Vernier stereotaxic instrument

Mice were anesthetized using intraperitoneal injection of 100 mg/kg ketamine + 10 mg/kg xylazine, and guide cannula (RWD Life Science Co., Ltd., Shenzhen, China) were bilaterally implanted into the prefrontal cortex region of mice brain using a stereotaxic apparatus (Vernier Stereotaxic Instrument, Leica Biosystems, Nussloch, Germany) by the following coordinates: 1.7 mm anteroposterior, ±0.25 mm dorsolateral, and depth –2.5 mm from the bregma [28 ]. After seven days of recovery, we infused L-AAA (100 µg/µl; Sigma) bilaterally using injection cannula and a microdriven pump (Pump 11 Elite Nanomite, Harvard Apparatus, Holliston, MA, USA). We administered infusion once daily for 2 days at a rate of 0.1 µl/min for 6 minutes.

Surgery was performed as previously described (Lim et al., 2010 (link), 2015a (link)). In brief, rats were anesthetized (2.5% isoflurane inhalation) and placed in a stereotactic frame (Vernier Stereotaxic Instrument, Leica Biosystems, Nussloch, Germany). A bilateral stimulating electrode was implanted in the vmPFC (AP: +2.70 mm; L: ±0.60 mm; V: 4.60 mm) based on the rat brain atlas of Paxinos and Watson (1998) . All animals were given a 2-week recovery period.
For stimulation, a bipolar stimulating electrode (Synergy, Singapore) was constructed using an inner platinum-iridium core wire with a gold-plated cannula (Technomed, Beek, Netherlands) (Lim et al., 2009 (link); Tan et al., 2010 (link)). A digital stimulator DS8000 and stimulus isolators DLS100 (World Precision Instruments, Sarasota, USA) were used to deliver the electrical stimuli. In the acute DBS experiment, either HFS (100 Hz) or LFS (10 Hz) with stimulation amplitudes of 50, 100, 200, and 400 μA was used. The pulse width was set at 100 μs. In the chronic DBS experiment, the stimulation parameter (HFS, 200 μA amplitude, and 100 μs pulse width), derived from the present acute DBS study (Figure 2) and previous findings (Hamani et al., 2010a (link); Lim et al., 2015b (link)), was used to test the hypothesis that chronic stimulation enhances both the short- and long-term memory functions.

Stereotaxic injection of siRNA was performed as described previously (Choi et al., 2015). In brief, diluted siRNA (50 ng/μl) was mixed with Neurofect transfection reagent (T800075; Genlantis, San Diego, CA, USA). The siRNA mix (1.8 μl of 7.5 ng/μl) was injected into each CA3 (stereotaxic coordinate: AP, −1.9; ML, ±3.0; DV, −2.1 mm) using a stereotaxic injection system (Vernier Stereotaxic Instrument, Leica Biosystems, Wetzlar, Germany).