Lars Terenius

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.

ARREST OF BETA -AMYLOID FIBRIL FORMATION BY A PENTAPEPTIDE LIGAND

Polymerization of amyloid β-peptide (Aβ) into amyloid fibrils is a critical step in the pathogenesis of Alzheimers disease. Here, we show that peptides incorporating a short Aβ fragment (KLVFF; Aβ) can bind full-length Aβ and prevent its assembly into amyloid fibrils. Through alanine substitution, it was demonstrated that amino acids Lys, Leu, and Phe are critical for binding to Aβ and inhibition of Aβ fibril formation. A mutant Aβ molecule, in which these residues had been substituted, had a markedly reduced capability of forming amyloid fibrils. The present data suggest that residues Aβ serve as a binding sequence during Aβ polymerization and fibril formation. Moreover, the present KLVFF peptide may serve as a lead compound for the development of peptide and non-peptide agents aimed at inhibiting Aβ amyloidogenesis in vivo.

The distribution of enkephalin-immunoreactive cell bodies in the rat central nervous system ☆

With the indirect immunofluorescence technique the distribution of methionine-enkephalin-immunoreactive cell bodies was studied in the central nervous system of rats pretreated with colchicine. The antiserum used did cross-react to 10% with leucine-enkephalin but to less than 0.1% with alpha-, beta-, and gamma-endorphine. Cell bodies with a specific immunofluorescence were observed in the tel-, di-, mes- and rhombencephalon and in the spinal cord.

Ibotenic acid-induced neuronal degeneration: A morphological and neurochemical study

SummaryPossible neurotoxic actions of intracerebral injections of ibotenic acid, a conformationally restricted analogue of glutamic acid, have been evaluated in rat brain and compared with those of kainic acid.Light microscopical analysis revealed that ibotenic acid produced a marked disappearance of nerve cells in all areas studied, namely striatum, the hippocampal formation, substantia nigra and piriform cortex. Lesions in areas distant to the injection site were not seen. Axons of passage and nerve terminals of extrinsic origin did not seem to be damaged, since, e.g., no apparent degeneration of the dopaminergic terminals in the neostriatum was observed except for a small area surrounding the cannula. In the neostriatum, enkephalin immunoreactive neuronal cell bodies as well as nerve terminals disappeared after injection of ibotenic acid into this nucleus. After injection into the substantia nigra tyrosine hydroxylase immunoreactive cell bodies in the zona compacta disappeared, whereas no certain effect could be seen on the enkephalin immunoreactive nerve fibers.In vitro experiments, conducted with striatal synaptosomal and membrane preparations, showed that ibotenic acid differed from kainic acid by being devoid of a significant inhibitory effect on high affinity glutamate uptake and by having a low affinity for 3H-kainic acid binding sites. Furthermore, ibotenic acid did not interfere with the binding of a number of radioligands for other transmitter receptors.As compared to kainic acid, ibotenic acid has the advantage of being less toxic to the animals and of producing more discrete lesions, possibly due to faster metabolism and/or other fundamental biochemical differences. Because of these special features, ibotenic acid seems to represent a valuable new tool in the morphological and functional analysis of central neuronal systems.

Effect of low- and high-frequency TENS on Met-enkephalin-Arg-Phe and dynorphin A immunoreactivity in human lumbar CSF

&NA; Transcutaneous nerve stimulation (TENS) treatment was given for 30 min to 37 patients divided into 3 groups of 10 patients and 1 group of 7 patients. Two groups received low‐frequency (2 Hz) and the other 2 groups high‐frequency (100 Hz) stimulation. A diagnostic lumbar cerebrospinal fluid (CSF) sample was obtained immediately before and after stimulation. The CSF samples were subjected to analysis of immunoreactive (ir) opioid peptides, Met‐enkephalin‐Arg‐Phe (MEAP) from preproenkephalin and dynorphin A (Dyn A) from preprodynorphin, respectively. Low frequency TENS applied on the hand and the leg resulted in a marked increase (367%, P < 0.05) of ir‐MEAP but not ir‐Dyn A, whereas high‐frequency (100 Hz) TENS produced a 49% increase in ir‐Dyn A (P < 0.01) but not ir‐MEAP. This is the first report in humans that 2 Hz and 100 Hz peripheral stimulation induces differential release of peptides from preproenkephalin and preprodynorphin, respectively.

Enkephalin immunoreactive nerve fibres and cell bodies in sympathetic ganglia of the guinea-pig and rat.

The occurrence and distribution of enkephalin-like immunoreactivity was studied by light microscopy, using an indirect fluorescent-labelled antibody technique, in the superior cervical ganglion, the inferior mesenteric ganglion and the coeliac-superior mesenteric ganglion complex of the guinea-pig and rat. In theguinea-pig a very dense network of enkephalin-positive fibres was observed in the inferior mesenteric ganglion and a less dense one in the coeliac-superior mesenteric ganglion complex. In both ganglia some ‘small intensely fluorescent’ cells were immunoreactive. In the superior cervical ganglion only few fluorescent fibres were seen but several ‘small intensely fluorescent’ cells were enkephalin-positive. In therat the inferior and coeliac-superior mesenteric ganglia contained medium-dense networks of enkephalin-positive fibres. An irregularly distributed network of fluorescent fibres was observed in the superior cervical ganglion, where also several principal ganglion cells were enkephalinimmunoreactive, particularly after colchicine treatment. These findings indicate the presence of several peripheral neuron systems containing enkephalin or a similar peptide. Several antisera raised to methionine- and leucine-enkephalin as well as to α- and β-endorphin were used. Some of these antisera were compared by incubating sections of the inferior mesenteric ganglion with increasing dilutions of antiserum as well as with antisera treated with increasing concentrations of methionine- and leucine-enkephalin, respectively. On the basis of these findings the problem of differentiating between methionine- and leucine-enkephalin is discussed.

A molecular model of Alzheimer amyloid beta-peptide fibril formation.

Polymerization of the amyloid beta (Aβ) peptide into protease-resistant fibrils is a significant step in the pathogenesis of Alzheimer’s disease. It has not been possible to obtain detailed structural information about this process with conventional techniques because the peptide has limited solubility and does not form crystals. In this work, we present experimental results leading to a molecular level model for fibril formation. Systematically selected Aβ-fragments containing the Aβ16–20sequence, previously shown essential for Aβ-Aβ binding, were incubated in a physiological buffer. Electron microscopy revealed that the shortest fibril-forming sequence was Aβ14–23. Substitutions in this decapeptide impaired fibril formation and deletion of the decapeptide from Aβ1–42 inhibited fibril formation completely. All studied peptides that formed fibrils also formed stable dimers and/or tetramers. Molecular modeling of Aβ14–23 oligomers in an antiparallel β-sheet conformation displayed favorable hydrophobic interactions stabilized by salt bridges between all charged residues. We propose that this decapeptide sequence forms the core of Aβ-fibrils, with the hydrophobic C terminus folding over this core. The identification of this fundamental sequence and the implied molecular model could facilitate the design of potential inhibitors of amyloidogenesis.

Nociceptin/Orphanin FQ Microinjected into Hippocampus Impairs Spatial Learning in Rats

The newly discovered peptide nociceptin/orphanin FQ has been found to increase reactivity to pain and to influence locomotor activity after intracerebroventricular administration. This study investigated the possible role of hippocampal nociceptin/orphanin FQ in spatial learning and in spontaneous locomotion. Male rats were trained in the Morris water task after microinjection of 10 nmol nociceptin/orphanin FQ or artificial cerebrospinal fluid (as control) into the CA3 region of the dorsal hippocampus. Nociceptin/orphanin FQ was found to severely impair spatial learning without interfering with swimming performance. Itrahippocampal injection of nociceptin/orphanin FQ markedly decreased exploratory locomotor activity including vertical movements (rearing). The data suggest that nociceptin/orphanin FQ is a potent modulator of synaptic plasticity within the hippocampus.

Dynorphin-immunoreactive neurons in the central nervous system of the rat

Abstract An antiserum specific for the C-terminal region of dynorphin 1–17 (DYN) was used to examine the distribution of this endogenous opioid peptide in the rat brain with the indirect immunofluorescence technique. DYN-positive nerve cell bodies and fibers were found in many nuclei in the spinal cord, medulla, mesencephalon, hypothalamus and forebrain. These findings indicate that a widespread system of DYN neurons is present in the brain distinct from the previously described enkephalin and endorphin systems.