疑核的研究進展

張學英,，艾洪濱

（山東師范大學生命科學學院生理學實驗室,，山東 濟南250014）

中國神經(jīng)科學雜志 2004年第20卷第2期目錄

摘要:  疑核是重要的內臟運動核和軀體運動核,，它與中樞神經(jīng)系統(tǒng)和外周器官建立了廣泛的神經(jīng)聯(lián)系。疑核,，迷走-孤束復合體,，最后區(qū)，腹外側延髓和中間網(wǎng)狀結構共同組成了延髓內臟帶,。疑核內含有大量的神經(jīng)遞質,，如乙酰膽堿，生物胺,，神經(jīng)肽,，氨基酸等及其受體。疑核在調控呼吸,、心血管活動,、免疫、消化和內分泌功能中起重要作用,。

關鍵詞:  疑核,； 細胞構筑； 神經(jīng)遞質,； 受體

基金項目：山東省自然科學基金(Y2002D18)　　通訊作者：艾洪濱　　作者簡介：張學英(1976-), 女, 山東青島人, 碩士研究生, 主要從事消化-神經(jīng)生理學研究　　E-mail：　　聯(lián)系電話：(0531)6185359  收稿日期：2003-10-28

Histogenesis defined that the dorsal motor nucleus of vagus (DMV) and the nucleus ambiguus (NA) were located together in the embryo and took apart with the generation development in the vertebrate. NA is situated in the dorsolateral of inferior olivary nucleus and ventromedial of spinal trigeminal tract. Nucleus tractus solitariis (NTS), DMV, area postrema (AP), NA, ventrolateral medulla (VLM) and the reticular formation between NTS and VLM form the medullary visceral zone (MVZ), which named‘life center’modulates the activities such as circulation, respiration, digestion, etc.[1, 2] In recent years, studies on the cytoarchitecture, fiber contacts, physiological functions have been widely done.

1  Morphology, cytoarchitecture and synaptic contacts of NA

The reports about the morphology and cytoarchitecture of NA were diverse because of the different species of animal and principle of classification. The rat NA, from rostral to caudal, was composed of the loose, which projects to laryngeal intrinsic muscles, the semicompact, to the pharyngeal muscles, the compact, to the esophageal muscles and peripheral airway, and the external formation, ventral to the upper three parts, to the cardiovascular organs. The studies on cytoarchitecture and ultrastructure of the rat revealed that the compact formation of NA (AmC) consists of mostly esophageal motoneurons and small neurons not projecting to the esophagus[3, 4]. The dendrites of esophageal motoneurons run rostrocaudally making bundles in the AmC. Most of the axon terminals in the AmC are Gray’s type I, that is, the terminal contains round synaptic vesicles with asymmetric synaptic contact, and a few are Gray’s II, that is, the terminal contains pleomorphic vesicles with symmetric synaptic contact . The fine structure of the semicompact formation of NA (AmS) of the rat, composed of mostly less-compact clusters of motoneurons whose dendrites extend radially into the reticular formation[5, 6],

and whose boundary is not as clear as that of the AmC, was investigated with retrograde tracing of pharyngeal neurons[7, 8]. The labeled pharyngeal neurons found throughout the AmS were large (29 µm×39 µm or 27.6 µm×44.1 µm) and polygonal, had 2-25 axosomatic synapses per somatic profile and contained many Nissl bodies and well-developed cell organelles with a prominent spherical nucleus. Besides pharyngeal neurons, two types were recognized in AmS, unlabeled medium-sized neurons and small neurons. The medium-sized neuron was dark and oval (19.3 µm×33.2 µm), contained many free ribosomes and much swollen rough endoplasmic reticulum with a distorted oval nucleus. The small was spindle-shaped (12.3 µm×20.2 µm) and had poorly developed cell organelles with an irregularly shaped nucleus. The fine structure of the laryngomotor loose formations of nucleus ambiguus of the rat was studied in single and serial sections by means of light and electron microscopy[7]. Laryngeal motoneurons measured 42 µm×30 µm with 6-33 synapses per profile. In both the pharyngomotor semicompact and laryngomotor loose formations, axon terminals that contained round vesicles and formed asymmetric junctions (Gray’s type I ) and that contained pleomorphic vesicles and formed symmetric junctions (Gray’s type II) were distributed in approximately equal proportions on somata and dendrites, forming over 90% of the synapse population. A small percentage (2%-8%) of synapses had a subsurface cistern situated below the axon terminal (C type). Small, atypical motoneurons measuring 15 µm×5 µm with an invaginated nucleus were also present in both subdivisions. Recent studies showed cell bodies of tracheal vagal preganglionic neurous were multipolar, contained abundant endoplasmic, reticulum, had a round nucleus with a prominent nucleolus, no satellite body and displayed somatic and dendritic spices, distinguishable from pharyngeal and laryngeal motoneurons in other divisions of the NA which lack somatic spines[9]. The NA of milk goat lamb was divided into the medial and the lateral. The medial consists of large multipolar (diameter>36 µm), medium-sized fusiform (25 µm -35 µm) and small fusiform (<25 µm). The nucleus of multipolar lies in the middle or a little eccentric, but that of fusiform eccentric. The lateral consists of small fusiform (<25 µm) and the nucleus lies in the middle or eccentric of the cells. The soma of the large multiform in the medial has spines and the dendrites have processes and spines in the Golgi-Cox brainstem slice. The frog NA was also studied. The labeled cell column extended from the upper part of the medulla to the rostral spinal cord over a distance of about 3500 µm, consisting of four subdivisions, pharyngomotor subdivision, visceromotor subdivision, laryngomotor subdivision and the accessory nerve subdivision[10]. By comparison with that of the rat, there are differences in nuclear organization, reflecting differences in peripheral structures.

A great variety of nuclei in the CNS form synapses with NA. There are several types of synaptic contacts both symmetric and asymmetric, axo-somatic, axo-dentritic, dendro-dendritic, somato-dendritic, somato-somatic[7-9, 11]. The different divisions of the NA are diverse in the synaptology, which may be related with specific physiological functions.

2  Fibrous contacts with other nuclei and peripheral organs

2.1  Contacts with peripheral organs  The NA is not only somatic but also visceral motor nucleus. The fibers from the NA innervate many organs, call muscles, palate muscles, esophageal, trachea, cardiovasculus, gastrointestinal tract, thyroid, thymus, pancreas, testis, ovaries, bladder, etc.

2.2  Contacts with the central nervous system (CNS)

2.2.1  Hypothalamus  Hypothalamus is a subcortical center in the autonomic nervous system. Anatomical studies indicate the pathways are presented between the hypothalamus, including the ventromedial area , lateral area (LHA) and paraventricular area (PVN), and the motor areas of both the sympathetic system in the spinal cord and the parasympathetic system in the brainstem (DMV and NA)[12~14]. The axons of arginine vasopressin (AVP) positive neurons in the PVN project to the locus coeruleus (LC), NA, NTS, AP, etc.

2.2.2  The limbic system  The limbic system is composed of limbic lobe,，insular lobe and subcortical nuclei. Immunohistochemical investigations have shown the central nucleus of amygdala (CeA) contains corticotropin-releasing factorergic and GABAergic neurons, which innervate many nuclei, i.e. parabranchial nucleus, DMV, NTS, NA, etc. concerning cardiovascular activities in the pontomeculla. Microinjection of CRF into rat CeA induced pressor. CeA and bed nucleus of stria terminalis contain somatostatin (SOM)ergic and neurotensinergic neurons whose terminals contact with MTZ.

2.2.3  The cerebellum   The cerebellum plays an important role in the regulation of somatic motor activity. Experimental studies have shown the involvement of the medial cerebellar nucleus in cardiovascular and behavioural responses. The afferents to the cerebellar flocculus in cats were observed using the HRP tracing technique. The flocculus receives large numbers of mossy fiber afferents from the vestibular and perihypoglossal nuclei, bilateral; and climbing fibers from the contralateral inferior olive. In addition, large numbers of HRP-labeled neurons have been identified within many nuclei, including the NA.

2.2.4  Other nuclei in the brainstem   The reticular formation of brain stem consists of reticular nerve fibers and a great variety of nuclei. According to the diversity in the cytoarchitecture and functions, the reticular formation is divided into three parts, medial motorial areas (giant cells), lateral sensory areas (mini cells) and raphe nuclei, which all have synaptic contacts with the NA. Various specific afferents from the midbrain periaqueductal gray matter (PAG), the parabrachial  nucleus (PB), Kölliker-Fuse nucleus (KF), the vestibular nuclei, the NTS, the paratrigeminal nucleus, a primary sensory nucleus have been demonstrated among subnuclei of the NA. The pre-Botzinger complex, a inhibitory intermediate nucleus forms synaptic contacts with the NA, modulating the activity of the NA. Moreover, NA was observed to receive monosynaptic afferent projection bilaterally, with a contralateral predominance, from the retroambiguus in the primate, and to receive the projections from gigantocellular reticular nucleus, raphe nuclei, etc. These results may be reflected in the cytology and synaptology of the NA at the ultrastructural level.

By anterograde or retrograde tracing, the NA was observed to send efferent projections to the lateral LC and also to the gigantocellular tegmental field, the rostral ventrolateral medulla, the dorsomedial medulla, the NTS, and the caudal ventrolateral medulla, the lateral facial nucleus, and to KF or the phrenic motor nucleus of the spinal cord. Physiological studies have suggested the presence of the interneurons to control swallowing activity in the NA. These studies suggest that there are several kinds of neurons in the NA.

3  Transmitters and receptors in the NA

  Studies confirm that the NA contains a great variety of transmitters of acetylcholine, biogenic amine, neuropeptide, amino acid, etc. and their receptors with the application and development of immunohistochemistry and fluorescence histochemical techniques.

3.1  Acetylcholine (ACh)    Studies on the distributions of ACh were done by using choline acetyltransferase (ChAT), an ACh specific marker enzyme. By ChAT immunocytochemical technique, the NA is confirmed to be cholinoceptive as well as cholinergic neurons[15]. Nicotinic and muscarinic cholimoceptors (mainly nicotinic), observed in NA induced spontaneous and miniature excitatory postsynaptic potentials, which indicate the existence of a cholinergic nicotinic and muscarinic synapses mediating fast transmission in brainstem vagal motoneurons. Studies demonstrate the endogenous ACh and nicotine may evoke presynapic facilitation of glutamate release and enhance GABAergic and glycinergic inputs to cardiac vagal neurons[16], and ACh was injected into the NA to enhance the cellular immunity. Therefore, as a transmitter in the NA ACh plays an important role in maintaining cell immunity, modulating barorecepor-cardiac vagal reflex and heart rate.

3.2  Biogenic amine

3.2.1  Catecholamine   Catecholaminergic neurons are observed to be distributed widely in the CNS, using immunocytochemical technique of displaying tyrosine hydroxylase (TH), a marker for catecholaminergic neurons. Massari et al. (1998) confirmed that TH nerve terminals formed axo-dendritic synapses on negative inotropic vagal motoneurons in the ventrolateral NA, which conduct the vagal control of left ventricular contractility. Microinjection of dopamine (DA) into the right NA caused a dose-dependent decrease in heart rate and the response was blocked by a specific DA2 receptor antagonist. And catecholaminergic neurons may modulate the barorecepor-vasopressin reflex and may augment the ventilation by the mechanism of decrementing the discharge of the respiratory neurons.

3.2.2  Serotonin (5-HT)  Serotonins are mainly secreted by the cells of medullary raphe nucleus. 5-HT containing terminals and 5-HT (1A, 2A, 3A, 5A) receptors were observed in the NA and the combination of 5-HT and its receptors may elicit bradycardia, hypotension and the decrease in phrenic nerve activity. These receptors play a fundamental role in the reflex regulation of parasympathetic outflow.

3.3  Neuropeptide

3.3.1  Thyrotropin-releasing hormone (TRH)   Studies suggest TRH-like immunoreactivity and receptors were presentedly high-concentrated in the NA by immunohischemistry technique or an in situ hybridization study. TRH or its analogue microinjected into the NA dependently stimulated gastric acid, pepsin and serotonin secretion, mucosal blood flow, contractility, emptying and ulceration in rats, rabbits, sheep, cats, and TRH action was abolished by vagotomy. TRH induced NA neurons to be depolarized, expressing enhanced postinhibitory rebound or exhibiting oscillations of the membrane potential using an in vitro brain stem slice preparation. These demonstrated that the NA is involved in the CNS action of TRH to influence gastrointestinal function and ulceration through vagal dependent pathways.

3.3.2  Opioid  Dynorphin and enkephalin peptide were observed in the NA . Studies on the distribution of mu-opioid receptor (MOR) was performed using the in situ hybridization technique or Fluoro-Gold following immunogold detection, and the results were that MOR1 labeling neurons were located in the DMV and NA and those in NA were more likely to be localized to plasma membrane sites[11, 17, 18]. Morphine hydrocholoride was injected into NA to inhibit the cellular immunity and the inhibitory effect could not be blocked by naloxone hydrocholoride, which show that enkephalinergic neurons in the NA may coordinate the immunity of the body. Studies also confirm that the MOR combining with ligands can modulate the activity of visceral premotor neurons by inhibiting voltage-gated calcium currents, including cardiac premotor neurons[18] and mu-selective opioids and nociceptin act on preceding neurons to decrease glycinergic inputs to cardiac vagal neurons in the NA[19].

3.3.3  Somatostatin (SOM)  SOM is a neuropeptide distributed over the areas of CNS and peripheral organs, modulating the heart rate and the release of transmitters, hormones, immunity and the motive of gut, the differentiation of epithelial cells. SOM-like fibers were found in the neurons of NA and colocalized with NOS in the phargngeal motor neurons, the AmS. SOMergic neurons may enhance the excitatory action of glutamate, but inhibit that of Ach in the NA.

3.3.4  Arginine vasopressin (AVP)  Studies have showed that AVP is an important neuropeptide that can modulate the reflex control of blood pressure and heart rate. The NA, where cardiac parasympathetic neurons are located, receives dense AVP projections. Experiments conducted using whole cell patch clamp recording in an in vitro slice preparation in rats, demonstrate that AVP increase the frequency and amplitude of GABAergic inhibitory post-synaptic currents in cardiac parasympathetic neurons, mediated by V(1a)-AVP receptors, which may be at least one mechanism by which central AVP may increase heart rate and inhibit reflex bradycardia.

  Studies also indicate that NA contains substance P, neurokinin, calcitonin gene-related peptide, galanin and vasoactive intestinal peptide, androgen and estrogen, etc.

3.4  Amino acid

3.4.1  Gamma amino butyric acid (GABA)  GABA is an amino acid transmitter present in large quantities in inhibitory neurons of the CNS. Anatomical researches show GABA(A, B) receptors present in both the soma and dendrite of the NA[13, 20-22]. GABA released from CeA, PAG and NTS activate the GABA receptors in the NA and induce to distend the airway, increase the heart rate, decrease intragastric pressure and pyloric motility, modulate respiration and vocalization[13, 21].

3.4.2              Glycine  Glycine is the other inhibitory transmitter, which is exclusively secreted in the spinal cord and brainstem. Investigations using immunohistochemistry indicate the neurons that are immunoreactive for glycine are located in DMV, NTS, hypoglossal nucleus and NA. Inhibitory glycinergic input to cardiac vagal neurons in the NA may increase the heart rate and inhibit reflex bradycardia and also regulate respiration.

3.4.3  Glutamate (Glu)    Glu is an excitatory transmitter in the CNS in the primates, which is relative to rapid excitatory synaptic transmitting, nerve growth and apoptosis, synaptic plasticity，and many neurological diseases. Glu receptors (GluRs) are divided into two populations, ionic and metabotropic (iGluRs and mGluRs respectively). The anterior contains NMDA and non-NMDA receptors. Stimulation of NTS activated glutamatergic currents in cardiac vagal neurons in the NA and post-synaptic responses were separated into NMDA and non-NMDA components. In vagal outflow nucleus, mGluR(2/3, 7, 8, 1a) subtypes immunoreactivity was observed in the cell bodies and processes of the DMV and the cell bodies and fibers of the dorsal and ventral division of the NA throughout the rostro-caudal extent. Microinjection of Glu into the NA and DMV, where the vagus nerve originates, produced marked bradycardia. These studies demonstrate that GluR in the NA may be involved in shaping synaptic transmission, and regulating the swallowing activity and cardiovascular function.

3.5  Nitric oxide (NO)  NO is a free radical, which is produced in several tissues of the body and is thought to be the first of a new class of neural messenger molecules and a retrograde modulator of synaptic transmission in the brain. NO plays an important modulatory role in the CNS, endocrine and immune system. Nitric oxide synthase (NOS) is the enzyme that produces NO from the substrate l-arginine. The distribution of neuronal isoform of NOS represented the localization of generating the NO. NOS positive neurons and processes were seen in the spinal trigeminal nucleus, NTS, NA, and other nuclei in the pontine. The majority of cholinergic, aminergic and serotonergic neurons in the pons are nitric oxide synthase-positive. Studies suggest that endogenous NO may reinforce the output activity of the medullary respiratory network, control the heart rate, modulate the differentiation and mature of T cells in thymus, coordinate the motility of esophagus and pharynx.

4  Physiologic functions of NA

Morphological studies have indicated the DMV and NA form the parasympathetic preganglionic neurons. The different divisions of NA represent the various visceral areas. Physiological investigations suggest that the NA has the functions of the modulation of heart, respiration, endocrine glands and gastrointestinal motility.

4.1  Modulating the respiratory activity  Studies confirm that the medulla is the essential center of respiratory rhythm in the mammals. Respiratory neurons are mainly divided into two groups according to the positions, dorsal respiratory group and ventral respiratory group. NA is a component of ventral respiratory group. A group of respiratory neurons in the rostral NA complex is involved in the generation of inspiratory and expiratory drives which enable spontaneous respiration[23-25]. Electrical stimulation of the rat NA inhibited the respiration in our study. Studies suggest that GABA(A) receptors in the ambiguus respiratory neurons may have an inhibitory role in the synaptic transmission for maintaining the respiratory oscillation in the NA and endogenous NO may reinforce the output activity of the medullary respiratory network[26, 27].

4.2  Modulating the cardiovascular activity  A great many of morphologic and physiological studies suggest that the parasympathetic preganglionic neurons of the heart are located in the DMV, NA and the intermediate zone[28, 29]. NA efferent fascicles contain more large fibers (presumably B-type), whereas the DMV issue more fine caliber fibers (presumably C-type), and vagal control of the heart involves the convergence and integration of distinct NA and DMV projections within the cardiac plexuses. Recently, more viewpoints are that the cardiac vagal preganglionic neurons are predominantly located in the NA. NA, together with DMV, the caudal ventrolateral medulla (CVLM), and the lateral tegmental field (FTL), is cardioinhibitory area. Chemical stimulation of NA induced bradycardic responses, which were abolished by injection of lidocaine into the rostral ventrolateral medulla[30, 31]. In our study, electrical stimulation of the rat NA also induced bradycardia. Studies also show that the synaptic interaction of negative inotropic vagal preganglionic neurons in the NA with TH immunoreactive[a1]  terminals would permit the vagal control of left ventricular contractility. GABAergic neurons and NO in the NA play important roles in the modulation of the HR and artery pressure[32].

The effects of NA on the heart are mediated by respiratory neurons. The cardiorespiratory pathway was studied by transsynaptic virus expressing GFP (green fluorescent protein), which provides a neural basis of respiratory sinus arrhythmias. Rentero et al. investigated the activity pattern of cardiac motoneurons in rat NA using extracellular recordings and found cardiac vagal motoneurons firing was modulated by the central respiratory cycle, showing peak activity during inspiration, which may be the mechanisms of respiratory sinus arrhythmias[33].

4.3  Effects on the gastrointestinal tract  Morphological studies suggest that the vagal preganglionic neurons innervating the stomach are largely located in DMV, partly in NA[34]. But the distributions of the efferent fibers from NA and the afferent fibers to the stomach were reported differently. Shapiro (1985) reported that the NA projecting to the stomach predominantly innervate the forestomach. The neurons sending from the rostral NA are distributed to the stomach. But Hsieh et al. (1998) found that parasympathetic preganglionic neurons innervating the gastrointestinal tract were located exclusively in the DMV. Pagani (1986) found electrical stimulation of the cat NA complex resulted in pronounced increases in gastroduodenal mobility, but had no effects on pepsinogen secretion or titratable acidity, and elicited obvious changes in electrical activity of the pylorus. And in our study,，electrical stimulation of the rat NA had not influence on the gastric acid secretion. These results suggest that the fibers sending from NA mainly innervate smooth muscles of the gastrointestinal tract.

4.4  Effects on the immunologic function  Investigations indicate that the NA plays an important role in modulation of cellular immunity. Morphological studies have shown the somas of the parasymthetic preganglionic neurons innervating the thymus are located in NA, DMV and the retrofacial nucleus. Physiological studies indicate the cholinergic neurons in the NA may increase the cellular immunity, but the noradrenergic decrease the cellular immunity, and the enkephalinergic may regulate the activities of the anterior two transmitters. The modulation of NA on the cellular immunity is mediated by Ach and muscarinic cholimoceptors. Studies also indicate the NA has functional connections with pancreas and thyroid. Insulin and thyroxin may modulate the immunologic function. Studies also demonstrate NA is a relay with relation to immunity, communicating cerebral cortex, LHA and PAG. In conclusion, the NA regulate the immunity not only through neuron pathway, but also through humour pathway by insulin, thyroxin and so on.

  In summary, NA is an important visceral and somatic motonucleus and is located in MVZ. NA plays a pivotal role in the modulation of respiration, cardiovascular activity, immunologic function, digestion and endocrine functions.

References:

[1]  Chen LW, Rao ZR, Yang ZJ, et al. Cytoarchitecture of medullary visceral zone of the rat [J]. J Fourth Milit Med Univ, 1998, 19(3): 244-248.

[2]  Yang ZJ, Rao ZR, Ju G. Evidence for the medullary visceral zone as a neural station of neuroimmunodulation [J]. J Neurosci Res, 2000, 38(3): 237-247.

[3]  Hopkins DA. Ultrastructure and synaptology of the nucleus ambiguus in the rat: the compact formation [J]. J Comp Neurol, 1995, 360:705-725.

[4]  Hayakawa T, Yajima Y, Zyo K. Ultrastuctural characterization of pharyngeal and esophageal motoneurons in the nucleus ambiguus of the rat [J]. J Comp Neurol, 1996, 370:135-146.

[5]  Bieger D, Hopkins DA. Viscerotopic representation of the upper alimentary tract in the medulla oblongata in the rat: the nucleus ambiguus [J]. J Comp Neurol, 1987, 262:546-562.

[6]  Altschuler SM, Bao X, Miselis RR. Dendritic architecture of nucleus ambiguus motoneurons projecting to the upper alimentary tract in the rat [J]. J Comp Neurol, 1991, 309:402-414.

[7]  Saxon DW, Robertson GN, Hopkins DA. Ultrastructure and synaptology of the nucleus ambiguus in the rat: the semicompact and loose formations [J]. J Comp Neurol, 1996, 375(1):109-127.

[8]  Hayakawa T, Zheng JQ, Seki M, et al. Fine structure of the semicompact formation of the nucleus ambiguus of the rat [J]. Anat Embryol, 1997, 196(6): 465-476.

[9]  Massari VJ, Haxhiu MA. Substance P afferent terminals innervate vagal preganglionic neurons projecting to the trachea of the ferret [J]. Auton Neurosci, 2002, 96(2): 103-112.

[10] Matesz C, Szekely G. Organization of the ambiguus nucleus in the frog (Rana esculenta) [J]. J Comp Neurol, 1996, 371(2):258-269.

[11] Aicher SA, Mitchell JL, Mendelowitz D. Distribution of mu-opioid receptors in rat visceral premotor neurons [J]. Neuroscience, 2002, 115(3): 851-860.

[12] Zheng JQ, Seki M, Hayakawa T, et al. Descending projections from the paraventricular hypothalamic nucleus to the spinal cord: anterograde tracing study in the rat [J]. Okajimas Folia Anat Jpn, 1995,72:119-136.

[13] Kostarcayk E, Zhang X, Giesler GJ Jr. Spinohypothalamic tract neurons in the cervical enlargement of rats: locations of antidromically identified ascending axons and their collateral branches in the contralateral brain [J]. J Neurophsiol, 1997, 77(1): 435-451.

[14] Ciriello J, McMurray JC, Babic T, et al. Collateral axonal projections from hypothalamic hypocretin neurons to cardiovascular sites in nucleus ambiguus and nucleus tractus solitarius[J]. Brain Res, 2003, 991(1-2):133-141.

[15] Kus L, Borys E, Ping Chu Y, et al. Distribution of high affinity choline transporter immunoreactivity in the primate central nervous system [J]. J Comp Neurol, 2003, 463(3):341-357.

[16] Wang J, Wang X, Irnaten M, et al. Endogenous acetylcholine and nicotine activation enhances GABAergic and glycinergic inputs to cardiac vagal neurons [J]. J Neurophysiol, 2003, 89(5):2473-2481.

[17] Minami M, Onogi T, Toya T, et al. Molecular cloning and in situ hybridization histochemistry for rat mu-opioid receptor [J]. Neurosci Res, 1994, 18(4):315-322.

[18] Irnaten M, Aicher SA, Wang J, et al. Mu-opioid receptors are located postsynaptically and endomorphin-1 inhibits voltage-gated calcium currents in premotor cardiac parasympathetic neurons in the rat nucleus ambiguus [J]. Neuroscience, 2003, 116(2):573-582.

[19] Venkatesan P, Baxi S, Evans C, et al. Glycinergic inputs to cardiac vagal neurons in the nucleus ambiguus are inhibited by nociceptin and mu-selective opioids [J]. J Neurophysiol. 2003, 90(3):1581-1588.

[20] Yajima Y, Hayashi Y. GABA(A) receptor-mediated inhibition in the nucleus ambiguus motoneuron [J]. Neuroscience, 1997, 79(4):1078-1088.

[21] Yajima Y, Hayashi Y. Ambiguus respiratory neurons are modulated by GABA(A) receptor-mediated inhibition [J]. Neuroscience,  1999, 90(1):249-257.

[22] Wang ZM, Li YQ. Immunohistochemical location of GABAB receptor 1 subtype in the brainstem motor nuclei of the rat [J]. Chin J Neurosci, 2001, 17(2):101-104.

[23] Nakazawa K, Umezaki T, Zheng Y, et al. Behaviors of bullar respiratory interneurons during fictive swallowing and vomiting. Otolaryngol [J]. Head Neck Surg, 1999, 120(3):412-418.

[24] Kunibe I, Nonaka S, Katada A, et al. The neuronal circuit of augmenting effects on intrinsic laryngeal muscle activities induced by nasal air-jet stimulation in decerebrate cats [J]. Brain Res, 2003, 978(1-2):83-90.

[25] Moore CT, Wilson CG, Mayer CA, et al. A GABAergic inhibitory microcircuit controlling cholinergic outflow to the airways [J]. J Appl Physiol, 2004, 96(1):260-270.

[26] Shintani T, Mori RL, Yates BJ. Locations of neurons with respiratory-related activity in the ferret brainstem [J]. Brain Res, 2003, 974(1-2):236-242.

[27] Pierrefiche O, Maniak F, Larnicol N. Rhythmic activity from transverse brainstem slice of neonatal rat is modulated by nitric oxide [J]. Neuropharmacology, 2002, 43(1):85-94.

[28] Cheng ZJ, Zhang H, Guo SZ, et al. differential control over vagal efferent postganglionic neurons in rat intrinsic cardiac ganglia by neurons in the nucleus ambiguus and the dorsal motor nucleus of the vagus: anatomical evidence[J]. Am J Physiol Regul Integr Comp Physiol, 2003,.

[29] Takanaga A, Hayakawa T, Tanaka K, et al. Immunohistochemical characterization of cardiac vagal preganglionic neurons in the rat [J]. Auton Neurosci, 2003, 106(2):132-137.

[30] Marchenko V, Sapru HN. Cardiovascular responses to chemical stimulation of the lateral tegmental field and adjacent medullary      reticular formation in the rat [J]. Brain Res, 2003, 977(2):247-260.

[31] Shaw FZ, Yen CT, Chen RF. Neural and cardiac activities are altered by injection of picomoles of glutamate into the nucleus ambiguus of the rat [J]. Chin J Physiol, 2002, 45(2):57-62.

[32] Wang J, Wang X, Irnaten M, et al. Endogenous acetylcholine and nicotine activation enhances GABAergic and glycinergic inputs to cardiac vagal neurons [J]. J Neurophysiol, 2003, 89(5):2473-2481.

[33] Rentero N, Cividjian A, Trevaks D, et al. Activity patterns of cardiac vagal motoneurons in rat nucleus ambiguous [J]. Am J Physiol, Regul Integr Comp Physiol, 2002, 283(6):R1327-1334.

[34] Chiocchetti R, Clavenzani P, Barazzoni AM, et al. Viscerotopic representation of the subdiaphragmatic tracts of the digestive apparatus within the vagus complex in the sheep [J]. Brain Res, 2003, 961(1):32-44.