In vivo microdialysis procedure

Concentric microdialysis probes were constructed with a 2-mm-long membrane. Following anesthesia with sodium pentobarbital (120 mg/kg i.p.), 9-month-old male mice were placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA), dialysis probes were implanted in the hippocampus and secured to the skull with anchor screws and dental cement. Stereotaxic coordinates from Bregma and skull surface were: AP −3.00 mm, L −3.00 mm, DV −4.5 mm; according to Franklin and Paxinos (Franklin, 2012). Microdialysis experiments were conducted 20–24 h after surgery in freely moving mice by continuously perfusing probes with an artificial cerebrospinal fluid (aCSF) containing 125 mM NaCl, 2.5 mM KCl. The aCSF was perfused at 1.6 μL/min with a Harvard model 22 syringe pump (Harvard Apparatus, South Natick, MA, USA) attached to an overhead liquid swivel (Instech, Plymouth Meeting, PA, USA). An initial sample of dialysate corresponding to the first 2.5 h was discarded and then, one sample of aCSF (16 μL) was collected every 10 minutes up to 3 samples, to establish a stable baseline level of glutamate before any pharmacological intervention. Next, dihydrokainate (DHK) was administered locally by reverse dialysis through the dialysis probes. We administered 0.1 mM, 0.3 mM, 1 mM and 3 mM doses and we collected one sample of dialysate every 10 minutes (4 samples for each DHK dose).

On the following day, mice were injected intraperitoneally with the pro-convulsive agent pentylenetetrazol (PTZ) in order to examine glutamate levels in a challenging condition involving an increase of the excitation/inhibition ratio. After a 2.5 h stabilization period, 4 baseline dialysate 10 min samples were collected before PTZ treatment (10 and 30 mg/kg i.p.). A total of six samples of dialysate per dose were collected.

At the completion of dialysis experiments, mice were sacrificed by cervical dislocation and brains were rapidly removed and frozen in dry ice for subsequent histological examination.