Species differences in cannabinoid receptor 2 and receptor responses to cocaine self-administration in mice and rats

Abstract

The discovery of functional cannabinoid receptors 2 (CB2Rs) in brain suggests a potential new therapeutic target for neurological and psychiatric disorders. However, recent findings in experimental animals appear controversial. Here we report that there are significant species differences in CB2R mRNA splicing and expression, protein sequences, and receptor responses to CB2R ligands in mice and rats. Systemic administration of JWH133, a highly selective CB2R agonist, significantly and dose-dependently inhibited intravenous cocaine self-administration under a fixed ratio (FR) schedule of reinforcement in mice, but not in rats. However, under a progressive ratio (PR) schedule of reinforcement, JWH133 significantly increased breakpoint for cocaine self-administration in rats, but decreased it in mice. To explore the possible reasons for these conflicting findings, we examined CB2R gene expression and receptor structure in the brain. We found novel rat-specific CB2C and CB2D mRNA isoforms in addition to CB2A and CB2B mRNA isoforms. In situ hybridization RNAscope assays found higher levels of CB2R mRNA in different brain regions and cell types in mice than in rats. By comparing CB2R-encoding regions, we observed a premature stop codon in the mouse CB2R gene that truncated 13 amino-acid residues including a functional autophosphorylation site in the intracellular C-terminus. These findings suggest that species differences in the splicing and expression of CB2R genes and receptor structures may in part explain the different effects of CB2R-selective ligands on cocaine self-administration in mice and rats.

Figure 1

Differential effects of JWH133 on cocaine self-administration in rats and mice. (a) Systemic administration (i.p.) of JWH133 significantly decreased cocaine self-administration under fixed ratio (FR2) reinforcement in mice, but not in rats, as assessed by mean numbers of cocaine infusions per hour. (b) Systemic administration of JWH133 significantly increased interinfusion intervals in mice, but not in rats. (c) Systemic administration of JWH133 significantly increased PR BP for cocaine self-administration in rats, but decreased PR BP in mice. (d) Intranasal (i.n.) administration of JWH133 (10, 25, or 50 μg/10 μl/side) produced biphasic effects—low doses increased whereas high doses decreased PR BP in rats. (e) Systemic administration of JWH133 significantly inhibited oral sucrose self-administration in WT mice, but not in rats or CB₂-KO mice. *P<0.05, compared with vehicle control group.

Figure 2

(a) Mouse CB₂ (mCB₂R, Cnr2, 4D3) and (b) rat CB₂ (rCB₂R, Cnr2, 5q36) genomic structures, alternatively spliced transcripts, and the locations that each probe targeted to detect brain CB₂ mRNA. Gene: open boxes represent exons, horizontal lines introns and black box mini-intron within the last exon. Transcripts: spliced exon numbers are indicated in the exons. The TaqMan probes were designed to fit the junctions of the spliced exons and are represented by horizontal black bars. The RNAscope probes hybridize 3′-UTR regions marked by gray boxes.

Figure 3

Agarose gel analysis results of PCR fragments, illustrating mCB_2A, rCB_2A, and rCB_2C (a), mCB_2B and rCB_2B (b), and rCB_2D (c) isoforms (ie, shown in Figure 2). When using a probe that targeted the gene-deleted region in CB₂-KO mice, mCB₂ mRNA signal was detectable only in the striatum and spleen of WT mice, but not in CB₂-KO mice (d). MW, molecular weight marker; SPL, spleen; ko-CB₂, knockout mice; wt, wild type; MID, midbrain; CTX, cortex; CER, cerebellum; HIP, hippocampus; PFC, prefrontal cortex; MID, midbrain; DST, dorsal striatum; NAC, nucleus accumbens; AMG, amygdala; LIV, liver; TES, testis; OVA, ovary; Prefix: m, mouse and r, rat.

Figure 4

RT-qPCR comparison of CB₂R isoform tissue expression in mice and rats. The white bars represent rCB₂ mRNA levels in rat tissues and the gray bars represent mCB₂ mRNA levels in mouse tissues. The y axis is fold change in CB₂ mRNA, in which rat cortical CB₂ mRNA level was used as a reference to quantify the levels in other brain regions, whereas for rat testis CB₂ level was used as a reference to quantify CB₂ mRNA in peripheral tissues. For rat-specific rCB_2C and rCB_2D isoforms, the cortical CB₂ mRNA level was used as a reference for both brain and peripheral tissues.

Figure 5

Brain CB₂ mRNA expression by RNAscope in situ hybridization (ISH) assays. (a) Brain section diagrams, illustrating the anatomic locations and the coordinates of PFC, DST, NAC, and VTA for the ISH images. (b, c) Quantification of CB₂R pixels or densities per cell in each brain region of mice and rats. (d) Representative RNAscope ISH microscope images of different brain regions. CB₂ mRNA signals are green, TH and DAT signals are red, and nuclear signals are blue (DAPI). Calibration bar is 20 μm. *P<0.05, compared with rats.

Figure 6

CB₂R 3D and 2D structures in rat and mouse. (a, b) In silico models of mCB₂R and rCB₂R 3D structures. (c, d) The corresponding amino-acid sequences of mCB₂R and rCB₂R. Ribbons represent seven transmembrane domains and bended lines represent intracellular and extracellular domains. The ‘DRY' motif is indicated by colored balls. The charged amino-acid exchanges in the extracellular and the intracellular loops between mCB₂R and rCB₂R are marked by colored narrow lines labeled with amino-acid codes and numbers in the 3D models. The truncated 13 amino acids of mCB₂R are marked with single amino-acid code in the intracellular C-terminal domain of rCB₂R. The autophosphorylation site S352 is marked in red. The charged amino acid substitutions and the C-terminal truncation of mCB₂R are marked in the mCB₂R with dark red coloration (c, d) and other amino acid substitutions between mCB₂R and rCB₂R are marked in light red coloration (d).

Figure 7

Computer models of JWH133 binding site of CB₂R. (a) JHW133 docking to the active site of hCB₂R. Green rings and lines represent JHW133 structure. (b) Complex conformations between JHW133 and hCB₂R. The JHW133 structure is represented by gray spheres and the amino-acid residues that contact with JHW133 are indicated by three-letter amino-acid symbols and numbers.