Influence of antipsychotics and serotonin antagonists on presynaptic receptors modulating the release of serotonin in synaptosomes of the nucleus accumbens of rats.

In the nucleus accumbens of rats the release of [3H]serotonin (5-HT) from superfused synaptosomes stimulated by 30 mM K+ was investigated. In the presence of 40 microM of the uptake inhibitor cocaine the release of [3H]5-HT was inhibited by 5-HT in a concentration-dependent manner (IC50 = 0.45 microM). The maximum inhibitory effect of 5-HT was 54% of controls. The inhibition of K+-stimulated release of [3H]5-HT induced by 5-HT was antagonized completely by methiothepine and clozapine, respectively, whereas methysergide had only a weak antagonizing effect in a concentration of 20 microM or less, haloperidol was ineffective. Furthermore, the synaptosomal K+-stimulated release of [3H]5-HT was also inhibited by dopamine (DA) in a concentration-dependent manner (IC50 = 0.1 microM). This inhibitory effect was antagonized by antipsychotic drugs, the rank order of antagonistic potencies was sulpiride greater than haloperidol greater than clozapine; methiothepine was ineffective. The experimental system (the K+-stimulated synaptosomal release of [3H]5-HT seems to be a suitable model for differentiating dopaminergic and/or serotonergic components of antipsychotics or other drugs on presynaptic receptors.

Influence of antipsychotics on presynaptic receptors modulating the release of dopamine in synaptosomes of the nucleus accumbens of rats.

The release of preloaded [3H]dopamine (DA) from superfused synaptosomes stimulated by 30 mM K+ was investigated in the nucleus accumbens of rats. Under conditions preventing the uptake of DA (presence of 40 microM cocaine) release of [3H]DA was inhibited by DA and apomorphine in a concentration-dependent manner (IC50s 0.65 and 0.3 microM, respectively). The maximal inhibitory effects of DA, as well as of apomorphine, were about 50% of the controls. The DA-induced inhibition was antagonized by antipsychotics completely; the rank order of antagonistic potencies was haloperidol greater than clozapine greater than sulpiride; methiothepine was ineffective. Furthermore, the K+-stimulated release of [3H]DA was inhibited by serotonin in a concentration-dependent manner (IC50 = 0.9 microM). This inhibitory effect was antagonized by methiothepine with a high efficiency, by clozapine and methysergide with moderate efficiencies; haloperidol and sulpiride were ineffective. The experimental system demonstrated appears to be suitable for characterizing the DA- and serotonin-antagonistic potencies of antipsychotics and other drugs on presynaptic autoreceptors as well as receptors modulating release of DA in the nucleus accumbens.

Characterization of synaptosomal dopamine release in rat and human brain after post-mortem storage and cryopreservation.

In preparing pathobiochemical studies on [(3)H]dopamine (DA) release in post-mortem human brain, freezing methods were tested regarding their applicability for cryopreservation of brain tissue. An optimal method was to incubate pieces of the nucleus accumbens of rats or humans in 0.32 M sucrose containing 5% dimethylsulfoxide before freezing the brain material. After cryopreservation of these pieces in liquid nitrogen the synaptosomal K(+)-stimulated [(3)H]DA release was found to be unchanged in comparison with the values obtained before freezing. Moreover, the inhibition of K(+)-stimulated [(3)H]DA release by extracellular DA, which is mediated by DA autoreceptors, was also detectable after using this freezing method. During post-mortem storage of rat brains in situ for up to 48 h it was found that [(3)H]DA release changes occurred in dependence on storage temperature: at 2 degrees C no alteration was noted, however, at 22 degrees C a relatively rapid, biphasic exponential decrease was found. Furthermore, subchronic haloperidol pretreatment of rats did not have any influence on post-mortem changes of [(3)H]DA release and its modulation by DA autoreceptors. In conclusion, it seems that post-mortem human brain is suitable for investigating synaptosomal K(+)-stimulated [(3)H]DA release and its autoreceptor-mediated inhibition. For this end a suitable cryopreservation method is presented and a correction for post-mortem delay is proposed which is based on changes of [(3)H]DA release at 22 degrees C in rats.

Influence of cholecystokinin on the synaptosomal dopamine uptake in the nucleus accumbens of rats.

The influence of the sulfated cholecystokinin octapeptide (CCK-8S) on the synaptosomal high-affinity [(3)H]dopamine (DA) uptake was investigated in the medial and lateral part of nucleus accumbens in rats. CCK-8S induced a concentration-dependent biphasic inhibition of [(3)H]-DA uptake in both subregions. After preincubation of CCK-8S with the synaptosomes the inhibitory effect was completely abolished. Kinetic analysis of the uptake influence suggests an uncompetitive inhibition by CCK-8S; this means that CCK-8S attacks only the DA-uptake carrier complex by inhibitory manner. The possible regulatory relevance of this mechanism is discussed.

Influence of psychotomimetics and lisuride on synaptosomal dopamine release in the nucleus accumbens of rats.

In superfusion experiments with a crude synaptosomal fraction from the nucleus accumbens of rats, lysergic acid diethylamide, mescaline and N,N-dimethyltryptamine (representing different chemical classes of psychotomimetics) produced a concentration-related inhibition of K+-evoked [3H]dopamine release whereas spontaneous release remained unchanged. The nonpsychotomimetic ergoline lisuride tested at the same concentration range did not modulate spontaneous or K+-evoked [3H]dopamine release. The inhibitory effects of the psychotomimetics tested were antagonized by equal concentrations of haloperidol or methiothepine. It is postulated that the effects of psychotomimetics on DA release were triggered via simultaneous action at presynaptic dopamine and 5-hydroxytryptamine receptors which are located at dopamine nerve endings in the nucleus accumbens.

Presynaptic dopamine and serotonin receptors modulating tyrosine hydroxylase activity in synaptosomes of the nucleus accumbens of rats.

Tyrosine hydroxylase (TH) activity was determined by measuring the formation of [3H]DOPA from [3,5-3H]tyrosine in the crude synaptosomal fraction of the nucleus accumbens under conditions preventing dopamine reuptake in 30 mM K+-containing medium. TH seems to be allosterically activated under depolarizing conditions: a 4.4 fold decrease of the Km value for tyrosine of the synaptosomal TH was observed. Synaptosomal TH activity was inhibited concentration dependently by dopamine and apomorphine resulting in IC50 values of 0.4 and 0.25 microM, respectively. The maximal inhibitory effects of dopamine as well as apomorphine were about 50% of the controls. The dopamine-induced inhibition was completely antagonized by neuroleptics. The rank order of antagonistic potencies was haloperidol greater than clozapine greater than sulpiride (with increasing EC50); methiothepine was ineffective. Moreover, synaptosomal TH activity was inhibited by serotonin in a concentration-dependent manner (IC50 = 0.8 microM). This inhibition was completely antagonized by methiothepine while, on the other hand, haloperidol was ineffective. The experimental system demonstrated here appears to be suitable for estimating the presynaptic dopamine and serotonin antagonistic potencies of drugs.

Putative interaction of presynaptic dopamine and serotonin receptors modulating synaptosomal tyrosine hydroxylase activity in the nucleus accumbens of rats.

Inhibition of synaptosomal tyrosine hydroxylase activity by dopamine (DA) autoreceptors and serotonin (5-HT) heteroreceptors was used as a functional measure for the receptor activity in the nucleus accumbens of rats. Kinetic analysis of the concentration dependence of inhibition with and without antagonists indicates that the sensitivity of the autoreceptor to DA is significantly increased by blockade of the 5-HT heteroreceptor and vice versa. The results provide evidence for autoreceptor-heteroreceptor interactions at the presynaptic membrane level on dopaminergic nerve terminals.

Studies in postmortem dopamine uptake. I. Kinetic characterization of the synaptosomal dopamine uptake in rat and human brain after postmortem storage and cryopreservation. Comparison with noradrenaline and serotonin uptake.

The effect of various post-mortem storage times and temperatures on the kinetic parameters of synaptosomal high-affinity dopamine (DA) uptake was studied in rat nucleus accumbens. After post-mortem storage up to 48 hours of rat heads in situ at 22 degrees C the KM data increase moderately in contrast to the Vmax data decreasing rapidly (t1/2 = 30 hours); at 2 degrees C similar changes were observed. The post-mortem changes of the kinetic parameters of noradrenaline (NA) and serotonin (5 HT) uptake were shown in this model to be similar. It is proposed to use this animal model for correcting the kinetic data of DA, NA and 5 HT uptake in human brains regarding the various post-mortem delays. Furthermore, in cryopreservation experiments using a two-step freezing procedure in liquid nitrogen of brain pieces preincubated with dimethylsulfoxide DA uptake of rat and human brain synaptosomes was unchanged after freezing-thawing. The same is true for both NA and 5 HT uptake as demonstrated in human brain tissue. Therefore, post-mortem human brain seems to be suitable for investigating synaptosomal DA, NA, and 5 HT uptake after cryopreservation and correcting for post-mortem delay, when determining kinetic parameters.

Studies in postmortem dopamine uptake. II. Alterations of the synaptosomal catecholamine uptake in postmortem brain regions in schizophrenia.

Kinetic parameters KM and Vmax of the synaptosomal high-affinity dopamine (DA) and noradrenaline (NA) uptake were measured in the nucleus (n.) accumbens, n. caudatus and frontal cortex from post-mortem brains of schizophrenic patients and matched controls. Additionally, the 5-hydroxytryptamine (5 HT) uptake was determined in the n. accumbens of the same specimens. The KM and Vmax data of DA uptake were significantly elevated in the n. accumbens (KM to 253%, Vmax to 271%) and in the n. caudatus (KM to 201%, Vmax to 174%) of schizophrenics in comparison with controls. The kinetic parameters of the NA uptake increased similarly in the n. accumbens and n. caudatus of schizophrenics in comparison with controls. The alterations in DA and NA uptake kinetics do not seem to be primarily dependent on neuroleptic medication. On the other hand, DA and NA uptake in the frontal cortex as well as 5 HT uptake in the n. accumbens was unchanged. The results are compatible with the hypothesis that both DA and NA transmission systems are changed in schizophrenia. They give a first indication of presynaptic functional alterations in schizophrenia.

Synaptosomal uptake and release of dopamine and 5-hydroxy-tryptamine in the nucleus accumbens in vitro following in vivo administration of lysergic acid diethylamide in rats.

The uptake and the depolarisation-induced release of dopamine (DA) and serotonin (5-HT) were investigated after systemic application of LSD on synaptosomes of the nucleus accumbens of rats. For the release experiments synaptosomes were prelabelled with [14C]-DA and [3H]-5-HT, respectively, and superfused with physiological and potassium-enriched (50 mM) solutions. Low doses of LSD (0.1 and 0.5 mg/kg i. p.) induced a dose-dependent inhibition of the DA-release and an increase of the DA-uptake, respectively. LSD inhibited both the release and the uptake of 5-HT significantly. The results are discussed with respect to a reliable characterization of the in vivo induced effects of LSD on the isolated synaptosomes.

Characterization of the synaptosomal high-affinity uptake of noradrenaline in the nucleus accumbens of rats.

In the nucleus (n.) accumbens, a predominantly dopaminergic innervated brain region, a high-affinity noradrenaline (NA) uptake exists indicating NA termination in this area. The affinity of the uptake carrier (Km) is in the range of that given for other regions. For characterizing the specificity of NA uptake, inhibitory experiments of NA and dopamine (DA) uptake were carried out with nomifensine and desipramine. The experiments with desipramine clearly show that the NA uptake is independent of the DA uptake into dopaminergic terminals occurring predominantly in this nucleus.

Synaptosomal uptake and release of dopamine in rat striatum after hypoxia.

Hypoxia induces alterations of central monoaminergic transmission and of behavior. We studied the effect of hypoxia on adult and newborn rats to obtain more information about long-lasting changes of dopamine (DA) transmission caused by neonatal hypoxia. One single exposure of adult rats to hypoxia leads to short-term alterations of DA uptake: decreased affinity of the uptake carrier to DA (Km, 269.5% versus control) and a sharp increase of Vmax up to 301.4% resulting in an increase of total uptake of DA into the striatum synaptosomes. The K+-evoked DA release decreased to 69.5%. After 1 week of recovery all parameters are normalized. Chronic postnatal hypoxia (postnatal day 2-11) caused long-lasting changes of DA release and uptake opposite to those observed in adult rats. Three months after hypoxia, the K+-stimulated DA release was enhanced (132% of control), and the uptake was reduced due to decreased affinity of the uptake carrier system for the substrate (Km, 187% of control value). In conclusion, the alterations observed after chronic postnatal hypoxia reflect special adaptive processes that are related to the high plasticity of the immature neonatal brain and contribute to an increased DA function in the nigrostriatal system.

Characterization of synaptosomal dopamine uptake in post-mortem brain regions of schizophrenics.

The drug sensitivity of synaptosomal high-affinity dopamine (DA) uptake was investigated in post-mortem brain regions of schizophrenics, in comparison to controls matched for age, sex, and post-mortem delay, and in model experiments in rats. DA uptake was inhibited by nomifensine in the investigated regions of rat brain in a concentration-dependent manner; the regional rank order of inhibitory potency was: nucleus (n) caudatus greater than n. accumbens greater than frontal cortex. Furthermore, it was shown that the inhibitory potency of nomifensine is unchanged after in situ storage of rat brain tissue for 48 h and after the cryopreservation method used. In post-mortem brain of human controls, nomifensine inhibited DA uptake with the same regional differences as in rats; however, the inhibitory potencies were three-fourfold weaker. In schizophrenia, on the other hand, synaptosomal DA uptake inhibition by nomifensine was significantly weaker than in the corresponding control brains for all regions studied. This suggests a decreased affinity of the DA uptake carrier to nomifensine, similar to DA shown in recent studies with schizophrenic patients. The possible relevance of investigating functional parameters for understanding patho-biochemical mechanisms in schizophrenia is discussed.

Carbon dioxide fixation in the brain: its relation to glucose synthesis.

The incorporation in vivo of radiocarbon from 14C-bicarbonate in blood into relevant metabolites in rat brain is described. The animals, partially hepatectomized and nephrectomized, received the tracer bicarbonate via the intravenous route. The time course of label was followed in CO2 of blood and brain, in the anionic and cationic fractions of brain extract, in aspartate, glutamate, glutamine and in free glucose and in glycogen. From the tracer kinetic data a flux of 0.08 microgram atom fixed carbon min-1.g-1 brain tissue was calculated. Substantial amounts of 14C were found in free glucose, only a few percent in glycogen. The flux of newly synthetized glucose was approximated to 0.5--1.0 percent of the steady state level of glucose in brain tissue. In special experiments the localization of 14C in the carbon chain of aspartate and glucose was examined. 5 min following the tracer injection a practically total randomization of 14C between C-1 and C-4 aspartate was seen. From the radioactivity in glucose 94 percent were found in C-3 and C-4, only 6 percent in residual carbon. This 14C-pattern is typical for the labelling of glucose by CO2 fixation and retrograde Embden-Meyerhof pathway.

[Regulation of 3H-dopamine release by presynaptic GABA and glutamate heteroreceptors in the synaptosomes of the rat nucleus accumbens]. / Reguliatsiia vysvobozhdeniia 3H-dofamina presinapticheskimi geteroretseptorami GAMK i glutamata v sinaptosomakh prilezhashchego iadra mozga.

The influence of GABA, muscimol, delta-aminolevulinic acid (DALA), baclofen and L-glutamate on K+-evoked release of 3H-dopamine (3H-DA) from the rat brain n. accumbens crude synaptosomal fraction was studied in superfusion experimental conditions. Both GABA-receptor agonists--GABA and muscimol (50 microM) depressed the 3H-DA release by bicuculline- and picrotoxin-sensitive manner. On the contrary, glutamate, DALA and baclofen led to the increase in 3H-DA efflux independently of the presence of GABA-receptor antagonists. While the action of glutamate was antagonized by glutamate-receptor blocker--glutamic acid diethyl ester (GDEE), the effects of DALA and baclofen were suppressed upon adding to superfusion medium of GABA uptake inhibitors (nipecotic acid and 2,4-diaminobutyric acid) but not GDEE. The data obtained demonstrate that 3-H-DA secretion from n. accumbens is inhibited by GABA- and stimulated by glutamate-heteroreceptors. At the same time the mechanism of interaction between baclofen, DALA and GABA-uptake blockers effects with 3H-DA release needs special investigations.

[Effect of increased plasma levels of glucose, adrenaline, and angiotensin upon glucose metabolism of totally ischemic and normally perfused rat brain]. / Untersuchung über die Wirkung eines erhöhten Gehaltes an Glukose, Adrenalin und Angiotensin im Plasma auf den Glukosestoffwechsel des total ischämischen und normal durchbluteten Grosshirns von Ratten

Tracer kinetic studies on the effect of i.v. infused adrenaline and angiotensin, and a hyperglycemia induced by glucose application, upon glucose metabolism of the rat brain under ischemic and normoxic conditions are reported. in the ischemic brain, the initial glycolytic rate proved dependent on the glucose content being kept at various levels by glucose administration or hormone infusion prior to the onset of ischemia. The typical saturation kinetics revealed a maximal glucose conversion only from a definite initial content of brain glucose, being equivalent to a glucose level of approximately 13 mumole/ml in plasma, and appeared to depend on the presence of glucose in the cellular space. The early cessation of anaerobic lactate formation even with high glucose in the cellular space. The early cessation of anaerobic lactate formation even with high glucose depot in the brain tissue is referred to inhibition of glycolytic key enzymes by increasing tissue azidosis. The aerobic glucose conversion, as calculated from the Cglucose flux in amino acids associated with the citrate cycle was unaffected by the cerebral glucose content (hyperglycemia by hormone or glucose application). During glucose infusion the cerebral levels of NH3, total NH2 and glutamine rose; the Cglucose flux into aspartate and glutamine was increased and almost proportionally reduced in glutamate and gamma-aminobutyrate. These flux shifts are interpreted as a switching of C-chains from pyruvate owing to increased CO2 fixation, and as a biochemical correlate of an increased irritation level of the experimental animals.

Diminished synaptosomal dopamine (DA) release and DA autoreceptor supersensitivity in schizophrenia.

Post-mortem brain regions of schizophrenics were investigated in comparison to matched controls regarding synaptosomal K(+)-stimulated [3H]DA release and its modulation by DA autoreceptors. Brain specimens were cryopreserved in liquid nitrogen until release experiments; synaptosomes from brain pieces preincubated with dimethylsulfoxide have unchanged ability for [3H]DA release and its autoreceptor-mediated inhibition after cryo-preservation. Release data were individually corrected for post-mortem delay back to the zero-time state based on [3H]DA release alterations after in situ preservation of rat heads. In schizophrenia, the K(+)-stimulated [3H]DA release from superfused synaptosomes of the nucleus (n.) accumbens and n. caudatus was diminished significantly. Furthermore, functional supersensitivity of the modulatory DA autoreceptors could be demonstrated in these regions. The alterations demonstrated seem not to be due primarily to medication since in neuroleptic-free patients similar changes were shown.

[Effect of the atypical neuroleptics carbidine and sulpiride on dopamine biosynthesis in the synaptosomes of the nucleus accumbens septi in rats]. / Vliianie atipichnykh neiroleptikov karbidina i sul'pirida na biosintez dofamina v sinaptosomakh prilezhashchego iada mozga krys.

Neurochemical mechanisms of actions of atypical neuroleptics carbidine (a derivative of gamma-carboline) and sulpiride on dopamine (DA) biosynthesis in synaptosomes of the nucleus accumbens septi of the rat brain were studied. Carbidine caused a dose-dependent decrease of the animals' locomotor hyperactivity induced by apomorphine (1 mg/kg). Both carbidine and sulpiride administered in a dose of 5 mg/kg increased the activity of tyrosine hydroxylase in synaptosomes of the nucleus accumbens septi. In vitro carbidine decreased and sulpiride exerted no effect on the activity of tyrosine hydroxylase of the nucleus accumbens septi synaptosomes. Carbidine as well as sulpiride in vitro failed to modify the release of 3H-DA from superfused synaptosomes. At the same time, both in vivo and in vitro, the two neuroleptics reduced the inhibitory effect of DA on the 3H-DA release from synaptosomes of the nucleus accumbens septi and on the activity of tyrosine hydroxylase.