Species differences in cannabinoid receptor 2 (CNR2 gene): identification of novel human and rodent CB2 isoforms, differential tissue expression and regulation by cannabinoid receptor ligands

Cannabinoids, endocannabinoids and marijuana activate two well-characterized cannabinoid receptors (CB-Rs), CB1-Rs and CB2-Rs. The expression of CB1-Rs in the brain and periphery has been well studied, but neuronal CB2-Rs have received much less attention than CB1-Rs. Many studies have now identified and characterized functional glial and neuronal CB2-Rs in the central nervous system. However, many features of CB2-R gene structure, regulation and variation remain poorly characterized in comparison with the CB1-R. In this study, we report on the discovery of a novel human CB2 gene promoter transcribing testis (CB2A) isoform with starting exon located ca 45 kb upstream from the previously identified promoter transcribing the spleen isoform (CB2B). The 5' exons of both CB2 isoforms are untranslated 5'UTRs and alternatively spliced to the major protein coding exon of the CB2 gene. CB2A is expressed higher in testis and brain than CB2B that is expressed higher in other peripheral tissues than CB2A. Species comparison found that the CB2 gene of human, rat and mouse genomes deviated in their gene structures and isoform expression patterns. mCB2A expression was increased significantly in the cerebellum of mice treated with the CB-R mixed agonist, WIN55212-2. These results provide much improved information about CB2 gene structure and its human and rodent variants that should be considered in developing CB2-R-based therapeutic agents.     

Density and function of central serotonin (5-HT) transporters, 5-HT1A and 5-HT2A receptors, and effects of their targeting on BTBR T+tf/J mouse social behavior

BTBR mice are potentially useful tools for autism research because their behavior parallels core social interaction impairments and restricted-repetitive behaviors. Altered regulation of central serotonin (5-HT) neurotransmission may underlie such behavioral deficits. To test this, we compared 5-HT transporter (SERT), 5-HT(1A) and 5-HT(2A) receptor densities among BTBR and C57 strains. Autoradiographic [(3) H] cyanoimipramine (1 nM) binding to SERT was 20-30% lower throughout the adult BTBR brain as compared to C57BL/10J mice. In hippocampal membrane homogenates, [(3) H] citalopram maximal binding (B(max) ) to SERT was 95 ± 13 fmol/mg protein in BTBR and 171 ± 20 fmol/mg protein in C57BL/6J mice, and the BTBR dissociation constant (K(D) ) was 2.0 ± 0.3 nM versus 1.1 ± 0.2 in C57BL/6J mice. Hippocampal 5-HT(1A) and 5-HT(2A) receptor binding was similar among strains. However, 8-OH-DPAT-stimulated [(35) S] GTPγS binding in the BTBR hippocampal CA(1) region was 28% higher, indicating elevated 5-HT(1A) capacity to activate G-proteins. In BTBR mice, the SERT blocker, fluoxetine (10 mg/kg) and the 5-HT(1A) receptor partial-agonist, buspirone (2 mg/kg) enhanced social interactions. The D(2) /5-HT(2) receptor antagonist, risperidone (0.1 mg/kg) reduced marble burying, but failed to improve sociability. Overall, altered SERT and/or 5-HT(1A) functionality in hippocampus could contribute to the relatively low sociability of BTBR mice.     

Differential modulation of dopaminergic systems in the rat brain by dietary protein

Rats that consume a diet 50% rich in protein exhibit hyperactivity and hyperresponsiveness to nociceptive stimuli, in which facilitation of dopaminergic activity has been implicated. We studied the regional changes in the concentrations of dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the brains of rats that were maintained on high-protein (HP, 50% casein), normal-protein (NP, 20% casein), and low-protein (LP, 8% casein) diets for 36 weeks. Brain nuclei that represented different DAergic systems were punchdissected and analyzed using HPLC. In the substantia nigra, the striatum, and the dentate gyrus, DA concentrations decreased and increased, respectively, with a decrease and increase in dietary protein (p<0.05 compared to the NP diet). Similar trends in the effect of the HP diet were observed in the ventral tegmental area, amygdala, frontal cortex, subiculum, centromedial nucleus (CM) of the thalamus, and inferior colliculi (IC), although the differences in DA concentrations were not statistically significant. These brain areas also showed a pattern of decreased DA concentration in association with the LP diet, and the differences were statistically significant (p<0.05) in the CM and IC. DA concentrations in most regions of the midbrain and brainstem were not different between the diet groups, nor were consistent trends observed in those regions. Also, there were no consistent relationships between DOPAC/DA and HVA/DA ratios and dietary protein level. These data suggest that only discrete dopaminergic neuronal circuits in the rat forebrain were sensitive to changes in dietary protein level.     

A low protein-high carbohydrate diet decreases D2 dopamine receptor density in rat brain.

The property of D2 dopamine receptors in the rat brain was evaluated after long-term dietary manipulation. Groups of rats were pair-fed with equicaloric diet containing low protein (8%)-high carbohydrate, high protein (52%)-low carbohydrate and normal protein (20%) for 36 weeks. The low protein-high carbohydrate fed rats exhibited a significant decrease in the density (Bmax) of D2 dopamine receptor in the striatum (28%) and the mesolimbic regions (36%) with no apparent change in the receptor affinity (Kd). These findings suggest that long-term consumption of a low protein-high carbohydrate diet, by decreasing D2 dopamine receptor density, may be an important determinant of central dopaminergic function.     

In-Vitro and In-Vivo Action of Cannabinoids

The discovery of endocannabinoids such as anandamide and the wide spread localization of cannabinoid receptors in the brain and peripheral tissues, suggests that the cannabinoid system represents a previously unrecognized ubiquitous net work in the nervous system, whose physiology and function is unfolding. In this study, we tested the hypothesis that some of the actions of anandamide are independent of a cannabinoid receptor mechanism. This was accomplished by the use of cannabinoid agonist and antagonist interaction in an in-vitro and in-vivo test systems. In-vitro, we used Xenopus laevis oocytes expression system and two-voltage clamp technique in combination with differential display polymerase chain reaction to determine whether the differential display of genes following treatment with anandamide may be linked to AMPA glutamate receptor. The differential expression of genes in vivo after the sub-acute administration of anandamide could not be directly linked with the AMPA glutamate receptor. In the voltage clamp studies we investigated the effects of anandamide on recombinant AMPA GluR3 sub-unit currents generated by kainic acid in oocytes expressing the AMPA glutamate receptor. In the in-vitro studies, we present evidence that anandamide inhibited the kainate activated currents in oocytes expressing AMPA glutamate receptor involves cAMP transduction via a cannabinoid receptor independent mechanism. In the in-vivo studies, SR141716A, the CB1 antagonist, induced anxiolysis, that was dependent on the mouse strain used in the anxiety model and blocked the anxiogenic effects of anandamide or methanandamide whereas SR141716A had no effect on the anandamide inhibition of kainate activated currents in-vitro.