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In vitro experiments were performed on brainstem-spinal cord preparations from mouse neonates to compare the noradrenergic regulations of the respiratory network in the control C3H/HeJ strain and the transgenic Tg8 strain which has been created from the C3H/HeJ strain by deletion of the gene encoding monoamine oxidase A (MAOA), the main enzyme for serotonin degradation. In both control and MAOA-deficient strains, we show: (i). that the pontine A5 area exerts a potent inhibitory modulation on the respiratory rhythm generator; (ii). that noradrenaline application induces a tonic phrenic activity; and (iii). that noradrenaline increases the respiratory rhythm. The latter effect is however delayed and weak in the Tg8 strain. Therefore, MAOA-deficiency has only slightly altered the noradrenergic regulations of the respiratory network.

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.

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.

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.

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.

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.

In order to investigate the mechanisms through which serotonin (5-HT) modulates the activity of the respiratory rhythm generator, respiratory activity was recorded from cervical ventral roots of the superfused isolated brainstem-spinal cord preparation of the newborn rat. Replacing the normal bathing medium by a medium containing 5-HT (30 microM) increased the respiratory frequency by 70% of the control value. Intact pontomedullary structures are necessary for this effect to take place, however, since the 5-HT-induced increases in respiratory frequency were no longer observed after elimination (section and electrolytic lesion) of the caudal ventro-lateral pons containing the A5 areas. Local applications of 5-HT (dual bath, microdialysis and microinjection experiments) revealed, however, that 5-HT acts at the medullary level and that its effects are not due to a diffuse action on all the neurons of the medullary respiratory centers but to a specific action focusing on structures located in the rostral ventro-lateral medulla.

Respiratory activity was recorded from the hypoglossal nerve and cervical ventral roots of the superfused isolated brainstem and spinal cord of the newborn rat in order to investigate the effects of serotonin (5-HT). Three experimental procedures were used: (i) brainstem superfusion by exogenous 5-HT (previously reported), (ii) electrical stimulation of pontine midline structures, and (iii) pressure microejection of L-glutamate at the same sites. Each of the experimental procedure increased respiratory frequency and depressed the amplitude of the discharge of the hypoglossal nerve more than that of the cervical roots. These results indicate that 5-HT acts centrally to modulate the activity of the respiratory generator and inhibits the hypoglossal activity. A possible involvement of 5-HT mediated inhibition in obstructive apnea is discussed.

The involvement of the dorsal part of the medulla (the so-called dorsal respiratory group: DRG) in the networks participating in respiratory function was investigated in newborn (in vitro) and adult (in vivo) rats. In the dorsal part of the medulla of the isolated brainstem of newborn rats, no respiratory neurons were found and stimulations or lesions neither modified nor suppressed the respiratory output. On the contrary, similar experiments suggest that sites in the ventral medulla have a fundamental importance for respiration. In adult rats, lesion of the DRG areas by electrocoagulation induced transient changes in respiratory timing, and resulted in a significant decrease in the amplitude of the contralateral phrenic output. These results suggest that the dorsal part of the medulla is not involved in controlling respiratory activity in the newborn rat. In adults, no definite conclusion can be reached, but the functional role of the DRG, if any, is probably restricted.

Newborn rat respiratory activity was recorded on hypoglossal nerve and ventral cervical roots during in vitro experiments performed on superfused brainstem spinal cord preparations. The addition of serotonin (5-HT) to the bathing medium increased the respiratory frequency and selectively depressed the hypoglossal activity. Any decreases in the amplitude of cervical recordings were always restricted and reversible, whereas the hypoglossal activity was abolished. Furthermore, on cervical roots, 5-HT induced a tonic activity superimposed on the respiratory one, which was never observed with the hypoglossal nerve. When 5-HT was applied on isolated hemispinal cord, a tonic activity could still be elicited. These results indicate that serotonin (i) modulates the activity of neurons involved in the generation of respiratory rhythm, (ii) depresses the activity of hypoglossal motoneurons, and (iii) evokes tonic activity in cervical motoneurons, probably as the result of direct spinal effects.

In anaesthetized, bivagotomized and artificially ventilated rats, the respiratory effects of systemic injection of MK-801, a non-competitive N-methyl-D-aspartate antagonist, were studied. In all the experiments (n = 11), the injection increased the inspiratory duration and decreased the expiratory duration. In 4 experiments, the inspiratory duration was drastically lengthened, resulting in an apneustic-like breathing pattern. These results demonstrate that apneusis is difficult but possible to induce in rats and suggest that termination of inspiration is controlled via central mechanisms in which NMDA-like receptors are involved.

Pontine influences on the breathing pattern were studied in rats using either in vivo experiments in anaesthetized adult animals or in vitro experiments in newborn preparations. In cats, after anaesthesia and bivagotomy, pontine transection has been described as evoking an apneustic pattern of breathing (long sustained inspiratory discharges interrupted by short expiratory pauses). In rats, after anaesthesia, bivagotomy and pontine transection, apneustic patterns of breathing were not observed, either in vivo or in vitro. These results suggest interspecies differences between rat and cat in the pontine influences on the medullary respiratory generator.

Activity of Renshaw cells evoked by electrical stimulation of either phrenic or internal intercostal axons was extracellularly recorded in anaesthetized spontaneously breathing cats. The response of all the studied units to antidromic invasion of the corresponding motoneurones was related to the respiratory cycle and some units displayed spontaneous respiratory activity. Recurrent IPSPs were recorded on phrenic and intercostal motoneurones.

Simultaneous recordings were made intracellularly from phrenic motoneurons (PMs) and extracellularly from their central drivers, the inspiratory bulbospinal neurons (IBSNs) of the dorsal respiratory nucleus. On the basis of their order of recruitment from the beginning of inspiration (as estimated by the phrenic discharge). PMs and IBSNs were classified as early (E) or late (L) units. Unitary excitatory postsynaptic potentials (EPSPs) evoked in the PM by the IBSN were frequently observed between IBSNs and PMs with a similar recruitment, both E, 11/28 or both L, 10/13, and were scarce between IBSNs and PMs with different recruitment. E and L, 8/28, or L and E, 2/11. Since measured membrane resistances of E and L PMs were not statistically different, the recruitment order of PMs may be considered as mainly determined by the timing (E or L) of the central drive that they received.