Chronic Intermittent Ethanol Exposure Enhances the Excitability and Synaptic Plasticity of Lateral Orbitofrontal Cortex Neurons and Induces a Tolerance to the Acute Inhibitory Actions of Ethanol

Abstract

Alcoholism is associated with changes in brain reward and control systems, including the prefrontal cortex. In prefrontal areas, the orbitofrontal cortex (OFC) has been suggested to have an important role in the development of alcohol-abuse disorders and studies from this laboratory demonstrate that OFC-mediated behaviors are impaired in alcohol-dependent animals. However, it is not known whether chronic alcohol (ethanol) exposure alters the fundamental properties of OFC neurons. In this study, mice were exposed to repeated cycles of chronic intermittent ethanol (CIE) exposure to induce dependence and whole-cell patch-clamp electrophysiology was used to examine the effects of CIE treatment on lateral OFC (lOFC) neuron excitability, synaptic transmission, and plasticity. Repeated cycles of CIE exposure and withdrawal enhanced current-evoked action potential (AP) spiking and this was accompanied by a reduction in the after-hyperpolarization and a decrease in the functional activity of SK channels. CIE mice also showed an increase in the AMPA/NMDA ratio, and this was associated with an increase in GluA1/GluA2 AMPA receptor expression and a decrease in GluN2B NMDA receptor subunits. Following CIE treatment, lOFC neurons displayed a persistent long-term potentiation of glutamatergic synaptic transmission following a spike-timing-dependent protocol. Lastly, CIE treatment diminished the inhibitory effect of acute ethanol on AP spiking of lOFC neurons and reduced expression of the GlyT1 transporter. Taken together, these results suggest that chronic exposure to ethanol leads to enhanced intrinsic excitability and glutamatergic synaptic signaling of lOFC neurons. These alterations may contribute to the impairment of OFC-dependent behaviors in alcohol-dependent individuals.

Figure 1

Chronic intermittent ethanol (CIE) exposure enhances current-induced spiking in lateral orbitofrontal cortex (lOFC) neurons and decreases the magnitude of the after-hyperpolarization (AHP). (a) Representative traces showing enhanced action potential spiking in all CIE-exposed groups as compared with air-treated controls. (b) Number (mean±SEM) of spikes from lOFC neurons plotted against a series of current injections (40–160 pA). In comparison with air control (n=12), basal firing rates of lOFC neurons were significantly enhanced in 3-day withdrawal (3D-WD; n=11), 7-day WD (7D-WD; n=14), and 10-day WD (10D-WD; n=11) CIE groups (two-way analysis of variance (ANOVA): main effect of CIE, F_{(3, 308)}=27.96, p<0.0001; post-hoc comparison; air vs CIE treated; ***p<0.0001). (c) Effects of CIE treatment on AHP. CIE induced a significant reduction in AHP amplitude (two-way ANOVA: main effect of CIE, F_{(3, 75)}=6.70, p<0.0005; post-hoc comparison, air vs CIE treated, **p<0.01) at 3D-WD, but not at the 7-day and 10-day time points.

Figure 2

Chronic intermittent ethanol (CIE) exposure reduces the amplitude of apamin-sensitive tail currents without changing small conductance calcium-activated potassium (SK) channel subunit expression. (a) Representative traces showing a decrease in total tail current amplitudes in CIE-exposed mice. In addition, shown are traces in the presence of the SK channel blocker apamin showing reduction in SK currents in CIE-exposed animals. (b) Comparison of the peak tail current amplitudes (mean±SEM) in response to depolarizing voltage steps (400 ms, from −20 to +30 mV with 10 mV between steps). There was a significant main effect of CIE treatment on total tail current amplitude (two-way analysis of variance (ANOVA): F_{(2, 150)}=34.76, p<0.0001) with amplitudes from both the 3-day withdrawal (WD; q=8.31, ****p<0.0001; n=12) and 7-day WD (q=3.93; ***p<0.001; n=5) CIE groups being significantly different from the control (n=11). The SK channel blocker apamin (100 nM) significantly reduced tail current amplitudes detected in lateral orbitofrontal cortex (lOFC) neurons from air control mice (c; two-way ANOVA: F_{(1, 72)}=33.76, ****p<0.0001; n=7) but did not significantly change the tail current amplitude in 3-day (d; two-way ANOVA: F_{(1, 36)}=3.217, p=0.0813; n=4) and 7-day (e; two-way ANOVA: F_{(1, 36)}=2.845, p=0.1003; n=4) WD groups. (f) Representative western blottings and optical density analysis of SK2 and SK3 subunits in lOFC neurons from air- and CIE-exposed mice. In comparison with air controls, there were no significant changes in SK2 or SK3 protein expression in CIE-treated mice at the 3-day WD time point (SK2 t-test, t(9)=0.51, p=0.62; SK3 t-test, t(20)=0.70, p=0.49).

Figure 3

Chronic intermittent ethanol (CIE) exposure enhances the AMPA/NMDA excitatory postsynaptic current (EPSC) ratio and alters glutamatergic receptor subunit expression. (a) Representative traces demonstrating stimulus-evoked AMPA and NMDA EPSCs in lateral orbitofrontal cortex (lOFC) neurons from air- and CIE-exposed mice. CIE exposure produced a trend toward an increase in stimulus-evoked AMPA EPSCs (b; mean±SEM) and a decrease in stimulus-evoked NMDA EPSCs (c), but these changes were not statistically different from the control. (d) In comparison with air control mice (n=16), CIE exposure produced a significant increase in AMPA/NMDA ratio at the 3-day withdrawal (WD) time point (one-way analysis of variance (ANOVA): F_{(2, 38)}=12.73, ****p<0.0001; n=14). (e) Representative western blottings and optical density (mean±SEM) of AMPA and NMDA receptor subunits in lOFC neurons from air (n=6) and 3-day WD (n=5) groups. A significant increase in AMPA GluA1 (t(9)=2.69, *p=0.03) and GluA2 (t(8)=5.00, *p=0.001) subunit expression was observed in CIE-exposed mice. CIE induced a significant decrease in NMDA GluN2B subunit expression (t(8)=2.34, *p=0.047). There were no differences in expression of NMDA GluN1 and GluN2A subunits between control and CIE-exposed groups.

Figure 4

Chronic intermittent ethanol (CIE) exposure increases the amplitude, but not frequency (expressed as mean of the inter-event interval), of spontaneous excitatory postsynaptic currents (sEPSCs) and inhibitory postsynaptic currents (IPSCs) in lateral orbitofrontal cortex (lOFC) neurons. Representative traces of sEPSCs (top) and IPSCs (middle) from air- and CIE-treated (3D-WD) neurons are shown. Calibration bars (x=50 pA; y=0.5 s). A significant increase in amplitude (a; mean±SEM) but not frequency (b; expressed as inter-event interval) of AMPA-mediated sEPSCs was observed in CIE-exposed mice (one-way analysis of variance (ANOVA): F_{(3, 39)}=2.889, p=0.048) and pairwise comparisons to control revealed significant increases at the 3-day (q=2.46, *p<0.05; n=17) and 10-day (q=2.55, **p<0.05; n=12) withdrawal (WD) time points. Increases in amplitude (c) but not frequency (d; expressed as inter-event interval) of sIPSCs were detected in 7-day (q=3.74, ** p<0.01; n=20) and 10-day (q=3.299, **p<0.01) WD animals. A trend toward an increase in the amplitude (e) but not frequency (f; expressed as inter-event interval) of miniature EPSCs (mEPSCs) was observed in CIE mice (one-way ANOVA: F_{(2, 18)}=2.858, p=0.08), but pairwise comparisons revealed no significant differences between groups.

Figure 5

Chronic intermittent ethanol exposure (CIE) enhances the duration of glutamatergic synaptic plasticity induced by a spike-timing-dependent plasticity (STDP) protocol. (a) Representative traces showing stimulus-evoked AMPA EPSPs before (left) and after (20–30 min; middle; 50–60 min; right) spike-timing protocol. Calibration bars (x=20 ms; y=5 mV). Graphs show amplitude of mean EPSPs (SEM bars not shown for clarity) before and after STDP induction (arrow) for air- (open circles, n=7) and CIE-treated mice at 3-day (closed circles, n=10) and 7-day (closed triangles, n=9) after withdrawal (WD). (b) Significant increases in EPSP amplitude (values normalized to pre-pairing baseline; mean±SEM) at 20–30 min post pairing were observed in both control (one sample t-test vs 100%, t(7)=4.662, **p=0.0023) and CIE-exposed mice (one sample t-test vs 100%, 3-day CIE, t(9)=8.678, ***p<0.0001; 7-day CIE, t(8)=5.675, **p=0.0005) with no difference in the magnitude of this effect between groups. At 50–60 min post pairing, the increase in EPSP amplitude in CIE-exposed mice persisted (one sample t-test vs 100%, 3-day CIE, t(9)=5.642, ***p=0.0003; 7-day CIE, t(8)=5.258, ***p=0.0008) and these values were significantly greater than the air control (one-way analysis of variance: F_{(2, 48)}=6.808, p=0.0025; post-hoc comparison 3-day CIE, q=4.594, ****p<0.0001; 7-day CIE, q=4.286, ***p<0.001). The amplitude of EPSP at 50–60 min post pairing in air-exposed group returned to control levels that were not different from the pre-STDP baseline (one sample t-test, t(7)=0.5634, p=0.5907).

Figure 6

Chronic intermittent ethanol (CIE) exposure suppresses the ability of acute ethanol to inhibit spike firing of lateral orbitofrontal cortex (lOFC) neurons. Representative traces show current-evoked spiking in the absence and presence of 33 mM ethanol applied to lOFC neurons from air control (left) and CIE-treated (right) mice. Graphs show summary of the effect of acute ethanol (33 mM, 10 min) on action potential (AP) spiking (mean±SEM) induced by a series of current injections (40–220 pA) in lOFC neurons obtained from air control (a, n=8) and CIE-treated mice at 3-day (b, n=7), 7-day (c, n=6), and 10-days (d, n=7) of withdrawal (WD). Similar to the results shown in Figure 1b, WD from CIE exposure produced an increase in AP firing (note different y axes between a and b–e). Acute ethanol significantly reduced spiking in lOFC neurons obtained from air control (a; two-way analysis of variance (ANOVA): F_{(1, 7)}=6.66, *p=0.036), but did not significantly change AP firing in 3-day (b; two-way ANOVA: F_{(1, 6)}=1.14, p=0.327), 7-day (c; two-way ANOVA: F_{(1, 5)}=3.68, p=0.113) or 10-day (d; two-way ANOVA: F_{(1, 6)}=0.583, p=0.474) CIE groups.

Figure 7

Chronic intermittent ethanol (CIE) exposure does not alter basal tonic currents mediated by GABA_A and glycine receptors in lateral orbitofrontal cortex (lOFC) neurons but blunts the ethanol-induced increase in holding current. Graphs show individual values and mean (±SEM) changes in tonic current shift from baseline following application of gabazine (GABA_A currents; a) or gabazine plus strychnine (Glycine currents; b) in lOFC neurons obtained from air- (n=13) and CIE-treated mice at 3-day (n=12) and 7-day (n=6) after withdrawal (WD). No significant differences in the magnitude of basal tonic currents were found between air control and CIE groups (gabazine; one-way repeated-measures analysis of variance (ANOVA): F_{(2, 28)}=0.115, p>0.05; gabazine plus strychnine, one-way repeated-measures ANOVA: F_{(2, 30)}=0.3, p>0.05). (c) Representative western blottings and optical density (mean±SEM) of glycine receptor α-subunits in lOFC neurons from air and 3-day WD groups. There was no difference in expression of glycine receptors between air- and CIE-exposed groups (two-tailed t-test, t(8)=1.459, p=0.17). (d) Mean changes in holding current in response to locally applied ethanol (33 and 66 mM) in lOFC neurons from air- (n=12) and CIE-treated mice 3 days (n=6) and 7 days (n=7) after WD. Ethanol induced a significant increase in holding current in control (one-sample t-test, t=4.57, *p<0.0008) but not CIE-treated neurons.

Figure 8

Chronic intermittent ethanol (CIE) exposure suppresses the ability of the GlyT1 transport inhibitor sarcosine to reduce spiking in lateral orbitofrontal cortex (lOFC) neurons. Representative traces showing current-evoked spike firing in lOFC neurons from air-treated (left panel) or CIE-exposed mice (right panel) in the absence and presence of sarcosine (100 μM). Summary graphs show inhibitory effect of sarcosine (100 μM) on action potential spiking (mean±SEM) induced by a series of current injections (40–220 pA) in lOFC neurons obtained from air control (a, n=10, two-way analysis of variance (ANOVA): F_{(1, 9)}=9.285, *p=0.0139) but not CIE-treated mice tested at 3 (b, n=6) and 7 days (c, n=7). (d) CIE exposure reduced the expression of the GlyT1 transporter as measured by western blotting. Data are mean (±SEM) of normalized optical density of GlyT1 immunoreactivity from air- (n=5) and CIE-treated mice withdrawn for 3 days (n=6). Two-way ANOVA shows significant effect of CIE treatment on GlyT1 expression (F_{(1, 18)}=5.63, *p=0.029) in pellet (P2) and supernatant (S2) fractions.