Bengt Falck

FLUORESCENCE OF CATECHOL AMINES AND RELATED COMPOUNDS CONDENSED WITH FORMALDEHYDE

The reaction under mild conditions between formaldehyde and phenylalanine and phenylethylamine derivatives has been studied. When the amines included in a dried protein film were exposed to formaldehyde vapour a very intense green to yellow fluorescence was give only by those that as well as being primary amines also have hydroxyl groups at the 3 and 4 positions (3,4-dihydroxyphenylalanine, dopamine, noradrenaline). The 3-OH group seems to be esssential for the reaction. The catechol amines, which are secondary amines (adrenaline, epinine), gave a much weaker fluorescence that developed more slowly. The results obtained on further examination of the reaction favour the view that the amines primarily condense with formaldehyde to 1,2,3,4-tetrahydroisoquinolines which are involved in a secondary reaction to become highly fluorescent and at the same time insoluble. This secondary reaction may be a binding to protein, and oxidation with the formation of double bonds in the heterocyclic ring, or both.

Autoradiography of some suspected neurotransmitter substances: GABA glycine, glutamic acid, histamine, dopamine, and pl-dopa

The uptake of GABA, glycine, glutamic acid, aspartic, l-DOPA, dopamine and histamine into the rabbit retina in vivo and in vitro was studied autoradiographically. GABA and glycine accumulated in cells that had in the main the position and spread of amacrine cells and also in some ganglion cells. Radioactivity appeared in Mu¨ller cells and retinal pigment cells after exposure of the retina to aspartic or glutamic acid in vivo. After exposure to histamine in vivo, radioactivity appeared in the retinal pigment cells and diffusely in the retina. l-DOPA and dopamine were taken up into cells with the position of the adrenergic retinal neurons. The experiments demonstrate that in central nervous tissue such as the retina, certain classes of cells preferentially accumulate glycine or GABA, presumably into two different sets of cells. For several reasons, it is less probable that ganglion cells operate with either glycine or GABA as neurotransmitter; the results thus suggest that the preferential uptake of either substance into a cell is not necessarily a sign of its being a neurotransmitter in the particular cell. However, reasons are given for presuming GABA to be the neurotransmitter of certain amacrine cells, and the results also suggest that a similar role for glycine is worthy of further consideration.

Identification and terminal distribution of the tubero-hypophyseal monoamine fibre systems in the rat by means of stereotaxic and microspectrofluorimetric techniques.

Summary The monoamine-containing fibres to the median eminence and the neurointermediate lobe have been studied by fluorescence microscopy in combination with microknife lesions. The origin and the course of the fibres were evaluated on the basis of monoamine accumulation proximal to the lesion and monoamine disappearance distal to the lesion. The nature of the monoamines stored in the various fibre systems was characterized with microspectrofluorimetry. Five groups of monoamine-containing axons were seen to enter the median eminence: (a) A large group of dopamine axons which originated in cells of the arcuate nuclei and the ventral parts of the anterior periventricular nuclei; they entered the fibre layer of the median eminence close to the infundibular recess. (b) A large group of noradrenaline axons which originated in areas outside the mediobasal hypothalamus; they entered the anterior median eminence from the lateral side, close to the brain surface, and intermingled with the arcuato-hypophyseal dopamine fibres in the internal layer and the deeper part of the external layer of the median eminence. (c) Two minor groups of catecholamine axons: one reached the median eminence from the anterior side, with an antero-posterior direction, probably mingled with the axons of the supraoptico-hypophyseal tract; the other ran into the arcuate nucleus with a dorso-ventral course along and close to the third ventricle; the fibres could not be traced further. (d) A small group of scattered axons containing an unidentified fluorigenic substance which differed microspectrofluorimetrically from the catecholamines and 5-hydroxytryptamine. Judging from the disappearance of monoamines distal to the lesion and from the chemical and microspectrofluorimetric analyses of the catecholamines in the median eminence and the neuro-intermediate lobe, the following conclusions concerning the terminal ramifications of the catecholamine fibre systems could be drawn: The arcuato-hypophyseal dopamine neurones give rise to one part of the terminals in the zona externa of the median eminence and to most, possibly all, terminals in the zona interna. The tubero-hypophyseal noradrenaline fibres also have their terminals in the zona externa, and they contribute to the catecholamine innervation of the neuro-intermediate lobe. Many studies on the functional role of aminergic mechanisms in the hypothalamic regulation of the pituitary functions have focussed on the presence of dopamine in the median eminence. The present investigation has demonstrated two prominent neurone systems in the hypothalamo-pituitary complex: one containing dopamine, the other containing noradrenaline. Moreover, the results suggest the involvement of a third neurone system, probably containing an indole derivative.

A method for differentiating dopamine from noradrenaline in tissue sections by microspectrofluorometry.

Fluorophores induced from noradrenaline and dopamine in tissue sections by treatment with formaldehyde can be separated by their different behavior upon HCl treatment. The noradrenaline fluorophore converts to a fully aromatic isoquinoline while the dopamine fluorophore remains in the form of a nonquinoidal dihydroisoquinoline. The two fluorophores have readly distinguishable excitation spectra. The procedure has been tested both in model systems and in tissue sections. The microspectrofluorometer used is described.

OBSERVATIONS ON THE APPEARANCE OF NOREPINEPHRINE IN THE SYMPATHETIC NERVOUS SYSTEM OF THE CHICK EMBRYO.

Abstract The appearance of norepinephrine in developing autonomic structures of the chick embryo has been investigated. A highly sensitive and specific method for the cellular localization of certain catecholamines and tryptamines was used in combination with chemical determination of catecholamines. Norepinephrine was found in the sympathetic chain at the very stage when this appears as a well defined organ (embryos incubated 3.5 days). Outgrowing adrenergic nerves may contain norepinephrine from a very early stage of their development. Migrating norepinephrine-containing cells could be followed into the aortic plexus and to the interrenal buds, where they form the adrenal medulla. Up to the hatching stage, all medullary cells seem to store norepinephrine. Some data are given on the appearance of adrenergic nerves in the pancreas, duodenum, rectum, and heart. DOPA decarboxylase activity appeared shortly before norepinephrine could be histochemically demonstrated.

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

SummaryA 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°C for 1h), embedded in paraffin 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.

Classification of monoamine neurones in the rat mesencephalon: Distribution of a new monoamine neurone system

Abstract The large monoamine-containing neurone systems in the rat mesencephalon have been characterized and classified by means of microspectrofluorometry in combination with various drug treatments. Previous fluorescence histochemical studies have demonstrated gree fluorescent and yellow fluorescent neurones in this region. The identity of the green fluorescent neurons as catecholamine-containing was confirmed in the present study. The yellow fluorescent neurones were found to be of two distinctly different types. One (the so-called C-type) was identified as 5-hydroxytryptamine (5-HT)-containing. This type had the characteristic 5-HT spectra with the excitation/emission maxima at 315 and 380–390/520–535 nm and showed a rapid photodecomposition. The fluorescence was abolished or strongly reduced by treatment with reserpine or the tryptophan-5-hydroxylase inhibitor, p -chlorophenylalanine, and was markedly increased after treatment with the MAO inhibitor, nialamide. The second yellow fluorescent neurone type (the so-called B-type) had characteristics clearly deviating from those of the 5-HT neurones: the excitation maxima were at 315, 370–380 and 420 nm, the emission maximum was at 495–510 nm, and the photodecomposition rate was rather slow. This B-type of yellow fluorophore was abolished or strongly reduced after the reserpine treatment but was not affected by the treatments with p -chloropheylalanine or nialamide. The B-type cell bodies were found intermingled with the 5-HT cell bodies in the raphe nuclei, most of the B-type cell bodies being localized in the nucleus linearis caudalis and the nucleus medianus raphe (B8) and scattered in a wing-like structure situated lateral to this latter nucleus. The B-type and the 5-HT type of fibres which could, at least partly, be traced back to the raphe nuclei were partially separated in the ventromedial tegmentum. Anteriorly, they ran into the medial forebrain bundle where they intermingled. It is concluded that the p -chlorophenylalanine-resistant B-type neurone is a morphologically and histochemically well-defined and separate entity. The possibility is discussed that a hitherto unknown indolamine is found in the B-type neurone.

On the presence of adrenergic nerves in the pars intermedia of the frog, Rana temporaria

Abstract By means of a highly specific and sensitive fluorescence method, a plexus of catecholamine-containing nerve terminals has been demonstrated in the pars intermedia of the frog, Rana temporaria L. This plexus, which synaptically encloses the intermedia cells, possesses the basic characteristics of an adrenergic ground-plexus. There is support for the hypothesis that these adrenergic fibers mediate the inhibitory control by the brain of the pars intermedia.

Possible axo-axonal synapses between peripheral adrenergic and cholinergic nerve terminals

SummaryThe relations between adrenergic and cholinergic terminals were studied in rat iris and rat heart with the electron microscope. Adrenergic terminals were identified by treating the animals with 5-hydroxydopamine, which produces dense-cored synaptic vesicles in adrenergic terminals in tissues fixed in glutaraldehyde and osmium. The specificity of this observation was verified. It was found that adrenergic and cholinergic nerve terminals often come in close contact with one another, the distance between the adjoining membranes being about 250 Å. At times, faint membrane thickenings could be observed in these places. The available pharmacological, physiological, and morphological evidence leaves little room for doubt that cholinergic terminal fibres can influence the adrenergic fibres. From mainly morphological evidence, it is also postulated that adrenergic terminals influence cholinergic ones.

Neuronal localization of dopamine and 5-Hydroxytryptamine in some mollusca

SummaryThe localization of biogenic monoamines in ganglionic tissues from Anodonta piscinalis, Helix pomatia, and Buccinum undatum has been studied by means of the histochemical fluorescence method of Falck and Hillarp.In cerebral, visceral, and pedal ganglia (besides nonfluorescent nerve cells) neurons emitting a green or yellow fluorescence were found. No other cell systems exhibiting a specific fluorescence were observed. An abundance of monoaminergic terminals were found in the central parts of these ganglia. Spectrophotofluorimetric determinations showed that there are large quantities of dopamine and 5-hydroxytryptamine in the tissues investigated. The amounts of dopamine and 5-hydroxytryptamine agree well with the distribution of green and yellow fluorescence, respectively, in the ganglia.There are many similarities between the vertebrate and the molluscan monoaminergic neurons. The morphology of the neurons is the same, the intraneuronal distribution of the monoamines is identical, depletion experiments with reserpine and denervation experiments give the same results, and the synaptic arrangement of monoaminergic fibres on non-adrenergic neurons has the same appearance. Apparently, however, dopamine and 5-hydroxytryptamine are the only monoamines acting as neuronal transmitters in the species investigated.