Isolation and thin-layer chromatographic identification of several peptides with NH2-terminal tryptophan and their histochemical demonstration in the ACTH cells of the rat hypophysis

Summary Methods for the isolation and thin-layer chromatographic identification of amino-terminal tryptophyl-peptides presumably responsible for histochemical tryptophyl-peptide reactions in the ACTH cells of the rat hypophysis are described. In the hypophyseal extract several tryptophylpeptide bands — depending on the homogenization solution — were demonstrated on thin-layer chromatograms. Tryptophyl-peptides were demonstrated from their fluorescence induced 1) with glyoxylic acid (glyoxylic acid introduced into the homogenization solution), 2) by exposure of the chromatographic plates to combined formaldehyde and chloral vapour or 3) by exposure to combined formaldehyde and acetyl chloride vapour. A positive PAS reaction was demonstrated in some tryptophyl-peptide bands. Thus, some tryptophyl-peptides seem to contribute to the observed PAS positivity of the ACTH cells.This investigation was supported by grants from the Jalmari and Rauha Ahokas Foundation and the J.K. Paasikivi Foundation     

Quantitative fluorescence histochemistry of combined formaldehyde-chloral-induced fluorescence of amino-terminal tryptophyl-peptide in model experiments and in the pars intermedia of the rat hypophysis

The relationship between the intensity of combined formaldehyde-chloral vapour-induced fluorescence and the concentration of amino-terminal tryptophyl-peptide in model experiments was found to be non-linear. At a certain concentration the intensity began to increase more slowly than the concentration, and when the concentration further increased the intensity even began to decrease. Based on the studies previously reported and on the above findings it seems that fluorescence induced by combined formaldehyde-chloral vapour, glyoxylic acid vapour and possibly also other combined formaldehyde and carbonyl compounds in the hypophyseal cells containing amino-terminal tryptophyl-peptides is quenched in normal conditions due to the high local concentration. Thus, small to moderate changes in the amounts of amino-terminal tryptophyl-peptides cannot be observed by measuring the fluorescence intensity. In tissue experiments the intensity of combined formaldehyde-chloral vapour-induced fluorescence in the rat pars intermedia was measured after reserpine treatment, which decreases the number of hormone storage granules as demonstrated electron microscopically. The fluorescence intensity measurements were combined with an estimation of the amounts of amino-terminal tryptophyl-peptides extracted from hypophyses and separated in thin-layer chromatography, and subsequently demonstrated by combined formaldehyde-chloral vapour and a protein stain (amido black). Reserpine treatment decreased the fluorescence intensity in the pars intermedia and in thin-layer chromatography, and the staining of the fluorescent band with amido black was also decreased. Amino-terminal tryptophyl-peptides appeared to be depleted from the pars intermedia cells together with endorphins and other hormones of the ACTH/MSH cells containing tryptophan.     

Quantitative fluorescence histochemistry of combined formaldehyde-chloral-induced fluorescence of amino-terminal tryptophyl-peptide in model experiments and in the pars intermedia of the rat hypophysis

Summary The relationship between the intensity of combined formaldehydechloral vapour-induced fluorescence and the concentration of amino-terminal tryptophyl-peptide in model experiments was found to be non-linear. At a certain concentration the intensity began to increase more slowly than the concentration, and when the concentration further increased the intensity even began to decrease. Based on the studies previously reported and on the above findings it seems that fluorescence induced by combined formaldehyde-chloral vapour, glyoxylic acid vapour and possibly also other combined formaldehyde and carbonyl compounds in the hypophyseal cells containing amino-terminal tryptophyl-peptides is quenched in normal conditions due to the high local concentration. Thus, small to moderate changes in the amounts of amino-terminal tryptophyl-peptides cannot be observed by measuring the fluorescence intensity. In tissue experiments the intensity of combined formaldehyde-chloral vapour-induced fluorescence in the rat pars intermedia was measured after reserpine treatment, which decreases the number of hormone storage granules as demonstrated electron microscopically. The fluorescence intensity measurements were combined with an estimation of the amounts of amino-terminal tryptophyl-peptides extracted from hypophyses and separated in thin-layer chromatography, and subsequently demonstrated by combined formaldehyde-chloral vapour and a protein stain (amido black). Reserpine treatment decreased the fluorescence intensity in the pars intermedia and in thin-layer chromatography, and the staining of the fluorescent band with amido black was also decreased. Amino-terminal tryptophyl-peptides appeared to be depleted from the pars intermedia cells together with endorphins and other hormones of the ACTH/MSH cells containing tryptophan.This study was supported by grant from J.K. Paasikivi Foundation.     

Reactivity of tissue tryptophyl units in histofluorescence methods using carbonyl compounds

Summary The contribution of tissue tryptophyl residues, with both amino and carboxyl groups linked to the peptide bonds, to visible fluorescence was studied following various histochemical methods. Tryptophan residues of chymotrypsinogen and trypsinogen exhibited visible fluorescence after (1) combined formaldehyde-HCl vapour, (2) combined formaldehyde and acetyl chloride vapour, and (3) glyoxylic acid vapour treatment.     

Histochemical observations on uptake of L-dopa into endocrine cells of the rat pituitary gland during the postnatal development

The uptake of L-dopa into the cells of the adenohypophysis of the rat was studied during the postnatal development and at adult age using the formaldehyde-induced fluorescence method (FIF). The cells taking up L-dopa were classified by Alcian blue-PAS-Orange G staining. The correlation between the cells taking up L-dopa and those containing tryptophyl-peptide was estimated during the postnatal period and in adult rats. The cells containing tryptophyl-peptide were demonstrated using fluorescence induced by treatment with combined formaldehyde and acetyl chloride vapour. The following observations were made: 1) Great majority of the cells taking up L-dopa did not contain tryptophyl-peptide. Thus the accumulation of L-dopa into the cells of pars distalis is not due to accumulation of L-dopa into the cells by the same transport mechanism as the amino acids for tryptophyl-peptide. 2) Of the cells taking up L-dopa in the adult rats 96% were chromophobes, 2.0% acidophilic cells (somatotrophs and cells producing prolactin), 0.9% R-mucoid cells (corticotrophs), and 1.2% S1- and S2-mucoid cells (gonadotrophs and thyrotrophs). At 10 and 25 days' age the relative numbers of the cells taking up L-dopa were about the same. 3) Pretreatment with nialamide caused only a slight increase in the number of the cells taking up L-dopa. The decrease in the number of the cells uptaking L-dopa of the pars distalis, which takes place after 5 weeks' age is thus not caused by the increased MAO-activity. 4) Strongly chromophilic cells did not take up L-dopa. At the light of our results it seems evident that L-dopa is taken up by the chromophobic cells when these differentiate into chromophilic cells. The accumulation of L-dopa may be a sign of an active transport of amino acids into the cells. The accumulation of L-dopa into the chromophobic stellate and follicular cells may reflect their metabolic activity. These cells probably have an important role in the production of the hormones of the pars distalis.     

Simultaneous fluorescence histochemical demonstration of catecholamines and tryptophyl-peptides in endocrine cells.

Simple and efficient fluorescence histochemical methods for the concomitant demonstration of tryptophyl-peptide-containing cells and dopamine-containing cells have been developed in this study. Combined formaldehyde and chloral vapour or solution of 5% glyoxylic acid monohydrate in n-butanol induced concomitantly strong yellow fluorescence in the tryptophyl-peptide-containing cells and moderate green fluorescence in the dopamine-containing cells in the sections of the freeze-dried adenohypophysis.     

Mechanisms of fluorophore formation in the histochemical glyoxylic acid method for monoamines

Summary Glyoxylic acid vapour is a most powerful reagent for the fluorescence histochemical visualization of biogenic monoamines. In the present investigation the mechanisms of fluorophore formation in the glyoxylic acid reaction has been studied in detail for tryptamine in histochemical models and in freeze-dried tissue, utilizing microspectrofluorometric, Chromatographic, and mass spectrometric techniques in combination with isotope measurements.The glyoxylic acid-tryptamine reaction proceeds through an initial Pictet-Spengler type cyclization to 1,2,3,4-tetrahydro--carboline-1-carboxylic acid, followed by two alternative fluorophore forming reactions yielding 3,4-dihydro--carboline, or the 2-carboxymethyl-3,4-dihydro--carbolinium and 2-methyl-3,4-dihydro--carbolinium salts, which are all strongly fluorescent. It is shown that the yield of fluorophores is considerably higher in the glyoxylic acid vapour reaction than in the formaldehyde vapour reaction of the standard Falck-Hillarp method, and that this higher efficiency of glyoxylic acid is due to the most favourable catalysing properties of the carboxylic group of the glyoxylic acid molecule.     

The use of glyoxylic acid for the fluorescence histochemical demonstration of peripheral stores of noradrenaline and 5-hydroxytryptamine in whole mounts.

The reactions of glyoxylic acid with peripheral stores of noradrenaline and 5-hydroxytryptamine to provide a fluorescence histochemical method for their localization have been investigated. Incubation in glyoxylic acid, followed by drying and heating of whole mount preparations gives an intense and well localized reaction. For incubation, a concentration of 2% glyoxylic acid, buffered to pH 7 at room temperature for 30 minutes gives ideal results. The method is equally good if the pH is varied in the range 6 to 9 or if the tissue is stored in the incubation mixture for up to 6 hours. Ideal development of the fluorophore requires an initial excess of moisture in the tissue, that this moisture is driven off during development, and that the tissue is protected from further moistening. A suitable method of achieving these ends is to heat partially dried tissue at 100 degrees C for 4 minutes and then cover it with paraffin oil. 5-hydroxytryptamine can be readily distinguished from noradrenaline because it forms a fluorophore after reaction at pH 3.5, whereas noradrenaline does not. Both amines can be visualized after incubation at neutral pH. Comparison with the formaldehyde vapour technique reveals three main advantages (and no disadvantages) of the glyoxylic acid method: (1) it gives a finer localization with higher fluorescence yield, (2) the glyoxylic acid method is less susceptible to variations in procedure and, (3) it is both simpler and quicker to apply.     

Isolation of formaldehyde and glyoxylic acid during the oxidation of acetate and glycolate by yeast cells in presence of phenylhydrazine

Summary During the oxidation of acetate and glycolate by the respiration of living yeast cells, in presence of phenylhydrazine, it has been possible to isolate glyoxylic acid, formaldehyde and formyl-compounds as intermediate products.     

Demonstration of catecholamines in peripheral adrenergic nerves in stretch preparations with fluorescence induced by aqueous solution of glyoxylic acid.

Fluorescence induced by aqueous solution of glyoxylic acid and formaldehyde-induced fluorescence of catecholamines were compared for the demonstration of peripheral adrenergic nerves in stretch preparations. Glyoxylic acid was better than formaldehyde for the demonstration of the adrenergic nerves. On the other hand, the formaldehyde was better than glyoxylic acid for the demonstration of biogenic amines in cell bodies.     

Immunocytohistochemical characterization of pituitary cells of the bluefin tuna, Thunnus thynnus L

In this paper we report the first complete mapping of the pituitary in a tuna species. The various different adenohypophysis cell types of the bluefin tuna, Thunnus thynnus L. have been identified and located using different antisera against mammalian and piscine hormones and various histochemical techniques: PAS, Alcian Blue pH 2.5 and lectins -ConA and WGA(Neutral and Acidic Glycoproteins); Bromophenol Blue (Proteins) and Tioglycollate-Ferric-Ferricianide-FeIII (-S-S- groups). Prolactin (PRL) and adrenocorticotrophic (ACTH) cells were located in the rostral pars distalis (RPD) of the pituitary, while the proximal pars distalis (PPD) displayed gonadotrophic (GTH), thyrotrophic (TSH), somatotrophic (GH) and also a few PRL cells. Moreover, somatolactin (SL) and melanotrophic (MSH) cells were identified inside the pars intermedia (PI). Interestingly, some SL-immunoreactive fibers were also detected in the neurohypophysis. Some GTH cells were also located on the exterior surface of the PI. Glycoproteins containing mannose (Man) and/or glucose (Glc); N-acetyl-glucosamine (GlcNAc) and/or sialic acid sugar residues, as well as -S-S- groups, were observed in GTH, TSH and SL cells. The Bromophenol Blue technique stained amphiphilic SL, acidophilic GH cells and weakly ACTH cells. GH and ACTH cells were unreactive to PAS, Alcian Blue, Tioglycollate-Ferric-Ferricianide-FeIII and lectin (Con A and WGA) techniques. Finally, PAS reaction was positive in amphiphilic SL cells, which were PbH unreactive, while MSH and ACTH cells were stained with PbH technique.     

Differentiation of two types of endocrine cells which take up amine precursors using their capacity to take up the fluorescent dihydroisoquinoline derivative of dopamine.

A study was made of the accumulation of the strongly fluorescent 2-carboxymethyl-6,7-dihydroxy-3,4-dihydroisoquinolinium compound (2-Carb. Me-DIQ) derived from the condensation reaction of dopamine with glyoxylic acid in endocrine cells possessing the capacity to take up and store biogenic monoamine precursors. Thin-layer chromatographic studies of urine showed that 2-Carb. Me-DIQ was metabolized into two strongly fluorescent metabolites, possessing at least one hydroxyl group in the phenol moiety of the molecule, which were excreted in urine together with the parent compound. Histochemical observations, however, indicated that the tissue fluorescence showing maximal emission at 480 nm was due to 2-Carb. Me-DIQ. Generally, the injection of 2-Carb. Me-DIQ induced a strong fluorescence in those tissue components possessing the extraneuronal uptake mechanism of catecholamines. In the endocrine cells strong fluorescence was seen in the pineal glandular cells and in some cells of the pars distalis of the hypophysis, of which some cells also took up DL-5-HTP, as was seen following formaldehyde vapour treatment. No accumulation of 2-Carb. Me-DIQ was observed in the pancreatic islet cells, the C cells of the thyroid gland or the tracheal enterochromaffin-like cells. These findings lead to the conclusion that biogenic monoamines in the cells of the pars distalis of the hypophysis might use the phenolic moiety of the molecule to bind to some intracellular receptor. Thus, the pars distalis cells may have an intracellular binding mechanism for biogenic monoamines that is different from other endocrine cells showing the uptake and storage of biogenic monoamines. On the other hand, the findings gave further support to the suggestion that in the pancreatic islet cells, the thyroidal C cells and the tracheal enterochromaffin-like cells biogenic monoamines are stored by a mechanism in which the basic, positively charged amino group of biogenic monoamines is bound electrostatically to the anionic, negatively charged carboxyl group of a hormone storage granule. The pars distalis cells and the pineal glandular cells seemed to take up amines and amine derivatives in a similar manner. This suggests that in the pars distalis cells, too, biogenic monoamines have an active metabolism and possibly some regulative role in hormone synthesis and/or secretion.     

Differentiation of two types of endocrine cells which take up amine precursors using their capacity to take up the fluorescent dihydroisoquinoline derivative of dopamine

Summary A study was made of the accumulation of the strongly fluorescent 2-carboxymethyl-6,7-dihydroxy-3,4-dihydroisoquinolinium compound (2-Carb. Me-DIQ) derived from the condensation reaction of dopamine with glyoxylic acid in endocrine cells possessing the capacity to take up and store biogenic monoamine precursors. Thin-layer chromatographic studies of urine showed that 2-Carb. Me-DIQ was metabolized into two strongly fluorescent metabolites, possessing at least one hydroxyl group in the phenol moiety of the molecule, which were excreted in urine together with the parent compound. Histochemical observations, however, indicated that the tissue fluorescence showing maximal emission at 480 nm was due to 2-Carb. Me-DIQ. Generally, the injection of 2-Carb. Me-DIQ induced a strong fluorescence in those tissue components possessing the extraneuronal uptake mechanism of catecholamines. In the endocrine cells strong fluorescence was seen in the pineal glandular cells and in some cells of the pars distalis of the hypophysis, of which some cells also took up DL-5-HTP, as was seen following formaldehyde vapour treatment. No accumulation of 2-Carb. Me-DIQ was observed in the pancreatic islet cells, the C cells of the thyroid gland or the tracheal enterochromaffin-like cells. These findings lead to the conclusion that biogenic monoamines in the cells of the pars distalis of the hypophysis might use the phenolic moiety of the molecule to bind to some intracellular receptor. Thus, the pars distalis cells may have an intracellular binding mechanism for biogenic monoamines that is different from other endocrine cells showing the uptake and storage of biogenic monoamines On the other hand, the findings gave further support to the suggestion that in the pancreatic islet cells, the thyroidal C cells and the tracheal enterochromaffin-like cells biogenic monoamines are stored by a mechanism in which the basic, positively charged amino group of biogenic monoamines is bound electrostatically to the anionic, negatively charged carboxyl group of a hormone storage granule. The pars distalis cells and the pineal glandular cells seemed to take up amines and amine derivatives in a similar manner. This suggests that in the pars distalis cells, too, biogenic monoamines have an active metabolism and possibly some regulative role in hormone synthesis and/or secretion.This work was supported by grants from the Jalmari and Rauha Ahokas Foundation and the J.K. Paasikivi Foundation     

Differentiation of the formaldehyde-induced fluorescent products of noradrenaline and dopamine by microspectrofluorometry

Synopsis The influence of concentration of dopamine and noradrenaline on the spectral characteristics of their formaldehyde-induced fluorophore, together with the influence of duration of reaction with formaldehyde, has been investigated in a model system. No substantial differences between fluorescence spectra of the amine fluorophores were observed. Accordingly, the influence of hydrochloric acid and ammonia vapours on the fluorophores was investigated. A shift to shorter wavelengths in the excitation maximum of each fluorophore was observed after a brief exposure to hydrochloric acid vapour; more prolonged exposure resulted in a pronounced shift back to longer wavelengths in the case of noradrenaline but no substantial change was observed with dopamine. Brief exposure to hydrochloric acid vapour resulted in a substantial increase in the rate of fading of the noradrenaline fluorophore on exposure to exciting radiation. It is suggested that this offers a convenient way of differentiating the amines.     

Mutagenicity of products formed by ozonation of naphthoresorcinol in aqueous solutions

The mutagenicity of products formed by ozonation of naphthoresorcinol in aqueous solution was assayed with Salmonella typhimurium strains TA97, TA98, TA100, TA102 and TA104 in the presence and absence of S9 mix from phenobarbital- and 5,6-benzoflavone-induced rat liver. Ozonated naphthoresorcinol was mutagenic in TA97, TA98, TA100 and TA104 without S9 mix. By the addition of S9 mix, the mutagenic activity of ozonated naphthoresorcinol was markedly suppressed in TA98 and TA100, but became positive in TA102. High-performance liquid chromatography (HPLC) after derivatization to 2,4-dinitrophenylhydrazones demonstrated the formation of glyoxal as an ozonation product of naphthoresorcinol. Ion chromatographic technique also demonstrated the formation of o-phthalic acid, muconic acid, maleic acid, mesoxalic acid, glyoxylic acid and oxalic acid as ozonation products. The mutagenicity assays of these identified products with five Salmonella showed that glyoxal and glyoxylic acid were directly mutagenic; the former in TA100, TA102 and TA104, the latter in TA97, TA100 and TA104. In the presence of S9 mix, glyoxylic acid gave a positive response of mutagenicity for TA102. The experimental evidence supported that glyoxal and glyoxylic acid may contribute to the mutagenicity of ozonated naphthoresorcinol.     

Ultrastructural and Fluorescence Microscopical Characterization of the Intestinal Endocrine Cells in a Cyclostome,Myxine glutinosa

Endocrine cells of so-called basal-granulated-open type in the intestinal epithelium of a cyclostome, the Atlantic hagfish (Myxine glutinosa), are characterized ultrastructurally and fluorescence microscopically. These cells regularly extend from the basal lamina to the gut lumen, ending in an apical process with microvilli and a filamentous surface coat. Fasting results in an accumulation of secretion granules in all cytoplasmic portions, except for the terminal web area. A similarity is recorded between the distribution of secretion granules and the finely granular fluorescamine-induced fluorescence, suggesting that the fluorescence is associated to some component(s) of the secretory granules. Granule release may take place at the base after an adequate stimulus (presence of food?) at the luminal portion of the cells. The formaldehyde condensation technique shows that insulin-containing hagfish islet parenchymal cells, but not intestinal endocrine cells, store dopamine after intestinal supply of the amine precursor. Acidification of formaldehyde vapour-fixed intestinal epithelium induces fluorescence in the granules of zymogen cells but not of endocrine cells, indicating a low concentration of tryptophyl-peptide(s) in the secretory granules of hagfish intestinal endocrine cells.     

Micromethod for the quantitative determination of leucine from biological experimental material using a dansylation technic]

A method is described allowing quantitative determination of leucine from biological material up to 1 - 10(-11) M by means of dansylation of amino acids and their thin-layer chromatographic separation on polyamide microfoils. By selecting [14C]-U-L-leucine as an 'internal' standard, which is added to the tissue during the homogenization procedure, it is possible to correct for data scattering related to variations of the conversion rate or the yield of the dansylation reaction, and to losses in the different steps of thin-layer chromatography that are difficult to standardize.     

Prostaglandin synthesis in rat adrenocortical cells.

The biosynthesis of prostaglandins by isolated rat adrenocortical cells has been studied by determinations of products formed during incubations with labeled arachidonic acid and by radioimmunoassays. Analysis by thin-layer chromatographic separation of silicic acid column fractions indicated that PGE2, PGA2, (B2) and PGF2 alpha were the predominant prostaglandins formed by rat adrenocortical cells. Approximately 75% of the incorporated isotope was associated with the prostaglandins of the PGE pathway [PGE2 + PGA2 (B2)]. This was a consistent finding whether cells were incubated directly with arachidonic acid or with cells prelabeled with the substrate prior to study. ACTH did not affect the uptake or oxidation of [1-14C]-arachidonate, but did significantly increase incorporation of labeled substrate into [14C]prostaglandins. Of the ACTH-induced increase, 92% was accounted for by an increase in prostaglandins of the E pathway. Studies with prelabeled cells indicated that 77% of the prostaglandins synthesized in both control and ACTH-stimulated adrenocortical cells was released into the incubation medium during the 2-hr study. These had the same composition [88% PGE2 + PGA2 (B2)] as did the intracellular prostaglandins. Analysis by radioimmunoassays gave comparable data on the distribution of E- and F-type prostaglandins in control cells and cells incubated with ACTH or dibutyryl cyclic AMP. Thus, with these techniques, 88-92% of the increased prostaglandin synthesis due to ACTH or cyclic AMP was produced by the PGE2 rather than the PGF2 alpha pathway.     

An improved histofluorescence procedure for freeze-dried paraffin-embedded tissue based on combined formaldehyde-glyoxylic acid perfusion with high magnesium content and acid pH.]

A technique is described for highly sensitive and precise visualization of central catecholamine systems in paraffin sections of freeze-dried tissue. The procedure is based on perfusion of the animal with a solution containing formaldehyde and/or glyoxylic acid, in the presence of a very high magnesium content (40 g MgSO4/150 ml solution) and acid pH. The perfused tissue is rapidly frozen, freeze-dried, treated with formaldehyde vapours (at +80 degrees C for 1 h), embedded in parffin in vacuo, and finally sectioned. The present technique has a sensitivity for the dopamine- and noradrenaline-containing systems that is comparable with that of the glyoxylic acid-Vibratome technique, which utilizes fresh, glyoxylic acid-perfused tissue. Thus, the preterminal axon pathways become fluorescent throughout their full extent and the several new terminal systems, discovered with the glyoxylic acid-Vibratome method, are well demonstrable. The method is also highly useful for the study of the cell bodies and their dendritic processes. The catacholamine fibre systems are visualized without any signs of diffusion and with a richness in detail. In animals pretreated with L-tryptophan and MAO-inhibitor the technique is also useful for studies on central indolamine-containing systems.     

New aspects on factors determining the sensitivity of the formaldehyde and glyoxylic acid fluorescence histochemical methods for monoamines

Summary The fluorophore and fluorescence yield from tryptamine and 3-methoxytyramine in histochemical protein models have been compared in the standard formaldehyde reaction, the acid-catalyzed formaldehyde reaction, the formaldehyde-ozone reaction, and the aluminum-formaldehyde reaction. In the standard formaldehyde reaction both the fluorophore and fluorescence yields are low. However, the other reactions give a dramatic increase in fluorescence intensity (18–20 times) from tryptamine and 3-methoxytyramine whereas only minor changes (up to 100% increase) in fluorophore yield are observed. It is concluded that the relative fluorescence intensity of each fluorophore molecule formed in the three modifications of the formaldehyde reaction is much higher than that of the molecules formed in the standard formaldehyde reaction. It has previously been demonstrated that the fluorophores formed from dopamine in the gaseous formaldehyde and glyoxylic acid reactions have a much higher (10 times) relative fluorescence intensity than the synthetic fluorophores. The present experiments show that if the histochemical models are dissolved in buffer after the reaction and new models are made from this solution, the fluorescence intensity of the fluorophores formed in the reaction is drastically reduced and becomes comparable to that of the synthetic ones. The results of this and our previous studies indicate that hitherto unknown fluorescence enhancing mechanisms play a major role for the fluorescence yield, i.e. the sensitivity, in the various formaldehyde and glyoxylic acid methods. One possible explanation to the high relative fluorescence intensity of the fluorophores formed in the histochemical reactions could be an energy transfer between, e.g. the non-fluorescent intermediary reaction products (the tetrahydro derivatives) and the fluorophores (the dihydroisoquinolines and dihydro--carbolines). Such an energy transfer is probably attenuated in the dissolved models, where the distances between and orientations of the various molecules have been changed.Abbreviations DA dopamine - FA formaldehyde - GA glyoxylic acid - 3-MT 3-methoxytyramine - 4-MT 3-hydroxy-4-methoxyphenylethylamine - T tryptamine - DHC dihydro--carbolines - THC tetrahydro--carboline - 2-Carb.Me-DHIQ 2-carboxymethyl-3,4-dihydroisoquinolinium compound - THIQ-1-COOH tetrahydroisoquinoline-1 carboxylic acid