OBJECTIVETo study the effect of baicalin on synaptosomal adenosine triphosphatase (ATPase) and lactate dehydrogenase (LDH) and its regulatory effect on the adenylate cyclase (AC)/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway in rats with attention deficit hyperactivity disorder (ADHD).

METHODSA total of 40 SHR rats were randomly divided into five groups: ADHD model, methylphenidate hydrochloride treatment (0.07 mg/mL), and low-dose (3.33 mg/mL), medium-dose (6.67 mg/mL), and high-dose (10 mg/mL) baicalin treatment (n=8 each). Eight WKY rats were selected as normal control group. Percoll density gradient centrifugation was used to prepare brain synaptosomes and an electron microscope was used to observe their structure. Colorimetry was used to measure the activities of ATPase and LDH in synaptosomes. ELISA was used to measure the content of AC, cAMP, and PKA.

RESULTSCompared with the normal control group, the ADHD model group had a significant reduction in the ATPase activity, a significant increase in the LDH activity, and significant reductions in the content of AC, cAMP, and PKA (P<0.05). Compared with the ADHD model group, the methylphenidate hydrochloride group and the medium- and high-dose baicalin groups had a significant increase in the ATPase activity (P<0.05), a significant reduction in the LDH activity (P<0.05), and significant increases in the content of AC, cAMP, and PKA (P<0.05). Compared with the methylphenidate hydrochloride group, the high-dose baicalin group had significantly greater changes in these indices (P<0.05). Compared with the low-dose baicalin group, the high-dose baicalin group had a significant increase in the ATPase activity (P<0.05); the medium- and high-dose baicalin groups had a significant reduction in the LDH activity (P<0.05) and significant increases in the content of AC, cAMP, and PKA (P<0.05). Compared with the medium-dose baicalin group, the high-dose baicalin group had a significant increase in the ATPase activity (P<0.05).

CONCLUSIONSBoth methylphenidate hydrochloride and baicalin can improve synaptosomal ATPase and LDH activities in rats with ADHD. The effect of baicalin is dose-dependent, and high-dose baicalin has a significantly greater effect than methylphenidate hydrochloride. Baicalin exerts its therapeutic effect possibly by upregulating the AC/cAMP/PKA signaling pathway.

Adenosine Triphosphatases ; metabolism ; Adenylyl Cyclases ; physiology ; Animals ; Attention Deficit Disorder with Hyperactivity ; drug therapy ; physiopathology ; Cyclic AMP ; physiology ; Cyclic AMP-Dependent Protein Kinases ; physiology ; Flavonoids ; pharmacology ; therapeutic use ; L-Lactate Dehydrogenase ; metabolism ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Signal Transduction ; drug effects ; Synaptosomes ; chemistry ; ultrastructure

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.
Chromatography, Liquid ; Dopamine ; In Vitro Techniques* ; Lisdexamfetamine Dimesylate* ; Rodentia ; Synaptosomes

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.
Chromatography, Liquid ; Dopamine ; In Vitro Techniques* ; Lisdexamfetamine Dimesylate* ; Rodentia ; 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.
Acetylglucosamine/*metabolism ; Amino Acid Sequence ; Animals ; Brain/*metabolism ; Chromatography, Affinity ; Glycosylation ; Mice ; Molecular Sequence Data ; Peptides/isolation & purification ; Phosphorylation ; Synapsins/chemistry/*metabolism ; Synaptosomes/*metabolism ; Tandem Mass Spectrometry ; tau Proteins/chemistry/*metabolism

Diverse subtypes of voltage-gated sodium channels (VGSCs) have been found throughout tissues of the brain, muscles and the heart. Neurotoxins extracted from the venom of the Asian scorpion Buthus martensi Karsch (BmK) act as sodium channel-specific modulators and have therefore been widely used to study VGSCs. α-type neurotoxins, named BmK I, BmK αIV and BmK abT, bind to receptor site-3 on VGSCs and can strongly prolong the inactivation phase of VGSCs. In contrast, β-type neurotoxins, named BmK AS, BmK AS-1, BmK IT and BmK IT2, occupy receptor site-4 on VGSCs and can suppress peak currents and hyperpolarize the activation kinetics of sodium channels. Accumulating evidence from binding assays of scorpion neurotoxins on VGSCs, however, indicate that pharmacological sensitivity of VGSC subtypes to different modulators is much more complex than that suggested by the simple α-type and β-type neurotoxin distinction. Exploring the mechanisms of possible dynamic interactions between site 3-/4-specific modulators and region- and/or species-specific subtypes of VGSCs would therefore greatly expand our understanding of the physiological and pharmacological properties of diverse VGSCs. In this review, we discuss the pharmacological and structural diversity of VGSCs as revealed by studies exploring the binding properties and cross-competitive binding of site 3- or site 4-specific modulators in VGSC subtypes in synaptosomes from distinct tissues of diverse species.
Animals ; Binding Sites ; Binding, Competitive ; Brain ; metabolism ; Heart ; physiology ; Humans ; Insect Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Insecta ; Ion Channel Gating ; drug effects ; physiology ; Kinetics ; Mammals ; Muscles ; metabolism ; Neurotoxins ; chemistry ; classification ; pharmacology ; Protein Binding ; Scorpions ; chemistry ; Sodium ; metabolism ; Sodium Channel Blockers ; pharmacology ; Sodium Channels ; classification ; genetics ; metabolism ; Synaptosomes ; drug effects ; metabolism

To explore novel monoamine reuptake inhibitor with antidepressant activity, a series of substituted aryl alkanol piperidine derivatives were designed and synthesized. All of them were new compounds, and their structures were confirmed with 1H NMR and HR-MS. The results showed that compounds 4, 5 and 8 displayed strong 5-HT, NA and DA reuptake inhibiting activities in vitro. Among the tested compounds, 4, 5 and 13 exhibited potent antidepressant activities in the mice forced swimming test. Compounds 4 and 5 have potent antidepressant activities and are worth further development.
Animals ; Antidepressive Agents ; chemical synthesis ; chemistry ; pharmacology ; Dopamine ; metabolism ; Male ; Mice ; Molecular Structure ; Motor Activity ; drug effects ; Neurotransmitter Uptake Inhibitors ; chemical synthesis ; chemistry ; pharmacology ; Norepinephrine ; metabolism ; Piperidines ; chemical synthesis ; chemistry ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Serotonin ; metabolism ; Structure-Activity Relationship ; Swimming ; Synaptosomes ; metabolism

OBJECTIVETo study the role of cell adhesion molecules Necl1 in synaptogenesis in primary cultured neurons.

METHODSSemi-quantitive reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression pattern of Necl1 in the neuronal differentiation cell model in vitro. Western blot was performed to detect the expression pattern of Necl1 in primary cultured rat neurons and in purified synaptosome. Immunofluoresence was used to detect the synapse formation in primary neurons and in 293 cells co-culture and to detect the density of synapses in primary neuron with ectopic expression of Necl1.

RESULTSNecl1 expression increased after retinoic acid (RA) induction in SH-SY5Y and P19 cells. The increase of Necl1 expression was consistent with the days of primary neurons culture in vitro, and Necl1 partly localized in synaptosome. The overexpression of Necl1 in 293 cells induced the synapse formation between cocultured 293 cells and neurons. Ectopic expression of Necl1 in primary neurons increased the density of synapses.

CONCLUSIONNecl1 plays an important role in neuronal synapse formation.

Animals ; Blotting, Western ; Cell Adhesion Molecules, Neuronal ; genetics ; metabolism ; Cell Differentiation ; drug effects ; genetics ; Cell Line ; Cells, Cultured ; Fluorescent Antibody Technique ; Humans ; Neurons ; cytology ; drug effects ; metabolism ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Synapses ; drug effects ; metabolism ; physiology ; Synaptosomes ; drug effects ; metabolism ; Tretinoin ; pharmacology

BACKGROUND: Formation of cholesterol oxidation products is a suggested mechanism of neurodegenerative disorders. Neuronal cell death is mediated by an increased release of excitotoxic glutamate from the presynaptic nerve endings. Tyrosine-specific protein kinases modulate neurotransmitter release at the nerve terminals. Tyrphostin AG126 has anti-inflammatory and cytoprotective effects. However, it remains uncertain whether tyrphostin AG126 has a preventive effect on the alteration of nerve terminal function induced by cholesterol oxidation products. METHODS: The present study was performed to assess the effect of cholesterol oxidation products against nerve terminal function using synaptosomes isolated from rat cerebrum. We determined the preventive effect of tyrphostin AG126 against oxysterol toxicity by measuring the effects on the glutamate release, depolarization of the membrane potential, changes in Ca2+ levels, and Na+/K+-ATPase activity. RESULTS: Synaptosomes treated with 7-ketocholesterol or 25-hydroxycholesterol exhibited a sustained release of glutamate, depolarization of membrane potential, early rapid increase in cellular Ca2+ levels and decrease in Na+/K+-ATPase activity. Those responses were concentration-dependent. Treatment of tyrphostin AG126 interfered with alteration of synaptosomal functions and decrease in Na+/K+-ATPase activity induced by 7-ketocholesterol or 25-hydroxycholesterol. CONCLUSIONS: The results show that 7-ketocholesterol and 25-hydroxycholesterol seem to cause the release of glutamate by inducing depolarization of the membrane potential and early rapid increase in cellular Ca2+ levels and by inactivating Na+/K+-ATPase in the cerebral synaptosomes. Treatment of tyrphostin AG126 may prevent the oxysterol-induced nerve terminal dysfunction.
Animals ; Brain ; Cell Death ; Cerebrum ; Cholesterol ; Glutamic Acid ; Hydroxycholesterols ; Ketocholesterols ; Membrane Potentials ; Neurodegenerative Diseases ; Neurons ; Neurotransmitter Agents ; Presynaptic Terminals ; Protein-Tyrosine Kinases ; Rats ; Synaptosomes ; Tyrphostins

OBJECTIVE: To explore the role of 5-HT and postsynaptic 5-HT1A receptors in the stress adaptation. METHODS: p-PCA was used to deplete the 5-HT in rats. The 5-HT1A agonist 8-OH-DPAT and antagonist WAY100635 were used to determine the effect of postsynaptic 5-HT1A receptors on the ratso behaviors in the Elevated Plus-Maze test, the Forced Swimming test, and the Morris Water Maze test. RESULTS: Compared with the intact rats, the 5-HT depleted rats showed more seriously anxious behaviors in the Elevated Plus-Maze test and more obvious learned helplessness in the Forced Swimming test. After having been stressed the 5-HT depleted rats showed significantly impaired learning and memory compared with the intact rats according to Morris Water Maze test. Activation of postsynaptic 5-HT1A receptors by 8-OH-DPAT in the 5-HT depleted rats or the 5-HT depleted stress rats significantly decreased the symptoms of anxiety and learned helplessness behaviors which were prevented by the treatment of WAY100635. The 8-OH-DPAT and WAY100635 had no obvious effect on the 5-HT depletion or 5-HT depleted stress rats in the Morris Water Maze test. CONCLUSION Deficiency of 5-HT in rats may suppress its ability to stress adaptation. Activation of post-synaptic 5-HT1A receptors can attenuate the anxiety and depressive behavior symptoms, and facilitate rats to adapt stress.
Affect ; physiology ; Animals ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptor, Serotonin, 5-HT1A ; physiology ; Recognition, Psychology ; physiology ; Restraint, Physical ; Serotonin ; physiology ; Stress, Psychological ; metabolism ; psychology ; Synaptosomes ; chemistry

OBJECTIVETo investigate the effect of microwave radiation on synaptic structure, characteristic of synaptosome, the contents and release of neurotransmitters in hippocampus in Wistar rats.

METHODSWistar rats were exposed to microwave radiation with average power density of 30 mW/cm(2). Electron telescope was used to study the change of the synaptic structure at 6 h after radiation and to identify synaptosome. Flow cytometry and electron spin resonance were used to study the change of the concentration of Ca(2+) in synapse and the fluidity of membrane proteins of synaptosome. High performance liquid chromatography (HPLC) and spectrophotometer were used to study the changes of contents and release of amino acids and acetylcholine in hippocampus.

RESULTSMicrowave radiation of 30 mW/cm(2) caused deposits of synapse vesicle, elongation of active zone, the increase of thickness of postsynaptic density (PSD) and curvature, and perforation of synapse. The concentration of Ca(2+) in synapse (P<0.01) and tc of membrane proteins (P<0.01) of synaptosome increased contents of glutamic acid and glycine (P<0.01) and release of GABA increased the increase of contents and release of acetylcholine, and activity of acetyl cholinesterase (P<0.01) increased.

CONCLUSIONMicrowave radiation can induce the injure of synaptic structure and function of hippocampus, and then induce the disorder of the ability of learning and memory in rats.

Animals ; Hippocampus ; metabolism ; pathology ; radiation effects ; Male ; Microwaves ; adverse effects ; Rats ; Rats, Wistar ; Synapses ; metabolism ; pathology ; radiation effects ; Synaptosomes ; metabolism ; radiation effects