Sr 6n

Animals were first anesthetized using isoflurane (5%) in 30% oxygen (O₂) and 70% nitrous oxide (N₂O). Animals were then transferred to a stereotaxic instrument (SR‐6N; Narishige Scientific Instrument Lab), with the skull being opened at the level between the bregma and lambda. At all time points during surgery, animals were administered 2%–3% isoflurane via a facial mask.
Bilateral CeA lesions were generated via conducting two small craniotomys just over the amygdala. A concentric electrode (CEA 200; MicroProbes) connected to the Lesion‐Making Device (53500; Ugo Basile) was inserted into the brain 2.4 mm caudal to bregma, 4.1 mm from the midline, and 7.5 mm in depth from the cortical surface (Paxinos & Watson, 2005). A constant current (400 µA; for 25 s) was used to generate lesions at the indicated points, with a clip attached to the tail serving as an indifferent electrode. For sham lesion animals, the same surgery and electrode positioning was conducted, but no current was administered to animals. Animals were given 3 days to recover from this operation.

Mice were anesthetized with urethane 1.5 g/kg i.p., and then we used the stereotaxic apparatus (SR-6N, Narishige Inc., Tokyo, Japan) to fix the mouse. The mouse scalp was cut, the hippocampal PP-DG (anterior penetrating fiber-dentate granule cell layer) position was located, a hole was drilled at the localization, the recording electrode was inserted into the DG (2.0 mm after the anterior fontanel, 1.4 mm beside the midline, and 1.5 mm under the subdural layer), the stimulating electrode was inserted into the PP (3.8 mm after the anterior fontanel, 3.0 mm beside the midline, and 1.5 mm under the subdural layer), and the reference electrode was clamped on the scalp. During the experiment, the environment was kept quiet. A population spike (PS, bandwidth: 100 μs, current intensity: 0.3 mA) was conducted, and the electrodes were modulated to achieve the best PS wave shape. After 30∼60 min, the stimulating intensity was decreased to half the PS wave and then was kept stable for 30 min and recorded as the baseline. Then, tetanic stimulation (TS) was given to induce long-term potentiation (LTP) and recode the PS wave for 60 min.

Offspring were anesthetized with a mixture (i.p.) of medetomidine hydrochloride (0.3 mg/kg; Wako), midazolam (4 mg/kg; Wako), and butorphanol tartrate (5 mg/kg; Wako), and then positioned between the ear bars of a stereotaxic frame (SR-6N; Narishige, Tokyo, Japan). Full-length Reelin (0.2 pmol/0.5 μL) protein or PBS (control) was delivered bilaterally with a Hamilton syringe at a rate of 0.1 μL/min for a total volume of 0.5 μL on each side. The needle was left in place for 5 min. Injection volume and concentration of recombinant Reelin protein was determined using previous studies (Rogers et al., 2011 (link), 2013 (link); Ishii et al., 2015 (link)). The following coordinates were used: -1.75 mm rostrocaudal, -2.0 mm dorsoventral, ±1.0 mm mediolateral from bregma (relative to dura). Immediately after removal of the needle, the skin was closed with tissue adhesive (Vetbond, 3M, St. Paul, MN). The injection point was confirmed by methylene blue microinjection (Supplementary Figure S1).

Surgical procedures were performed in an aseptic environment using sterilized surgical tools. Guinea pigs were anesthetized and their heads were fixed in stereotaxic apparatus (SR-6N; Narishige, Tokyo, Japan). A Plexiglas headstage (1.0 cm × 1.0 cm × 0.5 cm), designed to secure the animal’s head and to hold the airpuff pipe, was attached to anchoring screws using dental cement on the skull surface. A small nylon loop was sutured into the edge of the left upper eyelid. In the present study, this loop was utilized to attach the left upper eyelid to a movement-measuring device. After the surgery, guinea pigs were allowed 1 week of recovery. Post surgery, all guinea pigs gained weight normally and showed no abnormal behavior.

We anesthetized mice with a mixture of ketamine (90 mg/kg) and xylazine (10 mg/kg) and immobilized it in a stereotaxic frame (SR6N, Narishige). Following a midline incision of the skin covering the skull, we made a burrhole to open a cranial window at 0.48 mm posterior and 0.15 mm lateral to the bregma over the SCN-SPZ (Franklin and Paxinos, 2008 ). A fine glass capillary was used to inject 0.05 μl of AAV8-hSyn-hM3D(Gq)-mCherry (2.5 × 10e12 gc/ml, catalog #50474, Addgene; RRID:Addgene_50474) or AAV8-CAG-GFP (2 × 10e12 vm/ml, UNC GTC Vector Core, University of North Carolina, Chapel Hill, NC) with a speed of 0.1 μl/min targeting the bilateral SCN-SPZ (5.8 and 5.7 mm deep from the pia mater). After closing the skin covering the cranial window by suture, the mouse was allowed to recover in its home cage for at least 7 d. Then, we administered a solution of clozapine N-oxide (CNO; 1 mg/kg, BML-NS105-0005, Enzo Life Sciences) by intraperitoneal injection to activate cells expressing hM3D.