Buprenorphine

Mice (6- to 8-week-old) were anesthetized with isoflurane and placed in a stereotaxic apparatus (David Kopf). After a midline incision, a bipolar 75-µm-diameter twisted stainless steel wire (A&M Systems) was positioned in each dorsal hippocampus (2 mm posterior to Bregma, 2 mm lateral to the midline, 1.5 mm deep). Two jeweler’s screws (Braintree Scientific) were positioned in the skull, one over left frontal cortex (1 mm posterior to Bregma and 2 mm lateral to the midline) and one over right occipital cortex (1 mm anterior to Lambda and 2 mm lateral to the midline). A 6-pin holder (Braintree Scientific) was centered over the skull with dental cement (Grip cement). The skin around the cement was swabbed with Betadine. The animal was placed under a heat lamp until fully ambulatory. Buprenorphine (Henry Schein) was administered sc (0.05 mg/kg) once, and once again after 12 hr.
After 2 weeks, animals were attached to a commutator in a standard mouse cage with overhead video camera (Axis Systems) and recorded at 2 kHz (Sirenia Software; Pinnacle Systems). After 20 min, they were injected with KA (25 mg/kg) or pilocarpine (250 mg/kg) as described earlier and recorded for 24 hr. The dosage of pilocarpine was lower than the dose used for animals without EEG electrodes because we found that higher doses led to mortality. The sample size for each condition was seven mice.

3-Amino-1,2,4-triazole (ATZ) from Tokyo Chemical Industry Co., Ltd. (Portland, OR). Mercaptosuccinic acid (MSA) from Sigma-Aldrich (St. Louis, MO). Buprenorphine from Henry Schein (Dublin, OH). Isoflurane from Henry Schein (Dublin, OH). Tegaderm Film 1624W from 3M (Maplewood, MN).

On P7, pups underwent AAV injection surgery. We injected pAAV-CamKIIa-ChrimsonR-mScarlet-KV2.1 (Addgene, Watertown, MA, USA) into the VTA (location calculated from literature [24 (link)]) to achieve virus-mediated expression of ChrimsonR, which is a light-gated cation channel protein capable of activating dopaminergic neurons. The virus was injected at 0.1 µL/min for 10 min, and the virus required 7 days for full expression in the VTA.
P7 pups were anesthetized using isoflurane, and their head was fixed in a stereotaxic surgery station for rats (Narishige, Tokyo, Japan). An incision was made along the sagittal suture to expose the skull. A bur hole was made above the VTA, and the dura was broken using a glass capillary needle. The virus injection needle was then slowly inserted to the correct depth, and the injection began. Following the completion of the injection, the injection needle remained for an additional 5 min in the VTA to ensure complete administration. The needle was then slowly removed, and a suture (Ethicon, Raritan, NJ, USA) was used to close the incision. The pup was removed from isoflurane, administered buprenorphine (Henry Schein, Melville, NY, USA), and monitored until fully awake. We then returned the pup to its mother.

Annexin A1 tripeptide fragment (Ac-Gln-Ala-Trp) (ANXA1sp) was synthesized by GenScript Biotech (Piscataway, NJ) as previously described (Zhang et al., 2010 (link)) and was reconstituted in DMSO and placed in individual doses. Ketamine, xylazine, and buprenorphine were purchased from Henry Schein animal health (Dublin, OH).