3 ± 0.7 pA/pF, p < 0.01, n = 26), and the frequency and amplitude of sIPSCs were increased (p < 0.01, Figure 3A; p < 0.001, Figure 3F). There was no change in baclofen current density (11.3 ± 0.8 pA/pF, p = 0.57, n = 15). Therefore, spontaneous dopamine transmission, like GABA- and glutamate-dependent transmission, was increased by a single exposure to cocaine. In vitro, dopamine neurons fire Baf-A1 chemical structure action potentials in a regular, pacemaker pattern. Electrical stimulation causes a sulpiride-sensitive pause in firing, indicating that eIPSCs inhibit spontaneous firing (Beckstead and Williams, 2007; Beckstead et al., 2004; Courtney et al.,

2012). Loose cell-attached recordings were made from SN dopamine neurons from wild-type mice to assess whether sIPSCs can inhibit firing. A single electrical stimulus caused a pause (e-pause) in pacemaker firing (Figures 4A and 4C). Spontaneous pauses (s-pause) occurred approximately

1 event/min (Figures 4A and 4B). Exposure to L-DOPA (10 μM, 10 min) increased the frequency of s-pauses (p < 0.05, n = 13 cells, Figures 4A and 4B). The s-pauses were abolished by sulpiride (600 nM, p < 0.01, Figure 4A), indicating that the s-pauses were the result of D2 receptor activation. The duration of the pauses were calculated by subtracting the mean interspike interval (ISI) from the time between two action potentials (Figure 4C). and The duration of the s-pause was shorter (71%) than the e-pause (p < 0.01, n = 59 Regorafenib in vivo e-pauses and 50 s-pauses, Figure 4D). It is interesting to note that the difference in duration

is the same as that when comparing the evoked and spontaneous IPSCs (Figure 4D). After L-DOPA, the duration of the s-pause remained shorter than the e-pause (74% of e-pause, p < 0.001, n = 130 e-pauses and 255 s-pauses, Figure 4E). In a separate set of experiments, reserpine was used to verify that vesicular release of dopamine underlies the s-pauses. Application of reserpine (1 μM, 10 min) dramatically reduced the frequency of s-pauses (L-DOPA: 2.43 ± 0.8 per min; reserpine: 0.15 ± 0.1 per min; paired two-tailed t test, p = 0.03, n = 8 cells). Taken together, these results indicate that sIPSCs and spontaneous pauses result from spontaneous dopamine release. Thus, spontaneous release of dopamine in brain slices influences the firing pattern of dopamine neurons. The presence of spontaneous miniature GIRK-mediated IPSCs is the strongest evidence to date that signaling mediated by GPCRs can be similar to transmission mediated by ligand-gated ion channels. First, the results of this study demonstrate that despite the slow intrinsic signaling kinetics of GPCR activation, these receptors can signal in a point-to-point manner, in which the presynaptic site of release is located very close to postsynaptic receptors.