Post-hypoxic Frequency Decline Research Articles

Basal and Hypoxic Respiratory Behaviors in Aged 6‐OHDA‐Induced SN Lesion PD Rat Model: Effects of the 5‐HT1A Receptor Agonist Buspirone

Parkinson’s disease (PD) is progressive neurodegenerative disease, with the diagnosis of idiopathic PD occurring in humans at ~60 years of age. Most studies, however, use young adult (≤3 month old) rats to investigate PD‐related abnormalities in various physiological systems, with the most commonly used experimental rat PD model being generated by unilateral injection of the neurotoxin 6‐hydroxydopamine (6‐OHDA) into specific regions of the nigrostriatal pathway. While this model results in the loss of dopaminergic neurons and the subsequent changes in downstream neurotransmitter signaling that are characteristic of PD, it does not consider potential contributions of physical changes to the brain and neurotransmitter signaling that are part of normal aging on the progression of PD. To better understand age‐related influences and potential contributions of PD‐related abnormalities in serotonergic (5‐HT) neurotransmission on respiratory dysfunction (which is a common non‐motor symptom) in PD, we investigated basal and hypoxic (12% O2; 90s) respiratory behaviors before and after acute administration of the frequently prescribed anxiolytic 5‐HT1A receptor agonist buspirone (BUS; 0.5 mg/kg, iv) in an aged (~9 month old) female Sprague‐Dawley PD rat model produced by unilateral 6‐OHDA injection into the substantia nigra (SN). For these experiments, diaphragm EMG activity was recorded and quantified from spontaneously breathing urethane‐anesthetized rats at 2‐weeks after SN 6‐OHDA or vehicle (control) injections. We found that 6‐OHDA‐injected rats have a lower basal breathing frequency (P<0.05) due to shorter expiratory duration (TE; P<0.05) than vehicle‐injected rats, but both 6‐OHDA‐ and vehicle‐injected rats exhibit significant increases in breathing frequency (P≤0.001) and EMG amplitude (P≤0.023) during hypoxia; the magnitude of the peak frequency increase in 6‐OHDA‐injected rats was slightly greater (P=0.083). Both 6‐OHDA‐ and vehicle‐injected rats also showed similar post‐hypoxic frequency decline (P≤0.003), but only vehicle‐injected rats displayed a slight and sustained post‐hypoxic amplitude depression (P<0.1). In vehicle‐injected rats, acute BUS injection produced a progressive decrease in basal EMG amplitude (P=0.066) and a persistent increase in breathing frequency (P=0.064) by decreasing inspiratory duration (TI; P<0.001) while in 6‐OHDA‐injected rats, a transient initial increase in EMG amplitude (P=0.043) and a persistent decrease in TI (P=0.005) and TE (P=0.089) were noted. In both 6‐OHDA‐ and vehicle‐injected rats, BUS injection also slightly exaggerated the initial hypoxic frequency increase, but blunted the amplitude increase with amplitude progressively declining to near pre‐hypoxic levels before the end of the hypoxic exposure. BUS injection also shortened the duration of post‐hypoxic EMG amplitude depression in vehicle‐injected rats. These data demonstrate that some differences in basal and hypoxic respiratory behaviors exist between older 6‐OHDA‐ and vehicle‐injected rats, and that some respiratory behaviors of these rat models are differentially impacted by BUS‐induced effects on respiratory control. We suggest that this older PD rat model be further evaluated.

Cardiorespiratory plasticity in humans following two patterns of acute intermittent hypoxia.

What is the central question of this study? Do cardiorespiratory experience-dependent effects (EDEs) differ between two different stimulus durations of acute isocapnic intermittent hypoxia (IHx; 5-min vs. 90-s cycles between hypoxia and normoxia)? What is the main finding and its importance? There was long-term facilitation in ventilation and blood pressure in both IHx protocols, but there was no evidence of progressive augmentation or post-hypoxia frequency decline. Not all EDEs described in animal models translate to acute isocapnic IHx responses in humans, and cardiorespiratory responses to 5-min versus 90-s on/off IHx protocols are largely similar. Peripheral respiratory chemoreceptors monitor breath-by-breath changes in arterial CO2 and O2 , and mediate ventilatory changes to maintain homeostasis. Intermittent hypoxia (IHx) elicits hypoxic ventilatory responses, with well-described experience-dependent effects (EDEs), derived mostly from animal work involving intermittent 5-min cycles of hypoxia and normoxia. These EDEs include post-hypoxia frequency decline (PHxFD), progressive augmentation (PA) and long-term facilitation (LTF). Comparisons of these EDEs between animal models and humans using similar IHx protocols are lacking. In addition, it is unknown whether shorter bouts of hypoxia, which may be more relevant to clinical conditions, elicit EDEs of similar magnitudes in humans. Respiratory (frequency, tidal volume and minute ventilation ( ) and cardiovascular (heart rate and mean arterial pressure (MAP)) variables were measured during and following two patterns of acute isocapnic IHx in 14 healthy human participants (four female): (1) 5×5min and (2) 5×90s on/off hypoxia. Participants' end-tidal was clamped at 45Torr during hypoxia and 100Torr during normoxia. We found that (1) PHxFD and PA were not present in either IHx pattern (P>0.14), (2) LTF was present in following both 5-min (P<0.001) and 90-s isocapnic IHx trials (P<0.001), and (3) LTF was present in MAP following 5-min isocapnic IHx (P<0.001), and trended towards significance following 90-s IHx (P=0.058). We demonstrate that acute isocapnic IHx alone may not elicit all of the EDEs that have been described in animal models. Additionally, ventilatory LTF occurred regardless of the length of hypoxia-normoxia cycles.

Impact of aging on basal and hypoxic respiratory behaviors in short‐ and long‐term 6‐OHDA‐induced SN lesion PD rat models

Parkinson's Disease (PD) is a progressive neurodegenerative disease often associated with aging, as it typically occurs in individuals over the age of 50. As such, investigating features and mechanisms of PD becomes complicated since molecular changes to dopaminergic (DA) neurons, subsequent neuronal loss, and downstream alterations to non-DA neurotransmitter systems can progress over decades to cause the various motor and non-motor symptoms (NMS), which can be further compounded by changes caused by normal aging. Amongst the earliest symptoms to arise in PD patients, respiratory abnormalities are a common feature, and ongoing work in our laboratory has focused on characterizing respiratory dysfunction phenotypes in preclinical PD rat models produced by 6-hydroxydopamine (6-OHDA) neurotoxin-induced lesion of the nigrostriatal pathway. These studies are typically conducted in rats aged 3-4 months at 2-4 weeks following lesion. To begin to understand the impact of aging on PD-related respiratory abnormalities, the current study evaluated basal and hypoxic (12% O2; 90s) respiratory behaviors in ~38-week-old urethane-anesthetized spontaneously breathing female Sprague-Dawley rats that received a unilateral substantia nigra (SN) 6-OHDA injection at either 2-weeks (short-term lesion; STL) or 24-weeks (long-term lesion; LTL) before being studied; SN vehicle (STV; LTV) injections served as controls. We found that LTL rats had higher basal breathing frequencies and slightly reduced burst-to-burst frequency and amplitude variability than LTV rats (which is similar to our observations in the younger SN 6-OHDA vs vehicle injected rats) while STL rats had lower breathing frequencies with slightly increased burst-to-burst amplitude variability compared to STV rats, with STV rats exhibiting breathing frequencies similar to LTL rats. The overall burst-to-burst frequency variability was greater in LTL and LTV rats compared to STL and STV rats, but STL rats exhibited greater burst-to-burst inspiratory duration (TI) and expiratory duration (TE) variability compared to STV rats; LTL rats had reduced burst-to-burst variability in these behaviors compared to LTV rats. Evaluation of the hypoxic ventilatory response revealed that LTL rats exhibit a blunted increase in frequency and amplitude in response to hypoxia compared to LTV and STL rats, with STL rats exhibiting an exaggerated hypoxic frequency increase compared to STV rats. While the increases in frequency were primarily mediated by decreases in TE in all groups, LTV rats also had reduced TI and they exhibited a larger post-hypoxic frequency decline due to a substantial increase in TE. Regardless of lesion (or vehicle injection) time, all of the older rats had a substantial delay in recovery following hypoxia. In contrast, younger SN 6-OHDA (compared to vehicle) injected rats exhibited an exaggerated frequency but blunted amplitude response to hypoxia, which rapidly recovered to pre-hypoxic levels. These data demonstrate that in older rats, the timing of the lesion may differentially affect the components underlying PD-related abnormalities in respiratory behaviors. Additional studies are needed to identify the mechanisms responsible.

Acute Fluoxetine Administration Improves Intermittent Hypoxia‐induced Inspiratory Burst Amplitude Deficits During Peak LPS‐induced Neuroinflammatory Phase in Anesthetized Spontaneously Breathing Adult Male Rats

Studies examining the impact of neuroinflammation on various aspects of neural control of breathing typically use systemic administration of the bacterial endotoxin lipopolysaccharide (LPS) and evaluate alterations in ventilatory control during either the induction (~2–4 hr) or resolution (~24 hr) phases of the acute neuroinflammatory response. Changes in LPS‐regulated pro‐inflammatory gene/protein expression, however, have been shown to peak at ~4–6 hrs following LPS exposure, but little is known about the impact of LPS‐induced neuroinflammation on ventilatory control at this time point. Systemic LPS administration has also been shown to increase serotonin (5‐HT) transporter activity and reduce 5‐HT levels. Since numerous inspiratory motor behaviors are 5‐HT‐dependent, including some hypoxic ventilatory behaviors, LPS‐induced changes in 5‐HT could contribute to ventilatory deficits noted in LPS‐induced neuroinflammation studies. To begin to address these issues, we examined the impact of peak phase LPS‐induced neuroinflammation on inspiratory motor behaviors elicited by intermittent hypoxia (IH) in urethane‐anesthetized vagal intact spontaneously breathing adult male Sprague‐Dawley rats in both the absence and presence of acute administration of the SSRI fluoxetine hydrochloride (FLX). Beginning at ~4 hr after systemic LPS (3 mg/kg, ip) or saline (CON) treatment, basal (BL) diaphragm EMG activity was recorded for ~30 min (40% O2), FLX (10 mg/kg) or vehicle (VEH, 0.9% saline) was injected (iv) and allowed 30 min to exert steady‐state effects, and then IH exposure composed of 5 alternating 3‐min episodes of hypoxia (11% O2; 2%CO2) and hyperoxia (40% O2) was delivered, after which the rat was allowed to recover. We found that during the first 30–60s following FLX injection in both CON‐ and LPS‐treated rats, burst amplitude (by ~12%) and frequency (by ~40%) were increased, after which amplitude returned to BL levels and frequency attained a new steady‐state ~10% above BL levels; VEH injection was ineffective in altering either burst amplitude or frequency. As expected during IH exposure in CON‐treated VEH‐injected rats, a robust increase in burst frequency and amplitude were observed with the subsequent hypoxic exposures eliciting progressive augmentation of burst amplitude (PA; IH1, ~45%; IH5, ~65%). In contrast in CON‐treated FLX‐injected rats, IH‐induced amplitude effects were markedly attenuated (IH1‐IH5, ~11–21%), and similar to those seen during IH exposure in LPS‐treated VEH‐injected rats (~7–11%). In LPS‐treated FLX‐injected rats, however, an increase in both burst amplitude and PA (IH1, ~19%; IH5, ~35%) were noted. Similar IH‐induced increases in burst frequency and post hypoxic frequency decline were also observed in both LPS‐ and CON‐treated FLX‐ and VEH‐injected rats. These preliminary observations suggest that acute FLX administration may partially correct the blunted increase in burst amplitude and induce PA in response to IH exposure during peak phase in LPS‐treated rats albeit similar FLX treatment appears to impair IH‐induced amplitude effects in CON‐treated rats.Support or Funding InformationNIH NS101737; SBU Thomas Hartman Center for Parkinson’s Disease Research

Effects of Two‐Hit Lipopolysaccharide (LPS) Administration on Acute Intermittent Hypoxia (AIH)‐Induced Inspiratory Motor Behaviors in Urethane‐anesthetized Spontaneously Breathing Adult Male Sprague‐Dawley Rat

Ongoing studies designed to induce peripheral inflammation, and its accompanying neuroinflammation, have begun to examine various aspects of neural control of breathing. While most studies typically administer the bacterial endotoxin lipopolysaccharide (LPS) systemically or in an airway/lung targeted manner in order to produce models of systemic inflammatory response syndrome (SIRS)/sepsis and acute lung injury (ALI), a ‘two‐hit’ LPS administration protocol in which systemic LPS (ip, 3 mg/kg) is administered ~24 hr prior to an airway/lung targeted (intratracheal (IT); 0.5 mg/kg) LPS injection has been used to better reflect chronic (lung/airway) disease states. One of the most studied respiratory behaviors in which inflammation has been shown to negatively impact breathing is AIH‐induced inspiratory motor (phrenic) long‐term facilitation (pLTF), where in anesthetized, vagotomized, ventilated adult male rats, single‐hit systemic LPS administration attenuates AIH‐induced serotonin2‐dependent pLTF. Here, we examined the impact of two‐hit LPS administration in urethane‐anesthetized vagal intact spontaneously breathing adult male Sprague‐Dawley rats on AIH‐induced inspiratory motor behaviors using AIH composed of five 3‐min episodes of hypoxia (11% O2) separated by 3‐min exposures to hyperoxia (40% O2) with all rats being continuously supplied with 2% CO2. Similar experiments were performed in control rats that were either untreated or received two‐hit saline injections. We found that in control rats, exposure to the repetitive bouts of hypoxia elicited increases in both diaphragm EMG burst frequency (~18% increase) and amplitude (IH1, ~22% to IH5, ~31%) that resulted in progressive augmentation (PA) and LTF inspiratory burst amplitude that persisted for ~75 min post‐AIH (sustained ~12–18% increase above baseline levels); no frequency LTF and a robust post hypoxic frequency decline (PHFD) after each hypoxic exposure and for up to 25 min post‐AIH were also observed. In contrast, in LPS‐treated rats, exposure to the repetitive bouts of hypoxia elicited a more robust increase in diaphragm EMG burst frequency (~25–30% increase) and a blunted or absent amplitude response (IH1, ≤8%). Moreover, in these rats, PA of burst amplitude was absent, inspiratory burst amplitude LTF was highly variable with an overall amplitude increase of ~10% above baseline levels that subsided by 20 min post‐AIH, and PHFD after each hypoxic exposure slightly increased in magnitude and persisted for up to 50 min post‐AIH. These preliminary observations suggest that (compared to control rats) two‐hit LPS administration affects mechanisms underlying multiple components responsible for inspiratory burst frequency and amplitude plasticity in response to hypoxic exposure(s) and following AIH exposure. Additional experiments, including those using other AIH protocols, will be needed to confirm these observations and identify specific mechanisms underlying these differences.Support or Funding InformationNIH NS101737; Thomas Hartman Center for Parkinson's Disease Research at Stony Brook UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rett Syndrome mutation MeCP2 T158A affects hypoxic ventilatory response in mice

Rett syndrome (RTT), whose phenotype includes respiratory disorders, is caused by loss‐of‐function mutations in methyl‐CpG‐binding protein 2 (MeCP2). Mouse models of the syndrome that have been generated by deletion of Mecp2 have an altered ventilatory response to acute hypoxia (HVR) characterized by a minute ventilation (VE) that exceeds wild type (WT) and that persists in the early recovery period. Such large deletions, however, account for only ~10% of cases. Here we determine if mice with a common single point mutation retain this respiratory pattern. Knockin mice that carry a RTT‐associated threonine to alanine mutation at residue 158 (T158A) show RTT‐like behavioral phenotypes similar to Mecp2‐null mice (Nature Neuroscience 15:274,2011). Experiments were performed in heterozygous T158A female mice at 9 months of age. Baseline respiratory frequency, tidal volume and minute ventilation in T158A animals was similar to WT. Compared to WT littermates relative VE of T158A at 1 min of 8% O2 was twice as large. WT mice showed the expected post‐hypoxia frequency decline (224±9.5 to 203±12 bpm) while T158A had a sustained tachypnea (203±10 to 219±14.5). HVR in mice with a T158A mutation is similar to that with a null mutation of MeCP2.Support: IRSF, RSRT, NewLife Foundation (JMB); NIH, IRSF (ZZ)

Two weeks of buspirone protects against posthypoxic ventilatory pauses in the C57BL/6J mouse strain

The purpose was to determine if 2 weeks of buspirone suppressed post-hypoxic breathing instability and pauses in the C57BL/6J (B6) mouse. Study groups were vehicle (saline, n=8), low-dose (1.5mg/kg, n=8), and high-dose buspirone (5.0mg/kg, n=8). Frequency, measured by plethysmography, was the major metric, and a pause defined by breathing cessation >2.5 times the average frequency. Mice were tested after 16 days of ip injections of vehicle or drug. On day 17, 4 mice in each group were tested after buspirone and the 5-HT1A receptor antagonist, 4-iodo-N-{2-[4-(methoxyphenyl)-1-piperazinyl] ethyl}-N-2-pyridinyl-benzamide (p-MPPI, 5mg/kg). A post-hypoxic pause was present in 6/8 animals given vehicle and 1/16 animals given buspirone at either dose, but always present (8/8) with p-MPPI, regardless of buspirone dose. Post-hypoxic frequency decline was blunted by buspirone (−10% vehicle vs. −5% at both doses) and restored by p-MPPI; ventilatory stability as described by the coefficient of variation which was reduced by buspirone (p<0.04) was increased by p-MPPI (0.01). In conclusion, buspirone administration after 2 weeks acts through the 5-HT1A receptor to reduce post-hypoxic ventilatory instability in the B6 strain.

Ventilatory behavior and carotid body morphology of Brown Norway and Sprague Dawley rats

Differences in acute ventilatory behavior are associated with carotid body (CB) structural and immunohistologic profiles in some, but not all, reports. Brown Norway (BN) rats exhibit lower acute ventilatory responses to hypoxia and hypercapnia compared to Sprague Dawley (SD) rats. We hypothesized that BN rats possess CB with fewer glomus cells. Ventilation was recorded in 6-month-old BN and SD rats exposed to hypoxia-reoxygenation and hypercapnia. Extracted CBs were examined using H&E staining, and immunohistochemistry with antibodies specific for tyrosine hydroxylase (TH), neural nitric oxide synthase (nNOS), and pyruvate dehydrogenase (PD). Sections were analyzed for cell and immunostaining density. SD displayed greater hypoxic and hypercapnic responses, and post-hypoxic short term potentiation, whereas BN exhibited post-hypoxic frequency decline. Contrary to our hypothesis, BN demonstrated a denser arrangement of glomus cells with a larger TH stained area (31.7% BN, 22.6% SD; p<0.0001), and nNOS stained area (37.3% BN, 32.1%; SD; p=0.01). Hence, respiratory phenotype does not correlate intuitively with these anatomic features.

Morphological differences of the carotid body among C57/BL6 (B6), A/J, and CSS B6A1 mouse strains

The C57/BL6 (B6) mouse strain exhibits post-hypoxic frequency decline and periodic breathing, as well as greater amount of irregular breathing during rest in comparison to the A/J and to the B6a1, a chromosomal substitution strain whereby the A/J chromosome 1 is bred onto the B6 background (Han et al., 2002; Yamauchi et al., 2008a,b). The hypothesis was that morphological differences in the carotid body would associate with such trait variations. After confirming strain differences in post-hypoxic ventilatory behavior, histological examination (n=8 in each group) using hematoxylin and eosin (H&E) staining revealed equivalent, well-defined tissue structure at the bifurcation of the carotid arteries, an active secretory parenchyma (type I cells) from the supportive stromal tissue, and clustering of type I cells in all three strains. Tyrosine hydroxylase (TH) immunohistochemical staining revealed a typical organization of type I cells and neurovascular components into glomeruli in all three strains. Image analysis from 5 μm sections from each strain generated a series of cytological metrics. The percent carotid body composition of TH+ type I cells in the A/J, B6 and B6a1 was 20±4%, 39±3%, and 44±3%, respectively (p=0.00004). However, cellular organization in terms of density and ultrastructure in the B6a1 is more similar to the B6 than to the A/J. These findings indicate that genetic mechanisms that produce strain differences in ventilatory function do not associate with carotid body structure or tyrosine hydroxylase morphology, and that A/J chromosome 1 does not contribute much to B6 carotid body morphology.

Synchrony is altered in the respiratory pattern generator following acute hypoxic challenge

Arthur B. Otis stated in 1954 that the work of breathing accounts for a small fraction of basal metabolism and that inspiration, not expiration, consumes energy. In line with Otis’ findings, our hypothesis is that the respiratory control network uses synchrony, in addition to recruitment and firing rate changes, to generate an efficient respiratory pattern. We hypothesize that synchrony may have unique roles in respiration and that this property is malleable, adapting to maintain homeostasis. To test our hypotheses, we used the perfused in situ preparation of rat (SD/Harlan) and recorded (n=7) from the left phrenic nerve and ensembles of respiratory neurons (n=53) in the Bötzinger/pre‐Bötzinger region using a multielectrode array. Preparations were exposed to acute bouts (20–45s) of hypoxia and spike synchronization between neuronal pairs (n=240) was quantified during baseline and post‐hypoxic epochs (80s/epoch). Neuron pairs from populations which appeared homogenous with respect to firing pattern demonstrated consistent changes in coordinated firing after hypoxia with the exception of a subpopulation of expiratory neurons which showed both increasing and decreasing synchrony. We conclude that synchronization is malleable within populations of the respiratory pattern generator and may contribute to patterning changes during post hypoxic frequency decline. Funded by AHA SDG 0735037N and NIH HL090554

Acetazolamide protects against posthypoxic unstable breathing in the C57BL/6J mouse

Acetazolamide (Acz), a carbonic anhydrase inhibitor, is used to manage periodic breathing associated with altitude and with heart failure. We examined whether Acz would alter posthypoxic ventilatory behavior in the C57BL/6J (B6) mouse model of recurrent central apnea. Experiments were performed with unanesthetized, awake adult male B6 mice (n = 9), ventilatory behavior was measured using flow-through whole body plethysmography. Mice were given an intraperitoneal injection of either vehicle or Acz (40 mg/kg), and 1 h later they were exposed to 1 min of 8% O(2)-balance N(2) (poikilocapnic hypoxia) or 12% O(2)-3% CO(2)-balance N(2) (isocapnic hypoxia) followed by rapid reoxygenation (100% O(2)). Hypercapnic response (8% CO(2)-balance O(2)) was examined in six mice. With Acz, ventilation, including respiratory frequency, tidal volume, and minute ventilation, in room air was significantly higher and hyperoxic hypercapnic ventilatory responsiveness was generally lower compared with vehicle. Poikilocapnic and isocapnic hypoxic ventilatory responsiveness were similar among treatments. One minute after reoxygenation, animals given Acz exhibited posthypoxic frequency decline, a lower coefficient of variability for frequency, and no tendency toward periodic breathing, compared with vehicle treatment. We conclude that Acz improves unstable breathing in the B6 model, without altering hypoxic response or producing short-term potentiation, but with some blunting of hypercapnic responsiveness.

Altered respiratory pattern and hypoxic response in transgenic newborn mice lacking the tachykinin-1 gene

Substance P is known to be involved in respiratory rhythm and central pattern-generating mechanisms, especially during early development. We therefore studied respiratory responses in transgenic newborn mice (Tac1(-/-)) lacking substance P and neurokinin A (NKA). In vivo, the effects of intermittent isocapnic hypoxia (IH) and hypercapnia were studied using whole body flow plethysmography at P2-3 and P8-10. In vitro, anoxic responses and the effects of hypocapnic and hypercapnic conditions were studied in brain stem-spinal cord preparations (C4 activity) at P2. Hypoxic challenge considerably modified the respiratory activity in transgenic mice displayed in vivo as an attenuated increase in tidal volume during IH. Transgenic mice also showed a more prominent posthypoxic frequency decline in vivo, and posthypoxic neuronal arrests appeared more often in vitro. We recognized two types of sigh activity: with or without a following pause. During IH, the amount of sighs with a pause decreased and those without increased, a redistribution that became stronger with age only in controls. Intermittent anoxia induced long-term facilitation effects in controls, but not in Tac1(-/-) animals, manifested as an increase in burst frequency in vitro and by an augmentation of ventilation during posthypoxic periods in vivo. Thus our data demonstrate that a functional substance P/NKA system is of great importance for the generation of an adequate respiratory response to hypoxic provocation in newborn mice and during early maturation. It also indicates that substance P (and/or NKA) is involved in the development of the plasticity of the respiratory system.

Upper airway mechanics and post-hypoxic ventilatory decline during NREM sleep

Termination of hypoxia results in a transient ventilatory decline referred to as post-hypoxic ventilatory decline (PHVD). We wished to determine whether PHVD is due to changes in ventilatory motor output or upper airway mechanics. We studied 19 healthy normal subjects (15 men, 4 women) during stable non-REM (NREM) sleep. Subjects were exposed to multiple episodes of brief (3 min) hypoxia that terminated with one breath of 100% FI(O2). Minute ventilation (V (I)), tidal volume (V (T)), timing, and upper airway resistance (R (ua)) were measured during the control, hypoxia, and for the first six breaths immediately after cessation of hypoxia. In addition, we measured diaphragmatic electromyograms (EMGdia) via surface electrodes in four subjects. V (I) and V (T) decreased during the recovery period to a nadir of 81 and 83% of room air control, respectively. However, there was no significant change in respiratory frequency or upper airway resistance during the post-hypoxic recovery period. Decreased V (I) was associated with a comparable decrease in EMGdia. We conclude that: (1) PHVD occurs in normal humans during NREM sleep, (2) there is no evidence of post-hypoxic frequency decline in humans during NREM sleep, and (3) PHVD is centrally mediated and not driven by upper airway mechanics.

Ventilatory responses to isocapnic and poikilocapnic hypoxia in humans

We examined the hypoxic ventilatory response (HVR) including breathing frequency (f(R)) and tidal volume (V(T)) responses during 20 min of step isocapnic (IH) and poikilocapnic (PH) hypoxia (45 Torr). We hypothesized an index related to [Formula: see text] (pHPR) may be more robust during PH. Peak HVR was suppressed during PH (P<0.001), and mediated by V(T) during PH and both V(T) and f(R) during IH. The relative magnitude of HVD remained similar between conditions indicating a suppressive role of hypocapnia in development of the HVR unrelated to the degree of subsequent HVD, implying a primarily O(2) dependant mechanism. Post-hypoxic frequency decline was observed following both IH (3.4+/-3.7 bpm, P<0.05) and PH (3.6+/-3.1 bpm, P<0.01), despite no f(R) response during exposure to PH. Use of pHPR improved the signal to noise ratio during PH, though failed to detect the peak ventilatory response, and therefore may not be appropriate when describing peak responses.

Tidal volume and breathing frequency responses to poikilocapnic and isocapnic hypoxia in humans

The acute response to hypoxia in humans has been reported to arise predominately from increases in tidal volume (VT) with negligible changes in breathing frequency (fB). The effect of arterial CO2 on this response has not been described. We examined the ventilatory contributions of VT and fB to the hypoxic response under both poikilocapnic (PH) and isocapnic (IH) conditions in 10 normal adults (26 ± 4 yrs, mean ± SD). During IH, PETCO2 was held constant at 37 ± 2.0 Torr (1 Torr above resting values) while PETO2 was reduced stepwise to 45 Torr using the technique of dynamic end-tidal gas forcing. During PH, PETO2 was reduced stepwise to 45 Torr while PETCO2 was uncontrolled (i.e. zero inspired CO2). Hypoxia was maintained for 20 minutes, and tests were separated by 1 hour. Peak ventilation (VE), was significantly higher during IH (49.8 ± 14.3 L·min−1) compared to PH (24.1 ± 7.7 L·min−1, P < 0.001). During IH, peak VE was mediated by increases in both VT (1.0 ± 0.4 to 2.2 ± 0.5 L, P < 0.001) and fB (14.7 ± 4.6 to 19.2 ± 4.5 breaths·min−1, P < 0.05), while during PH peak VE was mediated by increased VT (0.8 ± 0.2 to 1.5 ± 0.4 L, P < 0.01) with no change observed in fB. At the termination of both IH and PH, a significant frequency undershoot was observed (20 ± 23 %, P < 0.05 and 22 ± 22 %, P < 0.01 respectively), previously documented only in animals. These data demonstrate a role of arterial CO2 in the development of a fB response to hypoxia, though whether this is due to CO2 per se or the magnitude of peak VE remains unknown. We have also documented a significant post hypoxic frequency decline not previously reported in humans occurring during both conditions. This study was approved by the local Ethics board and supported by AHFMR, HSFA and CIHR.

Specific carotid body chemostimulation is sufficient to elicit phrenic poststimulus frequency decline in a novel in situ dual-perfused rat preparation

Time-dependent ventilatory responses to hypoxic and hypercapnic challenges, such as posthypoxic frequency decline (PHxFD) and posthypercapnic frequency decline (PHcFD), could profoundly affect breathing stability. However, little is known about the mechanisms that mediate these phenomena. To determine the contribution of specific carotid body chemostimuli to PHxFD and PHcFD, we developed a novel in situ arterially perfused, vagotomized, decerebrate rat preparation in which central and peripheral chemoreceptors are perfused separately (i.e., a nonanesthetized in situ dual perfused preparation). We confirmed that 1) the perfusion of central and peripheral chemoreceptor compartments was independent by applying specific carotid body hypoxia and hypercapnia before and after carotid sinus nerve transection, 2) the PCO(2) chemoresponse of the dual perfused preparation was similar to other decerebrate preparations, and 3) the phrenic output was stable enough to allow investigation of time-dependent phenomena. We then applied four 5-min bouts (separated by 5 min) of specific carotid body hypoxia (40 Torr PO(2) and 40 Torr PCO(2)) or hypercapnia (100 Torr PO(2) and 60 Torr PCO(2)) while holding the brain stem PO(2) and PCO(2) constant. We report the novel finding that specific carotid body chemostimuli were sufficient to elicit several phrenic time-dependent phenomena in the rat. Hypoxic challenges elicited PHxFD that increased with bout, leading to progressive augmentation of the phrenic response. Conversely, hypercapnia elicited short-term depression and PHcFD, neither of which was bout dependent. These results, placed in the context of previous findings, suggest multiple physiological mechanisms are responsible for PHxFD and PHcFD, a redundancy that may illustrate that these phenomena have significant adaptive advantages.

Time-dependent modulation of carotid body afferent activity during and after intermittent hypoxia

The ventilatory response to several minutes of hypoxia consists of various time-dependent phenomena, some of which occur during hypoxia (e.g., short-term depression), whereas others appear on return to normoxia (e.g., posthypoxic frequency decline). Additional phenomena can be elicited by acute, intermittent hypoxia (e.g., progressive augmentation, long-term facilitation). Current data suggest that these phenomena originate centrally. We tested the hypothesis that carotid body afferent activity undergoes time-dependent modulation, consistent with a direct role in these ventilatory phenomena. Using an in vitro rat carotid body preparation, we found that 1) afferent activity declined during the first 5 min of severe (40 Torr Po(2)), moderate (60 Torr Po(2)), or mild (80 Torr Po(2)) hypoxia; 2) after return to normoxia (100 Torr Po(2)) and after several minutes of moderate or severe hypoxia, afferent activity was transiently reduced compared with prehypoxic levels; and 3) with successive 5-min bouts of mild, moderate, or severe hypoxia, afferent activity during bouts increased progressively. We call these phenomena sensory hypoxic decline, sensory posthypoxic decline, and sensory progressive augmentation, respectively. These phenomena were stimulus specific: similar phenomena were not seen with 5-min bouts of normoxic hypercapnia (100 Torr Po(2) and 50-60 Torr Pco(2)) or hypoxic hypocapnia (60 Torr Po(2) and 30 Torr Pco(2)). However, bouts of either normoxic hypercapnia or hypocapnic hypoxia resulted in sensory long-term facilitation. We suggest time-dependent carotid body activity acts in parallel with central mechanisms to shape the dynamics of ventilatory responses to respiratory chemostimuli.

Differences in time-dependent hypoxic phrenic responses among inbred rat strains

Hypoxic ventilatory responses differ between rodent strains, suggesting a genetic contribution to interindividual variability. However, hypoxic ventilatory responses consist of multiple time-dependent mechanisms that can be observed in different respiratory motor outputs. We hypothesized that strain differences would exist in discrete time-dependent mechanisms of the hypoxic response and, furthermore, that there may be differences between hypoglossal and phrenic nerve responses to hypoxia. Hypoglossal and phrenic nerve responses were assessed during and after a 5-min hypoxic episode in anesthetized, vagotomized, and ventilated rats from four inbred strains: Brown Norway (BN), Fischer 344 (FS), Lewis (LW), and Piebald-viral-Glaxo (PVG). During baseline, burst frequency was higher in PVG than LW rats (P < 0.05), phrenic burst amplitude was higher in PVG vs. other strains (P < 0.05), and hypoglossal burst amplitude was higher in PVG and BN vs. FS and LW (P < 0.05). During hypoxia, burst frequency did not change in BN or LW rats, but it increased in PVG and FS rats. The phrenic amplitude response was smallest in PVG vs. other strains (P < 0.05), and the hypoglossal response was similar among strains. Short-term potentiation posthypoxia was slowest in FS and fastest in LW rats (P < 0.05). Posthypoxia frequency decline was absent in PVG, but it was observed in all other strains. Augmented breaths were observed during hypoxia in FS rats only. Thus genetic differences exist in the time domains of the hypoxic response, and these are differentially expressed in hypoglossal and phrenic nerves. Furthermore, genetic diversity observed in hypoxic ventilatory responses in unanesthetized rats may arise from multiple neural mechanisms.

Chronic hypoxia abolishes posthypoxia frequency decline in the anesthetized rat.

In anesthetized rats, increases in phrenic nerve amplitude and frequency during brief periods of hypoxia are followed by a reduction in phrenic nerve burst frequency [posthypoxia frequency decline (PHFD)]. We investigated the effects of chronic exposure to hypoxia on PHFD and on peripheral and central O2-sensing mechanisms. In Inactin-anesthetized (100 mg/kg) Sprague-Dawley rats, phrenic nerve discharge and arterial pressure responses to 10 s N2 inhalation were recorded after exposure to hypoxia (10 +/- 0.5% O2) for 6-14 days. Compared with rats maintained at normoxia, PHFD was abolished in chronic hypoxic rats. Because of inhibition of PHFD, the increased phrenic burst frequency and amplitude after N2 inhalation persisted for 1.8-2.8 times longer in chronic hypoxic (70 s) compared with normoxic (25-40 s) rats (P < 0.05). After acute bilateral carotid body denervation, N2 inhalation produced a short depression of phrenic nerve discharge in both chronic hypoxic and normoxic rats. However, the degree and duration of depression of phrenic nerve discharge was smaller in chronic hypoxic compared with normoxic rats (P < 0.05). We conclude that after exposure to chronic hypoxia, a reduction in PHFD contributes to an increased duration of the acute hypoxic ventilatory response in anesthetized rats. Furthermore, after exposure to chronic hypoxia, the central network responsible for respiration is more resistant to the depressant effects of acute hypoxia in anesthetized rats.

Periodic breathing and genetics.

The episodic waxing and waning of ventilation is a fundamental event in sleep apnea syndromes. Post-hypoxic frequency decline (PHFD) and periodic breathing (PB) are evoked by brief hypoxic exposures in unanaesthetized and unrestrained inbred C57BL/6J mice, but not in A/J mice; and expression of PHFD differs not only among these mice strains but in among rat strains as well. These observations along with the current literature on genetic factors that operate on ventilatory behavior at rest and with chemosensory drive lead to the hypothesis that genetic factors infer some proportion of risk for the ventilatory instability observed in human sleep apnea syndromes.