Rapid Preparation of Synaptosomes from Mammalian Brain Using Nontoxic Isoosmotic Gradient Material (Percoll)

Nagy, Ágnes; Delgado‐Escueta, Antonio V.

doi:10.1111/j.1471-4159.1984.tb12851.x

Cited by 325 publications

(171 citation statements)

References 55 publications

Supporting

Mentioning

166

Contrasting

Order By: Relevance

“…Briefly, mice were anesthetized with halothane (2-bromo-2-chloro-1,1,1-trifluorothane), and brains were dissected and collected on ice. The cerebellum (ϳ50 mg) was homogenized in 20ϫ (w/v) medium I (0.32 M sucrose, 0.1 mM EDTA, and 5 mM HEPES, pH 7.5; 1 ml) (Nagy and Delgado-Escueta, 1984). The P2 fraction (synaptosome fraction) was suspended with 1 ml of medium I.…”

Section: Methodsmentioning

confidence: 99%

GABA Transporter Deficiency Causes Tremor, Ataxia, Nervousness, and Increased GABA-Induced Tonic Conductance in Cerebellum

Chiu

Brickley²,

Jensen³

et al. 2005

J. Neurosci.

220

170

View full text Add to dashboard Cite

GABA transporter subtype 1 (GAT1) knock-out (KO) mice display normal reproduction and life span but have reduced body weight (female, Ϫ10%; male, Ϫ20%) and higher body temperature fluctuations in the 0.2-1.5/h frequency range. Mouse GAT1 (mGAT1) KO mice exhibit motor disorders, including gait abnormality, constant 25-32 Hz tremor, which is aggravated by flunitrazepam, reduced rotarod performance, and reduced locomotor activity in the home cage. Open-field tests show delayed exploratory activity, reduced rearing, and reduced visits to the central area, with no change in the total distance traveled. The mGAT1 KO mice display no difference in acoustic startle response but exhibit a deficiency in prepulse inhibition. These open-field and prepulse inhibition results suggest that the mGAT1 KO mice display mild anxiety or nervousness. The compromised GABA uptake in mGAT1 KO mice results in an increased GABA A receptor-mediated tonic conductance in both cerebellar granule and Purkinje cells. The reduced rate of GABA clearance from the synaptic cleft is probably responsible for the slower decay of spontaneous IPSCs in cerebellar granule cells. There is little or no compensatory change in other proteins or structures related to GABA transmission in the mGAT1 KO mice, including GAT1-independent GABA uptake, number of GABAergic interneurons, and GABA A -, vesicular GABA transporter-, GAD65-, and GAT3-immunoreactive structures in cerebellum or hippocampus. Therefore, the excessive extracellular GABA present in mGAT1 KO mice results in behaviors that partially phenocopy the clinical side effects of tiagabine, suggesting that these side effects are inherent to a therapeutic strategy that targets the widely expressed GAT1 transporter system.

show abstract

Section: Methodsmentioning

confidence: 99%

GABA Transporter Deficiency Causes Tremor, Ataxia, Nervousness, and Increased GABA-Induced Tonic Conductance in Cerebellum

Chiu

Brickley²,

Jensen³

et al. 2005

J. Neurosci.

220

170

View full text Add to dashboard Cite

show abstract

“…Brain samples were processed according to the synaptosomal preparation described by Nagy and Delgado-Escueta (1984). Briefly, the amygdalae of each animal were homogenized in 2 ml of buffer [0.32 M sucrose, 1 mM EDTA, 1 mg/ml BSA, 5 mM HEPES (pH 7.4, reached using 1 M NaOH)] by use of a Dounce homogenizer (10 strokes and 520 rpm).…”

Section: Synaptosome Preparationmentioning

confidence: 99%

TrkB Modulates Fear Learning and Amygdalar Synaptic Plasticity by Specific Docking Sites

Musumeci

Sciarretta²,

Rodríguez‐Moreno³

et al. 2009

J. Neurosci.

View full text Add to dashboard Cite

Understanding the modulation of the neural circuitry of fear is clearly one of the most important aims in neurobiology. Protein phosphorylation in response to external stimuli is considered a major mechanism underlying dynamic changes in neural circuitry. TrkB (Ntrk2) neurotrophin receptor tyrosine kinase potently modulates synaptic plasticity and activates signal transduction pathways mainly through two phosphorylation sites [Y515/Shc site; Y816/PLC␥ (phospholipase C␥) site]. To identify the molecular pathways required for fear learning and amygdalar synaptic plasticity downstream of TrkB, we used highly defined genetic mouse models carrying single point mutations at one of these two sites (Y515F or Y816F) to examine the physiological relevance of pathways activated through these sites for pavlovian fear conditioning (FC), as well as for synaptic plasticity as measured by field recordings obtained from neurons of different amygdala nuclei. We show that a Y816F point mutation impairs acquisition of FC, amygdalar synaptic plasticity, and CaMKII signaling at synapses. In contrast, a Y515F point mutation affects consolidation but not acquisition of FC to tone, and also alters AKT signaling. Thus, TrkB receptors modulate specific phases of fear learning and amygdalar synaptic plasticity through two main phosphorylation docking sites.

show abstract

“…Synaptic vesicles were isolated from the striatum of C57BL/6J adult males (n ϭ 8) essentially as described (42). Presynaptic and postsynaptic protein samples were prepared from the striatum of adult male C57BL/6J mice (n ϭ 12); synaptosomes were prepared by Percoll gradient purification (43) and were solubilized by sequential extraction as reported (17). Protein samples were separated by SDS/PAGE and analyzed by Western blot.…”

Section: Animals Male and Female Homozygous Gpr37mentioning

confidence: 99%

GPR37 associates with the dopamine transporter to modulate dopamine uptake and behavioral responses to dopaminergic drugs

Marazziti

Mandillo

Pietro

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

100

106

View full text Add to dashboard Cite

The orphan G protein-coupled receptor 37 (GPR37) is a substrate of parkin; its insoluble aggregates accumulate in brain samples of Parkinson's disease patients. We report here that GPR37 interacts with the dopamine transporter (DAT) and modulates DAT activity. GPR37 and DAT were found colocalized in mouse striatal presynaptic membranes and in transfected cells and their interaction was confirmed by coimmunoprecipitation assays. Gpr37-null mutant mice showed enhanced DAT-mediated dopamine uptake in striatal membrane samples, with a significant increase in the number of plasma membrane DAT molecules. The null mutant mice also exhibited a decrease in cocaine-induced locomotor activity and in catalepsy induced by dopamine receptor antagonists. These results reveal the specific role of GPR37, a putative peptidergic G proteincoupled receptor, in modulating the functional expression of DAT and the behavioral responses to dopaminergic drugs.G protein-coupled receptor ͉ Parkinson's disease ͉ dopamine transporter T he orphan G protein-coupled receptor 37 (GPR37) is homologous to endothelin (ET B -R) and bombesin (GRP-R, NMB-R) receptors (1) and it is highly expressed in mammalian brain oligodendrocytes, Purkinje cells, and neurons belonging to the CA3 hippocampal region and to the substantia nigra (SN) pars compacta (2). GPR37 is a substrate of the ubiquitin-protein ligase parkin, and it has been named parkin-associated endothelin-like receptor (PAEL-R) (3). An insoluble form of GPR37 is accumulated in brain samples of Parkinson's disease (PD) patients, and the overexpression of GPR37 in cell cultures, in the absence of parkin, can lead to unfolded protein-induced cell death (3, 4). Little is known about the physiological function of the receptor in the brain and in dopaminergic neurons in particular, although it has been speculated that the aggregation of GPR37 in insoluble complexes is responsible for the preferential loss of SN neurons through the endoplasmic reticulum-specific apoptotic pathway (5, 6). Recent data reported an interaction between GPR37, the head activator neuropeptide and its binding protein (sorting protein-related receptor; SorLA), supporting the hypothesis that GPR37 is involved in neuronal cell survival (7). To investigate the receptor's function, we generated homozygous Gpr37-null mutant mice, which exhibit a reduction in striatal dopamine (DA) content, specific locomotor deficits, and enhanced sensitivity to amphetamine (8).Several binding partners for the DA transporter (DAT) have been identified, suggesting that a regulated multiprotein complex controls its synthesis, targeting, and expression at specific cellular membrane domains (9). Presynaptic DAT expression is of crucial importance in modulating the synaptic availability of DA at nigrostriatal synapses, and its regulation is dynamically controlled for the maintenance of normal dopaminergic neurotransmission. The increase or decrease of DAT expression in the presynaptic membranes results in decreased or increased synaptic DA concentration, ...

show abstract

Rapid Preparation of Synaptosomes from Mammalian Brain Using Nontoxic Isoosmotic Gradient Material (Percoll)

Cited by 325 publications

References 55 publications

GABA Transporter Deficiency Causes Tremor, Ataxia, Nervousness, and Increased GABA-Induced Tonic Conductance in Cerebellum

GABA Transporter Deficiency Causes Tremor, Ataxia, Nervousness, and Increased GABA-Induced Tonic Conductance in Cerebellum

TrkB Modulates Fear Learning and Amygdalar Synaptic Plasticity by Specific Docking Sites

GPR37 associates with the dopamine transporter to modulate dopamine uptake and behavioral responses to dopaminergic drugs

Contact Info

Product

Resources

About