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GABA Transporters and GABA,-Like Receptors on Catfish Cone- but Not Rod-Driven Horizontal Cells

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GABA Transporters and GABA,-Like Receptors on Catfish Cone- but Not Rod-Driven Horizontal Cells The Journal of Neuroscience, May 1994, 14(5): 2648-2658 GABA Transporters and GABA,-like Receptors on Catfish Cone- but Not Rod-driven Horizontal Cells CumJian Dong, Serge A. Picaud, and Frank S. Werblin Division of Neurobiology, Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720 The effects of GABA and related agents were studied in receptor current is most likely mediated by the GABA, re- solitary rod- and cone-driven horizontal cells, acutely iso- ceptor based on the above pharmacological profile. The rel- lated from the catfish retina using enzymatic and mechanical ative effectiveness of GABA, muscimol, Pans- and c&-C treatment. Both types of horizontal cells, which normally re- aminocrotonic acid (TACA and CACA) was determined at ceive glutamatergic input from photoreceptors, responded this GABA, receptor site after cells were bathed in choline to pressure ejection of the glutamate analog kainate (50 PM) Ringer to eliminate the transporter current and in the pres- with an inward current of 300-800 pA when voltage-clamped ence of 100 PM bicuculline methiodide to block GABA, re- at -70 mV using the whole-cell patch-clamp technique. But ceptor current. The order of effectiveness was GABA > TACA pressure ejection of GABA (500 PM) elicited an inward current > muscimol > CACA. Our results demonstrate the presence only in cone-driven horizontal cells. This current, ranging of GABA transporters as well as GABA, and GABA, recep- between 100 and 400 pA, consisted of two components: (1) tors in catfish cone- but not rod-driven horizontal cells. This a GABA receptor-gated chloride current that reversed near suggests that the GABA-mediated autofeedback and chem- the chloride equilibrium potential and was blocked by bath ical coupling described recently in an amphibian retina may application of picrotoxin (100-500 PM), and (2) a GABA trans- also exist in the catfish cone horizontal cell network. porter-mediated current that was picrotoxin resistant but [Key words: GABA, GABA, receptor, GABA transporter, was blocked by NO-71 1 (1 FM) and cis-4-hydroxynipecotic horizontal cell, retina, catfish, neurotransmission] acid (250 PM), two potent GABA transporter blockers. The GABA transporter current could also be eliminated when GABA is a major inhibitory neurotransmitter in the vertebrate sodium was replaced by either choline or lithium in the bath- CNS. Its action is believed to be mediated by a bicuculline- ing medium. The picrotoxin-sensitive receptor-gated cur- sensitive GABA, receptor, which gates Cl- channels and a bi- rent could not be elicited by the GABA, receptor agonist cuculline-resistant GABA, receptor, which modulates potassi- baclofen, nor could it be blocked by the potent GABA, re- um and calcium channels (Bormann, 1988; Sieghart, 1992). ceptor antagonist P-hydroxysaclofen. The picrotoxin-sen- There is now increasing evidence that there exists a third type sitive current could be divided into two components based of GABA receptor, namely GABA, receptor, which gatesa Cl- on their sensitivity to the specific GABA, receptor antagonist conductance but is not sensitive to conventional GABA, re- bicuculline methiodide. The bicuculline-sensitive compo- ceptor antagonistsor modulators, such as bicuculline and pen- nent was found only in some cells, whereas the bicuculline- tobarbital (Johnston, 1986; Feigenspanet al., 1993; Lukasiewicz resistant, picrotoxin-sensitive component was found in all et al., 1994; Qian and Dowling, 1993). cells tested. The bicuculline-resistant current was insensi- GABA is also used as a neurotransmitter in the luminosity- tive to pentobarbital, an allosteric modulator of GABA, re- type (L-type) cone horizontal cells that mediate lateral inter- ceptor. To confirm the effectiveness of the specific batch of actions in the outer retina. L-type horizontal cells play an im- bicuculline methiodide and pentobarbital, we tested both portant role in the formation of the antagonistic receptive field drugs in ganglion cells in the salamander retinal slice prep- surround in cone photoreceptors and bipolar cells through the aration, where the GABA-elicited current is almost exclu- feedback and feedforward synapses,respectively (Baylor et al., sively mediated by GABA, receptors. Bicuculline methiodide 1971; O’Bryan, 1973; Yang and Wu, 1991). There is strong almost completely blocked, while pentobarbital significantly evidence suggestingthat those synapsesare GABAergic. For enhanced, the GABA current recorded in ganglion cells. Thus, example, GABA is synthesized in L-type horizontal cells (Lam, in catfish cone horizontal cells the bicuculline-resistant GABA 1975; Lam et al., 1979) and can be releasedwhen these cells are depolarized (Schwartz, 1982; Ayoub and Lam, 1984; Schwartz, 1987). Furthermore, the strength of the antagonistic Received June 2, 1993; revised Sept. 14, 1993; accepted Oct. 14, 1993. surround in the cone photoreceptor and bipolar cell is greatly We thank Drs. Haohua Qian for many helpful discussions, John McReynolds altered in the presenceof GABA or GABA receptor antagonists for comments on the manuscript, and George Grant for developing the computer (Murakami at al., 1982; Wu, 1986, 1991). Autoradiographic software used in the data acquisition and analysis. This work was supported by an NRSA Postdoctoral Fellowship EY06482 to C.J.D., HFSP and NATO Fel- and electrophysiological studieshave demonstrated that GABA lowships to S.A.P., and an NIH Grant EY00561 to F.S.W. is releasedfrom and taken up into horizontal cells mainly via Correspondence should be addressed to Dr. Frank S. Werblin, Division of Neurobiology, Department of Molecular and Cell Biology, 145 LSA, University a calcium-independent,electrogenic transporter (Schwartz, 1982, of California at Berkeley, Berkeley, CA 94720. 1987; Yazulla and Kleinschmidt, 1983; Ayoub and Lam, 1984). Copyright 0 1994 Society for Neuroscience 0270-6474/94/142648-l 1$05.00/O To understand further the mechanismsof GABAergic trans- The Journal of Neuroscience, May 1994, 74(5) 2649 Figure 1. Morphology of acutely dis- sociatedcatfish rod andcone horizontal I cells. Top, A rod horizontal cell. Bot- tom, A conehorizontal cell. Scalebars, 50 urn. mission in the outer retina, we studied and characterized the NaCl, 4 KCl, 1 MgCl,, 15 glucose, 10 HEPES, 5 EGTA, and 5 L-cysteine to which was added 10 U/ml papain (Worthington Biochemical Corp., effects of GABA and related compounds in solitary rod- and Freehold, NJ). The pH of the solution was adjusted to 7.4 with NaOH. cone-driven horizontal cells, acutely isolated from the catfish Afterward, the retina pieces were isolated from the epithelium layer and retina. placed into the fresh papain solution for another 20 min. Following this procedure, the retina pieces were rinsed five times with the normal catfish bathing medium (see below) and kept for up to 6 hr at 4°C until Materials and Methods used in the normal bathing medium containing 1 mg/ml bovine serum Cell dissociation. Solitary rod and cone horizontal cells acutely disso- albumin (Sigma, St. Louis, MO). Cells used for electrical recordings ciated from the catfish (Zctalurus punctatus) retina were used in the were freshly dissociated from the stored retina pieces by mechanical present study to avoid possible changes in receptor subtypes and dis- trituration with a fire-polished glass pipette immediately before record- tribution that could occur durina the culture period. Cells were obtained ing (Linn and Christensen, 1992). through enzymatic treatment of the retinas modified from the proce- Morphological and electrophysiological identification. Both cone and dures described elsewhere (Tachibana. 198 1; Linn and Christensen, rod horizontal cells retained their characteristic morphology (Naka and 1992). Briefly, a catfish was decapitated and pithed. Both eyes were Carraway, 1975) after the dissociation procedures and could be iden- enucleated and excised. The excessive vitreous fluid was carefully re- tified easily under light microscopy. Rod horizontal cells were larger in moved using filter paper. The eyecups were then cut into four pieces size with broad, radiating branches and lacked an axon (Fig. 1, top), and incubated for 20 min in papain solution containing (in mM) 126 whereas cone horizontal cells were stellate and had an axon (Fig. 1, 2650 Dong et al. l GABA Transporters and Receptors on Horizontal Cells ROD HC CONE HC 6001 I PA 400- 200- I *--m , ,100 -50 N 100 -200 mV -400 -100' / 1 J 100 pA 2 200 pA 10 ms OmV II Figure 2. Differences in electrophysiological properties between rod (left) and cone (right) horizontal cells. The upper portions show current- voltage relations of the sustained whole-cell current. The lower portions show the sample current traces recorded when the membrane voltages were stepped from a holding potential of -70 mV to 0 mV and after the leak currents were subtracted. Signals were low-pass filtered at 3000 Hz. Most of the capacitive current was erased for clarity. bottom). Rod and cone horizontal cells also have strikingly different rate of local perfusion was l-2 ml/min. The pipette solution used in electrical properties, as illustrated in Figure 2. The upper part of the most whole-cell recordings contained (in mM) 130 KCl, 0.5 CaCl,, figure shows the current-voltage (Z-I’) relations of the sustained whole- 5 EGTA, 10 HEPES, 4 ATP-2Na+, and 0.5 GTP-MgZ+ adjusted to pH cell current obtained from a rod and a cone horizontal cell. The lower 7.2 with KOH. Exceptions are noted in the figure captions. Tram- and part shows two sample current traces recorded when the membrane cis-4-aminocrotonic acid (TACA and CACA) were purchased from To- voltages were stepped from a holding potential of -70 mV to 0 mV cris Neuramin (Bristol, UK); 2-hydroxysaclofen, from Research Bio- and after the leak currents were subtracted. The cone horizontal cell chemicals Inc. (Natick, MA); and GABA, muscimol, bicuculline meth- had three prominent voltage-dependent currents: a sustained inward- iodide, picrotoxin, and baclofen, from Sigma (St. Louis, MO).
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