Surgical implantation of microdialysis probes

Microdialysis probes were constructed as previously described²⁴ and their surgical implantation was conducted on each rat (n = 36) for pineal and/or cortical dialysate collection using a method modified from a published protocol and under strictly aseptic conditions²⁴. The rats were anesthetized lightly first using a combination of ketamine (10 mg//kg, i.m.) and xylazine (2 mg/kg, i.m.). The animal’s head was shaved and positioned in a stereotaxic instrument with the head flat. For the rest of the surgery, anesthesia was provided by 1.8% (1.5–2%) isoflurane. The skull was exposed by a 2 cm coronal incision between the two ears along the interaural line. Three stabilizing stainless steel screws 1 mm in diameter were placed to allow the positioning of the probes on the skull. Two small burr holes were created on both sides of the skull. The smaller hole on the right side was ~0.5 mm in diameter, which prevented the tip of the 25-gauge dialysis needle from penetrating the skull, whereas the larger hole on the left side was ~1 mm in diameter and allowed the probe to easily exit the skull during implantation. Next, the right probe was carefully pushed into the brain tissue through the pineal from the right side of the skull leaving the epoxy ball outside of the skull. Following the completion of probe insertion, the epoxy on the left side was removed using cautery and the tungsten rod was then carefully pulled out of the probe. The excess dialysis fiber was cut and the hollow fiber tip was then secured to the tip of the second part of the probe using epoxy resin. The probe setup was fixed to the anchor screws on top of the skull with dental cement. Finally, the skin was sutured, leaving two probes: one to introduce the perfusate, and the other end to collect dialysate. The entire procedure took less than two hours per animal. The animals were returned to their cages, housed individually, and allowed to recover from the surgery for 7 days before microdialysis recording proceeded. Target was verified via HPLC assessment of dialysate for melatonin, which emanates specifically from the pineal in the brain.