Midazolam

Sixteen Merino ewes (43.5 ± 6.2 kg) were fasted overnight for the study. Procedures involving animal care, data capture, and anesthetic and surgical techniques, including placement of microdialysis catheters and microdialysis, were done in accordance with previously described methods (13 (link)). At the commencement of the study, all animals were anesthetized with midazolam (0.5 mg/kg; Pfizer), buprenorphine (300 μg; Reckitt Benckiser Healthcare), and alfaxalone (3 mg/kg; Jurox). Anesthesia was maintained with an infusion of alfaxalone (6 mg/kg/h; Jurox), midazolam (0.25 mg/kg/h; Pfizer), fentanyl (15 μg/kg/h; Hameln Pharmaceuticals), and ketamine (10 mg/kg/h; Troy Laboratories). All anesthetic and analgesic medications were titrated to maintain adequate surgical anesthesia.
A central venous catheter (Arrow International), facial artery arterial catheter (Arterial Leadercath, Vygon), and pulmonary artery catheter (Swan-Ganz CCOmbo, Edwards Lifesciences) were inserted to facilitate drug administration and cardiovascular monitoring.
Microdialysis catheters (CMA 63 and 70 MD probes) were surgically inserted into the femoral artery, brain, heart, liver, and left kidney.
Throughout the study, all animals received protocolized ventilation to maintain an Sa_O2 of >94% and an end-tidal CO₂ of 35−45 mm Hg.

The treatment was conducted by adding a range of midazolam doses (50nM, 100 nM, 150 nM) (Hospira Inc., Lake Forest, Illinois, USA) into experiment wells, and incubating the wells at 37°C with 5% CO₂ in the incubator (Panasonic Healthcare, Wood Dale, IL, USA) at 9:00 am on 7DIV. Half of the media volume was replaced on 8DIV and on 9DIV, and a full dose of the drug was added to maintain the dissolved concentration. The experiment was ended by fixing the cells using 4% paraformaldehyde in PBS at 9:00 am on 10DIV (Figure 1B).

All animal protocols were approved by the University of California Davis Animal Use and Care Committee, and experiments were carried out in compliance with the National Institutes of Health Guide for the use and care of laboratory animals. Upon arrival to the vivarium, 7 week old mice (Swiss Webster mice, Charles River Laboratories, Wilmington, MA) were acclimated to their new housing environment for one week and kept under a 12 hour light/dark cycle with free access to food and water.
DZP (5 mg/mL, Hospira, Inc., Lake Forest, IL) was dissolved in 40% propylene glycol, 10 % ethanol and saline. TPPU (1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea) was synthesized [18 (link)], purified and characterized in-house (a kind gift of Dr Sing Lee from UC Davis) and was dissolved in PEG400 to obtain a clear solution. Mice were injected (i.p.) either with DZP alone at 1, 3 or 10 mg/kg or in combination with TPPU at 3 mg/kg i.p. one hour before administration of DZP at the doses indicated. Midazolam (Hospira, Inc., Lake Forest, IL) was administered at 1.8 and 10 mg/kg doses by intramuscular route using saline as vehicle. Animals that served as controls were given the corresponding vehicle solutions, PEG400 for TPPU or 40% propylene glycol, 10% ethanol in saline for DZP and saline for Midazolam injections.

Each rat (n=18) was anesthetized, placed in a stereotaxic frame, and a microdialysis probe was aimed for the amygdala as described above (Fig. 2B). Five microdialysis samples were collected during dialysis with Ringer’s solution (control). A CMA/110 liquid switch was then activated to perfuse the probe with Ringer’s solution alone, or Ringer’s solution containing midazolam (100 μM, Hospira, Lake Forest, IL), diazepam (100 μM, Sigma-Aldrich, St. Louis, MO), eszopiclone (100 μM, Toronto Research Chemicals, Toronto, Canada), zolpidem (100 μM, Sigma-Aldrich, St. Louis, MO), or tetrodotoxin (TTX, 1 μM). Five microdialysis samples were obtained during dialysis administration of BZ site agonists. This within-subjects design was used to test only one drug per rat (Fig. 2C). After collection of the last sample, the dialysis probe was removed from the brain, the scalp was closed, and delivery of isoflurane was discontinued.