Disruption of fear memory consolidation and reconsolidation by actin filament arrest in the basolateral amygdala
Introduction
Dynamic changes of actin filaments play a critical role in structural plasticity that is believed to underlie information storage in neural networks (Lamprecht and LeDoux, 2004, Matus, 2000). Presumably through trafficking of neurotransmitter receptors and cytoskeletal restructuring of pre- and post-synaptic sites, actin filaments help recently acquired fear memories to be transformed into a more lasting state, a process called memory consolidation. Indeed, changes of synaptic actin filaments and their effect on post-synaptic spines have been demonstrated in cultivated neuronal cells upon induction of synaptic plasticity (Honkura, Matsuzaki, Noguchi, Ellis-Davies, & Kasai, 2008). These match with observations made in vivo of post-synaptic actin filament assembly (Fukazawa et al., 2003) and long recognized morphological changes at the synapse (e.g., Fifkova & Van Harreveld, 1977). In fact, interfering with actin filament dynamics disturbs long-term potentiation of neuronal activity in the hippocampus (Kim and Lisman, 1999, Krucker et al., 2000) and local injections of the actin depolymerizing toxins cytochalasin D or lantrunculin A into the amygdala or hippocampus disturb the formation of auditory cued and contextual fear memory (Mantzur, Joels, & Lamprecht, 2009).
Classical fear conditioning is very well suited for the investigation of such memory consolidation processes. Subjects very quickly learn to associate a previously neutral stimulus (conditioned stimulus, CS, such as a tone) or context with a coinciding aversive stimulus (unconditioned stimulus, US, such as electric foot shock). After a single training session, animals form a robust fear memory and to subsequent exposures to the CS respond with species-specific defensive behaviors, such as freezing (Blanchard & Blanchard, 1969). It is now well established that the basolateral complex of the amygdala (BLA) provides a core structure and site of neural plasticity in such classical fear conditioning. The BLA, comprising the lateral and basal amygdala subnuclei, serves as a convergence site for uni- and multimodal sensory signals with ascending nociceptive pathways and affective information processed in limbic brain circuits (Maren, 2003, Maren and Quirk, 2004). Importantly, neural plasticity has been observed in this region following behavioral training as well as after electrical stimulation of thalamic, hippocampal or cortical afferences (Rogan et al., 1997, Sah et al., 2008). Molecular and cellular processes that are critical for the acquisition and consolidation of fear memories have been identified and ample evidence has accumulated for an involvement of actin cytoskeleton dynamics (Diana et al., 2007, Lamprecht et al., 2002, Lamprecht, Farb, et al., 2006, Mantzur et al., 2009, Stork et al., 2004). Fear conditioning has also been used to investigate the phenomenon of memory reconsolidation, i.e., the modification and renewed storage of prior memories upon their retrieval. It was shown that although consolidation and reconsolidation share principle characteristics, neurochemical and molecular details are only partially overlapping (Alberini, 2005, Tronson and Taylor, 2007).
In the current study we tested how an arrest of actin filaments in the mouse BLA through bilateral application of the death cap (amanita phalloides) toxin phalloidin affects the consolidation and reconsolidation of Pavlovian fear memory. We focused on the proposed role of actin filaments in the morphological changes in synaptic structures, which typically become evident ca. 0.5 h after stimulation and are thought to provide a long-lasting memory correlate (Bonhoeffer and Yuste, 2002, Korkotian and Segal, 2001). To begin to dissect these processes, phalloidin was applied to the BLA in different animals at time points either 0.5 h, 6 h and 24 h after conditioning or retrieval. Our results not only provide evidence for a similar role of amygdalar actin filament dynamics in consolidation and reconsolidation of auditory fear memory, but also suggest that a temporally distinct actin-dependent cellular process in the BLA is involved in responses to a familiarized background context upon fear memory re-activation.
Section snippets
Subjects
Eight-to-twelve-week old male C57BL/6JBomTac mice (M&B Taconic, Berlin, Germany) were used in all experiments. Mice were purchased at an age of 6 weeks and maintained in our facility in groups of 3–4 under a reversed 12 h day/12 h night cycle (lights on at 19:00) with food and water ad libitum. Two days before the experiments, animals were singled out and further-on kept individually in transparent cages, maintaining visual, auditory and olfactory contact to their conspecifics. The time course of
Probe localization
A total of 102 animals received bilateral injections of either phalloidin or vehicle to the BLA. Close histological examination of the injection sites revealed an intact cellular architecture beneath the injection site and cellular phalloidin-rhodamine staining. A summary of injection sites of animals, which were tested for consolidation and reconsolidation is given in Fig. 1B and for animals tested in elevated plus maze in Fig. 5D.
Fear behavior
To determine the role of actin filament dynamics in fear
Discussion
In the current study we demonstrate that Pavlovian fear memory is severely disturbed if actin filament disassembly in the mouse BLA is prevented through local application of the fungal toxin phalloidin at specific time points after its acquisition or its re-activation. Our data add to a number of recent molecular and pharmacological studies in that we (1) demonstrate the involvement of actin filament dynamics in both consolidation and reconsolidation of conditioned fear and (2) define a
Acknowledgments
We are grateful to C. Obst, F. Webers and A. Koffi von Hoff for expert technical assistance, and to A. Deters and T. Nawrath for excellent animal care. This work was supported by grants from the German Research Foundation (DFG, STO488/3 and GRK1167 to OS).
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These authors contributed equally to this work.