ChR2 was expressed in SNc DA neurons virally. (A) or genetically (B). (A) Overlay of fifteen consecutive light-evoked (1ms, 473 nm, 5 mW·mm−2; blue line) IPSCs recorded under control conditions at −70 mV from a SPN in the dorsal striatum of a Slc6a3-ires-Cre mouse previously injected in the SNc with an AAV encoding Cre-dependent ChR2-mCherry. The first trace obtained after break-in is in black and subsequent oIPSCs are shown in progressively lighter shades of gray. The magenta trace depicts the average waveform of the first five oIPSCs. Recordings were performed with NBQX (10 μM), R-CPP (10 μM), and CGP55845 (2 μM) in the perfusate. (B) As in (A) for a SPN recorded under control conditions in the dorsal striatum of a Slc6a3-ires-Cre;Ai32 mouse. (C) Plot of the amplitude of consecutive oIPSCs shown in (A; magenta) and (B; green) over time. (D) As in (C) for oIPSCs recorded in dorsal striatum SPNs from AAV-infected mice (n = 17; magenta), Ai32 mice (n = 8; green), or both (n = 25, black). Amplitude is normalized to the first oIPSC after break-in. Note how the amplitude of oIPSCs progressively decreases with each stimulus under control conditions, regardless of the method used for expressing ChR2. This decrement in oIPSC amplitude is specific to dopaminergic synapses, as oIPSCs recorded from the collaterals of iSPNs in Adora2a-Cre expressing ChR2-mCherry in the dorsal striatum remained maintained their amplitude for the duration of the recording (n = 8; gray). Data represent mean ± SEM. *p<0.001 vs black trace; Sidak's multiple comparison test. (E) Mean latency from flash onset to oIPSC onset in individual dorsal striatum SPNs recordings from AAV-infected (magenta) and Ai32 (green) mice. The difference in synaptic delay are likely due to differences in expression levels of ChR2. Mean (±SEM) shown in red. *p<0.001, Mann–Whitney test. (F) Mean standard deviation (SD) of the synaptic latency of oIPSCs in individual cells indicates little temporal jitter in both experimental models, in agreement with monosynaptic transmission. Mean (±SEM) shown in red. n.s., no significant difference between means (p=0.8; Mann–Whitney test). (G) Time course of oIPSC amplitude rundown starting at the time of drug application (or the 5th stimulus for control recordings in ACSF). The number of SPN recordings in each condition are: ACSF, 25; picrotoxin, 19; SR95531, 11; SKF 89976A, 9; SKF 89976A+SNAP-5114, 8. Changes in the decay kinetics of oIPSCs shown in Figure 2 were quantified 3–4 min after initiating the perfusion of SKF89976A, just as the effects of the GABAA receptor antagonists begin to significantly inhibit oIPSCs. Note that acute application of GAT antagonists does not immediately affect the amplitude of oIPSCs in SPNs. Data represent mean ± SEM. *p<0.01, picrotoxin or SR95531 vs black trace; Tukey's multiple comparison test.