PHN were prepared from embryonic day 18 (E18) rat hippocampus. Tissues were acquired from a commercial vendor (Brainbits LLC, Springfield, Illinois). To promote growth and adhesion, glass bottom 35 mm culture dishes (Cat# P35GC-0-10-C, MatTek, Inc., Ashland, Massachusetts) were coated with concentration of poly-D-lysine (Sigma, St. Louis, Missouri) solution for approximately 24 hr at . Prior to use, the dishes were rinsed with sterile 18 MΩ deionized water and allowed to dry. In order to culture PHN, the hippocampus was dissociated by repeated triturating with a 1 mL pipette tip. Following a resting period, the supernatant, containing dispersed cells, was transferred to a 15 mL tube, centrifuged at for 1 min and removed. The remaining cell pellet was resuspended with 1 mL of NbActiv1™ (Cat# NbActiv1, Brainbits LLC, Illinois) with 25 μM glutamate (Sigma, Missouri). Cells were plated at approximately , and incubated at 37°C, 5% . After 4 days, half of the medium was exchanged with fresh, warm NbActiv4™ (Cat# NbActiv4, Brainbits LLC, Illinois) medium with 25 μM glutamate. This process was repeated every three to five days throughout the culture lifetime.