Improved Method for Isolating Synaptosomes from 11 Regions of One Rat Brain: Electron Microscopic and Biochemical Characterization and Use in the Study of Drug Effects on Nerve Terminal γ‐Aminobutyric Acid in Vivo
- 1 September 1985
- journal article
- Published by Wiley in Journal of Neurochemistry
- Vol. 45 (3), 879-889
- https://doi.org/10.1111/j.1471-4159.1985.tb04076.x
Abstract
A procedure is described for the rapid preparation of nerve ending particles (synaptosomes) from 11 regions of one rat brain. The synaptosomal fractions have been characterized by electron microscopy and determination of four marker enzymes, i.e., glutamate decarboxylase (GAD), acetylcholinesterase, succinate dehydrogenase, and glycerol 3‐phosphate dehydrogenase. Comparison with a much lengthier standard (Ficoll‐sucrose) preparation showed that the synaptosomal yield of the new procedure was substantially better as judged by both morphological evaluation and protein recovery. The improved synaptosome preparation was used for determination of regional γ‐aminobutyric acid (GABA) levels in synaptosomal fractions. The postmortem increase in GABA level during removal and dissection of brain tissue and homogenization and fractionation procedures could be minimized by rapid processing of the tissue at low temperatures and inclusion of the GAD inhibitor 3‐mercaptopropionic acid (3‐MP; 1 mM) in the homogenizing medium. The addition of GABA (0.2 mM) to the homogenizing medium did not alter the GAGA levels in the synaptosomes, indicating that no significant redistribution of GABA occurred during subcellular fractionation in sodium‐free media. Synaptosomal GABA levels determined in the 11 rat brain areas showed the same regional distribution as the GABA‐synthesizing enzyme GAD. On the basis of these findings, it was suggested that the synaptosome preparation could be used to evaluate the in vivo effects of drugs on nerve terminal GABA. Treatment of rats with a convulsant dose of 3‐MP (50 mg/kg i.p.) 3 min before decapitation significantly lowered synaptosomal GABA levels in olfactory bulb, hippocampus, thalamus, tectum, and cerebellum. The 3‐MP‐induced seizures and reduction of GABA levels could be prevented by administration of valproic acid (200 mg/kg i.p.) 15 min before the 3‐MP injection. The data indicate that the improved synaptosome preparation offers a convenient method of preparing highly purified synaptosomes from a large number of small tissue samples and can provide useful information on the in vivo effects of drugs on regional GABA levels in nerve terminals.Keywords
This publication has 35 references indexed in Scilit:
- In vivo effects of anticonvulsant drugs on nerve terminal (synaptosomal) GABA levels in 11 brain regions of the ratJournal of Neural Transmission, 1985
- Amino Acid Content of Nerve Endings (Synaptosomes) in Different Regions of Brain: Effects of Gabaculine and Isonicotinic Acid HydrazideJournal of Neurochemistry, 1984
- Interactions of Di‐n‐Propylacetate, Gabaculine, and Aminooxyacetic Acid: Anticonvulsant Activity and the γ‐Aminobutyrate SystemJournal of Neurochemistry, 1981
- GABA in plasma and cerebrospinal fluid of different species. Effects of γ‐acetylenic GABA, γ‐vinyl GABA and sodium valproateJournal of Neurochemistry, 1979
- THE EFFECT OF THE CONVULSANT 3‐MERCAPTOPROPIONIC ACID ON ENZYMES OF THE γ‐AMINO‐BUTYRATE SYSTEM IN THE RAT CEREBRAL CORTEXJournal of Neurochemistry, 1973
- ALTERATION OF GABA SYSTEM AND PURKINJE CELLS IN RAT CEREBELLUM BY THE CONVULSANT 3‐MERCAPTOPROPIONIC ACIDJournal of Neurochemistry, 1972
- Isolierung und Kristallisation der Glycerin-3-phosphat-Dehydrogenase aus KaninchenleberHoppe-Seyler´s Zeitschrift Für Physiologische Chemie, 1972
- MERCAPTOPROPIONIC ACID: A CONVULSANT THAT INHIBITS GLUTAMATE DECARBOXYLASE1Journal of Neurochemistry, 1970
- SUBCELLULAR DISTRIBUTION OF ENDOGENOUS AND [3H] γ‐AMINOBUTYRIC ACID IN RAT CEREBRAL CORTEXJournal of Neurochemistry, 1969