2.3.1 Subjects
Sprague-Dawley rats (250-300 grams, from Harlan) were used for breeding at the University of Texas at Austin. For the first experiment, offspring from 17 breeding pairs were used (n=99). All breeding pairs were left together for approximately two weeks before the male breeder was removed and the female was left housed individually until pups were born. Females were checked daily at 10AM to determine if births had occurred, on the first day pups were present this was marked as the date of birth and considered P0. When pups reached either P17 or P25, all rats except controls that remained naïve, received either paired or unpaired presentations of the CS (white noise) and US (footshock). Only one male per litter was used per group in order to avoid litter effects. All animals were weaned at 21 days of age and housed two or three animals per cage on a 12h/12h light/dark cycle with food and water ad libitum. Our basic principle regarding housing for this project was to match the cagemates on their adult experimental treatment condition. As such, at weaning, the rats were housed with cagemates that received a different treatment at P17 or P25 (naïve, paired, and unpaired). For the first experiment, rats from a given litter were used for either the P17 or P25 experiment (not both).
For the second experiment, offspring from six breeding pairs were used to assess long term memory 24-hours after paired conditioning at either P17 or P25 (n=24). All rats were conditioned using paired conditioning procedures detailed below or remained
naïve. All groups included one rat from each breeding pair. Upon weaning, rats were housed with a brother until termination of the experiment. Behavioral training and testing were performed during the rats’ dark phase under dim light. The studies were approved by the IACUC of the University of Texas at Austin.
2.3.2 Apparatus and procedures
Four identical conditioning chambers (Coulbourn Instruments, Allentown, PA) were used to fear condition the rats. The chambers were equipped with stainless-steel rod floors (at .5cm thickness, designed for mice such that infant rats could not slip through the bars into the lower pan of the chamber) connected to a shock-generator (Model H13-15; Coulbourn Instruments). Each chamber was enclosed in a sound attenuated box. All experimental procedures were controlled by a computer program (Freezeframe;
Coulbourn Instruments) which allowed to precisely administer paired or unpaired white noise and footshock presentations. To provide a different context for the extinction phase, the inside of conditioning chamber was modified with black flat plastic floors (rather than metal grids) and the outside of the plexiglass walls of the conditioning box were covered with paper with alternating black and white lines.
2.3.3 Training
For the first experiment, training was as follows. Pups were conditioned to a CS (white noise 80dB, 10s) and US (0.6mA, 1s) at P17 or P25. After 2-minutes of
adaptation, one group received four trials of paired training, where the CS co-terminated with the US. The other group received four trials of unpaired training, where the CS and US did not overlap. The inter-trial interval (ITI) was varied from 85s to 135s with a mean
ITI of 110s. For the unpaired condition, the total duration spent in the conditioning apparatus was kept the same as the paired group. The ITI between stimuli (CSs and USs) varied between 35 and 85, with a mean ITI of 55. During P17 conditioning, vanilla extract (McCormick) was put on the mother’s nose to prevent her from discriminating between pups that were removed for training and pups who remained with her. Rats rely on their olfaction to identify their pups and we have found that when applying a small amount of vanilla on the mother’s nose, she does not interact differently with the pups that were taken away and those that remained with her. After each session, the
conditioning chamber was rinsed with water, followed by 70% ethanol (EtOH). A naïve group of rats were separated from their mother in an opaque plastic box (30
cm20cm15 cm) for 10-minutes, the same amount time that animals in other conditions spent for conditioning procedure. Naïve rats did not receive CS or US presentations. At two months of age, each group of animals received either another two trials of CS-US paired or un-paired training, and were tested for fear extinction the next day. For the fear extinction phase, after a 2-minute adaptation period, the 10s CS (80 dB, White Noise) was presented 18 times with a 60s ITI in a different context chamber (Figure 2.1A).
For the second experiment, all rats were tested for long term memory 24-hours after paired conditioning or removal from the homecage (naïve condition). Long-term memory was assessed in the same context half the rats were conditioned in the previous day (Figure 2.1B), using a 5-minute adaptation period followed by four, 10s CS (80 dB, White Noise) presentations with a varied ITI, mean 110s and ranging between 85s to
135s, similar to that used for the conditioning session the previous day, but in a reverse presentation.
Figure 2.1 Schematic diagrams of the experimental procedures. (A) In experiment 1 pups were conditioned using either paired or unpaired procedures, or remained naïve at P17 or P25. At two months of age, each group was tested for memory to the context and the cue followed by either paired or un-paired CS-US training, 24-hours later rats went through cued fear extinction, and finally tested for LTM the next day. (B) Experiment 2 consisted of pups receiving paired conditioning or naïve procedures at P17 or P25 followed by LTM testing the following day. LTM=Long-term Memory, CS=
Conditioned Stimulus and US= Unconditioned Stimulus.
2.3.4 Freezing measurement
Freezing behavior was defined as the absence of all bodily movement, excluding respiration related movement, and was quantified as total percent time during the 10-seconds for each CS presentation. Baseline freezing was collected during the 10-10-seconds before the 1st CS.
2.3.5 Statistical analysis
All freezing data were analyzed with Prism6 (Graphpad software, La Jolla, CA).
All data are presented as mean±SEM. Early fear memory retention was tested with one-way ANOVA, and adult fear extinction freezing was tested with a single-factor repeated-measures ANOVA using 3 different groups as a between subjects factor for each adult training condition (e.g. NP vs. UP vs. PP or NU vs. UU vs. PU; the 1st character
represents infant conditioning type, and 2nd represents adult conditioning type: N, Naïve;
P, Paired; U, Un-paired). If there were main effects or interactions, Tukey’s test were used for post-hoc analysis. The null hypothesis was rejected at the p < 0.05 level.