SUPPLEMENTARY METHODS
Subjects: Sprague-Dawley rats from (Charles River Laboratories, Maine, USA) were
used in all experiments. Rats were pair-housed in plastic cages with ad libitum access
to food and water. Animals were kept on a 12hr:12hr light:dark cycle (lights on at 7
a.m. and off at 7 p.m.).
Surgery: Rats were implanted with bilateral cannulae (Plastics One, USA) directed at
the basolateral amygdala (AP: -3.0, ML:+5.0, DV:-7.9) (10). Surgical anaesthesia was
Equithesin, and Tylenol was given for post-surgical analgesia. All animals were
allowed 10 days to recover before fear conditioning.
Drugs: 6-benzoyl-cAMP, (6-BNZ-cAMP; Biolog, USA) the cAMP analog and
specific PKA activator (6) was dissolved in 1XPBS for a concentration of 1mM, and
1L was infused per side for a total of 1nM of 6-BNZ-cAMP per side, per day. PBS
was infused as the vehicle control, with 1L infused per side per day.
RP-cAMPS was dissolved in 1XPBS for a concentration of 36g/L (4,5),
and 0.5L was infused per side for a total of 18g of RP-cAMPS per side. PBS was
infused as the vehicle control, with 0.5L infused per side per day.
All drugs were infused at a rate of 0.25L/minute over four minutes (Harvard
Apparatus, USA), and infusion cannulae (Plastics One, USA) were removed 2
minutes after the completion of the infusion to allow time for drug diffusion.
Fear Conditioning: All animals were trained in Context A, a dark, quiet, operant
chamber (MedAssociates, USA) with standard bar floors wiped with 70% EtOH in a
dark room.
Training for the reconsolidation experiment and control groups consisted of a
five-minute habituation period followed by a single 30-second tone co-terminating
with a 1-second, footshock. Since low initial levels of freezing were required to allow
the possibility of enhancements, animals in the 6-BNZ-cAMP received single 30second tone paired with a 1-second, 1.3mA footshock. Training for the extinction
experiment was identical except for the addition of a second tone-shock pairing two
minutes after the first.
Conversely, high levels of freezing were required to see disruptions, and
animals in the RP-cAMPS experiment received a single 30-second tone paired with a
1-second, 2.0mA footshock. Animals were returned to their homecages 30-seconds
following offset of the final tone.
All stimuli were controlled by programs in MED-PC IV (MedAssociates,
USA).
Reconsolidation: The reconsolidation procedures were modified from that described
by Nader et al., (11). Reactivation (retrieval) trials were conducted in Context B, a
light 5-choice box (MedAssociates, USA) with fan background noise, and mesh
floors, wiped with a 1% lemon-extract solution (McClintock, USA). This context shift
was done to ensure specificity of the effect to memory for the cue (tone) as opposed
to the context.
Reactivation sessions consisted of a five-minute habituation period followed
by presentation of the 30-second tone. No shock was presented during these sessions.
Rats were removed to a different room 30-seconds after the tone offset, and postsession infusions were done immediately.
For the 6-BNZ-cAMP experiment examining enhancement of reconsolidation,
this session and infusion were repeated for four consecutive days, and a fifth
reactivation session was presented 72-hours after the fourth day.
For the Rp-cAMPS experiment, a single reactivation trial followed by infusion
was given only once, and freezing to the tone was assessed in the same context the
following day.
No Reactivation controls were given identical exposure to context B as their
respective experimental groups, however no tone CS was presented. Rats were placed
in Context B for 6-minutes before being removed to the adjacent room and given
PBS/6-BNZ-cAMP, or PBS/Rp-cAMPS infusions.
Delayed [6-hour] infusion controls were treated identically to the experimental
groups for the respective experiments, except that infusions were given 6 hours after
reactivation trials and not immediately after. Animals were returned to homecages in
the time interval between reactivation and infusions.
No training controls never received fear conditioning, and were instead placed
directly into Context B for a “reactivation” session of a novel tone, identical to those
described above. At the conclusion of the session, animals were removed from the
boxes and given infusions of PBS, RP-cAMPS, or 6-BNZ-cAMP either immediately,
or 6-hours after exposure to the tone. Groups received the same number of
“reactivation” sessions and infusions as in the respective experimental groups.
Extinction: Extinction sessions were conducted in Context B (as described above).
After a 5-minute habituation period, seven 30-second tone CS’s were presented, with
a 2-minute ITI. Animals were removed from context B 30 seconds after the final CS
presentation and were immediately given infusions of PBS or 6-BNZ-cAMP.
Behavioral Scoring: All reactivation and extinction sessions were video-recorded
using Sony Hi-8 equipment, and tapes were later scored for freezing by someone
blind to treatment condition. Scoring (freezing or not) was recorded every other
second in the 30-seconds prior to tone onset (pre-tone freezing) and throughout the
30-second tone, allowing a Percent Freezing score to be calculated for each pre-tone
and tone period throughout each session.
Statistical Analysis: Repeated measures ANOVA was used to analyze the effect of
treatment across days, and post-hoc t-tests were used to further investigate
differences.