Bertil Hamberger

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.

Distribution of noradrenaline nerve terminals in cortical areas of the rat

With the help of the histochemical fluorescence method for the demonstration of catecholamines it has been possible to establish that there exists a diffuse network of very fine, varicose noradrenaline nerve terminals in practically all parts of the cerebral cortex of the rat. The noradrenaline terminals probably mainly make axodendritic contacts preferentially in the terminal branches of the apical shaft, and are usually less frequent in the efferent layers of the cortex. These noradrenaline afferents probably originating from the lower brain stem may influence the activity of cortical areas of diverse functions, e.g. motor and sensory functions.

STANDARDIZATION OF PARAFORMALDEHYDE AND OF CERTAIN PROCEDURES FOR THE HISTOCHEMICAL DEMONSTRATION OF CATECHOLAMINES

Adrenergic mechanisms can now be studied directly at cellular and subcellular levels with the help c)f the sensitive fluorescence method c)f Falck and Hillarp for the histochemica! demonstration of certain catecholamines, e.g. the adrenergic transmitter. Briefly this method inve)lves the treatment of freeze-dried or air-dried tissues with formaldehyde gas derived from paraformaldehyde at 80#{176}C(for details, see Dahlstr#{246}m and Fuxe, Ada Physiol. Scand. 62: Suppl. 232, 1964; Norberg and Hamberger, ibid. 63: Suppl. 238, 1964). During this treatment catecholaniines are converted to intensely fluorescent 3, 4-dihydro isoquinolines (Corrodi and Hil!arp, Helv. Chini. Ada 46: 2425, 1963; 47: 911, 1964). This reaction requires the presence of water, but if too much water is present the amines or their fluorescent products can diffuse. Water for the reaction is derived frem three main sources: the tissue, the air initially enclosed in the reaction vessels, and the paraformaldeiiyde used. It is of great importance to standardize the freeze-drying (or air-drying) in such a way that. the pieces will have a low and fairly constant content of water. The dried pieces readily adsorb water from the air and must be handled in a dry atmosphere. Paraformaldehyde under these condi tions becomes the most important source of water. This water comes partly from pyrolysis of the polymer, but of greater importance is adsorbed water, which varies considerably depending for example on the storage of the paraformaldehyde. The present work reports the principles of a simple procedure for standardization of paraformaldehyde based on the finding that this substance takes up or loses water to constant levels when incubated at room temperature (20-22#{176}C)in an atmosphere of constant relative humidity. Incubations were performed in closed vessels containing aqueous solutions of sulfuric acid of varying density. Any relative humidity between 10 amid 90% can be obtained in this way (Handbook of Chemistry and Physics. 44th ed. Chemical * Supported by USPHS Grant (NB 02854-04), National Institute of Neurological Diseases and Blindness, and a grant from the Swedmsh Medical Research Council. Rubber Pumbhishiing Co., Cleveland, 1963). Amounts of adsorbed water were deterniiuied by using the Karl Fischer reagent (Mitchell and South , 4 quametry, Interscience, New York , 1948). A full account of these experiments will be pubhished in a forthcoming paper (Hamnberger, to l)e

Studies on central and peripheral noradrenaline neurons using a new dopamine-β-hydroxylase inhibitor

Abstract The effects of a new dopamine-β-hydroxylase inhibitor, FLA63, have been studied on central and peripheral noradrenaline (NA) neurons using histochemical and biochemical analysis of monoamines. FLA63 caused a rapid and selective depletion of central NA stores without affecting dopamine (DA) and 5-hydroxytryptamine (5-HT) stores. The histochemical findings indicated that practically all the NA cell bodies in the lower brain stem were localized to the medulla oblongata and pons whereas the DA cell bodies were localized to the mesencephalon. FLA63 induced NA depletion was nerve impulse dependent. NA formation from dopa was markedly blocked by FLA63 treatment. The results with dopa suggest that DA cannot accumulate in high amounts in the NA storage granules. The in vitro studies showed neither NA uptake blocking activity nor any inhibitory effects on monoamine oxidase activity by FLA63. Studies on cold stress and FLA63 indicated that central NA neurons were not of critical importance in thermoregulation.

Drug‐induced changes in the release of [3H]‐noradrenaline from field stimulated rat iris

1 Isolated rat irides were incubated with [3H]‐noradrenaline [3H‐NA] (10−7m), supervised with buffer and then stimulated by an electrical field. The effect of desipramine, clonidine, phentolamine, phenoxybenzamine, GD131, normetanephrine and 4‐tropolone‐acetamide on the stimulation‐induced overflow of [3H]‐NA was tested by adding the drug to the super‐fusing buffer. The effect of pretreatment with phentolamine or phenoxybenzamine on the stimulation‐induced overflow of [3H]‐NA was also studied. 2 The effect of desipramine, clonidine, phentolamine, phenoxybenzamine and GD131 on uptake of [3H]‐NA in isolated irides was determined. 3 Desipramine moderately increased the stimulation‐induced overflow at concentrations which almost completely inhibited neuronal uptake. It was calculated that in the isolated rat iris 30–40% of the released [3H]‐NA is inactivated by reuptake into the nerve terminal. This figure may represent the true reuptake percentage in this preparation. Desipramine‐induced inhibition of [3H]‐NA release from the nerve terminal, possibly via a negative feed‐back mechanism, may also contribute to this low figure. 4 Phentolamine and phenoxybenzamine, in concentrations or doses which did not inhibit neuronal uptake of [3H]‐NA, consistently increased the stimulation‐induced overflow. This increase was further augmented when neuronal uptake was inhibited. 5 The α‐adrenoceptor stimulating drug clonidine decreased the stimulation‐induced overflow. 6 GD131, normetanephrine and 4‐tropolone‐acetamide did not greatly affect the stimulation‐induced overflow of [3H‐NA]. 7 It is concluded that the increased [3H]‐NA overflow obtained after α‐adrenoceptor blockade is due to an increased [3H]‐NA release from the nerve terminals.

ADRENERGIC SYNAPTIC TERMINALS AND NERVE CELLS IN BLADDER GANGLIA OF THE CAT.

Abstract A histochemical investigation of the intramural bladder ganglia of the cat, using a method specific for the adrenergic transmitter, has shown that the ganglia are composed of both adrenergic and non-adrenergic, probably cholinergic, nerve cells. Like certain other autonomic ganglia, the bladder ganglin contain typical adrenergic nerve terminals which form synaptic structures around the non-adrenergic ganglion cells. These systems of synaptic terminals constitute a new mechanism for adrenergic influence on ganglionic transmission.

Dopamine and noradrenaline releasing action of amantadine in the central and peripheral nervous system: A possible mode of action in Parkinson's disease

Abstract Histochemical, biochemical and functional analyses of the effects of amantadine on the monoamine neurons in the peripheral and central nervous system of rat after various pretreatments have shown that amantadine, both in vivo and in vitro, is capable of releasing DA and NA from extragranular stores in central DA and NA neurons and in peripheral NA neurons. The functional findings on rotational behaviour, reflex activity and locomotor behaviour show that the increased release of DA and NA induced by amantadine in the brain also results in increased DA and NA receptor activity. Thus, both from a chemical and functional viewpoint amantadine has an amphetamine-like action. The potency of amantadine, however, is weak compared to that of amphetamine. A direct action on DA receptors can be excluded, since presynaptic stores of CA are required for amantadine to increase CA receptor activity.

Uptake and accumulation of 3H-noradrenaline in adrenergic nerves of rat iris. Effect reserpine, monoamine oxidase and tyrosine hydroxylase inhibition

Abstract Isolated rat irides were incubated in Krebs-Ringer bicarbonate buffer containing 3 H-noradrenaline ( 3 H-NA) and the 3 H-NA taken up was determined. The extracellular space determined with 14 C-sorbitol was about 40% of the iris. The equilibration and clearing of the extracellular space occured within 15 min incubation time. Four pharmacologically different uptake models for NA were used: irides from untreated rats, nialamide pretreated rats, reserpine + nialamide pretreated rats and H44/68 (synthesis inhibitor) pretreated rats. Specific chemical determinations disclosed that very small amounts of metabolites are formed in the iris preparation after an in vitro incubation. It was concluded that there is an efficient uptake and accumulation of NA in adrenergic nerves of rat iris. The membrane pump seemed to be the dominant and most important mechanism for the initial uptake while the granular uptake mechanism was of importance for the total storage capacity and intraneuronal retention. Reserpine did not affect the axonal membrane uptake to any significant degree. After pretreatment with a synthesis inhibitor, H44/68, the uptake and accumulation of 3 H-NA in the iris was only increased 10–20% compared to the untreated iris, indicating that the depleted NA stores were not completely refilled. The extraneuronal uptake was very small provided that the medium concentration of NA was not too high (10 −6 M or lower). This uptake increased considerably in relation to the neuronal uptake when NA concentrations of 10 −5 M of higher were used, although there was no true accumulation of amine in the tissue compared with the medium.

Combined fluorescence histochemistry and 3h-noradrenaline measurements of adrenergic nerves

SummaryA method is described which combines the histochemical fluorescence technique of Falck and Hillarp with isotope measurements in the same pieces of tissue. Tissue pieces incubated in isotope solutions were treated for fluorescence microscopy and examined. They were then removed from the microscopical slides, and the radioactivity determined. It was shown that NA1 content and estimated fluorescence intensity were well correlated. The procedure devised is of special value when isotope measurements are needed of structures which can be safely identified only in the fluorescence microscope, and it has been used for quantitative estimations of adrenergic innervation.

Expanding Role of Fine-needle Aspiration Cytology in Thyroid Diagnosis and Management

In non-iodine-deficient areas, 4% to 7% of the population are reported to have thyroid abnormalities. Prophylactic operations of these nodules in the thyroid are not indicated and not cost-effective, as at least four of five nodules are colloid goiter and only a few are malignant. The need for a reliable preoperative diagnosis is great, and fine-needle aspiration (FNA) is now considered the first choice during workup for thyroid nodules. The steps in the FNA procedure are clinical examination and localization of the target lesion, aspiration, preparation of smears, and collecting material for ancillary microscopy techniques. All these steps must be exercised to allow optimal use of FNA. It can also be combined with various other methods, such as immunohistochemistry of thyroglobulin and calcitonin, analysis of nuclear DNA, DNA preparation for molecular biology analyses, and magnetic resonance spectra. The accuracy of the clinical routine in our unit was evaluated by studying the 5-year outcomes of almost 4000 FNAs of the thyroid. The results were good, with only a few false-negative and false-positive results; but the problem of differentiating follicular adenoma from follicular carcinoma remains a significant problem. It is now well established that FNA biopsy and cytology is the best modality available for the workup of thyroid nodules and is widely utilized in endocrine surgical centers worldwide.