Interactions between postnatal sustained hypoxia and intermittent hypoxia in the adulthood to alter brainstem structures and respiratory function.

Neural plasticity is defined as a persistent change in the morphology and/or function based on prior experiences. Plasticity is well evident when the triggering experience occurs early in life, but in the case of respiratory control plasticity, it also can be triggered in adult life. We have combined a 10 days postnatal hypoxic (PH) (0-10 days of age;11% O(2)) and a 15 days intermittent hypoxia (IH) exposures in the adulthood (90-105 days of age; 5% O(2), 40 s/20% O(2), 80 s; 8 h/day) to test if early PH interacts with IH of the adulthood to generate detrimental plastic changes. After recording of ventilatory parameters, the brains were studied immunocytochemically for localization of the organization pattern of non-phosphorylated subunit of neurofilament H (NFH) and tyrosine hydroxylase (TH) expression in the nucleus tractus solitarius (Sol) and caudal (CVL) and rostral ventrolateral reticular (RVL) nuclei, areas related to central cardio-respiratory regulation. In comparison to control, PH male rats (but not females) at 1 month of age hyperventilated at rest, in response to moderate hypoxia (12% O(2)) and 5% CO(2), the effect being due to increased tidal volume. At 3.5 months sex differences in ventilation disappeared and it was indistinguishable between control and PH. IH tended to decrease ventilation in both control (C) and PH animals. PH augmented PENH values in air and in hypoxic conditions when compared with C group. IH in both groups, tended to decrease the PENH value, being statistically different in PH+IH. Results also show an increment of disorganization of NFH-positive labeled structures at the level of Sol and CVL/RVL nuclei in PH, IH and HP+HI groups. PH rats showed differences in the number of TH-positive neurons at the level of CVL/RVL nuclei, which was increased in the PH and PH+IH groups with respect to C one. In conclusion, PH alters the central morpho-physiological organization and the catecholaminergic components of cardio-respiratory nuclei, whose effects were enhanced after a period of IH in the adulthood.

Co-localization of nitric oxide synthase and choline acetyltransferase in the brain of the goldfish (Carassius auratus).

This work investigates the nitrergic and cholinergic systems in the brain and spinal cord of the goldfish (Carassius auratus). We studied the immunohistochemical localization of antibodies against the neuronal nitric oxide synthase (nNOS) and choline acetyltransferase (ChAT) by bright-field and confocal microscopy. Nitrergic and cholinergic cells were segregated within the telencephalon, in both dorsal and ventral areas, and co-distributed in some nuclei of the diencephalon, mesencephalon, rhombencephalon, and spinal cord. Double-labeling experiments revealed nNOS/ChAT-positive cells in (1) the diencephalon: the preoptic and suprachiasmatic nuclei, the habenula, the dorsal thalamus, and the nucleus of the medial longitudinal fasciculus; (2) the mesencephalon: the optic tectum, the mesencephalic portion of the trigeminal nucleus, the oculomotor and trochlear nuclei, and the Edinger-Westphal nucleus; and (3) the rhombencephalon: the secondary gustatory nucleus, the nucleus isthmi, the lateral lemniscus nucleus, the cerebellum, the reticular formation, different nuclei of the octaval column, the motor zone of the vagal lobe, and the trigeminal, facial, abducens, glosso-pharyngeal, vagal, and hypobranchial motor nuclei. Double-labeled cells were also observed in the spinal motor column. The percentage of double-labeled cells was different in each studied nucleus, indicating a selective distribution pattern. Because double-labeled cells were more abundant in those nuclei involved with sensory and motor physiological processes, we suggest the involvement of both nitric oxide and acetylcholine in these neural functions in fish.

Respiratory response to systemic inhibition of the Na+/H+ exchanger type 3 in intact rats.

The Na+/H+ exchangers (NHEs) are a family of antiporters involved in the maintenance of neural steady-state intracellular pH. The NHE3 seems to be the predominant subtype in central chemosensitive cells. We aimed to analyze the effect of a selective NHE3 inhibition on the respiratory pattern in spontaneously breathing rats with intact vagi. Rats were intravenously infused for 10 min with the selective NHE3 inhibitor AVE1599 (Aventis Pharma Deustchland, 0.5 and 2 mg/kg) or with phosphate buffer. Whole-body plethysmography was used to monitor breathing pattern before, during, and up to 30 min after the drug infusion. Immunohistochemistry for the c-Fos protein was performed in the animal brains and c-Fos-positive cells were counted along the brainstem. Selective NHE3 inhibition induced a significant increase in the respiratory frequency and in the number of c-Fos immunopositive cells in the lateral parabrachial nucleus, the pre-Bötzinger complex and a rostral extension of the retrotrapezoid nucleus/parapyramidal region (p<0.05, ANOVA). We conclude that systemic administration of AVE1599 increases respiratory frequency and activates ponto-medullary areas implicated in the central control of breathing and chemoreception.

Prenatal activation of 5-HT2A receptor induces expression of 5-HT1B receptor in phrenic motoneurons and alters the organization of their premotor network in newborn mice.

In newborn mice of the control [C3H/HeJ (C3H)] and monoamine oxidase A-deficient (Tg8) strains, in which levels of endogenous serotonin (5-HT) were drastically increased, we investigated how 5-HT system dysregulation affected the maturation of phrenic motoneurons (PhMns), which innervate the diaphragm. First, using immunocytochemistry and confocal microscopy, we observed a 5-HT(2A) receptor (5-HT(2A)-R) expression in PhMns of both C3H and Tg8 neonates at the somatic and dendritic levels, whereas 5-HT(1B) receptor (5-HT(1B)-R) expression was observed only in Tg8 PhMns at the somatic level. We investigated the interactions between 5-HT(2A)-R and 5-HT(1B)-R during maturation by treating pregnant C3H mice with a 5-HT(2A)-R agonist (2,5-dimethoxy-4-iodoamphetamine hydrochloride). This pharmacological overactivation of 5-HT(2A)-R induced a somatic expression of 5-HT(1B)-R in PhMns of their progeny. Conversely, treatment of pregnant Tg8 mice with a 5-HT(2A)-R antagonist (ketanserin) decreased the 5-HT(1B)-R density in PhMns of their progeny. Second, using retrograde transneuronal tracing with rabies virus injected into the diaphragm of Tg8 and C3H neonates, we studied the organization of the premotor network driving PhMns. The interneuronal network monosynaptically connected to PhMns was much more extensive in Tg8 than in C3H neonates. However, treatment of pregnant C3H mice with 2,5-dimethoxy-4-iodoamphetamine hydrochloride switched the premotoneuronal network of their progeny from a C3H- to a Tg8-like pattern. These results show that a prenatal 5-HT excess affects, via the overactivation of 5-HT(2A)-R, the expression of 5-HT(1B)-R in PhMns and the organization of their premotor network.

Distribution of NADPH-diaphorase and nitric oxide synthase reactivity in the central nervous system of the goldfish (Carassius auratus).

The nitrergic system has been inferred from cells positive to nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry and/or to the neuronal isoform of nitric oxide synthase (nNOS) immunohistochemistry in different species of vertebrates. The aim of the present work was to systematically study the distribution of cell producing nitric oxide in the goldfish (Carassius auratus) brain. To reach this goal, we firstly studied co-localization for NADPHd and nNOS techniques and demonstrated an extensive double labeling. Then, we studied the distribution through the brain by the two separate methods and found labeled cells widely distributed in brain and spinal cord. In the telencephalon, such cells were in both dorsal and ventral areas. In the diencephalon, the cells were found in some nuclei of the preoptic area and hypothalamus, habenula, pretectum, and dorsal and ventral thalamic regions. In the midbrain, cells were observed in the optic tectum, torus longitudinalis, and tegmental nuclei. In the rhombencephalon, cells were found in the cerebellum, the reticular formation, the locus coeruleus, the raphe nuclei, and the nuclei of the cranial nerves. Labeled cells were also observed in the gray area of the spinal cord. Cognizing that a direct comparison of the present results with those reported in other vertebrates is not clear-cut because of homologies; we conclude that the nitrergic system is roughly similar from fish to mammals.

Enhanced c-Fos expression in the rostral ventral respiratory complex and rostral parapyramidal region by inhibition of the Na+/H+ exchanger type 3.

Previous studies have shown that selective inhibition of Na+/H+ exchanger type 3 (NHE3) induces intracellular acidification and activates CO2/H+-sensitive medullary neurons, mimicking the responses evoked by hypercapnic stimuli. In addition, NHE3 blockers administration decreases the duration of apnoea induced by laryngeal stimulation, presumably by means of central chemoreceptor activation. To test the hypothesis that the central chemoreceptor network may be affected by NHE3 inhibition, brainstem c-Fos immunoreactive cell counting was performed after systemic administration of the NHE3 blocker AVE1599 (Aventis Pharma Deutschland GmbH) (2 mg/kg). The rostro-caudal quantitative c-Fos analysis showed a significant increase in the number of c-Fos positive cells in the rostral part of the ventral respiratory complex (VRC) as well as in the rostral part of the parapyramidal (Ppy) region. The VRC activated region (-4.2 to -3.2mm interaural) included the pre-Bötzinger complex, the rostral ventral respiratory group and the rostral ventrolateral medulla, all of them involved in cardiorespiratory control. The activated Ppy region corresponded with the rostral chemosensitive area, which elicits the strongest ventilatory response upon ventral medullary surface stimulation with H+/CO2. Most cells activated in Ppy after NHE3 inhibition were serotonergic. Hence, systemic application of NHE3 blockers may induce central chemoreceptors activation and an increase in the respiratory network activity in a similar way to known physiological stimuli such as hypercapnia. On the other hand, selective NHE3 blockers could be excellent tools for treatment of pathological states where central chemoreceptor function is diminished or absent, such as central hypoventilation syndrome or sudden infant death syndrome.

Microalgae as a safe food source for animals: nutritional characteristics of the acidophilic microalga Coccomyxa onubensis.

BACKGROUND: Edible microalgae are marine or fresh water mesophilic species. Although the harvesting of microalgae offers an abundance of opportunities to the food and pharmaceutical industries, the possibility to use extremophilic microalgae as a food source for animals is not well-documented. OBJECTIVE: We studied the effects of dietary supplementation of a powdered form of the acidophilic microalga Coccomyxa onubensis on growth and health parameters of laboratory rats. METHOD: Four randomly organized groups of rats (n=6) were fed a standard diet (Diet 1, control) or with a diet in which 0.4% (Diet 2), 1.25% (Diet 3), or 6.25% (Diet 4) (w/w) of the standard diet weight was substituted with dried microalgae powder, respectively. The four groups of animals were provided ad libitum access to feed for 45 days. RESULTS: C. onubensis biomass is rich in protein (44.60% of dry weight) and dietary fiber (15.73%), and has a moderate carbohydrate content (24.8%) and a low lipid content (5.4%) in which polyunsaturated fatty acids represent 65% of the total fatty acid. Nucleic acids are present at 4.8%. No significant difference was found in growth rates or feed efficiency ratios of the four groups of rats. Histological studies of liver and kidney tissue revealed healthy organs in control and C. onubensis-fed animals, while plasma hematological and biochemical parameters were within healthy ranges for all animals. Furthermore, animals fed a microalgae-enriched diet exhibited a statistically significant decrease in both blood cholesterol and triglyceride levels. The blood triglyceride content and very low density lipoprotein-cholesterol levels decreased by about 50% in rats fed Diet 4. CONCLUSIONS: These data suggest that C. onubensis may be useful as a food supplement for laboratory animals and may also serve as a nutraceutical in functional foods. In addition, microalgae powder-supplemented diets exerted a significant hypocholesterolemic and hypotriglyceridemic effect in animals.

Characterization of efferent projections of chemosensitive neurons in the caudal parapyramidal area of the rat brain.

The caudal parapyramidal area of the rat brain contains a population of neurons that are highly sensitive to an increase in the extracellular hydrogen ion concentration ([H+]o). Some of them fire synchronously with respiration when [H+]o is increased. These chemosensitive neurons are located in the caudal ventrolateral medulla in a medial region, closest to the pyramidal tract, and a lateral region, beneath the lateral reticular nucleus. To assess the nature of medullary connections, biotinylated dextran amine injections were performed after recordings from the neurons had been completed. The injections were located within the areas containing serotonergic neurons of the caudal parapyramidal area. The injections within the medial and lateral parts of the caudal parapyramidal region revealed bilateral terminal fields of varicosities within the nucleus of the solitary tract and the ventral respiratory column. Efferent bilateral projections to the lateral paragigantocellular, lateral reticular, and inferior olive nuclei, as well as ipsilateral projections to medial and lateral caudal parapyramidal regions were also identified. Efferent projections towards the raphe obscurus from both medial and lateral caudal parapyramidal regions were found. Medial caudal parapyramidal regions also sent efferent projections towards the raphe pallidus, B1-B3 region, and to the dorsal and ventral parts of the medullary reticular nuclei. The detection of H(+)-sensitive neurons in the caudal parapyramidal area and their projections towards the nucleus of the solitary tract and to the ventral respiratory column, associated with respiratory regulation, indicate that this region could be an excellent candidate for central chemoreception.

Identification of central nervous system neurons innervating the respiratory muscles of the mouse: a transneuronal tracing study.

In recent years, the central control of breathing in mammals has been the subject of numerous studies. The aim of the present one was to characterize the neuronal network projecting to the main respiratory motoneurons, in adult mice. To this end, the morphology and location of the respiratory motoneurons and their sequential connections with other neurons were revealed using a transneuronal tracing technique by means of the rabies virus infection. The injections of the rabies virus in the respiratory muscles resulted in labeling the motoneurons and their serially connected interneurons at multiple levels of the mouse central nervous system: spinal cord, pons and medulla, cerebellum, mesencephalon, diencephalon, and telencephalon. Most of these labeled areas have been previously identified in the control of cardiorespiratory regulation, as well as in other autonomic functions. These anatomical data provide support for the integration of respiratory-related activities in complex behavioral responses. Furthermore, these data suggest similarities in the evolution of central respiratory networks in mammals.

Cholinergic and nitrergic neuronal networks in the goldfish telencephalon.

The general organization of cholinergic and nitrergic elements in the central nervous system seems to be highly conserved among vertebrates, with the involvement of these neurotransmitter systems now well established in sensory, motor and cognitive processing. The goldfish is a widely used animal model in neuroanatomical, neurophysiological, and behavioral research. The purpose of this study was to examine pallial and subpallial cholinoceptive, cholinergic and nitrergic populations in the goldfish telencephalon by means of histochemical and immunohistochemical techniques in order to identify neurons containing acetylcholinesterase (AChE), choline acetyltransferase (ChAT), NADPH-diaphorase (NADPHd), and neuronal nitric oxide synthase (nNOS), and to relate their distribution to their putative functional significance. Regions containing AChE-labeled neurons represented terminal fields of cholinergic inputs as well as a widespread distribution of AChE-related enzymes; these regions also usually contained NADPHd-labeled neurons and often contained small numbers of nNOS-positive cells. However, the ventral subdivisions of the medial and lateral parts of the dorsal telencephalic area, and the ventral and lateral parts of the ventral telencephalic area, were devoid of nNOS-labeled cells. ChAT-positive neurons were found only in the lateral part of the ventral telencephalic area. ChAT- and nNOS-positive fibers exhibited a radial orientation, and were seen as thin axons with en-passant boutons. The distribution of these elements could help to elucidate the role of cholinergic and nitrergic neuronal networks in the goldfish telencephalon.

Neonatal caffeine treatment up-regulates adenosine receptors in brainstem and hypothalamic cardio-respiratory related nuclei of rat pups.

While neonatal caffeine treatment is commonly used to alleviate apnea of prematurity in neonates and to improve neurological outcomes, its effects on adenosine A1 and A(2A) receptors (A1-R and A(2A)-R) are poorly known. We hypothesized that the central pharmacological action of caffeine is mediated by modification of the postnatal development of the adenosinergic system during a critical period. On postnatal days 2-6 (P2-P6) two groups of newborn rats were orally administered water plus glucose and/or caffeine at therapeutic doses to mimic the clinical use of caffeine in human neonates. Cardio-respiratory parameters were measured and the presence of A1-R and A(2A)-R and c-Fos protein was identified immunohistochemically in animals sacrificed from P2 to P11. % Haemoglobin saturation, and hearth and breath rates were significantly increased in caffeine-treated group (P5-P6). Significant differences were identified in the relative gene expression of A1-R and A(2A)-R, with an increase of A1-R labeling in the anterior hypothalamic area, ventromedial hypothalamic nucleus, parabrachial complex and ventrolateral medulla of the caffeine-treated group at P6. A moderate increase in A(2A)-R labeling was observed in ponto-medullary nuclei and other hypothalamic areas. An increase in c-Fos-positive labeled cells was found in the caffeine-treated group at P5-P6 within the same areas described above, with the most clear-cut increase seen in the arcuate nucleus. Indeed, increased A1-R and A(2A)-R gene expression was observed in both the brainstem and hypothalamus at P5. Up-regulation of adenosinergic maturation in central cardio-respiratory areas in caffeine-treated neonatal rats could explain the pharmacological effects of caffeine observed in premature infants.

Genesis and control of the respiratory rhythm in adult mammals.

The neural mechanisms responsible for respiratory rhythmogenesis in mammals were studied first in vivo in adults and subsequently in vitro in neonates. In vitro data have suggested that the pacemaker neurons are the kernel of the respiratory network. These data are reviewed, and their relevance to adults is discussed.