Determinants of functional coupling between astrocytes and respiratory neurons in the pre-Bötzinger complex

Respiratory neuronal network activity is thought to require efficient functioning of astrocytes. Here, we analyzed neuron-astrocyte communication in the pre-Bötzinger Complex (preBötC) of rhythmic slice preparations from neonatal mice. In astrocytes that exhibited rhythmic potassium fluxes and glutamate transporter currents, we did not find a translation of respiratory neuronal activity into phase-locked astroglial calcium signals. In up to 20% of astrocytes, 2-photon calcium imaging revealed spontaneous calcium fluctuations, although with no correlation to neuronal activity. Calcium signals could be elicited in preBötC astrocytes by metabotropic glutamate receptor activation or after inhibition of glial glutamate uptake. In the latter case, astrocyte calcium elevation preceded a surge of respiratory neuron discharge activity followed by network failure. We conclude that astrocytes do not exhibit respiratory-rhythmic calcium fluctuations when they are able to prevent synaptic glutamate accumulation. Calcium signaling is, however, observed when glutamate transport processes in astrocytes are suppressed or neuronal discharge activity is excessive.     

The pre-Bötzinger oscillator in the mouse embryo

Studies of the sites and mechanisms involved in mammalian respiratory rhythm generation point to two clusters of rhythmic neurons forming a coupled oscillator network within the brainstem. The location of these oscillators, the pre-Bötzinger complex (preBötC) at vagal level, and the para-facial respiratory group at facial level, probably result from regional patterning schemes specifying neural types in the hindbrain during embryogenesis. Here, we report evidence that the preBötC oscillator (i) is first active at embryonic stages, (ii) originates in the post-otic hindbrain neural tube and (iii) requires the glutamate vesicular transporter 2 for rhythm generation.     

Increased GABA(A) receptor ε-subunit expression on ventral respiratory column neurons protects breathing during pregnancy

GABAergic signaling is essential for proper respiratory function. Potentiation of this signaling with allosteric modulators such as anesthetics, barbiturates, and neurosteroids can lead to respiratory arrest. Paradoxically, pregnant animals continue to breathe normally despite nearly 100-fold increases in circulating neurosteroids. ε subunit-containing GABA_ARs are insensitive to positive allosteric modulation, thus we hypothesized that pregnant rats increase ε subunit-containing GABA_AR expression on brainstem neurons of the ventral respiratory column (VRC). In vivo, pregnancy rendered respiratory motor output insensitive to otherwise lethal doses of pentobarbital, a barbiturate previously used to categorize the ε subunit. Using electrode array recordings in vitro, we demonstrated that putative respiratory neurons of the preBötzinger Complex (preBötC) were also rendered insensitive to the effects of pentobarbital during pregnancy, but unit activity in the VRC was rapidly inhibited by the GABA_AR agonist, muscimol. VRC unit activity from virgin and post-partum females was potently inhibited by both pentobarbital and muscimol. Brainstem ε subunit mRNA and protein levels were increased in pregnant rats, and GABA_AR ε subunit expression co-localized with a marker of rhythm generating neurons (neurokinin 1 receptors) in the preBötC. These data support the hypothesis that pregnancy renders respiratory motor output and respiratory neuron activity insensitive to barbiturates, most likely via increased ε subunit-containing GABA_AR expression on respiratory rhythm-generating neurons. Increased ε subunit expression may be critical to preserve respiratory function (and life) despite increased neurosteroid levels during pregnancy.     

Neural Mechanisms Underlying Breathing Complexity

Breathing is maintained and controlled by a network of automatic neurons in the brainstem that generate respiratory rhythm and receive regulatory inputs. Breathing complexity therefore arises from respiratory central pattern generators modulated by peripheral and supra-spinal inputs. Very little is known on the brainstem neural substrates underlying breathing complexity in humans. We used both experimental and theoretical approaches to decipher these mechanisms in healthy humans and patients with chronic obstructive pulmonary disease (COPD). COPD is the most frequent chronic lung disease in the general population mainly due to tobacco smoke. In patients, airflow obstruction associated with hyperinflation and respiratory muscles weakness are key factors contributing to load-capacity imbalance and hence increased respiratory drive. Unexpectedly, we found that the patients breathed with a higher level of complexity during inspiration and expiration than controls. Using functional magnetic resonance imaging (fMRI), we scanned the brain of the participants to analyze the activity of two small regions involved in respiratory rhythmogenesis, the rostral ventro-lateral (VL) medulla (pre-Bötzinger complex) and the caudal VL pons (parafacial group). fMRI revealed in controls higher activity of the VL medulla suggesting active inspiration, while in patients higher activity of the VL pons suggesting active expiration. COPD patients reactivate the parafacial to sustain ventilation. These findings may be involved in the onset of respiratory failure when the neural network becomes overwhelmed by respiratory overload We show that central neural activity correlates with airflow complexity in healthy subjects and COPD patients, at rest and during inspiratory loading. We finally used a theoretical approach of respiratory rhythmogenesis that reproduces the kernel activity of neurons involved in the automatic breathing. The model reveals how a chaotic activity in neurons can contribute to chaos in airflow and reproduces key experimental fMRI findings.     

Anatomical and functional development of the pre-B?tzinger complex in prenatal rodents.

Developmental anomalies of central respiratory neural control contribute to newborn mortality and morbidity. Elucidation of the cellular, molecular, trophic, and genetic mechanisms involved in the formation and function of respiratory nuclei during prenatal development will provide a foundation for understanding pathologies. The pre-B?tzinger Complex (pre-B?tC) is a specific group of neurons located in the ventrolateral medulla that is critical for respiratory rhythmogenesis. Thus it has become a major focus of research. Here, we provide an overview of current knowledge regarding the anatomical and functional emergence of the rodent pre-B?tC during the prenatal period.     

The uncoupling of respiratory-chain phosphorylation by 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole

1. 30mmum-4,5,6,7-Tetrachloro-2-trifluoromethylbenzimidazole (TTFB) uncouples respiratory-chain phosphorylation. The respiration rate of uncoupled mitochondria is significantly greater than the state 3 rate. Neither added orthophosphate nor ATP is required to sustain this rate. The oligomycin- and octylguanidine-induced inhibitions of respiration are relieved by uncoupling concentrations of TTFB. 2. Uncoupling concentrations of TTFB elicit a high adenosinetriphosphatase activity and inhibit the ATP-dependent succinate-linked reduction of NAD catalysed by submitochondrial particles from ox-heart mitochondria. 3. The action of TTFB is similar to that of 2,4-dinitrophenol. Evidence is presented which shows that it is the anionic forms of benzimidazoles and imidazoles, having the negative charge localized on the ring nitrogen atom, which are the effective uncoupling agents.     

Assessment of Intraocular Measurements in Neonatal Foals and Association with Gender,Laterality, and Body Weight: A Clinical Study

Objective of this study was to describe intraocular measurements in newly born foals (1–7 days of age) and assess the association between globe measurements and gender, laterality, and body weight. B-scan ultrasonographic biometry was performed on both eyes of 22 healthy foals (44 eyes) ages 1–7 days using a 10-MHz transducer. Intraocular measurements (anterior chamber depth, central lens thickness, vitreous chamber depth, axial globe length, longitudinal globe length, lens poles distance) were carried out using the ultrasound internal calipers. The influence of gender (male or female), laterality (right or left eye), and body weight (“light” <48 kg; “heavy” ≥48 kg) on ocular measurements was analysed by the Student t test. Values of P<0.05 were accepted as significant for all analyses. Mean anterior chamber depth was 2.2±0.5 mm (Standard Deviation); central lens thickness was 9.9±0.8 mm; vitreous chamber depth was 15.5±1.1 mm; axial globe length was 27.6±1.6 mm; longitudinal globe length was 35.8±1.2 mm, and lens poles distance was 16.4±1.0 mm. Intraocular measurements were not influenced by gender, laterality nor body weight. This study provides reference values for intraocular measurements in neonatal foals and may be useful in the diagnosis and treatment of congenital and acquired pathologies involving the globe.     

The respiration of isolated rat hepatic cells in suspension

1. Rat-hepatic cells in suspension have been shown to have an endogenous respiration of 5·6±0·17 when suspended in 0·1 m-sucrose and 0·02 m-tris–hydrochloric acid buffer. The respiration in 0·25 m-sucrose and 0·02 m-tris–hydrochloric acid buffer is 30–40% less. 2. Potassium chloride (0·05 m) is slightly inhibitory and calcium chloride (0·0025 m) highly inhibitory to endogenous respiration of the hepatic cells in suspension. The cells do not respire in Krebs–Ringer phosphate buffer. 3. The respiration of the hepatic cells in suspension is stimulated by pyruvate, citrate, isocitrate, oxoglutarate, succinate, fumarate, malate and glutamate; there is no significant stimulation (or inhibition) by glucose, fructose, acetate and butyrate. In almost all the cases where stimulation was observed, it was found that the higher the endogenous respiration the lower is the stimulation.     

Cytokine and Nitric Oxide Levels in Patients with Sepsis – Temporal Evolvement and Relation to Platelet Mitochondrial Respiratory Function

Background

The levels of nitric oxide (NO) and various cytokines are known to be increased during sepsis. These signaling molecules could potentially act as regulators and underlie the enhancement of mitochondrial function described in the later phase of sepsis. Therefore, we investigated the correlation between observed changes in platelet mitochondrial respiration and a set of pro- and anti-inflammatory cytokines as well as NO plasma levels in patients with sepsis.

Methods and Results

Platelet mitochondrial respiration and levels of TNFα, MCP-1 (monocyte chemotactic protein-1), INFγ (interferon-γ), IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-17 and NO were analyzed in 38 patients with severe sepsis or septic shock at three time points during one week following admission to the ICU. Citrate synthase, mitochondrial DNA and cytochrome c were measured as markers of cellular mitochondrial content. All mitochondrial respiratory states increased over the week analyzed (p<0.001). IL-8 levels correlated with maximal mitochondrial respiration on day 6–7 (p = 0.02, r² = 0.22) and was also higher in non-survivors compared to survivors on day 3–4 and day 6–7 (p = 0.03 respectively). Neither NO nor any of the other cytokines measured correlated with respiration or mortality. Cytochrome c levels were decreased at day 1–2 by 24±5% (p = 0.03) and returned towards values of the controls at the last two time points. Citrate synthase activity and mitochondrial DNA levels were similar to controls and remained constant throughout the week.

Conclusions

Out of ten analyzed cytokines and nitric oxide, IL-8 correlated with the observed increase in mitochondrial respiration. This suggests that cytokines as well as NO do not play a prominent role in the regulation of platelet mitochondrial respiration in sepsis. Further, the respiratory increase was not accompanied by an increase in markers of mitochondrial content, suggesting a possible role for post-translational enhancement of mitochondrial respiration rather than augmented mitochondrial mass.     

Expression of phospho‐Ca2+/calmodulin‐dependent protein kinase II in the pre‐Bötzinger complex of rats

The pre‐Bötzinger complex (pre‐BötC) in the ventrolateral medulla oblongata is a presumed kernel of respiratory rhythmogenesis. Ca²⁺‐activated non‐selective cationic current is an essential cellular mechanism for shaping inspiratory drive potentials. Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII), an ideal ‘interpreter’ of diverse Ca²⁺ signals, is highly expressed in neurons in mediating various physiological processes. Yet, less is known about CaMKII activity in the pre‐BötC. Using neurokinin‐1 receptor as a marker of the pre‐BötC, we examined phospho (P)‐CaMKII subcellular distribution, and found that P‐CaMKII was extensively expressed in the region. P‐CaMKII‐ir neurons were usually oval, fusiform, or pyramidal in shape. P‐CaMKII immunoreactivity was distributed within somas and dendrites, and specifically in association with the post‐synaptic density. In dendrites, most synapses (93.1%) examined with P‐CaMKII expression were of asymmetric type, occasionally with symmetric type (6.9%), whereas in somas, 38.1% were of symmetric type. P‐CaMKII asymmetric synaptic identification implicates that CaMKII may sense and monitor Ca²⁺ activity, and phosphorylate post‐synaptic proteins to modulate excitatory synaptic transmission, which may contribute to respiratory modulation and plasticity. In somas, CaMKII acts on both symmetric and asymmetric synapses, mediating excitatory and inhibitory synaptic transmission. P‐CaMKII was also localized to the perisynaptic and extrasynaptic regions in the pre‐BötC.     

Reference Equation for Respiratory Pressures in Pediatric Population: A Multicenter Study

Previous studies have proposed only one prediction equation for respiratory muscle strength without taking into consideration differences between ages in pediatric population. In addition, those researches were single-center studies. The objective of this study was to establish reference equations for maximal inspiratory pressure (PImax) and maximal expiratory pressure (PEmax) in children and teenagers. In a multicenter study, 450 healthy volunteers were evaluated (aged 6–18yrs). There were included volunteers with normal lung function. We excluded volunteers who could not perform the tests; participated in physical activity more than twice a week; were born prematurely; smokers; chronic respiratory, cardiologic, and/or neurologic diseases; had acute respiratory disease during the prior three weeks. The volunteers were divided into two groups: Group 6–11 (6–11yrs) and Group 12–18 (12–18yrs). PImax and PEmax were measured according to statement. The mean PImax value was 85.6 (95%IC 83.6–87.6 cmH₂O), and PEmax 84.6 (95%IC 85.5–86.2 cmH₂O). The prediction equations for PImax and PEmax for Group 6–11 were 37.458–0.559 + (age * 3.253) + (BMI * 0.843) + (age * gender * 0.985); and 38.556 + 15.892 + (age * 3.023) + (BMI * 0.579) + (age * gender * 0.881), respectively (R² = 0.34 and 0.31, P<0.001). The equations for Group 12–18 were 92.472 + (gender * 9.894) + 7.103, (R² = 0.27, P = 0.006) for PImax; and 68.113 + (gender * 17.022) + 6.46 + (BMI * 0.927), (R² = 0.34, P<0.0001) for PEmax. This multicenter study determined the respiratory muscle strength prediction equations for children and teenagers.     

Opioid-induced quantal slowing reveals dual networks for respiratory rhythm generation

Current consensus holds that a single medullary network generates respiratory rhythm in mammals. Pre-B?tzinger Complex inspiratory (I) neurons, isolated in transverse slices, and preinspiratory (pre-I) neurons, found only in more intact en bloc preparations and in vivo, are each proposed as necessary for rhythm generation. Opioids slow I, but not pre-I, neuronal burst periods. In slices, opioids gradually lengthened respiratory periods, whereas in more intact preparations, periods jumped nondeterministically to integer multiples of the control period (quantal slowing). These findings suggest that opioid-induced quantal slowing results from transmission failure of rhythmic drive from pre-I neurons to preB?tC I networks, depressed below threshold for spontaneous rhythmic activity. Thus, both I (in the slice), and pre-I neurons are sufficient for respiratory rhythmogenesis.     

TEMPERATURE CHARACTERISTIC FOR PRODUCTION OF CO2 BY PHASEOLUS SEEDLINGS

The temperature characteristic for respiratory production of CO₂ by young seedlings of Phaseolus aureus (Roxb.) is µ = 16,500 calories, 12–21°C., even when the analyses depend upon the use of many seedlings crowded in a small respiration chamber, provided reasonable precautions are taken to avoid injury and to permit proper thermal adaptation. There is evidence of a definite critical temperature at 20–21°. These findings agree quantitatively with those obtained with other similar seedlings, and contradict the results reported by Kurbatov and Leonov (1930); the reasons for this are analysed.     

Synaptic relationship between somatostatin- and neurokinin-1 receptor-immunoreactive neurons in the pre-Bötzinger complex of rats

J. Neurochem. (2012) 122, 923-933. ABSTRACT: The pre-B?tzinger complex (pre-B?tC) in the ventrolateral medulla oblongata is critical for the generation of respiratory rhythm in mammals. Somatostatin (SST) and neurokinin 1 receptor (NK1R) immunoreactivity have been used as markers of the pre-B?tC. SST immunoreactivity almost completely overlaps with small fusiform NK1R-immunoreactive (ir) neurons, the presumed rhythmogenic neurons, but not with large multipolar NK1R-ir neurons. Understanding the neurochemical characteristics, especially the synaptic relationship of SST/NK1R-ir neurons within the pre-B?tC network is essential in providing cellular and structural bases for understanding their physiological significance. This work has not been documented so far. We found that SST immunoreactivity was highly expressed in terminals, somas, and primary dendrites in the pre-B?tC. Besides the small fusiform neurons, a small population of medium-sized NK1R-ir neurons also colocalized with SST. Large NK1R-ir neurons were not SST-ir, but received somatostatinergic inputs. SST-ir terminals were glutamatergic or GABAergic, and synapsed with NK1R-ir neurons. Most of synapses between them were of the symmetric type, indicating their inhibitory nature. Asymmetric synapses were evident between SST-ir terminals and NK1R-ir dendrites, strongly suggesting an excitatory innervation from the presumed rhythmogenic neurons as these neurons are glutamatergic. We speculate that SST-mediated excitatory and inhibitory synaptic transmission onto NK1R-ir rhythmogenic and follower neurons synchronizes their activity to contribute to respiratory rhythmogenesis and control.     

Determination of Femoral Neck Angle and Torsion Angle Utilizing a Novel Three-Dimensional Modeling and Analytical Technology Based on CT Datasets

Introduction

Exact knowledge of femoral neck inclination and torsion angles is important in recognizing, understanding and treating pathologic conditions in the hip joint. However, published results vary widely between different studies, which indicates that there are persistent difficulties in carrying out exact measurements.

Methods

A three dimensional modeling and analytical technology was used for the analysis of 1070 CT datasets of skeletally mature femurs. Individual femoral neck angles and torsion angles were precisely computed, in order to establish whether gender, age, body mass index and ethnicity influence femoral neck angles and torsion angles.

Results

The median femoral neck angle was 122.2° (range 100.1–146.2°, IQR 117.9–125.6°). There are significant gender (female 123.0° vs. male 121.5°; p = 0.007) and ethnic (Asian 123.2° vs. Caucasian 121.9°; p = 0.0009) differences. The median femoral torsion angle was 14.2° (-23.6–48.7°, IQR 7.4–20.4°). There are significant gender differences (female 16.4° vs. male 12.1°; p = 0.0001). Femoral retroversion was found in 7.8% of the subjects.

Conclusion

Precise femoral neck and torsion angles were obtained in over one thousand cases. Systematic deviations in measurement due to human error were eliminated by using automated high accuracy morphometric analysis. Small but significant gender and ethnic differences were found in femoral neck and torsion angles.     

THE RATE OF OXYGEN UTILIZATION BY YEAST AS RELATED TO TEMPERATURE

Suspensions of the yeast Saccharomyces cerevisiae gave reproducible rates of O₂ uptake over a period of 6 months. The relation of rate of consumption of O₂ to temperature was tested over a wide range of temperatures, and the constant in the formulation of the relationship is found to be reproducible. The values of this constant (µ) have been obtained for five separate series of experiments by three methods of estimation. The variability of µ has the following magnitudes: the average deviation of a single determination expressed as per cent of the mean is ±2 per cent in the range 30–15°, and ±0.8 per cent in the range 15–3°C. This constancy of metabolic activity measured as a function of temperature can then be utilized for more precise investigations of processes controlling the velocity of oxidations of substrates, and of respiratory systems controlled by intracellular respiratory pigments. The data plotted according to the Arrhemus equation give average values of the constant µ as follows: for the range 35–30°, µ = 8,290; 30–15°, µ = 12,440 ±290; 15–3°, µ = 19,530 ±154. The critical temperatures are at 29.0° and 15.7°C. A close similarity exists between these temperature characteristics (µ) and values in the series usually obtained for respiratory activities in other organisms. This fact supports the view that a common system of processes controls the velocities of physiological activities in yeast and in other organisms.     

Physical activity,dietary intake and quality of life during COVID-19 lockdown in patients awaiting transcatheter aortic valve implantation

BackgroundThe COVID-19 pandemic has led to a national lockdown in the Netherlands, which also affected transcatheter aortic valve implantation (TAVI) patients. The objective of the study was to describe physical activity, dietary intake and quality of life (QoL) in patients on the waiting list for TAVI pre-lockdown and during lockdown.MethodsConsecutive patients awaiting TAVI at the Amsterdam University Medical Centers, the Netherlands were included. Measurements were self-reported effect of lockdown, physical activity, dietary intake and QoL.ResultsIn total, 58 patients (median age 80, interquartile range (IQR) 76–84, 45% female) were observed pre-lockdown and 16 patients (median age 78, IQR 76–82, 25% female) during lockdown. Ten of the 16 patients during lockdown reported a decline in physical activity. However, we observed a median number of 5861 steps a day (IQR 4579–7074) pre-lockdown and 8404 steps a day (IQR 7653–10,829) during lockdown. Median daily protein intake was 69 g (IQR 59–82) pre-lockdown and 90 g (IQR 68–107) during lockdown. Self-rated health on a visual analogue scale was 63 points (IQR 51–74) pre-lockdown and 73 points (IQR 65–86) during lockdown.ConclusionsMore than half of the patients during lockdown reported less physical activity, while we observed a higher number of steps a day, a similar dietary intake and a higher QoL. Therefore, patients on the TAVI waiting list appeared to be able to cope with the lockdown measures.     

Emergency medical services evaluations for chest pain during first COVID-19 lockdown in Hollands-Midden,the Netherlands

ObjectiveTo assess whether the COVID-19 lockdown in 2020 had negative indirect health effects, as people seem to have been reluctant to seek medical care.MethodsAll emergency medical services (EMS) transports for chest pain or out-of-hospital cardiac arrest (OHCA) in the Dutch region Hollands-Midden (population served > 800,000) were evaluated during the initial 6 weeks of the COVID-19 lockdown and during the same time period in 2019. The primary endpoint was the number of evaluated chest pain patients in both cohorts. In addition, the number of EMS evaluations of ST-elevation myocardial infarction (STEMI) and OHCA were assessed.ResultsDuring the COVID-19 lockdown period, the EMS evaluated 927 chest pain patients (49% male, age 62 ± 17 years) compared with 1041 patients (51% male, 63 ± 17 years) in the same period in 2019, which corresponded with a significant relative risk (RR) reduction of 0.88 (95% confidence interval (CI) 0.81–0.96). Similarly, there was a significant reduction in the number of STEMI patients (RR 0.52, 95% CI 0.32–0.85), the incidence of OHCA remained unchanged (RR 1.23, 95% CI 0.83–1.83).ConclusionDuring the first COVID-19 lockdown, there was a significant reduction in the number of patients with chest pain or STEMI evaluated by the EMS, while the incidence of OHCA remained similar. Although the reason for the decrease in chest pain and STEMI consultations is not entirely clear, more attention should be paid to the importance of contacting the EMS in case of suspected cardiac symptoms in possible future lockdowns.Supplementary InformationThe online version of this article (10.1007/s12471-021-01545-y) contains supplementary material, which is available to authorized users.     

Impairment of Central Chemoreception in Neonatal Rats Induced by Maternal Cigarette Smoke Exposure during Pregnancy

It has been postulated that prenatal cigarette smoke exposure (CSE) increases the risk for sudden infant death syndrome. The victims of infant death syndrome suffer from respiratory abnormalities, such as central apnea, diminished chemoreflex and alteration in respiratory pattern during sleep. However, no experimental evidence on CSE model exists to confirm whether prenatal CSE gives rise to reduction of neonatal central chemoreception in in vitro preparations in absence of peripheral sensory feedback. The aim of the present study was to test the hypothesis that maternal CSE during pregnancy depresses central chemoreception of the neonatal rats. The pregnant rats were divided into two groups, control (n = 8) and CSE (n = 8). Experiments were performed on neonatal (0–3days) rat pups. Fictive respiratory activity was monitored by recording the rhythmic discharge from the hypoglossal rootlets of the medullary slices obtained from the neonatal rats. The burst frequency (BF) and integrated amplitude (IA) of the discharge were analyzed. Their responses to acidified artificial cerebrospinal fluid (aCSF) were tested to indicate the change of the central chemosensitivity. Under condition of perfusing with standard aCSF (pH 7.4), no significant difference was detected between the two groups in either BF or IA (P>0.05). Under condition of perfusing with acidified aCSF (pH 7.0), BF was increased and IA was decreased in both groups (P<0.01). However, their change rates in the CSE group were obviously smaller than that in the control group, 66.98 ± 10.11% vs. 143.75 ± 15.41% for BF and −22.38 ± 2.51% vs. −44.90 ± 3.92% for IA (P<0.01). In conclusion, these observations, in a prenatal CSE model, provide important evidence that maternal smoking during pregnancy exerts adverse effects on central chemoreception of neonates.