Metabolomic profiling of preterm birth in pregnant women living with HIV.

BACKGROUND: Preterm birth is a leading cause of death in children under the age of five. The risk of preterm birth is increased by maternal HIV infection as well as by certain antiretroviral regimens, leading to a disproportionate burden on low- and medium-income settings where HIV is most prevalent. Despite decades of research, the mechanisms underlying spontaneous preterm birth, particularly in resource limited areas with high HIV infection rates, are still poorly understood and accurate prediction and therapeutic intervention remain elusive. OBJECTIVES: Metabolomics was utilized to identify profiles of preterm birth among pregnant women living with HIV on two different antiretroviral therapy (ART) regimens. METHODS: This pilot study comprised 100 mother-infant dyads prior to antiretroviral initiation, on zidovudine monotherapy or on protease inhibitor-based antiretroviral therapy. Pregnancies that resulted in preterm births were matched 1:1 with controls by gestational age at time of sample collection. Maternal plasma and blood spots at 23-35 weeks gestation and infant dried blood spots at birth, were assayed using an untargeted metabolomics method. Linear regression and random forests classification models were used to identify shared and treatment-specific markers of preterm birth. RESULTS: Classification models for preterm birth achieved accuracies of 95.5%, 95.7%, and 80.7% in the untreated, zidovudine monotherapy, and protease inhibitor-based treatment groups, respectively. Urate, methionine sulfone, cortisone, and 17α-hydroxypregnanolone glucuronide were identified as shared markers of preterm birth. Other compounds including hippurate and N-acetyl-1-methylhistidine were found to be significantly altered in a treatment-specific context. CONCLUSION: This study identified previously known as well as novel metabolomic features of preterm birth in pregnant women living with HIV. Validation of these models in a larger, independent cohort is necessary to ascertain whether they can be utilized to predict preterm birth during a stage of gestation that allows for therapeutic intervention or more effective resource allocation.

Approaches to Preventing Intrapartum Fetal Injury.

Electronic fetal monitoring (EFM) was introduced into obstetric practice in 1970 as a test to identify early deterioration of fetal acid-base balance in the expectation that prompt intervention ("rescue") would reduce neonatal morbidity and mortality. Clinical trials using a variety of visual or computer-based classifications and algorithms for intervention have failed repeatedly to demonstrate improved immediate or long-term outcomes with this technique, which has, however, contributed to an increased rate of operative deliveries (deemed "unnecessary"). In this review, we discuss the limitations of current classifications of FHR patterns and management guidelines based on them. We argue that these clinical and computer-based formulations pay too much attention to the detection of systemic fetal acidosis/hypoxia and too little attention not only to the pathophysiology of FHR patterns but to the provenance of fetal neurological injury and to the relationship of intrapartum injury to the condition of the newborn. Although they do not reliably predict fetal acidosis, FHR patterns, properly interpreted in the context of the clinical circumstances, do reliably identify fetal neurological integrity (behavior) and are a biomarker of fetal neurological injury (separate from asphyxia). They provide insight into the mechanisms and trajectory (evolution) of any hypoxic or ischemic threat to the fetus and have particular promise in signaling preventive measures (1) to enhance the outcome, (2) to reduce the frequency of "abnormal" FHR patterns that require urgent intervention, and (3) to inform the decision to provide neuroprotection to the newborn.

Early pregnancy metabolites predict gestational diabetes mellitus: implications for fetal programming.

BACKGROUND: Aberrant fetal programming in gestational diabetes mellitus seems to increase the risk of obesity, type 2 diabetes, and cardiovascular disease. The inability to accurately identify gestational diabetes mellitus in the first trimester of pregnancy has thwarted ascertaining whether early therapeutic interventions reduce the predisposition to these prevalent medical disorders. OBJECTIVE: A metabolomics study was conducted to determine whether advanced analytical methods could identify accurate predictors of gestational diabetes mellitus in early pregnancy. STUDY DESIGN: This nested observational case-control study was composed of 92 gravidas (46 in the gestational diabetes mellitus group and 46 in the control group) in early pregnancy, who were matched by maternal age, body mass index, and gestational age at urine collection. Gestational diabetes mellitus was diagnosed according to community standards. A comprehensive metabolomics platform measured 626 endogenous metabolites in randomly collected urine. Consensus multivariate criteria or the most important by 1 method identified low-molecular weight metabolites independently associated with gestational diabetes mellitus, and a classification tree selected a subset most predictive of gestational diabetes mellitus. RESULTS: Urine for both groups was collected at a mean gestational age of 12 weeks (range, 6-19 weeks' gestation). Consensus multivariate analysis identified 11 metabolites independently linked to gestational diabetes mellitus. Classification tree analysis selected a 7-metabolite subset that predicted gestational diabetes mellitus with an accuracy of 96.7%, independent of maternal age, body mass index, and time of urine collection. CONCLUSION: Validation of this high-accuracy model by a larger study is now needed to support future studies to determine whether therapeutic interventions in the first trimester of pregnancy for gestational diabetes mellitus reduce short- and long-term morbidity.

Maternal Central Blood Pressure Is Associated with Fetal Middle Cerebral Artery Dopplers.

Cardiovascular adaptations to pregnancy involve physiological mechanisms that increase cardiac output, decrease total vascular resistance, and decrease both systolic and diastolic blood pressure (BP). These maternal hemodynamic changes modulate uteroplacental blood flow and fetal-placental Doppler indices. Our objective was to create maternal cardiac profiles of pregnant women using non-invasive measurements of central BP to identify changes in maternal-fetal hemodynamics as a surrogate to fetal status. This was a prospective cohort study of all singleton pregnancies in a perinatal referral center between January and April 2018. Central BP was measured non-invasively using the BP+ device. The BP+ device is a supra-systolic oscillometric central BP device, which measures BP waveforms peripherally and calculates central BP. We compared various BP+ values for peripheral BP with central BP and stratified by gestational age. We investigated the correlations between peripheral BP, central BP, estimated fetal weight (EFW), and the pulsatility indices (PI) of Doppler velocimetry and demonstrate that both central systolic and diastolic BP correlated to peripheral systolic and diastolic BP. Linear regression analysis confirmed that central BP predicts the middle cerebral artery (MCA) PI. The MCA PI correlated with EFW, specifically higher central systolic BP is associated with a lower MCA PI, implying a possible etiology of fetal brain shunting with poor placental perfusion. Future studies using predictors and markers of fetal outcomes from maternal cardiac parameters should consider maternal cardiovascular measurements to peripheral arterial BP.

Maternal Fontan procedure is a predictor of a small-for-gestational-age neonate: a 10-year retrospective study.

BACKGROUND: Women with single ventricle cardiac physiologic condition who have undergone Fontan procedures are surviving well into reproductive age and historically have been discouraged from pregnancy, despite the paucity of data regarding maternal and neonatal outcomes. OBJECTIVE: Our primary objective was to investigate, in a large cohort, the maternal and neonatal outcomes of pregnant women who have undergone the Fontan procedure and to understand maternal and neonatal sequelae of their pregnancies. STUDY DESIGN: This single-center retrospective cohort study involves pregnant women with a Fontan palliation who delivered at UCLA Medical Center over a 10-year period (2007-2017). All pregnancies were evaluated for differences in maternal and neonatal characteristics. RESULTS: We identified 37 distinct pregnancies in 24 women with a Fontan procedure. The physiologic pregnancy-related increase in cardiac output is blunted substantially in Fontan circulation. Third-trimester cardiac index positively correlated to birthweight z-score (R2=0.48; P=.038) but not to small for gestational age (R2=0.13; P=.339). The most common cardiac complications in pregnancies of >24 weeks gestation were sustained arrhythmia (37.5%) and decompensated heart failure (21%). The 37 pregnancies comprised 25 live births (67.6%), 1 fetal death (2.7%), 9 spontaneous abortions (24%), and 2 pregnancy terminations (5.4%). Of the live births, 60% were preterm at an average gestational age of 34.9±3.7 weeks. Newborn infants were delivered via cesarean in 53%, operative vaginal delivery in 28%, and spontaneous vaginal delivery in 20%. Forty percent of neonates were born small (<10th percentile) for gestational age; 44.0% of all neonates were admitted to the neonatal intensive care unit. CONCLUSION: Women with a single ventricle and Fontan circulation can have a successful pregnancy, although they are at increased risk for arrhythmias and heart failure. The decreased cardiac reserve in these pregnancies blunts the normal increase in maternal cardiac output, which is associated with preterm delivery and small-for-gestational-age neonates. Further studies are needed to determine to what extent the impaired rise in maternal cardiac output reduces uteroplacental perfusion, placental exchange, fetal growth, and onset of parturition.

Management of Pregnancy in Patients With Complex Congenital Heart Disease: A Scientific Statement for Healthcare Professionals From the American Heart Association.

Today, most female children born with congenital heart disease will reach childbearing age. For many women with complex congenital heart disease, carrying a pregnancy carries a moderate to high risk for both the mother and her fetus. Many such women, however, do not have access to adult congenital heart disease tertiary centers with experienced reproductive programs. Therefore, it is important that all practitioners who will be managing these women have current information not only on preconception counseling and diagnostic evaluation to determine maternal and fetal risk but also on how to manage them once they are pregnant and when to refer them to a regional center with expertise in pregnancy management.

Cardiovascular and Neonatal Outcomes in Pregnant Women With High-Risk Congenital Heart Disease.

Congenital heart disease (CHD) increases the risk of adverse maternal and neonatal outcomes. However, previous studies have included mainly women with low-risk features. A single-center, retrospective analysis of pregnant women with CHD was performed. Inclusion criteria were the following high-risk congenital lesions and co-morbidities: maternal cyanosis; New York Heart Association (NHYA) functional class >II; severe ventricular dysfunction; maternal arrhythmia, single ventricle (SV) physiology, severe left-sided heart obstruction and severe pulmonary arterial hypertension. Multivariate analyses for predictors of adverse maternal cardiovascular and neonatal outcomes were performed. Forty-three women reported 61 pregnancies. There were no maternal or neonatal deaths. Maternal cardiac (31%) and neonatal (54%) complications were frequent. The most frequent cardiac events were pulmonary edema, arrhythmia, and reduced NYHA class. Previous arrhythmia conferred a 12-fold increase in the odds of experiencing at least one major cardiac complication. Maternal SV physiology was an independent risk factor for low birth weight, risk of neonatal intensive care unit admission and lower gestational age. Maternal cyanosis and severe pulmonary arterial hypertension also predicted adverse neonatal outcomes. In conclusion, mothers without antepartum arrhythmia or functional incapacity are unlikely to experience arrhythmias or a decrease in NYHA class during pregnancy. In addition, SV physiology is a robust predictor of neonatal complications. Antepartum counseling and assessment of maternal fitness are crucial for the woman with CHD.

Thalamic mediation of hypoxic respiratory depression in lambs.

Immaturity of respiratory controllers in preterm infants dispose to recurrent apnea and oxygen deprivation. Accompanying reductions in brain oxygen tensions evoke respiratory depression, potentially exacerbating hypoxemia. Central respiratory depression during moderate hypoxia is revealed in the ventilatory decline following initial augmentation. This study determined whether the thalamic parafascicular nuclear (Pf) complex involved in adult nociception and sensorimotor regulation (Bentivoglio M, Balerecia G, Kruger L. Prog Brain Res 87: 53-80, 1991) also becomes a postnatal controller of hypoxic ventilatory decline. Respiratory responses to moderate isocapnic hypoxia were studied in conscious lambs. Hypoxic ventilatory decline was compared with peak augmentation. Pf and/or adjacent thalamic structures were destroyed by the neuron-specific toxin ibotenic acid (IB). IB lesions involving the thalamic Pf abolished hypoxic ventilatory decline. Lesions of adjacent thalamic nuclei that spared Pf and control injections of vehicle failed to blunt hypoxic respiratory depression. Our findings reveal that the thalamic Pf region is a critical controller of hypoxic ventilatory depression and thus a key target for exploring molecular concomitants of forebrain pathways regulating hypoxic ventilatory depression in early development.

Fetal breathing movements and changes at birth.

The fetus, which develops within a fluid-filled amniotic sac, relies on the placenta for respiratory gas exchange rather than the lungs. While not involved in fetal oxygenation, fetal breathing movements (FBM) nevertheless have an important role in lung growth and in development of respiratory muscles and neural regulation. FBM are regulated differently in many respects than postnatal respiration, which results from the unique intrauterine environment. Prominent distinctions of FBM include its episodic nature and apnea-sensitivity to hypoxia. The latter characteristic is the basis for using FBM in the assessment of fetuses at risk for hypoxic injury. At birth, the transition to continuous postnatal respiration involves a fall in temperature, gaseous distention of the lungs, activation of the Hering-Breuer reflexes, and functional connectivity of afferent O2 chemoreceptor activity with respiratory motoneurons and arousal centers. Importantly, exposure to drugs or adverse conditions in utero not only can change patterns of FBM but also can lead to epigenetic dysregulation in postnatal respiration. Such changes, can blunt respiratory and arousal defenses against hypoxic challenges in sleep. Thus, fetal hypoxia and/or drug exposure may in later life dispose sleeping infants, children, and adults to hypertension, diabetes mellitus, brain injury, and sudden death.

Adenosine A2a receptors and O2 sensing in development.

Reduced mitochondrial oxidative phosphorylation, via activation of adenylate kinase and the resulting exponential rise in the cellular AMP/ATP ratio, appears to be a critical factor underlying O2 sensing in many chemoreceptive tissues in mammals. The elevated AMP/ATP ratio, in turn, activates key enzymes that are involved in physiologic adjustments that tend to balance ATP supply and demand. An example is the conversion of AMP to adenosine via 5'-nucleotidase and the resulting activation of adenosine A(2A) receptors, which are involved in acute oxygen sensing by both carotid bodies and the brain. In fetal sheep, A(2A) receptors associated with carotid bodies trigger hypoxic cardiovascular chemoreflexes, while central A(2A) receptors mediate hypoxic inhibition of breathing and rapid eye movements. A(2A) receptors are also involved in hypoxic regulation of fetal endocrine systems, metabolism, and vascular tone. In developing lambs, A(2A) receptors play virtually no role in O2 sensing by the carotid bodies, but brain A(2A) receptors remain critically involved in the roll-off ventilatory response to hypoxia. In adult mammals, A(2A) receptors have been implicated in O2 sensing by carotid glomus cells, while central A(2A) receptors likely blunt hypoxic hyperventilation. In conclusion, A(2A) receptors are crucially involved in the transduction mechanisms of O2 sensing in fetal carotid bodies and brains. Postnatally, central A(2A) receptors remain key mediators of hypoxic respiratory depression, but they are less critical for O2 sensing in carotid chemoreceptors, particularly in developing lambs.

Adenosine A1 and A2a receptors modulate insulinemia, glycemia, and lactatemia in fetal sheep.

Adenosine A(1) and A(2A) receptor subtypes modulate metabolism in adult mammals. This study was designed to determine the role of these receptors in regulating plasma levels of insulin, glucose, and lactate in 20 chronically catheterized fetal sheep (>0.8 term). In normoxic fetuses (Pa(O(2)) approximately 24 Torr), systemic blockade of A(1) receptors with DPCPX (n = 6) increased plasma concentrations of insulin, glucose, and lactate, but antagonism of A(2A) receptors with ZM-241385 (n = 5) had no significant effects. Intravascular administration of adenosine (n = 9) reduced insulin concentrations and elevated glucose and lactate levels. DPCPX (n = 6) augmented the glycemic and lactatemic responses of adenosine. In contrast, ZM241385 (n = 5) virtually abolished adenosine-induced hyperglycemia and hyperlactatemia. Isocapnic hypoxia (Pa(O(2)) approximately 13 Torr) suppressed insulinemia and enhanced glycemia and lactatemia, but only the hyperglycemia was blunted by blockade of A(1) (n = 6) or A(2A) (n = 6) receptors. We conclude that 1) endogenous adenosine via A(1) receptors depresses plasma concentrations of insulin, glucose, and lactate; 2) exogenous adenosine via A(2A) receptors increases glucose and lactate levels, but these responses are dampened by stimulation of A(1) receptors; and 3) hypoxia, which increases endogenous adenosine concentrations, induces hyperglycemia that is partly mediated by activation of A(1) and A(2A) receptors. We predict that adenosine, via A(1) receptors, facilitates at least 12% of glucose uptake and utilization in normoxic fetuses.

Increased senescence and reduced functional ability of fetal endothelial progenitor cells in pregnancies complicated by preeclampsia without intrauterine growth restriction.

OBJECTIVE: The aim of this study was to investigate the number and functional ability of fetal endothelial progenitor cells in pregnancies complicated by preeclampsia without intrauterine growth restriction. STUDY DESIGN: Fetal endothelial progenitor cells were isolated, and counted from 17 women with preeclampsia without intrauterine growth restricion and 30 normal women. Colony-forming assay and differentiation time assay were performed to detect functional activity of the cells. To assess cellular senescence, senescence-associated beta-galactosidase staining was performed for endothelial progenitor cells. RESULTS: Compared with normal pregnancy, the number of endothelial progenitor cells was significantly lower, differentiation time from endothelial progenitor cell into outgrowing cell was longer, and the number of colonies after differentiation was smaller in preeclampsia (P< .001), respectively. The intensity of senescence-associated beta-galactosidase staining was higher in preeclamptic pregnancy (P < .001). CONCLUSION: The number and functional ability of fetal endothelial progenitor cells from preeclampsia without intrauterine growth restriction are significantly decreased and they are more senescent compared with those of normal pregnancy.

Ultrasound and fetal diagnosis.

PURPOSE OF REVIEW: The purpose of this review is to highlight publications from the last year that have advanced the use of ultrasound in obstetrics. RECENT FINDINGS: Anatomic examination of the fetus in the first trimester has been emphasized because it allows for early diagnosis of many conditions. The prevalence of absent nasal bone, a marker for trisomy 21, in euploid fetuses depends on ethnicity. Nasal bone hypoplasia is another marker for Down syndrome. Studies on genetic screening in the first trimester have involved various serum analytes, adjustments in timing and calculations, use in multiple gestations, and the association of extreme measurements with adverse outcomes. A first-trimester integrated screening approach, which incorporates nuchal translucency, nasal bone, crown-rump length, pregnancy-associated plasma protein-A, and free beta-human chorionic gonadotropin, has the potential to maximize detection rates of Down syndrome and trisomy 18 and minimizes the screen-positive rate. The value of combining first and second-trimester results in sequential, contingent, or integrated screening protocols has been assessed. Isolated mild ventriculomegaly (10-12 mm) may prove to be a normal variant, and the role of 'soft' ultrasound markers in genetic counseling continues to be debated. Anomaly or high-risk status detection in the second trimester has been enhanced by the use of Doppler, 3D/4D ultrasound, and magnetic resonance imaging. SUMMARY: Imaging techniques have been critical in the development of screening methods for Down syndrome or trisomy 18 and for euploid fetuses at high risk for adverse outcomes.

Adenosine A2A-receptor blockade abolishes the roll-off respiratory response to hypoxia in awake lambs.

Adenosine (ADO) receptor antagonists (aminophylline, caffeine) blunt the respiratory roll-off response to hypoxia in the newborn. This study was designed to determine the ADO receptor subtype involved in the respiratory depression. Chronically catheterized lambs of 7-16 days of age breathed via face mask a gas mixture with a fraction of inspired O2 of 0.21 (normoxia) or 0.07 (hypoxia), while being infused intravascularly with 9-cyclopentyl-1,3-dipropylxanthine (DPCPX; ADO A1-receptor antagonist, n=8), ZM-241385 (ADO A2A-receptor antagonist, n=7), or vehicle. Ventilation was measured at 20 degrees C by a turbine transducer flowmeter. In normoxia [arterial Po2 (PaO2) of approximately 83 Torr], infusion of vehicle did not alter cardiorespiratory measurements, whereas hypoxia (PaO2 of approximately 31 Torr, 15 min) elicited biphasic effects on mean arterial pressure (transient increase), heart rate (HR; diminishing tachycardia), and minute ventilation. In the latter, hypoxia increased ventilation to a peak value of approximately 2.5 times control within the first 3 min, which was followed by a significant (P<0.05) decline to approximately 50% of the maximum increment over the subsequent 7 min. ZM-241385 abolished the hypoxic ventilatory roll-off and blunted the rate of rise in HR without affecting mean arterial pressure or rectal temperature responses. In normoxia, DPCPX increased ventilation and mean arterial pressure but did not change HR. Compared with vehicle, DPCPX did not significantly affect cardiorespiratory responses to hypoxemia (PaO2 of approximately 31 Torr, 10 min). It is concluded that 1) ADO A2A receptors are critically involved in the ventilatory roll-off and HR responses to hypoxia, and 2) ADO A1 receptors, which are tonically active in cardiorespiratory control in normoxia, appear to have little impact on hypoxic ventilatory depression.

Induced fetal depressor or pressor responses associated with c-fos by intravenous or intracerebroventricular losartan.

Previous fetal studies have indicated depressor responses of intravenous (i.v.) administration of angiotensin antagonists. However, little is known of central effects of angiotensin blockers on fetal cardiovascular controlling. The cardiovascular effects of central administration of the angiotensin-1 (AT(1)) and angiotensin-2 (AT(2)) receptor antagonists, losartan and PD123319, were investigated in the chronically catheterized near-term ovine fetuses. Intravenous losartan produced within 1.5 min a significant and persistent depressor response [maximum Delta mean arterial pressure (MAP)=9 mm Hg] without altering fetal heart rate. Intracerebroventricular (i.c.v.) administration of losartan (1-5 mg/kg) increased fetal arterial pressures (Delta MAP=9-14 mm Hg). Central application of losartan (1 mg/kg) also increased fetal heart rate (maximum Delta heart rate=33 beat per minute). Losartan increased c-fos expression in the median preoptic nucleus and paraventricular nuclei in the forebrain, and the tractus solitarius nuclei, the lateral parabrachial nuclei, and the ventrolateral medullabrain. These brain sectors are with abundant AT(1) receptors and have been demonstrated in the involvement in cardiovascular regulation. In contrast, intracerebroventricular injection of the AT(2) receptor antagonist PD123319 had no effect on fetal arterial pressure and heart rate. The results demonstrate strikingly functional differences of losartan on the fetal cardiovascular regulation in central and peripheral sides.

Electrical stimulation of the posteromedial thalamus modulates breathing in unanesthetized fetal sheep.

Having previously shown that lesions in the posteromedial group of thalamic nuclei abolish hypoxic inhibition of fetal breathing, we devised this study to identify thalamic loci that depress breathing by focal stimulation of specific sectors of the caudal thalamus and adjacent structures. Multipolar electrode arrays consisting of a series of eight stimulation contacts at 1.25-mm intervals were implanted vertically through guide cannulae into the caudal diencephalon of 12 chronically catheterized fetal sheep (>0.8 term), and central neural tissue was stimulated between adjacent contacts. Each site was stimulated repeatedly with increasing current searching for spatial and stimulus strength parameters for a reliable alteration in respiratory rate. Respiratory period increased when stimulation involved areas of the parafascicular nuclear complex (Pf), which more than doubled the mean period compared with the baseline of 0.90 +/- 0.19 s. The change in respiratory period was due to an increase in expiratory time, whereas inspiratory time and breath amplitude were not significantly affected. Breathing period and expiratory time were also increased when the stimulations involved the intralaminar wing surrounding the mediodorsal nucleus, the rostral central gray, zona incerta, and ventral tegmental area. Reductions in respiratory frequency occurred less consistently, with stimulation involving surrounding zones including the sub-Pf, ventromedial nucleus, and ventrobasal nuclear complex. These findings support the hypothesis that a restricted area of the posteromedial thalamus (principally Pf) constitutes part of a neuronal circuitry that modulates respiratory motoneurons.

Adenosine A(2A) receptors mediate hypoxic inhibition of fetal breathing in sheep.

OBJECTIVE: Hypoxia inhibits fetal breathing through activation of central adenosine (ADO) receptors that modulate fetal behavioral state. This study was designed to determine whether adenosine A(1) and/or A(2A)receptor subtypes mediate the depressant effects of hypoxia. STUDY DESIGN: In 14 chronically catheterized fetal sheep (>0.8 term), hypoxemia was induced by having the ewe breathe a gas mixture of 9% oxygen for 1 hour. During hypoxia, the fetus was infused intra-arterially with a vehicle or an antagonist for adenosine A(1) or A(2A) receptors. Statistical analysis was performed by using analysis of variance with Tukey's least significant difference criterion. RESULTS: Fetal isocapnic hypoxemia (PaO(2): control, approximately 24 mm Hg; hypoxia, approximately 14 mm Hg) virtually eliminated rapid eye movements and breathing when the fetus was infused with vehicle or the A(1) receptor antagonist. In contrast, adenosine A(2A) receptor blockade abolished the hypoxia-induced arrest of rapid eye movements and breathing. CONCLUSION: Hypoxic inhibition of rapid eye movements and breathing is critically dependent on activation of adenosine A(2A) receptors.