Binding of gold-protein ligand complexes to neurokinin receptors. visualization with the electron microscope on pig coronary artery and rat portal vein.

Active neurokinin (NK) receptors were visualized with 5-nm colloidal gold-protein-substance P (GPSP) and -senktide (GPSenk) complexes on vascular tissues. Electron micrographs of pig coronary strips incubated with GPSP showed gold particles either bound to the plasmalemma or inside intracytoplasmic vesicles of endothelial cells. Preincubation with SP or the NK1 receptor antagonist L-703606 prevented GPSP marking. No gold particles were seen after incubation with GPSenk. On coronary strips in vitro, which had been precontracted with U46619, GPSP induced relaxations similar to those produced by equimolar concentrations of SP, both relaxations being inhibited by L-703606. Analogous to senktide, GPSenk was totally inactive on arterial strips. Incubation of rat portal veins with GPSenk showed gold particles bound to the plasmalemma or inside intracytoplasmic vesicles of smooth muscle cells. Preincubation with senktide or the NK3 receptor antagonist R-820 prevented GPSenk marking. On portal veins in vitro GPSenk induced contractions similar to those induced by equimolar concentrations of senktide or NKB; these effects were inhibited by R-820. Our results show that colloidal gold-protein complexes present biological activity and selectivity similar to those of their respective native ligand and detect the presence of active receptors; in addition, they suggest the presence of NK-receptor-mediated endocytosis in endothelial cells of coronary artery and in smooth muscle cells of the portal vein.

Effect of a dipeptide inhibiting ubiquitin-mediated protein degradation nerve-dependent limb regeneration in the newt.

The dipeptide Leu-Ala, which inhibits ubiquitin-mediated protein degradation, has been shown to act in vitro as an inhibitor of neurite outgrowth of PC12 cells (Hondermarck et al. [1992] Biochem. Biophys. Res. Commun. 189:280). Using agarose beads as vehicles, we tested, in vivo, the effect of this dipeptide (and the inactive inverse, Ala-Leu, as a control) on limb regeneration in the newt (Triturus cristatus), a nerve-dependent developmental process. Leu-Ala inhibited the growth of mid-bud blastemas without altering blastema differentiation, while Ala-Leu had no effect. Cytological observations of dipeptide-treated blastemas using Bodian staining or neurofilament antibodies showed that all the blastema tissues were unmodified except with regard to innervation. Leu-Ala-treated blastemas were devoid of nerve fibers in the epidermal cap, while the mesenchyme distal to the dipeptide impregnated bead exhibited fewer nerve fibers than did Ala-Leu-treated blastemas, which were similar to the control nontreated blastemas. Thus, Leu-Ala, in reducing blastema innervation, inhibits its growth in the same manner as surgical denervation.

Microwave-aided binding of gold-protein-ligand (GPL) complexes. Light microscopic observations in the rat brain.

We describe a rapid method for the preparation and binding site labeling of cryostat sections for use in light microscopy. Instead of using antibodies to bind to specific sites, substance P, delta-sleep-inducing peptide, oxytocin, and dopamine were covalently attached to BSA and then the BSA-ligand complex was adsorbed on 5-nm colloidal gold particles. Bioassays carried out on isolated organs indicated that the physiological activity of the ligand GPL complex was maintained. Most of the technical steps included use of an ordinary microwave oven (MWO), with tissues exposed for less than 1 min in any given step. Cryostat sections of unfixed rat brain were pre-incubated for 50 sec in the MWO in a Tris-buffered solution (pH 7.4) containing 1.5% BSA, then further incubated for 50 sec in the MWO in Tris-buffered solution containing 1% gelatin and the diluted colloidal gold suspension. After washing, the preparations were postfixed for 30 sec in the MWO in 5% formaldehyde solution, pH 7.4. Finally, the cell-bound gold particles were enlarged by a silver-enhancing process and counterstained. Preparations observed at high magnification provided excellent resolution of the cell binding sites. Positive and negative controls performed by addition of BSA-conjugated ligands to the pre-incubation and incubation medium, and displacement of the markers by an excess of unbound ligand in the pre-incubation or the incubation medium, showed the specificity of the tissue labeling.

Cyclic nucleotide fluctuations during newt limb regeneration depend on injury and nerve action.

Impressive cyclic GMP accumulations have been previously observed in stump tissues of amputated newt forelimbs. The aim of the present study was to measure cGMP and cAMP levels in innervated and in denervated stumps, in order to detect a possible nervous influence on the cyclic nucleotide accumulation of stump tissues. Denervation experiments were carried out in amputated newt forelimbs. Tissue sampling was made at several intervals (3, 9, 14 and 20 days) and cyclic nucleotide levels were measured by RIA. Cyclic nucleotide levels of innervated unamputated animals served as controls. Cyclic GMP and cyclic AMP contents of innervated regenerating stumps were compared with cGMP and cAMP contents of contralateral denervated stumps. Results showed clearly that cGMP accumulation was sharply reduced by denervation while cAMP levels were less modified. We suggest that dedifferentiating cells are the main source for the stump accumulated cGMP which increases under nervous control.

[Immunity, neuropeptides and psychiatry]. / Immunité, neuropeptides et psychiatrie.

The author proposes and defends new lines in psychiatric research, aimed not primarily at the study of etiology but of pathogeny. Two systems recognize self from non self: the nervous and the immune systems. These communicate with each other by means of hormones, growth factors, neurotransmitters and neuromodulators. The biochemical family of the neuropeptides therefore plays an important role in the transmission of this information and participates in the complex regulatory mechanisms which insure the physical and psychical equilibrium of the individual. Disorders of such regulation lead to or participate in the psychosomatic diseases and to psychiatric pathology. The author proposes that, in order to elucidate the normal complex regulatory mechanisms, one should first carry out studies on neuropeptides in primitive living organisms and observe the functional capacities of lymphocytes and thymocytes from psychiatric patients.

Mapping of enkephalins in human brain. An immunohistofluorescence study on brains from patients with senile and presenile dementia.

The results of this study of the distribution of enkephalin-like immunoreactivity in four human "senile" and "presenile" brains by immunofluorescence microscopy (Coons' Method) showed specifically fluorescing varicosities containing fibres in the following areas: nucleus accumbens, nucleus caudatus, pallidum (mainly the external segment), septal nuclei, substantia innominata, hypothalamus, hypophysis, substantia nigra, nucleus interpeduncularis, locus coeruleus and other nuclei of the brain stem, most of the nuclei of the cranial nerves (mainly the sensitive) and spinal cord (mainly the substantia gelatinosa of the posterior horn). Fibres were observed surrounding cell bodies in the substantia nigra, in the nucleus raphe and in the anterior horn of the spinal cord (motor cells). Cylindrical ("pipe-shaped") structures formed by enkephalin-like immunoreactive fibres were seen in the pallidum, between the pallidum and the nucleus accumbens, and in the substantia nigra. A complete map of enkephalin-like immunoreactivity, based on Riley's Atlas of the human brain, is included. The distribution of enkephalin-like immunoreactivity showed many similarities to that in animal species that have been studied by immunohistochemistry (rat, primate) except for a lack of detectable enkephalin immunoreactivity in the amygdala in our material. No conclusions about the possible relationship of this finding to the clinical condition of dementia can be drawn without further work.

Distribution of substance P-like immunoreactivity in the brain of the newt (Triturus cristatus).

The distribution of immunoreactive substance P (sP)-containing structures in the newt brain and spinal cord was explored with an indirect immunofluorescence method. Five sP-positive elements were detected: perikarya, dots, fibers, pericellular appositions, and pipe-shaped structures. Perikarya were seen at the levels of the spinal ganglia, spinal cord, raphe nucleus, interpeduncular nucleus, mesencephalon, preoptic area, infundibulum, dorsocaudal part of the ventral hypothalamus, habenula, and corpus striatum. Pericellular terminals were observed in periventricular areas, known to be rich in catecholaminergic cells; pipe-shaped structures were observed from the corpus striatum to diencephalon, and in mesencephalon. The olfactory nerve and nuclei were devoid of sP-positive elements. Six sP-immunofluorescent pathways were detected. One of them is composed of axons with huge varicosities and extends from the lateral spinal cord area to the mesencephalon. This pathway has not been described as yet in other animals and could be peculiar to the newt.

Delta sleep-inducing peptide in the rat brain: an immunohistological microscopic study.

The authors have developed a method which makes it possible, for the first time, to visualize the delta sleep-inducing peptide in histological preparations and study it under the light and fluorescence microscope. Their research builds on Monnier 's discovery, in 1963, of a humoral hypnogenic factor in rabbits which was subsequently isolated and identified as a nonapeptide. Dubbed delta sleep-inducing peptide (DSIP), this factor was later detected in rat brain by radioimmunoassay but has eluded histological visualization until recently. In their work, the authors used an anti-DSIP antiserum suitable for immunohistological purposes. Two indirect immunohistological methods (PAP and immunofluorescence) allowed them to visualize, for the first time, structures containing specific DSIP-like immunoreactivity in some areas of the rat brain: indusium griseum, nucleus septi lateralis, hippocampus, striae longitudinales of Lancisi , bandeletta diagnalis of Broca, pallidum, hypothalamus, hypophysis and neocortex. Some DSIP pathways seem likely: (1) indusium griseum - striae longitudinales - hippocampus; (2) nucleus septi lateralis - striae longitudinales , bandeletta diagonalis - hippocampus; (3) neurons of the pyramidal layer of the hippocampus - gyrus dentatus; (4) pallidum - commissura of Ganser - hypothalamus. The possible correlations between DSIP neurons and neurons with other neurotransmitters are discussed. In preliminary clinical trials, DSIP has shown promise for the treatment of insomnia and the opiate and alcohol withdrawal syndromes.(ABSTRACT TRUNCATED AT 250 WORDS)

[Neuropeptides and psychiatry report presented at the French-Swiss Psychiatric meeting in Bel-Air, Geneva, 4 May 1980]. / Neuropeptides et psychiatrie. (texte du rapport présenté à la réunion Franco-Suisse de Psychiatrie, à Bel-Air, Genève, le 4 mai 1980).

In this mini-review the definition, some localizations and effects of 18 neuropeptides (as known at the beginning of 1980) are recalled, as well as some of the methods used. The hypothesis that neuropeptides may modify both functions and structures is presented. After a brief comment on the neuropeptides/monoamines relations and on some pharmacological results, the possible implications of neuropeptides dysfunctions in various psychiatric disorders are discussed. Some facts leading to the suspicion that both substance P and endorphines are increased in some psychoses are mentioned. The results of therapeutic trials are discussed. The importance of neuropeptides for the maintenance of internal homeostasis and behavioural adjustments is stressed.

Localization of substance P-like immunoreactivity in Hydra.

Substance P-like immunoreactivity was found in Hydra attenuata mainly but not exclusively in the nerve and interstitial cells, localized in the cytoplasm and on the cell surface membranes.

Histofluorescence of monoamines in newt forelimb regenerates.

Newt forelimb regenerates were studied at various stages of development using the histofluorescent method of Falck and Hillarp. A green formaldehyde-induced fluorescence was found in nerve fibres, large dendritic cells, skin gland cells and skin gland cell secretions. To ascertain the nature of the fluorescent material, animals were submitted to treatments with L-dopa, nialamide, benserazide and reserpine, used separately or in combination and administered before cutting off the regenerates. The modifications of the fluorescence after the various treatments confirmed the monoaminic nature of the fluorophores. Catecholaminic fibres were numerous in tissues of fast-growing stages while in dedifferentiated cell areas as well as in prochondral cell condensations and in cartilage they were completely absent. Fluorescent dendritic cells that have never been described before in regenerating limbs were observed and, from their localisation and cytological appearance, classed as promelanophores (or melanoblasts).

Production of retarded, albino, regenerates in newts by alpha-methyl-p-tyrosine.

Inhibition of tyrosine hydroxylase with alpha-methyl-p-tyrosine retarded growth and prevented melanization of limb regenerate in adult newts (Triturus cristatus).