Although much has been learned about the neurobiological mechanisms underlying Pavlovian fear conditioning at the systems and cellular levels, relatively little is known about the molecular mechanisms underlying fear memory consolidation. The present experiments evaluated the role of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/ MAPK) signaling cascade in the amygdala during Pavlovian fear conditioning. We first show that ERK/MAPK is transiently activated-phosphorylated in the amygdala, specifically the lateral nucleus (LA), at 60 min, but not 15, 30, or 180 min, after conditioning, and that this activation is attributable to paired presentations of tone and shock rather than to nonassociative auditory stimulation, foot shock sensitization, or unpaired tone-shock presentations. We next show that infusions of U0126, an inhibitor of ERK/MAPK activation, aimed at the LA, dose-dependently impair long-term memory of Pavlovian fear conditioning but leaves short-term memory intact. Finally, we show that bath application of U0126 impairs long-term potentiation in the LA in vitro. Collectively, these results demonstrate that ERK/MAPK activation is necessary for both memory consolidation of Pavlovian fear conditioning and synaptic plasticity in the amygdala. Key words: amygdala; fear conditioning; ERK; MAPK; learning; LTPConsiderable evidence has implicated the lateral and basal nuclei of the amygdala (LBA) in the plastic changes underlying acquisition and retention of Pavlovian fear conditioning. Lesion, tract tracing, and electrophysiological studies suggest that fear conditioning involves transmission of sensory information to the lateral nucleus of the amygdala (LA) where alterations in synaptic transmission are thought to encode key aspects of the learning (Fendt and Fanselow, 1999;Maren, 1999;LeDoux, 2000). However, although fear conditioning has received much attention at the systems and cellular levels, relatively little is known about the molecular mechanisms that underlie consolidation of fear memory in the LA.One relatively recent discovery is the role of the mitogenactivated protein (MAP) family of kinases in synaptic plasticity and memory. These include the p38 MAP kinase (MAPK) and Jun (or stress-activated protein) kinase members, which have been implicated in stress-related cellular responses to injury or inflammation, and also the extracellular signal-regulated kinase (ERK), which has been implicated in cellular growth and differentiation (Kornhauser and Greenberg, 1997;Impey et al., 1999;Oruban et al., 1999). In neurons, ERK/MAPK has been shown to be potently activated by phosphorylation after synaptically driven increases in intracellular Ca 2ϩ (Rosen et al., 1994;Impey et al., 1999). Furthermore, ERK/MAPK has been shown to be activated-phosphorylated in the hippocampus after long-term potentiation (LTP) induction in the Schaffer collateral pathway, an effect that is blocked, along with LTP, by pretreatment with inhibitors of ERK/ MAPK activation Sweatt, 1996, 1997;Imp...

Long-term potentiation (LTP) at sensory input synapses to the lateral amygdala (LA) is a candidate mechanism for memory storage during fear conditioning. We evaluated the effect of L-type voltage-gated calcium channel (VGCC) and NMDA receptor (NMDAR) blockade in LA on LTP at thalamic input synapses induced by two different protocols in vitro and on fear memory in vivo. When induced in vitro by pairing weak presynaptic stimulation with strong (spike eliciting) postsynaptic depolarization, LTP was dependent on VGCCs and not on NMDARs, but, when induced by a form of tetanic stimulation that produced prolonged postsynaptic depolarization (but not spikes), LTP was dependent on NMDARs and not on VGCCs. In behavioral studies, bilateral infusions of NMDAR antagonists into the LA impaired both short-term and long-term memory of fear conditioning, whereas VGCC blockade selectively impaired long-term memory formation. Collectively, the results suggest that two pharmacologically distinct forms of LTP can be isolated in the LA in vitro and that a combination of both contribute to the formation of fear memories in vivo at the cellular level.

Fear conditioning is a form of associative learning in which subjects come to express defense responses to a neutral conditioned stimulus (CS) that is paired with an aversive unconditioned stimulus (US). Considerable evidence suggests that critical neural changes mediating the CS-US association occur in the lateral nucleus of the amygdala (LA). Further, recent studies show that associative long-term potentiation (LTP) occurs in pathways that transmit the CS to LA, and that drugs that interfere with this LTP also disrupt behavioral fear conditioning when infused into the LA, suggesting that associative LTP in LA might be a mechanism for storing memories of the CS-US association. Here, we develop a detailed cellular hypothesis to explain how neural responses to the CS and US in LA could induce LTP-like changes that store memories during fear conditioning. Specifically, we propose that the CS evokes EPSPs at sensory input synapses onto LA pyramidal neurons, and that the US strongly depolarizes these same LA neurons. This depolarization, in turn, causes calcium influx through NMDA receptors (NMDARs) and also causes the LA neuron to fire action potentials. The action potentials then back-propagate into the dendrites, where they collide with CS-evoked EPSPs, resulting in calcium entry through voltage-gated calcium channels (VGCCs). Although calcium entry through NMDARs is sufficient to induce synaptic changes that support short-term fear memory, calcium entry through both NMDARs and VGCCs is required to initiate the molecular processes that consolidate synaptic changes into a long-term memory.

While learning to fear stimuli that predict danger promotes survival, the inability to inhibit fear to inappropriate cues leads to a pernicious cycle of avoidance behaviors. Previous studies have revealed large inter-individual variations in fear responding with clinically anxious humans exhibiting a tendency to generalize learned fear to safe stimuli or situations. To shed light on the origin of these inter-individual variations, we subjected rats to a differential auditory fear conditioning paradigm in which one conditioned auditory stimulus (CSϩ) was paired to footshocks whereas a second (CSϪ) was not. We compared the behavior of rats that received pretraining excitotoxic lesions of the bed nucleus of the stria terminalis (BNST) to that of sham rats. Sham rats exhibit a continuum of anxious/fearful behaviors. At one end of the continuum were rats that displayed a poor ability to discriminate between the CSϩ and CSϪ, high contextual freezing, and an anxiety-like trait in the elevated plus maze (EPM). At the other end were rats that display less fear generalization to the CSϪ, lower freezing to context, and a nonanxious trait in the EPM. Although BNST-lesioned rats acquired similarly high levels of conditioned fear to the CSϩ, they froze less than sham rats to the CSϪ. In fact, BNST-lesioned rats behaved like sham rats with high discriminative abilities in that they exhibited low contextual fear and a nonanxious phenotype in the EPM. Overall, this suggests that inter-individual variations in fear generalization and anxiety phenotype are determined by BNST influences on the amygdala and/or its targets.