in adults, causing a difference in hypercapnic response of ∼50% after neuron perturbation through effects on respiratory amplitude only. The suppressed respiratory frequency upon perturbation of Egr2-neurons thus may stem from non-serotonergic neurons within the Egr2 domain. Perturbation of Egr2-neurons did not affect body temperature, even on exposure to ambient 4°C. These findings support a model in which Egr2-neurons are a critical component of the respiratory chemoreflex into adulthood. Methodologically, these results highlight how pharmacogenetic approaches allow neuron function to be queried in unanesthetized adult animals, reaching beyond the roadblocks of developmental lethality and compensation as well as the anatomical disturbances associated with invasive methods. This article is part of a Special Issue entitled Optogenetics (7th BRES).

Subtle alterations in breathing and heart rate control in the 5-HT1A receptor knockout mouse in early postnatal development.

We hypothesized that absence of the 5-HT(1A) receptor would negatively affect the development of cardiorespiratory control. In conscious wild type (WT) and 5-HT(1A) receptor knockout (KO) mice, we measured resting ventilation (Ve), oxygen consumption (Vo(2)), heart rate (HR), breathing and HR variability, and the hypercapnic ventilatory response (HCVR) at postnatal day 5 (P5), day 15 (P15), and day 25 (P25). In KO mice compared with WT, we found a 17% decrease in body weight at only P5 (P < 0.01) and no effect on Vo(2). Ve was significantly (P < 0.001) lower at P5 and P25, but there was no effect on the HCVR. Breathing variability (interbreath interval), measured by standard deviation, the root mean square of the standard deviation (RMSSD), and the product of the major (L) and minor axes (T) of the Poincaré first return plot, was 57% to 187% higher only at P5 (P < 0.001). HR was 6-10% slower at P5 (P < 0.001) but 7-9% faster at P25 (P < 0.001). This correlated with changes in the spectral analysis of HR variability; the low frequency to high frequency ratio was 47% lower at P5 but 68% greater at P25. The RMSSD and (L × T) of HR variability were ~2-fold greater at P5 only (P < 0.001; P < 0.05). We conclude that 5-HT(1A) KO mice have a critical period of potential vulnerability at P5 when pups hypoventilate and have a slower respiratory frequency and HR with enhanced variability of both, suggesting abnormal maturation of cardiorespiratory control.

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.

Respiration and autonomic regulation and orexin.

Orexin, a small neuropeptide released from neurons in the hypothalamus with widespread projections throughout the central nervous system, has broad biological roles including the modulation of breathing and autonomic function. That orexin activity is fundamentally dependent on sleep-wake state, and circadian cycle requires consideration of orexin function in physiological control systems in respect to these two state-related activity patterns. Both transgenic mouse studies and focal orexin receptor antagonism support a role for orexins in respiratory chemosensitivity to CO2 predominantly in wakefulness, with further observations limiting this role to the dark period. In addition, orexin neurons participate in the regulation of sympathetic activity, including effects on blood pressure and thermoregulation. Orexin is also essential in physiological responses to stress. Orexin-mediated processes may operate at two levels: (1) in sleep-wake and circadian states and (2) in stress, for example, the defense or "fight-or-flight" response and panic-anxiety syndrome.