Fig. 8. ACh produces biphasic nonsynaptic response in RPeD1 isolated in CM. RPeD1 was cultured alone for 18 hr in either DM or CM before testing the electrophysiological response to neurotransmitter. For neurons cultured in DM, exogenous application of 1 μm ACh (solid line) induced an inhibitory and hyperpolarizing response in a spontaneously firing cell (A, i) or if the cell were held at constant membrane potential (−56 mV; A, ii). MLA (1 μm) blocked the inhibitory response of ACh in RPeD1 neurons cultured in DM (B, i, ii). Cholinergic responses in RPeD1 were partially reduced but not completely blocked by mec (C, i, ii). Co-perfusion of both MLA and mec also completely blocked the ACh-induced nonsynaptic response in RPeD1 (D, i, ii). The hyperpolarizing response to ACh was restored immediately (1–5 min) after drug washout with normal DM (E, i, ii). For neurons cultured in CM, exogenous application of 1 μm ACh (solid line) onto RPeD1 produced a biphasic response in CM (F). Perfusion with the nAChR antagonist blocked these responses (B, C) in the same manner as described for the synapse in Figure 6. That is, MLA (1 μm) blocked the hyperpolarizing component of the biphasic response in CM (G), whereas mec (1 μm) abolished the depolarizing phase of the biphasic response (H). Co-perfusion of these two nAChR antagonists completely blocked the ACh-induced nonsynaptic response in RPeD1 (I). The biphasic response of RPeD1 to ACh recovered immediately after washout with normal DM (J). The action of the nAChR blockers was specific to the ACh response. HMC failed to block the hyperpolarizing response of exogenously applied 1 μmserotonin (5-HT; solid line), seen in this trace as inhibiting spontaneous action potentials in RPeD1 (K). The effect of CM was specific to the postsynaptic RPeD1 neuron. The application of 1 μmACh onto VD4 had the same inhibitory effect regardless of whether VD4 was cultured in DM (L) or CM (M). Solid lines indicate the duration of application ACh, except where noted for 5-HT. RPeD1 neurons were held at a constant membrane potential of −56 mV (A, ii–E, ii, F–J) or allowed to fire spontaneously with no current injection (A, i–E, i, K–M).