GABAB Receptor-Activated Inwardly Rectifying Potassium Current in Dissociated Hippocampal CA3 Neurons

The Journal of Neuroscience, October 15, 1996, 16(20):6374–6385 GABAB Receptor-Activated Inwardly Rectifying Potassium Current in Dissociated Hippocampal CA3 Neurons Deborah L. Sodickson and Bruce P. Bean Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201, and Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115 GABA and the GABAB receptor agonist baclofen activated a constant of ;1 sec. GABA activated the potassium conduc- potassium conductance in acutely dissociated hippocampal tance with a half maximally effective concentration (EC50)of1.6 CA3 neurons. Baclofen-activated current required internal GTP, mM, much lower than that for activation of GABAA receptor- was purely potassium selective, and showed strong inward activated chloride current in the same cells (EC50 ;25 mM). At rectification. As with acetylcholine-activated current in atrial low GABA concentrations, activation of the GABAB current had 1 myocytes, external Cs blocked inward but not outward cur- a Hill coefficient of 1.4–2.1, suggesting cooperativity in the 21 rent in a highly voltage-dependent manner, whereas Ba receptor-to-channel pathway. Although the maximal conduc- blocked with no voltage dependence. Unlike the cardiac cur- tance activated by GABAB receptors is much smaller than that rent, however, the baclofen-activated current showed no intrin- activated by GABA receptors, its higher sensitivity to GABA sic voltage-dependent relaxation. With fast solution exchange, A and slower time course make it well suited to respond to low current was activated by baclofen or GABA with a lag of ;50 concentrations of extra-synaptic GABA. msec followed by an exponential phase (time constant ;225 msec at saturating agonist concentrations); deactivation was Key words: baclofen; G-protein; GIRK; GABA; GABAA; chlo- preceded by a lag of ;150 msec and occurred with a time ride current; cesium; barium Two major postsynaptic conductances activated by GABA exist in has been described (Dascal et al., 1993; Kubo et al., 1993; Lesage central neurons. One is a chloride conductance activated by et al., 1994; Doupnik et al., 1995; Krapivinsky et al., 1995b). Both GABAA receptors and is present in virtually all central neurons. native channels and cloned channels seem to be activated by The second is a less widely distributed potassium conductance binding of bg subunits of G-proteins (Logethetis et al., 1987; activated by GABAB receptors (Ga¨hwiler and Brown, 1985; New- Reuveny et al., 1994; Wickman et al., 1994; Huang et al., 1995; berry and Nicoll, 1985; Dutar and Nicoll, 1988). In neurons that Inanobe et al., 1995; Krapivinsky et al., 1995b; Oh et al., 1995). possess both receptors postsynaptically, the requirements for ac- G-protein a subunits may modulate the activation (Schreibmayer tivation are often strikingly different, with GABAB-mediated in- et al., 1996) or help mediate coupling to specific transmitter hibitory postsynaptic potentials requiring stronger or more sus- receptors (Huang et al., 1995). tained stimulation than GABA -mediated responses (Dutar and A Detailed characterization of the GABA -activated potassium Nicoll, 1988; Otis and Mody, 1992). The reasons for this are not B channels in hippocampal neurons has been hindered by lack of a clearly understood. suitable single-cell preparation. Experiments using tissue slices In hippocampal pyramidal neurons, the GABA -activated po- B are limited by the difficulty of making rapid solution changes. For tassium conductance is inwardly rectifying (Ga¨hwiler and Brown, 1985) and mediated by G-proteins (Andrade et al., 1986; Thal- unknown reasons, the conductance is absent or minimal in con- mann, 1988; Thompson and Ga¨hwiler, 1992a) and can be blocked ventional hippocampal tissue culture preparations (Harrison, by external Ba21 ions (Newberry and Nicoll, 1985; Misgeld et al., 1990; Yoon and Rothman, 1991; Pfrieger et al., 1994), although it 1989). These properties are shared by transmitter-activated po- is preserved in organotypic cultures (Ga¨hwiler and Brown, 1985). tassium currents present in various other neurons (North et al., Even such basic properties as the dose–response relationship for 1987; North, 1989; Nicoll et al., 1990) as well as in cardiac atrial GABA and kinetics of current activation are unknown. Knowl- myocytes, where detailed characterization has been possible edge of such properties should help in understanding the differ- (Hartzell, 1988). A family of cDNAs encoding subunits of ences in synaptic activation of GABAA and GABAB receptors. G-protein-activated inward-rectifier potassium (GIRK) channels Acutely isolated cells have been used previously to study serotonin-activated potassium current in dorsal raphe neurons (Penington et al., 1993a). Stimulated by this, we found that robust Received May 31, 1996; revised July 26, 1996; accepted July 30, 1996. GABA receptor-activated potassium currents can be recorded in This work was supported by National Institutes of Health Grant HL35034. We B thank David Cardozo for advice, and Gabriela Greif, Craig Jahr, Stefan a preparation of acutely dissociated hippocampal CA3 neurons McDonough, Indira Raman, Gary Westbrook, and John Williams for comments on that permits rapid solution changes. We characterized the kinet- this manuscript. Correspondence should be addressed to Deborah Sodickson, Volen Center, ics, rectification, ionic block, and GABA dependence of the cur- Brandeis University, 415 South Street, Waltham, MA 02254. rent. The results show that the slow kinetics of GABAB-mediated Bruce Bean’s present address: Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115. inhibitory postsynaptic currents are primarily attributable to ki- Copyright q 1996 Society for Neuroscience 0270-6474/96/166374-12$05.00/0 netics of receptor-to-channel coupling and that activation of • Sodickson and Bean GABAB-Activated Potassium Current J. Neurosci., October 15, 1996, 16(20):6374–6385 6375 GABAB receptors requires very low levels of extracellular GABA, perhaps near background levels. MATERIALS AND METHODS Preparation of freshly dissociated neurons. Hippocampi from 7- to 12-d-old Long Evans rats were dissected in ice-cold, oxygenated dissociation solution containing (in mM): 82 Na2SO4,30K2SO4, 5 MgCl2, 10 HEPES, 10 glucose, and 0.001% phenol red indicator, pH 7.4. Slices were cut 400 mM thick and incubated for 9 min at 378C in dissociation solution containing 3 mg/ml protease (Type XXIII, Sigma, St. Louis, MO). En- zyme solution was then replaced with dissociation solution containing 1 mg/ml trypsin inhibitor and 1 mg/ml bovine serum albumin, and the slices were allowed to cool to room temperature under an oxygen atmosphere. As cells were needed, slices were withdrawn, and the CA3 region was dissected out and triturated to release individual cells. Cells were placed in the recording chamber in Tyrode’s solution containing (in mM): 150 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, 10 glucose, and 10 HEPES, pH 7.4 with NaOH. Cells were used within 6–8 hr of slice preparation. CA3 pyramidal neurons were identified morphologically, based on size and shape. Cells identified as pyramidal neurons had a large pyramidal- shaped cell body (12–16 m width, 20–36 m length) with a thick apical dendritic stump (4–6 m width, 18–24 m length) and, in some neurons, one to four basal dendritic stumps (1–4 m width, 6–14 m length). Average cell capacitance was 23 6 5pF(n5125; range, 15–38 pF). Despite the presence of dendritic stumps, the cells behaved as if they were electro- tonically compact: capacity transients settled with a single time constant of 200–350 msec. Whole-cell voltage-clamp recordings. Patch pipettes were pulled from 100 ml Boralex micropipettes (Dynalab, Rochester, NY.). Pipette resis- Figure 1. Dose-dependent activation of current by baclofen applied to tances ranged from 2 to 5 MV when filled with internal solution contain- hippocampal CA3 neurons. A, Inward current elicited at a holding poten- ing (in mM): 108 KH PO , 4.5 MgCl , 9 HEPES, 9 EGTA, 14 mM creatine 2 4 2 tial of 292 mV by successive application of 500 nM,1mM,5mM, and 100 phosphate (Tris salt), 4 mM Mg-ATP, and 0.3 mM GTP (Tris salt), pH 1 1 mM baclofen, with 60 mM external K and 243 mM internal K . B, adjusted to 7.4 with 135.4 mM KOH. The creatine phosphate ATP and Dose–response relationship for baclofen activation of current. Symbols GTP were added from 103 concentrated aliquots stored at 2708C. To and error bars represent mean 6 SEM for determinations in nine cells for prevent nucleotide hydrolysis, the final internal solution was kept on ice 500 nM to1mMbaclofen and four cells for 100 nM baclofen. The response after phosphates were added. at each baclofen concentration was normalized to the maximal response The liquid junction potential between the internal solution and Ty- obtained with 100 mM or1mMbaclofen. The full series of seven or eight rode’s solution (in which the current was zeroed before obtaining a seal) agonist concentrations was preceded and followed by applications of 50 was 12 mV, measured as described by Neher (1992). Membrane poten- 2 mM baclofen, and results were used only if these responses differed by tials were corrected for this junction potential. ,20%. The line is the best least-squares fit to 1/(1 1 EC50 /[baclofen]), Seals were formed and the whole-cell configuration was obtained in where EC is the half-maximally effective concentration of baclofen. EC bath Tyrode’s solution. The cell was then bathed by a gravity-driven, 50 50 5 3 mM. External solution: 60 mM KCl, 94 mM NaCl, 2 mM CaCl ,10mM constant stream of external solution flowing through microcapillary per- 2 HEPES, 2 mM MgCl ,10mMglucose, pH 7.4 with KOH, 3 mM TTX. fusion pipes positioned directly in front of the cell. The perfusion pipes 2 consisted of a linear array of 12 microcapillary tubes of internal diameter 200 m or 250 m, glued together side by side and fed from separate neurons, baclofen inhibits voltage-dependent calcium current as well as reservoirs.

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