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A software framework is described which was designed to process EEG, video of one eye, and head movement in real time, towards achieving early detection of microsleeps for prevention of fatal accidents, particularly in transport sectors. The framework is based around a pipeline structure with user-replaceable signal processing modules. This structure can encapsulate a wide variety of feature extraction and classification techniques and can be applied to detecting a variety of aspects of cognitive state. Users of the framework can implement signal processing plugins in C++ or Python. The framework also provides a graphical user interface and the ability to save and load data to and from arbitrary file formats. Two small studies are reported which demonstrate the capabilities of the framework in typical applications: monitoring eye closure and detecting simulated microsleeps. While specifically designed for microsleep detection/prediction, the software framework can be just as appropriately applied to (i) other measures of cognitive state and (ii) development of biomedical instruments for multi-modal real-time physiological monitoring and event detection in intensive care, anaesthesiology, cardiology, neurosurgery, etc. The software framework has been made freely available for researchers to use and modify under an open source licence.

Mice that are deficient in the transcription factor methyl-CpG-binding protein 2 (MeCP2) have a depressed hypercapnic ventilatory response (HCVR). The expression of MeCP2 can be selectively removed from astrocytes or neurons, thus offering a tool to dissect the role of this transcription factor in astrocytes from that in neurons. Studies were carried out in the progeny of mice that were a cross between those harboring a tamoxifen (TAM)-inducible Cre recombinase transgene driven by the human astrocytic glial fibrillary acidic protein (hGFAP) promoter, or Cre recombinase under control of the synapsin promoter, with mice containing a Cre-excisable exon III in the Mecp2 gene. The TAM-conditional excision of the Mecp2 exon allowed the respiratory CO2 response to be studied in the same animals before and after selective depletion of MeCP2 in astrocytes. Immunohistochemistry showed that following TAM treatment only ∼20% of GFAP-labeled cells in the retrotrapazoid nucleus and in the raphé magnus were positive for MeCP2. The slope of the relative increase in minute ventilation as a function of 1, 3, and 5% inspired CO2 was depressed in mice with depleted astrocyte MeCP2 compared with wild-type littermates. In contrast, selective depletion of MeCP2 in neurons did not significantly affect slope. While neurons which constitute the respiratory network ultimately determine the ventilatory response to CO2, this study demonstrates that loss of MeCP2 in astrocytes alone is sufficient to result in a dramatic attenuation of the HCVR. We propose that the glial contribution to HCVR is under the control of the MeCP2 gene., (Copyright © 2015 the American Physiological Society.)

Disturbances in respiration are common and debilitating features of Rett syndrome (RTT). A previous study showed that the 5-HT1a receptor agonist (R)-(+)-8-hydroxy-dipropyl-2-aminotetralin hydrobromide (8-OH-DPAT) significantly reduced the incidence of apnea and the irregular breathing pattern in a mouse model of the disorder. 8-OH-DPAT, however, is not available for clinical practice. Sarizotan, a full 5-HT1a agonist and a dopamine D2-like agonist/partial agonist, has been used in clinical trials for the treatment of l-dopa-induced dyskinesia. The purpose of this study was to evaluate the effects of sarizotan on respiration and locomotion in mouse models of RTT. Studies were performed in Bird and Jaenisch strains of methyl-CpG-binding protein 2--deficient heterozygous female and Jaenisch strain Mecp2 null male mice and in knock-in heterozygous female mice of a common nonsense mutation (R168X). Respiratory pattern was determined with body plethysmography, and locomotion was determined with open-field recording. Sarizotan or vehicle was administered 20 minutes before a 30-minute recording of respiratory pattern or motor behavior. In separate studies, a crossover design was used to administer the drug for 7 and for 14 days. Sarizotan reduced the incidence of apnea in all three RTT mouse models to approximately 15% of their pretreatment levels. The irregular breathing pattern was corrected to that of wild-type littermates. When administered for 7 or 14 days, apnea decreased to 25 to 33% of the incidence seen with vehicle. This study indicates that the clinically approved drug sarizotan is an effective treatment for respiratory disorders in mouse models of RTT.

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., (Copyright © 2014 IBRO. All rights reserved.)

Rett syndrome is a neurological disorder caused by loss of function mutations in the gene that encodes the DNA binding protein methyl-CpG-binding protein 2 (Mecp2). A prominent feature of the syndrome is disturbances in respiration characterized by frequent apnea and an irregular interbreath cycle. 8-Hydroxy-2-dipropylaminotetralin has been shown to positively modulate these disturbances (Abdala AP, Dutschmann M, Bissonnette JM, Paton JF, Proc Natl Acad Sci U S A 107: 18208-18213, 2010), but the mode of action is not understood. Here we show that the selective 5-HT1a biased agonist 3-chloro-4-fluorophenyl-(4-fluoro-4-{[(5-methylpyrimidin-2-ylmethyl)-amino]-methyl}-piperidin-1-yl)-methanone (F15599) decreases apnea and corrects irregularity in both heterozygous Mecp2-deficient female and in Mecp2 null male mice. In whole cell voltage-clamp recordings from dorsal raphe neurons, F15599 potently induced an outward current, which was blocked by barium, reversed at the potassium equilibrium potential, and was antagonized by the 5-HT1a antagonist WAY100135. This is consistent with somatodendritic 5-HT1a receptor-mediated activation of G protein-coupled inwardly rectifying potassium channels (GIRK). In contrast, F15599 did not activate 5-HT1b/d receptors that mediate inhibition of glutamate release from terminals in the nucleus accumbens by a presynaptic mechanism. Thus F15599 activated somatodendritic 5-HT1a autoreceptors, but not axonal 5-HT1b/d receptors. In unanesthetized Mecp2-deficient heterozygous female mice, F15599 reduced apnea in a dose-dependent manner with maximal effect of 74.5 ± 6.9% at 0.1 mg/kg and improved breath irrregularity. Similarly, in Mecp2 null male mice, apnea was reduced by 62 ± 6.6% at 0.25 mg/kg, and breathing became regular. The results indicate respiration is improved with a 5-HT1a agonist that activates GIRK channels without affecting neurotransmitter release.

Mice deficient in the transcription factor methyl-CpG-binding protein 2 (Mecp2), a mouse model of Rett syndrome, display reduced CO2 chemosensitivity, which may contribute to their breathing abnormalities. In addition, patients with Rett syndrome and male mice that are null for Mecp2 show reduced levels of brain serotonin (5-HT). Serotonin is known to play a role in central chemosensitivity, and we hypothesized that increasing the availability of 5-HT in this mouse model would improve their respiratory response to CO2. Here we determined the apnoeic threshold in heterozygous Mecp2-deficient female mice and examined the effects of blocking 5-HT reuptake on the CO2 response in Mecp2-null male mice. Studies were performed in B6.129P2(C)-Mecp2(τm1.1Bird) null males and heterozygous females. In an in situ preparation, seven of eight Mecp2-deficient heterozygous females showed arrest of phrenic nerve activity when arterial CO2 was lowered to 3%, whereas the wild-types maintained phrenic nerve amplitude at 53 ± 3% of maximal. In vivo plethysmography studies were used to determine CO2 chemosensitivity in null males. These mice were exposed sequentially to 1, 3 and 5% CO2. The percentage increase in minute ventilation in response to increased inspired CO2 was less in Mecp2(-/y) than in Mecp2(+/y) mice. Pretreatment with citalopram, a selective 5-HT reuptake inhibitor (2.5 mg kg(-1) i.p.), 40 min prior to CO2 exposure, in Mecp2(-/y) mice resulted in an improvement in CO2 chemosensitivity to wild-type levels. These results suggest that decreased 5-HT in Mecp2-deficient mice reduces CO2 chemosensitivity, and restoring 5-HT levels can reverse this effect.

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., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

The fetal respiratory response to acute hypoxia is characterized by depression, often to apnea. This study examined the effect of hypoxia on the electromyogram (EMG) of the thyroarytenoid (TA) muscle. Under anesthesia catheters were placed in the fetal sheep carotid artery, fourth cerebral ventricle, trachea and amniotic fluid and wires sewn into the diaphragm and TA muscle. During normoxic episodes of slow fetal breathing (<40 breaths per min) TA EMG activity was phasic beginning immediately after diaphragmatic EMG bursts and ending well before the next burst. This timing is consistent with the post-inspiratory (post-I) phase of the respiratory cycle. Lowering fetal arterial Pa O(2) from approximately 20mm Hg to approximately 13 mm Hg resulted in arrest of diaphragm EMG and tonic TA activity. Instillation of the (R,S)- -amino-3- hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) ionotrophic glutamate receptor antagonist 2,3-dihydro-6-nitro-7-sulphamoyl-benzo(f) quinoxaline (NBQX) into the cerebrospinal fluid (CSF) of the fourth ventricle abolished tracheal pressure deflections and diaphragmatic EMG activity. Tonic TA activity, however, could still be evoked by hypoxia. These results indicate that fetal post-I motoneurons are not inhibited by moderate hypoxia and that their tonic activity may be due to a loss of inhibitory input., ((c) 2010 Elsevier B.V. All rights reserved.)

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (Mecp2) that encodes a DNA binding protein involved in gene silencing. Periodic breathing (Cheyne-Stokes respiration) is commonly seen in RTT. Freely moving mice were studied with continuous recording of pleural pressure by telemetry. Episodes of periodic breathing in heterozygous Mecp2 deficient (Mecp2(+/-)) female mice (9.4 +/- 2.2 h(-1)) exceeded those in wild-type (Mecp2(+/+)) animals (2.5 +/- 0.4 h(-1)) (P = 0.010). Exposing Mecp2(+/-) animals to 40% oxygen increased the amount of periodic breathing from 118 +/- 25 s/30 min in air to 242 +/- 57 s/30 min (P = 0.001), and 12% oxygen tended to decrease it (67 +/- 29 s/30 min, P = 0.14). Relative hyperoxia and hypoxia did not affect the incidence of periodic breathing in Mecp2(+/+) animals. The ventilation/apnea ratio (V/A) was less at all levels of oxygen in heterozygous Mecp2(+/-) females compare with wild type (P = 0.003 to P < 0.001), indicating that their loop gain is larger. V/A in Mecp2(+/-) fell from 2.42 +/- 0.18 in normoxia to 1.82 +/- 0.17 in hyperoxia (P = 0.05) indicating an increase in loop gain with increased oxygen. Hyperoxia did not affect V/A in Mecp2(+/+) mice (3.73 +/- 0.28 vs. 3.5 +/- 0.28). These results show that periodic breathing in this mouse model of RTT is not dependent on enhanced peripheral chemoreceptor oxygen sensitivity. Rather, the breathing instability is of central origin.

Methyl-CpG-binding protein 2 is a transcription factor that is involved in gene silencing. It is mutated in the majority of cases of Rett syndrome. This X-linked neurodevelopmental disorder is reported to involve abnormalities in autonomic cardiovascular regulation. As an initial step in understanding the basis for these abnormalities we have characterized autonomic cardiovascular function in Mecp2 deficient mice. Arterial pressure waves were recorded in freely moving animals using telemetry. Baseline blood pressure and pulse interval (PI) as well as indices of heart rate variability (HRV): standard deviation of PI (SDNN), range encompassing 90% of PIs (PI90) and standard deviation of adjacent PIs (SDSD) were similar in Mecp2(+/+) and Mecp2(+/-) animals. Spectral analysis of mean arterial pressure (MAP) and PI in the frequency domain showed similar relative power in low frequency 1 (LF1, 08-0.4 Hz), low frequency 2 (LF2, 0.4-1.0 Hz), middle frequency (MF, 1-3 Hz) and high frequency (HF, 3.0-10.0 Hz) bands. Autonomic blockade with atropine or propranolol as well as elevation in ambient temperature to 32 degrees C resulted in changes in blood pressure, PI and HRV that did not differ between the strains. Atropine, propranolol and elevated temperature resulted in similar changes in both MAP and PI spectral power. Baroreceptor function was tested using intravenous injections of nitroprusside followed by phenylephrine. Maximum gain was not different. These results do reveal any disturbance of autonomic cardiovascular regulation in the Mecp2 deficient mouse genotype.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2) that encodes a DNA binding protein involved in gene silencing. Selective deletion of Mecp2 in post-mitotic neurons in mice results in a Rett-like phenotype characterized by disturbances in motor activity and body weight, suggesting that these symptoms are exclusively caused by neuronal deficiency. Included in the RTT phenotype are episodes of respiratory depression that follow hyperventilation. Here we show that the respiratory phenotype depends on the organ distribution of Mecp2 deficiency. Both female mice heterozygous for a null mutation in Mecp2 (Mecp2+/-) and those with selective deletion of the protein in neurons (Mecp2+/nestin-Cre lox), showed an initial response to hypoxia that exceeded that in wild type (WT). However, marked respiratory depression following hypoxic hyperventilation was only seen in Mecp2+/- animals. Addition of carbon dioxide to the hypoxic exposure eliminated the respiratory depression. Tidal volume and lung volume were larger in Mecp2+/- and respiratory depression was directly related to tidal volume. Taken together these results indicate that the depression is due to hypocapnia. Respiratory depression in this mouse model of Rett Syndrome is seen in with ubiquitous deficiency in Mecp2 but not when it is confined to neurons.

Background: Recent reports have shown that there are developmental changes in the ventilatory response to hypercapnia in the rat. These are characterized by an initial large response to carbon dioxide immediately after birth followed by a decline with a trough at one week of age, followed by a return in sensitivity. A second abnormality is seen at postnatal day 5 (P5) rats in that they cannot maintain the increase in frequency for 5 min of hypercapnia. In mice lacking GAD65 the release of GABA during sustained synaptic activation is reduced. We hypothesized that this developmental pattern would be present in the mouse which is also less mature at birth and that GABA mediates this relative respiratory depression., Methods: In awake C57BL/6J and GAD65-/- mice the ventilatory response to 5% carbon dioxide (CO2) was examined at P2, P4, P6, P7, P12.5, P14.5 and P21.5, using body plethysmography., Results: Minute ventilation (VE) relative to baseline during hypercapnia from P2 through P7 was generally less than from P12.5 onwards, but there was no trough as in the rat. Breaking VE down into its two components showed that tidal volume remained elevated for the 5 min of exposure to 5% CO2. At P6, but not at other ages, respiratory frequency declined with time and at 5 min was less that at 2 and 3 min. GAD65-/- animals at P6 showed a sustained increase in respiratory rate for the five mins exposure to CO2., Conclusion: These results show, that in contrast to the rat, mice do not show a decline in minute ventilatory response to CO2 at one week of age. Similar to the rat at P5, mice at P6 are unable to sustain an increase in CO2 induced respiratory frequency and GAD65 contributes to this fall off.

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.

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.

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)

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 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.

The respiratory responses to theophylline during normoxia and hypoxia were determined in 13 unanesthetized fetal sheep. Theophylline (plasma levels approximately 111 mumol/L) increased the incidence of fetal breathing movements measured over 120 min from 37.7 +/- 4.8% to 61.1 +/- 5.7% (SEM) in normoxic fetuses. In isocapnic hypoxia (arterial O2 tension approximately 1.86 kPa), theophylline increased the incidence from 20.0 +/- 6.3 to 52.0 +/- 6.1%. Theophylline also resulted in an increase in the slope of inspiration during both normoxia and hypoxia. We conclude that adenosine modulates fetal respiratory drive during normoxia and hypoxia.