In Vivo Voltammetric Monitoring of Catecholamine Metabolism in the A1 and A2 Regions of the Rat Medulla Oblongata

Catecholaminergic metabolism was estimated in A1 and A2 noradrenergic nuclei of the rat medulla oblongata using differential normal pulse voltammetry combined with electrochemically treated carbon fiber microelectrodes. In both areas an oxidation peak appearing at +50 mV was recorded. Electrochemical data and pharmacological experiments indicated that 3,4-dihydroxyphenylacetic acid (DOPAC) synthesized by noradrenergic neurons was the major contributor to this signal. Indeed, alpha-methyl-p-tyrosine, by inhibiting tyrosine hydroxylase, and pargyline, by inhibiting monoamine oxidase, both totally suppressed the peak appearing at +50 mV in A1 and A2 areas. Conversely, FLA-63, an inhibitor of dopamine-beta-hydroxylase, increased it. Moreover, a local and unilateral injection of catecholaminergic neurotoxin (6-hydroxydopamine) in the vicinity of A1 induced a 60% decrease in the peak height. This effect was prevented by pretreatment with desipramine, an inhibitor of noradrenaline reuptake, which is known to protect noradrenergic neurons against the action of 6-hydroxydopamine. Finally, specific drugs acting on alpha-2-noradrenergic receptors (clonidine and piperoxane) modulated the peak height recorded from both structures. Thus, as previously shown in the locus ceruleus, the variations in the extracellular DOPAC levels reflect the metabolic activity of A1 and A2 noradrenergic neurons.     

A fluorescence histochemical study of the degeneration and regeneration of noradrenergic nerves in the chick following treatment with 6-hydroxydopamine

Summary The fluorescence histochemical method of Falck and Hillarp for the cellular localisation of biogenic amines was used to follow the disappearance and reappearance of peripheral noradrenergic nerves in the chick after treatment with 6-hydroxydopamine. With certain exceptions, it seemed that the disappearance and reappearance of the nerves was due to their degeneration and subsequent regeneration. However, there was not a uniform destruction of the noradrenergic nerves associated with different effector tissues. The extent of degeneration was greater in older than in younger chicks following equivalent doses of the drug, possibly indicating a greater uptake efficiency of the older nerves. It appeared that the noradrenergic nerves in the younger chicks regenerated more rapidly than those of older chicks, but in none of the animals studied were there any obvious morphological modifications of the regenerating nerves.The major part of this work was carried out in the Department of Zoology, Melbourne University, Australia.We are grateful to Professor G. Burnstock for use of laboratory facilities, and to the National Heart Foundation of Australia for financial support.     

Diminished Noradrenergic Stimulation Reduces the Activity of Rolipram-Sensitive, High-Affinity Cyclic AMP Phosphodiesterase in Rat Cerebral Cortex

Abstract: The present study examined the in vivo regulation of rolipram-sensitive, high-affinity cyclic AMP phosphodiesterase (PDE4) in rat cerebral cortex. The hydrolysis of cyclic AMP, formed by stimulation of β-adrenergic receptors, was measured in cerebral cortical slices. Hydrolysis of cyclic AMP formed under these conditions was inhibited by the PDE4-selective inhibitor rolipram but not by selective inhibitors of other PDE families. Intraventricular infusion of 6-hydroxydopamine (6-OHDA; 200 µg) decreased the rate constant of cyclic AMP hydrolysis and increased the cyclic AMP half-life 17 days, but not 1 or 7 days, following the treatment. A reduction in norepinephrine (NE) content occurred first; the NE level was reduced to 42, 24, and 6% of control at 1, 7, and 17 days after 6-OHDA infusion, respectively. This was followed by the development of supersensitivity of β-adrenergic receptor-linked adenylyl cyclase, which occurred 7 days after the infusion. The reduction in PDE4 activity occurred last. When a higher dose of 6-OHDA (300 µg) was used, the reduction in the rate constant of cyclic AMP hydrolysis occurred by 7 days; at this time NE content was depleted to 6% of control. Similar to 6-OHDA treatment, continuous blockade of β-adrenergic receptors, produced by chronic propranolol infusion, decreased the rate constant of cyclic AMP hydrolysis. Therefore, the current results indicate that diminished stimulation of β-adrenergic receptors, either by loss of noradrenergic innervation or by receptor blockade, reduces the activity of PDE4. This suggests that PDE4 regulation may contribute in the homeostasis of the noradrenergic receptor-effector system in the brain.     

Fluorescence histochemical and ultrastructural observations on the effects of intravenous injections of vinblastine on noradrenergic nerves

Summary Fluorescence histochemical and ultrastructural observations were made on noradrenergic nerves in chicks, following intravenous injections of vinblastine. The drug caused a reversible blockade of the axoplasmic transport of catecholamines in non-terminal axons. Some terminal fibres showed a loss of fluorescence 48 hours after injections of animals with 10 mg/kg vinblastine, and ultrastructurally there were signs of neural necrosis.Some of the work described in this paper was carried out in the Department of Zoology, Melbourne University. We are grateful to professor G. Burnstock for use of facilities, and the National Heart Foundation of Australia for financial assistance.     

An autoradiographic study of α1-adrenergic receptors in the brain of the Japanese quail (Coturnix coturnix japonica)

Summary The neuroanatomical distribution of ₁-adrenergic receptors was studied in Japanese quail by quantitative in vitro autoradiography using the specific antagonist [³H]prazosin as the ligand. The presence of saturable (B_max <200 fmol/mg protein) high affinity (K_d < 0.12 nM) binding sites was detected by saturation analysis. High concentrations of [³H]prazosin binding sites were detected in the archistriatum/pars ventralis, the hippocampus, the cortex piriformis, the area corticoidea dorsolateralis, the dorsal thalamus, and the nucleus praetectalis. Lower concentrations were seen in the intercollicular nucleus, the lateral septum, and the posterior and tuberal hypothalamus. Very little binding was seen in the preoptic and anterior hypothalamic areas. The relatively high number of binding sites identified in the telencephalic structures agrees well with previous mammalian studies. This is in contrast with the pattern in the anterior hypothalamus where, in mammals, a number of nuclei have been reported to contain a high receptor density.     

Neurochemical Characterization of the Alterations in the Noradrenergic Afferents to the Cerebellum of Adult Rats Exposed to X-Irradiation at Birth

Abstract: A single dose of x-irradiation was applied on the cephalic end of newborn rats, and the alterations in the noradrenergic afferents to the cerebellum were studied 180 days later. A net increase in the noradrenaline content of cerebellum was found (122% of nonirradiated controls). The response of noradrenaline content to reserpine injection (0.9 mg/kg, i.p.) was similar in exposed and control rats. Likewise, the ³H release induced by Ro 4-1284 from cerebellar cortex slices labeled with [³H]noradrenaline was unmodified by x-rays, although a mild increase in the spontaneous efflux of ³H was found. The retention of ³H by the slices was reduced in exposed animals (58% of controls). Both the in vitro activity of tyrosine hydroxylase and the accumulation of L-3,4-dihydroxyphenylalanine (L-DOPA) were not significantly different between x-treated rats and controls. In contrast, monoamine oxidase activity was markedly reduced in x-irradiated cerebellum (38% of controls). The x-ray-induced decrease in cerebellar weight (—60%) resulted in marked increases in noradrenaline concentration (223%), tyrosine hydroxylase activity per milligram of protein (206%), and ³H retention (50%). The accumulation of L-DOPA per gram of tissue was also increased at every time considered. These data indicate that x-irradiation at birth produces a cerebellar loss not completely shared by the noradrenergic afferents, and a permanent imbalance between the noradrenergic afferent input and its target cells might eventually result. In spite of the enhanced noradrenaline content, the lack of increase in maximal tyrosine hydroxylase activity and ³H retention seems to indicate that a long-term sprouting of the noradrenergic terminals in the cerebellum induced by the ionizing treatment is unlikely.     

Uptake of H3-5-hydroxytryptophan by noradrenergic nerves in the mouse pancreas studied with electron microscopic autoradiography

Summary The uptake and distribution of radioactivity in vascular adrenergic nerves in the mouse pancreas following the injection of tritiated 5-hydroxytryptophan was studied by means of electron microscopic autoradiography. Autoradiographic silver grains were found selectively accumulated over axonal profiles. Quantitative analysis revealed a characteristic intraneuronal distribution of the silver grains, most of which probably represent 5-hydroxytryptamine formed by decarboxylation from the labeled precursor. Thus, the grain density over adrenergic nerve terminals, containing a mixed population of vesicles and granules, was about 5 times higher than the grain density recorded over non-terminal axonal parts and at least 20 times higher than the grain density found over surrounding adventitial tissue and smooth muscle cells. This was interpreted as an evidence that 5-hydroxytryptamine was taken up and stored in adrenergic terminals.     

The innervation of the myometrium of the cynomolgus monkey (Macaca fascicularis)

Summary The autonomic innervation of the myometrium of Macaca fascicularis consists of bundles of unmyelinated nerve fibres running between the smooth muscle cells, and is therefore considered to be of the fascicular (= unitary) type. Close contacts between nerve fibres and smooth muscle fibres were not found. Modification of the chromaffin method according to Tranzer and Richards made it possible to visualize the heterogeneity of the nerve fibres in a single bundle. The following fibre types were found to coexist: (1) noradrenergic fibres containing synaptic vesicles with a dense granule, (2) cholinergic fibres containing empty synaptic vesicles, and (3) non-adrenergic noncholinergic (NANC) fibres containing only or predominantly large dense-cored vesicles, which do not react with this method. Noradrenergic fibres are the most numerous (around 60%), followed by NANC fibres (30%) and cholinergic elements (around 10%). The distribution of these three types is similar in the cervix, the isthmus and the body of the uterus in pregnant and non-pregnant females.     

Effects of Chronic and Subchronic Nicotine on Tyrosine Hydroxylase Activity in Noradrenergic and Dopaminergic Neurones in the Rat Brain

Chronic nicotine (0.8 mg/kg by daily subcutaneous injection) over a 7 to 28-day period was found to increase the activity of tyrosine hydroxylase in predominantly noradrenergically innervated regions but not in dopaminergic projection areas. Increases in tyrosine hydroxylase activity were observed in dopaminergic cell body regions only after nicotine treatment for 3 to 5 days. The increase in tyrosine hydroxylase activity in noradrenergic neurones was evident first in the cell bodies in the locus coeruleus from 3 to 7 days, reaching 223% of control activities, and was followed by increases of up to 205% in the terminals up to 3 weeks later. It was then established that nicotine for 7 days was sufficient to increase the activity of the enzyme to the same extent in the terminals at 21 days even without further nicotine administration. This is consistent with axonal transport preceded by induction of the enzyme in noradrenergic cell bodies, whereas "delayed activation" might account for the transient effect seen in dopaminergic cell body regions. The response in the locus coeruleus to nicotine for 7 days was completely blocked by daily preinjection with mecamylamine but not with hexamethonium, which is consistent with the effect of nicotine on tyrosine hydroxylase being mediated by central nicotinic receptors.     

Differential Interaction of 1-Methyl-4-Phenylpyridinium Ion with the Putatively Vesicular Binding Site of [3H]Tyramine in Dopaminergic and Nondopaminergic Brain Regions

Abstract: The neurotoxin 1-methyl-4-phenylpyridinium ion (MPP⁺) in the brain striatum has recently been shown to bind at a putatively vesicular site labeled by [³H]tyramine ([³H]TY). Whereas in the rat and mouse striatum MPP⁺ antagonized TY binding competitively, in the cerebellum there was a mixed-type antagonism, which suggests the simultaneous occupancy of two different sites. K _i values from displacement curves revealed a fourfold difference in the affinity of MPP⁺ for TY sites in the two brain regions. The degeneration of central noradrenergic terminals induced by an intraperitoneal injection of the toxin N -(2-chloroethyl)- N -ethyl-2-bromobenzylamine in rats decreased by 80% the maximal number of cerebellar TY binding sites, while not affecting striatal binding. Furthermore, guanethidine, a marker for noradrenaline (NA) vesicles, potently inhibited TY binding in NA-innervated regions, such as the cerebellum and the parietal cortex, and poorly in the striatum. It is concluded (a) that both MPP⁺ and TY may also label NA vesicles and (b) that the vesicular carriers for dopamine and NA have different characteristics, which may underlie a regional specificity in the rate of endovesicular sequestration of MPP⁺, with either neurodegenerative or neuroprotective consequences, depending on the brain area involved.