Ethanol blocks long-term potentiation of GABAergic synapses in the ventral tegmental area involving mu-opioid receptors

Abstract

It is well documented that ethanol exposure alters GABA (gamma-aminobutyric acid)-releasing synapses, and ethanol addiction is associated with endogenous opioid system. Emerging evidence indicates that opioids block long-term potentiation in the fast inhibitory GABA(A) receptor synapses (LTP(GABA)) onto dopamine-containing neurons in the ventral tegmental area (VTA), a brain region essential for reward-seeking behavior. However, how ethanol affects LTP(GABA) is not known. We report here that in acute midbrain slices from rats, clinically relevant concentrations of ethanol applied both in vitro and in vivo prevents LTP(GABA), which is reversed, respectively, by in vitro and in vivo administration of naloxone, a mu-opioid receptor (MOR) antagonist. Furthermore, the blockade of LTP(GABA) induced by a brief in vitro ethanol treatment is mimicked by DAMGO ([D-Ala(2), N-MePhe(4), Gly-ol]-enkephalin), a MOR agonist. Paired-pulse ratios are similar in slices, 24 h after in vivo injection with either saline or ethanol. Sp-cAMPS, a stable cAMP analog, and pCPT-cGMP, a cGMP analog, potentiates GABA(A)-mediated inhibitory postsynaptic currents in slices from ethanol-treated rats, indicating that a single in vivo ethanol exposure does not maximally increase GABA release, instead, ethanol produces a long-lasting inability to generate LTP(GABA). These neuroadaptations to ethanol might contribute to early stage of addiction.

Figure 1

In vitro exposure to ethanol blocks LTP_GABA in VTA dopamine neurons involving μ-opioid receptors. (a) LTP_GABA in a dopamine neuron using whole-cell recording methods. High-frequency stimulation (HFS) was delivered at the point specified by the arrow. Inset: averaged IPSCs before (gray) and 20 min after HFS (black). In this and all other figures, 10 consecutive IPSCs from each condition were averaged for illustration. The dashed line in this and other figures is an extrapolation of the mean response before HFS. (b) Bath-applied ethanol blocks LTP_GABA. After recording the baseline IPSCs in normal ACSF for 10 min, ethanol (EtOH, 40 mM) was bath applied for 15 min and then washed out (for 6 min) before the delivery of HFS. Inset: averaged IPSCs before (gray) and 20 min after HFS (black). (c) Average of 11 experiments from dopamine neurons (Control LTP_GABA, open circles; ethanol cells, filled circles). (d) Normalized PPR at 20 min after HFS under a variety of experimental conditions. The PPR at 20 min was normalized to the pre-HFS PPR values. (^*P<0.05, Student's t-test for PPR after HFS compared with pre-HFS values). (e) Bath-applied naloxone blocked the inhibition of LTP_GABA induced by bath-applied EtOH. After recording the baseline IPSCs in normal ACSF for 10 min, naloxone (5 μM) was bath applied for 5 min before the application of the mixture containing naloxone (5 μM) and ethanol (40 mM) for 15 min. HFS was delivered 6 min after washout of the mixture (at the arrow). Inset: averaged IPSCs before (gray) and 20 min after HFS (black). (f) Average of 9 experiments from dopamine neurons (Naloxone+EtOH, open circles; EtOH alone, filled circles). (g) Bath-applied DAMGO blocks LTP_GABA. After recording the baseline IPSCs in normal ACSF for 10 min, DAMGO (1 μM) was bath applied for 15 min and then washed out (for 6 min) before the delivery of HFS. Inset: averaged IPSCs before (gray) and 20 min after HFS (black). (h) Average of six experiments from dopamine neurons.

Figure 2

In vivo exposure to ethanol blocks LTP_GABA involving the activation of MORs. (a) LTP_GABA was blocked in dopamine neurons in slices prepared 24 h after a single in vivo ethanol exposure. Insets: averaged IPSCs before (gray) and 20 min after HFS (black). (b) Average of six experiments from slices prepared 24 h after either saline (open circles) or ethanol (filled circles) treatment. (c) In vivo exposure to naloxone prevents in vivo ethanol-induced blockade of LTP_GABA in slices prepared 24 h later. Insets: averaged IPSCs before (gray) and 20 min after HFS (black). (d) Average of six experiments from slices prepared 24 h after ethanol (filled circles) or naloxone+ethanol (open circles) injection. (e) Bar chart illustrating the magnitude of LTP_GABA 20 min after HFS in various experimental conditions (^*P<0.05 compared with pre-HFS values). (f) Normalized Paired-pulse ratio (PPR) under a variety of experimental conditions (^*P<0.05 compared with pre-HFS values). (g) PPRs are similar in slices 24 h after in vivo injection with either saline (open circles, n=14) or ethanol (filled circles, n=14) treatment. Each symbol represents the PPR value for one animal (mean basal PPR values (black horizontal bars) from saline-treated rats: 1.20±0.05, n=14; ethanol-treated rats: 1.09±0.05, n=14, P=0.14).

Figure 3

Sp-cAMPS and pCPT-cGMP further potentiate GABAergic IPSCs in slices prepared 24 h after in vivo ethanol treatment (2 g/kg, i.p.). (a) Bath-applied Sp-cAMPS (20 μM) increased the amplitude and reduced the paired-pulse ratio (PPR) of GABAergic IPSCs on dopamine neurons in slices from ethanol-treated rats. Insets: averaged IPSCs before (gray) and 20 min after Sp-cAMPS (black). (b) Average of 16 experiments from slices prepared 24 h after ethanol treatment. Bath-applied Sp-cAMPS (20 μM) significantly potentiated GABAergic IPSCs (P<0.05). (c) Bath-applied pCPT-cGMP (100 μM) increased the amplitude and reduced the PPR of GABAergic IPSCs on dopamine neurons in slices from ethanol-treated rats. Insets: averaged IPSCs before (gray) and 20 min after pCPT-cGMP (black) treatment. (d) Average of eight experiments from slices prepared 24 h after ethanol treatment. Bath-applied pCPT-cGMP (100 μM) increased the amplitude of GABAergic IPSCs. (e) The bar chart shows that at 20 min after the application of Sp-cAMPS, or pCPT-cGMP, the amplitude of IPSCs recorded from slices prepared 24 h after ethanol treatment was significantly increased (^*P<0.05, compared with baseline before application of Sp-cAMPS, n=16, or pCPT-cGMP, n=8). (f) At 20 min after application of Sp-cAMPS, or pCPT-cGMP, the PPR of IPSCs recorded from slices prepared 24 h after ethanol treatment was significantly increased (^*P<0.05, compared with baseline before application of Sp-cAMPS, n=16, or pCPT-cGMP, n=8).