Low Basal CB2R in Dopamine Neurons and Microglia Influences Cannabinoid Tetrad Effects

Abstract

There are two well-characterized cannabinoid receptors (CB1R and CB2R and other candidates): the central nervous system (CNS) enriched CB1R and peripheral tissue enriched CB2R with a wide dynamic range of expression levels in different cell types of human tissues. Hepatocytes and neurons express low baseline CB1R and CB2R, respectively, and their cell-type-specific functions are not well defined. Here we report inducible expression of CB1R in the liver by high-fat and high sugar diet and CB2R in cortical neurons by methamphetamine. While there is less controversy about hepatocyte CB1R, the presence of functional neuronal CB2R is still debated to date. We found that neuron CB2R basal expression was higher than that of hepatocyte CB1R by measuring mRNA levels of specific isoform CB2A in neurons isolated by fluorescence-activated cell sorting (FACS) and CB1A in hepatocytes isolated by collagenase perfusion of liver. For in vivo studies, we generated hepatocyte, dopaminergic neuron, and microglia-specific conditional knockout mice (Abl-Cnr1^Δ, Dat-Cnr2^Δ, and Cx3cr1-Cnr2^Δ) of CB1R and CB2R by crossing Cnr1^f/f and Cnr2^f/f strains to Abl-Cre, Dat-Cre, and Cx3cr1-Cre deleter mouse strains, respectively. Our data reveals that neuron and microglia CB2Rs are involved in the "tetrad" effects of the mixed agonist WIN 55212-2, CB1R selective agonist arachidonyl-2'-chloroethylamide (ACEA), and CB2R selective agonist JWH133. Dat-Cnr2^Δ and Cx3cr1-Cnr2^Δ mice showed genotypic differences in hypomobility, hypothermia, analgesia, and catalepsy induced by the synthetic cannabinoids. Alcohol conditioned place preference was abolished in DAT-Cnr2^Δ mice and remained intact in Cx3cr1-Cnr2^Δ mice in comparison to WT mice. These Cre-loxP recombinant mouse lines provide unique approaches in cannabinoid research for dissecting the complex endocannabinoid system that is implicated in many chronic disorders.

Keywords: CB1 receptor; CB2 receptor; Cre-loxP system; cannabinoids; hepatocytes; in situ hybridization; microglia; neurons; tetrad effects.

Figure 1

Comparison of low baseline SD (n = 6) and high-fat/high sugar diets (HFHS) (n = 6) liver cannabinoid receptors (CB1R) and cortex (n = 6) CB2R to high baseline cortex CB1R and spleen (n = 3) CB2R. SD represents standard diet and HFHS high-fat and high sugar diet. (A) using SD liver CB1A as a reference and (B) using cortex CB2A (n = 6) as a reference.

Figure 2

Fluorescence-activated cell sorting (FACS) of neurons and microglia. (A) The Y-axis is the fluorescent intensity of rat cortex neurons labeled with R-phycoerythrin (PE) conjugated NeuN antibody in logarithmic plots, and X-axis is the forward-scatter in linear scale representing cell sizes. (B) The Y-axis is the fluorescence intensity of transgenic Cx3c1-eGFP mouse cortex microglia in logarithmic plots, and the X-axis is the forward-scatter logarithmic plots representing cell sizes. (C) Comparison of low baseline CB1R expression in liver (n = 4), hepatocytes (n = 4, RT-preAmp-qPCR), and neurons (n = 6) using liver CB1A as a reference. (D) Comparison of low baseline CB2R expression in mouse cortex (n = 6), neurons (n = 6) and microglia (n = 4) using liver (n = 4) CB2A as a reference. (E) Comparison of CB2A expression in rat cortex neurons of saline (n = 6) and methamphetamine (n = 6) treatments.

Figure 3

RNAscope in situ hybridization (ISH) for CB1A (A) brain anatomical expression in glutamatergic neurons of thalamus. Yellow arrows represent CB1A and vGluT2 co-expression in medium thalamic nucleus and green arrows represent the absence of CB1A in vGluT2 neurons. (B) Corresponding Allen Brain Atlas conventional CB1R ISH in mouse lateral thalamus (box). RNAscope ISH for CB1A (C) brain anatomical expression in dopaminergic and glutamatergic neurons of lateral VTA. Yellow arrows represent CB1A, and vGluT2 co-expression and green arrows represent the absence of CB1A in dopaminergic neurons. (D) Corresponding Allen Brain Atlas conventional CB1R ISH in mouse VTA (box). Scale bar is 20 μm.

Figure 4

Diagrams of Cnr1^f/f (A) Cnr2^f/f (B) alleles. Open boxes represent exons and Neo-footprint, gray boxes open reading frames of Cnr1 and Cnr2, solid line introns and genomic flanking regions, triangle lines alternative splicing patterns, numbers base pairs (bp), black forward arrowheads loxP sites, and black ovals remnant FRT sites in Neo-footprint, solid bars RNAscope ISH probes, and upward arrows polyA sites. (C) Validation of CB1R-specific deletion in Alb-Cnr1^Δ hepatocytes by preAmp-RT-qPCR; Q represents QuantaBio preamp and real-time PCR mixes and T Thermo Fisher preamp and real-time PCR mixes. (D) Agarose gel of Cnr2 floxed mice: mutant is 545 bp, wild type 386 bp, and the heterozygous 545 bp and 386 bp. (E) Agarose gel of Cx3cr1-Cre mice: mutant is 380 bp, wild type 816 bp, and the heterozygous 380 bp and 816 bp. MW represents the molecular weight marker.

Figure 4

Diagrams of Cnr1^f/f (A) Cnr2^f/f (B) alleles. Open boxes represent exons and Neo-footprint, gray boxes open reading frames of Cnr1 and Cnr2, solid line introns and genomic flanking regions, triangle lines alternative splicing patterns, numbers base pairs (bp), black forward arrowheads loxP sites, and black ovals remnant FRT sites in Neo-footprint, solid bars RNAscope ISH probes, and upward arrows polyA sites. (C) Validation of CB1R-specific deletion in Alb-Cnr1^Δ hepatocytes by preAmp-RT-qPCR; Q represents QuantaBio preamp and real-time PCR mixes and T Thermo Fisher preamp and real-time PCR mixes. (D) Agarose gel of Cnr2 floxed mice: mutant is 545 bp, wild type 386 bp, and the heterozygous 545 bp and 386 bp. (E) Agarose gel of Cx3cr1-Cre mice: mutant is 380 bp, wild type 816 bp, and the heterozygous 380 bp and 816 bp. MW represents the molecular weight marker.

Figure 5

CB2R-mediated behaviors in the tetrad effects in DAT-Cnr2^Δ mice. The effects of CB1R, CB2R and mixed CB1R and CB2R ligand, WIN 55212-2 (3.0 mg/kg), ACEA (1.0 mg/kg) and JWH133 (20 mg/kg) in the tetrad effects. (A) Hypomobility; (B) hypothermia; (C) analgesia (tail-flick nociception test and a similar result in the hot plate test); (D) catalepsy. Asterisks represent p < 0.05.

Figure 6

CB2R-mediated behaviors in the tetrad effects in WT, Cx3cr1-Cnr2 mice. The effects of the selected dose of the mixed CB1R and CB2R ligand WIN 55212-2 (3.0 mg/kg) in the tetrad effects. (A) Hypomobility; (B) hypothermia; (C) analgesia (tail-flick nociception test and a similar result in the hot plate test); (D) catalepsy. Asterisks represent p < 0.05.

Figure 7

CB2R modulation of alcohol preference in Alcohol 8%-induced conditioned place preference in WT and Cx3cr1-Cnr2^Δ mice, but not in DAT-Cnr2^Δ mice. (A) WT and DAT-Cnr2^Δ mice; (B) WT and Cx3cr1-Cnr2^Δ mice. Asterisks represent p < 0.05.