In the LC of line 3.1, 97% ± 1% of neurons that expressed ChR2-YFP also expressed TH, while 72% ± 6% of neurons that expressed TH also expressed ChR2-YFP (n = 122 for VTA, n = 86 for SN, n = 63 for LC, where n refers to counted cells; Figure 1A). To further characterize these lines, we quantified the copy number of Cre in Th::Cre rats with digital PCR ( Supplemental Experimental Procedures). Across multiple
sublines (Th::Cre 3.1, 3.5, and 4.4), we observed a single copy number of Cre in the genome ( Table S1). We performed a systematic in vitro electrophysiological study of the cellular and optogenetic properties of ChR2-YFP-expressing VTA neurons in Th::Cre+ rats, along with a comparison http://www.selleckchem.com/products/byl719.html of the same properties of neurons in Th::Cre+ littermates injected with a control virus that expressed only
YFP. Figure 2A shows a sample trace from a ChR2-expressing Th::Cre neuron in response to current injection steps, demonstrating the classical “sag” response induced by the hyperpolarizing pulse, the result of a hyperpolarization-activated cation current (Ih) that is present in many TH+ VTA neurons ( Margolis et al., 2006, Lammel et al., 2008, Lammel et al., 2011 and Neuhoff et al., 2002). Given that the VTA TH+ neurons are heterogeneous and do not all express a prominent Ih ( Lammel et al., 2008 and Lammel et al., 2011), in addition to analyzing light responses and intrinsic properties of neurons with a prominent Ih current (Ih/large neurons), we have also included in this http://www.selleckchem.com/products/s-gsk1349572.html analysis cells without a prominent Ih (Ih/small neurons), in either case comparing the properties of neurons that express ChR2-YFP to neurons
that express YFP only. Continuous blue light elicited large inward currents (peak photocurrent: −2950 ± 1574 pA for Ih/large neurons, steady-state photocurrent: −756.5 ± 225.5 pA; n = 7 Ih/large neurons, Figure 2B), and optical stimulation trains produced neural responses that were similar to those evoked by isothipendyl electrical stimulation in both ChR2-expressing or YFP-only-expressing neurons (Figure 2C for pulse trains, Figure S2D for individual waveforms); notably, the amplitude of both optically and electrically evoked spike trains attenuated during the course of the pulse train. Th::Cre ChR2-expressing neurons reliably responded to light-induced spike trains over a range of frequencies from 5 to 40 Hz ( Figure 2D for Ih/large neurons, Figure S2B for Ih/small neurons). Multiple spikes in response to a single light pulse were never observed during the presentation of pulse trains under these expression, illumination, and opsin (ChR2) conditions. We also confirmed that light stimulation at various frequencies (5 to 40 Hz) failed to evoke neural responses in YFP-only-expressing neurons ( Figure S2C).