Figure 8. D1 receptor-dependent reduction of the threshold of LTP induction requires calcium influx, and induction of the cAMP-PKA and MEK-MAPK signaling cascades. A) Inclusion of the calcium chelator, BAPTA (10 mM) in the patch pipette (n = 5) abolished the GBR12783 mediated LTP induction by 2xHFS. B) Inclusion of the PKA inhibitor, KT5720 (100 nM) (n = 5) also blocked the GBR12783 mediated LTP induction by 2xHFS. C) Bar chart summarizing the group effects of GBR12783 on LTP induction using 2xHFS. As illustrated, pretreatment with SCH23390 (black bar), BAPTA (hooped bar), KT5720 (diagonal bar), and U0126 (hatched bar) all fully blocked the GBR12783-facilitated LTP (n = 5 in each case). ** P,0.01. Error bars indicate SEM. doi:10.1371/journal.pone.0026065.g008 Because both D1- and TrkB receptor activation lowered the threshold for LTP induction in the BLA, and D1 receptor antagonism completely abolished LTP in response to 5xHFS, we next investigated whether these two transmitter systems may act synergistically to facilitate LTP induction. Here, the D1 receptor antagonist, SCH23390, was applied 10 min before 7, 8-dihydrox- yflavone, which was applied 20 min prior to 2xHFS. Prior application of SCH23390 fully blocked the ability of the TrkB receptor agonist to lower the LTP threshold in response to 2xHFS (109 68% of baseline, n = 6, 5 animals, Figure 10A, C), suggesting that a synergistic activation of D1 and TrkB receptors was necessary to reduce LTP threshold. To examine if the reverse Dopamine and BDNF Modulate LTP in the BLA relationship applied, we next examined the effects of prior application of the BDNF scavenger, TrkB-Fc, on the facilitation of LTP induced by 2xHFS in the presence of the dopamine transport inhibitor, GBR12783. A 10 min pretreatment with TrkB-Fc fully blocked the GBR12783-induced lowering of LTP threshold (11469% of baseline, n = 5, 4 animals, Figure 10B, C), further supporting the hypothesis that a synergistic interaction between these two transmitter systems was required to reduce LTP threshold in BLA principal neurons. Finally, we ran immunohistochemical- and single-cell RT-PCR studies to confirm that all of the proteins predicted by our physiological studies were expressed in the BLA. We have shown previously that D1 receptors are found in the spines of BLA principal neurons [7], where they form a close association with NMDA receptors [8], and the Allen Brain Atlas shows moderate to high mRNA expression levels for the matrix metalloproteinase isoforms, MMP16 and MMP17, and also the TrkB receptor. Here, we extend these observations using dual-immnuofluorescence microscopy to show that protein for MMP16, MMP17, and the TrkB receptor are extensively co-localized with CaMKIIa, a major component of excitatory synapses in presumed BLA principal neurons (Figure 11A–C). To further confirm that neurons of the BLA express the machinery necessary to mediate the synergistic modulation of LTP induction by D1- and TrKB receptors, we extracted cytosolic mRNA from physiologically identified BLA principal neurons and performed single-cell RT–PCR to examine their mRNA expres- sion profile. As expected, all cells sampled (n = 15) expressed mRNA transcripts for CaMKIIa. However, 7/15 neurons sampled expressed only D1 receptor mRNA transcripts, 5/15 neurons expressed both D1 and D5 receptor mRNA transcripts, and 3/15 neurons expressed only D5 receptor mRNA transcripts. Intriguingly, all of the neurons that expressed D1 receptor mRNA transcripts also expressed mRNA transcripts for the TrkB receptor as well as ERK1/2. Moreover 8/15 neurons expressed MMP17 transcripts and 3/15 neurons expressed MMP16 mRNA tran- scripts. Significantly, those neurons that expressed both D1 and D5 receptor mRNA transcripts expressed all of the proteins necessary to facilitate a synergistic interaction between the D1 and BDNF signaling cascades (Figure 11D). Discussion To the best of our knowledge, this study represents the first of its kind to show that a) endogenous dopamine release and subsequent D1 receptor activation is prerequisite for long-term potentiation of glutamatergic input onto BLA principal neurons from cortical afferents, b) that endogenous release of dopamine, zinc, and BDNF results in a synergistic activation of D1 and TrkB receptors, which together function to lower the threshold for LTP induction, and that c) convergent activation of the MEK-ERK signaling cascade may play a critical role in lowering the threshold for LTP induction. We believe that the results of our study offer a plausible mechanism by which the results of several previously independent studies can be unified into a common model of affective memory formation in the BLA. For example, previous studies have shown that the majority of dopaminergic terminals in the BLA make synaptic contacts onto the spines of principal neurons [8], that these spines co-localize D1- and NMDA receptors, which function to modulate excitatory synaptic transmission [7], and that local dopamine release and subsequent activation of D1 receptors is necessary for the acquisition of fear memory [3]. Similarly, Bolshakov, Shumyatsky and co-workers have shown that synap- PLoS ONE | www.plosone.org 9 October 2011 | Volume 6 | Issue 10 | e26065