The effect of prenatal nicotine exposure on PDGFR-mediated anti-apoptotic mechanism in the caudal brainstem of developing rat

Maternal cigarette smoking is a major risk factor for sudden infant death syndrome (SIDS); however, the mechanism underlying this association is currently unknown. Prenatal nicotine exposure is accompanied by a decrease in the magnitude of hypoxic ventilatory depression, the component of hypoxic ventilatory response that activates the PDGF-β receptor (PDGFR) and its downstream anti-apoptotic cascade in the caudal brainstem (CB) of developing rats. In this study, we evaluated the effect of prenatal nicotine exposure on PDGFR activation and the subsequent activation of downstream anti-apoptotic processes through the Akt/BAD pathway. The 5-day timed-pregnant Sprague–Dawley rats underwent surgical implantation of an osmotic pump containing either normal saline (control) or a solution of nicotine tartrate. The CB was harvested from 5-day-old rat pups (n = 8–10 for each time point) in each group after exposure to normoxia or hypoxic challenges with 10% O₂ for 5, 15, 30, 60 or 120 min. Immunoprecipitation and immunoblots of CB lysates revealed phosphorylation of PDGFR, Akt and BAD-136 during hypoxia in control pups. Prenatal nicotine exposure was associated with attenuation of these responses at all time points. Analysis of an early apoptotic marker in the CB revealed that activation of cleaved caspase-3 occurred only at 120 min of hypoxic exposure in the control. Prenatal nicotine exposure accelerated this response, causing early activation at 30 and 60 min. We conclude that prenatal nicotine exposure attenuates the phosphorylation of PDGFR, Akt and Bad-136 during hypoxia in the CB of developing rats. This modulation of anti-apoptotic cascades accelerates activation of the early apoptotic marker. We speculate that prenatal nicotine exposure affects apoptosis in the CB of developing animals and may increase the vulnerability of neural cells in the respiratory control area, a process that may underlie the association between maternal smoking and SIDS.     

Prenatal nicotine exposure alters the response of the mouse in vitro respiratory rhythm to hypoxia

Prenatal nicotine exposure is associated with deficiencies in the ability to respond to life threatening stressors such as hypoxia. Although many of these deficiencies appear to originate from defects in the brainstem respiratory network, the specific effects of prenatal nicotine exposure on the brainstem respiratory network are not well understood. We have tested the effects of prenatal nicotine exposure on the respiratory rhythm using an in vitro mouse brainstem slice preparation containing the pre-Bötzinger Complex, a region of the ventral respiratory group that is the postulated site of inspiratory rhythm generation. We found that nicotine exposure during pre- and early postnatal development led to a lower frequency of baseline fictive respiratory discharges from rhythmic slices and a reduction in the ability of the slice to maintain a respiratory rhythm during exposure to severe hypoxia compared to controls. These impairments of the central respiratory rhythm could potentially affect the ability to survive a period of exposure to severe hypoxia in vivo.     

Prenatal nicotine exposure alters glycinergic and GABAergic control of respiratory frequency in the neonatal rat brainstem-spinal cord preparation

Bath application of GABA-A receptor agonists in neonatal rat brainstem-spinal cord preparations (BSSC) reduces respiratory frequency, an effect that is enhanced by prenatal nicotine exposure. Here we test the hypothesis that these effects can be reproduced by microinjection of GABAergic and glycinergic agonists into the pre-Botzinger complex region (PBC). We recorded the activity of phrenic motor axons from the fourth cervical ventral root in 1-3 days old BSSC that were exposed to either nicotine (6 mg/(kg day)) or saline prenatally. Microinjection of glycine or muscimol into the PBC caused abrupt, reversible apnea in all experiments. Apnea duration with glycine averaged 50.3+/-5 s in saline-exposed (N=12), and 95.7+/-9.9 s in nicotine-exposed (N=12) neonates (P<0.001). Apnea duration with muscimol averaged 51+/-5.1 s in saline-exposed (N=10), and 86+/-10.6 s in nicotine-exposed (N=12) neonates (P<0.05). These data show that prenatal nicotine exposure alters development of central ventilatory control, and that neurons in the PBC region are involved.     

Acute hypoxic ventilatory response and exercise-induced arterial hypoxemia in men and women

Recent studies claim a higher prevalence of exercise-induced arterial hypoxemia (EIAH) in women relative to men and that diminished peripheral chemosensitivity is related to the degree of arterial desaturation during exercise in male endurance athletes. The purpose of this study was to determine the relationship between the acute ventilatory response to hypoxia (AHVR) and EIAH and the potential influence of gender in trained endurance cyclists and untrained individuals. Healthy untrained males (n = 9) and females (n = 9) and trained male (n = 11) and female (n = 10) cyclists performed an isocapnic AHVR test followed by an incremental cycle test to exhaustion. Oxyhemoglobin saturation (Sa(O(2)) was lower in trained men (91.4 +/- 0.9%) and women (91.3 +/- 0.9%) compared to their untrained counterparts (94.4 +/- 0.8% versus 94.3 +/- 0.7%) (P < 0.05). AHVR and maximal O(2) consumption were related for all subjects (r = -0.46), men (r = -0.45) and women (r = -0.53) (P < 0.05) but AHVR was unrelated to Sa(O(2)) for any groups (P > 0.05). We conclude that resting AHVR does not have a significant role in maintaining Sa(O(2)) during sea-level maximal cycle exercise in men or women.     

Prenatal hyperoxia blunts the hypoxic ventilatory chemosensitivity of the 1-day old chicken hatchling

Hypoxia sustained during the prenatal period lowers the ventilatory (V˙(E)) response to hypoxia of the newborn. This phenomenon probably results from a disturbance in the normal development of the peripheral chemoreceptors, as shown to be the case postnatally after sustained period of low or high oxygen. To test the possibility that prolonged prenatal hyperoxia may have a similar effect, the breathing pattern and the V˙(E) responses to hypoxia or hypercapnia were measured by a modification of the barometric technique in 1-day old chicken hatchlings exposed to 40% O2 or 60% O2 (N=16 each) during the last week of incubation (hatching included), and in controls incubated in normoxia (N=16). During air breathing and moderate hypoxia (15% O2), neither group differed from controls. However, the V˙(E) response to 10% O2 was reduced to less than half normal in both groups of prenatal-hyperoxia hatchlings. The hypoxic drop in oxygen consumption V˙(O2) was more marked than in controls, which probably helped to limit the degree of hypoxemia and to sustain the hyperventilation (increase in V˙(E)-V˙(O2) ratio). The V˙(E) response to hypercapnia was almost normal, suggesting that there was no mechanical limitation on V˙(E). The degree of blunting in the V˙(E) response to hypoxia was very similar to that previously measured in hatchlings exposed to hypoxia during the last week of incubation. The results support to the view that sustained changes in oxygenation during the prenatal period reduce the newborn's V˙(E) response to hypoxia probably because of a major dysfunction of the carotid bodies.     

Neonatal maternal separation and neuroendocrine programming of the respiratory control system in rats

Neonatal maternal separation (NMS) disrupts central nervous system (CNS) development. Although the consequences of NMS are typically linked with abnormal psychological and behavioural development, there is growing evidence indicating that NMS affects maturation of the respiratory control system. This review discusses results from animal studies in which ventilatory responses to chemical stimuli were measured either in unrestrained rats or in an anesthetised preparation. Data show that NMS interferes with development of ventilatory chemoreflexes in a persistent, sex-specific fashion by affecting both the central and peripheral components of the respiratory control system. NMS likely disrupts the balance between inhibitory (GABAergic) and excitatory modulation within key integrative structures involved in respiratory regulation. Because enhancement of ventilatory chemoreflexes is a hallmark of several cardio-respiratory disorders in humans these results raise important questions concerning the impact of the neonatal of environment on the emergence of respiratory disease related to neural control dysfunction later in life.     

Dopaminergic mechanisms of neural plasticity in respiratory control: transgenic approaches

Data supporting the hypothesis that dopamine-2 receptors (D(2)-R) contribute to time-dependent changes in the hypoxic ventilatory response (HVR) during acclimatization to hypoxia are briefly reviewed. Previous experiments with transgenic animals (D(2)-R 'knockout' mice) support this hypothesis (J. Appl. Physiol. 89 (2000) 1142). However, those experiments could not determine (1) if D(2)-R in the carotid body, the CNS, or both were involved, or (2) if D(2)-R were necessary during the acclimatization to hypoxia versus some time prior to chronic hypoxia, e.g. during a critical period of development. Additional experiments on C57BL/6J mice support the idea that D(2)-R are critical during the period of exposure to hypoxia for normal ventilatory acclimatization. D(2)-R in carotid body chemoreceptors predominate under control conditions to inhibit normoxic ventilation, but excitatory effects of D(2)-R, presumably in the CNS, predominate after acclimatization to hypoxia. The inhibitory effects of D(2)-R in the carotid body are reset to operate primarily under hypoxic conditions in acclimatized rats, thereby optimizing O(2)-sensitivity.     

Age-dependent involvement of ATP-sensitive potassium channel Kir6.2 in hypoxic ventilatory depression of mouse

In order to examine whether ATP-sensitive potassium channel Kir6.2 is involved in hypoxic ventilatory responses, especially in hypoxic ventilatory depression (HVD), and whether the involvement shows age-dependence, we measured the hypoxic ventilatory response in the Kir6.2-knockout mouse (Kir6.2-/-) in an unanesthetized unrestrained state by means of pressure plethysmography in the 2nd and 4th postnatal weeks, and compared the response with that of its wild type counterpart, the C57BL6/J mouse. In the 4th postnatal week, but not in the 2nd week, the Kir6.2-/- exhibited a larger and longer initial augmentation and a weaker subsequent depression of respiratory frequency and ventilation in response to hypoxia (FIO(2)=0.12 in N(2)). These findings suggest that Kir6.2 is involved in HVD of the mouse at a certain point during the postnatal development.     

Ventilatory chemosensitivity and thermogenesis of the chicken hatchling after embryonic hypercapnia

Hypoxia during incubation results in hatchlings with a reduced thermogenic capacity and a blunted ventilatory (V (E)) chemosensitivity (Szdzuy, K., Mortola, J.P., 2007b. Ventilatory chemosensitivity of the 1-day-old chicken hatchling after embryonic hypoxia. Am. J. Physiol. (Regul. Integr. Comp. Physiol.) 293, R1640-R1649). We asked if similar effects occurred with embryonic hypercapnia, that is, with a non-hypoxic sustained stimulation of the chemoreceptors. White Leghorn chicken eggs were incubated at 38 degrees C either in air (controls, C) or in 4% CO(2) from embryonic day 5 (4% CO(2)), hatching included. The 4% CO(2) embryos hatched about 12h later than C, with similar body weight. On the day of hatching the thermogenic capacity, assessed from the changes in oxygen consumption (.V(O2)) during 1h at 30 degrees C, increased from the early (about 3h old) to the late hours (about 20 h old), and was similar between 4% CO(2) and C. Ventilatory chemosensitivity was evaluated from the changes in (.V(E)) and in ventilatory equivalent (.V(E)/.V(O2)) during acute hypoxia (15 and 10% O(2), 20 min each) or hypercapnia (2 and 4% CO(2), 20 min each). Both at the early and late hours (.V(E)) chemosensitivity was lower in 4% CO(2) than in C. The .V(E)/.V(O2) responses of 4% CO(2) in hypoxia and hypercapnia averaged, respectively, about 45 and 60% of C. A separate set of eggs incubated in 2% CO(2) gave results qualitatively intermediate between C and 4% CO(2). We conclude that prenatal hypercapnia does not compromise the newborn's thermogenesis, but, like hypoxia, affects the development of respiratory control, resulting in a blunted chemosensitivity.     

Effects of dexmedetomidine on respiratory mechanics and control of breathing in normal rats

Dexmedetomidine is a highly selective and specific alpha(2)-adrenergic agonist, with sedative, analgesic, and sympatholytic activities. The aim of the present study was to define the effects of DMED in respiratory mechanics in normal rats. In addition, lung morphometry was studied to determine whether the physiological changes reflected underlying morphological changes defining the sites of action of dexmedetomidine. Arterial blood gases were also determined. Twelve adult Wistar rats were randomly assigned to two groups of six animals each: PENTO and DMED. In PENTO group animals were sedated (diazepam, 5mg, i.p.) and anaesthetised with pentobarbital sodium (20mgkg(-1) i.p.). The rats of the DMED group received dexmedetomidine (250mugkg(-1) i.p. followed by intravenous infusion of 0.5mugkg(-1)h(-1)). In spontaneously breathing rats, minute ventilation, respiratory frequency, and neuromuscular inspiratory drive were lower in dexmedetomidine group, which also presented hypercapnia, whereas tidal volume, inspiratory, expiratory, and total respiratory cycle times were higher in dexmedetomidine group compared to the PENTO group. During mechanical ventilation, respiratory mechanical parameters were similar in both groups. These findings were supported by the absence of histological changes. In conclusion, under the conditions studied, dexmedetomidine did not change respiratory mechanical parameters and lung histology, but induced ventilatory depression.     

Prenatal nicotine exposure and development of nicotinic and fast amino acid-mediated neurotransmission in the control of breathing

There is mounting evidence that neonatal animals exposed to nicotine in the prenatal period exhibit a variety of anatomic and functional abnormalities that adversely affect their respiratory and cardiovascular control systems, but how nicotine causes these developmental alterations is unknown. The principle that guides our work is that PNE impairs the ability of nicotinic acetylcholine receptors (nAChRs) to modulate the pre-synaptic release of both inhibitory (particularly GABA) and excitatory (glutamate) neurotransmitters, leading to marked alterations in the density and/or function of receptors on the (post-synaptic) membrane of respiratory neurons. Such changes could lead to impaired ventilatory responses to sensory afferent stimulation, and altered breathing patterns, including central apneic events. In this brief review we summarize the work that lead to the development of this hypothesis, and introduce some new data that support and extend it.     

The role of the pontine respiratory complex in the response to intermittent hypoxia

These experiments were designed to determine the effects of EEG state on the response of rats to intermittent hypoxia and to test the hypotheses that short-term potentiation (STP) and ventilatory long term facilitation (vLTF) are state dependent; and that neurons with NMDA receptors in the dorso-ventral pontine respiratory group (dvPRG) modulate the development of STP and vLTF in rats. Low-doses of urethane anaesthesia (<1.3 g/kg) that do not cause significant respiratory depression or reductions in sensitivity to hypoxia result in cycling between EEG states that superficially resemble wake and slow wave sleep in rats and are accompanied by changes in breathing pattern that closely resemble those seen when unanaesthetized rats cycle between wake and SWS. When changes between these states were accounted for, intermittent, poikilocapnic hypoxia did not produce a significant vLTF. However, there was a persistent STP of tidal volume and vLTF did develop after blockade of NMDAr in the region of the PBrKF complex by microinjection of MK-801. Blockade of NMDA-type glutamate receptor-mediated processes in the dorsal pons also caused animals to cycle into State III, but did not alter the response to either continuous or intermittent hypoxia indicating that the response to hypoxia was not state dependent. This shows that neurons in the region of the PRG inhibit STP and vLTF, but no longer do so if PRG NMDA receptor activation is blocked.     

Prenatal and lactation nicotine exposure affects morphology and function of brown adipose tissue in male rat offspring

The aim of this study was to investigate the effects of prenatal and lactation nicotine exposure on the morphology and function of brown adipose tissue (BAT) in male rat offspring. We conducted a morphological assay and gene expression study of interscapular BAT (iBAT) in male rat offspring. The male offspring from nicotine-exposed dams exhibited higher body weight and iBAT weight. Hematoxylin and eosin staining and transmission electron microscopy showed that iBAT from nicotine-exposed male offspring presented a “whitening” phenotype characterized by lipid droplet accumulation and impaired mitochondria with a randomly oriented and fractured cristae. The expression of the iBAT structure and function-related genes all decreased in nicotine-exposed male offspring. These data indicate that prenatal and lactation nicotine exposure affects morphology and function of iBAT in male rat offspring.     

Functional genomics and proteomics in control of breathing

New genomic and proteomic techniques offer remarkable promise as tools to address longstanding questions regarding molecular mechanisms involved in the control of breathing. Here, these techniques are described. Additionally, recent examples of the application of these and related molecular techniques are provided-particularly including observations regarding the role of S-nitrosylation signaling reactions in regulating ventilatory responses.     

Intermittent hypoxia and plasticity of respiratory chemoreflexes in metamorphic bullfrog tadpoles

We tested the hypothesis that intermittent hypoxia elicits plasticity in respiratory chemoreflexes in bullfrog tadpoles. Metamorphic tadpoles (Taylor-Kollros stages XVI-XX) were subjected to intermittent hypoxia (PW(O(2))=45 Torr; 12 h/day) or constant normoxia (PW(O(2))=156 Torr) for 2 weeks before ventilatory responses to hypoxia and hypercarbia were measured. Buccal pressure changes were used to quantify the frequency and amplitude of movements associated with gill and lung ventilation. Morphometric assessment showed that intermittent hypoxia delayed development in comparison with controls. Oxygen consumption was enhanced in tadpoles subjected to intermittent hypoxia; however, this increase was not sufficient to affect basal ventilatory activity or the hypoxic ventilatory response. During acute hypercarbic exposure, tadpoles subjected to intermittent hypoxia showed (1) a greater decrease in gill ventilation frequency and (2) a greater increase in lung ventilation frequency than tadpoles maintained under control conditions. We conclude that intermittent hypoxia augments the responsiveness to hypercarbia, thereby promoting lung ventilation when animals face this stimulus. This manifestation of respiratory plasticity may reflect uncoupling between physiological and morphological development in the bi-modally breathing bullfrog tadpole.     

Effect of hypercapnia on sleep and breathing in unanesthetized cats

OBJECTIVES: In this study, we looked at the effect of hypercapnia on sleep architecture and breathing. We characterized the effect of hypercapnia on duration, frequency, and latency of NREM and REM sleep. We described state-specific patterns of breathing as well. This study is relevant to understand possible treatments for sleep disordered breathing. METHODS: Four cats were studied during 3-hour sessions while breathing 0%, 2%, 4%, and 6% CO2 in room air. Each animal was studied 4 days per week for a period of 4 weeks. The animals breathed through a tube inserted into the trachea via a surgically created fistula. Respiration was measured using pneumotachography, and brain activity was recorded from implanted electrodes to discriminate states of sleep and wakefulness. RESULTS: Two percent inspired CO2 increased sleep duration and decreased time awake. On the other hand, 6% CO2 induced a worsening of sleep parameters: the duration of wakefulness increased by 24.2%. As a response to hypercapnia, tidal volume (V(T)), minute ventilation (V(E)), and respiratory effort (V(T)/T(I)) increased proportionally in all states with increasing levels of CO2. With 6% CO2, breathing tended to become similar in all states of consciousness. All breathing parameters converged towards a common value independently of the states. CONCLUSION: We conclude that a mild hypercapnic stimulus can stimulate both breathing and sleep, and it may be useful in treatment of sleep disordered breathing.     

Prenatal stress affects the rate of sexual maturation and attractiveness in bank voles

Field studies reveal that bank vole females' mobility and aggression increase during pregnancy. Here we investigated the reaction of pregnant females to social stress evoked by short but frequent meetings with another female at the same stage of pregnancy. The stress neither evoked pregnancy termination nor affected pregnancy duration but had a long-term effect on the reproductive activity of the offspring. Prenatal stress reduced the rate of sexual maturation of voles as estimated at the age of 20 days. Uterine weights of prenatally stressed females and testes weights of prenatally stressed males were significantly lower than in offspring born to nonstressed mothers. Olfactory signals are known to be important in the sexual preferences of bank voles. Adult prenatally stressed females were more attractive to other adult females than were nonstressed animals. For bank vole males, however, prenatal stress decreased the attractiveness of females; adult males selected nonstressed females over stressed partners, by odor. This study shows that prenatal conditions evoked by short but frequent encounters with another pregnant female lead to delayed puberty in females and males, and decrease sexual attractiveness in adult offspring. These two negative effects may significantly limit the reproduction of prenatally stressed offspring.     

Sex steroid hormones and the neural control of breathing

We review evidence that sex steroid hormones including estrogen, progesterone and testosterone are involved in the central neural control of breathing. Sex hormones may exert their effects on respiratory motoneurons via neuromodulators, in particular, the serotonergic system. Recent studies have shown that levels of serotonin (5HT) in the hypoglossal and phrenic nuclei are greater in female than in male rats. Serotonin-dependent plasticity in hypoglossal and phrenic motor output also differs in male and female rats. Changing levels of gonadal hormones throughout the estrus cycle coincide with changing levels of 5HT in respiratory motor nuclei, and gonadectomy in male rats results in a decrease in 5HT-dependent plasticity in respiratory motor output. We speculate that sex steroid hormones are critically involved in adaptations in the neural control of breathing throughout life, and that decreasing levels of these hormones with increasing age may have a negative influence on the respiratory control system in response to challenge.