STEREOTAXIC SURGERY
- Anesthetize animal (mouse or rat) with 5% isoflurane (Aerrane, Baxter, Deerfield, IL) in air using the V-10 Anesthesia system (VetEquip, Inc., Pleasanton, CA). Following induction of anesthesia, reduce the level of isoflurane and maintain it at 1.5%. Provide thermoregulation through a thermostat regulated heating pad (Fine Science Tools Inc., Foster City, CA).
- Shave the hair and clean with iodine before incision. Place the animal onto the stereotaxic apparatus (Kopf 900) by mounting the ear bars into the ear canals. Cut the skin (2 cm long for rats, 1 cm long for mice) and remove all soft tissue from the surface of the skull.
- Establish stereotaxic coordinates as Paxinos and Watson, 2006 (The Rat Brain in Stereotaxic Coordinates, Academic Press) or Franklin and Paxinos, 1997 (The Mouse Brain in Stereotaxic Coordinates, Academic Press). Drill a hole through the skull with a battery-operated driller designed for rodent surgery (Fine Science Tools, Inc.). Insert in-house constructed vertical microdialysis probe (cut-off, 5,000 Da) or (and) EEG electrode (Plastic One, UK).
- Drill one additional hole to tighten the screw. Immobilize implants by dental cement (Duralay, Keliance).
- House animal individually with food and water available ad libitum.
Experimental procedures
Perform brain dialysis or (and) EEG recording on freely moving animal 24 hours after surgery.
Microdialysis protocol
- Pump an artificial cerebrospinal fluid (147 mM NaCl, 4 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, pH adjusted to 7.0) through the dialysis probe at a constant rate via a microinjection pump (CMA/100, Carnegie Medicine, Stockholm, Sweden). Choose flow rate and sampling time to collect small volumes (5 to 20 μL).
- Stabilize probe for 2 hours.
- Collect samples for basal levels.
- Inject the drug systemically or by probe and continue to collect samples for a predetermined time.
- At the end of the experiment sacrifice the animal with a letal dose of anaesthetic.
- Remove and fix the brain in 4% paraformaldehyde, and then store it at -80°C for next histological confirmation of probe placement.
EEG recording protocol
- Transfer animal to a EEG plastic cage with an aluminum floor covered with tissue paper moistened with saline to ensure that the animal was earthed. Plastic cage is in a Faraday cage to reduce electrical interference from external sources.
- Amplify (BioAmp ML 136)and filter (100 Hz) EEG signal; digitize and store it using a PowerLab 2/20 running Chart v4.2 software (ADInstruments, Hastings, UK) (www.adinstruments.com/labtutor-medical). The EEG data sets were pre-processed by a 50 Hz notch filter and a high pass filter at 0.3 Hz.
- Record data to establish basal level.
- Inject the drug systemically or by probe and continue to collect data for a predetermined time interval.
- Power spectrum analysis (Fourier transform)
HPLC analysis
- Analize microdialysis sample by reverse-phase high-performance liquid chromatography (Jasco Europe) (Hypersil ODS column: 5 µm, 150x3 mm Chrompack, London, UK).
- Aminoacidic neurotransmitters are derivatizate with ophthaldialdehyde / mercaptopropionic acid reagent and separated by fluorometric detection (wavelengths: emission, 450 nm; excitation, 370 nm).
- Mobile phase gradient: 50 mM sodium acetate buffer pH 6.95, with methanol increasing linearly from 2 to 30% (v/v) over 27 min.
- Flow rate 0.5 ml min-1. Detection limit 1nM/sample.
