BLA-dependent fear learning and memory. Intriguingly, in striatal neurons D1 receptor stimulation can trans-activate TrkB receptors [18], and in the hippocampus dopamine-mediated persistence of long-term memory (LTM) is reported to be mediated by BDNF [19], further suggesting that a synergistic interaction between the dopamine and BDNF systems could play a similar role in BLA- dependent fear memory formation. While synaptic plasticity underlying fear memory formation is assumed to occur in BLA principal neurons, to date no studies have directly addressed the role of D1 receptor activation on LTP in the BLA, or the role of TrkB receptor activation on LTP, specifically in this cell population. The present whole-cell patch clamp recording study was designed to address these knowledge gaps and determine whether these two systems act independently or synergistically to regulate synaptic plasticity in principal neurons of the BLA. Results LTP induction in BLA principal neurons Most in vitro studies that have examined the cellular mechanisms underlying LTP formation in the BLA have used bath application of GABAA receptor antagonists to block fast inhibitory synaptic transmission and isolate evoked EPSPs. However, we have shown that global reduction of GABAA receptor-mediated synaptic transmission in the BLA can result in profound changes in excitatory drive that would confound the interpretation of any subsequent analysis of LTP [20]. To circumvent this potential confound, we evoked monosynaptic EPSPs (eEPSPs) in BLA principal neurons following stimulation of the external capsule (EC, Figure 1A) by including picrotoxin (50 mM) in the patch pipette to block GABAA receptor-mediated synaptic transmission only in the principal neuron under study. Multi-electrode recording studies in freely moving animals have shown that coherent theta frequency oscillations occur between the BLA and mPFC [21] and between the BLA and hippocampus [22] during fear memory acquisition and recall. Hence, we reasoned that theta frequency modulation of synaptic transmission onto BLA principal neurons may play a significant role in fear memory formation. Consequently, we first examined the effects of theta- burst stimulation of the EC (TBS, see Methods) on the amplitude of eEPSPs in BLA principal neurons. Here, TBS consisted of 40ms bursts of high frequency stimulation (HFS) applied to the EC at 5Hz for 5 seconds. As illustrated in Figure 1B, TBS of the EC induced a LTP of the eEPSP that persisted for .45 minutes (blue diamonds). When measured 30 min following TBS, the peak EPSP amplitude was significantly greater than that of the baseline eEPSP amplitude (144619%, n = 6, 4 animals, P,0.001). Two-way ANOVA revealed no significant effect of treatment at any time point for either the access resistance or the membrane input resistance (F (10, 110) = 0.57, P = 0.98). We next examined the effects of spaced 5xHFS (5x100Hz/1s trains at 20s intervals) of the EC on LTP induction in BLA principal neurons. In the hippocampus, TBS and 5xHFS induce LTP by activating the same cAMP second messenger cascade [23,24,25], but the response to 5xHFS was reported to be more robust. Like TBS, 5xHFS of the EC induced a robust LTP of the eEPSP in BLA principal neurons (15563% of baseline, n = 5, 3 animals, P,0.001, Figure 1B, red squares). Two-way ANOVA revealed no effect at any time point of treatment for either the access resistance or the membrane input resistance (F (10, 110) = 0.24, P = 0.99). Thus, both TBS and 5xHFS induced a significant LTP of the eEPSP amplitude in BLA principal neurons. We next determined whether an abbreviated HFS protocol (26100Hz/1s trains at 20s intervals; 2xHFS) could induce LTP in BLA principal neurons. In control ACSF, 2xHFS caused a brief Dopamine and BDNF Modulate LTP in the BLA post-tetanic potentiation of the ePSP (PTP; 141611% of baseline) that rapidly decayed back to baseline after 20 min (113612% of baseline, n = 8, 5 animals; Figure 1B, green triangles). Hence, 2xHFS was used in subsequent experiments as a sub-threshold protocol to examine the effects of drug treatment on the sensitivity of BLA principal neurons to LTP induction. Two-way ANOVA revealed no significant effect of treatment at any time point for either the access resistance or the membrane input resistance (F (9, 100) = 0.26, P = 0.9825). Moreover, because LTP induction by TBS was more variable than the response to 5xHFS in most subsequent experiments we used 5xHFS to study the mechanisms underlying LTP induction in BLA principal neurons. To examine any potential effects of treatment on presynaptic release we also examined the paired-pulse ratio of eEPSCs (PPR, see Methods), a measure of short-term presynaptic plasticity, before, immediately after, as well as 10 min and 30 min after each of the LTP induction protocols (TBS, 5xHFS and 2xHFS). Typical examples of the paired-pulse response before and after each of the stimulation protocols are illustrated in Figure 1C. Group data is illustrated in Figure 1D. As expected all three stimulation protocols elicited a significant reduction in PPR during the PTP response immediately following the stimulation (P,0.05, n = 6, 4 animals), whereas the PPR was not significantly different from control values 10 or 30 min after the stimulation. These data suggest that the LTP induced by either TBS or 5xHFS was most likely postsynaptic and not presynaptic in origin. Consistent with previous reports [26], inclusion of the calcium chelator, BAPTA (10 mM) in the patch pipette, fully blocked the induction of LTP by either TBS (10667% of baseline, n = 6, 4 animals; Figure 2A) or 5xHFS (10466% of baseline, n = 5, 3 animals; Figure 2B), further supporting a postsynaptic origin for this response. Moreover, intracellular application of BAPTA caused no enduring change in input resistance at any time point measured (F (16, 136) = 1275, P = 1.27, Two-way ANOVA). Finally, previous studies had reported that LTP induction in the lateral amygdala was pathway specific [27,28]. Hence, in a parallel study we examined the response of BLA principal neurons to 5xHFS of the thalamic input. In contrast to the response to 5xHFS of the EC, stimulation of the thalamic input induced only a transient increase in the eEPSP amplitude in BLA principal neurons that was not significantly different from baseline when measured 30 min after 5xHFS (11968% of baseline amplitude, n = 6, 4 animals, P,0.05; data not shown). Dopamine-dependency of LTP induction in BLA principal neurons In vivo studies have shown that fear learning is dependent on dopamine release in the BLA, and subsequent activation of dopamine D1 receptors [3]. Consequently, we next examined the role of D1 receptor activation on the induction of LTP in BLA. Here, slices were pretreated with the D1 receptor antagonist, SCH23390 (10 mM), for 10 min prior to, and during TBS. Bath application of SCH23390 caused a significant attenuation of TBS- induced LTP in BLA neurons (98612% of baseline, n = 6, 4 animals, Figure 2C). These data not only suggested that TBS induced the release of endogenous dopamine, but also that subsequent activation of D1 receptors was necessary for the induction of LTP in cortical pathways onto BLA principal neurons. Similarly, prior application of SCH23390 also blocked 5xHFS-induced LTP in BLA principle neurons (11664% of baseline, n = 6, 5 animals, Figure 2D), Application of SCH23390 alone caused no enduring change in input resistance at any time point measured (F (10, 88) = 0.68, P = 0.7366, Two-way ANOVA). PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e26065