J Physiolo<w (Paris) (1995) 89, 137-145 137 ©Elsevier. Paris Overexpression of choline acetyltransferase reconstitutes discrete acetylcholine release in some but not all synapse formation-defective neuroblastoma cells ZG Zhong a, H Misawa b, S Furuya c, Y Kimura a, M Noda a, S Yokoyama a, H Higashida a* aDepartmenl q( Biophysics. Kanazawa Unieetwity School ql" Medicine, 13 I Takara-machi, Kamtz, wa 920: hDepartment of Neurology, Tokyo Metropolitan Institute for Neuroscience, Tokyo 183: CNational hzslitute .[br Physiological Sciences, Okazaki 444, Japan (Received 29 December 1994: accepted 2 February 1995) Summary -- Secretion of acetylcholine (ACh) in neuroblastoma cells overexpressingcholine acctyltransferase(CHAT)was exalnined. With transient transfection of ChAT eDNA, neuroblastoma cells, which have no endogenous ChAT and either adhere to myotubes or not. failed to form functional synapses, and thus no evidence for release of ACh was detected. Stable neurohlastoma cell lines overexpressing ChAT accumulated ACh inside the cell. and slowly released ACh to the outside of the cell in a calcium independent fashion. However, after co-culturing them with rat muscle cells, these transformed cells adhered to myotubes and ACh was secreted in a discrete fashion into the synaptic cleft efficiently in some neuroblastomacell lines but rather inefficientlyin another cell line. The results show that the latent secretion machinery of ChAT overexpressing neuroblastoma cells either is competent or possess defect(s) in ACh release. synapse formation / neuromuscularjunction / vesicular acetylcholine secretion / eDNA Introduction Cholinergic neurons which secrete acetylcholine (ACh) play an important role in muscle contraction (Katz, 1966) as well as in learning, memory and sleep (Bartus et al, 1982; Hasselmo and Bower, 1993; Sarter and Bruno, 1994). Choline acetyltransferase (CHAT; EC 2.3.1.6) is the enzyme that is responsible tbr the synthesis of ACh (Wu and Hersh. 1994). Full-length complementary DNAs (cDNAs) encoding mammalian ChAT have been cloned and sequenced (see table I of Wu and Hersh, 1994). Regu- lation of this gene has been studied (Hersh el al, 1993; Misawa el d, 1992, 1993, 1994; Quirin-Stricker et aJ. 1994; see also Wu and Hersh, 1994). Furthermore, re- cently, possible co-ordinate expression of ChAT with ve- sicular ACh transporter has been reported (Bejanin el a/. 1994; Efickson et al, 1994). ChAT cDNAs have been expressed in CHO cells (Ishii el a/, 1990; Oda e/al, 1992), insect cells (Habert e¢ d, 1992), fibroblast cells (Schin- stine e¢ a/, 1992; Misawa et al, 1994) and neuroblastoma cells (Ishii et al, 1990; Kimum e¢ a/. 1992; Misawa et al, 1994; Zhong e/d, 1995). These genetically engineered cells, specially neuroblastoma cells overexpressing CHAT, would be useful for the study of cholinergic phenotype expression, such as ACh release into the synaptic cleft. To monitor discrete ACh output from cells, skeletal muscle cells which possess nicotinic ACh receptors are frequently used as a biological and chemical sensor, since the time resolution is in the millisecond range (Sakmann, *Correspondence and reprints 1992). There are two ways to detect ACh released from presynaptic cells in the in vitro culture condition by elec- trical recording of nicotinic ACh responses in muscle cells. One is Nelson's method of co-culturing cells with fused myotubes to facilitate synaptic contacts (Nelson el al, [976; Nirenberg et d, 1983b). The other is to approach the cell closely with an excised outside-out patch of muscle membrane at the tip of a patch pipette to the surtitce of secreting cells (Hume et al, 1983; Yong and Poo, 1983). The former synapse formation experiments in co-culture may have one disadwmtage in that synaptic contact is greatly influenced by the factor of extracellular matrix (Chow, 1990). However, it may have an advantage in that ACh release can be exanlined under a more physiological condition. Furthermore, the co-culture method provides to study developmental changes of cell-cell interaction (Fu- mya and Fumya, 1983: Fumya et d, 1983). Here, we examined whether ACh can be secreted into the synaptic cleft from neuroblastoma cells tran- siently and stably transfected with CHAT. We demon- strate two typical results in recording synaptic responses in co-cultured myotubes. By transient trans- fection of neuroblastoma cells with CHAT, transformed cells are unable to produce miniature end-plate poten- tials (MEPPs) in myotubes, in stably transformed cells up-regulation of MEPP frequency is efficiently or less efficiently obse~,ed in a cell line-specific manner, un- veiling latent structure of secetou machinery utilized in neuroblastoma cells. Three factors, synthesis and re- lease of ACh and adherence to myotubes, are used for dissecting synapse formation processes in neu- roblastoma cells.