the endocannabinoid system is composed of cannabinoid receptor type 1 ( cb1r ) , cannabinoid receptor type 2 ( cb2r ) , their endogenous ligands ( endocannabinoids ) , and their synthetizing and metabolizing enzymes . the physiological and psychological effects of cannabinoids can be detected almost everywhere in the body due to the abundance of cannabinoid receptors . expression patterns of cb1r and cb2r are well documented in the retina of numerous species , including rodents and primates [ 16 ] . in rodents , cb1r and cb2r are expressed in many retinal cell types , particularly cone and rod photoreceptors , horizontal cells , amacrine cells , bipolar cells , and ganglion cells [ 1 , 7 ] . in vervet monkeys , cb1r is mainly found in cones of the central retina , rod spherules with very low expression , horizontal cells , bipolar cells , and amacrine and ganglion cells . cb2r , on the other hand , is strictly expressed in primate glial mller cells . beyond the retina , the expression pattern of cb1r has been observed in the dorsal lateral geniculate nucleus   and primary visual cortex   of primates . most of our knowledge on the role of cannabinoids in human vision comes from reports , anecdotes , and studies with cannabis consumers ( for review see ) . besides the well - known  red eye effect ( vasodilation ) of marijuana and reduction of intraocular pressure ( iop ) [ 1113 ] , the functional effects of endocannabinoids on the visual system are not yet well defined . nevertheless , the administration of cannabinoids produces some known alterations in the human visual system . indeed , case studies suggested the existence of cannabis - mediated visual effects in humans , particularly an increase in glare recovery at low contrast , a reduction in vernier and snellen acuities [ 16 , 17 ] , improvement in night vision [ 18 , 19 ] , blurred vision , changes in color discrimination , and an increase in photosensitivity . most of the latter ( psychophysical ) effects may have a retinal component , which might be due to neurochemical changes induced by the retinal endocannabinoid system . indeed , many physiological effects of cannabinoids were reported for every retinal cell type in bovines , guinea pigs , rodents , and fishes ( for review see [ 10 , 22 ] ) . in the bovine retina , the activation of cb1r increases monoamine oxidase . in the guinea pig retina , stimulation of cb1r results in the inhibition of dopamine release , and in the rat retina , the activation of cannabinoid receptors modulates [ 35s ] gtp s - binding and voltage - dependent membrane currents in photoreceptors , bipolar cells , and ganglion cells [ 3 , 2528 ] . in addition , cannabinoid agonists increase the cone response to light offset in the goldfish retina . the electroretinogram ( erg ) is a useful tool for assessing retinal function by measuring the electrical responses of all populations of retinal cells , mainly photoreceptors ( cones and rods ) , bipolar cells , amacrine cells , and mller cells [ 3032 ] . the erg waves include two main components : the negative amplitude ( a - wave ) and the positive one ( b - wave ) . traditionally , the a - wave reflects the response of rods and cones to light [ 33 , 34 ] . the generation of the b - wave , the second major component of the erg , is attributed to the inner retina , mainly the depolarization of bipolar and mller cells [ 3032 , 3539 ] . specific stimuli and recording environments are selected to isolate the components of the erg and target particular populations of retinal cells . for instance , rod function is assessed in dark - adapted eyes , under scotopic conditions , while cone responses are better assessed with high intensity flashes , under photopic conditions . in this study , we investigated the changes in normal retinal function as measured by electroretinography in adult vervet monkeys after blockade of cb1r or cb2r by their antagonists am251 and am630 , respectively . vervet monkeys are becoming the preferred animal model used in biomedical research second only to the rhesus macaque . vervets are very similar in physiology and behavior to macaques , and they are more accessible and disease - free with less health and safety risks . vervet monkeys have a foveal binocular vision with a high cone density that decreases with eccentricity , trichromatic color vision , and a six - layered dorsal lateral geniculate nucleus [ 42 , 43 ] . recently , we have standardized a noninvasive , painless erg method for vervet monkeys   that showed highly comparable recordings to macaques   and humans . six of those monkeys were injected with am251 , and another six were injected with am630 . an additional 4 monkeys were injected with the vehicle ( dmso ) used to dilute of our antagonists in order to provide control values . the animals were fed with primate chow ( harlan teklad high protein monkey diet ; harlan teklad , madison , wi , usa ) and fresh local fruits , with water available ad libitum . all experiments were performed according to the guidelines of the canadian council on animal care ( ccac ) and the association for research in vision and ophthalmology ( arvo ) statement for the use of animals in ophthalmic and vision research . the experimental protocol was also reviewed and approved by the local animal care and use committee ( university of montreal , protocol # 14 - 007 ) and the institutional review board of the behavioral science foundation . briefly , all animals received an intramuscular injection of ketamine ( 10  mg / kg ; troy laboratories , glendenning , new south wales , australia ) and xylazine ( 1  mg / kg ; lloyd laboratories , shenandoah , ia , usa ) to maintain an adequate level of sedation that prevents the animals from moving and blinking . this drug mixture has no effect on the erg recordings . with 1% tropicamide ( mydriacyl ) and 2.5% phenylephrine hydrochloride ( mydfrin ) ( alcon laboratories , fort worth , tx , usa ) , the pupils were fully dilated ( approximately 9  mm in diameter ) , with the accommodation paralyzed . the cornea was anesthetized with 0.5% proparacaine hydrochloride ( alcaine ; alcon laboratories , fort worth , tx , usa ) . to prevent corneal drying , the eyes were moisturized frequently with 2.5% methylcellulose ( gonak ; akorn , inc . , buffalo grove , il , usa ) . after a recording session that lasted about 2 hours , the animals were sent back to their prior natural settings after a recovery period in isolation . the cb1r antagonist am251 was purchased from cayman chemicals ( ann arbor , mi , usa ) . the cb2r antagonist am630 was purchased from tocris ( tocris bioscience , ellisville , mo , usa ) . both antagonists were diluted in dmso under sterile conditions . assuming no leakage , the final concentration was 1.5% v / v for dmso , 0.01  mg/l for am251 , and 0.003  mg/l for am630 . to factor out any effects of the vehicle ( dmso ) , we subtracted the erg recordings of the dmso - injected animals from the erg recordings of the drug injected animals . in this way , the effects that we report are only those above and beyond effects of the vehicle . after inspection and examination of the eyes , a drop of the topical anesthetic , proparacaine , was then applied over the injection site . the conjunctival and corneal surfaces were further moistened with methylcellulose ( moisture eyes , bausch & lomb canada , vaughan , on , canada ) . the cornea was protected with sterile coatings while placing the barraquer eye speculum ( 1.75 inches , 10  mm wide small blades ) . a total of 50  l of drug solution was injected 2  mm posterior to the corneal limbus into the vitreous cavity . upon removal of the needle , the injection site was compressed for about one minute using a sterile cotton swab to avoid reflux . the back of the eye was inspected using an ophthalmoscope before and after the intravitreal injection to verify the integrity of the retina . no substantial differences were observed in intraocular pressure before and after the intravitreal administration . as a follow - up , the animals ' eyes were checked every day for seven days following injection , and a topical antibiotic ointment was administered ( tobrex , 0.3% tobramycin ophthalmic ointment , alcon canada , mississauga , canada ) . full - field stimulation was produced by a ganzfeld light source ( utas e-3000 electrophysiology equipment ; lkc technologies , inc . , gaithersburg , md , usa ) that was placed in front of the animal 's face . the ergs were evoked by < 5  ms white flashes delivered in full - field conditions . xenon flash luminance of 2.5 to 800  cdsm ( 0  db to 20  db in lkc units ) was used for photopic recordings and led flash luminance of 2.5  10 to 6  cdsm ( 40  db to 4  db in lkc units ) for scotopic recordings . for light - adapted ergs a steady background - adapting field ( 30  cdm ) was presented inside the ganzfeld to saturate the rod system . interstimulus intervals of at least 20 seconds were used at high intensities in the dark - adapted eyes . flash intensities and background luminance were calibrated using a research radiometer ( il1700 photometer , international light inc . , newburyport , ma , usa ) with a sed033 detector placed at 36  cm from the source . all erg procedures followed the iscev guidelines and the recently published standardized erg protocol of vervet monkeys . erg responses were recorded separately between corneal contact lens electrodes ( jet electrodes , diagnosys llc , lowell , ma , usa ) lying across the center of the cornea of each eye . the jet electrodes were equipped with four small posts on the convex surface in order to keep the eyelids open . reference and ground gold disc electrodes ( model f - e5gh ; grass technologies , astro - med , inc . , west warwick , ri , usa ) were , respectively , placed to the external canthi and forehead with adhesive paste ( ten20 conductive eeg paste , kappa medical , prescott , az , usa ) . for the analysis of the waveforms , the a - wave amplitude was measured from the baseline to the trough of the a - wave . the amplitude of the b - wave was measured from the trough of the a - wave to the peak of the b - wave . the peak latency was defined from the onset of the flash to the trough or peak . baselines and postinjection photopic amplitudes and latencies were calculated as averages to minimize the noise inherent in the erg signals and improve power , allowing for robust parametric statistical analysis . since there was only one baseline recording of the injected eye , the baseline value was calculated from an average across both eyes ( when available ) since ergs do not vary considerably across eyes . for the postinjection values , we had several recordings from the injected eye ( one every 10 minutes for 40 minutes ) . visual inspection revealed the peak effect to be present at both the 30 and 40 minutes of postinjection recordings . retinal response diagrams were drawn using adobe illustrator and processed in adobe indesign ( adobe systems canada , ottawa , on , canada , software version cs5 ) . the recording protocol for assessing the effect of the drugs the absolute trough ( a - wave ) and peak ( b - wave ) of the erg curves were obtained at each light intensity value . when the erg curve for low light intensities ( < 2  log  cdsm ) did not return to baseline 350  ms after the stimulus , the amplitudes of the a- and b - wave were corrected to account for the baseline shift . when no a - wave , or no wave at all , was detected , an amplitude of 0 was given and the latency was left blank for that specific stimulus intensity . postinjection amplitudes and latencies were expressed as percent of change from preinjection amplitudes ( postinjection minus preinjection , divided by preinjection ) . the delta change percentages of injecting am251 and am630 were then subtracted from the delta change percent for the control injection , the vehicle dmso . thus , positive normalized effects indicate an increase as a result of injecting the drug , greater than the change that results from injecting the vehicle alone . to assess the statistical significance of the observed increase , we analyzed the amplitudes of the a- and b - waves using general estimating equations ( gee ) with flash intensity as within subject factor , because each monkey was repeatedly measured ( at each flash intensity ) . these main effects , and their interaction , were used to estimate the normalized effects of retinal injection of am251 and am630 on retinal function . significant effects were followed up , when appropriate , with pairwise comparisons , significant values indicated with stars in the relevant figures . retinal function was evaluated using electroretinography in light - adapted conditions following injection of the vehicle dmso , the cb1r antagonist am251 , or the cb2r antagonist am630 ( figure 2 ) . our results show that the amplitudes of the b - wave after injection maintained a normal photopic hill shape indicating that the functional integrity of the retina was not impaired ( figure 3(a ) ) . gee analysis revealed a significant main effect of flash intensity ( p < .001 ) , and a significant interaction between the flash intensity and injection group ( p = .001 ) . am251 was not significantly different from dmso at any of the flash intensities ( figure 3(b ) ) . in contrast , am630 caused a significant increase in amplitude , relative to the control injection , across several flash intensities , from 0.6 to 1.6  log  cdsm ( 0.6 : 55% increase in amplitude relative to the control , p = .041 ; 0.9 : 53% increase , p = .038 ; 1.4 : 63% increase , p = .003 ; 1.6 : 60% increase , p = .011 ; significant effects indicated with  in figure 3(c ) ) . the main effect of am251 is , on average , a 6% increase , which is not significantly different from the vehicle alone , represented by zero on the y - axis of figures 3(b)3(d ) ( p = .713 ) . the main effect of am630 , averaged across all flash intensities , is a nonsignificant , but trending , 34% increase in responsiveness of the retina compared to the vehicle alone ( p = .067 , figure 3(d ) ) . latencies were also analyzed with the same gee model and the interaction ( p < .05 ) was followed up as above . pairwise comparisons revealed no significant differences , between the drugs and the vehicle , at any of the flash intensities ( not shown ) . our results show that , after injection , the amplitude of the a - wave followed the normal curve ( figure 4(a ) ) . gee analysis revealed a significant main effect of flash intensity ( p < .001 ) , and a significant interaction between the flash intensity and drug group ( p < .001 ) . this interaction indicates that the effect of the drugs was not the same across all flash intensities . am251 caused a significantly higher amplitude than dmso at the highest flash intensities of 2.4 and 2.9  log  cdsm ( 2.4 : 36% increase in amplitude relative to the vehicle , p = .040 ; 2.9 : 32% increase , p = .038 ; significant effects indicated with  in figure 4(b ) ) . for its part , am630 caused a significant increase in amplitude , relative to the control injection , across a larger set of flash intensities , from 0.9 to 2.9  log  cdsm ( ( 0.9 : 30% increase , p < .001 ; 1.4 : 38% increase , p = .002 ; 1.6 : 32% increase , p = .001 ; 2.4 : 39% increase , p = .015 ; 2.9 : 35% increase , p = .006 ) , significant effects indicated with  in figure 4(c ) ) . am251 caused an increase in the a - wave amplitude only at the highest flash intensities , while am630 increases the a - wave amplitude across a wider set of flash intensities . the main effect of am251 is , on average , a 12% increase , which is not significant relative to the vehicle alone ( p = .428 ) . conversely , the main effect of am630 , averaged across all flash intensities , is a nonsignificant but trending 26% increase in the responsiveness of the retina compared to the vehicle alone ( p = .080 , figure 4(d ) ) . latencies were also analyzed with the same gee model and the interaction ( p < .05 ) was followed up as above . pairwise comparisons at each intensity value revealed no significant differences between the drugs and the vehicle ( not shown ) . to assess the effect of the cannabinoid receptor antagonists in scotopic conditions , erg responses of the dark - adapted retina however , the amplitudes of the b - wave were increased for both treatment groups , while the drugs did not reliably alter the pattern of the a - waves ( figure 5 ) . after injection , the amplitude of the scotopic b - waves had the normal shape : increasing amplitudes for increasing flash intensities ( figure 6(a ) ) . gee analysis revealed a significant main effect of injection group ( p < .001 ) . there was no main effect of flash intensity ( p = .842 ) nor was there an interaction of group with flash intensity ( p = .953 ) . due to a lack of interaction , pairwise comparisons at each flash intensity were not justified , but mean and standard errors are plotted in figure 6(b ) ( am251 ) and figure 6(c ) ( am630 ) . following up on the main effect of drug , pairwise comparisons between groups revealed significantly higher amplitudes following the injection of am251 compared to the vehicle alone ( 20% increase , p < .001 ) and a similar increase in amplitude following injection of am630 ( 18% increase , p = .002 ) ( figure 6(d ) ) . both pharmacological agents led to a significant increase in the latency relative to the vehicle ( am251 : 8% , p = .036 ; am630 : 12% , p = .001 ) . after injection , the amplitude of the scotopic a - wave had the normal shape : increasing amplitudes for increasing flash intensities , beginning at 1  log  cdsm ( figure 7(a ) ) . therefore , the statistical analysis for the scotopic a - wave only involved the values obtained from the flashes at 1 to 1.4  log  cdsm . gee analysis revealed a significant main effect of flash intensity ( p = .001 ) and a significant interaction between the intensity of the flash and the drug injected ( p < .001 ) . the interaction was followed up with pairwise comparisons , between the drugs and the vehicle alone , at each flash intensity value . this revealed no significant differences , between the drugs and the vehicle , at any of the intensities ( figures 7(b ) and 7(c ) ) . thus , while the two agonists cause varied effects at the different flash intensities , the effects of each drug relative to the vehicle alone , at a given flash intensity , were not reliable enough to be significant . the overall effect of am251 and am630 , averaged across the flash intensities , was not significantly different from the vehicle alone ( figure 7(d ) ) . latencies were also analyzed with the same gee model and the interaction ( p < .05 ) was followed up as above . pairwise comparisons revealed one significant difference at flash intensity 0.6 ; following injection of am251 , the latency to peak was 21% slower compared to the injection of the vehicle alone ( p = .011 , not shown ) . cb2 in the normal monkey retina . the abundance of cb1r and cb2r expression in the retina already pointed to an important role of these receptors in normal vision . we analyzed changes in photopic and scotopic erg responses after blocking these receptors with their respective antagonists . the experimental design used dmso as the control , rather than preinjection values , to control for any effects of the vehicle . we demonstrated that , in photopic conditions , only the blockade of cb2r increased the amplitude of the b - wave , above the standard flash intensity value , while blocking cb1r or cb2r increased the amplitude of the a - wave , at high flash intensity values . in scotopic conditions , however , blockade of either cb1r or cb2r increased only the amplitude of the b - wave irrespective of flash intensity . the amplitude of the main component of the erg , the photopic b - wave , represents primarily the activation of depolarization on - bipolar cells measured as a positive retinal potential on the corneal surface [ 36 , 4850 ] . in addition , the b - wave is attributed to the interaction of on - bipolar cells and mller glial cells [ 36 , 37 ] . in the vervet monkey , cb1r is expressed mainly in cones and in the other retinal components , while cb2r is exclusively present in the glial mller cells , leading to a complementary relationship between neurons and glia regarding endocannabinoid action . the light - induced potassium increase in the outer and inner plexiform layers ' cells , which are depolarized by light stimulation , mller cells , via kcnj10 ( kir4.1 ) channels and potassium siphoning of the excess potassium ions into the vitreous [ 52 , 53 ] , control the light - mediated potassium increase in retinal extracellular space . the depolarization of the mller cells contributes to the erg b - wave through the buffering of potassium channels [ 35 , 55 , 56 ] . thus , the blockade of potassium channels should result in a decrease of the erg b - wave . our results revealed a significant increase of the photopic b - wave amplitude following the blockade of cb2r , which supports our previously proposed model . pka is a positive modulator of potassium channels and therefore , the blockade of cb2r via an increase of pka activity will increase the activity of k channels in mller cells , and thus an increased photopic b - wave amplitude ( figure 8) . it may also be possible that am630 affected the off cone pathway that originates from the dendritic contacts of bipolar cells with cones , which could partially explain the increase of the photopic b - wave amplitude only at the middle intensity flash values . another potential interpretation is that since cb2r is not expressed on cones , am630 may have modulated other non - cb2 receptors located on cone photoreceptors . stimulation of cones by light inhibits retinal dark currents through phototransduction signals that take place in the cone outer segments as seen in the a - wave of the erg . the early portion of the a - wave represents the activity of the cone photoreceptors [ 59 , 60 ] , while the later portion reflects the contribution of hyperpolarizing bipolar cells , proximal amacrine cells , and ganglion cells [ 6163 ] . stimulation of cones may activate the cb1r in its pedicles , which in turn leads to the inhibition of glutamate release in the synaptic cleft . this increase mimics the effect of a bright light that contributes to larger amplitude of the photopic a - wave . blocking cb2r on the other hand has an even larger effect on the amplitude of the a - wave , which can be explained by a similar mechanism that involves additional potassium buffering by mller cells   ( figure 8(a ) ) . other receptors that contribute to the photopic b - wave may also explain how am630 could affect the photopic a - wave . indeed , the increase of the photopic b - wave might cause a large change in the a - wave ( a result of cone modulation ) , which would be independent of the action of mller cells . the a - wave measured under scotopic conditions represents rod function . in the dark - adapted retina , blockade of either cb1r or cb2r this null effect can be explained by the small quantity of cb1r expressed in the rod spherules in primates [ 3 , 5 ] . in contrast , the large quantity of putative cannabinoid receptor ( gpr55 ) , found exclusively in rods , has a significant effect on the scotopic erg . thus , we can not rule out that the increase of the scotopic b - wave amplitude following the injection of am251 might be due to gpr55 activity . however , cb1r is found in large quantities in rod bipolar cells   and , in conjunction with cb2r in mller cells , likely contributes to the large increase of the scotopic b - wave amplitude . differential effects between cb1r and cb2r might be explained by the nature of the ion channels involved . the potassium buffering role of mller cells leads to the increase of the scotopic b - wave following cb2r blockade . the calcium increase in postsynaptic rod bipolar cells results from cb1r blockade ( see figure 8(b ) ) . since cb1r agonists induce a reduction in the amplitude of calcium channel currents in retinal bipolar cells , it is not surprising , as shown here , that the cb1r antagonist am251 had the opposite effect : mainly , an increase in rod bipolar cells activity . these findings might be helpful for the development of new pharmacological targets for the treatment of retinal intoxication [ 67 , 68 ] and diseases . these retinal pathologies are generally associated with a decrease in the amplitude of the electroretinographic waves . we show here that pharmacological agents that block the retinal cannabinoid receptors can induce an increase in the amplitude of the erg response profiles . manipulating the endocannabinoid system might therefore serve as a therapy to restore normal vision and protect the retina .
the expression patterns of the cannabinoid receptor type 1 ( cb1r ) and the cannabinoid receptor type 2 ( cb2r ) are well documented in rodents and primates . in vervet monkeys 
 , cb1r is present in the retinal neurons ( photoreceptors , horizontal cells , bipolar cells , amacrine cells , and ganglion cells ) and cb2r is exclusively found in the retinal glia ( mller cells ) . 
 however , the role of these cannabinoid receptors in normal primate retinal function remains elusive . 
 using full - field electroretinography in adult vervet monkeys , we recorded changes in neural activity following the blockade of cb1r and cb2r by the intravitreal administration of their antagonists ( am251 and am630 , resp . ) in photopic and scotopic conditions . 
 our results show that am251 increases the photopic a - wave amplitude at high flash intensities , whereas am630 increases the amplitude of both the photopic a- and b - waves . in scotopic conditions , both blockers increased the b - wave amplitude but did not change the a - wave amplitude . 
 these findings suggest an important role of cb1r and cb2r in primate retinal function .