5-HT at hypoglossal motor nucleus and respiratory control of genioglossus muscle in anesthetized rats

Serotonin (5-HT) from medullary raphe neurons excites hypoglossal motoneurons innervating genioglossus (GG) muscle. Since some raphe neurons also show increased activity in hypercapnia, we tested the hypothesis that serotonergic mechanisms at the hypoglossal motor nucleus (HMN) modulate GG activity and responses to CO2. Seventeen urethane-anesthetized, tracheotomized and vagotomized rats were studied. Microdialysis probes were used to deliver mianserin (5-HT receptor antagonist, 0 and 0.1 mM) or 5-HT (eight doses, 0-50 mM) to the HMN during room air or CO2-stimulated breathing. Mianserin decreased respiratory-related GG activity during room air and CO2-stimulated breathing (P<0.001), and also suppressed GG responses to CO2 (P=0.05). In contrast, GG activity was increased by 5-HT at the HMN, and was further increased in hypercapnia (P<0.02). However, 5-HT increased respiratory-related GG activity at levels lower (1 mM) than those eliciting tonic GG activity (10-30 mM 5-HT). The results show that 5-HT at the HMN contributes to the respiratory control of GG muscle.     

CO2 retention in lung disease; could there be a pre-existing difference in respiratory physiology

Some patients with lung disease retain CO₂, while others with similar lung function do not. This could be explained if CO₂ retainers had a pre-existing low hypercapnic ventilatory response (HCVR) and, from this, a tendency to retain CO₂. To test if such a phenomenon exists in healthy people, we determined the change in end-tidal P_CO2 (ΔPET_CO2) produced by the addition of a dead-space (DS), during wakefulness and sleep, and related this to the HCVR measured awake. The group mean (n=14) HCVR slope was 2.2±1.1 (S.D.) L min⁻¹ mmHg⁻¹. The ΔPET_CO2 with the application of DS was 1.6±1.6 mmHg awake and 2.6±2.2 mmHg asleep. During wakefulness the ?PET_CO2 with DS did not correlate with the HCVR slope. However, during sleep, four subjects had a marked increase in the ΔPET_CO2 (3.7, 4.3, 6.2, 8.0 mmHg) and a relatively low HCVR (slope 1.5, 1.7, 1.5, 1.7 L min⁻¹ mmHg⁻¹, respectively). We speculate that such individuals, should they develop lung disease, may be predisposed to retain CO₂.     

The effects of ventilation pattern on carbon dioxide transfer in three computer models of the airways

We investigate the effects on arterial P(CO(2)) and on arterial-end tidal P(CO(2)) difference of six different ventilation patterns of equal tidal volume, and also of various combinations of tidal volume and respiratory rate that maintain a constant alveolar ventilation. We use predictions from three different mathematical models. Models 1 (distributed) and 2 (compartmental) include combined convection and diffusion effects. Model 3 incorporates a single well-mixed alveolar compartment and an anatomical dead-space in which plug flow occurs. We found that: (i) breathing patterns with longer inspiratory times yield lower arterial P(CO(2)); (ii) varying tidal volume and respiratory rate so that alveolar ventilation is kept constant may change both PA(CO(2)) and the PA(CO(2))-PET(CO(2)) difference; (iii) the distributed model predicts higher end-tidal and arterial P(CO(2)) than the compartmental models under similar conditions; and (iv) P(CO(2)) capnograms predicted by the distributed model exhibit longer phase I and steeper phase II than other models.     

Associative conditioning of the exercise ventilatory response in humans

Repeated hypercapnic exercise augmented the ventilatory response to subsequent trials of exercise alone in running goats and in humans performing arm exercise, suggesting a form of associative conditioning or 'long-term modulation' had taken place. These studies did not include 'control' single stimulus conditioning paradigms. This study demonstrated that ten repeated trials of familiar leg bicycling exercise with dead-space induced hypercapnia also elicited similar significant increases in inspired ventilation (+ 22%; P < 0.009) and tidal volume (VT; + 255 +/- 73 ml(BTPS); mean +/- S.E.M.; P = 0.004) within the first 20 sec of subsequent exercise only trials. Long-term modulation of the early ventilatory response to cycling was not fully replicated by ten trials of 'control' paradigms involving either repeated exercise alone or resting dead space alone. This study thus demonstrated that long term modulation of the early ventilatory response exercise was due to an explicit effect of associative conditioning and not simply sensitisation to repeated trials of a single stimulus.     

Muscimol dialysis into the caudal aspect of the Nucleus tractus solitarii of conscious rats inhibits chemoreception

We studied the effects on chemoreception of bilateral focal inhibition of the caudal Nucleus tractus solitarii (cNTS) by microdialysis of muscimol (0.5 mM) in rats during wakefulness and NREM sleep at two temperatures, 24 degrees C and 30 degrees C, just below and within the thermoneutral zone, respectively. Body temperature and VO2 did not differ at these two temperatures. The CO2 response (% increase in V(E)/VO2) did not differ at 24 degrees C vs. 30 degrees C and muscimol inhibited the CO2 response equally at both temperatures. In contrast, the hypoxic response (% increase in V(E)/VO2) was greater at 30 degrees C than at 24 degrees C and muscimol inhibited it only at 30 degrees C. These effects were similar in wakefulness and NREM sleep. We conclude that: (1) ambient temperature can affect the V(E)/VO2 response to hypoxia but not hypercapnia and (2) at 24 degrees C muscimol in the cNTS affects the CO2 response but not the hypoxic response providing indirect support for the presence of chemoreception within the NTS.     

Fluoxetine augments ventilatory CO2 sensitivity in Brown Norway but not Sprague Dawley rats

The Brown Norway (BN; BN/NHsdMcwi) rat exhibits a deficit in ventilatory CO₂ sensitivity and a modest serotonin (5-HT) deficiency. Here, we tested the hypothesis that the selective serotonin reuptake inhibitor fluoxetine would augment CO₂ sensitivity in BN but not Sprague Dawley (SD) rats. Ventilation during room air or 7% CO₂ exposure was measured before, during and after 3 weeks of daily injections of saline or fluoxetine (10 mg/(kg day)) in adult male BN and SD rats. Fluoxetine had minimal effects on room air breathing in BN and SD rats (p > 0.05), although tidal volume (V_T) was reduced in BN rats (p < 0.05). There were also minimal effects of fluoxetine on CO₂ sensitivity in SD rats, but fluoxetine increased minute ventilation, breathing frequency and V_T during hypercapnia in BN rats (p < 0.05). The augmented CO₂ response was reversible upon withdrawal of fluoxetine. Brain levels of biogenic amines were largely unaffected, but 5-HIAA and the ratio of 5-HIAA/5-HT were reduced (p < 0.05) consistent with selective and effective 5-HT reuptake inhibition. Thus, fluoxetine increases ventilatory CO₂ sensitivity in BN but not SD rats, further suggesting altered 5-HT system function may contribute to the inherently low CO₂ sensitivity in the BN rat.     

Unevenness of ventilation assessed by the expired CO(2) gas volume versus V(T) curve in asthmatic patients

Recently, we have shown that the expired CO2 gas volume versus tidal volume (VCO2-VT) curve is a useful tool for assessing unevenness of ventilation because it allows the separation of tidal volume into three functional compartments: (a) the CO2-free expired air (V0), (b) the transitional volume (Vtr), (c) the alveolar volume (VA) and the measurement of alveolar FCO2 during resting breathing in normal subjects and patients with COPD. In this paper, we have investigated whether changes pertaining to unevenness of ventilation taking place immediately after the administration of methacholine can be assessed using the VCO2-VT curve in asthmatic patients. The VCO2-VT curve was obtained during tidal breathing from 16 stable asthmatic patients who underwent a methacholine challenge test. It has been found that the Vtr, and hence Bohr's dead space (VD,Bohr = V0 + Vtr), over tidal volume ratios were significantly increased immediately after the methacholine administration, whilst the V0 over tidal volume ratio was not affected. The change of the above ratios was not related to the percentage decrease of FEV1.0 following methacholine administration.     

GABAA receptors mediate postnatal depression of respiratory frequency by barbiturates

We tested the hypothesis that barbiturates depress respiratory motor output by actions on the GABAA receptor. We examined the influence of pentobarbital sodium on nerve activity recorded from a fourth cervical (C4) ventral root (phrenic motoneuron output) in the in vitro brainstem-spinal cord preparation of neonatal rats aged 1-3 days. Bath application of pentobarbital slowed the respiratory rhythm but this effect could be reversed by drug washout or by simultaneous application of 8 microM bicuculline methiodide, a GABAA receptor antagonist. Pentobarbital up to a concentration of 80 microM (or 20 mg/l) did not change the magnitude of C4 nerve bursts. The GABAA receptor agonist muscimol evoked similar changes. The results support the hypothesis that respiratory depression by barbiturates is due to GABAA receptor-mediated inhibition, with the principal effects on rhythm generation. In the light of recent studies suggesting that GABAA receptors may be excitatory in the early neonatal period, we examined postnatal changes in the GABAergic slowing of respiratory rhythm. Stimulation of GABAA receptors slowed respiratory rhythm from the first postnatal day, with no change in efficacy over the first 3 days of life.     

Elimination of central chemosensitivity by coagulation of a bilateral area on the ventral medullary surface in awake cats

Breathing and respiratory response to CO₂ were observed in 6 awake cats and 1 control before and after bilateral coagulation of the formerly described area S (Schläfke and Loeschcke, 1967) on the ventral medullary surface under hyperoxic conditions. Ventilation decreased,PCO₂ rose and CO₂ response was almost or completely abolished in 4 cats, and moderately reduced in 2 cats. Inhalation of CO₂ had an inhibitory effect on ventilation in two cases. In some instances the respiratory frequency was increased by CO₂. Periodic breathing as well as spotaneous hyperventilatron elicited by arousal indicate parallels to the Pickwickian or Ondine's curse syndrome. No respiratory changes were produced by a lesion on the pyramidal tract medial to the area S. It is concluded that central chemosensitivity can be eliminated within the superficial layer of the area S. The loss of CO₂ response seems to be correlated with complete destruction of the superficial nerve cells located within the area S (Petrovický, 1968) and degeneration within the ventral part of the nucleus paragigantocellularis.Supported by the Deutsche Forschungsgemeinschaft (SFB 114, Bionach)     

Effects of clonidine on breathing during sleep and susceptibility to central apnoea

We hypothesized that administration of clonidine would decrease the hypocapnic apnoeic threshold (HAT) and widen the CO₂ reserve during non-REM sleep.     

Variation in human ventilatory control—genetic influence on the hypoxic ventilatory response

Studies in human subjects have shown considerable variation in the ventilatory response to hypoxia among individuals. The potential influence of genetic factors was initially suggested by the finding of clusters of low responses in families of patients with unexplained hypoventilation and endurance athletes. Further evidence of a genetic effect was evident in studies which found greater similarity of hypoxic ventilatory response in monozygotic than in dizygotic twins. The apparently selective effect on the hypoxic versus the hypercapnic response suggests that the genetic influence is related to peripheral chemosensitivity. Studies in cats and rats also point to a genetic effect on the carotid body.     

Increased ventilation and CO2 chemosensitivity in acetylcholinesterase knockout mice

To investigate the effects of a permanent excess of acetylcholine (AChE) on respiration, breathing and chemosensitivity were analyzed from birth to adulthood in mice lacking the AChE gene (AChE-/-), in heterozygotes, and in control wild-type (AChE+/+) littermates. Breathing at rest and ventilatory responses to brief exposures to hypoxia (10% O2) and hypercapnia (3-5% CO2) were measured by whole-body plethysmography. At rest AChE-/- mice show larger tidal volumes (VT, + 96% in adults), overall ventilation (VE, + 70%), and mean inspiratory flow (+270%) than wild-type mice, with no change in breathing frequency (fR). AChE-/- mice have a slightly blunted response to hypoxia, but increased VE and fR responses to hypercapnia. Heterozygous animals present no consistent alterations of breathing at rest and chemosensitivity is normal. Adult AChE-/- mice have an increased VE/VO2 and a marginally higher normalized VO2. The results suggest that the hyperventilation and altered chemosensitivity in AChE-/- mice largely reflect alterations of central respiratory control.     

Effect of alteration of peripheral blood flow on the central circulation in man during supine cycling in different ambient temperatures

Summary Whether the alteration of peripheral circulation caused by changing ambient temperature (T_a) affects central circulatory changes in man during supine cycling was investigated in four well-trained men, who exercised at two levels (117.7 or 176.6 W). Exercise metabolic rate (VO₂) in cold (0 C or 10 C) was the same as it was at 20 C, whereas the cardiac output (CO; CO₂ rebreathing technique) and heart rate were significantly lower (e.g., 176.6 W at 0 C, both p<0.01). In heat (30 C or 40 C), the VO₂ reduced with falling CO and mean arterial blood pressure from those at 20 C (e.g., 176.6 W at 40 C, all cases p<0.01), whereas the peak post-exercise calf blood flow (CBF_p) increased (p<0.01). The VO₂ and stroke volume (SV) were inversely proportional to the ratio of CBF_p to CO/kg body weight (CBF_p/CO) (r>–0.78, p<0.001). Total peripheral resistance (TPR) was related to arteriovenous oxygen difference (A-VO₂ difference) (r>0.78, p<0.001). The TPR and A-VO₂ difference decreased as T_a rose, while CBF_p/CO was almost the same. As CBF_p/CO had exceeded 50 and further progressed, however, the two parameters elevated until the same level as that at 0 C. The present results suggest that during moderately prolonged (16–60 min) supine cycling in different T_a's the central circulatory changes are mainly affected by the altered peripheral blood flow in competing between skin and muscle for blood flow.     

Effect of medroxyprogesterone on inspiratory flow shapes during sleep in postmenopausal women

We previously showed that medroxyprogesterone acetate (MPA) effectively decreases the arterial CO(2) levels in postmenopausal women with partial upper airway obstruction. The aim of the present study was to analyze the effects of MPA on the inspiratory flow shapes during sleep. Eight postmenopausal women with hypoxemia and partial upper airway obstruction during sleep (patients) received MPA 60 mg daily for 14 days. Four matched postmenopausal women without MPA treatment served as controls. Sleep and nasal pressure were recorded on each visit. Each breath was analyzed for duration, volume and inspiratory flow shape class. MPA shortened inspiration and prolonged expiration. The inspiratory volumes increased consistently in all flow shape classes. The inspiratory shapes with single late peak were transformed to those with double peak. MPA also decreased shapes with mid-peak or mid-plateau. MPA did not have an effect on sleep. Sleep modified the flow shape distribution only in patients but in a similar fashion in stages S2, SWS and REM. The results suggest that postmenopausal women present with a significant proportion breaths with poor initial inspiratory flow, which is reversed with MPA-induced respiratory stimulation.     

Ventral medulla pHi measured in vivo by 31P NMR is not regulated during hypercapnia in anesthetized rat

Chemoreceptors in the ventral medulla contribute to the respiratory response to hypercapnia. Do they ‘sense’ intracellular pH (pH_i)? We measured pH_i in the ventral medulla or cortex (control) using ³¹P-NMR obtained via a novel 3×5 mm² surface coil in anesthetized rats breathing air or 7% CO₂. During air breathing over 240 min, pH_i decreased slightly from 7.13±0.02 to 7.05±0.02 (SEM; n=5; 2 cortex, 3 ventral medulla). During 180 min of hypercapnia, cortical pH_i (n=4) decreased from 7.17±0.02 to 6.87±0.01 by 90 min and recovered by 150 min. Ventral medulla pH_i showed no such regulation. It decreased from 7.11±0.02 to 6.88±0.02 at 90 min and recovered only after cessation of hypercapnia (n=5), results consistent with pH_i being the chemoreceptor stimulus. However, non-chemoreceptor neurons that contribute to our medullary NMR signal also do not appear to regulate pH_i in vitro. Regional differences in pH_i regulation between cortex and ventral medulla may be due to both chemosensitive and non-chemosensitive neurons.     

Lack of gender difference in ventilatory chemoresponsiveness and post-hypoxic ventilatory decline

Altered chemoresponsiveness has been postulated to explain the gender difference in the incidence of sleep disordered breathing (SDB). The purpose of this investigation was to ascertain a gender difference in the effect of hypocapnic hypoxia on ventilation. Hypocapnic hypoxia was induced in stable NREM sleep for 3 min periods. In the first analysis, hypoxic ventilatory response in a steady state (SHVR) was defined as the amount of change in minute ventilation (VI) between mean room air (RA) and hypoxia divided by the change in Sa O2 between RA and hypoxia (DeltaVI/DeltaSa O2). The mean group SHVR values were 0.23+/-0.15 and 0.20+/-0.10 L/min per %SaO2, for men and women, respectively (P = ns). In the second analysis, we analyzed the decline in ventilatory parameters after the cessation of hypoxia. There was no difference in VI between the genders (men, 5.6+/-1.7 L/min vs. women, 4.9+/-1.9 L/min, P = ns). We conclude that the gender difference in SDB is not explained by a difference in the ventilatory response to hypocapnic hypoxia.     

Ventilatory pattern and chemosensitivity in M1 and M3 muscarinic receptor knockout mice

Acetylcholine (ACh) acting through muscarinic receptors is thought to be involved in the control of breathing, notably in central and peripheral chemosensory afferents and in regulations related to sleep-wake states. By using whole-body plethysmography, we compared baseline breathing at rest and ventilatory responses to acute exposure (5 min) to moderate hypoxia (10% O(2)) and hypercapnia (3 and 5% CO(2)) in mice lacking either the M(1) or the M(3) muscarinic receptor, and in wild-type matched controls. M(1) knockout mice showed normal minute ventilation (V(E)) but elevated tidal volume (V(T)) at rest, and normal chemosensory ventilatory responses to hypoxia and hypercapnia. M(3) knockout mice had elevated V(E) and V(T) at rest, a reduced V(T) response slope to hypercapnia, and blunted V(E) and frequency responses to hypoxia. The results suggest that M(1) and M(3) muscarinic receptors play significant roles in the regulation of tidal volume at rest and that the afferent pathway originating from peripheral chemoreceptors involves M(3) receptors.     

Connexin36 distribution in putative CO2-chemosensitive brainstem regions in rat

Recent work from our laboratory has demonstrated that the gap junction proteins connexin26 (Cx26) and connexin32 (Cx32) are expressed in neurons in putative CO2-chemosensitive brainstem regions in both neonatal and adult rats. Whether the recently identified neuron-specific gap junction protein connexin36 (Cx36) is also present in these brainstem regions remains to be determined. Therefore, in the current experiments, immunoblot and immunohistochemical protocols were used to investigate the regional distribution and cellular localization of Cx36 in putative CO2-chemosensitive brainstem regions in both neonatal and adult rats. Immunoblot analyses revealed Cx36 expression in putative CO2-chemosensitive brainstem regions in each of the age groups examined, although both regional and developmental differences in the relative expression levels were detected. Immunohistochemical analyses confirmed Cx36 expression in neurons in each of the putative CO2-chemosensitive brainstem regions and revealed both somal and dendritic labeling patterns. These findings provide additional morphological evidence supporting the potential for gap junctional communication in these regions in both neonatal and adult rats. We propose that the gap junction protein Cx36 also contributes to the neuroanatomical substrate for gap junctional communication, which is hypothesized to play a role in central CO2 chemoreception.     

Lessons from chronic intermittent and sustained hypoxia at high altitudes

Recurrent sleep apnea (RSA), mimicking chronic intermittent hypoxia (CIH), may trigger unique adaptations in oxygen sensing in the carotid body, and consequent cellular functions unlike the effects of sustained hypoxia (SH). As a mechanism, an augmented generation of reactive oxygen species (ROS) in CIH has been invoked at the exclusion of SH effects. The ROS might act at hypoxia inducible factors (HIF-1s), giving rise to various genes whose function is to restore the tissue P(O(2)) close to the original. In a spate, review articles on the CIH effects at sea level have appeared but little on high altitude (HA). Their views have been reexamined with the primary focus on the peripheral chemoreception. At HA, RSA is more common in the lowlanders because of a high ventilatory sensitivity to hypoxia (with the consequent effects) unlike the high altitude natives (HAN). Undoubtedly, the HIF-1s play a central role at HA, the mechanisms of which are unknown and explorable.     

Commentary on the definition of eupnea and gasping

Despite clear qualitative differences, it has proven difficult to identify criteria that reliably differentiate eupnea and gasping--particularly when multiple species or experimental preparations are considered. From a motor control perspective, this is unsurprising. Three organizational rules are common to nearly all rhythmic activities: (1) the basic rhythm is produced by a small network of cells, (2) the activity of this network in isolation often differs dramatically from the behavior of the whole animal, and (3) the rhythmogenic networks responsible for related behaviors are not fixed and independent but dynamically modifiable and overlapping. In this context, it becomes clear that the definition of a particular pattern and the investigation of the mechanisms underlying its production are inseparable. Rather than attempting to rigidly apply criteria developed using any one experimental preparation, the classification of respiratory patterns must evolve alongside our understanding of how each pattern is produced-a process that is only aided by investigations using a variety of experimental preparations.