Carotid Sinus Nerve Responses Research Articles

A comparative study of carotid body chemosensitivity in lean and obese mice: Implication for obesity induced hypertension

Obesity is associated with increased circulating level of the hormone leptin. Previously we have shown in mice that, leptin induces hypertension acting on transient receptor potential melastatin 7 (Trpm7) channels in the carotid body (CB), a multi-modal peripheral chemoreceptor organ that controls breathing and drives sympathetic tone. Further, in mice leptin augments hypoxic ventilatory response which is abolished by CB denervation. This suggests a potential involvement of CB chemosensitivity. However, no comparative study of CB chemosensory responses has been conducted in lean and obese mice to support this notion. Based on these premises, we hypothesize that obese mice should have higher leptin induced CB chemosensory responses during normoxia and hypoxia compared to lean mice, and Trpm7 receptor antagonist should abolish/reduce these responses. To prove this, we measured afferent carotid sinus nerve (CSN) activity, using a novel ex vivo perfused mouse (male) CB preparation from lean (b.w.; 35 ± 0.5 gm) and diet induced obese (DIO) mice (b.w.; 53 ± 1.5gm) with C57BL/6J background. We found that compared to lean mice; (a) CSN response to hypoxia (PO2 =60 Torr) was 50% more in obese mice, (b) in presence of leptin (50nM), CSN responses to normoxia (PO2=100Torr) and hypoxia were augmented 50% and 150% more respectively in obese mice. Finally, leptin induced increase in CSN responses were partially reduced by Trpm7 antagonist FTY720 (3μM) in obese compared to lean mice. In conclusion, CB chemosensitivity is enhanced in obese compared to lean mice and CB Trpm7 channel could be a therapeutic target for obesity related hypertension. Grant support: NIH R01 HL1331000. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Accelerator and Brake Modulatory System of Carotid Body Sensitivity

The carotid body (CB) is the primary sensor of arterial gases in the human body that through reflex pathways maintains cardiovascular homeostasis. It is known that CB chemosensitivity increases upon repeated stimulation as well as disease states; however, the molecular basis for this remains elusive. Objectives: To investigate the effect of excitatory and inhibitory amino acids in mediating CB afferent activity. Hypothesis: We hypothesised that glutamate and γ-aminobutyric acid (GABA) act to modulate CB sensitivity. Methods: Electrophysiological recordings of carotid sinus nerve (CSN) afferent discharge were made from an ex vivo arterially perfused common carotid artery bifurcation preparation. Effects of agents on CB sensitivity were assessed from responses evoked by cyanide (CN−) (1.23 μmol bolus). Results: The CSN response to CN− consisted of a rapid high amplitude discharge that was quenched abruptly, leading to a low amplitude tail. Glutamate administration augmented the CSN response to CN− by 2-fold compared to baseline ( p=0.0005). In contrast, GABA administration suppressed the CN− response by 2-fold ( p = 3.71 x 10−5). An N-methyl-D-aspartic acid (NMDA) receptor antagonist (MK801; 100 μM) increased the response amplitude resulting in one single high amplitude discharge (160% increase from baseline), abolishing the low amplitude tail. Such an effect was also observed following GABAA receptor blockade with bicuculline (500 μM), when an 80% increase was observed. The CN− response was blocked by PPADS (a P2X receptor antagonist; 100 μM) (242 ± 154 vs 4.82 ± 2.23 % change from baseline; p=0.03), suggesting it to be solely mediated by purinergic transmission. Summary of the data: We propose that CN-evoked excitation of CB chemosensory cells leads to the co-release of ATP and glutamate. ATP activates directly P2X receptors on petrosal chemosensory afferents, while glutamate acting via NMDA receptors on chemosensory cells leads to activity-induced potentiation of ATP release and co-release of GABA to quench ATP-mediated CSN discharge in a time-controlled manner. This is supported by our finding that blockade of either excitatory (NMDAR) or inhibitory (GABAA) receptors both heighten the CN− evoked CB response. A statement of the conclusions: Our results suggest an intrinsic ‘accelerator and brake’ paracrine mechanism within the CB involving glutamate and GABA controlling CB afferent sensitivity. Research was supported by the Health Research Council of New Zealand and the Sidney Taylor Trust. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Erythropoietin Produces a Dual Effect on Carotid Body Chemoreception in Male Rats

Erythropoietin (EPO) regulates respiration under conditions of normoxia and hypoxia through interaction with the respiratory centers of the brainstem. Here we investigate the dose‐dependent impact of EPO in the CB response to hypoxia and hypercapnia. We show, in isolated “en bloc” carotid body (CB) preparations containing the carotid sinus nerve (CSN) from adult male Sprague Dawley rats, that EPO acts as a stimulator of CSN activity in response to hypoxia at concentrations below 0.5 IU/ml. Under hypercapnic conditions, EPO did not influence the CSN response. EPO concentrations above 0.5 IU/ml decreased the response of the CSN to both hypoxia and hypercapnia, reaching complete inhibition at 2 IU/ml. The inhibitory action of high‐dose EPO on the CSN activity might result from an increase in nitric oxide (NO) production. Accordingly, CB preparations were incubated with 2 IU/ml EPO and the unspecific NO synthase inhibitor (L‐NAME), or the neuronal‐specific NO synthase inhibitor (7NI). Both NO inhibitors fully restored the CSN activity in response to hypoxia and hypercapnia in presence of EPO. Our results show that EPO activates the CB response to hypoxia when its concentration does not exceed the threshold at which NO inhibitors masks EPO’s action.

Erythropoietin Produces a Dual Effect on Carotid Body Chemoreception in Male Rats.

Erythropoietin (EPO) regulates respiration under conditions of normoxia and hypoxia through interaction with the respiratory centers of the brainstem. Here we investigate the dose-dependent impact of EPO in the CB response to hypoxia and hypercapnia. We show, in isolated “en bloc” carotid body (CB) preparations containing the carotid sinus nerve (CSN) from adult male Sprague Dawley rats, that EPO acts as a stimulator of CSN activity in response to hypoxia at concentrations below 0.5 IU/ml. Under hypercapnic conditions, EPO did not influence the CSN response. EPO concentrations above 0.5 IU/ml decreased the response of the CSN to both hypoxia and hypercapnia, reaching complete inhibition at 2 IU/ml. The inhibitory action of high-dose EPO on the CSN activity might result from an increase in nitric oxide (NO) production. Accordingly, CB preparations were incubated with 2 IU/ml EPO and the unspecific NO synthase inhibitor (L-NAME), or the neuronal-specific NO synthase inhibitor (7NI). Both NO inhibitors fully restored the CSN activity in response to hypoxia and hypercapnia in presence of EPO. Our results show that EPO activates the CB response to hypoxia when its concentration does not exceed the threshold at which NO inhibitors masks EPO’s action.

Fail-safe aspects of oxygen supply.

A fall in oxygen supply releases a remedial response that is otherwise prevented when the oxygen supply is sufficient; for example, the remedial function of HIF-1α is released when oxygen levels fall. the physiological responses initiated when oxygen supply is compromised operate in a fail-safe manner. This concept is applied to two cases: the control of cerebral blood flow, and the detection of hypoxia by the carotid body. The fail-safe oxygen supply concept was tested with simple computer simulations to demonstrate its function and verify the ability to reproduce measured data. The computer model reproduced published observations, suggesting that the fail-safe concept can be considered as a principle that provides novel insight into the physiology of oxygen supply in these cases. An engineered fail-safe system automatically prevents or mitigates the consequences of a system failure. This operational concept can be applied both to the delivery of oxygen to the brain during hypoxia and anaemia, and to the carotid body response to hypoxia and hypercapnia. I aimed to develop simple mathematical models of these fail-safe processes and examine their ability to replicate experimental observations. The intent is to demonstrate the validity of applying the fail-safe concept, not to reveal the details of the physiology involved. The model calculations are based on a single compartment of the relevant tissue in each case that is challenged with a decrease in oxygen supply. The model equation parameters were adjusted to reproduce experimental observations. The fail-safe model of cerebral blood flow control yielded results similar in form to published experimental observations of the cerebral blood flow responses to hypoxia and anaemia. The fail-safe model of carotid body glomus cell control of intracellular hydrogen ion concentration also yielded results similar in form to observations of carotid sinus nerve responses to hypoxia and hypercapnia. The ability of these simple models to simulate experimental observations demonstrates the applicability of the fail-safe concept to oxygen delivery. I suggest that a fail-safe view of oxygen delivery provides novel physiological insight.

Feasibility of kilohertz frequency alternating current neuromodulation of carotid sinus nerve activity in the pig

Recent research supports that over-activation of the carotid body plays a key role in metabolic diseases like type 2 diabetes. Supressing carotid body signalling through carotid sinus nerve (CSN) modulation may offer a therapeutic approach for treating such diseases. Here we anatomically and histologically characterised the CSN in the farm pig as a recommended path to translational medicine. We developed an acute in vivo porcine model to assess the application of kilohertz frequency alternating current (KHFAC) to the CSN of evoked chemo-afferent CSN responses. Our results demonstrate the feasibility of this approach in an acute setting, as KHFAC modulation was able to successfully, yet variably, block evoked chemo-afferent responses. The observed variability in blocking response is believed to reflect the complex and diverse anatomy of the porcine CSN, which closely resembles human anatomy, as well as the need for optimisation of electrodes and parameters for a human-sized nerve. Overall, these results demonstrate the feasibility of neuromodulation of the CSN in an anesthetised large animal model, and represent the first steps in driving KHFAC modulation towards clinical translation. Chronic recovery disease models will be required to assess safety and efficacy of this potential therapeutic modality for application in diabetes treatment.

Induction of asthma causes sensitization of the carotid bodies to lysophosphatidic acid

Previously, we demonstrated an important role for the carotid bodies in asthmatic bronchoconstriction. Specifically, we showed that an allergen‐induced increase in circulating lysophosphatidic acid (LPA) in asthmatic rats activates LPA receptors (LPAr) in the carotid bodies and post‐ganglionic TRPV1 channels, provoking vagal activity and thereby triggering acute bronchoconstriction (Jendzjowsky et al. 2018 Nat Comm. 9, 4030). Here we report that induction of asthma causes molecular and functional changes to the carotid bodies that sensitize them to LPA.Experiments were carried out using the standard ovalbumin‐sensitized brown Norway rat model of asthma. Carotid bodies and petrosal ganglia were extracted 3 hours after the third ovalbumin challenge in asthmatic (OVA) or third saline challenge in naïve (N) rats. Using qPCR (Δctt OVA vs N), LPAr expression was increased in the carotid body (LPAr1: 556x; LPAr2: 20x; LPAr3: 2x; LPAr4: 150x; LPAr5 only detected in OVA: LPAr6: 175x; TRPV1 not detected, as demonstrated previously Roy et al. 2012 J Appl Physiol 112:212–224) whereas LPAr 1–6 and TRPV1 were similarly expressed in petrosal ganglia of OVA and N rats. Using the perfused ex vivo carotid body preparation, baseline normalized carotid sinus nerve responses to increasing doses of LPA activity, were significantly augmented in OVA compared to N rats (2.5uM‐ OVA: 1.12 ± 0.01 N: 1.05 ± 0.02, p=0.162; 5uM: OVA: 1.23 ± 0.03 N:1.10 ± 0.02, p=0.009; 10uM: OVA: 1.40 ± 0.07 N: 1.17 ± 0.03, p<0.001). In contrast, carotid body activity in response to hypoxia was not different between OVA (2.54 ± 0.47) and N (1.95 ± 0.10) rats (p=0.243).Our data suggest that asthmatic carotid bodies have enhanced responsiveness to LPA, independent of hypoxic sensitivity. These changes likely contribute to the acute allergen‐induced bronchoconstriction which is the hallmark of asthma that appears to be mediated, in part, by a novel carotid body‐vagal‐lung neuronal pathway. Targeting carotid body LPA signalling may reduce asthmatic bronchoconstriction and provide new pharmaceutical targets for inflammatory lung disease.Support or Funding InformationThis project was funded by The Lung Association and Canadian Institute for Health Research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sensorimotor control of breathing in the mdx mouse model of Duchenne muscular dystrophy.

Respiratory failure is a leading cause of mortality in Duchenne muscular dystrophy (DMD), but little is known about the control of breathing in DMD and animal models. We show that young (8weeks of age) mdx mice hypoventilate during basal breathing due to reduced tidal volume. Basal CO2 production is equivalent in wild-type and mdx mice. We show that carotid bodies from mdx mice have blunted responses to hyperoxia, revealing hypoactivity in normoxia. However, carotid body, ventilatory and metabolic responses to hypoxia are equivalent in wild-type and mdx mice. Our study revealed profound muscle weakness and muscle fibre remodelling in young mdx diaphragm, suggesting severe mechanical disadvantage in mdx mice at an early age. Our novel finding of potentiated neural motor drive to breathe in mdx mice during maximal chemoactivation suggests compensatory neuroplasticity enhancing respiratory motor output to the diaphragm and probably other accessory muscles. Patients with Duchenne muscular dystrophy (DMD) hypoventilate with consequential arterial blood gas derangement relevant to disease progression. Whereas deficits in DMD diaphragm are recognized, there is a paucity of knowledge in respect of the neural control of breathing in dystrophinopathies. We sought to perform an analysis of respiratory control in a model of DMD, the mdx mouse. In 8-week-old male wild-type and mdx mice, ventilation and metabolism, carotid body afferent activity, diaphragm muscle force-generating capacity, and muscle fibre size, distribution and centronucleation were determined. Diaphragm EMG activity and responsiveness to chemostimulation was determined. During normoxia, mdx mice hypoventilated, owing to a reduction in tidal volume. Basal CO2 production was not different between wild-type and mdx mice. Carotid sinus nerve responses to hyperoxia were blunted in mdx, suggesting hypoactivity. However, carotid body, ventilatory and metabolic responses to hypoxia were equivalent in wild-type and mdx mice. Diaphragm force was severely depressed in mdx mice, with evidence of fibre remodelling and damage. Diaphragm EMG responses to chemoactivation were enhanced in mdx mice. We conclude that there is evidence of chronic hypoventilation in young mdx mice. Diaphragm dysfunction confers mechanical deficiency in mdx resulting in impaired capacity to generate normal tidal volume at rest and decreased absolute ventilation during chemoactivation. Enhanced mdx diaphragm EMG responsiveness suggests compensatory neuroplasticity facilitating respiratory motor output, which may extend to accessory muscles of breathing. Our results may have relevance to emerging treatments for human DMD aiming to preserve ventilatory capacity.

Is the Carotid Body a Metabolic Monitor?

The carotid body is a multi-modal sensor and it has been debated if it senses low glucose. We have hypothesized that the carotid body is modified by some metabolic factors other than glucose and contributes to whole body glucose metabolism. This study examined the roles of insulin, leptin and transient receptor potential (TRP) channels on carotid sinus nerve (CSN) chemoreceptor discharge. In agreement with other studies, CSN activity was not modified by low glucose. Insulin did not affect the CSN hypoxic response. Leptin significantly augmented the CSN response to hypoxia and nonspecific Trp channel blockers (SKF96365, 2-APB) reversed the effect of leptin. Gene expression analysis showed high expression of Trpm3, 6, and 7 channels in the carotid body and petrosal ganglion. The results suggest that the adult mouse carotid body does not sense glucose levels directly. The carotid body may contribute to neural control of glucose metabolism via leptin receptor-mediated TRP channel activation.

Metabolic monitoring by the carotid body (873.8)

The carotid body is a multimodal sensor. Recently, it has been debated whether the CB also senses glucose and is excited by low glucose. In spite of the conflicting data resulted from different preparations, in vivo data have consistently shown that the CB is likely to be involved in the glucose metabolism. For example, stimulation of the CB by NaCN increased systemic blood glucose (Alvarez-Buylleet al.); carotid sinus denervation eliminated an increase in blood glucose which was induced by chronic intermittent hypoxia (Shin et al.). We found that after 6 hours fasting, blood glucose in young mice (<P14) decreased and hypoxic ventilatory response (HVR) was extremely depressed in spite of low glucose (Shirahata et al.). In addition, leptin deficient mice showed an attenuated HVR (Tankersley et al.). Thus, we have hypothesized that CB is a metabolic monitor and modified by substances related to whole body glucose metabolism. In this study we used adult DBA/2J and C57BL/6J mice. CB function was monitored in vitro by the hypoxic response of the carotid sinus nerve (CSN) which was continually superfused with Krebs solution. Changing glucose at different levels from 10 to 0 mM (replaced by sucrose) did not influence the basal CSN activity nor the CSN response to hypoxia. Insulin did not change the basal and hypoxic CSN activity. Leptin (25-50 ng/mL) significantly increased the basal CSN activity and the response to hypoxia was synergetically augmented. The effect of leptin was partially inhibited by putative trip channel inhibitors (2-APB, SKF). The expression of leptin receptor genes were confirmed in the CB and the petrosal ganglion by RT-PCR analysis. These data suggest that the adult mouse CB does not sense glucose levels directly. Leptin may be a link between metabolic states and CB function, and TRP channels may play a role in leptin-induced excitation of the CB. Grant Funding Source: Supported by HL081345 (MS), HL080105 (VYP), AHA 12POST118200001 (M-KS)

Chronic Caffeine Intake in Adult Rat Inhibits Carotid Body Sensitization Produced by Chronic Sustained Hypoxia but Maintains Intact Chemoreflex Output

Sustained hypoxia produces a carotid body (CB) sensitization, known as acclimatization, which leads to an increase in carotid sinus nerve (CSN) activity and ensuing hyperventilation greater than expected from the prevailing partial pressure of oxygen. Whether sustained hypoxia is physiological (high altitude) or pathological (lung disease), acclimatization has a homeostatic implication because it tends to minimize hypoxia. Caffeine, the most commonly ingested psychoactive drug and a nonselective adenosine receptor antagonist, alters CB function and ventilatory responses when administered acutely. Our aim was to investigate the effect of chronic caffeine intake on CB function and acclimatization using four groups of rats: normoxic, caffeine-treated normoxic, chronically hypoxic (12% O₂, 15 days), and caffeine-treated chronically hypoxic rats. Caffeine was administered in drinking water (1 mg/ml). Caffeine ameliorated ventilatory responses to acute hypoxia in normoxic animals without altering the output of the CB (CSN neural activity). Caffeine-treated chronically hypoxic rats exhibited a decrease in the CSN response to acute hypoxia tests but maintained ventilation compared with chronically hypoxic animals. The findings related to CSN neural activity combined with the ventilatory responses indicate that caffeine alters central integration of the CB input to increase the gain of the chemoreflex and that caffeine abolishes CB acclimatization. The putative mechanisms involved in sensitization and its loss were investigated: expression of adenosine receptors in CB (A(2B)) was down-regulated and that in petrosal ganglion (A(2A)) was up-regulated in caffeine-treated chronically hypoxic rats; both adenosine and dopamine release from CB chemoreceptor cells was increased in chronic hypoxia and in caffeine-treated chronic hypoxia groups.

The progesterone receptor antagonist mifepristone abolishes carotid sinus nerve response to hypoxia in rat pups.

We tested the hypothesis that endogenous progesterone enhances peripheral chemoreceptors function in rat pups. Newborn rats have been treated with the progesterone receptor antagonist mifepristone (Mif) between postnatal days 3‐12 (70μg/g/day) and compared to control (Cont). We used the in‐vitro carotid body/carotid sinus nerve (CSN) preparation to record CSN activity (impulses/sec) in 12‐day‐old rats under normoxia (perfusion medium bubbled with 21%O2‐5%CO2), in response to hypoxia (5%O2‐5%CO2), or nicotine (mediator between carotid body type I cells and CSN terminals, 100μM) supefusion for 3‐4 minutes each. Baseline CSN firing frequency was similar between groups, at 2.7±0.3 in Cont and 2.6±0.3 Hz in Mif pups. At steady state hypoxic CSN activity increased to 7.5±0.9 Hz in Cont, but was left unchanged in Mif pups (3.0±0.5 Hz, p&lt;0.0002 between groups). In response to nicotine superfusion, CSN firing activity increased to 10.8±1.9 Hz in Cont and to 6.1±3.3 Hz in Mif (p&lt;0.04 between groups). We conclude that endogenous progesterone plays an important role in carotid body during postnatal development in rats. Founded by SickKids Foundation (XG07‐006)

Respiratory plasticity after perinatal hyperoxia is not prevented by antioxidant supplementation

Perinatal hyperoxia attenuates the hypoxic ventilatory response in rats by altering development of the carotid body and its chemoafferent neurons. In this study, we tested the hypothesis that hyperoxia elicits this plasticity through the increased production of reactive oxygen species (ROS). Rats were born and raised in 60% O(2) for the first two postnatal weeks while treated with one of two antioxidants: vitamin E (via milk from mothers whose diet was enriched with 1000 IU vitamin E kg(-1)) or a superoxide dismutase mimetic, manganese(III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP; via daily intraperitoneal injection of 5-10 mg kg(-1)); rats were subsequently raised in room air until studied as adults. Peripheral chemoreflexes, assessed by carotid sinus nerve responses to cyanide, asphyxia, anoxia and isocapnic hypoxia (vitamin E experiments) or by hypoxic ventilatory responses (MnTMPyP experiments), were reduced after perinatal hyperoxia compared to those of normoxia-reared controls (all P<0.01); antioxidant treatment had no effect on these responses. Similarly, the carotid bodies of hyperoxia-reared rats were only one-third the volume of carotid bodies from normoxia-reared controls (P <0.001), regardless of antioxidant treatment. Protein carbonyl concentrations in the blood plasma, measured as an indicator of oxidative stress, were not increased in neonatal rats (2 and 8 days of age) exposed to 60% O(2) from birth. Collectively, these data do not support the hypothesis that perinatal hyperoxia impairs peripheral chemoreceptor development through ROS-mediated oxygen toxicity.

Carotid sinus nerve responses and ventilatory acclimatization to hypoxia in adult rats following 2 weeks of postnatal hyperoxia

Adult rats have decreased carotid body volume and reduced carotid sinus nerve, phrenic nerve, and ventilatory responses to acute hypoxic stimulation after exposure to postnatal hyperoxia (60% O2, PNH) during the first 4 weeks of life. Moreover, sustained hypoxic exposure (12%, 7 days) partially reverses functional impairment of the acute hypoxic phrenic nerve response in these rats. Similarly, 2 weeks of PNH results in the same phenomena as above except that ventilatory responses to acute hypoxia have not been measured in awake rats. Thus, we hypothesized that 2-week PNH-treated rats would also exhibit blunted chemoafferent responses to acute hypoxia, but would exhibit ventilatory acclimatization to sustained hypoxia. Rats were born into, and exposed to PNH for 2 weeks, followed by chronic room-air exposure. At 3-4 months of age, two studies were performed to assess: (1) carotid sinus nerve responses to asphyxia and sodium cyanide in anesthetized rats and (2) ventilatory and blood gas responses in awake rats before (d0), during (d1 and d7), and 1 day following (d8) sustained hypoxia. Carotid sinus nerve responses to i.v. NaCN and asphyxia (10 s) were significantly reduced in PNH-treated versus control rats; however, neither the acute hypoxic ventilatory response nor the time course or magnitude of ventilatory acclimatization differed between PNH and control rats despite similar levels of PaO2 . Although carotid body volume was reduced in PNH rats, carotid body volumes increased during sustained hypoxia in both PNH and control rats. We conclude that normal acute and chronic ventilatory responses are related to retained (though impaired) carotid body chemoafferent function combined with central neural mechanisms which may include brainstem hypoxia-sensitive neurons and/or brainstem integrative plasticity relating both central and peripheral inputs.

Carotid chemoafferent plasticity in adult rats following developmental hyperoxia

Developmental hyperoxia impairs carotid chemoreceptor development and induces long-lasting reduction in carotid sinus nerve (CSN) responses to hypoxia in adult rats. Studies were carried out to determine if CSN responses to acute hypoxia would exhibit hypoxia-induced plasticity in adult 3-5-months-old rats previously treated with postnatal hyperoxia (60% O2, PNH) of 1, 2, or 4 weeks duration. CSN responses to acute hypoxia were assessed in adult rats exposed to 1 week of sustained hypoxia (12% O2, SH). In normal adult rats and adult rats treated with 1 week of PNH, CSN responses to acute hypoxia were significantly increased in urethane-anesthetized rats when studied 3-5 h after SH. Apparent increases in CSN responses to hypoxia were not significant in rats treated with 2 weeks of PNH and were clearly absent after 4 weeks of PNH, but exponential analysis suggests a PNH duration-dependent plasticity of the CSN response to acute hypoxia after SH. In a second study rats exposed to 2 weeks of PNH were treated with SH for 1 week as adults and acute hypoxic responses were tested 4-5 months later. CSN responses in these rats were unaffected by SH suggesting a lack of persistent SH-induced functional plasticity. We conclude that rats treated with 1 week of PNH retain the capacity for hypoxia-induced plasticity of carotid chemoafferent function and some potential for plasticity may be present after 2 weeks of PNH, whereas 4 weeks of PNH impairs the capability of rats to exhibit plasticity following 1 week of SH.

Adult carotid chemoafferent responses to hypoxia after 1, 2, and 4 wk of postnatal hyperoxia.

Exposing newborn rats to postnatal hyperoxia (60% O2) for 1-4 wk attenuates the ventilatory and phrenic nerve responses to acute hypoxia in adult rats. The goal of this research was to increase our understanding of the carotid chemoreceptor afferent neural input in this depressed response with different durations of postnatal hyperoxic exposure. Rats were exposed from a few days before birth to 1, 2, or 4 wk of 60% O2 and studied after 3-5 mo in normoxia. The rats were anesthetized with urethane. Whole carotid sinus nerve (CSN) responses to NaCN (40 microg/kg iv), 10 s of asphyxia and acute isocapnic hypoxia (arterial Po2 45 Torr) were determined. Mean CSN responses to stimuli after postnatal hyperoxia were reduced compared with controls. Responses in rats exposed to 1 wk of postnatal hyperoxia were less affected than those exposed to 2 and 4 wk of hyperoxia, which were equivalent to each other. These studies illustrate the importance of normoxia during the first 2 wk of life in development of carotid chemoreceptor afferent function.

Life-long impairment of hypoxic phrenic responses in rats following 1 month of developmental hyperoxia.

Hypoxic ventilatory and phrenic responses are reduced in adult rats (3-5 months old) exposed to hyperoxia for the first month of life (hyperoxia treated). We previously reported that hypoxic phrenic responses were normal in a small sample of 14- to 15-month-old hyperoxia-treated rats, suggesting slow, spontaneous recovery. Subsequent attempts to identify the mechanism(s) underlying this spontaneous recovery of hypoxic phrenic responses led us to re-evaluate our earlier conclusion. Experiments were conducted in two groups of aged Sprague-Dawley rats (14-15 months old) which were anaesthetized, vagotomized, neuromuscularly blocked and ventilated: (1) a hyperoxia-treated group raised in 60 % O2 for the first 28 postnatal days; and (2) an age-matched control group raised in normoxia. Increases in minute phrenic activity and integrated phrenic nerve amplitude (integral Phr) during isocapnic hypoxia (arterial partial pressures of O2, 60, 50 and 40 +/- 1 mmHg) were greater in aged control (n = 15) than hyperoxia-treated rats (n = 11; P < or = 0.01). Phrenic burst frequency during hypoxia was not different between groups. To examine the central integration of carotid chemoafferent inputs, steady-state relationships between carotid sinus nerve (electrical) stimulation frequency and phrenic nerve activity were compared in aged control (n = 7) and hyperoxia-treated rats (n = 7). Minute phrenic activity, integral Phr and burst frequency were not different between groups at any stimulation frequency between 0.5 and 20 Hz. Carotid body chemoreceptor function was examined by recording whole carotid sinus nerve responses to cessation of ventilation or injection of cyanide in aged control and hyperoxia-treated rats. Electrical activity of the carotid sinus nerve did not change in five out of five hyperoxia-treated rats in response to stimuli that evoked robust increases in carotid sinus nerve activity in five out of five control rats. Estimates of carotid body volume were lower in aged hyperoxia-treated rats (4.4 (+/- 0.2) x 10(6) microm3) compared to controls (17.4 (+/- 1.6) x 10(6) microm3; P <0.01). We conclude that exposure to hyperoxia for the first month of life causes life-long impairment of carotid chemoreceptor function and, consequently, blunted phrenic responses to hypoxia.

Barium-stimulated chemosensory activity may not reflect inhibition of background voltage-insensitive K + channels in the rat carotid body

To test the hypothesis that the voltage-insensitive background leak K + channel is responsible for the oxygen-sensitive properties of glomus cells in the rat carotid body (CB) we used Ba 2+, a non-specific inhibitor of K + currents. In vitro changes in cytosolic calcium ([Ca 2+] c) and chemosensory discharge were studied to measure the effect of Ba 2+. In normal Tyrode buffer, Ba 2+ (3 and 5 mM) significantly increased carotid sinus nerve (CSN) discharge over baseline firing rates under normoxia (P o 2∼120 Torr) from ∼150 to ∼600 imp/0.5 s. However, addition of 200 μM Cd 2+ which completely blocked increase in CSN activity stimulated by hypoxia (P o 2∼30 Torr), hypercapnia (P co 2∼60 Torr, P o 2∼120 Torr) and high CO (P co∼550 Torr, P o 2∼120 Torr) did not significantly inhibit Ba 2+-stimulated CSN discharge. The response to hypoxia is abolished with Ca 2+-free tyrode buffer containing 10 mM EGTA. Yet, in the same buffer, Ba 2+ increased CSN discharge from ∼2 to ∼180 imp/0.5 s. With 200 μM Cd 2+ and 10 mM EGTA, Ba 2+ still increased CSN discharge from ∼2 to ∼150 imp/0.5 s. Oligomycin (2 μg) abolished the hypoxic response. However, in the presence of oligomycin CSN response to Ba 2+ was significant. Since Ba 2+ increased neural discharge under conditions where hypoxia stimulated CSN discharge is completely abolished, we suggest that the effect of Ba 2+ on CSN discharge may not have anything to do with the oxygen sensing mechanism in the CB.

P O 2–P CO 2 stimulus interaction in [Ca 2+] i and CSN activity in the adult rat carotid body

Since glomus cell intracellular calcium ([Ca 2+] i) plays a key role in generating carotid sinus nerve (CSN) discharge, we hypothesized that glomus cell [Ca 2+] i would correspond to CSN discharge rates during P O 2 –P CO 2 stimulus interaction in adult rat carotid body (CB). Accordingly, we measured steady state P O 2 –P CO 2 interaction in CSN discharge rates during hypocapnia (P CO 2 =8–10 Torr), normocapnia (P CO 2 =33–35 Torr) and hypercapnia (P CO 2 =68–70 Torr) in normoxia (P O 2 ∼130 Torr) and hypoxia (P O 2 ∼36 Torr). The results showed P O 2 –P CO 2 stimulus interaction in CSN responses. [Ca 2+] i levels were measured in isolated type I cells (2–3 cells/field), using Ca 2+ sensitive fluoroprobe indo-1AM. The [Ca 2+] i responses increased with increasing P CO 2 in normoxia. In hypoxia, [Ca 2+] i did not increase during hypocapnia but increased during normocapnia, showing P O 2 –P CO 2 interaction. However, CSN response during hypoxia was far greater than that for [Ca 2+] i response, particularly during hypocapnic hypoxia. Thus, the [Ca 2+] i interaction cannot account for the whole CSN interaction. The origin of this CSN P O 2– P CO 2 interaction must have occurred in part beyond cellular [Ca 2+] i interaction. Interactions at both sites (glomus cell membrane and sinus nerve endings) are reminiscent of reversible O 2–heme protein reaction with a Bohr effect.

Dynamic ventilatory responses in rats: normal development and effects of prenatal nicotine exposure

Infants of smoking mothers are at increased risk of SIDS, one cause of which is thought to be due to impaired ventilatory responses. We tested the hypotheses that prenatal nicotine exposure impairs the development of dynamic carotid chemoreceptor-driven ventilatory responses, and reduces the ability to lower metabolic rate in hypoxia. Osmotic minipumps were implanted into 20 pregnant rats at day 3 of gestation to deliver nicotine (6 mg/kg per day free base) or saline for 4 weeks. Minute ventilation was recorded breath by breath in rat pups at 3, 8 and 18 days ( n=6, 8 and 6) postnatal in response to 5-sec challenges of 100% O 2 (Dejours test) and 5% O 2+5% CO 2. Carotid sinus nerve (CSN) responses to hypoxia and CO 2 were recorded from 22 control and 17 nicotine-exposed preparations at ages between 3–20 days. Oxygen consumption ( V ̇ O 2 ) was measured in groups of pups at 3 days ( n=7 each for nicotine and control) and 8 days ( n=5 each for nicotine and control) in room air and 10% O 2. There was no detectable effect of nicotine exposure on the development of CSN responses. Ventilatory responses to 5% O 2–5% CO 2 increased with age but did not differ between nicotine and control groups. Ventilatory responses to 100% O 2 were unaffected by nicotine exposure at 8 and 18 days. However, the 3-day nicotine group showed no significant response to 100% O 2 whereas V e was significantly reduced in the control group by 100% O 2. There was no significant effect of nicotine exposure on the ability to reduce oxygen consumption in hypoxia at 3 or 8 days, but at 3 days, baseline (room air) variability in oxygen consumption was greater in the nicotine group. We conclude that nicotine exposure appears to result in abnormal ventilatory responses to withdrawal of baseline peripheral chemoreceptor drive during a period of early postnatal life. We speculate that a transient abnormality could contribute to a period of instability and increased vulnerability to challenges.