Optimal neuroprotection by erythropoietin requires elevated expression of its receptor in neurons.

Erythropoietin receptor (EpoR) binding mediates neuroprotection by endogenous Epo or by exogenous recombinant human (rh)Epo. The level of EpoR gene expression may determine tissue responsiveness to Epo. Thus, harnessing the neuroprotective power of Epo requires an understanding of the Epo-EpoR system and its regulation. We tested the hypothesis that neuronal expression of EpoR is required to achieve optimal neuroprotection by Epo. The ventral limbic region (VLR) in the rat brain was used because we determined that its neurons express minimal EpoR under basal conditions, and they are highly sensitive to excitotoxic damage, such as occurs with pilocarpine-induced status epilepticus (Pilo-SE). We report that (i) EpoR expression is significantly elevated in nearly all VLR neurons when rats are subjected to 3 moderate hypoxic exposures, with each separated by a 4-day interval; (ii) synergistic induction of EpoR expression is achieved in the dorsal hippocampus and neocortex by the combination of hypoxia and exposure to an enriched environment, with minimal increased expression by either treatment alone; and (iii) rhEpo administered after Pilo-SE cannot rescue neurons in the VLR, unless neuronal induction of EpoR is elicited by hypoxia before Pilo-SE. This study thus demonstrates using environmental manipulations in normal rodents, the strict requirement for induction of EpoR expression in brain neurons to achieve optimal neuroprotection. Our results indicate that regulation of EpoR gene expression may facilitate the neuroprotective potential of rhEpo.

Breathing at high altitude.

Acclimatization to long-term hypoxia takes place at high altitude and allows gradual improvement of the ability to tolerate the hypoxic environment. An important component of this process is the hypoxic ventilatory acclimatization (HVA) that develops over several days. HVA reveals profound cellular and neurochemical re-organization occurring both in the peripheral chemoreceptors and in the central nervous system (in brainstem respiratory groups). These changes lead to an enhanced activity of peripheral chemoreceptor and re-inforce the central translation of peripheral inputs to efficient respiratory motor activity under the steady low O(2) pressure. We will review the cellular processes underlying these changes with a particular emphasis on changes of neurotransmitter function and ion channel properties in peripheral chemoreceptors, and present evidence that low O(2) level acts directly on brainstem nuclei to induce cellular changes contributing to maintain a high tonic respiratory drive under chronic hypoxia.

Brain heparanase expression is up-regulated during postnatal development and hypoxia-induced neovascularization in adult rats.

Heparanase is an endo-beta-d-glucuronidase which specifically cleaves extracellular and cell surface heparan sulphates at intra-chain sites. Its enzymatic activity is strongly implicated in cell dissemination associated with tumor metastasis and inflammation. Indeed, heparanase gene is expressed in various tumors and its over-expression is correlated with increased tumor vascularity and metastatic potential of tumor cells. However, heparanase expression in non-invasive and non-immune tissue, including brain, has received less attention. Using RT-qPCR, western blot and histological analysis, we demonstrate in the adult rat that heparanase transcript is differentially expressed according to brain area, and that heparanase protein is mainly detected in neurons. Furthermore, we provide evidence that heparanase transcript and protein reach their greatest levels at early postnatal stages, in particular within the neocortex characterized by intensive structural plasticity. Using the in vitro model of PC12-induced neuronal differentiation, we suggest that developmental regulation of heparanase may coincide with axonal and dendritic pathfinding. At adulthood, we demonstrate that the increased heparanase transcript level correlates in the hippocampus with enhanced angiogenesis following repeated hypoxia exposures. Taken together, our results emphasize the potential importance of heparanase in brain homeostasis, both during development and adaptative responses to severe environmental challenges.

VEGF overexpression in the astroglial cells of rat brainstem following ozone exposure.

Ozone, a major photochemical pollutant, produces rapid damages in the pulmonary airway tract and in the central nervous system. This study focused on the neural mechanisms underlying the adaptive responses to an acute ozone exposure. Vascular endothelial growth factor (VEGF) is a factor associated with cellular recovery following brain injury. The aim of this study was to assess and localize the cellular expression of VEGF, since the central respiratory areas show a neuroplasticity in response to ozone. Adult rats were subjected to 0.5ppm ozone for 3h and then recovered for further 3h. The expression of VEGF was evaluated by immunocytochemistry in the central respiratory areas, i.e., the nucleus tractus solitarius (NTS) and the ventrolateral medulla (VLM). The data show a VEGF overexpression at the end of ozone exposure, which persisted during the 3-h recovery. Interestingly, using confocal analysis the bulk of VEGF labeling was observed in astroglial cell bodies and branches, while neuronal labeling was hardly noticed. Moreover, VEGF colocalized with IL-6 and TNFalpha in astrocytes closely apposed to blood vessel walls. The vasculature area was markedly increased (+58%) during post-ozone recovery. The data show that an acute ozone exposure affects primarily glial cells in the central nervous system. The VEGF up-regulation which persists after ozone exposure may contribute to brain repair and consecutive functional adaptations.

Prenatal nicotine alters maturation of breathing and neural circuits regulating respiratory control.

While perinatal nicotine effects on ventilation have been widely investigated, the prenatal impact of nicotine treatment during gestation on both breathing and neural circuits involved in respiratory control remains unknown. We examined the effects of nicotine, from embryonic day 5 (E5) to E20, on baseline ventilation, the two hypoxic ventilatory response components and in vivo tyrosine hydroxylase (TH) activity in carotid bodies and brainstem areas, assessed at postnatal day 7 (P7), P11 and P21. In pups prenatally exposed to nicotine, baseline ventilation and hypoxic ventilatory response were increased at P7 (+48%) and P11 (+46%), with increased tidal volume (p<0.05). Hypoxia blunted frequency response at P7 and revealed unstable ventilation at P11. In carotid bodies, TH activity increased by 20% at P7 and decreased by 48% at P11 (p<0.05). In most brainstem areas it was reduced by 20-33% until P11. Changes were resolved by P21. Prenatal nicotine led to postnatal ventilatory sequelae, partly resulting from impaired maturation of peripheral chemoreceptors and brainstem integrative sites.

Localization of L-DOPA uptake and decarboxylating neuronal structures in the cat brain using dopamine immunohistochemistry.

The present study examined dopamine-immunoreactive neuronal structures using immunohistochemistry in conjunction with an anti-dopamine antiserum, following injection of l-3,4-dihydroxyphenylalanine (L-DOPA) with or without an inhibitor of monoamine oxidase (Pargyline) in the cat brain. L-DOPA injection made it possible to detect dopamine immunoreactivity in presumptive serotonergic and noradrenergic cell bodies and axons. Weak to moderate dopamine immunoreactivity was observed in non-aminergic cells (possibly so-called "D" cells containing aromatic L-amino acid decarboxylase (AADC)) in several hypothalamic, midbrain, pontine and medullary nuclei. Intense dopamine immunoreactivity became visible in a large number of cells and axons (possibly containing AADC) with wide distribution in the brain following administration of L-DOPA with Pargyline. AADC is most likely active in cells and axons that take up L-DOPA, where it decarboxylates the L-DOPA to dopamine. However, newly synthesized dopamine in such cells is rapidly oxidized by monoamine oxidase.

Unexpected expression of orexin-B in basal conditions and increased levels in the adult rat hippocampus during pilocarpine-induced epileptogenesis.

Orexin-A (OX-A) and -B (OX-B) peptides present in the hippocampus are considered to be exclusively contained in fibers arising from hypothalamus neurons, which were established as the only source of orexins (OXs). Because OX-A is known to exert excitatory actions in the hippocampus, we hypothesized that the level of OXs targeted toward the hippocampus may be increased following status-epilepticus (SE)-induced epileptogenesis in the rat pilocarpine model of temporal lobe epilepsy. We found that tissue concentration of prepro-OX mRNA, which encodes for both peptides, rapidly decreased in the hypothalamus of rats having experienced pilocarpine-induced SE (Pilo-SE) followed by a reduced density of OX-A and OX-B immunopositive fibers arising from these neurons. By contrast, it was unexpected to detect within the hippocampus the presence of prepro-OX mRNA in basal conditions and to evidence its up-regulation during the 1- to 3-day period following Pilo-SE. The number of prepro-OX mRNA copies determined by real-time RT-PCR was approximately 50-fold lower in the hippocampus than that in the hypothalamus, precluding the use of in situ hybridization to localize the cells which synthesize the transcript within the hippocampus. The increase in prepro-OX mRNA level within the hippocampus was accompanied by the detection of OX-B-like immunoreactivity 2-3 days post-SE, not only in pyramidal neurons, granule cells and cell bodies resembling interneurons, but also in some astrocytes scattered throughout the hippocampus. The present data suggest that the gene encoding OXs can be activated in the hippocampus, which may play a role in the pathogenesis of epilepsy.

Dopamine transporters are involved in the onset of hypoxia-induced dopamine efflux in striatum as revealed by in vivo microdialysis.

Although many studies have revealed alterations in neurotransmission during ischaemia, few works have been devoted to the neurochemical effects of mild hypoxia, a situation encountered during life in altitude or in several pathologies. In that context, the present work was undertaken to determine the in vivo mechanisms underlying the striatal dopamine efflux induced by mild hypoxaemic hypoxia. For that purpose, the extracellular concentrations of dopamine and its metabolite 3,4-dihydroxyphenyl acetic acid were simultaneously measured using brain microdialysis during acute hypoxic exposure (10% O(2), 1h) in awake rats. Hypoxia induced a +80% increase in dopamine. Application of the dopamine transporters inhibitor, nomifensine (10 microM), just before the hypoxia prevented the rise in dopamine during the early part of hypoxia; in contrast the application of nomifensine after the beginning of hypoxia, failed to alter the increase in dopamine. Application of the voltage-dependent Na(+) channel blocker tetrodotoxin abolished the increase in dopamine, whether administered just before or after the beginning of hypoxia. These data show that the neurochemical mechanisms of the dopamine efflux may change over the course of the hypoxic exposure, dopamine transporters being involved only at the beginning of hypoxia.

Tyrosine hydroxylase expression and activity in the rat brain: differential regulation after long-term intermittent or sustained hypoxia.

Tyrosine hydroxylase, a hypoxia-regulated gene, may be involved in tissue adaptation to hypoxia. Intermittent hypoxia, a characteristic feature of sleep apnea, leads to significant memory deficits, as well as to cortex and hippocampal apoptosis that are absent after sustained hypoxia. To examine the hypothesis that sustained and intermittent hypoxia induce different catecholaminergic responses, changes in tyrosine hydroxylase mRNA, protein expression, and activity were compared in various brain regions of male rats exposed for 6 h, 1 day, 3 days, and 7 days to sustained hypoxia (10% O(2)), intermittent hypoxia (alternating room air and 10% O(2)), or normoxia. Tyrosine hydroxylase activity, measured at 7 days, increased in the cortex as follows: sustained > intermittent > normoxia. Furthermore, activity decreased in the brain stem and was unchanged in other brain regions of sustained hypoxia-exposed rats, as well as in all regions from animals exposed to intermittent hypoxia, suggesting stimulus-specific and heterotopic catecholamine regulation. In the cortex, tyrosine hydroxylase mRNA expression was increased, whereas protein expression remained unchanged. In addition, significant differences in the time course of cortical Ser(40) tyrosine hydroxylase phosphorylation were present in the cortex, suggesting that intermittent and sustained hypoxia-induced enzymatic activity differences are related to different phosphorylation patterns. We conclude that long-term hypoxia induces site-specific changes in tyrosine hydroxylase activity and that intermittent hypoxia elicits reduced tyrosine hydroxylase recruitment and phosphorylation compared with sustained hypoxia. Such changes may not only account for differences in enzyme activity but also suggest that, with differential regional brain susceptibility to hypoxia, recruitment of different mechanisms in response to hypoxia will elicit region-specific modulation of catecholamine response.

Central and peripheral changes in catecholamine biosynthesis and turnover in rats after a short period of ozone exposure.

We investigated in rat the effects of ozone exposure (0.7 ppm) for 5 h on the catecholamine biosynthesis and turnover in sympathetic efferents and various brain areas. For this purpose, the activity of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, was assessed in superior cervical ganglia and in two major noradrenergic cell groups, A2 and A6 (locus coeruleus). Tyrosine hydroxylase activity was estimated in vivo by measuring the accumulation of l-dihydroxyphenylalanine after pharmacological blockade of L-aromatic acid decarboxylases by NSD-1015 (100 mg/kg i.p.). The catecholamine turnover rate was measured after inhibition of tyrosine hydroxylase by alpha-methyl-para-tyrosine (AMPT, 250 mg/kg, i.p., 2.5 h) in peripheral sympathetic target organ (heart and lungs) as well as in some brain catecholamine terminal areas (cerebral cortex, hypothalamus and striatum). Ozone caused differential effects according to the structure. Catecholamine biosynthesis was stimulated in superior cervical ganglia (+44%, P < 0.05) and caudal A2 subset (+126%, P < 0.01), whereas catecholamine turnover was increased in heart (+183%, P < 0.01) and cortex (+22%, P < 0.05). On the other hand, catecholamine turnover was inhibited in lungs (-53%, P < 0.05) and striatum (-24%, P < 0.05). A brief exposure to ozone, at a concentration chosen to mimic pollution level encountered in urban areas, can modulate catecholamine biosynthesis and utilization rate in the sympathetic and central neurones.

Behavioral and neurochemical effects induced by subchronic combined exposure to toluene at 40 ppm and noise at 80 dB-A in rats.

We investigated whether exposure to noise, in addition to its well-known potentiating effect on toluene-induced ototoxicity, may also exacerbate behavioral disturbances and brain neurochemical alterations produced by subchronic exposure to low toluene concentration. To test this hypothesis, we evaluated whether subchronic combined exposure (16 weeks, 104 h per week) to noise at 80 dB-A and toluene at 40 ppm potentiates the recently reported neurotoxic effects of subchronic exposure to 40 ppm toluene. Locomotor and rearing activities, sensitization to narcosis induced by acute toluene at high concentration, and tyrosine and tryptophan hydroxylase activities in the caudate-putamen and hippocampus were investigated in both male and female rats. Our results confirm that subchronic exposure to 40 ppm toluene significantly decreases rearing activity and leads to a sensitization to toluene-induced narcosis, as evaluated by loss of righting reflex, but fails to demonstrate any adverse effect of noise, alone or in combination with toluene. Given that toluene has addictive properties, the lack of potentiating behavioral and neurochemical effect of noise is discussed with regards to a recent study that has shown that methamphetamine neurotoxicity is potentiated by exposure to loud noise.

Breathing pattern and hypoxic sensitivity during ageing in a new model of obesity-resistant rat.

The Lou/C rat, an inbred strain of Wistar origin, is presented both as an obesity-resistant rat and as a relevant model of successful ageing. In the present study, we examined throughout ageing the breathing pattern and the ventilatory response to hypoxia in Lou/C and age-matched Wistar rats. Ventilation was assessed by whole body plethysmography in resting condition and during a hypoxic challenge (10% O(2)) at 2, 6, 12 and 24 months of age. Lou/C rats maintained constant their resting ventilation rate throughout ageing while Wistar rats tended to decrease their ventilation with advancing age. Elderly Lou/C as well as Wistar rats increased their frequency rather than their tidal volume in response to hypoxia. As Lou/C rats did not develop obesity, ageing per se surpasses the effect of fat accumulation and prevents old rats to mobilise their tidal volume in response to hypoxia.

Neurochemical perspectives on the control of breathing during sleep.

A specific depression of minute ventilation occurs during sleep in normal subjects. This sleep-related ventilatory depression is partially related to mechanical events and upper airway atonia but some data also indicate that it is likely to be centrally mediated. This paper reviews the anatomical and neurochemical connections between sleep/wake- and respiratory-related areas in an attempt to identify the potential implication of sleep-related neurochemicals (serotonin, catecholamines, GABA, acetylcholine) in the sleep-related hypoventilation. The review of available data suggests that the sleep-related ventilatory depression depends upon the enhanced GABAergic activity together with a loss of suprapontine influence depending on the cessation of activity of the reticular formation. During REM sleep, an additional inhibitory activity emerges from the pontine cholinergic neurons, which contributes to the breathing irregularities and the associated depression of minute ventilation and ventilatory response to chemical stimuli. This model may contribute to a better understanding of the neurochemical environment of respiratory neurons during sleep, which remains a question of importance regarding the numerous pathological states that are linked to specific perturbations of breathing control during sleep.

Behavioral and neurochemical effects induced by subchronic exposure to 40 ppm toluene in rats.

Chronic toluene inhalation at concentrations above occupational exposure limits (e.g., 100 ppm; NIOSH) has been repeatedly shown to induce neurotoxic effects. In contrast, although few clinical and experimental data are available on the effects of toluene exposure at concentrations below occupational exposure standards, some of these data may support adverse effects of long-term exposure to low toluene concentrations. To test this hypothesis, we investigated the neurobehavioral and neurochemical effects of 40 ppm inhaled toluene in a rat model of 16-week subchronic exposure, examining locomotor and rearing activities; adaptation/sensitization to narcosis produced by acute exposure to toluene at high concentration; and tyrosine hydroxylase and tryptophan hydroxylase activities, and dopamine (DA) and serotonin (5-HT) turnovers in the caudate-putamen, nucleus accumbens, hippocampus, prefrontal cortex, and cerebellum. Our results mainly show that subchronic exposure to 40 ppm toluene significantly resulted in a sensitization to toluene-induced narcosis, a decrease in rearing activity, and alterations in DA and 5-HT transmissions. This demonstrates that subchronic toluene exposure at a low concentration may lead to adverse changes in neurobehavioral and neurochemical functioning, and further questions in a public health perspective the actual neurotoxic potential of toluene and other organic compounds, because deficits in functioning are generally viewed as precursors of more serious adverse effects.

Sympathetic and brain monoaminergic regulation of energy balance in obesity-resistant rats (Lou/C).

In contrast to the Wistar rat, the Lou/C rat does not develop obesity with age. To determine the role of sympathetic output and brain monoamines in the different energy balance of Lou/C rats, the monoamine contents and activity of rate-limiting enzymes in catecholamine and serotonin biosynthesis were assessed in brain structures involved in energy balance regulation, i.e., brainstem noradrenergic (A6, A5, A2) and serotonergic cell groups (dorsal raphe, and median raphe), and two hypothalamic nuclei (ventromedial nucleus and paraventricular nucleus). In vivo tyrosine hydroxylase activity and noradrenaline content were measured in sympathetic target organs storing fuel substrates, the liver, white adipose and brown adipose tissues in the Lou/C rat and compared to the Wistar rat. In Lou/C rats, indirect calorimetric measurements showed a higher energy expenditure despite a reduced food intake. The Lou/C rat displayed selective monoamine features. The catecholaminergic activity was higher in the white adipose tissue and interscapular brown adipose tissue but lower in the liver and adrenal gland of Lou/C rats. The noradrenergic activity in A2, A6 and ventromedial nucleus, and the serotonergic pattern in A6, dorsal raphe and median raphe were lower in Lou/C. The metabolic particularities of Lou/C rats are associated with (i) a selectively enhanced sympathetic activity restricted to the white adipose tissue and brown adipose tissue, (ii) a reduced noradrenergic activity in selective brainstem and hypothalamic areas, which control the energy expenditure and food intake.

Biological, hormonal, and psychological parameters in professional soccer players throughout a competitive season.

We examined changes in the haematological, metabolic, immunological, hormonal, and psychological fields using selected variables in 20 professional soccer players. over the course of a competitive season. The team performance was assessed by computing the winning percentage. A symptom checklist was used to assess the severity of upper respiratory tract infections. A high-intensity training programme induced a significant increase in cortisol and uric acid concentrations. Despite lower glutamine concentrations than the normal range throughout the study, infection occurred only in two of the soccer players. Moreover, the levels of immunological factors IgA, IgG, and IgM, and the haematological parameters were unaltered. Subsequent decreased performance coincided with changes in specific mood states of the team. Our results show some alterations on the metabolic, hormonal, and psychological variables over the five fields studied, suggesting that combined psychological and physiological changes during training are of primary interest to monitor the training stress in relation to performance in team sport.