The Journal of Neuroscience, April 15, 1999, 19(8):2938–2944 The Metabotropic Receptor mGluR6 May Signal Through Go, But Not Phosphodiesterase, in Retinal Bipolar Cells Scott Nawy Departments of Ophthalmology and Visual Science, and Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461 Bipolar cells are retinal interneurons that receive synaptic input with cells dialyzed with cGMP alone. Comparable results were from photoreceptors. Glutamate, the photoreceptor transmitter, obtained with the PDE inhibitor 3-isobutyl-1-methyl-xanthine hyperpolarizes On bipolar cells by closing nonselective cation (IBMX) or with 8-pCPT-cGMP and IBMX together, indicating channels, an effect mediated by the metabotropic receptor that PDE is not required for mGluR6 signal transduction. Addi- a mGluR6. Previous studies of mGluR6 transduction have sug- tion of the G-protein subunit Go to the pipette solution sup- gested that the receptor couples to a phosphodiesterase (PDE) pressed the cation current and occluded the glutamate re- a bg that preferentially hydrolyzes cGMP, and that cGMP directly gates sponse, whereas dialysis with Gi or with transducin G had the nonselective cation channel. This hypothesis was tested by no significant effect on either the cation current or the response. a dialyzing On bipolar cells with nonhydrolyzable analogs of cGMP. Dialysis of an antibody directed against Go also reduced the Whole-cell recordings were obtained from On bipolar cells in slices glutamate response, indicating a functional role for endoge- a of larval tiger salamander retina. Surprisingly, On bipolar cells nous Go . These results indicate that mGluR6 may signal dialyzed with 8-(4-chlorophenylthio)-cyclic GMP (8-pCPT-cGMP), through Go , rather than a transducin-like G-protein. or 8-bromo-cyclic GMP (8-Br-cGMP) responded normally to glu- tamate or L-2-amino-4-phosphonobutyrate (L-APB). Response Key words: cGMP; 8-pCPT-cGMP; mGluR6; phosphodiester- amplitudes and kinetics were not significantly altered compared ase; retinal bipolar cell; Go ; phosphodiesterase The synapse between photoreceptors and On bipolar cells is the the channel, reminiscent of the phototransduction cascade. Sup- first in the visual system. Glutamate, the photoreceptor transmit- port for this model comes from the observation that the G-protein ter, hyperpolarizes On bipolar cells via activation of a G-protein- linking the receptor to the effector enzyme is sensitive to both coupled receptor (Nawy and Jahr, 1990; Shiells and Falk, 1990). pertussis and cholera toxin (Shiells and Falk, 1992a), as is rod This receptor was first cloned in rat and classified as mGluR6 transducin, but other studies instead suggest a role for Go in the a (Nakajima et al., 1993), one of the group III metabotropic recep- mGluR6 cascade. Antibodies directed against Go , but not trans- tors that are distinguished by their high affinity for L-2-amino-4- ducin, label dendrites of On bipolar cells (Vardi et al., 1993; phosphonobutyrate (L-APB) (Nakanishi, 1994; Pin and Duvoisin, Vardi, 1998), and mGluR6 couples more efficiently to Go than to 1995), confirming results of pharmacological studies in retina transducin in transfected cells (Weng et al., 1997). If Go is part of (Shiells et al., 1981; Slaughter and Miller, 1981, 1985). Receptor the cascade, then there might be an additional step linking Go to activation by L-APB, or the photoreceptor transmitter, suppresses PDE, because Go is not known to couple directly to phosphodi- a cation current (Shiells et al., 1981; Attwell et al., 1987; Nawy esterases. Alternatively, PDE may not have any role in the cas- and Copenhagen, 1987). Introduction of cGMP into the cell cade. The present study was undertaken to distinguish between through a patch pipette increases the amplitude of this current, these possibilities. leading to the suggestion that the current is generated by a cyclic One prediction of the PDE model is that analogs of cGMP that nucleotide-gated channel (Nawy and Jahr, 1990; Shiells and Falk, are resistant to hydrolysis should be able to support the cation 1990; de la Villa et al., 1995), although this has yet to be con- current but should prevent glutamate from shutting off this cur- firmed by molecular cloning or direct activation of the channel in rent. Here it is shown that dialysis with 8-bromo-cyclic GMP isolated patches. The channel may additionally be regulated by (8-Br-cGMP) or 8-(4-chlorophenylthio)-cyclic GMP (8-pCPT- 1 Ca2 /calmodulin-dependent kinase (Walters et al., 1998). cGMP), or with the phosphodiesterase inhibitor 3-isobutyl-1- The mGluR6 receptor is thought to signal through a G-protein methyl-xanthine (IBMX), does not significantly inhibit the re- to a cGMP-preferring phosphodiesterase (PDE) (Nawy and Jahr, sponse to glutamate, compared with cells dialyzed with cGMP a 1990; Shiells and Falk, 1990; Thoreson and Miller, 1994). Acti- alone. Furthermore, introduction into the cell of either Go or an a vation of PDE would lead to hydrolysis of cGMP and closure of antibody directed against Go interferes with mGluR6 transduc- tion. Thus neither PDE nor a transducin-like G-protein appear to Received Aug. 31, 1998; revised Jan. 13, 1999; accepted Feb. 1, 1999. be essential elements in this transduction pathway. This work was supported by National Institutes of Health Grant EY10254, and by an unrestricted grant from Research to Prevent Blindness Inc. I thank Drs. Alejan- MATERIALS AND METHODS dro Peinado and Eric Wexler for helpful discussions. Correspondence should be addressed to Dr. Scott Nawy, Kennedy Center Room Materials. Slices of retina from larval tiger salamanders (Kons Scientific, 525, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, NY Germantown, WI) were prepared as described previously (Nawy and 10461. Jahr, 1990; Walters et al., 1998). Briefly, salamanders were anesthetized Copyright © 1999 Society for Neuroscience 0270-6474/99/192938-07$05.00/0 with 3-aminobenzoic acid ethyl ester and decapitated, and the eyes were Nawy • Glutamate Responses in On Bipolar Cells J. Neurosci., April 15, 1999, 19(8):2938–2944 2939 enucleated. Whole retinas were isolated and placed on a 0.65 mm cellu- lose acetate/nitrate membrane filter (Millipore, Bedford, MA) that was secured with vacuum grease to a glass slide adjacent to the recording chamber. Slices were then cut to a thickness of 150–200 mm with a tissue slicer (Stoelting, Wood Lane, IL), transferred to the recording chamber while remaining submerged, and viewed with a Zeiss (Thornwood, NY) Axioskop equipped with a water-immersion 403 objective with Hoffman modulation contrast (Modulation Contrast, Greenvale, NY). The extra- cellular solution contained (in mM): 108 NaCl, 2 CaCl2 , 2.5 KCl, 10 HEPES, 10 glucose, 0.1 picrotoxin, pH 7.6, and was perfused continu- ously through the chamber at ;1 ml/min. The internal solution was composed of 75 KH2PO4 , 10 KCl, 10 HEPES, 10 EGTA, 4 MgATP, 1 cGMP, and 0.5 NaGTP, pH 7.4 with KOH. In the experiments summa- 1 rized in Figure 6, K -gluconate (90 mM), was used instead of KH PO . 1 2 4 To block K currents during measurements of I–V plots, 20 mM tetra- ethylammonium (TEA) Cl was substituted for NaCl in the extracellular solution, and for KH2PO4 in the pipette solution on an equimolar basis. ATP, GTP, cGMP, 8-Br-cGMP, and 8-pCPT-cGMP were dissolved in water as 1003 stocks, aliquoted for single experiments, and stored frozen. A concentrated stock (200–500 mM in DMSO) of IBMX was stored frozen and added to the internal or external solution on the day of the experiment. Cyclosporin A was stored at 4°C in ethanol asa2mM a a 2 bg stock. Go and Gi were aliquoted and stored in buffer at 80°C. G 2 was stored at 20°C in 10 mM HEPES, pH 7.0, 2 mM MgCl2 ,1mM b-mercaptoethanol, and 50% glycerol, and was added to the pipette a solution at a dilution of 1:400. Anti-Go (1 mg/ml) was stored at 4°C and diluted to a final concentration of 30 mg/ml in pipette solution immedi- ately before use. All compounds listed above were obtained from Sigma (St. Louis, MO) except 8-Br-cGMP and 8-pCPT-cGMP (Biolog, La a a bg Jolla, CA), Go and Gi (Calbiochem, San Diego, CA), transducin G (a gift of Dr. Thomas Sakmar, Rockefeller University), and anti-Goa (Chemicon, Temecula CA). Electrophysiology. Patch pipettes were fabricated from borosilicate glass (WPI, Sarasota, FL) using a two-stage vertical puller (Narishige, Sea Cliff, NY) and were fire-polished to resistances of 2–3 MV. Whole- cell recordings were obtained with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA), and had input and series resistance values of ;1GV and 10–19 MV, respectively. Cells were discarded if the series resistance exceeded 20 MV, if the holding current changed suddenly, or if the holding current during the first application of agonist exceeded Figure 1. The glutamate response is potentiated by cGMP even when 2 20 pA (i.e., current measured while the sustained inward current was hydrolysis of cGMP is inhibited. A, Whole-cell recording of an On bipolar suppressed). Drugs were applied via two polymer-coated fused silica cell in the tiger salamander retinal slice. The pipette solution contains m m tubes (outer diameter 350 m, inner diameter 250 m; Polymicro Tech- ATP and GTP but not cGMP. The amplitude of the response to 1 mM nologies, Phoenix, AZ) containing external control solution, or agonist, glutamate is unchanged during the first 3 min of recording. Thick trace is m usually glutamate (1 mM), or L-APB (2 M). The tubes were mounted to 30 sec response; thin trace is 3 min response. B, Recording of another On a computer-controlled piezo-bimorph (Morgan-Matroc, Bedford, OH).