Increased pulmonary vascular permeability and oedema induced by intrathecally injected endothelins in rat.

The intrathecal (i.t.) injection of endothelins to conscious rats was found to cause respiratory arrest. To gain some insights into this central phenomenon, peripheral vascular permeability and lung oedema were measured after i.t. and i.v. injections of these peptides. When injected at T-8 spinal cord level, endothelin-1 (65 and 650 pmol) and endothelin-3 (650 pmol) enhanced vascular permeability in the lungs by 22-fold and 7-fold, respectively, and caused sudden death at the highest dose. Less prominent increases (between 1.4- and 2.2-fold) of vascular permeability were observed in other tissues (trachea, kidney, ears, skin of hind paws and back skin) with endothelin-1. Endothelin-1 (650 pmol) caused a similar increase (27-fold) in lung vascular permeability when injected at T-2, although the response was significantly less (P < 0.05) if injected at the L-4 (15-fold) spinal cord level. Only endothelin-1 produced lung oedema when injected at the T-2 or T-8 level. In contrast, intravenous injection of endothelins-1 and -3 (650 pmol) did not produce lung oedema and the lung vascular permeability was increased by only 1.4-1.6-fold and all rats survived. The prior i.t. injection of 6.5 nmol BQ-123 (cyclo[D-Trp, D-Asp, L-Pro, D-Val, L-Leu]), a selective endothelin ET(A) receptor antagonist, prevented the increases of lung vascular permeability and oedema and the mortality induced by i.t. endothelin-1 (650 pmol). Whereas i.v. treatment with phentolamine (2 mg/kg) or pentolinium (25 mg/kg + 50 mg/kg per h x 15 min) abolished the lung vascular permeability changes evoked by endothelin-1 (650) pmol), atropine (1 mg/kg), NG-nitro-L-arginine (50 mg/kg) or indomethacin (5 mg/kg) had no effect. Moreover, the effects of endothelin-1 were attenuated in capsaicin pretreated rats (125 mg/kg, 10 days earlier) and almost abolished in rats subjected to sympathectomy with 6-hydroxydopamine (100 mg/kg, 24-48 h earlier). All these treatments except atropine and NG-nitro-L-arginine prevented the endothelin-1-induced lung oedema and reduced the lethality by around 50%. These results suggest that the increases of pulmonary vascular permeability and oedema induced by i.t. endothelin-1 are due to an intense pulmonary vasoconstriction mediated by alpha-adrenoceptors following the release of catecholamines in response to the activation of endothelin ET(A) receptor in the spinal cord. This central phenomenon seems to be reflexogenic, including the involvement of primary afferent C-fibers and spinal cord ascending fibers to the brain. Thus, endothelin-1 could play a role in neurogenic pulmonary oedema through a central mechanism.

Receptor and mechanism that mediate endothelin- and big endothelin-1-induced phosphoinositide hydrolysis in the rat spinal cord.

In rat spinal cord slices, endothelin-1 and endothelin-3 enhanced [3H]inositol phosphate production between 1 nM and 10 microM (endothelin-1 > endothelin-3) while sarafotoxin 6c and the endothelin ETB receptor agonist IRL-1620 (Suc-[Glu9,Ala11,15]endothelin-1-(8-21)) were almost ineffective. BQ-123 (cyclo(D-Trp,D-Asp,L-Pro,D-Val,L-Leu), a selective endothelin ETA receptor antagonist, reduced the endothelin-1- and endothelin-3-induced [3H]inositol phosphate production, with similar inhibition constants (IC50: 16.7 +/- 3.4 and 8.0 +/- 1.6 microM, respectively). The inhibition of endothelin-1 was enhanced when BQ-123 was preincubated for 30 min instead of 15 min. BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D -1-methoxy- carbonyltryptophanyl-D-Nle), a selective ETB receptor antagonist, did not modify the endothelin-1-induced [3H]inositol phosphate production. Big endothelin-1 (1 nM to 1 microM) was slightly less potent than endothelin-1 in enhancing [3H]inositol phosphate production. This response was sensitive to phosphoramidon and [Phe22]big endothelin-1-(19-37), two inhibitors of endothelin-converting enzyme. Pretreatment of slices with pertussis toxin, indomethacin or PN 200-110 ((-)-isradipine, a dual inhibitor of L- and R-type Ca2+ channels) did not alter the response to 1 microM endothelin-1 while this response was abolished by tetrodotoxin. Finally, endothelin-1 enhanced [3H]inositol phosphate production with an identical EC50 (2.1 nM) in spinal cord slices of Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) although the maximal response was reduced in SHR. These data indicate that endothelins stimulated [3H]inositol phosphate production in the rat spinal cord through the activation of an endothelin ETA receptor that trigger the release of an unidentified neurotransmitter. This effect does not appear to be associated to activation of a Gi/G(o)-type of G-protein, dihydropyridine-sensitive L-type Ca2+ channels or to the production of prostaglandins. Furthermore, the findings support the presence of a phosphoramidon-sensitive endothelin-converting enzyme in the spinal cord.

Cardiovascular responses to intrathecal neuropeptide gamma in conscious rats: receptor characterization and mechanism of action.

1. In the conscious rat, cardiovascular responses to intrathecally (i.t.) administered neuropeptide gamma (NP gamma) were studied prior to and after the i.t. pretreatment with selective antagonists at NK1 ((+/-)-CP 96345 and RP 67580), NK2 (SR 48968) and NK3 (R 486) receptors. Pretreatment with a mixture of peptidase inhibitors (phosphoramidon, captopril, bacitracin, phenanthroline) was also tested to ascertain whether or not the effect of NP gamma was mediated by a metabolite. The involvement of peripheral catecholamines was examined with intravenous injection of alpha-adrenoceptor (phentolamine) and beta-adrenoceptor (propranolol) antagonists. 2. NP gamma (0.078-78 nmol) induced dose-dependent increases in heart rate (HR) and mean arterial blood pressure (MAP). The highest dose of 78 nmol did not induce an increase of MAP greater than that with 7.8 nmol but was preceded by a transient decrease of MAP (1-3 min). No desensitization was observed when three injections of 7.8 nmol NP gamma were given at 90 min intervals. 3. Cardiovascular and behavioural (biting/scratching) effects evoked by 0.78 nmol NP gamma were significantly reduced by the NK1 antagonists, (+/-)-CP 96345 (65 nmol) or RP 67580 (7.8 and 78 nmol). However, cardiovascular responses to NP gamma were not affected by (+/-)-CP 96345 (6.5 nmol), SR 48968 (7.8 and 78 nmol) or R 486 (25 nmol). Pretreatment with peptidase inhibitors significantly enhanced the cardiovascular and behavioural responses to NP gamma. 4. The pressor response to 7.8 nmol NP gamma was converted to a vasodepressor response by pretreatment with phentolamine (2 mg kg-1, i.v.) while the chronotropic response was markedly reduced by propranolol (2 mg kg-1, i.v.). 5. These results suggest that the cardiovascular responses to i.t. NP gamma are mediated by NK1 receptors in the spinal cord leading to the peripheral release of catecholamines from sympathetic fibres or the adrenal medulla. It is unlikely that the spinal action of NP gamma results from its metabolic conversion into neurokinin A or another major metabolite.

Characterization of the tachykinin receptors involved in spinal and supraspinal cardiovascular regulation.

The pharmacological characterization of the tachykinin receptors involved in spinal and supraspinal cardiovascular regulation is reviewed in this report. In conscious rats, substance P (SP), neurokinin A (NKA), neurokinin B (NKB), neuropeptide K (NPK), and neuropeptide gamma (NP gamma) were injected either intrathecally (i.t.) or intracerebroventricularly (i.c.v.), and their effects were assessed on mean arterial blood pressure (MAP) and heart rate (HR). Moreover, selective antagonists for NK1 ((+/-)-CP-96045 and RP-67580), NK2 (SR-48968), and NK3 (R-486) receptors were tested against the agonists. I.t. tachykinins elicited dose-dependent increases in MAP and HR (NPK > NP gamma > SP > NKA > NKB). The cardiovascular response to i.t. SP, NPK, and NP gamma was significantly attenuated by the prior i.t. administration of (+/-)-CP-96345 and RP-67580 but not by SR-48968 and R-486. By the i.c.v. route, tachykinins also elicited pressor and tachycardiac responses dose dependently (NPK > NP gamma > SP > NKA > NKB). Senktide and [MePhe7]NKB, two NK3-selective agonists, were slightly more potent than NKB on both parameters. Whereas the cardiovascular response to NPK was largely blocked by (+/-)-CP-96345 and RP-67580, that to SP was reduced by 40-50%. This treatment had no effect on the cardiovascular response to NKA and [MePhe7]NKB. Conversely, SR-48968 reduced by 40-50% the NKA-induced cardiovascular changes without affecting the central mediated effects of NPK, SP, and [MePhe7]NKB. However, when coadministered, RP-67580 and SR-48968 abolished the effects to SP and NKA while leaving untouched those induced by [MePhe7]NKB. Finally, the central effects mediated by [MePhe7]NKB, senktide, and NKB were blocked by R-486. These findings suggest that the i.t. action of tachykinins on the rat cardiovascular system is mediated by a NK1 receptor in the spinal cord, while NK1, NK2, and NK3 receptors are likely involved in the supraspinal (hypothalamus) effects of these neuropeptides. It is also concluded that NPK is a pure and powerful NK1 agonist, in contrast to SP and NKA, which are not selective for NK1 and NK2 receptors, respectively.

Endothelins stimulate phosphoinositide breakdown in the rat spinal cord through ETA receptors.

In rat spinal cord slices, endothelin-1 (ET-1) and -3 (ET-3), sarafotoxin 6c (STX-6c), and the ETB receptor agonist IRL-1620 produced increases of [3H]inositol phosphate (IP) accumulation with the following rank order of potency: ET-1 > ET-3 >> STX-6c = IRL-1620. The ET-1- and ET-3-induced IP accumulations were blocked by BQ-123, a selective ETA antagonist, with a similar inhibition constant (IC50 16.7 +/- 3.4 microM and 8.0 +/- 1.6 microM, respectively) whereas BQ-788, a selective ETB antagonist, was inactive. These data indicate that ET-induced IP accumulation in rat spinal cord is mediated by an ETA receptor. The present study is in agreement with ETA receptor-mediated cardiovascular changes induced by intrathecal injection of ET-1 in the conscious rat.

Cardiovascular effects of intrathecally administered endothelins and big endothelin-1 in conscious rats: receptor characterization and mechanism of action.

In conscious rats, the intrathecal (i.t.) injection of endothelin-1 (ET-1; 65-650 pmol) and endothelin-3 (ET-3; 162-650 pmol) produced dose-dependent increases of mean arterial blood pressure (MAP) accompanied by either a tachycardia or a bradycardia. A number of animals died by a sudden respiratory arrest. ET-3 was less toxic and less potent than ET-1 on MAP and heart rate (HR) while BQ-3020, a selective ETB agonist, had no toxic effect and exhibited only a weak pressor effect on blood pressure. The prior i.t. injection of 65 nmol BQ-123, a selective ETA receptor antagonist, blocked both the cardiovascular and toxic effects of ET-1 but failed to modify the cardiovascular effect evoked by i.t. substance P (6.5 nmol) or to cause intrinsic cardiovascular and toxic effects. While the pressor response to ET-1 was significantly inhibited after i.v. injection of phentolamine, the bradycardia was blocked by pentolinium. The cardiovascular response to ET-1 was, however, unaffected in rats either sympathectomized with 6-hydroxydopamine or pretreated with capsaicin. Furthermore, big ET-1 (100 pmol) caused toxic effects and delayed cardiovascular changes which were prevented by the prior i.t. administration of either BQ-123 (65 nmol) or 100 nmol phosphoramidon, an endothelin-converting enzyme (ECE) inhibitor. These results suggest: (1) that the cardiovascular and toxic effects of i.t. endothelins are mediated by ETA receptors in the rat spinal cord; (2) that the pressor response and bradycardia are likely due to the activation of the sympatho-adrenal nervous system and to a vagal reflex mechanism, respectively; and (3) that a phosphoramidon-sensitive ECE converts big ET-1 to ET-1 in the rat spinal cord.

Spinal cord serotonin receptors in cardiovascular regulation and potentiation of the pressor response to intrathecal substance P after serotonin depletion.

The aim of this study was to characterize, in conscious rats, the spinal cord 5-hydroxytryptamine (5-HT) receptors involved in mean arterial pressure (MAP) and heart rate (HR) regulation as well as to examine the influence of bulbospinal 5-HT fibers on cardiovascular responses to intrathecal (i.t.) substance P (SP). The i.t. injection of 5-HT or 5-carboxamidotryptamine (5-CT) (5-HT1A,1B,1D agonist) reduced MAP and increased HR in a dose-dependent manner. In contrast, the agonists 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 5-HT1A agonist) and alpha-CH3-5-HT (5-HT1C and 5-HT2) only caused a decrease in HR, while the agonist 2-CH3-5-HT (5-HT3) was devoid of cardiovascular effects. The vasodepressor response to 5-CT was antagonized by methiothepin but not affected by mesulergine, ketanserin, propranolol, or yohimbine. However, all five antagonists reduced the HR increase to 5-CT. Ketanserin, propranolol, mesulergine, yohimbine, and methylsergide were without effect on resting MAP, while methiothepin reduced MAP. Methiothepin, ketanserin, and methylsergide increased resting HR, yet the other antagonists had no effect on this parameter. Rats treated with p-chlorophenylalanine or 5,7-dihydroxytryptamine, but not with 6-hydroxydopamine, exhibited higher resting HR than that of control rats. Although the resting MAP was unaffected, the pressor response to i.t. SP was significantly enhanced by either 5-HT toxin. The results suggest that the receptor mediating the depressor response to 5-HT and 5-CT conforms with the broad pharmacological profile of a 5-HT1-like receptor and is unlikely to be of the 5-HT2 or 5-HT3 subtype. Since the HR response evoked by 5-CT was blocked by antagonists that exhibit affinities for various 5-HT receptor subtypes, it is suggested that a nonspecific blockade or, alternatively, that more than one receptor contributes to this cardiac effect. In addition, the results raise the possibility that a spinal 5-HT input, likely mediated by 5-HT2 receptors, tonically inhibits HR. Hence, an antagonistic interaction between 5-HT and SP is proposed to play a role in the control of arterial blood pressure in the spinal cord.

Thienylphencyclidine protection for the spinal cord of adult rats against extension of lesions secondary to a photochemical injury.

The purpose of this study was to evaluate treatment with the N-methyl-D-aspartate antagonist thienyl-phencyclidine (TCP) after spinal cord injury for its behavioral, electrophysiological, morphological, and immunohistochemical effects. Five minutes after a photochemical lesion was produced in rats at the T-8 level, the animals received TCP (1 mg/kg, intravenously) or TCP vehicle (saline). The animals were evaluated on Day 18 for neurological recovery by testing motor and sensory functions. The TCP-treated group showed less neurological impairment than the untreated group (p < 0.05 for inclined-plane stability and withdrawal reflex to extension). Somatosensory evoked potential testing was performed on Days 21 to 23 and the wave amplitude between the onset and P1 in the TCP-treated group was higher than in the untreated group (p < 0.05). Mean arterial blood pressure was not significantly modified after TCP injection. Morphometric studies of the lesion area in cross section revealed a significantly reduced spinal cord infarction in the TCP-treated group (p < 0.05). Immunohistochemical evaluation of the spinal cord in lumbar area showed an increased level of serotonin immunoreactivity in the dorsal horn of animals treated by TCP. These results demonstrate the efficacy of TCP in reducing secondary lesions after spinal cord injury in rats.

Effects of neonatal adrenalectomy on 5-HT and TRH-like immunoreactivity in the rat spinal cord.

This study investigated the effects of neonatal unilateral adrenalectomy on the serotonin- (5-HT) and thyrotropin-releasing hormone-like immunoreactivities (TRH-LI) in the intermediolateral cell column (IML) of the rat spinal cord where the sympathetic preganglionic neurons innervating the adrenal medulla are located. The density of the innervation was measured by a computer-assisted image analysis. Two weeks after the lesion, only one rat (1/3) showed a 30% decrease of 5-HT-LI in the ipsilateral IML, although no modifications were observed for TRH-LI. At 1 month, we observed a variable decrease of 19% to 30% for either 5-HT- or TRH-LI in the ipsilateral IML. However, one animal (1/3) showed nonsignificant modifications. At 3 months, we showed a 17% mean loss of 5-HT-LI and TRH-LI in the ipsilateral IML. However, the decreases of 5-HT-LI and TRH-LI did not always appear similar. These results appear different from those obtained after neonatal removal of the superior cervical ganglion.

Pre- and post-natal ontogeny of thyrotropin-releasing-hormone in the rat spinal cord: an immunocytochemical study.

This work aimed at providing by means of immunocytochemical techniques a detailed study of the ontogeny of thyrotropin-releasing hormone (TRH) in the spinal cord of the rat. We report the first appearance of TRH-immunoreactive fibers in the ventral funiculus of thoracic and lumbar levels at embryonic day 17. At embryonic day 18, fibers penetrated the ventral gray matter towards the central canal. At embryonic day 19, the first immunoreactive fibers were seen in the intermediolateral cell column at upper thoracic levels. This region was invaded at lower thoracic levels on the day of birth. At this time, TRH-immunoreactive axodendritic synapses were observed in the ventral horn and in the intermediolateral cell column. Immunoreactivity increased in these regions until post-natal day 21 when the adult pattern of TRH immunoreactivity was established in the sympathetic nuclei and in the ventral horn. However, a transient TRH-like immunoreactivity was detected in lamina IIi of the dorsal horn between post-natal days 14 and 30: at ultrastructural level, immunoreactive varicosities were seen to establish axodendritic synapses. In conclusion, TRH is one of the earliest peptidergic systems established in the spinal cord and it presents extensive temporal and topographical similarities with the serotonergic system with which it could be colocalized.

Immunocytochemical mapping of noradrenergic projections to the rat spinal cord with an antiserum against noradrenaline.

The mapping of noradrenergic innervation was performed in transverse and longitudinal sections of the adult rat spinal cord using noradrenaline immunocytochemistry. Noradrenergic fibres and terminals were distributed in the dorsal horn (mainly in the superficial part), in the vicinity of the different groups of motoneurons, and concentrated in the intermediolateral cell column and around the central canal. The ultrastructural study showed principally axodendritic synapses in the ventral horn and in the intermediolateral cell column. Fewer axosomatic synapses were detected. In the dorsal horn, noradrenaline-innervation was predominantly non-synaptic. It is hypothesized that the noradrenergic modulation of nociception is not mediated through classical synapses. The concept of 'volume transmission' can explain such an influence. Conversely, noradrenaline may be involved in the control of locomotion and automatic functions through conventional synapses.

Fate of B1-B2 and B3 rhombencephalic cells transplanted into the transected spinal cord of adult rats: light and electron microscopic studies.

Embryonic cell suspensions (14-day embryos) containing either B3 or B1-B2 serotonergic cell groups were obtained by microdissection of specific rhombencephalic regions and transplanted into the transected spinal cord of adult male Sprague-Dawley rats. After 3 months of survival, the animals were sacrificed and the spinal cords processed for the immunocytochemical detection of serotonin (5-HT). 5-HT-immunoreactive fibers from B1-B2-grafted cells were selectively distributed in the ventral horn and the intermediolateral cell column (IML) where they established conventional synaptic contacts. However, B3 5-HT cells grew and extended their processes into the dorsal horn where in addition we observed scarce synaptic contacts as in the normal spinal cord. These results suggest that the specificity of the 5-HT innervation of the spinal cord by grafted neurons is due, at least partly, to the presence of local mechanisms mediating guidance and cell recognition, possibly operating in conjunction with preexisting substrate pathways.

Pre- and postnatal development of noradrenergic projections to the rat spinal cord: an immunocytochemical study.

Using immunocytochemistry with a specific antiserum against noradrenaline, the pre- and postnatal development of noradrenergic (NA) projections to the rat spinal cord was studied from embryonic day 16 (E16) to adulthood (the day following nocturnal mating being considered as E0). In this study, pregnant animals were pre-treated with the MAO inhibitor pargyline (200 mg/kg i.p.), whereas postnatal animals received 100 mg/kg. In vibratome sections, noradrenaline-immunoreactive (NA-IR) axons were seen to invade the spinal cord at E16, at cervical and upper thoracic levels, from the ventral funiculus. At E18, small caliber NA-IR fibers were present in the ventral horn at all cord levels, and some fibers were seen in the intermediolateral cell column (IML) at thoracic level. The growth of axons towards the dorsal horn became noticeable by postnatal day 0 (P0). At P3, fine beaded and radially orientated NA-IR fibers were observed at all levels. The pattern of NA innervation of the dorsal horn was similar to that of the adult by P7. The segregation of noradrenaline immunoreactivity in the ventral and dorsal horns, the IML and the periependymal area was more obvious at all levels by P14 and P20. From P30 the NA innervation was similar to that found in the adult spinal cord. Thus, noradrenaline, like serotonin, was present early in the spinal cord before the onset of specific functions. In addition to and prior to its transmitter function, it might play a trophic role in the neurogenesis of the spinal cord.

Serotoninergic, noradrenergic, and peptidergic innervation of Onuf's nucleus of normal and transected spinal cords of baboons (Papio papio).

We have investigated with light and electron microscope immunocytochemistry the aminergic and peptidergic innervation of Onuf's nucleus in adult baboons. This nucleus, located in the ventrolateral part of the sacral spinal cord (S2 and S3), is considered to control urethral and anal sphincters and penile muscles. By comparison of intact and transected spinal cords, we have found that serotoninergic innervation has two origins: first, supraspinal, innervating the whole nucleus, with a possible predominance in the dorsal half; and second, intraspinal, corresponding to the ventral half of the nucleus. Thyrotropin-releasing hormone innervation appears largely coincident with serotonin, both in intact and transected spinal cords. Noradrenaline is exclusively of supraspinal origin, as attested by its disappearance below the level of the section. Substance P, calcitonin gene-related peptide, and Leu- and Met-enkephalin, which profusely innervate Onuf's nucleus, are on the contrary not affected by the transection. They most likely originate from the cord itself or the dorsal root ganglia. Thus, Onuf's nucleus innervation in the baboon arises both from supraspinal and intraspinal sources. The present study provides an anatomical basis for both voluntary and reflex controls of excretory and sexual functions in a primate. The same neurotransmitter (serotonin) according to its cell origin and discrete topography could exert different influences upon the same effector system.

Distribution of thyrotropin-releasing hormone in the rat spinal cord with special reference to sympathetic nuclei: a light- and electron-microscopic immunocytochemical study.

This paper deals with the distribution of thyrotropin-releasing hormone-like immunoreactivity in the spinal cord of the rat, and particularly in the sympathetic nuclei, at light and electron microscopic levels. In the dorsal horn, the inner part of laminae II and III displayed thin thyrotropin-releasing hormone immunoreactive profiles. Electron microscopy revealed small immunoreactive varicosities which made synaptic contact with small dendrites or dendritic spines. Dense thyrotropin-releasing hormone-like immunoreactivity was observed in all sympathetic nuclei (nucleus intermediolateralis pars fascicularis and principalis, nucleus intercalatus and dorsal commissural nucleus) except the nucleus intercalatus pars ependymalis. Electron microscopy showed many immunoreactive varicosities which were often in synaptic contact with dendrites (proximal or distal), rarely with perikarya and never with axons. Sometimes, the same immunoreactive varicosity made axodendritic contacts with two dendrites and, conversely one dendrite was sometimes synaptically contacted by two or more immunoreactive varicosities. The ventral horn displayed a diffuse thyrotropin-releasing hormone-like immunoreactivity except for the cremaster nucleus (at lumbar level) which was densely outlined by immunoreactive profiles. Occasionally a large cell body in lamina IX (a putative motoneuron) was outlined by immunoreactive profiles but ultrastructural studies revealed very few immunoreactive axosomatic synapses, while immunoreactive symmetrical or asymmetrical axodendritic synapses were observed. The present study clearly confirms the existence of thyrotropin-releasing hormone immunoreactive synapses, thus substantiating the physiological role of this hormone in the spinal cord.

Transplantation of embryonic serotonin immunoreactive neurons into the transected spinal cord of adult monkey (Macaca fascicularis).

Five adult monkeys (Macaca fascicularis) underwent a total section of the spinal cord at the thoracic level (T6). Four of them received a daily treatment with cyclosporin (10 mg/kg). Ten days later, two animals treated with cyclosporin and one without cyclosporin received at T8 and T10 levels an injection of a cell suspension prepared from the rhombencephalon of a 40-day-old macaque embryo. Two control animals received one injection of Hank's balanced salt solution. The animals were sacrificed after 2 months (one grafted and one control) and 3 months (two grafted and one control), and their spinal cord was processed for the immunocytochemical detection of serotonin using light and electron microscopy. After 2 months of survival, serotonergic neurons had survived and developed within the transplant. Three months after transplantation, in the animal treated with cyclosporin, serotonergic neurons were found to survive with their axons growing into the host grey matter and establishing axosomatic and axodendritic synapses in the ventral horn. If the graft was isolated in the white matter no fibers were seen invading the grey matter.

Plasticity of the serotonergic innervation of the dorsal horn of the rat spinal cord following neonatal capsaicin treatment.

Neonatal capsaicin treatments (25 or 50 mg/kg, 12, 24, or 48 hr after birth given subcutaneously) were applied in order to follow by immunocytochemical techniques the postnatal development and plasticity of the serotonergic system in the dorsal horn of the rat spinal cord. Two markers of the lesions of C primary afferents induced by capsaicin were tested by immunocytochemical detection: substance P and calcitonin gene-related peptide (CGRP). We show that the internal part of substantia gelatinosa (lamina Ili) which does not contain serotonergic fibers in intact or vehicle-treated rats is invaded within a few days after capsaicin treatment by serotonergic fibers apparently sprouting from the deepest laminae. Moreover, these fibers often establish axodendritic synapses while synapses are rare in intact animals in the whole dorsal horn. This reorganization is stable whatever the dose of capsaicin used or the moment chosen for its injection. On the other hand, while lesions of substance P-ergic fibers appeared quite stable, partial recovery of CGRP innervation was found after 3 to 6 months, especially with the low dose of capsaicin. We discuss the ability of the serotonergic system innervating the dorsal cord either to find new targets or to fill vacated sites when one of its putative targets is removed.

5-Hydroxytryptamine, substance P and thyrotropin-releasing hormone synapses in the intermediolateral cell column of the rat thoracic spinal cord.

Serotonin-, substance P-, and thyrotropin-releasing hormone-immunoreactive profiles were studied in the intermediolateral cell column at the thoracic level of the rat spinal cord with light- and electron-microscopic immunocytochemistry. For each transmitter, a dense immunoreactive deposit was observed with the light microscope. At ultrastructural level, morphologically identified synapses amounted to 47% of all serotonergic varicosities, to 49% for substance P and 50% for thyrotropin-releasing hormone. Synapses appeared both symmetrical and asymmetrical. In each case, these synapses were mainly axodendritic (98%). These synaptic connections could mediate the physiological influence of these 3 substances in the spinal cord on the cardiovascular system.

Effects of neonatal removal of superior cervical ganglion on serotonin and thyrotropin-releasing hormone immunoreactivity in the intermediolateral cell column of the rat spinal cord.

In this study, we investigated the effects of the neonatal removal of the right superior cervical ganglion on the serotonin-like and thyrotropin-releasing-hormone-like immunoreactivities (5-HT-LI and TRH-LI) in the intermediolateral cell column (IML) of the spinal cord by quantitative image analysis. Two weeks after the lesion, we observed a 60% reduction in 5-HT-LI, while TRH-LI was not significantly reduced, in the right IML (lesioned side) at T1-2 levels. One month after the lesion, 5-HT-LI and TRH-LI were significantly reduced by 60% in the right IML at T1-2 levels. After 3 months, this decrease persisted at this level. In addition, we observed a 30% loss of the 5-HT-LI in the right IML at T3-4 levels, whereas TRH-LI did not decrease significantly at T3-4 levels. These findings are discussed and compared with those of other experimental studies on serotonergic reorganization in the rat spinal cord.

Immunohistochemical evidence for the coexistence of substance P, thyrotropin-releasing hormone, GABA, methionine-enkephalin, and leucin-enkephalin in the serotonergic neurons of the caudal raphe nuclei: a dual labeling in the rat.

By means of dual immunohistochemical labeling on the same brain section examined with a light microscope, the present study reports the presence with serotonin (5-hydroxytryptamine; 5-HT) of gamma-aminobutyric acid (GABA), substance P (SP), thyrotropin-releasing hormone (TRH), leucin-enkephalin (LEU-enk), or methionine-enkephalin (MET-enk), within the same neuron in the nuclei raphe magnus, raphe obscurus, and raphe pallidus of the rat. On the one hand, peptides or GABA are detected with specific rabbit antibodies by indirect peroxidase labeling using peroxidase-conjugated Fab fragments, and on the other, 5-HT is detected with a rabbit antibody against the BSA-serotonin conjugate by radio-immunocytochemistry using [125I]-labeled protein A. The possible coexistence of TRH and SP in these neurons is also investigated by using peroxidase labeling and radio-immunocytochemical detection, respectively. In the whole caudal raphe nuclei the proportion of each coexisting peptide with 5-HT appears in decreasing order as: TRH greater than SP greater than MET-enk # LEU-enk greater than GABA. In all instances the level of coexistence differs considerably in B1-B2 vs. B3 cell groups. No SP/TRH dually labeled cells have ever been found in any of the serotonergic nuclei of the caudal raphe. Given the evidence that these raphe nuclei project possibly to the spinal cord, these data constitute an anatomical substrate for the several distinct physiological functions presumably subserved by 5-HT in the cord, namely the modulation of nociception, motor, and autonomic functions.