Localisation of agonist stimulated CMP-phosphatidate accumulation in single mammalian neurones

Abstract

Phosphatidylinositol bisphosphate (PtdIns(4,5)P2) hydrolysis by receptor stimulated phospholipase C (PLC) generates the ubiquitous second messengers, inositol msphosphate and diacylglycerol. The components of this signalling pathway are especially prominent in the mammalian central nervous system [1,2], but available assays of this response provide no information on the anatomical location of the responding cells, nor do they distinguish between, for example, responding neurons or glial cells. Both products of PLC activity are actively recycled to replenish inositol phospholipid pools and previous work has established that, under appropriate circumstances, CMP-phosphatidate (CMP-PA; the specific lipid precursor of inositol phospholipids), is a convenient and sensitive marker of the membranes supporting agonist-stimulated inositol lipid hydrolysis. Specifically, CMP-PA accumulates in stimulated cells provided that lithium is present to prevent resynthesis of inositol from inositol phosphates [3,4]. In the absence of lithium, inositol combines with CMP-PA to resynthesise phosphatidly inosi tol.CMP-PA is the only lipid which becomes labelled when cells are incubated with [3H]-cytidine [3,4]. Thus the accumulation of [3H]-CMP-PA was recently exploited to map the distribution of agonist stimulated inositol lipid hydrolysis in rat brain slice preparations using standard autoradiographic procedures [5]. We now report substantial modifications to the published procedures which have allowed us to visualise [3H]-CMP-PA accumulation in single neurones; combining biochemical and autoradiographic methods we demonstrate a muscarinic cholinagic receptor-stimulated increase in [3H]-CMP-PA in single hippocampal pyramidal neurones.Hippocampi from adult male Wistar rats (200-2508) were dissected on ice and chopped laterally using a McIlwain tissue chopper. The slices were recuperated in Krebs Ringer bicarbonate (KRB) for 60 min at 37OC with replenishment of fresh oxygenated KRB every 15 min. The slices were then t r a n s f e d to 2Oml flat bottomed glass bottles containing 5pCi of [3H]-cytidine in 590p1 fresh KRB and constantly flushed with 02/C02 (95/5). The slices were incubated for 30 min. before the addition of lop1 of LiCl (final concentration, 10mM) or LiCl plus carbachol (10mM and lmM, respectively) from stock solutions.Incubations were terminated after 60 min. by the addition of ice cold fixative; 2% paraformaldehyde, 2.5% glutaraldehyde in 0.1M phosphate buffered saline (PBS), pH 7.4. Cryoprotectant (20% sucrose, 8% glycerol in 0.1M PBS) was added after 45 min. and the samples were frozen in liquid nitrogen and thawed at room temperature. Ribonuclease treatment was carried out in 0.005% saponin, 3% polyethyleneglycol, 2mM EDTA, 50mM Tris-HC1, pH 7.4 for 60 min. at 3 P C and terminated by the addition of ice cold buffer. The slices were then postfixed in l%OsO4 in 0.1M PBS, dehydrated in increasing concentrations of ethanol with a final treatment in propylenoxide, infiltrated overnight and embedded in Ducurpan resin. Semit...