Excess extracellular AChE decreases the association between neurexin and neuroligin and inhibits neuroligin-induced presynaptic specialization. A. Cell lysates were prepared from HEK293 cells transfected with neurexin-1β-1’-His (Nrxn-1β-1’) alone (lane 1) or neurogligin-1 (Nlgn-1) alone (lane 2) or both (lanes 3 and 4). Neurexin-1β-1’ was proved by blotting His (top panel), and neuroligin-1 was confirmed by a neuroligin antibody (middle panel). Immunoprecipitation of His co-precipitated a heavy band of neuroligin from the lysate of cells expressing both proteins (upper band in lane 3 of bottom panel). Immunoprecipitating His co-precipitated a smaller band of neuroligin from the lysate of cells cultured in AChE-conditioned medium (upper band in lane 4 of bottom panel). B. Images of synapsin in primary neurons co-cultured with HEK293 cells transfected with or without neuroligin-1. Left column, first row: HEK293 cells transfected with GFP alone; second row: Cells transfected with GFP and neuroligin-1; third row: Cells transfected with GFP and neuroligin-1 and treated with AChE-conditioned medium. Middle column: Axonal neurites of hippocampal neurons were identified by immunostaining of synapsin. No synapsin clusters were seen in axonal neurits passing through GFP-expressing cells (Control; top row); whereas dense synapsin-clusters were formed on neuroligin-transfected cells (Nlgn; middle row). Less synapsin-clusters were formed on neuroligin-transfected cells grown in AChE-conditioned medium (Nlgn + AChE; bottom row). Right column; Overlay images of GFP-expressing cells and synapsin-positive structures. B’. Insets illustrate synapsin-clusters associated with control cells (top), Nlgn-expressing cells (mid) and Nlgn-expressing cells in AChE-conditioned medium (bottom). C. Number of synapsin-positive clusters in single cells transfected with vector (Ctrl, 0.8 ± 0.2, n = 36 cells), with neuroligin-1 (Nlgn, 22.5 ± 2, n = 19) or with neuroligin-1 and in AChE-conditioned medium (Nlgn + AChE, 8.7 ± 1.3, n = 22, * P < 0.05, relative to conditions without AChE).