December 2013
Volume 54, Issue 13
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Retina  |   December 2013
Roles of Cannabinoid Receptors Type 1 and 2 on the Retinal Function of Adult Mice
Author Affiliations & Notes
  • Bruno Cécyre
    Laboratoire des neurosciences de la vision, École d'optométrie, Université de Montréal, Québec, Canada
    Laboratoire de neuropharmacologie, École d'optométrie, Université de Montréal, Québec, Canada
  • Nawal Zabouri
    Laboratoire des neurosciences de la vision, École d'optométrie, Université de Montréal, Québec, Canada
    Laboratoire de neuropharmacologie, École d'optométrie, Université de Montréal, Québec, Canada
  • Frédéric Huppé-Gourgues
    Laboratoire de neurobiologie de la cognition visuelle, École d'optométrie, Université de Montréal, Montréal, Québec, Canada
  • Jean-François Bouchard
    Laboratoire de neuropharmacologie, École d'optométrie, Université de Montréal, Québec, Canada
  • Christian Casanova
    Laboratoire des neurosciences de la vision, École d'optométrie, Université de Montréal, Québec, Canada
  • Correspondence: Jean-François Bouchard, Laboratoire de neuropharmacologie, École d'optométrie, Université de Montréal, C.P. 6128 Succursale Centre-Ville, Montréal, QC, Canada H3C 3J7; jean-francois.bouchard@umontreal.ca
Investigative Ophthalmology & Visual Science December 2013, Vol.54, 8079-8090. doi:10.1167/iovs.13-12514
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      Bruno Cécyre, Nawal Zabouri, Frédéric Huppé-Gourgues, Jean-François Bouchard, Christian Casanova; Roles of Cannabinoid Receptors Type 1 and 2 on the Retinal Function of Adult Mice. Invest. Ophthalmol. Vis. Sci. 2013;54(13):8079-8090. doi: 10.1167/iovs.13-12514.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Endocannabinoids are important modulators of synaptic transmission and plasticity throughout the central nervous system. The cannabinoid receptor type 1 (CB1R) is extensively expressed in the adult retina of rodents, while CB2R mRNA and protein expression have been only recently demonstrated in retinal tissue. The activation of cannabinoid receptors modulates neurotransmitter release from photoreceptors and could also affect bipolar cell synaptic release. However, the impact of CB1R and CB2R on the retinal function as a whole is currently unknown.

Methods.: In the present study, we investigated the function of cannabinoid receptors in the retina by recording electroretinographic responses (ERGs) from mice lacking either CB1 or CB2 receptors (cnr1−/− and cnr2−/− , respectively). We also documented the precise distribution of CB2R by immunohistochemistry.

Results.: Our results showed that CB2R is localized in cone and rod photoreceptors, horizontal cells, some amacrine cells, and bipolar and ganglion cells. In scotopic conditions, the amplitudes of the a-wave of the ERG were increased in cnr2−/− mice, while they remained unchanged in cnr1−/− mice. The analysis of the velocity-time profile of the a-wave revealed that the increased amplitude was due to a slower deceleration rather than an increase in acceleration of the waveform. Under photopic conditions, b-wave amplitudes of cnr2−/− mice required more light adaptation time to reach stable values. No effects were observed in cnr1−/− mice.

Conclusions.: The data indicated that CB2R is likely to be involved in shaping retinal responses to light and suggest that CB1 and CB2 receptors could have different roles in visual processing.

Introduction
Cannabinoids (CBs) are the principal psychoactive components of marijuana plant ( Cann abis sativa ). The endocannabinoid (eCB) system is involved in a variety of neurobiological functions such as signal processing, nociception, learning and memory, and motor coordination. Endocannabinoids are generally associated with the modulation of neuronal transmission and, more recently, with developmental processes. In the last decade, several studies have shown that CB receptors (CBRs) are present in the retina of most mammals including humans, 1 suggesting that the eCB system could be involved in some aspects of visual processing. 
The cannabinoid receptor type 1 (CB1R) is ubiquitously expressed in the nervous system. In the retina, its presence has been shown in several species, ranging from fishes to primates 29 (see Yazulla 10 for review). Briefly, CB1R is present in cones, horizontal cells, some bipolar cells, and amacrine and ganglion cells. Patch-clamp studies have demonstrated that CB1R activation differentially modulates ion channels in photoreceptors and glutamate synaptic release. 1113 In bipolar cells, CB1R activation inhibits calcium 2 and potassium rectifying 14 currents. Functionally, these effects could lead to a decreased synaptic release and changes in the temporal aspects of bipolar cells' response. In addition, CB1R activation can also modulate gamma-aminobutyric acid (GABA)ergic release from amacrine cells 8 and inhibit high-voltage–activated calcium channel in cultured ganglion cells, which impacts the cells' excitability. 9 It is not known how the reported effects are reflected at the global functional level. 
Although cannabinoid receptor type 2 (CB2R) expression in neurons remains controversial, several authors have reported a sparse expression in neurons in several structures such as the cerebellum, brainstem, hippocampus, and prefrontal cortex (see Atwood and Mackie 15 for review). Only two studies 16,17 have demonstrated the presence of CB2R in the rodent retina. From its mRNA distribution, Lu et al. 16 have localized CB2R in photoreceptors, inner nuclear layer, and ganglion cell layer in the adult rat retina. While this study provides valuable information, the results do not confirm the presence of CB2R in the retina, since G protein–coupled receptor mRNA and protein expression at the cell surface do not necessarily correlate. 18 Using cells' morphology and position, CB2R protein has been more precisely localized by Lopez et al. 17 in the same animal model: it was found in the inner segment of photoreceptors and in horizontal, amacrine, and ganglion cells. However, the CB2R antibody used by these authors was not fully characterized. Thus, the exact localization of CB2R in retinal cells remains an open question. To our knowledge, no studies have investigated the effect of CB2R activation at the retinal level. 
The objective of this study was to evaluate the roles of CBRs in the retinal function in a mammalian in vivo model. To achieve this goal, electroretinograms (ERGs, representing the global evoked response of the retina) of mice lacking CB1 or CB2 receptors (cnr1−/− and cnr2−/− , respectively) and their wild-type (WT) controls were compared under different light conditions. Given that the exact expression of pattern CB2R in the retina remains debatable, the presence of CB2R in the mice retina was confirmed and precisely assessed with a specific CB2R antibody and specific retinal markers. Our results indicate that CB2R is largely expressed by retinal neurons of mice and suggest that this receptor is likely to play a greater role in retinal processing than CB1R. 
Methods
Animals
All procedures were performed in accordance with the guidelines from the Canadian Council on Animal Care and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and were approved by the Ethics Committee on Animal Research of the Université de Montréal. The cnr1−/− and cnr2−/− transgenic mice were obtained from Beat Lutz (Institute of Physiological Chemistry and Pathobiochemistry, University of Mainz, Germany) and Jackson Laboratory (Bar Harbor, ME), respectively. The cnr1−/− and cnr2−/− mice were on a C57BL/6N and C57BL/6J genetic background, respectively. Both transgenic mice were compared to background and age-matched WT controls from separate colonies. All animals were maintained in-house, under a 12-hour dark/light cycle. Male and female adult mice (3–4 months old) were used for the experiments. 
Tissue Preparation
Mice were euthanized by an overdose of isoflurane. One eye was immediately removed for Western blot analysis. The retina was dissected on ice, promptly frozen, and kept at −80°C until further processing. Simultaneously, a transcardiac perfusion was conducted with phosphate-buffered 0.9% saline (PBS; 0.1 M, pH 7.4), followed by phosphate-buffered 4% paraformaldehyde (PFA), until the head was lightly fixed. The nasal part of the second eye was marked with a suture and removed. Two small holes were made in the cornea before a first postfixation in 4% PFA for a period of 30 minutes. The cornea and lens were removed and the eyecups were postfixed for 30 minutes in 4% PFA. The eyecups were then washed in PBS, cryoprotected in 30% sucrose overnight, embedded in Neg 50 tissue Embedding Media (Fisher Scientific, Ottawa, ON, Canada), flash-frozen and kept at −80°C. Sections (14 μm thick) were cut with a cryostat (Leica Microsystems, Concord, ON, Canada) and placed on gelatin/chromium-coated slides. 
Western Blot
Retinas were homogenized in RIPA buffer (150 mM NaCl, 20 mM Tris, pH 8.0, 1% NP-40, 0.1% SDS, 1 mM EDTA), supplemented with a protease inhibitor mixture (aprotinin, leupeptin, pepstatin [1 μg/mL] and phenylmethylsulfonyl fluoride [0.2 mg/mL]; Roche Applied Science, Laval, QC, Canada). Thirty micrograms of protein per sample of the homogenate were resolved by electrophoresis on a 10% SDS-polyacrylamide gel, transferred onto a nitrocellulose membrane, blocked with 5% skim milk, and incubated overnight with antibodies directed against CB2R, CB1R, N-acyl phosphatidyethanolamine phospholipase D (NAPE-PLD), diacylglycerol lipase α (DAGLα), fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MGL), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the latter serving as a loading control. The blots were exposed to the appropriate horseradish peroxidase–coupled secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA). Detection was carried out by using homemade ECL Western blot detection reagents (final concentrations: 2.5 mM luminol, 0.4 mM p-coumaric acid, 0.1 M Tris-HCl pH 8.5, 0.018% H2O2). 
Immunohistochemistry
Frozen sections were washed in PBS, postfixed for 10 minutes in cold acetone, washed, and then blocked in 1% bovine serum albumin, bovine gelatin, and 0.5% Triton X-100 in PBS for 1 hour. Some sections from WT animals were incubated overnight in rabbit anti-CB2R solution with an antibody against various retinal markers. In addition, sections from the three genotypes were incubated with retinal markers (see Table 1) to compare the distribution and morphology of retinal cells. The sections were then washed in PBS, blocked for 30 minutes, incubated for 1 hour with Alexa Fluor 647 donkey anti-rabbit for CB2R and Alexa Fluor 488 donkey anti-rabbit or Alexa Fluor 488 donkey anti-mouse for cell type markers (Molecular Probes, Eugene, OR). After washes, the sections were mounted with homemade PVA-Dabco mounting medium. The CB2R/recoverin, CB2R/cone-arrestin, 1921 and CB2R/calbindin combinations required serial incubations, as the retinal marker host was rabbit, as previously performed on retinal tissues. 6 The dilution factors, the immunogens, and provenance of the antibodies are provided in Table 1
Table 1
 
Antibodies Used in This Study
Table 1
 
Antibodies Used in This Study
Antibody Target Immunogen Dilution Host Provenance
CB2R Cannabinoid receptor type 2 N-terminus 14 aa of human CB2R (20–33 residues) I, 1:200 W, 1:2000 Rabbit 101550; Cayman Chemical, Ann Arbor, MI
CB1R Cannabinoid receptor type 1 N-terminus 77 aa of rat CB1R (1–77 residues) W, 1:1000 Rabbit C1233; Sigma-Aldrich, Oakville, ON, Canada
NAPE-PLD N-acyl phosphatidyethanolamine phospholipase D N-terminus 13 aa of mouse NAPE-PLD (9–21 residues) W, 1:500 Rabbit NB110-80070; Novus Biologicals, Oakville, ON, Canada
DAGLα Diacylglycerol lipase α C-terminus 42 aa of mouse DAGLα (1003–1044 residues) W, 1:200 Rabbit DGLa-Rb-Af380; Frontier Institute, Ishikari, Hokkaido, Japan
FAAH Fatty acid amide hydrolase C-terminus 19 aa rat FAAH (561–579 residues) W, 1:3000 Rabbit 101600; Cayman Chemical
MGL Monoacylglycerol lipase N-terminus 35 aa of mouse MGL (1–35 residues) W, 1:200 Rabbit MGL-Rb-Af200; Frontier Institute
GAPDH Loading control The full-length rabbit muscle GAPDH protein W, 1:20,000 Mouse G8795; Sigma-Aldrich
Mouse cone-arrestin (LUMIj) Cone photoreceptors C-terminus 13 aa of the mCAR protein (369–381 residues) I, 1:1000 Rabbit Cheryl M. Craft, Mary D. Allen Laboratory for Vision Research, Doheny Eye Institute, USC, Los Angeles, CA
Recoverin Rod photoreceptors, and ON and OFF cone bipolar cells The full-length recombinant human recoverin I, 1:2000 Rabbit AB5585; EMD Milipore Corporation, Billerica, MA
Calbindin (D-28K) Horizontal and amacrine cells Recombinant rat calbindin D-28K full length I, 1:1000 Rabbit CB-38a; Swant, Marly, Switzerland
PKC (clone H7) Rod bipolar cells C-terminus 28 aa of human protein (645–672 residues) I, 1:500 Mouse Sc-8393; Santa Cruz Biotechnology, Santa Cruz, CA
Syntaxin (clone HPC1) Amacrine cells Synaptosomal plasma fraction of rat hippocampus I, 1:500 Mouse S0664; Sigma-Aldrich
Brn-3a (clone 5A3.2) Ganglion cells Amino acids 186–224 of Brn-3a fused to the T7 gene 10 protein I, 1:100 Mouse MAB1585; EMD Millipore Corporation
Glutamine synthetase (GS, clone GS-6) Müller cells The full protein purified from sheep brain I, 1:3000 Mouse MAB302; EMD Millipore Corporation
Antibody Characterization
The CB2R antibody has been characterized in mice renal tissue 22 ; consequently, we tested its specificity in the mice retina by Western blot and immunohistochemistry. The anti-CB2R reacted with a robust band at 45 kDa (Fig. 1A), which was absent with the addition of the immunizing peptide. No unspecific signal could be observed on retinal sections when CB2R antibody was preincubated with its immunizing peptide (Fig. 1B). Moreover, no unspecific signal was visible in the cnr2−/− mice (Fig. 1C), whereas the CB2R staining in the WT mouse yielded a strong signal in the retina (Fig. 1D). It should be noted that this antibody showed inconsistent results from one lot to another. These results display the specificity of lot No. 0424681-1. 
Figure 1
 
Characterization of an antibody directed against CB2R in adult mice retina. Immunoblots of CB2R immunoreactivity in a WT mouse retina and in presence of its blocking peptide (BP) (A). Specific band was seen at around 45 kDa in WT group only. GAPDH was used as a loading control. CB2R immunoreactivity in the retina of a WT mouse in presence of its appropriate BP (B), in cnr2−/− mice (C), and in WT mouse (D). All images were taken with identical microscopy settings. Scale bar: 50 μm.
Figure 1
 
Characterization of an antibody directed against CB2R in adult mice retina. Immunoblots of CB2R immunoreactivity in a WT mouse retina and in presence of its blocking peptide (BP) (A). Specific band was seen at around 45 kDa in WT group only. GAPDH was used as a loading control. CB2R immunoreactivity in the retina of a WT mouse in presence of its appropriate BP (B), in cnr2−/− mice (C), and in WT mouse (D). All images were taken with identical microscopy settings. Scale bar: 50 μm.
Confocal Microscopy
Images of the central retina (within 200 μm of the optic nerve head) were taken with a laser scanning confocal microscope (TCS SP2; Leica Microsystems), with an ×40 oil immersion objective. Image stacks (1024 × 1024 pixels × 0.5 μm per stack) were captured by using the Leica confocal (LCS) software (version 2.6.1; Leica Microsystems). Offline processing was done with the Fiji software (version 1.47g; www.fiji.sc). 23 The stack images were taken sequentially to ensure no “bleed-through” between channels. Gaussian noise from images was partially removed by using the PureDenoise plugin for Fiji 24 and stacks were collapsed to projection images. 
Electroretinography
The ERG recordings were performed as previously reported. 25 Briefly, after an overnight dark adaptation (at least 12 hours of complete darkness), mice were anesthetized (ketamine 85 mg/kg, xylazine 5 mg/kg). Pupils were dilated with a drop of 1% cyclopentolate hydrochloride. The mice were then positioned in a Ganzfeld dome that housed a photostimulator (PS33plus; Grass Instruments, Middleton, WI). The ERGs were recorded with a silver wire electrode inserted in a custom-made corneal lens adapted for mice. 26 Reference and ground electrodes (E2 subdermal electrode; Grass Instruments) were inserted subcutaneously at the base of the head and in the tail. Broadband ERGs (bandwidth, 1–1000 Hz; 10,000×) (P511; Grass Instruments) and oscillatory potentials (bandwidth, 100–1000 Hz; 50,000×) were recorded simultaneously. Signals were fed to an analog-digital interface (1401; CED, Cambridge, UK) and were acquired by using the software Signal (v.3.01x; CED). Scotopic luminance-response functions were obtained in response to progressively brighter stimuli spanning a 3.97 log-unit range (interstimulus interval: 10 seconds; average: 5 flashes; luminance interval −2.3 to 1.67 log scot cd-s/m2). The photopic (cone-mediated) signal was recorded thereafter: Electroretinograms were recorded every 5 minutes for 20 minutes under a photopic background of 30 cd/m2 (flash luminance: 1.36 log photo cd-s/m2; interstimulus interval: 1 second, averaged over 20 flashes). 
Analysis
Analysis of the ERG waveforms was performed according to standard practice. 27 The amplitude of the a-wave was measured from baseline to the most negative trough, whereas the b-wave amplitude was measured from the trough of the a-wave to the highest positive peak of the retinal response. The kinetics of the a-wave was also analyzed by calculating the velocity-time profile at a photoreceptor saturating luminance 28 computed as the derivative of the waveform. All the points of the a-wave were taken into account. The last point of the waveform was set as the first derivative value equal or superior to 0. Implicit times were measured from flash onset to the peak of the waves. Scotopic luminance-response function curves were obtained by plotting the amplitude of the b-wave against the luminance of the flash used to evoke each response. 
The mixed V max referred to the highest evoked amplitude of the b-wave. The amplitude of oscillatory potentials (OPs) was measured and reported as the sum of the OPs (OP1/2 + OP3 + OP4 + OP5 in scotopic conditions and OP1 + OP2 + OP3 in photopic conditions). In a randomly chosen subgroup of animals, a fast Fourier transform analysis was performed with Matlab (The Mathworks, Nattick, MA) to confirm that no changes occurred in the power/frequency distribution of the ERG waveform. Statistical analysis was performed by using repeated measures ANOVA followed by one-way ANOVA and Dunnett post hoc test (SPSS 20; IBM, Somers, NY). 
Quantification of Retinal Layer Thickness
Frozen sections were washed in PBS, and their nuclei were labeled with Sytox Green Nucleic Acid Stain (1:100,000; Molecular Probes) for 1 hour. The thickness of each retinal layer was measured at ×40 magnification, including the outer nuclear layer (ONL), the outer plexiform layer (OPL), the inner nuclear layer (INL), the inner plexiform layer (IPL), and the ganglion cell layer (GCL). The thickness of each layer was measured with LCS software (version 2.6.1; Leica Microsystems) and was normalized with respect to the total thickness of the retina to correct for local and interanimal variations. Statistical analysis was performed by using one-way ANOVA. 
Results
Localization of CB2R in the Mice Retina
Our data indicated that the CB2 receptor is widely distributed in the retina. CB2R immunoreactivity in the photoreceptor layer was mostly found in cones, but rods were also labeled. CB2R was present in the outer and inner segments, in the cell body, but not in the pedicles of cones (Fig. 2A). It was also expressed in the inner and outer segments and cell body of rod photoreceptors (Fig. 2B). 
Figure 2
 
Double-label immunofluorescence illustrating colocalization of CB2R throughout the mouse retina. CB2R (magenta) is present in inner (arrowheads) and outer (arrows) segments of cones (mouse cone-arrestin), but not in pedicles (circles; [A]), rod photoreceptors (recoverin; [B]), horizontal cells (calbindin; arrows) and their dendrites (arrowheads; [C]), cone (recoverin; [D]) and rod (PKC) bipolar cells' soma (arrows; [E]), their presynaptic connections (arrowheads; [E]) and axons (arrows) but not in postsynaptic connections (arrowheads; [F]), amacrine cells (syntaxin; [G]), ganglion cells (Brn-3a; [H]), and Müller cells (glutamine synthetase; [I]). Scale bar: 10 μm.
Figure 2
 
Double-label immunofluorescence illustrating colocalization of CB2R throughout the mouse retina. CB2R (magenta) is present in inner (arrowheads) and outer (arrows) segments of cones (mouse cone-arrestin), but not in pedicles (circles; [A]), rod photoreceptors (recoverin; [B]), horizontal cells (calbindin; arrows) and their dendrites (arrowheads; [C]), cone (recoverin; [D]) and rod (PKC) bipolar cells' soma (arrows; [E]), their presynaptic connections (arrowheads; [E]) and axons (arrows) but not in postsynaptic connections (arrowheads; [F]), amacrine cells (syntaxin; [G]), ganglion cells (Brn-3a; [H]), and Müller cells (glutamine synthetase; [I]). Scale bar: 10 μm.
Horizontal cells showed expression of CB2R at the membrane of the soma and in their dendrites (Fig. 2C). Both rod and cone bipolar cells were CB2R immunoreactive at the membrane of the soma (Figs. 2D, 2E). Rod bipolar cells also expressed CB2R at their dendrites and axons (Figs. 2E, 2F). Some amacrine cells showed also CB2R immunoreactivity at the membrane of the soma (Fig. 2G). 
CB2R staining was detected in the GCL and was present in the ganglion cells' soma (Fig. 2H). CB2R was not expressed at the membrane of the Müller cells' soma or in their inner and outer processes (Fig. 2I). 
The Dark-Adapted Retinal Function
We recorded dark-adapted retinal responses in WT, cnr1−/− , and cnr2−/− mice at different light intensities. Representative examples of scotopic ERGs from WT, cnr1−/− , and cnr2−/− mice are shown in Figure 3. The averaged a- and b-wave amplitudes are presented as a function of flash luminance in Figures 4A and 4B, respectively. A significant change in the a-wave amplitude was observed in cnr2−/− but not in cnr1−/− mice (repeated measures ANOVA with Dunnett post hoc test, for intensities 0.13 to 1.67 log scot cd-s/m2, P = 0.798 for cnr1−/− and P = 0.037 for cnr2−/− mice). The amplitude of the b-wave (Fig. 4C) was not significantly altered in cnr1−/− or cnr2 −/− mice. To better understand the change in the a-wave, the velocity-time profile was calculated at a saturating luminance (1.3 log scot cd-s/m2). The averaged a-wave for all three groups is presented in Figure 4C. One can observe that the earliest part of the waveforms overlay and only separate in the latest segment. The averaged velocity-time profiles for all three groups are presented in Figure 4D. No differences were found in the accelerating (descending) portion of the curve (repeated measures ANOVA with Dunnett post hoc test from 0 to 7.4 ms). We confirmed those results by analyzing the a-wave amplitude at 7 ms 29 and found no significant differences between the groups. The deceleration (ascending) portion, however, showed significant changes between groups as cnr2−/− mice maintained higher velocities than WT animals (repeated measures ANOVA with Dunnett post hoc test from 7.4 to 15 ms, P = 0.729 for cnr1−/− and P = 0.018 for cnr2−/− mice). Fourier analysis was also performed on both a- and b-waves and revealed no other significant changes (data not shown). 
Figure 3
 
Example of scotopic ERGs in the different genotypes. Representative ERGs recorded in WT, cnr1−/− , and cnr2−/− mice. The luminance-response function of each animal was established by presenting progressively brighter flashes (bottom to top) indicated to the right of the traces as the log luminance (scot cd-s/m2).
Figure 3
 
Example of scotopic ERGs in the different genotypes. Representative ERGs recorded in WT, cnr1−/− , and cnr2−/− mice. The luminance-response function of each animal was established by presenting progressively brighter flashes (bottom to top) indicated to the right of the traces as the log luminance (scot cd-s/m2).
Figure 4
 
Amplitudes of scotopic ERG a- and b-waves plotted as a function of flash luminance. Scotopic ERG a-wave (A) and b-wave (B) amplitudes are displayed by genotype. The values are means ± SEM from all animals in each group (WT: open circles, n = 20; cnr1−/− : grey circles, n = 20; cnr2−/− : closed circles, n = 18; #repeated measures ANOVA, P ≤ 0.05, *one-way ANOVA with Dunnett post hoc test, P ≤ 0.05). (C) The averaged a-wave for each group is plotted as a function of time. (D) The velocity-time profile of the a-wave for each group displays differences between cnr2−/− and WT mice in the deceleration segment on the waveform (repeated measures ANOVA, P ≤ 0.05; *one-way ANOVA with Dunnett post hoc test, P ≤ 0.05).
Figure 4
 
Amplitudes of scotopic ERG a- and b-waves plotted as a function of flash luminance. Scotopic ERG a-wave (A) and b-wave (B) amplitudes are displayed by genotype. The values are means ± SEM from all animals in each group (WT: open circles, n = 20; cnr1−/− : grey circles, n = 20; cnr2−/− : closed circles, n = 18; #repeated measures ANOVA, P ≤ 0.05, *one-way ANOVA with Dunnett post hoc test, P ≤ 0.05). (C) The averaged a-wave for each group is plotted as a function of time. (D) The velocity-time profile of the a-wave for each group displays differences between cnr2−/− and WT mice in the deceleration segment on the waveform (repeated measures ANOVA, P ≤ 0.05; *one-way ANOVA with Dunnett post hoc test, P ≤ 0.05).
Furthermore, the maximal ERG response evoked in scotopic conditions (the mixed rod-cone response) was also analyzed. The amplitude of the b-wave at maximal b-wave amplitude remained unchanged in all experimental groups (Table 2). No changes were observed in the luminance needed to evoke the maximal response in either group. No differences were observed in the latency of the a- and b-waves in both knockout (KO) groups, suggesting no involvement of CBRs in the kinetics of the response onset (data not shown). 
Table 2
 
Group Data Reporting the Amplitudes of Mixed V max as Well as the Light Intensities Evoking Them
Table 2
 
Group Data Reporting the Amplitudes of Mixed V max as Well as the Light Intensities Evoking Them
WT cnr1−/− cnr2−/−
Mixed V max, μV 369 ± 29 371 ± 26 446 ± 22
Light luminance mixed V max, log scot cd-s/m2 1.27 ± 0.04 1.13 ± 0.05 1.06 ± 0.09
The OPs were also analyzed, and representative examples of recordings for WT and cnr2−/− groups are presented in Figure 5A.The sum of all dark-adapted OPs was computed and compared. The averaged total amplitude is presented as a function of flash luminance in Figure 5B. We observed a tendency for the amplitude of the OPs to be larger between −0.13 to 1.30 log scot cd-s/m2 in cnr2−/− than in WT animals. However, this tendency did not reach statistical significance. No changes were observed in the response of the cnr1−/− or cnr2−/− mice (repeated measures ANOVA, for intensities 0.13 to 1.67 log scot cd-s/m2, P = 0.345). Fourier analysis was also performed and revealed no differences between the three genotypes (data not shown). No differences were observed in the latency of any of the analyzed OPs for either mice strain (results not shown). 
Figure 5
 
Example of scotopic OPs in the different groups. (A) Representative OPs recorded in WT and cnr2−/− mice. (B) The total sum of the amplitudes of all OPs is plotted as a function of flash luminance to compute the luminance-response function curve. The values are means ± SEM from all animals in each group (WT: open circles, n = 17; cnr1−/− : grey circles, n = 19; cnr2−/− : closed circles, n = 12).
Figure 5
 
Example of scotopic OPs in the different groups. (A) Representative OPs recorded in WT and cnr2−/− mice. (B) The total sum of the amplitudes of all OPs is plotted as a function of flash luminance to compute the luminance-response function curve. The values are means ± SEM from all animals in each group (WT: open circles, n = 17; cnr1−/− : grey circles, n = 19; cnr2−/− : closed circles, n = 12).
The Light-Adapted Retinal Function
To evaluate the adaptation abilities of the retina, the ERG was recorded under photopic conditions at different time points. The averaged amplitude ± SEM of the photopic b-wave is displayed as a function of time in Figure 6. While the responses observed in cnr1−/− and WT mice are comparable, those in cnr2−/− mice have higher amplitudes at intermediate time points (one-way ANOVA, P = 0.021 and P = 0.018 for 10 and 15 minutes, respectively). The latency of the photopic b-wave did not vary across groups (data not shown). Light-adapted OPs were also analyzed and no significant changes were observed for any of the experimental groups (summed amplitude values; results not shown). 
Figure 6
 
The amplitudes of photopic ERG b-wave are plotted as a function of time. Repeated ERGs were taken at different times and were used to evaluate the retina ability to adapt to light from a dark-adapted state. The cnr2−/− mice displayed significantly higher amplitudes at 10- and 15-minute time points (means ± SEM; WT: open circles, n = 19; cnr1−/− : grey circles, n = 18; cnr2−/− : closed circles, n = 18; *one-way ANOVA with Dunnett post hoc test, P < 0.05).
Figure 6
 
The amplitudes of photopic ERG b-wave are plotted as a function of time. Repeated ERGs were taken at different times and were used to evaluate the retina ability to adapt to light from a dark-adapted state. The cnr2−/− mice displayed significantly higher amplitudes at 10- and 15-minute time points (means ± SEM; WT: open circles, n = 19; cnr1−/− : grey circles, n = 18; cnr2−/− : closed circles, n = 18; *one-way ANOVA with Dunnett post hoc test, P < 0.05).
Retinal Structure in cnr1 /− and cnr2−/− Mice
To ensure that there were no major structural changes in KO mice, we compared the basic retinal anatomy across groups by examining retinal layering and thickness. Figure 7A shows the typical retinal layering in all genotypes. First-look observations of the retinas showed no obvious changes in retinal structures. Then, the total retinal thickness (Fig. 7B), the thickness of each layer (Fig. 7C), and the number of cell rows for each nuclear layer (results not shown) were precisely measured. No significant differences were found for all parameters. The overall retinal layering was thus preserved in all genotypes. In addition, the distribution and morphology of retinal cells were compared for all genotypes to verify if they were affected. No obvious changes were observed in any retinal cell type for all genotypes (Figs. 8A–X). 
Figure 7
 
Deletion of cnr1 or cnr2 does not change retinal morphology. (A) Nuclei staining of retinas of WT, cnr1−/− , and cnr2−/− mice with Sytox. Normal retinal layer structures were preserved in all mice. Scale bar: 20 μm. (B) Total thickness calculated from ONL-GCL in the three genotypes. (C) Mean thickness of each retinal layer in the three genotypes. White, grey, and black bars correspond to the WT, cnr1−/− , and cnr2−/− mice, respectively.
Figure 7
 
Deletion of cnr1 or cnr2 does not change retinal morphology. (A) Nuclei staining of retinas of WT, cnr1−/− , and cnr2−/− mice with Sytox. Normal retinal layer structures were preserved in all mice. Scale bar: 20 μm. (B) Total thickness calculated from ONL-GCL in the three genotypes. (C) Mean thickness of each retinal layer in the three genotypes. White, grey, and black bars correspond to the WT, cnr1−/− , and cnr2−/− mice, respectively.
Figure 8
 
Absence of CB1R or CB2R does not produce any obvious changes in the distribution and morphology of cone photoreceptors (mouse cone-arrestin; [AC]), rod photoreceptors (recoverin; [DF]), horizontal cells (calbindin; [GI]), cone bipolar cells (recoverin; [JL]), rod bipolar cells (PKC; [MO]), amacrine cells (HPC; [PR]), ganglion cells (Brn-3a; [SU]), and Müller cells (glutamine synthetase; [VX]) in WT, cnr1−/− , and cnr2−/− mice. Normal morphology and distribution of every retinal cell types were preserved in all mice. Scale bars: 10 μm.
Figure 8
 
Absence of CB1R or CB2R does not produce any obvious changes in the distribution and morphology of cone photoreceptors (mouse cone-arrestin; [AC]), rod photoreceptors (recoverin; [DF]), horizontal cells (calbindin; [GI]), cone bipolar cells (recoverin; [JL]), rod bipolar cells (PKC; [MO]), amacrine cells (HPC; [PR]), ganglion cells (Brn-3a; [SU]), and Müller cells (glutamine synthetase; [VX]) in WT, cnr1−/− , and cnr2−/− mice. Normal morphology and distribution of every retinal cell types were preserved in all mice. Scale bars: 10 μm.
Furthermore, we wanted to establish if the elimination of one receptor affected other elements of the eCB system. We measured the total amounts of CB2R, CB1R, synthesis enzymes NAPE-PLD and DAGLα, as well as degradative enzymes FAAH and MGL in the WT, cnr1−/− , and cnr2−/− mice retina. No differences were found in the total amount of those proteins across groups (Figs. 9A–D). 
Figure 9
 
Total retinal concentration of proteins from the eCB system. (A, B) Representative examples of FAAH, NAPE-PLD, DAGLα, MGL, CB1R, and CB2R expression in WT, cnr1−/− , and cnr2−/− mice retina lysate. (C, D) Averaged ratio ± SEM relative to WT of FAAH, NAPE-PLD, DAGLα, MGL, CB1R, and CB2R expression in WT (white), cnr1−/− (grey), and cnr2−/− (black) mice. Specific bands were seen at around 53 kDa for CB1R, 45 kDa for CB2R, 120 kDa for DAGLα, 66 kDa for FAAH, 37 kDa for MGL, and 46 kDa for NAPE-PLD.
Figure 9
 
Total retinal concentration of proteins from the eCB system. (A, B) Representative examples of FAAH, NAPE-PLD, DAGLα, MGL, CB1R, and CB2R expression in WT, cnr1−/− , and cnr2−/− mice retina lysate. (C, D) Averaged ratio ± SEM relative to WT of FAAH, NAPE-PLD, DAGLα, MGL, CB1R, and CB2R expression in WT (white), cnr1−/− (grey), and cnr2−/− (black) mice. Specific bands were seen at around 53 kDa for CB1R, 45 kDa for CB2R, 120 kDa for DAGLα, 66 kDa for FAAH, 37 kDa for MGL, and 46 kDa for NAPE-PLD.
Discussion
This study investigated the functional and anatomical consequences of knocking out CB1R or CB2R in the mouse retina. Using ERG, we showed that under scotopic conditions, the removal of CB1R did not change the response, while the absence of the CB2R affected the amplitude of the a-wave, without affecting the b-wave or amplitude and latency of the OPs. Under photopic conditions, removal of CB1R did not affect the retinal response, but the elimination of CB2R yielded a different light adaptation pattern. This study is the first evidence that CB2R affects global retinal function. 
The CB2R Is Widely Distributed in the Mice Retina
The present results confirm the expression of CB2R in the rodent retina. To our knowledge, this study is the first to precisely localize CB2R in the mice retina by using an antibody controlled for specificity. Barutta et al. 22 have also used the same CB2R antibody and verified its specificity in cnr2−/− mice renal sections. Our results are in agreement with a study using RT-PCR and in situ hybridization, which shows that CB2R is present in the retinal ganglion cell layer, the INL, and the inner segments of photoreceptors cells. 16 A recent study has reported a similar distribution in the rat retina, with CB2R being localized in retinal pigmentary epithelium, inner photoreceptor segments, horizontal and amacrine cells, neurons in GCL, and fibers of the IPL. 17 Our results provided further confirmation of CB2R expression in mammalian retina. More specifically our data showed the presence of CB2R in cone and rod photoreceptors, horizontal cells, cone and rod bipolar cells, some amacrine cells, and ganglion cells. 
CB1R Removal Does Not Affect the ERGs
Our data indicated that CB1R deletion does not have an impact on the retinal activity as measured with the ERG. This absence of functional changes observed in cnr1−/− mice was unexpected, given the wide distribution of CB1R expression in the rodent retina. 3,5,6 In addition, several authors 1113 have reported that CBR activation differentially modulates calcium and potassium currents in rod and cone photoreceptors. These authors suggest that the net effect of CBR activation on calcium and potassium currents in rod photoreceptors would be a decrease in the light sensitivity. From these findings, we expected to observe increased amplitude in the a-wave in mice lacking CB1 receptor. As no effects were detected, this could indicate that the modulation of these currents by CB1R is too weak to be measured with the ERG. 
The overall effect of CBR activation on cones is difficult to predict, as changes in ionic currents would lead to opposing outcomes. 13 Our data did not provide any more cues about this, since we did not observe an effect of CB1R removal on cone response. 
Electroretinograms reflect essentially the activity of photoreceptors and bipolar cells. Consequently, our data do not preclude a role for CB1R in the response of other retinal neurons such as horizontal, amacrine, and ganglion cells. The latter is further supported by several studies demonstrating that CB1R activity modulates the excitability of amacrine and ganglion cells. 8,9,30 Nevertheless, altogether our results suggest that the absence of CB1R does not affect the global response of the retina. However, this receptor could still affect other visual structures, as we 31 recently have reported that CB1R exerts a regulatory action on the neurovascular coupling and functional organization of the mice visual cortex along the azimuth axis. 
Effect of CB2R Removal on the ERGs
Under scotopic conditions, our data indicated that the amplitude of the a-wave is larger in cnr2−/− than in WT mice at several intensities. The a-wave is thought to originate from photoreceptors; hence, this effect may reflect the consequences of CB2R removal on these cells. As mentioned earlier in the introduction, the activation of CBRs by the administration of an agonist can decrease the sensitivity of rod photoreceptors. 13 Provided that we report no changes in the a-wave of cnr1−/− mice and an increased a-wave in cnr2−/− mice, we suggest that the alteration of rod sensitivity reported in Straiker and Sullivan 13 could be mediated by CB2R rather than CB1R. Analysis of the velocity-time profile at saturating luminance revealed no differences in the accelerating segment of the a-wave (the leading edge) between groups. However, this analysis indicated that the cnr2−/− mice maintained higher velocities than their WT counterparts in the decelerating segment of the a-wave. One may propose that CB2R deletion decreases the postreceptoral contribution to the a-wave. This hypothesis is unlikely as it has been shown that the a-wave profile is not modified by the pharmacologic elimination of the b-wave at saturating intensities. 32 Since it has been shown that the decelerating segment could be caused by the recovery of the a-wave, 33 our results may indicate that photoreceptors recuperate more slowly in cnr2−/− mice. 
Anatomical studies showed that CB2R was expressed in the bipolar cells of mice (this article and Lu et al. 16 ). Moreover, Yazulla et al. 14 have demonstrated that CBRs modulate potassium currents in retinal bipolar cells. Based on these findings and given that bipolar cells contribute, in part, to the genesis of the b-wave, one can expect that CB2R elimination will affect the b-wave amplitude and/or latency. Our present results showed only a trend in which all the b-wave data points have higher amplitude in cnr2−/− than in WT or cnr1−/− mice. No such tendencies were observed for the b-wave latency. These results indicate a limited effect of CB2R on the b-wave, while the effect on the a-wave is larger. 
Under photopic conditions, an enhanced b-wave was observed at intermediate recording time points (10 and 15 minutes) in cnr2−/− mice. Among the roles most commonly attributed to cannabinoid receptors are adaptive mechanisms such as long-term-depression 3436 and downregulation of synaptic release 11 in the visual system and elsewhere. Thus, these results suggest that CBRs are likely to be involved in mechanisms subtending retinal adaptation. 
We showed that functional changes observed in cnr1−/− and cnr2−/− mice are not due to abnormal retinal cell distribution or compensation of the eCB system activity. Previous studies have shown that the eCB system interacts with GABA, glutamate, and dopamine systems. 3740 For example, CB1R agonists stimulate dopamine release from the guinea pig retina. 37 Hence, the changes in retinal response in cnr2−/− mice, and/or lack thereof in cnr1−/− mice, could be partly due to compensation mechanisms in GABA, glutamate, or dopamine systems. 
In conclusion, this study demonstrated that CB2R is expressed in several cell types in the mice retina. Our results also suggest that CB1R and CB2R contribute differently to visual functions: CB1R does not seem to be involved in global retinal responses, while CB2R appears to be implicated in rod and cone sensitivity and light adaptation. 
Acknowledgments
We thank Pierre Lachapelle (McGill University, Montréal, Quebec, Canada) for his constructive comments on this manuscript, and Vasil Diaconu (University of Montreal, Montréal, Quebec, Canada) for light measurements. We thank the Mary D. Allen Laboratory for Vision Research, Doheny Eye Institute, and Cheryl M. Craft for generously supplying the antibody against cone arrestin. We also thank Alexandra Gagné and Sébastien Thomas for their excellent assistance. 
Supported by a joint Fonds de recherche du Québec-Santé (FRQS) Vision Network grant – Fondation des maladies de l'oeil and Canadian Institutes of Health Research (CIHR) Grant MOP 130337 (J-FB and CC), by NSERC Grant 194670-2009 (CC), and by CIHR Grant MOP 177796 and Natural Sciences and Engineering Research Council of Canada (NSERC) Grant 311892-2010 (J-FB). Also supported by a CIHR-E.A. Baker Foundation studentship (NZ), a Réseau FRQS de recherche en santé de la vision studentship (BC), and a Chercheur-Boursier Junior 2 from the FRQS (J-FB). 
Disclosure: B. Cécyre, None; N. Zabouri, None; F. Huppé-Gourgues, None; J.-F. Bouchard, None; C. Casanova, None 
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Footnotes
 BC and NZ contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Figure 1
 
Characterization of an antibody directed against CB2R in adult mice retina. Immunoblots of CB2R immunoreactivity in a WT mouse retina and in presence of its blocking peptide (BP) (A). Specific band was seen at around 45 kDa in WT group only. GAPDH was used as a loading control. CB2R immunoreactivity in the retina of a WT mouse in presence of its appropriate BP (B), in cnr2−/− mice (C), and in WT mouse (D). All images were taken with identical microscopy settings. Scale bar: 50 μm.
Figure 1
 
Characterization of an antibody directed against CB2R in adult mice retina. Immunoblots of CB2R immunoreactivity in a WT mouse retina and in presence of its blocking peptide (BP) (A). Specific band was seen at around 45 kDa in WT group only. GAPDH was used as a loading control. CB2R immunoreactivity in the retina of a WT mouse in presence of its appropriate BP (B), in cnr2−/− mice (C), and in WT mouse (D). All images were taken with identical microscopy settings. Scale bar: 50 μm.
Figure 2
 
Double-label immunofluorescence illustrating colocalization of CB2R throughout the mouse retina. CB2R (magenta) is present in inner (arrowheads) and outer (arrows) segments of cones (mouse cone-arrestin), but not in pedicles (circles; [A]), rod photoreceptors (recoverin; [B]), horizontal cells (calbindin; arrows) and their dendrites (arrowheads; [C]), cone (recoverin; [D]) and rod (PKC) bipolar cells' soma (arrows; [E]), their presynaptic connections (arrowheads; [E]) and axons (arrows) but not in postsynaptic connections (arrowheads; [F]), amacrine cells (syntaxin; [G]), ganglion cells (Brn-3a; [H]), and Müller cells (glutamine synthetase; [I]). Scale bar: 10 μm.
Figure 2
 
Double-label immunofluorescence illustrating colocalization of CB2R throughout the mouse retina. CB2R (magenta) is present in inner (arrowheads) and outer (arrows) segments of cones (mouse cone-arrestin), but not in pedicles (circles; [A]), rod photoreceptors (recoverin; [B]), horizontal cells (calbindin; arrows) and their dendrites (arrowheads; [C]), cone (recoverin; [D]) and rod (PKC) bipolar cells' soma (arrows; [E]), their presynaptic connections (arrowheads; [E]) and axons (arrows) but not in postsynaptic connections (arrowheads; [F]), amacrine cells (syntaxin; [G]), ganglion cells (Brn-3a; [H]), and Müller cells (glutamine synthetase; [I]). Scale bar: 10 μm.
Figure 3
 
Example of scotopic ERGs in the different genotypes. Representative ERGs recorded in WT, cnr1−/− , and cnr2−/− mice. The luminance-response function of each animal was established by presenting progressively brighter flashes (bottom to top) indicated to the right of the traces as the log luminance (scot cd-s/m2).
Figure 3
 
Example of scotopic ERGs in the different genotypes. Representative ERGs recorded in WT, cnr1−/− , and cnr2−/− mice. The luminance-response function of each animal was established by presenting progressively brighter flashes (bottom to top) indicated to the right of the traces as the log luminance (scot cd-s/m2).
Figure 4
 
Amplitudes of scotopic ERG a- and b-waves plotted as a function of flash luminance. Scotopic ERG a-wave (A) and b-wave (B) amplitudes are displayed by genotype. The values are means ± SEM from all animals in each group (WT: open circles, n = 20; cnr1−/− : grey circles, n = 20; cnr2−/− : closed circles, n = 18; #repeated measures ANOVA, P ≤ 0.05, *one-way ANOVA with Dunnett post hoc test, P ≤ 0.05). (C) The averaged a-wave for each group is plotted as a function of time. (D) The velocity-time profile of the a-wave for each group displays differences between cnr2−/− and WT mice in the deceleration segment on the waveform (repeated measures ANOVA, P ≤ 0.05; *one-way ANOVA with Dunnett post hoc test, P ≤ 0.05).
Figure 4
 
Amplitudes of scotopic ERG a- and b-waves plotted as a function of flash luminance. Scotopic ERG a-wave (A) and b-wave (B) amplitudes are displayed by genotype. The values are means ± SEM from all animals in each group (WT: open circles, n = 20; cnr1−/− : grey circles, n = 20; cnr2−/− : closed circles, n = 18; #repeated measures ANOVA, P ≤ 0.05, *one-way ANOVA with Dunnett post hoc test, P ≤ 0.05). (C) The averaged a-wave for each group is plotted as a function of time. (D) The velocity-time profile of the a-wave for each group displays differences between cnr2−/− and WT mice in the deceleration segment on the waveform (repeated measures ANOVA, P ≤ 0.05; *one-way ANOVA with Dunnett post hoc test, P ≤ 0.05).
Figure 5
 
Example of scotopic OPs in the different groups. (A) Representative OPs recorded in WT and cnr2−/− mice. (B) The total sum of the amplitudes of all OPs is plotted as a function of flash luminance to compute the luminance-response function curve. The values are means ± SEM from all animals in each group (WT: open circles, n = 17; cnr1−/− : grey circles, n = 19; cnr2−/− : closed circles, n = 12).
Figure 5
 
Example of scotopic OPs in the different groups. (A) Representative OPs recorded in WT and cnr2−/− mice. (B) The total sum of the amplitudes of all OPs is plotted as a function of flash luminance to compute the luminance-response function curve. The values are means ± SEM from all animals in each group (WT: open circles, n = 17; cnr1−/− : grey circles, n = 19; cnr2−/− : closed circles, n = 12).
Figure 6
 
The amplitudes of photopic ERG b-wave are plotted as a function of time. Repeated ERGs were taken at different times and were used to evaluate the retina ability to adapt to light from a dark-adapted state. The cnr2−/− mice displayed significantly higher amplitudes at 10- and 15-minute time points (means ± SEM; WT: open circles, n = 19; cnr1−/− : grey circles, n = 18; cnr2−/− : closed circles, n = 18; *one-way ANOVA with Dunnett post hoc test, P < 0.05).
Figure 6
 
The amplitudes of photopic ERG b-wave are plotted as a function of time. Repeated ERGs were taken at different times and were used to evaluate the retina ability to adapt to light from a dark-adapted state. The cnr2−/− mice displayed significantly higher amplitudes at 10- and 15-minute time points (means ± SEM; WT: open circles, n = 19; cnr1−/− : grey circles, n = 18; cnr2−/− : closed circles, n = 18; *one-way ANOVA with Dunnett post hoc test, P < 0.05).
Figure 7
 
Deletion of cnr1 or cnr2 does not change retinal morphology. (A) Nuclei staining of retinas of WT, cnr1−/− , and cnr2−/− mice with Sytox. Normal retinal layer structures were preserved in all mice. Scale bar: 20 μm. (B) Total thickness calculated from ONL-GCL in the three genotypes. (C) Mean thickness of each retinal layer in the three genotypes. White, grey, and black bars correspond to the WT, cnr1−/− , and cnr2−/− mice, respectively.
Figure 7
 
Deletion of cnr1 or cnr2 does not change retinal morphology. (A) Nuclei staining of retinas of WT, cnr1−/− , and cnr2−/− mice with Sytox. Normal retinal layer structures were preserved in all mice. Scale bar: 20 μm. (B) Total thickness calculated from ONL-GCL in the three genotypes. (C) Mean thickness of each retinal layer in the three genotypes. White, grey, and black bars correspond to the WT, cnr1−/− , and cnr2−/− mice, respectively.
Figure 8
 
Absence of CB1R or CB2R does not produce any obvious changes in the distribution and morphology of cone photoreceptors (mouse cone-arrestin; [AC]), rod photoreceptors (recoverin; [DF]), horizontal cells (calbindin; [GI]), cone bipolar cells (recoverin; [JL]), rod bipolar cells (PKC; [MO]), amacrine cells (HPC; [PR]), ganglion cells (Brn-3a; [SU]), and Müller cells (glutamine synthetase; [VX]) in WT, cnr1−/− , and cnr2−/− mice. Normal morphology and distribution of every retinal cell types were preserved in all mice. Scale bars: 10 μm.
Figure 8
 
Absence of CB1R or CB2R does not produce any obvious changes in the distribution and morphology of cone photoreceptors (mouse cone-arrestin; [AC]), rod photoreceptors (recoverin; [DF]), horizontal cells (calbindin; [GI]), cone bipolar cells (recoverin; [JL]), rod bipolar cells (PKC; [MO]), amacrine cells (HPC; [PR]), ganglion cells (Brn-3a; [SU]), and Müller cells (glutamine synthetase; [VX]) in WT, cnr1−/− , and cnr2−/− mice. Normal morphology and distribution of every retinal cell types were preserved in all mice. Scale bars: 10 μm.
Figure 9
 
Total retinal concentration of proteins from the eCB system. (A, B) Representative examples of FAAH, NAPE-PLD, DAGLα, MGL, CB1R, and CB2R expression in WT, cnr1−/− , and cnr2−/− mice retina lysate. (C, D) Averaged ratio ± SEM relative to WT of FAAH, NAPE-PLD, DAGLα, MGL, CB1R, and CB2R expression in WT (white), cnr1−/− (grey), and cnr2−/− (black) mice. Specific bands were seen at around 53 kDa for CB1R, 45 kDa for CB2R, 120 kDa for DAGLα, 66 kDa for FAAH, 37 kDa for MGL, and 46 kDa for NAPE-PLD.
Figure 9
 
Total retinal concentration of proteins from the eCB system. (A, B) Representative examples of FAAH, NAPE-PLD, DAGLα, MGL, CB1R, and CB2R expression in WT, cnr1−/− , and cnr2−/− mice retina lysate. (C, D) Averaged ratio ± SEM relative to WT of FAAH, NAPE-PLD, DAGLα, MGL, CB1R, and CB2R expression in WT (white), cnr1−/− (grey), and cnr2−/− (black) mice. Specific bands were seen at around 53 kDa for CB1R, 45 kDa for CB2R, 120 kDa for DAGLα, 66 kDa for FAAH, 37 kDa for MGL, and 46 kDa for NAPE-PLD.
Table 1
 
Antibodies Used in This Study
Table 1
 
Antibodies Used in This Study
Antibody Target Immunogen Dilution Host Provenance
CB2R Cannabinoid receptor type 2 N-terminus 14 aa of human CB2R (20–33 residues) I, 1:200 W, 1:2000 Rabbit 101550; Cayman Chemical, Ann Arbor, MI
CB1R Cannabinoid receptor type 1 N-terminus 77 aa of rat CB1R (1–77 residues) W, 1:1000 Rabbit C1233; Sigma-Aldrich, Oakville, ON, Canada
NAPE-PLD N-acyl phosphatidyethanolamine phospholipase D N-terminus 13 aa of mouse NAPE-PLD (9–21 residues) W, 1:500 Rabbit NB110-80070; Novus Biologicals, Oakville, ON, Canada
DAGLα Diacylglycerol lipase α C-terminus 42 aa of mouse DAGLα (1003–1044 residues) W, 1:200 Rabbit DGLa-Rb-Af380; Frontier Institute, Ishikari, Hokkaido, Japan
FAAH Fatty acid amide hydrolase C-terminus 19 aa rat FAAH (561–579 residues) W, 1:3000 Rabbit 101600; Cayman Chemical
MGL Monoacylglycerol lipase N-terminus 35 aa of mouse MGL (1–35 residues) W, 1:200 Rabbit MGL-Rb-Af200; Frontier Institute
GAPDH Loading control The full-length rabbit muscle GAPDH protein W, 1:20,000 Mouse G8795; Sigma-Aldrich
Mouse cone-arrestin (LUMIj) Cone photoreceptors C-terminus 13 aa of the mCAR protein (369–381 residues) I, 1:1000 Rabbit Cheryl M. Craft, Mary D. Allen Laboratory for Vision Research, Doheny Eye Institute, USC, Los Angeles, CA
Recoverin Rod photoreceptors, and ON and OFF cone bipolar cells The full-length recombinant human recoverin I, 1:2000 Rabbit AB5585; EMD Milipore Corporation, Billerica, MA
Calbindin (D-28K) Horizontal and amacrine cells Recombinant rat calbindin D-28K full length I, 1:1000 Rabbit CB-38a; Swant, Marly, Switzerland
PKC (clone H7) Rod bipolar cells C-terminus 28 aa of human protein (645–672 residues) I, 1:500 Mouse Sc-8393; Santa Cruz Biotechnology, Santa Cruz, CA
Syntaxin (clone HPC1) Amacrine cells Synaptosomal plasma fraction of rat hippocampus I, 1:500 Mouse S0664; Sigma-Aldrich
Brn-3a (clone 5A3.2) Ganglion cells Amino acids 186–224 of Brn-3a fused to the T7 gene 10 protein I, 1:100 Mouse MAB1585; EMD Millipore Corporation
Glutamine synthetase (GS, clone GS-6) Müller cells The full protein purified from sheep brain I, 1:3000 Mouse MAB302; EMD Millipore Corporation
Table 2
 
Group Data Reporting the Amplitudes of Mixed V max as Well as the Light Intensities Evoking Them
Table 2
 
Group Data Reporting the Amplitudes of Mixed V max as Well as the Light Intensities Evoking Them
WT cnr1−/− cnr2−/−
Mixed V max, μV 369 ± 29 371 ± 26 446 ± 22
Light luminance mixed V max, log scot cd-s/m2 1.27 ± 0.04 1.13 ± 0.05 1.06 ± 0.09
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