A thyrotropin-releasing hormone-containing system in the rat dorsal horn separate from serotonin

In this study, we report the identification of a thyrotropin-releasing hormone (TRH)-containing system in the dorsal horn of the rat spinal cord. This system is distinct from the TRH and serotonin (5-hydroxytryptamine, 5-HT) cotransmitter supraspinal system that has projections to the intermediolateral (IML) and ventral columns. Spinal cord sections from untreated rats, and those treated with colchicine or 5,7-dihydroxytryptamine (5,7-DHT) were processed using peroxidase-antiperoxidase (PAP) immunocytochemistry with nickel intensification. Results of the 5,7-DHT treatment were verified by quantifying TRH and 5-HT by radioimmunoassay (RIA) and high performance liquid chromatography (HPLC), respectively. Prominent immunocytochemical staining for TRH in the dorsal horn was seen in varicose fibers mainly in lamina II and superficial lamina III of the dorsal horn of the spinal cord of control rats. A few fibers were seen ascending into lamina I. A moderate number of fibers that were immunoreactive for 5-HT were primarily in laminae I and II. The distribution of TRH- and 5-HT-containing neurites in the IML and the ventral horn agreed with previously published reports. Rats treated with colchicine showed many small round TRH immunoreactive cells that were limited to laminae II/III of the dorsal horn. TRH immunoreactivity in the dorsal horn and IML was resistant to the effects of the selective serotonin neurotoxin, 5,7-DHT, while the ventral horn was depleted of TRH staining. Serotonin was almost completely eliminated in all spinal cord laminae. Quantitative biochemical studies showed significant, but non-parallel reductions of TRH and 5-HT in cervical, thoracic and lumbar spinal cord. These studies demonstrate the existence of TRH-containing cell bodies and terminals in the dorsal horn of the rat spinal cord. These findings provide evidence that a TRH-containing system exists in the dorsal horn of the rat and that it is distinct from the descending medullary raphe system that contains 5-HT; suggest that a population of TRH-containing fibers that project to the IML may not contain 5-HT; and confirm previously published results that 5-HT and TRH coexist in terminals in the ventral horn of the spinal cord.     

Chronic immobilization stress: evidence for decreases of 5-hydroxy-tryptamine immunoreactivity and for increases of glucocorticoid receptor immunoreactivity in various brain regions of the male rat

Summary Male rats were exposed to severe 14 day immobilization stress. Body weight, body temperature, food and water intake, behavioral parameters, and serum corticosterone levels were measured during and after the stress period. On the 7th day after cessation of stress the experimental animals together with the control rats were taken to immunocytochemical analysis involving morphometry and microdensitometry of tyrosine hydroxylase (TH), 5-hydroxytryptamine (5-HT), various neuropeptide, and glucocorticoid receptor (GR) immunoreactivities (IRs) in a large number of regions of the central nervous system. In addition, adrenocorticotropic hormone (ACTH) IR was analyzed in the pituitary gland.Seven days following cessation of the chronic stress food intake, total locomotion and forward locomotion had been restored to normal. Serum corticosterone levels appeared to remain increased even 6 days following cessation of the chronic immobilization stress, probably caused by increased release of ACTH. Paraventricular corticotropin releasing hormone (CRF) IR was negatively correlated with the pituitary ACTH IR, indicating that the increase in ACTH release was produced by an increased release of CRF from the hypothalamus.The major immunocytochemical change observed 7 days after cessation of stress was a disappearance of 5-HT IR in the 5-HT cell groups B 1, B 2, B 3, and B 7. 5-HT IR in nerve terminals was only affected in the dorsal horn, where 5-HT IR was increased in the substantia gelatinosa. GR IR was found to be significantly increaed in monoaminergic cell groups: serotoninergic B 7, dopaminergic A 12, and noradrenergic A 1, A 2, and A 6. A trend for a reduction of TH IR was observed in nigral DA cells associated with significant reductions in TH IR in striatal DA nerve terminals. Finally, increases in 5-HT and substance P (SP) IR were found in the nerve terminals of the substantia gelatinosa of the cervical spinal cord in the stress group.In the present experimental model evidence has been obtained for a maintained activation of the hypothalamic-pituitary-adrenal axis as evaluated 7 days after cessation of severe chronic immobilization stress. The reduction of 5-HT IR in various 5-HT cell groups indicates a reduction of 5-HT synthesis, which may also be associated with reduced 5-HT release from the nerve terminals, since no depletion was observed in terminal regions and in one case an increase in 5-HT IR was noted (substantia gelatinosa). The increase in GR IR, demonstrated in the NA and 5-HT cell groups in the presence of a maintained hypersecretion of corticosterone may represent signs of an upregulation of GR synthesis and/or increased translocation, which take place in the presence of maintained hypersecretion of corticosterone. Thus, 5-HT and NA neurons may respond more effectively to circulating glucocorticoids after severe chronic stress. In this way glucocorticoids may protect against stress-induced exhaustion of neurons leading to impairment of transmission. Studies on TH IR suggest a deficit in the DA transmission line of the nigrostriatal DA neurons, but of no other CA neurons studied. Such effects may contribute to behavioral suppression. Finally, the stress-induced increases in 5-HT and SP IR in the substantia gelatinosa may in part underlie the phenomenon of stress-induced analgesia.     

运动性疲劳后脊髓前角5-羟色胺和色胺酸羟化酶表达的变化

目的 探讨运动性疲劳后脊髓前角5-羟色胺(5-hydroxytryptamine,5-HT)及其合成关键酶色胺酸羟化酶(tryptophan hydroxylase,TPH)表达的变化,阐释在脊髓水平运动性疲劳的发生机制.方法 健康成年雄性Wistar大鼠16只随机分为运动性疲劳组和对照组,采用免疫组化方法和计算机图像分析技术,检测大鼠脊髓前角的5-HT及TPH免疫反应阳性纤维及终末的平均积分光密度值.结果 运动性疲劳后,脊髓前角5-HT及TPH阳性反应纤维和终末减少,染色较浅,平均积分光密度值明显低于对照组(P＜0.05).结论 脊髓前角5-HT及TPH的含量在运动性疲劳后降低,提示脊髓前角5-HT与运动性疲劳的发生有关.     

5-Hydroxytryptamine, substance P, and thyrotropin-releasing hormone in the adult cat spinal cord segment L7: immunohistochemical and chemical studies.

The terminal projections of the descending 5-hydroxytryptamine (5-HT) bulbospinal pathway and the coexistence among 5-HT-, substance P (SP)-, and thyrotropin-releasing hormone (TRH)-like immunoreactivities (LI) in fibers innervating the L7 segment in the cat spinal cord were studied quantitatively and semiquantitatively by use of the indirect double-staining immunofluorescence technique. The content of 5-HT, SP, and TRH in different parts of the spinal cord was determined by use of radioimmunoassay (RIA) (SP and TRH) and high-performance liquid chromatography with electrochemical detection (HPLC-ECD) (5-HT). For all three substances studied, immunoreactive (IR) axon terminals were found in all parts of the gray matter, but with clear regional variation in the density of innervation. Thus, all three substances showed a dense innervation in the motor nucleus, particularly in the ventral part of the nucleus, while the superficial dorsal horn was very densely innervated by SP-IR fibers (laminae I and II) and TRH-IR fibers (laminae II and III). In the motor nucleus, the studied substances coexisted to a very high degree, but some 5-HT-IR fibers (about 10%) lacked peptide-LI and some SP-IR fibers (about 10%) lacked 5-HT-LI while virtually all TRH-IR fibers also contained 5-HT-LI. In the superficial dorsal horn (laminae I-III), no coexistence was detected, while other parts of the gray matter displayed various degrees of coexistence in between those found in the motor nucleus and laminae I-III. The quantitative analysis of IR varicosities in the motor nucleus suggested that the unilateral L7 motor nucleus is innervated by about 55-110 x 10(6) 5-HT-IR nerve terminals, which may indicate as many as 4,000 boutons per descending 5-HT cell body in the brain stem only with this restricted projection. When combing these results with the biochemical data, it could be calculated that the concentration of 5-HT in IR varicosities is about 3-6 x 10(-3) M, while the corresponding figures for SP and TRH was 0.3-0.5 x 10(-3) M and 0.1-0.2 x 10(-3) M, respectively. In cats subjected to spinal cord transection at the lower thoracic level, all 5-HT-IR fibers in the L7 segment had disappeared 44 days after the lesion, indicating a strict suprasegmental origin of 5-HT-IR fibers in this segment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased 5-hydroxytryptamine immunoreactivity in traumatized spinal cord

Summary The possibility that serotonin (5-hydroxytryptamine, 5-HT) is involved in the early tissue reactions occurring in spinal cord trauma was examined in a rat model using an immunocytochemical technique. The injury was made in the form of a 5-mm long and 2.5-mm wide lesion of the right dorsal horn at the level of T_10–11. Injured rats, pretreated with the 5-HT synthesis blocking agent, p-chlorophenyl alanine (p-CPA) were compared with untreated injured controls and the animals were allowed to survive for 5 h. The distribution of 5-HT was examined in proximal and distal cross-sections of the cord, located 2 and 5 mm away from the injury. Normal rats showed immunoreactive material in nerve cell processes and in a few nerve cell bodies of the ventral horns. The trauma to the spinal cord caused a marked increase in 5-HT immunoreactivity in the segments located 2 mm proximal and distal to the injury, particularly in the ipsilateral ventral horn. The segment located 5 mm distal to the lesion showed a similar increase in immunoreactivity but it was apparently less pronounced in the corresponding proximal segment. Treatment with p-CPA markedly reduced the trauma-induced increase in 5-HT immunoreactivity in all the segments. These immunohistochemical findings were in line with the changes in the contents of 5-HT measured biochemically in corresponding spinal cord segments. At the onset of the trauma to the spinal cord 5-HT is thus present in the tissue, mainly in the form of 5-HT-containing nerve cell processes. Biochemical determinations also revealed that there is an increased amount of 5-HT in the traumatized spinal cord. The present study indicates that this is at least partly due to an increased amount of 5-HT in neurons and nerve cell processes of the perifocal region. The pathophysiollogical significance of the observed 5-HT-reaction in spinal cord injury is not known in all its details. However, 5-HT might be implicated in such tissue reaction, such as increased microvascular permea bility and edema formation occurring in the early period after trauma.Supported by grants from the Swedish Medical Research Council, projects 02710 and 03020, 1987 Års Stiftelse för Strokeforskning, Riksbankens Jubileumsfond, RTP, Folksam, Wallenius Line, Trygg-Hansa, the Karolinska Institute Research Funds and from the Torsten and Ragnar Söderberg Foundation     

Denervation supersensitivity to 5-hydroxytryptamine in the rat spinal cord is not due to the absence of 5-hydroxytryptamine

D,L-5-Hydroxytryptophan and 5-HT agonists administered systemically, stimulate motoneuronal discharges as measured by the spontaneous EMG activity of the hindlimbs in paraplegic rats. Denervation supersensitivity is observed after surgical section of the spinal cord or after treatment with 5,7-dihydroxytryptamine (5,7-DHT). Such denervation supersensitivity, however, cannot be reproduced by equivalent depletion of 5-HT by synthesis inhibition or reversed by chronic intrathecal administration of 5-HT agonists. These results suggest that in the anterior horn of the spinal cord, the trigger of denervation supersensitivity to serotonin is not the absence of the neurotransmitter itself but the absence of the terminals or some other compound contained therein.     

Immunohistochemical localization of seven different peptides in the human spinal cord

It is necessary to study the normal chemical contents in the human spinal cord in order to understand neurochemical changes that might occur under pathological conditions. In the present study, the comparative distribution of seven peptides was examined immunohistochemically in four levels (cervical, C; thoracic, T; lumbar, L; sacral, S) of the human spinal cord by means of the peroxidase-antiperoxidase technique. The peptides examined included bombesin (BOM), substance P (SP), cholecystokinin (CCK), somatostatin (SOM), methionine-enkephalin (M-ENK), vasoactive intestinal polypeptide (VIP), and thyrotropin releasing hormone (TRH). Among the seven peptides examined, four (BOM, CCK, SOM, and TRH) have never been described in the human spinal cord and the present work clearly demonstrates their existence in specific patterns. The terminals that were immunostained for BOM and CCK were localized in high concentration in the superficial dorsal horn (laminae I-II), in moderate amounts in the lateral part of laminae V and VII, and lesser amounts in the intermediate gray (lamina VII) and the dorsal part of the central gray (lamina X). Whereas BOM showed a similar distribution pattern at all spinal levels, CCK was mainly found in thoracic and lumbar levels. The SOM terminals were localized in the superficial dorsal horn (the highest density in lamina II but very few in lamina I), the intermediolateral cell column, intermediate gray, and central gray. This peptide was more widely distributed in the sacral cord with its terminal field extending into the ventral horn. The TRH terminals were mainly located in the ventral horn. Frequently, TRH terminals were seen adjacent to large ventral horn neurons. Furthermore, many neurons in the ventral and intermediate gray and Clarke's column demonstrated TRH immunoreactivity. The other three peptides (SP, M-ENK, and VIP) have been previously demonstrated in the human spinal cord and the present study confirmed their general spinal distribution with minor differences.     

The effect of p-chloroamphetamine and p-chlorophenylalanine on the level of thyrotropin-releasing hormone and its receptors in some brain structures and lumbar spinal cord of the rat.

The concentration of thyrotropin-releasing hormone (TRH) and the density and affinity of TRH receptors were examined in the ventral and dorsal lumbar spinal cord, nucleus accumbens and striatum of rats with the 5-hydroxytryptamine (5-HT) nerve terminal destroyed with p-chloroamphetamine (PCA), or in animals treated with the inhibitor of 5-HT synthesis p-chlorophenylalanine (PCPA). PCA (2 x 10 mg/kg i.p., 9 and 8 d before killing) and PCPA (3 x 300 mg/kg i.p., 72, 48 and 24 h before killing)--either of them dramatically diminishing the 5-HT and 5-HIAA concentrations in all the examined structures--reduced the TRH level and increased the density of TRH receptors in the ventral lumbar spinal cord. PCPA also reduced the TRH content in the nucleus accumbens. The PCA-induced reduction in the TRH level and increase in the density of TRH receptors in the ventral lumbar spinal cord were significantly attenuated by citalopram (2 x 20 mg/kg i.p., 30 min before PCA), a selective inhibitor of 5-HT uptake. Our results constitute a further proof that coexistence of TRH and 5-HT takes place in the ventral lumbar spinal cord and then indicate that other form(s) of relationship between 5-HT and TRH may exist in some parts of the central nervous system. They also suggest that an up-regulation of TRH receptors occurs in the spinal cord as a result of TRH depletion.     

Serotonergic and peptidergic inputs to the primate ventral spinal cord as visualized with multiple chromagens on the same tissue section

Primate spinal cord tissue sections were sequentially incubated in serotonin (5-HT), substance P (SP) and methionine-enkephalin antisera and reacted immunocytochemically. The findings indicate that virtually all SP-like immunoreactivity coexists with 5-HT in the ventral horn, while an additional group of 5-HT terminals do not contain SP immunoreactivity.     

Decreased immunoreactive (IR) calcitonin gene-related peptide correlates with sprouting of IR-peptidergic and serotonergic neuronal processes in spinal cord and brain nuclei from the Wobbler mouse during motoneurons disease

The Wobbler mouse possesses an inherited form of motoneuron disease that expresses itself most dramatically in the forelimbs. Previous immunocytochemical (ICC) studies have shown that neuronal processes containing substance P (SP), thyrotropin releasing hormone (TRH) and serotonin (5-HT) seem to sprout in the ventral horn of the cervical spinal cord taken from the Wobbler mouse. By radioimmunoassay, increased concentrations of spinal SP, TRH, and 5-HT, as well as leucine and methionine enkephalins (LE, ME) have been documented. The present ICC study quantifies the numbers of neuronal processes in the Wobbler cervical spinal cord and brainstem which contain SP, 5-HT, LE, ME and other neuropeptides (cholecystokinin, CCK; neuropeptide Y; galanin; calcitonin gene-related peptide, CGRP). It is proposed that those processes that sprout early in the mononeuron disease (5-HT, LE, ME, CCK and also TRH according to other studies) may be involved in the etiology. In addition, it is hypothesized that the loss of CGRP within the ventral horn may represent the loss of a trophic factor that is important to the survival motoneurons and may influence the increase of fiber densities around the dying motoneurons.     

The effect of fenfluramine on the thyrotropin-releasing hormone (TRH) content in the rat brain structures and lumbar spinal cord

The effect of fenfluramine (20 mg/kg i.p.) was studied on the thyrotropin-releasing hormone (TRH) content in several brain structures and the ventral part of the lumbar spinal cord of the rat. The effect of fenfluramine on the concentration of 5-hydroxytryptamine (5-HT) in some brain structures and the lumbar spinal cord was also examined. It was found that fenfluramine had no effect on the TRH level in the hypothalamus, hippocampus, occipital cortex, septum, nucleus accumbens and ventral part of the lumbar spinal cord, though the drug produced a profound depletion (by more than 60%) of 5-HT in the hypothalamus, nucleus accumbens, striatum and lumbar spinal cord. On the other hand, fenfluramine significantly increased the TRH content in the striatum, this effect was completely abolished by citalopram (20 mg/kg i.p.) or metergoline (10 mg/kg i.p.) Citalopram also prevented the fenfluramine-induced depletion of the striatal 5-HT. These results indicate a separate neuronal storage of TRH and 5-HT in the structures (ventral part of the lumbar spinal cord, nucleus accumbens, septum) in which the peptide and indoleamine coexist in 5-HT neurons. They also suggest that the fenfluramine-induced increase in the striatal TRH concentration is due to 5-HT release and stimulation of 5-HT receptors.     

Distribution of 125I-galanin binding sites, immunoreactive galanin, and its coexistence with 5-hydroxytryptamine in the cat spinal cord: biochemical, histochemical, and experimental studies at the light and electron microscopic level.

The distribution of galanin-like immunoreactivity (GAL-LI) in the spinal cord of the cat was studied by use of indirect histochemistry and the peroxidase-antiperoxidase (PAP) technique. In the ventral horn GAL-immunoreactive (IR) axonal fibers and terminals were most frequent in the ventral part of the motor nucleus. The GAL-IR axons also contained 5-hydroxytryptamine (5-HT)-LI, and they disappeared after spinal cord transection. It was concluded that these GAL-IR fibers belong to the serotoninergic bublospinal pathway. In the medulla oblongata from normal cats, scattered GAL-IR cell bodies were encountered within the nucleus raphe obscurus and nucleus raphe pallidus. Electron microscopic observations revealed that the fine structure of the GAL-IR axonal boutons in the motor nucleus was similar to that of 5-HT-IR boutons with a varying number of immunoreactive large dense core vesicles. The postsynaptic element in all cases studied was a dendrite. A dense GAL-IR axonal plexus was found in the superficial laminae I-II of the dorsal horn. Coexistence was found between the GAL- and substance P-LI in fibers within the dorsal horn plexus. Spinal cord transection did not alter the pattern of GAL-LI in the dorsal horn, while the vast majority of GAL-IR axonal swellings disappeared following dorsal root sectioning. Electron microscopic observations in lamina II (substantia gelatinosa) revealed that the GAL-IR axonal terminals could be divided into two main groups. One with small to medium-sized axonal boutons formed synaptic contacts with both dendritic and axonal profiles. The other formed the central axon terminals of glomeruli, suggesting that GAL-LI may be present in C-type primary afferents. Numerous small GAL-IR cell bodies were encountered in laminae II and III. GAL-IR cell bodies were also observed in lamina X. The dorsal root ganglia contained a low but consistent number of small to medium-sized GAL-IR cell bodies, which all contained immunoreactive calcitonin gene-related peptide (CGRP). Following peripheral sciatic nerve transection, the number and the labeling intensity of GAL-IR cell bodies in the corresponding dorsal root ganglia showed a moderate increase. Radioimmunoassay revealed that the concentration of GAL-LI increased along the rostrocaudal axis of the normal spinal cord, and was about three times higher in the dorsal than in the ventral regions. The concentration in the dorsal root ganglia was intermediate to those seen in the corresponding dorsal and ventral cord regions.(ABSTRACT TRUNCATED AT 400 WORDS)     

The coexistence of serotonin- and substance P-like immunoreactivity in the spinal cord of the rat as shown by immunofluorescent double labeling

This study surveyed the coexistence of serotonin-like immunoreactivity (5-HT-IR) with substance P-like immunoreactivity (SP-IR) in fibers and terminals within various portions of the spinal cord of the rat. A previously characterized technique of immunofluorescent double labeling was used to stain 5-HT-IR red and SP-IR green, and a search was then made for single fibers that fluoresced both colors. These were found to be most common in the ventral horn, wherein 99% of 5-HT-IR fibers were also immunoreactive for SP. Coexistence of these substances was very rare in the superficial dorsal horn: fewer than 3% of 5-HT-IR fibers were immunoreactive for SP. In the region surrounding the central canal and in the intermediolateral cell column (IML), over half of all 5-HT-IR fibers and terminals were doubly labeled. In the white matter, doubly labeled fibers were most common in the ventral funiculus and somewhat less common in the lateral funiculus. They were rare in the dorsal columns. It is concluded that the coexistence of 5-HT and SP in nerve fibers and terminals is associated with somatic and sympathetic autonomic motoneurons. The role of the coexistence of 5-HT and SP in somatosensation, including pain, is unclear.     

Evidence for 5-hydroxytryptamine, substance P, and thyrotropin-releasing hormone in neurons innervating the phrenic motor nucleus

Retrograde tracing with a fluorescent dye (Fast Blue) combined with immunohistochemistry was used to identify putative neurotransmitter(s) at the phrenic motor nucleus in the cat. Fast Blue was injected bilaterally into the diaphragm of five cats, where each phrenic nerve enters the muscle. Seven days later the animals were perfusion fixed and tissue sections from the fourth, fifth, and sixth cervical spinal cord segments were analyzed using a fluorescence microscope. Retrogradely labeled fluorescent phrenic motor neuron cell bodies appeared in all of the segments but primarily in sections from the fifth segment. The same or adjacent transverse sections were then used for the demonstration of the distribution of the neurotransmitters 5-hydroxytryptamine (5-HT), substance P, and thyrotropin-releasing hormone (TRH) in the area of the phrenic motor nucleus using the indirect immunofluorescence technique. The most conspicuous neurotransmitters found at the phrenic motor nucleus were 5-HT and substance P. We observed dense and diffuse fiber networks throughout the ventral horn which contains the phrenic motor nucleus. These fibers contained varicosities in close proximity to phrenic motor neurons. In addition to 5-HT- and substance P-containing nerve endings, some fibers containing TRH were also found in the area of the phrenic motor nucleus. These results are consistent with earlier physiological data suggesting that 5-HT, substance P, and TRH are important neurotransmitters and/or neuromodulators involved in central control of respiration.     

Influences of both thyroid and bovine growth hormones on substance P, thyrotropin-releasing hormone, serotonin and 5-hydroxyindoleacetic acid contents in the lumbar spinal cord of developing rats

Neonatal hypothyroidism was induced by injection of 131I on the first living day whilst neonatal hyperthyroidism was induced by daily administration of high doses of thyroxine (T4). Following decapitation, segments of the lumbar spinal cord were microdissected by a punch technique. We measured serotonin and 5-hydroxyindoleacetic acid (5-HIAA) contents by high performance liquid chromatography and both substance P and thyrotropin-releasing hormone (TRH) levels by radioimmunoassay. We demonstrated that: (1) neonatal hyperthyroidism decreased substance P and TRH levels in the dorsal and ventral horns respectively, without modifying serotonin and 5-HIAA contents; (2) neonatal hypothyroidism increased the concentration of substance P in dorsal horn, of TRH in ventral horn (confirming our previous work), of serotonin in ventral horn, and of 5-HIAA in both ventral and dorsal horns; (3) T4-replacement therapy abolishes hypothyroid effects on substance P, TRH, and 5-HIAA, but not on 5-HT accumulation; and (4) bovine growth hormone-replacement therapy has no therapeutic action on the hypothyroid-induced accumulation of substance P, TRH, serotonin and 5-HIAA.     

Altered responsiveness to substance P and 5-hydroxytryptamine in cat dorsal horn neurons after 5-HT depletion with p-chlorophenylalanine

The responsiveness of functionally identified cat spinal dorsal horn neurons to iontophoretically applied substance P (SP) and 5-hydroxytryptamine (5-HT) has been investigated by means of extracellular recording after 5-HT depletion with p-chlorophenylalanine (p-CPA). In addition, the spinal levels of 5-HT, SP, cholecystokinin octapeptide, neurotensin, and vasoactive intestinal polypeptide have been measured in intact and p-CPA-pretreated cats. In the present study we have demonstrated an altered responsiveness of dorsal horn neurons to locally applied SP and 5-HT. We found in p-CPA-pretreated cats that the proportion of neurons responding with excitation to SP and 5-HT was significantly increased. At the same time, depression induced by 5-HT in the dorsal horn cells was virtually absent in p-CPA-pretreated animals. Our finding that spinal level of 5-HT was significantly decreased in p-CPA-treated animals is consistent with previous studies. No convincing alteration in the spinal levels of 4 analyzed peptides was found in p-CPA-treated animals. The present study has shown that pharmacological depletion of 5-HT has two major effects: (1) it increases significantly the proportion of dorsal horn neurons excited by SP and 5-HT; and (2) it is ineffective in inducing 5-HT supersensitivity. Further work is needed to explain mechanisms involved in these effects.     

Thyrotropin-releasing hormone (TRH)-like immunoreactivity in the grey monkey (Macaca fascicularis) spinal cord and medulla oblongata with special emphasis on the bulbospinal tract.

The distribution of thyrotropin-releasing hormone (TRH)-like immunoreactivity (LI) has been studied in the grey monkey (Macaca fascicularis) spinal cord and medulla oblongata by the use of indirect immunofluorescence and the peroxidase-antiperoxidase (PAP) technique. Furthermore, double-labeling experiments were performed in order to study colocalization of 5-hydroxytryptamine (5-HT)- and substance P-LI. A dense innervation of TRH-immunoreactive (IR) varicose fibers was found in the ventral horn motor nuclei, in the region surrounding the central canal, in the intermediolateral cell column, and in the dorsal horn laminae II and III. In addition, cell bodies harboring TRH-LI were found in the dorsal horn laminae II-IV. In the ventral horn, many of the large cell bodies and their proximal dendrites were totally encapsulated by TRH-IR fibers. From double-labeled sections a high degree of coexistence could be established between TRH-/5-HT-LI, TRH-/substance P-LI, and 5-HT-/substance P-LI in fibers in the motor nuclei; as a consequence, a large proportion of these fibers should harbor TRH-/5-HT-/substance P-LI. A coexistence between TRH-/5-HT-LI could also be demonstrated in the intermediolateral cell column. However, no unequivocal coexistence could be found between TRH-/substance P-LI and 5-HT-/substance P-LI in this region. In the dorsal horn, no clear coexistence could be encountered for any of the above indicated combinations. Electron microscopic analysis of material from the lumbar lateral motor nucleus demonstrated TRH-IR terminals making synapses with large cell bodies and dendrites. In addition, contacts lacking synaptic specializations could also be verified. In the medulla oblongata, with the use of the PAP technique, a large number of cell bodies containing TRH-LI were encountered in the midline raphe nuclei and in nucleus reticularis lateralis. A similar distribution pattern could be found for 5-HT-LI, but no cell bodies containing substance P-LI could be seen in these regions. Chemical analysis of specimens from cervical, thoracic, and lumbar spinal cord revealed higher concentrations of TRH- and 5-HT-LI in the ventral quadrants, whereas substance P-LI dominated in the dorsal quadrants. Thus, the concentrations of TRH-, 5-HT-, and substance P-LI was in accordance with the observed regional variation in density of IR-fibers and varicosities found in the spinal cord. We have shown that TRH-LI has a distribution in the monkey spinal cord and medulla oblongata similar to that previously demonstrated in other species.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Recovery of 5-HT Immunoreactivity in Lumbosacral Spinal Cord and Locomotor Function after Thoracic Hemisection

To determine the role of serotonin (5-HT) in recovery from spinal cord injury, we examined spinal cord 5-HT immunohistologically and assessed locomotor recovery after thoracic (T8) spinal cord hemisection in 68 rats. Forty eight rats had laminectomy and hemisection, while the remaining 20 rats received laminectomy only. All rats were evaluated every other day for 4 weeks, using a 0–14 point scale open field test. Hemisection markedly reduced mean hindlimbs scores from 14 to 1.5 ± 0.32 and 5.6 ± 0.31 (mean ± standard error of mean) in the ipsilateral and contralateral side, respectively. The rats all recovered apparently normal walking by 4 weeks. The 5-HT immunohistological study revealed a marked reduction of 5-HT-containing terminals in the ipsilateral but not the contralateral lumbosacral cord by 1 week after hemisection. By 4 weeks after hemisection, 5-HT immunoreactive fibers and terminals returned to the ipsilateral lumbosacral cord, with many 5-HT fibers crossing over the central canal at thoracic level. We estimated the recovery of 5-HT neural elements in lumbosacral ventral horn by ranking 5-HT staining intensity and counting 5-HT terminals. The return of 5-HT immunoreactivity of the lumbosacral ventral horn correlated with locomotor recovery. Locomotory recovery invariably occurred when the density of 5-HT terminals approached 20% of control values. These results indicate that return of 5-HT fibers and terminals predict the time course and extent of locomotory recovery after thoracic spinal cord hemisection.     

Immunohistochemical demonstration of some putative neurotransmitters in the lamprey spinal cord and spinal ganglia: 5-hydroxytryptamine-, tachykinin-, and neuropeptide-Y-immunoreactive neurons and fibers

The distribution of some putative neurotransmitters was investigated in the spinal cord and spinal ganglia of the lamprey, a primitive vertebrate, by using immunohistochemical methods. In the spinal cord a midline row of 5-hydroxytryptamine (5-HT)-immunoreactive neurons was present immediately ventral to the central canal over the entire length of the spinal cord. The ventral processes of these neurons formed a dense ventromedial plexus of varicosities. In the dorsal, lateral, and ventral spinal axon columns, several longitudinal 5-HT fibers were present. After chronic spinal transections the distribution of 5-HT fibers was unchanged; it is therefore concluded that there was no substantial descending 5-HT contribution and that the spinal 5-HT neurons supplied the regional 5-HT innervation. The spinal 5-HT cells sent fibers into the dorsal and ventral roots; 5-HT cell bodies and fibers were also present in the spinal dorsal root ganglia, in their dorsal, ventral, and lateral nerve branches, and in the dorsal and ventral branches of the ventral roots. Neurons and fibers containing peptides of the tachykinin (TK) family (to which, amongst others, substance P belongs) were found in the spinal cord. TK neurons in the spinal cord supplied the local TK innervation, as well as TK fibers in the dorsal and ventral roots. Fibers have been found containing either TK, or 5-HT, or both compounds. Neurons containing neuropeptide-Y (NPY)-immunoreactive material were present in a medial column just dorsal to the central canal. The NPY neurons have longitudinal, mainly descending, fibers that provide the local NPY innervation of the lamprey spinal cord. The present results provide evidence for local spinal systems containing 5-HT, TK, 5-HT and TK, or NPY, but in contrast to mammals, these compounds do not seem to arise from supraspinal neurons.     

The 5-HT2A receptor is widely distributed in the rat spinal cord and mainly localized at the plasma membrane of postsynaptic neurons

Serotonin (5-HT) plays a major role at the spinal level by modulating most spinal functions through several receptor subtypes including the 5-HT2A receptor. To gain further insight into the cellular role of this receptor, we performed an immunocytochemical study of 5-HT2A receptors in the rat spinal cord, at light and electron microscope levels. The results showed that 5-HT2A receptors were widely distributed in the spinal cord at all segmental levels. Immunolabeling was particularly dense in lamina IX and in the dorsal horn lamina IIi. Immunoreactive cell bodies were numerous in lamina IX, where many but not all motoneurons were labeled, as shown by double labeling with choline acetyltransferase antibodies. Stained cell bodies were also observed in the gray matter. The study at the ultrastructural level focused on the lumbar dorsal horn (laminae I-II) and ventral horn (lamina IX). At both levels, 5-HT2A immunoreactivity was mainly postsynaptic on dendrites and cell bodies. However, a little presynaptic labeling was also observed in axon and axon terminals, some of them containing large granular vesicles attesting to their peptidergic nature. The main result of our study was the "nonsynaptic" plasma membrane localization of 5-HT2A receptors covering a large surface of cell bodies and dendrites, suggesting a paracrine form of action of serotonin. These observations are consistent with a double role (pre- and postsynaptic) for serotonin on these receptors on various cellular targets.