Gösta Jonsson

THE FORMALDEHYDE FLUORESCENCE METHOD FOR THE HISTOCHEMICAL DEMONSTRATION OF BIOGENIC MONOAMINES A REVIEW ON THE METHODOLOGY

The histochemical fluorescence method of Falck and Hillarp for the demonstration of biogenic monoamines is based on the finding that the amines can be condensed with formaldehyde to yield strongly fluorescent compounds, provided that they are enclosed in a dried protein layer, as in freeze-dried or air-dried tissues. This review deals mainly with certain principal features of the method: its chemistry, sensitivity, specificity and possibilities for histochemical differentiation between the various amines. Some comments are made on certain of the results obtained with this method.

Ibotenic acid-induced neuronal degeneration: A morphological and neurochemical study

SummaryPossible neurotoxic actions of intracerebral injections of ibotenic acid, a conformationally restricted analogue of glutamic acid, have been evaluated in rat brain and compared with those of kainic acid.Light microscopical analysis revealed that ibotenic acid produced a marked disappearance of nerve cells in all areas studied, namely striatum, the hippocampal formation, substantia nigra and piriform cortex. Lesions in areas distant to the injection site were not seen. Axons of passage and nerve terminals of extrinsic origin did not seem to be damaged, since, e.g., no apparent degeneration of the dopaminergic terminals in the neostriatum was observed except for a small area surrounding the cannula. In the neostriatum, enkephalin immunoreactive neuronal cell bodies as well as nerve terminals disappeared after injection of ibotenic acid into this nucleus. After injection into the substantia nigra tyrosine hydroxylase immunoreactive cell bodies in the zona compacta disappeared, whereas no certain effect could be seen on the enkephalin immunoreactive nerve fibers.In vitro experiments, conducted with striatal synaptosomal and membrane preparations, showed that ibotenic acid differed from kainic acid by being devoid of a significant inhibitory effect on high affinity glutamate uptake and by having a low affinity for 3H-kainic acid binding sites. Furthermore, ibotenic acid did not interfere with the binding of a number of radioligands for other transmitter receptors.As compared to kainic acid, ibotenic acid has the advantage of being less toxic to the animals and of producing more discrete lesions, possibly due to faster metabolism and/or other fundamental biochemical differences. Because of these special features, ibotenic acid seems to represent a valuable new tool in the morphological and functional analysis of central neuronal systems.

A sensitive method for the determination of plasma catecholamines using liquid chromatography with electrochemical detection

Abstract Simple and sensitive methods for the determination of plasma catecholamines are of great interest since the level of catecholamines in plasma reflects the activity of the sympatho-adrenal system. In the present work a previously described procedure based on high pressure liquid chromatography with electrochemical detection has been adapted for assay of plasma catecholamines. This method permits simultaneous detection of noradrenaline, adrenaline and dopamine in concentrations down to 0.1 nmol/1 in less than one ml plasma.

FLUORESCENCE METHODS FOR THE HISTOCHEMICAL DEMONSTRATION OF MONOAMINES. 3. SODIUM BOROHYDRIDE REDUCTION OF THE FLUORESCENT COMPOUNDS AS A SPECIFICITY TEST

It has been shown in previous papers that catecholamines and 5-hydroxytryptamine can under certain conditions be converted to highly fluorescent 6,7 - dihydroxy - 3,4 - dihydroisoquinolines and 6-hydroxy-3,4-dihydro-β-carboline respectively, and that this can be used as a highly sensitive and specific method for the histochemical demonstration of the monoamines at the cellular level. In the present paper it is shown that the fluorescent compounds are very readily reduced by sodium borohydride to the corresponding, non-fluorescent 1,2,3,4-tetrahydro-compounds—even if they are present in a non-extractable state in dried serum albumin spots or in tissue sections—and that the fluorescence can be regenerate by renewed formaldehyde treatment. The non-specific fluorescence (e. g. autofluorescence) in tissue sections was never observed to undergo any changes on borohydride treatment. On the basis of these findings a very simple histochemical test has been worked out to check directly in the tissue section whether or not an observed fluorescence is due to the presence of the reacting monoamines.

Neurochemical and histochemical characterization of neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine on brain catecholamine neurones in the mouse

Systemic administration of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) caused a rapid and long‐lasting reduction of both 3,4‐dihydroxyphenylalanine (dopamine, DA) and noradrenaline (NA) in mouse brain, as observed histo‐ and neurochemically. The depleting effects were more pronounced after repeated MPTP administration and the most marked reductions were observed after 2 × 50 mg MPTP/kg s.c., when DA in striatum and NA in frontal cortex were reduced by > 90% 1 week after MPTP. Mice with such catecholamine depletions were markedly sedated and almost completely immobilized. The behavioural syndrome after MPTP resembled that seen after reserpine, a monoamine‐depleting drug. MPTP also caused a long‐lasting reduction of catecholamine uptake in striatal DA and cortical NA nerve terminals and reduced tyrosine hydroxylase activity in these regions. There was no evidence that MPTP caused any marked DA and NA cell body death. MPTP given acutely transiently elevated serotonin levels. The results are compatible with a neurotoxic action of MPTP on both DA and NA nerve terminals. The nigro‐striatal DA and the locus coeruleus NA neurone systems appeared to be most susceptible. Synthesis and utilization of residual striatal DA and cortical NA were increased, as often observed in partially denervated monoamine‐innervated brain regions. Both DA and NA showed a gradual recovery, which took months to become complete and may have been related to a regrowth of catecholamine nerve terminals.

The effect of imipramine-like drugs and antihistamine drugs on uptake mechanisms in the central noradrenaline and 5-hydroxytryptamine neurons☆

Abstract The effect of antidepressant drugs of the imipramine type and of the antihistamine drugs chlorpheniramine and brompheniramine on the amine uptake-accumulation mechanism of the central noradrenaline (NA) and 5-hydroxytryptamine (5-HT) neurons were studied in vivo and in vitro in the rat. The in vitro experiments were performed on isolated nerve ending particles following the uptake and accumulation of tritiated NA and 5-HT in the presence of the psychoactive drugs in the incubation medium. The in vivo experiments were analyzed by a fluorescence histochemical method for NA and 5-HT and involved an examination of the effect of the psychoactive drugs on the uptake and accumulation of intraventricularly administered NA or 5-HT and of the accumulation of endogenously synthesized 5-HT aftei monoamine oxidase inhibition in rats pretreated with reserpine. The present results support the view that tertiary amines of the imipramine-type of drugs, such as amitriptyline, imipramine and chlorimipramine, which are well-known and frequently used antidepressant drugs, preferentially block 5-HT uptake in the central 5-HT neurons, whereas secondary amines of the imipramine tppe, such as desipramine, preferentially block NA uptake in the central NA neurons. Functional studies using the extensor and flexor reflex model to evaluate the influence on 5-HT and NA receptor activity, respectively, by the psychoactive drugs further support this view. Thus, the antidepressant action of these drugs may be related to their ability to increase 5-HT receptor activity by blocking 5-HT reuptake. The antihistamine drugs, chlorpheniramine and especially brompheniramine, were found to be very potent blockers of 5-HT uptake as revealed both chemically and functionally. They also had, in contrast to the imipramine-like drugs of the tertiary amine type, a potent blocking action on NA reuptake in the central NA neurons. Drugs with these properties may be of new importance in the management of mental depression.

Evidence for a neurotrophic role of noradrenaline neurons in the postnatal development of rat cerebral cortex

SummaryThe effects of neonatal administration of the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA; 1–4 doses of 100 mg/kg body weight s.c.) on the postnatal development of pyramidal neurons in several cortical regions of the rat was studied using a Golgi-Cox neuronal impregnation technique. Rats were sacrificed in the adult stage (eight weeks) and the following regions were studied: anterior frontal cortex, posterior frontal cortex (including motor cortex), anterior parietal cortex (including sensory cortex), posterior parieto-occipital cortex and cingulate cortex. Significant alterations were seen in animals which received four doses of 6-OHDA. These alterations can be summarized as follows: (1) a decreased length and branching of basolateral dendrites of pyramidal cells, with loss of dendritic spines, which were found in both the internal pyrimidal layer (layer V) and the external pyramidal layer (layer III), most abundantly in the frontal cortex and cingulate cortex; (2) an increased number of pyramidal cells of layer V with premature apical dendritic termination in layer III rather than the usual termination in layers I and II. This was most abundant in the cingulate cortex; (3) occasional disorientation of pyramidal cell apical dendrites away from the normal vertical plane by 15 or more degrees, seen in frontal, parietal and cingulate cortex; (4) an increased number of pyramidal cells with rounded somatic contours, found in frontal, anterior parietal and cingulate cortex. These phenomena were occasionally seen in normal cortex, but were significantly increased in their occurrence after four doses of 6-OHDA. Such alterations were not significant in rats treated with one or three doses of 6-OHDA. The extent and severity of morphological alterations correlate with reductions in endogenous noradrenaline (NA) in cerebral cortex, which was found to average 50% of control levels after one dose of 6-OHDA, an 80% reduction after three doses, and a 97–98% reduction after four doses, suggesting that the NA denervation must be almost complete to result in readily detectable significant morphological changes in the development of cortical pyramidal cells. No consistent changes in endogenous dopamine (DA) levels were observed, except for an increase in the cingulate cortex. The anatomical alterations in pyramidal cells described in the present study suggest that NA neurons which project into the cerebral cortex have a neurotrophic role in the postnatal development of cortex.

Neurotoxicity of the meperidine analogue N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine on brain catecholamine neurons in the mouse

The effect of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (NMPTP) on central monoamine neurons in mice was investigated using histo- and biochemical techniques. NMPTP (2 X 10 mg/kg i.v.) produced a rapid and long-lasting reduction (-30%) of striatal dopamine, while the dopamine levels were only transiently reduced in mesencephalon and frontal cortex. HVA and DOPAC were initially markedly reduced (-50 to -70%) in striatum while a marked recovery was found in the chronic stage. NMPTP also induced a long-term reduction of noradrenaline in striatum and frontal cortex while 5-hydroxytryptamine and 5-HIAA levels were essentially unaltered. The data indicate a neurotoxic action of NMPTP on both dopamine and noradrenaline nerve terminals in mouse brain.

Quantitation of Fluorescence of Biogenic Monoamines: Demonstrated with the Formaldehyde Flourescence Method

The present review deals with the possibilities of using the FALCK-HILLARP fluorescence technique for quantitation of biogenic motzoamines present in tissue cells structures, with special reference to neuronally stored monoamines. Since a well standardized technique is of utmost importance for quantitative work, the basic chemistry and the different factors affecting the histochemical reaction and the fluorescence intensity are thoroughly discussed. The main problem when using the present method for monoamine quantitation is that the local intrancuronal concentration of monoamine is generally so high that a concentration dependent quenching of the fluorescence exists. The situation is furthermore complicated by the fact that the fluorescence yield is affected by the subcellular localization of monoamine; the fluorescence yield being higher when the monoamine is extragranularly distributed. It can be concluded that, if changes in the fluorescence intensity are recorded as compared with control, this reflects true changes in amine concentration in most cases. On the other hand, changes in amine concentration may escape detection when the remaining amine concentration is so high that quenching occurs or when a redistribution of amine and/or fluorophor from the storage granules to the extragranular space has taken place. Therefore, in doubtful cases the fluorescence intensity estimations should be correlated with chemical-analytical determinations of monoamine content in comparable pieces of tissue.

Effects of 6-hydroxydopamine on the uptake and storage of noradrenaline in sympathetic adrenergic neurons

Abstract The time-course of 6-hydroxydopamine induced disappearance of endogenous noradrenaline in mouse heart and iris, its reappearance and changes in uptake-storage properties of the adrenergic nerves for noradrenaline have been investigated using biochemical, isotope and histochemical techniques. There was a very rapid depletion of endogenous noradrenaline. Thus 1 hr after the injection of 6-hydroxydopamine (20 mg/kg i.v.) only 20% of the endogenous noradrenaline was left in mouse heart; after 8 hr, only 6%. Subcellular distribution studies showed that soon after injection 6-hydroxydopamine caused a change in the intraneuronal distribution of previously taken up 3H-noradrenaline and that there was a preferential release from the microsomal fraction, which contains the amine storage granules. The ability of the adrenergic nerves to take up and accumulate exogenously administered noradrenaline was also lost after 1 hr, probably due to severe damage of the uptake-storage mechanisms. Previous electronmicroscopic investigations have revealed that 6-hydroxydopamine causes a selective destruction of the adrenergic nerve terminals. This effect seems to be dependent on a critical concentration of 6-hydroxydopamine acting upon the adrenergic nerves. There was a gradual restoration of the adrenergic ground-plexus and noradrenaline content with parallel recovery of the uptake capacity for noradrenaline, which for heart was complete after about 8 wk. This was probably due to regeneration of the adrenergic nerves. A low dose of 6-hydroxydopamine (1 mg/kg i.v.) resulted in a small temporary drop of the noradrenaline level and also of the noradrenaline uptake capacity. Almost complete recovery of the transmitter content was observed in a few days, whereas the noradrenaline uptake returned to normal values after a few hours. Studies with 3H-6-hydroxydopamine showed that the adrenergic nerves take up this amine, since the uptake was considerably reduced by sympathetic denervation. Desmethylimipramine of 0°C, which efficiently block the ‘membrane pump’ of the adrenergic nerves, also strongly inhibited the uptake. The ability of the microsomal fraction to take up 3H-6-hydroxydopamine was markedly blocked by reserpine, but a granular uptake, at least via the Mg2+-ATP dependent mechanism, did not seem to be a prerequisite for the degeneration effects.