Synapse-specific reconsolidation of distinct fear memories in the lateral amygdala

doi:10.1038/nprot.2007.181

Method Article

Synapse-specific reconsolidation of distinct fear memories in the lateral amygdala

https://doi.org/10.1038/nprot.2007.181

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Neuroscience

Biological techniques

amygdala

auditory thalamus

fear conditioning

plasticity

retrieval

reconsolidation

Pavlovian fear conditioning is a paradigm in which an initially neutral stimulus such as a tone (conditioned stimulus, CS) acquires aversive properties and comes to elicit fear responses through its pairing with an aversive unconditioned stimulus (US), such as an electrical foot shock. In a typical reconsolidation experiment, a single CS is presented the day after training in the presence of a pharmacological agent aimed at disrupting the cellular machinery involved in reconsolidation processes. Post-reactivation short-term memory (PR-STM) is then tested shortly after the retrieval (within 1-4 hours) and post-reactivation long-term memory (PR-LTM) is tested 24 hours later. Intact performance at PR-STM but impaired at PR-LTM indicates disruption of time-dependent reconsolidation processes. Here we developed a new protocol in which two conditioned stimuli (CS) were used, one to be reactivated (CSr), the other not (CSn).

> behavioural chamber for auditory fear conditioning

> infusion pump

> electrophysiological setup for implantation and recordings in the awake animal

Surgery:

Implant rats (male Sprague-Dawley) with LA cannulas and a stimulation electrode aimed at the left auditory thalamus [medial geniculate nucleus/posterior intralaminar nucleus (MGm/PIN)].
Attach an insulated stainless steel recording wire (1-2 MΩ) to the left LA cannula, extending ~0.5 mm from the base of the infusion cannula. Determine the final depth using stimulation of the MGm/PIN as a guide. Place two cortical silver balls contralaterally to serve as a reference and ground.
Leave animals at least one week before starting the experiment.

Behavioural procedure:

Choose two CSs, either made of pure tone or FM tone, to minimize generalization. Our pure tone CS consisted of a 20-s series of acoustic tone pips (1 kHz, 50-ms duration, 1-ms ramp, intensity 20dB higher than the background noise, delivered at 1 Hz). The FM tone CS consisted of a 20-s series of frequency modulation tone sweeps (12.5-kHz carrier frequency, 50-Hz modulation frequency, 2.5-kHz modulation depth, 250-ms duration, 1-ms ramp, intensity 20 dB higher than background noise, delivered at 1 Hz).
Reactivate one of the tones (CSr) in a counterbalanced manner in each experiment (Supplementary Fig. 1a from Nature Neuroscience article). For the US, we used a 0.5-s (0.75-mA) electric foot shock.

Habituation to context (Day 1 & 2):

Place rats in the conditioning chamber for 30 min on 2 consecutive days. The purpose of these sessions is to minimize the development of conditioning to the context once the US is introduced.

CS pre-exposure (Day 3 & 4):

Give rats three pre-exposures of one of the CS followed by three pre-exposures of the other CS (variable intertrial interval (ITI), 157 s on average) in the chamber. Reverse the order on the next day.

Conditioning (Day 5):

Place the rats in the chamber. After a 120-s acclimation, give three CSr–US and three CSn-US pairings intermixed (variable ITI =130 s on average). Deliver the US immediately after the end of each CS.

Memory reactivation (Day 6):

Acclimate the rats to the chamber for 120s in a modified context with a black peppermint scented floor, and present a single CSr.
Thirty minutes before reactivation, infuse rats bilaterally with 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)-butadiene (U0126) (1 µg; 0.5 µl in 50% DMSO) or an equivalent volume of vehicle (50% DMSO). This dose of U0126 was chosen based on findings showing that it impairs fear memory consolidation and reconsolidation. Further, a recent study has shown that this dose of U0126 impairs the consolidation of neurophysiological changes associated with fear conditioning, while no impairment of acquisition at STM was produced by vehicle solution (50% DMSO) or U0126.

Post-reactivation short-term memory-retention tests (PR-STM):

Three hours after the reactivation trial, place rats in the chamber and test for PR-STM. After a 120 s acclimation period, present four presentations of CSr, followed 180 s later by four presentations of CSn.

Post-reactivation long-term memory retention tests (PR-LTM) (Day 7)

Approximately 24 hr after the reactivation trial, give rats a PR-LTM test. Present nine CSr and nine CSn following the same order as in the PR-STM test (CSr, followed by CSn).

Recordings and data analysis:

Record rats’ freezing behavior onto videotape during all sessions for off-line scoring. During pre-exposure and retention tests, auditory-evoked field potentials (AEFPs) should be recorded from both the LA and MGm/PIN (through the tip of the stimulation electrode), and analyzed using DataWave. Because a single trial does not provide stable enough electrophysiological recordings, no data can be collected during the reactivation trial. AEFPs can be averaged across all tone-pip presentations within a trial and collapsed across trials. The amplitude of the short latency negative component can be measured for each potential (_{12–16 ms for the LA;}7–9 ms for the MGm/PIN). As in our previous study on consolidation, we assessed retention of behavioral and electrophysiological changes by expressing the change during the last four trials of the PR-LTM test as a percentage of that during the four trials of the PR-STM test. A 100% value in PR-LTM results means no change from the PR-STM test.

Three weeks

Disruption of long-term memory selective to the reactivated memory, and its electrophysiological correlates in two neural structures.

Schafe, G.E., Doyère, V. & LeDoux, J.E. J Neurosci 25, 10010-10014 (2005).

Dębiec, J., Doyère, V., Nader, K. & LeDoux, J.E. Proc Natl Acad Sci USA 103, 3428-3433 (2006).

Doyère, V., Schafe, G.E., Sigurdsson, T. & LeDoux, J.E. Eur J Neurosci 17, 2703-2715 (2003).

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Synapse-specific reconsolidation of distinct fear memories in the lateral amygdala

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Version 1

Introduction

Equipment

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Anticipated Results

References

Associated Publications

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Synapse-specific reconsolidation of distinct fear memories in the lateral amygdala

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Version 1

Introduction

Equipment

Procedure

Timing

Anticipated Results

References

Associated Publications

Status:

Version 1

Privacy Policy

Terms of Service

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