5-HT-dependent synaptic plasticity of the prefrontal cortex in postnatal development

Abstract

Important functions of the prefrontal cortex (PFC) are established during early life, when neurons exhibit enhanced synaptic plasticity and synaptogenesis. This developmental stage drives the organization of cortical connectivity, responsible for establishing behavioral patterns. Serotonin (5-HT) emerges among the most significant factors that modulate brain activity during postnatal development. In the PFC, activated 5-HT receptors modify neuronal excitability and interact with intracellular signaling involved in synaptic modifications, thus suggesting that 5-HT might participate in early postnatal plasticity. To test this hypothesis, we employed intracellular electrophysiological recordings of PFC layer 5 neurons to study the modulatory effects of 5-HT on plasticity induced by theta-burst stimulation (TBS) in two postnatal periods of rats. Our results indicate that 5-HT is essential for TBS to result in synaptic changes during the third postnatal week, but not later. TBS coupled with 5-HT_2A or 5-HT_1A and 5-HT₇ receptors stimulation leads to long-term depression (LTD). On the other hand, TBS and synergic activation of 5-HT_1A, 5-HT_2A, and 5-HT₇ receptors lead to long-term potentiation (LTP). Finally, we also show that 5-HT dependent synaptic plasticity of the PFC is impaired in animals that are exposed to early-life chronic stress.

Figure 1

5-HT reduces the amplitude of postsynaptic responses. (A) Scheme showing the prelimbic region of medial PFC on a coronal slice and the position of the stimulation electrode and recorded neurons. (B) Current–voltage (I–V) plots of PFC layer 5 neurons at P14-16. (C) Example of action potentials obtained by current injection in pyramidal neurons from layer 5. (D) The graph shows the cumulative relative spike intervals of neurons recorded from layer 5 at P14-16. The interval values were normalized with respect to the first spike time (t = 0). (E) The graph shows the frequency histogram for the interspike intervals of neurons recorded from layer 5 at P14-16. (F) Scheme showing the position of the stimulation electrode and recorded neurons in the prelimbic region of medial PFC. (G) The graph shows the EPSPs recorded in P14-16 animals before and during 5-HT (50 µM) bath application. The EPSPs were normalized to the mean of responses recorded during the baseline. The black bar represents the period of application of 5-HT in the bath. The EPSPs were normalized to the mean of responses recorded during the baseline and the bars represent the SEM. Traces show the results of a representative experiment the average of postsynaptic responses recorded before (1: 5 to 10 min, black line) and after (2: 25 to 30 min, magenta line) application of 5-HT. (H) Graph showing the paired pulse ratio of EPSPs in P14-16 animals. Two different conditions are compared: control (white inserts) and 5-HT application (gray inserts). Postsynaptic responses were normalized to the response caused by the first pulse. The EPSPs were normalized to the mean of responses recorded during the baseline and the bars are respect to the SEM. Traces show the results of a representative experiment with the average of postsynaptic responses recorded before the baseline and 20 min after the bath application of 5-HT.

Figure 2

5-HT_2A, 5-HT_1A, and 5-HT₇ receptors antagonists inhibit the effects of 5-HT on synaptic transmission. (A) The graph shows the EPSPs recorded in P14-16 animals before and during 5-HT (50 µM) bath application, in the presence or not of the 5-HT_2A receptor antagonist. Two different conditions are compared: 5-HT (dark grey inserts, 5-HT, 50 µM) and 5-HT_2A receptor antagonist + 5-HT (blue inserts; Ketanserin, 10 µM, + 5-HT, 50 µM). The EPSPs were normalized to the mean of responses recorded during the baseline and the bars represent the SEM. Traces show the results of a representative experiment with the average of postsynaptic responses recorded before (1: 5 to 10 min, black line) and after (2: 25 to 30 min, magenta line) application of 5-HT. (B) The graph shows the EPSPs recorded in P14-16 animals before and after 5-HT (50 µM) application in the presence or not of 5-HT_1A receptor antagonist. Two different conditions are compared: 5-HT (dark grey inserts, 5-HT) and 5-HT_1A receptor antagonist + 5-HT (red inserts, WAY-100635, 1 µM, + 5-HT, 50 µM). The EPSPs were normalized to the mean of responses recorded during the baseline and the bars indicate the SEM. Traces show the results of a representative experiment with the average of postsynaptic responses recorded before (1: 5 to 10 min, black line) and after (2: 25 to 30 min, magenta line) application of 5-HT. (C) The graph shows the EPSPs recorded in P14-16 animals before and after 5-HT (50 µM) application, with two different conditions being compared: 5-HT (dark gray inserts, 5-HT) and 5-HT₇ receptor antagonist + 5-HT (green inserts, SB-269970, 10 µM, + 5-HT, 50 µM). The EPSPs were normalized to the mean of responses recorded during the baseline and the bars represent the SEM. Traces show the results of a representative experiment with the average of postsynaptic responses recorded before (1: 5 to 10 min, black line) and after (2: 25 to 30 min, magenta line) application of 5-HT. (D) Summary of the depressive effects of 5-HT. The graph bars show the EPSP values recorded during two different periods of the experiment: 15–20 min and 25–30 min.

Figure 3

5-HT_1A, 5-HT_2A and 5-HT₇ receptors agonists reduce the amplitude of synaptic responses. (A) The graph shows the EPSPs recorded in P14-16 animals before and during 5-HT_2A receptor agonist TCB-2 (10 µM) bath application. The EPSPs were normalized to the mean of responses recorded during the baseline. Traces show the results of a representative experiment with the average of postsynaptic responses recorded before (1: 5 to 10 min, black line) and after (2: 25 to 30 min, magenta line) application of TCB-2. (B) The graph shows the EPSPs recorded in P14-16 animals before and during 5-HT_1A receptor agonist 8-OH-DPAT (1 µM) bath application. The EPSPs were normalized to the mean of responses recorded during the baseline. Traces show the results of a representative experiment with the average of postsynaptic responses recorded before (1: 5 to 10 min, black line) and after (2: 25 to 30 min, magenta line) application of 8-OH-DPAT. (C) The graph shows the EPSPs recorded in P14-16 animals before and during 5-HT₇ receptor agonist LP-44 (2 µM) bath application. The EPSPs were normalized to the mean of responses recorded during the baseline. Traces show the results of a representative experiment with the average of postsynaptic responses recorded before (1: 5 to 10 min, black line) and after (2: 25 to 30 min, magenta line) application of LP-44. (D) Graph showing the effects of 5-HT and 5-HT_1A/2A/7 agonists. The averaged synaptic responses recorded during the 25–30 min period are reported. EPSPs were normalized to the mean of responses recorded during the baseline.

Figure 4

TBS induction causes LTP in P14-16 animals slices under the modulation of 5-HT. (A) The graph shows the EPSPs recorded in P14-16 animals before and after TBS induction. The EPSPs were normalized to the mean of responses recorded during the baseline. Traces show the average of postsynaptic responses recorded before (1: baseline, − 5 to 0 min, black line) and after the TBS induction (2: post induction, 35–40 min, black line). (B) The graph shows the EPSPs recorded in P14-16 animals before and after TBS induction in the presence or not of 5-HT. The EPSPs were normalized to the mean of responses recorded during the baseline. Two different conditions are compared: control (white insert) and 5-HT (gray inserts, 50 µM). Traces show the results of a representative experiment with the average of postsynaptic responses recorded before (1: baseline, − 5 to 0 min, black line) and after the TBS induction (2: post induction, 35–40 min, black line). (C) Graph showing the paired pulse ratio of 4 pulses (10 Hz) EPSPs in slices treated with 5-HT (50 µM). Two different conditions are compared: before (white inserts) and after TBS induction (gray inserts). Postsynaptic responses were normalized to the response caused by the first pulse. Traces show the results of a representative experiment with the average of postsynaptic responses recorded before the baseline and 40 min after the TBS induction.

Figure 5

5-HT modulation inhibited by one of the 5-HT_1A/2A/7 receptors antagonists shift synaptic plasticity from LTP to LTD. (A) The graph shows the EPSPs recorded in P14-16 animals before and after TBS induction. The EPSPs were normalized to the mean of responses recorded during the baseline. Two different conditions are compared: control (light grey inserts) and 5-HT (50 µM) together with the 5-HT_2A receptor antagonist (blue inserts, Ketanserin, 10 µM). Traces show the results of a representative experiment with the average of postsynaptic responses recorded before (1: baseline, − 5 to 0 min, black line) and after the TBS induction (2: post induction, 35–40 min, black line). (B) The graph shows the EPSPs recorded in P14-16 animals before and after TBS induction. The EPSPs were normalized to the mean of responses recorded during the baseline. Two different conditions are compared: control (light grey inserts) and 5-HT (50 µM) together with the 5-HT_1A receptor antagonist (red inserts, WAY-100635, 1 µM). Traces show the results of a representative experiment with the average of postsynaptic responses recorded before (1: baseline, − 5 to 0 min, black line) and after the TBS induction (2: post induction, 35–40 min, black line). (C) The graph shows the EPSPs recorded in P14-16 animals before and after TBS induction. The EPSPs were normalized to the mean of responses recorded during the baseline. Two different conditions are compared: control (light grey inserts, 50 µM) and 5-HT together with the 5-HT₇ receptor antagonist (green inserts, SB-269970, 10 µM). Traces show the results of a representative experiment with the average of postsynaptic responses recorded before (1: baseline, − 5 to 0 min, black line) and after the TBS induction (2: post induction, 35–40 min, black line). (D) Scatter graph showing the EPSPs recorded during the 35–40 min period after TBS induction, for individual experiments (small dots) and for the mean values of the experimental group (large dots, with bars representing the SEM). Five experimental groups are compared based on the condition of drug incubation (Control, 5-HT, Ketanserin + 5-HT, WAY-100635 + 5-HT and SB-269970 + 5-HT). EPSPs values were normalized to the mean of responses recorded during the baseline. (E) The graph shows the percentage of cells exhibiting LTP, LTD or no change in five experimental groups (Control, 5-HT, Ketanserin + 5-HT, WAY-100635 + 5-HT and SB-269970 + 5-HT).

Figure 6

Application of 5-HT together with the three 5-HT_1A/2A/7 receptors antagonists has no effect on TBS synaptic plasticity. (A) The graph shows the EPSPs recorded in P14-16 animals before and after TBS induction. The EPSPs were normalized to the mean of responses recorded during the baseline. Two different conditions are compared: control (white inserts) and 5-HT together with the three 5-HT_1A/2A/7 receptor antagonists (dark grey inserts: 5-HT, 50 µM, Ketanserin, 10 µM, WAY-100635, 1 µM, SB-269970, 10 µM). Traces show the results of a representative experiment with the average of postsynaptic responses recorded before (1: baseline, − 5 to 0 min, black line) and after the TBS induction (2: post induction, 35–40 min, black line). (B) Scatter graph showing the averaged EPSPs recorded during the 35–40 min period after TBS induction, for individual experiments (small dots) and for the mean values of the experimental group (large dots, with bars representing the SEM). Two experimental groups are compared based on the condition of drug incubation: control and 5-HT + 5-HT_1A/2A/7 receptors antagonists (5-HT, 50 µM, Ketanserin, 10 µM, WAY-100635, 1 µM, SB-269970, 10 µM). EPSPs values were normalized to the mean of responses recorded during the baseline. (C) The graph shows the percentage of cells exhibiting LTP, LTD or no change in two experimental groups: control and 5-HT + 5-HT_1A/2A/7 receptors antagonists (5-HT, 50 µM, Ketanserin, 10 µM, WAY-100635, 1 µM, SB-269970, 10 µM).

Figure 7

TBS coupled to activation of 5-HT_2A receptor induces LTD. (A) The graph shows the EPSPs recorded in P14-16 animals before and after TBS induction. Two different conditions are compared: control (light grey inserts) and application of 5-HT_2A receptor agonist (blue inserts, TCB-2, 10 µM). The EPSPs were normalized to the mean of responses recorded during the baseline. Traces show the results of a representative experiment with the average of postsynaptic responses recorded before (1: baseline, − 5 to 0 min, black line) and after the TBS induction (2: post induction, 35–40 min, black line). (B) Graph showing the paired pulse ratio of 4 pulses (10 Hz) EPSPs in slices treated with TCB-2. Two different conditions are compared: before (white inserts) and after TBS induction (blue inserts). Postsynaptic responses were normalized to the response caused by the first pulse. Traces show the results of a representative experiment with the average of postsynaptic responses recorded before the baseline and 40 min after the TBS induction. (C) The graph shows the EPSPs recorded in P14-16 animals before and after TBS induction. Two different conditions are compared: control (light grey inserts) and application of 5-HT_2A receptor agonist (red inserts, 8-OH-DPAT, 1 µM). The EPSPs were normalized to the mean of responses recorded during the baseline. Traces show the results of a representative experiment with the average of postsynaptic responses recorded before (1: baseline, − 5 to 0 min, black line) and after the TBS induction (2: post induction, 35–40 min, black line). (D) The graph shows the EPSPs recorded in P14-16 animals before and after TBS induction. Two different conditions are compared: control (light grey inserts) and application of 5-HT_2A receptor agonist (red inserts, LP-44, 2 µM). The EPSPs were normalized to the mean of responses recorded during the baseline. Traces show the results of a representative experiment with the average of postsynaptic responses recorded before the average of postsynaptic responses recorded before (1: baseline, − 5 to 0 min, black line) and after the TBS induction (2: post induction, 35–40 min, black line). (E) Scatter graph showing the averaged EPSPs recorded during the 35–40 min period after TBS induction, for individual experiments (small dots) and for the mean values of the experimental group (large dots, with bars representing the SEM). Five experimental groups are compared based on the condition of drug incubation (Control, TCB-2, 8-OH-DPAT and LP-44). EPSPs values were normalized to the mean of responses recorded during the baseline. (F) The graph shows the percentage of cells exhibiting LTP, LTD or no change in five experimental groups (Control, TCB-2, 8-OH-DPAT and LP-44).

Figure 8

Application of three 5-HT_1A/2A/7 receptors agonists induces LTD. (A) The graph shows the EPSPs recorded in P14-16 animals before and after TBS induction. The EPSPs were normalized to the mean of responses recorded during the baseline. Two different conditions are compared: control (white inserts) and 5-HT_1A/2A/7 receptor agonists (dark grey inserts: TCB-2, 10 µM, 8-OH-DPAT, 1 µM LP-44, 2 µM). Traces show the results of a representative experiment with the average of postsynaptic responses recorded before (1: baseline, − 5 to 0 min, black line) and after the TBS induction (2: post induction, 35–40 min, black line). (B) Scatter graph showing the averaged EPSPs recorded during the 35–40 min period after TBS induction, for individual experiments (small dots) and for the mean values of the experimental group (large dots, with bars representing the SEM). Two experimental groups are compared based on the condition of drug incubation: control and 5-HT_1A/2A/7 receptors agonists (TCB-2, 10 µM, 8-OH-DPAT, 1 µM LP-44, 2 µM). EPSPs values were normalized to the mean of responses recorded during the baseline. (C) The graph shows the percentage of cells exhibiting LTP, LTD or no change in two experimental groups: control and 5-HT_1A/2A/7 receptors agonists (TCB-2, 10 µM, 8-OH-DPAT, 1 µM LP-44, 2 µM).

Figure 9

5-HT and the 5-HT_1A/2A/7 receptors agonists have no effect on neuronal excitability. (A) Graph showing the averaged membrane resting potential recorded in the control condition (before the drug application, for 5 min) and under the effect of 5-HT (50 µM, from 25 to 30 min after the drug application). (B) Graph showing the averaged membrane resting potential recorded in the control condition (before the drug application, for 5 min) and under the effect of TCB-2 (10 µM, from 25 to 30 min after the drug application). (C) Graph showing the averaged membrane resting potential recorded in the control condition (before the drug application, for 5 min) and under the effect of 8-OH-DPAT (1 µM, from 25 to 30 min after the drug application). (D) Graph showing the averaged membrane resting potential recorded in the control condition (before the drug application) and under the effect of LP-44 (2 µM, from 25 to 30 min after the drug application). (E) Graph showing the averaged membrane resting potential recorded under the effect of 5-HT and 5-HT_1A/2A/7 agonists (from 25 to 30 min after the drug application). Four experimental groups are compared: 5-HT (50 µM), TCB-2 (10 µM), 8-OH-DPAT (1 µM) and LP-44 (2 µM). EPSPs values were normalized to the mean of membrane resting potential values recorded before the drug application, for 5 min. (F) Graph showing the averaged input resistance recorded in the control condition (before the drug application, for 5 min) and under the effect of 5-HT (50 µM, from 25 to 30 min after the drug application). (G) Graph showing the averaged input resistance recorded in the control condition (before the drug application, for 5 min) and under the effect of TCB-2 (10 µM, from 25 to 30 min after the drug application). (H) Graph showing the averaged input resistance recorded in the control condition (before the drug application, for 5 min) and under the effect of 8-OH-DPAT (1 µM, from 25 to 30 min after the drug application). (I) Graph showing the averaged input resistance in the control condition (before the drug application, for 5 min) and under the effect of LP-44 (2 µM, from 25 to 30 min after the drug application). (J) Graph showing the averaged input resistance recorded under the effect of 5-HT and 5-HT_1A/2A/7 agonists (from 25 to 30 min after the drug application). Four experimental groups are compared: 5-HT (50 µM), TCB-2 (10 µM), 8-OH-DPAT (1 µM) and LP-44 (2 µM). EPSPs values were normalized to the mean of input resistance values recorded before the drug application, for 5 min.

Figure 10

TBS induction fails to induce synaptic plasticity in slices from P24-26 animals. (A) The graph shows the EPSPs recorded in P24-46 animals before and during 5-HT bath application. The EPSPs were normalized to the mean of responses recorded during the baseline. The black bar represents the period of application of 5-HT (50 µM) in the bath. Traces show the results of a representative experiment with the average of postsynaptic responses recorded before (1: 5 to 10 min, black line) and after (2: 25 to 30 min, magenta line) application of 5-HT. (B) Graph showing the effects of 5-HT on the averaged post synaptic responses for the 25–30 min period in P14-16 and P24-26 animals. EPSPs were normalized to the mean of responses recorded during the baseline. (C) The graph shows the EPSPs recorded in P24-26 animals before and after TBS induction. The EPSPs values were normalized to the mean of responses recorded during the baseline, with two different conditions being compared: control (yellow inserts) and 5-HT (brown inserts, 50 µM). Traces show the results of a representative experiment with the average of postsynaptic responses recorded before (1: baseline, − 5 to 0 min, black line) and after the TBS induction (2: post induction, 35–40 min, black line). (D) Graph showing the averaged EPSPs recorded during the 35–40 min period after TBS induction. Four experimental groups are compared (P14-16 TBS, P14-16 TBS + 5-HT, P24-26 TBS, P24-16 TBS + 5-HT).

Figure 11

5-HT reduces the amplitude of postsynaptic responses in MS animals. (A) The graph shows the time of immobility measured in the Forced Swim Test performed on P24-26 animals. Two groups of animals are compared: MS and non-MS. Bars represent the average of normalized (%) time of immobility with SEM. (B) The graph shows the EPSPs recorded in P14-16 animals before and during 5-HT bath application. The EPSPs were normalized to the mean of responses recorded during the baseline. Two different conditions are compared: non-MS (dark grey inserts) and MS (light violet) animals. The black bar represents the period of application of 5-HT (50 µM) in the bath. Traces show the results of a representative experiment with the average of postsynaptic responses recorded before (1: 5 to 10 min, black line) and after (2: 25 to 30 min, magenta line) application of 5-HT. (C) Graph showing the effects of 5-HT on the averaged post synaptic responses recorded during the 25–30 min period in P14-16 MS animals. EPSPs were normalized to the mean of responses recorded during the baseline. (D) The graph shows the EPSPs recorded in P24-26 animals before and during 5-HT bath application. The EPSPs were normalized to the mean of responses recorded during the baseline. Two different conditions are compared: non-MS (brown inserts) and MS (dark brown inserts) animals. The black bar represents the period of application of 5-HT (50 µM) in the bath. Traces show the results of a representative experiment with the average of postsynaptic responses recorded before (1: 5 to 10 min, black line) and after (2: 25 to 30 min, magenta line) application of 5-HT. (E) Graph showing the effects of 5-HT on the averaged post synaptic responses for the 25–30 min period in P24-26 MS animals. EPSPs were normalized to the mean of responses recorded during the baseline.

Figure 12

TBS induction fails to induce synaptic plasticity in slices from MS animals. (A) The graph shows the EPSPs recorded in MS P14-16 animals before and after TBS induction. The EPSPs were normalized to the mean of responses recorded during the baseline, with two different conditions being compared: control (light violet) and 5-HT (dark violet, 50 µM). Traces show the results of a representative experiment with the average of postsynaptic responses recorded before (1: baseline, − 5 to 0 min, black line) and after the TBS induction (2: post induction, 35–40 min, magenta line). (B) Graph showing the averaged EPSPs recorded during the 35–40 min period after TBS induction in P14-16 animals. Four experimental groups are compared (non-MS TBS, non-MS TBS + 5-HT, MS TBS, MS TBS + 5-HT). (C) The graph shows the EPSPs recorded in MS P24-26 animals before and after TBS induction. The EPSPs were normalized to the mean of responses recorded during the baseline, with two different conditions being compared: control (beige inserts) and 5-HT (dark brown inserts, 50 µM). Traces show the results of a representative experiment with the average of postsynaptic responses recorded before (1: baseline, − 5 to 0 min, black line) and after the TBS induction (2: post induction, 35–40 min, magenta). (D) Graph showing the averaged EPSPs recorded during the 35–40 min period after TBS induction in P24-26 animals. Four experimental groups are compared (non-MS TBS, non-MS TBS + 5-HT, MS TBS, MS TBS + 5-HT).

Figure 13

5-HT Modulation of Synaptic Plasticity. The figure summarizes the model proposed for the 5-HT modulation of synaptic plasticity induced by TBS. Limited 5-HT receptor types stimulation result in LTD expression, while the synergistic recruitment of multiple 5-HT receptor types activation allows the occurrence of LTP. The metaplastic interaction between TBS and 5-HT is age dependent and absent in the P24-26 period. (A) The expression of LTP requires the interaction between TBS and 5-HT. (B) TBS induction coupled with activation of 5-HT_2A receptor decreases the synaptic strength. (C) TBS induction coupled with activation of the 5-HT_1A and 5-HT₇ receptors decreases the synaptic strength. (D) TBS induction coupled with 5-HT do not cause changes of synaptic efficacy in P24-26 animals.