Amino Acid Neurotransmitters in Nucleus Tractus Solitarius: An In Vivo Microdialysis Study

Amino acid neurotransmitters in the nucleus tractus solitarius (NTS) are thought to play a key role in the mediation of visceral reflexes and glutamate has been proposed as the neurotransmitter of visceral afferent nerves projecting to this region. The present studies sought to characterize the use of in vivo microdialysis to examine extracellular fluid levels of amino acids in the NTS of anesthetized rats. Using a microdialysis probe that was 450 μm in length and a sensitive HPLC assay for amino acids, amino acids could be measured in dialysate samples collected from the NTS. Perfusion of the microdialysis probe with 60 mM K^±, to elicit depolarization of nerve terminals in the vicinity of the probe, resulted in increased dialysate fluid levels of aspartate, glutamate, glycine, taurine, and GABA. In contrast, glutamine and tyrosine were decreased and other amino acids were not significantly affected. Prior removal of the ipsilateral nodose ganglion did not alter the K^±-evoked changes in dialysate levels of any of these amino acids. Electrical stimulation of the vagus nerves, using a variety of stimulus parameters, did not significantly alter dialysate levels of glutamate or any of the other amino acids that were measured. Blockade of glutamate uptake with dihydrokainate increased dialysate levels of glutamate, aspartate, and GABA, but in the presence of dihydrokainate vagal stimulation did not alter dialysate levels of these amino acids. The results show that in vivo microdialysis can be used to examine amino acid efflux in the rat NTS and provide further evidence for amino acidergic neural transmission in the NTS. However, these studies fail to support the hypothesis that vagal afferents release glutamate or aspartate.     

Neurotransmitter and Neuromodulator Activity in the Gustatory Zone of the Nucleus Tractus Solitarius

The rostral nucleus of the solitary tract (rNST) is the firstcentral relay in the gustatory pathway. While previous investigationshave provided a wealth of information on the pattern of centralterminations of gustatory afferent fibers, the morphology ofsynaptic connections of rNST neurons and responses of secondorder neurons to taste stimuli applied to the tongue, littleis known regarding the neurophysiological characteristics ofsynaptic transmission in rNST. We have used an in vitro brainslice preparation of the rNST to study the intrinsic biophysicalproperties, neuropharmacology and synaptic responses of rNSTneurons. These experiments have revealed that rNST neurons respondto the excitatory amino acid neurotransmitter glutamate, aswell as the inhibitory amino acid neurotransmitter     

A Diffusion Barrier Between the Area Postrema and Nucleus Tractus Solitarius

The blood–brain barrier (BBB) is a structural and functional barrier that prevents free exchange of circulating substances with the brain, where the endothelial cells of microvessels are joined by tight junctions. The circumventricular organs (CVOs), by contrast, lack tight junctions and exhibit more direct communication with the circulating blood and cerebrospinal fluid. Despite many outstanding morphological studies at the electron microscopic level, there remain misconceptions that the CVOs provide direct passage of blood-borne substances to the rest of the brain. This study will show the structure of the anatomical borders of the dorsal vagal complex in the brainstem. A distinct diffusion barrier between the area postrema (AP, a CVO) and the nucleus tractus solitarius (NTS) was illustrated by immunohistochemistry at both the light and electron microscopic levels. The border zone between the AP and NTS was underlined by a continuous monolayer of columnar cells that were immunopositive for both the tight junction protein zona occludin-1 and the astrocyte marker glial fibrillary acidic protein. This observation of a diffusion barrier between the AP and NTS resolves a long-standing dispute about whether the NTS is a structural extension of the AP with a leaky BBB. Special issue article in honor of Dr. Ji-Sheng Han.     

Serotonin2 Receptors in the Nucleus Tractus Solitarius: Characterization and Role in the Baroreceptor Reflex Arc

1. There is a general agreement concerning the key role of the baroreceptor reflex in blood pressure homeostasis. It is also well accepted that baroreceptor afferent messages are first integrated within the nucleus tractus solitarius (NTS) and that an excitatory amino acid, probably glutamate, is the principal neurotransmitter of corresponding afferents fibers. However, important points concerning the processing of baroreceptor messages within the NTS remain to be clarified, in particular the possible modulatory role of other neuroactive substances at this particular level in the medulla oblongata.2. In this context, the present review focuses on serotonin, and the possible facilitatory influence of NTS serotonergic afferents and receptors on the baroreceptor reflex arc. Relevant pharmacological, electrophysiological, immunohistochemical, and biochemical data, are presented and discussed. They can be summarized as follows.3. The selective destruction of the nodose ganglion-NTS serotonergic pathway produces a long-term increase in blood pressure variability, similar to that caused by baroreceptor denervation.4. Microinjection of picomolar doses of 5-HT into the NTS elicits the typical responses of baroreceptor activation.5. The cardiovascular effects elicited by local microinjections of specific agonists and antagonists into the NTS of intact rats and of animals that underwent nodose ganglionectomy indicate that the baroreceptor-like effects of locally administered 5-HT are mediated by the activation of postsynaptic 5-HT₂ receptors.6. The medullary pathways which mediate NTS 5-HT₂ receptor-evoked responses are similar to those involved in the baroreceptor reflex arc.7. Pharmacological and electrophysiological studies suggest that the cardiovascular effects of intra-NTS 5-HT involve the 5-HT_2A receptor subtype expressed by NTS barosensitive neurons that receive polysynaptic vagal afferents.8. Intra-NTS microinjection of a subthreshold dose of DOI, a 5-HT₂ receptor agonist, which, on its own, does not produce any cardiovascular changes, significantly enhances the bradycardiac component of the baroreflex.9. Altogether, the data summarized above show that, in the NTS, 5-HT acting at 5-HT_2A receptors exerts a facilitatory influence on the baroreceptor reflex, especially on the cardiac component of this reflex.10. Convergent pharmacological and electrophysiological data indicate that, in the NTS, functional interactions between NMDA- and 5-HT_2A-receptors coexpressed by the same neurons probably underlie the facilitatory influence of 5-HT upon the baroreceptor reflex.11. Under physiological conditions, the 5-HT_2A receptor-mediated facilitatory modulation of the cardiovagal component of the baroreflex might be triggered by 5-HT released from nodose ganglion-NTS serotoninergic afferent neurons and/or for serotoninergic projections originating in raphe nuclei. The latter possibility might notably occur during recovery after physical exercise and/or during the freezing reaction in stressed animals.     

Electrophysiological Responses of Nucleus Tractus Solitarius Neurons to CCK and Gastric Distension in Newborn Lambs

The effect of cervical vagus nerve stimulation, gastric distension and CCK-8S administration was studied on the activity of 120 neurons located in the nucleus tractus solitarius (NTS) of anesthetized newborn lambs. One hundred cells responded to the three different inputs.The distribution of the cells in the NTS was from 3 mm rostral to 3 mm caudal to the obex, the major responsive cells being located at the level of the obex. Neurons were either excited or inhibited by gastric distension and CCK-8S, and the responses to these two stimuli were always in the same direction. A small number of cells responded to gastric distension and CCK-8S but not to vagus nerve stimulation.Injection of the CCK-A receptor antagonist 2-NAP abolished both the responses to CCK-8S and to gastric distension. The results are consistent with the idea that CCK-8S acts directly on vagal mechanoreceptive endings in the gastric corpus close to duodenum.These results from lambs may reflect the pathway by which gastric distension and peripheral CCK-8S modulate NTS cells activity during colostrum ingestion, which could in turn activate structures related to learning and memory processes involved in the development of mother preference.     

Hypoglycemia-Activated GLUT2 Neurons of the Nucleus Tractus Solitarius Stimulate Vagal Activity and Glucagon Secretion

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Respiratory Reactions in Rats to Microinjections of Bombesin into the Region of the Nucleus Tractus Solitarius

In experiments on 35 mongrel albino rats, we studied the effects of a neuropeptide, bombesin (0.2 l, concentrations from 10^-13 to 10^-4), which was injected into the nucleus tractus solitarius, on the pattern of respiration and on the electrical activity of the main inspiratory muscles. The local influence of bombesin on this region of the respiratory center led to the expressed intensification of respiration. The respiratory volume and the amplitude of respiratory EMG bursts (oscillatory volleys in the diaphragmatic and external intercostal muscles), as well as the integral intensity of pulmonary ventilation, increased. The maximum deviations of these indices developed under the influence of the peptide in the intermediate concentrations (10^-10 to 10^-7 M). The observed reactions were characterized by short latencies, rapid development (2 to 5 min long), and relatively short duration. The data obtained are indicative of the possibility of direct involvement of the tested neuropeptide (bombesin) in the control of respiration and confirm concepts on the important role of the dorsal structures of the respiratory center in realization of respiratory effects of neuropeptides.     

Immunohistochemical Localization of Cardio-Active Neuropeptides in the Heart of a Living Fossil, Nautilus pompilius L. (Cephalopoda, Tetrabranchiata)

Neuropeptides play an important role in modulating the effects of neurotransmitters such as acetylcholine and noradrenaline in the heart and the vascular system of vertebrates and invertebrates. Various neuropeptides, including substance P (SP), vasoactive intestinal polypeptide (VIP) and FMRFamide, have been localized in the brain in cephalopods and the neurosecretory system of the vena cava. Previous studies involving cephalopods have mainly focussed on the modern, coleoid cephalopods, whereas little attention was paid to the living fossil Nautilus. In this study, the distributions of the peptides related to tachykinins (TKs) and the high affinity receptor for the best characterized TK substance P (tachykinin NK-1), VIP, as well as FMRFamide were investigated in the heart of Nautilus pompilius L. by immunohistochemistry. TK-like immunoreactivity (TK-LI) was seen associated to a sub-population of hemocytes, VIP-LI glial cells in larger nerves entering the heart, whereas FMRFamide immunoreactivity was distributed throughout the entire heart, including the semilunar atrioventricular valves. The pattern of FMRFamide immunoreactivity matched that of Bodian silver staining for nervous tissue. The NK-1-LI receptor was located on endothelial cells, which were also positive for endothelial nitric oxide synthase-LI (eNOS). The results indicate that neuropeptides may be involved in the regulation of the Nautilus heart via different mechanisms, (1) by direct interaction with myocardial receptors (FMRFamide), (2) by interacting with the nervus cardiacus (VIP-related peptides) and (3) indirectly by stimulating eNOS in the endothelium throughout the heart (TK-related peptides).     

Immunohistochemical Localization of Periostin in Human Gingiva

The periostin is a matricellular protein expressed in collagen-rich tissues including some dental and periodontal tissues where it is regulated by mechanical forces, growth factors and cytokines. Interestingly the expression of this protein has been found modified in different gingival pathologies although the expression of periostin in normal human gingiva was never investigated. Here we used Western blot and double immunofluorescence coupled to laser-confocal microscopy to investigated the occurrence and distribution of periostin in different segments of the human gingival in healthy subjects. By Western blot a protein band with an estimated molecular mass of 94 kDa was observed. Periostin was localized at the epithelial-connective tissue junction, or among the fibers of the periodontal ligament, and never co-localized with cytokeratin or vimentin thus suggesting it is an extracellular protein. These results demonstrate the occurrence of periostin in adult human gingiva; its localization suggests a role in the bidirectional interactions between the connective tissue and the epithelial cells, and therefore in the physiopathological conditions in which these interactions are altered.Key words: Periostin, matricellular proteins, human gingiva     

血管紧张素II在小鼠卵母细胞中的免疫组化定位(英文)

本文研究了血管紧张素II在小鼠卵母细胞中的免疫组织化学定位。结果表明血管紧张素II不仅分布在卵巢内的黄体细胞、卵泡的膜细胞、基质和血管,在卵母细胞的细胞质和细胞膜上也见有阳性分布。颗粒细胞和卵丘细胞上未见着色。在恢复减数分裂过程中,处于生发泡破裂和第一极体排放期的卵母细胞内也检测到血管紧张素II[(\265\304\303\342\322\337\321\364\320\324\316\357\241\243)238.1(\322\362\264\313)],血管紧张素II有可能在卵泡的生长发育和卵母细胞的成熟过程中起着重要作用。     

小鼠卵巢促性腺激素受体的免疫组化定位

目的探明小鼠两种促性腺激素受体(FSHr、LHr)在卵巢的位置分布,揭示促性腺激素(GTH)调节卵巢机制及与卵泡发育分化的关系。方法运用免疫组化ABC法对小鼠卵巢FSHr、LHr分别进行定位染色,结合图像分析系统处理分析阳性切片。结果①FSHr阳性物质主要见于GC、TC、卵母细胞及间质细胞。随卵泡的发育,FSHr、LHr阳性细胞数量呈增长趋势,卵泡早期与中期之间阳性细胞数量差异显著,平均吸光度变化差异不显著。②LHr阳性物质主要见于卵泡TC、间质细胞、GC、卵母细胞,阳性物质着色以卵泡中、晚期较强,阳性细胞数量以卵泡中期与晚期之间差异显著。平均吸光度变化差异不显著。结论卵泡颗粒细胞、膜细胞上受体是接受促性腺激素的主要调节部位,受体数量与卵泡大小和发育程度有一定的正相关。     

Immunocytochemical Localization of Serotonin and Neuropeptides in the Neuroendocrine Paraneurons of Teleost and Lungfish Gills*

Neuroendocrine (NE) cells in the gills of six fish species were identified by serotonin and peptide immunohistochemistry. They occur either as single cells or as cell clusters within the filamental epithelium. Two populations of NE cells were identified that exhibit an immunoreactivity either for serotonin alone or for met-enkephalin and leu-enkephalin co-distributed in the same cells. We demonstrate for the first time the occurrence of open-type endocrine cells in the fish gill epithelium and confirm the presence of only serotonin-immunopositive cells of the closed type. The NE cells of the species studied do not react positively to other neuroendocrine markers occurring in the mammalian lung. Great differences probably exist in the expression of these epitopes on antigenic structures among various animal species. Although the physiological role of neuropeptides and serotonin in the fish gill NE cells is unknown, the presence of these molecular markers is of interest not only from an evolutionary aspect, but also in terms of a potential animal model to study the supposed functions of the airway neuroendocrine system in the vertebrates.     

Exercise Training Attenuates Hypertension and Cardiac Hypertrophy by Modulating Neurotransmitters and Cytokines in Hypothalamic Paraventricular Nucleus

Aims

Regular exercise as an effective non-pharmacological antihypertensive therapy is beneficial for prevention and control of hypertension, but the central mechanisms are unclear. In this study, we hypothesized that chronic exercise training (ExT) delays the progression of hypertension and attenuates cardiac hypertrophy by up-regulating anti-inflammatory cytokines, reducing pro-inflammatory cytokines (PICs) and restoring the neurotransmitters balance in the hypothalamic paraventricular nucleus (PVN) in young spontaneously hypertensive rats (SHR). In addition, we also investigated the involvement of nuclear factor-κB (NF-κB) p65 and NAD(P)H oxidase in exercise-induced effects.

Methods and results

Moderate-intensity ExT was administrated to young normotensive Wistar-Kyoto (WKY) and SHR rats for 16 weeks. SHR rats had a significant increase in mean arterial pressure and cardiac hypertrophy. SHR rats also had higher levels of glutamate, norepinephrine (NE), phosphorylated IKKβ, NF-κB p65 activity, NAD(P)H oxidase subunit gp91^phox, PICs and the monocyte chemokine protein-1 (MCP-1), and lower levels of gamma-aminobutyric acid (GABA) and interleukin-10 (IL-10) in the PVN. These SHR rats also exhibited higher renal sympathetic nerve activity (RSNA), and higher plasma levels of PICs, and lower plasma IL-10. However, ExT ameliorates all these changes in SHR rats.

Conclusion

These findings suggest that there are the imbalances between excitatory and inhibitory neurotransmitters and between pro- and anti-inflammatory cytokines in the PVN of SHR rats, which at least partly contributing to sympathoexcitation, hypertension and cardiac hypertrophy; chronic exercise training attenuates hypertension and cardiac hypertrophy by restoring the balances between excitatory and inhibitory neurotransmitters and between pro- and anti-inflammatory cytokines in the PVN; NF-κB and oxidative stress in the PVN may be involved in these exercise-induced effects.     

Immunohistochemical Localization of Nerve Fibers in the Pseudocapsule of Fibroids

The pseudocapsule surrounding fibroids consists of compressed myometrium containing nerves and blood vessels that continue into adjacent myometrium. Oxytocin (OXT) is thought to affect wound healing after myomectomy. We determined the presence of OXT and protein gene product 9.5 (PGP9.5) immunoreactive nerve fibers in pseudocapsule compared to adjacent myometrium. Samples (N=106) of pseudocapsule and adjacent myometrium were collected from 57 women with uterine fibroids undergoing myomectomy, and stained with anti-OXT and PGP 9.5 antibodies to demonstrate the presence of nerve fibers. Nerve fibers in the pseudocapsule stained positively with OXT (89/106, 84.0%) and PGP 9.5 (94/106, 88.7%). The densities of nerve fibers staining with PGP 9.5 and OXT in the pseudocapsule were highest in the isthmus (23.68±22.45/mm² and 43.35±40.74/mm², respectively). There were no significant differences in the density of nerve fibers, stained with either OXT or PGP 9.5, between the pseudocapsule, and adjacent normal myometrium regardless of the fibroid location in the uterus (P>0.05). These results suggest that the pseudocapsule should avoid to be damaged during the myomectomy procedure.Key words: fibroid pseudocapsule, nerve fibers, oxytocin, myomectomy, protein gene product 9.5, immunohistochemistry     

Structural Types of Spinal Cord Marginal (Lamina I) Neurons Projecting to the Nucleus of the Tractus Solitarius in the Rat

The structural types of spinal cord marginal (lamina I) neurons projecting to the nucleus of the tractus solitarius (NTS) were studied. Upon injections of cholera toxin subunit B (CTb) into the caudal part of the NTS, including its lateral and medial portions, labeled cells occurred bilaterally in laminae I, IV-VII, and X, and the lateral spinal nucleus (LSN). After injections into the lateral portion alone, only a few cells were labeled in laminae V, VII, and X, and the LSN, and none in the superficial dorsal horn. Of 1882 labeled marginal cells, 38% belonged to the flattened type, 37% to the pyramidal type, and 25% to the fusiform type. Flattened and pyramidal cells were labeled in considerably greater numbers than those reported when other supraspinal targets of these cells were injected with CTb. Since cells in the NTS are known to be under marked 7-aminobutyric acidergic (GABA-ergic) inhibition, it is possible that only strong input conveyed by great numbers of flattened and pyramidal cells is capable of overcoming that barrier. Fusiform cells were labeled in numbers similar to those observed previously after tracer injections into the two other targets of this neuronal type, the parabrachial nuclei and the lateral reticular nucleus. Considering that these regions, as well as the NTS, control cardiovascular and respiratory functions, it is suggested that fusiform cells transmit noxious input that will influence autonomic reflexes processed in the three nuclei.     

Immunohistochemical Localization of Calmodulin-Dependent Cyclic Phosphodiesterase in the Human Brain

The amplification of cyclic nucleotide second messenger signals within neurons is controlled by phosphodiesterases which are responsible for their degradation. Calmodulin-dependent phosphodiesterase (CaMPDE) is an abundant enzyme in brain which carries out this function. For the first time, we have localized CaMPDE in the normal human brain at various ages, using a monoclonal antibody designated A6. This antibody was generated using standard techniques, purified, and applied to tissue sections. Autopsy specimens of human brain with no neuropathological abnormalities were selected representing a range of pre- and postnatal ages. Sections of various brain regions were evaluated for immunoreactivity, graded as nil, equivocal, or definite. We demonstrated definite CaMPDE immunohistochemical staining in neocortex, especially in neurons in layers 2 and 5. There was definite neuronal immunoreactivity in the hippocampus, and in the subiculum. The striatum had definite patchy neuronal staining. Definite terminal staining in the globus pallidus externa and substantia nigra pars reticulata outlined resident neurons, interpreted as axonal terminal staining. Cerebellar Purkinje cells showed definite immunoreactivity. In the developing brain, definite immunohistochemical staining was seen in the cerebellar external granular layer. The expression of CaMPDE in specific subsets of neurons suggests they may correlate with cells having dopaminergic innervation and/or high levels of neuronal integration.