Digital lab standard stereotaxic instrument

Callosal and thalamic (ventroposterior medial nucleus, VPM) injections were performed using stereotaxic guided injection. Postnatal day (P)5 pups were anesthetized on ice. Heads were fixed in a Digital Lab Standard Stereotaxic Instrument (Stoelting 51,900). A small incision was performed on the top of the skull to visualize the bregma for stereotaxic references. For retrograde labeling, red Retrobeads (Lumafluor, Inc.) were loaded in a glass capillary mounted on a Nanoinjector (Nanoject II Auto-Nanoliter Injector, Drummond Scientific Company 3-000-204) and injected as 10 × 18 nL injections. S1 coordinates were (from bregma); X: + 1.1, Y: −0.9, Z: -0.9; VPM coordinates were X: + 1.2, Y: −0.9, Z: −2.5. Spinal cord injections were performed at P2 under ultrasound-guided injections using a Vevo 770 ultrasound backscatter microscopy system (Visual Sonics, Canada) as previously described^{15 (link)}.

On the day of surgery, the rats were anesthetized using a combination of xylazine and ketamine (in a ratio of 0.15:0.85). A microinjection needle with a diameter of 33 gauge was connected to a 10-μl syringe (Hamilton, United States). The needle was inserted unilaterally into the NAc (coordinates: AP: +1.75, ML: 1.48, DV: −6.2). A purified adeno-associated virus (AAV) (2 × 10⁹ units; 1 μL volume) encoding hChR2 (H134R)-m-Cherry under the control of the human synapsin 1 gene promoter was injected for 5 min, with the syringe left in place for an additional 7 min. The injections were performed using a Digital Lab Standard™ Stereotaxic instrument (Stoelting Co. IL, United States). Following the surgery, the rats were housed individually in separate cages for 2 weeks to allow for recovery and complete expression of the viral vector. After 2 weeks of injecting the channel rhodopsin-containing virus into the NAc, the animals underwent another surgery in which an optic fiber was implanted above the VTA (coordinates: AP: -5.1, ML: 0.7, DV: −8.1). The rats were given 1 week to recover before the initiation of the behavioral experiments.