Respiratory phenotypes are distinctly affected in mice with common Rett syndrome mutations MeCP2 T158A and R168X.

Respiratory disturbances are a primary phenotype of the neurological disorder, Rett syndrome (RTT), caused by mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). Mouse models generated with null mutations in Mecp2 mimic respiratory abnormalities in RTT girls. Large deletions, however, are seen in only ∼10% of affected human individuals. Here we characterized respiration in heterozygous females from two mouse models that genetically mimic common RTT point mutations, a missense mutation T158A (Mecp2(T158A/)(+)) or a nonsense mutation R168X (Mecp2(R168X/+)). MeCP2 T158A shows decreased binding to methylated DNA, while MeCP2 R168X retains the capacity to bind methylated DNA but lacks the ability to recruit complexes required for transcriptional repression. We found that both Mecp2(T158A/+) and Mecp2(R168X/+) heterozygotes display augmented hypoxic ventilatory responses and depressed hypercapnic responses, compared to wild-type controls. Interestingly, the incidence of apnea was much greater in Mecp2(R168X/+) heterozygotes, 189 per hour, than Mecp2(T158A/+) heterozygotes, 41 per hour. These results demonstrate that different RTT mutations lead to distinct respiratory phenotypes, suggesting that characterization of the respiratory phenotype may reveal functional differences between MeCP2 mutations and provide insights into the pathophysiology of RTT.

Acute intermittent hypoxia-induced expression of brain-derived neurotrophic factor is disrupted in the brainstem of methyl-CpG-binding protein 2 null mice.

Rett syndrome is a neurodevelopmental disorder caused by loss-of-function mutations in the gene encoding the transcription factor methyl-CpG-binding protein 2 (MeCP2). One of its targets is the gene encoding brain-derived neurotrophic factor (bdnf). In vitro studies using cultured neurons have produced conflicting results with respect to the role of MeCP2 in BDNF expression. Acute intermittent hypoxia (AIH) induces plasticity in the respiratory system characterized by long-term facilitation of phrenic nerve amplitude. This paradigm induces an increase in BDNF protein. We hypothesized that AIH leads to augmentation of BDNF transcription in respiratory-related areas of the brainstem and that MeCP2 is necessary for this process. Wild-type and mecp2 null (mecp2(-/y)) mice were subjected to three 5-min episodes of exposure to 8% O(2)/4% CO(2)/88% N(2), delivered at 5-min intervals. Normoxia control wild-type and mecp2 null mice were exposed to room air for the total length of time, that is, 30 min. Following a recovery in room air, the pons and medulla were rapidly removed. Expression of BDNF protein and transcripts were determined by ELISA and quantitative PCR, respectively. AIH induced a significant increase in BDNF protein in the pons and medulla, and in mRNA transcript levels in the pons of wild-type animals. In contrast, there were no significant changes in either BDNF protein or transcripts in the pons or medulla of mice lacking MeCP2. The results indicate that MeCP2 is required for regulation of BDNF expression by acute intermittent hypoxia in vivo.

Developmental changes in the hypoxic ventilatory response in C57BL/6 mice.

C57BL/6 mice are the strain into which most null mutations for neurotransmitters or their receptors are backcrossed. A number of these transgenic mice have recently been shown to have an abnormal respiratory phenotype; however, the postnatal development of the ventilatory response to hypoxia has not been characterized in C57BL/6 mice. The effect of 8% oxygen for 5 min was examined in mice at five periods from P1 to P30 using a body plethysmograph. Neonatal and juvenile animals from P7 to P30 showed a biphasic pattern in hypoxia in which the increase in minute ventilation achieved in the first min declined towards baseline by the fifth minute and was decreased below baseline in the first minute of return to air breathing. In contrast P1-P3 C57BL/6 mice had a sustained increase in both respiratory frequency and tidal volume and their minute volume remained above baseline on return to air. The decline in oxygen consumption, measured in the fifth minute of hypoxia, was not different in P1-P3 mice compared to P8-P10. These results suggest that the earliest response to hypoxia of the respiratory system in this strain is not characterized by a time dependent depression as seen in older animals and in species whose motor systems are relatively more developed at birth.

Respiration and parturition affected by conditional overexpression of the Ca2+-activated K+ channel subunit, SK3.

In excitable cells, small-conductance Ca2+-activated potassium channels (SK channels) are responsible for the slow after-hyperpolarization that often follows an action potential. Three SK channel subunits have been molecularly characterized. The SK3 gene was targeted by homologous recombination for the insertion of a gene switch that permitted experimental regulation of SK3 expression while retaining normal SK3 promoter function. An absence of SK3 did not present overt phenotypic consequences. However, SK3 overexpression induced abnormal respiratory responses to hypoxia and compromised parturition. Both conditions were corrected by silencing the gene. The results implicate SK3 channels as potential therapeutic targets for disorders such as sleep apnea or sudden infant death syndrome and for regulating uterine contractions during labor.

Mechanisms regulating hypoxic respiratory depression during fetal and postnatal life.

Selected topics in the respiratory response to acute hypoxia in the fetus and newborn are reviewed. Peripheral chemoreceptors acting through ionotrophic glutamate receptors play an important role in affecting the initial augmentation phase. Whether fall off in peripheral chemoreceptor activity contributes to the secondary depressive phase remains controversial. A number of approaches including permanent electrolytic and reversible cooling lesions, Fos protein activation, and double-labeling immunohistochemistry has converged to show that an area in and around the locus ceruleus in the rostral pons affects the central depression. There is evidence that this is mediated by catecholamines acting at alpha(2)-adrenergic receptors. Tonic activity in early expiratory (postinspiratory) neurons may contribute to hypoxia-induced apneic episodes in the fetus and newborn. Desensitization of alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid receptors has been demonstrated in respiratory-related neurons both in vivo and in vitro. The role that this process might play in the depressive phase of the hypoxic ventilatory response has not been established. In vitro experiments with isolated brain stem-spinal cord preparations or transverse brain stem slices usually involve anoxia, whereas whole animal experiments use 8-15% O(2). Therefore, caution must be exercised in attempting to construct a unifying framework from these two approaches.

Restructuring residency training in obstetrics and gynecology.

This essay presents a brief review of the history advocating a restructuring of residency training to increase the flexibility of the experience. The advantages for both the general obstetrician-gynecologist and the subspecialist are reviewed.

Non-NMDA receptors modulate respiratory drive in fetal sheep.

1. Experiments were carried out in unanaesthetized fetal sheep to evaluate the significance of non-N-methyl-D-aspartate (non-NMDA) receptor neurotransmission in the expression of fetal breathing movements. Catheters placed in the trachea and amniotic fluid and electrodes beneath the parietal bones and in the nuchal muscle were used to monitor breath amplitude and frequency and fetal behavioural state. 2. Experiments were carried out by instillation of neurotransmitter agonists, antagonists or receptor modulators into the cerebrospinal fluid (CSF) of the fourth ventricle by means of a chronic catheter introduced through the foramen magnum. 3. The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) decreased respiratory rate in a dose dependent manner by lengthening both inspiratory time (T1) and expiratory time (T0). 4. Kainate and (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) increased breath amplitude. Instillation of the antagonist 2,3-dihydro-6-nitro-7-sulphamoyl-benzo(f) quinoxaline (NBQX) prior to administering AMPA resulted in apnoea, which was not overcome by the agonist. 5. Cyclothiazide, which has been shown to prevent desensitization of AMPA receptors, caused an increase in both breath amplitude (152 +/- 73%; mean +/- S.D.; P = 0.004) and frequency (46 +/- 37%; P = 0.049). 6. These data suggest that glutamate acting at non-NMDA receptors is an essential component for the expression of fetal breathing movements, and that under resting conditions these non-NMDA receptors are desensitized following glutamate synaptic release.

GABAergic and glutamatergic effects on behaviour in fetal sheep.

1. Studies were carried out in unanaesthetized fetal sheep at 125-135 days gestation to investigate neurotransmitters involved in behavioural state. 2. Catheters and electrodes were chronically placed to record tracheal and arterial pressure, electrocortical activity (ECoG), nuchal muscle activity and to instill drugs into the cerebrospinal fluid (CSF) of the fourth ventricle. 3. Administration of the N-methyl-D-aspartate receptor antagonists DL-2-amino-5-phosphopentanoic acid (AP5) or (+)-5-methyl-10,11-dihydro-5H-dibenzol[a,d]cyclohepten-5,10- iminemoleate (MK-801) increased the incidence of fetal behaviour characterized by low voltage ECoG, nuchal muscle activity and an increase in mean arterial blood pressure from 4.1 +/- 6 to 60.6 +/- 6.2% (mean +/- S.E.M.) (AP5; P = 0.003) and from 7.6 +/- 3.6 to 50.8 +/- 7.0% (MK-801; P = 0.004; values are expressed as the percentage of each 60 min period in which the state was present). 4. The incidence of fetal breathing during periods of low voltage (LV)-ECoG and nuchal muscle activity was 83.1 +/- 5.6%. The incidence of fetal breathing during LV-ECoG associated with nuchal muscle atonia was 63.1 +/- 5.0% before AP5 or MK-801 and 64.4 +/- 9.8% after instillation of these drugs. The amplitude of fetal breaths increased from 4.0 +/- 0.3 mmHg in low voltage ECoG periods to 6.7 +/- 0.8 mmHg (P = 0.006) during periods of low voltage with nuchal muscle activity. There was no significant change in breath timing during these periods.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of central adenosine on brainstem blood flow in fetal sheep.

The effect of the adenosine analogue R-N6-(phenylisopropyl)adenosine (R-PIA) on blood flow to the medulla and pons was examined in unanaesthetized fetal sheep. Microspheres labelled with isotopes were used to determine blood flow before and after instillation of 0.2 or 0.5 microgram R-PIA into the cerebrospinal fluid of the fourth ventricle. Blood flow to the medulla, which had a mean value (+/- S.E.M.) of 285 +/- 41 ml min-1 (100 g)-1 during the control period, was not changed by the central instillation of R-PIA. Blood flow to the pons was also not affected. These data indicate that central adenosine, which depresses respiratory drive in fetal sheep, acts by mechanisms independent of removal of carbon dioxide from the brainstem.

A cholinergic mechanism involved in fetal breathing during the high voltage ECoG state.

The effects of muscarinic cholinergic neurotransmission on fetal breathing was determined by administration of carbachol or carbachol plus a muscarinic receptor antagonist into the cerebrospinal fluid of the fourth ventricle in unanesthetized fetal sheep. In the hour following the instillation of carbachol (1.0 microgram), the incidence of fetal breathing in high voltage electrocortical state (ECoG) increased to 63 +/- 11.7 (SEM) percent compared to 1.2 +/- 0.9 after instillation of vehicle (Ringer solution). The cholinergic agonist increased breath amplitude from 4.5 +/- 0.4 to 10.6 +/- 1.4 mmHg. These effects were eliminated when the M1 receptor antagonist pirenzepine (50-100 micrograms) was administered with carbachol but not by antagonists which are relatively selective for M2 or M3 receptors. When administered alone, muscarinic receptor antagonists did not effect the incidence or amplitude of fetal breathing in low voltage. These data indicate that the apnea which occurs during high voltage in the sheep fetus involves an inhibition of acetylcholine acting at M1 muscarinic receptor bearing neurons.

The effects of centrally administered adenosine on fetal sheep heart rate accelerations.

OBJECTIVE: We examined the effect of administering a long-acting adenosine analog, L-2-N6-(phenylisopropyl) adenosine, into the cerebrospinal fluid of the fourth ventricle on fetal sheep heart rate accelerations. STUDY DESIGN: Pregnant ewes between 123 and 130 days' gestation were anesthetized, and the fetal head was exteriorized. Catheters were placed in the fourth cerebral ventricle through the foramen magnum and in the brachial artery to record fetal heart rate. Studies were performed in unanesthetized fetuses 4 to 7 days after surgery. Accelerations were defined as a 10 beats/min rise in heart rate for at least 5 seconds. RESULTS: The mean number of accelerations before administration of L-2-N6- (phenylisopropyl) adenosine was 3.9 +/- 0.7 (mean +/- SE) per 10-minute epoch, decreasing to 2.0 +/- 0.7 and 1.4 +/- 0.8 after instillation of 0.2 microgram and 0.5 micrograms of L-2-N6-(phenylisopropyl) adenosine, respectively (p < 0.05). Increasing the L-2-N6-(phenylisopropyl) adenosine dose to 10.0 micrograms resulted in loss of heart rate accelerations. Accelerations were not reduced when theophylline, an adenosine receptor blocker, was given before L-2-N6-(phenylisopropyl) adenosine. CONCLUSION: Increasing amounts of a centrally administered adenosine analog progressively decreased the number of fetal sheep heart rate accelerations, most probably by suppression of brainstem sympathetic outflow.

Unidirectional transport of glucose and lactate into brain of fetal sheep and guinea-pig.

The first-passage multiple-indicator dilution method was used to measure blood to brain transport of D- and L-glucose, D- and L-lactate and sucrose relative to 22Na, an impermeable reference tracer, in fetal sheep. Fractional extraction for D-glucose was 0.315 +/- 0.051 (S.E.M.) at normal glucose levels and fell to 0.198 +/- 0.041 at 5.2 +/- 0.4 mM-glucose. Fractional extractions for L-glucose, D- and L-lactate and sucrose were not different from zero. No specific blood-brain transport system was detected for L-lactate in fetal sheep in vivo (fractional extraction = -0.024 +/- 0.019). Uptake of L-lactate into isolated microvessels from fetal sheep cerebrum in vitro showed a slightly higher rate (32.2 +/- 8.9 pmol min-1 (mg protein)-1) than that for D-lactate (22.6 +/- 5.6). In fetal guinea-pigs, the carotid arterial injection method with tritiated water as the permeable reference was used to measure the brain uptake index (BUI). BUI was determined for D-glucose (0.304 +/- 0.065) sucrose (0.008 +/- 0.001), L-lactate (0.418 +/- 0.112) and D-lactate (0.071 +/- 0.024). Unidirectional influx calculated from these measurements and estimates of cerebral blood flow showed that transport would be rate-limiting for cerebral glucose utilization at arterial glucose levels below 0.5 mM in fetal sheep and 1.7 mM in fetal guinea-pig. In fetal sheep, but not in fetal guinea-pigs, lactate efflux may be limited by brain-blood transport.

Autoradiographic localization of adenosine A1 receptors in brainstem of fetal sheep.

Quantitative autoradiographic techniques were used to localize adenosine A1 receptors at the light microscopic level with the antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine [( 3H]DPCPX) in the brainstem of fetal sheep. Since adenosine has been proposed as a neuromodulator, which effects the depression of fetal breathing movements during hypoxia, attention was directed to respiratory neuronal areas. The highest density of A1 receptors in respiratory related groups was found in an area of the rostral ventrolateral medulla, which is ventral to the facial nucleus, caudal to the superior olive and lateral to the rostral inferior olive. Intermediate densities were seen in the medial and lateral parabrachial nuclei. Adenosine A1 receptor density was low in the areas of the nucleus of the solitary tract and the nucleus ambiguous. These data suggest that moderate hypoxia in the fetus may depress respiration by withdrawing a tonic stimulus at the level of the ventral lateral medullary chemoreceptors.

Vascular lactic acid infusions do not alter the incidence of fetal breathing movements or their inhibition by acute hypoxemia.

Hypoxemia transiently inhibits the incidence of fetal breathing movements (FBM), but their incidence returns to normal after several hours despite maintained hypoxemia. We hypothesized that the lactic acidosis associated with prolonged systemic hypoxemia might mediate the adaptation of the hypoxemic inhibition of FBM. In sheep fetuses, the incidence of FBM was measured in a control hour and during 6 h of i.v. infusion of L-lactic acid, which raised the blood lactate concentrations to levels seen with moderate hypoxemia. FBM were observed at the same incidence as during control during each of the first 4 h (all approximately 40%). In the 5th h of lactic acid infusion, fetal hypoxemia was induced by lowering maternal inspired oxygen fraction and FBM occurred only 8 +/- 1% (SEM) of that hour. In a subsequent normoxemic recovery hour, the incidence of FBM remained below control levels. In the same animals on a different day, a similar hypoxemia induced without the acid infusion caused a comparable inhibition of FBM, but the incidence of FBM returned to the control level in a subsequent recovery hour. A moderate peripheral lactic acidosis does not blunt the inhibition of FBM evoked by acute hypoxemia and is not a likely explanation for the return of FBM during prolonged hypoxemia but actually might mediate some of the inhibition.

Fetal cerebral blood flow and metabolism during oligemia and early postoligemic reperfusion.

The early time period following ischemia may be of pathogenetic importance in hypoxic-ischemic brain injury. Global cerebral oligemia was induced in ten late gestation fetal sheep by inflation of a balloon occluder around the brachiocephalic artery. Cerebral blood flow, oxygen, glucose, and lactate net flux, and oxygen delivery were measured by the Fick principle following 1 h of oligemia and at 5, 30, and 60 min of postoligemic reperfusion. During oligemia, cerebral blood flow decreased by 74 +/- 10% (mean +/- SD) and oxygen consumption decreased by 34 +/- 24%. The glucose:oxygen quotient was elevated throughout the oligemic period. In the early (5 min) reperfusion period, blood flow and oxygen delivery were not different from control but oxygen consumption was persistently depressed by 27 +/- 32%; fractional extraction of oxygen was 0.38 +/- 0.10 during control and 0.24 +/- 0.09 during early reperfusion. The venous oxygen tension increased modestly from 15.2 +/- 2.4 to 18.0 +/- 1.7 mm Hg; the postoligemic venous pO2 was limited by the lack of reactive hyperemia combined with the low arterial pO2 of the intrauterine environment. Postoligemic carbohydrate fluxes could not be differentiated from control possibly due to blood-brain barrier limitations. These factors may be related to the relative resistance of the fetal brain to hypoxic-ischemic injury.

The effect of centrally administered adenosine on fetal breathing movements.

The central effects of the adenosine analogue L-2-N6-(phenylisopropyl) adenosine (L-PIA) on breathing movements was determined by making injections into the fourth ventricle in unanesthetized fetal sheep. Administration of 0.5 micrograms L-PIA reduced the percent time during which fetal breathing occurred from 48.0 +/- 5.2 (SEM) to 19.5 +/- 6.1. Inspiratory slope was reduced to 62 +/- 5.5 and to 43 +/- 5.7 percent of the control values when 0.2 and 0.5 micrograms L-PIA were given respectively. The effects of L-PIA on the percent time fetal breathing movements occurred and on inspiratory slope were prevented by the prior systemic administration of theophylline (plasma concentrations approximately 15 micrograms/ml). When the vehicle for L-PIA, dimethyl sulfoxide in Ringer solution was given into the fourth ventricle or when 0.5 micrograms L-PIA was given systemically, there was no effect on fetal breathing. None of these protocols resulted in a change in sagittal sinus blood pH, PO2 or, PCO2. These data indicate adenosine acts at the brain stem to depress fetal respiratory drive.