Cannabinoids affect the mouse visual acuity via the cannabinoid receptor type 2

www.nature.com/scientificreports OPEN Cannabinoids affect the mouse visual acuity via the cannabinoid receptor type 2 Bruno Cécyre1,2, Ismaël Bachand1, François Papineau1, Chloé Brochu1, Christian Casanova2 & Jean‑François Bouchard1* Recently, there have been increasing indications that the endocannabinoid (eCB) system is involved in vision. Multiple research teams studied the cannabinoid receptor type 2 (CB2R) expression and function in the mouse retina. Here, we examined the consequence of CB2R modulation on visual acuity using genetic and pharmacologic tools. We found that Cnr2 knockout mice show an enhanced visual acuity, CB2R activation decreased visual acuity while CB2R blockade with the inverse agonist AM630 increased it. The inhibition of 2-arachidonylglycerol (2-AG) synthesis and degradation also greatly increased and decreased visual acuity, respectively. No differences were seen when the cannabinoid receptor type 1 (CB1R) was deleted, blocked or activated implying that CB2R exclusively mediates cannabinoid modulation of the visual acuity. We also investigated the role of cannabinoids in retinal function using electroretinography (ERG). We found that modulating 2-AG levels affected many ERG components, such as the a-wave and oscillatory potentials (OPs), suggesting an impact on cones and amacrine cells. Taken together, these results reveal that CB2R modulates visual acuity and that eCBs such as 2-AG can modulate both visual acuity and retinal sensitivity. Finally, these findings establish that CB2R is present in visual areas and regulates vision-related functions. In the last years, there have been increasing indications that the endocannabinoid (eCB) system is involved in vision. The cannabinoid receptor type 1 (CB1R) is found in the majority of retinal neurons, including photoreceptors, horizontal cells, bipolar cells, amacrine cells and ganglion cells (for review s ee1). Many studies, based on patch-clamp recordings from retinal slices, found that cannabinoids affected potassium, chloride and calcium currents (for review s ee1). Most of these studies were carried out using compounds such as WIN55,212-2, a synthetic cannabinoid with similar affinity to both CB1R and CB2R. Nowadays, synthetic cannabinoid agonists and inverse agonists with a better selectivity for CB1R or CB2R were developed. Among them, ACEA (CB1R agonist), HU308 (CB2R agonist), AM251 (CB1R inverse agonist) and AM630 (CB2R inverse agonist) were shown to be very s elective2,3. There is mounting evidence that CB2R is expressed in neuronal tissues, such as cerebellum, brainstem, hippocampus, cortex, and retina1. Indeed, the retinal expression of CB2R was reported in many animals including monkeys4, though it remains controversial because of the lack of specificity of antibodies directed against CB2R5. However, many studies confirmed the presence of a functional CB2R in the retina with pharmacological and genetic tools. For instance, CB2R was found to be involved in retinal ganglion cell (RGC) axon guidance during development6; in vivo recording of electroretinogram (ERG) responses demonstrated that mice lacking CB2R (Cnr2-/-) exhibited an increased a-wave amplitude under scotopic conditions, reflecting an enhanced sensitivity of photoreceptors7. Furthermore, a recent report studying the retinal function of Cnr2-/- mice confirmed that ERG responses are altered in these mice, and found that a prolonged treatment with CB2R inverse agonist AM630 mimics the effects seen in Cnr2-/- mice8. These accumulating facts demonstrate the modulator effect of CB2R, thus suggesting its functional presence in the retina. Until now, almost all studies regarding CB2R expression and function in visual structures were realized on the retina. Since vision does not rely strictly on the retina, other tests need to be conducted to evaluate the impact of CB2R on the visual function. One of these assays takes advantage of the optomotor response (OMR), consisting of a stereotyped head movement in response to movement in the surrounding environment. This reflex is highly 1 Laboratoire de Neuropharmacologie, École d’optométrie, Université de Montréal, Succursale Centre-Ville, C.P. 6128, Montréal, Québec H3C 3J7, Canada. 2Laboratoire des Neurosciences de la Vision, École d’optométrie, Université de Montréal, Montréal, Québec, Canada. *email: jean‑ Scientific Reports | (2020) 10:15819 | https://doi.org/10.1038/s41598-020-72553-y 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1.  CB2R deletion enhances visual acuity in adults and through postnatal development. (A,B) The Cnr2-/mice showed a better spatial frequency threshold compared to Cnr2+/+ animals. No differences were observed between Cnr1-/- and Cnr1+/+ mice. For (A) and (B) respectively, Cnr1+/+, n = 14 and 38; Cnr1-/-, n = 30 and 38; Cnr2+/+, n = 32 and 37; Cnr2-/-, n = 18 and 32. Statistical analysis was performed by one-way (A) or two-way (B) ANOVA with Tukey post hoc test. The values are mean ± SEM. *** p = 0.0002, **** p < 0.0001 compared to Cnr2+/+. (c/d: cycles per degree). conserved among many species and does not require a training process. Hence, it does not require constrains to animals, which are allowed to move freely on a platform. Compared to ERGs, the OMR can assess deficits in RGCs, optic nerve transmission or brain visual integration. We investigated the visual acuity and retinal function of mice after genetic manipulation or pharmacological modulation. The results reported here show that transgenic mice lacking CB2R displayed an increased visual acuity. Similarly, the administration of CB2R agonists and inverse agonists respectively decreased and increased visual acuity. Furthermore, the modulation of 2-AG levels affected retinal sensitivity, confirming the functional presence of cannabinoid receptors in the retina and suggesting that eCBs could be implicated in the retinal homeostasis. These results are consistent with the hypothesis that CB2R is expressed in the retina and strengthen the current knowledge of cannabinoids in visual function. Results The deletion of Cnr2 enhances the visual acuity during the development through adult‑ hood. Our data indicate that the absence of CB2R enhanced the visual acuity in adults. Indeed, the spa- tial frequency threshold of Cnr2-/- mice was significantly higher than the one of Cnr2+/+ mice (Fig. 1A; oneway ANOVA, p = 0.0002). No differences were observed between Cnr1-/- and Cnr1+/+ mice (Fig. 1A; one-way ANOVA, p = 0.2013). The important role of cannabinoids during visual system development has been extensively studied in the literature6,9. In order to better evaluate the impact of Cnr2 deletion on visual acuity, we measured the spatial frequency threshold of the same wildtype and mutant mice during their postnatal development, with regular tests between eye opening and adulthood. Similarly to adult animals, the visual acuity of Cnr2-/- pups was globally enhanced compared to Cnr2+/+ mic (...truncated)