In vertebrates, cannabinoids modulate neuroimmune interactions through two cannabinoid receptors (CNRs) conservatively expressed in the brain (CNR1, syn. CB1) and in the periphery (CNR2, syn. CB2). Our comparative genomic analysis indicates several evolutionary losses in the CNR2 gene that is involved in immune regulation. Notably, we show that the CNR2 gene pseudogenized in all parrots (Psittaciformes). This CNR2 gene loss occurred because of chromosomal rearrangements. Our positive selection analysis suggests the absence of any specific molecular adaptations in parrot CNR1 that would compensate for the CNR2 loss in the modulation of the neuroimmune interactions. Using transcriptomic data from the brains of birds with experimentally induced sterile inflammation we highlight possible functional effects of such a CNR2 gene loss. We compare the expression patterns of CNR and neuroinflammatory markers in CNR2 -deficient parrots (represented by the budgerigar, Melopsittacus undulatus and five other parrot species) with CNR2 -intact passerines (represented by the zebra finch, Taeniopygia guttata ). Unlike in passerines, stimulation with lipopolysaccharide resulted in neuroinflammation in the parrots linked with a significant upregulation of expression in proinflammatory cytokines (including interleukin 1 beta ( IL1B ) and 6 ( IL6 )) in the brain. Our results indicate the functional importance of the CNR2 gene loss for increased sensitivity to brain inflammation.

In vertebrates, an ancient duplication in the genes for cannabinoid receptors (CNRs) allowed the evolution of specialised endocannabinoid receptors expressed in the brain (CNR1) and the periphery (CNR2). While dominantly conserved throughout vertebrate phylogeny, our comparative genomic analysis suggests that certain taxa may have lost either the CNR1 regulator of neural processes or, more frequently, the CNR2 involved in immune regulation. Focussing on conspicuous CNR2 pseudogenization in parrots (Psittaciformes), a diversified crown lineage of cognitively-advanced birds, we highlight possible functional effects of such a loss. Parrots appear to have lost the CNR2 gene at at least two separate occasions due to chromosomal rearrangement. Using gene expression data from the brain and periphery of birds with experimentally-induced sterile inflammation, we compare CNR and inflammatory marker (interleukin 1 beta, IL1B) expression patterns in CNR2-deficient parrots (represented by the budgerigar, Melopsittacus undulatus and five other parrot species) with CNR2-intact passerines (represented by the zebra finch, Taeniopygia guttata). Though no significant changes in CNR expression were observed in either parrots or passerines during inflammation of the brain or periphery, we detected a significant up-regulation of IL1B expression in the brain after stimulation with lipopolysaccharide (LPS) only in parrots. As our analysis failed to show evidence for selection on altered CNR1 functionality in parrots, compared to other birds, CNR1 is unlikely to be involved in compensation for CNR2 loss in modulation of the neuroimmune interaction. Thus, our results provide evidence for the functional importance of CNR2 pseudogenization for regulation of neuroinflammation.