Cunningham mouse adapter

Mice were anesthetized with isoflurane (3% for induction, 0.7–1% for maintenance) and mounted in a stereotaxic frame (David Kopf Instruments) fitted with a Cunningham mouse adapter (Stoelting Co.). Small cranial windows were drilled over the recording regions (versus bregma), prelimbic prefrontal cortex (PL): AP + 1.8; ML ± 0.3; DV −2.1 mm, and microelectrode arrays (MEAs) were inserted into this region. Glutamate dynamics were assessed on a subsecond timescale by MEA recordings with two of four electrode sites coated with L-glutamate oxidase enzyme, which breaks down L-glutamate into α-ketoglutarate and peroxide (H₂O₂) as previously described^{67 (link),68 (link)}. By using constant voltage amperometry with the application of a fixed potential, the H₂O₂ was oxidized, with electron loss, and the resulting current was recorded using a Fast Analytical Sensing Technology-16 (FAST-16 MKII) electrochemistry instrument (Quanteon). Depolarization-induced glutamate release was induced by an isotonic solution of 70 mM KCl ejected for 1 s at 1 min intervals through a glass micropipette positioned at a distance of 50–100 μm from the MEA recording sites. A MATLAB graphic interface was used to calculate concentrations of glutamate from an average of 3–5 amplitudes per mouse. The maximum amplitude of tonic and evoked glutamate (exclude amplitude less than 0.5 μM) were measured.

Infection of mice with MCMV was performed as previously described 33. Briefly, female mice (6–8 week old) were anesthetized using a combination of Ketamine and Xylazine (100 mg and 10 mg/kg body weight, respectively) and immobilized on a small animal stereotactic instrument equipped with a Cunningham mouse adapter (Stoelting Co., Wood Dale, IL). The skin and underlying connective tissue were reflected to expose reference sutures (sagittal and coronal) on the skull. The sagittal plane was adjusted such that bregma and lambda were positioned at the same coordinates on the vertical plane. Virulent, salivary gland‐passaged MCMV RM461 (1 × 10⁵ TCID50 units in 10 µl), was injected into the right lateral ventricle at 0.9 mm lateral, 0.5 mm caudal, and 3.0 mm ventral to bregma using a Hamilton syringe (10 µl) fitted to a 27 G needle. The injection was delivered over a period of 3–5 min. The opening in the skull was sealed with bone wax and the skin was closed using 4–0 silk sutures with a FS‐2 needle (Ethicon, Somerville NJ).