Role of MyD88 in IL-1β and Ethanol Modulation of GABAergic Transmission in the Central Amygdala

Abstract

Myeloid differentiation primary response protein (MyD88) is a critical neuroimmune adaptor protein in TLR (Toll-like receptor) and IL-1R (Interleukin-1 receptor) signaling complexes. These two pro-inflammatory families play an important role in the neurobiology of alcohol use disorder, specifically MyD88 regulates ethanol drinking, ethanol-induced sedation, and ethanol-induced deficits in motor coordination. In this study, we examined the role of MyD88 in mediating the effects of IL-1β and ethanol on GABAergic transmission in the central amygdala (CeA) of male mice using whole-cell patch-clamp recordings in combination with pharmacological (AS-1, a mimetic that prevents MyD88 recruitment by IL-1R) and genetic (Myd88 knockout mice) approaches. We demonstrate through both approaches that IL-1β and ethanol's modulatory effects at CeA GABA synapses are not dependent on MyD88. Myd88 knockout potentiated IL-1β's actions in reducing postsynaptic GABA_A receptor function. Pharmacological inhibition of MyD88 modulates IL-1β's action at CeA GABA synapses similar to Myd88 knockout mice. Additionally, ethanol-induced CeA GABA release was greater in Myd88 knockout mice compared to wildtype controls. Thus, MyD88 is not essential to IL-1β or ethanol regulation of CeA GABA synapses but plays a role in modulating the magnitude of their effects, which may be a potential mechanism by which it regulates ethanol-related behaviors.

Keywords: GABA; IL-1R1; Myd88 knockout; alcohol; interleukin-1; neuroimmune; sIPSC.

Figure 1

Basal spontaneous GABAergic transmission is similar in the CeA of Myd88 KO and wildtype (WT) mice. (A) Representative traces of sIPSCs from WT and Myd88 KO mice. (B–E) There were no significant differences in the basal sIPSC frequencies (B), amplitudes (C), rise times (D), and decay times (E) of CeA neurons from WT (n = 41 neurons) and Myd88 KO (n = 45 neurons) mice. The scattergrams represent values for each cell. Statistical significance was calculated by an unpaired t-test, and significance was set at p < 0.05.

Figure 2

MyD88 deletion dampens IL-1β’s effects on postsynaptic GABA_A receptor function. (A–B) IL-1β had dual effects on the sIPSC frequencies (A) and amplitudes (B) in both WT and KO mice. The scattergrams on the left show the normalized effects of IL-1β (50 ng/mL) in individual cells (WT: n = 13 cells; KO: n = 15 cells), and the right panels show the percentage of the CeA neurons responding to IL-1β with an increase, no change or decrease in the sIPSC frequencies and amplitudes. (C) Representative traces of CeA neurons responding to IL-1β with decreased sIPSC frequencies and amplitudes. (D–E) While there were no differences in the predominant effect of IL-1β on the mean sIPSC frequency in WT (n = 11 cells) and KO (n = 13 cells) mice (D), Myd88 KO mice showed an IL-1β-induced decrease in the sIPSC amplitude (E). The statistical significance for the IL-1β effects was calculated by one-sample t-test (** p < 0.01, and *** p < 0.001), and for the comparison of the IL-1β effects between the WT and KO mice, an unpaired t-test was used (# p < 0.05).

Figure 3

The effects of the MyD88 mimetic (AS-1) in WT mice. (A–B) The scattergrams represent the responses of individual CeA cells to the acute application of the AS-1 (50 mM) and subsequent co-application of IL-1β, where panel A shows the effects of AS-1 on the sIPSC frequencies (WT: n = 7 cells), and panel B shows its effects on the sIPSC amplitude. (C–D). In five out of six WT neurons pretreated with AS-1, there was an IL-1β-induced decrease in the mean sIPSC frequency, and the magnitude of this effect was not significantly different from the IL-1β-induced decrease in the Myd88 KO mice (data from Figure 2D). (D) IL-1β in the presence of AS-1 had no significant effects on the mean sIPSC amplitudes of WT cells, and the extent of this effect was not significantly different from the IL-1β’s effects in the Myd88 KO mice (data from Figure 2E). The statistical significance for the AS-1 and IL-1β effects was calculated by one-sample t-test (** p < 0.01 and *** p < 0.0001), and for the comparison of the IL-1β’s effects between the WT, following AS-1 pretreatment, and KO mice, an unpaired t-test was used.

Figure 4

Facilitation of the presynaptic GABA release by 100 mM ethanol is more robust in Myd88 KO than in the WT mice. (A,B). Ethanol (44 mM) facilitated presynaptic GABA release across both genotypes, as represented by the increase in the mean sIPSC frequencies in 10 out of 14 neurons from WT and 7 out of 16 cells from KO mice. (C) Representative recordings of the CeA neurons responding to 44 mM ethanol with the increase in the sIPSC frequencies. (D) There was no significant difference in the magnitude of the ethanol-induced potentiation of the GABA release between the WT and KO mice. (E) Representative traces of the CeA neurons responsive to 100 mM ethanol. (F) The magnitude of the 100 mM ethanol-induced potentiation of the GABA release was significantly stronger in the KO mice (n = 4 out of 5 cells) compared to the WT mice (n = 5 out of 5 cells) mice. The statistical significance for the ethanol effects was calculated by one-sample t-test (* p < 0.05, ** p < 0.01, and *** p < 0.001), and an unpaired t-test (# p < 0.05) was used for the comparison of the magnitude of the ethanol effects between the WT and KO mice.