Reduction of dopamine synthesis inhibition by dopamine autoreceptor activation in striatal synaptosomes with in vivo reserpine administration.

In an attempt to clarify the mechanisms by which dopamine (DA) autoreceptor activation inhibits DA synthesis, the efficacy and potency of the D2 DA agonists bromocriptine, lisuride, and pergolide, and the D1-D2 DA agonist apomorphine were studied in rat striatal synaptosomes, in which the rate of DA synthesis (formation of 14CO2 from L-[1-14C]tyrosine) was increased 103% by treating the animals from which the synaptosomes were obtained with reserpine (5 mg/kg i.p. twice, 24 and 2 h before they were killed), using the striatal total homogenate as the standard synaptosomal preparation. The increase in DA synthesis evoked by reserpine was additive with that produced by treatment of the synaptosomes with dibutyryl cyclic AMP, suggesting that, not a cyclic AMP-dependent, but possibly a Ca(2+)-dependent mechanism was involved. The DA agonists showed a concentration-dependent inhibition of DA synthesis in the control synaptosomes, which was antagonized by the selective D2 DA antagonist (-)-sulpiride. In the synaptosomes with increased rate of DA synthesis obtained from the rats treated with reserpine, the concentration-response curves of DA synthesis inhibition for the other DA agonists were shifted to the right, and the effect of bromocriptine was completely eliminated, whereas bromocriptine antagonized the effect of apomorphine. The increased rate of DA synthesis was not preserved in the striatal P1 + P2 fraction obtained from the reserpine-treated rats, but the effects of the DA agonists were still reduced to the same degree as those in the total homogenate. (-)-Sulpiride did not enhance DA synthesis in synaptosomes from the reserpine-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Synaptosomal dopamine autoreceptors: sensitivity changes after in vitro and in vivo treatments.

Dopamine (DA) synthesis in rat striatal synaptosomes was approximately doubled either by treating the animals from which the synaptosomes were obtained with reserpine, or by treating the preparations in vitro with d-amphetamine, ouabain or dibutyryl cyclic AMP. The concentration-response curve of DA synthesis inhibition by apomorphine was shifted to the right after treatment with all these compounds. The inhibitory effect of bromocriptine on DA synthesis was reduced completely after treatment with all the above compounds with the exception of dibutyryl cyclic AMP. When the inhibitory effect of bromocriptine was eliminated by treatment with reserpine or d-amphetamine, bromocriptine antagonized the inhibitory effect of apomorphine. This indicates that bromocriptine could still be bound to the DA autoreceptors and that the reduced sensitivity was due to a reduced functioning of the DA autoreceptors. The reduced sensitivity to apomorphine observed after all the above treatments was possibly due both to a reduced function of and/or to a reduced binding to the DA autoreceptors. The increase in DA synthesis produced by treatment with reserpine in vivo or with d-amphetamine or ouabain in vitro was additive to that produced by a maximally effective concentration of dibutyryl cyclic AMP in vitro, and thus mediated by a presumably non-cyclic AMP-dependent mechanism. Our results obtained with bromocriptine suggest that stimulation of the DA autoreceptors may inhibit DA synthesis by diminishing Ca2+-dependent and not cyclic AMP-dependent phosphorylation of tyrosine hydroxylase.

Autoreceptors mediate the inhibition of dopamine synthesis by bromocriptine and lisuride in rats.

The administration of bromocriptine and lisuride to rats caused a decrease in striatal dopamine (DA) synthesis, as measured by 3,4-dihydroxy-phenylalanine (DOPA) accumulation after decarboxylase inhibition. DOPA formation was inhibited by a maximum of about 60% of control values by bromocriptine and lisuride, 5.0 and 0.3 mg/kg, respectively. Both compounds showed very similar time-courses for the effect and failed to modify DOPA accumulation during the first 30 min. Pretreatment with (-)-sulpiride (50 mg/kg i.p.), a specific D2-receptor blocker, completely prevented the inhibitory effect of bromocriptine and lisuride on DOPA accumulation. Finally, both compounds significantly reversed the gamma-butyrolactone (GBL) (700 mg/kg i.p.)-induced DOPA accumulation at doses (0.25 and 0.015 mg/kg, respectively) that were inactive in normal rats. The data suggest that bromocriptine and lisuride act as agonists on D2-presynaptic autoreceptors which have different sensitivity to the agonist according to the basal firing rate of DA neurons.

Inhibition of dopamine synthesis in striatal synaptosomes by lisuride: stereospecific reversal by (-)-sulpiride.

Lisuride, an ergot D2 dopamine receptor agonist inhibited dopamine synthesis in striatal synaptosomes concentration-dependently. Significant inhibition was detected at 10(-8) M, and the inhibition by 10(-4) M lisuride was 50%. The inhibitory effect of lisuride was reversed by more than 50% not only by the D1-D2 dopamine receptor blocker haloperidol but also by the D2 dopamine receptor blocker(-)-sulpiride. The effect of sulpiride was stereospecific. Under the same test conditions a similar inhibition of dopamine synthesis by apomorphine was reversed by the neuroleptics almost completely. The results suggest that there are dopamine autoreceptors controlling dopamine synthesis in synaptosomes and these receptors resemble D2 dopamine receptors according to the nomenclature of Kebabian and Calne (1979).

Increase of dopamine synthesis in synaptosomes from rats treated with neuroleptics or reserpine.

Dopamine (DA) synthesis in rat striatum was increased three- to four-fold by in vivo treatment with gammabutyrolactone (GBL), reserpine, haloperidol and (-)sulpiride. DA synthesis in striatal synaptosomes (measured by formation of 14CO2 from labelled tyrosine) did not change after GBL and only doubled after reserpine and neuroleptic administration. The increase of synaptosomal DA synthesis was proportional to and probably due to kinetic activation of tyrosine hydroxylase which, after neuroleptic drugs, remained activated for at least 15 min in synaptosomal incubations at 37 degree C.

Ultrastructure of neurosecretosomes sedimented in the nuclear fraction from the posterior pituitary of the rat.

Subcellular fractions of the posterior pituitary of the rat were isolated by differential and density gradient centrifugation, and microsamples prepared for electron miscroscopy by KMnO4 or glutaraldehyde-OsO4 fixation. The nuclear fraction, P1 (1000 g x 7 min), was the main neurosecretosome (NSS) fraction and contained nuclei and mitochondria in addition to NSS (60%). The crude mitochondrial fraction, P2 (10,000 g x 20 min), contained free mitochondria (70%), NSS, unidentified membrane particles, fat droplets derived from pituicytes, neurosecretory granules (NSG) and synaptosomes, identified by the presence of synaptic membrane thickenings. Fraction P3 (32,900 g x 20 min) contained mainly free NSG, while fraction P4 (118,000 g x 30 min) had a high microsome content.

Foot-shock stress accelerates non-striatal dopamine synthesis without activating tyrosine hydroxylase.

Electric foot-shock stress (20 min) increases DOPAC content in the frontal cortex (by about 80%) and in the nucleus accumbens (by 35%) but not in the striatum. However, foot shock stress failed to modify the kinetic properties of tyrosine hydroxylase (Vmax, Km for DMPH4 cofactor) in any of the above areas. Similar results were obtained in rats in which noradrenergic terminals in the n. accumbens and in the frontal cortex had been eliminated by injection of 6-OH-dopamine into the ascending dorsal noradrenergic bundle. The results support the hypothesis that limbic and cortical DA is involved in emotional states and indicate that DA synthesis may be regulated independently from changes in the kinetics properties of tyrosine hydroxylase.

Ultrastructure of synaptosomes from fetal rat brain.

The crude mitochondrial fraction P2 and subfractions of P2 were prepared from the brain stem, hemispheres and whole brain of 19-day-old fetal rats. Samples were fixed in glutaraldehyde-osmium, NaMnO4 or by Tranzer's triple fixation method (aldehydr-chromate-dichromate-osmium) and examined by electron microscopy. The C-fraction from whole brain was the main synaptosome fraction, containing 3.2% presynaptic terminals as counted from all membrane bound particles. The brain stem showed more presynaptic terminals than the hemisphere (2.8% versus 0.9%) suggesting a caudal-rostral maturation gradient for synaptogenesis. The maturity of the nerve endings obtained was very variable in contrast to the rather uniform synaptosomes derived from adult tissue. They varied from profiles without any substructures to mature synaptosomes displaying asymmetric synaptic junctions. Monoamine synaptosomes containing small granular vesicles were not detected in the present study, suggesting immaturity of the granular monoamine pool at this stage of development.

Ultrastructural identification of monoaminergic synaptosomes from one day old rat brain.

Synaptosomes from one day old and adult rat brain were studied. Specific cytochemical methods for demonstrating monoaminergic (MA) nerve endings were used. Permanganate fixation after preincubation with 5-OHDA or alpha-methyl-NA demonstrated MA synaptosomes. Their number was small in the adult (less than 1 %) and still smaller in the one day old rat brain. The MA synaptosomes from developing rats were different from the adult ones. The large amount of endoplasmic reticulum in developing synaptosomes suggests that granular vesicles are formed from endoplasmic reticulum in nerve endings.

Synaptosomes containing large agranular vesicles isolated from developing rat brain.

Using the subcellular fractionation technique the fine structure of the isolated nerve endings (synaptosomes) from the hemispheres and brain stem of the 1-day old and adult rat was examined. In the synaptosomal fractions of the brain of 1-day old rats we observed a new type of nerve endings containing predominantly large agranular vesicles about 1,000 A in diameter. After incubation with alpha-methylnoradrenaline or 5-hydroxydopamine these vesicles remained agranular. It is assumed that the new type of large vesicles represent developing synaptic vesicles.

Pharmacological and ultrastructural maturation of serotonergic synapses during ontogeny.

Studies were made on the course of maturation of serotonergic synapses during ontogeny in rat brain. Mature synaptosomes containing the same five types of synatic vesicles as in the adult, including small dense core vesicles, could be isolated in low proportion from the brain of 1-day-old rats. Although the buoyant density of these synaptosomes varied more than in the adult, the 5-hydroxytryptamine (5-HT) synaptosomes at the two age groups had similar sedimentation characteristics. Ouabain and imipramine, which block active transport of 5-HT, and reserpine, which blocks its granular storage, resulted respectively in a similar slight inhibition of uptake and accumulation of (-14C)5-HT in synaptosomes of 19-day foetuses. Transport and storage of (-14C)5-HT in the brain stem matured simultaneously with endogenous 5-HT content. In subcellular fractionation of the brain 5-HT content, the percentage of 5-HT in the supernatant was significantly lower in neonatal than in adult rats. After treatments with monoamine oxidase inhibitors (MAOIs) and reserpine plus MAOI, respectively, the maximum brain content and subcellular distribution of 5-HT were similar in 1-day-old and adult rats; the result suggests the existence of some other binding mechanism besides the Mg++-ATP-dependent granular storage. Imipramine and N,N-dimethyltryptamine, which cause stimulation of 5-HT receptors, decreased the turnover of brain 5-HT by 40% in adults but had no effect in neonatal rats. Immobilization increased the turnover of brain 5-HT by 35% in adults but had no effect in neonatal rats, whereas fasting increased it by 20% in adults and by 150% in neonatal rats. At 3 weeks of age the responses to imipramine, immobilization and fasting resembled those seen in adults. These responses occurred later than the appearance of the endogenous content, transport and storage of 5-HT and may require maturation of synaptic junctions, the latest neuronal structures to develop.