The Journal of Neuroscience, May 15, 2002, 22(10):4163–4174 ␳ Elimination of the 1 Subunit Abolishes GABAC Receptor Expression and Alters Visual Processing in the Mouse Retina Maureen A. McCall,1,2 Peter D. Lukasiewicz,4 Ronald G. Gregg,2,3 and Neal S. Peachey5,6,7 Departments of 1Psychological and Brain Sciences, 2Ophthalmology and Visual Sciences, and 3Biochemistry and Molecular Biology, University of Louisville, Louisville, Kentucky 40292, 4Department of Ophthalmology and Visual Sciences, Washington University, St. Louis, Missouri 63110, 5Cleveland Veterans Affairs Medical Center, Cleveland, Ohio 44106, 6Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, and 7Department of Neurosciences, Case Western Reserve University, Cleveland, Ohio 44106 Inhibition is crucial for normal function in the nervous system. In receptor function in the retina because GABAC receptors are the CNS, inhibition is mediated primarily by the amino acid highly expressed on the axon terminals of rod bipolar cells and GABA via activation of two ionotropic GABA receptors, GABAA because this site modulates the visual signal to amacrine and ␳ and GABAC. GABAA receptor composition and function have ganglion cells. In GABAC 1 null mice, GABA-evoked re- been well characterized, whereas much less is known about sponses, normally mediated by GABAC receptors, were elimi- native GABAC receptors. Differences in molecular composition, nated, and signaling from rod bipolar cells to third order cells anatomical distributions, and physiological properties strongly was altered. These data demonstrate that elimination of the ␳ suggest that GABAA receptors and GABAC receptors have GABAC 1 subunit, via gene targeting, results in the absence of distinct functional roles in the CNS. To determine the functional GABAC receptors in the retina and selective alterations in nor- role of GABAC receptors, we eliminated their expression in mice mal visual processing. using a knock-out strategy. Although native rodent GABAC receptors are composed of ␳1 and ␳2 subunits, we show that Key words: knock-out; rod bipolar cell; electroretinogram; after ␳1 subunit expression was selectively eliminated there IPSC; whole-cell patch clamp; TPMPA; ionotropic receptor; was no GABAC receptor expression. We assessed GABAC chloride channel; inhibition In the CNS, inhibition is mediated primarily by GABA, which Lukasiewicz, 1996; Koulen et al., 1997; Boue-Grabot et al., 1998; activates two ionotropic GABA receptors, GABAA and GABAC Feigenspan and Bormann, 1998; Fletcher et al., 1998; Wassle et (Sivilotti and Nistri, 1991). GABAA and GABAC receptors are al., 1998). molecularly distinct, differing in both subunit composition and Both GABAA and GABAC receptors modulate inhibition in ␣ ␤ ␥ ␦ ⑀ ␲ subunit complexity (GABAA: 1–6, 1–3, 1–3, , , and the retina (Lukasiewicz and Shields, 1998). In particular, the ␳ subunits; GABAC: 1–3 subunits) (for review, see Enz, 2001; GABAC receptors are thought to modulate the response proper- Moss and Smart, 2001; Zhang et al., 2001). In addition, each ties of retinal ganglion cells, such as their center-surround antag- receptor type or subtype forms a chloride channel whose phar- onism, the sustained or transient nature of their responses, and macology, current kinetics, and affinities for GABA differ. Most contrast gain (Thibos and Werblin, 1978; Lukasiewicz and Werb- CNS neurons express GABAA receptors, and many express more lin, 1994; Pan and Lipton, 1995; Cook and McReynolds, 1998a,b; than one subtype (Persohn et al., 1992; Wisden and Seeburg, Dong and Werblin, 1998; Bloomfield and Xin, 2000; Flores-Herr 1992; Fritschy et al., 1998). In contrast, GABA receptors are C et al., 2001). The relatively restricted expression of GABAC simpler (Enz and Cutting, 1999a; Zhang et al., 2001), and their receptors and their late expression in postnatal development expression is more restricted (Johnston, 1994; Boue-Grabot et al., (Greka et al., 2000) make them particularly amenable for study 1998; Rozzo et al., 1999), primarily to the terminals of a class of using gene targeting. retinal interneurons, the bipolar cells (Enz et al., 1996; ␳ We inactivated the gene encoding the GABAC 1 subunit in mice and investigated the effects of the elimination of this subunit Received Jan. 2, 2002; revised Feb. 19, 2002; accepted Feb. 28, 2002. in the retina. We assessed the specific roles of GABAC receptors This work was supported by National Science Foundation (NSF) Grant IBN in the inhibitory circuit mediated by the rod bipolar cell. Overall 007388 (M.A.M.), National Institutes of Health Grants EY12354 (R.G.G), EY08922, and EY02687 (P. D. L.), the Medical Research Service, Department of retinal and rod bipolar cells to cell morphology were intact, and Veterans Affairs (N.S.P.), NSF-Experimental Program to Stimulate Competitive both electrophysiological and immunohistochemical experiments Research cooperative agreement OS-9452895 (University of Louisville), and the showed that eliminating expression of the ␳1 subunit eliminated Research to Prevent Blindness (University of Louisville and Washington Univer- sity). We thank Aaron DenDekker, Sherry Willer, Rita Hackmiller, Angela Mitchell expression of the GABAC receptor. Electroretinogram (ERG) (University of Louisville), and My Tran (Washington University) for technical measurements showed that only inner retinal function was altered assistance. In addition, we thank Drs. Ralf Enz and Heinz Wa¨ssle for the gift of the ␳ in GABA ␳1 null mice, a result consistent with an alteration in GABAC 1 antibody, Dr. Rachel Wong for advice with the immunohistochemistry, C and Dr. Botir Sagdullaev for critically reading this manuscript. the balance of excitation and inhibition between second and third Correspondence should be addressed to Dr. Maureen A. McCall, Department of order retinal neurons. These data show that the GABAC receptor Psychological and Brain Sciences, University of Louisville, Life Sciences Building, ␳ Louisville, KY 40292. E-mail: [email protected]. is not expressed in the retina of GABAC 1 null mice and repre- Copyright © 2002 Society for Neuroscience 0270-6474/02/224163-12$15.00/0 sent the first use of gene targeting to alter inhibition in a specific • 4164 J. Neurosci., May 15, 2002, 22(10):4163–4174 McCall et al. GABAC Receptor Elimination Alters Retinal Processing retinal circuit. These mice will allow us to enhance our under- The composition of the PCR solution and cycling conditions are de- scribed above. standing of the role of GABAC receptor-mediated inhibition in visual processing. Histology Light microscopy MATERIALS AND METHODS Retinal tissue was prepared as described previously (McCall et al., Production of GABA ␳1 null mice 1996b). Mice were killed by anesthetic overdose, and their eyes were C removed and fixed by immersion in 4% paraformaldehyde in 0.1 M Targeting vector phosphate buffer (PB), pH 7.4, for 18 min, and the fixed eyecups were A P1 clone was identified using a PCR strategy with two primers within rinsed (three times for 10 min each) in 0.1 M PB. For plastic sections, Ј ␳ eyecups were dehydrated through a graded series of alcohols (70, 80, 90, the 3 end of the cDNA encoding the GABAC 1 subunit gene and was purchased from IncyteGenomics. From this genomic clone, two frag- and 95%) for 1 hr each and infiltrated overnight in JB-4 (Electron ␳ Microscopy Sciences, Fort Washington, PA). The eyecups were then ments of the GABAC 1 subunit gene were isolated: an 18 kbp HindIII fragment containing the promoter, exon 1, and a portion of intron 1, and submerged in JB-4 plus polymerizer in BEEM capsules. After the plastic ␮ a 2.2 kbp EcoRI–XhoI fragment containing exon 10 and part of the 3Ј had hardened, 1 m transverse sections were cut on an ultramicrotome, untranslated region (see Fig. 1A). These fragments were used to con- stained with cresyl violet, and coverslipped. Images were captured on a struct the targeting vector (see Fig. 1B), which consisted of four cassettes: Nikon Optiphot-2 with a cooled CCD camera and associated software Ј ␳ (Spot 2; Diagnostic Instruments, Sterling Heights, MI). a 4.4 kbp 5 homology unit, which included 4.0 kbp of the GABAC 1 promoter, exon 1 (220 bp) and 210 bp of intron 1; a 1.4 kbp 3Ј homology unit from the 3Ј untranslated region of the gene, and two selectable Immunohistochemistry markers, a neomycin resistance gene (neo) and a thymidine kinase gene Eyecups were prepared and fixed as described above. The retinas were (TK) in pBluescript (Stratagene, La Jolla, CA). When homologous dissected out of the eyecup, cryoprotected through a graded series of recombination occurs (see Fig. 1A, dashed Xs), exons 2–9 are replaced sucrose, and frozen in OCT–20% sucrose (at a ratio of 2:1) (Barthel and with the neo cassette, yielding the modified locus (see Fig. 1C). Raymond, 1990). For most experiments the retinas from WT and ␳ ␳ GABAC 1 null littermates were sandwiched together, embedded in the Production of targeted embryonic stem cells and GABAC 1 same mold, and when cut and mounted onto slides were separated at null mice most by 150 ␮m. Sixteen micrometer sections were cut on a cryostat and Ϫ The general strategy for the production of targeted embryonic stem mounted onto slides and stored at 70°C. Before immunohistochemistry (ES) cells has been described previously (McCall et al., 1996a). Five the slides were warmed to 37°C and washed in 0.1 M PB three times for ␳ 15 min. Sections were incubated in 0.5% Triton X-100 in 0.1 M PB (PBX), micrograms of the GABAC 1 targeting vector was linearized by diges- tion with SalI and introduced via electroporation into 5 ϫ 10 6 HM-1 followed by incubation at room temperature (RT) for 1 hr in a blocking ES cells. Two hundred and thirty-two G418 and 1-(2Ј-deoxy-2Ј-fluoro- solution consisting of 10% normal goat serum in PBX. Sections were ␤-D-arabinofuranosyl)-5-iodouracil-resistant ES cell clones were ana- incubated overnight at RT in one of the following primary antibodies lyzed by PCR to determine which were correctly targeted on the 3Ј end.