Ethanol induces persistent potentiation of 5-HT3 receptor-stimulated GABA release at synapses on rat hippocampal CA1 neurons

Abstract

Several studies have shown that ethanol (EtOH) can enhance the activity of GABAergic synapses via presynaptic mechanisms, including in hippocampal CA1 neurons. The serotonin type 3 receptor (5-HT₃-R) has been implicated in the neural actions of ethanol (EtOH) and in modulation of GABA release from presynaptic terminals. In the present study, we investigated EtOH modulation of GABA release induced by 5-HT₃-R activation using the mechanically isolated neuron/bouton preparation from the rat CA1 hippocampal subregion. EtOH application before and during exposure to the selective 5-HT₃ receptor agonist, m-chlorophenylbiguanide (mCPBG) potentiated the mCPBG-induced increases in the peak frequency and charge transfer of spontaneous GABAergic inhibitory postsynaptic currents. Interestingly, the potentiation was maintained even after EtOH was removed from the preparation. A protein kinase A inhibitor reduced the magnitude of EtOH potentiation. Fluorescent Ca²⁺ imaging showed that Ca²⁺ transients in the presynaptic terminals increased during EtOH exposure. These findings indicate that EtOH produces long-lasting potentiation of 5-HT₃-induced GABA release by modulating calcium levels, via a process involving cAMP-mediated signaling in presynaptic terminals.

Keywords: Alcohol; GABAergic synapse; Hippocampus; Neuron/bouton; Serotonin.

Fig. 1.. 5-HT increases the frequency and amplitude of spontaneous IPSCs.

A. Representative sIPSC traces before, during and after exposure to different concentrations of 5-HT. Scale bar = 100 pA, 5 s. B. Bar graph showing peak amplitude of IPSCs evoked by the different 5-HT concentrations (n = 3) C. Bar graph showing peak frequency of IPSCs evoked by the different 5-HT concentrations (n = 3) D. Bar graph showing charge transfer of IPSCs evoked by the different 5-HT concentrations (n = 3). (unpaired t-tests *P < 0.05, **P < 0.001). Cumulative probability distributions for E. IPSC amplitude, F. Rise time, and G. Decay time.

Fig. 2.. 5-HT transiently increases the frequency of GABAergic sIPSCs through the activation of 5-HT₃ receptors and ethanol potentiates the increase induced by a selective 5-HT₃ agonist.

A. The GABA_A receptor antagonist gabazine suppressed all sIPSCs as well as the response to 5-HT. Scale bar = 100 pA, 30 s. B, C. The response to 5-HT was blocked in the presence of the selective 5-HT₃R antagonist MDL 72222 while baseline sIPSCs remained (n = 5). Scale bar = 200 pA, 30 s (unpaired t-tests *P < 0.05). D. Representative sIPSC waveforms in response to 5-HT in the absence and presence of 80 mM EtOH. sIPSCs induced by 5-HT. Scale bar = 200 pA, 30 s. E, F. No difference was observed in the charge transfer and peak frequency of sIPSC responses to 5-HT before and during EtOH incubation (n = 5). G. Representative sIPSC waveforms in response to the selective 5-HT₃ receptor agonist, mCPBG in the absence and presence of 80 mM EtOH. Scale bar = 200 pA, 30 s. H, I. EtOH potentiates the charge transfer and peak frequency of the 5-HT₃ agonist-induced increase in sIPSCs (paired t-tests *P < 0.05, n = 10).

Fig. 3.. The sIPSC response to mCPBG depends on EtOH exposure and the exposure time.

A, B. The charge transfer and peak frequency of sIPSCs were not potentiated until EtOH was applied (paired t-tests *P <0.05, n = 5). C. EtOH potentiation of sIPSC charge transfer with different EtOH incubation times (unpaired t-tests *P <0.05, n = 4 for 0, 60 s conditions, n = 10 for 210 s condition). D. When a second mCPBG application was given at different time points after EtOH exposure, the potentiation of the mCPBG response was smaller at longer EtOH wash-out times (unpaired t-tests *P <0.05, **P <0.001, n = 6 for 90s condition, n = 9 for other conditions). E, F. The charge transfer and peak frequency of sIPSC responses to high K⁺ were not potentiated by EtOH exposure (n = 5).

Fig. 4.. Potentiation of the response to mCPBG was maintained even after EtOH washout.

A. Representative waveforms of sIPSCs in response to 10 μM mCPBG before, during and after washout of 80 mM EtOH. Scale bar = 400 pA, 10 s. B. Plots of sIPSC frequency (paired t-tests *P < 0.05, n = 6). C. EtOH potentiation of sIPSC charge transfer and peak frequency was maintained even after EtOH washout (paired t-tests *P < 0.05, n = 6). D. EtOH increases the charge transfer of sIPSC responses to mCPBG in a concentration-dependent manner. The post-EtOH-washout potentiation was also more distinct with higher EtOH concentrations. (paired t-tests *P < 0.05, n = 9, 9, 8 and 6 for 10, 20, 40 and 80 mM EtOH, respectively).

Fig. 5.. Voltage-gated calcium channels and voltage-gated sodium channels are involved in 5-HT₃ receptor-mediated IPSC increases.

A. Ca_V2.2 N-type voltage-gated Ca²⁺ channel blocker ω-conotoxin-GVIA (ω-Ctx) suppressed baseline sIPSCs as well as the mCPBG response. Scale bar = 400 pA, 10 s. B, C. ω-Ctx significantly decreased the charge transfer and peak frequency of the mCPBG-induced increase in sIPSCs (paired t-tests **P < 0.01, n = 5). D. Ca_V2.1 P/Q-type voltage-gated Ca²⁺ channel blocker, ω-agatoxin-IVA (ω-AgaTx) had little effect on sIPSC responses to mCPBG. Scale bar = 400 pA, 10 s. E, F. No difference was observed in the charge transfer and peak frequency of sIPSC responses to mCPBG in the presence of ω-AgaTx (n = 7). G. Representative waveforms of mIPSC responses to mCPBG before and during ethanol exposure in the presence of TTX. Scale bar = 200 pA, 10 s. H, I. In the presence of TTX, EtOH did not potentiate mIPSC charge transfer during mCPBG application (n = 4).

Fig. 6.. Ethanol potentiation involves the cAMP-dependent protein kinase.

A. Representative waveforms of sIPSC responses to mCPBG and mCPBG + EtOH in the presence of the competitive PKA inhibitor, Rp-cAMP. Scale bar = 400 pA, 10s. B. Plots of sIPSC frequency in the presence of Rp-cAMP (n = 6) before, during and after EtOH exposure. C. EtOH potentiation of the 5-HT₃ receptor-induced increase in charge trransfer was inhibited in the presence of Rp-cAMP (n = 6).

Fig. 7.. Calcium imaging shows that calcium entry is increased in presynaptic boutons after EtOH incubation.

A. Fluorescence image after calcium dye loading in a mechanically-dissociated neuron. B. Wide-field image of patch pipette with giga-ohm seal on the neuron. C. Fluorescence image 2 min after opening the membrane for whole-cell recording. D. Calcium transients were observed from a subpopulation of dye-loaded puncta (white arrowheads) after diluting the dye in the postsynaptic neuron. Scale bar = 10 μm. E. The spontaneous calcium transients were suppressed by TTX. F. Representative spontaneous normalized Ca²⁺ transients before and during EtOH incubation. G. The spontaneous calcium entry increased in the presence of 80 mM EtOH (paired t-tests *P < 0.05, n = 12). H. In control experiments without EtOH exposure, no increase of calcium entry was observed. The small decrease in transients in the control condition is likely due to photo bleaching (n = 12).