Ultrastructural cytochemistry of biogenic amines in nervous tissue: methodologic improvements.

A new fixation method has been developed for localizing biogenic amines in nervous tissue. The method is a modification of the chromaffin reaction in which all fixation steps are buffered with mixtures of sodium chromate and potassium dichromate. In this way the fixation and cytochemical reaction are carried out almost simultaneously. Using the rat vas deferens as a model tissue, it was found that the preservation of electron dense (chromaffin) cores in the vesicles of adrenergic nerve terminals depended on several factors: a short primary fixation using low concentrations of aldehydes, the presence of the chromate/dichromate buffer during all fixation steps and, finally, a long incubation period in a slightly acidic (pH 6.0) solution of this buffer before postfixation in osmium tetroxide. Using this method it was possible to identify not only small and large dense-cored vesicles as storage sites for amines but also a tubular reticulum (neuronal endoplasmic reticulum), the latter especially in nerve terminals of mesenteric arteries and iris. Biogenic amines were also visualized in sympathetic ganglion cells and in the central nervous system e.g., supraependymal nerve terminals, tissues that up to now proved the most difficult in terms of amine localization. In all the tissues examined the cytochemical reaction was highly selective and present in well preserved tissue, which is a significant advance over previously available techniques. It therefore offers new opportunities for further studies on the role of biogenic amines as neurotransmitters.

Lipid composition of membranes of amine-storage organelles.

1. The lipid composition of the membranes from isolated 5-hydroxytryptamine-storage organelles of blood platelets of rabbits and of those from chromaffin granules of bovine adrenal medulla was compared. 2. In contrast with the membranes of the chromaffin granules, those of the 5-hydroxytryptamine organelles did not contain lysophosphatidylcholine (lysolecithin). 3. Both the cholesterol/phospholipid ratio and the relative proportions of phosphatidylethanolamine (kephalin), phosphatidylinositol and phosphatidylserine were about the same in both membranes, whereas phosphatidylcholine (lecithin) and sphingomyelin showed somewhat higher values in the membranes of the 5-hydroxytryptamine organelles. 4. In conclusion, the release of 5-hydroxytryptamine from blood platelets is probably not correlated with the presence of lysophosphatidylcholine in the membranes of the storage organelles and may thus differ from the mechanism of catecholamine release in adrenal medulla.

Storage of ATP and 5-hydroxytryptamine in blood platelets of guinea-pigs.

1. By density gradient centrifugation of homogenized blood platelets of guinea-pigs, subcellular organelles have been isolated which exhibit much higher concentrations (mumole/mug protein) of ATP than the whole platelets.2. The organelles also store endogenous 5-HT whose concentration is, however, considerably inferior to that of ATP.3. Investigated by electron microscopy most of the isolated organelles look empty and only very few show dense osmiophilic cores indicating the presence of 5-HT. Accordingly the occurrence of dense osmiophilic organelles in the whole platelets is also very rare.4. Exogenous 5-HT taken up by the platelets markedly accumulates in the subcellular fraction containing the organelles storing ATP. Concomitantly, the platelets show a rise in the number of osmiophilic organelles and the frequency of osmiophilic cores in the isolated organelles is increased.5. From these and previous in vitro experiments it is concluded that the so called 5-HT storage organelles of blood platelets primarily store ATP and only subsequently accumulate 5-HT.