Ump3 microsyringe pump

Male and female mice C57BL/6J mice at 7 weeks of age were anesthetized with an intraperitoneal injection of a cocktail containing xylazine (10 mg/kg) and ketamine (100 mg/kg) diluted in saline and mounted onto a stereotaxic frame, as described elsewhere ^{36 (link), 44 (link)}. Cre-dependent AAV5-hSyn-DIO-hM3D(Gq)-mCherry (subsequently referred to as AAV-DIO-hM3Dq-mCherry; titer 8.2 × 10¹² viral genomes per ml), AAV5-hSyn-DIO-hM4D(Gi)-mCherry (referred to as AAV-DIO-hM4Di-mCherry; titer 5.3 × 10¹² viral genomes per ml) and AAV5-hSyn-DIO-mCherry (referred to as AAV-DIO-mCherry; titer 1.8 × 10¹² viral genomes per ml, a gift from Bryan Roth (Addgene viral prep # 44361-AAV5, # 50475-AAV5 and # 50459-AAV5, respectively) ^{27 (link)} were injected into the ARC (coordinates: AP −1.4 mm, ML ± 0.2 mm, DV −5.8 mm from bregma). A total volume of 0.4 μL AAV vectors was delivered bilaterally into the ARC at a rate of 0.1 μL/min with a 33-gauge stainless steel injector connected to a UMP3 micro syringe pump (World Precision Instruments, Sarasota, FL). Additional 5 min were allowed for diffusion and prevention of backflow. Behavioral procedures were conducted 14 days after AAV injection. The injection sites were verified by examining the mCherry fluorescence in each animal at the end of the experiments. The animals with mis-injections were excluded from statistical analysis.

Adult mice (>P50) were anesthetized with 2–3% isoflurane. Under the stereotaxic frame (David Kopf Instruments), the skull was exposed in aseptic conditions and the virus was injected bilaterally into the EP (coordinates: −1.0mm A/P, +/− 2.1mm M/L, and 4.2mm D/V, from bregma) through a pulled glass pipette at a rate of 50 nl/min with a UMP3 microsyringe pump (World Precision Instruments). 150 nl was infused per injection site. At least 4 weeks passed after virus injection before experiments were performed.

To identify cell specific expression of GluN2C-containing NMDA receptors in GPe and nRT, and for mapping output pathways, we used Grin2C^{tm1(EGFP/cre/ERT2)Wtsi} reporter mice (Wellcome Trust Sanger Institute; (Ravikrishnan et al., 2018 (link))) which allows expression of tamoxifen-inducible Cre. For the virus injections, mice were anaesthetized, and a small hole was drilled above the GPe (− 0.4 mm posterior, − 1.75 mm lateral, and − 3.75 mm ventral with respect to bregma). The virus particles AAV5/EF1αa-DIO-mCherry (UNC vector core, Chapel Hill, NC, USA) were injected (100nl) using a microliter syringe (NanoFil; World Precision Instruments, Sarasota, FL) with 33-gauge needle (NF33BV-2; World Precision Instruments) at a rate of 1 nl/s using a UMP3 micro-syringe pump (World Precision Instruments). The coordinates used for injection into nRT were − 0.2 mm posterior, 1.6 mm lateral, and − 3.5 mm ventral. After 2 weeks of surgery, these mice were injected with tamoxifen (75 mg/kg, ip) once-in-a day for 5 days. The animals were sacrificed 1 month after the last dose of tamoxifen and brains were processed for immunohistochemistry.

For GCaMP3 imaging, AAV1-Cre-mCherry and AAV8-DFI-Gcamp3.8 were packaged at University of North Carolina Gene Therapy Center Virus Core Facility. For Figures 4B–D, P16–P21 pups with the genotype GCaMP3^f/f were anesthetized with isofluorane and placed on a small stereotaxic frame (David Kopf Instruments). To target the hippocampus, coordinates of posterior 2.8 mm and lateral 3.0 mm relative to Bregma, 2.2 mm from the pia were used. Unilateral injections of 1 μl of AAV1-Cre-mCherry (2×10¹² genome copy/ml) were made into the right hemispheres at a rate of 100 nl/min through a UMP3 micro syringe pump (World Precision Instruments). After injection, pups were returned to their home cage with their mother, weaned around P21–P23, and expression was allowed to occur for at least 11 days post infection. For Figures S2C–D, AAV1-Cre-mCherry (2×10¹² genome copy/ml) and AAV-DFI-Gcamp3.8 (1.39X10¹² virus molecules/ml) were injected into P0/P1 hippocampus according to previously published procedures (Lu et al., 2009 (link)), and mice were imaged at P15–P19.