Animals
Mice were male C57BL/6J mice (000664) and male Vglut1-cre mice (023527) mice obtained from The Jackson Laboratory (Bar Harbor) and male Glt1-G-CaMP740 mice obtained from RIKEN BioResource Research Center (G7NG817, RBRC09650). Mice were at least 8 weeks old at the time of surgery, before which they were group-housed in a temperature (72 ± 5 °F) and humidity (45 ± 15%) controlled vivarium under a 12 h–12 h light–dark cycle (lights on 06:00). After surgery, the mice were single housed to prevent cage mates from damaging intracranial implants. Experimental procedures were approved by the NIAAA and National Institute of Natural Sciences Animal Care and Use Committees and followed the NIH guidelines outlined in ‘Using Animals in Intramural Research’ and the local Animal Care and Use Committees.
General surgical and histological procedures
Surgery
To target injection of viral constructs, optic fibres and GRIN lenses, mice were placed in a stereotaxic alignment system (Kopf Instruments) under isoflurane anaesthesia. Unless stated otherwise, viral constructs (see the relevant sections for details) were unilaterally (one-photon imaging, fibre photometry) or bilaterally (behavioural experiments, single-unit recordings) infused into the BLA in a volume of 0.36 µl over 10 min using a pulled-glass capillary (Drummond Scientific, 2-000-001; tip diameter, ~20 μm) connected to a Nanoject syringe (Drummond Scientific) at the coordinates: anteroposterior (AP) +1.42, mediolateral (ML) +3.27, dorsoventral (DV) −5.15, −4.95 and −4.75 (1 × 0.12 µl injection at each of the three DV coordinates) relative to bregma. After each injection, the glass capillary was left in place for 10 min to ensure diffusion. Testing began no sooner than 4 weeks after surgery, to allow for recovery and virus expression.
For behavioural experiments, the scalp wound was closed with tissue adhesive (GLUture) and a layer of topical antibiotic ointment applied. For fibre photometry experiments, optic fibres (MFC-400/430-0.66-6mm_SM3-FLT, B280-4653.6, Doric Lenses) were unilaterally implanted into the BLA at the coordinates AP +1.40, ML +3.25, DV −4.8 relative to bregma, and affixed to the skull with screws and acrylic dental cement (Coralite Dental Products). For one-photon and two-photon Ca2+ imaging experiments, a GRIN lens (ProView GRIN lenses; diameter, 0.6 mm; length, 7.3 mm; 1050-005442, Inscopix) was unilaterally implanted into the BLA at the coordinates AP +1.40, ML +3.25, DV −4.8 relative to bregma, and affixed to the skull with C&B-Metabond Quick Adhesive Cement System (Parkell). For single-unit recordings, a 16-tungsten microelectrode array (35 µm diameter, 150 µm spacing, configured in a semi-circle surrounding the tip of an optic fibre, Innovative Neurophysiology) was unilaterally implanted into the BLA, with the centre of the array at the coordinates: AP −1.50 mm, ML ±3.20 mm, DV −4.95 mm, relative to bregma. Moreover, a ferrule-fibre assembly (Thorlabs) was implanted at a 30° angle, such that the tip was positioned 0.5 mm above the microelectrode tips, with the array and optic fibre affixed to the skull with screws and dental cement (Coralite Dental Products).
Histology
On completion of testing, animals were terminally anaesthetized with sodium pentobarbital (50–60 mg per kg) and transcardially perfused with ice-cold PBS followed by ice-cold 4% paraformaldehyde in phosphate buffer. Brains were removed and suspended in 4% PFA overnight and then 4 °C 0.1 M PB for 1–2 days. For fibre photometry and imaging, the head was suspended in 4% PFA overnight before brain extraction. Coronal sections (50 μm) were cut with a vibratome (Leica VT1000 S, Leica Biosystems) in 0.1 M phosphate buffer and/or stored in 2% sodium azide in PBS, then cover slipped with mowiol-based mounting medium (17951, Polysciences) with Hoechst (1 μg ml−1, Thermo Fisher Scientific).
Unless stated otherwise, BLA containing sections were imaged at ×5 (HCX PL FLUOTAR objective, ×5/0.15) using either a Leica slide scanner with an ANDOR Zyla Monochrome camera and Leica DM6 motorized platform controlled by Aperio VERSA software (Leica Biosystems), an Olympus BX41 fluorescence microscope (Olympus America) or a laser-scanning confocal microscope (LSM700, Carl Zeiss) at ×20 (Plan-Apochromat air objective ×20/0.8 NA) and ×63 (Plan-Apochromat oil objective ×63/1.4 NA). Mice with absent/mistargeted viral expression, fibre optic or GRIN lens placement were removed from the analyses. Exceptions to these general histological procedures are described in the relevant sections below.
Immunohistochemistry
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