Vasoactive intestinal peptide-like immunoreactivity in nerves associated with the cardiovascular system of guinea-pigs

The distribution of nerves with vasoactive intestinal peptide (VIP)-like immunoreactivity has been examined in the heart and vascular system of guinea-pigs. There was a very sparse supply of fibres to the heart. No immunoreactive cell bodies were found in the intrinsic cardiac ganglia; however, positive nerve cell bodies were seen along the superior vena cava near the right atrium. There were immunoreactive fibres with most arteries; these fibres were located at the media-adventitia junction. The supply to major distributing arteries, such as the aorta, subclavian, carotid and femoral arteries as well as to the pulmonary arteries, was sparse. Of the individual vascular beds, the most densely supplied arteries were the mesenteric and uterine (or in the male deferential) arteries. Arteries running to other organs or tissues, such as skeletal muscle, kidney, pancreas, spleen and heart were less densely supplied. There were clear differences in the innervation of different cerebral vessels. The greatest density was associated with the anterior and middle cerebral arteries. Fewer nerves accompanied the posterior cerebral, cerebellar and meningeal arteries. There was a sparse innervation of the rostral part of the basilar artery. Throughout the body, veins were sparsely supplied. The distribution of nerves with VIP-like immunoreactivity was not changed when noradrenergic nerves were degenerated by 6-hydroxydopamine or when substance P nerves were disrupted by capsaicin. It is concluded that VIP containing nerves innervating the heart and blood vessels form a population distinct from the substance P-containing and the noradrenergic nerves. It is suggested that the VIP fibres might be efferent vasodilator nerves to the blood vessels.     

Neurons with 5-hydroxytryptamine-like immunoreactivity in the enteric nervous system: Their projections in the guinea-pig small intestine

Changes in the distribution of 5-hydroxytryptamine-like immunoreactivity have been examined in enteric neurons at various times after microsurgical lesions of the enteric plexuses. In the myenteric plexus, varicose immunoreactive nerve fibres disappeared or were reduced in number in ganglia anal to an interruption of the myenteric plexus. Up to about 2 mm on the anal side, all varicose immunoreactive fibres disappeared from the ganglia. At about 14–16 mm below an interruption, there were about 50% of the normal number of fibres in the myenteric ganglia and at about 24 mm the innervation was normal. In the submucosa, fibres immunoreactive for 5-hydroxytryptamine were absent from an area on the anal side following interruption of the myenteric plexus. From consideration of the pattern of disappearance, it is deduced that some myenteric nerve cell bodies send immunoreactive axons in an anal direction to supply submucous ganglia. The axons run for about 8 mm in the myenteric plexus, enter the submucosa and then run for a further 4 mm approximately.Thus, varicose fibres immunoreactive for 5-hydroxytryptamine, which occur around the enteric ganglion cells of both plexuses arise from nerve cell bodies in the myenteric ganglia that send their axons in an anal direction.     

Interaction between noradrenergic and cholinergic systems in the rat brain: behavioural function in locomotor activity.

Anticholinergic drugs such as scopolamine and atropine induce a mild locomotor stimulation when given intraperitoneally to rats. This effect is usually ascribed to interaction between dopaminergic and cholinergic transmission in the striatum or nucleus accumbens. However, an interaction of acetylcholine with noradrenergic systems is also apparent from biochemical data and the results reported here indicate that at least part of the locomotor activity induced by scopolamine or atropine involves a noradrenergic component. Depletion of forebrain noradrenaline by injection of 4 μg of the selective neurotoxin 6-hydroxydopamine into the dorsal bundle was found to potentiate the locomotor activation induced by various doses of scopolamine or atropine. This was a central effect since methylscopolamine, which does not pass the blood-brain barrier, failed to induce locomotor activity and was not affected by the noradrenergic lesion. The noradrenergic interaction was restricted to cholinergic drugs since locomotor activity induced by the indirect dopamine agonist amphetamine was not affected by noradrenaline depletion.These studies show that the interaction between noradrenergic and cholinergic transmission, which has previously been indicated by biochemical analysis, influences behaviour and they also cast some light on the functions of the central noradrenergic system itself.     

Substance P-like immunoreactivity in the human trigeminal ganglion.

Presence of substance P-like immunoreactive neurons and nerve fibers is demonstrated in the trigeminal ganglion of newborn and adult human subjects by the indirect immunofluorescence technique. Two populations of neurons containing high and low densities of immunoreactive material, respectively, are identified. Morphometric analyses indicate that (i) most of positive neurons are medium and small sized; (ii) immunoreactive perikarya grow in size from newborns to adults, with up to a 50% increase in diameter. Percent frequency of positive perikarya, on the other hand, is higher in newborns (23.6%) and decreases in adults (16.7%).     

Substance P-like immunoreactivity at the frog neuromuscular junction.

It has been recently shown that frog motoneurons and their nerve terminals contain calcitonin gene-related peptide-like immunoreactivity in large dense-core vesicles (Matteoli et al., Proc. natn. Acad. Sci. U.S.A. 85, 7366-7370, 1988). We report here by an immunofluorescence approach that the same neurons and nerve terminals also contain substance P-like immunoreactivity. The demonstration of substance P-like immunoreactivity in the frog motor nerve endings supports previous data suggesting a physiological role for this peptide in the modulation of cholinergic transmission.     

Neurons with 5-hydroxytryptamine-like immunoreactivity in the enteric nervous system: their visualization and reactions to drug treatment

Immunoreactive nerve cell bodies and fibres in the intestine have been examined using three antibody preparations raised against 5-hydroxytryptamine. Cross reactivity studies indicate that the substance localized was an hydroxylated indoleamine. In the guinea-pig small intestine, nerve cell bodies were located in the myenteric plexus and varicose fibres were found in the ganglia of the myenteric and submucous plexus. The nerve cell bodies had prominent short, broad processes and a single long process. Similar nerve cells and fibres were found in the guinea-pig stomach and large intestine and areas of intestine that were examined in mice, rabbits and rats. Properties of the neurons were examined in the small intestine of the guinea-pig. The immunoreactive material was depleted by treatment with reserpine, but not by guanethidine or 6-hydroxydopamine in dose sufficient to deplete noradrenaline stores in axons in the intestine. No depletion of 5-hydroxytryptamine by the neurotoxin 5, 7-dihydroxytryptamine was observed. After depletion by reserpine, immunoreactivity of the neurons could be restored by application in vitro of 5-hydroxytryptamine, 5,7-dihydroxytryptamine or 5-hydroxytryptophan. The restoration by 5-hydroxytryptophan was prevented by the inhibitor of L-aminoacid decarboxylase, benserazide. After reserpine treatment, immunoreactivity was not restored by tryptophan. Uptake of 5, 7-dihydroxytryptamine into the nerves was antagonized by fluoxetine. The distribution of neurons with 5-hydroxytryptamine-like immunoreactivity was compared with the distribution of enteric amine-handling neurons that take up and decarboxylate L-dopa. This comparison indicated that there are two classes of aromatic amine neuron in the guinea-pig small intestine, the enteric 5-HT neurons and enteric, non-5-HT, amine handling neurons.     

Substance P modifies the 6-hydroxydopamine induced alteration of postnatal development of central noradrenaline neurons

Systemic treatment of new-born rats with the catecholamine neurotoxin 6-hydroxydopamine leads to a permanent and selective alteration of the postnatal development of the central noradrenaline neurons, in particular of the locus coeruleus system. The changes involve a pronounced and permanent degeneration of distant nerve terminal projections (e.g. in the cerebral cortex and spinal cord) and a hyperinnervation of regions close to the noradrenaline perikarya (e.g. in the cerebellum and pons-medulla). Substance P administered intracisternally was found to counteract significantly both the 6-hydroxydopamine-induced denervation and hyperinnervation, as monitored by measuring endogenous noradrenaline levels and [3H]noradrenaline uptake in vitro. The counteracting effect of substance P disclosed a clear dose-response relationship and was most effective when injected on postnatal days one and two, while practically no effects were observed after injection on postnatal days three and four. Substance P treatment alone of new-born rats had no effect on the postnatal development of the regional monoamine levels. Binding studies employing radioligand technique showed substance P treatment to abolish the 6-hydroxydopamine-induced increase in beta-receptor binding in the frontal cortex, suggesting the 'spared' noradrenaline terminals to be functionally active. Substance P was shown to increase the utilization of noradrenaline in the neonatal stage. The results indicate that the counteracting effect of substance P may be due to a prevention of degeneration, growth stimulation and/or trophic influences on central noradrenaline neurons, possibly related to an excitatory effect of substance P on noradrenaline neurons.     

Collateral sprouting and reduced activity of the rat mesocortical dopaminergic neurons after selective destruction of the ascending noradrenergic bundles

The properties of the mesocortical dopaminergic neurons projecting to the pregenual and anterior supragenual cortices were examined 3–6 months after the degeneration of ascending noradrenergic pathways caused by bilateral multiple or single microinjections of 6-hydroxydopamine made laterally to the pedunculus cerebellaris superior. In all rats and in all cortical areas examined, noradrenaline levels were reduced by more than 75%. A similar decrease in noradrenaline levels was obtained in the ventral tegmental area. As indicated by the increases in cortical levels of dopamine and in [³H]dopamine specific uptake sites as well as by histochemical analysis, these lesions induced a collateral sprouting of the mesocortical dopaminergic neurons. The intensity of the effect varied from one animal to another and even from one anteromedial hemicortex to another. When present, the increase in dopamine levels was observed in all the cortical areas investigated. As suggested by the decreased ratio of the amount of dihydroxyphenylacetic acid to dopamine in the cortex, the activity of the mesocortical dopaminergic neurons was reduced in the rats with lesions. This effect was even seen in rats in which the cortical levels of dopamine were only slightly increased. Both the collateral sprouting and the reduced activity of the mesocortical dopaminergic neurons were related to the degeneration of the noradrenergic neurons and not to a non-specific effect of 6-hydroxydopamine, since both phenomena did not occur in rats pretreated with desipramine, a treatment which prevented the decline in noradrenaline levels.Thus, a lesion of the ascending noradrenergic pathways can lead to sprouting of dopaminergic neurons in the cortex and a reduced activity of these dopaminergic neurons. The respective role of the disappearance of the noradrenergic innervation in the cerebral cortex and in the ventral tegmental area in the collateral sprouting and in the reduced activity of the mesocortical dopaminergic neurons is discussed.     

Norepinephrine-containing terminals in kitten visual cortex: Laminar distribution and ultrastructure

The laminar distribution of monoamine-containing terminals in the visual cortex (areas 17 and 18) of 6- to 8-week-old kittens was studied with a modified glyoxylic acid histofluorescence method. The ultrastructure of monoamine-containing terminal boutons in cortical tissues fixed with glyoxylic acid perfusion followed by immersion in potassium permanganate was also studied in serial sections. In both coronal and sagittal planes, rich innervation of catecholamine-containing fibers and terminals was found throughout all 6 cortical layers. Intensely fluorescent stem fibers, which are preterminal fibers in the white matter, entered layer VI from the white matter after veering abruptly towards the cortical surface. While the stem fibers ran towards the surface, many collateral terminal fibers branched out in all directions in the middle layers; a dense plexus of catecholamine fibers and terminals was found mostly in layers II and III. Reaching the superficial layer, the stem fibers gave off terminal fibers which ran parallel to the surface.Monoamine-containing terminal boutons were identified by electron microscopy as small bulges which contained dense-cored vesicles. About 10% of the monoamine-containing boutons showed the usual synaptic membrane specialization suggesting synapse formation. Another 10% of the monoamine boutons showed only some characteristic features such as widening of the associated intermembrane space, and aggregation and contact of cored vesicles with the presynaptic membrane. The remaining 80% of the monoamine boutons failed to show any of the above described properties. The 2 types of monoamine boutons with classical synaptic features were seen in all layers, particularly II and III, but not in layer VI. The most frequent target of monoamine boutons in all 6 layers seemed to be dendritic shafts. The few axo-spiny and axo-somatic contacts observed occurred in the superficial and deep layers, respectively. The circumstantial evidence suggested that the monoamine visualized in the current study of either histofluorescence or electron microscopy was in fact norepinephrine rather than dopamine and serotonin.The rich innervation of all cortical layers with catecholamine-containing boutons is consistent with noradrenaline being a factor in modifying the plasticity of neurons in the visual cortex.     

Substance P-like immunoreactivity in cultured spinal ganglia from chick embryos

Summary The localization of substance P (SP) or a SP-like peptide in cultured spinal ganglia from chick embryos was studied by the indirect immunofluorescence technique. Ganglia from 8–16 days old chick embryos and from newly hatched chickens were cultured in a control medium or in the presence of nerve growth factor (NGF). Addition of colchicine and exposure to different explanted peripheral tissues were also tried. Ganglia from the younger embryos (8–12 days) cultured for 24 h with added NGF showed a weak SP-like immunoreactivity (SPLI) in some cell bodies and strong specific immunofluorescence in nerve fibres growing out from the ganglia. In spinal ganglia of the older embryos (14 and 16 days) and newly hatched chickens cultured with and without NGF the concentration of SPLI in the cell bodies was considerably higher. Addition of colchicine to spinal ganglia cultured 12 h in NGF-medium, resulted in retraction of nerve fibres and strongly fluorescent, expanded nerve fibres were observed in peripheral parts of the ganglia. Explants of skin placed near the spinal ganglia stimulated the outgrowth of fibres, some of them containing SPLI. A few fluorescent fibres were also seen within the skin explants. Also heart tissue explants stimulated outgrowth of nerve fibres, but innervation of these explants with SPLI-containing nerves could not be observed. Nerve fibre-extension from the spinal ganglia was not stimulated by spinal cord explants. The present results support the existence of SP-containing primary sensory neurons in chickens.     

Substance P-like immunoreactivity in the retina and optic lobe of the squid

The distribution of substance P-like immunoreactivity within the squid retina and brain was studied by immunofluorescence. Positive immunoreactivity was observed as a single layer of fibres in the retina. The retina was devoid of tyrosine-hydroxylase, serotonin, gamma-aminobutyric acid, cholecystokinin, neuropeptide Y, somatostatin, enkephalin and vasoactive intestinal peptide immunoreactivities. Substance P immunoreactivity was particularly abundant in the optic lobe. The optic lobe had a distinct layer of substance P fibres near the periphery. Immunoreactive cell bodies, fibres and varicosities were additionally present in various areas of the optic lobe. Substance P immunoreactivity in the other ganglia of the brain was restricted to a few scattered fibres.     

Projections of substance P-containing neurons within the guinea-pig small intestine

The origins of substance P immunoreactive axons in the small intestine of the guinea-pig were investigated with an immunohistochemical technique in whole mount preparations. Nerve pathways were interrupted either in vitro or in vivo to detect the accumulation of substance P proximal to the lesion and the disappearance of immunoreactive fibres resulting from the degeneration of the severed axons. Various operations, namely, extrinsic denervation, interruption of the myenteric plexus (myotomy) or removal of the myenteric plexus with the longitudinal muscle (myectomy), were performed prior to examination of substance P-containing neurons.There are several projections of substance P-containing neurons which supply the intestine. Extrinsic neurons are the sources of two projections, one to submucosal blood vessels and one to the submucous ganglia. Intrinsic neurons located in the submucous ganglia supply the villi. Five projections arise from the myenteric plexus, a very short projection ending either within the same row of ganglia or within the adjacent rows of ganglia on both sides, a longer projection within the myenteric plexus, a very short projection to the circular muscle, a projection to the submucous ganglia where the axons surround most of submucous nerve cell bodies, and a projection to the villi.It is likely that the highly organised patterns of innervation by different substance P-containing neurons have specific roles in the intestine. Some of these neurons may act as sensory neurons, others as interneurons, and yet others as motor neurons in nerve pathways within the enteric nervous system.     

Substance P-like immunoreactivity in the gracile nucleus and fasciculus in old mice

Substance P-like immunoreactivity (SPI) was observed in many axonal profiles within the gracile nucleus and fasciculus of 13-month-old mice. No SPI, however, was detected in the gracile nucleus and fasciculus in 3- and 6-month-old mice. The results suggest that the accumulation of SPI elements in axons of the gracile nucleus and fasciculus is one of the age-related changes in the central nervous system.     

Noradrenergic and non-noradrenergic nerves containing small granular vesicles in Auerbach's plexus of the guinea-pig: evidence against the presence of noradrenergic synapses

The appearance and distribution of varicosities containing small granular vesicles in Auerbach's plexus of the guinea-pig ileum, distal colon and rectum has been studied with the electron-microscope. Two types of varicosity were recognised. The first type was located predominantly at the surface of the plexus and did not form synapses on intrinsic neurons. This type became labelled with 5-hydroxydopamine, a specific marker for noradrenergic axons, and was destroyed by 6-hydroxydopamine and extrinsic denervation, procedures which lead to degeneration of noradrenergic nerves in the gut. The second type formed axodendritic and axosomatic synapses on intrinsic neurons and the morphology of its synaptic vesicles differed subtly from that of the first type. The second type was unaffected by 5-hydroxydopamine, 6-hydroxydopamine, or extrinsic denervation. It is concluded that the two types of small granular vesicle-containing varicosities belong to different neurons and that the first type is noradrenergic. Noradrenergic varicosities do not, therefore, form synapses in Auerbach's plexus. This conclusion is in accord with the electrophysiological findings. The second type of small granular vesicle-containing varicosity is not noradrenergic although it was formerly thought to be so. It is intrinsic to the gut and is resistant to the serotoninergic neurotoxin, 5,6-dihydroxytryptamine.     

Ibotenic acid lesions of the lateral hypothalamus: comparison with 6-hydroxydopamine-induced sensorimotor deficits

Three groups of rats received unilateral injections of ibotenic acid, 6-hydroxydopamine or vehicle control into the lateral hypothalamic area, and were given a range of tests of sensorimotor capacity. As expected from previous reports, the 6-hydroxydopamine injections induced a marked sensorimotor impairment to the contralateral side of the body. By contrast, the ibotenic acid injections produced no detectable sensorimotor changes, although the parameters and histological extent of the lesion were identical to those which produce aphagia, adipsia and sustained regulatory impairments when administered bilaterally. These results dissociate the classic electrolytic lesion of the lateral hypothalamus into homeostatic impairments following damage to intrinsic hypothalamic neurones, and sensorimotor impairments dependent only on damage to passing catecholamine fibre systems.     

重复注射6-羟多巴胺建立帕金森病动物模型

目的探讨通过大鼠中脑内重复注射 6-羟多巴胺 (6-hydroxydopamine,6-OHDA)建立高效、稳定、可靠的帕金森病动物模型。方法将 60 只雄性 SD 大鼠随机分为一次打击组和二次打击组,经腹腔注射 4%水合氯醛(40 mg / 100g)麻醉,脑立体定位仪固定,于左侧黑质致密部(SNC)和中脑腹侧被盖区(VTA)分别注射 6-羟多巴胺 (6-OHDA,2 g / L,2μl),二次打击组一周后在相同位置重复注射同剂量的 6-OHDA,建立帕金森模型,观察其行为改变,通过 HE、TH 和 DIG-dUTP 染色观察其细胞形态的改变及凋亡情况。结果经过二次打击的大鼠,模型成功率(旋转周数 >7 r / min)为 86.7%,明显多于一次打击组的 33.3%;HE 染色显示,二次打击组凋亡细胞的阳性率(37.12%)明显多于一次打击组(21.25%);DIG-dUTP 染色显示,一次打击组大鼠左侧中脑黑质区神经细胞肿胀的数量多 ,凋亡数量少,凋亡细胞阳性率为 20.73%,二次打击组细胞凋亡数显著增多,凋亡细胞阳性率达 36.03%;TH 染色显示,二次打击组 TH 阳性细胞数明显减少,TH 细胞阳性率(18.61%)显著低于一次打击组(36.55%)。结论通过二次打击建立帕金森病动物模型成功率高于一次打击。     

The anatomical substrate of the turning behaviour seen after lesions in the nigrostriatal dopamine system

Studies of the fibre connections of the substantia nigra suggest that the behavioural results of changes in the activity in the striatal dopamine-containing system are mediated by a pathway from the striatum to the substantia nigra and thence to the thalamus. Small discrete electrolytic lesions in the appropriate part of the crus cerebri interrupt the striatonigral axons without damage to the nigrostriatal system. Such lesions inhibit turning induced by activation of striatal dopamine receptors.Similarly, turning induced by apomorphine in rats lesioned with 6-hydroxydopamine is inhibited by damage to the ipsilateral ventromedial area of thalamus which receives fibres from the substantia nigra.     

天麻对帕金森病大鼠黑质凋亡细胞表达及酪氨酸羟化酶阳性神经元的影响

目的研究和探讨帕金森病（PD）的发病机理及天麻防治PD的作用机制。方法采用6-羟基多巴胺（6-OHDA）大鼠模型,通过免疫组化技术观察天麻对PD大鼠黑质酪氮酸羟化酶阳性（TH^＋）神经元及Caspase-3阳性（Caspase-3^＋）神经元表达水平的影响。结果PD大鼠脑组织黑质致密部（SNpc）及腹侧被盖区（VTA）TH^＋神经元表达水平组间比较差异有统计学意义（F值分别为13．29、5．04,P〈0．01）。与正常组大鼠脑组织SNpc和VTA区TH^＋神经元表达水平[（32．13±4．84）,（30．23±3．21）]比较,模型组大鼠TH^＋神经元表达水平[（18．89±3．73）和（24．20±6．35）]明显下降（P〈0．01）;经天麻和美多巴治疗后TH^＋神经元丢失减少,天麻小剂量组TH^＋神经元表达水平分别为（26．24±3．06）和（26．31±6．1）,中剂量组分别为（22．44±3．81）和（26．91±6．28）;大剂量绀分别为（20．95±4．71）和（27．3±7．1）;美多巴组分别为（27．89±4．56）和（25．974±5．14）;其中,天麻小剂量组和美多巴组SNpc区TH^＋神经元表达水平和模型组比较差异有统计学意义（P〈0．05）。PD大鼠脑组织SNpc及VTA区Caspase-3阳性细胞表达水平组间比较差异有统计学意义（F值分别为6．09、5．53,P〈0．01）。与正常组Caspase-3阳性细胞表达水平[（9．83±3．03）,（7．04±1．76）]比较,模型组火鼠Caspase-3阳性细胞表达水平[（14．53±2．33）和（12．84±2．58）]明显升高（P〈0．01）;经天麻和美多巴治疗后Caspase-3阳性细胞表达水平下降,天麻小剂量组分别为（10．68±1．83）和（7．72±1．92）,中剂量组分别为（11．29±2．8）和（10．38±3．79）;大剂量组分别为（11．89±2．97）和（11．37±1．86）;美多巴组分别为（9．99±3．3）和（10．69±3．11）;其中,美多巴组SNpc区和天麻小剂量组VTA区Caspase-3阳性细胞表达水平和模型组比较差异有统计学意义（P〈0．05）。天麻和美多巴治疗各组TH^＋神经元表达与Caspase-3^＋神经元表达呈相反趋势。结论细胞凋亡可能是PD大鼠中腑多巴胺神经元丢失的主要方式。天麻可通过调节Caspase-3减少细胞凋亡,其中,小剂量天麻对治疗PD具有较好的作用。本研究结果提示天麻可不同程度地保护多巴胺神经元。     

Modulation of cortical release of acetylcholine by noradrenaline released from nerves arising from the rat locus coeruleus

The release of acetylcholine was studied in isolated cerebral cortex slices of the rat. There was no significant difference in the release between right and left sides of the cerebral cortex. Noradrenaline reduced the ouabain-stimulated release of acetylcholine and phentolamine prevented its action. The spontaneous and evoked release of acetylcholine was higher in those slices where noradrenergic input was somehow impaired: 6-hydroxydopamine pretreatment or locus coeruleus lesion ipsilaterally resulted in a higher release. Following a locus coeruleus lesion the spontaneous evoked release of acetylcholine from slices dissected from the ipsilateral side was higher in comparison to the contralateral side. Noradrenaline significantly reduced the resting release of acetylcholine only in those cases where the noradrenergic control has been previously removed.It is suggested that the release of acetylcholine is continously controlled by noradrenaline released from nerves arising from the locus coeruleus. The removal of this inhibitory system results in an increase of acetylcholine release.