Neurotransmitter Release Assay

The animals received microinjections of a mixture of NpHR and ChR2 vectors, as described in surgical procedure. For the assay, the mice were anesthetized with 15% halothane in mineral oil, and brains were rapidly removed and placed into ice-cold phosphate-buffered saline (PBS), pH 7.4. One-mm-thick coronal slices were made using a mouse brain matrix (Brain Research Laboratories, Waban, MA), and BLA was dissected with custom-made tissue punchers (1-mm diameter) using visual landmarks aided by a stereotaxic atlas.²⁷ The excised tissues were minced to increase surface area of the slices for radioactive-tagged glutamate uptake and oxygen/glucose supply. Synaptosome preparation was conducted as described previously by Lonart et al.²⁸ Briefly, the excised tissues were placed in ice-cold isotonic sucrose solution (0.32 M sucrose, 100 µM EDTA, 5 mM HEPES, pH 7.4) and homogenized at 900 rpm with a motor-driven homogenizer. Debris and nuclei were pelleted by differential centrifugation at 900×g at 4°C for 10 min, and the supernatant was then centrifuged at 11,500×g at 4°C for 20 min to pellet the synaptosomes. The synaptosomes were resuspended in ice-cold aerated (95% O_2, 5% CO₂) Krebs-bicarbonate-HEPES buffer (KBH; 118 mM NaCl, 13.5 mM KCl, 1.25 mM CaCl₂, 1.2mM MgSO₄, 1.2 mM KH₂PO₄, 25 mM NaCO₃, 5 mM HEPES-NaOH (pH 7.4), 11.5 mM D-glucose) and equilibrated on ice for at least 30 min.

Neurotransmitter release assay was conducted as described previously by Lonart et al.²⁸ Briefly, slice preparations were incubated in a solution containing 3.8 µM ¹⁴C-glutamate (L-[¹⁴C (U)]-glutamic acid, 260 mCi/mmol specific activity, PerkinElmer, Boston, MA) for 30 min at 35^oC in freshly bubbled KBH, whereas synaptosome preparations were incubated with 115 nM ³H-glutamate (L-[3, 4-³H]-glutamic acid, 52.0 Ci/mmol specific activity, PerkinElmer) for 5 min at 35^oC in freshly bubbled KBH. Afterward, they were transferred to a superfusion chamber containing a glass fiber filter (GF/B) covered with 25 µl of 50% Sephadex slurry to superfuse with continuously bubbled KBH at 35^oC. After a 45-min washing period to remove any un-incorporated radioisotopes, 3-min fractions of the superfusate were then collected from the slices to determine radioactivity efflux as a measure of glutamate release. Studies with synaptosomes were identical except that the washing period was 12 min and fractions of the superfusate were collected for 1 min. Release was optogenetically induced by exposing the superfusion chambers to either blue light (473 nm) alone or a combination of blue and green lights (532 nm) produced by 100–200 mW lasers (Shanghai Laser & Optics Century Co., Ltd, Shanghai, China). Radioactivity in the eluted fractions and in the slices/synaptosomes remaining at the end of the experiment were determined with a liquid scintillation spectrometer (LS 3801, Beckman Instruments, Inc., Fullerton, CA). Release was expressed as the fractional release rate, calculated as the fraction of radioactivity released, divided by the amount remaining in the slice or synaptosome preparation at that particular time point.