Anandamide centrally depresses the respiratory rhythm generator of neonatal mice.

Endogenous cannabinoid receptors are widely distributed throughout the CNS, including the brainstem, and modulate a variety of functions, including breathing. In adult rats, activation of cannabinoid 1 receptors has been shown to depress breathing. Here in neonatal mice, we used in vitro electrophysiology, pharmacology, and immunohistochemistry to analyse the central effects of the endocannabinoid anandamide (AEA) on the activity of the medullary respiratory rhythm generator (RRG). First of all, in vitro electrophysiology on medullary preparations has revealed that bath application of AEA (30 µM, 15 min) significantly depressed respiratory activity. Secondly, applying pre-treatments with alpha-1 (Prazosin, 5 µM, 10 min) and alpha-2 (Yohimbine, 5 µM, 10 min) adrenoceptor antagonists prior to AEA application abolished the AEA-induced depression of the RRG. Finally, immunostaining revealed a dense network of fibres positive for the cannabinoid 1 receptor in the ventrolateral medulla (VLM), a region known to contain both the RRG and the modulatory A1/C1 catecholaminergic group. Moreover, cannabinoid 1 receptor positive fibres were found in close apposition with A1/C1 catecholaminergic cells, identified by the presence of tyrosine hydroxylase. In regard of our electrophysiological, pharmacological and immunostaining results, we conclude that AEA has a central depressive effect on the neonatal RRG, probably via the medullary A1/C1 catecholaminergic neurons which are already known to modulate the respiratory rhythm generator.

Early abnormalities of post-sigh breathing in a mouse model of Rett syndrome.

Rett syndrome is a neurodevelopmental disease accompanied by complex, disabling symptoms, including breathing symptoms. Because Rett syndrome is caused by mutations in the transcriptional repressor methyl-CpG-binding protein 2 (MeCP2), Mecp2-deficient mice have been generated as experimental model. Males of Mecp2-deficient mice (Mecp2(-/y)) breathe normally at birth but show abnormal respiratory responses to hypoxia and hypercapnia from postnatal day 25 (P25). After P30, Mecp2(-/y) mice develop breathing symptoms reminiscent of Rett syndrome, aggravating until premature death at around P60. Using plethysmography, we analyzed the sighs and the post-sigh breathing pattern of unrestrained wild type male mice (WT) and Mecp2(-/y) mice from P15 to P60. Sighs are spontaneous large inspirations known to prevent lung atelectasis and to improve alveolar oxygenation. However, Mecp2(-/y) mice show early abnormalities of post-sigh breathing, with long-lasting post-sigh apnoeas, reduced tidal volume when eupnoea resumes and lack of post-sigh bradypnoea which develop from P15, aggravate with age and possibly contribute to breathing symptoms to come.

Ret deficiency in mice impairs the development of A5 and A6 neurons and the functional maturation of the respiratory rhythm.

Although a normal respiratory rhythm is vital at birth, little is known about the genetic factors controlling the prenatal maturation of the respiratory network in mammals. In Phox2a mutant mice, which do not express A6 neurons, we previously hypothesized that the release of endogenous norepinephrine by A6 neurons is required for a normal respiratory rhythm to occur at birth. Here we investigated the role of the Ret gene, which encodes a transmembrane tyrosine kinase receptor, in the maturation of norepinephrine and respiratory systems. As Ret-null mutants (Ret-/-) did not survive after birth, our experiments were performed in wild-type (wt) and Ret-/- fetuses exteriorized from pregnant heterozygous mice at gestational day 18. First, in wt fetuses, quantitative in situ hybridization revealed high levels of Ret transcripts in the pontine A5 and A6 areas. Second, in Ret-/- fetuses, high-pressure liquid chromatography showed significantly reduced norepinephrine contents in the pons but not the medulla. Third, tyrosine hydroxylase immunocytochemistry revealed a significantly reduced number of pontine A5 and A6 neurons but not medullary norepinephrine neurons in Ret-/- fetuses. Finally, electrophysiological and pharmacological experiments performed on brainstem 'en bloc' preparations demonstrated impaired resting respiratory activity and abnormal responses to central hypoxia and norepinephrine application in Ret-/- fetuses. To conclude, our results show that Ret gene contributes to the prenatal maturation of A6 and A5 neurons and respiratory system. They support the hypothesis that the normal maturation of the respiratory network requires afferent activity corresponding to the A6 excitatory and A5 inhibitory input balance.

Phox2a gene, A6 neurons, and noradrenaline are essential for development of normal respiratory rhythm in mice.

Although respiration is vital to the survival of all mammals from the moment of birth, little is known about the genetic factors controlling the prenatal maturation of this physiological process. Here we investigated the role of the Phox2a gene that encodes for a homeodomain protein involved in the generation of noradrenergic A6 neurons in the maturation of the respiratory network. First, comparisons of the respiratory activity of fetuses delivered surgically from heterozygous Phox2a pregnant mice on gestational day 18 showed that the mutants had impaired in vivo ventilation, in vitro respiratory-like activity, and in vitro respiratory responses to central hypoxia and noradrenaline. Second, pharmacological studies on wild-type neonates showed that endogenous noradrenaline released from pontine A6 neurons potentiates rhythmic respiratory activity via alpha1 medullary adrenoceptors. Third, transynaptic tracing experiments in which rabies virus was injected into the diaphragm confirmed that A6 neurons were connected to the neonatal respiratory network. Fourth, blocking the alpha1 adrenoceptors in wild-type dams during late gestation with daily injections of the alpha1 adrenoceptor antagonist prazosin induced in vivo and in vitro neonatal respiratory deficits similar to those observed in Phox2a mutants. These results suggest that noradrenaline, A6 neurons, and the Phox2a gene, which is crucial for the generation of A6 neurons, are essential for development of normal respiratory rhythm in neonatal mice. Metabolic noradrenaline disorders occurring during gestation therefore may induce neonatal respiratory deficits, in agreement with the catecholamine anomalies reported in victims of sudden infant death syndrome.

Altered respiratory activity and respiratory regulations in adult monoamine oxidase A-deficient mice.

The abnormal metabolism of serotonin during the perinatal period alters respiratory network maturation at birth as revealed by comparing the monoamine oxidase A-deficient transgenic (Tg8) with the control (C3H) mice (Bou-Flores et al., 2000). To know whether these alterations occur only transiently or induce persistent respiratory dysfunction during adulthood, we studied the respiratory activity and regulations in adult C3H and Tg8 mice. First, plethysmographic and pneumotachographic analyses of breathing patterns revealed weaker tidal volumes and shorter inspiratory durations in Tg8 than in C3H mice. Second, electrophysiological studies showed that the firing activity of inspiratory medullary neurons and phrenic motoneurons is higher in Tg8 mice and that of the intercostal motoneurons in C3H mice. Third, histological studies indicated abnormally large cell bodies of Tg8 intercostal but not phrenic motoneurons. Finally, respiratory responses to hypoxia and lung inflation are weaker in Tg8 than in C3H mice. dl-p-chlorophenyl-alanine treatments applied to Tg8 mice depress the high serotonin level present during adulthood; the treated mice recover normal respiratory responses to both hypoxia and lung inflation, but their breathing parameters are not significantly affected. Therefore in Tg8 mice the high serotonin level occurring during the perinatal period alters respiratory network maturation and produces a permanent respiratory dysfunction, whereas the high serotonin level present in adults alters the respiratory regulatory processes. In conclusion, the metabolism of serotonin plays a crucial role in the maturation of the respiratory network and in both the respiratory activity and the respiratory regulations.

Perinatal changes of I(h) in phrenic motoneurons.

The hyperpolarization-activated cationic current (I(h)) was characterized and its maturation studied on phrenic motoneurons (PMNs), from reduced preparations of foetal (E18 and E21) and newborn (P0-P3) rats, using the whole-cell patch-clamp technique. In voltage-clamp mode, 2-s hyperpolarizing steps (5-mV, -50 to -110 mV) elicited a noninactivating inward current, blocked by external application of Cs+ or ZD 7288. At -110 mV, Ih current density averaged 0.67 +/- 0.41 pA/pF at E18, reached a transient peak at E21 (1.38 +/- 0.11 pA/pF) and decreased at P0-P3 (0.77 +/- 0.22 pA/pF). V1/2 was similar at E18 and E21 (-79 mV) but was significantly hyperpolarized at P0-P3 (-90 mV). The time constant of activation was voltage-dependent, and significantly faster at E21. Reversal potential was similar at all ages when estimated by extrapolation or tail current procedures. It was positively shifted by 25 +/- 6 mV when external potassium was raised from 3 to 10 m M, suggesting a similar sensitivity to K+ from E18 to P0-3. Cs(+) or ZD 7288 applications on PMNs at rest in current-clamp mode, in a partitioned chamber, induced a 10 +/- 2 mV hyperpolarization at E18 and E21, and an 8 +/- 2 mV hyperpolarization at P0-3. The area of the central respiratory drive potential or current was increased by 33 and 31%, respectively, at E21, but was not significantly modified at E18 and P0-3. Our data suggest a critical period during the perinatal maturation of Ih during which it is transiently upregulated and attenuates the influence of the central respiratory drive on PMNs just prior to birth.

5-Hydroxytryptamine(2A) and 5-hydroxytryptamine(1B) receptors are differently affected by the monoamine oxidase A-deficiency in the Tg8 transgenic mouse.

In brainstem-spinal cord preparations of neonatal control C3H and transgenic Tg8 mice where deletion of the gene encoding monoamine oxidase-A results in serotonin (5-hydroxytryptamine (HT)) excess, whole cell recordings of identified phrenic motoneurons (Phr Mns) were performed to study the modulation of their activity by 5-HT. In C3H mice, a dual effect was observed: (i) a facilitation via 5-HT(2A) receptors and (ii) a decrease of the transmission of the central inspiratory drive via 5-HT(1B) receptors. In Tg8 mice, the 5-HT(2A)-mediated facilitation was present but the 5-HT(1B)-mediated decrease was lacking. Therefore, the conservation of the 5-HT(2A) response vs. the loss of the 5-HT(1B) one suggest that the two types of receptors respond differently to 5-HT level changes.

Abnormal phrenic motoneuron activity and morphology in neonatal monoamine oxidase A-deficient transgenic mice: possible role of a serotonin excess.

In rodent neonates, the neurotransmitter serotonin (5-HT) modulates the activity of both the medullary respiratory rhythm generator and the cervical phrenic motoneurons. To determine whether 5-HT also contributes to the maturation of the respiratory network, experiments were conducted in vitro on the brainstem-spinal cord preparation of neonatal mice originating from the control strain (C3H) and the monoamine oxidase A-deficient strain, which has a brain perinatal 5-HT excess (Tg8). At birth, the Tg8 respiratory network is unable to generate a respiratory pattern as stable as that produced by the C3H network, and the modulation by 5-HT of the network activity present in C3H neonates is lacking in Tg8 neonates. In addition, the morphology of the phrenic motoneurons is altered in Tg8 neonates; the motoneuron dendritic tree loses the C3H bipolar aspect but exhibits an increased number of spines and varicosities. These abnormalities were prevented in Tg8 neonates by treating pregnant Tg8 dams with the 5-HT synthesis inhibitor p-chlorophenylalanine or a 5-HT(2A) receptor antagonist but were induced in wild-type neonates by treating C3H dams with a 5-HT(2A) receptor agonist. We conclude that 5-HT contributes, probably via 5-HT(2A) receptors, to the normal maturation of the respiratory network but alters it when present in excess. Disorders affecting 5-HT metabolism during gestation may therefore have deleterious effects on newborns.

Locomotor network maturation is transiently delayed in the MAOA-deficient mouse.

In vivo and in vitro experiments were performed in control (C3H) and monoamine oxidase A (MAOA)-deficient (Tg8) neonatal mice to determine whether MAOA deficiency affected spinal locomotor network maturation. Comparing the swimming behaviors at birth in C3H mice with those in Tg8 mice revealed a delayed role for the hindlimbs in Tg8 swimming, even though adult swimming behavior was acquired at postnatal day 14 (P14) in both strains. Analyzing the locomotor network activity in vitro showed that serotonin (5-HT) induced and modulated locomotor-like discharges in hindlimb ventral roots of C3H but not Tg8 neonates. The Tg8 network began, however, to be affected by 5-HT at P11. Thus both in vivo and in vitro results argue for a transient delay of locomotor network maturation in the Tg8 strain.

Cellular and synaptic effect of substance P on neonatal phrenic motoneurons.

Experiments were carried out on the in vitro brainstem-spinal cord preparation of the newborn rat to analyse the effects of substance P (SP) on phrenic motoneuron (PMN) activity. In current-clamp mode, SP significantly depolarized PMNs, increased their input resistance, decreased the rheobase current and shifted the firing frequency-intensity relationships leftwards, but did not affect spike frequency adaptation or single spike configuration. The neurokinin receptor agonist NK1 had SP-mimetic effects, whereas the NK3 and NK2 receptor agonists were less effective and ineffective, respectively. In a tetrodotoxin-containing aCSF, only SP or the NK1 receptor agonist were still active. No depolarization was observed when the NK1 receptor agonist was applied in the presence of muscarine. In voltage-clamp mode, SP or the NK1 receptor agonist produced an inward current (ISP) which was not significantly reduced by extracellular application of tetraethylammonium, Co2+, 4-aminopyridine or Cs+. In aCSF containing tetrodotoxin, Co2+ and Cs+, ISP was blocked by muscarine. No PMN displayed any M-type potassium current but only a current showing no voltage sensitivity over the range -100 to 0 mV, reversing near the expected EK +, hence consistent with a leak current. SP application to the spinal cord only (using a partitioned chamber) significantly increased the phrenic activity. Pretreatment with the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5) decreased the C4 discharge duration and blocked the effect of SP, thus exhibiting an NMDA potentiation by SP. In conclusion, SP modulates postsynaptically the response of phrenic motoneurons to the inspiratory drive through the reduction of a leak conductance and the potentiation of the NMDA component of the synaptic input.

Pulmonary stretch receptor discharges and vagal regulation of respiration differ between two mouse strains.

1. Experiments were performed on adult pentobarbitone-anaesthetized mice of the OF1 and the C3H/HeJ (C3H) strains, to analyse the regulation of respiration by pulmonary stretch receptors (PSRs). 2. Although the mean respiratory period, inspiratory and expiratory durations, and tidal volume did not differ significantly between the two strains, the inspiratory onset was drastically inhibited in OF1 mice but only slightly inhibited in C3H mice in response to tracheal occlusion performed at the very end of inspiration. 3. Low current electrical stimulation of the vagus nerve induced inspiratory onset inhibition in both strains, suggesting that the weak inspiratory onset inhibition elicited by tracheal occlusion in C3H mice did not originate from a low sensitivity of the respiratory centres to PSRs. 4. During normal respiration, PSR firing rate increased with tidal volume, but reached significantly higher values in OF1 than C3H mice. During tracheal occlusion, PSR firing rate was significantly higher at the end of inspiration and during the first third of the occlusion period in OF1 than C3H mice. 5. The airway pressure resistance was significantly higher in OF1 than C3H mice. After abolishing the tracheo-bronchial muscle tone with atropine in OF1 mice, tracheal occlusions induced weak inspiratory onset inhibitions resembling the C3H mouse responses. 6. The possibility that differences in tracheo-bronchial tone between OF1 and C3H mice may lead to a greater PSR discharge and thus to a powerful inhibition on the OF1 medullary respiratory centres during tracheal occlusion is discussed.

Central respiratory effects of substance P in neonatal mice: an in vitro study.

Experiments were performed on neonatal mice to know whether substance P (SP) modified the rhythm and the amplitude of the phrenic bursts generated in vitro in brainstem-cervical cord preparations. In OF1 and C3H neonatal preparations, SP or the tachykinin NK1 receptor agonist [Sar9,Met(O2)11] substance P both increased significantly phrenic burst amplitude (10(-7) M) but had no significant effect on respiratory rhythm unless used at concentrations 10 times larger. In neonates from the monoamine oxidase-A deficient transgenic Tg8 line, SP increased phrenic burst amplitude but had no effect on the respiratory rhythm at the tested concentrations. The role of SP in regulating neonatal respiratory activity is discussed on the basis of rat and mouse results.

Maturation of the mammalian respiratory system.

In this review, the maturational changes occurring in the mammalian respiratory network from fetal to adult ages are analyzed. Most of the data presented were obtained on rodents using in vitro approaches. In gestational day 18 (E18) fetuses, this network functions but is not yet able to sustain a stable respiratory activity, and most of the neonatal modulatory processes are not yet efficient. Respiratory motoneurons undergo relatively little cell death, and even if not yet fully mature at E18, they are capable of firing sustained bursts of potentials. Endogenous serotonin exerts a potent facilitation on the network and appears to be necessary for the respiratory rhythm to be expressed. In E20 fetuses and neonates, the respiratory activity has become quite stable. Inhibitory processes are not yet necessary for respiratory rhythmogenesis, and the rostral ventrolateral medulla (RVLM) contains inspiratory bursting pacemaker neurons that seem to constitute the kernel of the network. The activity of the network depends on CO2 and pH levels, via cholinergic relays, as well as being modulated at both the RVLM and motoneuronal levels by endogenous serotonin, substance P, and catecholamine mechanisms. In adults, the inhibitory processes become more important, but the RVLM is still a crucial area. The neonatal modulatory processes are likely to continue during adulthood, but they are difficult to investigate in vivo. In conclusion, 1) serotonin, which greatly facilitates the activity of the respiratory network at all developmental ages, may at least partly define its maturation; 2) the RVLM bursting pacemaker neurons may be the kernel of the network from E20 to adulthood, but their existence and their role in vivo need to be further confirmed in both neonatal and adult mammals.

Serotonin levels are abnormally elevated in the fetus of the monoamine oxidase-A-deficient transgenic mouse.

Developmental changes in levels of serotonin, L-tryptophan and 5-hydroxyindol acetic acid (5-HIAA) were measured by high pressure liquid chromatography (HPLC) in the forebrain, brainstem and cervical cord of fetal, neonatal and adult mice from the wild strain C3H and the transgenic strain Tg8, created from the C3H line by the disruption of the gene encoding monoamine oxidase A. The results indicated that the absence of monoamine oxidase A activity in Tg8 mice results in abnormally high 5-hydroxytryptamine (5-HT) levels in all the central nervous structures and at all the studied developmental ages. Since serotonin levels were 4-5 times larger in Tg8 than in C3H mice at gestational day 20, comparing the central network function at birth of C3H and Tg8 neonates should shed some light on the role of serotonin in prenatal network maturation.

Serotonergic inhibition of phrenic motoneuron activity: an in vitro study in neonatal rat.

In vitro experiments were conducted on neonatal rat brainstem-spinal cord preparations to test the hypothesis of an inhibitory modulation of phrenic activity by serotonin (5-HT) via non-5-HT2A receptors [Lindsay, A.D. and Feldman, J.L., Modulation of respiratory activity of neonatal rat phrenic motoneurones by serotonin, J. Physiol., 461 (1993) 213-233]. The changes induced by 5-HT and related agents on phrenic root discharges and membrane currents in identified phrenic motoneurons were analysed after blockade of spinal 5-HT2A receptors. Spinal application of 5-HT1B (but not 5-HT1A) receptor agonists depressed the phrenic activity and the effect was prevented by pretreatment with 5-HT1B (but not 5-HT1A, 5-HT2A and 5-HT3) receptor antagonists. Results from phrenic motoneuron whole cell recordings do not reject a presynaptic location of the 5-HT receptors responsible for this depression.

Serotonergic modulation of central respiratory activity in the neonatal mouse: an in vitro study.

In order to determine whether the serotonergic modulation of the central respiratory activity previously reported in neonatal rats occurs in species other than the rat, we performed identical in vitro experiments on the neonatal mouse to those performed on the neonatal rat. The effects of adding serotonin (5-hydroxytryptamine, 5-HT) and related agents to the superfusate suggested that the respiratory rhythm generator undergoes an excitatory modulation via medullary 5-HT1A receptors. Upon applying the drugs to the spinal cord alone, 5-HT was found to have a dual effect on phrenic motoneuron firing: (i) a facilitatory effect mediated by 5-HT2A receptors and (ii) a depressive effect on their inspiratory discharge mediated by non-5-HT1A, non-5-HT2A, non-5-HT3 receptors, possibly of the 5-HT1B subtype. It was therefore concluded that serotonin modulates the neonatal central respiratory activity in mice as well as in rats, and that similar 5-HT receptor subtypes are involved in this process in both species.

Rostral ventrolateral medulla and respiratory rhythmogenesis in mice.

To compare the mechanisms governing perinatal respiratory rhythmogenesis in mice and rats, we adapted to the neonatal mouse the in vitro brainstem-spinal cord preparation of the neonatal rat. In mouse preparations retaining the pons, phrenic root did not show any rhythmic activity. Elimination of the pons induced phrenic rhythmic bursts which (1) induced respiratory chest movements (rib cage kept attached to the spinal cord), (2) were abolished by spinal cord transection, (3) could be prematurely induced by rostral ventro-lateral medulla (RVLM) stimulation, (4) occurred in phase with the bursting firing of RVLM neurons, and (5) were abolished by RVLM lesion. Then, the RVLM appears crucial for respiratory rhythmogenesis in both species; some results suggest however that vagal and pontine respiratory controls might not be identical in mice and rats.

Tachykinins and central respiratory activity: an in vitro study on the newborn rat.

The newborn rat brainstem-spinal cord preparation was used to study the effects of tachykinins on the activity of the respiratory rhythm generator in vitro and to characterize the receptors involve. Substance P and tachykinin NK1 and NK3 receptor agonists induced a concentration-dependent increase in the respiratory frequency (10(-9)-10(-7) M), whereas the respiratory frequency was only slightly affected by the tachykinin NK2 receptor agonist. Pre-treatments with tachykinin NK1 receptor antagonists (SR140333, (S)1-¿2-[3-(3.4-dichlorophenyl) -1-(3-isopropoxyphenylacetyl)piperidin-n-3-yl]ethyl¿-4-ph eny l-1-azoniabicyclo [2,2,2]octane chloride; GR82334, pGlu-Ala- Asp-Pro-Asn-Lys-Phe-Tyr-(S-S)Pro-Leu(spiro-gamma-lactam)-Trp-NH2) reduced the substance P-induced increases in the respiratory frequency but the tachykinin NK2 receptor antagonist (SR48968, ((S)-N-methyl-N-[4-4-acetylamino-4-phenylpiperidine)-2-(3,4-dichlorop hen yl) butyl]benzamide); MEN 10376, Asp-Tyr-D-Trp-Val-D-Trp-Lys-NH2) had no effect; the increase in the respiratory frequency induced by the tachykinin NK3 receptor agonist was not affected by a pre-treatment with tachykinin NK1 and NK2 receptor antagonists. These result indicate that tachykinin NK1 and NK3 receptors may be involved in the control of the respiratory frequency.

Perinatal developmental changes in respiratory activity of medullary and spinal neurons: an in vitro study on fetal and newborn rats.

Experiments were performed in vitro on fetal and newborn rat brainstem-spinal cord preparations to analyse the perinatal developmental changes in inspiratory motor output. The amplitude of the inspiratory bursts of the whole C4 ventral root (global extracellular recording), the firing patterns of 80 medullary inspiratory neurons (unitary extracellular recording) and the firing and membrane properties of 71 respiratory neurons in the C4 ventral horn (whole-cell recording) were analysed at embryonic day 18 (E18), 21 (E21) and post natal days 0 to 3 (P0-3). At E18, the amplitude of the C4 bursts was weak and variable from one respiratory cycle to the next, as well as the discharge pattern of most of the medullary inspiratory neurons. C4 motoneurons were immature, very excitable and displaying variable inspiratory discharges, but already able to deliver sustained bursts of potentials when depolarised. At E21 and P0-3, the amplitude of the C4 bursts was increased and stable, most of the medullary inspiratory neurons already were able to generate a stable firing pattern and C4 motoneurons showed maturational changes in terms of the resting potential, spike amplitude and input membrane resistance. This work suggests that the short period extending from E18 to E21 is a critical maturational period for the medullary respiratory network which becomes able to elaborate a stable respiratory motor output.

Effects of ethanol on respiratory activity in the neonatal rat brainstem-spinal cord preparation.

Ethanol (1-12 mM) added to the superfusion medium of the isolated brainstem-spinal cords of newborn rats did not affect phrenic activity but significantly reduced hypoglossal activity by 54%, 67% and 55% at 3, 6 and 12 mM, respectively. Although the reasons for the suppression of hypoglossal activity remain unknown, this preparation may be a useful model for determining why cranial motoneurons are more vulnerable than phrenic motoneurons to various agents and, more generally, how ethanol impairs neural function.