Protein kinase Mzeta maintains fear memory in the amygdala but not in the hippocampus.

Long-term potentiation (LTP) is a persistent, activity-dependent increase in synaptic strength that is believed to be a neural substrate for some forms of memory formation and storage (Martin, Grimwood, & Morris, 2000; Moser, Krobert, Moser, & Morris, 1998; Whitlock, Heynen, Shuler, & Bear, 2006). While the induction of LTP is fairly well characterized, the maintenance of established LTP has historically received less attention. Recent work indicates that protein kinase Mzeta (PKMζ), an atypical isoform of protein kinase C, is both necessary and sufficient to maintain hippocampal LTP (Ling et al., 2002; Sajikumar, Navakkode, Sacktor, & Frey, 2005). PKMζ consists of a catalytic domain that is independent of the regulatory domain that accompanies all other PKC isoforms, giving it the unique ability to constantly maintain LTP by increasing the number of active postsynaptic AMPA receptors (Hernandez et al., 2003; Ling, Bernado, & Sacktor, 2006; Ling et al., 2002; Muslimov et al., 2004; Serrano, Yao, & Sacktor, 2005). To the extent that LTP represents a physiological substrate for long term memory in the behaving animal, inhibition of this kinase would be expected to disrupt the retention or storage of memory. In vivo studies have demonstrated that inhibition of PKMζ appears to erase certain forms of established memories (Pastalkova et al., 2006; Shema, Hazvi, Sacktor, & Dudai, 2009; Shema, Sacktor, & Dudai, 2007). Spatial memory can be disrupted if PKMζ is inhibited in the hippocampus, a brain area known to play a role in spatial and contextual learning (Pastalkova et al., 2006; Serrano et al., 2008). Similarly if PKMζ is inhibited in the insular cortex, which plays a critical role in the learning of conditioned taste aversion, 1-month and 3-month-old taste aversion memories can be effectively erased (Shema et al., 2007; 2009). It is critically important to determine precisely which forms of memory require PKMζ activity. Recently, it was suggested that PKMζ is necessary to maintain specific associations but is not required for general contextual or procedural memory (Serrano et al., 2008). Consistent with this hypothesis, inhibition of PKMζ activity in the dorsal hippocampus disrupted the maintenance of specific spatial memory in the radial arm maze and water maze but was not effective in disrupting working memory or procedural strategies in these tasks (Serrano et al., 2008). Spatial memory in inhibitory avoidance and active avoidance tasks requiring similar contextual cues, however, were fully impaired by PKMζ inhibition (Pastalkova et al., 2006, Serrano et al., 2008). The distinction between spatial memories that require PKMζ and those that are independently maintained remains unclear. The role of PKMζ in the maintenance of context fear associations is central to this debate. There is currently general agreement that the acquisition and long-term retention of Pavlovian fear conditioning, in which neutral cues are arranged to predict an aversive outcome such as foot shock, critically depends on processes occurring within the amygdala (Fanselow & LeDoux, 1999; Helmstetter, Parsons, & Gafford, 2008; Maren, 2001). Exposing rats to the training protocol during fear conditioning results in altered gene expression in amygdala neurons (Levenson et al., 2004; Ressler, Paschall, Zhou, & Davis, 2002; Stork, Stork, Pape & Obata, 2001), induction of LTP at local synapses (Rogan & LeDoux, 1995) and the activation of intracellular signaling pathways involved in long–term synaptic modification (Parsons, Gafford, & Helmstetter, 2006). If amygdala neurons are prevented from making new mRNA or protein during the period immediately after training, no new memories are formed (Bailey, Kim, Sun, Thompson, & Helmstetter, 1999; Parsons et al., 2006). Disruption of local protein synthesis in the amygdala during the period after memory retrieval is also sufficient to disrupt the “reconsolidation” of that memory (Nader, Schafe, & LeDoux, 2000). Contextual fear conditioning requires involvement of the hippocampus in addition to the amygdala. It is believed that the hippocampus is responsible for providing a configural representation of individual context cues to the amygdala, where it becomes associated with the footshock (Matus-Amat, Higgins, Barrientos, & Rudy, 2004; Rudy & O'Reilly, 1999, 2001). Posttraining lesions of the hippocampus can prevent the recall of recent contextual fear without disrupting fear to a discrete CS, such as a tone (Kim & Fanselow, 1992). The involvement of the hippocampus in contextual but not cued fear learning allows for the study of two memories for the same training experience in a single animal. Both auditory and contextual fear memory formation can be disrupted by inhibiting general kinase activity or protein synthesis in the amygdala (Bailey et al., 1999; Goosens, Holt, & Maren, 2000; Maren, Ferrario, Corcoran, Desmond, & Frey, 2003; Parsons et al., 2006; Schafe & Le Doux, 2000). Injections of similar inhibitors in the hippocampus are effective in disrupting context fear memory formation without affecting fear to the discrete auditory CS (Fischer, Sananbenesi, Schrick, Spiess, & Radulovic, 2004; Gafford, Parsons, & Helmstetter, 2005). Importantly, these inhibitors are only effective if applied within a few hours of the acquisition trial (Schafe & LeDoux, 2000) and thus affect memory formation, rather than memory storage. PKMζ inhibition, however, is uniquely able to reverse some forms of established memory after the period of consolidation has passed (Pastalkova et al., 2006; Sacktor, 2008; Shema et al., 2007; Serrano et al., 2008). Based on this past research, it might be expected that PKMζ inhibition in the amygdala would disrupt both contextual and auditory fear memory while inhibition of PKMζ in the hippocampus would selectively impair contextual fear memory. Surprisingly, initial work indicates that inhibiting PKMζ in the hippocampus fails to disrupt context fear memory while its inhibition in the amygdala following a slightly different training protocol is sufficient to impair both context and auditory fear memories (Serrano et al., 2008). The purpose of this study was to further investigate the role of PKMζ in the maintenance of context and auditory fear memory using a standard Pavlovian fear conditioning procedure for all animals. Both the training context and an auditory cue were used as conditional stimuli (CS) and electric shocks were used as the biologically significant outcome. After training but before memory retrieval we applied the selective PKMζ inhibitor, ζ-pseudosubstrate inhibitory peptide (ZIP) (Ling et al., 2002; Pastalkova et al., 2006) to the basolateral complex of the amygdala or the dorsal hippocampus. Animals were tested in the training context 2 hours after ZIP injection to assess the strength of their context fear memory. Following this initial test, a number of follow-up tests were performed to ensure that any observed memory deficits were long-lasting and not attributable to tissue damage. These results will indicate whether long-term memory storage mechanisms in the hippocampus and amygdala use similar or different intra-cellular principles to maintain an identical associative fear memory.