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(The 0.00 MAC groups were arbitrarily assigned a concentration value of 0.01 MAC so that logarithms of the concentrations could be calculated for these groups.) A pooled EC 50was calculated for each training procedure (three-trial tone, three-trial context, one-trial tone, and one-trial context) after determining that within each training procedure there was no significant statistical difference between the EC 50values of the increased, maintained, or decreased posttraining treatments.
#Anterograde amnesia software#
24Logistic regression analysis was then applied to provide the EC 50and standard error values for suppression of fear conditioning to tone and to context for each of the posttraining isoflurane treatments (BMDP Statistical Software University of California Press, Berkeley, CA). In addition, the freezing score of each animal was dichotomously classified as either freezing or nonfreezing, using the previously determined classification thresholds of 19 and 5% for tone and context training, respectively. Figure 1diagrams the sequence of tone-shock pairs for the three- and one-trial training procedures. An additional one-trial tone conditioned group was trained during 0.00 MAC, and the posttraining isoflurane concentration rapidly increased (as described for three-trial training) and maintained at 1.0 MAC for 5 h. For context conditioning, the identical procedures were used except that no tone was administered. Within 5 s after the shock, the posttraining isoflurane concentration was (1) rapidly increased by injecting 10 ml of liquid isoflurane into the inflow port and delivering 3.0 MAC isoflurane in 20 l/min oxygen until the equilibrated chamber concentration reached 2.0 MAC, after which the chamber concentration was brought to and held at 1.0 MAC for 1 h or (2) decreased to zero by returning the animal to its home cage (purged of isoflurane). For one-trial tone conditioning, animals received a single tone-shock pair as described above for the three-trial study. Within 30 s after the third shock, the posttraining isoflurane concentration was (1) increased by delivering 3.0 MAC isoflurane in 20 l/min oxygen until the concentration in the chamber reached 1.0 MAC, after which isoflurane was delivered at 1.0 MAC for 1 h (2) maintained at the target concentration for 1 h or (3) decreased to zero by returning the animal to its home cage (purged of isoflurane). Training chambers were cleaned with 2% ammonium hydroxide before and after each animal occupied it.įor three-trial tone conditioning, animals received three tone-shock pairs consisting of a 30-s tone (90 dB, A-scale, 2,000 Hz) coterminating with a 2-s electric shock (11 Hz bipolar square wave, 2 mA for oxygen groups and 3 mA for groups receiving isoflurane) shock pairs were 90 s apart. 24A speaker was mounted on the rear wall of each training chamber. During 0.50 MAC isoflurane administration, the floor consisted of 18 more narrowly spaced bars to prevent the feet of obtunded animals from falling beneath the bars and thereby avoiding the foot shocks. For concentrations of isoflurane less than 0.5 MAC, the floor of each training chamber consisted of 14 stainless steel rods (6-mm diameter), spaced 1.8 cm center to center wired to a shock scrambler (Gemini Avoidance System San Diego Instruments, San Diego, CA). Carbon dioxide was removed with a soda lime canister, and gas concentrations were sampled from a port in the circle system. Fresh gas inflow was provided by a 5-l/min oxygen flow through a calibrated isoflurane vaporizer. A circular flow through the four chambers was maintained by a fan producing a background noise of 70 dB (A-scale Sound Level Meter Radio Shack, Ft. Inlet and outlet ports allowed continuous ventilation through the chambers. The top of each training chamber contained an 8-cm diameter port sealed with a rubber cork. Training was accomplished in four identical rectangular box-shaped chambers (25 × 20 × 17 cm) constructed of clear acrylic and located in a well-lit room.