Ibotenic Acid Lesion Surgery

Ibotenic acid (10 mg/ml in saline) will be infused over 5 min for a total volume of 0.1 µl.

Rats will be stereotaxically injected with ibotenic acid (Natural Products Co., Vashon, WA) to destroy cell bodies within specific brain subnuclei while sparing fibers of passage. Under isoflurane anesthesia and following aseptic surgical preparation, the rat's head is immobilized in a stereotaxic apparatus. An incision is made longitudinally along the midline of the head, to expose the skull. A 1.8 mm hole is drilled in the skull with a Dremel motortool and a Fine Science Tool trephine bit. Injections will be made through a blunt 33 gauge needle attached to a 2 µl Hamilton microsyringe under the control of a syringe pump. Ibotenic acid (10 mg/ml in saline) will be infused over 5 min for a total volume of 0.1 µl. The injector needle will be left in place for 10 min after the infusion and before withdrawal to minimize back flow along the needle track. Sham lesions will be produced by injection of phosphate-buffered saline (0.1 µl) into the same areas, thus controlling for non-specific damage from lowering the injector through overlying tissue. All lesions will be produced bilaterally.

Note on Infusion Pump: The KD Scientific 200P pump has a table of syringe sizes, but the settings for Hamilton microsyringes only go down to 10ul. To get 0.2 microliters from a 1 ul syringe, set the pump to a Hmailton 10 ul syringe, but put in the volume of 2.2 ul at 0.44 ul per minute, which will dispense 0.2 microliters over 5 minutes from the 1 ul syringe. (Should probably recalibrate these settings every so often.)

The skull hole is packed with gel foam, and the incision closed with wound clips. Wound clips are removed 7-10 days after surgery. Antibiotics (dose to be determined in consultation with the veterinary staff) are administered 1 h prior to cranial surgery, and buprenorphine (0.2 mg/kg i.p.) is given immediately post-surgery as an analgesic. Antibiotics are given once daily for 7 days after surgery. Body weight and water intake are measured daily, along with other signs of stress, irritability, or pain. Reduced water intake or the appearance of dehydration will be treated with continued antibiotics, and replacement and maintenance of body fluids with warmed 0.15M NaCl or 0.3 M dextrose as recommended by the veterinary staff.

Prior to experimental testing, coordinates for injector placement will be determined in a group of test animals based on Paxinos and WatsonInjector placement will be verified by injections of Sudan Black (0.4 µl). A series of preliminary studies will be conducted to determine the extent of cell damage by cresyl violet staining. Animals will have injections performed, then be euthanized. Sections will be cut through the remainder of the brain and stained with Weil myelin stain to visualize degenerating neurons that may project through the lesion site. Based on these preliminary studies, the concentration and volume of ibotenic acid injections will be adjusted to maximize cell death without damaging fibers of passage.

Verification of Lesion Site

Following Jongen-Relo, A. L., & Feldon, J. (2002) PMID 12126979 and in the gustatory cortex, Geddes et al. (2008) PMID 18823161, we will determine loss of neurons using a mouse monoclonal anti-NeuN, clone A60, from Millipore, catalog MAB377, price $259.00. Jongen-Relo used a primary concentration of 1:5000 with a 48-h incubation at 4 C.