Figure 1.
Stimulus dependent induction of LTP in BLA principal neurons.
A) A schematic illustrating the position of the stimulation (Scortical and Sthalamic) and recording (R) electrodes. EC, external capsule. B) Stimulation of the EC elicits long term synaptic plasticity in BLA afferents following TBS (n = 6, blue diamonds) and 5×100 Hz (n = 5, red squares), but not 2×100 Hz (n = 8, green triangles). C) Raw data show the effects of different stimulation protocols on the PPR of EPSCs. EPSC amplitude was measured using paired stimuli pulses separated by 50 ms, before, immediately after, and 10 min and 30 min post stimulation. D) A bar chart showing the group data for the effects of stimulation protocol on PPR. Note the large decrease (P <0.05) immediately post-stim, indicating the presynaptic component of post-tetanic-potentiation. * P<0.05. Error bars indicate S.E.M.
Figure 2.
Calcium influx, activation of D1 receptors, and induction of the cAMP-PKA cascade are all required for the induction and maintenance of LTP in BLA principal neurons.
A, B) Inclusion of the calcium chelator, BAPTA (10 mM) in the patch pipette, fully blocked the induction of LTP by either TBS (n = 6) or 5xHFS (n = 5). C, D) Endogenous dopamine was released during TBS and 5xHFS and was necessary for the induction of LTP in BLA principal neurons. Incubation with SCH23390 (10 µM) before and during TBS (n = 6) or 5xHFS (n = 6) completely abolished the induction of LTP. E, F) The induction LTP by either TBS or 5xHFS was dependent on activation of the cAMP-PKA signaling cascade. Intracellular application of PKA inhibitor KT5720 (100 nM) fully blocked TBS-induced LTP (n = 5), as well as 5xHFS-induced LTP (n = 5).
Figure 3.
Application of either the dopamine transport inhibitor, GBR12783, or the D1 receptor agonist, SKF38393 facilitated 5xHFS induced LTP in BLA principal neurons.
A, B) Bath application of either the selective dopamine uptake inhibitor, GBR12783 (10 µM, n = 6, blue diamonds), or the selective D1 family receptor agonist, SKF38393 (50 µM, n = 5, blue diamonds), before and during 5xHFS enhanced the magnitude of LTP in principal neurons. The time of drug application is marked by the horizontal bar below the line plots. C) Raw data showing the effects of drug application on the PPR of evoked EPSCs in BLA principal neurons. D) Bar chart showing the group data for the effects of drug application on the PPR. ** P<0.01. Error bars indicate S.E.M.
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 µM) 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.
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.
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 µM), 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 µg/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 µM, n = 5, hooped bar), for 30 min before recording abolished the induction of LTP. Similarly, bath application of the MMP inhibitor, GM6001 (Gallardin, 10 µM, n = 4, diagonal hashed bar), or the copper-zinc chelator (DETC, 20 µM, n = 5, stippled bar) significantly attenuated LTP in BLA principal neurons. ** P<0.01, * P<0.05. Error bars indicate SEM.
Figure 7.
D1 receptor activation acts to reduce the threshold for LTP induction in BLA principal neurons.
A) Application of the dopamine reuptake inhibitor GBR12783 (10 µM, n = 5), or B) the D1 receptor agonist SKF38393 (50 µM, n = 5) induced a robust and stable LTP in BLA principal neurons following 2xHFS. C) Raw data showing the effects of GBR12783 and SKF38393 on the PPR in BLA principal neurons. D) A bar chart illustrating the group effects of drug application on the PPR of EPSCs before, immediately after, and at 10 min and 30 min post 2xHFS. ** P<0.01. Error bars indicate S.E.M.
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.
Figure 9.
TrkB receptor activation lowers the threshold of LTP induction in the BLA.
A) Application of the TrkB receptor agonist, 7, 8-dihydroxyflavone (500 nM, n = 6) facilitated the induction of LTP by 2xHFS. B) Left, raw data showing a typical PPR response before, and at 10 and 30 min after 2xHFS in the presence of the TrkB agonist. Right, a bar chart illustrating the group data. C) Bar chart summarizing the group effects of TrkB receptor activation on LTP threshold using 2xHFS. Application of BDNF (100 ng/ml, n = 4) for 30 min, mimicked the induction of LTP by 2xHFS induced by 7, 8-dihydroxyflavone. Intracellular application of calcium chelator, BAPTA (10 mM, n = 5, hooped bar) as well as the PKA inhibitor, KT5720 (100 nM, n = 5, diagonal bar) or the selective MEK inhibitor, U0126 (10 µM, n = 4, hatched bar) fully blocked the 7, 8-dihydroxyflavone facilitated LTP. However, incubation with DETC (20 µM, n = 6, striped Bar) did not block this effect. ** P<0.01, * P<0.05. Error bars indicate SEM.
Figure 10.
Synergistic activation of D1- and TrkB receptors lowers the threshold for LTP induction in BLA principal neurons.
A) Preincubation with SCH23390 (10 µM) blocked the induction of LTP by 2xHFS in the presence of 7, 8-dihydroxyflavone (500 nM). B) Preincubation with TrkB/Fc blocked the induction of LTP by 2xHFS in the presence of GBR12783. C) Bar chart illustrating the synergistic interaction between D1 and TrkB receptors to reduce LTP threshold in BLA principal neurons. ** P<0.01. Error bars indicate S.E.M.
Figure 11.
BLA projection neurons co-express mRNA and protein for D1- and TrkB receptors, as well as MMP17.
A-A″) Photomicrographs showing the high level of co-localization for TrkB (A, red) and CaMKIIa- (A′, green) immunoreactivity in putative principal neurons of the BLA (A″, merged, double arrows). Note, a subpopulation of TrkB-positive neurons do not co-localize CaMKIIa (A″, open arrows, merged). B-B″) Photomicrographs showing the high level of co-localization for MMP17 (B, red) and CaMKIIa- (B′, green) immunoreactivity in BLA neurons (B″, merged, double arrows). Note, a subpopulation of MMP17-positive neurons do not co-localize CAMKIIa. C-C″) Photomicrographs showing the co-localization of MMP16 (C, red) and CaMKIIa (C′, green) immunoreactivity in BLA neurons (C″, merged, double arrows). Note, a subpopulation of CAMKIIa-positive neurons do not co-localize MMP16. All photomicrographs were taken from the basolateral nucleus of the BLA. In all images magnification = 63x, scale bar = 10 mm. C) Photograph of an agarose gel showing the typical mRNA expression pattern for a D1- and D5- mRNA-positive principal neuron. The associated table shows the relative expression of D1, D5, TrkB, MMP17 and ERK1/2 mRNA expression in single BLA principle neurons.