Involvement of delta opioid receptors in alcohol withdrawal-induced mechanical allodynia in male C57BL/6 mice

Abstract

Background: As a legal drug, alcohol is commonly abused and it is estimated that 17 million adults in the United States suffer from alcohol use disorder. Heavy alcoholics can experience withdrawal symptoms including anxiety and mechanical allodynia that can facilitate relapse. The molecular mechanisms underlying this phenomenon are not well understood, which stifles development of new therapeutics. Here we investigate whether delta opioid receptors (DORs) play an active role in alcohol withdrawal-induced mechanical allodynia (AWiMA) and if DOR agonists may provide analgesic relief from AWiMA.

Methods: To study AWiMA, adult male wild-type and DOR knockout C57BL/6 mice were exposed to alcohol by a voluntary drinking model or oral gavage exposure model, which we developed and validated here. We also used the DOR-selective agonist TAN-67 and antagonist naltrindole to examine the involvement of DORs in AWiMA, which was measured using a von Frey model of mechanical allodynia.

Results: We created a robust model of alcohol withdrawal-induced anxiety and mechanical allodynia by orally gavaging mice with 3g/kg alcohol for three weeks. AWiMA was exacerbated and prolonged in DOR knockout mice as well as by pharmacological blockade of DORs compared to control mice. However, analgesia induced by TAN-67 was attenuated during withdrawal in alcohol-gavaged mice.

Conclusions: DORs appear to play a protective role in the establishment of AWiMA. Our current results indicate that DORs could be targeted to prevent or reduce the development of AWiMA during alcohol use; however, DORs may be a less suitable target to treat AWiMA during active withdrawal.

Keywords: Alcohol use disorder; Allodynia; Delta opioid receptor; G protein-coupled receptor; Hyperalgesia; Withdrawal.

Figure 1. Endogenous activity at delta opioid receptors attenuate alcohol withdrawal-induced mechanical allodynia in a model of voluntary alcohol consumption

Wild-type (WT) or DOR knockout (KO) C57BL/6 mice were trained to drink in a limited-access, two-bottle choice paradigm for three weeks, and consumption of the 10% alcohol solution was measured over a 4-h period (A). The mechanical sensitivity measured by using von Frey filaments was assessed on days 1, 2, 4, 7 and 14 after alcohol withdrawal. Significance between groups was determined by two-way ANOVA (B). The cumulative alcohol withdrawal-induced mechanical allodynia (AWiMA) (expressed in arbitrary units (AU)) in WT and DOR KO mice was calculated using the trapezoidal rule (C). *p < .05 normalized versus day 0 (BL, baseline).

Figure 2. Mice daily gavaged with 3 g/kg alcohol develop induced mechanical allodynia during alcohol withdrawal

Timeline for chronic alcohol consumption and measurement of mechanical nociception of Figure 2B and 2D (A). Wild-type C57BL/6 mice were given either 20% alcohol solution (2 g/kg or 3 g/kg) or water via oral gavage (o.g.) once daily for five consecutive days for a duration of three weeks, and mechanical sensitivity was measured using von Frey filaments. Significance between groups was determined by two-way ANOVA (B). The cumulative alcohol withdrawal-induced mechanical allodynia (AWiMA) (expressed in arbitrary units (AU)) in 2 and 3 g/kg gavaged alcohol mice was calculated using the trapezoidal rule (C). 3 g/kg alcohol-withdrawn mice (24 hours after last exposure) were injected intrathecally (i.t.) with 10 nmol/10 ul of α2A-adrenergic receptor agonist clonidine, a medication used for treatment of alcohol withdrawal symptoms. Ten minutes after injection, mechanical antinociception was measured by using von Frey filaments. Significance was determined by one-way ANOVA (D). ***p < .001; **p < .01 normalized versus water-treated group.

Figure 3. Mice daily gavaged with 3 g/kg alcohol demonstrate anxiolytic-related behaviors during alcohol withdrawal

Wild-type C57BL/6 mice were given 3 g/kg alcohol or water via oral gavage once daily for five consecutive days for a duration of three weeks, and anxiety-like behaviors were measured using an open field exploration test (A-D) and light-dark transition box (E-H). Significance between groups was determined by unpaired t-test; **p < .01; *p < .05.

Figure 4. Endogenous activity at delta opioid receptors attenuate alcohol withdrawal-induced mechanical allodynia in a model of gavaged heavy alcohol intake

Timeline for chronic alcohol consumption and measurement of mechanical nociception of Figure 4B (A). Wild-type (WT) or DOR knockout (KO) C57BL/6 mice were given 3 g/kg alcohol or water via oral gavage (o.g.) once daily for five consecutive days for a duration of three weeks, and mechanical nociceptive sensitivity was examined on days 1, 2, 4, 7, 14, 21 and 28 after alcohol withdrawal using von Frey filaments. Significance between groups was determined by two-way ANOVA (B). The cumulative alcohol withdrawal-induced mechanical allodynia (AWiMA) (expressed in arbitrary units (AU)) in WT and DOR KO mice was calculated using the trapezoidal rule (C). **p < .01 normalized versus water-treated group.

Figure 5. Chronic naltrindole administration prior to each alcohol exposure prolongs duration of alcohol withdrawal-induced mechanical allodynia

The acute subcutaneous (s.c.) injection of the DOR-selective antagonist naltrindole (NTI) dose-dependently caused mechanical allodynia in naïve mice (A). The chronic administration of 2 mg/kg NTI also led to prolonged hyperalgesia in naïve mice (B). Timeline for chronic alcohol consumption and measurement of mechanical nociception of Figure 4B (C). Twenty-two wild-type C57BL/6 mice were divided into three groups (n = 7-8), and were s.c. injected with 0.2 mg/kg NTI or saline. Thirty minutes after injection, each group was given 3 g/kg alcohol or water by oral gavage once daily for five consecutive days/week for two weeks. The mechanical sensitivity of all animals measured using von Frey filaments was examined on days 1, 2, 4, 7, 14, 21, 28, 35 and 49 after alcohol withdrawal (D). The cumulative alcohol withdrawal-induced mechanical allodynia (AWiMA) (expressed in arbitrary units (AU)) was calculated using the trapezoidal rule (E). Data was normalized to the mechanical sensitivity response in saline/water-treated group. **p < .01; *p < .05. BL, baseline.

Figure 6. Potency of the DOR-selective agonist TAN-67 is reduced in mice withdrawn from gavaged alcohol but not volitional alcohol

Naïve or alcohol-withdrawn C57BL/6 mice (n = 9-10) subjected to either voluntary (A) or gavage (B) administration were intrathecally injected with increasing doses of the DOR-selective agonist TAN-67, and mechanical antinociception was measured using von Frey filaments. Data are represented as a percentage of maximum possible effect (% MPE), which is defined as [(measurement − baseline) / (cutoff − baseline)] × 100.