Synaptosomes Preparation

There is some evidence that receptors for other neurotransmitters on 5-HT nerve terminals also modify release of 5-HT. These include nicotinic receptors (increase release from striatal synaptosomes), a2A-adrenoceptors (depress cortical release) and H3-receptors (cortical depression). Because changes in 5-HT release on activation of these receptors is evident in synaptosomal preparations, it is likely that these are true heteroceptors . 

Preliminary experiments to delineate the optimum conditions for [ H]MDA and [ HJMDMA binding to rat brain membranes indicated that the best signal-to-noise ratio was found using a crude synaptosomal preparation incubated at 4 °C. While most experiments employed 50 mM Tris-HCl (pH 7.1) as the incubation medium, we found that the addition of 0.27 M sucrose to the incubation medium increased the specific binding approximately fivefold. Subsequent experiments were carried out under both conditions. Other preliminary experiments suggested that [ H]MDA and... 

H]MDMA interacted with multiple sites in rat brain. A low affinity pH]MDA binding site (apparent Kd>1.0 mM) was found to be resistant to boiling of the synaptosomal preparation for 15 minutes. This site was saturable, as indicated by a 30 pereent inhibition of [ H]MDA binding to boiled synaptosomes by 1.0 mM MDA and a 56 pereent inhibition of the binding by 0.1 mM of the serotonin uptake bloeker paroxetine. The indication of a saturable, nonspecific binding site for [ H]MDA in boiled membranes necessitated that we use boiled tissue to assess nonspecific binding in all subsequent experiments. 

Smith, R.C. Meltzer, H.Y. Arora, R.C. and,Davis, J.M. Effects of phencyclidine on 3H-catecholamine and 3H-serotonin uptake in synaptosomal preparations from rat brain. Biochem Pharmacol... 

Facilitation of dopaminergic activity by A9-THC may result from multiple mechanisms. A9-THC increases DA synthesis149 and release150 in synaptosomal preparations. In addition, using in vivo... 

Nicotine increased DA levels both in vivo11,193 and in vitro. 94 196 Nicotine197 and its metabolites198 were found to both release and inhibit the reuptake of DA in rat brain slices, with uptake inhibition occurring at a lower concentration than that required for DA release. In addition, the (-) isomer was more potent than the (+) isomer.197 However, the effects of nicotine upon DA release and uptake were only apparent when brain slices were utilized because nicotine was unable to affect DA when a synaptosomal preparation was utilized.197 These results indicate that nicotine exerts its effects upon the DAT indirectly, most likely via nicotine acetylcholine receptors. This finding was supported by the results of Yamashita et al.199 in which the effect of nicotine on DA uptake was examined in PC 12 and COS cells transfected with rat DAT cDNA. Nicotine inhibited DA uptake in PC 12 cells that possess a nicotine acetylcholine receptor. This effect was blocked by the nicotinic antagonists hexamethonium and mecamylamine. Additionally, nicotine did not influence DA uptake in COS cells, which lack nicotinic acetylcholine receptors. 

Grady, S., Marks, M.J., Wonnacott, S., Collins, A.C. Characterization of nicotinic receptor-mediated [3H]dopamine release from synaptosomes prepared from mouse striatum. J. Neurochem. 59 848,1992. 

Koe B. Molecular geometry of inhibitors of the uptake of catecholamines and serotonin in synaptosomal preparations of rat brain. J. Pharmacol. Exp. Ther. 199 649, 1976. 

Gleitz J, Beile A, Peters T. (1995). (+/-)-Kavain inhibits veratridine-activated voltage-dependent Na(+)-channels in synaptosomes prepared from rat cerebral cortex. Neuropharmacology. 34(9) 1133-38. 

Gylys KH, Fein JA, Yang F, Cole CM. 2004. Enrichment of presynaptic and postsynaptic markers by size-based gating analysis of synaptosome preparations from rat and human... 

Fonlupt P, Rey C, Pacheco H. 1987. Comparison of basal and noradrenaline stimulated methylation of chloroform-extractable products in synaptosomal preparations from the rat brain. Biochem Pharmacol 36 1527-1729. 

F. Matsumura, sponsored by the National Institute of Environmental Health Sciences (NIEHS), plans to study the toxic effects of chlorinated and pyrethroid pesticides primarily on calcium and sodium regulating processes in the nervous system. To examine the interactions of the pesticides with calcium regulating processes, researchers will use synaptosomal preparations from the brains of rats and the central nervous systems of squid. To examine the interactions of the pesticides with sodium regulating processes, they will collect antibodies directed against sodium channel proteins. 

So far we have discussed the effect of CCK peptides on serotonin brain concentrations. Some evidence also exist on the action of serotonin on the CCK system. Raiteri and colleagues (1993a) looked at the effects of serotonin on the release of cholecystokinin-like immunoreactivity (CCK-LI) in synaptosomes prepared from rat cerebral cortex and nucleus accumbens. In both areas, serotonin increased the calcium-dependent depolarization-evoked CCK-LI release in a dose-related fashion. This effect was antag-... 

EGb inhibits the uptake of [3H]norepinephrine ([3H]NE) and [3H]dopamine and [3H]5-hydroxytryptamine ([3H]5-HT) into in vitro synaptosomes prepared from the striatum and cortex in a concentration-dependent manner. The rank order of potency for the inhibition of amine uptake is NE > dopamine > 5-HT [173]. Similar results were obtained by Ramassamy et al. [174]. These workers showed that EGb decreased the specific uptakes of [3H]dopamine, [3H]5-HT and [3H]choline by synaptosomes prepared from the striatum of mice in a concentration-dependent manner. The IC,0 values were 637 pg/rol for [3H]dopamine uptake, 803 pg/ml for [3H]5-HT uptake, >2000 pg/ml for [3H]choline uptake. However, they concluded that the inhibition of amine uptake caused by EGb appears to be non-specific, since EGb also prevents the specific binding of the dopamine uptake inhibitor [3H]GBR12783 to membranes prepared from striatum. 

EGb in vitro modifies the [3H]5-HT uptake by synaptosomes prepared from mice cerebral cortex in a biphasic manner. As mentioned above, the uptake of [3H]5-HT is inhibited by a high concentration of EGb [174]. On the other hand, low concentrations of EGb (4-16 pg/ml) significantly increase [3H]5-HT uptake. A similar increase was also obtained when synaptosomes were prepared from the cortices of mice treated orally with EGb, either acutely (100 mg/kg, 14 hours and 2 hours before death) or semi-chronically (2 x 100 mg/kg/day, for 4 days). Furthermore, such an increment in the [3H]5-HT uptake is attributed to the flavonoid constituents of EGb [175], and may be associated with the mechanism of its antidepressant activity. 

White, T.D., Downie, J. W., Leslie, R. A. Characteristics of fC- and veratridine-induced release of ATP from synaptosomes prepared from dorsal and ventral spinal cord, Brain Res. 1985, 334, 372-374. 

Lynch M. A. and Voss K. L. (1990). Arachidonic acid increases inositol phospholipid metabolism and glutamate release in synaptosomes prepared from hippocampal tissue. J. Neurochem. 55 215-221. 

Scheer HW (1989) Interactions between a-latrotoxin and trivalent cations in rat striatal synaptosomal preparations. J Neurochem 52 1590-7... 

Grady SR, Murphy KL, Cao J, Marks MJ, McIntosh JM, Collins AC (2002) Characterization of nicotinic agonist-induced [(3)H]dopamine release from synaptosomes prepared from four mouse brain regions. J Pharmacol Exp Ther 301 651-60 Graham B, Redman S (1994) A simulation of action potentials in synaptic boutons during presynaptic inhibition. J Neurophysiol 71 538 49... 

Fig. 6. (A) Representative scheme illustrating the heterotransporter mechanisms. (B) Effects of glycine (3—3000 /tM) on the release of [3H] GABA from gliosomes and synaptosomes prepared from rat cerebral cortex. (C) Effects of strychnine, 5,7-dichlorokynurenic add (5,7-DCK), glycyldodecylamide (GDA), 7V-[3-(4 -fluorophenyl)-3-(4 -phenylphenoxy)propyl]sarcosine (NFPS), and 4-benzoyl-3,5-dimethoxy-jV -[l-(dimethylaminodclopenthyl)methyl]benzamide (ORG25543B) on [3H]GABArelease...

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