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While barotrauma, decompression sickness, and drowning-related injuries are common morbidities associated with diving and decompression from depth, it remains unclear what impact rapid decompression has on mitochondrial function. In vitro diving simulation was performed with human dermal fibroblast cells subjected to control, air, nitrogen, and oxygen dive conditions. With the exception of the gas mixture, all other related variables, including absolute pressure exposure, dive and decompression rates, and temperature, were held constant. High-resolution respirometry was used to examine key respiratory states. Mitochondrial dynamic function, including net movement, number, and rates of fusion/fission events, was obtained from fluorescence microscopy imaging. Effects of the dive conditions on cell cytoskeleton were assessed by imaging both actin and microtubules. Maximum respiration was lower in fibroblasts in the air group than in the control and nitrogen groups. The oxygen group had overall lower respiration when compared with all other groups. All groups demonstrated lower mitochondrial motility when compared with the control group. Rates of fusion and fission events were the same between all groups. There were visible differences in cell morphology consistent with the actin staining; however, there were no appreciable changes to the microtubules. This is the first study to directly assess mitochondrial respiration and dynamics in a cell model of decompression. Both hyperbaric oxygen and air dive conditions produce deleterious effects on overall mitochondrial health in fibroblasts.

The cold-sensitive cation channel TRPM8 is a target for menthol, which is used routinely as a cough suppressant and as an additive to tobacco and food products. Given that cold temperatures and menthol activate neurons through gating of TRPM8, it is unclear how menthol actively suppresses cough. In this study we describe the antitussive effects of (-)-menthol in conscious and anesthetized guinea pigs. In anesthetized guinea pigs, cough evoked by citric acid applied topically to the tracheal mucosa was suppressed by menthol only when it was selectively administered as vapors to the upper airways. Menthol applied topically to the tracheal mucosa prior to and during citric acid application or administered continuously as vapors or as an aerosol to the lower airways was without effect on cough. These actions of upper airway menthol treatment were mimicked by cold air delivered to the upper airways but not by (+)-menthol, the inactive isomer of menthol, or by the TRPM8/TRPA1 agonist icilin administered directly to the trachea. Subsequent molecular analyses confirmed the expression of TRPM8 in a subset of nasal trigeminal afferent neurons that do not coincidently express TRPA1 or TRPV1. We conclude that menthol suppresses cough evoked in the lower airways primarily through a reflex initiated from the nose.

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

Although central to the susceptibility of adult diseases characterized by abnormal rhythmogenesis, characterizing the genes involved is a challenge. We took advantage of the C57BL/6J (B6) trait of hypoxia-induced periodic breathing and its absence in the C57BL/6J-Chr 1(A/J)/NaJ chromosome substitution strain to test the feasibility of gene discovery for this abnormality. Beginning with a genetic and phenotypic analysis of an intercross study between these strains, we discovered three quantitative trait loci (QTLs) on mouse chromosome 1, with phenotypic effects. Fine-mapping reduced the genomic intervals and gene content, and the introgression of one QTL region back onto the C57BL/6J-Chr 1(A/J)/NaJ restored the trait. mRNA expression of non-synonymous genes in the introgressed region in the medulla and pons found evidence for differential expression of three genes, the highest of which was apolipoprotein A2, a lipase regulator; the apo a2 peptide fragment (THEQLTPLVR), highly expressed in the liver, was expressed in low amounts in the medulla but did not correlate with trait expression. This work directly demonstrates the impact of elements on mouse chromosome 1 in respiratory rhythmogenesis.

People with Rett syndrome (RTT) have breathing instability in addition to other neuropathological manifestations. The breathing disturbances contribute to the high incidence of unexplained death and abnormal brain development. However, the cellular mechanisms underlying the breathing abnormalities remain unclear. To test the hypothesis that the central CO(2) chemoreception in these people is disrupted, we studied the CO(2) chemosensitivity in a mouse model of RTT. The Mecp2-null mice showed a selective loss of their respiratory response to 1-3% CO(2) (mild hypercapnia), whereas they displayed more regular breathing in response to 6-9% CO(2) (severe hypercapnia). The defect was alleviated with the NE uptake blocker desipramine (10 mg·kg(-1)·day(-1) ip, for 5-7 days). Consistent with the in vivo observations, in vitro studies in brain slices indicated that CO(2) chemosensitivity of locus coeruleus (LC) neurons was impaired in Mecp2-null mice. Two major neuronal pH-sensitive Kir currents that resembled homomeric Kir4.1 and heteromeric Ki4.1/Kir5.1 channels were identified in the LC neurons. The screening of Kir channels with real-time PCR indicated the overexpression of Kir4.1 in the LC region of Mecp2-null mice. In a heterologous expression system, an overexpression of Kir4.1 resulted in a reduction in the pH sensitivity of the heteromeric Kir4.1-Kir5.1 channels. Given that Kir4.1 and Kir5.1 subunits are also expressed in brain stem respiration-related areas, the Kir4.1 overexpression may not allow CO(2) to be detected until hypercapnia becomes severe, leading to periodical hyper- and hypoventilation in Mecp2-null mice and, perhaps, in people with RTT as well.

The peroxisome proliferator-activated receptor gamma 2 (PPARγ2) genotypes are related to obesity and the metabolic syndrome (MetS). A low level of cardiorespiratory fitness is also a strong determining factor in the development of MetS. This cross-sectional study was performed to investigate the influence of the interaction between the PPARγ2 genotype and cardiorespiratory fitness on the risk of MetS. Healthy Japanese men (n = 211) and women (n = 505) participated in this study. All subjects were divided into 8 groups according to sex, fitness level (high and low fitness groups), and age (younger, age < 40 yr; middle-aged/older, age ≥ 40 yr). The PPARγ2 genotypes (Pro12Ala and C1431T) were analyzed by real-time PCR with Taq-Man probes. Two-way ANCOVA with adjustment for age as a covariate indicated that fitness and the CC genotype of C1431T in the PPARγ2 gene interacted to produce a significant effect on MetS risk in younger men and that the risk of MetS in the CC genotype group with low cardiorespiratory fitness was significantly higher than that in the corresponding CT+TT genotypes or in the high fitness groups. There was no significant interaction between fitness and genotype in determining MetS risk in middle-aged/older men or in women in any group. With regard to the Pro12Ala genotype of the PPARγ2 gene, there were no significant differences in fitness or genotype effects nor were there any interactions between measurement variables. We concluded that the CC genotype of C1431T in the PPARγ2 gene together with low cardiorespiratory fitness may increase the risk of MetS in younger men (age < 40 yr), even with adjustment for age.

The long-term effects of uncoupled mitochondrial respiration by uncoupling protein-2 (UCP2) in mammalian physiology remain controversial. Here we show that increased mitochondrial uncoupling activity of different tissues predicts longer lifespan of rats compared with mice. UCP2 reduces reactive oxygen species (ROS) production and oxidative stress throughout the aging process in different tissues in mice. The absence of UCP2 shortens lifespan in wild-type mice, and the level of UCP2 positively correlates with the postnatal survival of superoxide dismutase-2 mutant animals. Thus UCP2 has a beneficial influence on cell and tissue function leading to increased lifespan.

Mild reductions in ambient temperature dramatically increase the mortality of neonatal mice deficient in pituitary adenylate cyclase-activating polypeptide (PACAP), with the majority of animals succumbing in the second postnatal week. During anesthesia-induced hypothermia, PACAP(-/-) mice at this age are also vulnerable to prolonged apneas and sudden death. From these observations, we hypothesized that before the onset of genotype-specific mortality and in the absence of anesthetic, the breathing of PACAP-deficient mice is more susceptible to mild reductions in ambient temperature than wild-type littermates. To test this hypothesis, we recorded breathing in one group of postnatal day 4 PACAP+/+, (+/-), and (-/-) neonates (using unrestrained, flow-through plethysmography) and metabolic rate in a separate group (using indirect calorimetry), both of which were exposed acutely to ambient temperatures slightly below (29 degrees C), slightly above (36 degrees C), or at thermoneutrality (32 degrees C). At 32 degrees C, the breathing frequency of PACAP(-/-) neonates was significantly less than PACAP+/+ littermates. Reducing the ambient temperature to 29 degrees C caused a significant suppression of tidal volume and ventilation in both PACAP+/- and (-/-) animals, while the tidal volume and ventilation of PACAP+/+ animals remained unchanged. Genotype had no effect on the ventilatory responses to ambient warming. At all three ambient temperatures, genotype had no influence on oxygen consumption or body temperature. These results suggest that during mild reductions in ambient temperature, PACAP is vital for the preservation of neonatal tidal volume and ventilation, but not for metabolic rate or body temperature.

Apart from enhancing red blood cell production, erythropoietin (Epo) has been shown to modulate the ventilatory response to reduced oxygen supply. Both functions are crucial for the organism to cope with increased oxygen demand. In the present work, we analyzed the impact of Epo and the resulting excessive erythrocytosis in the neural control of normoxic and hypoxic ventilation. To this end, we used our transgenic mouse line (Tg6) that shows high levels of human Epo in brain and plasma, the latter leading to a hematocrit of approximately 80%. Interestingly, while normoxic and hypoxic ventilation in Tg6 mice was similar to WT mice, Tg6 mice showed an increased respiratory frequency but a decreased tidal volume. Knowing that Epo modulates catecholaminergic activity, the altered catecholaminergic metabolism measured in brain stem suggested that the increased respiratory frequency in Tg6 mice was related to the overexpression of Epo in brain. In the periphery, higher response to hyperoxia (Dejours test), as well as reduced tyrosine hydroxylase activity in carotid bodies, revealed a higher chemosensitivity to oxygen in transgenic mice. Moreover, in line with the decreased activity of the rate-limiting enzyme for dopamine synthesis, the intraperitoneal injection of a highly specific peripheral ventilatory stimulant, domperidone, did not stimulate hypoxic ventilatory response in Tg6 mice. These results suggest that high Epo plasma levels modulate the carotid body's chemotransduction. All together, these findings are relevant for understanding the cross-talk between the ventilatory and erythropoietic systems exposed to hypoxia.

Differences in breathing pattern between awake C57BL/6J (B6) and A/J mice are such that A/J mice breathe slower, deeper, and with greater variability than B6. We theorized that urethane anesthesia, by affecting cortical and subcortical function, would test the hypothesis that strain differences require a fully functional neuroaxis. We anesthetized B6 and A/J mice with urethane, placed them in a whole-body plethysmograph, and measured the durations of inspiration and expiration, respiratory frequency (Fr), and peak amplitude during exposure to room air (21% O2), hyperoxia (5 min, 100% O2), hypoxia (5 min, 8% O2), and posthypoxic reoxygenation (5 min, 100% O2). Breathing variability was assessed by calculating the coefficient of variation (CV) and by applying spatial statistics to Poincaré plots constructed from the timing and amplitude data. Even though Fr in anesthetized B6 and A/J mice was greater than that for unanesthetized animals, anesthetized A/J mice still breathed slower, deeper, and with greater variability than B6 mice at rest and during hyperoxia. During the fourth minute of hypoxia, Fr and its CV were not significantly different between strains. Even though Fr was similar between strains immediately after hypoxia, its CV was significantly greater for B6 than A/J mice. Posthypoxic Fr was significantly less than baseline Fr in B6 but not A/J mice, and the CV for posthypoxic Fr was greater for B6 but less for AJ mice compared with baseline CV. This difference in patterning was confirmed by spatial statistical analysis. We conclude that strain-specific differences in respiratory pattern and its variability are robust genetic traits. The neural substrate for these differences, at least partially, exists within subcortical structures generating the breathing pattern.

The hypothesis that BHE/Cdb rats with mutations in their mitochondrial genome might accommodate this mutation by changing their food intake patterns was tested. Four experiments were conducted. Experiments 1 and 2 examined food intake patterns of BHE/Cdb rats fed a stock diet or BHE/Cdb and Sprague-Dawley rats fed a high-fat diet from weaning. Experiment 3 examined the daily rhythms of respiration and heat production in these rats at 200 days of age. Experiment 4 examined the effects of diet composition on these measurements at 50-day intervals. The Sprague-Dawley rats, regardless of diet, had the typical day-night rhythms of feeding and respiration. In contrast, the BHE/Cdb rats fed the high-fat diet showed normal rhythms initially, but with age, these rhythms were attenuated. The changes in rhythms preceded the development of glucose intolerance.

Acutely lowering ambient O(2) tension increases ventilation in many mammalian species, including humans and mice. Inheritance patterns among kinships and between mouse strains suggest that a robust genetic influence determines individual hypoxic ventilatory responses (HVR). Here, we tested specific genetic hypotheses to describe the inheritance patterns of HVR phenotypes among two inbred mouse strains and their segregant and nonsegregant progeny. Using whole body plethysmography, we assessed the magnitude and pattern of ventilation in C3H/HeJ (C3) and C57BL/6J (B6) progenitor strains at baseline and during acute (3-5 min) hypoxic [mild hypercapnic hypoxia, inspired O(2) fraction (FI(O(2))) = 0.10] and normoxic (mild hypercapnic normoxia, FI(O(2)) = 0.21) inspirate challenges in mild hypercapnia (inspired CO(2) fraction = 0.03). First- and second-filial generations and two backcross progeny were also studied to assess response distributions of HVR phenotypes relative to the parental strains. Although the minute ventilation (VE) during hypoxia was comparable between the parental strains, breathing frequency (f) and tidal volume were significantly different; C3 mice demonstrated a slow, deep HVR relative to a rapid, shallow phenotype of B6 mice. The HVR profile in B6C3F(1)/J mice suggested that this offspring class represented a third phenotype, distinguishable from the parental strains. The distribution of HVR among backcross and intercross offspring suggested that the inheritance patterns for f and VE during mild hypercapnic hypoxia are consistent with models that incorporate two genetic determinants. These results further suggest that the quantitative genetic expression of alleles derived from C3 and B6 parental strains interact to significantly attenuate individual HVR in the first- and second-filial generations. In conclusion, the genetic control of HVR in this model was shown to exhibit a relatively simple genetic basis in terms of respiratory timing characteristics.

The purpose of the present study was to determine the genetic control of baseline breathing pattern by examining the mode of inheritance between two inbred murine strains with differential breathing characteristics. Specifically, the rapid, shallow phenotype of the C57BL/6J (B6) strain is consistently distinct from the slow, deep phenotype of the C3H/HeJ (C3) strain. The response distributions of segregant and nonsegregant progeny were compared with the two progenitor strains to determine the mode of inheritance for each ventilatory characteristic. The BXH recombinant inbred (RI) strains derived from the B6 and C3 progenitors were examined to establish strain distribution patterns for each ventilatory trait. To establish the mode of inheritance, baseline breathing frequency (f), tidal volume, and inspiratory time (TI) were measured five times in each of 178 mature male animals from the two progenitor strains and their progeny by using whole body plethysmography. With respect to f and TI, the two progenitor strains were consistently distinct, and segregation analyses of the inheritance pattern suggest that the most parsimonious genetic model for response distributions of f and TI is a two-loci model. In similar experiments conducted on 82 mature male animals from 12 BXH RI strains, each parental phenotype was represented by one or more of the RI strains. Intermediate phenotypes emerged to confirm the likelihood that parental strain differences in f and TI were determined by more than one locus. Taken together, these studies suggest that the phenotypic difference in baseline respiratory timing between male B6 and C3 mice is best explained by a genetic model that considers at least two loci as major determinants.