Western blot analysis of neuronal tissues taken from fear-conditioned rats showed a selective activation of phosphatidylinositol 3-kinase (PI-3 kinase) in the amygdala. PI-3 kinase was also activated in response to long-term potentiation (LTP)-inducing tetanic stimulation. PI-3 kinase inhibitors blocked tetanus-induced LTP as well as PI-3 kinase activation. In parallel, these inhibitors interfered with long-term fear memory while leaving short-term memory intact. Tetanus and forskolin-induced activation of mitogen-activated protein kinase (MAPK) was blocked by PI-3 kinase inhibitors, which also inhibited cAMP response element binding protein (CREB) phosphorylation. These results provide novel evidence of a requirement of PI-3 kinase activation in the amygdala for synaptic plasticity and memory consolidation, and this activation may occur at a point upstream of MAPK activation.

Memory consolidation is mediated by new protein synthesis. However, the transcriptional pathways induced in neurons by behavioral training that activate gene responses have yet to be fully delineated. We have previously shown that nuclear factor B (NF-B) is activated in the amygdala after fear conditioning. Here we report that fear conditioning resulted in an increase in histone acetyl-transferase activity, the association between NF-B p65 and CBP, and the increase in acetylated p65. Pretreating animals with histone deacetylase (HDAC) inhibitors prolonged the nuclear expression of acetyl-p65 and increased its DNA binding activity. Consistent with these results, HDAC inhibitors enhanced long-term but not short-term fear memory, and this effect was attenuated by B decoy DNA, whereas scrambled DNA was without effect. This study provides evidence that HDAC-mediated deacetylation functions as an intranuclear molecular switch culminating in the termination of NF-B transcriptional response that is involved in the formation of fear memory.

Memory extinction refers to a gradual decrease of the previously acquired response when exposed to conditional stimulus without pairing with unconditional stimulus. Here we show for the first time that fear training-induced phosphorylation of specific substrates in the rat amygdala is reduced after extinction trials and is accompanied by an increase in the protein level and enzymatic activity of calcineurin. In parallel, calcineurin inhibitors prevented extinction-induced protein dephosphorylation as well as extinction of fear memory. Thus, extinction training increased phosphatase activity likely via an expression of calcineurin. Calcineurin then created a negative-feedback loop and directly or indirectly dephosphorylated specific substrates, which, in their phosphorylated state, were required for memory consolidation. Accordingly, in our experimental condition, extinction could be ascribed at least in part to a weakening of the original signaling.

This study is designed to characterize the signal cascades by which brain-derived neurotrophic factor (BDNF) modulates long-term memory of fear conditioning. Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis of tissue homogenates taken from fear-conditioned rats showed an increase in the amygdala of BDNF protein levels and its receptor TrkB phosphorylation. Bilateral administration of a TrkB ligand scavenger TrkB IgG and a Trk-specific tyrosine kinase inhibitor K252a to the amygdala impaired fear memory, as measured with fear-potentiated startle. Fear conditioning resulted in the association of Shc and TrkB, Shc and Ras, the increase in active Ras and phosphorylation of mitogen-activated protein kinase (MAPK). Treatment of amygdala slices with BDNF for 15 min increased the levels of active Ras, and MAPK and Akt phosphorylation. BDNF-induced MAPK phosphorylation was completely abolished by MEK inhibitors, and was partially inhibited by farnesyltransferase or phosphatidylinositol-3 kinase (PI-3 kinase) inhibitors. On the other hand, BDNF-induced Akt phosphorylation was unaffected by farnesyltransferase or MEK inhibitors, but could be blocked by PI-3 kinase inhibitors. Together, these data suggest a requirement of BDNF for fear learning. The memory-enhancing effect of BDNF involves the activation of MAPK and PI-3 kinase. BDNF-induced MAPK phosphorylation in the amygdala is mediated via TrkB and the Shc-binding site. Shc binding to TrkB leads to activation of Ras, Raf, and MEK. In addition, BDNF could induce phosphorylation of MAPK via activation of PI-3 kinase.

Much evidence indicates that extinction training does not erase memory traces but instead forms inhibitory learning that prevents the expression of original memory. Fear conditioning induces long-term potentiation and drives synaptic insertion of AMPA receptors into the amygdala. Here we show that extinction training applied 1 h after training reversed the conditioning-induced increase in surface glutamate receptor subunit 1 (GluR1) in parallel with the inhibition of startle potentiation. However, if applied 24 h after training, extinction training reduced startle potentiation without influencing the GluR1 increase. We infused D-cycloserine (DCS), a partial agonist of the glycine site on the NMDA receptor, bilaterally into the amygdala 30 min before extinction training. This augmented the extinction training-elicited reduction in startle and reversed the conditioning-induced increase in GluR1. Delivery of five sets of tetanic stimulation (TS) to the external capsule produced a robust enhancement of synaptic responses in the lateral amygdala neurons that persisted for Ͼ2 h. Low-frequency stimulation applied 1 h after TS had no long-lasting effect on synaptic responses. The same treatments, however, induced depotentiation in the presence of DCS and reversed TS-induced increase in surface GluR1. Together, this study has two important findings: (1) whether a memory trace remains intact or is erased depends on the interval between conditioning and extinction training and (2) DCS facilitates the reversal of memory trace. DCS-induced augmentation of extinction and reversal of GluR1 surface expression are likely mediated by DCS-facilitated endocytosis of AMPA receptors.

Understanding the mechanism of how fear memory can be extinguished could provide potential therapeutic strategies for the treatment of posttraumatic stress disorders. Here we show that infusion of CB1 receptor antagonist into the infralimbic (IL) subregion of the medial prefrontal cortex (mPFC) retarded cue-alone-induced reduction of fear-potentiated startle. Conversely, cannabinoid agonist WIN55212-2 (WIN) facilitated the extinction. Unexpectedly, administration of WIN without cue-alone trials reduced startle potentiation in a dose-dependent manner. The effect of cannabinoid agonists was mimicked by endocannabinoid uptake or fatty acid amide hydrolase inhibitors. Rats were trained with 10 conditioned stimulus (CS(+)) (yellow light)-shock pairings. Extinction training with CS(+) (yellow light)-alone but not CS(-) (blue light)-alone trials decreased fear-potentiated startle. Intra-IL infusion of WIN before CS(-)-alone trials decreased startle potentiation, suggesting that the cannabinoid agonist decreased conditioned fear irrespective of whether the rats underwent CS(+)- or CS(-)-alone trials. Cannabinoid agonists activated extracellular signal-regulated kinases (ERKs) in mPFC slices, and ERK inhibitor blocked the effect of cannabinoid agonists on fear-potentiated startle. These results suggest that CB1 receptors acting through the phosphorylation of ERK are involved not only in the extinction of conditioned fear but also in the adaptation to aversive situations in general.

The effects of selective beta-adrenergic receptor agonist isoproterenol (Iso) on neuronal excitability and synaptic transmission were investigated in brain slices of rat amygdala. Iso (15 microM) produced a long-lasting enhancement of the EPSP that was not blocked by pretreatment with 20 microM D-2-amino-5-phosphonovalerate (D-APV) alone or D-APV in combination with kynuretic acid (1 mM). The sensitivity of postsynaptic neurons to the glutamate receptor agonist AMPA was unchanged by Iso pretreatment. Superfusion of Iso reversibly blocked the after-hyperpolarization (AHP) that followed a depolarizing current pulse and caused more action potential firing. Intracellular application of a selective inhibitor of the catalytic subunit of cAMP-dependent protein kinase A blocked the effect of Iso on the AHP, whereas Iso-induced potentiation was entirely normal in the same neuron. In addition, Iso decreased the magnitude of paired-pulse facilitation, which is consistent with a presynaptic mode of action. Substituting the Mg2+ for Ca2+ in the medium completely abolished the Iso-induced enhancement of the EPSP. The effect of Iso also was blocked by low concentrations of omega-agatoxin-IVA, but not by nifedipine or omega-conotoxin-GVIA. These results suggest that Iso enhances synaptic transmission in the amygdala via a presynaptic site of action: the mechanism underlying the potentiating effect likely is attributable to an increased Ca2+ influx through P- and/or Q-type Ca2+ channels.

Fear conditioning has been ascribed to presynaptic mechanisms, particularly presynaptic facilitation of transmission at thalamo-and cortico-amygdala synapses. Here, by labeling surface receptors with biotin or using membrane fractionation approaches, we report that fear conditioning resulted in an increase in surface expression of GluR1 subunit of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in the amygdala, whereas total GluR1 mRNA and protein levels were unchanged. The control group that received conditioned stimulus (CS) and unconditioned stimulus in an unpaired fashion did not present any increase, indicating that GluR1 increase was specific to the learning component of the task. Conditioninginduced increase in surface expression of GluR1 depended on the activation of N-methyl-D-aspartate receptors and protein kinases and required the synthesis of new proteins. CS-alone trials applied 24 h before training attenuated fear-potentiated startle and prevented conditioning-induced increase in surface expression of GluR1. Increase in GluR1 was also observed in the amygdala slices after delivery of tetanic stimulation that elicited long-term potentiation of synaptic transmission. Proteasome inhibitor increased surface expression of GluR1 in a time-and dose-dependent manner. Furthermore, pretraining administration of proteasome inhibitor into the amygdala facilitated the fear-potentiated startle. These results suggest that long-term memory formation is correlated with the change in synaptic expression of GluR1, and trafficking of GluR1 to the synaptic sites contributes at least in part to the expression of fear memory.Fear conditioning, an animal model of emotional learning and post-traumatic stress disorder, is initiated by a cue (conditioned stimulus, CS) that is previously paired with an aversive stimulus (unconditioned stimulus, US) such as footshock. Neuronal changes mediating the association between the CS and US occur in the lateral (LA) and basolateral (BLA) amygdala (McKernan and Shinnick-Gallagher, 1997;Rogan et al., 1997;Pare, 2003). It is generally recognized that consolidation of long-term memory in vertebrate and invertebrate brains requires transcription and translation of new proteins (Dudai, 1996;McGaugh, 2000). Newly synthesized proteins are believed to deposit at the synapses that encode the persistent changes in synaptic strength. Despite the signal cascades that have been identified to be responsible for the consolidation of fear memory, little is known about the downstream effectors leading to the expression of memory. Previous study has showed that conditioned fear is associated with a reduction in paired-pulse facilitation and an enhancement in the probability of transmitter release in the thalamo-amygdala pathway (McKernan and Shinnick-Gallagher, 1997). Quantal analysis of unitary synaptic responses in cortico-amygdala pathway after induction of long-term potentiation (LTP) revealed a marked decrease in the fraction of failures, with no change in potency of CV of the...