成年大鼠脑突触体的制备、刺激及其他用途

Jigar Modi; Howard Prentice; Jang-Yen  Wu

doi:10.21769/BioProtoc.2664

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Peer-reviewed

Preparation, Stimulation and Other Uses of Adult Rat Brain Synaptosomes

成年大鼠脑突触体的制备、刺激及其他用途

JM Jigar Modi

HP Howard Prentice email

JW Jang-Yen Wu email

发布: 2017年12月20日第7卷第24期 DOI: 10.21769/BioProtoc.2664 浏览次数: 6909

评审: Anca Flavia SavulescuAnonymous reviewer(s)

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Cover of Molecular Neurobiology, featuring study using the protocol.

Mar 2017

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Abstract

In this paper, our protocol for preparation of brain synaptosomes is described. Synaptosomes are a valuable model system for analysis of structural components of the synapse as well as for investigation of synaptic function. Synaptosomal preparations are necessary for understanding molecular changes at synapses where critical post-translational modifications of synaptic proteins may occur. Not only are synaptosomes rich in synaptic proteins, but they can be used for analyzing uptake of neurotransmitters into synaptic vesicles and for analysis of the involvement of neurotransmitter synthesis and release. Synaptosomes can be stimulated with increased calcium influx to release neurotransmitters. Synaptosomal preparations have been used in characterizing calcium dependent phosphorylation and activation of the GABA synthesizing enzyme GAD65 (L-glutamic acid decarboxylase with molecular weight of 65 kDa). By examining protein complexes on the membrane of synaptic vesicles obtained from synaptosomal preparations, it was possible to characterize the role of GAD65 in the coupled synthesis and vesicular uptake of GABA (γ-aminobutyric acid) culminating in GABA vesicular release, which contributes in an important way to fine-tuning of GABAergic neurotransmission.

Keywords: GAD65 (GAD65)

Synaptic Vesicle (SV) (突触囊泡（SV）)

GABA (GABA)

Synaptosomes (突触体)

Background

Synaptosomal preparation methods were established in neuroscience research laboratories over 40 years ago and have been of great value in scientific innovations relating to neurotransmitter release with respect to elevated extracellular potassium as well as responses to increased intracellular calcium. In addition to elucidating the process of neurotransmitter release, synaptosomal preparations have been a valuable source of synaptic vesicles. Studies on synaptic vesicles have for example been used in the characterization of protein components that participate in coupled neurotransmitter synthesis and vesicular release. Synaptosomal preparations have also been very valuable as an intermediate in isolation of synaptic vesicles which are then analyzed in terms of protein complexes located on the vesicular membrane that include neurotransmitter synthesizing enzymes. Of key significance in this regard is the vesicular membrane GAD65 complex which includes CSP (Cystine-String Protein), VGAT (Vesicular GABA transporters) and CaMKII (calcium/calmodulin-dependent protein kinase II). Regulation of GAD65 activity has been further characterized in terms of GAD65 cleavage by calpain and activation of membrane bound GAD through phosphorylation as well as accelerated anchoring of GAD65 to SV by palmitoylation.

The basic methodology for vesicular preparation presented in this paper does not significantly differ from the originally established technology from the 1970s but the applications have been greatly extended over this time and therefore expanded to include analytical investigations on neurotransmitter synthesizing enzymes, on signaling complexes associated with these enzymes, on mechanisms of regulation of these enzymes and on functional outcomes of enzyme activation including vesicular neurotransmitter release in response to high extracellular potassium levels or to increased calcium influx.

Materials and Reagents

Pipette tips (VWR, catalog number: 82028-570 )
Centrifuge tubes (round bottom–50 ml) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 3118-0050 )
15 ml Falcon tubes (VWR, catalog number: 10026-076 )
Glass tube (DWK Life Sciences, Kimble, catalog number: 885451-0022 )
Scintillation vial (Fisher Scientific, catalog number: 03-337-20 )
Experiments involving animals:
The use of animals and animal tissues in this protocol was approved by and in accordance with the requirements of Institutional Animal Care and Use Committee, Florida Atlantic University. The Public Health Service animal welfare assurance number is A3883-01. For all experiments, Adult 250-300 g Sprague-Dawley male rats (Harlan, IL, USA) were used. Two animals per cage were housed and maintained at 22 °C with an alternating 12-h light/dark cycle. The animals were allowed a minimum stabilization period of at least 3 days after which they were utilized for experimentation.
Scintillation cocktail (RPI, catalog number: 111175 )
Sucrose (Fisher Scientific, catalog number: BP220-212 )
HEPES-NaOH (RPI, catalog number: H2393 )
Tris-HCl (Fisher Scientific, catalog number: BP152-1 )
Calcium chloride dihydrate (CaCl₂·2H₂O) (Fisher Scientific, catalog number: C79-500 )
Disodium phosphate (Na₂HPO₄) (Sigma-Aldrich, catalog number: 255793-50G )
Monopotassium phosphate (KH₂PO₄) (Fisher Scientific, catalog number: P286-1 )
Sodium bicarbonate (NaHCO₃) (Sigma-Aldrich, catalog number: S5761 )
Sodium chloride (NaCl) (VWR, catalog number: BDH0286 )
Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: EDS-100G )
Triton X-100 (Sigma-Aldrich, catalog number: X100-100ML )
Mammalian Protease Inhibitor (MPI) (Sigma-Aldrich, catalog number: P8340 )
Phosphatase inhibitor (PhI) (Halt^TM) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 78426 )
2-Aminoethylisothio-uronium bromide (AET) (Sigma-Aldrich, catalog number: A5879 )
Pyridoxal 5’ Phosphate (PLP) (Sigma-Aldrich, catalog number: P9255-25G )
Dipotassium phosphate (K₂HPO₄) (Sigma-Aldrich, catalog number: P8281 )
Pyruvate kinase (Sigma-Aldrich, catalog number: P9136-25KU )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P3911-500G )
Phospho(enol) pyruvate (Sigma-Aldrich, catalog number: P7127-250MG )
Adenosine 5’-triphosphate (ATP) (Sigma-Aldrich, catalog number: A26209-10G )
Magnesium sulfate (MgSO₄) (Fisher Scientific, catalog number: M65-500 )
γ-Amino-n-Butyric acid (GABA) (Sigma-Aldrich, catalog number: A5835 )
Glutamate (Glu) (Sigma-Aldrich, catalog number: G5889-100G )
[³H] Glutamate (Amersham Biosciences, catalog number: TRK445 )
[¹⁴C] GABA (PerkinElmer, catalog number: NEC290E250UC )
Oxirane acrylic beads (Sigma-Aldrich, catalog number: O9754 )
HEPES buffered sucrose solution (see Recipes)
Krebs ringer phosphate (KRP) buffer (see Recipes)
Brain lysis buffer (see Recipes)
Standard GAD buffer (see Recipes)
GPBS buffer (see Recipes)
ATP mixture (see Recipes)

Equipment

Pipettes (VWR, catalog number: 89079-976 )
Guillotine (Stoelting, catalog number: 51330 )
Con-torque homogenizer (Eberbach, model: E2355 , catalog number: 7265)
Avanti J-25 centrifuge (Beckman Coulter, model: Avanti J-25 , catalog number: 363102)
Optima L-90 K centrifuge (Beckman Coulter, model: Optima L-90K , catalog number: 365672)
Tri-Carb 2900TR Liquid Scintillation Analyzer (PerkinElmer, model: Tri-Carb 2900TR )

Software

ImageJ software (ImageJ)
Prism software (GraphPad Software)
SPSS software

Procedure

English

中文翻译

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如何引用

Modi, J., Prentice, H. and Wu, J. (2017). Preparation, Stimulation and Other Uses of Adult Rat Brain Synaptosomes. Bio-protocol 7(24): e2664. DOI: 10.21769/BioProtoc.2664.