Molecular Vision 2006; 12:1160-1166 <http://www.molvis.org/molvis/v12/a132/> Received 31 January 2006 | Accepted 20 July 2006 | Published 6 October 2006

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Cannabinoid receptor-mediated inhibition of calcium signaling in rat retinal ganglion cells

Mélanie R. Lalonde,¹ Christine A.B Jollimore,² Kelly Stevens,¹ Steven Barnes,^1,3 Melanie E.M. Kelly²
 
 

Departments of ¹Physiology and Biophysics, ²Pharmacology, and ³Ophthalmology and Visual Sciences, Dalhousie University, Halifax, Nova Scotia, Canada

Correspondence to: Melanie E.M. Kelly, PhD., Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada, B3H 4H7; Phone: 902-494-3325; FAX: (902) 494-6309; email: mkelly@dal.ca

Abstract

Purpose: The physiological actions of CB₁ cannabinoid receptors (CB₁Rs) in mammalian retina have yet to be fully described in all cell types. Here we investigate the actions of CB₁R activation on high-voltage-activated (HVA) Ca²⁺ channel currents in purified cultures of rat retinal ganglion cells (RGCs).

Methods: Reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry were used to determine the presence of CB₁R mRNA and protein in a purified RGC culture generated from neonatal rats using a two-step panning procedure. Ruptured-patch whole-cell voltage clamp was used to test the effect of CB₁R agonists (WIN 55,212-2) and antagonists (SR141716A, AM281) on HVA Ca²⁺ channel currents.

Results: RT-PCR analysis confirmed CB₁R mRNA in cultured RGCs and immunocytochemistry for CB₁R protein revealed labeling in both the cell body and neurites of isolated RGCs. Patch-clamp recording from cultured rat RGCs showed that the CB₁R agonist WIN 55,212-2 inhibited HVA Ca²⁺ channel currents up to 50% in a concentration-dependent manner (0.5, 1, and 5 μM). The Ca²⁺ channel current inhibition by WIN 55,212-2 was blocked by CB₁R antagonists AM281 and SR141716.

Conclusions: Activation of CB₁Rs in cultured RGCs inhibits HVA Ca²⁺ channel currents. These data show that cannabinoids can modify the excitability of RGCs and could affect retinal output. This finding has implications for retinal signal processing as it suggests that endogenous cannabinoids have inhibitory effects on RGCs and that exogenous cannabinoids could modulate retinal function by this pathway as well.

Introduction

Cannabinoids, the main psychoactive components of marijuana and hashish, act on endogenous G protein-coupled receptors. Two cannabinoid receptor subtypes have been cloned, CB₁R and CB₂R, and are activated by endogenous endocannabinoid ligands such as anandamide (N-arachidonoylethanolamide, AEA), 2-arachidonoyl glycerol (2-AG) and 2-arachidonoyl glyceryl ether [1-4]. Evidence that non-CB₁/CB₂ cannabinoid receptors also contribute to endocannabinoid action in specific tissues has recently emerged [5].

CB₁Rs are conserved among vertebrate species and are abundantly expressed in the mammalian nervous system. CB₂Rs are expressed in peripheral tissues, specifically in immune cells [6], but have also been identified in areas of the brain [7,8] and the eye [9]. CB₂R mRNA has been reported in the retina [10]. Both CB₁Rs and CB₂Rs couple to G proteins of the G_i/o subtype, although coupling to Gs [11] and Gq [12] has been reported for the CB₁R. Activation of CB₁Rs results in inhibition of adenylyl cyclase and a decrease in levels of cAMP [13] as well as activation of the mitogen activated protein kinase pathway [14]. CB₁R activation also has been reported to inhibit both N-type [15-19] and P/Q-type voltage-dependent Ca²⁺ channels [17,20]. Consistent with the inhibitory effects of cannabinoids on Ca²⁺ channel currents, several studies have now shown that cannabinoids can reduce the presynaptic release of neurotransmitters, including glutamate [21,22] and inhibit postsynaptic voltage-dependent Ca²⁺ channels, to modulate neuronal excitability and synaptic transmission [23].

CB₁R immunoreactivity has been detected throughout the retina of a number of species including rat, with immunoreactivity detected in the ganglion cell layer and ganglion cell axons [24]. However, the cellular effects of cannabinoids on RGCs have not been investigated. The aims of the present study were to determine whether CB₁Rs are expressed in isolated purified RGC cultures and to determine whether exogenous cannabinoids, acting via CB₁Rs, can modulate neuronal Ca²⁺ signaling via alterations in Ca²⁺ channel activity.

Methods

Reverse transcriptase polymerase chain reaction

All procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Dalhousie University Committee for the Use of Laboratory Animals. To obtain RNA, adult Long-Evans rats (Charles River, Montreal, Quebec) were deeply anesthetized using sodium pentobarbital, decapitated, and the eyes enucleated. The anterior segment and lens were removed and discarded. The retina was then dissected out from the posterior eyecups and immersed in liquid nitrogen and stored at -70 °C prior to RNA extraction using TRIZOL^TM according to the manufacturer's instructions (Invitrogen). cDNA was obtained from 2 μg of total RNA using random primers, oligo(dt)15-18, and moloney murine leukemia virus (M-MLV) reverse transcriptase (Life Technologies, Burlington, Ontario). The primers for amplification of the CB₁R are shown in Table 1. Using cDNA from rat retina as a template, PCR conditions for amplification of the CB₁R included: denaturation at 94 °C for 1 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 65 °C for 1 min and extension at 72 °C for 1 min, followed by final extension at 72 °C for 10 min. PCR products were visualized by 1.0% agarose gel containing ethidium bromide. The amplicons were verified as previously described [25,26], using restriction enzyme analysis as well as sequencing (DNA Sequencing Facility, Dalhousie University).

Immunocytochemistry

Coverslips with cultured RGCs were fixed in 4% paraformaldehyde for 2 h at 4 °C. Following fixation, cells were subjected to three 5 min washes with phosphate-buffered solution (PBS) and followed by 20 min incubation with 0.3% Triton X-100. After three additional 5 min washes with PBS, a solution of PBS containing 10% goat serum was applied to the cells for 1.5 h at room temperature. The PBS/goat serum was removed, and the fixed cells were incubated overnight at 4 °C with a fresh solution of PBS containing the primary antibody (1:200; rabbit anti-CB₁R; Cayman Chemical Company, Ann Arbor, MI). The next day, the cells were rinsed five times with fresh PBS for 5 min per wash and then incubated with the secondary antibody (1:200; AlexaFluor 546 goat anti-rabbit; Invitrogen Canada Inc., Burlington, Ontario) at room temperature for 1 h. The cells were then washed with distilled water once, followed by three washes in PBS at 5 min per wash, coverslipped and visualized with a 60X oil immersion lens using a laser-scanning confocal microscope (Nikon C1, Nikon Instruments, Mississauga, Ontario). Control experiments were performed, as per the manufacturer's instructions, by preabsorption of the primary antibody with the antigen peptide (1:1) for 1 h with agitation before application to the cells and repeating the staining protocol.

Purified retinal ganglion cell culture

Natural litters (one to ten pups for each experiment) of Long-Evans rats (Charles River, Montreal, Quebec) were sacrificed at postnatal 7 to 8 days by overexposure to halothane and decapitation. Following enucleation, the anterior segment and lens were removed and the posterior eyecups were immersed in dissection medium consisting of Hibernate-A (BrainBits, Springfield, IL) with 2% B27 supplements and 10 μg/ml gentamicin. The retinas were dissected and maintained in the dissection medium until all retinas were isolated.

The retinal tissue was incubated for 30 min at 37 °C in a papain (Worthington Biochemicals, Lakewood, NJ) solution (165 units in 10 ml Ca²⁺/Mg²⁺-free DPBS) containing 1 mM L-cysteine and 0.004% DNase. The papain-treated retinas were then triturated sequentially in DPBS (with Ca²⁺ and Mg²⁺) containing 1.5 mg/ml ovomucoid (Roche Diagnostics, Laval, Quebec), 1.5 mg/ml bovine serum albumin (BSA), and 0.004% DNase. In addition, this solution contained the rabbit anti-rat macrophage antibodies (1:75; Axell brand, Accurate Chemical, Westbury, NY) for the macrophage-panning step. The suspension was centrifuged, rewashed in a high concentration ovomucoid/BSA solution (10 mg/ml each in DPBS), and the dissociated cells were resuspended in DPBS with 0.2 mg/ml BSA and 5 μg/ml insulin.

The two-step panning procedure to purify the RGCs has been previously described [27-29]. Briefly, macrophages were first removed by incubating the mixed retinal cell suspension on petri dishes coated with affinity-purified goat anti-rabbit IgG (H+L) antibodies (Jackson Immuno Research Laboratories Inc., West Grove, PA). The remaining cells were then transferred to a petri dish that had first been coated with affinity-purified goat anti-mouse IgM (μ chain) antibodies (Jackson Immuno Research Laboratories Inc., West Grove, PA) and second with anti-Thy-1.1 monoclonal IgM antibodies (cell line T11D7e2; number TIB-103, American Type Culture Collection, Manassas, VA). After repeatedly rinsing the plate with DPBS, the adherent RGCs were released by first incubating the cells in a 0.125% trypsin solution, and then by manually pipetting an enzyme inhibitor solution (30% FBS in Neurobasal-A) along the surface of the dish.

Purified RGCs were plated onto poly-D-lysine/laminin-coated Biocoat glass coverslips (12 mm round; BD Biosciences, Bedford, MA) in 24-well tissue culture plates at a density of 2.5x10⁴ cells per well. The cells were cultured in 600 μl of serum-free culture medium consisting of Neurobasal-A with 2% B27 supplements, 1 mM glutamine, 50 ng/ml BDNF, 10 ng/ml CNTF, 5 mM forskolin, and 10 mg/ml gentamicin. Cultures were maintained at 37 °C in a humidified 5% CO₂-air atmosphere. All experiments on the isolated RGCs were performed on the second day following cell dissociation and panning.

Electrophysiological recordings from isolated retinal ganglion cells

Whole-cell recordings of Ca²⁺ channel currents were obtained from individual RGCs in the purified culture using the ruptured-patch technique. The standard bath solution to isolate Ca²⁺ channel currents, using barium as a charge carrier, contained (in mM): 115 NaCl, 2.5 KCl, 5 CsCl, 10 BaCl₂, 15 TEACl, 10 glucose, and 15 HEPES, adjusted to pH 7.6 with NaOH, while the intracellular pipette solution contained (in mM): 140 CsCl, 0.8 MgCl₂, 0.1 CaCl₂, 1:4 EGTA:CsOH, 10 HEPES, 1 Mg²⁺-ATP, and 0.2 Na⁺-GTP, adjusted to pH 7.2 with CsOH. Tetrodotoxin (TTX; 1 μM) was added to the bath solution in order to eliminate any contribution from sodium channels to the whole-cell current. WIN 55,212-2 was added to the superfusing standard bath solution, which was applied at room temperature (between 21 and 25 °C).

Patch electrodes were pulled from fire polished micro-hematocrit capillary tubes (VWR Scientific, West Chester, PA) using a two-step vertical pipette puller (Kopf model 730, David Kopf Instruments, Tujunga, CA). Pipette tips were first dipped in standard intracellular solutions and then back-filled with the same solution. Filled pipettes had 5-10 MΩ tip resistances, measured in the standard bath. The bath reference electrode consisted of a bath solution-filled agar bridge with an AgCl wire (World Precision Instruments, Sarasota, FL). Offset currents were nulled before seals were made. Whole-cell voltage was clamped with an Axopatch-1D amplifier (Axon Instruments, Foster City, CA) using whole-cell capacitance and series resistance compensation to reduce capacitive artifacts. The current signal was filtered at 0.5-1 kHz (Ithaco 4302 Dual 24dB/octave filter, Ithaca, NY) and digitized at 1 kHz with an Indec Systems interface (Sunnyvale, CA) for storage on the hard disk of a computer running BASIC-FASTLAB acquisition software. BASIC-FASTLAB generated the voltage-clamp commands, and provided some data analysis. Holding potential was set at -60 mV to isolate high-voltage activated Ca²⁺ channel activity, and voltage steps in 10 mV increments of 100 ms duration from -70 mV to +40 mV were applied every 1 s. Current-voltage (I-V) relations were constructed by calculating the mean current about 10 ms before the end of each voltage step.

Data analysis

All data are reported as the means±SEM and statistical analysis was performed using SigmaStat software (SigmaStat 2.03; SPSS Science, Chicago, IL). For the electrophysiology experiments, statistical analyses of drug effect were performed on raw data using Student's paired t-tests. Differences between mean current remaining from two different samples were compared with the non-parametric Mann-Whitney Rank Sum Test. Probability (p) values of less than 0.05 were considered statistically significant (* p<0.05).

Results

CB₁Rs in the rat retina and in cultured retinal ganglion cells

RT-PCR with cDNA templates from rat retina and purified cultured RGCs resulted in a PCR product of the expected size (521 bp) for the CB₁ receptor (Figure 1A). Subsequent sequencing of the PCR product confirmed that the sequence of the PCR product corresponded to the sequence for the rat CB₁ receptor (GenBank number NM_012784).

Immunocytochemical staining of purified cultured RGCs with CB₁R antibodies showed localization of CB₁R protein in isolated RGCs, with punctate labeling seen throughout the cell soma area and neurites (Figure 1B). Staining for CB₁Rs was suppressed by antibody preabsorption with the peptide antigen (Figure 1C).

CB₁R-mediated inhibition of HVA Ca²⁺ channel currents in retinal ganglion cells

The effect of CB₁R agonist, WIN 55,212-2, on high voltage-activated (HVA) Ca²⁺ channel activity was assessed under voltage clamp in purified rat RGCs. A holding potential of -60 mV was used to inactivate low-voltage-activated Ca²⁺ channels and isolate HVA Ca²⁺ channels. When tested in 10 mM Ba²⁺ solution, RGC Ca²⁺ channel currents, which were completely blocked by cadmium (100 μM, -0.2±0.8% remaining; n=12; data not shown), were significantly inhibited by 5 μM WIN 55,212-2 (p<0.05; Figure 2). The time course of the drug application (Figure 2A) demonstrates that the Ca²⁺ channel currents decrease after 2 min of WIN 55,212-2 superfusion, and that this inhibition was generally not reversible after 4 min of washing. Following WIN 55,212-2 superfusion, current inhibition could be recovered in only 2 of 5 cells, possibly reflecting the lipophilic nature of the cannabinoid agonist. Figure 2B shows sample current traces elicited by a depolarizing voltage step to 0 mV from a holding potential of -60 mV with the inhibition by WIN 55,212-2 (WIN, 5 μM) evident. Current-voltage relations generated from the same cell show that WIN 55,212-2 reduced the amplitude of HVA Ca²⁺ channel currents with no apparent voltage dependence (Figure 2C).

The Ca²⁺ channel current inhibition seen in the presence of 5 μM WIN 55,212-2 was blocked by both CB₁R antagonists, AM281 and SR141716. The time course of WIN application during superfusion with SR141716 (0.1 μM; SR) in Figure 3A shows no effect of WIN in this cell. Figure 3B shows sample current traces elicited by a depolarizing voltage step to 0 mV from a holding potential of -60 mV with no apparent inhibition by WIN 55,212-2 (5 mM). Current-voltage (I-V) relations generated from the same cell show that in the presence of SR141716, WIN 55,212-2 had no effect on the amplitude or voltage dependence of HVA Ca²⁺ channel currents (Figure 3C).

Data summarized in Figure 4 indicate that HVA Ca²⁺ channel current in RGCs was significantly (p<0.05) reduced by 20±6% (n=5), 25±6% (n=4), and 51±9% (n=5) in the presence of 0.5, 1, and 5 μM WIN 55,212-2, respectively. The inhibition caused by 5 μM WIN 55,212-2 was blocked by CB₁R antagonists, AM281 (0.1 μM, 83±7% remaining, n=4) and SR141716 (0.1 μm, 95±9% remaining, n=5).

Discussion

Our results demonstrate the presence of CB₁R mRNA in purified RGC cultures. CB₁R protein in isolated RGCs was also confirmed by positive labeling using a CB₁R antibody. Patch-clamp recordings showed that voltage-gated Ca²⁺ channel currents in isolated rat RGCs are inhibited in a dose-dependent manner by the CB₁R agonist, WIN 55,212-2. This inhibition was blocked by the CB₁R antagonists AM281 and SR141716. Taken together, these results further establish the presence of CB₁Rs in RGCs and show that cannabinoids activate signaling pathways leading to inhibition of HVA Ca²⁺ channels in these cells.

In the retinas of monkey, rat, mouse, chick and salamander, CB₁R immunoreactivity has been identified in both outer and inner plexiform layers, the inner nuclear layer and the ganglion cell layer [24,30]. In fish retina, CB₁R immunoreactivity was also seen in Müller cells and while labeling was present in bipolar cells it was absent in the ganglion cells [24,31]. The wide distribution of cannabinoid receptors in the vertebrate retina suggest that an endocannabinoid system may be of functional importance in the regulation of retinal activity and the processing of visual signals. In accordance with this, the endogenous receptor ligands, anandamide and 2-AG have been detected in the mammalian retina [24,32-34] together with AEA synthetase and hydrolase activity [32,33]. Fatty acid amide hydrolase (FAAH), the microsomal enzyme that hydrolyses the endocannabinoid anandamide to arachidonic acid and ethanolamine, as well as 2-AG, has also been described in rat RGCs [32]. Thus, endogenous cannabinoid ligands, released from retinal neurons, may function as neurotransmitters and neuromodulators to activate cannabinoid receptors and modulate retinal function.

Activation of CB₁Rs in the retina has been reported to have a number of physiological actions. The cannabinoid receptor agonist, WIN 55212-2 was reported to inhibit K⁺ currents in salamander photoreceptors, while it enhanced the Ca²⁺ currents of rod photoreceptors and suppressed Ca²⁺ currents of large single cones in this species [35]. These differential effects of CB₁R activation in salamander rods and cones may be attributable to the presence of different L-type Ca²⁺ channel α subunits. In goldfish cones, however, biphasic modulation of Ca²⁺, K⁺ and Cl currents was observed with WIN 55212-2 [36]. Low doses of the cannabinoid agonist (<1 μM) enhanced currents via a cholera toxin-sensitive Gs pathway, and higher doses of WIN 55212-2 acting via pertussis toxin (PTX)-sensitive Gi/o pathway, inhibited these currents. These effects were all blocked by the CB₁R antagonist, SR141716A. Thus, the biphasic effects of WIN 55212-2 in goldfish cones were attributed to the coupling of CB₁Rs to two distinct signal transduction pathways with different efficiencies, as has been previously reported for other G protein coupled receptors, including β_2- and α_2-adrenoreceptors [37,38]. In salamander bipolar cells, CB₁R activation did not produce an enhancement of L-type Ca²⁺ current, rather Ca²⁺ channel currents were inhibited [35]. In other cell types of the CNS, activation of CB₁Rs results in inhibition of N- and P/Q-type Ca²⁺ channels [19,39,40]. Suppression of N-type Ca²⁺ channel currents following CB₁R activation has been reported to occur via a PTX-sensitive Gi/o-coupled pathway that is independent of cAMP metabolism [15,16].

At many central synapses, the release of endocannabinoids by postsynaptic cells provides a rapid retrograde signal that activates presynaptic CB₁Rs, decreases presynaptic Ca²⁺ influx and inhibits neurotransmitter release [3,4,18,40,41]. In the retina, CB₁Rs have previously been identified at several sites including rod and cone photoreceptors and bipolar cells. The inhibition of presynaptic Ca²⁺ and K⁺ channels in these cells would be expected to modulate synaptic transmission and alter visual processing. For example, in photoreceptors, CB₁R-mediated inhibition of Ca²⁺ channels would reduce the amplitude of synaptic signals and subsequently reduce signal strength propagated throughout the retina. In contrast, facilitation of Ca²⁺ channels, as reported at lower concentration of WIN 55212-2 in goldfish cones [36] and in salamander rods [35] would strengthen signaling at subsequent levels of retinal processing. Inhibition of K⁺ current in photoreceptors by CB₁R activation in photoreceptors would be expected to depolarize the cells, offsetting the effects of Ca²⁺ channel inhibition. Inhibition of K channels might also alter the temporal response properties of photoreceptors.

Since RGCs carry the output of the retina, our data could imply that endocannabinoids, released in a Ca²⁺-dependent manner from presynaptic neurons and/or from RGCs themselves, can act to inhibit RGC somatic or dendritic HVA Ca²⁺ channels and subsequently modulate postsynaptic integrative processing. Alternatively, it is possible that in these purified cultures of RGCs, CB₁Rs that are normally expressed presynaptically at the axon terminals of RGCs projecting into the brain, find expression throughout the soma and neuritic processes of the cultured cells. The presence of atypical cannabinoid receptors (for review see [5]) or binding to other non-cannabinoid receptors could also contribute to the postsynaptic actions of cannabinoid ligands [42]. However, as SR141716A and AM281, both selective CB₁R antagonists [43-45], abrogated the inhibitory effects of WIN 55,212-2 on Ca²⁺ currents in RGCs, this would argue for a CB₁R -mediated effect.

Rat RGCs have been reported to express several different types of HVA Ca²⁺ channel, including ω-conotoxin-sensitive N-type (40-50%), nifedipine-sensitive L-type (15-30%), but not ω-agatoxin-sensitive P/Q type [46-49]. In RGCs, inhibition of HVA Ca²⁺ channels would be expected to alter all Ca²⁺-sensitive responses of the cells, including activation of Ca²⁺-sensitive ion channels, Ca²⁺-mediated neurotransmitter release, and other Ca²⁺-regulated processes.

Acknowledgements

We thank Mr. Jianing Yu and Drs. Andrew Hartwick and William Baldridge of the Department of Anatomy & Neurobiology at Dalhousie University for providing the purified cultures of retinal ganglion cells. This work was supported by grants from the Canadian Institutes for Health Research (CIHR; MT-10968 to SB; IAO-13484 to MEMK; MGC-57078 to SB, MEMK, and others). MRL was supported by CIHR/CNIB E.A. Baker Studentship Award.

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