A, Strategy for induction of bidirectional gene expression of ChR2 tagged with mCherry and HaloR tagged with EGFP under tetracycline-transactivator (tTA) control. Bi-tetO, bidirectional tet promoter; pA, poly adenylation signal. BTR mice were bred with αCaMKII-tTA mice to confer Str-specific expression. B, EGFP epifluorescence in BTR:: αCaMKII-tTA bigenic mice atlow magnification, in a fixed 100 μm sagittal section. EGFP fluorescence revealed expression in the dorsal Str and the projections. Str, striatum; GP, globus pallidus; SN, substantia nigra. C–E, Epifluorescence of EGFP and mCherry in an acute 400 μm sagittal slice. C, Lower magnification images of the Str and the GP with EGFP (C1) and mCherry (C2) showed Str-specific expression and expression in the axon terminals of MSNs in the GP. D, Lower magnification images of the SN with EGFP (D1) and mCherry (D2) showed expression in the axon terminals of MSNs in the SN. E, High magnification images of EGFP in the Str showed many small puncta, presumably HaloR aggresomes (E1). In contrast, mCherry fluorescence (E2) was seen in fewer but clearly identifiable cells, revealing ChR2 in the plasma membrane.

A, ChR2 response in mCherry MSNs under voltage clamp (A1) and current clamp (A2). Excitation of ChR2 robustly activated mCherry MSNs (left traces); with intracellular QX314 to block Na⁺ currents, the underlying current or depolarization was evident (right traces). B, Light-evoked responses in mCherry MSNs were not blocked by gabazine (10 μM, red trace) or CNQX (40 μM, orange trace). C, Current-voltage relationship of the ChR2 response. C1, Sample traces of the ChR2 responses at different membrane potentials. Averages of 10 traces are shown. C2, Current-voltage relationship for ChR2 responses. n = 6 cells. D, E, Optimizing photostimulation protocol for synaptic studies. D, Duration was varied from 1 to 100 ms. D1, Typical responses recorded under voltage clamp (left) and current clamp (right). D2, Peak amplitude of the response (relative to 2 ms illumination) under voltage clamp (open circles) and current clamp (closed circles) for each illumination duration. n = 6 cells. E, Stimulus frequency ranged from 0.1 to 5 Hz. E1, Typical traces for the first (red), second (gray) and fiftieth (black) traces at 0.1 Hz (top) and 2 Hz (bottom) stimulation. E2, Peak amplitudes of ChR2 responses during 50 pulse stimulation at 0.1 Hz (open circles; n = 6 cells), 0.5 Hz (closed circles; n = 6 cells) and 2 Hz (open triangles; n = 6 cells). Amplitudes are expressed as the ratio of the initial response (red markers). (E3) Reduction of peak amplitude of ChR2 response at the 50th stimulation. Data are shown as the ratio of the first pulse for each stimulus frequency. n = 6 cells.

Light-evoked IPSCs in the dorsal Str from a non-mCherry (dark) MSN (left), TAN (tonically active neuron, middle) and FSI (fast spiking interneuron, right). A1, Schematics of the Str circuitry with an electrode indicating the cell type recorded. mCherry MSNs expressing ChR2 are red. Non-mCherry MSNs with projection fibers, larger cells, identified as TANs (which are the large cholinergic interneurons), and other interneurons without projection fibers, mainly FSIs, are in black. Blue ovals indicate the area of photostimulation. A2, A3, Cell types were identified by their spontaneous firing pattern (A2) and response to current injection (200–300 pA) under current clamp (A3). TANs fired spontaneously (A2, middle). A4, A5, Light-evoked IPSCs. Individual IPSC traces (3 traces superimposed; A4) and averages of 10 traces (A5) are shown by cell type. Blue bars indicate timing of photostimulation (EGFP filter, 10 msec, 0.1 Hz). Short illumination evoked synaptic responses in MSNs and TANs, which were completely blocked by gabazine (10 μM; gray traces), while no apparent response was observed on FSIs. B, Summary of the amplitudes of recorded light-evoked IPSCs in each cell type. Average amplitudes for cells with IPSCs (with connection, C+; open bars) and average amplitudes from cells without significant IPSCs (no connection, C-; closed bars) are shown. The number of cells recorded is shown in parentheses. No FSIs showed light-evoked IPSCs.

A, Light-evoked IPSCs in the GP. A1, Schematic shows GABAergic inputs to the GP. mCherry MSNs are indicated in red; other MSNs and target neurons are indicated in black. A2, A3, Target neurons were classified by their spontaneous firing pattern (A2) and response to hyperpolarizing current injection (A3). A4, A5, Individual light-evoked IPSCs (3 traces superimposed, A4) and averages of 10 traces (A5) are shown by cell type. Type B/C neurons in the GP showed light-evoked IPSCs, which were blocked completely by gabazine 10 μM (gray traces, A5). B, Summary of the amplitudes of recorded light-evoked IPSCs in GP cells. Average amplitudes for cells with IPSCs (C+; open bars) and average amplitudes from cells without significant IPSCs (C-; closed bars) are shown; the number of cells recorded is shown in parentheses. Light-evoked IPSCs were mainly seen in Type B/C cells. C, Light-evoked IPSCs in the SN. C1, Schematic of the circuitry. C2, C3. SNr GABA and SNc DA neurons were identified by their location (SNr or SNc), spontaneous firing patterns (C2) and response to hyperpolarization (C3). C4, C5, Individual light-evoked IPSCs (3 traces superimposed, C4) and averages of 10 traces (C5) are shown by cell type. SNr GABA neurons showed light-evoked IPSCs, which were blocked completely by gabazine 10 μM (gray traces, C5). D, Summary of the amplitudes of recorded light-evoked IPSCs in SN cells. Light-evoked IPSCs were seen in all but one SNr GABA neuron, while none were seen in SNc neurons.

Schematic diagrams show the putative location of GABAergic synaptic inputs in the GP (left), SNr (middle) and SNc (left), with inputs from mCherry MSNs in red. The bar graph shows the comparison in the three cell types of light-evoked IPSCs in recordings with K⁺-based pipette solution (white bars) to recordings with Cs⁺-based pipette solution (gray bars). The number of cells recorded is shown in parentheses; * indicates p < 0.05 (un-paired t-test). In the GP, Cs+ increased IPSC amplitude (left; so in the schematic MSN inputs are shown more on distal dendrites). In the SNr, Cs+ had no effect on IPSC amplitude (middle; so in the schematic MSN inputs are shown on more proximal dendrites). While, in the SNc, no light-evoked ISPCs were seen, even with the increased space clamp with Cs+ (right; so in the schematic only black GABAergic inputs are shown).

A1, Schematic diagram showing indirect pathway MSN projection to the GP (lower red Str neuron), and direct pathway MSN (upper red Str neuron) projection through the GP to the SN, with collaterals in the GP. Recordings were obtained from putative Type B/C GP neurons identified by light-evoked IPSCs, and DA modulation tested. A2, The D1 agonist SKF83822 (2 μM) had no effect on the light-evoked IPSC. A3, The D2 agonist quinpirole (2 μM) inhibited the light-evoked IPSC, which was reversed by the D2 antagonist sulpiride (10 μM). B, Summary of effects in the GP for D1 agonist (SKF), D2 agonist (quin) and D2 antagonist (sul) on light-evoked IPSCs, expressed as the percent of the preceding control IPSC amplitude in saline. The dashed line (100%) indicates no effect. The number of cells recorded is shown in parentheses; ** indicates p < 0.01 (one-sample t-test). C1, Schematic shows direct MSN projections to SNr GABA neurons. C2, SKF83822 (2 μM) facilitated the light-evoked IPSC, which was reversed by the D1 antagonist SCH23390 (10 μM). C3, The D2 agonist quinpirole had no effect on the light-evoked IPSC. D, Summary of effects in the SNr for D1 agonist (SKF), D1 antagonist (SCH) and D2 agonist (quin). * indicates p < 0.05.

Identified neuron populations are shown as circles within the black outlines indicating the dorsal Str (dStr), GP and SN. mCherry MSNs expressing ChR2 are shown in the center (solid gray circle without text). Connections to target neurons are shown as gray arrows. The relative strengths of the connections are indicated by the thickness of the arrows and tone of gray (black for 0% and white for 100%) of circles showing postsynaptic neuron types; arrows were not drawn if there were no connections. In each cell, the top number indicates the percent of target neurons showing detectable light-evoked IPSCs, and the bottom number the average amplitude of the IPSCs. For example, in the dStr, 63% of non-mCherry MSNs showed significant light-evoked synaptic responses (of average amplitude 104 pA), no FSIs showed responses, and 75% of TANs showed responses (of average amplitude 17 pA). Since only about 10% of MSNs expressed ChR2 in BTR::αCaMKII-tTA bigenic mice, the amplitudes shown should be scaled about ten-fold to obtain the actual strength of the connections.