Effect of substance P on the 5,7-dihydroxytryptamine induced alteration of the postnatal development of central serotonin neurons

Systemic treatment with the serotonin neurotoxin 5,7-dihydroxytryptamine [5,7-HT]in the neonatal stage leads to a permanent alteration of the postnatal development of the serotonin neurons in rat brain with denervation of distant nerve terminal projections and hyperinnervation in regions close to the serotonin perikarya. Intracisternal administration of substance P was found to counteract both the denervation and the hyperinnervation, as evaluated by measuring endogenous serotonin levels and [3H]-serotonin uptake in vitro. Furthermore, substance P was found to potentiate the reduction of serotonin induced by tryptophan hydroxylase inhibition with alpha-propyldopacetamide, indicating that substance P can produce an increase in serotonin utilization and turnover. The results suggest that substance P has a degeneration preventing and/or regrowth stimulatory effect on damaged serotonin neurons during ontogeny.     

Activity of neurons of the red nucleus after destruction of the inferior olive in the rat

The spontaneous frequency of discharge of the red nucleus neurones has been evaluated in the rat before and after total and bilateral destruction of the inferior olive by 3-acetylpyridine. The suppression of the inferior olive increasing the cerebellar inhibition, produces, as a consequence, a consequence, a disfacilitation of the activity of the red nucleus neurones. The process persists for a few days, then a progressive compensation takes place. A month later the average frequency of discharge is recovered but a normal unit activity and motor behaviour are not restaured.     

Development of ionic conductances in neurons of the inferior olive in the rat: an in vitro study

Neurons of the inferior olive of the rat were studied at different stages of their postnatal (PN) development by using the current clamp technique in slices maintained in vitro. Antidromic and synaptic activation of inferior olivary neurons could be achieved in preparations as young as PN day 2. Neurons at this age already exhibited a variety of ionic conductances which included fast sodium-dependent spikes, high-threshold and low-threshold calcium spikes, potassium-dependent currents, Ca-dependent after-hyperpolarizing potentials (AHPS), and both instantaneous and time-dependent inward rectification at hyperpolarized levels of membrane potential. The two types of Ca-dependent responses recorded in olivary neurons during the first postnatal week were graded with the magnitude of the depolarization imposed on the cells. Furthermore, the high-threshold Ca spikes were only clearly observed during this early period when K conductances were depressed by the injection of caesium into the cells or by bath application of 4-aminopyridine. In contrast, the high-threshold Ca spikes could be obtained without suppression of K currents and were all-or-none in character in some neurons after PN day 8 and in all neurons after PN day 11. The observations suggest that the balance between K and Ca currents changes throughout maturation and is largely in favour of the K current until about the end of the first PN week. At all ages studied, the low-threshold Ca spikes were much less sensitive to the Ca channel blocker cadmium than were the high-threshold Ca spikes. Finally, spontaneous, regular oscillations of the membrane potential were observed for the first time at PN day 16 and were only commonly observed after PN day 19, suggesting a late development of electrotonic coupling between olivary neurons.     

Immunocytochemical study of substance P containing nerve terminals in rat spinal cord

Summary The subcellular localization of substance P (SP) in the dorsal horn of the rat spinal cord was studied using the unlabelled antibody procedure of Sternberger with different fixatives (4% paraformaldehyde alone or with varying amounts of glutaraldehyde), buffer systems for the immunohistochemical incubations, and the presence or absence of the detergent, Trition X-100. Hand-sliced tissues were compared with Vibratome sections, and showed adequate results which are described below. Labelled terminals of two types could be seen in all samples incubated with anti-SP sera. The two types of positive terminals can be described as those which contained mostly immunoreactive clear vesicles, and those which contained both immunoreactive clear and dense core vesicles. Brief fixation during pressure perfusion with increased concentrations of glutaraldehyde (up to 2%) improved the tissue preservation and, as a result, the intensity and definition of the SP immunoreaction products. The use of Tris or phosphate buffer for the immunohistochemical incubations maintained the intensity of staining in well-fixed tissues. However, Tris incubations contributed to a diffusion of immunoreaction products and increased the number of broken membranes in the labelled processes as well as those of myelin. These phenomena were not observed in phosphate buffer, which preserved the tissue better than Tris. Like Tris, pretreatment with the detergent Triton X-100 (TX) contributed further to the diffusion of the immunoreaction products, and increased the number of broken membranes. For example,without TX, the outer membrane and envelope of the mitochondria became intensely and clearly labelled when phosphate buffer was used for incubations;with TX pretreatment, the staining was far more diffuse, and the intensity of staining became reduced such that only the mitochondrial outer surface appeared somewhat immunopositive. Using phosphate buffer alone, we observed well-defined immunoreaction products around the microtubules of many-containing processes. This finding was less clear in other preparations, especially those pretreatedwith TX. We therefore submit that the conditions of tissue fixation and incubation may influence greatly the data amassed by the technique of immunocytochemistry. Results must be evaluated in view of the methods chosed for each immunocytochemical study. Optimal technical conditions encourage new morphological findings, as shown below concerning the circuitry of SP neurons in the dorsal horn.This work was initiated while Dr. K. Kakudo was a postdoctoral Fellow in Anatomic Pathology, Medical College of Georgia, from Osaka University Medical School, JapanPresently at the Department of Anatomy, Tulane Medical School, New Orleans, Louisiana, USAThe work, presented at the Annual Meeting of the 31st Annual Meeting of the Histochemical Society held in New Orleans, April 11–15, 1980, was partially supported by BRSG 10-16-04-3611-23, CCHD10-12-04-3600-00 (LLV) and the Department of Pathology (address reprint requests to LLV)     

Enkephalin, substance P, and serotonin axonal input to c-fos-like immunoreactive neurons of the rat spinal cord

Mustard oil, which stimulates small diameter afferents, was used to evoke the expression of the oncogene c-fos in the lumbar spinal cord. C-fos-like immunoreactivity was concentrated in, but not limited to, neuronal nuclei of laminae I and II of the lumbar dorsal horn. Double-label immunocytochemistry was used to determine if neurons which expressed c-fos-like immunoreactivity received axonal input from enkephalin-, substance P- or serotonin-immunoreactive neurons. The analysis of vibratome and semithin plastic-embedded tissue sections demonstrated that the majority of c-fos-like immunoreactive neurons received input from enkephalin-, substance P- or serotonin-immunoreactive axonal varicosities.     

Depletion of tryptophan hydroxylase by 5,6-dihydroxytryptamine in rat brain. Time course and regional differences

—Wistar rats were injected intraventricularly with 75 μg 5,6-dihydroxytryptamine. Tryptophan hydroxylase was assayed in seven regions of the brain as well as spinal cord at intervals following injection. Spinal cord was depleted to 1 per cent of control by 12 days. Tectum was depleted to 32 per cent of control by 9 days. The time course of depletion in most regions was biphasic, with an early reduction of activity 1 h after injection with continued reduction of activity 1-2 days following injection, and a recovery of activity at 4-6 days. The activity again drops at 9-12 days, and this reduction of activity persists to varying degrees to 60 days, with slight recovery at this time in certain regions.     

P物质对大鼠骶髓后连合核神经元甘氨酸激活的氯电流的调控

采用制霉菌素穿孔膜片箍技术,研究了Ｐ物质对急性分离的大鼠骶髓后的核神经元士的宁敏感性甘氨酸反应的调控作用。在箍制电压为－４０ｍＶ时,ＳＰ时１ｍｍｏｌ／Ｌ－１μｍｏｌ／Ｌ之间呈浓度依赖性地增强３０μｍｏｌ／Ｌ甘氨酸激活的氯电流。ＳＰ既不改变ＩＧｌｙ的翻转电位,也不是影响Ｇｌｙ与其受体的亲和力。Ｓｐａｎｔｉｄｅ和选择性Ｎ中受体拮抗剂,Ｌ－６６８,１６９,可阻断ＳＰ的增强作用,而选择性ＮＫ２受体拮抗剂,     

De- and regeneration of the bulbospinal serotonin neurons in the rat following 5,6-or 5,7-Dihydroxytryptamine treatment

Summary In an attempt to determine the conditions which permit central 5-HT neurons to respond to a chemical injury of their axons by sprouting and regeneration, the pattern and time-course of recovery of 5-HT concentrations and regrowth of bulbospinal 5-HT axons were evaluated in rats subjected to intraventricular treatment with either 75 g 5,6- or 150 g 5,7-DHT. While 5,6-DHT treatment is followed by a significant recovery of 5-HT concentrations in the telodiencephalon, brainstem and upper part of the spinal cord within 3 months, there is no significant restoration of the severely depleted 5-HT levels in the telodiencephalon and spinal cord, and only limited recovery in 5-HT content of the brainstem preparation after 5,7-DHT.These differences conform to the observation of widespread and effective regrowth and regeneration of the bulbospinal 5-HT neurons in the 5,6-DHT treated lower brainstem and upper spinal cord but restricted and localized sprouting efforts in the 5,7-DHT treated lower medulla oblongata. This could be explained by a cell body near lesion of the non-terminal indoleamine axons by 5,7-DHT which results in a late retrograde, irreversible degeneration of most of the indoleamine pericarya from group B1 and many of group B3.It is concluded that the preservation of a critical length of the main axon and part of its collaterals is necessary for the neuron's survival, and that the individual pattern of the neuropil architecture of brain centres which are invaded by the axonal sprouts may significantly influence their growth characteristics and thus either favour or impede their chance to reestablish connections with their original effector. Aberrant, localized, intense sprouting of drug-damaged axons may in itself reflect the need of the neuron—deprived of most of its axonal tree—to reestablish its original total axonal length by multiple branching.Supported by grants from the Deutsche Forschungsgemeinschaft. The authors are indebted to Rolf Franck for his technical assistance.Supported by grants from the Swedish Medical Research Council (No. 04 X-3874 and 04 X-56).     

Immunocytochemical identification of axons containing coexistent serotonin and substance P in the cat lumbar spinal cord

Axons containing both serotonin-like (5-HT)-LI and substance P-like (SP)-LI immunoreactivity were identified in all laminae of the cat spinal cord at the level of the lumbar enlargement. Using an immunologically-specific, double immunofluorescence method, coexistent 5-HT-LI and SP-LI immunoreactivity could be visualized in the same tissue section with appropriate FITC and rhodamine fluorescent filter sets. The fewest number of coexistent axons were observed in the superficial laminae of the dorsal horn, while their number increased in the more ventral dorsal horn laminae. Numerous coexistent axons were observed in the area adjacent to the central canal. The greatest number of coexistent axons was found in the ventral horn, especially in the motoneuronal cell groups. This study demonstrates that axons containing coexistent 5-HT-LI and SP-LI immunoreactivity are found in all laminae of the cat lumbar spinal cord and are thus involved in both sensory and motor functions. Their more frequent occurrence in the ventral horn suggests a greater role for coexistent 5-HT and SP in motor function. Since axons containing coexistent 5-HT and SP, and those containing only 5-HT, likely originate from different populations of neurons, our observations provide evidence for a diverse origin of descending 5-HT afferents to the different spinal laminae.     

Origin and development of VIP and substance P containing neurons in the embryonic avian gut

The development of substance P (SP) and VIP containing structures of the quail and chick guts was studied by immunocytochemistry. The appearance of VIP and substance P nerves follows a rostrocaudal pattern from day 9 in the quail and day 10 in the chick embryo. Immunoreactive fibres are first visible in the oesophagus and at 12 days they extend over the whole length of the intestine. VIP and substance P ganglionic cells are first localized in the foregut (day 9 for VIP containing neurons and day 13 for SP ones) and observed in the mid- and hind-gut just before hatching. Transplantation on the chorioallantoic membrane (CAM) of fragments of various parts of the digestive tract were carried out to see whether in such circumstances the pattern of VIP and SP containing nerves was comparable to normal. The explants contained numerous SP and VIP immunofluorescent nerve fibres. In addition, cell bodies with VIP and SP immunoreactivity appeared brightly fluorescent in the enteric ganglia of the graft showing that these peptidergic nerve cells belong to the intrinsic innervation of the gut.     

Origin and development of VIP and substance P containing neurons in the embryonic avian gut

Summary The development of substance P (SP) and VIP containing structures of the quail and chick guts was studied by immunocytochemistry. The appearance of VIP and substance P nerves follows a rostrocaudal pattern from day 9 in the quail and day 10 in the chick embryo. Immunoreactive fibres are first visible in the oesophagus and at 12 days they extend over the whole length of the intestine. VIP and substance P ganglionic cells are first localized in the foregut (day 9 for VIP containing neurons and day 13 for SP ones) and observed in the mid- and hind-gut just before hatching. Transplantation on the chorioallantoic membrane (CAM) of fragments of various parts of the digestive tract were carried out to see whether in such circumstances the pattern of VIP and SP containing nerves was comparable to normal. The explants contained numerous SP and VIP immunofluorescent nerve fibres. In addition, cell bodies with VIP and SP immunoreactivity appeared brightly fluorescent in the enteric ganglia of the graft showing that these peptidergic nerve cells belong to the intrinsic innervation of the gut.     

Activation of the neurokinin-1 receptor by substance P triggers the release of substance P from cultured adult rat dorsal root ganglion neurons

Hypoxia and ischemia occur in the spinal cord when blood vessels of the spinal cord are compressed under pathological conditions such as spinal stenosis, tumors, and traumatic spinal injury. Here by using spinal cord slice preparations and patch-clamp recordings we investigated the influence of an ischemia-simulating medium on dorsal horn neurons in deep lamina, a region that plays a significant role in sensory hypersensitivity and pathological pain. We found that the ischemia-simulating medium induced large inward currents in dorsal horn neurons recorded. The onset of the ischemia-induced inward currents was age-dependent, being onset earlier in older animals. Increases of sensory input by the stimulation of afferent fibers with electrical impulses or by capsaicin significantly speeded up the onset of the ischemia-induced inward currents. The ischemia-induced inward currents were abolished by the glutamate receptor antagonists CNQX (20 μM) and APV (50 μM). The ischemia-induced inward currents were also substantially inhibited by the glutamate transporter inhibitor TBOA (100 μM). Our results suggest that ischemia caused reversal operation of glutamate transporters, leading to the release of glutamate via glutamate transporters and the subsequent activation of glutamate receptors in the spinal dorsal horn neurons.     

Cholinergic neurons and the cardiovascular response produced by central injection of substance P in the normotensive rat

The role of cholinergic neurons in central cardiovascular regulation is not well understood, however, activation of brain cholinergic neurons in several species evokes a hypertensive response. As with central cholinergic stimulation, intracerebroventricular (i.c.v.) injection of substance P (sP) elicits a pressor response in unanesthetized rats. The purpose of this study was to determine whether the cardiovascular effects following i.c.v. injection of this neuropeptide are mediated by central cholinergic neurons. Therefore, the cardiovascular response to sP was examined in control rats, and in animals pretreated centrally with classical pre- and post-synaptic cholinergic antagonists. Drugs were administered directly into the lateral ventricle while rats were freely-moving in their home cages. I.c.v. injection of sP produced a dose - dependent increase in arterial pressure and heart rate. The hypertensive response was significantly reduced by pretreatment with hemicholinium-3. This dose (20 ug) of hemicholinium-3 is capable of producing a maximal depletion of brain acetylcholine levels. The increase in heart rate to substance P was not as sensitive to hemicholinium-3 pretreatment as was blood pressure. Central pretreatment with the nicotinic receptor antagonist, hexamethonium was more effective than the muscarinic antagonist, atropine in blocking the pressor response to sP. Hexamethonium did not significantly alter the tachycardic response to the peptide, but atropine produced a small, but significant reduction in the response. Therefore, the pressor response to central injection of sP may be mediated to a large extent through cholinergic pathways involving nicotinic receptors.     

Coexistence of substance P and neurotensin-like peptides in single neurons of the rat hypothalamus

The coexistence of substance P with neurotensin-like immunoreactivity in certain neurons of the hypothalamus were demonstrated by the double immunofluorescence method. Substance P and neurotensin-like immunoreactivity coexisted within single neurons of some hypothalamic areas such as the medial preoptic area, perifornical area, anterior hypothalamic area, lateral hypothalamic area, periventricular nucleus and posterior hypothalamic nucleus, although they did not coexist in the majority of immunoreactive cells.     

Effects of serotonin depletion byp-chlorophenylalanine,p-chloroamphetamine or 5,7-dihydroxytryptamine on central dopaminergic neurons: Focus on tuberoinfundibular dopaminergic neurons and serum prolactin

Three serotonin (5-HT) neurotoxins,p-chlorophenylalanine (PCPA, 125 and 250 mg/kg, i.p.),p-chloroamphetamine (PCA, 10 mg/kg, i.p.) and 5,7-dihydroxytryptamine (5,7-DHT, 200 µg/rat, i.c.v.) were used to examine whether depletion of central 5-HT has an effect on central dopaminergic (DA) neuronal activities or on prolactin (PRL) secretion. Adult ovariectomized Sprague-Dawley rats primed with estrogen (polyestradiol phosphate, 0.1 mg/rat, s.c.) were treated with one of three neurotoxins and then decapitated in the morning after 3–7 days. Blood sample and brain tissues were collected. The acute effect of PCA (from 30 to 180 min) was also determined. The concentrations of 5-HT, DA and their metabolites, 5-hydroxyindoleacetic acid and 3,4-dihydroxyphenylacetic acid, in the median eminence, striatum and nucleus accumbens were determined by HPLC-electrochemical detection. All three toxins significantly depleted central 5-HT stores by 11–20%. Except for PCPA, neither PCA nor 5,7-DHT had any significant effect on basal DA neuronal activities or PRL secretion. PCA also exhibited an acute effect on the release and reuptake of 5-HT and DA. In summary, depletion of central 5-HT stores to a significant extent for 3–7 days did not seem to affect basal DA neuronal activity and PRL secretion.     

Pharmacologic actions of capsaicin: apparent involvement of substance P and serotonin.

The pharmacologic actions of capsaicin in the cardiovascular, respiratory, sensory, thermoregulatory, and gastrointestinal systems were reviewed as were the ultrastructural, neurophysiological, and neurochemical effects produced by this drug. Present evidence strongly suggests that substance P and serotonin are both involved in the pharmacological activity of capsaicin. Capsaicin apparently produces no direct effects on GABA-ergic, enkephalinergic or catecholaminergic systems.