Picrotoxin

Corticosterone, picrotoxin, and ketoprofen were purchased from Cayman Chemical. FITC-AffiniPure Donkey Anti-Mouse IgG (H + L) and AlexaFluor-594-IgG Fraction Monoclonal Mouse Anti-Biotin were purchased from Jackson ImmunoResearch Laboratories. TTX was purchased from the Research Institute of the Aquatic Products of Hebei. All other regents and drugs were purchased from Sigma Millipore, Tocris Bioscience, or BBI Life Sciences.

The generation of TrkA-KFG mutant mice has been described elsewhere [20 (link)]. All tests were conducted on at least 3-month-old, age-matched, male mutants, or C57BL/6CR mice. Male mice were used to exclude the effects of sexual dimorphism and estrous phase on behavior. Animals were kept in the NCI vivarium in colony cages at an ambient temperature of 25 ± 2 °C and 45–55% relative humidity with 12 h light: dark cycle. The mice had free access to standard rodent pellet diet and drinking water. Donepezil (3 mg/kg i.p) was obtained from Sigma-Aldrich (USA). Picrotoxin (0.5 mg/kg i.p) and midazolam (1.25 mg/g i.p) were from Cayman Chemicals (USA). The drug dosage was based on results of pilot experiments and earlier publications [21 (link)–23 (link)]. TMT (2, 3, 5-Trimethyl-3-thiazoline) was obtained from PheroTech, Canada. All procedures were approved by NCI-Frederick ACUC committee and followed the National Institutes of Health Guidelines for animal care and use.

(2R)-amino-5-phosphonovaleric acid (AP5) and picrotoxin were obtained from Cayman Chemicals. CNQX was obtained from Tocris. APV was initially dissolved in ddH2O to a stock concentration of 10 mM; the final concentration applied was 50 μM. CNQX was dissolved in ddH2O to a stock concentration of 10 mM; the final concentration applied was 20 μM. picrotoxin at 100 μM was added directly to the Mg2+-free ACSF and was dissolved with an extensive period of stirring. Briefly, baseline recordings were made under standard ACSF. Then, the bath solution was switched to Mg2+-free ACSF containing 100 μM picrotoxin while stimulating the Schaffer collateral axons every 30 sec to maximize glutamatergic activations and responses. Epileptiform responses with long lasting phase with multiple spikes started to appear after 15 min. AP5 was added to inhibit NMDAR-mediated responses, once the AMPAR only component of the response stabilized for > 5 sweeps, CNQX was added to inhibit all responses.