Potential for Dependence on Lisdexamfetamine - In vivo and In vitro Aspects

Although lisdexamfetamine is used as a recreational drug, little research exists regarding its potential for dependence or its precise mechanisms of action. This study aims to evaluate the psychoactivity and dependence profile of lisdexamfetamine using conditioned place preference and self-administration paradigms in rodents. Additionally, biochemical techniques are used to assess alterations in the dopamine levels in striatal synaptosomes following administration of lisdexamfetamine. Lisdexamfetamine increased both conditioned place preference and self-administration. Moreover, after administration of the lisdexamfetamine, dopamine levels in the striatal synaptosomes were significantly increased. Although some modifications should be made to the analytical methods, performing high performance liquid chromatography studies on synaptosomes can aid in predicting dependence liability when studying new psychoactive substances in the future. Collectively, lisdexamfetamine has potential for dependence possible via dopaminergic pathway.

Potential for Dependence on Lisdexamfetamine - In vivo and In vitro Aspects

Although lisdexamfetamine is used as a recreational drug, little research exists regarding its potential for dependence or its precise mechanisms of action. This study aims to evaluate the psychoactivity and dependence profile of lisdexamfetamine using conditioned place preference and self-administration paradigms in rodents. Additionally, biochemical techniques are used to assess alterations in the dopamine levels in striatal synaptosomes following administration of lisdexamfetamine. Lisdexamfetamine increased both conditioned place preference and self-administration. Moreover, after administration of the lisdexamfetamine, dopamine levels in the striatal synaptosomes were significantly increased. Although some modifications should be made to the analytical methods, performing high performance liquid chromatography studies on synaptosomes can aid in predicting dependence liability when studying new psychoactive substances in the future. Collectively, lisdexamfetamine has potential for dependence possible via dopaminergic pathway.

Bilirubin-induced calcium overload in synaptosomes isolated from brainstem neurons of rats / 上海交通大学学报（医学版）

Objective· To observe real-time changes of calcium concentration ([Ca2+]i) exposure to bilirubin in synaptosomes isolated from brainstem nucleus of rats. Methods · Forty P7-14 SD rats were randomly assigned to three groups: control group, bilirubin group (with levels of 0.1, 1 and 10 μmol/L) and bulirubin plus glycoursodeoxycholic acid (GUDCA) group. The synaptosomes were purified from brainstem nucleus by sucrose density gradient centrifugation. After loading OG-BAPTA in synaptosomes, two dimensional image of intracellular calcium and analysis of fluorescence intensity were achieved by Confocal laser scanning microscopy. Results · Synaptosomes with well biological activity were obtained from brainstem of the SD rats. In the control group, a progressive increase in fluorescent intensity of [Ca2+]I was detected. In the bilirubin group, acuter increases in fluorescent intensity were observed in all levels of bilirubin, with a manner of both concentration and time-dependent (P<0.05). Fluorescent intensity of [Ca2+]I was reduced in the present of GUDCA, which was not significant compared with the control group (P=0.656). However, GUDCA could abate the increase of fluorescent intensity of [Ca2+]I induced by bilirubin exposure, of which showing significant decrease in 10 μmol/L bilirubin exposure (P=0.000). Conclusion · Bilirubin could induce calcium overload in synaptosomes. GUDCA could abate bilirubin-induced calcium overload in synaptosomes, possibly explaining its protection effect of neurons from bilirubin neurotoxicity.

An optimized extraction protocol of prefrontal cortical and striatal synaptosomes from SHR rat / 中国比较医学杂志

Objective To introduce an improved extraction method of prefrontal cortical and striatal synaptosomes from SHR rat. Methods Synaptosomes were prepared from SHR rat brain tissue by Percoll density gradient centrifugation.Transmission electron microscopy was used to assess the morphology and structural integrity of the synaptosomes.Results The obtained synaptosomes showed oval structures surrounded by an intact membrane.Presynaptic components contained one or more mitochondria and a large number of synaptic vesicles.The synaptic clefts were clearly visible, and prominent part of the characteristic compact structure was clear, complete and with higher electron-density. The synaptosome presynaptic membrane, synaptic cleft, and postsynaptic membrane were well preserved, and the synaptosomes were densely distributed, showing typical morphological characteristics of synaptosomes.Conclusions The results of our study improved the traditional preparation method and provide a less time-consuming, highly productive protocol for preparation of structurally typical and intact synaptosomes, suitable for further research on neuroscience and neurological diseases.

Synapsin-1 and tau reciprocal O-GlcNAcylation and phosphorylation sites in mouse brain synaptosomes

O-linked N-acetylglucosamine (O-GlcNAc) represents a key regulatory post-translational modification (PTM) that is reversible and often reciprocal with phosphorylation of serine and threonine at the same or nearby residues. Although recent technical advances in O-GlcNAc site-mapping methods combined with mass spectrometry (MS) techniques have facilitated study of the fundamental roles of O-GlcNAcylation in cellular processes, an efficient technique for examining the dynamic, reciprocal relationships between O-GlcNAcylation and phosphorylation is needed to provide greater insights into the regulatory functions of O-GlcNAcylation. Here, we describe a strategy for selectively identifying both O-GlcNAc- and phospho-modified sites. This strategy involves metal affinity separation of O-GlcNAcylated and phosphorylated peptides, beta-elimination of O-GlcNAcyl or phosphoryl functional groups from the separated peptides followed by dithiothreitol (DTT) conjugation (BEMAD), affinity purification of DTT-conjugated peptides using thiol affinity chromatography, and identification of formerly O-GlcNAcylated or phosphorylated peptides by MS. The combined metal affinity separation and BEMAD approach allows selective enrichment of O-GlcNAcylated peptides over phosphorylated counterparts. Using this approach with mouse brain synaptosomes, we identified the serine residue at 605 of the synapsin-1 peptide, 603QASQAGPGPR612, and the serine residue at 692 of the tau peptide, 688SPVVSGDTSPR698, which were found to be potential reciprocal O-GlcNAcylation and phosphorylation sites. These results demonstrate that our strategy enables mapping of the reciprocal site occupancy of O-GlcNAcylation and phosphorylation of proteins, which permits the assessment of cross-talk between these two PTMs and their regulatory roles.

Effect of Propofol on Glutamate and γ-aminobutyric Acid Release from Rat Hippocampal Synaptosomes / 华中科技大学学报（医学）（英德文版）

To investigate the effect of propofol on the release of glutamate and γ-aminobutyric acid (GABA) from rat hippocampal synatosomes, synaptosomes was made from hippocampus and incubated with artificial cerebrospinal fluid (aCSF). With the experiment of Ca2+-dependent release of glutamate and GABA, dihydrokainic acid (DHK) and nipectic acid were added into aCSF. For the observation of Ca2+-independent release of glutamate and GABA, no DHK, nipectic acid and Ca2+were added from aCSF. The release of glutamate and GABA were evoked by 20μmol/L veratridine or 30 mmol/L KCl. The concentration of glutamate and GABA in aCSF was measured by using high-performance liquid chromatography (HPLC). 30, 100 and 300 μmol/L propofol significantly inhibited veratridine-evoked Ca2+-dependent release of glutamate and GABA (P＜0.01 or P＜0.05). However, propofol showed no effect on elevated KCl-evoked Ca2+-dependent release of glutamate and GABA (P＞0.05). Veratridine or elevated KCl evoked Ca2+ -independent release of glutamate and GABA was not affected significantly by propofol (P＞0.05). Propofol could inhibit Ca2+-dependent release of glutamate and GABA. However, it has no effect on the Ca2+-independent release ofglutamate and GABA.

Effects of sodium magnesium fructose diphosphate on free calcium concentration and nitric oxide synthase activity of ischemic synaptosome / 药学学报

Aim To study the effects of sodium magnesium fructose diphosphate (SMFD) on free calcium concentration and nitric oxide synthase activity of ischemic synaptosome, so as to explore the protective mechanisms of SMFD on cerebral ischemia. Methods The synaptosomes from normal rat brain were prepared by phase partition and cultured with oxygen-glucose deprivation to establish ischemic synaptosome model. The intrasynaptosomal free calcium concentration and nitric oxide synthase activity were detected separtately after the synaptosomes were co-incubated with SMFD (1.3 mmol*L-1) or fructose-1,6-diphosphate (FDP, 4.0 mmol*L-1) for 60 min. Results SMFD decreased the free calcium concentration and reduced the activity of nitric oxide synthase (NOS) of ischemic synaptosomes. Its effects were more powerful than those of FDP. Conclusion SMFD may protect neurons from ischemic injury by preventing intracellular Ca2+ overload and inhibiting the activity of nitric oxide synthase.

Effect of Ca2+-channel Blockers on Norepinephrine Release in the Rat Hippocampal Slice and Synaptosome

The aim of this study was to investigate the role of Ca2+-channel blockers in norepinephrine (NE) release from rat hippocampus. Slices and synaptosomes were incubated with [3H]-NE and the releases of the labelled products were evoked by 25 mM KCl stimulation. Nifedipine, diltiazem, nicardipine, flunarizine and pimozide did not affect the evoked and basal release of NE in the slice. But, diltiazem, nicardipine and flunarizine decreased the evoked NE release with a dose-related manner without any change of the basal release from synaptosomes. Also, a large dose of pimozide produced modest decrement of NE release. omega-conotoxin (CTx) GVIA decreased the evoked NE release in a dose-dependent manner without changing the basal release. And omega-CTxMVIIC decreased the evoked NE release in the synaoptosomes without any effect in the slice, but the effect of decrement was far less than that of omega-CTxGVIA. In interaction experiments with omega-CTxGVIA, omega-CTxMVIIC slightly potentiated the effect of omega-CTxGVIA on NE release in the slice and synaptosomal preparations. These results suggest that the NE release in the rat hippocampus is mediated mainly by N-type Ca2+-channels, and that other types such as L-, T- and/or P/Q-type Ca2+-channels could also be participate in this process.

Lithium-induced increase of synaptosomal uptake of norepinephrine in rat brain

Lithium remains the most widely used therapeutic agent for bipolar affective disorder, particularly mania. Although many investigators have studied the effects of lithium on abnormalities in monoamine neurotransmitter as a pathophysiological basis of affective disorder, the action mechanism of lithium ion remains still unknown. To explore the action mechanism of lithium in the brain, we examined the effects of lithium on the extrasynaptosomal concentrations of catecholamines and their metabolites. Synaptosomes were prepared from the rat forebrains and assays of catecholamines and metabolites were made using HPLC with an electrochemical detector. Lithium of 1mM decreased the extrasynaptosomal concentrations of NE from the control group of 3.07+/-1.19 to the treated group of 0.00+/-0.00 (ng/ml of synaptosomal suspension) but not that of DHPG. It can be suggested that lithium increases synaptosomal uptake of NE. Increased intraneuronal uptake of NE would decrease neurotransmission and extraneuronal metabolism of NE. Because increased brain NE metabolism and neurotransmission have been suggested as important components in the pathophysiology of bipolar affective disorder, especially mania, lithium-induced increase of intraneuronal NE uptake can be suspected as an action mechanism of therapeutic effect of lithium in manic patient, possibly in bipolar affective disorder.

EFFECT OF TOTAL GLYCOSIDE OF CYNANCHUM OTOPHYLLUM SCHNEID ON SYNAPTOSOMAL AMINO ACID CONTENTS AND WHOLE BRAIN ENZYME ACTIVITIES IN MICE / 中国药理学通报

The total glycoside extracted from the root of cynanchum oiophy-llum schneid ( COS ) has anticonvulsant activity. Its effect on synap-tosomal amino acid contents and brain enzyme activities in mice were studied in this paper. After the ip administration of COS, the content of GABA was increased, and that of Glu and Asp decreased, in synaptosomes of both normal and TSC induced convulsant mice, while the content of Gin remained unchanged. A fall in the activities of GDH, glutaminase and asparaginase in whole brain was seen. No changes were observed in the activities of GPT, GOT and glutamine synthetase.