Oxytocinergic innervation to the upper thoracic sympathetic preganglionic neurons in the rat

A combination of retrograde cell body labeling and immunohistochemistry was employed to elucidate how oxytocinergic fibers make contact with sympathetic preganglionic neurons (SPNs) in the rat spinal cord from T1 to T4. SPNs were labeled retrogradely using cholera toxin subunit B (CTb) or horseradish peroxidase-conjugated CTb. Oxytocin-immunoreactive (ir) fibers were found in the intermediate zone, including the sympathetic preganglionic subnuclei. In the central autonomie nucleus and the intercalated nucleus, brown-stained oxytocin-ir varicosities or terminals were frequently observed to stud black-stained dendrites of SPNs. Electron microscopical observations showed that oxytocin-ir terminals form synapses with dendrites or soma of the sympathetic preganglionic neurons. The terminals contained numerous small clear round vesicles and a few large, cored vesicles. These results clearly show that a large proportion of SPNs are innervated by oxytocin-containing fibers. The origin of these fibers is discussed, and it is concluded that they are probably descending fibers from the paraventricular nucleus of the hypothalamus.     

Confocal imaging of autonomic preganglionic neurons in the spinal cord of the caecilian Typhlonectes natans (Amphibia: Gymnophiona)

Little is known about the spinal sympathetic organization in the caecilian amphibians. We examined for the first time the location of sympathetic preganglionic neurons (SPNs) in the spinal cord using a panel of specific markers expressed in SPNs. The SPNs of anuran amphibians form two cell columns segregated mainly in the lateral and medial marginal areas of the central gray matter. In the caecilian Typhlonectes natans immunoreactivity for galanin and ChAT is found in most laterally arranged neurons lying in spinal segments 2–7. They are encircled by TH- and nNOS-immunoreactive nerve fibers. These neurons might project specifically to a population of adrenergic sympathetic postganglionic neurons in paravertebral ganglia and/or non-adrenergic sympathetic postganglionic neurons in the celiac ganglia. However the segmental restriction and target specificity of the neurons of the species studied are not known. As mucous and granular glands in the dermis may represent one of the peripheral targets of the adrenergic ganglion cells and reflect the prominent preganglionic cell columns, an immunohistochemical study was done also on these glands. Retrograde-tracing studies are, however, needed to study the segmental localization of the preganglionic neurons and their projections to the postganglionic neurons in sympathetic ganglia.     

Cholecystokinin-8-like-immunoreactive fibers in rat lumbosacral autonomic regions are sexually dimorphic and altered by a reduction of androgen receptors

Cholecystokinin-8-like-immunoreactive (CCK-8-LI) fibers in laminae VII and X of the rat lumbosacral spinal cord demarcate the position of preganglionic autonomic neurons. This investigation reveals that adult male Sprague-Dawley, or King-Holtzman/Sprague-Dawley rats contain more CCK-8-LI fibers in lumbosacral laminae VII and X than adult females. Furthermore, testicular feminization mutation male rats (which lack 85-90% of their functional androgen receptors) contain fewer CCK-8-LI fibers than normal male or female rats, with the amount of CCK-8-LI being reduced to a greater extent in the sympathetic vs. the parasympathetic regions of the lumbosacral spinal cord. Thus, CCK-8-LI in testicular feminization mutation male rats has a distinctly female-like pattern. These results suggest that testosterone is a regulatory factor for CCK-8-LI fibers found in laminae VII and X of the lumbosacral spinal cord. Sexual dimorphism in lumbosacral CCK-8-LI fibers may contribute to modulating the final common pathway which differentially regulates the reproductive organs and stereotypic reproductive behavior, and may be involved with the sex differences described for pain.     

Morphological change of substance P receptor immunoreactive dendrites of preganglionic sympathetic neurons

Substance P (SP)-containing terminals and SP receptors (SPRs) are found on the dendrites of preganglionic sympathetic neurons (PSNs) in the intermedio-lateral nucleus (IML) of the spinal cord. The SP-containing fibers were thought to be of supraspinal origin. However, the primary sensory nerve fibers terminated around PSNs, and some of them directly on PSNs. We observed approximately 150 SPR-immunoreactive (ir) varicosities on the dendrites of PSNs in slices of the first thoracic segment (T1) in control rats. The number of varicosities decreased to 41% 14 days after hemisection of the spinal cord at the fourth cervical segment (C4), and to 55% 14 days after sectioning the spinal dorsal roots at the C8 and T1 levels. The number of varicosities decreased by 33% in 8-week-old rats which had been administered capsaicin subcutaneously within 24 hours after birth to eradicate unmyelinated sensory fibers. However, varicosities increased by 15% 15 minutes after injection of capsaicin into the plantar surface of the front paw to stimulate somatosensory nerve fibers in adult rats. The results demonstrate that SPR-ir varicosities on the dendrites of PSNs were modulated not only by the supraspinal nervous system but also directly by the primary sensory nerve terminals.     

Secretoneurin-immunoreactivity in nerve terminals apposing identified preganglionic sympathetic neurons in the rat: colocalization with substance P and enkephalin

Preganglionic sympathetic neurons projecting to the superior cervical ganglion are innervated by nerve fibers containing classical neurotransmitters as well as neuropeptides. In this study we examined the possible participation of a novel peptide, secretoneurin (a cleavage product of secretogranin II), in regulation of sympathetic outflow to head and neck by using a retrograde labelling-technique combined with immunohistochemistry. In addition, the coexistence of secretoneurin with substance P and leu-enkephalin, peptides known to innervate preganglionic neurons, was investigated. The majority of retrogradely labeled neurons were localized in the nucleus intermediolateralis of spinal cord segments T1–T3 (maximum at T2). Nearly all of Fast Blue positive neuronal perikarya were apposed by nerve fibers and terminals exhibiting immunoreactivity to secretoneurin. The main secretoneurin-immunoreactive form found in the upper thoracic segments corresponded to the free peptide secretoneurin as revealed by chromatography and radioimmunoassay. More than half of labeled neurons were surrounded by nerve endings containing in addition substance P or leu-enkephalin which were also, however, less frequently colocalized. Our results suggest that secretoneurin influences the activity of preganglionic sympathetic neurons projecting to the superior cervical ganglion. Regarding their frequent colocalization with substance P and leu-enkephalin, functional interactions of these peptides on preganglionic sympathetic nerve activity have to be considered.     

Synergistic effect of serotoninergic and cholinergic nerve fibers

The mechanisms of interactions between various compartments of the autonomic nervous system were evaluated by studying sympathetic potentiation of vagal cardioinhibitory action. It was found that this effect is realized through preganglionic serotoninergic nerve fibers making synaptic contacts with intracardiac serotoninergic neurons. Our results indicate that cardiac activity is modulated by the vagus nerve and serotoninergic nerve fibers of the sympathetic nerve acting synergistically.     

Age-related changes of enkephalinergic innervation of human sympathetic neurons

The age-related changes in the distribution of enkephalin-immunoreactive (ENK-IR) nerve fibers in relation to the principal neurons were studied in 23 human sympathetic ganglia from patients aged between 22 and 98 years. There were no age-related changes in the diameter of nerve cells and the packing density of the neurons did not change. There was an age-related decrease in the proportion of neurons innervated by enkephalin-containing fibers. In all age groups the neurons innervated by ENK-IR nerve fibers were larger than those neurons which were not surrounded by ENK-IR nerves. The pigment material in the enkephalin-innervated neurons was non-osmiophilic. The small neurons containing dark neuromelanin type of age pigment were not innervated by ENK-IR nerves. It is speculated that ENK-IR preganglionic nerves play a role in maintaining neuronal integrity of sympathetic neurons.     

Varied effects of experimental diabetes on the autonomic nervous system of the rat

Disorders of the autonomic nervous system are a frequent late complication of human diabetes. They have been extensively studied clinically, yet their pathologic aspects are still poorly understood. Also, reports on the autonomic nervous system in animal models for diabetes are scanty. Therefore we have investigated sympathetic preganglionic and postganglionic nerve fibers, vagal fibers, as well as sympathetic and enteric neurons of male Wistar rats 1 year after streptozotocin or vehicle injection. By light and electron microscopic morphology we observed: various degenerative changes in sympathetic neurons and in Schwann cells of the sympathetic fibers; intraaxonal glycogen deposits in all fiber types; and a large amount of lipoid material in intraganglionic and endoneural mesenchymal cells. By morphometry, the cytoplasmic area and perimeter as well as the cytoplasmic to nuclear ratio were significantly reduced in the sympathetic neurons of diabetic rats. Further we found axonal dwindling, enlarged myelin-axons space and an increased number of Schwann cell pi-granules in the sympathetic preganglionic fibers of diabetic animals. Axonal glycogenosomes were absent in the vagus of control and were present in that of diabetic rats. By stereology, the mitochondria and smooth endoplasmic reticulum were reduced in the sympathetic neurons of diabetic rats, whereas in the same animals the volume density of the Golgi complex was increased in the sympathetic neurons and decreased in the enteric neurons. In conclusion, relevant changes occur in the sympathetic preganglionic nerve fibers which suggest a causal relationship between fiber and neuronal lesions. Further, the stereologic findings imply decreased cellular activity and imbalance between cellular synthesis and secretion in the sympathetic neurons.     

Norepinephrine depolarizes lateral horn cells of neonatal rat spinal cord in vitro

Superfusion of norepinephrine (NE) (1-50 microM) onto lateral horn cells, including antidromically identified sympathetic preganglionic neurons (SPNs), situated in thin transverse neonatal rat thoracolumbar spinal cord slices caused a membrane depolarization and repetitive cell discharges. The NE depolarization was associated with an increase in membrane resistance, and the response became smaller upon conditioning hyperpolarization; a clear reversal of polarity, however, was not observed. Pretreating the slices with phentolamine and prazosin but not yohimbine or propranolol prevented the depolarizing effect of NE. This finding, in conjunction with the evidence of the presence of noradrenergic fibers in the spinal cord, suggests that NE may serve as an excitatory neurotransmitter to neurons of the lateral horn.     

Biophysical and histological determinants underlying natural firing behaviors of splanchnic sympathetic preganglionic neurons in neonatal rats

Isolated thoracic spinal cords of neonatal rats spontaneously generate splanchnic sympathetic nerve discharge (SND) with a quasiperiodic rhythm 1-Hz. Using in vitro nerve-cord preparations that retained T6–T12 spinal segments, we investigated whether the natural firing behavior of sympathetic preganglionic neurons (SPNs) encoded the SND rhythm and what were the main biophysical and histological determinants of SPN firing. Under extracellular recording conditions, electrical stimulation of splanchnic nerves elicited antidromic responses in 212 SPNs. Among them, 92 SPNs were quiescent; 120 active SPNs had an average firing rate of 0.72±0.04 Hz, which was close to the quasiperiodic rhythm of SND. SPNs with rhythmic burst firing were rare. Probability plots of interspike intervals were constructed to extract mathematical features underlying SPN firing. Most active SPNs (88%) had a firing well described by unimodal Gaussian, suggesting a predominantly tonic pattern with normal variations. Biophysical properties of 112 SPNs were measured under whole-cell recording conditions. The charging time constant, τ, is positively correlated with the average firing rate. Histological properties were examined in 45 SPNs with intracellular diffusion of Lucifer Yellow or biocytin. SPNs with pyramidal somata and multipolar dendrites tend to be spontaneously active. In contrast, those with bipolar somata and fewer dendritic branches were quiescent in firing. These observations suggest that activity levels of SPNs are correlated with their capacity for temporal and spatial summation of synaptic inputs. How the seemingly tonic firing of individual SPNs is integrated into whole-nerve SND with quasiperiodic rhythms is discussed.     

Neuropeptide Y-immunoreactive synapses in the intermediolateral cell column of rat and rabbit thoracic spinal cord

Neuropeptide Y (NPY)-immunoreactive nerve fibers in the intermediolateral cell column of rat spinal cord segments T2-T3 and T8-T10 and rabbit segments T3-T6 were studied with light and electron microscopic immunocytochemistry. Plexuses of NPY-immunoreactive nerve fibers were found by light microscopy. NPY-positive synapses were present electron microscopically but non-immunoreactive synapses greatly outnumbered NPY-immunoreactive ones. In the lateral horn of rat T9, 30% of the vesicle-containing NPY-positive axon profiles formed synapses, 95% of which were axodendritic. These synaptic connections may mediate the effects of brainstem NPY neurons on the activity of sympathetic preganglionic neurons.     

Remodelling of connections in pelvic ganglia after hypogastric nerve crush

Pelvic ganglia innervate the urogenital organs and contain both sympathetic and parasympathetic neurons. Previous studies have shown that within days of cutting either the lumbar or sacral preganglionic axons that innervate pelvic ganglia, many axon collaterals grow and appear to form specific connections with denervated pelvic neurons. Here we have examined the longer term consequences of partial deafferentation by studying pelvic ganglia up to 7 weeks after hypogastric nerve (HGN) crush, a lesion which also allows faster regeneration of spinal axons. Noradrenergic neurons were denervated by HGN crush, as demonstrated by loss of varicosities immunostained for the synaptic proteins, synaptophysin and synapsin. A week after HGN crush, axon collaterals grew from parasympathetic pelvic ganglion neurons, shown by the presence of numerous varicose fibers immunostained for vasoactive intestinal peptide (VIP). These VIP fibers were poorly stained or unstained for synaptophysin, even after 7 weeks. At early post-operative times the VIP fibers grew irregularly; however, with longer post-operative times they appeared to target particular VIP-negative, noradrenergic neurons. Our results also indicate that some lumbar preganglionic axons regenerated during the post-operative period, although this only affected a minority of sympathetic neurons. These reinnervated sympathetic neurons were not associated with VIP fibers, suggesting that the new intrinsic connections may have precluded regeneration or targeting of preganglionic axons. Together these results demonstrate that there is considerable remodelling within pelvic ganglia after partial deafferentation. This occurs under conditions where spinal preganglionic axons can regenerate. New intra-ganglionic connectivity may be permanent and may impact on this regeneration.     

Antidromic discharges of sympathetic preganglionic neurons located outside of the spinal cord lateral horns

Responses arising in thin ventral rootlets and antidromic discharges of single sympathetic preganglionic neurons (SPNs) in T₃ and L₂ segments of the cat spinal cord were recorded simultaneously during stimulation of the white rami communicantes in the same segments. It was shown that in addition to the classical lateral horn SPN with B-axons (conduction velocities 3—10 m/sec) there are also ventral horn SPNs which have B-axons with faster conduction velocities (10–20 m/sec) and intermediate zone SPNs with C-axons (conduction velocities 0.98–1.7 m/sec).     

Both synaptic and antidromic stimulation of neurons in the rat superior cervical ganglion acutely increase tyrosine hydroxylase activity

Electrical stimulation of the preganglionic cervical sympathetic trunk produces an acute increase in the rate of DOPA synthesis in the rat superior cervical ganglion. The present study was designed to test the possibility that this acute transsynaptic stimulation of catechol biosynthesis could be, at least in part, a consequence of an increase in the firing rate of the postganglionic sympathetic neurons. For this purpose, the effect of stimulation in vitro of the preganglionic cervical sympathetic trunk was compared to that of stimulation of the predominantly postganglionic internal and external carotid nerves. Stimulation of the cervical sympathetic trunk at 10 Hz for 30 min produced a 4.6-fold increase in DOPA synthesis, while simultaneous stimulation of the two postganglionic trunks produced a 3.1-fold increase. The internal carotid nerve is known to contain a small population of preganglionic fibers that synapse on principal neurons in the ganglion before entering this nerve trunk. To eliminate the possibility that the effect of stimulation of the internal carotid nerve is mediated by synaptic stimulation via these preganglionic "through fibers", the effect of stimulation of previously decentralized ganglia was examined. While decentralization reduced the magnitude of the effect of stimulation of the internal and external carotid nerves, a 2.0-fold increase in DOPA synthesis was still seen. In addition, when these nerve trunks were stimulated in control ganglia that had been maintained in organ culture for 48 h to allow time for the degeneration of afferent nerve terminals, DOPA synthesis increased 4.1-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

Descending input from the hypothalamic paraventricular nucleus to sympathetic preganglionic neurons in the rat

Summary The descending projection of the hypothalamic paraventricular nucleus (PVN) to the sympathetic preganglionic neurons (SPNs) in the upper thoracic cord of the rat was studied. PVN-fibers were labeled by anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L), while SPNs were retrogradely labeled with cholera toxin subunit B (CTb) which was injected into the superior cervical ganglion. SPNs labeled with CTb were mainly observed in the nucleus intermediolateralis (IML) pars principalis and pars funicularis, and a small number of them were in the nucleus intercalatus (IC) and central autonomic nucleus (CA). SPNs found in the IML had dendrites that projected in various directions. Five types of dendritic projections were noted: medial, rostral, caudal, lateral (including dorsolateral) and ventral. Longitudinal dendritic bundles interconnected each cell cluster in the IML. Medial dendrites of the IML, together with dendrites of the IC and CA, formed transverse dendritic bundles extending from the IML to the central canal. The transverse dendritic bundles disentangled near the midline and formed a loose dendritic plexus in the region just dorsal to the central canal. PVN-fibers labeled with PHA-L were observed primarily in lamina I and intermediate gray (lamina VII). Although varicose PVN-fibers and SPNs coexisted in the IML, the tight packing of the dendritic bundles prevented any clear demonstration of direct contacts between them. On the other hand, PVN-fibers were occasionally found to appose and wind around the primary or secondary dendrites of some SPNs of the CA and IC. These dendrites were studded with varicosities of PVN-fibers for a short length, and terminal boutons of PVN-fibers were also seen to make contact directly with the dendrites. The results of this study substantiated a direct connection between the PVN and SPNs, using a combination of immunohistochemical techniques for PHA-L and CTb. The possible involvement of a direct pathway from the PVN to SPNs in cardiovascular regulation is discussed.Abbreviations AF anterior funiculus - CA central autonomic nucleus - CC central canal - CTb cholera toxin subunit B - HRP horseradish peroxidase - IC nucleus intercalatus - IMf nucleus intermediolateralis pars funicularis - IML nucleus intermediolateralis - IMp nucleus intermediolateralis pars principalis - LDB longitudinal dendritic bundle - LF lateral funiculus - PF posterior funiculus - PHA-L Phaseolus vulgaris leucoagglutinin - PVN hypothalamic paraventricular nucleus - SPNs sympathetic preganglionic neurons - TDB transverse dendritic bundle     

Increase in protein kinase-C-beta-like immunoreactivity (PKC-beta-LI) in the rat superior cervical ganglion after decentralization.

The effect of decentralization on protein kinase-C-beta-like immunoreactivity (PKC-beta-LI) in the rat superior cervical ganglion (SCG) was studied. The cervical sympathetic trunk was transected and at different time points (0 h-14 d) after transection the rats were decapitated and the SCGs examined immunohistochemically. In the control ganglia only faint PKC-beta-LI was seen in the principal neurons, whereas several strongly immunolabelled nerve fibers were observed. In the principal ganglionic neurons PKC-beta-LI was found to be increased 1-8 days after denervation, with the maximum accumulation occurring at 4 days. Fourteen days post-transection PKC-beta-LI in the neuronal perikarya was back to the initial level, but a decrease in the number of PKC-beta-immunoreactive nerve fibers was observed. The results suggest that the amount of PKC-beta in the principal neurons of the rat SCG may be partly regulated transsynaptically by preganglionic innervation.     

Morphology of sympathetic preganglionic neurons innervating the superior cervical ganglion in the chicken: an immunohistochemical study using retrograde labeling of cholera toxin subunit B

Summary Preganglionic sympathetic neurons (SPNs) in the chicken were demonstrated immunohistochemically using cholera toxin subunit B (CTb) as a retrograde tracer. After injection of CTb-solution into the superior cervical ganglion, labeled SPNs were mainly found in the ipsilateral sympathetic preganglionic column of Terni (the column of Terni), with only a few in the intermediate zone. They were observed from the caudal half of the 15th cervical segment to the rostral tip of the 3rd thoracic segment. Cell somata of SPNs were loosely packed with-in the column of Terni, where they had an elliptic shape with the long axis oriented rostrocaudally. In the horizontal plane three kinds of dendrites could be discriminated on the basis of their orientation. Longitudinally oriented dendrites emanated from the rostral and the caudal poles of the SPNs. Medially oriented dendrites were observed to cross the midline and enter the contralateral column of Terni, where they further branched to form a loose dendritic plexus; some extended beyond the lateral limit of the contralateral column of Terni to reach the intermediate zone. Laterally oriented dendrites formed periodically arranged dendritic bundles projecting into the intermediate zone. The present findings provide a detailed account of the dendritic organization of SPNs in the chicken, and suggest that avian SPNs share certain structural features in common with mammalian SPNs.     

Coexistence of serotonin- and substance P-like immunoreactivity in nerve fibers apposing identified sympathoadrenal preganglionic neurons in rat intermediolateral cell column

In this study we examined the possibility that serotonin (5-HT) and substance P (SP) coexist in fibers and terminals afferent to sympathoadrenal preganglionic (SAP) neurons in the intermediolateral cell column (IML) of the spinal cord. SAP neurons in the IML were identified by retrograde labeling with either Fast Blue or True Blue injected into the adrenal medulla of rats. A simultaneous immunofluorescent double labeling technique was used to identify both 5-HT- and SP-like immunoreactivity in single tissue sections. Labeled SAP neurons were observed which were apposed by fibers immunoreactive for either neurotransmitter, as well as SAP neurons apposed by neither 5-HT- nor SP-like immunoreactive structures. In addition, 5-HT- and SP-like immunoreactivity were observed in separate fibers apposing the same labeled neuron and coexisting in fibers and terminal appearing in apposition to labeled SAP neurons. These data suggest a complex interaction by these neurotransmitters in regulating sympathetic outflow and may provide a model for interpreting conflicting observations concerning the effects of local 5-HT administration on sympathetic nerve activity.     

猫空回肠交感节后神经元和副交感节前神经元的局部定位

本文应用ＨＲＰ逆行标记法研究猫空回肠的植物神经支配，发现猫空回肠交感节后神经纤维来自腹腔节和肠系膜前节以及双侧Ｔ９～Ｌ５交感于神经节，但大多数来自椎前节。交感节神经元大多数呈簇状集中分布在右侧腹腔节的前下部和肠系膜前节，而在左侧腹腔节和右侧腹腔节后上部仅有少数散在分布的标记细胞，具有明显的局部定位分布的特征。猫空回肠的交感节后神经元在双侧交感于神经节具有基本相同的分布范围，即Ｔ９～Ｌ５节段。其集中分布节段是Ｔ１１～Ｌ３，符合“既分散又集中的分布模式”。腹腔节和肠系膜前节内的标记细胞呈椭圆形、圆形或不规则形。交感干神经节内的标记细胞呈椭圆形和梭形。猫空回肠的副交感书前神经元主要分布在双侧迷走神经前核的闩以上４ｍｍ到闩以下２ｍｍ的范围内．但多数分布在此核的背外侧部靠近闩的水平上，细胞呈椭圆形。     

内脏大神经初级传入神经元向中枢投射的光镜和电镜研究——CT-HRP跨神经节运送法

用猫20只,于内脏大神经注射CT—HRP,对初级传入在中枢内的投射进行了光、电镜观察。光镜下,内脏初级传入神经元的中枢投射纤维主要经Lissauer's束到脊髓后角边缘,部分可能终止于Ⅰ层,大部分分为内、外侧束包绕灰质后角边缘由浅部板层向深部板层进入Ⅴ、Ⅶ、Ⅹ层。内侧束可能有部分上升到薄束核。电镜观察,在Ⅰ、Ⅴ、Ⅶ、Ⅹ层看到标记的轴突末梢且可直接与交感节前神经元形成突触。本文还对内脏传入通路进行了讨论并认为交感传入纤维可能经脊髓后角神经元中继上传至孤束核。