These cells are capable of differentiating into post-mitotic neurons and astrocytes, designated NT2.N and NT2.A cells respectively. NT2 neurons exhibit virtually all the functional competencies of central nervous system neurons and they have cholinergic, GABAergic and glutamate receptor and neurotransmitter systems. These cells generate action potentials, as well as sustaining neurotransmitter release and response, as well as expressing functional synapses. They also AG 556 express the high voltage activated L, N, P/Q and R calcium channel currents and calcium activated BK channels which are involved in neuronal hyperpolarisation following action potential firing. Our group has extensive experience of these cells, demonstrating that NT2 neuronal networks signal to NT2 astrocytes in co-culture, and that 3-AQC astrocytic networks communicate via gap junction-mediated and gliotransmitter signalling. In order for an in vitro model to be applicable for the pharmacological study of anticholinergic effects, it must express functional muscarinic acetylcholine receptors. This family of five G Protein-coupled receptor subtypes are widely distributed on multiple organs and tissues and are critical to the maintenance of central and peripheral cholinergic neurotransmission. All five mAChR subtypes have been identified in the brain and their regional expression varies between species. Regarding the intrinsic suitability of the NT2.N/A coculture model, functional mAChRs have previously been identified in NT2.N cells. Additionally, cultured astrocytes exhibit a wide variety of functional neurotransmitter receptors, including all five mAChRs. Astrocytes are now known to respond to signals from cholinergic neurons, with the activation of cellular mAChRs being coupled to an increase in astrocytic intracellular calcium i and with their blockage having the opposite effect. Information processing and brain storage was classically thought to rely solely on neurons. However, it has been demonstrated that astrocytic i increase as a result of cholinergic transmission is crucial to the mechanisms of learning and memory. Thus it was decided to focus on the mAChR- induced calcium responses of NT2 astrocytes in order to develop an in vitro predictive model of the anticholinergic impact of polypharmacy on the CNS. In this study, the confirmation of cholinergic activity in NT2.N/A cultures was achieved via calcium imaging by exposing them to oxotremorine and using the fluorescent calcium dye fluo- 4 to detect Ca2+ mediated network responses in terms of changes to astrocytic i. Blockage of these responses by increasing concentrations of clinically relevant drugs with anticholinergic functionality was examined and the resulting dose-response curves used to rank these drugs and combinations thereof for anticholinergic potential, in comparison with the existing ACB scale as well as with some published SAA values for amitriptyline and dicycloverine.