A.A.J. Verhofstad

Immunohistochemical support for three putative transmitters in one neuron: Coexistence of 5-hydroxytryptamine, substance p- and thyrotropin releasing hormone-like immunoreactivity in medullary neurons projecting to the spinal cord

Abstract Using indirect immunofluorescence histochemistry, in part combined with the elution and restaining technique of Tramu , Pillez & Leonardelli (1978) , the distribution of 5-hydroxytryptamine (5-HT), thyrotropin releasing hormone (TRH) and substance P immunoreactive neurons has been studied in the medulla oblongata and spinal cord of normal and colchicine-treated rats. Evidence was obtained that at least some cell bodies in the medullary raphe nuclei and adjacent areas contained all three compounds, 5-HT, TRH and substance P. Other cell bodies in the same areas may contain two or only one of these three putative transmitters. Alternatively, the intraneuronal levels of one or two of the substances may be too low to be detected with the present technique, in spite of the fact that colchicine treatment was used to elevate peptide levels in the cell somata. In a quantitative evaluation the proportion of 5-HT, TRH and substance P neurons was calculated at different levels and in different nuclei of the medulla oblongata. Out of all immunoreactive neurons, there were approximately twice as many 5-HT (56%) as TRH (23%) and substance P (21%) cells respectively, and this relation was also found in several major subnuclei, such as the nucleus raphe magnus and nucleus raphe obscurus. In the ‘arcuate’ region very high proportions of 5-HT cells (about 60–80%) were observed with only few substance P cells (2–12%). The ‘parapyramidal’ and ‘paraolivar’ regions, which include the nucleus interfascicularis hypoglossi, had more substance P (26–36%) than TRH (15–17%) cells. The most ‘even’ distribution was observed in the nucleus raphe pallidus (5-HT: 43%; TRH: 32%; substance P: 25%). The evaluation also indicated how the respective cell type (5-HT, TRH and substance P cells) distributed between the different subnuclei. Thus, at rostral levels the ‘suprapyramidal’ region contained a large proportion (about 30%) of the total numbers of counted 5-HT, TRH and substance P cells, respectively. Furthermore, the nucleus raphe magnus contained a large part (about 30%) of the TRH and substance P cells, but a smaller fraction (about 20%) of the 5-HT cells. Analysis of adjacent sections at regular intervals confirmed the overall quantitative evaluation. Generally, the distribution of 5-HT, TRH and substance P cells were roughly parallel. An exception was the midportion of the rostral medulla oblongata, where 5-HT cells were very numerous. Of particular interest was the fact that, especially in the nucleus raphe pallidus, there were in several series almost the same number of 5-HT, TRH and substance P cells, supporting the view that many cells in this nucleus contained all these compounds. In the spinal cord overlapping networks of 5-HT, TRH and substance P immunoreactive fibres were observed in the ventral horn. The number of 5-HT immunoreactive fibres seemed higher than the TRH and substance P immunoreactive ones. After treatment with the neurotoxins 5,6- or 5,7-dihydroxytryptamine there was an almost complete disappearance of all three types of fibres in the ventral horn, further supporting the occurrence of the two peptides in 5-HT neurons, either both of them together in the same 5-HT neuron or each of them in separate 5-HT neurons. It is, however, important to note that there are, in all probability, 5-HT neurons in the lower medulla oblongata which contain neither TRH nor substance P. Furthermore, in other brain regions there is no certain correlation between the distribution patterns of 5-HT, TRH and substance P immunoreactive cells. The results are consistent with the coexistence of 5-HT, TRH and substance P in neurons of the medulla oblongata that project to the spinal cord. Some neurons may contain detectable levels only of 5-HT and substance P, others only of 5-HT and TRH, while others contain all three substances. It can, however, not be excluded that some neurons contain only one of these compounds or that other combinations exist.

The distribution and cells of origin of serotonergic inputs to the paraventricular and supraoptic nuclei of the rat.

The distribution of serotonin-immunoreactive varicosities in the paraventricular (PVH) and supraoptic (SO) nuclei was charted in normal immunohistochemical material and the probable cells of origin of these projections were identified using a combined retrograde transport-immunohistochemical method. The density of serotonergic fibers in the PVH and the SO is quite low relative to that in the immediately surrounding neuropil, in striking contrast to noradrenergic inputs to the nuclei. Immunoreactive fibers are concentrated in specific parts of the parvocellular division of the PVH, whereas in the magnocellular division of the nucleus, and in the SO, they are found mostly in regions where oxytocinergic cells predominate. These projections appear to arise from 3 distinct serotonergic cell groups (B7, B8 and B9) in the midbrain.

An immunohistochemical procedure to detect patients with paraganglioma and phaeochromocytoma with germline SDHB, SDHC, or SDHD gene mutations: a retrospective and prospective analysis.

BACKGROUND Phaeochromocytomas and paragangliomas are neuro-endocrine tumours that occur sporadically and in several hereditary tumour syndromes, including the phaeochromocytoma-paraganglioma syndrome. This syndrome is caused by germline mutations in succinate dehydrogenase B (SDHB), C (SDHC), or D (SDHD) genes. Clinically, the phaeochromocytoma-paraganglioma syndrome is often unrecognised, although 10-30% of apparently sporadic phaeochromocytomas and paragangliomas harbour germline SDH-gene mutations. Despite these figures, the screening of phaeochromocytomas and paragangliomas for mutations in the SDH genes to detect phaeochromocytoma-paraganglioma syndrome is rarely done because of time and financial constraints. We investigated whether SDHB immunohistochemistry could effectively discriminate between SDH-related and non-SDH-related phaeochromocytomas and paragangliomas in large retrospective and prospective tumour series. METHODS Immunohistochemistry for SDHB was done on 220 tumours. Two retrospective series of 175 phaeochromocytomas and paragangliomas with known germline mutation status for phaeochromocytoma-susceptibility or paraganglioma-susceptibility genes were investigated. Additionally, a prospective series of 45 phaeochromocytomas and paragangliomas was investigated for SDHB immunostaining followed by SDHB, SDHC, and SDHD mutation testing. FINDINGS SDHB protein expression was absent in all 102 phaeochromocytomas and paragangliomas with an SDHB, SDHC, or SDHD mutation, but was present in all 65 paraganglionic tumours related to multiple endocrine neoplasia type 2, von Hippel-Lindau disease, and neurofibromatosis type 1. 47 (89%) of the 53 phaeochromocytomas and paragangliomas with no syndromic germline mutation showed SDHB expression. The sensitivity and specificity of the SDHB immunohistochemistry to detect the presence of an SDH mutation in the prospective series were 100% (95% CI 87-100) and 84% (60-97), respectively. INTERPRETATION Phaeochromocytoma-paraganglioma syndrome can be diagnosed reliably by an immunohistochemical procedure. SDHB, SDHC, and SDHD germline mutation testing is indicated only in patients with SDHB-negative tumours. SDHB immunohistochemistry on phaeochromocytomas and paragangliomas could improve the diagnosis of phaeochromocytoma-paraganglioma syndrome. FUNDING The Netherlands Organisation for Scientific Research, Dutch Cancer Society, Vanderes Foundation, Association pour la Recherche contre le Cancer, Institut National de la Santé et de la Recherche Médicale, and a PHRC grant COMETE 3 for the COMETE network.

Immunohistochemical studies on the effect of capsaicin on spinal and medullary peptide and monoamine neurons using antisera to substance P, gastrin/CCK, somatostatin, VIP, enkephalin, neurotensin and 5-hydroxytryptamine

SummaryAfter neonatal treatment of rats with capsaicin, the spinal cord, the spinal trigeminal nucleus and spinal and trigeminal ganglia were analysed with immunohistochemistry using antisera to several peptides and 5-hydroxytryptamine. A marked decrease was observed in substance P-, cholecystokinin-, somatostatin- and VIP-like immunoreactivity present in the central branches of primary sensory neurons in the spinal cord and in substance P- and somatostatin-like immunoreactivity in sensory ganglion cells. No definite depleting effect of capsaicin could be established on 5-hydroxytryptamine and peptides, such as enkephalin and neurotensin, present in centrally originating fibres in the dorsal horn of the spinal cord. The results demonstrate that the effects of capsaicin are not confined to substance P immunoreactive primary sensory neurons. The possibility is discussed that capsaicin effects specifically functioning rather than chemically specific primary sensory neurons.

The effects of monoamine neurotoxins on peptides in the rat spinal cord

The coexistence of two neuronally-localised peptides, substance P and thyrotropin-releasing hormone (TRH), in descending serotoninergic nerve fibres to the spinal cord was investigated using immunocytochemical and biochemical methods. Substance P-like material in the spinal cord was shown to be identical to the undecapeptide substance P by the criteria of gel filtration, high performance liquid chromatography and behaviour in substance P specific radioimmunoassays. Immunocytochemical staining for 5-hydroxytryptamine, substance P, and TRH showed that all three substances had a similar distribution in nerve fibres and terminals in the ventral and lateral grey matter of the spinal cord. After treatment with the serotonin neurotoxin 5,7-dihydroxytryptamine, neuronal elements containing 5-hydroxytryptamine, substance P and TRH degenerated and disappeared from these parts of the spinal cord in parallel with one another. Biochemical measurements of 5-hydroxytryptamine, substance P and TRH in the spinal cord after treatment with 5,7-dihydroxytryptamine confirmed that these three substances were all depleted from the ventral horn and, in addition, showed that there was a small depletion of substance P from the dorsal horn. Two other neuropeptides, somatostatin and methionine-enkephalin were not depleted from the spinal cord by treatment with 5,7-dihydroxytryptamine nor was substance P in other parts of the brain. Substance P in the spinal cord was unaffected by 6-hydroxydopamine, a drug known to destroy catecholamine-containing neurones. These results are consistent with coexistence of substance P and TRH together with 5-hydroxytryptamine in the descending axons and terminals of bulbospinal neurones.

Immunohistochemical evidence for colocalization of γ-aminobutyric acid and serotonin in neurons of the ventral medulla oblongata projecting to the spinal cord

Fluorescence immunohistochemistry was used to analyze the medulla oblongata of colchicine-treated rats that had been incubated with guinea pig antibodies to serotonin (5-HT) and either rabbit or sheep antibodies to glutamic acid decarboxylase (GAD). Numerous cells in the rostral ventrolateral medulla in the region of nucleus raphe magnus were immunostained for either 5-HT or GAD. A substantial number of neurons showed positive immunoreactivity for both substances, and were most frequently observed in the lateral aspect of nucleus raphe magnus. In addition, a number of the 5-HT/GAD-containing neurons were retrogradely labelled with Fast blue dye that had been injected into the thoracic spinal cord. This work provides evidence for colocalization of the classical neurotransmitters 5-HT and GABA in single cells of the ventral medulla oblongata, some of which project to the spinal cord.

Combined quantitative dynamic contrast-enhanced MR imaging and (1)H MR spectroscopic imaging of human prostate cancer.

To differentiate prostate carcinoma from healthy peripheral zone and central gland using quantitative dynamic contrast‐enhanced (DCE) magnetic resonance (MR) imaging and two‐dimensional 1H MR spectroscopic imaging (MRSI) combined into one clinical protocol.

Serotonin-induced stimulation of cortisol secretion from human adrenocortical tissue is mediated through activation of a serotonin4 receptor subtype

The occurrence of serotonin in the human adrenal gland was demonstrated both by immuno-histochemical and biochemical approaches. Using specific polyclonal antibodies to serotonin, the presence of numerous immunoreactive cells was revealed by means of the peroxidase-antiperoxidase technique. These cells exhibited the morphological characteristics of mast cells. Combination of high performance liquid chromatography and electrochemical detection showed the presence of substantial amounts of both serotonin and its metabolite 5-hydroxyindolacetic acid in adrenocortical extracts. The role of serotonin in the regulation of steroidogenesis from human adrenocortical slices was studied in vitro using a perifusion system technique coupled to a specific radioimmunoassay for cortisol. Graded doses of serotonin (from 10(-8) M to 3 x 10(-7) M) increased cortisol production in a dose-dependent manner. Prolonged exposure of adrenal fragments to serotonin (10(-7) M) induced a biphasic response, i.e. a rapid and transient increase in cortisol secretion followed by a plateau phase, suggesting the existence of a desensitization phenomenon. The stimulatory effect of serotonin (10(-7) M) was not altered during infusion of the serotonin1 and/or serotonin2 receptor antagonists methysergide (10(-6) M) and ketanserin (10(-6) M), respectively. In contrast, ICS 205 930 (10(-6) M), a non-selective serotonin3/serotonin4 antagonist, totally abolished the response of adrenal slices to serotonin (10(-7) M). The benzamide derivative zacopride, considered as a serotonin4 agonist, induced a robust stimulation of cortisol secretion. In addition, the corticotropic effects of serotonin (10(-7) M) and zacopride (10(-6) M) were not additive. Incubation of adrenocortical fragments with zacopride (10(-6) M) or serotonin (10(-6) M) caused a significant increase in cAMP formation. Taken together, these data suggest that serotonin, locally released by intra-adrenal mast-like cells, may act as a paracrine factor to stimulate cortisol secretion in man. Our results also indicate that serotonin-induced corticosteroid production is mediated through activation of a serotonin4 receptor subtype positively coupled to adenylate cyclase.

Sprouting of striatal serotonin nerve terminals following selective lesions of nigro-striatal dopamine neurons in neonatal rat

The effects of neonatal intracisternal 6-hydroxydopamine (6-OHDA; 50 micrograms) treatment on striatal serotonin (5-HT) nerve terminals in rat have been characterized using histo- and neurochemical methods. The 6-OHDA lesion caused a 60% reduction of striatal dopamine (DA) concentration when analyzed in the adult stage, while 5-HT levels were increased by about 40% and 3H-5-HT uptake in vitro was increased by about 60%. Using computerized image analysis, a marked increase in 5-HT-like immunoreactive terminal density was found in both rostral (+200%) and caudal (+50%) striatum. Pretreatment with the DA uptake blocker amfolenic acid completely counteracted the 6-OHDA-induced alterations in both DA and 5-HT neurons in the striatum, while pretreatment with the noradrenaline uptake blocker desipramine had no significant effects. Regional analysis of 5-HT levels in the CNS after neonatal 6-OHDA treatment or the combined desipramine + 6-OHDA treatment showed no significant effect in any of the brain areas analyzed, apart from the observed 5-HT increase in striatum. It was furthermore observed that the striatal 5-hydroxyindoleacetic acid (5-HIAA)/5-HT ratio was decreased, while the 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratio was increased following the 6-OHDA lesion, indicating compensatory mechanisms in turnover of transmitters. These alterations were completely reversed after pretreatment with amfolenic acid. The present results support the view that the 5-HT hyperinnervation following neonatal 6-OHDA treatment is a collateral sprouting response induced by lesioning of the striatal DA neurons.

Serotonergic and non-serotonergic projections from the nucleus raphe dorsalis to the caudate-putamen complex in the rat, studied by a combined immunofluorescence and fluorescent retrograde axonal labeling technique

Abstract Immunofluorescence, induced in serotonergic neurons, was combined with retrograde axonal tracing by propidium iodide. Evidence is presented for the existence of serotonergic as well as non-serotonergic projections from the nucleus raphe dorsalis to the caudate-putamen complex.