David D. Kline

Induction of sensory long-term facilitation in the carotid body by intermittent hypoxia: Implications for recurrent apneas

Reflexes from the carotid body have been implicated in cardiorespiratory disorders associated with chronic intermittent hypoxia (CIH). To investigate whether CIH causes functional and/or structural plasticity in the carotid body, rats were subjected to 10 days of recurrent hypoxia or normoxia. Acute exposures to 10 episodes of hypoxia evoked long-term facilitation (LTF) of carotid body sensory activity in CIH-conditioned but not in control animals. The magnitude of sensory LTF depended on the length of CIH conditioning and was completely reversible and unique to CIH, because conditioning with a comparable duration of sustained hypoxia was ineffective. Histological analysis revealed no differences in carotid body morphology between control and CIH animals. Previous treatment with superoxide anion (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\cdot}-}\end{equation*}\end{document}) scavenger prevented sensory LTF. In the CIH-conditioned animals, carotid body aconitase enzyme activity decreased compared with controls. These observations suggest that increased generation of reactive oxygen species contribute to sensory LTF. In CIH animals, carotid body complex I activity of the mitochondrial electron transport is inhibited, suggesting mitochondria as one source of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\cdot}-}\end{equation*}\end{document} generation. These observations demonstrate that CIH induces a previously uncharacterized form of reactive oxygen species-dependent, reversible, functional plasticity in carotid body sensory activity. The sensory LTF may contribute to persistent reflex activation of sympathetic nerve activity and blood pressure in recurrent apnea patients experiencing CIH.

Defective carotid body function and impaired ventilatory responses to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1α

To investigate whether the transcriptional activator hypoxia-inducible factor 1 (HIF-1) is required for ventilatory responses to hypoxia, we analyzed mice that were either wild type or heterozygous for a loss-of-function (knockout) allele at the Hif1a locus, which encodes the O2-regulated HIF-1α subunit. Although the ventilatory response to acute hypoxia was not impaired in Hif1a+/− mice, the response was primarily mediated via vagal afferents, whereas in wild-type mice, carotid body chemoreceptors played a predominant role. When carotid bodies isolated from wild-type mice were exposed to either cyanide or hypoxia, a marked increase in sinus nerve activity was recorded, whereas carotid bodies from Hif1a+/− mice responded to cyanide but not to hypoxia. Histologic analysis revealed no abnormalities of carotid body morphology in Hif1a+/− mice. Wild-type mice exposed to hypoxia for 3 days manifested an augmented ventilatory response to a subsequent acute hypoxic challenge. In contrast, prior chronic hypoxia resulted in a diminished ventilatory response to acute hypoxia in Hif1a+/− mice. Thus partial HIF-1α deficiency has a dramatic effect on carotid body neural activity and ventilatory adaptation to chronic hypoxia.

Exogenous Brain-Derived Neurotrophic Factor Rescues Synaptic Dysfunction in Mecp2-Null Mice

Postnatal deficits in brain-derived neurotrophic factor (BDNF) are thought to contribute to pathogenesis of Rett syndrome (RTT), a progressive neurodevelopmental disorder caused by mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2). In Mecp2-null mice, a model of RTT, BDNF deficits are most pronounced in structures important for autonomic and respiratory control, functions that are severely affected in RTT patients. However, relatively little is known about how these deficits affect neuronal function or how they may be linked to specific RTT endophenotypes. To approach these issues, we analyzed synaptic function in the brainstem nucleus tractus solitarius (nTS), the principal site for integration of primary visceral afferent inputs to central autonomic pathways and a region in which we found markedly reduced levels of BDNF in Mecp2 mutants. Our results demonstrate that the amplitude of spontaneous miniature and evoked EPSCs in nTS neurons is significantly increased in Mecp2-null mice and, accordingly, that mutant cells are more likely than wild- type cells to fire action potentials in response to primary afferent stimulation. These changes occur without any increase in intrinsic neuronal excitability and are unaffected by blockade of inhibitory GABA currents. However, this synaptopathy is associated with decreased BDNF availability in the primary afferent pathway and can be rescued by application of exogenous BDNF. On the basis of these findings, we hypothesize that altered sensory gating in nTS contributes to cardiorespiratory instability in RTT and that nTS is a site at which restoration of normal BDNF signaling could help reestablish normal homeostatic controls.

Altered respiratory responses to hypoxia in mutant mice deficient in neuronal nitric oxide synthase

1 The role of endogenous nitric oxide (NO) generated by neuronal nitric oxide synthase (NOS‐1) in the control of respiration during hypoxia and hypercapnia was assessed using mutant mice deficient in NOS‐1. 2 Experiments were performed on awake and anaesthetized mutant and wild‐type control mice. Respiratory responses to varying levels of inspired oxygen (100, 21 and 12 % O2) and carbon dioxide (3 and 5 % CO2 balanced oxygen) were analysed. In awake animals, respiration was monitored by body plethysmograph along with oxygen consumption (VO2), CO2 production (VCO2) and body temperature. In anaesthetized, spontaneously breathing mice, integrated efferent phrenic nerve activity was monitored as an index of neural respiration along with arterial blood pressure and blood gases. Cyclic 3′,5′‐guanosine monophosphate (cGMP) levels in the brainstem were analysed by radioimmunoassay as an index of nitric oxide generation. 3 Unanaesthetized mutant mice exhibited greater respiratory responses during 21 and 12 % O2 than the wild‐type controls. Respiratory responses were associated with significant decreases in oxygen consumption in both groups of mice, and the magnitude of change was greater in mutant than wild‐type mice. Changes in CO2 production and body temperature, however, were comparable between both groups of mice. 4 Similar augmentation of respiratory responses during hypoxia was also observed in anaesthetized mutant mice. In addition, five of the fourteen mutant mice displayed periodic oscillations in respiration (brief episodes of increases in respiratory rate and tidal phrenic nerve activity) while breathing 21 and 12 % O2, but not during 100 % O2. The time interval between the episodes decreased by reducing inspired oxygen from 21 to 12 % O2. 5 Changes in arterial blood pressure and arterial blood gases were comparable at any given level of inspired oxygen between both groups of mice, indicating that changes in these variables do not account for the differences in the response to hypoxia. 6 Respiratory responses to brief hyperoxia (Dejours test) and to cyanide, a potent chemoreceptor stimulant, were more pronounced in mutant mice, suggesting augmented peripheral chemoreceptor sensitivity. 7 cGMP levels were elevated in the brainstem during 21 and 12 % O2 in wild‐type but not in mutant mice, indicating decreased formation of nitric oxide in mutant mice. 8 The magnitude of respiratory responses to hypercapnia (3 and 5 % CO2 balanced oxygen) was comparable in both groups of mice in the awake and anaesthetized conditions. 9 These observations suggest that the hypoxic responses were selectively augmented in mutant mice deficient in NOS‐1. Peripheral as well as central mechanisms contributed to the altered responses to hypoxia. These results support the idea that nitric oxide generated by NOS‐1 is an important physiological modulator of respiration during hypoxia.

Adaptive Depression in Synaptic Transmission in the Nucleus of the Solitary Tract after In Vivo Chronic Intermittent Hypoxia: Evidence for Homeostatic Plasticity

The respiratory system is highly pliable in its adaptation to low-oxygen (hypoxic) environments. After chronic intermittent hypoxia (CIH), alterations in the regulation of cardiorespiratory system become persistent because of changes in the peripheral chemoreceptor reflex. We present evidence for the induction of a novel form of homeostatic plasticity in this reflex pathway in the nucleus tractus solitarius (NTS), the site of termination of the chemosensory afferent fibers. CIH induces an increase in NTS postsynaptic cell activity initiated by spontaneous presynaptic transmitter release that is counterbalanced by a reduction in evoked synaptic transmission between sensory afferents and NTS second-order cells. This is accomplished via presynaptic mechanisms involving changes in neurotransmitter release and calcium/calmodulin-dependent kinase II activation.

Ventilatory changes during intermittent hypoxia: importance of pattern and duration.

Intermittent hypoxia is encountered in life more often than sustained hypoxia. The purpose of this article is to summarize the long-term effects of intermittent hypoxia on control of breathing. Emphasis is given to intermittent hypoxia associated with recurrent apneas and with brief repeated ascents to high altitude. Reported responses to chronic recurrent apneas include both depressed and enhanced hypoxic ventilatory responses (HVR). In addition, recurrent apneas are often associated with depression of the hypercapnic ventilatory response (HCVR). On the other hand, intermittent hypoxia associated with repeated ascents to high altitude augments HVR with little or no influence on the HCVR. In a rat model, prolonged exposure to intermittent hypoxia simulating recurrent apneas selectively enhances carotid body sensitivity to acute hypoxia and induces long-lasting activation of baseline activity, whereas intermittent hypoxia simulating repeated ascents to high altitude has little or no effect on peripheral chemoreceptor activity. Thus the mechanisms by which episodic hypoxia alter HVR appear to differ and seem to depend on the paradigm of intermittent hypoxia. Prolonged episodic hypoxia also leads to long-term facilitation (LTF) of respiratory motor output in humans and in experimental animals. Recent studies on experimental animals suggest the involvement of HIF-1 transcription factor in inducing enhanced HVR in response to chronic recurrent apnea pattern. Future studies are needed to identify the molecular and cellular signaling pathways associated with intermittent hypoxia and their impact on ventilatory control during hypoxia and hypercapnia.

Endogenous Brain-Derived Neurotrophic Factor in the Nucleus Tractus Solitarius Tonically Regulates Synaptic and Autonomic Function

Brain-derived neurotrophic factor (BDNF) and its receptor, TrkB, are highly expressed in the nucleus tractus solitarius (nTS), the principal target of cardiovascular primary afferent input to the brainstem. However, little is known about the role of BDNF signaling in nTS in cardiovascular homeostasis. We examined whether BDNF in nTS modulates cardiovascular function in vivo and regulates synaptic and/or neuronal activity in isolated brainstem slices. Microinjection of BDNF into the rat medial nTS (mnTS), a region critical for baroreflex control of sympathetic outflow, produced dose-dependent increases in mean arterial pressure (MAP), heart rate (HR), and lumbar sympathetic nerve activity (LSNA) that were blocked by the tyrosine kinase inhibitor K252a. In contrast, immunoneutralization of endogenous BDNF (anti-BDNF), or microinjection of K252a alone, decreased MAP, HR, and LSNA. The effects of anti-BDNF were abolished by blockade of ionotropic glutamate receptors, indicating a role for glutamate signaling in the response to BDNF. In vitro, BDNF reduced the amplitude of miniature EPSCs as well as solitary tract (TS) evoked EPSC amplitude and action potential discharge (APD) in second-order nTS neurons. BDNF effects on EPSCs were independent of GABAergic signaling and abolished by AMPA receptor blockade. In contrast, K252a increased spontaneous EPSC frequency and TS evoked EPSC amplitude. BDNF also attenuated APD evoked by injection of depolarizing current into second-order neurons, indicating reduced intrinsic neuronal excitability. Our data demonstrate that BDNF signaling in mnTS plays a tonic role in regulating cardiovascular function, likely via modulation of primary afferent glutamatergic excitatory transmission and neural activity.

Mutant mice deficient in NOS-1 exhibit attenuated long-term facilitation and short-term potentiation in breathing

The objective of the present study is to examine the potential role of nitric oxide (NO) in short‐term potentiation (STP) and long‐term facilitation (LTF) of breathing. Experiments were performed in wild‐type (WT) and mutant mice deficient in nitric oxide synthase‐1 (NOS‐1), as well as in WT mice administered the NOS‐1 inhibitor 7‐nitroindazole (7‐NI; 50 mg kg−1; i.p.). Respiratory responses following either single or recurrent episodes of hypoxia (7 % O2, balance N2) were analysed in unanaesthetised animals by body plethysmography along with rate of O2 consumption (V̇O2) and CO2 production (V̇CO2). After a single hypoxic challenge, respiration in WT mice remained elevated for 5 min, suggesting STP in ventilation. Following termination of three consecutive hypoxic challenges, respiration remained elevated during normoxia for as long as 30 min, indicating LTF in breathing under awake conditions. STP and LTF were significantly attenuated or absent in WT mice after 7‐NI. A similar attenuation or absence of STP and LTF was also seen in NOS‐1 mutant mice. Changes in V̇O2 and V̇CO2 were comparable among mice during the post‐hypoxic period, suggesting that the absence of STP and LTF was not due to alterations in body metabolism. These results suggest endogenous NO is an important physiological modulator of ventilatory STP and LTF.

Chronic intermittent hypoxia enhances carotid body chemoreceptor response to low oxygen

Episodic or intermittent hypoxia occurs in many pathophysiological situations, including sleep apneas and recurrent apneas in premature infants. In addition, intermittent hypoxia is not all uncommon in humans who have lung diseases such as chronic obstructive pulmonary disease (COPD), asthma or pulmonary fibrosis. Thus mammals experience episodic hypoxia, often in life, even perhaps more so than sustained hypoxia that occurs in situations like high altitude.

Hypoxia activates nucleus tractus solitarii neurons projecting to the paraventricular nucleus of the hypothalamus

Peripheral chemoreceptor afferent information is sent to the nucleus tractus solitarii (nTS), integrated, and relayed to other brain regions to alter cardiorespiratory function. The nTS projects to the hypothalamic paraventricular nucleus (PVN), but activation and phenotype of these projections during chemoreflex stimulation is unknown. We hypothesized that activation of PVN-projecting nTS neurons occurs primarily at high intensities of hypoxia. We assessed ventilation and cardiovascular parameters in response to increasing severities of hypoxia. Retrograde tracers were used to label nTS PVN-projecting neurons and, in some rats, rostral ventrolateral medulla (RVLM)-projecting neurons. Immunohistochemistry was performed to identify nTS cells that were activated (Fos-immunoreactive, Fos-IR), catecholaminergic, and GABAergic following hypoxia. Conscious rats underwent 3 h normoxia (n = 4, 21% O(2)) or acute hypoxia (12, 10, or 8% O(2); n = 5 each). Hypoxia increased ventilation and the number of Fos-IR nTS cells (21%, 13 ± 2; 12%, 58 ± 4; 10%, 166 ± 22; 8%, 186 ± 6). Fos expression after 10% O(2) was similar whether arterial pressure was allowed to decrease (-13 ± 1 mmHg) or was held constant. The percentage of PVN-projecting cells activated was intensity dependent, but contrary to our hypothesis, PVN-projecting nTS cells exhibiting Fos-IR were found at all hypoxic intensities. Notably, at all intensities of hypoxia, ∼75% of the activated PVN-projecting nTS neurons were catecholaminergic. Compared with RVLM-projecting cells, a greater percentage of PVN-projecting nTS cells was activated by 10% O(2). Data suggest that increasing hypoxic intensity activates nTS PVN-projecting cells, especially catecholaminergic, PVN-projecting neurons. The nTS to PVN catecholaminergic pathway may be critical even at lower levels of chemoreflex activation and more important to cardiorespiratory responses than previously considered.