5-Hydroxytryptamine induces a protein kinase A/mitogen-activated protein kinase-mediated and macromolecular synthesis-dependent late phase of long-term potentiation in the amygdala

Abstract

The amygdala is a critical site for the acquisition of learned fear memory in mammals, and the formation and long-term maintenance of fear memories are thought to be associated with changes of synaptic strength in the amygdala. Here we report that serotonin (5-hydroxytryptamine; 5-HT), a modulatory neurotransmitter known to be linked to learned fearful and emotional behavior, has dual effects on excitatory synaptic transmission in the basolateral amygdala. There is an early depression of synaptic transmission lasting 30-50 min, mediated by 5-HT1A, and a late, long-lasting facilitation lasting >5 h in slice recordings, mediated by the 5-HT4 receptor. 5-HT late phase long-term potentiation (L-LTP) is blocked by inhibitors of either protein kinase A (PKA) and/or mitogen-activated kinase (MAPK) and requires new protein synthesis and gene transcription. Moreover, the 5-HT-induced L-LTP in neurons of amygdala is blocked by the actin inhibitor cytochalasin D, suggesting that 5-HT stimulates a cytoskeletal rearrangement. These results show, for the first time, that 5-HT can produce long-lasting facilitation of synaptic transmission in the amygdala and provides evidence for the possible synaptic role of 5-HT in long-term memory for learned fear.

Figure 1.

5-HT induces L-LTP in the BL of amygdala. A, Schematic illustration of stimulating and recording sites in a coronal brain slice. The stimulating electrode (Stim) was placed in the LA of amygdala. The recording electrode (Rec) was placed in the BL of amygdala. B, Top, The synaptic potential recorded from the LA–BL pathway is blocked completely by the glutamate antagonist CNQX. Calibration: 0.5 mV, 3 ms. B, Bottom, The synaptic potential follows a 50 Hz tetanus in a one-for-one manner without failure. Calibration: 0.5 mV, 10 ms. C, Bath application of 5-HT (300 μm) induces an early phase of synaptic depression, followed by a late phase of long-lasting synaptic potentiation in the BL. 5-HT, Filled circles; baseline control, unfilled circles. Representative field potentials 10 min before and 5 h after the application of 5-HT are shown in the insets. Calibration: 0.5 mV, 5 ms. D, Histograms showing dose-dependent early synaptic depression and late synaptic potentiation. D1, Early synaptic depression induced by different doses of 5-HT measured 30 min after 5-HT application. D2, L-LTP induced by different doses of 5-HT measured 3.5 h after 5-HT application. Error bars indicate the mean ± SEM.

Figure 2.

5-HT-induced L-LTP in the BL is mediated by the 5-HT₄ receptor. A, 5-HT₄ receptor antagonist RS 23597 (50 μm; perfused 30 min before, during 5-HT, and 90 min after washout of 5-HT) blocks the L-LTP induced by 5-HT (300 μm). 5-HT, Unfilled circles; RS 23597 alone, unfilled squares; RS 23597 plus 5-HT, filled circles. B, 5-HT_1A receptor antagonist NAN-190 (20 μm; perfused 60 min before and during 5-HT application) reduces the early synaptic depression but does not alter the late phase synaptic potentiation. 5-HT, Unfilled circles; NAN-190 alone, unfilled squares; 5-HT plus NAN-190, filled circles. C, 5-HT₄ receptor agonist RS 67333 (50 μm) induces L-LTP in the BL in the presence of the 5-HT reuptake inhibitor zimelidine. RS 67333 alone, Unfilled circles; zimelidine, filled triangles; RS 67333 plus zimelidine, filled circles; vehicle control, filled squares. Representative field potentials 10 min before and 3.5 h after 5-HT or a 5-HT agonist in each group are shown in the insets. Calibration: 1 mV, 3 ms. Error bars indicate the mean ± SEM.

Figure 3.

5-HT-induced L-LTP in the BL depends on the activation of PKA/MAPK. A, PKA inhibitor KT5720 (2 μm) blocks L-LTP induced by 5-HT (300 μm). 5-HT, Unfilled circles; KT5720 alone, unfilled squares; KT5720 plus 5-HT, filled circles. B, MAPK inhibitor U0126 (10 μm) blocks the late synaptic potentiation induced by 5-HT but does not block the early synaptic depression. 5-HT, Unfilled circles; U0126 alone, unfilled squares; U0126 plus 5-HT, filled circles. Representative field potentials 10 min before and 3.5 h after 5-HT in each group are shown in the insets. Calibration: 1 mV, 5 ms. Error bars indicate the mean ± SEM.

Figure 4.

5-HT-induced L-LTP is independent of NMDA receptors. A, In the presence of NMDA receptor antagonist d-APV (50 μm) 5-HT (300 μm) still induces L-LTP, which is not significantly different from that in control experiments. 5-HT, Unfilled circles; 5-HT plus d-APV, filled circles. B, In the presence of the L-VDCC blocker nifedipine (15 μm) L-LTP induced by 5-HT was reduced. 5-HT, Unfilled circles; 5-HT plus nifedipine, filled circles. C, 5-HT (300 μm) still induces L-LTP in the presence of the GABAergic antagonist picrotoxin. Control, Unfilled circles; 5-HT, filled circles. Representative field potentials 10 min before and 3.5 h after 5-HT in each group are shown in the insets. Calibration: 1 mV, 5 ms. Error bars indicate the mean ± SEM.

Figure 5.

Gene transcription and actin cytoskeletal regulation are required for 5-HT L-LTP. A, Protein synthesis inhibitor anisomycin (25 μm; applied 60 min before, during, and 60 min after washout of 5-HT) blocks the late synaptic potentiation induced by 5-HT. 5-HT, Unfilled circles; anisomycin alone, unfilled squares; anisomycin plus 5-HT, filled circles. Insets show the representative field potentials 10 min before and 3.5 h after 5-HT in each group. Calibration: 1 mV, 3 ms. B, Transcription inhibitor ACTD (40 μm; perfused 30 min before, during, and 60 min after washout of 5-HT) does not affect the synaptic depression but blocks the L-LTP induced by 5-HT (300 μm). 5-HT, Unfilled circles; 5-HT plus ACTD, filled circles. Insets show the representative field potentials 10 min before and 3.5 h after 5-HT in each group. C, ACTD applied 2 h after the application of 5-HT does not alter the L-LTP induced by 5-HT. Insets show the representative field potentials 10 min before (left) and 5 h after (right) 5-HT. D, Actin polymerization inhibitor cytochalasin D (Cyto D; 10 μm; perfused into slices 60 min before, during, and 90 min after washout of 5-HT) blocks the L-LTP induced by 5-HT (300 μm). 5-HT, Unfilled circles; 5-HT plus Cyto D, filled circles. Insets show representative field potentials 10 min before and 3.5 h after 5-HT in each group. E, Cytochalasin D applied 2 h after the application of 5-HT does not alter the L-LTP induced by 5-HT. Insets show representative field potentials 10 min before (left) and 5 h after (right) 5-HT. Calibration: 1 mV, 5 ms. Error bars indicate the mean ± SEM.

Figure 6.

5-HT-induced L-LTP occludes the LTP induced by electrical tetanus. A, 5-HT L-LTP occludes subsequent L-LTP induced by HFS. At 3 h after the application of 5-HT (300 μm) the stimulus intensity was reduced to match the amplitude of baseline potential before 5-HT, and eight trains of tetanus (4 × 2 trains of 100 Hz of stimulation in 3 min intervals) were applied. These tetanus trains failed to induce LTP in 5-HT-treated slices. LTP induced by eight trains of tetanus in control experiments, Unfilled circles; LTP in 5-HT-treated slices, filled circles. Insets, Representative field potentials before (left) and 40 min after (right) tetanus in control and in 5-HT-treated slices. Calibration: 1 mV, 5 ms. B, 5-HT₄ receptor antagonist RS 23597 (50 μm) blocks the L-LTP induced by HFS (4 × 2 trains of 100 Hz). Control, Unfilled circles; RS 23597, filled circles. C, PKA inhibitor KT5720 (2 μm) and protein synthesis inhibitor anisomycin (25 μm) block L-LTP induced by HFS. Control, Unfilled circles; KT5720, unfilled squares; anisomycin, filled circles. Error bars indicate the mean ± SEM.