Roger Monteau

Possible modulation of the medullary respiratory rhythm generator by the noradrenergic A5 area:an in vitro study in the newborn rat

Respiratory activity was recorded on hypoglossal nerve or ventral cervical roots during in vitro experiments performed in the superfused brainstem-cervical cord preparation of newborn rats. Section and coagulation experiments revealed that the medullary respiratory generator was tonically inhibited by a structure located in the caudal ventrolateral pons. Electrical and pharmacological stimulations located this structure more precisely between the superior olivary nuclei and the sensory nucleus of the Vth nerve, i.e. in an area containing the A5 noradrenergic nucleus. Norepinephrine and alpha 2-antagonists (yohimbine, idazoxan) added to the bathing medium modified the respiratory frequency. Norepinephrine decreased respiratory frequency whereas norepinephrine antagonists increased respiratory rate. The electrical stimulation of the caudal ventrolateral pons which inhibited the respiratory rhythm under normal bathing medium became ineffective after alpha 2-antagonist. The results herein suggest that a noradrenergic inhibitory drive, originating from the A5 area or surrounding structures modulates the activity of the medullary respiratory generator. This hypothesis is discussed in relation to A5 involvement in cardiovascular regulation.

Noradrenergic modulation of the medullary respiratory rhythm generator in the newborn rat: an in vitro study.

1. Superfused brain stem‐spinal cord preparations of newborn rats, which continue to show a rhythmic respiratory activity in vitro, were used to analyse the mechanisms whereby the A5 noradrenergic area modulates the activity of the medullary respiratory rhythm generator in the newborn. 2. In preparations including the pons (ponto‐medullary preparations), noradrenaline (NA, 25‐100 microM) added to the bathing medium either increased (n = 29/50) or decreased (n = 21/50) the respiratory frequency and elicited a tonic discharge in the cervical ventral roots in 50% of the experiments. Double‐bath experiments showed that the increases in respiratory frequency were due to NA acting on the pons, whereas the decreases in respiratory frequency were due to NA acting on the medulla. The NA‐induced increases in respiratory frequency were attributed to inhibition of A5 neurons by NA and therefore to withdrawal of A5 inhibition on the medullary rhythm respiratory generator. The NA‐induced decreases in respiratory frequency seemed to mimic the effects of endogenous NA on the A5 medullary targets. 3. Noradrenaline‐induced tonic activity (i) could be induced after elimination of the pons but not on isolated spinal cord, (ii) could be elicited by alpha 1‐ but not alpha 2‐agonists, (iii) could be blocked by alpha 1‐ but not alpha 2‐antagonists. The tonic activity therefore originated from activation of alpha 1 receptors located in the medulla but its importance in respiratory function is doubtful. 4. In medullary preparations (elimination of the pons by transection), the effects of NA agonists and antagonists on respiratory frequency were analysed. Significant decreases in respiratory frequency were induced by NA, adrenaline, phenylephrine and alpha‐methyl‐NA, but not by the agonists classified as alpha 2 (clonidine and guanfacine), alpha 1 (6‐fluoro‐NA) and beta (isoprenaline). Since yohimbine, idazoxan and piperoxane (alpha 2 antagonists) blocked the NA‐induced decreases in respiratory frequency whereas prazosin (alpha 1‐antagonist) did not, it is postulated that alpha 2‐receptors may be involved in modulating respiratory frequency. 5. Stimulation, lesion and NA microejection experiments showed the complexity of the mechanisms mediating NA‐induced changes in respiratory activity but suggested that the main site of NA action is located in the rostral ventrolateral medulla, where electrical stimulations triggered inspiration prematurely, lesions suppressed the NA‐induced decrease in respiratory frequency, and localized application of NA led to an immediate decrease in the respiratory frequency.(ABSTRACT TRUNCATED AT 400 WORDS)

Serotonergic influences on central respiratory activity: an in vitro study in the newborn rat

The in vitro brainstem-spinal cord of the newborn rat has been used to study the central effects of serotonin (5-HT) on the brainstem respiratory motor control system. Brainstem superfusion with a medium containing 5-HT (30 microM) induced a short latency increase of respiratory frequency, often (60% of the experiments) followed by delayed tonic activity. Weaker concentrations of 5-HT (10-20 microM) were ineffective but prior application of drugs limiting 5-HT inactivation (pargyline and fluoxetine) revealed 5-HT effects. Changes in respiratory frequency are: (1) completely antagonized by methysergide; (2) not suppressed by 5-HT2 (ketanserine) and 5-HT3 (zacopride, GR3832F) antagonists; and (3) induced by 5-HT1 agonists (RU24969, buspirone). Since 5-HT2 agonists (DOI, alpha-methyl-5-HT) only evoked minor changes in frequency, the central action of 5-HT on the respiratory rhythm generator seems to depend on activation of 5-HT1 receptors. Tonic activity induced by 5-HT is: (1) antagonized by methysergide or ketanserine but not 5-HT3 antagonists; (2) induced by 5-HT2 but not 5-HT1 agonists; (3) still induced in the isolated spinal cord by 5-HT superfusion or 5-HT microinjection in the cervical ventral horn; and (4) sometimes replaced by rhythmic activity at a frequency different from that of respiration. Tonic activity does not involve the central circuitry responsible for respiration but depends on 5-HT2 receptors linked to spinal networks. These results suggest that 5-HT exerts a facilitory modulation on the respiratory rhythm generator through 5-HT1 medullary receptors and on motoneurons through 5-HT2 spinal receptors.

Permanent release of noradrenaline modulates respiratory frequency in the newborn rat: an in vitro study.

1. Respiratory activity was recorded on ventral cervical roots during in vitro experiments performed on superfused newborn rat brain stem‐cervical cord preparations. 2. Eliminating the pontine structures by performing a transection at the level of the ponto‐medullary junction resulted in a sustained increase in respiratory frequency, which suggests the existence of a pontine inhibitory drive impinging on the medullary rhythm generator. 3. Noradrenaline (NA) and drugs affecting NA efficiency were added to the bathing medium and the resulting changes in respiratory frequency were analysed. NA decreased the respiratory frequency, and this effect was potentiated by pargyline (an inhibitor of the NA degradation by monoamine oxidases) and blocked by yohimbine (an alpha 2‐antagonist). 4. Yohimbine or piperoxane (which blocks the alpha 2‐adrenoceptors) increased the resting respiratory frequency to the level reached after ponto‐medullary transection, whereas pargyline or desipramine (which potentiates NA efficiency) decreased the respiratory rate. Since these effects were no longer observed after elimination of the pons, it is suggested that a permanent release of endogenous NA by pontine areas may modulate the activity of the medullary respiratory rhythm generator. 5. When alpha‐methyltyrosine (an inhibitor of NA biosynthesis) was applied to the pons, the respiratory frequency was increased, whereas when tyrosine (a precursor of NA) was applied, the respiratory frequency decreased. This decrease was enhanced by pargyline, suppressed by alpha‐methyltyrosine and blocked by piperoxane. 6. To conclude, it is suggested that the mechanisms underlying NA biosynthesis (i) continue to function under these in vitro experimental conditions and (ii) are responsible for a permanent release of endogenous NA, which slows down the respiratory frequency. These results are discussed as regards the possibility that the medullary respiratory rhythm generator may be modulated via the noradrenergic area A5 in the newborn rat.

Compared effects of serotonin on cervical and hypoglossal inspiratory activities: an in vitro study in the newborn rat.

1. Experiments were performed on the brain stem‐spinal cord preparation of newborn rats, in which the phrenic and hypoglossal nerves continue to show rhythmic respiratory activity in vitro, in order to compare the effects of serotonin (5‐HT) on both activities and to analyse the mechanisms responsible for the depression by 5‐HT of the hypoglossal activity. 2. Under control conditions, simultaneous recordings of the inspiratory discharges of hypoglossal and cervical roots showed that the two bursts did not start simultaneously and had different patterns (time‐to‐peak and peak values); this suggests that both pools of motoneurons did not share the same central drive(s). 3. Adding 5‐HT and related agents to the bathing medium delayed and depressed the hypoglossal inspiratory discharge via activation of 5‐HT2 receptors since these effects were elicited by 5‐HT2 agonists (alpha‐methyl‐5‐HT and 1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane‐HCl (DOI)) but not by 5‐HT1 agonists (RU 24969 and (+/‐)‐8‐hydroxy‐2‐(di‐N‐propylamino)tetralin hydrobromide (8‐OH‐DPAT)). The 5‐HT depression of the hypoglossal discharge was prevented by applying a pretreatment with a specific 5‐HT2 antagonist (ketanserin). Parallel to the hypoglossal discharge decrease, 5‐HT elicited a permanent cervical root discharge along with a persistent inspiratory bursting. Adding the 5‐HT precursor L‐tryptophan to the bathing medium depressed the hypoglossal (XII) discharge without affecting the cervical one. 4. Local application of 5‐HT within the hypoglossal motor nucleus decreased the hypoglossal output, revealing that the 5‐HT depression of the hypoglossal discharge was at least partly mediated by the 5‐HT effects at the level of the motoneurons. Local application of 5‐HT within the cervical motor nucleus elicited a permanent firing in the cervical root with a persistent inspiratory bursting. 5. Intracellular analysis confirmed the existence of differences in central respiratory drive between cervical and hypoglossal motoneurons under control conditions, as well as differences in response to 5‐HT. All the hypoglossal motoneurons became silent under 5‐HT bathing, and showed no change in the input membrane resistance, a moderate depolarization, and a delayed central respiratory drive with a decreased amplitude. The cervical motoneurons became more active during inspiration, despite a decrease in the amplitude of the central respiratory drive, which was compensated for by a large depolarization and an increased input membrane resistance. Some cervical motoneurons even fired at a low rate during expiration.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro study of central respiratory-like activity of the fetal rat

SummaryA fetal rat brain stem-spinal cord in vitro preparation (15–20 days) which retains for several hours respiratory-like discharges on cervical and cranial ventral roots has been developed for analysing fetal central respiratory activity. Two different patterns of easily distinguishable rhythmic activity were recorded. The first, of spinal origin, appeared every 2–10 min as long bursts of potentials (3–30 s) on cervical, but not hypoglossal, roots. The second pattern corresponded to brief bursts (1 s) of potentials occurring on both cervical and hypoglossal roots at a frequency ranging from 3–4 cycles min-1. The second type of activity was likely to be respiratory since it originated from the medulla, and behaved similarly to the respiratory activity recorded in vitro from newborn rats. The fetal respiratory-like activity was never observed at day 15, appeared at day 16 in 30% of the preparations with fluctuating frequency and amplitude bursts, and stabilised at day 20 where it was usually present and resembled newborn rat respiratory activity: its frequency was stable but was reduced by withdrawal of CO2 and pH stimuli and modulated by a pontine noradrenergic influence. This fetal preparation offers many advantages for studying the ontogeny of the central respiratory activity because of the background knowledge available on the adult and newborn rat respiratory centers and the possibility of performing electrophysiological, morphological and pharmacological fetal studies directly at the central level without any feedback from the periphery.

Acetylcholine and central chemosensitivity: in vitro study in the newborn rat.

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.

Serotonergic modulation of the respiratory rhythm generator at birth : an in vitro study in the rat

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.

Differential effects of serotonin on respiratory activity of hypoglossal and cervical motoneurons : an in vitro study on the newborn rat

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.

Functional significance of the dorsal respiratory group in adult and newborn rats : in vivo and in vitro studies

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.