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.