Mitragynine (Kratom)-Induced Cognitive Impairments in Mice Resemble Δ9-THC and Morphine Effects: Reversal by Cannabinoid CB1 Receptor Antagonism

Abstract

Kratom is a widely abused plant-based drug preparation with a global interest in recent years, well beyond its native grounds in Southeast Asia. Mitragynine, its major psychoactive constituent is known to exhibit opioid-like behavioral effects with resultant neuroplasticity in the brain reward system. Its chronic administration is associated with cognitive impairments in animal studies. However, the underlying molecular mechanism for such a deficit remains elusive. In this study, the involvement of cannabinoid type-1 (CB₁) receptors in cognitive deficits after chronic mitragynine exposures was investigated for 28 days (with incremental dose sensitization from 1 to 25 mg/kg) in adult male Swiss albino mice using the IntelliCage^® system. Chronic high-dose mitragynine exposure (5-25 mg/kg, intraperitoneal [i.p.]), but not low-dose exposure (1-4 mg/kg, i.p.), induced hyperlocomotion, potentiated the preference for sucrose reward, increased resistance to punishment, and impaired place learning and its reversal. Comparable deficits were also observed after chronic treatments with Δ-9-tetrahydrocannabinol (THC, 2 mg/kg, i.p.) or morphine (5 mg/kg, subcutaneous). Mitragynine-, morphine-, and THC-induced learning and memory deficits were reversed by co-treatment with the CB₁ receptor antagonist, NIDA-41020 (10 mg/kg, i.p.). A significant upregulation of CB₁ receptor expression was found in the hippocampal CA1 region and ventral tegmental area after chronic high-dose mitragynine and morphine, whereas a downregulation was observed after chronic THC. In conclusion, the present study suggests a plausible role of the CB₁ receptor in mediating the dose-dependent cognitive deficits after chronic high-dose mitragynine exposure. This also highlights the potential of CB₁ receptor antagonism in ameliorating the cognitive deficits associated with long-term kratom/mitragynine consumption in humans.

Keywords: cannabinoid receptor 1 (CB1); cognition; kratom; mitragynine; morphine; Δ9-tetrahydrocannabinol (THC).

FIGURE 1

Summary of the IntelliCage experimental protocol.

FIGURE 2

Effects of drug intervention on exploratory activities. Baseline exploratory activity in (A) novel and (B) familiar environment. The mean number of visits to all corners per test group during the first hour in the novel IntelliCage and 3 consecutive days in the familiar IntelliCage environment throughout the baseline phase. Without any drug intervention, all groups in (A) and (B) showed no significant differences in novelty-induced exploration and baseline daily activity (p > 0.05; one-way ANOVA). (C) Effects of drug sensitization to daily activity in the familiar IntelliCage environment. The mean number of visits to all corners during 3 days of baseline phase vs 3 days of sensitization phase (Day 5–7). Morphine and high-dose mitragynine–treated mice showed a significant increment in their general familiar vs baseline activities, in which the increments are comparable (not significant) between mitragynine vs morphine. By contrast, THC-treated mice showed a significant decrease in general activity. *p < 0.05 vs baseline; **p < 0.01 vs baseline (two-way repeated measures ANOVA followed by post-hoc Tukey test). ^#includes animals from drug only and drug + receptor antagonist groups (i.e., each group n = 10).

FIGURE 3

Effects of drug intervention on preference for natural reward (sucrose) and persistence for sucrose seeking. (A) Preference for sucrose reward. The percentage of licks at the natural reward (10% sucrose) corner during the 2-day sucrose phase vs pre-sucrose (*p < 0.05, **p < 0.01, ***p < 0.001 vs pre-sucrose; a: p < 0.05 vs Tween 20; ns = p > 0.05 vs morphine; two-way repeated measures ANOVA with post-hoc Tukey test). (B) Persistence of sucrose seeking. The percentage of reward licks when paired with air-puff punishment measured during the 3-day phase vs without air-puffs. *p < 0.05; **p < 0.01 vs reward licks without punishment; a: p < 0.05 vs Tween 20 (two-way repeated measures ANOVA with post-hoc Tukey). ^#includes animals from drug only and drug + receptor antagonist groups (i.e., each group n = 10).

FIGURE 4

Effects of drug intervention on place learning and reversal learning, and NIDA-41020 antagonism. Data are shown as the percentage of preference for the correct (water-reinforced) corner and number of trials (the mean number of all visits with nose pokes are shown in the table below the graph) (A) during the 5 days of place learning phase, as well as (B) 6 consecutive days of the reversal learning phase. Data revealed highly significant learning and reversal learning in untreated and Tween 20 control groups and the low-dose mitragynine group. The morphine-, THC-, and high-dose mitragynine-treated groups failed to attain place learning and reversal learning. The cannabinoid CB₁ receptor antagonist NIDA-41020 significantly reversed morphine-, THC-, and mitragynine-induced place learning and reversal learning impairment in mice. *p < 0.05; **p < 0.01 vs Tween 20 (two-way ANOVA with post-hoc Tukey; n = 10/group).

FIGURE 5

Immunohistochemistry of positive and negative controls at ×400 magnification to validate staining specificity. (A) Immunoreactive fibers within the mouse cerebellar molecular layer served as the positive control for CB₁ receptor. The brown deposits confirm the presence of CB₁ receptors (OD = 0.28). (B) No stain was detected in the neurons and surrounding fibers of the CA1 hippocampal region (negative control; OD = 0.00). GL = granular layer; PCL = Purkinje cell layer; ML = molecular layer; Bars = 50 µm.

FIGURE 6

Effects of drug intervention on CB₁ receptor staining in the (A) CA1 pyramidal region of the mice hippocampus (×100 magnification). The micrographs represent (B) untreated, (C) Tween 20, (D) morphine, (E) morphine + NIDA-41020, (F) THC, (G) THC + NIDA-41020, (H) high-dose mitragynine, (I) high-dose mitragynine + NIDA-41020, (J) low-dose mitragynine, and (K) low-dose mitragynine + NIDA-41020 groups at ×400 magnification. Arrowheads indicate CA1 pyramidal neurons surrounded by dense plexus of CB₁ receptor immunoreactive fibers. (L) Densitometric analysis are shown as the mean + SEM of 15 replicates per group. ****p < 0.0001 vs Tween 20; a: p < 0.05 vs drug + NIDA-41020 groups (one-way ANOVA followed by post-hoc Tukey test). Bars = 50 µm.

FIGURE 7

Effects of drug intervention on CB₁ receptor staining in (A) mice VTA (×100 magnification). The micrographs represent (B) untreated, (C) Tween 20, (D) morphine, (E) morphine + NIDA-41020, (F) THC, (G) THC + NIDA-41020, (H) high-dose mitragynine, (I) high-dose mitragynine + NIDA-41020, (J) low-dose mitragynine, and (K) low-dose mitragynine + NIDA-41020 groups at ×400 magnification. Arrowheads indicate CB₁ immunoreactive neurons and the surrounding fibers. (L) Densitometric analysis are shown as mean + SEM of 15 replicates per group. ****p < 0.0001 vs Tween 20; a: p < 0.05 vs drug + NIDA-41020 groups (one-way ANOVA followed by post-hoc Tukey test). Bars = 50 µm.

FIGURE 8

CB₁ receptor (A, B) protein and (C) gene levels in mice brain mesolimbic area after 28 days of drug intervention. Data are shown as the mean + SEM of three biological replicates per group. The CB₁ receptor values have been normalized to β-actin controls. Data revealed that high-dose mitragynine and morphine groups significantly increased, whereas THC decreased; CB₁ receptor expression in the brain mesolimbic area vs controls and low-dose mitragynine group. NIDA-41020 reversed the drug-induced CB₁ receptor alteration. ***p < 0.001, ****p < 0.0001 vs Tween 20; a: p < 0.05 vs drug + NIDA-41020 groups (one-way ANOVA followed by post-hoc Tukey test).