Figure 4. The D1 receptor antagonist SCH23390 fully blocked the induction of LTP by 5xHFS, as well as the facilitation of LTP by GBR12783 and SKF38393. The bar chart summarizes the group effects of different dopaminergic manipluations on the magnitude of LTP induced by 5xHFS. Prior application of SCH23390 (10 mM) blocked 5xHFS induced LTP (grey bar) as well as GBR12783 (n = 5, hooped bar) or SKF38393 (n = 5, stippled bar) facilitated LTP, ** P,0.01, * P,0.05. Error bars indicate S.E.M. doi:10.1371/journal.pone.0026065.g004 to HFS [29], and exogenous BDNF has been shown to facilitate field potential LTP in the BLA [15]. Hence, we next determined whether local release of endogenous BDNF may also contribute to 5xHFS-induced LTP in BLA principal neurons. As TrkB receptors are members of a family of receptor tyrosine kinases [30], we first examined the effects of bath application of the tyrosine kinase inhibitor, genistein (100 mM) on 5xHFS-induced LTP in BLA principal neurons. As shown in Figure 6A, bath application of genistein for 30 min prior to 5xHFS prevented the induction of LTP (11866% of baseline, n = 5, 4 animals, P,0.01; Figure 6A, red squares, Figure 6C, grey bar), which is consistent with previous studies in the hippocampus [31]. Application of genistein caused no enduring change in input resistance at any time point measured (F (14, 150) = 0.31, P = 0.9921, Two-way ANOVA). However, the genistein effect could result from an action that was independent of BDNF - TrkB receptor activation. Hence, we next examined the effects on LTP induction of prior application of the TrkB-immunoglobulin-G fusion protein, TrkB-Fc (1 mg/ml), which scavenges BDNF and prevents TrkB receptor activation [32]. As shown in Figure 6B, prior application of TrkB-Fc prevented 5xHFS-induced LTP in BLA principal neurons (11768% of baseline, n = 6, 4 animals, P,0.01; Figure 6C, black bar). Application of TrkB-Fc caused no enduring change in input resistance at any time point (F (12, 104) = 1.17, P = 0.3169, Two-way ANOVA). Together these data suggest that 5xHFS of cortical inputs resulted, not only in the release of endogenous dopamine, but also endogenous BDNF release. TrkB receptors are coupled to three main signaling pathways, including the mitogen-activated protein (MAP) kinase kinase (MEK) cascade, phospho-inositide-3 kinase, and phospholipase C (PLC). In aplysia, activation of the MEK cascade is necessary for the induction of LTP and memory formation [33]. To explore whether activation of the MEK cascade downstream from BDNF was required for the induction of LTP in the BLA, we included the MEK inhibitor, U0126 (10 mM), in the patch recording pipette and allowed it to equilibrate for 30 min prior to LTP induction. Intracellular perfusion of U0126 prevented the induction of LTP in all neurons tested (11665% of base line, n = 5, 4 animals; P,0.01; Figure 6C, hooped bar). Dopamine and BDNF Modulate LTP in the BLA Figure 5. Facilitation of LTP by GBR12783 and SKF38393 are dependent on activation of the cAMP - PKA signaling cascade. A) Intracellular application of the PKA inhibitor, KT5720 (100 nM), blocked the GBR12783 facilitated LTP (n = 6) B) Application of KT5720 also fully blocked 5xHFS induced LTP in the presence of SKF38393 (n = 5) C) Bar chart showing the group data for drug effects 30 minutes post stimulation. *** P,0.001, Error bars indicate S.E.M. doi:10.1371/journal.pone.0026065.g005 Application of U0126 caused no enduring change in input resistance at any time point (F (10, 110) = 0.08, P = 0.9999, Two- way ANOVA).These results further suggested that BDNF release and TrkB receptor activation were also critically involved in the induction of LTP in BLA principal neurons, possibly acting via activation of the MEK cascade. However, BDNF is synthesized as a precursor protein, preproBDNF, and proteolytic cleavage is required to generate mature BDNF (mBDNF) [34]. ProBDNF is processed into mBDNF by either matrix metalloproteinases (MMPs) [35] or by tissue plasminogen activator (tPA)-dependent activation of plas- PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e26065