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A retrograde and transneuronal infection with rabies virus was performed in mouse neonates to locate the central nervous structures involved in the motor command of the spinal respiratory motoneurons and to discriminate the location and hierarchical organization of the neurons in and between these infected central nervous structures.

Abnormally high brain 5-HT levels in monoamine oxidase-A knockout (MAO-A KO) mouse neonates raise the question of whether the distribution and density of the 5-HT1A receptors (5-HT1AR) expressed in the brain by postnatal day P7 are affected and, if so, whether the 5-HT1A autoreceptors in the dorsal raphe are modified in the same way as the postsynaptic 5-HT1AR present in raphe target structures. [3H]8-OH-DPAT binding and quantitative autoradiography were performed to answer these questions. Binding specificity was first confirmed in adult wild-type mice and rat brain sections. 5-HT1AR binding was then analyzed in four MAO-A mutant vs. five wild-type neonatal brains, from olfactory bulb to cervical cord. Among 12 structures expressing postsynaptic 5-HT1AR in wild-type neonates, the highest densities involved the retrosplenial cortex, entorhinal cortex, and septum (52-46 fmol/mg tissue); low densities occurred in the hippocampus and spinal cord (24 fmol/mg tissue); in addition, the raphe autoreceptor density was only 20 fmol/mg tissue. In mutants, the distribution of postsynaptic 5-HT1AR was unchanged, but an overall decrease in density occurred (-32% to -63%); the raphe autoreceptors decreased in mutants by at least -79%. Data are discussed with reference to the ectopic 5-HT uptake and accumulation reported to occur during the first 10 postnatal days in wild-type and MAO-A KO mice. As previously suggested to explain the raphe autoreceptor loss in 2-month-old MAO-A KO mice, the overall 5-HT1AR down-regulation in mutant pups probably results from extracellular 5-HT excess in both raphe and target structures. The greater the 5-HT excess, the more the functional receptor density decreases., (Copyright 2006 Wiley-Liss, Inc.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

Previous experiments performed in the in vitro newborn rat brainstem-spinal cord preparation reported that the addition of serotonin (5-HT, 30-50 microM) to the bathing medium induced increases in the respiratory frequency and a large tonic discharge on all the cervical ventral roots. The aim of the present work was to define whether the 5-HT-induced tonic discharge involved respiratory or non-respiratory motoneurones. Intracellular recordings demonstrated that cervical (C2) motoneurones (n = 27) were depolarized by 5-HT but that the 5-HT-induced tonic discharge was mainly due to recruitment of silent motoneurones (n = 18) which fired permanently (15/18; 17 +/- 3 Hz) under 5-HT. The respiratory motoneurones (n = 9) retained a phasic inspiratory discharge (5/9), even if some (4/9) occasionally exhibited a few spikes during expiration. Therefore, it is concluded that the 5-HT-induced tonic discharge is unlikely to have functional significance in respiration.

In vitro experiments were performed in the superfused brainstem-spinal cord preparation of newborn rats in order to analyse the central respiratory effects of acetylcholine. The central motor output was assessed from recording electrical activity in nerves supplying respiratory muscles. Acetylcholine added to the bathing medium induced dose-dependent increases in respiratory frequency which were blocked by muscarinic (but not nicotinic) antagonists and enhanced by physostigmine. These effects originated from the medullary ventral surface where chemosensitive structures have been previously located. The respiratory central chemosensitivity of the isolated brainstem was analysed using a CO2 free, pH 7.9 medium instead of the normal medium (bubbled with 5% CO2, pH 7.3). Decreases at the H+ and CO2 stimuli led to decreased inspiratory activity, resulting mainly from a decrease in the amplitude of the motor output. These responses were enhanced by atropine and diminished by physostigmine. These results obtained in vitro on the newborn rat suggest that cholinergic synapses are not directly involved in the genesis of respiratory rhythmicity but confirm previous results obtained in vivo in adult animal revealing that acetylcholine is implicated in the central respiratory chemosensitivity.

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.

In anesthetized spontaneously breathing cats (C4-C5 deafferentation), recurrent inhibition of phrenic motoneurons was analyzed by studying either recurrent IPSPs in phrenic motoneurons, or Renshaw cell discharges evoked by C5 phrenic nerve stimulation. Of 90 intracellularly recorded phrenic motoneurons, 7 motoneurons showed evoked recurrent IPSPs with stimulation of C5 phrenic axons subthreshold for eliciting antidromic activation of the motoneuron from which intracellular recording was done. These IPSPs could be reversed by imposed hyperpolarization of the motoneuron, and were of greater amplitude during inspiration than during expiration. Within the phrenic nucleus, interneurons were classified as Renshaw cells if they responded to C5 phrenic axon stimulation with a typical high frequency burst of potentials. Reactivity of these Renshaw cells was related to the respiratory cycle, number of spikes in the burst being greater during inspiration than during expiration. Injection of a nicotinic cholinergic blocker (mecamylamine) decreased responses of Renshaw cells but the respiratory modulation was still present. Some Renshaw cells (18/33) were spontaneously active during inspiration. Their activity was generally maximal during the last third of inspiration. Since: spontaneous activity of Renshaw cells is related to the respiratory drive; persists after C7 spinal transection and after mecamylamine poisoning of the axonal recurrent pathway; and might appear before sustained phrenic activity, the assumption of a central respiratory drive impinging on the Renshaw cells has to be retained.