T. Hökfelt

Distribution of substance P-like immunoreactivity in the central nervous system of the rat--I. Cell bodies and nerve terminals.

Abstract The distribution of substance P-immunoreactive structures in the central nervous system of young (1–3 weeks old), adult and colchicine-treated adult male rats has been studied, using the indirect immunofluorescence technique of Coons and collaborators. Substance P-positive cell bodies were observed in more than thirty areas including the spinal cord and many parts of the brain stem. Extensive networks of substance P-positive nerve terminals of varying densities were found in most areas of the central nervous system. The cerebral and cerebellar cortices contained only few substance P-positive structures. It was difficult to identify substance-P immunoreactive axons in the rats studied. Some pathways could, however, be described but further experimental studies are necessary to elucidate the projections of the substance P-immunoreactive neurons in the rat central nervous system.

Substance p: localization in the central nervous system and in some primary sensory neurons.

Antibodies to substance P with a high titer have been produced and used in immunohistochemical studies on the peripheral and central nervous system of the rat and the cat. Evidence was obtained for the localization of substance P in a certain population of primary sensory neurons, probably small nerve cells with unmyelinated processes. Substance P or a peptide similar to it was also observed in cell bodies in the medial habenula and in probable nerve terminals in many brain areas. The results give morphological support for a transmitter (or modulator) role of substance P in the nervous system.

Differential co-existence of neuropeptide Y (NPY)-like immunoreactivity with catecholamines in the central nervous system of the rat

The distribution of neuropeptide Y immunoreactive cell bodies in relation to various types of catecholamine-containing cell bodies in the rat brain was analyzed immunohistochemically using antisera to tyrosine hydroxylase, dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase. Coexistence of the peptide in catecholamine cell bodies was established by using an elution-restaining procedure. Neuropeptide Y-like immunoreactivity was observed in most noradrenergic cell bodies of the Al/Cl cell groups in the ventro lateral medulla oblongata. Similarly this peptide immunoreactivity was also observed in the majority of the adrenergic cell bodies of the C2 group. In the dorsal and dorsal-lateral part of the nucleus of the solitary tract, where a group of small adrenergic cells is present, several small neuropeptide Y immunoreactive cells were also observed. The possibility of coexistence of adrenaline and neuropeptide Y in these cells remains to be established. The majority of the noradrenergic cell bodies of the A2 group, as well as the presumptive dopaminergic cells within its ventromedial part, seemed to lack neuropeptide Y-like immunoreactivity. Many noradrenergic cell bodies of the A6 group in the locus coeruleus proper were neuropeptide Y-immunoreactive, whereas the peptide could not be observed in the subcoeruleus group. Neither the A5 and A7 noradrenergic cells in the pons, nor any of the dopaminergic cell groups in the mesencephalon and forebrain (A8-A15) seemed to contain a neuropeptide Y-like peptide. The findings indicate that central catecholamine neurons can be subdivided into distinct sub-groups based upon the coexistence of a specific peptide.

Distribution of peptide- and catecholamine-containing neurons in the gastro-intestinal tract of rat and guinea-pig: Immunohistochemical studies with antisera to substance P, vasoactive intestinal polypeptide, enkephalins, somatostatin, gastrin/cholecystokinin, neurotensin and dopamine β-hydroxylase

Abstract The distribution of peptide-containing neurons in the oesophagus, stomach and small and large intestine of the rat and the guinea-pig has been studied with the indirect immunofluorescence technique of Coons & Co-workers (1958) using antisera to substance P, vasoactive intestinal polypeptide (VIP), enkephalin, somatostatin, gastrin and neurotensin. (The gastrin antiserum is to the C-terminal portion and consequently reacts also with cholecystokinin (CCK)-like peptides.) For comparison, the noradrenergic innervation was visualized with antiserum to dopamine β-hydroxylase. For improved visualization of peptide-containing cell bodies, a mitotic inhibitor (colchicine or vinblastine) was applied locally on the different parts of the gastro-intestinal tract of several animals. Substance P-, VIP-, enkephalin- and somatostatin-like immunoreactivity was observed in all parts of the gastro-intestinal tract studied. Gastrin/CCK had a more limited distribution, especially in the guinea-pig and neurotensin was seen only in certain regions and layers of the rat gastro-intestinal tract. Immunoreactivity to all peptides except neurotensin was observed both in cell bodies and fibres; immunoreactivity to neurotensin has so far only been seen in nerve fibres. Substance P and enkephalin immunoreactive cells were often numerous in the myenteric plexus, whereas VIP and somatostatin immunoreactive cells were preferentially located in the submucous plexus. Some VIP immunoreactive cells were observed in the lamina propria. Large numbers of especially substance P-, VIP- and enkephalin-containing fibres were often seen in the circular muscle layer and in the two ganglionic plexuses. Substance P immunoreactive fibres formed the densest network in the ganglionic plexuses, whereas VIP immunoreactive fibres constituted the most impressive network in the lamina propria and often extended into the most superficial parts of the mucosa. Enkephalin immunoreactive structures were mainly confined to the circular and longitudinal muscle layers and the myenteric plexus. Somatostatin immunoreactive fibres were mainly found in the ganglionic plexuses. Peptide-containing fibres, particularly these containing substance P and VIP were often seen along blood vessels, but never with such a density as the noradrenergic (dopamine β-hydroxylase immunoreactive) fibres. No somatostatin or neurotensin immunoreactive fibres were observed in relation to clearly identifiable blood vessels. The possible coexistence of two peptides in one neuron was studied. For this part of the study the proximal colon and five antisera, namely substance P, VIP, enkephalin. somatostatin and gastrin/CCK antisera were selected. Evidence was obtained for the occurrence of a somatostatin-like and a gastrin/ CCK-like peptide in the same neurons. This may indicate a common precursor for the two peptides in these particular neurons. Each of the substance P-, VIP- and enkephalin-like peptides. on the other hand, seem to be present in different neuronal populations, which were themselves distinct from the somatostatin-gastrin/CCK immunoreactive neurons. In addition, somatostatin immunoreactive neurons different from the gastrin/CCK immunoreactive ones seem to exist. The gastrin/CCK immunoreactive fibres around blood vessels may represent a further, separate population of fibres, since no somatostatin immunoreactive fibres were seen at this location. The findings indicate the existence of numerous subpopulations of enteric neurons, each characterized by its content of a certain peptide (or peptides). The axons of most of these neurons probably terminate in the wall of the gastro-intestinal tract, but some seem to project to other organs. In addition, some peptide-containing fibres in the gastro-intestinal wall may have an extrinsic origin. The relationship between these peptide-containing neurons and the cholinergic enteric neurons and any of the other non-cholinergic. non-adrenergic inhibitory and excitatory neurons present in the enteric nervous system is not known. It is, however, noteworthy that a somatostatin-like peptide seems to be present in noradrenergic neurons of prevertebral ganglia that project to the intestine. The possibility must be kept in mind that one or more of the peptides in the gut could be localized in neurons that contain other potential transmitters, e.g. acetylcholine. The wide variety of pharmacological actions of these neuronal peptides on smooth muscle and neurons in the gut and on its blood vessels raises the possibility that some of them may be neurotransmitters.

Immunohistochemical studies using antibodies to leucine-enkephalin: Initial observations on the nervous system of the rat

Enkephalins are peptides which have pharmacological properties similar to those of morphine. Guinea pigs were immunized with a leucine-enkephalin/thyroglobulin conjugate. Immunofluorescence histochemistry with antiserum revealed a widely distributed system of axons and their terminals in the nervous system of the rat. Prominent networks of enkephalin-like immunoreactivity were found in some brainstem nuclei and in portions of the limbic forebrain. The myenteric plexus in the gastrointestinal tract also contained fluorescent fibers. The distribution of the positive immunofluorescence parallels the occurrence of enkephalin as revealed by biochemical techniques. Some areas known to have a high opiate receptor density were also shown to contain striking networks of enkephalin-like immunoreactivity. Such findings provide morphological support for the hypothesis that enkephalins are contained in nerve terminals close to opiate receptors.

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.

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry

SummaryIn the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using35S-labelled and alkaline phosphatase-conjugated probes, in order to develop a reproducible double-labelling procedure. We have compared the total exclusion of tissue fixation with tissue sections fixed by immersion in formalin. In addition, the effect of dithiothreitol was assessed both when combining radiolabelled and non-radioactive probes on a single tissue section and when the probes were used separately. Hybridization of unfixed tissue resulted in stronger specific labelling and lower background both for radiolabelled and alkaline phosphatase-conjugated probes. No loss in tissue preservation was seen at the light microscopic level after hybridization of unfixed tissue. High concentrations (200 mM) of dithiothreitol strongly suppressed background when using35S-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (<1 mM). This incompatibility led to a protocol using unfixed tissue sections and a sequential hybridization procedure, with the radiolabelled probe and high concentrations of dithiothreitol in the first step and the alkaline phosphatase-conjugated probe without dithiothreitol in the second step.

High levels of neuropeptide Y in peripheral noradrenergic neurons in various mammals including man

Using a highly specific radioimmunoassay for neuropeptide Y (NPY), levels in the peripheral nervous system of guinea-pig, cat, pig and man were measured. In all species very high levels (up to 800 pmol X g-1) were found in sympathetic ganglia and in tissues which receive a dense sympathetic innervation, such as vas deferens, heart atrium, blood vessels and spleen. By immunocytochemistry, NPY-immunoreactive (-IR) principal ganglion cells in sympathetic ganglia and the pelvic plexus were also found to contain dopamine-beta-hydroxylase (DBH) and tyrosine hydroxylase (TH), strongly suggesting that the NPY-IR cells are noradrenergic. NPY- and DBH-IR nerves had a roughly parallel occurrence in the heart, spleen, kidney, respiratory and urogenitary tracts, around blood vessels and within visceral smooth muscle. Considerably more NPY-IR than DBH-IR nerve fibres were seen in the gastrointestinal tract from the oesophagus to the anal sphincter. In addition, NPY-IR local ganglion cells were observed in the submucous and myentric plexuses. NPY-like immunoreactivity was also observed in the adrenal medulla of guinea-pig and cat. NPY thus seems to be a major peptide in the sympathetic nervous system, supporting its proposed role in sympathetic neurotransmission.

Immunoreactive calcitonin gene-related peptide and substance P coexist in sensory neurons to the spinal cord and interact in spinal behavioral responses of the rat

Using immunohistochemistry evidence was obtained for the coexistence of calcitonin gene-related peptide (CGRP)- and substance P (SP)-like immunoreactivity in spinal sensory neurons. Analysis of caudally directed biting and scratching (CBS) behavior was carried out after intrathecal administration of CGRP and SP alone or in combination. Thus, SP (up to 20 micrograms) alone caused CBS only for a few minutes after injection, whereas SP (10 micrograms) plus CGRP (20 micrograms) caused a response with a duration up to 40 min. CGRP (20 micrograms) alone had no effects in this model. These findings provide support for a possible interaction of the two peptides at synapses in the dorsal horn of the spinal cord.

Substance P-immunoreactive sensory nerves in the lower respiratory tract of various mammals including man

SummaryThe occurrence and origin of substance P (SP)-immunoreactive (IR) nerves in the lower respiratory tract was studied by means of immunohistochemistry in the guinea-pig, rat, cat and man. In addition, biopsies from human material were also analysed by radioimmunoassay. SP-IR nerves were seen in four principal locations: 1) under or within the lining epithelium, 2) around blood vessels, 3) within the bronchial smooth muscle layer, and 4) around local tracheobronchial ganglion cells. Ligation experiments combined with capsaicin pretreatments indicated that all SP-IR nerves in the respiratory tract are sensory. The trachea seems to be mainly supplied by the vagal nerves, while intrapulmonary bronchi and blood vessels receive SP-IR nerves of both vagal and non-vagal (spinal) origin. SP-IR nerves were also found in the human bronchi with principally similar location as in the guinea-pig. The levels of SP-IR in the trachea and peripheral bronchi of man were about 3–4 pmol/g, which is in the same range as the content of corresponding tissues from the guinea-pig.In conclusion, the present experimental findings of SP-IR nerves in the lower respiratory tract in both experimental animals and man support the functional evidence for the importance of SP in the vagal and non-vagal (spinal) control of bronchial smooth muscle tone and vascular permeability.