ETUDE DES RELAIS CENTRAUX IMPLIQUES DANS L'ADAPTATION RESPIRATOIRE A L'HYPOXIE. APPROCHE ELECTROPHYSIOLOGIQUE IN VIVO ET IN VITRO, ET IMMUNOHISTOCHIMIQUE

PARIS-BIUSJ-Physique recherche (751052113) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocSudocFranceF

Postnatal changes in the respiratory response of the conscious rat to serotonin 2A/2C receptor activation are reflected in the developmental pattern of fos expression in the brainstem.

The influence on the breathing pattern of the activation of serotonin receptors belonging to the subtypes 2(A) and 2(C) (5-HT(2A/2C)) has been assessed in newborn and adult conscious rats. Rats were given an acute intraperitoneal dose of the agonist DOI (1-(2.5-dimethoxy-4-iodophenyl)-2-aminopropane; 5 mg/kg). In newborns, DOI elicited a long-lasting respiratory depression by decreasing both tidal volume and respiratory frequency. In adults, DOI retained a depressant influence, although attenuated, on tidal volume. In contrast, it elicited an increase in respiratory frequency. In separate subsets of newborn and adult rats, immunohistochemistry has been used to monitor c-fos expression induced by DOI in the medullary and pontine regions involved in respiratory control. Counts of immunoreactive neurons indicated a marked increase in the neuronal populations activated in the adult compared to the newborn rat. The response to both experimental factors (newborn vs. adult controls) and drug (injected vs. control age-matched rats) were more pronounced in mature animals. Among developmental changes in the pattern of labeling, DOI elicited Fos expression in the adult but not in the neonate in the ventrolateral subnucleus of the nucleus of the solitary tract, the parabrachial area and the K?ker-Fuse nucleus. This finding suggested that changes in the respiratory response to DOI might at least partly depend on maturational events within networks involved in the modulation of respiratory timing

Effet de l'exposition in utero au diazépam sur la fonction respiratoire et la réponse à l'hypoxie du rat nouveau-né (impact sur les systémes GABAergiques et adénosinergiques)

Le diazépam (DZP) est une benzodiazépine thérapeutique qui potentialise l effet inhibiteur du GABA endogène lié aux récepteurs GABAA et qui peut être administrée à la femme enceinte. Le comportement du nouveau-né est perturbé par l exposition in utero au DZP. Notre objectif a été d apprécier les conséquences de cette exposition sur la fonction respiratoire et de déterminer les mécanismes impliqués chez le rat âgé de 0-2 jours. Des enregistrements in vivo par pléthysmographie chez l animal non contraint montrent que l exposition in utero au DZP modifie le pattern respiratoire en eupnée (diminution de la fréquence et augmentation du volume courant) mais pas la ventilation. Elle majore l effet dépresseur de l hypoxie alvéolaire sur le volume courant. Elle induit une augmentation de la fréquence de la commande respiratoire centrale, évaluée in vitro sur des préparations de tronc cérébral. Elle atténue les effets dépresseurs de l hypoxie tissulaire sur cette commande. La PCR quantitative en temps réel et une étude pharmacologique montrent selon les régions une régulation négative de l expression des gènes codant pour les sous-unités a1 et a2 du récepteur GABAA et/ou à une diminution de la réponse de ces récepteurs au DZP. De plus, l expression des récepteurs de l adénosine de type A1 et A2A et la réponse à leurs agonistes sont modifiées par l exposition au DZP. Enfin, l HPLC suggère une altération de la biosynthèse du glutamate et du GABA dans le tronc cérébral. Ces données montrent que l exposition prénatale au diazépam affecte des systèmes de neuromodulation qui ont un rôle majeur dans le contrôle respiratoire et que ses conséquences à plus long terme méritent d être évaluées.AMIENS-BU Santé (800212102) / SudocSudocFranceF

Evaluation des conséquences d'une exposition à la caféine in utero sur la commande centrale respiratoire de rats nouveau-nés (approches électrophysiologique et anatomique sur des préparations de système nerveux central isolées)

Le travail réalisé dans le cadre de ce doctorat a pour objectif de mieux apprécier les conséquences d'une exposition à la caféine in utero sur la commande respiratoire des nouveau-nés. Les expériences ont été réalisées sur un modèle animal qui permet de s'affranchir des problèmes liés aux études cliniques : l'hétérogénéité des doses de caféine et de sa consommation conjointe avec d'autres substances. Les comparaisons de la commande centrale respiratoire (CCR) et de l'expression de c-fos ont été réalisées sur des préparations de système nerveux central isolées de rats nouveau-nés exposés ou non à la caféine. Nos résultats 1/ confirment l'hypothèse selon laquelle une exposition à la caféine in utero est à l'origine de perturbations respiratoires chez le nouveau-né et 2/ suggèrent un mécanisme neurochimique pour expliquer ces modificationsAMIENS-BU Santé (800212102) / SudocSudocFranceF

Autonomic response and Fos expression in the NTS following intermittent vagal stimulation: importance of pulse frequency.

Chronic intermittent stimulation of the vagus nerve (VNS) is an approved adjunctive therapy of refractory epilepsy. Nevertheless, the circuits triggered by VNS under the variable conditions used in patients are not well understood. We analyzed the effect of increasing pulse frequency on physiological variables (intragastric pressure, cardiac and respiratory frequencies) and neuronal activation in the solitary tract nucleus (NTS), the entry level of peripheral vagal afferents, in the rat. For this purpose, we compared the subnuclear distribution of Fos-like immunoreactivity within the NTS following VNS at frequencies selected for their low (1 Hz) or high (10 Hz) therapeutic efficacy. In addition, NADPH diaphorase histochemistry was conducted in double-labeling experiments to check whether activated neurons may express nitric oxide (NO). We demonstrated that increasing pulse frequency had a major influence on the cardiorespiratory response to VNS and on the amount of activated neurons within NTS subdivisions engaged in cardiorespiratory control. These data, in line with clinical observations, suggested that within the range of therapeutic frequency, VNS may favor the regulation by vagal inputs of cortical activities within limbic areas involved in both epileptogenesis and cardiorespiratory afferent control. Furthermore, we did not find any evidence that anticonvulsant VNS might trigger NOergic neurons in the NTS

Immunohistochemical Studies of the Reactions of the Nucleus Tractus Solitarius and Parabrachial Complex Neurons to the Vagus Nerve Stimulation

Proceedings of the 9th International Multidisciplinary Conference «Stress and Behavior» Saint-Petersburg, Russia, 16–19 May 2005.It is well known that vagal stimulation may be an alternative to therapeutic treatment of inoperable and drug-resistant forms of epilepsy. However the mechanisms of its antiepileptic effects, and the regularity of the transmission of the interoceptive information to the forebrain structures, are poorly understood. Besides it is necessary to carry out the physiological experimental studies to optimize vagal stimulation parameters, in order to reduce side-effects (such as respiratory distress and cardiac abnormalities). The present study analysed spatial organization of the nucleus tractus solitarius and parabrachial complex nitroergic neurons expressing c-Fos proteins (product of the immediate early gene c-fos) in response to the electrical stimulation of the left cervical central cut end of the vagus nerve. The experiments were performed on the Sprague-Dawley adult rats. Double labelling for c-Fos proteins and NADPH-diaphorase was conducted on fixed brain tissue. Serial coronal brainstem sections (40 mkm) across the nucleus tractus solitarius and parabrachial complex areas were collected. Fos immunohistochemistry was conducted according to ABC procedure, using a primary polyclonal antibody against Fos (Santa-Cruz, sc-52) and detection kits including solutions of secondary biotinylated antibody, streptavidin and biotinylated horseradish peroxidase (Novocastra). NADPH-diaphorase was revealed according to standard procedures in the presence of beta-NADPH and nitro blue tetrazolium as substrates. The light microscope for detection of c-Fos and NADPH positive cells was used. Peculiarities of the reflectory changes of intragastric pressure, breathing and heart rates were studied as «autonomic indicators» of efficacy of vagal stimulation. Parameters of the nerve stimulation were related to those used in clinical practice for the treatment of epilepsy (100–300 mA, 0.5–1.0 ms, 1 or 10 Hz for 10 s). Without stimulation, NADPH-positive neurons were identified and the peculiarities of their localization in the commissural, medial and ventrolateral (the area postrema level) subnuclei of the nucleus tractus solitarius and caudal and rostral parts of the lateral and medial subnuclei of the parabrachial complex were studied. In neurons of the same areas the spontaneous c-Fos protein expression was investigated. After the vagus nerve stimulation by restangular pulses of 0.5 ms at 10 Hz and 200–300 mA for 10 s, the amount of c-Fos positive cells in commissural, medial and ventrolateral subnuclei of the nucleus tractus solitarius rised in 75, 100 and 220 %, accordingly. The quantity of these neurons rose in parabrachial complex: in rostral parts of the lateral and medial subnuclei in 130 and 80 %, accordingly and in caudal parts of the same subnuclei (40 and 210 %). Under the same parameters of the vagus nerve stimulation, the intragastric pressure decreased and the breathing stopped (apnoea during the period of stimulation). The heart rate inhibited under these conditions by 10–50 % vs. the baseline. Thus, here we identified nitroergic neurons in the nucleus tractus solitarius (the first synapse level for interoceptive impulses) and the parabrachial complex (the second synapse level) responding by c-Fos protein expression to electrical stimulation of the left cervical vagus nerve. Therefore, the participation of mentioned neurons in mechanisms of the therapeutic action of vagal stimulation has been demonstrated. Also we showed that vagal stimulation is accompanied by the changes in intragastric pressure, heart and breathing rates. Reflectory modifications of the breathing rate are the most sensitive «autonomic indicator» of the vagus nerve stimulation efficacy. The study was supported in part by grant 04–04-48710 from the Russian Foundation for Basic Research and by NATO fellowship (402349H to V.O.)

Immunohistochemical Studies of the Reactions of the Nucleus Tractus Solitarius and Parabrachial Complex Neurons to the Vagus Nerve Stimulation

Proceedings of the 9th International Multidisciplinary Conference «Stress and Behavior» Saint-Petersburg, Russia, 16–19 May 2005.It is well known that vagal stimulation may be an alternative to therapeutic treatment of inoperable and drug-resistant forms of epilepsy. However the mechanisms of its antiepileptic effects, and the regularity of the transmission of the interoceptive information to the forebrain structures, are poorly understood. Besides it is necessary to carry out the physiological experimental studies to optimize vagal stimulation parameters, in order to reduce side-effects (such as respiratory distress and cardiac abnormalities). The present study analysed spatial organization of the nucleus tractus solitarius and parabrachial complex nitroergic neurons expressing c-Fos proteins (product of the immediate early gene c-fos) in response to the electrical stimulation of the left cervical central cut end of the vagus nerve. The experiments were performed on the Sprague-Dawley adult rats. Double labelling for c-Fos proteins and NADPH-diaphorase was conducted on fixed brain tissue. Serial coronal brainstem sections (40 mkm) across the nucleus tractus solitarius and parabrachial complex areas were collected. Fos immunohistochemistry was conducted according to ABC procedure, using a primary polyclonal antibody against Fos (Santa-Cruz, sc-52) and detection kits including solutions of secondary biotinylated antibody, streptavidin and biotinylated horseradish peroxidase (Novocastra). NADPH-diaphorase was revealed according to standard procedures in the presence of beta-NADPH and nitro blue tetrazolium as substrates. The light microscope for detection of c-Fos and NADPH positive cells was used. Peculiarities of the reflectory changes of intragastric pressure, breathing and heart rates were studied as «autonomic indicators» of efficacy of vagal stimulation. Parameters of the nerve stimulation were related to those used in clinical practice for the treatment of epilepsy (100–300 mA, 0.5–1.0 ms, 1 or 10 Hz for 10 s). Without stimulation, NADPH-positive neurons were identified and the peculiarities of their localization in the commissural, medial and ventrolateral (the area postrema level) subnuclei of the nucleus tractus solitarius and caudal and rostral parts of the lateral and medial subnuclei of the parabrachial complex were studied. In neurons of the same areas the spontaneous c-Fos protein expression was investigated. After the vagus nerve stimulation by restangular pulses of 0.5 ms at 10 Hz and 200–300 mA for 10 s, the amount of c-Fos positive cells in commissural, medial and ventrolateral subnuclei of the nucleus tractus solitarius rised in 75, 100 and 220 %, accordingly. The quantity of these neurons rose in parabrachial complex: in rostral parts of the lateral and medial subnuclei in 130 and 80 %, accordingly and in caudal parts of the same subnuclei (40 and 210 %). Under the same parameters of the vagus nerve stimulation, the intragastric pressure decreased and the breathing stopped (apnoea during the period of stimulation). The heart rate inhibited under these conditions by 10–50 % vs. the baseline. Thus, here we identified nitroergic neurons in the nucleus tractus solitarius (the first synapse level for interoceptive impulses) and the parabrachial complex (the second synapse level) responding by c-Fos protein expression to electrical stimulation of the left cervical vagus nerve. Therefore, the participation of mentioned neurons in mechanisms of the therapeutic action of vagal stimulation has been demonstrated. Also we showed that vagal stimulation is accompanied by the changes in intragastric pressure, heart and breathing rates. Reflectory modifications of the breathing rate are the most sensitive «autonomic indicator» of the vagus nerve stimulation efficacy. The study was supported in part by grant 04–04-48710 from the Russian Foundation for Basic Research and by NATO fellowship (402349H to V.O.)

Consequences of prenatal exposure to diazepam on the respiratory parameters, respiratory network activity and gene expression of alpha1 and alpha2 subunits of GABA(A) receptor in newborn rat.

International audienceDiazepam (DZP) enhances GABA action at GABA(A) receptor. Chronic prenatal administration of DZP delays the appearance of neonatal reflexes. We examined whether maternal intake of DZP might affect respiratory control system in newborn rats (0-3 day-old). This study was conducted on unrestrained animals and medulla-spinal cord preparations. In addition, the level of expression of the genes encoding for the alpha1 and alpha2 subunits of the GABA(A) receptor was assessed by quantitative real-time RT-PCR. In rats exposed to DZP, the respiratory frequency was significantly lower and the tidal volume higher than in controls with no significant alteration of the minute ventilation. The recovery from moderate hypoxia was delayed compared to controls. The respiratory-like frequency of medullary spinal cord preparation from DZP-exposed neonates was higher than in the control group. Acute applications of DZP (1 microM) to these preparations increased respiratory-like frequency in both groups, but this facilitation was attenuated following prenatal DZP exposure. The present data indicate that prenatal exposure to DZP alters both eupneic breathing and the respiratory response to hypoxia. These effects might partly be ascribed to the down-regulation of the expression of genes encoding GABA(A) receptor subunits. On the other hand, the effects of DZP exposure on reduced preparations suggested changes in the GABA(A) receptor efficiency and/or disruption of the normal development of the medullary respiratory network

Consequences of prenatal exposure to diazepam on the respiratory parameters, respiratory network activity and gene expression of alpha1 and alpha2 subunits of GABA(A) receptor in newborn rat.

International audienceDiazepam (DZP) enhances GABA action at GABA(A) receptor. Chronic prenatal administration of DZP delays the appearance of neonatal reflexes. We examined whether maternal intake of DZP might affect respiratory control system in newborn rats (0-3 day-old). This study was conducted on unrestrained animals and medulla-spinal cord preparations. In addition, the level of expression of the genes encoding for the alpha1 and alpha2 subunits of the GABA(A) receptor was assessed by quantitative real-time RT-PCR. In rats exposed to DZP, the respiratory frequency was significantly lower and the tidal volume higher than in controls with no significant alteration of the minute ventilation. The recovery from moderate hypoxia was delayed compared to controls. The respiratory-like frequency of medullary spinal cord preparation from DZP-exposed neonates was higher than in the control group. Acute applications of DZP (1 microM) to these preparations increased respiratory-like frequency in both groups, but this facilitation was attenuated following prenatal DZP exposure. The present data indicate that prenatal exposure to DZP alters both eupneic breathing and the respiratory response to hypoxia. These effects might partly be ascribed to the down-regulation of the expression of genes encoding GABA(A) receptor subunits. On the other hand, the effects of DZP exposure on reduced preparations suggested changes in the GABA(A) receptor efficiency and/or disruption of the normal development of the medullary respiratory network