A model for studying activation of PLA2 in adult mouse hippocampal neurons

A model for studying activation of PLA2 in adult mouse hippocampal neurons

Field: Development and regeneration

Authors: Gisselsson, Lennart
Ekström, Per

Address of presenting
author: Lennart Gisselsson
Helgona vägen 3b
22362 Lund

Hämtställe 17

E-mail: lennart.gisselsson@zoofys.lu.se

Phone: 070-2116983

Fax: 046-2224539

Text of abstract: The enzyme group of phospholipase A2 (PLA2) has an essential role in the metabolism of membrane phospholipids, which for instance can lead to the production of arachidonic acid (AA). In turn AA is used in the formation of proinflammatory mediators such as prostaglandins and leukotrienes. PLA2 enzymes, which occur in both cytosolic and secreted forms, appear to be present in all type of tissues, including neurons. In the latter, PLA2 may be involved in apotosis, cell survival and axonal outgrowth. We are interested in the function of PLA2 in hippocampal neurons and have therefore looked for a way of studying PLA2 activation in hippocampal primary cultures. In the present model mouse hippocampi are dissociated with papain in Hibernate A medium after which cells are fractionated using a gradient of Optiprep. Cell fractions containing neurons are then separated and grown serum free in Neurobasal A medium supplemented with B27. Seven days after establishing the cultures they form a monolayer of cells with neurite like extensions, which can be stained positive for the neuronal marker beta III tubulin. At this point the cells are incubated overnight with radioactive AA. After removing unincorporated label with several washes, the release of radioactivity to the medium is followed by liquid scintillation counting. The released radioactivity, representing liberated AA, can be taken as a measure of PLA2 activity, which thus can be readily studied and manipulated by the addition of various compounds and factors to the culture medium. The effect of the manipulations on neuronal growth and survival are followed in parallel sister cultures. In conclusion, we have established a model that allows studies of the role of PLA2 activity in cultured hippocampal neurons from the adult mouse.

Keywords: phospholipase A2 (PLA2), hipocampus, neurons, primary cultures, mice

	A model for studying activation of PLA2 in adult mouse hippocampal neurons
Field:	Development and regeneration
Authors:	Gisselsson, Lennart Ekström, Per
Address of presenting author:	Lennart Gisselsson Helgona vägen 3b 22362 Lund Hämtställe 17
E-mail:	lennart.gisselsson@zoofys.lu.se
Phone:	070-2116983
Fax:	046-2224539
Text of abstract:	The enzyme group of phospholipase A2 (PLA2) has an essential role in the metabolism of membrane phospholipids, which for instance can lead to the production of arachidonic acid (AA). In turn AA is used in the formation of proinflammatory mediators such as prostaglandins and leukotrienes. PLA2 enzymes, which occur in both cytosolic and secreted forms, appear to be present in all type of tissues, including neurons. In the latter, PLA2 may be involved in apotosis, cell survival and axonal outgrowth. We are interested in the function of PLA2 in hippocampal neurons and have therefore looked for a way of studying PLA2 activation in hippocampal primary cultures. In the present model mouse hippocampi are dissociated with papain in Hibernate A medium after which cells are fractionated using a gradient of Optiprep. Cell fractions containing neurons are then separated and grown serum free in Neurobasal A medium supplemented with B27. Seven days after establishing the cultures they form a monolayer of cells with neurite like extensions, which can be stained positive for the neuronal marker beta III tubulin. At this point the cells are incubated overnight with radioactive AA. After removing unincorporated label with several washes, the release of radioactivity to the medium is followed by liquid scintillation counting. The released radioactivity, representing liberated AA, can be taken as a measure of PLA2 activity, which thus can be readily studied and manipulated by the addition of various compounds and factors to the culture medium. The effect of the manipulations on neuronal growth and survival are followed in parallel sister cultures. In conclusion, we have established a model that allows studies of the role of PLA2 activity in cultured hippocampal neurons from the adult mouse.

Keywords:	phospholipase A2 (PLA2), hipocampus, neurons, primary cultures, mice

Index

Created 2000-03-14

Department of Physiological Sciences, Lund University

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