Serotonin receptor subtypes involved in vagus nerve stimulation-induced phrenic long-term facilitation in rats

Episodic vagus nerve stimulation (VNS) induces phrenic long-term facilitation (LTF, a persistent augmentation of phrenic nerve activity after the stimulation ends), sensitive to the serotonin 5-HT(1,2,5,6,7) receptor antagonist methysergide and similar to that elicited by episodic hypoxia or carotid sinus nerve stimulation. This study examined the effect of ketanserin (5-HT(2) antagonist) or clozapine (5-HT(2,6,7) antagonist) on VNS-induced LTF in anesthetized, vagotomized, paralyzed and ventilated rats to determine which receptor subtype(s) is involved. Three episodes of 5 min VNS (50 Hz, 0.1 ms, approximately 500 microA) with 5 min intervals elicited phrenic LTF in control (amplitude: 38% above baseline at 60 min post-VNS) and ketanserin (2 mg x kg(-1), i.p.) pre-treated rats (45%), but not clozapine (3 mg x kg(-1)) rats (8%). These data suggest that unlike hypoxia-induced LTF (5-HT(2) receptor-dependent), VNS-induced LTF requires non-5-HT(2) serotonin receptors, perhaps 5-HT(6) and/or 5-HT(7) subtype(s).     

Propofol abolished the phrenic long-term facilitation in rats

The aim was to investigate the effect of propofol anesthesia on the phrenic long-term facilitation (pLTF) in rats. We hypothesized that pLTF would be abolished during propofol-compared with urethane anesthesia.     

Episodic hypoxia induces long-term facilitation of upper airway muscle activity in spontaneously breathing anaesthetized rats

We performed these experiments to determine if repeated exposure to episodic hypoxia induces long term facilitation (LTF) in anaesthetized spontaneously breathing rats. A previous study in spontaneously breathing rats was unable to demonstrate evidence of LTF with repeated hypoxia, but this may have been due to the low number of hypoxic episodes used. We hypothesized that with sufficient exposure, episodic hypoxia LTF of genioglossus (GG), hyoglossus (HG) and diaphragm (Dia) activities would be elicited. Experiments were performed in 24 anaesthetized spontaneously breathing rats with intact vagi. Peak and tonic GG, HG and Dia EMG activities were recorded before, during and for 1 h following exposure to eight ( n = 8) or three ( n = 8) episodes of isocapnic hypoxia (     = 0.1) each of 3 min duration. A third time control series was also performed with exposure to normoxia alone (     = 0.28, n = 8). Short-term potentiation of GG and HG muscle activity developed during the early period after repeated exposure to eight and three hypoxic episodes. LTF, however, occurred only after eight hypoxic episodes. This manifested as an increase in peak GG and Dia inspiratory muscle activity and tonic HG activity. LTF of respiratory breathing frequency was also induced, reflected by a reduction in inspiratory and expiratory time. These findings support our initial hypothesis that LTF in the anaesthetized, spontaneously breathing rat is dependent on the number of exposures to hypoxia and show that the responses to repetitive hypoxia are composed of both short and long-term facilitatory changes.     

Phrenic long-term facilitation requires NMDA receptors in the phrenic motonucleus in rats

Exposure to episodic hypoxia induces a persistent augmentation of respiratory activity, known as long-term facilitation (LTF). LTF of phrenic nerve activity has been reported to require serotonin receptor activation and protein syntheses. However, the underlying cellular mechanism still remains poorly understood. NMDA receptors play key roles in synaptic plasticity (e.g. some forms of hippocampal long-term potentiation). The present study was designed to examine the role of NMDA receptors in phrenic LTF and test if the relevant receptors are located in the phrenic motonucleus. Integrated phrenic nerve activity was measured in anaesthetized, vagotomized, neuromuscularly blocked and artificially ventilated rats before, during and after three episodes of 5 min isocapnic hypoxia ( P _a,O2= 30–45 mmHg), separated by 5 min hyperoxia (50% O₂). Either saline (as control) or the NMDA receptor antagonist MK-801 (0.2 mg kg⁻¹, i.p. ) was systemically injected ∼1 h before hypoxia. Phrenic LTF was eliminated by the MK-801 injection (vehicle, 32.8 ± 3.7% above baseline in phrenic amplitude at 60 min post-hypoxia; MK-801, −0.5 ± 4.1%, means ± s.e.m. ), with little change in both the CO₂-apnoeic threshold and the hypoxic phrenic response (HPR). Vehicle (saline, 5 × 100 nl) or MK-801 (10 μ m ; 5 × 100 nl) was also microinjected into the phrenic motonucleus region in other groups. Phrenic LTF was eliminated by the MK-801 microinjection (vehicle, 34.2 ± 3.4%; MK-801, −2.5 ± 2.8%), with minimal change in HPR. Collectively, these results suggest that the activation of NMDA receptors in the phrenic motonucleus is required for the episodic hypoxia-induced phrenic LTF.     

Episodic hypoxia evokes long-term facilitation of genioglossus muscle activity in neonatal rats

The aim of this study was to determine if episodic hypoxia evokes persistent increases of genioglossus muscle (GG) activity, termed long-term facilitation (LTF), in neonatal rats in vivo . Experiments were performed on anaesthetized, spontaneously breathing, intubated neonatal rats (postnatal days (P) 3–7), divided into three groups. The first group ( n = 8) was subjected to three 5-min periods of hypoxia (5% O₂–95% N₂) alternating with 5 min periods of room air. The second group ( n = 8) was exposed to 15 min of continuous hypoxia. The third ( n = 4) group was not exposed to hypoxia and served as a control. GG EMG activity and airflow were recorded before, during and for 60 min after episodic and continuous hypoxic exposure. During hypoxia, GG EMG burst amplitude and tidal volume ( V _T ) significantly increased compared to baseline levels (episodic protocol: mean ± s.e.m ; 324 ± 59% of control and 0.13 ± 0.007 versus 0.09 ± 0.005 ml, respectively; continuous protocol: 259 ± 30% of control and 0.16 ± 0.005 versus 0.09 ± 0.007 ml, respectively; P < 0.05). After the episodic protocol, GG EMG burst amplitude transiently returned to baseline; over the next 60 min, burst amplitude progressively increased to levels significantly greater than baseline (238 ± 40% at 60 min; P < 0.05), without any significant increase in V _T and respiratory frequency ( P > 0.05). After the continuous protocol, there was no lasting increase in GG EMG burst amplitude. We conclude that LTF of upper airway muscles is an adaptive respiratory behaviour present from birth.     

Attenuated phrenic long-term facilitation in orexin neuron-ablated mice

We examined phrenic long-term facilitation (LTF) in urethane-anesthetized, vagotomized, paralyzed, and artificially ventilated orexin neuron-ablated mice and their wild-type littermates. Effect of isocapnic single hypoxic episode (SHE, for 45 s) and intermittent hypoxia (IH, 5 times of SHE separated by 5 min) on phrenic nerve activity (PNA) was measured for 1–2 h. In wild-type mice, amplitude of PNA gradually increased after cessation of IH and reached 55 ± 15% above the baseline (n = 7, p < 0.05) whereas the burst rate of PNA did not change. Qualitatively similar but significantly attenuated response (16 ± 8%) was observed in orexin neuron-ablated mice. SHE did not affect amplitude nor frequency in both animals. We conclude that orexin contributes to eliciting phrenic LTF at least in part in mice. This study also showed, for the first time, phrenic LTF following IH in WT mice. Characteristics of phrenic and ventilatory LTF in mice were similar to those in rats.     

Microinjection of methysergide into the raphe nucleus attenuated phrenic long-term facilitation in rats

Exposure to acute intermittent hypoxia (AIH) evokes persistent increase in respiratory activity that lasts up to 60 min after hypoxic episodes have ceased. This persistent increase in phrenic nerve activity (PNA) is known as phrenic long-term facilitation (LTF). AIH-induced phrenic LTF in anesthetized rats is serotonin dependant. The present study was performed to determine whether microinjection of methysergide (4 mM, 20 ± 5 nl), a broad spectrum 5-HT receptor antagonist, into the caudal raphe nuclei influences phrenic LTF. Peak integrated PNA and respiratory frequency were recorded at 15, 30, and 60 min after five 3-min episodes of normocapnic hypoxia in urethane-anesthetized, vagotomized, paralyzed and ventilated male Sprague–Dawley rats. In control animals, phrenic nerve amplitude was elevated 66.7 ± 8.6% from baseline 1 h after episodic hypoxia, indicating phrenic LTF. Experimental microinjections of methysergide prior to AIH exposure attenuated phrenic LTF (amplitude increase 2.62 ± 2.9% over baseline). We conclude that methysergide microinjections into the caudal raphe region attenuated phrenic LTF induced by AIH, indicating involvement of 5-HT receptor activation at a supraspinal level.     

Lipopolysaccharide attenuates phrenic long-term facilitation following acute intermittent hypoxia

Lipopolysaccharide (LPS) induces inflammatory responses, including microglial activation in the central nervous system. Since LPS impairs certain forms of hippocampal and spinal neuroplasticity, we hypothesized that LPS would impair phrenic long-term facilitation (pLTF) following acute intermittent hypoxia (AIH) in outbred Sprague-Dawley (SD) and inbred Lewis (L) rats. Approximately 3h following a single LPS injection (i.p.), the phrenic response during hypoxic episodes is reduced in both rat strains versus vehicle treated, control rats (SD: 84 ± 7% vs. 128 ± 14% baseline for control, p < 0.05; L: 62 ± 10% vs. 90 ± 9% baseline for control, p < 0.05). At 60 min post-AIH, pLTF is also diminished by LPS in both strains: (SD: 22 ± 5% vs. 73.5 ± 14% baseline for control, p < 0.05; L: 18 ± 15% vs. 56 ± 8% baseline for control, p < 0.05). LPS alone does not affect phrenic burst frequency in either rat strain, suggesting that acute LPS injection has minimal effect on brainstem respiratory rhythm generation. Thus, systemic LPS injections and (presumptive) inflammation impair pLTF, a form of spinal neuroplasticity in respiratory motor control. These results suggest that ongoing infection or inflammation must be carefully considered in studies of respiratory plasticity, or during attempts to harness spinal plasticity as a therapeutic tool in the treatment of respiratory insufficiency, such as spinal cord injury.     

大鼠迷走神经和膈神经的解剖相关性实验研究

目的 为大鼠迷走神经移位膈神经重建高位颈髓损伤大鼠的膈肌功能提供显微解剖学依据。 方法　10只健康雌性SD大鼠在10倍手术显微镜下解剖双侧膈神经、迷走神经及其分支。用数显卡尺测量迷走神经与膈神经在“膈神经主干起始平面”、“锁骨上平面”、“入膈肌平面”的相对距离,用读数显微镜测量各平面迷走神经和膈神经的直径。 结果　在颈部,迷走神经直径为（0.3284±0.0247）mm,膈神经直径为（0.2267±0.0164）mm,二者的相对距离很接近,无论是在“膈神经主干起始平面”还是“锁骨上平面”,平均都不超过2.5 mm;在“入膈肌平面”平面,迷走神经直径为（0.2912±0.0326）mm,膈神经直径为（0.2794±0.0282）mm,二者的相对距离较颈部远,左侧为（8.71±0.804）mm,右侧为（6.203±0.952） mm。 结论　(1)在颈部,迷走神经与膈神经的直径相差不大,相对距离很接近,二者可直接无张力缝合。(2)在入膈肌平面,迷走神经与膈神经的直径大致相同,相对距离稍远,但将膈神经和迷走神经向上游离一段距离后仍可实现二者的直接无张力缝合。     

Electrical stimulation of the phrenic nerve in dogs

N. N. Burdenko Institute of Neurosurgery, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. N. Konovalov.) Translated from Byullenten' Éksperimental'noi Biologii i Meditsiny, Vol. 106, No. 7, pp. 19–21, July, 1988.     

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.     

Episodic spinal serotonin receptor activation elicits long-lasting phrenic motor facilitation by an NADPH oxidase-dependent mechanism

Phrenic long-term facilitation (pLTF) is a serotonin (5-HT)-dependent augmentation of phrenic motor output induced by acute intermittent hypoxia (AIH). AIH-induced pLTF requires spinal NADPH oxidase activity and reactive oxygen species (ROS) formation. Since 5-HT receptor activation stimulates NADPH oxidase activity in some cell types, we tested the hypothesis that episodic spinal 5-HT receptor activation (without AIH) is sufficient to elicit an NADPH oxidase-dependent facilitation of phrenic motor output (pMF). In anaesthetised, artificially ventilated adult male rats, episodic intrathecal 5-HT injections (3 × 6 μl injections at 5 min intervals) into the cerebrospinal fluid (CSF) near cervical spinal segments containing the phrenic motor nucleus elicited a progressive increase in integrated phrenic nerve burst amplitude (i.e. pMF) lasting at least 60 min post-5-HT administration. Hypoglossal (XII) nerve activity was unaffected, suggesting that effective doses of 5-HT did not reach the brainstem. A single 5-HT injection was without effect. 5-HT-induced pMF was dose dependent, but exhibited a bell-shaped dose–response curve. Activation of different 5-HT receptor subtypes, specifically 5-HT₂ versus 5-HT₇ receptors, may underlie the bell-shaped dose–response curve via a mechanism of 'cross-talk' inhibition. Pre-treatment with NADPH oxidase inhibitors, apocynin or diphenylenodium (DPI), blocked 5-HT induced pMF. Thus, episodic spinal 5-HT receptor activation is sufficient to elicit pMF by an NADPH oxidase-dependent mechanism, suggesting common mechanisms of ROS formation with AIH-induced pLTF. An understanding of the mechanisms giving rise to AIH-induced pLTF and 5-HT induced pMF may inspire novel therapeutic strategies for respiratory insufficiency in diverse conditions, such as sleep apnoea, cervical spinal injury or amyotrophic lateral sclerosis.     

Spectral parameters of phrenic nerve activity in mature rats during electrical stimulation of retrotrapezoid nucleus

In mature rats, electrical stimulation of the retrotrapezoid nucleus increased the amplitude and frequency of the high-frequency peak in the firing spectrum of the phrenic nerve, while the amplitude of medium-frequency peak and the amplitude ratio of medium- to high-frequency peaks decreased. These changes in spectral parameters were associated with accelerated increase in central inspiratory activity, decreased amplitude of phrenic nerve firing, and increased frequency of respiratory rhythm. It is hypothesized that being a relay structure of central chemosensitive mechanism, the retrotrapezoid nucleus regulates parameters of medium-and high-frequency spectral peaks of efferent electrical activity in the respiratory center together with the dorsal and ventral respiratory groups. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 138, No. 8, pp. 139–143, August, 2004     

迷走神经电刺激对大鼠肠缺血再灌注后肠损伤的影响

目的:观察迷走神经电刺激对大鼠肠缺血再灌注（I/R）后肠损伤的影响。 方法:30只雄性Wistar大鼠，双侧颈迷走神经切断后，随机分为3组（n=10）：（1）肠缺血再灌注（I/R）组：暴露腹腔后夹闭肠系膜上动脉（SMA）1 h，开放再灌注2 h；（2）迷走神经刺激（VNS）组：在夹闭前及再灌注开始均以5 V、2 ms和1 Hz强度的电能持续刺激左颈部迷走神经远端20 min；（3）假手术对照(SC)组：仅暴露腹腔，不行SMA夹闭及电刺激。颈动脉插管监测平均动脉压（MAP）。所有动物在再灌注2 h后处死，取小肠组织行光学、电子显微镜观察肠粘膜损伤程度并行改良的Chiu’s评分；检测血浆丙二醛（MDA）、肿瘤坏死因子（TNFα）含量。 结果:各组大鼠MAP在缺血期基本保持平稳。而在再灌注期，I/R组的MAP随时间延长明显低于SC组（P＜0.05），而VNS组能明显拮抗MAP下降（P＜0.05）。光学、电子显微镜观察显示I/R后肠组织出现不同程度的损伤，I/R组最为严重，而VNS组相对较轻；但两组的改良Chiu’s评分值显著高于SC组（P＜0.01），VNS组的评分值明显低于I/R组（P＜0.01）。I/R组血浆MDA、TNFα含量明显高于SC组和VNS组（P＜0.05，P＜0.01）；VNS组血浆MDA含量高于SC组（P＜0.05），VNS组血浆TNFα与SC组无显著差异（P＞0.05）。 结论:电刺激迷走神经能显著减轻I/R肠粘膜结构的病理改变并使再灌注期的循环血压得到一定改善，其保护效应可能与减少脂质过氧化、下调TNFα生成有关。     

NADPH oxidase activity is necessary for acute intermittent hypoxia-induced phrenic long-term facilitation

Phrenic long-term facilitation (pLTF) following acute intermittent hypoxia (AIH) is a form of spinal, serotonin-dependent synaptic plasticity that requires reactive oxygen species (ROS) formation. We tested the hypothesis that spinal NADPH oxidase activity is a necessary source of ROS for pLTF. Sixty minutes post-AIH (three 5-min episodes of 11% O₂, 5 min intervals), integrated phrenic and hypoglossal (XII) nerve burst amplitudes were increased from baseline, indicative of phrenic and XII LTF. Intrathecal injections (∼C₄) of apocynin or diphenyleneiodonium chloride (DPI), two structurally and functionally distinct inhibitors of the NADPH oxidase complex, attenuated phrenic, but not XII, LTF. Immunoblots from soluble (cytosolic) and particulate (membrane) fractions of ventral C₄ spinal segments revealed predominantly membrane localization of the NADPH oxidase catalytic subunit, gp91^phox, whereas membrane and cytosolic expression were both observed for the regulatory subunits, p47^phox and RAC1. Immunohistochemical analysis of fixed tissues revealed these same subunits in presumptive phrenic motoneurons of the C₄ ventral horn, but not in neighbouring astrocytes or microglia. Collectively, these data demonstrate that NADPH oxidase subunits localized within presumptive phrenic motoneurons are a major source of ROS necessary for AIH-induced pLTF. Thus, NADPH oxidase activity is a key regulator of spinal synaptic plasticity, and may be a useful pharmaceutical target in developing therapeutic strategies for respiratory insufficiency in patients with, for example, cervical spinal injury.     

Effects of anti-inflammatory vagus nerve stimulation in endotoxemic rats on blood and spleen lymphocyte subsets

Background

Anti-inflammatory cytokine effects of vagus nerve stimulation in sepsis syndromes are well established. Effects on immune cells are less clear. Therefore, we studied changes in peripheral and spleen leukocyte subsets in an endotoxic rat sepsis model.

Methods

Ventilated and sedated adult male SD rats received 5 mg/kg b.w. lipopolysaccharide intravenously to induce endotoxic sepsis. Controls and a group with both-sided vagotomy were compared to animals with both sided vagotomy and left distal vagus nerve stimulation. 4.5 h after sepsis induction immune cell counts and types in the peripheral blood and spleen were determined [T-lymphocytes (CD3+), T-helper cells (CD3+ CD4+), activated T-helper cells (CD3+ CD4+ CD134+), cytotoxic T-cells (CD3+ CD8+), activated cytotoxic T-cells (CD3+ CD8+ CD134+), B-lymphocytes (CD45R+ CD11cneg-dim), dendritic cells (CD11c+ OX-62 +), natural killer cells (CD161+ CD3neg) and granulocytes (His48 +)] together with cytokine and chemokine plasma levels (IL10; IFN-g, TNF-a, Cxcl5, Ccl5).

Results

Blood cell counts declined in all LPS groups. However, vagus nerve stimulation but not vagotomy activated cytotoxic T-cells. Vagotomy also depleted natural killer cells. In the spleen, vagotomy resulted in a strong decline of all cell types which was not present in the other septic groups where only granulocyte numbers declined.

Conclusion

Vagotomy strongly declines immune cell counts in the septic spleen. This could not be explained by an evasion or apoptosis of cells. A marginalisation of spleen immune cells into the peripheral microcirculation might be therefore most likely. Further studies are warranted to clear this issue.     

Enhanced recognition memory following vagus nerve stimulation in human subjects

Neuromodulators associated with arousal modulate learning and memory, but most of these substances do not freely enter the brain from the periphery. In rodents, these neuromodulators act in part by initiating neural messages that travel via the vagus nerve to the brain, and electrical stimulation of the vagus enhances memory. We now extend that finding to human verbal learning. We examined word-recognition memory in patients enrolled in a clinical study evaluating the capacity of vagus nerve stimulation to control epilepsy. Stimulation administered after learning significantly enhanced retention. These findings confirm in humans the hypothesis that vagus nerve activation modulates memory formation similarly to arousal.