Studies on reaction and binding of monoamines after fixation and processing for electron microscopy with special reference to fixation with potassium permanganate

Summary In the present paper certain properties of potassium permanganate (KMnO₄), a fixative used for electron microscopical investigations, have been studied in model test tube experiments and on tissues. Evidence was obtained that KMnO₄ reacts with different types of biogenic monoamines resulting in a formation of a precipitate. In addition, also various monoamine analogues, precursors and metabolites reacts with KMnO₄. The reaction taking place may be an oxidation-reduction-reaction in which KMnO₄ is reduced, probably mainly to manganese dioxide by hydroxyl groups of the amines and related compounds. This is corroborated by the fact that no reaction takes place between KMnO₄ and -phenylethylamine or amphetamine, two substances, which lack hydroxyl groups.Using labelled monoamines evidence was obtained that the amine partly is retained within the precipitate formed after the reaction with KMnO₄ and also in tissues fixed with KMnO₄, indicating a possibility to perform autoradiographic studies on KMnO₄ fixed tissue.Electron microscopic studies on tissues fixed under various conditions revealed that fixation with low concentrations (0.6 and 1.0%) of KMnO₄ and at high temperatures (about 20° C) leads to inferior results as to general morphology and as to the visualization of intraneuronal amine stores.Different types of permanganates were tested as fixatives. These results show that fixation with permanganates with monovalent metallic ions (K⁺, Li⁺ and Na⁺) give good results of comparable quality, whereas fixation with zinc permanganate results in seriously destroyed tissues. However, tissue fixed with calcium permanganate reveals very distinct membranes. Furthermore, evidence was obtained that fixation with high concentrations of LiMnO₄ (6 and 9%) and NaMnO₄ (6 and 9%) was more sensitive as to the demonstration of monoamines at the ultrastructural level as compared to 3% KMnO₄. Thus, with e.g. 6 and 9% LiMnO₄ small granular vesicles could be seen in slices from the caudate nucleus after incubation with -methyl-dopamine. This was not possible when using 3% KMnO₄ as a fixative.     

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Release of ( 3 H)noradrenaline and ( 3 H)dopamine from field stimulated cerebral cortex slices. Effect of tyrosine hydroxylase and dopamine- -hydroxylase inhibition

Rat cerebral cortex slices were incubated in vitro with [³H]dopamine (DA) or [³H]noradrenaline (NA) (10^?7M), superfused by fresh buffer and stimulated by an electric field. The stimulation-induced overflow of [³H]DA and [³H]NA was determined. In slices from untreated rats about 16 ng [³H]NA/g tissue was formed from [³H]DA, corresponding to about 5 per cent of the endogenous NA concentration. Stimulation markedly enhanced the overflow of [³H]NA. The [³H]NA newly formed from [³H]DA was overflowing to a greater extent than [³H]NA previously taken up from the incubation medium, indicating a preferential release of newly synthesized transmitter. The stimulation-induced overflow of [³H]DA and [³H]NA was increased in slices of rats pretreated with a tyrosine hydroxylase inhibitor (H44/68). It seems that depletion of the endogenous NA stores of central NA neurons by tyrosine hydroxylase inhibition makes the [³H]cate-cholamines more available for release. Pretreatment of the rats with the DA-β-hydroxylase inhibitors FLA63 or FLA69 considerably diminished the formation of [³H]NA from [³H]DA. Stimulation markedly enhanced the overflow of [³H]DA indicating that DA can act as a ‘false transmitter’ in central NA neurons after DA-β-hydroxylase inhibition.     

Enzyme-linked immunosorbent assay. II. Quantitative assay of protein antigen, immunoglobulin G, by means of enzyme-labelled antigen and antibody-coated tubes

Alkaline phosphatase from calf intestinal mucosa has been conjugated to a protein antigen, rabbit IgG. Such conjugates, prepared by glutardialdehyde, have been used in a competitive solid phase immunoassay. In this test native antigen inhibits the binding of the conjugate to homologous antibodies adsorbed to plastic tubes. Using this assay 1-100 ng/ml of the antigen could be determined.     

Trans‐generational maternal effect: temperature influences egg size of the offspring in Atlantic salmon Salmo salar

Effect of increased temperature during egg maturation on the mass of single eggs produced by the offspring was investigated experimentally in Atlantic salmon Salmo salar. Mass of eggs produced by next‐generation females was larger when their mothers experienced warmer water during the last two months of egg maturation, relative to those that experienced unheated river water. There was no similar trans‐generational paternal effect on offspring egg mass.