Figure 6. Activation of the BDNF and TrkB pathway is critical for the induction of LTP in BLA principal neurons. A) Bath application of of the non-selective receptor tyrosine kinase inhibitor, genistein (100 mM), before 5xHFS (arrow) blocked the induction of LTP in BLA principal neurons (n = 5, red squares). B) Bath application of the BDNF scavenger molecule, TrkB/Fc (1 mg/ml, n = 6), for 30 min before 5xHFS prevented the induction of LTP in BLA. C) Bar chart summarizing the group effects of modulating the BDNF and TrkB cascade on the induction of LTP. Intracellular application of MEK inhibitor, U0126 (10 mM, n = 5, hooped bar), for 30 min before recording abolished the induction of LTP. Similarly, bath application of the MMP inhibitor, GM6001 (Gallardin, 10 mM, n = 4, diagonal hashed bar), or the copper- zinc chelator (DETC, 20 mM, n = 5, stippled bar) significantly attenuated LTP in BLA principal neurons. ** P,0.01, * P,0.05. Error bars indicate SEM. doi:10.1371/journal.pone.0026065.g006 minogen [36]. A recent study reported that tPA was almost completely absent in the BLA [37]. Hence, we reasoned that if LTP induction in the BLA was dependent on TrkB receptor activation, then the conversion of proBDNF to mBDNF by MMPs Dopamine and BDNF Modulate LTP in the BLA could play a significant role in this process. Thus, blockade of MPPs would be predicted to attenuate LTP. Consistent with this prediction, application of the non-selective MMP inhibitor, gallardin (10 mM), 30 min before 5xHFS significantly reduced the magnitude of LTP to 13167% of baseline (n = 4, 3 animals, P,0.05; Figure 6C, diagonal bar), which is consistent with previous studies in MMP-2 knockout mice [38]. Application of gallardin caused no enduring change in input resistance at any time point (F (12, 104) = 0.6, P = 0.8370, Two-way ANOVA). Significantly, MMPs are only activated in the presence of extracelluar zinc [39], and cortical afferents to the BLA are known to contain vesicular zinc [40]. Hence, zinc released from presynaptic terminals could directly activate MMPs in the BLA following 5xHFS and trigger the conversion of proBDNF to mBDNF. Consistent with this observation, inclusion of the copper- zinc chelator (DETC, 20 mM) in the ACSF significantly attenuated 5xHFS-induced LTP in BLA principal neurons (12366% of base line, n = 5, 4 animals, P,0.01, Figure 6C, hatched bar). Whereas application of DETC alone caused no enduring change in input resistance at any time point measured (F (13, 112) = 0.70, P = 0.6963, Two-way ANOVA). Moreover, as shown below, DETC failed to block the facilitating effects of exogenous BNDF application on 2xHFS induced LTP, suggesting that DETC application was not affecting LTP-induction independently of TrkB receptor activation. Together these data suggested that 5xHFS of cortical inputs into the BLA may cause a concomitant release of dopamine, zinc, and BDNF resulting in the subsequent activation of postsynaptic D1 and TrkB receptors, both of which act to enhance the induction and maintenance of LTP. Gain-control of LTP induction in BLA principal neurons Elsewhere in the brain, D1 receptor activation or BDNF release has been shown to lower the threshold for LTP induction [41,42]. Hence, we next determined whether activation of D1- or TrkB receptors could affect the threshold for LTP induction in the BLA using the 2xHFS stimulation protocol, which could not induce LTP in principal neurons (see Figure 1B). Significantly, in the presence of either the dopamine transport inhibitor, GBR12783 or the D1 receptor agonist, SKF38393, 2xHFS induced a robust and stable LTP in BLA principal neurons (GBR12783: 15368% of baseline, n = 5, 4 animals, P,0.01; Figure 7A, red squares, and Figure 8C, grey bar; SKF38393:14866% of baseline, n = 5, 4 animals, P,0.01; Figure 7B). Moreover, in all BLA principal neurons tested pretreating slices for 10 min with the D1 receptor antagonist SCH23390 (10 mM) fully blocked the GBR12783 (11164% of baseline, n = 5, 3 animals, P,0.001, Figure 8C, black bar) and SKF38393-mediated facilitation of LTP induced by 2xHFS (10765% of baseline, n = 5, 3 animals, P,0.001, data not shown). However, as illustrated in Figure 7C, analysis of the PPR showed no significant difference from control values at 10 or 30min after 2xHFS LTP induction in the presence of either GBR12783 or SKF38393 (Figure 7C, D) suggesting that facilitation of LTP was not dependent on alterations in presynaptic release mechanisms. Consistent with this observation, inclusion of either BAPTA (10 mM) (11968% of baseline, n = 5, 3 animals, P,0.05, Figure 8A, C, hooped bar) or the PKA inhibitor KT5720 (100 nM) (106610% of baseline, n = 5, 3 animals, P,0.001, Figure 8B, C, diagonal bar) in the patch pipette fully blocked the induction of LTP by 2xHFS in the presence of GBR12783. Together these data suggested that the postsynaptic mechanisms underlying the induction of LTP by 2xHFS in the presence of GBR12783 were similar to those mediating the response to 5xHTS. Significantly, application of the MEK inhibitor, U0126, PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e26065