Responses of early and late onset phrenic motoneurons to lung inflation

In anesthetized or decerebrate cats that were paralyzed and ventilated with a cycle-triggered pump, we produced changes in activity of the whole phrenic nerve and of individual phrenic motoneurons (fibers or cells in the spinal cord) by withholding lung inflation during the inspiratory (I) phase. The neurons were classified into early- and late-onset types (discharge onset less or greater than 80 msec, respectively, after whole phrenic onset). Both unit and whole phrenic activity exhibited a variety of responses to inflation (excitation, depression, or no effect); but there were no consistent differences between responses of early- and late-onset neurons. The distribution of responses was quite different from that of dorsal respiratory group (DRG) I neurons (Cohen and Feldman, 1984); in particular there was no group of phrenic neurons corresponding to the late-onset I-beta neurons (I neurons excited by inflation). We conclude that the inputs to late-onset phrenic neurons are not predominantly or exclusively from late-onset DRG neurons.     

The role of neural connections crossed at the cervical level in determining rhythm and amplitude of respiration in cats and rabbits

The part played by crossed cervical pathways in determining respiratory rhythm and phrenic nerve amplitude was studied in rabbits and cats. The animals were anesthetized with halothane, paralyzed, vagotomized and mechanically ventilated. All decussating brain stem pathways were surgically interrupted. This resulted in asynchronous firing of the respiratory nerves on the left and right side of the body. However, both frequency and amplitude of the respiratory output depended on the mutual phase relationships due to neural connections crossed at the cervical level. Analysis of the mutual phase and amplitude relationships lead to the following conclusions. a. Phrenic motor neurons receive during inspiration a considerable amount of excitation via diffused pathways crossing the midline at C4-C6 level. b. Phrenic motor neurons are actively inhibited via crossed cervical pathways. c. The level of tonic bias transmitted via descending bulbo-spinal pathways significantly influences the magnitude of PhN output. d. Activity of phrenic motor neurons is transmitted back to the bulbar respiratory centers via an ascending spino-bulbar pathway.     

Functions of the retrofacial nucleus in chemosensitivity and ventilatory neurogenesis

The hypothesis was evaluated that neurons within the retrofacial nucleus of medulla integrate afferent stimuli from the central chemoreceptors. In decerebrate, vagotomized, paralyzed and ventilated cats, activity of the phrenic nerve was monitored. Peak integrated phrenic activity increased in hypercapnia; the frequency of phrenic bursts typically declined slightly. The retrofacial nucleus was ablated by radio-frequency lesions or neurons within this nucleus were destroyed by microinjections of kainic acid. Results were similar following lesions or injections. Following unilateral ablations, peak phrenic activity was greatly reduced at normocapnia and hypercapnia; the frequency of phrenic bursts typically rose. Both frequency and peak phrenic activity fell further after the contralateral destruction with a cessation of all phasic phrenic discharge being observed in most animals. Injections of kainic acid in regions rostral, caudal or medial to the retrofacial nucleus produced no consistent changes in phrenic activity. We conclude that neuronal activities in the region of the retrofacial nucleus are important both in the integration of stimuli from the central chemoreceptors and in defining the discharge patterns of respiratory neurons.     

Respiratory resetting induced by activation of inspiratory bulbo-spinal neurons

The respiratory effects elicited by spinal (C2-C3) stimulation at the level of descending inspiratory axons were studied in paralysed, non-vagotomized and artificially ventilated cats anaesthetized with urethane-chloralose. The activation of inspiratory bulbospinal axons in the ventrolateral quadrant was confirmed by recording the ipsilateral phrenic excitation following a single pulse. Brief stimulus trains delivered at the same locus during expiration elicited short- and long-term phrenic activations. The short-term activation consisted of a tetanic orthodromic response. The long-term activation, of central origin, exhibited the same pattern as a spontaneous inspiration and consisted of an inspiratory resetting which necessitated weak anaesthesia and light hypocapnia. Control experiments (restricted lesions of the medulla and the cervical cord, recording of afferent activity in thalamic sensory nuclei, medullary stimulation) revealed that this inspiratory resetting could not be related to appreciable activation of either non-respiratory efferents or spinal afferent pathways studied but was likely to depend on the activation of the descending inspiratory axons. We conclude that the respiratory resetting obtained by spinal stimulation resulted from mass antidromic activation of the inspiratory bulbospinal neurons which thus appear to be involved in the generation of the respiratory rhythm.     

Properties of postganglionic sympathetic neurons with axons in phrenic nerve.

The aim of the study was to test the reflex and resting properties of postganglionic sympathetic neurons with axons located in the right phrenic nerve. The experiments have been performed on chloralose-anesthetized cats with both vago-aortic nerves cut. The somata or the postganglionic sympathetic neurons were located in the stellate ganglion. Axons of these neurons passed through the upper and lower phrenic nerve roots and through the phrenic nerve itself. The presence of cardiac and respiratory rhythmicities was detected in the activity of the phrenic postganglionic sympathetic neurons. Hyperventilation, which abolished burst discharges of the phrenic nerve, decreased the sympathetic activity by 14%. Systemic hypoxia (ventilating the animals for 2 min with 8% O2 in N2) increased the sympathetic activity threefold. The results of our experiments suggest that axons of the sympathetic neurons located in the right phrenic nerve could possibly be diaphragmatic muscle vasoconstrictors.     

A novel inhibitor of the Na+/H+ exchanger type 3 activates the central respiratory CO2 response and lowers the apneic threshold.

Cultured CO2-sensitive neurons from the ventrolateral medulla of newborn rats enhanced their bioelectric activity upon intracellular acidification induced by inhibition of the Na+/H+ exchanger type 3 (NHE3). Now we detected NHE3 also in the medulla oblongata of adult rabbits. Therefore, this animal model was employed to determine whether NHE3 inhibition also affects central respiratory chemosensitivity in vivo. Seven anesthetized (pentobarbital), vagotomized, paralyzed rabbits were artificially ventilated with O2-enriched air. From the phrenic nerve compound discharge, integrated burst amplitude (IPNA), respiratory rate (fR), and phrenic minute activity (IPNA. fR) were taken as measures of central respiratory rhythm and drive. Effects of potent NHE3 inhibition with the novel brain permeant substance S8218 were studied by comparing respiratory characteristics before and after up to 9.2 +/- 1.1 mg/kg cumulative drug application, yielding average plasma concentrations of 0.9 +/- 0.2 microg/ml. In response to S8218, the baseline level of IPNA. fR was significantly enhanced by an average of 51.0 +/- 6.4% (n = 27, p < 0.0001). The influence of NHE3 inhibition on the respiratory CO2 response was studied at plasma concentrations of S8218 maintained in the range of 0.3 microg/ml (10(-6) M). Although the metabolic acid-base status thereby remained widely unchanged, the group mean apneic threshold PaCO2 was significantly lowered by 0.45 +/- 0.11 kPa (n = 7, p < 0.01), whereby in four of seven animals even strong hyperventilation failed to suppress phrenic nerve rhythmicity completely. Likewise, S8218 significantly augmented IPNA. fR, in the range of PaCO2 between 1 and 6 kPa above threshold, by an average of 38.0 +/- 8.5% (n = 35, p < 0.0001). These in vivo results are compatible with the effects of NHE3 inhibition on chemosensitive brainstem neurons in vitro. Moreover, rhythmogenesis is supported through NHE3 inhibition by lowering the threshold PCO2 for central apnea.     

Phrenic afferent input to the lateral medullary reticular formation of the cat

The afferent inputs from phrenic nerve stimulation to the lateral reticular formation of the lower brain stem were studied in anesthetized spontaneously breathing cats. The activity of reticular neurons was recorded by means of extracellular tungsten microelectrodes. Electrical stimulation of the central end of the right phrenic nerve evoked excitatory or inhibitory responses in the lateral reticular nucleus (LRN), in the nucleus ambiguus (AMB) and in a region dorsal to the AMB of ipsi- and contralateral sides. Phrenic afferents belonging to the flexor reflex afferent group were involved in these responses. The discharge pattern of the respiratory related units (RRU) of the AMB were exceptionally affected by phrenic nerve stimulations. It is concluded that high threshold phrenic afferents relay in the LRN before projecting to the cerebellar cortex. The overlapping of respiratory and non-respiratory afferents in the reticular formation may participate to the adaptations of respiratory and somatomotor functions during specific behaviors.     

Action of intercostal muscle afferents on the respiratory rhythm of anesthetized cats.

Experiments designed to reveal an action of intercostal afferent stimulation on the rhythm of breathing were carried out on barbiturate-anesthetized, paralyzed cats, ventilated by a positive pressure respirator. Tetanic stimuli were applied to midthoracic, external intercostal nerves of intact animals and of animals displaying an apneustic pattern consequent to vagotomy and pontine lesions. In intact animals, the respiratory rhythm, as indicated by phrenic efferent activity, could be paced by intercostal nerve stimuli when they were timed to occur in a particular relationship to the lung volume cycle. Apparently, the action of the stimuli supplemented the phasic, volume-related afferent input. The response required considerable spatial summation and resulted from the action of intercostal afferents to either shorten inspiration or prolong expiration. These reflex effects could be attributed to activation of group II afferents. Although the actions of these afferents were relatively weak in the intact preparation, they proved potent in terminating an apneustic breath; brief stimuli, if they activated group II afferents, cut short phrenic discharge and initiated an expiratory phase, thereby restoring a reasonably normal respiratory rhythm.     

Respiration-related neurons in the medial nuclear complex of the solitary tract of the cat

Respiration-related (RR) neuronal activity was systematically searched for in the nuclear complex of the solitary tract (nTS) with special interest focused on regions located medially to the ventrolateral subnucleus (vlnTS) which is widely considered to be the anatomical location of the dorsal respiratory group (DRG). The experiments were carried out on cats under pentobarbitone anaesthesia, paralyzed and artificially ventilated. RR activity was recorded consistently not only in the vlnTS, but also in more medial regions. R alpha-type neurons were often encountered in ventral and medial aspects of the nTS. R beta-type neurons and expiration-related neurons were found also in ventral regions of the nTS. A small number of 'late onset' and 'early burst' inspiration-related neurons was found respectively in ventral and ventromedial regions of the nTS. The activity of neurons presenting a rhythmic pattern completely determined by lung volume changes due to the respirator was consistently recorded both in ventral and medial areas of the nTS. Most of these neurons were excited by lung inflation and displayed a discharge pattern characteristic of P-neurons. A few neurons, designated as 'inverse' P-neurons displayed an opposite behaviour, being inhibited by lung inflation. The results are consistent with both histological and neurophysiological studies showing that pulmonary stretch receptor afferents terminate also on the medial side of the solitary tract. It is suggested that the DRG is not restricted to the vlnTS, but comprises also neurons located in more medial regions of the nTS.     

Determinants of breathing frequency during walking

Breathing frequency has been shown to depend on metabolic demand, and also to be affected by stepping frequency during walking. To assess the influence of stepping frequency on breathing frequency, we recorded the timing of breathing and stepping in ten naive subjects walking on a treadmill. Five of the subjects showed periods of breathing in rhythm with their stepping (rhythmical entrainment); five subjects never showed rhythmical entrainment. In all subjects, respiratory frequency tended to increase with increases in estimated metabolic rate (EMR) produced by increases in walking speed or treadmill inclination. Breathing frequency was also affected by stepping frequency independent of EMR, both during rhythmical entrainment and in the absence of rhythmical entrainment. The dependence of breathing frequency on stepping frequency may reflect an excitatory influence of voluntary movement on the respiratory center. We conclude that stepping importantly influences breathing frequency during walking whether or not the two are rhythmically linked. Such lability of respiratory frequency probably reflects the small metabolic cost of departure from mechanically optimal frequencies.     

Neuroimaging evidence implicating cerebellum in the experience of hypercapnia and hunger for air

Recent neuroimaging and neurological data implicate cerebellum in nonmotor sensory, cognitive, vegetative, and affective functions. The present study assessed cerebellar responses when the urge to breathe is stimulated by inhaled CO(2). Ventilation changes follow arterial blood partial pressure CO(2) changes sensed by the medullary ventral respiratory group (VRG) and hypothalamus, entraining changes in midbrain, pons, thalamus, limbic, paralimbic, and insular regions. Nearly all these areas are known to connect anatomically with the cerebellum. Using positron emission tomography, we measured regional brain blood flow during acute CO(2)-induced breathlessness in humans. Separable physiological and subjective effects (air hunger) were assessed by comparisons with various respiratory control conditions. The conjoint physiological effects of hypercapnia and the consequent air hunger produced strong bilateral, near-midline activations of the cerebellum in anterior quadrangular, central, and lingula lobules, and in many areas of posterior quadrangular, tonsil, biventer, declive, and inferior semilunar lobules. The primal emotion of air hunger, dissociated from hypercapnia, activated midline regions of the central lobule. The distributed activity across the cerebellum is similar to that for thirst, hunger, and their satiation. Four possible interpretations of cerebellar function(s) here are that: it subserves implicit intentions to access air; it provides predictive internal models about the consequences of CO(2) inhalation; it modulates emotional responses; and that while some cerebellar regions monitor sensory acquisition in the VRG (CO(2) concentration), others influence VRG to adjust respiratory rate to optimize partial pressure CO(2), and others still monitor and optimize the acquisition of other sensory data in service of air hunger aroused vigilance.     

Respiratory-associated thalamic activity is related to level of respiratory drive.

We recorded phrenic nerve activity and thalamic single unit firing in unanesthetized, suprathalamically decerebrated, paralyzed and ventilated cats, in which vagi and carotid sinus nerves (CSN) had been ablated. Seventy-six (14%) of 545 neurons in regions of the thalamus related to the ascending reticular system, which had been tonically firing at low respiratory drives, developed rhythmic increases of firing associated with each respiration when drive had been increased by CSN stimulation or hypercapnia. The increases of neuronal firing occurred in late inspiration/post-inspiration but sometimes lasted into expiration; the magnitude of change was graded according to the magnitude of respiratory activity. Thalamic neurons also fired with a rhythm related to ventilator-induced chest expansion, some units showing both the respiratory-associated and the ventilator-related rhythms. Simultaneously recorded mesencephalic and thalamic neurons developed similar rhythms when drive was increased. We suggest that these neuronal activities reflect the conveyance of information about respiration to the cortex, where it may lead to the sensation of dyspnea and perhaps to arousal.     

Reflex inhibition of crural diaphragmatic activity by esophageal distention in cats

Distention of the esophagus has been shown to result in selective inhibition of phasic inspiratory activity in the crural portion of the diaphragm, with no effect on costal diaphragmatic activity. The purpose of this study was to determine rigourously the afferent pathways that mediate this response. Bipolar EMG electrodes were placed in the costal and crural portions of the diaphragm in decerebrate, spontaneously breathing cats. Distention of the esophagus by inflation of a Foley catheter balloon with 20 ml of air resulted in a selective inhibition of crural hiatal EMG activity, while costal EMG activity was maintained at predistention levels. The distention was accompanied by a reduction in respiratory frequency. Transection of the spinal cord at the C8-T1 level did not obliterate the crural inhibition produced by inflation. Section of the C4-C8 dorsal roots also failed to abolish the response. However, after bilateral cervical vagotomy, esophageal distention no longer influenced diaphragmatic EMG activity. These results indicate that the crural inhibition observed with esophageal distention is vagally mediated and is not influenced importantly by intercostal or phrenic afferents. Records of activity of the phrenic nerve branch innervating the crural portion of the diaphragm showed a similar response pattern, confirming that the inhibition is central in origin and that the crural fibers inhibited by distention are only a fraction of the total population of crural phrenic motoneurons.     

Entrainment of the respiratory rhythm by periodic lung inflation during vagal cooling

The aim of this study was to determine whether pulmonary receptors other than slowly adapting stretch receptors are capable of entraining the respiratory rhythm when periodically stimulated during artificial ventilation. Experiments were performed on anaesthetised (urethane, 1.5 g/kg) and paralysed (pancuronium bromide, 0.1 mg/kg) rabbits. Vagi were cooled in order to block conduction in the myelinated fibres innervating slowly adapting receptors. The effectiveness of this cooling was assimilated to the absence of the Hering-Breuer inflation reflex and the presence of the deflation reflex. Our results indicate that under such conditions: (1) harmonic entrainment (one phrenic burst for one pump period) can be observed, (2) the range of harmonic entrainment is more limited when the vagi are cooled, and (3) during harmonic entrainment the inspiratory duration and phrenic activity are similar to what is observed at the same period with intact vagi, whereas vagal cooling modifies the phase difference between the phrenic burst and the pump. We have concluded that periodic input from rapidly adapting receptors and/or vagal C-fibres can entrain the respiratory rhythm as does input from slowly adapting receptors but with different patterns as evidenced by phase relationship.     

大剂量阿托品对氧化乐果中毒大鼠膈肌功能的影响

目的探讨大剂量阿托品对膈肌功能的影响。方法使用MS-302生理药理分析仪测定10只大鼠在两种不同条件下（对照组用生理盐水皮下注射,中毒组用氧化乐果皮下注射）,大鼠反复加入阿托品后对膈肌功能的影响情况。结果两组大鼠膈肌功能随阿托品累积剂量增至0.6mg时,与阿托品0.2mg时的膈肌收缩功能相比均明显减弱(P<0.01);并随着阿托品累积量增至0.8mg时,膈肌功能接近完全抑制;当膈肌中阿托品清除约50min后,两组膈肌收缩功能几乎完全恢复正常。结论反复给予大剂量阿托品可以使大鼠膈肌功能减退,导致外周呼吸肌麻痹。     

COPD对大鼠膈神经放电和呼吸运动的影响探讨

目的探讨COPD对大鼠膈神经放电和呼吸运动的影响。方法随机将60只Wistar大鼠分为两组,30只/组。实验组:熏香烟联合气管内滴注猪胰蛋白酶建立大鼠COPD模型;对照组:不给予熏香烟,d15气管内滴注生理盐水20 u/100 g。结果实验组膈神经放电幅度增强且具有统计学意义(P0.05);两组吸气相时间和膈神经放电活动均呈正相关。结论 COPD可影响大鼠膈神经放电和呼吸运动,使膈神经放电活动增强,出现呼吸中枢吸气驱动增高的现象,提示大鼠呼吸中枢的活动可能发生了改变。     

Stimulating fastigial nucleus alters central mechanisms regulating phrenic activity

In paralyzed, mechanically ventilated, anesthetized cats, 30-sec trains of electrical stimulation in the rostral fastigial nucleus caused either respiratory excitation or inhibition (apnea) of respiration. The response elicited by stimulation was dependent on the frequency of stimulation: the duration of apnea decreased and respiratory excitation occurred more frequently at lower frequencies of stimulation. Excitation was characterized by increases of f and the rate of rise and peak (TNA) integrated activity of the phrenic nerve. TI and TE decreased. TNA and f remained elevated for at least three minutes after excitatory respiratory responses. Also, respiration was frequently elevated after an inhibitory response. Brief stimulation (100-200 ms) administered during expiration or inspiration altered either TE or TI and TNA, respectively. In addition, brief stimulation elicited short-latency inhibition or excitation of phrenic nerve activity. These effects were often unassociated with other phase changes. We conclude that activation of neurons or axons within the rostral fastigial nucleus can modulate activity of the phrenic nerve by altering the activity of at least three separate central mechanisms.     

Effects of DA-9701, a Novel Prokinetic Agent,on Phosphorylated Extracellular Signal-Regulated Kinase Expression in the Dorsal Root Ganglion and Spinal Cord Induced by Colorectal Distension in Rats

Background/Aims

DA-9701, a standardized extract of Pharbitis Semen and Corydalis Tuber, is a new prokinetic agent that exhibits an analgesic effect on the abdomen. We investigated whether DA-9701 affects visceral pain induced by colorectal distension (CRD) in rats.

Methods

A total of 21 rats were divided into three groups: group A (no CRD+no drug), group B (CRD+no drug), and group C (CRD+DA-9701). Expression of pain-related factors, substance P (SP), c-fos, and phosphorylated extracellular signal-regulated kinase (p-ERK) in the dorsal root ganglion (DRG) and spinal cord was determined by immunohistochemical staining and Western blotting.

Results

The proportions of neurons in the DRG and spinal cord expressing SP, c-fos, and p-ERK were higher in group B than in group A. In the group C, the proportion of neurons in the DRG and spinal cord expressing p-ERK was lower than that in group B. Western blot results for p-ERK in the spinal cord indicated a higher level of expression in group B than in group A and a lower level of expression in group C than in group B.

Conclusions

DA-9701 may decrease visceral pain via the downregulation of p-ERK in the DRG and spinal cord.     

Synchronized rhythms in chemosensitive neurons of the locus coeruleus in the absence of chemical synaptic transmission.

The activity of locus coeruleus (LC) neurons was examined in the en bloc isolated brainstem-spinal cord of the neonatal rat using paired whole cell or whole cell plus extracellular recording. In artificial cerebrospinal fluid (ACSF) LC neurons were synchronized by their respiratory innervation and in some neurons showing tonic or burst patterns of discharge these patterns of discharge could also be synchronized. Replacing ACSF with low Ca(2+)-high Mg(2+) generated synchronized rhythmic bursts which remained synchronized at high CO(2) (up to 20%). This rhythm was suppressed by TTX. Substitution of Ba(2+) for Ca(2+) in ACSF generated a synchronized rhythm which was TTX-insensitive. The frequency of this rhythm increased by 31+/-16% on raising CO(2) concentration from 2 to 10%. We conclude that the capacity of chemosensitive LC neurons to generate a synchronized rhythm depends on their electrical coupling, but not on chemical synaptic transmission.     

Lesions of regions for in vitro ventilatory genesis eliminate gasping but not eupnea

Medullary regions, termed `pre-inspiratory' and `pre-Bötzinger', are considered critical for the neurogenesis of rhythmic ventilatory activity of in vitro preparations of the neonatal rat. We examined the influence of destruction of neurons in these regions, by microinjections of kainic acid, upon eupnea and gasping in vivo. Decerebrate, vagotomized, paralyzed and ventilated rats of age 8–15 days were used; the phrenic nerve activity was recorded. Eupnea was not consistently altered following destruction of neurons in any region. However, in the majority of animals, anoxia-induced gasping was not observed following injections of kainic acid into the `pre-inspiratory' region, `pre-Bötzinger' complex or lateral tegmental field; the latter region is important for the neurogenesis of gasping in adults. Injections into other regions did not prevent the elicitation of gasping. These results do not support the possibility that neuronal activities which are responsible for respiratory rhythm generation in vitro underlie the neurogenesis of eupnea in vivo.