Ryan W. Bavis

BDNF is necessary and sufficient for spinal respiratory plasticity following intermittent hypoxia

Intermittent hypoxia causes a form of serotonin-dependent synaptic plasticity in the spinal cord known as phrenic long-term facilitation (pLTF). Here we show that increased synthesis of brain-derived neurotrophic factor (BDNF) in the spinal cord is necessary and sufficient for pLTF in adult rats. We found that intermittent hypoxia elicited serotonin-dependent increases in BDNF synthesis in ventral spinal segments containing the phrenic nucleus, and the magnitude of these BDNF increases correlated with pLTF magnitude. We used RNA interference (RNAi) to interfere with BDNF expression, and tyrosine kinase receptor inhibition to block BDNF signaling. These disruptions blocked pLTF, whereas intrathecal injection of BDNF elicited an effect similar to pLTF. Our findings demonstrate new roles and regulatory mechanisms for BDNF in the spinal cord and suggest new therapeutic strategies for treating breathing disorders such as respiratory insufficiency after spinal injury. These experiments also illustrate the potential use of RNAi to investigate functional consequences of gene expression in the mammalian nervous system in vivo.

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 (∫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⩽ 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, ∫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) × 106μm3) compared to controls (17.4 (± 1.6) × 106μm3; 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.

Developmental plasticity of the hypoxic ventilatory response in rats induced by neonatal hypoxia.

Neonatal hypoxia alters the development of the hypoxic ventilatory response in rats and other mammals. Here we demonstrate that neonatal hypoxia impairs the hypoxic ventilatory response in adult male, but not adult female, rats. Rats were raised in 10% O2 for the first postnatal week, beginning within 12 h after birth. Subsequently, ventilatory responses were assessed in 7‐ to 9‐week‐old unanaesthetized rats via whole‐body plethysmography. In response to 12% O2, male rats exposed to neonatal hypoxia increased ventilation less than untreated control rats (mean ±s.e.m. 35.2 ± 7.7%versus 67.4 ± 9.1%, respectively; P= 0.01). In contrast, neonatal hypoxia had no lasting effect on hypoxic ventilatory responses in female rats (67.9 ± 12.6%versus 61.2 ± 11.7% increase in hypoxia‐treated and control rats, respectively; P > 0.05). Normoxic ventilation was unaffected by neonatal hypoxia in either sex at 7–9 weeks of age (P > 0.05). Since we hypothesized that neonatal hypoxia alters the hypoxic ventilatory response at the level of peripheral chemoreceptors or the central neural integration of chemoafferent activity, integrated phrenic responses to isocapnic hypoxia were investigated in urethane‐anaesthetized, paralysed and ventilated rats. Phrenic responses were unaffected by neonatal hypoxia in rats of either sex (P > 0.05), suggesting that neonatal hypoxia‐induced plasticity occurs between the phrenic nerve and the generation of airflow (e.g. neuromuscular junction, respiratory muscles or respiratory mechanics) and is not due to persistent changes in hypoxic chemosensitivity or central neural integration. The basis of sex differences in this developmental plasticity is unknown.

Differential expression of respiratory long-term facilitation among inbred rat strains

We tested the hypotheses that: (1) long-term facilitation (LTF) following acute intermittent hypoxia (AIH) varies among three inbred rat strains: Fischer 344 (F344), Brown Norway (BN) and Lewis rats and (2) ventral cervical spinal levels of genes important for phrenic LTF (pLTF) vary in association with pLTF magnitude. Lewis and F344, but not BN rats exhibited significant increases in phrenic and hypoglossal burst amplitude 60min post-AIH that were significantly greater than control experiments without AIH, indicating strain differences in phrenic (98%, 56% and 20%, respectively) and hypoglossal LTF (66%, 77% and 5%, respectively). Ventral spinal 5-HT(2A) receptor mRNA and protein levels were higher in F344 and Lewis versus BN, suggesting that higher 5-HT(2A) receptor levels are associated with greater pLTF. More complex relationships were found for 5-HT(7), BDNF and TrkB mRNA. BN had higher 5-HT(7) and TrkB mRNA versus F344; BN and Lewis had higher BDNF mRNA levels versus F344. Genetic variations in serotonergic function may underlie strain differences in AIH-induced pLTF.

Ventilation and phrenic output following high cervical spinal hemisection in male vs. female rats.

Female sex hormones influence the neural control of breathing and may impact neurologic recovery from spinal cord injury. We hypothesized that respiratory recovery after C2 spinal hemisection (C2HS) differs between males and females and is blunted by prior ovariectomy (OVX) in females. Inspiratory tidal volume (VT), frequency (fR), and ventilation (VE) were quantified during quiet breathing (baseline) and 7% CO2 challenge before and after C2HS in unanesthetized adult rats via plethysmography. Baseline breathing was similarly altered in all rats (reduced VT, elevated fR) but during hypercapnia females had relatively higher VT (i.e. compared to pre-injury) than male or OVX rats (p<0.05). Phrenic neurograms recorded in anesthetized rats indicated that normalized burst amplitude recorded ipsilateral to C2HS (i.e. the crossed phrenic phenomenon) is greater in females during respiratory challenge (p<0.05 vs. male and OVX). We conclude that sex differences in recovery of VT and phrenic output are present at 2 weeks post-C2HS. These differences are consistent with the hypothesis that ovarian sex hormones influence respiratory recovery after cervical spinal cord injury.

Time course of alterations in pre- and post-synaptic chemoreceptor function during developmental hyperoxia

Postnatal hyperoxia exposure reduces the carotid body response to acute hypoxia and produces a long-lasting impairment of the ventilatory response to hypoxia. The present work investigated the time course of pre- and post-synaptic alterations following exposure to hyperoxia (Fl(O2) = 0.6) for 1, 3, 5, 8 and 14 days (d) starting at postnatal day 7 (P7) as compared to age-matched controls. Hyperoxia exposure for 1d enhanced the nerve response and glomus cell calcium response to acute hypoxia, but exposure for 3-5d caused a significant reduction in both. Hypoxia-induced catecholamine release and nerve conduction velocity were significantly decreased by 5d hyperoxia. We conclude that hyperoxia exerts pre-synaptic (glomus cell calcium and secretory responses) and post-synaptic (afferent nerve excitability) actions to initially enhance and then reduce the chemoreceptor response to acute hypoxia. The parallel changes in glomus cell calcium response and nerve response suggest causality between the two and that environmental hyperoxia can affect the coupling between acute hypoxia and glomus cell calcium regulation.

Chronic hyperoxia alters the expression of neurotrophic factors in the carotid body of neonatal rats

Chronic exposure to hyperoxia alters the postnatal development and innervation of the rat carotid body. We hypothesized that this plasticity is related to changes in the expression of neurotrophic factors or related proteins. Rats were reared in 60% O(2) from 24 to 36h prior to birth until studied at 3d of age (P3). Protein levels for brain-derived neurotrophic factor (BDNF) were significantly reduced (-70%) in the P3 carotid body, while protein levels for its receptor, tyrosine kinase B, and for glial cell line-derived neurotrophic factor (GDNF) were unchanged. Transcript levels in the carotid body were downregulated for the GDNF receptor Ret (-34%) and the neuropeptide Vgf (-67%), upregulated for Cbln1 (+205%), and unchanged for Fgf2; protein levels were not quantified for these genes. Immunohistochemical analysis revealed that Vgf and Cbln1 proteins are expressed within the carotid body glomus cells. These data suggest that BDNF, and perhaps other neurotrophic factors, contribute to abnormal carotid body function following perinatal hyperoxia.

Chronic hyperoxia and the development of the carotid body.

Preterm infants often experience hyperoxia while receiving supplemental oxygen. Prolonged exposure to hyperoxia during development is associated with pathologies such as bronchopulmonary dysplasia and retinopathy of prematurity. Over the last 25 years, however, experiments with animal models have revealed that moderate exposures to hyperoxia (e.g., 30-60% O(2) for days to weeks) can also have profound effects on the developing respiratory control system that may lead to hypoventilation and diminished responses to acute hypoxia. This plasticity, which is generally inducible only during critical periods of development, has a complex time course that includes both transient and permanent respiratory deficits. Although the molecular mechanisms of hyperoxia-induced plasticity are only beginning to be elucidated, it is clear that many of the respiratory effects are linked to abnormal morphological and functional development of the carotid body, the principal site of arterial O(2) chemoreception for respiratory control. Specifically, developmental hyperoxia reduces carotid body size, decreases the number of chemoafferent neurons, and (at least transiently) diminishes the O(2) sensitivity of individual carotid body glomus cells. Recent evidence suggests that hyperoxia may also directly or indirectly impact development of the central neural control of breathing. Collectively, these findings emphasize the vulnerability of the developing respiratory control system to environmental perturbations.

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.

Respiratory plasticity after perinatal hypercapnia in rats

Environmental conditions during early life may have profound effects on respiratory control development. We hypothesized that perinatal hypercapnia would exert lasting effects on the mammalian hypercapnic ventilatory response, but that these effects would differ between males and females. Rats were exposed to 5% CO2 from 1 to 3 days before birth through postnatal week 2 and ventilation was subsequently measured by whole-body plethysmography. In both male and female rats exposed to perinatal hypercapnia, a rapid, shallow breathing pattern was observed for the first 2 weeks after return to normocapnia, but ventilation was unchanged. Acute hypercapnic ventilatory responses (3% and 5% CO2) were reduced 27% immediately following perinatal hypercapnia, but these responses were normal after 2 weeks of recovery in both sexes and remained normal as adults. Collectively, these data suggest that perinatal hypercapnia elicits only transient respiratory plasticity in both male and female rats. This plasticity appears similar to that observed after chronic hypercapnia in adult animals and, therefore, is not unique to development.