Charlotte Sachs

Degeneration of adrenergic nerves produced by 6-hydroxydopamine

Abstract 6-Hydroxydopamine (6-OH-DA) has been shown to deplete adrenergic nerves very rapidly of endogenous noradrenaline (NA). Its action is probably more rapid than any other known drug. The depletion is more pronounced in the terminals than in the main axons and nerve cell bodies. The iris was found to be a very sensitive organ, while the vas deferens was less sensitive. The depletion may be due partially or solely to destruction of the nerves by 6-OH-DA or some metabolite, taken up by the axon membrane pump into the adrenergic nerves. The destruction of the nerves would thus make the nerves unable to retain endogenous NA. At the same time the nerves cannot take up and store α-methyl-NA. This inhibition of uptake is not due to direct interference by 6-OH-DA with the uptake mechanisms of the cell membrane, but the inhibition of uptake occurs simultaneously with the disappearance of endogenous NA. Therefore, our data indicate a degenerative destruction of the sympathetic nerves, and suggest that 6-OH-DA might be used in achieving a chemical sympathectomy.

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

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.

CHANGES IN THE DEVELOPMENT OF CENTRAL NOR ADRENALINE NEURONES FOLLOWING NEONATAL ADMINISTRATION OF 6-HYDROXYDOPAMINE1

Abstract— The effects of the neurotoxic compound 6‐hydroxydopamine on central noradrenaline (NA) neurones have been investigated in the adult rat after systemic administration of the drug at birth. This treatment produced a permanent and selective reduction in endogenous noradrenaline, [3H]noradrenaline uptake in vitro and the number of histochemically demonstrable noradrenaline nerve terminals in the forebrain, certainly related to neuroneal degeneration. The fluorescence morphology of the noradrenaline perikarya in the locus coeruleus was not notably affected. In the pons‐medulla region, the 6‐hydroxydopamine treatment led to an almost two‐fold increase in endogenous noradrenaline with a similar increase in [3H]noradrenaline uptake and formation of 3H‐catecholamines from [3H]tyrosine. Fluorescence histochemistry revealed an increased number of noradrenaline nerve terminals which in addition showed an increased fluorescence intensity. Subcellular distribution studies of endogenous noradrenaline in pons—medulla disclosed the highest relative noradrenaline increase in the microsomal fraction after 6‐hydroxydopamine at birth. Sucrose gradient centrifugations disclosed that the pons‐medulla synaptosomes from 6‐OH‐DA treated rats sedimented at a higher sucrose concentration than those from untreated controls. It is concluded that treatment of neonate rats with 6‐hydroxydopamine produces a selective degeneration of noradrenaline nerve terminals in the forebrain, especially in the cerebral cortex, whereas in the pons‐medulla this treatment leads to an increased intraneuronal noradrenaline concentration due to accumulation of noradrenaline in collateral systems not affected by 6‐hydroxydopamine and probably also to an increased outgrowth of noradrenaline nerve terminals.

DEGENERATION OF CENTRAL AND PERIPHERAL NORADRENALINE NEURONS PRODUCED BY 6‐HYDROXY‐DOPA

—The effects of systemically administered 2,4,5‐trihydroxyphenylalanine (6‐OH‐DOPA) on endogenous noradrenaline, [3H]amine uptake and fluorescence morphology has been investigated in mouse brain, heart and iris. 6‐OH‐DOPA in a dose of 100 mg/kg intraperitoneally caused practically no changes in these parameters. Pretreatment with a potent monoamine oxidase inhibitor (nialamide) led to a pronounced long‐lasting 6‐OH‐DOPA induced reduction in endogenous noradrenaline, [3H]amine uptake and nerve density of noradrenaline nerve terminals both in the central and peripheral nervous system. Histochemically accumulations of noradrenaline were observed in non‐terminal axons. These results strongly support the view that 6‐OH‐DOPA can produce degeneration of both central and peripheral noradrenaline neurons. The degeneration is mediated by decarboxylation of 6‐OH‐DOPA to 6‐OH‐DA, since the effects could be abolished by decarboxylase inhibition. The effect of 6‐OH‐DOPA was selective on noradrenaline neurons in the brain, since neither 5‐hydroxytryptamine nor dopamine neurons were affected, opening up new possibilities for studies on central noradrenaline transmitter mechanisms. In the brain there were pronounced accumulations of noradrenaline in the ascending noradrenaline axons making 6‐OH‐DOPA a powerful tool in the mapping of central noradrenaline pathways.

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.

DEVELOPMENT OF THE BLOOD‐BRAIN BARRIER FOR 6‐HYDROXYDOPAMINE

The postnatal development of the blood‐brain barrier for the neurotoxic action of 6‐hydroxydopamine on central noradrenaline neurons has been investigated by recording the in vitro uptake of [3H]noradrenaline in slices from cerebral cortex, hypothalamus and spinal cord in rats treated with large doses of 6‐hydroxydopamine at different ages. The [3H]noradranaline uptake was permanently and markedly reduced in all regions when the animals were treated at birth, certainly related to degeneration of noradrenaline neurons, caused by 6‐OH‐DA. In the cerebral cortex and hypothalamus an efficient protection against the effects of 6‐OH‐DA on [3H]noradrenaline uptake developed postnatally, while in the spinal cord this protection was never seen to become complete. The results obtained indicate a rapid formation of a blood‐brain barrier for 6‐OH‐DA in the cerebral cortex between the 7th and 9th day after birth. In the hypothalamus the development of this barrier seemed to have a more gradual time‐course, but appeared to be fully developed already at day 5 postnatally. Also in the spinal cord the barrier developed more gradually from birth to the adult age. It was observed, however, that both in the cerebral cortex and in the spinal cord, the blood‐brain barrier developed, could not completely protect the central noradrenaline neurons from the neurotoxic actions of large doses of 6‐OH‐DA administered systemically to adult rats. Furthermore, the results obtained support the view that 6‐OH‐DA does not seem to apparently affect the outgrowth of remaining NA neurons which have not been destroyed by the 6‐OH‐DA treatment.

Autonomic cardiovascular responses in parkinsonism: effect of levodopa with dopa-decarboxylase inhibition

ABSTRACT – Autonomically mediated cardiovascular responses to certain manoeuvres were studied in 20 parkinson patients, 24 h off levodopa‐decarboxylase inhibitor medication and again one h after medication. Results were compared with 15 healthy control subjects. The heart rate at rest was higher in parkinson, the respiratory sinus arrhythmia was lower, while the Valsalva ratio, the heart rate and blood pressure responses during an orthostatic test and the heart rate response to a dive reflex test were normal. These findings indicate a normal function of peripheral autonomic nerves and a possible central parasympathetic dysfunction.

Mapping of central noradrenaline pathways with 6-Hydroxy-DOPA

Abstract 6-Hydroxy-DOPA (6-OH-DOPA) was used as a tool to map out central noradrenaline pathways in the mouse with the histochemical fluorescence method of Falck and Hillarp. After inhibition of the monoamine oxidase, 6-OH-DOPA treatment at a dose of 100 mg/kg, results in the appearance of strongly fluorescent, longish accumulations of endogenous noradrenaline within the nerve fibers, while the dopamine and 5-hydroxytryptamine neurons were unaffected. However, this specificity is not absolute, since a higher dose, 400 mg/kg of 6-OH-DOPA also affected the nigro-striatal DA neurons. Mainly the noradrenaline fibers from the locus coeruleus area were demonstrated and could be traced into the cortical areas of the telencephalon and cerebellum, the geniculate bodies, the thalamus, the lateral hypothalamic area and the rhombencephalon. The ventral noradrenaline bundle was not markedly affected by 6-OH-DOPA. The 6-OH-DOPA technique makes it possible to map out many noradrenaline pathways without disturbing their topographical pattern and also to study them in areas containing dopamine fibers.

Uptake and accumulation of 3H-6-hydroxydopamine in adrenergic nerves☆

The uptake and accumulation of radioactivity in mouse atrium in vitro in a medium containing 3H-6-hydroxydopamine (6-OH-DA) has been investigated. Sympathetic denervation resulted in a marked decrease of uptake of radioactivity. Reserpine also caused a considerable reduction in the accumulation of radioactivity, which could be counteracted by the monoamine oxidase (MAO) inhibitor, nialamide; MAO inhibition alone resulted in an increased accumulation of radioactivity. 6-OH-DA acted as a competitive inhibitor of 3H-noradrenaline (NA) uptake in atria, although the affinity of 6-OH-DA for the uptake sites was much lower than of NA and related amines (dopamine, 5-hydroxydopamine and metaraminol). he results obtained support the view that the adrenergic nerves are able to take up and accumulate 6-OH-DA by the membrane pump mechanism and that 6-OH-DA behaves in many respects as a catecholamine. Quantitation of the adrenergic nerve terminals demonstrated by fluorescence histochemistry showed mouse iris to contain about 2 × 105 varicosities. The endogenous NA content per varicosity was calculated to be about 7 × 10−3 pg, corresponding to an average intraneuronal NA concentration of 4000–14,000 μg/g. The intraneuronal concentration of 6-OH-DA (derivatives included) after in vitro incubation in 10−4 M 3H-6-OH-DA (30 min) was found to be of the same order of magnitude as the normal endogenous NA concentration. On the basis of the present findings and results presented elsewhere, it is concluded that the critical intraneuronal concentration of 6-OH-DA needed to produce degeneration of the adrenergic nerves is about 10,000 μg/g.