Mni glutamate

All experiments were performed blind to drug treatment or CSDS behavioral phenotype. Animals were perfused with ice-cold artificial cerebrospinal fluid (ACSF; see Supplementary Methods). Coronal slices (200 μm thick) containing NAc were cut with a microslicer, transferred for 1 hour to a holding chamber containing sucrose-ACSF (where 254 mM sucrose replaced NaCl) at 34°C and subsequently maintained at room temperature (20–22°C) until use. Individual slices were transferred to a recording chamber mounted on an upright microscope (Olympus BX61WI) and continuously perfused (2–3 ml per minute) with oxygenated ACSF also containing 3.5 mM MNI-glutamate (4-methoxy-7-nitroindolinyl-caged-L-glutamate; Tocris) and 10 μM D-serine (Sigma). Recordings were performed at room temperature. Whole-cell voltage-clamp recordings were made from D1-MSNs (EGFP-) and D2-MSNs (EGFP+) in the NAc shell region. Previous studies have established that EGFP⁻ cells in Drd2-EGFP mice reliably represent D1 MSNs in dorsal striatum (25 (link)), although we recognize that a clean separation of these two cell types is more ambiguous in the NAc (26 (link); 27 (link)). Cells were held at −70 mV. Recordings from EGFP⁺ and EGFP⁻ cells were interleaved.

All drugs unless otherwise noted were purchased from Sigma. All drugs in the slice experiments were bath applied. In dextran-treated experiments, the solution was applied after the rise and decay of the slow EPSC became stable for ~ 7 minutes. Receptor antagonists used this study contained: GYKI-53655 (AMPAR; Tocris), JNJ-16259685 (mGluR1; Tocris), LY-341495 (group II mGluR; Tocris), MK-801 (NMDAR; Sigma), picrotoxin (GABA_AR; Sigma), strychnine (GlyR; Sigma). MNI-glutamate was purchased from Tocris. Dextran (MW 35000 – 45000) was purchased from Sigma.

MNI-glutamate (2 mM, Tocris Biosciences) and RuBi-GABA (5–10 μM, R&D Systems) were puffed onto the tissue from a glass pipette. Photolysis of MNI-glutamate and RuBi-GABA was performed with light from a mercury arc lamp (X-cite, Excelitas Technologies). Light was delivered to the entire tissue in 50 ms pulses through the data-acquisition software (Symphony, Symphony-DAS, Seattle, USA). MGCs were identified based on their spike responses as shown in Figure 1 for fovea and periphery. Light-evoked responses were blocked by perfusing with a solution containing cadmium chloride (200 μM). Responses to uncaged GABA and glutamate were measured at the excitatory and inhibitory reversal potentials. A limitation in quantitative interpretation of these experiments is that previous studies have shown that MNI-glutamate can inhibit GABA receptors (Fino et al., 2009 (link); Maier et al., 2005 (link)). This could explain why the amplitude of GABA-mediated current is smaller than expected from the immunohistochemical analysis for both foveal and peripheral MGCs. Nonetheless, the relative amplitude of inhibitory to excitatory current is larger for peripheral MGCs compared to their foveal counterparts, consistent with the rest of our experiments.