Experiment 1 investigated the effect of preexposure on conditioning, extinction, and recovery of fear responses. Rats were preexposed to one stimulus (designated A) and were conditioned to fear this stimulus, as well as a novel stimulus (designated B), and then responses to both CSs were extinguished. Following extinction training, rats received a US-alone exposure and were tested for reinstatement of conditioned responding to both CSs. Following a 5-day retention interval, rats were tested for recovery of conditioned responding to both CSs. The experiment was conducted and then replicated (n = 16 rats in each). There were no differences between the results of the two replications. Therefore, for convenience of exposition, the two replications are reported as a single experiment (n = 32).
Subjects were 32 experimentally naïve adult male Wistar rats (Rattus norvegicus) weighing between 400 and 470 g at the beginning of the experiment. They were obtained from a commercial supplier (Animal Research Centre, Perth, Australia) and were housed in groups of 8 in opaque plastic boxes (67 cm length × 40 cm width × 22 cm height). The boxes were kept in an air-conditioned colony room maintained on a 12:12-h light:dark cycle (lights on at 7:00 a.m.). Food and water were continuously available in the home cage during all phases of the experiment. Each rat was handled for 2–3 min each day for 4 days prior to the start of the experiment. All experimental procedures occurred between 9 a.m. and 6 p.m. The procedures were consistent with the ethical guidelines established by the American Psychological Association and were approved by the Animal Care and Ethics Committee of the University of New South Wales.
All phases of the experiment occurred in four identical chambers (23.5 cm depth × 20.5 cm width × 19.5 cm height). The front and rear walls of these chambers, as well as the hinged lid, were constructed of clear Perspex, and the sidewalls were constructed of stainless steel. The floor of each chamber was composed of 2-mm-wide stainless steel rods spaced 13 mm apart (center to center). The floor of each chamber was 3 cm above a tray of paper pellet bedding material (Fibrecycle, Mudgeeraba, Australia) that was changed between rats. After each rat had been removed from the chambers, the floors, walls, and lids were cleaned with a 25% white vinegar (Coles Supermarkets, Australia) solution to eliminate any residue and provide a distinctive odor to the chambers.
Each chamber was enclosed in a sound- and light-attenuating shell. The inside walls of the shells were painted black. A white fluorescent tube and a speaker mounted at the back of each shell were used, respectively, for the presentation of a 30-s light CS (approximately 57 lux measured at the center of the chamber), flashing at a rate of 2.5/s during the otherwise dark session, and a 30-s 1-kHz spike wave tone CS with a rise/fall time of 10 ms, measuring 75 dB (a scale) against a background noise of approximately 45 dB, measured by a digital sound level meter (Dick Smith Electronics, Australia). In this and the subsequent experiments, the physical identity of all the CSs was fully counterbalanced, with half of the subjects preexposed to the tone and half to the flashing light. A constant-current shock generator, capable of delivering unscrambled AC 50 Hz to the floor of each chamber, was used for the presentation of a 0.5-mA, 0.5-s shock US. Illumination of each chamber was provided by an infrared light source (940 ± 25 nm). A camera mounted on the back wall of each shell was used to record the behavior of each rat. Each camera was connected to a monitor and a DVD recorder located in another room of the laboratory. This room contained the computer that controlled stimulus presentations via appropriate software (LabView, National Instruments, Austin, TX).
The experiment involved seven phases: exposure to context, preexposure to the CS designated A, conditioning, extinction, US reexposure, reinstatement testing, and recovery testing.
Exposure to context
On day 1, rats received a 30-min exposure to the experimental chambers in order to familiarize them with the context, thereby potentially reducing context conditioning.
Pre-exposure to A
On days 2–7, rats received daily sessions consisting of 20 nonreinforced exposures to the tone or flashing light (designated A). The first stimulus presentation in each session occurred 3.5 min after each rat had been placed in the chamber. Each stimulus presentation lasted 30 s, and the average interval between stimulus presentations (defined as stimulus offset to stimulus onset) was 1.5 min (range: 30–150 s). After the final stimulus presentation in each session, rats remained in the chamber for a further 2 min before they were returned to their home cages. Thus, each preexposure session lasted 44 min, and there were 120 exposures to the stimulus designated A across the 6 days of this phase.
On days 8 and 9 rats received daily sessions consisting of four conditioning trials to each of the two CSs (i.e., eight trials in total to each CS over the 2 days of conditioning). The first CS was presented 2 min after each rat had been placed in the chamber. For half the rats, the session started with a familiar CS (designated A), while the remaining rats started the session with a novel CS (designated B). Thereafter, each CS was presented in an intermixed, pseudorandom order, with the constraint that the same CS could not occur on more than two consecutive trials. Each presentation of a CS lasted 30 s and coterminated with a footshock US (0.5-mA intensity and 0.5-s duration). The average interval between trials was 4.5 min (range: 2.5–6.5 min). After the final CS presentation in each session, rats remained in the chamber for a further 2 min before they were returned to their home cages. The average trial position for each CS was counterbalanced across the two conditioning sessions.
On days 10–13, rats received daily sessions consisting of 10 nonreinforced presentations of each CS (a total of 20 trials per session). The first CS presentation in each session occurred 3.5 min after the rats had been placed in the chambers. Each CS presentation lasted for 30 s, and the average interval between CS presentations was 1.5 min (range: 30–150 s). The order in which the two CSs were presented during extinction was subject to the same constraints as those described for conditioning.
On day 14, rats were placed in the chambers, and after 5 min, a footshock was delivered that was identical to that used during conditioning. Rats remained in the chambers for an additional 5 min before being returned to their home cages (the session lasted for a total of 10.5 min). On day 15, rats were tested for freezing to A and B under conditions of extinction. The two CSs were presented in the same order and with the same constraints as during extinction.
Following the reinstatement test, rats remained in the colony room for the next 4 days, where they were handled for 2–3 min every second day. On day 20, rats were tested for recovery to both CSs. The details for this test session were identical to those described above for the reinstatement test.
Scoring and statistical analyses
The behavior of each rat over conditioning, extinction, reinstatement, and recovery test was videotaped and then scored to determine the levels of freezing behavior, which was defined as absence of all movements except those related to breathing (Fanselow, 1980). The levels of freezing were measured using a time-sampling procedure in which each rat’s behavior was scored as freezing or not every 2 s. Scoring began 30 s prior to stimulus onset (pre-CS interval), to provide indication of the fear to context, and terminated 30 s post-stimulus-onset. The number of occasions on which the behavior was scored as freezing was expressed as a percentage of the total number of observations (i.e., 15 samples for a 30-s CS). All the data were scored by the experimenter, who was blind to the rat’s condition, and 25% of the test data were scored by an observer who was naïve as to the allocation of rats to counterbalanced conditions. The correlation between their scores was high (r > .90). Any discrepancies in scoring were resolved in favor of the naïve observer.
Freezing levels to the two CSs (A and B) in all phases of this and the subsequent experiments were analyzed using planned orthogonal contrasts which controlled the per-contrast error rate at α = .05 (Hays, 1973). With degrees of freedom ν1 = 1 and ν2 = 31, the F
critical(1, 31) value was 4.16 throughout all the phases of Experiment 1. A multivariate approach to repeated measures data was used (MANOVA; O’Brien & Kaiser, 1985). For clarity, conditioning data were analyzed in single trials, whereas the data for extinction, reinstatement, and recovery were presented and analyzed in blocks of two trials. Paired t tests were used to verify recovery of freezing between extinction and recovery test sessions (averaged over CS type), and where relevant, they were also used to investigate the source of significant interactions (trend × CS type).
Exposure to context, preexposure, and conditioning proceeded uneventfully. On the last conditioning session, the levels of freezing to A and B and the context were 53.33%, 57.71%, and 60.52%, respectively. Statistical analysis revealed that there was no difference in freezing to A and B across conditioning trials, F(1, 31) = 1.84, p = .19.
The mean percentage of freezing during each CS presentation, as well as 30 s prior to CS onset, over extinction training are shown in Fig. 1a. Inspection of this panel suggests that freezing to both CSs and the context decreased across extinction sessions. Inspection also suggests that, across extinction training, there was greater freezing during the presentation of the CSs (A and B) than during the context (pre-CS period). This was confirmed by statistical analysis, F(1, 31) = 309.63, p < .05. Statistical analysis also revealed a significant main effect of CS type, F(1, 31) = 9.54, p < .05, indicating that, across extinction, there was less freezing to A than to B. Analysis also revealed a significant linear decline in freezing across extinction blocks, F(1, 31) = 185.03, p < .05 indicating that responding to both CSs decreased across presentations. The linear trend × CS type interaction was not significant, F(1, 31) < 1.
The reinstatement data for both CSs and the pre-CS period are shown in Fig. 1b. Inspection of the panel also suggests that the unsignaled US presentation produced a reinstatement of conditioned responding, as compared with the level of freezing observed at the end of extinction, t(31) = 9.76, p < .05. Inspection of the data suggests that, across the reinstatement test, there was greater freezing to the CSs than to the context (pre-CS period). This was confirmed by statistical analysis, F(1, 31) = 117.02, p < .05. There was a significant linear trend, F(1, 31) = 24.99, p < .05, indicating that freezing responses to both CSs decreased in a linear fashion across the reinstatement test session. However, the main effect of CS type, F(1, 31) < 1, p > .35, and the linear trend × CS type interaction, F(1, 31) = 2.21, p > .35, were not significant.
The mean percentage of freezing during the presentation of both CSs and the pre-CS period in recovery tests are shown in Fig. 1c. Inspection suggests that there was substantial recovery of freezing to both CSs, as compared with the end of the reinstatement test, t(31) = 3.05, p < .05. Furthermore, over the course of the recovery test, there was significantly greater freezing to the CSs than to the context (pre-CS period), F(1, 31) = 123.508, p < .05. Statistical analysis between the CSs revealed a significant main effect of CS type, F(1, 31) = 13.33, p < .05, confirming that the levels of freezing were greater for A than for B. Furthermore, the level of freezing showed a linear decrease over the course of the test, F(1, 31) = 27.55, p < .05. The CS type × linear trend interaction was not significant, F(1, 31) < 1, indicating that there were no differences in the rate of decrease of responding as a function of CS type.
Experiment 1 failed to show an effect of preexposure on acquisition of conditioned freezing responses. This is likely due to high overall levels of freezing during fear conditioning that obscured differences in conditioning strength between the preexposed (A) and nonpreexposed (B) CSs. However, less freezing to A than B (i.e., latent inhibition) was observed during extinction once overall levels of freezing decreased. Preexposure did not affect the magnitude of reinstatement, with an unsignaled US presentation reinstating conditioned responding to both CSs to an equal level. During the delayed test, there was a reversal of the latent inhibition pattern observed during extinction, with greater recovery of conditioned responses to A than to B.
The differential recovery to the CSs in the delayed test confirms and extends Leung and Westbrook’s (2010) findings in showing that responding to a preexposed and extinguished CS recovers to a higher terminal level than does a CS just extinguished. However, as in the Leung and Westbrook study, it is not clear whether the differential recovery can be attributed to the passage of time alone, or whether the greater recovery to A than to B was precipitated by experience of unsignaled shock between the start of extinction and test. Accordingly, the following experiments were designed to identify the importance of unsignaled shock presentation in producing greater recovery to the preexposed than to the nonpreexposed CS.