Receptors From Rods Through Activation of A2-Like ...

Endogenous Adenosine Reduces Glutamatergic Output From Rods Through Activation of A2-Like Adenosine Receptors Salvatore L. Stella, Jr., Eric J. Bryson, Lucia Cadetti and Wallace B. Thoreson J Neurophysiol 90:165-174, 2003. ; doi: 10.1152/jn.00671.2002 You might find this additional info useful... This article cites 58 articles, 21 of which you can access for free at: http://jn.physiology.org/content/90/1/165.full#ref-list-1 This article has been cited by 7 other HighWire-hosted articles: http://jn.physiology.org/content/90/1/165#cited-by Updated information and services including high resolution figures, can be found at: http://jn.physiology.org/content/90/1/165.full

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J Neurophysiol 90: 165–174, 2003; 10.1152/jn.00671.2002.

Endogenous Adenosine Reduces Glutamatergic Output From Rods Through Activation of A2-Like Adenosine Receptors Salvatore L. Stella, Jr., Eric J. Bryson, Lucia Cadetti, and Wallace B. Thoreson Department of Pharmacology and Department of Ophthalmology, University of Nebraska Medical Center, Omaha, Nebraska 68198-5540 Submitted 13 August 2002; accepted in final form 10 March 2003

INTRODUCTION

Adenosine is a neuromodulator that is widely distributed throughout the CNS and is generated primarily by the catabolism of ATP through the ectonucleotidase cascade (Cunha et al. 1992, 1998; Dunwiddie et al. 1997). Adenosine exerts its effects by activating a distinct class of four G protein-coupled cell-surface receptors classified as A1, A2A, A2B, and A3 (Ralevic and Burnstock 1998). Generally it is thought that the inhibitory actions of adenosine are mediated by A1 receptors, whereas A2 receptors and particularly A2A receptors have been considered to mediate excitatory effects in the CNS (Ralevic Present address and address for reprint requests: S. L. Stella, Jr., Neurobiology Department, University of California Los Angeles, 10833 Le Conte Blvd., Box 951763 CHS, Los Angeles, California 90095-1763 (E-mail: [email protected]). www.jn.org

and Burnstock 1998; Sebastiao and Ribeiro 1996; van Calker et al. 1979). However, few studies demonstrate direct excitatory actions of A2 receptors on transmission; instead, the observed excitability produced by activation of A2 receptors appears to be mediated by reduced release of inhibitory transmitters (e.g., GABA) produced by presynaptic inhibition of voltage-gated Ca2⫹ channels (see review by Edwards and Robertson 1999). A2 receptors have been shown to inhibit calcium currents (ICa) in a number of preparations (Chen and van den Pol 1997; Kobayashi et al. 1998, 2000; Park et al. 1998) but other than the photoreceptor synapse considered in the present study, A2-mediated inhibition of ICa has not been reported at a glutamatergic synapse. Adenosine is selectively accumulated by rod-driven horizontal cells in the goldfish retina (Studholme and Yazulla 1997) and receptors for adenosine have been localized to the outer nuclear layer and photoreceptors (Kvanta et al. 1997; Paes de Carvalho et al. 1990, 1992). Adenosine release occurs in the dark (Blazynski and Perez 1991), when photoreceptors are depolarized and L-glutamate release from photoreceptors is at its greatest (Schmitz and Witkovsky 1997). We have previously shown that the L-type ICa in rod photoreceptors that mediates synaptic release of L-glutamate is inhibited by A2 but not A1 or A3 agonists (Stella et al. 2002). Therefore we hypothesized that activation of A2 receptors should diminish transmitter release from rod photoreceptors. The present study explores the postsynaptic consequences of activating A2 adenosine receptors on rod photoreceptors and expands the pharmacological profile of adenosine receptors to include effects of selective adenosine receptor antagonists. To accomplish this, we compared the effects of adenosine receptor antagonists and agonists on both ICa and depolarization-evoked increases of [Ca2⫹]i in rod photoreceptors measured using Ca2⫹ imaging and electrophysiological recording techniques. To assess the effects of adenosine on transmission, we examined postsynaptic currents in second-order retinal neurons and tested whether inhibition of ICa by A2 receptor activation in rod photoreceptors results in the inhibition of L-glutamate release. Our results indicate that selective A2 and A2A agonists cause a reduction in the release of L-glutamate onto second-order neurons from rods, an effect that is blocked by the selective A2A antagonist, ZM-214385. In accordance with these results, ZM214385 alone enhanced postsynaptic light responses from horizontal and bipolar cells. ZM-214385 also prevented both The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

0022-3077/03 $5.00 Copyright © 2003 The American Physiological Society

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Stella, Jr., Salvatore L., Eric J. Bryson, Lucia Cadetti, and Wallace B. Thoreson. Endogenous adenosine reduces glutamatergic output from rods onto second-order neurons through activation of A2-like adenosine receptors. J Neurophysiol 90: 165–174, 2003; 10.1152/jn.00671.2002. Adenosine is released from retina in darkness; photoreceptors possess A2 adenosine receptors, and A2 agonists inhibit L-type Ca2⫹ currents (ICa) in rods. We therefore investigated whether A2 agonists inhibit rod inputs into second-order neurons and whether selective antagonists to A1, A2A, or A3 receptors prevent Ca2⫹ influx through rod ICa. [Ca2⫹]i changes in rods were assessed with fura-2. ICa in rods and light responses of rods and second-order neurons were recorded using perforated patch-clamp techniques in the aquatic tiger salamander retinal slice preparation. Consistent with earlier results using the A2 agonist N6-[2-(3,5-dimethoxyphenyl)-2(2-methylphenyl)-ethyl]adenosine (DPMA), the A2A agonist CGS21680 significantly inhibited ICa and depolarization-evoked [Ca2⫹]i increases in rods. The A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and A2A antagonist, ZM-241385, but not the A3 antagonist, VUF-5574, inhibited effects of adenosine on Ca2⫹ influx in rods. DPCPX and ZM-241385 also inhibited effects of CGS-21680, suggesting they both act at A2A receptors. Both A2 agonists, CGS21680 and DPMA, reduced light-evoked currents in second-order neurons but not light-evoked voltage responses of rods, suggesting that activation of A2 receptors inhibits transmitter release from rods. The inhibitory effects of CGS-21680 on both depolarization-evoked Ca2⫹ influx and light-evoked currents in second-order neurons were antagonized by ZM-241385. By itself, ZM-241385 enhanced the light-evoked currents in second-order neurons, suggesting that endogenous levels of adenosine inhibit transmitter release from rods. The effects of these drugs suggest that endogenous adenosine activates an A2-like adenosine receptor on rods leading to inhibition of ICa, which in turn inhibits L-glutamate release from rod photoreceptors.

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adenosine and the A2A agonist, CGS-21680, from inhibiting depolarization-evoked Ca2⫹ increases in rod photoreceptors. This study establishes a novel A2-mediated role for adenosine at a glutamatergic synapse and suggests that as illumination levels vary they could be accompanied by changing levels of adenosine, which can modulate neurotransmission at the first synapse in vision. METHODS

Tissue preparation

Solutions and perfusion Solutions were applied by a single-pass, gravity-feed perfusion system, which delivered medium to the slice chamber (chamber volume: approximately 0.5 ml) at a rate of 1.0 ml/min. The normal amphibian superfusate that bathed the slices contained the following (in mM): 111 NaCl, 2.5 KCl, 1.8 CaCl2, 0.5 MgCl2, 10 HEPES, and 5 glucose. When measuring light-evoked responses, the superfusate was supplemented with 100 ␮M picrotoxin, 1 ␮M strychnine, and 0.1 mM glutamine. For photoreceptor recordings of ICa, the superfusate was switched to a Ba2⫹ solution to enhance Ca2⫹ currents. The Ba2⫹ solution contained the following (in mM): 99 NaCl, 2.5 KCl, 10 BaCl2, 0.5 MgCl2, 10 HEPES, 5 glucose, 0.1 picrotoxin, and 0.1 niflumic acid. The pH of all solutions was adjusted to 7.8 with NaOH. The osmolarity measured with a vapor pressure osmometer (Wescor) was 242 ⫾ 5 mOsm. Solutions were continuously bubbled with 100% O2.

Electrophysiology Patch pipettes were pulled on a Narashige PB-7 vertical puller from borosilicate glass pipettes (1.2 mm OD, 0.95 mm ID, omega dot) and had tips of approximately 1 ␮m OD with tip resistances of 10 to 15 M⍀. For bipolar and horizontal cells, pipettes were filled with a solution containing the following (in mM): 54 KCl, 61.5 KCH3SO4, 3.5 NaCH3SO3, 10 HEPES. For ICa recordings, pipettes were filled with a solution containing the following (in mM): 54 CsCl, 61.5 CsCH3SO3, 3.5 NaCH3SO4, 10 HEPES. The pH of these solutions was adjusted to 7.2 with KOH or CsOH, respectively, and the osmolarity was adjusted, if necessary, to 242 ⫾ 5 mOsm. To maintain endogenous second messenger signaling pathways and avoid the rundown that accompanies conventional whole-cell recording, we used the perforated patch method of whole-cell recording with the pore forming antibiotic, nystatin (Rae et al. 1991), or gramicidin (Kyrozis and Reichling 1995). Nystatin was mixed in DMSO at a J Neurophysiol • VOL

Measurement of [Ca2⫹]i transients Intracellular Ca2⫹ changes were assessed using the ratiometric dye, fura-2, on retinal slices (Stella et al. 2002). Briefly, retinal slices were incubated for 45 min in the dark with 0.5 ml of 10 ␮M fura-2/AM ⫹ 5 ␮M pluronic F-127 (Molecular Probes, Eugene, OR), followed by an additional incubation in fura-2/AM alone for 1.5 h in a slice chamber at 4°C. Images were acquired with a cooled CCD camera (SensiCam, Cooke) mounted on an upright fixed stage microscope (Nikon E600FN) equipped with a ⫻60 (1.0 N.A.) water immersion objective. Epifluorescent illumination was provided by 150 W Xe lamp (Opti-

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Larval tiger salamanders (Ambystoma tigrinum; Kons, Germantown, WI or Charles Sullivan, TN, 7–10 in.) were cared for according to institutional guidelines. Retinal slices were prepared according to the methods of Werblin (1978) and Wu (1987); exact procedures are described in further detail by Thoreson et al. (1997). Briefly, salamanders were killed by decapitation and pithed; the eyes were enucleated, and the anterior portion of an eye including the lens was removed. The resulting eyecup was cut into sections and placed vitreal side down onto a piece of filter paper (Millipore 2 ⫻ 5 mm, Type GS, 0.2-␮m pores). After the retina adhered to the filter paper, the retina was isolated under chilled amphibian superfusate and cut into 100- to 150-␮m slices using a razor blade tissue chopper (Stoelting). Retinal slices were rotated 90o for viewing of the retinal layers when placed under a water immersion objective (⫻40, 0.7 NA or ⫻60, 1.0 NA) and viewed on an upright fixed stage microscope (Olympus BHWI or Nikon E600FN). Dissections were usually performed under infrared light using Gen III image intensifiers (Nitemate NavIII, Litton Industries).

concentration of 120 mg/ml, vortexed briefly, and then added to the pipette electrolyte solution to achieve a final concentration of 480 ␮g/ml. Gramicidin was dissolved in ethanol at a concentration of 5 mg/ml, vortexed briefly, and then added to the pipette electrolyte solution to achieve a final concentration of 5 ␮g/ml. The final working stock was vortexed vigorously for 20 to 30 s and stored in the refrigerator. Fresh antibiotic solutions were made every 3 h. In successful recordings, seals ⬎1 G⍀ were obtained in 30 s or less, and cells were usually fully perforated within 5 min of sealing. Cells were voltage clamped using an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA). Currents were acquired and analyzed using Clampex 7.0 software (Axon Instruments). From a random sample of cells, the input resistance (RN) of rods averaged 595 ⫾ 25.6 M⍀ (n ⫽ 15); bipolar cell RN averaged 960 ⫾ 138.7 M⍀ (n ⫽ 7), and horizontal cell RN averaged 117 ⫾ 22.7 M⍀ (n ⫽ 7). Charging curves for bipolar cells and rods were typically well fit by a single exponential, indicating a compact electrotonic structure (⬍0.1 ␭). Rods were voltage clamped at ⫺70 mV; bipolar cells were voltage clamped at ⫺60 mV, and horizontal cells were voltage clamped at ⫺40 mV. Cells in which the access resistance exceeded 40 M⍀ were excluded from analysis. To assess ICa, voltage ramps (0.5 mV/ms) from ⫺90 to ⫹60 mV were applied every 30 s. Voltage ramps yield a similar current-voltage profile as a step protocol but cause less current rundown and provide more data points for fitting and analysis (Thoreson and Stella 2000). Drug solutions were applied after ICa appeared stable for ⱖ90 s. The leak conductance was assumed to be ohmic and equal to the minimum conductance between ⫺75 and ⫺55 mV and then digitally subtracted. Leak subtraction by blocking ICa with 0.1 mM Cd2⫹ yields almost identical voltage profiles (Thoreson and Stella 2000). Recordings of ICa were corrected off-line for series resistance. Light stimuli were generated by a tungsten light source and reflected into the microscope condenser pathway using a beam splitter. Light intensity was controlled by neutral density filters (Wratten gel) and wavelength was controlled by interference filters. The intensity of unattenuated light measured with a laser power meter (Metrologic) was 1.3 ⫻ 106 photon s⫺1 ␮m⫺2 at 580 nm. A flash duration of 1 s was generally used. Pipettes were positioned under visual control using an infraredsensitive CCD camera (Watec 902H) attached to the trinocular head of the microscope. Rods were identified by their characteristic outer segments. Horizontal cell somas are in the most distal region of the outer nuclear layer and horizontal cells exhibit large outward lightevoked currents, a low input resistance, and large inwardly rectifying currents. OFF bipolar cells also show outward light-evoked currents, but exhibit a higher input resistance and a current/voltage profile dominated by outwardly rectifying currents. ON bipolar cells show a similar outwardly rectifying current/voltage profile but exhibit inward light-evoked currents. The intensity required to produce a half-maximal response was determined for both 580 and 680 nm light in a sample of six cells (3 horizontal cells, 2 OFF bipolar cells, and 1 ON bipolar cell). Consistent with mixed rod and cone inputs, these cells were 1.2 ⫾ 0.36 (n ⫽ 6) log units more sensitive to 580 than 680 nm light.

A2 ADENOSINE RECEPTORS INHIBIT ROD TRANSMISSION

Drug solutions CGS-21680, N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine (DPMA), ryanodine, and 3,7-dimethyl-l-propargylxanthine (DMPX) were obtained from Sigma Chemical (St. Louis, MO). VUF-5574, 1,3-dipropyl-8-cyclopentylxathine (DPCPX), and ZM-241385 were obtained from Tocris Cookson (Ballwin, MO). DPMA and CGS-21680 were dissolved in DMSO as 50 mM stock solutions. All other solutions were prepared as 100 mM stocks with either distilled H2O or DMSO. Superfusion with 0.1% DMSO alone was not found to affect any of the properties of ICa or Ca2⫹ imaging responses that we studied. RESULTS

Effects of an A2A agonist CGS-21680 on rod ICa and depolarization-evoked increases in [Ca2⫹]i Voltage-dependent Ca2⫹ influx in rods has previously been shown to be mediated by dihydropyridine (DHP)-sensitive L-type calcium channels (Kourennyi and Barnes 2000; Stella et al. 2002; Thoreson et al. 1997, 2000). In this study, ICa was recorded with 10 mM Ba2⫹ as the charge carrier in rods. Depolarizing voltage ramps evoked inward currents that activated above –50 mV, peaked near –10 mV, and reversed between ⫹45 and ⫹60 mV [Fig. 1, control current-voltage (I-V) plot]. The average peak amplitude of ICa in rods was –356.9 ⫾ 41.6 pA (n ⫽ 9) and the voltage at which the current was half-maximal (V50) averaged – 30.3 ⫾ 1.9 mV. Previous work in our laboratory showed that DPMA, an A2 receptor agonist, inhibited ICa in rods but A1, N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl] adenosine (R-PIA) and A3, N6-2-(4-aminophenyl)ethyladenosine (APNEA) agonists were without effect (Stella et al. 2002). In situ hybridization has revealed the presence A2A receptors in the outer J Neurophysiol • VOL

FIG. 1. Effect of the A2A selective agonist CGS-21680 on voltage-dependent Ca2⫹ influx in rod photoreceptors: A: rod ICa was inhibited by 2 ␮M CGS-21680. The graph shows current voltage (I-V) relationships of voltage ramps obtained in control superfusate (solid trace) and test solution (dashed trace). The voltage was ramped from ⫺90 to ⫹60 mV at 0.5 mV/ms. B: effect of CGS-21680 on [Ca2⫹]i increases evoked by elevated [K⫹]o in rod photoreceptors and measured with fura-2. Depolarization-evoked [Ca2⫹]i increases in a rod were produced by 1-min applications of 50 mM [K⫹]o in control superfusate, CGS-21680 (2 ␮M), and wash. CGS-21680 was applied 3 min prior to the elevated [K⫹]o stimulus.

nuclear layer (Kvanta et al. 1997). We therefore tested effects of an A2A selective receptor agonist, CGS-21680, on rod ICa. Similar to effects of DPMA (Stella et al. 2002), application of CGS-21680 (2 ␮M) caused a reduction in the peak amplitude of ICa in rods by an average of 16.8 ⫾ 2.4% (n ⫽ 9, P ⫽ 0.001) without any apparent shift in the current-voltage relationship along the voltage axis (Fig. 1A). CGS-21680 also significantly inhibited the increases in [Ca2⫹]i generated by depolarizing rods with high K⫹ solution by an average of 29.3 ⫾ 3.2% (Fig. 1B, n ⫽ 33, P ⬍ 0.001). In some cells, we found that adenosine (50 ␮M) produced little or no inhibition. This may be due in part to the presence of endogenous adenosine as suggested by results presented later in the paper. It is also possible that release from Ca2⫹ stores on depolarization (Krizaj et al. 1999) could diminish the magnitude of inhibitory effects of adenosine on [Ca2⫹]i changes in rods. We therefore examined 10 cells in which adenosine failed to inhibit depolarization-evoked [Ca2⫹]i increases (104.8 ⫾ 2.6%, n ⫽ 10). In these cells, following block of calcium-induced calcium release (CICR) with ryanodine (100 ␮M), adenosine inhibited K⫹-evoked [Ca2⫹]i increases by 23.5 ⫾ 2.8% (P ⬍ 0.0001), suggesting that Ca2⫹ release from stores can obscure the effects of adenosine on rod voltage-dependent Ca2⫹ influx. Therefore for the antagonist studies described below, we excluded cells in which adenosine applied in control conditions inhibited depolarization-evoked increases in [Ca2⫹]i by ⬍10%.

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Quip) attached to a Sutter Lambda 10-2 filter wheel equipped with 340 and 380 nm interference filters and coupled to the microscope by a liquid light guide (Sutter). The camera, filter wheel, and image acquisition parameters were controlled with Axon Imaging Workbench (AIW 2.2). Binning of images (2 ⫻ 2) was used to increase rate of acquisition. Images were subtracted for background camera noise but no averaging or masking was performed. To activate voltage-dependent Ca2⫹ channels, cells were depolarized by increasing [K⫹]o from 2.5 to 50 mM for 1 min. Elevated KCl applications were performed at 15-min intervals. Images were acquired at 5- to 10-s intervals during elevated [K⫹]o applications. A pseudocolor fluorescence image of rods loaded with fura-2 in the retinal slice and their responses to high K⫹ is illustrated in Stella et al. (2002). For analysis, a region of interest was drawn over the soma and the change in fluorescence ratio (340/380 nm) was interpreted as reflecting changes in [Ca2⫹]i. The ratio change produced by elevated [K⫹]o applied in the presence of the test solution was compared with the ratio change produced in control conditions. Ratio values for control conditions were determined by averaging the prior control and subsequent wash responses for each drug application. Multiple cells were analyzed in each preparation; each experiment was performed on at least three different slice preparations. In simultaneous Ca2⫹ imaging and whole-cell recording experiments, it was found that the 380-nm light flashes used for imaging experiments often evoked light responses of ⱕ20 pA in rods initially placed in the recording chamber. However, these responses diminished quickly with repeated exposure to 340/380 ratio pairs. Statistical analysis was performed using paired and unpaired Student’s t-test (GraphPad Prism 3.0); significance was chosen at P ⬍ 0.05, and variability is reported as ⫾ SE.

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Effects of adenosine antagonists on depolarization-evoked [Ca2⫹]i increase in rods

A2 agonists inhibit light-evoked responses of second-order neurons but not photoreceptors A hallmark of synaptic transmission from photoreceptors to second-order neurons is the continuous release of L-glutamate, which is regulated by the activity of DHPsensitive calcium channels present at the inner segment and terminals of rods (Schmitz and Witkovsky 1997; Thoreson

FIG. 2. Effects of adenosine antagonists on [Ca2⫹]i increases evoked by elevated [K⫹]o in rod photoreceptors. Bar graph representing the effects of the A1 antagonist DPCPX (100 nM, 1 ␮M), A2A antagonist ZM-241385 (100 nM, 1 ␮M), and A3 antagonist VUF-5574 (1 ␮M). Depolarizationevoked [Ca2⫹]i increases in a rod were stimulated by 1-min applications of 50 mM [K⫹]o in control superfusate, adenosine (50 ␮M) plus the antagonist, and adenosine alone (50 ␮M). The fractional response represents the K⫹-evoked [Ca2⫹]i increase measured in test solution divided by the average of the K⫹evoked [Ca2⫹]i increases obtained in control conditions before and after application of the test solution. Adenosine applied in the presence of A3 antagonist VUF5574 (1 ␮M) did not prevent inhibition of Ca2⫹ increases (P ⫽ 0.6486, n ⫽ 14, paired t-test), but adenosine produced significantly less inhibition in the presence of both the A1 antagonist DPCPX (100 nM, P ⫽ 0.12, n ⫽ 12; 1 ␮M, P ⬍ 0.001, n ⫽ 17; paired t-test) and the A2A antagonist ZM-241385 (100 nM, P ⫽ 0.85, n ⫽ 19; 1 ␮M, P ⫽ 0.05, n ⫽ 15; paired t-test) at concentrations above 100 nM. Each bar represents the mean ⫾ SE.

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Consistent with the hypothesis that the observed effects are mediated by adenosine activation of A2 receptors, co-application of an A2 selective antagonist, DMPX (50 ␮M), with adenosine (50 ␮M) prevented adenosine from inhibiting K⫹evoked [Ca2⫹]i increases in rod photoreceptors (paired t-test, n ⫽ 14, P ⫽ 0.0018). After washout of both DMPX and adenosine, adenosine (50 ␮M) reversibly inhibited the K⫹evoked [Ca2⫹]i increase (35.1 ⫾ 5.2%, n ⫽ 14, P ⬍ 0.0001). It has been suggested that DMPX may have actions at both A1 and A2 receptors at the concentration tested (Seale et al. 1988). We therefore tested DPCPX, a putatively selective antagonist for A1 receptors, and ZM-241385, a selective antagonist for A2A receptors. Both DPCPX and ZM-241385 diminished the ability of adenosine to inhibit K⫹-evoked [Ca2⫹]i increases at a concentration of 1 ␮M but not 100 nM (Fig. 2). The A3 antagonist, VUF-5574, failed to prevent adenosine-mediated inhibition of the K⫹-evoked [Ca2⫹]i increase in rods at a concentration of 1 ␮M (Fig. 2), which is in agreement with previous observations showing no effect of the A3 selective agonist, APNEA, on the voltage-dependent Ca2⫹ influx into rods (Stella et al. 2002). In further support of a role for A2A receptors, the A2A selective antagonist ZM-241385 (10 ␮M, n ⫽ 8) eliminated

inhibition of the K⫹-evoked Ca2⫹ increase produced by the A2A agonist, CGS-21680 (2 ␮M) (Fig. 3A). ZM-241385 applied in the presence of CGS-21680 increased the K⫹-evoked Ca2⫹ increase by 41.0 ⫾ 14.6% above control. This augmentation of the K⫹-evoked Ca2⫹ increase is consistent with results of light response experiments presented below, suggesting that there is an endogenous source of adenosine in and around the synapse that is tonically suppressing depolarizationevoked Ca2⫹ influx into rods. 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (10 ␮M) also antagonized the inhibitory effect of CGS-21680 (2 ␮M) on the K⫹-evoked Ca2⫹ increase (Fig. 3B). Since the A1 agonist, R-PIA, did not inhibit either the rod ICa or the K⫹-evoked Ca2⫹ increase (Stella et al. 2002) and since DPCPX antagonized the effects of an A2A agonist, it seems likely that the effects of DPCPX are due to its actions on A2 receptors rather than A1 receptors.

A2 ADENOSINE RECEPTORS INHIBIT ROD TRANSMISSION

et al. 1997; Wilkinson and Barnes 1996; reviewed by Thoreson and Witkovsky 1999). Since inhibition of photoreceptor ICa by DHP antagonists reduces light-evoked responses in second-order neurons (Rieke and Schwartz 1994; Thoreson et al. 1997), we tested whether the A2A agonist CGS-21680, which inhibits rod ICa (Fig. 1), similarly inhibits synaptic transmission from photoreceptors onto second-order neurons. In the tiger salamander retina, rods are approximately 2.2 log units more sensitive to 580 nm than 680 nm light, whereas red-sensitive cones are approximately 0.25 log units more sensitive to 680 nm light (Makino and Dodd 1996). To assess transmitter release from rods, dark-adapted retinal slices were stimulated with 580 nm light flashes. Figure 4 illustrates the voltage responses of a rod and the current responses of horizontal and bipolar cells evoked by 580-nm light flashes before, during, and after superfusion with CGS-21680 (2 ␮M). In control conditions, the peak rod light-evoked voltage responses averaged 9.6 ⫾ 2.4 mV (n ⫽ 6). The amplitude of rod voltage responses was not significantly reduced by CGS-21680 (⫺0.1 ⫾ 0.1 mV, n ⫽ 6, P ⫽ 0.3808). In contrast, light-evoked currents in second-order neurons were suppressed by application of CGS-21680. In control superfusate, horizontal cell light responses averaged J Neurophysiol • VOL

347 ⫾ 145.4 pA (n ⫽ 4); ON bipolar cell responses averaged 48.5 ⫾ 7.4 pA (n ⫽ 3) and OFF bipolar cell responses averaged 53.1 ⫾ 11.1 pA (n ⫽ 4). Light-evoked currents of all three types of second-order neurons were inhibited by CGS-21680 by an average of 35.3 ⫾ 5.1% (n ⫽ 20, P ⬍ 0.0001). CGS-21680 inhibited horizontal cells by 39.3 ⫾ 10.8% (n ⫽ 9, P ⫽ 0.0067); inhibited OFF bipolar cells by 29.4 ⫾ 3.9 (n ⫽ 6, P ⫽ 0.0007), and inhibited ON bipolar cells by 35.2 ⫾ 6.1% (n ⫽ 5, P ⫽ 0.0047). The A2 selective agonist, DPMA (10 ␮M), produced a similar 43.0 ⫾ 5.3% inhibition of light-evoked currents of second-order neurons (n ⫽ 17, P ⬍ 0.0001). DPMA inhibited horizontal cells by 68.0 ⫾ 14.5% (n ⫽ 4, P ⫽ 0.0182), inhibited OFF bipolar cells by 34.4 ⫾ 5.1% (n ⫽ 5, P ⫽ 0.0025), and inhibited ON bipolar cells by 34.9 ⫾ 4.8% (n ⫽ 8, P ⫽ 0.0002). The ability of both A2 receptor agonists to inhibit light-evoked currents in all three types of second-order neurons is consistent with the hypothesis that A2-mediated inhibition of rod ICa in turn inhibits glutamate release from rods. L-glutamate release from rods is maximal in the dark. Therefore in darkness, inhibition of photoreceptor ICa by adenosine should suppress tonic glutamate-activated inward currents in horizontal cells and OFF bipolar cells and glutamate-gated outward currents in ON bipolar cells. Consistent with this prediction, the A2 agonists, DPMA and CGS-21680, produced a large net outward current in horizontal cells (e.g., see the baseline shift in Figs. 4, 5, and 6). These drugs produced smaller DC current changes in ON and OFF bipolar cells, consistent with the smaller light-evoked currents in these cells. Light-evoked currents of horizontal cells are larger than those of bipolar cells because they make a far greater number of synaptic contacts with rods (Oyster 1999). The amplitude of the dark current change induced by the A2 agonist is correlated with the change in the amplitude of the light response (horizontal cells n ⫽ 7, P ⫽ 0.0002, r2 ⫽ 0.95; OFF bipolar cells: n ⫽ 7, P ⫽ 0.001, r2 ⫽ 0.91; ON bipolar cells: n ⫽ 8, P ⫽ 0.0127, r2 ⫽ 0.67). Moreover, the reversal potentials of the DC currents produced by the A2 agonists were near 0 mV (CGS-21680: 1.6 ⫾ 6.4 mV, n ⫽ 7; DPMA: ⫺14.4 ⫾ 9.2 mV, n ⫽ 10). These results are consistent with the conclusion that the changes in dark current were largely due to suppression of tonic glutamate release from photoreceptors. Inhibitory actions of CGS-21680 on Ca2⫹ influx were antagonized by the A2A selective antagonist, ZM-241385 (10 ␮M). We likewise tested whether ZM-241385 (10 ␮M) could antagonize the inhibitory effect of CGS-21680 on light response of second-order neurons. Figure 5 illustrates the lightevoked current responses of horizontal and bipolar cells evoked by 580-nm light flashes before, during the application of CGS-21680 (2 ␮M), and in the presence of CGS-21680 and ZM-241385 (10 ␮M). As observed previously in Fig. 4, lightevoked currents in second-order neurons were suppressed by application of CGS-21680 (2 ␮M). In this set of experiments, light-evoked currents of all three types of second-order neurons were inhibited by CGS-21680 by an average of 37.3 ⫾ 7.0% (n ⫽ 7, P ⫽ 0.0018, Fig. 5B). The application of ZM-241385 reversed the inhibition of light-evoked currents produced by CGS-21680 (Fig. 5A) (n ⫽ 7, P ⫽ 0.0156, paired t-test, Fig. 5B) and in some cases caused an enhancement (e.g., the horizontal cell shown in Fig. 5A). This is similar to the effects

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FIG. 3. Both ZM-241385 (10 ␮M, A) and DPCPX (10 ␮M, B) antagonized the inhibitory effects of CGS-21680 (2 ␮M) on depolarization-evoked [Ca2⫹]i increases in rods. The A2A antagonist, ZM-241385 (n ⫽ 8), significantly inhibited the reduction produced by CGS-21680 (P ⫽ 0.013, paired t-test, df ⫽ 7; P ⬍ 0.0001, unpaired t-test, df ⫽ 39) and increased K⫹-evoked [Ca2⫹]i responses relative to control (141.6 ⫾ 14.6%, n ⫽ 8). DPCPX (n ⫽ 12) also significantly inhibited the reduction produced by CGS-21680 (P ⬍ 0.0001, paired t-test, df ⫽ 11; P ⬍ 0.0001, unpaired t-test, df ⫽ 43) and increased K⫹-evoked [Ca2⫹]i responses relative to control (115.4 ⫾ 6.9%, n ⫽ 12).

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of CGS-21680 and ZM-241385 on K⫹-evoked Ca2⫹ increases on rods (Fig. 2). The ability of ZM-241385 to reverse the inhibition of light-evoked currents by CGS-21680 in all three types of second-order neurons is consistent with the hypothesis that A2-mediated inhibition of rod ICa in turn inhibits glutamate release from rods. An A2A antagonist enhances light-evoked currents of secondorder neurons Since application of the A2A antagonist, ZM-241385, in the presence of CGS-21680 produced an average overall increase in both the K⫹-evoked Ca2⫹ influx into rods (Fig. 2) and the amplitude of light-evoked currents in second-order neurons (Fig. 5), it appeared likely that there is endogenous adenosine present at the rod synapse. To evaluate further the impact of endogenous adenosine on transmission from photoreceptors to second-order neurons, we tested the effects of ZM-241385 alone on light responses in photoreceptors and second-order neurons. Figure 6 shows rod voltage responses recorded in current clamp and light-evoked currents recorded under voltage clamp from various second-order neurons in the outer retina before, during, and after superfusion with ZM-241385 (10 ␮M). In control conditions, the rod light-evoked voltage responses averaged 11.0 ⫾ 2.7 mV (n ⫽ 4). The amplitude of J Neurophysiol • VOL

the rod voltage response was unchanged by ZM-241385, indicating that the photoresponse is not altered by antagonizing the adenosine receptors (⫺0.13 mV, P ⫽ 0.70). However, lightevoked currents in second-order neurons were enhanced by the application of ZM-241385 (Fig. 6). In control superfusate, horizontal cell light responses averaged 77.7 ⫾ 47.6 pA (n ⫽ 6) and ranged from 7.1 to 280.5 pA. ON bipolar cell responses averaged 33.8 ⫾ 7.5 pA (n ⫽ 9) and ranged from 12.4 to 87.4 pA. OFF bipolar cell responses averaged 35.4 ⫾ 1.7 pA (n ⫽ 5) and ranged from 29.6 to 38.6 pA. In ZM-241385, light-evoked currents of second-order neurons were increased to 169.7 ⫾ 17.4% of control conditions (n ⫽ 21, P ⫽ 0.0007, Fig. 6). Similar to CGS-21680, the antagonist ZM-241385 caused a greater shift in the DC current and larger enhancement of light responses in horizontal cells compared with ON or OFF bipolar cells (Fig. 6). Horizontal cells light-evoked currents were increased to 216.6 ⫾ 46.8% of control conditions (n ⫽ 7, P ⫽ 0.0471). ON and OFF bipolar cells showed an enhancement of light-evoked currents over control conditions of 148.4 ⫾ 10.2% (n ⫽ 9, P ⫽ 0.0014) and 142.2 ⫾ 14.4% (n ⫽ 5, P ⫽ 0.0428), respectively. The ability of the A2A antagonist ZM241386 to enhance light-evoked currents of second-order neurons implies that endogenous adenosine is present at the photoreceptor synapse and provides some tonic inhibition of synaptic transmission.

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FIG. 4. Light-evoked voltage responses from a rod and light-evoked currents from a horizontal cell, OFF bipolar cell, and ON bipolar cell. Control responses are shown at left. In the center panel responses were recorded in the presence of the A2A agonist, CGS-21680 (2 ␮M). For all cells, the drug was superfused for ⱖ3 min. At the right responses are recorded after washing for 5 min with control medium. Rod photoreceptors were recorded in current clamp. Horizontal cells were recorded in voltage clamp and held at – 40 mV; bipolar cells were held at – 60 mV. *CGS-21680 (2 ␮M) inhibited light-evoked currents in second-order neurons by an average of 34.4 ⫾ 7.1% (n ⫽ 13, P ⫽ 0.0004). CGS-21680 inhibited horizontal cells by 39.3 ⫾ 10.8% (n ⫽ 9, P ⫽ 0.0067); inhibited OFF bipolar cells by 29.4 ⫾ 3.9 (n ⫽ 6, P ⫽ 0.0007), and inhibited ON bipolar cells by 35.2 ⫾ 6.1% (n ⫽ 5, P ⫽ 0.0047). *N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA; 10 ␮M) inhibited light-evoked currents in second-order neurons by 43.0 ⫾ 5.3% (n ⫽ 17, P ⬍ 0.0001). DPMA inhibited horizontal cells by 68.0 ⫾ 14.5% (n ⫽ 4, P ⫽ 0.0182); inhibited OFF bipolar cells by 34.4 ⫾ 5.1% (n ⫽ 5, P ⫽ 0.0025), and inhibited OFF bipolar cells by 34.9 ⫾ 4.8% (n ⫽ 8, P ⫽ 0.0002).

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DISCUSSION

Our results reveal a novel inhibitory action of adenosine in the retina indicating that adenosine or its analogues inhibit the synaptic release of L-glutamate by inhibiting Ca2⫹ influx through rod ICa. As considered further below, this adenosinemediated inhibition of Ca2⫹ influx through L-type ICa in rods appears to be primarily due to activation of presynaptic A2-like receptors. This result contrasts with previous reports indicating that activation of A2 receptors enhance P-type ICa in hippocampal CA3 (Mogul et al. 1993) and rat brain stem neurons (Umemiya and Berger 1994). In addition, this study demonstrates the impact of endogenous adenosine on glutamate release from rods. Pharmacology of adenosine receptors in rod photoreceptors The pharmacological profile of adenosine’s actions in rods is most consistent with the presence of an A2-like receptor. CGS-21680 (Figs. 1 and 2), the A2A selective agonist, and DPMA, the A2 receptor agonist, were effective at inhibiting both ICa and the K⫹-evoked Ca2⫹ increase in rods, whereas A1 (R-PIA) and A3 (APNEA) agonists were ineffective (Stella et al. 2002). The A2 and A2A selective agonists also inhibited J Neurophysiol • VOL

light responses of second-order neurons (Fig. 4). Inhibitory effects of the A2A agonist, CGS-21680, on depolarizationevoked Ca2⫹ increases in rods and light-evoked currents in second-order neurons were antagonized by the A2A antagonist, ZM-241385 (Figs. 2 and 5). Similarly, antagonists for A2 (DMPX) and A2A (ZM-241385) adenosine receptors prevented adenosine-mediated inhibition of voltage-dependent Ca2⫹ influx in rods (Fig. 3). The A1 antagonist, DPCPX, also inhibited depolarization-evoked [Ca2⫹]i increases in rods. Similar to our results, 1 ␮M DPCPX was also found to antagonize adenosine agonist effects on cone myoid elongation in the fish retina (Rey and Burnside 1999). In salamander rods, the ineffectiveness of A1 agonists and the ability of DPCPX to antagonize the A2Aselective agonist, CGS-21680, suggest that DPCPX is acting on A2 receptors. Thus DPCPX appears to be less selective for A1 receptors in nonmammalian preparations than it is in mammalian preparations. Both A1 and A2 receptors have been detected in the retina. In mouse and rabbit retina [3H]-NECA, 5⬘-(N-ethylcarboxamido)adenosine, which labels both A1 and A2 receptors, mainly labeled photoreceptors (Blazynski 1990). A1 receptor agonist binding sites have been localized primarily to inner retinal layers (Blazynski 1990), suggesting that [3H]-NECA binding

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FIG. 5. Light-evoked currents from a horizontal cell, OFF bipolar cell, and ON bipolar cell. A: control responses were shown at left. In the center panel responses are recorded in the presence of the A2A agonist, CGS-21680 (2 ␮M). For all cells, the drug was superfused for ⱖ3 min. At the right responses were recorded after 3 min with CGS-21680 (2 ␮M) and ZM-214385 (10 ␮M). Horizontal cells were recorded in voltage clamp and held at – 40 mV; bipolar cells were held at – 60 mV. B: bar graphs summarizing the inhibition of light-evoked currents in second-order neurons (horizontal cells, ON and OFF bipolar cells) produced by the A2A agonist, CGS-21680, or the A2A agonist, CGS-21680 and the A2A antagonist, ZM-214385 (10 ␮M). The fractional response represents the light-evoked current obtained in the present of the test solution divided by the average of the control responses obtained before and after application of test solution. For these experiments using ZM-214385 to antagonize the A2A-mediated inhibition of the light-evoked currents, *CGS21680 (2 ␮M) in this sample of second-order neurons inhibited light-evoked currents by an average of 37.2 ⫾ 6.9% (n ⫽ 7, P ⫽ 0.0018). ZM-241385 (10 ␮M) reversed the CGS-21680 mediated inhibition of the light-evoked currents in second-order neurons to 200.9 ⫾ 74.2% of the control responses (**n ⫽ 7, P ⫽ 0.0156, paired t-test; horizontal cells: 422.0 ⫾ 214.8%, n ⫽ 2; OFF bipolar cells: 131.0 ⫾ 10.0%, n ⫽ 2; ON bipolar cells: 100.0 ⫾ 14.4%, n ⫽ 3). Each bar represents the mean ⫾ SE.

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in photoreceptors was mainly due to its affinity for A2 receptors. In situ hybridization in the rat retina localized A1 receptor mRNA entirely in the ganglion cell layer and A2A receptor mRNA in both the inner and the outer nuclear layers of the rat, whereas A2B receptor mRNA was absent from the retina (Kvanta et al. 1997). These findings are consistent with the physiological data of the present study, suggesting the presence of an A2-like receptor mediating adenosine’s effect at the rod synapse. Endogenous adenosine inhibits glutamate release from rods via A2-like adenosine receptor activation The light responses of second-order neurons are generated by the light-modulated release of L-glutamate from photoreceptors (Thoreson and Witkovsky 1999). Inhibition of ICa in photoreceptors by DHP antagonists inhibits glutamate release, thereby reducing light responses in second-order neurons (Rieke and Schwartz 1994; Schmitz and Witkvosky 1997; Thoreson et al. 1997). Therefore inhibition of ICa in rods by A2 receptor activation should likewise inhibit synaptic glutamate release. Consistent with this prediction, A2 receptor agonists significantly inhibited light-evoked currents in second-order neurons without altering the light-evoked voltage responses of rods (Fig. 4). The ability of A2 adenosine receptor agonists to reduce light responses in all three postsynaptic cell types is consistent with a common presynaptic site of action. Although we cannot exclude the possibility that there may be additional postsynaptic effects, the findings that CGS-21680 and DPMA produced dark current changes that reversed near 0 mV and were correlated in amplitude with light-evoked currents are consistent with the hypothesis that the primary effect of these drugs on second-order neurons is to inhibit glutamate release from rods. Furthermore, the present findings support the hyJ Neurophysiol • VOL

pothesis that endogenously released adenosine can inhibit release by inhibiting Ca2⫹ influx through ICa via activation of an A2-like adenosine receptor. This conclusion is supported by the following: 1) the similarity between the effects of adenosine, DPMA, and CGS-21680 on ICa and transmitter release from rods (Figs. 1, 2, and 4; Stella et al. 2002); 2) the finding that the antagonist ZM-241385 not only blocked the effects of adenosine and CGS-21680 (Figs. 2, 3, and 5) but also elicited a slight enhancement (see Figs. 2 and 5), suggesting that the synapse contained some basal level of adenosine; and 3) the ability of ZM-241385 alone to enhance transmitter release (Fig. 6), presumably by preventing adenosine binding to the A2-like receptor on rods. In the CNS A2 receptor activation has been shown to inhibit GABA release (Edwards and Robertson 1999), but to our knowledge this is the first example of a synapse at which activation of A2-like adenosine receptors appears to inhibit glutamate release. L-glutamate

Sources of adenosine and adenosine’s role at the photoreceptor synapse Adenosine levels reflect an equilibrium between processes that lead to the appearance of adenosine (e.g., formation from adenine nucleotides by nucleotidases and efflux from transporters) and mechanisms that reduce adenosine levels (e.g., uptake of adenosine and conversion to inosine by adenosine deaminase) (Gu and Geiger 1992; Roth et al. 1997). Adenosine formation is linked to the balance between energy supply and demand. In darkness, ATP is metabolized by the highly active Na⫹/K⫹ ATPase in the photoreceptor inner segment to counter the steady influx of Na⫹ entering through cyclic nucleotidegated channels in the outer segment (Shimazaki and Oakley 1986; Torre 1982). Increased ATP turnover can elevate intracellular levels of adenosine (Poulsen and Quinn 1998) and thus

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FIG. 6. Light-evoked voltage responses from a rod and light-evoked currents from a horizontal cell, OFF bipolar cell, and ON bipolar cell. A: control responses are shown at left. In the center panel responses were recorded in the presence of the A2A antagonist, ZM-214385 (10 ␮M). For all cells, the drug was superfused for ⱖ3 min. At the right responses were recorded after washing for 5 min with control medium. Rod photoreceptors were recorded in current clamp. Horizontal cells were recorded in voltage clamp and held at – 40 mV; bipolar cells were held at – 60 mV. ZM-214385 (10 ␮M) enhanced light-evoked currents in second-order neurons by an average of 169.7 ⫾ 17.3% of the control responses (n ⫽ 21, P ⫽ 0.0007). ZM-214385 enhanced horizontal cells by 216.0 ⫾ 46.8% of the control response (n ⫽ 7, P ⫽ 0.0471), OFF bipolar cells were enhanced by 142.2 ⫾ 14.4% of the control response (n ⫽ 5, P ⫽ 0.0428), and ON bipolar cells were enhanced by 148.4 ⫾ 10.1% of the control response (n ⫽ 9, P ⫽ 0.0014).

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This study was supported by National Eye Institute Grant EY-10542, Gifford Foundation, Lion’s Clubs, and Research to Prevent Blindness. W. Thoreson is the recipient of a Career Development Award from Research to Prevent Blindness. S. L. Stella was supported by the Gifford Young Investigator Award.

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adenosine is released tonically from retinas in the dark when photoreceptors are metabolically most active (Blazynski and Perez 1991; Paes de Carvalho et al. 1990; Perez et al. 1986). Extracellular adenosine is taken up by rod horizontal cells (Studholme and Yazulla 1997) and ATP is released during Ca2⫹ waves propagating through retinal glial cells (Newman 2001). A discrete population of photoreceptors in the primate retina but not rodent retina stain for adenosine immunoreactivity (Braas et al. 1987). Adenosine formation may also be more directly related to neurotransmission by the release of adenosine or adenine nucleotides together with glutamate via exocytosis (Robertson et al. 2001). Adenine nucleotides can be catabolized into adenosine by membrane-bound ectonucleotidases (Cunha et al. 1998; Zimmermann and Braun 1996) and by soluble nucleotidases present in vesicles (Todorov et al. 1997). Consistent with vesicular exocytosis of ATP in retinal neurons, the release of ATP from cultured retinal amacrine-like neurons is Ca2⫹ dependent and sensitive to botulinum neurotoxin (Santos et al. 1999). It seems unlikely that extracellular ATP is converted to adenosine in significant amounts at the rod synapse since application of 50 –75 ␮M ATP to the retinal slice did not inhibit ICa or depolarization-evoked Ca2⫹ increases in rods (Stella et al. 2002). However, this does not eliminate the possibility that ADP, AMP, or adenosine might be released in vesicles. Consistent with a Ca2⫹-dependent release of adenosine, K⫹-evoked depolarization stimulates adenosine release from isolated retinas (Blazynski and Perez 1991). The present results support the hypothesis that endogenously released adenosine in the retina can regulate transmission from rods onto second-order neurons by activating presynaptic A2like receptors to inhibit Ca2⫹ influx through L-type ICa. Increased adenosine levels in darkness, whether arising from synaptic or metabolic adenosine, would therefore be expected to inhibit synaptic transmission from rods. The inhibition of glutamate release from rods may contribute to the neuroprotective effects of adenosine in retina (Ghiardi et al. 1999; Roth et al. 1997) by limiting the potentially excitotoxic actions of glutamate at second-order neurons. Another important feature of adenosine may lie in its ability to serve as a brake on tonic transmitter release from rods. Adenosine is likely to act in concert with other neuromodulators such as dopamine (Stella and Thoreson 2000), somatostatin (Akopian et al. 2000), nitric oxide (Kurenny et al. 1994), pH (Barnes at al. 1993), and insulin (Stella et al. 2000). The apparent presence of adenosine in the rod synapse raises the possibility that it may play a role in dynamic control of synaptic transmission under constantly changing conditions of illumination at this critical first synapse in vision.

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