Hypoxia evokes a sequence of raphe-pontomedullary network operations for inspiratory drive amplification and gasping.

Hypoxia can trigger a sequence of breathing-related behaviors, from augmentation to apneusis to apnea and gasping. Gasping is an autoresuscitative behavior that, via large tidal volumes and altered intrathoracic pressure, can enhance coronary perfusion, carotid blood flow, and sympathetic activity, and thereby coordinate cardiac and respiratory functions. We tested the hypotheses that hypoxia-evoked gasps are amplified through a disinhibitory microcircuit within the inspiratory neuron chain and that this drive is distributed via an efference copy mechanism. This generates coordinated gasplike discharges concurrently in other circuits of the raphe-pontomedullary respiratory network. Data were obtained from six decerebrate, vagotomized, neuromuscularly blocked, and artificially ventilated adult cats. Arterial blood pressure, phrenic nerve activity, end-tidal CO2, and other parameters were monitored. Hypoxia was produced by ventilation with a gas mixture of 5% O2 in nitrogen. Neuron spike trains were recorded at multiple pontomedullary sites simultaneously and evaluated for firing rate modulations and short-timescale correlations indicative of functional connectivity. Experimental perturbations evoked reconfiguration of raphe-pontomedullary circuits during initial augmentation, apneusis and augmented bursts, apnea, and gasping. Functional connectivity, altered firing rates, efference copy of gasp drive, and coordinated incremental blood pressure increases support a distributed brain stem network model for amplification and broadcasting of inspiratory drive during autoresuscitative gasping. Gasping begins with a reduction in inhibition by expiratory neurons and an initial loss of inspiratory drive during hypoxic apnea and culminates in autoresuscitative efforts. NEW & NOTEWORTHY Severe hypoxia evokes a sequence of breathing-related behaviors culminating in gasping. We report firing rate modulations and short-timescale correlations in spike trains recorded simultaneously in the raphe-pontomedullary respiratory network during hypoxia. Our findings support a disinhibitory microcircuit and a distributed efference copy mechanism for amplification of gasping. Coordinated increments in blood pressure lead to a model for autoresuscitative bootstrapping of peripheral chemoreceptor reflexes, breathing, and sympathetic activity, complementing and extending prior work.

Hypoxia evokes a sequence of raphe-pontomedullary network operations for inspiratory drive amplification and gasping.

Hypoxia can trigger a sequence of breathing-related behaviors, from tachypnea to apneusis to apnea and gasping, an autoresuscitative behavior that, via large tidal volumes and altered intrathoracic pressure, can enhance coronary perfusion, carotid blood flow, and sympathetic activity, and thereby coordinate cardiac and respiratory functions. We tested the hypothesis that hypoxia-evoked gasps are amplified through a disinhibitory microcircuit within the inspiratory neuron chain and a distributed efference copy mechanism that generates coordinated gasp-like discharges concurrently in other circuits of the raphe-pontomedullary respiratory network. Data were obtained from 6 decerebrate, vagotomized, neuromuscularly-blocked, and artificially ventilated adult cats. Arterial blood pressure, phrenic nerve activity, end-tidal CO2, and other parameters were monitored. Hypoxia was produced by ventilation with a gas mixture of 5% O2 in nitrogen (N2). Neuron spike trains were recorded at multiple pontomedullary sites simultaneously and evaluated for firing rate modulations and short-time scale correlations indicative of functional connectivity. Experimental perturbations evoked reconfiguration of raphe-pontomedullary circuits during tachypnea, apneusis and augmented bursts, apnea, and gasping. The functional connectivity, altered firing rates, efference copy of gasp drive, and coordinated step increments in blood pressure reported here support a distributed brain stem network model for amplification and broadcasting of inspiratory drive during autoresuscitative gasping that begins with a reduction in inhibition by expiratory neurons and an initial loss of inspiratory drive during hypoxic apnea.

The O2-sensitive brain stem, hyperoxic hyperventilation, and CNS oxygen toxicity.

Central nervous system oxygen toxicity (CNS-OT) is a complex disorder that presents, initially, as a sequence of cardio-respiratory abnormalities and nonconvulsive signs and symptoms (S/Sx) of brain stem origin that culminate in generalized seizures, loss of consciousness, and postictal cardiogenic pulmonary edema. The risk of CNS-OT and its antecedent "early toxic indications" are what limits the use of hyperbaric oxygen (HBO2) in hyperbaric and undersea medicine. The purpose of this review is to illustrate, based on animal research, how the temporal pattern of abnormal brain stem responses that precedes an "oxtox hit" provides researchers a window into the early neurological events underlying seizure genesis. Specifically, we focus on the phenomenon of hyperoxic hyperventilation, and the medullary neurons presumed to contribute in large part to this paradoxical respiratory response; neurons in the caudal Solitary complex (cSC) of the dorsomedial medulla, including putative CO2 chemoreceptor neurons. The electrophysiological and redox properties of O2-/CO2-sensitive cSC neurons identified in rat brain slice experiments are summarized. Additionally, evidence is summarized that supports the working hypothesis that seizure genesis originates in subcortical areas and involves cardio-respiratory centers and cranial nerve nuclei in the hind brain (brainstem and cerebellum) based on, respectively, the complex temporal pattern of abnormal cardio-respiratory responses and various nonconvulsive S/Sx that precede seizures during exposure to HBO2.

Exogenous ketone ester delays CNS oxygen toxicity without impairing cognitive and motor performance in male Sprague-Dawley rats.

Hyperbaric oxygen (HBO2) is breathing >1 atmosphere absolute (ATA; 101.3 kPa) O2 and is used in HBO2 therapy and undersea medicine. What limits the use of HBO2 is the risk of developing central nervous system (CNS) oxygen toxicity (CNS-OT). A promising therapy for delaying CNS-OT is ketone metabolic therapy either through diet or exogenous ketone ester (KE) supplement. Previous studies indicate that KE induces ketosis and delays the onset of CNS-OT; however, the effects of exogeneous KE on cognition and performance are understudied. Accordingly, we tested the hypothesis that oral gavage with 7.5 g/kg induces ketosis and increases the latency time to seizure (LSz) without impairing cognition and performance. A single oral dose of 7.5 g/kg KE increases systemic ß-hydroxybutyrate (BHB) levels within 0.5 h and remains elevated for 4 h. Male rats were separated into three groups: control (no gavage), water-gavage, or KE-gavage, and were subjected to behavioral testing while breathing 1 ATA (101.3 kPa) of air. Testing included the following: DigiGait (DG), light/dark (LD), open field (OF), and novel object recognition (NOR). There were no adverse effects of KE on gait or motor performance (DG), cognition (NOR), and anxiety (LD, OF). In fact, KE had an anxiolytic effect (OF, LD). The LSz during exposure to 5 ATA (506.6 kPa) O2 (≤90 min) increased 307% in KE-treated rats compared with control rats. In addition, KE prevented seizures in some animals. We conclude that 7.5 g/kg is an optimal dose of KE in the male Sprague-Dawley rat model of CNS-OT.

Exogenous ketone salts inhibit superoxide production in the rat caudal solitary complex during exposure to normobaric and hyperbaric hyperoxia.

The use of hyperbaric oxygen (HBO2) in hyperbaric and undersea medicine is limited by the risk of seizures [i.e., central nervous system (CNS) oxygen toxicity, CNS-OT] resulting from increased production of reactive oxygen species (ROS) in the CNS. Importantly, ketone supplementation has been shown to delay onset of CNS-OT in rats by ∼600% in comparison with control groups (D'Agostino DP, Pilla R, Held HE, Landon CS, Puchowicz M, Brunengraber H, Ari C, Arnold P, Dean JB. Am J Physiol Regu Integr Comp Physiol 304: R829-R836, 2013). We have tested the hypothesis that ketone body supplementation inhibits ROS production during exposure to hyperoxygenation in rat brainstem cells. We measured the rate of cellular superoxide ([Formula: see text]) production in the caudal solitary complex (cSC) in rat brain slices using a fluorogenic dye, dihydroethidium (DHE), during exposure to control O2 (0.4 ATA) followed by 1-2 h of normobaric oxygen (NBO2) (0.95 ATA) and HBO2 (1.95, and 4.95 ATA) hyperoxia, with and without a 50:50 mixture of ketone salts (KS) dl-ß-hydroxybutyrate + acetoacetate. All levels of hyperoxia tested stimulated [Formula: see text] production similarly in cSC cells and coexposure to 5 mM KS during hyperoxia significantly blunted the rate of increase in DHE fluorescence intensity during exposure to hyperoxia. Not all cells tested produced [Formula: see text] at the same rate during exposure to control O2 and hyperoxygenation; cells that increased [Formula: see text] production by >25% during hyperoxia in comparison with baseline were inhibited by KS, whereas cells that did not reach that threshold during hyperoxia were unaffected by KS. These findings support the hypothesis that ketone supplementation decreases the steady-state concentrations of superoxide produced during exposure to NBO2 and HBO2 hyperoxia.NEW & NOTEWORTHY Exposure of rat medullary tissue slices to levels of O2 that mimic those that cause seizures in rats stimulates cellular superoxide ([Formula: see text]) production to varying degrees. Cellular [Formula: see text] generation in the caudal solitary complex is variable during exposure to control O2 and hyperoxia and significantly decreases during ketone supplementation. Our findings support the theory that ketone supplementation delays onset of central nervous system oxygen toxicity in mammals, in part, by decreasing [Formula: see text] production in O2-sensitive neurons.

Tethered Liquid Perfluorocarbon Coating for 72 Hour Heparin-Free Extracorporeal Life Support.

Coagulopathic complications during extracorporeal life support (ECLS) result from two parallel processes: 1) foreign surface contact and shear stress during blood circulation and 2) administration of anticoagulant drugs to prevent circuit thrombosis. To address these problems, biocompatible surfaces are developed to prevent foreign surface-induced coagulopathy, reducing or eliminating the need for anticoagulants. Tethered liquid perfluorocarbon (TLP) is a nonadhesive coating that prevents adsorption of plasma proteins and thrombus deposition. We examined application of TLP to complete ECLS circuits (membranes, tubing, pumps, and catheters) during 72 hours of ECLS in healthy swine (n = 5/group). We compared TLP-coated circuits used without systemic anticoagulation to standard of care: heparin-coated circuits with continuous heparin infusion. Coagulopathic complications, device performance, and systemic effects were assessed. We hypothesized that TLP reduces circuit thrombosis and iatrogenic bleeding, without impeding gas exchange performance or causing untoward effects. No difference in bleeding or thrombotic complication rate was observed; however, circuit occlusion occurred in both groups (TLP = 2/5; CTRL = 1/5). TLP required elevated sweep gas rate to maintain normocapnia during ECLS versus CTRL (10-20 vs. 5 L/min; p = 0.047), suggesting impaired gas exchange. Thrombus deposition and protein adhesion on explanted membranes were comparable, and TLP did not preserve platelet or blood cell counts relative to controls. We conclude that neither TLP nor standard of care is an efficacious solution to prevent coagulation disturbances during ECLS. Further testing of promising biomaterials for ECLS utilizing the model outlined here is warranted.

Seizures Caused by Exposure to Hyperbaric Oxygen in Rats Can Be Predicted by Early Changes in Electrodermal Activity.

Hyperbaric oxygen (HBO2) is breathed during undersea operations and in hyperbaric medicine. However, breathing HBO2 by divers and patients increases the risk of central nervous system oxygen toxicity (CNS-OT), which ultimately manifests as sympathetic stimulation producing tachycardia and hypertension, hyperventilation, and ultimately generalized seizures and cardiogenic pulmonary edema. In this study, we have tested the hypothesis that changes in electrodermal activity (EDA), a measure of sympathetic nervous system activation, precedes seizures in rats breathing 5 atmospheres absolute (ATA) HBO2. Radio telemetry and a rodent tether apparatus were adapted for use inside a sealed hyperbaric chamber. The tethered rat was free to move inside a ventilated animal chamber that was flushed with air or 100% O2. The animal chamber and hyperbaric chamber (air) were pressurized in parallel at ~1 atmosphere/min. EDA activity was recorded simultaneously with cortical electroencephalogram (EEG) activity, core body temperature, and ambient pressure. We have captured the dynamics of EDA using time-varying spectral analysis of raw EDA (TVSymp), previously developed as a tool for sympathetic tone assessment in humans, adjusted to detect the dynamic changes of EDA in rats that occur prior to onset of CNS-OT seizures. The results show that a significant increase in the amplitude of TVSymp values derived from EDA recordings occurs on average (±SD) 1.9 ± 1.6 min before HBO2-induced seizures. These results, if corroborated in humans, support the use of changes in TVSymp activity as an early "physio-marker" of impending and potentially fatal seizures in divers and patients.

Nociceptive pulmonary-cardiac reflexes are altered in the spontaneously hypertensive rat.

KEY POINTS: We investigated the cardiovascular and respiratory responses of the normotensive Wistar-Kyoto (WKY) rat and the spontaneously hypertensive (SH) rat to inhalation and intravenous injection of the noxious stimuli allyl isothiocyanate (AITC). AITC inhalation evoked atropine-sensitive bradycardia in conscious WKY rats, and evoked atropine-sensitive bradycardia and atenolol-sensitive tachycardia with premature ventricular contractions (PVCs) in conscious SH rats. Intravenous injection of AITC evoked bradycardia but no tachycardia/PVCs in conscious SHs, while inhalation and injection of AITC caused similar bradypnoea in conscious SH and WKY rats. Anaesthesia (inhaled isoflurane) inhibited the cardiac reflexes evoked by inhaled AITC but not injected AITC. Data indicate the presence of a de novo nociceptive pulmonary-cardiac reflex triggering sympathoexcitation in SH rats, and this reflex is dependent on vagal afferents but is not due to steady state blood pressure or due to remodelling of vagal efferent function. ABSTRACT: Inhalation of noxious irritants/pollutants activates airway nociceptive afferents resulting in reflex bradycardia in healthy animals. Nevertheless, noxious pollutants evoke sympathoexcitation (tachycardia, hypertension) in cardiovascular disease patients. We hypothesize that cardiovascular disease alters nociceptive pulmonary-cardiac reflexes. Here, we studied reflex responses to irritants in normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive (SH) rats. Inhaled allyl isothiocyanate (AITC) evoked atropine-sensitive bradycardia with atrial-ventricular (AV) block in conscious WKY rats, thus indicating a parasympathetic reflex. Conversely, inhaled AITC in conscious SH rats evoked complex brady-tachycardia with both AV block and premature ventricular contractions (PVCs). Atropine abolished the bradycardia and AV block, but the atropine-insensitive tachycardia and PVCs were abolished by the ß1 -adrenoceptor antagonist atenolol. The aberrant AITC-evoked reflex in SH rats was not reduced by acute blood pressure reduction by captopril. Surprisingly, intravenous AITC only evoked bradycardia in conscious SH and WKY rats. Furthermore, anaesthesia reduced the cardiac reflexes evoked by inhaled but not injected AITC. Nevertheless, anaesthesia had little effect on AITC-evoked respiratory reflexes. Such data suggest distinct differences in nociceptive reflex pathways dependent on cardiovascular disease, administration route and downstream effector. AITC-evoked tachycardia in decerebrate SH rats was abolished by vagotomy. Finally, there was no difference in the cardiac responses of WKY and SH rats to vagal efferent electrical stimulation. Our data suggest that AITC inhalation in SH rats evokes de novo adrenergic reflexes following vagal afferent activation. This aberrant reflex is independent of steady state hypertension and is not evoked by intravenous AITC. We conclude that pre-existing hypertension aberrantly shifts nociceptive pulmonary-cardiac reflexes towards sympathoexcitation.

CNS function and dysfunction during exposure to hyperbaric oxygen in operational and clinical settings.

Hyperbaric oxygen (HBO2) is breathed during hyperbaric oxygen therapy and during certain undersea pursuits in diving and submarine operations. What limits exposure to HBO2 in these situations is the acute onset of central nervous system oxygen toxicity (CNS-OT) following a latent period of safe oxygen breathing. CNS-OT presents as various non-convulsive signs and symptoms, many of which appear to be of brainstem origin involving cranial nerve nuclei and autonomic and cardiorespiratory centers, which ultimately spread to higher cortical centers and terminate as generalized tonic-clonic seizures. The initial safe latent period makes the use of HBO2 practical in hyperbaric and undersea medicine; however, the latent period is highly variable between individuals and within the same individual on different days, making it difficult to predict onset of toxic indications. Consequently, currently accepted guidelines for safe HBO2 exposure are highly conservative. This review examines the disorder of CNS-OT and summarizes current ideas on its underlying pathophysiology, including specific areas of the CNS and fundamental neural and redox signaling mechanisms that are thought to be involved in seizure genesis and propagation. In addition, conditions that accelerate the onset of seizures are discussed, as are current mitigation strategies under investigation for neuroprotection against redox stress while breathing HBO2 that extend the latent period, thus enabling safer and longer exposures for diving and medical therapies.

Delaying latency to hyperbaric oxygen-induced CNS oxygen toxicity seizures by combinations of exogenous ketone supplements.

Central nervous system oxygen toxicity (CNS-OT) manifests as tonic-clonic seizures and is a limitation of hyperbaric oxygen therapy (HBOT), as well as of recreational and technical diving associated with elevated partial pressure of oxygen. A previous study showed that ketone ester (1,3-butanediol acetoacetate diester, KE) administration delayed latency to seizures (LS) in 3-month-old Sprague-Dawley (SD) rats. This study explores the effect of exogenous ketone supplements in additional dosages and formulations on CNS-OT seizures in 18 months old SD rats, an age group correlating to human middle age. Ketogenic agents were given orally 60 min prior to exposure to hyperbaric oxygen and included control (water), KE (10 g/kg), KE/2 (KE 5 g/kg + water 5 g/kg), KE + medium-chain triglycerides (KE 5 g/kg + MCT 5 g/kg), and ketone salt (Na+ /K+ ßHB, KS) + MCT (KS 5 g/kg + MCT 5 g/kg). Rats were exposed to 100% oxygen at 5 atmospheres absolute (ATA). Upon seizure presentation (tonic-clonic movements) experiments were immediately terminated and blood was tested for glucose and D-beta-hydroxybutyrate (D-ßHB) levels. While blood D-ßHB levels were significantly elevated post-dive in all treatment groups, LS was significantly delayed only in KE (P = 0.0003), KE/2 (P = 0.023), and KE + MCT (P = 0.028) groups. In these groups, the severity of seizures appeared to be reduced, although these changes were significant only in KE-treated animals (P = 0.015). Acetoacetate (AcAc) levels were also significantly elevated in KE-treated animals. The LS in 18-month-old rats was delayed by 179% in KE, 219% in KE + MCT, and 55% in KE/2 groups, while only by 29% in KS + MCT. In conclusion, KE supplementation given alone and in combination with MCT elevated both ßHB and AcAc, and delayed CNS-OT seizures.

Carotid chemoreceptors tune breathing via multipath routing: reticular chain and loop operations supported by parallel spike train correlations.

We tested the hypothesis that carotid chemoreceptors tune breathing through parallel circuit paths that target distinct elements of an inspiratory neuron chain in the ventral respiratory column (VRC). Microelectrode arrays were used to monitor neuronal spike trains simultaneously in the VRC, peri-nucleus tractus solitarius (p-NTS)-medial medulla, the dorsal parafacial region of the lateral tegmental field (FTL-pF), and medullary raphe nuclei together with phrenic nerve activity during selective stimulation of carotid chemoreceptors or transient hypoxia in 19 decerebrate, neuromuscularly blocked, and artificially ventilated cats. Of 994 neurons tested, 56% had a significant change in firing rate. A total of 33,422 cell pairs were evaluated for signs of functional interaction; 63% of chemoresponsive neurons were elements of at least one pair with correlational signatures indicative of paucisynaptic relationships. We detected evidence for postinspiratory neuron inhibition of rostral VRC I-Driver (pre-Bötzinger) neurons, an interaction predicted to modulate breathing frequency, and for reciprocal excitation between chemoresponsive p-NTS neurons and more downstream VRC inspiratory neurons for control of breathing depth. Chemoresponsive pericolumnar tonic expiratory neurons, proposed to amplify inspiratory drive by disinhibition, were correlationally linked to afferent and efferent "chains" of chemoresponsive neurons extending to all monitored regions. The chains included coordinated clusters of chemoresponsive FTL-pF neurons with functional links to widespread medullary sites involved in the control of breathing. The results support long-standing concepts on brain stem network architecture and a circuit model for peripheral chemoreceptor modulation of breathing with multiple circuit loops and chains tuned by tegmental field neurons with quasi-periodic discharge patterns. NEW & NOTEWORTHY We tested the long-standing hypothesis that carotid chemoreceptors tune the frequency and depth of breathing through parallel circuit operations targeting the ventral respiratory column. Responses to stimulation of the chemoreceptors and identified functional connectivity support differential tuning of inspiratory neuron burst duration and firing rate and a model of brain stem network architecture incorporating tonic expiratory "hub" neurons regulated by convergent neuronal chains and loops through rostral lateral tegmental field neurons with quasi-periodic discharge patterns.

Normobaric hyperoxia stimulates superoxide and nitric oxide production in the caudal solitary complex of rat brain slices.

Central CO2-chemosensitive neurons in the caudal solitary complex (cSC) are stimulated not only by hypercapnic acidosis, but by hyperoxia as well. While a cellular mechanism for the CO2 response has yet to be isolated, previous data show that a redox-sensitive mechanism underlies neuronal excitability to hyperoxia. However, it remains unknown how changes in Po2 affect the production of reactive oxygen and nitrogen species (RONS) in the cSC that can lead to increased cellular excitability and, with larger doses, to cellular dysfunction and death. To this end, we used fluorescence microscopy in real time to determine how normobaric hyperoxia increases the production of key RONS in the cSC. Because neurons in the region are CO2 sensitive, we also examined the potential effects of CO2 narcosis, used during euthanasia before brain slice harvesting, on RONS production. Our findings show that normobaric hyperoxia (0.4 → 0.95 atmospheres absolute O2) increases the fluorescence rates of fluorogenic dyes specific to both superoxide and nitric oxide. Interestingly, different results were seen for superoxide fluorescence when CO2 narcosis was used during euthanasia, suggesting long-lasting changes in superoxide production and/or antioxidant activity subsequent to CO2 narcosis before brain slicing. Further research needs to distinguish whether the increased levels of RONS reported here are merely increases in oxidative and nitrosative signaling or, alternatively, evidence of redox and nitrosative stress.

Acute hypercapnic hyperoxia stimulates reactive species production in the caudal solitary complex of rat brain slices but does not induce oxidative stress.

Central CO2 chemoreceptive neurons in the caudal solitary complex (cSC) are stimulated by hyperoxia via a free radical mechanism. Hyperoxia has been shown to increase superoxide and nitric oxide in the cSC, but it remains unknown how changes in Pco2 during hyperoxia affect the production of O2-dependent reactive oxygen and nitrogen species (RONS) downstream that can lead to increased levels of oxidative and nitrosative stress, cellular excitability, and, potentially, dysfunction. We used real-time fluorescence microscopy in rat brain slices to determine how hyperoxia and hypercapnic acidosis (HA) modulate one another in the production of key RONS, as well as colorimetric assays to measure levels of oxidized and nitrated lipids and proteins. We also examined the effects of CO2 narcosis and hypoxia before euthanasia and brain slice harvesting, as these neurons are CO2 sensitive and hypothesized to employ CO2/H+ mechanisms that exacerbate RONS production and potentially oxidative stress. Our findings show that hyperoxia ± HA increases the production of peroxynitrite and its derivatives, whereas increases in Fenton chemistry are most prominent during hyperoxia + HA. Using CO2 narcosis before euthanasia modulates cellular sensitivity to HA postmortem and enhances the magnitude of the peroxynitrite pathway, but blunts the activity of Fenton chemistry. Overall, hyperoxia and HA do not result in increased production of markers of oxidative and nitrosative stress as expected. We postulate this is due to antioxidant and proteosomal removal of damaged lipids and proteins to maintain cell viability and avoid death during protracted hyperoxia.

Functional connectivity in raphé-pontomedullary circuits supports active suppression of breathing during hypocapnic apnea.

Hyperventilation is a common feature of disordered breathing. Apnea ensues if CO2 drive is sufficiently reduced. We tested the hypothesis that medullary raphé, ventral respiratory column (VRC), and pontine neurons have functional connectivity and persistent or evoked activities appropriate for roles in the suppression of drive and rhythm during hyperventilation and apnea. Phrenic nerve activity, arterial blood pressure, end-tidal CO2, and other parameters were monitored in 10 decerebrate, vagotomized, neuromuscularly-blocked, and artificially ventilated cats. Multielectrode arrays recorded spiking activity of 649 neurons. Loss and return of rhythmic activity during passive hyperventilation to apnea were identified with the S-transform. Diverse fluctuating activity patterns were recorded in the raphé-pontomedullary respiratory network during the transition to hypocapnic apnea. The firing rates of 160 neurons increased during apnea; the rates of 241 others decreased or stopped. VRC inspiratory neurons were usually the last to cease firing or lose rhythmic activity during the transition to apnea. Mayer wave-related oscillations (0.04-0.1 Hz) in firing rate were also disrupted during apnea. Four-hundred neurons (62%) were elements of pairs with at least one hyperventilation-responsive neuron and a correlational signature of interaction identified by cross-correlation or gravitational clustering. Our results support a model with distinct groups of chemoresponsive raphé neurons contributing to hypocapnic apnea through parallel processes that incorporate disfacilitation and active inhibition of inspiratory motor drive by expiratory neurons. During apnea, carotid chemoreceptors can evoke rhythm reemergence and an inspiratory shift in the balance of reciprocal inhibition via suppression of ongoing tonic expiratory neuron activity.

Female rats are more susceptible to central nervous system oxygen toxicity than male rats.

Abstract Tonic-clonic seizures typify central nervous system oxygen toxicity (CNS-OT) in humans and animals exposed to high levels of oxygen, as are encountered during scuba diving. We previously demonstrated that high doses of pseudoephedrine (PSE) decrease the latency to seizure (LS) for CNS-OT in young male rats. This study investigated whether female rats respond similarly to PSE and hyperbaric oxygen (HBO). We implanted 60 virgin stock (VS) and 54 former breeder (FB) female rats with radio-telemetry devices that measured brain electrical activity. One week later, rats were gavaged with saline or PSE in saline (40, 80, 120, 160, or 320 mg/kg) before diving to five atmospheres absolute in 100% oxygen. The time between reaching maximum pressure and exhibiting seizure was LS. Vaginal smears identified estrus cycle phase. PSE did not decrease LS for VS or FB, primarily because they exhibited low LS for all conditions tested. VS had shorter LS than males at 0, 40, and 80 mg/kg (-42, -49, and -57%, respectively). FB also had shorter LS than males at 0, 40, and 80 mg/kg (-60, -86, and -73%, respectively). FB were older than VS (286 ± 10 days vs. 128 ± 5 days) and weighed more than VS (299 ± 2.7 g vs. 272 ± 2.1 g). Males tested were younger (88 ± 2 days), heavier (340 ± 4.5 g), and gained more weight postoperatively (7.2 ± 1.6 g) than either VS (-0.4 ± 1.5 g) or FB (-1.6 ± 1.5 g); however, LS correlated poorly with age, body mass, change in body mass, and estrus cycle phase. We hypothesize that differences in sex hormones underlie females' higher susceptibility to CNS-OT than males.

Substance P differentially modulates firing rate of solitary complex (SC) neurons from control and chronic hypoxia-adapted adult rats.

NK1 receptors, which bind substance P, are present in the majority of brainstem regions that contain CO2/H(+)-sensitive neurons that play a role in central chemosensitivity. However, the effect of substance P on the chemosensitive response of neurons from these regions has not been studied. Hypoxia increases substance P release from peripheral afferents that terminate in the caudal nucleus tractus solitarius (NTS). Here we studied the effect of substance P on the chemosensitive responses of solitary complex (SC: NTS and dorsal motor nucleus) neurons from control and chronic hypoxia-adapted (CHx) adult rats. We simultaneously measured intracellular pH and electrical responses to hypercapnic acidosis in SC neurons from control and CHx adult rats using the blind whole cell patch clamp technique and fluorescence imaging microscopy. Substance P significantly increased the basal firing rate in SC neurons from control and CHx rats, although the increase was smaller in CHx rats. However, substance P did not affect the chemosensitive response of SC neurons from either group of rats. In conclusion, we found that substance P plays a role in modulating the basal firing rate of SC neurons but the magnitude of the effect is smaller for SC neurons from CHx adult rats, implying that NK1 receptors may be down regulated in CHx adult rats. Substance P does not appear to play a role in modulating the firing rate response to hypercapnic acidosis of SC neurons from either control or CHx adult rats.

Therapeutic ketosis with ketone ester delays central nervous system oxygen toxicity seizures in rats.

Central nervous system oxygen toxicity (CNS-OT) seizures occur with little or no warning, and no effective mitigation strategy has been identified. Ketogenic diets (KD) elevate blood ketones and have successfully treated drug-resistant epilepsy. We hypothesized that a ketone ester given orally as R,S-1,3-butanediol acetoacetate diester (BD-AcAc(2)) would delay CNS-OT seizures in rats breathing hyperbaric oxygen (HBO(2)). Adult male rats (n = 60) were implanted with radiotelemetry units to measure electroencephalogram (EEG). One week postsurgery, rats were administered a single oral dose of BD-AcAc(2), 1,3-butanediol (BD), or water 30 min before being placed into a hyperbaric chamber and pressurized to 5 atmospheres absolute (ATA) O2. Latency to seizure (LS) was measured from the time maximum pressure was reached until the onset of increased EEG activity and tonic-clonic contractions. Blood was drawn at room pressure from an arterial catheter in an additional 18 animals that were administered the same compounds, and levels of glucose, pH, Po(2), Pco(2), ß-hydroxybutyrate (BHB), acetoacetate (AcAc), and acetone were analyzed. BD-AcAc(2) caused a rapid (30 min) and sustained (>4 h) elevation of BHB (>3 mM) and AcAc (>3 mM), which exceeded values reported with a KD or starvation. BD-AcAc(2) increased LS by 574 ± 116% compared with control (water) and was due to the effect of AcAc and acetone but not BHB. BD produced ketosis in rats by elevating BHB (>5 mM), but AcAc and acetone remained low or undetectable. BD did not increase LS. In conclusion, acute oral administration of BD-AcAc(2) produced sustained ketosis and significantly delayed CNS-OT seizures by elevating AcAc and acetone.

A potential early physiological marker for CNS oxygen toxicity: hyperoxic hyperpnea precedes seizure in unanesthetized rats breathing hyperbaric oxygen.

Hyperbaric oxygen (HBO(2)) stimulates presumptive central CO2-chemoreceptor neurons, increases minute ventilation (V(min)), decreases heart rate (HR) and, if breathed sufficiently long, produces central nervous system oxygen toxicity (CNS-OT; i.e., seizures). The risk of seizures when breathing HBO(2) is variable between individuals and its onset is difficult to predict. We have tested the hypothesis that a predictable pattern of cardiorespiration precedes an impending seizure when breathing HBO2. To test this hypothesis, 27 adult male Sprague-Dawley rats were implanted with radiotelemetry transmitters to assess diaphragmatic/abdominal electromyogram, electrocardiogram, and electroencephalogram. Seven days after surgery, each rat was placed in a sealed, continuously ventilated animal chamber inside a hyperbaric chamber. Both chambers were pressurized in parallel using poikilocapnic 100% O(2) (animal chamber) and air (hyperbaric chamber) to 4, 5, or 6 atmospheres absolute (ATA). Breathing 1 ATA O(2) initially decreased V(min) and HR (Phase 1 of the compound hyperoxic ventilatory response). With continued exposure to normobaric hyperoxia, however, V(min) began increasing toward the end of exposure in one-third of the animals tested. Breathing HBO2 induced an early transient increase in V(min) (Phase 2) and HR during the chamber pressurization, followed by a second significant increase of V(min) ≤8 min prior to seizure (Phase 3). HR, which subsequently decreased during sustained hyperoxia, showed no additional changes prior to seizure. We conclude that hyperoxic hyperpnea (Phase 3 of the compound hyperoxic ventilatory response) is a predictor of an impending seizure while breathing poikilocapnic HBO(2) at rest in unanesthetized rats.