Xylazine reduces methamphetamine-induced hyperlocomotion in rats through κ-opioid and α2-adrenoceptor mechanisms

1. Introduction

Xylazine is an α2-adrenoreceptor agonist that acts at peripheral nerve terminals to cause bradycardia, hypotension, and sedation and at presynaptic CNS terminals to decrease norepinephrine release. Recently, xylazine, unlike other α2-adrenoreceptor agonists, was shown to be a full agonist at κ-opioid receptors and competitive antagonist at α7-nicotinic acetylcholine receptors [1, 2]. For the combination of xylazine with illicit substances, most information is related to opioids and the ‘tranq dope’ threat. In human users, the addition of xylazine to fentanyl enhances and prolongs euphoria and causes hypothermia and brain hypoxia that exacerbates fentanyl-induced respiratory depression and mortality [3]. In rodents, xylazine also worsens opioid-induced respiratory depression [4, 5] but does not enhance rewarding and motivational effects of opioids [6-10].

Xylazine misuse with methamphetamine (METH) is also increasing. In a recent toxicology study, xylazine was detected in 34% of human METH samples [11]. Additionally, in an analysis of drug overdose deaths in West Virginia, METH was the most common co-intoxicant among xylazine-positive decedents, second only to fentanyl [12]. Only one study (1994) has examined xylazine's effects on amphetamine, finding that 30 mg/kg attenuated amphetamine-induced seizures in rats [13]. Given the rising co-use of xylazine and METH and limited data on their interaction [10, 12], we examined how xylazine affects METH-related behaviors in rats. We selected the endpoint of hyperlocomotion, a hallmark behavioral effect of METH in rats. Because xylazine is a full κ-opioid receptor agonist [1], we assessed whether its effects on METH-evoked hyperlocomotion involve α2-adrenoceptors and κ-opioid receptors.

2. Methods

3. Results

4. Discussion

Xylazine caused inhibition of METH-induced hyperlocomotion that was sensitive to κ-opioid receptor and α2-adrenoceptor antagonists. Yohimbine abolished the inhibitory effects of xylazine on METH-evoked hyperlocomotion, suggesting that an α2-adrenoceptor mechanism facilitated the xylazine-METH interaction. METH causes hyperlocomotion in part by increasing central and peripheral levels of norepinephrine and enhancing sympathetic activity. Clonidine, an α2-adrenoceptor agonist and FDA-approved antihypertensive drug, reduces hyperlocomotion caused by acute METH or cocaine and inhibits cocaine-induced behavioral sensitization in rats [16]. In contrast, α2-adrenoceptor antagonists such as yohimbine and idazoxan enhance cocaine-induced hyperlocomotion in rats [17]. These results indicate that hyperlocomotion induced by psychostimulant exposure is inhibited by α2-adrenoceptor activation and enhanced by α2-adrenoceptor inhibition. The downstream mechanisms following α2-adrenoceptor activation that mediate xylazine's inhibition of METH-evoked hyperlocomotion remain unclear. Xylazine may have activated α2-adrenoceptor autoreceptors in the CNS to reduce sympathetic output, thereby dampening METH-induced hyperactivity. Another possibility is that xylazine, by activating α2-adrenoceptors on dopamine terminals, counteracted a METH-induced elevation of extracellular dopamine in the nucleus accumbens (NAC) and striatum that facilitated locomotor activation. α2-adrenoceptor agonists do reduce dopamine release from presynaptic terminals in slices from the rat NAC and rabbit caudate nucleus [18].

LY2456302, in a delayed manner, attenuated inhibitory effects of xylazine on METH-evoked hyperlocomotion, suggesting κ-opioid receptor involvement. Xylazine is a full agonist at κ-opioid receptors with a binding affinity of 0.47 μM [1]. Xylazine is also highly lipophilic and readily penetrates the brain, reaching brain concentrations greater than 4 μM for at least 2 h after a 10 mg/kg IP injection in rats [19]. These binding data and pharmacokinetic results indicate that xylazine, at the dose range (1-5 mg/kg, IP) used in our study, attains brain concentrations required to activate κ-opioid receptors. Previous work shows that U50,488H, a κ-opioid agonist, attenuates METH-induced hyperlocomotion in rats and reduces METH-induced circling behavior in mice [20]. κ-opioid receptors are also expressed on dopaminergic neurons in the VTA and terminals in the NAC [21], and K-opioid receptor agonists reduce stimulant-induced extracellular dopamine levels in the NAC of rats [22].

In summary, we identified xylazine-METH interactions in rats by showing that the acute locomotor stimulant responses to METH involve κ-opioid and α2-adrenoceptors. The greater efficacy of adrenergic antagonism throughout the entire 90 min following xylazine and METH co-administration, together with a more delayed effect of κ-opioid receptor blockade, suggest temporally distinct contributions of adrenergic and kappa opioid signaling. Since we have now established a functional xylazine-METH interaction, future studies with greater translational relevance will explore effects of xylazine on METH reward and seeking using conditioned place preference and self-administration assays.