Our results showing a gain of function effect, i.e. reduced LTP threshold, in the synaptic response to sub-threshold stimulation following application of BDNF or the dopamine transport inhibitor, GBR12783, are also consistent with previous studies in the prefrontal cortex and hippocampus. Here, endogenous DA and BDNF have been shown to amplify LTP via activation of D1 receptors [10], and TrkB receptors [56] respectively. A similar dopamine D1 receptor-dependent gain of sensitivity has also been reported for spike-timing-dependent plasticity in CA1 hippocam- pal neurons [57]. However, our study extends these observations to demonstrate that, in the BLA, the gain of function effect in response to DA or BDNF is dependent on the concurrent activation of both receptor-effector cascades. Moreover, our results further suggest that concurrent activation of D1 and TrkB receptors may trigger second messenger cascades that converge at the level of the MEK-ERK pathway. We have shown that the D1 receptor-mediated facilitation of LTP induction in BLA principal neurons is critically dependent on the activation of the AC – cAMP - PKA signaling cascade. Recently, BDNF-dependent learning in the BLA was shown to be mediated by activation of the MAPK and PI3K signaling pathways, and abolished in the presence of MEK inhibitors [15]. Consistent with this observation, we have shown that the MEK inhibitor, U0126, abolished both 5xHFS-induced LTP as well as the gain of function effect induced by either exogenous TrkB agonist or GBR12783 application. Significantly, Gean and colleagues have reported that PI3K acts as a critical intermediary for PKA-induced MAPK activation in the amygdala, and is required for amygdala LTP and fear memory formation [58]. TrkB receptor activation can activate PI3K and, hence, concurrent activation of D1 and TrkB receptors may lower the threshold of LTP induction by convergent activation of the MEK-MAPK signaling cascade. Although our study did not examine PLCc activation, Musumeci and colleagues have recently shown that mutation of the PLCc docking site of the TrkB receptor also impairs acquisition of fear conditioning, suggesting that synergism with the PLC cascade may also lower the threshold for LTP induction [17]. In summary, our results show that following HFS synaptically released zinc facilitates the synergistic interaction between endogenously released dopamine and BDNF to control the magnitude of LTP in BLA principal neurons. Aberrant activity of the dopaminergic- and BDNF systems have been implicated in the psychopathology of psychiatric disorders such as post- traumatic stress disorder (PTSD) and depression [59], as well as drug abuse [60]. Our observations on the synergistic interaction between dopamine and BDNF and their ability to amplify sensory input may provide important clues to the molecular mechanisms underlying the development of sensory generalization in PTSD, and sensitization in addiction. Methods Animals Male Sprague–Dawley rats (5–7 weeks old, Charles River, Raleigh, NC, USA) were used in this study. Every effort was used to minimize both animal suffering and the number of animals necessary to complete the study. All experimental protocols strictly conform to National Institutes of Health guidelines for the Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee of Emory University. Slice preparation Slices of 350 mm thickness containing the BLA were obtained as described previously [61]. Briefly, the brains of isoflurane (Fisher Dopamine and BDNF Modulate LTP in the BLA Scientific, Hanoverpark, IL, USA) anesthetized animals were rapidly dissected and immersed in a cold (4uC) 95–5% oxygen/ carbon dioxide oxygenated ‘‘cutting solution’’ with the following composition (in mM): NaCl (130), NaHCO3 (30), KCl (3.50), KH2PO4 (1.10), MgCl2 (6.0), CaCl2 (1.0), glucose (10), supplemented with kynurenic acid (2.0). Slices containing the BLA were cut using a Leica VTS-1000 Vibratome (Leica Microsystems Inc., Bannockburn, IL, USA). After cutting, slices were maintained at 37uC in oxygenated ‘‘cutting solution’’ for at least 50 min before transferring to regular artificial cerebrospinal fluid (ACSF) containing (in mM): NaCl (130), NaHCO3 (30), KCl (3.50), KH2PO4 (1.10), MgCl2 (1.30), CaCl2 (2.50), and glucose (10). Slices were kept in the regular ACSF for at least 30 min before recording. Patch clamp recording Individual slices were then transferred to a submersion-type recording chamber mounted on the fixed stage of a Leica DMLFS microscope (Leica Microsystems Inc., Bannockburn, IL, USA), and continuously perfused by gravity-fed oxygenated 32uC ACSF at a flow rate of 1–2 ml/min. Slices were viewed using differential interference contrast (DIC) optics and infrared (IR) illumination with an IR sensitive CCD camera (Orca ER, Hamamatsu, Tokyo Japan). Thin-walled borosilicate glass patches electrodes (WPI, Sarasota, FL, USA) which had a resistance of 4–6 MV were filled with (in mM): 130K-gluconate, 2 KCl, 10 HEPES, 3 MgCl2, 2 K- ATP, 0.2 NaGTP, and 5 phosphocreatine, adjusted to pH 7.3 with KOH, and having an osmolarity of 280–290 mOsm. Individual BLA projection neurons were visualized in situ using DIC microscopy in combination with a 40x water immersion objective and displayed in real time on a computer monitor. Projection neurons were identified according to their characteristic size and shape [62], and were normally located between 50 and 120 mM beneath the surface of the slice. Data acquisition and analysis were performed using a MultiClamp700B amplifier in conjunction with pClamp10.0 software and a DigiData 1320A AD/DA interface (Molecular Devices, Burlingame, CA, USA). Whole cell patch clamp recordings were obtained and recorded voltages were low-pass filtered at 5 kHz and digitized at 10– 20 kHz. At the start of each experiment, a series of standardized current clamp protocols were performed to further validate the identity of BLA projection neurons [63]. Excitatory postsynaptic potentials (EPSPs) onto BLA projection neurons were evoked as previously described [64]. In brief, a concentric bipolar stimulation electrode (FHC, Bowdoinham, ME, USA) was placed approximately 500 mm from the recorded neuron, close to the fiber tract of the external capsule immediately adjacent to the BLA. In all experiments, 50 mM picrotoxin was added to the patch solution to block GABAA currents exclusively in the recorded neuron. Slices were continuously perfused with oxygenated ACSF (32uC) containing the GABAB receptor antagonist CGP36742 (5 mM). This recording configuration allowed stable recording of isolated EPSPs without contamination from epileptiform, recurrent EPSPs. EPSPs (adjusted to 30% of maximal response) were evoked at 0.05 Hz, a 10 min baseline period was recorded in each experiment, and recordings continued for at least 40min after LTP induction. Initial EPSP amplitudes were normalized to the average of the baseline EPSP amplitude. 5x high frequency stimulation (HFS) LTP , an NMDA-receptor-dependent form of LTP [65] consisted of five trains of stimulation at 100 Hz for 1-s duration, applied with 20 s intervals; 2xHFS consisted of only two, 1-s trains of 100 Hz stimulation. Theta burst stimulation (TBS) consisted of 40 ms duration, 100 Hz bursts delivered at 5 Hz for PLoS ONE | www.plosone.org 13 October 2011 | Volume 6 | Issue 10 | e26065