Simultaneous immunohistochemical demonstration of intra-axonally transported markers and neuropeptides in the peripheral nervous system of the guinea pig

Summary Projections and peptide neurotransmitter/neuromodulator content of autonomic and visceral afferent neurons of the guinea pig were studied after application of the subunit B of cholera toxin (CTB) with or without horseradish peroxidase (HRP) as retrograde and anterograde tracers and subsequent immunohistochemical processing for double staining using antibodies raised to CTB, HRP and various neuropeptides. The results demonstrate that substance P (SP)- and calcitonin gene-related peptide (CGRP)-containing dorsal root ganglion cells project to the pylorus as well as to the celiac superior mesenteric and stellate ganglia as demonstrated with both retrograde and anterograde transport methodology. Binding studies revealed that a small number of the CTB-binding dorsal root ganglion cells contains immunoreactivity to SP and CGRP. The majority of the CTB-binding cells is SP- and CGRP-negative and terminate in the deeper parts of the dorsal horn. After injection of CTB conjugated to HRP (B-HRP) into the nodose ganglion, both motor and sensory elements were labeled in the medulla oblongata. Some of the CTB labeled vagal sensory nerve fibers in the nucleus tractus solitarii (NTS) were also found to contain immunoreactivity to SP or CGRP. The tracer was also transported through the peripheral branch of the nodose ganglion cells and labeled terminals in the esophagus.     

Simultaneous immunohistochemical demonstration of intra-axonally transported markers and neuropeptides in the peripheral nervous system of the guinea pig

Projections and peptide neurotransmitter/neuromodulator content of autonomic and visceral afferent neurons of the guinea pig were studied after application of the subunit B of cholera toxin (CTB) with or without horseradish peroxidase (HRP) as retrograde and anterograde tracers and subsequent immunohistochemical processing for double staining using antibodies raised to CTB, HRP and various neuropeptides. The results demonstrate that substance P (SP)- and calcitonin gene-related peptide (CGRP)-containing dorsal root ganglion cells project to the pylorus as well as to the celiac superior mesenteric and stellate ganglia as demonstrated with both retrograde and anterograde transport methodology. Binding studies revealed that a small number of the CTB-binding dorsal root ganglion cells contains immunoreactivity to SP and CGRP. The majority of the CTB-binding cells is SP- and CGRP-negative and terminate in the deeper parts of the dorsal horn. After injection of CTB conjugated to HRP (B-HRP) into the nodose ganglion, both motor and sensory elements were labeled in the medulla oblongata. Some of the CTB labeled vagal sensory nerve fibers in the nucleus tractus solitarii (NTS) were also found to contain immunoreactivity to SP or CGRP. The tracer was also transported through the peripheral branch of the nodose ganglion cells and labeled terminals in the esophagus.     

功能性ghrelin受体在内脏迷走和脊髓传入神经通路中的表达

本文在mRNA和蛋白水平观察了功能性ghrelin受体(growth hormone secretagogue receptor type la,GHS-Rla)在大鼠内脏迷走及脊髓传入神经通路中的表达.结果显示:(1)GHS-Rla免疫反应阳性神经元及GHS-Rla mRNA分布于背根神经节(dorsal root ganglion,DRG)及结状神经节(nodose ganglion,NG).(2)应用免疫双标技术观察到DRG和NG中都有一些GHS-Rla免疫反应阳性神经元,同时降钙素基因相关肽(calcitonin gene-related peptide,CGRP)染色呈阳性,显示GHS-Rla和CGRP共存于同一神经元,表明内脏传入神经元存在许多亚核群.(3)应用荧光金(fluorogold)标记的神经逆行追踪技术对从胃投射到DRG和NG的神经元进行免疫组织化学染色,观察到一些表达CGRP的GHS-Rla免疫反应阳性神经元也被荧光金染色.上述实验结果证实了GHS-Rla在迷走神经和脊髓传入神经元中的表达,提示ghrelin参与了胃.脑轴的调节.     

Placodal sensory neurons in culture: nodose ganglion neurons are unresponsive to NGF, lack NGF receptors but are supported by a liver-derived neurotrophic factor

Explant and dissociated neuron-enriched cultures of nodose ganglia (inferior or distal sensory ganglion of the Xth cranial nerve) were established from chick embryos taken between embryonic Day 4 (E4) and Day 16 (E16). The response of each type of culture to nerve growth factor (NGF) was examined over this developmental range. At the earliest ages taken (E4-E6), NGF elicited modest neurite outgrowth from ganglion explants cultured in collagen gel for 24 hr, although the effect of NGF on ganglia taken from E4 chicks was only marginally greater than spontaneous neurite extension from control ganglia of the same developmental age. The response of nodose explants to NGF was maximal at E6-E7, but declined to a negligible level in ganglia taken from E9-E10 or older chick embryos. In dissociated neuron-enriched cultures, nodose ganglion neurons were unresponsive to NGF throughtout the entire developmental age range between E5 and E12. In contrast to the lack of effect of NGF, up to 50% of nodose ganglion neurons survived and produced extensive neurites in dissociated cultures, on either collagen- or polylysine-coated substrates, in the presence of extracts of late embryonic or early posthatched chick liver (E18-P7). Antiserum to mouse NGF did not block the neurotrophic activity of chick (or rat or bovine) liver extracts. Whether cultured with chick liver extract alone or with chick liver extract plus NGF, nodose ganglion neurons taken from E6-E12 chick embryos and maintained in culture for 2 days were devoid of NGF receptors, as assessed by autoradiography of cultures incubated with 125I-NGF. Under similar conditions 70-95% of spinal sensory neurons (dorsal root ganglion--DRG) were heavily labeled. 2+     

Neurotrophin and GDNF family ligand receptor expression in vagal sensory nerve subtypes innervating the adult guinea pig respiratory tract

We combined retrograde tracing techniques with single-neuron RT-PCR to compare the expression of neurotrophic factor receptors in nodose vs. jugular vagal sensory neurons. The neurons were further categorized based on location of their terminals (tracheal or lungs) and based on expression of the ionotropic capsaicin receptor TRPV1. Consistent with functional studies, nearly all jugular neurons innervating the trachea and lungs expressed TRPV1. With respect to the neurotrophin receptors, the TRPV1-expressing jugular C-fiber neurons innervating both the trachea and lung compartments preferentially expressed tropomyosin-receptor kinase A (TrkA), with only a minority of neurons expressing TrkB or TrkC. The nodose neurons that express TRPV1 (presumed nodose C-fibers) innervate mainly intrapulmonary structures. These neurons preferentially expressed TrkB, with only a minority expressing TrkA or TrkC. The expression pattern in tracheal TRPV1-negative neurons, nodose tracheal presumed Aδ-fiber neurons as well as the intrapulmonary TRPV1-negative presumed Aβ-fiber neurons, was similar to that observed in the nodose C-fiber neurons. We also evaluated the expression of GFRα receptors and RET (receptors for the GDNF family ligands). Virtually all vagal sensory neurons innervating the respiratory tract expressed RET and GFRα1. The jugular neurons also categorically expressed GFRα3, as well as ～50% of the nodose neurons. GFRα2 was expressed in ～50% of the neurons irrespective of subtype. The results reveal that Trk receptor expression in vagal afferent neurons innervating the adult respiratory tract depends more on the location of the cell bodies (jugular vs. nodose ganglion) than either the location of the terminals or the functional phenotype of the nerve. The data also reveal that in addition to neurotrophins, the GDNF family ligands may be important neuromodulators of vagal afferent nerves innervating the adult respiratory tract.     

Subtype Identification in Acutely Dissociated Rat Nodose Ganglion Neurons Based on Morphologic Parameters

Nodose ganglia are composed of A-, Ah- and C-type neurons. Despite their important roles in regulating visceral afferent function, including cardiovascular, pulmonary, and gastrointestinal homeostasis, information about subtype-specific expression, molecular identity, and function of individual ion transporting proteins is scarce. Although experiments utilizing the sliced ganglion preparation have provided valuable insights into the electrophysiological properties of nodose ganglion neuron subtypes, detailed characterization of their electrical phenotypes will require measurements in isolated cells. One major unresolved problem, however, is the difficulty to unambiguously identify the subtype of isolated nodose ganglion neurons without current-clamp recording, because the magnitude of conduction velocity in the corresponding afferent fiber, a reliable marker to discriminate subtypes in situ, can no longer be determined. Here, we present data supporting the notion that application of an algorithm regarding to microscopic structural characteristics, such as neuron shape evaluated by the ratio between shortest and longest axis, neuron surface characteristics, like membrane roughness, and axon attachment, enables specific and sensitive subtype identification of acutely dissociated rat nodose ganglion neurons, by which the accuracy of identification is further validated by electrophysiological markers and overall positive predictive rates is 89.26% (90.04%, 76.47%, and 98.21% for A-, Ah, and C-type, respectively). This approach should aid in gaining insight into the molecular correlates underlying phenotypic heterogeneity of nodose ganglia. Additionally, several critical points that help for neuron identification and afferent conduction calibration are also discussed.     

Microinjection of ANG II into paraventricular nucleus enhances cardiac sympathetic afferent reflex in rats

The aims of present study were to determine whether angiotensin II (ANG II) in the paraventricular nucleus (PVN) is involved in the central integration of the cardiac sympathetic afferent reflex and whether this effect is mediated by the ANG type 1 (AT(1)) receptor. While the animals were under alpha-chloralose and urethane anesthesia, mean arterial pressure, heart rate, and renal sympathetic nerve activity (RSNA) were recorded in sinoaortic-denervated and cervical-vagotomized rats. A cannula was inserted into the left PVN for microinjection of ANG II. The cardiac sympathetic afferent reflex was tested by electrical stimulation (5, 10, 20, and 30 Hz in 10 V and 1 ms) of the afferent cardiac sympathetic nerves or epicardial application of bradykinin (BK) (0.04 and 0.4 microg in 2 microl). Microinjection of ANG II (0.03, 0.3, and 3 nmol) into the PVN resulted in dose-related increases in the RSNA responses to electrical stimulation. The percent change of RSNA response to 20- and 30-Hz stimulation increased significantly at the highest dose of ANG II (3 nmol). The effects of ANG II were prevented by pretreatment with losartan (50 nmol) into the PVN. Microinjection of ANG II (0.3 nmol) into the PVN significantly enhanced the RSNA responses to epicardial application of BK, which was abolished by pretreatment with losartan (50 nmol) into the PVN. These results suggest that exogenous ANG II in the PVN augments the cardiac sympathetic afferent reflex evoked by both electrical stimulation of cardiac sympathetic afferent nerves and epicardial application of BK. These central effects of ANG II are mediated by AT(1) receptors.     

Effect of intestinal inflammation on capsaicin-sensitive afferents in the ileum of<Emphasis Type="Italic"> Schistosoma mansoni</Emphasis>-infected mice

In the present study, the effect of intestinal schistosomiasis on the extrinsic sensory innervation of the murine ileum was investigated. Immunocytochemical techniques to localize calcitonin gene-related peptide (CGRP), substance P (SP), and vanilloid receptor 1 (VR1) were combined with retrograde tracing techniques and capsaicin treatment. Neurochemical characterization of extrinsic primary afferent neurons (EPANs) in normal and capsaicin-treated mice, revealed that CGRP and VR1, but not SP, were expressed in extrinsic afferents. Immunocytochemical analysis using the above-mentioned antibodies yielded three different populations of neurons in both dorsal root and nodose ganglia, namely CGRP/--, SP/--, and CGRP/SP-expressing neurons. Retrograde tracing revealed that only CGRP/--expressing neurons projected to the ileum. Intestinal schistosomiasis resulted in an upregulation of the number of CGRP-immunoreactive (ir) nerve fibers in the lamina propria of the villi, coinciding with an increase in mucosal mast cells in acutely and chronically infected animals. In infected animals, mucosal mast cells were found closely associated with a dense mucosal CGRP-ir fiber network. Neonatal capsaicin treatment led to a 70% reduction in the number of mucosal mast cells. In conclusion, the present study provides evidence that CGRP is a valid marker for EPANs in the mouse ileum, which are involved in the recruitment of mucosal mast cells. Morphological evidence is provided of a neuroimmune interaction between mucosal mast cells and EPANs in schistosoma-infected mice.     

脂多糖通过诱导白细胞介素-1的生成引起迷走传入神经活动

为探讨脂多糖（liopoplysaccharide,LPS）引起迷走传入神经活动是否可能通过白细胞介素-1（interleukin-1,IL-1）的作用,将Wistar大鼠随机分为LPS实验组和生理盐水对照组,用免疫组织化学方法检测迷走神经结状神经节c-Fos及CD14的表达以及腹腔迷走神经周围Mac-1阳性巨噬细胞（macrophage,Mφ）。用L929细胞增殖法检测LPS刺激Mφ上清IL-1的生物活性。用原位杂交的方法检测迷走神经结状神经节I型白细胞介素-1受体（IL-1R I）mRNA的表达。结果显示,LPS组迷走神经结状神经节神经元c-Fos蛋白表达为阳性,而对照组迷走神经结状神经节神经元c-Fos蛋白表达为阴性。LPS注射后1h,见腹腔迷走神经周围Mφ数量明显增多。Mφ在LPS刺激后45min、1h和2h时,IL-1生成明显增高,LPS组迷走神经结状神经节IL-1R I mRNA表达为阳性。以上结果提示,LPS引起迷走传入神经活动可能通过IL-1的作用。     

Role of TRPV1 and intracellular Ca2+ in excitation of cardiac sensory neurons by bradykinin

Bradykinin is an important mediator produced during myocardial ischemia and infarction that can activate and/or sensitize cardiac spinal (sympathetic) sensory neurons to trigger chest pain. Because a long-onset latency is associated with the bradykinin effect on cardiac spinal afferents, a cascade of intracellular signaling events is likely involved in the action of bradykinin on cardiac nociceptors. In this study, we determined the signal transduction mechanisms involved in bradykinin stimulation of cardiac nociceptors. Cardiac dorsal root ganglion (DRG) neurons in rats were labeled by intracardiac injection of a fluorescent tracer, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine percholate (DiI). Whole cell current-clamp recordings were performed in acutely isolated DRG neurons. In DiI-labeled DRG neurons, 1 microM bradykinin significantly increased the firing frequency and lowered the membrane potential. Iodoresiniferatoxin, a highly specific transient receptor potential vanilloid type 1 (TRPV1) antagonist, significantly reduced the excitatory effect of bradykinin. Furthermore, the stimulating effect of bradykinin on DiI-labeled DRG neurons was significantly attenuated by baicalein (a selective inhibitor of 12-lipoxygenase) or 2-aminoethyl diphenylborinate [an inositol 1,4,5-trisphosphate (IP(3)) antagonist]. In addition, the effect of bradykinin on cardiac DRG neurons was abolished after the neurons were treated with BAPTA-AM or thapsigargin (to deplete intracellular Ca(2+) stores) but not in the Ca(2+)-free extracellular solution. Collectively, these findings provide new evidence that 12-lipoxygenase products, IP(3), and TRPV1 channels contribute importantly to excitation of cardiac nociceptors by bradykinin. Activation of TRPV1 and the increase in the intracellular Ca(2+) are critically involved in activation/sensitization of cardiac nociceptors by bradykinin.     

Small-sized neurons of trigeminal ganglia express multiple voltage-sensitive calcium channels: a qualitative immunohistochemical study

The cell bodies of pseudounipolar neurons of the trigeminal ganglia have been presumed to play a supportive role to neurites, which transmit various sensations like pain from the periphery to the brain stem. However, several studies have recently shown that these neuronal cell bodies could modulate the afferent stimuli by up-regulating various ion channels and also by increasing the synthesis of neuropeptides like calcitonin gene-related peptide (CGRP). Since voltage-sensitive calcium ion channels (VSCCs) determine neuropeptides/neurotransmitters released by neurons, the aim of the present study was to localize the various VSCCs (N-, P/Q-, L-, T- and R-types) in the trigeminal ganglia neurons by immunohistochemistry. The results showed that all the VSCCs are expressed by the cell bodies of neurons though the small-sized neurons showed higher expression of these channels. The small-sized neurons were identified by immunohistochemical localization of CGRP, the most common neuropeptide for pain transmission in the trigeminal ganglia neurons. Some of these channels (N, P/Q and T types) were also expressed on the cell surface though previous electrophysiological studies have shown the expression of all the channels on the cell surface. It is suggested that the cell bodies could play a more active role than hereto ascribed to these, in the modulation of sensory stimuli.     

Allergic inflammation-induced neuropeptide production in rapidly adapting afferent nerves in guinea pig airways

In the vagal-sensory system, neuropeptides such as substance P and calcitonin gene-related peptide (CGRP) are synthesized nearly exclusively in small-diameter nociceptive type C-fiber neurons. By definition, these neurons are designed to respond to noxious or tissue-damaging stimuli. A common feature of visceral inflammation is the elevation in production of sensory neuropeptides. Little is known, however, about the physiological characteristics of vagal sensory neurons induced by inflammation to produce substance P. In the present study, we show that allergic inflammation of guinea pig airways leads to the induction of substance P and CGRP production in large-diameter vagal sensory neurons. Electrophysiological and anatomical evidence reveals that the peripheral terminals of these neurons are low-threshold Adelta mechanosensors that are insensitive to nociceptive stimuli such as capsaicin and bradykinin. Thus inflammation causes a qualitative change in chemical coding of vagal primary afferent neurons. The results support the hypothesis that during an inflammatory reaction, sensory neuropeptide release from primary afferent nerve endings in the periphery and central nervous system does not require noxious or nociceptive stimuli but may also occur simply as a result of stimulation of low-threshold mechanosensors. This may contribute to the heightened reflex physiology and pain that often accompany inflammatory diseases.     

Musings on the wanderer: what's new in our understanding of vago-vagal reflexes?: II. Integration of afferent signaling from the viscera by the nodose ganglia

To understand vago-vagal reflexes, one must have an appreciation of the events surrounding the encoding, integration, and central transfer of peripheral sensations by vagal afferent neurons. A large body of work has shown that vagal afferent neurons have nonuniform properties and that distinct subpopulations of neurons exist within the nodose ganglia. These sensory neurons display a considerable degree of plasticity; electrophysiological, pharmacological, and neurochemical properties have all been shown to alter after peripheral tissue injury. The validity of claims of selective recordings from populations of neurons activated by peripheral stimuli may be diminished, however, by the recent demonstration that stimulation of a subpopulation of nodose neurons can enhance the activity of unstimulated neuronal neighbors. To better understand the neurophysiological processes occurring after vagal afferent stimulation, it is essential that the electrophysiological, pharmacological, and neurochemical properties of nodose neurons are correlated with their sensory function or, at the very least, with their specific innervation target.     

Cranial sensory neuron development in the absence of brain-derived neurotrophic factor in BDNF/Bax double null mice

To investigate the role of brain-derived neurotrophic factor (BDNF) in differentiation of cranial sensory neurons in vivo, we analyzed development of nodose (NG), petrosal (PG), and vestibular (VG) ganglion cells in genetically engineered mice carrying null mutations in the genes encoding BDNF and the proapoptotic Bcl-2 homolog Bax. In bax(-/-) mutants, ganglion cell numbers were increased significantly compared to wild-type animals, indicating that naturally occurring cell death in these ganglia is regulated by Bax signaling. Analysis of bdnf(-/-)bax(-/-) mutants revealed that, although the Bax null mutation completely rescued cell loss in the absence of BDNF, it did not rescue the lethality of the BDNF null phenotype. Moreover, despite rescue of BDNF-dependent neurons by the bax null mutation, sensory target innervation was abnormal in double null mutants. Vagal sensory innervation to baroreceptor regions of the cardiac outflow tract was completely absent, and the density of vestibular sensory innervation to the cristae organs was markedly decreased, compared to wild-type controls. Moreover, vestibular afferents failed to selectively innervate their hair cell targets within the cristae organs in the double mutants. These innervation failures occurred despite successful navigation of sensory fibers to the peripheral field, demonstrating that BDNF is required locally for afferent ingrowth into target tissues. In addition, the bax null mutation failed to rescue expression of the dopaminergic phenotype in a subset of NG and PG neurons. These data demonstrate that BDNF signaling is required not only to support survival of cranial sensory neurons, but also to regulate local growth of afferent fibers into target tissues and, in some cells, transmitter phenotypic expression is required.     

Compensatory responses and development of the nodose ganglion following ablation of placodal precursors in the embryonic chick (Gallus domesticus)

The nodose ganglion is the distal cranial ganglion of the vagus nerve which provides sensory innervation to the heart and other viscera. In this study, removal of the neuronal precursors which normally populate the right nodose ganglion was accomplished by ablating the right nodese placode in stage 9 chick embryos. Subsequent histological evaluation showed that in 54% of lesioned embryos surviving to day 6, the right ganglion was absent. Most embryos surviving to day 12, however, had identifiable right ganglia. In day 12 embryos, the right ganglion which developed was abnormal, with ganglion volume and ganglion cell diameter reduced by 50% and 20%, respectively, compared to control ganglia. To investigate the source of the neuron population in the regenerated ganglion, we combined nodose placode ablation with bilateral replacement of chick with quail cardiac neural crest (from mid-otic placode to somite 3). These cells normally provide only non-neuronal cells to the nodose ganglion, but produce neurons in other regions. At day 9, quail-derived neurons were identified in the right nodose ganglia of these chimeras, indicating that cardiac neural crest cells can generate neurons in the ganglion when placode-derived neurons are absent or reduced in number. On the other hand, we found that sympathetic neural crest (from somites 10 to 20) does not support ganglion development, suggesting that only neural crest cells normally present in the ganglion participate in reconstituting its neuronal population. Our previous work has shown that right nodose placode ablation produces abnormal cardiac function, which mimics a life-threatening human heart condition known as long QT syndrome. The present results suggest that the presence of neural crest-derived neurons in the developing right nodose ganglion may contribute to the functional abnormality in long QT syndrome.This work was supported by grant PO1 HL 36059     

125I]-galanin binding sites in the human nodose ganglion

The cell bodies of centrally-projecting vagal afferent neurons are contained in the inferior vagal (nodose) ganglion. Although binding sites for a number of different neuropeptides/modulators have been detected in the human nodose ganglion, the presence of galanin binding sites has not been reported. In vitro receptor autoradiography using [125I]-galanin enabled visualisation of binding sites for galanin in the human nodose ganglion. The presence of such binding sites suggests a potential role for galanin in the neuromodulation of vagal transmission in humans.     

Formation of Neuromuscular Junctions and Synthesis of Sensory Neuropeptides in the Co-cultures of Dorsal Root Ganglion and Cardiac Myocytes

Aim The interactions between primary sensory neurons and cardiac myocytes are still unclear. In the present study, the co-culture model of dorsal root ganglion (DRG) explant and cardiac myocytes was used to characterize the morphological relationship between primary sensory nerve endings and cardiac myocytes and to investigate whether cardiac myocytes could induce substance P (SP) and calcitonin gene-related peptide (CGRP) synthesis in DRG neurons and release from DRG neurons in the neuromuscular co-cultures. Methods The formation of neuromuscular junctions was observed with scanning electron microscopy (SEM). SP and CGRP expression were detected by immunocytochemistry. Basal SP and CGRP release and capsaicin-evoked SP and CGRP release were analyzed by radioimmunoassay (RIA). Results In this study, neuromuscular junctions were observed in the co-cultures of DRG explant and cardiac myocytes. SP-immunoreactive (IR) and CGRP-IR neurons were detected in both neuromuscular co-cultures and DRG explant cultures, but the number of SP-IR and CGRP-IR neurons migrating from DRG explant was significantly increased in neuromuscular co-cultures. Capsaicin-evoked SP and CGRP release but not basal SP and CGRP release in neuromuscular co-cultures increased significantly as compared with that in the cultures of DRG explant alone. Conclusions The results implicated that the morphological relationship between sensory nerve terminal and cardiac myocyte is much more close in vitro than it is in vivo. Cardiac myocytes may induce sensory neuropeptide synthesis and capsaicin-evoked neuropeptide release in neuromuscular co-cultures. Further experiment needs to be performed about the significance of neuropeptide synthesis and capsaicin-evoked neuropeptide release induced by target cardiac myocytes. Zhen Liu and Huaxiang Liu contributed equally to this article.     

Ion channel modifying agents influence the electrical activity generated by canine intrinsic cardiac neurons in situ

This study was designed to establish whether agents known to modify neuronal ion channels influence the behavior of mammalian intrinsic cardiac neurons in situ and, if so, in a manner consistent with that found previously in vitro. The activity generated by right atrial neurons was recorded extracellularly in varying numbers of anesthetized dogs before and during continuous local arterial infusion of several neuronal ion channel modifying agents. Veratridine (7.5 microM), the specific modifier of Na+-selective channels, increased neuronal activity (95% above control) in 80% of dogs tested (n = 25). The membrane depolarizing agent potassium chloride (40 mM) reduced neuronal activity (43% below control) in 84% of dogs tested (n = 19). The inhibitor of voltage-sensitive K+ channels, tetraethylammonium (10 mM), decreased neuronal activity (42% below control) in 73% of dogs tested (n = 11). The nonspecific potassium channel inhibitor barium chloride (5 mM) excited neurons (47% above control) in 13 of 19 animals tested. Cadmium chloride (200 microM), which inhibits Ca2+-selective channels and Ca2+-dependent K+ channels, increased neuronal activity (65% above control) in 79% of dogs tested (n = 14). The specific L-type Ca2+ channel blocking agent nifedipine (5 microM) reduced neuronal activity (52% blow control in 72% of 11 dogs tested), as did the nonspecific inhibitor of L-type Ca2+ channels, nickel chloride (5 mM) (36% below control in 69% of 13 dogs tested). Each agent induced either excitatory or inhibitory responses, depending on the agent tested. It is concluded that specific ion channels (I(Na), I(CaL), I(Kv), and I(KCa)) that have been associated with intrinsic cardiac neurons in vitro are involved in their capacity to generate action potentials in situ.     

The cardiovascular response to epicardial application of neurotensin in guinea pigs

Topical application of picomoles of neurotensin (NT) on the surface of the left ventricle (epicardial application) of anesthetized guinea pigs evoked dose-dependent pressor effects and tachycardia. The pressor response to epicardial NT was attenuated by pentolinium, a mixture of phentolamine and propranolol, or by guanethidine. However it was not affected by indomethacin, atropine or by a mixture of mepyramine and cimetidine. The tachycardia caused by epicardial NT was not modified by any of the aforementioned drugs. Both the pressor effects and tachycardia elicited by epicardial application of NT were markedly inhibited by chronic treatment of guinea pigs with capsaicin, and by topical application of lidocaine or tetrodotoxin to the surface of the left ventricle. Epicardial application of calcitonin gene-related peptide (CGRP), substance P (SP) or capsaicin also elicited tachycardia and either a decrease (CGRP and SP) or increase of blood pressure (capsaicin) in anesthetized guinea pigs. Epicardial application of NT, CGRP, or capsaicin in isolated, perfused hearts of guinea pigs also caused tachycardia. Together, these results suggest that the pressor responses to topical application of NT on the surface of the left ventricle in anesthetized guinea pigs are partially reflex in nature and likely to result from the stimulation by NT of cardiac sympathetic, capsaicin-sensitive, sensory nerve endings, whereas the tachycardia caused by epicardial NT appears to be due both to direct and indirect effects of NT on ventricular muscle cells. The possible participation of CGRP and/or SP in the chronotropic effect of NT applied on the epicardium, and their putative role as neurotransmitter of cardiac, capsaicin-sensitive, sensory neurons are discussed.