Thyroliberin blocks potassium A-current in neurons of the respiratory center of adult rats in vitro

TRH is a well-known respiratory active neuropeptide. To study neuronal mechanisms of its activity, we have tested the effects of TRH on the potassium A-current in neurons of the ventrolateral solitary tract nucleus and pre-Botzinger complex in voltage-clamp experiments on adult rat brain slices. A-current was present in the neurons and it was partially and reversibly blocked by administration of THR (10(-8) M) to the bath solution. The significant decrease in amplitude of A-current was accompanied by the increase in inactivation constant (t). The effect of TRH on A-current amplitude was simulated by 5 mM 4-aminopyridine. The results presented here indicate that the stimulatory effects of TRH on neurons of the respiratory centre can be at least partially explained by its ability to block the potassium A-current.     

The effect of leu-enkephalin on membrane potential and activity of the rat respiratory center neurons in vitro

In frontal brainstem slices of Wistar rats, the whole-cell patch-clamp recordings showed the effect of opioid peptide leu-enkephalin (10 nM-1 microM) on membrane potential and spontaneous activity pattern of neurons in two divisions of the respiratory center, ventro-lateral area of the solitary tract nucleus, and the pre-B?tzinger complex. Leu-enkephalin induced a membrane hyperpolarization of the respiratory centre neurons and reduction of the spike activity level in spontaneously active units. After administration of leu-enkephalin, a decrease in frequency of bursts was found in bursting cells of the pro-B?tzinger complex; in two cases, there was a transition of bursting activity to tonic one. The data suggest that the mechanism of the central respiratory activity of leu-enkephalin is based on its direct action at the level of membrane of the respiratory centre neurons.     

Kinetics and subunit composition of NMDA receptors in respiratory-related neurons

NMDA receptors are involved in a variety of brainstem functions. The excitatory postsynaptic NMDA currents of pre-Botzinger complex interneurons and hypoglossal motoneurons, which are located in the medulla oblongata, show remarkably fast deactivation kinetics of approximately 30 ms compared with NMDA receptors in other types of neurons. Because structural heterogeneity might be the basis for physiological properties, we examined the expression of six NMDA receptor subunits (NMDAR1, NR2A-2D, and NR3A) plus eight NMDR1 splice variants in pre-Botzinger complex, hypoglossal and, for comparison, neurons from the nucleus of the solitary tract in young rats using single cell multiplex RT-PCR. Expression of NR2A, NR2B, and NR2D was observed in all three cell types while NR3A was much more abundant in pre-Botzinger complex interneurons, which belong to the rhythm generator of respiratory activity. In hypoglossal neurons, the NMDAR1 splice variants NMDAR1-4a and NMDAR1-4b were found. In neurons of the nucleus of the solitary tract, instead of NMDAR1-4b, the NMDAR1-2a splice variant was detected. This differential expression of modulatory splice variants might be the molecular basis for the characteristic functional properties of NMDA receptors, as neurons expressing a special NMDAR1 splice variant at the mRNA level show fast kinetics compared with neurons lacking this splice variant.     

Effect of leu-enkephalin on potassium currents in neurons of the respiratory center of rats in vitro

Action of opioid peptide: leu-enkephalin (10 nM - 1 microM), on K+A-current and inward rectifier in neurons of two divisions of the respiratory center: ventrolateral area of the solitary tract nucleus and the pre-Botzinger complex, was investigated in brainstem slices of Wistar rats by whole-cell voltage-clamp recordings. A-current and inward rectifier were found in all the neurons under study. A-current did not change after the application of leu-enkephalin to the bath solution while the amplitude of inward rectifier did reversibly increase. The data obtained suggest that the inhibitory effect of leu-enkephalin on neurons of the respiratory center, at least in part, can be based on its ability to modulate inward rectifier.     

Respiratory responses to microinjections of gastrin-releasing peptide into the solitary tract nucleus

In acute experiments on urethane-anesthetized rats, the respiratory effects ofmicroinjections of 10(-5), 10(-8) and 10(-10) M gastrin-releasing peptide (GRP) into the solitary tract nucleus were investigated. It was found that microinjections of the neuropeptide induced an increase in tidal volume, amplitude of diaphragm and external intercostal muscles firing activity and in expiratory duration. The most obvious respiratory responses observed when 10(-8) M GRP was used, while 10(-10) M GRP appeared to be sub-threshold and didn't alter the breathing pattern and activity of inspiratory muscles. In some experiments, where the blood pressure and the heart rate was monitored alone with breathing pattern, these parameters did not change after GRP microinjections into the solitary tract nucleus. The obtained data together with particularities of the distribution of GRP receptors in the brainstem suggest the possibility of GRP involvement into the respiratory control mechanisms at the level of solitary tract nucleus.     

Neuroanatomical and functional organization of the pre-Bötzinger complex in the cat

The technique of horseradish peroxidase retrograde axonal transport and local electrical stimulation of the pre-Botzinger complex was used to study the connections between neurones of the bulbar respiratory nucleus and descending pathways from bulbar nuclei in the cat spinal cord. A possible role of the nuclei under study for rhythmogenesis of breathing and respiratory control, is discussed.     

Orexin stimulates breathing via medullary and spinal pathways.

A central neuronal network that regulates respiration may include hypothalamic neurons that produce orexin, a peptide that influences sleep and arousal. In these experiments, we investigated 1) projections of orexin-containing neurons to the pre-Botzinger region of the rostral ventrolateral medulla that regulates rhythmic breathing and to phrenic motoneurons that innervate the diaphragm; 2) the presence of orexin A receptors in the pre-Botzinger region and in phrenic motoneurons; and 3) physiological effects of orexin administered into the pre-Botzinger region and phrenic nuclei at the C3-C4 levels. We found orexin-containing fibers within the pre-Botzinger complex. However, only 0.5% of orexin-containing neurons projected to the pre-Botzinger region, whereas 2.9% of orexin-containing neurons innervated the phrenic nucleus. Neurons of the pre-Botzinger region and phrenic nucleus stained for orexin receptors, and activation of orexin receptors by microperfusion of orexin in either site produced a dose-dependent, significant (P <0.05) increase in diaphragm electromyographic activity. These data indicate that orexin regulates respiratory activity and may have a role in the pathophysiology of sleep-related respiratory disorders.     

Respiratory effects of bombesin at the level of pre-Botzinger complex in rats

Respiratory effects of 0.1 pM - .1 mM bombesin microinjected to the pre-Botzinger complex were studied in anaesthetised rats. Bombesin induced an increase in minute ventilation, respiratory frequency, a decrease in expiratory duration and shortening of inter-burst intervals on the EMG of diaphragm and external intercostal muscles. The responses to bombesin characterised by short latency, quick development (with the maximum in 3-minutes after microinjection) and found to be reversible. The effects of bombesin on membrane potential, input resistance and pattern of spontaneous activity ofpre-Botzinger neurons were investigated in brainstem slices. 1 nM bombesin introduced into the perfusion solution in most cases (68%) induced membrane depolarisation, an increase in input resistance and in spike activity of spontaneously active cells. The data obtained suggest that the respiratory effects ofbombesin at the level ofpre-Botzinger complex are caused by its action on the membrane of neurons.     

Respiratory and hemodynamic responses to microinjections of opioids into the solitary tract nucleus

Microinjections of morphin and leu-enkephaline into the solitary tract nucleus of anaesthetised rats induced a dose-dependent decrease in tidal volume and in external intercostal muscle activity. In addition, leu-enkephaline and -endorphine decreased the respiration frequency. The respiratory effects were accompanied by a decrease in the mean blood pressure and heart rate. Naloxone administration exerted an opposite effect. The data obtained suggests an involvement of opioid peptides in respiratory and circulatory control via the solitary tract nucleus.     

respiratory responses to microinjections of bombesin into the solitary tract nucleus, and the mechanism of their realization

We investigated the respiratory effects of 10-(13)-10(-4) M bombesin microinjected into the solitary tract nucleus of adult anaesthetized rats. Bombesin markedly increased ventilation, tidal volume and electric activity of inspiratory muscles. The respiratory response was most pronounced when bombesin administered in mean concentrations (10(-10)-10(-7) M). We found that the respiratory effects ofbombesin could be based on its capacity for inhibition of Hering-Breuer inspiratory termination reflex at the level of the solitary tract nucleus. These results in aggregate with pattern of the distribution of endogenous bombesin and its receptors in the solitary tract nucleus area suggest the involvement of bombesin in the respiratory control via dorsal structures of the respiratory center.     

Postnatal developmental expressions of neurotransmitters and receptors in various brain stem nuclei of rats.

Previously, we reported that the expression of cytochrome oxidase in a number of brain stem nuclei exhibited a plateau or reduction at postnatal day (P) 3-4 and a dramatic decrease at P12, against a general increase with age. The present study examined the expression of glutamate, N-methyl-D-aspartate receptor subunit 1 (NMDAR1), GABA, GABAB receptors, glycine receptors, and glutamate receptor subunit 2 (GluR2) in the ventrolateral subnucleus of the solitary tract nucleus, nucleus ambiguus, hypoglossal nucleus, medial accessory olivary nucleus, dorsal motor nucleus of the vagus, and cuneate nucleus, from P2 to P21 in rats. Results showed that 1) the expression of glutamate increased with age in a majority of the nuclei, whereas that of NMDAR1 showed heterogeneity among the nuclei; 2) GABA and GABAB expressions decreased with age, whereas that of glycine receptors increased with age; 3) GluR2 showed two peaks, at P3-4 and P12; and 4) glutamate and NMDAR1 showed a significant reduction, whereas GABA, GABAB receptors, glycine receptors, and GluR2 exhibited a concomitant increase at P12. These features were present but less pronounced in hypoglossal nucleus and dorsal motor nucleus of the vagus and were absent in the cuneate nucleus. These data suggest that brain stem nuclei, directly or indirectly related to respiratory control, share a common developmental trend with the pre-Botzinger complex in having a transient period of imbalance between inhibitory and excitatory drives at P12. During this critical period, the respiratory system may be more vulnerable to excessive exogenous stressors.     

Comparative study of the activity of medullary respiratory neurones during polypnea triggered by hypothalamic electrical stimulation or by hypothalamic heating (author's transl)

We have compared in "encéphale isolé bas" cats the activity of medullary respiratory neurones during polypnea triggered by electrical stimulation (PSt) or by heating (PTh) of the hypothalamus. The medullary respiratory neurones are classified according to:--their anatomical localization (dorsal or ventral respiratory nucleus);--their axon destination (spinal : bulbo-spinal respiratory neurones; non spinal : propriobulbar neurones);--their discharge pattern;--the correlation coefficient between the number of spikes delivered in each burst and the duration of the corresponding respiratory phase (HILAIRE et MONTEAU, 1975). 1. During the two polypneas (PSt and PTh), we observe:--a reduction of activity that preferentially affects some groups of neurones (propriobulbar neurones) (fig. 3);--an inversion of the discharge firing rate, which increases during inspiration in normopnea and decreases in polypnea (fig. 1; fig. 6);--a decrease of the maximal discharge firing rate for the neurones of different groups (Table V). 2. However, two differences exist : during PSt, the maximal discharge firing rate increases for the inspiratory bulbo-spinal neurones of the dorsal nucleus and for the early-burster inspiratory propriobulbar neurones. The recruitment of the bulbo-spinal inspiratory neurones seems to be different; they are activated earlier during PSt than during PTh (Table VI). 3. Some of the observed differences are probably quantitative and we think that polypnea triggered by hypothalamic electrical stimulation is a good model for thermal polypnea.     

The effect of tachykinins microinjections into the solitary tract nucleus on respiration and blood circulation in rats

Administration of substance P and kassinin into the solitary tract nucleus of anesthetized rats induced a dose-dependent increase in ventilation, tidal volume, inspiratory muscle activity, and a decrease in the mean blood pressure and heart rate. Microinjections of peptides caused a decrease in ventilatory response to hypoxia and an inhibition of the Breuer-Hering reflex. The data obtained suggest involvement of tachykinins in the respiratory and circulatory control via the solitary tract nucleus.     

Neural control of deglutition and esophageal motility in mammals

Swallowing is a complex motor sequence involving the coordinated contraction of many muscles of the buccopharyngeal cavity, the larynx and the oesophagus. Most of the muscles are striated except those of the distal oesophagus which, in human and some other species, are of the smooth type. During swallowing, usually divided into a buccopharyngeal and an oesophageal stage (peristalsis), the sequential activity of the muscles results from motor orders programmed by a rhombencephalic swallowing centre and conveyed to the periphery by efferent fibres belonging to various pairs of cranial nerves (Vth, VIIth, IXth, Xth). Apart from the motor nuclei of the cranial nerves, the swallowing centre contains an nuclei of the cranial nerves, the swallowing centre contains an interneurone network responsible for the programming of deglutition. During swallowing, these interneurones (INs) exhibit a sequential activity quite parallel to that of muscles, and persisting in the absence of sensory feedback. The "swallowing INs" are located in two medullary regions: a dorsal region including the nucleus of the solitary tract and the adjacent reticular formation, a ventral region corresponding to the reticular formation surrounding the nucleus ambigus. The dorsal INs are involved in the initiation and the programming of swallowing. The ventral INs receive their swallowing input from the dorsal neurones and are probably switching neurones that distribute the swallowing excitation to the various pools of motoneurones. The swallowing program can be triggered by inputs originating from either the peripheral reflexogenic areas or the supramedullary structures (cerebral cortex in particular). Under physiological circumstances, the swallowing program is continuously modified by peripheral afferents (especially muscular) that adjust the force and the timing of contractions to the size of the swallowed bolus. In addition, an important operating feature of the programming network consists of a functional polarization so that the activity of proximal portions of the swallowing tract inhibits that of distal portions. This polarization implies the existence of inhibitory connections between interneurones, that could constitute "time-lag lines" responsible for the series of delays typical of the swallowing contractile sequence. Lastly, although the smooth muscle oesophagus contains its own programming system (intramural nervous system), motility of this area during deglutition also depends on the medullary program that combines with the intramural program by ways not yet elucidated.     

Differential effects of prostaglandin F2 alpha on oxytocinergic and non-oxytocinergic neurones in the paraventricular nucleus of the lactating rat

The effects of the prostaglandin F2 alpha (PGF2 alpha) given into the third cerebral ventricle on the unit activity of neurosecretory neurones in the paraventricular nucleus (PVN) were studied in urethane-anesthetized rats. The firing activity of PVN neurones was recorded extracellularly and 50 neurones were antidromically identified as neurosecretory neurones. Thirty of them were classified oxytocinergic neurones because they gave a burst of action potential 12-15 sec before reflex milk ejection and the remaining twenty PVN neurones which showed no response prior to reflex milk ejections were regarded as non-oxytocinergic ones. Twenty-five (83%) of the30 oxytocinergic neurones increased in the firing rate following the intraventricular (IVT) injection of PGF2 alpha (500ng in 1 microliter of isotonic saline) and the responses lasted for about 20-30 min. The remaining 5 (17%) oxytocinergic neurones showed no response in the firing rate to IVT PGF2 alpha. Fifteen (75%) of the 20 nonoxytocinergic neurones decreased in the firing activity in response to IVT PGF2 alpha, and the remaining 5 (25%) of them showed no response. IVT injection of isotonic saline (1 microliter) did not affect the firing activity of both the oxytocinergic and nonoxytocinergic cells. The intramammary pressure was slightly increased by the IVT administration of PGF2 alpha. These findings indicate that IVT PGF2 alpha has a differential action on oxytocinergic and non-oxytocinergic neurones in rats.     

Electrophysiological Evidence for Oxytocin Receptors on Neurones Located in the Dorsal Motor Nucleus of the Vagus Nerve in the Rat Brainstem

Abstract

Intracellular recordings were obtained from vagal neurones and their response to oxytocin was investigated in slices from the rat brainstem. Following recording, Lucifer Yellow was injected into the cells in order to verify their localization within the dorsal motor nucleus of the vagus nerve. Virtually all neurones throughout the rostro-caudal extent of the nucleus increased their rate of firing in the presence of 10-1000 nM oxytocin and their membrane depolarized in a reversible, concentration-dependent manner. This excitation was probably exerted directly on the impaled cells rather than being synaptically mediated, since it persisted in a low calcium-high magnesium medium or in the presence of tetrodotoxin. These data provide evidence for a direct membrane effect of oxytocin on a defined population of neurones in the rat brain.     

Respiratory responses to microinjections of leptin into the solitary tract nucleus

Regulatory polypeptide leptin, apart from its well-known hypothalamic effects, stimulates ventilation. The present study on anaesthetised rats was undertaken to elucidate the respiratory effects of 10(-10)-10(-4) M leptin microinjected into the solitary tract nucleus, containing a high concentration of leptin receptors. Injections of 10(-8)-10(-4) M leptin induced dose-dependent increase in ventilation, tidal volume and electric activity of inspiratory muscles; 10(-6) M leptin additionally induced a short-term increase in respiratory frequency and a shortening of both inspiratory and expiratory duration. The respiratory responses to leptin is also characterised by appearance of sighs: deep and prolonged inspirations associated with an augmented burst in the activity of the inspiratory muscles and prolonged post-sigh inter-burst interval. The results taken together with evidence of high concentration of specific leptin ObRb-receptor in the solitary tract nucleus suggest involvement of endogenous leptin in the control of breathing via dorsal structures of the respiratory center.     

Motor nucleus of the V-th nerve and the control of breathing. Breuer-Hering reflexes and apneustic breathing

Earlier studies from this department have demonstrated that neurones of the V-th nerve motor nucleus (NVmt) have oligosynaptic, inhibitory output to the inspiratory motoneurone themselves under the influence of a polysynaptic input from vagal afferents. To check the hypothesis that NVmt is a part of the pneumotaxic mechanism, we studied the effects of pharmacological microblockade of the NVmt on Breuer-Hering reflexes in halothane-anesthetized, paralyzed and artificially ventilated rabbits. Activities of NVmt neurones and phrenic nerve firing were recorded. Acid-base balance was controlled and histologic examinations were routinely performed. Expiratory activities were regulatory found in NVmt. Its blockade elicited a typical apneustic breathing. During the blockade the Breuer-Hering reflexes gave "paradoxical" effects: an increase in central respiratory frequency following inflation, inspiratory apneusis in response to deflation. We conclude that the NVmt is an important component of the pneumotaxic mechanism or even the anatomical substrate of the pneumotaxic "centre".     

Effect of substance P and thyrotropin-releasing hormone on sympathetic preganglionic neurones in the upper thoracic intermediolateral nucleus of the cat

When applied by iontophoresis onto single sympathetic preganglionic neurones in he intermediolateral nucleus of spinal segments T1-T3 in the cat, substance P and thyrotropin-releasing hormone (TRH) each had a weak excitatory effect. Two-thirds of the neurones studied were excited by substance P while one-fifth were excited by TRH. The time courses of the responses to substance P and to TRH were similar, and consisted of an increase in the rate of discharge with a latency of approximately 30 s from the onset of application. They were also prolonged (30-320 s) in afterdischarge following termination of application. These results indicate that substance P and TRH exert excitatory effects on single sympathetic pregnanglionic neurones, and support the possibility that they may be chemical mediators of synaptic transmission in the intermediolateral nucleus.     

家兔脑干呼吸相关结构向斜方体后的轴突投射—CB—HRP逆行示踪研究

实验在10只成年家兔上进行,斜方体后核（RTN）内微量注入霍乱毒素β亚单位耦合辣根过氧化酶（CB－HRP）后,在脑桥Koelliker-Fuse 核,臂旁内侧核及臂旁外侧核观察到大量HRP标记神经元,在延髓孤束核腹外侧区,疑核和后疑核,面神经后核的腹侧及内侧区观察到少数HRP标记神经元,在面神经后核,疑核及后疑核区域观察到大量HRP顺行标记末梢纤维,实验结果表明,RTN和脑桥及延髓的呼吸相关结构之间存在纤维联系。