Visualization of agonist-stimulated inositol phospholipid turnover in individual neurons of the rat cerebral cortex and hippocampus

Abstract

A novel autoradiographic method to identify individual neurons responding to neurotransmitter stimulation with increased phosphoinositide turnover is described. When phosphoinositide-coupled receptors are activated, phosphatidylinositol 4, 5-bisphosphate is hydrolysed by phospholipase C generating the two second messengers, inositol 1, 4, 5-trisphosphate and diacylglycerol. During prolonged receptor stimulation, both second messengers are actively recycled to maintain the effective intracellular levels of agonist-sensitive phosphoinositides. Lithium ions inhibit this recycling pathway by blocking the recovery of free inositol from inositol 1, 4, 5-trisphosphate thus leading to the accumulation of phosphatidyl cytidine monophosphate, a membrane bound molecule which is the activated precursor of the synthesis of phosphoinositides. Therefore, addition of excess myo-inositol reverts the effects of lithium inhibition. Thus, taking advantage of this fact and using [ 3H]cytidine as precursor, phosphatidyl [ 3H]cytidine monophosphate accumulation was induced in rat neocortical and hippocampal slices after muscarinic or metabotropic glutamate receptor stimulation. The labelled slices were then fixed, dehydrated and embedded in Durcupan resin. Semithin sections (1 μm thick) were cut and exposed to autoradiographic emulsion for several weeks. Biochemical analysis of the incorporation of [ 3H]cytidine into the chloroform extracted (containing lipids) and the alkali-solubilized (containing nucleic acids and proteins) fractions were carried out in parallel with morphological studies. The stimulation of both receptor types induced labelling of neurons in neocortex and hippocampus. In labelled cells silver grains were characteristically observed over the cytoplasm surrounding the nucleus and main dendritic processes. The anatomical location and distribution of labelled cells as well as the levels of response obtained in both brain regions studied, was found to be receptor specific. Inclusion of 30 mM myo-inositol in the incubation media reversed completely both the accumulation of phosphatidyl [ 3H]cytidine monophosphate and the labelling of cells, thus demonstrating that the label detected autoradiographically corresponds to phosphatidyl [ 3H]cytidine monophosphate. It is concluded that the method is sensitive and specific, allowing identification of individual neurons in both neocortical and hippocampal slices and after stimulation of both muscarinic and metabotropic glutamate receptor subtypes. The method may open a new means to study the phosphoinositide second messenger signalling pathway and the cells in which it takes place.