Stimulation of the rat medullary raphe nuclei induces differential responses in respiratory muscle activity

Neural control circuits that coordinate the motor activity of the diaphragm (DIA) and the geniohyoid muscle (GH) are potentially involved in pathological conditions such as various forms of sleep apnea. Here we investigated a differential role of the raphe magnus (RMg), pallidus (RPa) and the obscurus (ROb) nuclei in the neural control of DIA and GH muscle activity in rats under volatile anesthesia. In order to characterize a topographical organization of the raphe nuclei we analyzed changes in DIA and GH during high-frequency stimulation (HFS, 10–130 Hz, 60 μs pulse width, 40–160 μA, 30 s). HFS of the RMg and the ROb induced apnea, in the latter case apnea was associated with massive tonic discharge in the GH. By contrast, HFS of the RPa induced tachypnea. At caudal stimulation sites the tachypnea was accompanied by tonic DIA activity and cessation of GH. These data suggest a differential distribution of inhibitory and excitatory drives of DIA and GH muscles within distinct raphe nuclei.     

Anoxic disturbance of the isolated respiratory network of neonatal rats

Tissue oxygen (PO₂), K⁺ (aK_e), pH (pH_e) and Ca²⁺ ([Ca²⁺]_e) were measured in the region of the ventral respiratory group (VRG) in the in vitro brainstem-spinal cord preparation of neonatal rats. During tissue anoxia, elicited by superfusion of N₂-gassed solutions, an initial increase in the frequency of respiratory activity, lasting between 2 and 12 min, turned into a frequency depression. During anoxia periods of up to 60 min, respiratory activity persisted in solutions containing CO₂/bicarbonate, whereas a complete blockade was observed after 15–25 min in N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid- (Hepes)-buffered salines. After such anoxic apnea, respiratory rhythmicity could be reactivated by superfusion of hypoxic, CO₂/bicarbonate-buffered solutions. In both types of hypoxic solutions, aK_e increased by maximally 1.5mM, whereas an initial increase of pH_e by up to 0.05 pH units turned, after 2–4 min, into an acidification which could exceed 0.5 pH units. In contrast, [Ca²⁺]_e remained unaffected by anoxia. Addition of 2–5 mM cyanide (CN^-) to oxygenated Hepes-buffered saline evoked an increase in PO₂ in the VRG from 100 to more than 300 mmHg. The effects of CN^- on respiratory activity, aK_e and pH_e were almost identical to those during anoxia. In oxygenated, CO₂/bicarbonatefree solutions of different pH, however, an increase in pH_e in the VRG led to a decrease in respiratory frequency, whereas a fall of pH_e produced a frequency acceleration. A rise of aK_e in the VRG by more than 2 mM as induced by superfusion of a 7 mM K⁺ solution led to a sustained increase of respiratory frequency. The results indicate that blockade of aerobic metabolism does not severely perturb K⁺ and Ca²⁺ homeostasis and that the biphasic response to anoxia is not directly related to the observed changes in PO₂, aK_e, pH_e, or [Ca²⁺]_e. In the respiratory network of neonatal mammals, CO₂ might provide a stimulus for long-term maintenance of respiratory activity under oxygen depletion.     

Effect of lingual paralysis on swallowing and breathing coordination in rats

Background

The tongue plays an important role in both swallowing and breathing. Lingual motor deficits with consequences for swallowing are often observed in many neurological disorders. However, the impact of such deficits on swallowing and breathing coordination is unknown.

Objective

Our objective was to study the swallowing and ventilatory patterns in rats with unilateral lingual paralysis.

Methods

Our study was carried out on 10 Wistar rats. Respiratory variables in unrestrained and healthy animals were measured at rest and during water swallowing using whole-body plethysmography and a video camera. The procedure was repeated after unilateral sectioning of the hypoglossal nerve (XII). Swallowing frequency and occurrence during inspiration and expiration, tidal volume, total time of the ventilatory cycle and respiratory drive were assessed.

Results

Unilateral sectioning of the hypoglossal nerve led to a swallowing deficit and drooling. Respiratory rhythm and ventilatory drive (16.39 ± 2.13 ml/s vs 13.67 ± 1.28 ml/s) during swallowing decreased after hypoglossal nerve sectioning but were unaffected during rest without swallowing, while swallowing rate (17 ± 5/15 s) and occurrence during respiratory cycle phases did not change.

Conclusions

A swallowing deficit induced by lingual paralysis decreased the ventilatory drive during swallowing. This may be a mechanism to reduce or prevent pulmonary aspiration.     

成年大鼠延髓薄片制备的改良方法

目的:建立一种适用于膜片钳记录研究的成年大鼠延髓薄片制备方法。方法:用改制的注射器将延髓和上颈髓段从离断的椎管中直接吹出;采用水平切的方式制备延髓薄片;记录三叉神经脊束核尾侧亚核(Vc)神经元的自发放电和诱发放电活动。结果:分离得到的延髓和上颈髓标本光滑完整;水平切的延髓薄片较好地保持了Vc的形态学结构和神经元活性,可较好地记录到Vc神经元自发的兴奋性突触后电流和诱发的双脉冲抑制活动。结论:本方法操作简单,取材快速,延髓薄片外形完整且能保持神经元活性,适用于成年大鼠Vc的膜片钳研究。     

Effects of midbrain raphe destruction on sleep and locomotor activity in rats.

This study deals with the question whether the often reported sleep loss in animals with lesions in the rostral midbrain raphe system (the dorsal and medial raphe nuclei) might be explained by the hyperactivity shown by the animals when confronted with unfamiliar environments. Rats with electrolytic raphe lesions were compared with sham-operated rats as to both sleep and activity parameters under 3 conditions differing in familiarity. In spite of a reduction of forebrain serotonin and 5-hydroxy-indoleacetic acid of about 60–70%, raphe-lesioned rats could not be distinguished from sham-operated rats as tosleep parameters such as slow-wave sleep and REM sleep under different conditions. Thus, structural integrity of serotonin-containing neurons was not found to be a necessary condition for the normal production of sleep in rats. Locomotor activity, however, was significantly increased in raphe-lesioned rats, but this increase showed no relationship with sleep production.     

Connections between respiratory neurones in the neonatal rat transverse medullary slice studied with cross-correlation

In the transverse medullary slice prepared from neonatal rats the hypoglossal nerve rootlets exhibit a bursting 'respiratory' rhythm as do neurones in the pre-Bötzinger complex (PBC). We used cross-correlation analysis of the rhythmic multiunit discharges recorded from hypoglossal nerve rootlets, hypoglossal nucleus neurones and PBC neurones to investigate the connections between these groups. All cross-correlograms computed between left and right hypoglossal nerves, and between hypoglossal neurones and contralateral hypoglossal nerves, displayed central peaks with broad half-amplitude widths (mean ± s.d. of 29.6 ± 10.4 and 37.3 ± 6.0 ms, respectively), which we interpreted as evidence for activation from a common source. Five of the 18 cross-correlograms computed between left and right PBC neurones displayed peaks either side of time zero with narrower half-amplitude widths (mean ± s.d. of 9.3 ± 1.9 ms) superimposed on broader central peaks, which we interpreted as evidence for mutual excitation and common activation, respectively. Cross-correlograms computed between PBC neurones and contralateral hypoglossal neurones or nerves did not display consistent features, but some of those computed between PBC and ipsilateral hypoglossal neurones (two of eight) or nerves (two of five) displayed peaks with broad half-amplitude widths (mean ± s.d. of 36.8 ± 6.9 ms), offset from time zero by 6 ms (except for one at 18 ms), which we interpreted as evidence for excitation of hypoglossal neurones and motoneurones by PBC neurones. We concluded that rhythm is synchronised between left and right sides by mutual excitatory connections between left and right PBC neurones. The rhythm is transmitted to ipsilateral hypoglossal neurones by a paucisynaptic pathway. Both hypoglossal neurones and PBC neurones receive a common activation from as yet unidentified sources.     

The rhythmic,transverse medullary slice preparation in respiratory neurobiology: Contributions and caveats

Our understanding of the sites and mechanisms underlying rhythmic breathing as well as the neuromodulatory control of respiratory rhythm, pattern, and respiratory motoneuron excitability during perinatal development has advanced significantly over the last 20 years. A major catalyst was the development in 1991 of the rhythmically-active medullary slice preparation, which provided precise mechanical and chemical control over the network as well as enhanced physical and optical access to key brainstem regions. Insights obtained in vitro have informed multiple mechanistic hypotheses. In vivo tests of these hypotheses, performed under conditions of reduced control and precision but more obvious physiological relevance, have clearly established the significance for respiratory neurobiology of the rhythmic slice preparation. We review the contributions of this preparation to current understanding/concepts in respiratory control, and outline the limitations of this approach in the context of studying rhythm and pattern generation, homeostatic control mechanisms and murine models of human genetic disorders that feature prominent breathing disturbances.     

Acetazolamide and respiratory chemosensitivity to CO(2) in the neonatal rat transverse medullary slice

Hypoglossal nerve rootlets in the transverse medullary slice prepared from neonatal rats exhibit a bursting 'respiratory' rhythm that increases in frequency with CO(2), presumably due to activation of chemosensitive cells such as the central chemoreceptors. Carbonic anhydrase is associated with areas of central chemoreception and we propose a hypothesis for its involvement in the chemoreception process. We tested this hypothesis by blocking its activity with acetazolamide in six slice preparations. However, the addition of 1 mM acetazolamide dissolved in dimethyl sulphoxide to the superfusing bathing solution produced no alteration in the bursting frequency response of the slice to CO(2). We concluded that the chemoreception process producing the CO(2) response of the superfused, transverse medullary slice does not involve carbonic anhydrase.     

Bilateral vagotomy differentially alters the magnitude of hypoglossal and phrenic long-term facilitation in anesthetized mechanically ventilated rats

Acute intermittent hypoxia elicits long-term increases in respiratory motor output (long-term facilitation, LTF). Most investigators study LTF in mechanically ventilated, bilaterally vagotomized, and anesthetized animals. Vagotomy blocks inhibitory lung-volume feedback that could diminish the magnitude of LTF. However, the effects of vagotomy on LTF may not be so straight forward. In cats, vagotomy increases LTF of upper airway muscles but may decrease LTF of accessory pump muscles. The effects of vagotomy on LTF in rats are unknown. We hypothesized that the magnitude of hypoglossal and phrenic LTF would be differentially regulated by vagal afferent feedback in anesthetized and mechanically ventilated rats. Hypoglossal and phrenic motor outputs were recorded from vagotomized and vagally intact anesthetized mechanically ventilated adult Sprague-Dawley rats before, during, and up to 60-min after intermittent hypoxia. Ventilator frequency (f), pump volume, and peak tracheal pressure were not different between groups. The effects of vagotomy on the magnitude of LTF depended on the motoneuron population in question. The magnitude of hypoglossal LTF increased after vagotomy (vagi intact, -5+/-10%; vagotomy, 66+/-11% above baseline; p<0.05); whereas, the magnitude of phrenic LTF decreased after vagotomy (vagi intact, 135+/-24%; vagotomy, 40+/-13% above baseline; p<0.05). These data support previous work in anesthetized cats, and suggest that the expression of hypoglossal and phrenic respiratory motor plasticity is differentially regulated by vagal afferent feedback.     

Endogenous hydrogen sulfide is involved in regulation of respiration in medullary slice of neonatal rats

The purpose of the present study was to verify our assumption that rhythmic respiratory activity may be regulated by endogenous hydrogen sulfide (H(2)S) in the medullary slices of neonatal rats. We found that a moderate concentration of donor of H(2)S, NaHS, mainly induced diphasic respiratory responses indicated by changes of discharge frequency (DF) of hypoglossal rootlets, an initial inhibitory stage followed by a later excitatory one. Cystathionine beta-synthase (CBS) substrate, cysteine (CYS), exerted similar effects. CBS inhibitor, NH(2)OH, could eliminate both inhibitory and excitatory effects in the two stages induced by CYS. K(ATP) channel blocker, glibenclamide (Gl), could eliminate the decrease in DF in the initial stage, but not the increase in the later one. On the other hand, adenyl cyclase (AC) inhibitor, SQ-22536 (SQ) could eliminate the increase in DF in the later stage, but not the decrease in the initial one, of the rootlets caused by NaHS. Co-application of Gl and SQ eliminated both inhibitory and excitatory effect induced by NaHS. The cAMP level was increased in the later stage but not in the initial one by NaHS, and the increase in the cAMP level could be eliminated by SQ. It can be concluded that the endogenous H(2)S could be produced through the CBS-H(2)S pathway and could be involved in the control of the central rhythmic respiration in the in vitro medullary slices of neonatal rats by opening K(ATP) channels and activating AC-cAMP pathway of the neurons.     

Serotonergic projections from the caudal raphe nuclei to the hypoglossal nucleus in male and female rats

The respiratory control system is sexually dimorphic. In many brain regions, including respiratory motor nuclei, serotonin (5HT) levels are higher in females than in males. We hypothesized that there could be sex differences in 5HT input to the hypoglossal nucleus, a region of the brainstem involved in upper airway control. Adult Fischer 344 rats were anesthetized and a retrograde transsynaptic neuroanatomical tracer, Bartha pseudorabies virus (PRV), was injected into the tongue. Sections through the medulla were reacted immunocytochemically for the presence of (i) PRV, (ii) tryptophan hydroxylase (TPH; marker of 5HT neurons), (iii) PRV combined with TPH, and (iv) 5HT. Sex hormone levels were measured in female rats and correlated with TPH immunoreactivity, as hypoglossal 5HT levels vary with the estrous cycle. The number of PRV neurons was comparable in male and female rats. The number and distribution of TPH immunoreactive neurons in the caudal raphe nuclei were similar in male and female rats. The subset of 5HT neurons that innervate hypoglossal motoneurons was also similar in male and female rats. With the exception of the ventrolateral region of the hypoglossal nucleus, 5HT immunoreactivity was similar in male and female rats. These data suggest that sex differences in 5HT modulation of hypoglossal motoneurons in male and female rats are not the result of sex differences in TPH or 5HT, but may result from differences in neurotransmitter release and reuptake, location of 5HT synaptic terminals on hypoglossal motoneurons, pre- and postsynaptic 5HT receptor expression, or the distribution of sex hormone receptors on hypoglossal or caudal raphe neurons.     

Involvement of nitric oxide in regulation of the medullary respiratory rhythm in neonatal rats

The NADPH-diaphorase (as a neuronal NO-synthase) reactivity in the medullary structures of the respiratory rhythm (RR) generator and the role of NO in the regulation of respiratory activity in the phrenic nerve of artificially superfused semi-isolated medulla-spinal cord preparations were investigated in newborn rats. NADPH-diaphorase positive neurons were found in all nuclei of both dorsal and ventral respiratory groups of neurons. The maximal density of stained cells was present within the rostral part of the ventrolateral medulla (VLM), in the region of the lateral paragigantocellular reticular nucleus. It was found that endogenous NO mediates the mechanism of tonic inhibitory control of the RR frequency located in the rostral VLM under normal and hypoxic conditions, and appears to be involved in generation of the basic RR by the more caudal structures of VLM. It was shown that NO biosynthesis mediates the effect of NMDA receptors activation on the RR.     

Maturational changes in pontine and medullary alpha-adrenoceptor influences on respiratory rhythm generation in neonatal rats

We examined developmental changes in α-adrenoceptor influences and descending pontine inputs on the medullary respiratory network in the neonatal rat in vitro brainstem-spinal cord preparation. Using a split bath preparation to isolate the pons from the medulla, antagonists for α1 and α2 adrenoreceptors were applied to only the medulla at postnatal days 0, 2 and 4, before and after transection of the pons. Blocking α1 and α2 receptors in the medulla in the absence of a pons reduced burst frequency at all ages with a more pronounced effect in younger animals. At all ages the presence of a pons diminished the effect of blocking α2 receptors in the medulla and eliminated the effect of blocking α1 receptors. These results indicate that there is a tonic release of catecholamines within the medulla that is under influence from the pons. Additionally, transection experiments indicated that during development, the net influence of the pons changed from one of excitation to one of inhibition.     

Ondansetron and fluoxetine reduce sleep apnea in mice lacking monoamine oxidase A

Prospective clinical trials addressing the role of serotonin (5-HT) in sleep apnea have indicated that the 5-HT uptake inhibitor fluoxetine is beneficial to some patients with obstructive apnea, whereas the 5-HT₃ receptor antagonist ondansetron seems of little value despite its efficacy in rat and dog models of sleep apnea (central and obstructive). Here, we examined the effect of these drugs in transgenic mice lacking monoamine oxidase A (Tg8), which exhibit 3-fold higher rates of central sleep apnea than their wild-type counterparts (C3H), linked to their enhanced 5-HT levels. Acute ondansetron (2 mg kg⁻¹, intraperitoneal), acute fluoxetine (16 mg kg⁻¹) and 13-day chronic fluoxetine (1 or 16 mg kg⁻¹) decreased by 80% the total (spontaneous and post-sigh) apnea index in Tg8 mice during non-rapid eye movement sleep, with no statistically significant effect on apnea in C3H mice. Our study shows that both drugs reduce the frequency of apneic episodes attributable to increased monoamine levels in this model of MAOA deficiency, and suggests that both may be effective in some patients with central sleep apneas.     

Electrophysiological and behavioral effects of electrical stimulation of the raphe nuclei in cats

Electrical stimulation of n. raphe dorsalis in adult cats produced the following effects: (1) suppression of ponto-geniculate-occipital cortex (PGO) waves during REM sleep; (2) mild arousal and orienting responses; and, (3) suppression of feeding in an animal deprived of food for three days. Contrary to these effects, stimulation of n. raphe pontis and magnus triggered the occurrence of PGO waves, produced no arousal and failed to suppress the feeding of food deprived animals. No synchronogenic or sleep-inducing effects were produced by stimulation of any of these raphe nuclei. Stimulation of the midbrain reticular formation produced responses similar to those seen following dorsalis stimulation.     

Opioid μ-receptors in medullary raphe region affect the hypoxic ventilation in anesthetized rats

Opioids can attenuate the peripheral chemoreceptor-mediated hypoxic ventilatory response (HVR) by acting on central μ-type opioid receptors. Since the medullary raphe region (MRR) expresses abundant μ-receptors and participates in modulating HVR, we tested the role of μ-receptors within the caudal, medial, and rostral MRR (cMRR, mMRR, and rMRR) in modulating the HVR. We recorded cardiorespiratory activities and their responses to isocapnic hypoxia in anesthetized rats before and after local microinjection of DAMGO into the MRR, and intravenous administration of DAMGO (100 μg/kg) alone or coupled with a previous local injection of CTAP. Microinjecting DAMGO into the cMRR or mMRR but not the rMRR significantly attenuated the HVR. However, systemic DAMGO-induced HVR attenuation was not significantly affected by pretreating the cMRR and mMRR with CTAP. Our data suggest that cMRR and mMRR μ-receptors are capable of depressing the HVR, while their contribution to the attenuated HVR by systemic DAMGO is limited.     

Heme oxygenase–carbon monoxide pathway is involved in regulation of respiration in medullary slice of neonatal rats

Carbon monoxide (CO) is a novel biological messenger molecule. It is well known that CO can be synthesized in mammalian cells. In addition, CO is also demonstrated to participate in many physiological processes, such as vasomotion, thermoregulation and respiratory regulation. The purpose of our present study was to investigate the role of heme oxygenase–carbon monoxide (HO–CO) pathway in central regulation of respiration. The experiments were carried out on the medullary slices of neonatal Sprague–Dawley rats. The discharge activity of the hypoglossal rootlets was recorded to indicate the central rhythmic respiratory activity and its duration (DD), interval (DI), frequency (DF) and integrated amplitude (IA) were analyzed. The slices were perfused with ZnPP-9 (a potent inhibitor of heme oxygenase), CO and hemin (substrate of heme oxygenase), respectively, to observe their effects on respiratory activity. The results obtained were as follows: ZnPP-9 could decrease DD, DI and IA, and increase DF (P < 0.05); exogenous CO caused a decrease in DD and DF, and an increase in DI and IA (P < 0.05); in response to hemin, DI and IA decreased, DF increased (P < 0.05), and DD did not change significantly (P > 0.05); administration of both ZnPP-9 and hemin could decrease DI, and increase DF (P < 0.05), but did not affect DD and IA significantly (P > 0.05). It can be concluded from the results above that the HO–CO pathway may be involved in the regulation of rhythmic respiration at the level of medulla oblongata.     

Papain effects on rat hippocampal neurons in the slice preparation

Intracellular recordings were made from the CA1 stratum pyramidale region of rat hippocampal slices. Papain was applied to the cells via bath perfusion, and its effects on membrane properties, synaptic potentials and responses to pressure application of gamma-aminobutyric acid (GABA) were assessed. Papain did not markedly affect neuronal input resistance, resting potential or action potentials with treatment times lasting over one hour. Synaptic potentials were initially enhanced and then gradually abolished, with the fast inhibitory postsynaptic potential being the most sensitive and the late, potassium-dependent hyperpolarization being the most resistant to enzyme. Responses to GABA were enhanced by papain, the GABA-activated conductance increased, and a slow depolarizing wave appeared which resembled the effect caused by pentobarbital on these neurons. This study indicates that the use of papain in the acutely dissociated neuron preparation is not responsible for the dramatic increase in resting input resistance seen in these neurons. The GABA-activated conductance may be affected by the enzyme.     

Gender and age differences in symptoms' profile in sleep apnea syndrome: A possible cause of gender bias in diagnosis

Summary Objective   To compare the age-dependent severity and clinical profile of men and women with polysomnographically-documented obstructive sleep apnea syndrome (OSAS). Method   A retrospective comparison of polysomnographic and clinical data obtained from 358 carefully age- and RDI-matched men and women with OSAS diagnosed in the Technion Sleep Laboratories. Results   We found that women complained significantly more on "difficulties falling asleep", "early morning awakenings", "morning fatigue", "mid-sleep awakenings" and "morning headaches". Nevertheless, stepwise regression analysis revealed that subjective complaints were significant predictors of RDI in men only. Conclusions   We conclude that the clinical profile of OSAS in women is different from that in men, and that these differences should be taken into account when evaluating women suspected of having OSAS.