Application of 4-aminopyridine (4-AP) along with EAA) receptor antagonists produces γ-aminobutyric acid (GABAA) receptor-dependent synchronized activity in interneurons. This results in waves of activity propagating through upper cortical layers. Because interneurons in the neocortex are excited by nicotinic acetylcholine receptor (nAChR) agonists, ACh may influence synchronization of these local neocortical interneuronal networks. To study this possibility, we have used voltagesensitive dye imaging using the fluorescent dye RH 414 (30 μM) in rat neocortical slices. Recordings were obtained in the presence of 4-AP (100 μM) and the EAA receptor antagonists D-2-amino-5-phosphonvaleric acid (20 μM) and 6-cyano-7-nitro-quinoxaline-2,3-dione (10 μM). In response to intracortical stimulation, localized or propagated activity restricted to upper cortical layers was seen. Bath application of the ACh esterase inhibitor neostigmine (10 μM) and the nAChR agonist 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP; 10 μM) increased the response amplitude, the extent of spread, and the duration of this activity. These changes were seen in 13 of 16 slices tested with neostigmine (10 μM) and 4 of 7 slices tested with DMPP (10 μM). Application of the muscarinic AChR antagonist atropine (1 μM) did not block the enhancement of activity by neostigmine (n = 7). Application of dihydro-β-erythroidine (10 μM), known, at this concentration, to selectively antagonize α4β2-like nAChRs, blocked the effect of neostigmine (n = 5). The selective α7-like nAChR antagonist methyllycaconitine (50 nM) was ineffective (n = 5). These results suggest that activation of α4β2-like nAChRs by endogenously released ACh can enhance synchronized activity in local neocortical inhibitory networks. Copyright © 2006 The American Physiological Society.