Characterization of serotonin-induced inhibition of excitatory synaptic transmission in the anterior cingulate cortex

Abstract

Excitatory synaptic transmission in central synapses is modulated by serotonin (5-HT). The anterior cingulate cortex (ACC) is an important cortical region for pain perception and emotion. ACC neurons receive innervation of projecting serotonergic nerve terminals from raphe nuclei, but the possible effect of 5-HT on excitatory transmission in the ACC has not been investigated. In the present study, we investigated the role of 5-HT on glutamate neurotransmission in the ACC slices of adult mice. Bath application of 5-HT produced dose-dependent inhibition of evoked excitatory postsynaptic currents (eEPSCs). Paired pulse ratio (PPR) was significantly increased, indicating possible presynaptic effects of 5-HT. Consistently, bath application of 5-HT significantly decreased the frequency of spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs). By contrast, amplitudes of sEPSCs and mEPSCs were not significantly affected. After postsynaptic application of G protein inhibitor GDP-β-S, 5-HT produced inhibition of eEPSCs was significantly reduced. Finally, NAN-190, an antagonist of 5-HT_1A receptor, significantly reduced postsynaptic inhibition of 5-HT and abolished presynaptic inhibition. Our results strongly suggest that presynaptic as well as postsynaptic 5-HT receptor including 5-HT1A subtype receptor may contribute to inhibitory modulation of glutamate release as well as postsynaptic responses in the ACC.

Keywords: Adenylyl cyclase; Anterior cingulate cortex; Excitatory postsynaptic currents; Glutamatergic neurotransmission; Serotonin.

Fig. 1

Bath application of 5-HT reduced the amplitude of evoked EPSC. a Schematic diagram of a slice illustrating the placement of a whole-cell patch recording and stimulating electrode in anterior cingulate cortex. b Current-clamp recordings to identify pyramidal neurons (left) and interneurons (right) by step current injection. c An example showing the time course of recorded pyramidal neurons in layer II/III of ACC after application of 5-HT (5 μM) and washout. The insets showed averaged trace of six eEPSCs about baseline, application of 5-HT and washout. d The averaged time course of recorded neurons (n = 9 neurons/6 mice) in ACC. Bath application of 5-HT (5 μM) led to a gradual reduction of eEPSC amplitude and washing out 5-HT with ACSF caused the rise of eEPSC amplitude again. e Summary of the effect of 5-HT (5 μM) and washout on the amplitude of eEPSC. Bath application of 5-HT significantly reduced eEPSCs amplitude and washing the system with fresh ACSF partially but evidently reversed the decreased eEPSC (n = 9 neurons/6 mice). ^** p < 0.01 compared to baseline; ^# p < 0.05 compared to the group of 5-HT

Fig. 2

Bath application of 5-HT increased paired-pulse ratio. a Paired-pulse ratio (the ratio of EPSC2/EPSC1) was recorded with a 50 ms interval. One example showing the time course of PPR recorded in the ACC layer II/III neurons. The insets showed averaged trace of six sweeps about baseline, application of 5-HT and washout.b Summary of the effect of 5-HT (5 μM) and washout on the PPR. 5-HT increased the PPR notably (n = 10 neurons/7 mice). ^* p < 0.05 compared to baseline

Fig. 3

Effect of 5-HT on sEPSC recorded in neurons of the ACC. a Representative trace of sEPSC recorded in pyramidal neurons of the ACC layer II/III at a holding potential of −60 mV. b Cumulative probability plot showing the distribution of sEPSC amplitude in the phase of baseline, 5-HT application (5 μM) and washout.c Cumulative inter-events interval plot of recorded sEPSC in the phase of baseline, 5-HT application (5 μM) and washout. Black fill line indicated the phase of baseline, green fill line indicted the phase of 5-HT application and deep red dashed line indicated the phase of washout. d Summary result of averaged sEPSC amplitude (n = 15 neurons/7 mice). (E) Summary result of averaged sEPSC frequency (n = 12 neurons/7 mice). 5-HT (5 μM) application significantly reduced the frequency of sEPSC and washout partially reversed the reduction. ^** p < 0.01 compared to baseline; ^# p < 0.05 compared to the phase of 5-HT application

Fig. 4

Effect of 5-HT on mEPSC recorded in neurons of the ACC. a Representative mEPSC recorded in pyramidal neurons of the ACC layer II/III at a holding potential of −60 mV. b Cumulative plot of mEPSC amplitude of the phase of baseline, 5-HT application (5 μM) and washout. c Cumulative inter-event interval plot of recorded mEPSC in the phase of baseline, 5-HT application (5 μM) and washout. Black fill line indicated the phase of baseline, green fill line indicted the phase of 5-HT application and deep red dashed line indicated the phase of washout. d Summary plots of mEPSC amplitude (n = 12 neurons/6 mice). e Summary plots of mEPSC frequency (n = 10 neurons/6 mice). 5-HT (5 μM) application significantly reduced the frequency of mEPSC and washout partially reversed the reduction. ^** p < 0.01 compared to baseline; ^# p < 0.05 compared to the phase of 5-HT application

Fig. 5

5-HT- mediated reduction of eEPSC amplitude was inhibited by GDP-β-S. a The averaged time course showing the change of eEPSC amplitude after bath application of 5-HT (5 μM) in the recorded ACC neurons with or without the internal solution containing GDP-β-S (1 mM). The red dots indicated the recording with GDP-β-S. The grey dots indicated the recording without GDP-β-S (data from Figure 1). b Summary result of effect of 5-HT on eEPSC in the presence of GDP-β-S (n = 9 neurons/5 mice) or absence of GDP-β-S in the recording internal solution (n = 9 neurons/6 mice). ^* p < 0.05 compared to baseline; ^# p < 0.05 compared to the phase of 5-HT application with GDP-β-S in the internal solution of recording electrodes

Fig. 6

5-HT_1A receptor was involved in the inhibiting effect of 5-HT on synaptic transmission. a One sample neuron showing the time course of the change of eEPSC amplitude after 5-HT (5 μM) application in the presence of 5-HT_1A receptor antagonist, NAN-190 (5 μM). The insets showed averaged trace of six eEPSCs. b The averaged time course of recorded neurons (n = 9 neurons/6 mice) in ACC after application of 5-HT in the presence or absence of NAN-190. The red dots indicated the recording in the presence of NAN-190. The grey dots indicated the recording in the absence of NAN-190 (control, data from Figure 1). c One sample neuron illustrating the time course of the change of PPR after 5-HT application in the presence of NAN-190. The insets showed averaged trace of six sweeps. d Summary plot showing the effect of 5-HT (5 μM) on eEPSC amplitude (left) and PPR (right) when NAN-190 (5 μM) was added into the system beforehand. NAN-190 partially inhibited the reduction of eEPSC caused by 5-HT, and there was no big difference between the PPR before and after the application of 5-HT in the presence of NAN-190. n = 9 neurons/6 mice; ^* p < 0.05 compared to baseline. ^# p < 0.05 compared to the phase of 5-HT application in the recording with NAN-190 prior adding to the system

Fig. 7

Effect of 5-HT on sEPSC in the presence of NAN-190. a Representative sEPSC traces recorded in the presence of NAN-190 (5 μM). b Cumulative probability plots showing the distribution of sEPSC amplitude (left) and interval-events interval (right) in the phase of baseline (black line) and 5-HT application (dashed green line). c Summary plots of sEPSC data. Averaged values of sEPSC parameters: mean amplitude (left) and peak frequency (right) (n = 7 neurons/5 mice). In the presence of NAN-190, 5-HT had no evident effect on the frequency and amplitude of sEPSC

Fig. 8

Effect of 5-HT on mEPSC in the presence of NAN-190. a Representative mEPSC traces recorded in the presence of NAN-190 (5 μM). b Cumulative probability plots showing the distribution of mEPSC amplitude (left) and interval-events interval (right). Black line represented the phase of baseline and dashed green line represented the phase of 5-HT application. c Summary plots of mEPSC data. Averaged values of mEPSC parameters: mean amplitude (left) and peak frequency (right) (n = 8 neurons/6 mice). In the presence of NAN-190, one antagonist of 5-HT_1A receptor, both the frequency and amplitude did not change evidently after bath application of 5-HT