Quintessential stereotaxic injector

To retrogradely label RGCs, adult mice were anesthetized with ketamine/xylazine (100/10 mg/kg i.p.) and placed in a stereotaxic frame. A small incision was made in the scalp to expose the skull and a focal craniotomy was performed with a microdrill. A Hamilton syringe with 33 gauge needle attached to a Quintessential Stereotaxic Injector (Stoelting, Wood Dale, IL, USA) was lowered into the SC at an angle of 30° to the vertical and to a depth of 1–1.5 mm. Approximately 1 μL of fluorescently-conjugated cholera toxin subunit B (CTB-488, Invitrogen; 10 mg/mL in PBS) was injected at a rate of 0.5 mL/min. The scalp was sutured and mice were allowed to recover in their home cage on a warming plate. After 2 days, mice were sacrificed and intracardially perfused with PBS and PFA. Eyes were dissected out and prepared for imaging as described above.

The hydroxypyrone-based MMP-12 inhibitor was prepared as previously published [15 (link)]. 24 h after SE, rats were anaesthetized with isoflurane and placed on a stereotaxic apparatus equipped with a micro-injector (Quintessential Stereotaxic Injector, Stoelting C.O., Dublin, Ireland). Animals received 1 µL of the MMP-12 inhibitor (2 mM in 10% dimethyl sulfoxide (DMSO, Sigma-Aldrich), in PBS) through the cannula. More precisely, MMP-12 inhibitor was injected in the right hippocampal CA3 radiatum, while the left hippocampal CA3 radiatum served as an internal control. The duration of the injection was 5 min. The cannula was left in place for another 2 min to prevent spill-over. After the injection, the cannula was removed, and animals were left to recover from the anesthesia. These procedures were repeated twice/day for two days.

To knockout ErbB4 in PV-expressing interneurons specifically, the rAAV-fPV-CRE-pAs (titers: 3.38 × 10¹² VG/ml, BrainVTA, China) was injected into spinal dorsal horn of Erbb4^f/f mice. The mice were anesthetized with 1.25% Avertin (0.2 ml/10 g body weight, i.p.) and fixed in the stereotaxic apparatus by attaching clamps to the vertebral column (RWD Instruments, China). Before the laminectomy, shave the fur from the lower back and disinfect the skin with 75% ethanol. Then, incise the skin and separate the fascia covering the spine. After removing the dorsal portion of the vertebra and expose the spinal cord, the virus (0.5 μl/injection) was injected into the dorsal horn (500 μm lateral and 250 μm deep) using a glass micropipette connected to a Quintessential Stereotaxic Injector (Stoelting, Wood Dale, IL, United States). Mice were allowed to recover for at least 3 weeks after virus injection before experiments.

Stereotaxic surgeries to deliver viral constructs were performed as previously described in ref. ^{43 (link)}. Briefly, mice were anesthetized with a ketamine/xylazine cocktail (100 and 10 mg/kg, respectively), and their heads were affixed to a stereotaxic apparatus. Viral vectors were delivered through a 0.5 µL syringe (Neuros Model 7000.5 KH, point style 3; Hamilton, Reno, NV, USA) mounted on a motorized stereotaxic injector (Quintessential Stereotaxic Injector; Stoelting, Wood Dale, IL, USA) at a rate of 40 nL/min. Viral preparations were made by the Baylor NeuroConnectivity Core with the sterotype DJ8 and titers more than 10¹² vg/mL. Viral delivery was targeted to the Arc or PVH through four local injections with two to each side (200 nL/side, anteroposterior (AP): −1.4 and −1.6 mm, mediolateral (ML):±0.2 mm, and dorsoventral (DV): −5.9 mm). AAV-EF1a-Flex-EGFP-P2A-mNachBac, AAV-EF1a-Flex-Kir2.1-P2A-dTomato, AAV-CAG-Flex-mPOMC-P2A-EGFP, AAV-CAG-Flex-Alpha-MSH, or AAV-CAG-Flex-Beta-endorphin, or AAV-CAG-Flex-MC4R-P2A-GFP were delivered bilaterally or unilaterally into the Arc of POMC-Cre mice, POMC-cre:ob/ob mice, or the PVH of MC4R-cre mice. Two mutations (E224G and Y242F) were made in the Kir2.1 construct so that the channel will be more effective in reducing neuron activity^{43 (link)}. AAV-Flex-GFP or AAV-Flex-mCherry injections were used as a control group.