Innate immune factors modulate ethanol interaction with GABAergic transmission in mouse central amygdala

Abstract

Excessive ethanol drinking in rodent models may involve activation of the innate immune system, especially toll-like receptor 4 (TLR4) signaling pathways. We used intracellular recording of evoked GABAergic inhibitory postsynaptic potentials (eIPSPs) in central amygdala (CeA) neurons to examine the role of TLR4 activation by lipopolysaccharide (LPS) and deletion of its adapter protein CD14 in acute ethanol effects on the GABAergic system. Ethanol (44, 66 or 100mM) and LPS (25 and 50μg/ml) both augmented eIPSPs in CeA of wild type (WT) mice. Ethanol (44mM) decreased paired-pulse facilitation (PPF), suggesting a presynaptic mechanism of action. Acute LPS (25μg/ml) had no effect on PPF and significantly increased the mean miniature IPSC amplitude, indicating a postsynaptic mechanism of action. Acute LPS pre-treatment potentiated ethanol (44mM) effects on eIPSPs in WT mice and restored ethanol's augmenting effects on the eIPSP amplitude in CD14 knockout (CD14 KO) mice. Both the LPS and ethanol (44-66mM) augmentation of eIPSPs was diminished significantly in most CeA neurons of CD14 KO mice; however, ethanol at the highest concentration tested (100mM) still increased eIPSP amplitudes. By contrast, ethanol pre-treatment occluded LPS augmentation of eIPSPs in WT mice and had no significant effect in CD14 KO mice. Furthermore, (+)-naloxone, a TLR4-MD-2 complex inhibitor, blocked LPS effects on eIPSPs in WT mice and delayed the ethanol-induced potentiation of GABAergic transmission. In CeA neurons of CD14 KO mice, (+)-naloxone alone diminished eIPSPs, and subsequent co-application of 100mM ethanol restored the eIPSPs to baseline levels. In summary, our results indicate that TLR4 and CD14 signaling play an important role in the acute ethanol effects on GABAergic transmission in the CeA and support the idea that CD14 and TLR4 may be therapeutic targets for treatment of alcohol abuse.

Keywords: Alcohol; Drug abuse; Extended amygdala; Inflammation; LPS; Neuroimmune; Toll-like receptor.

Figure 1. Ethanol concentration-response analysis in CeA of WT control and CD14 KO mice

A) Representative traces from intracellular recordings of evoked GABA_A-IPSPs (eIPSPs) in CeA neurons from control WT (top panel) and CD14 KO mice (bottom panel). B) Ethanol at concentrations of 44 (n = 12), 66 (n = 8) and 100 mM (n = 4) significantly increased mean eIPSP amplitudes in WT mice. In CD14 KO mice, with superfusion of 44 mM (n = 12), 66 mM (n = 12) and 100 mM (n = 8), only the supramaximal (in WT mice) ethanol concentration of 100 mM increased mean eIPSP amplitudes. C) Ethanol effects on paired-pulse facilitation (PPF) in CeA of WT and CD 14 KO mice. Values represent mean ± SEM, and statistical significance (* and ^) was set at p < 0.05. (^) was calculated by ANCOVA and (*) by Bonferroni post-hoc test. Note that only the 44 mM ethanol concentration in WT CeA significantly affected PPF, suggesting a presynaptic increase in GABA release in this case.

Figure 2. A possible role for TLR4 in ethanol effects on GABAergic transmission

A) Time-course of effects of (+)-naloxone (10 µM) pre-treatment on ethanol-induced increases in eIPSP amplitudes in WT and CD 14 KO mice. Ethanol 44 mM, a maximally effective concentration in controls, was added to the (+)-naloxone superfusing CeA slices from WT mice (see bars, left panel), and 100 mM ethanol (maximal in CD14 CeA) added to the CeA slices of CD14 KO mice (right panel). Time points represent 4 min bins of mean evoked IPSP amplitudes following (+)-naloxone, (+)-naloxone+ethanol, and washout. B) There was no effect of (+)-naloxone alone on the mean amplitudes of eIPSPs in CeA of WT mice (n = 6), but (+)-naloxone delayed and weakened the ethanol potentiation of the mean amplitude of eIPSPs (left panel) compared to untreated slices (see Figure 1B). (+)-naloxone alone significantly decreased the mean eIPSP amplitudes in CeA slices of CD14 KO (n = 7) mice, but did not prevent the 100 mM ethanol augmentation of eIPSP amplitudes back to baseline levels (right panel). Statistics: *compares effects of (+)-naloxone and (+)-naloxone+ethanol to baseline; p < 0.05. # indicates significant differences between effects of (+)-naloxone and (+)-naloxone+ethanol on eIPSPs with p < 0.05.

Figure 3. LPS augments eIPSPs in WT CeA: concentration-response analysis

A) Representative recordings from CeA neurons of WT and CD 14 KO mice following 25 µg/ml LPS superfusion. B) LPS at 25 (n = 8) and 50 (n = 6), but not 10 µg/ml (n = 6) significantly increased mean amplitudes of eIPSPs in WT mice, whereas the tested concentrations were ineffective in CeA of CD14 KO mice (25 µg/ml: n = 13; 50 µg/ml: n = 8). D) LPS had no significant effect on the PPF ratios in CeA of WT or CD 14 KO mice. Statistical significance was set at ^ p < 0.05, and values correspond to mean ± SEM.

Figure 4. LPS effects on mIPSCs suggest postsynaptic mechanisms of action

A) Representative whole-cell current recording of mini IPSCs in a mouse CeA neuron (Vh = −60 mV) superfused with LPS (25 µg/ml). B) Cumulative fractions calculated by Kolmogorov-Smirnov sample test show that LPS did not change mIPSC frequency fractions. C) However LPS significantly shifted the amplitude fractions to the right, representing an increase in amplitude (p < 0.05). D) Summed data: LPS had no significant effect on mean GABAergic mIPSC frequency, but E) increased the mean amplitude of mIPSCs . Statistical significance * was set at p < 0.05 and calculated by Student’s t-test.

Figure 5. LPS effects on eIPSPs are likely mediated by the TLR4-MD-2 complex

A) Representative multiple overlapping recordings from an individual CeA neuron from WT mouse following superfusion of ACSF only (baseline), (+)-naloxone (10 µM) and co-application of (+)-naloxone and LPS (25 µg/ml). B) Summed data: (+)-naloxone (10 µM), a selective TLR4 inhibitor, had no effect on mean eIPSP amplitudes but blocked the usual (compare to Figure 3A and B) 25 µg/ml LPS-induced increase in mean amplitudes of eIPSPs in WT mice (n = 6). C) Representative traces of PPF from an individual neuron, showing no change after LPS. D) (+)-naloxone and subsequent co-application of LPS did not alter PPF ratio significantly. Dashed horizontal lines represent an average of 4 minutes of recorded baseline

Figure 6. LPS and ethanol interactions in CeA of WT and CD14 KO mice

A) Time-course of the influence of LPS (25 µg/ml) pre-treatment on ethanol (44 mM) effects on evoked GABA_A-IPSPs in CeA neurons from WT (n = 7) and CD14 KO (n = 13) mice. Statistical significance: *represents p < 0.05 and was calculated by one-sample t-test. B) Summed data showing that LPS alone increased mean eIPSPs and potentiated the 44 mM ethanol-induced increase in mean eIPSP amplitudes in WT mice and restored ethanol effects in the CeA of CD 14 KO mice, without showing a direct of LPS. Statistical significance: *LPS pre-treatment and LPS+ethanol co-application effects compared to baseline; #LPS+ethanol effects compared to LPS pre-treatment and washout. *represents p < 0.05, and **is p < 0.01. C) Time-course of the effects of ethanol pre-treatment and ethanol+LPS co-application on eIPSPs, showing a robust increase in eIPSP amplitudes by ethanol followed by an apparent occlusive effect on LPS action in WT mice; both effects are abolished in the CD 14 KO. Statistical significance *represents p < 0.05. D) Summed data: ethanol 44 mM pre-treatment occluded LPS-induced increases in evoked IPSP amplitudes in WT (n = 8) mice but neither ethanol nor ethanol plus LPS had any effect in CD14 KO (n = 10) mice. Statistical significance: * and ** are p < 0.05 and p < 0.01, respectively; effects compared to baseline; # and ^ are p < 0.05 and represent differences in the effects of ethanol and ethanol+LPS application compared to washout.