The Journal of Neuroscience, June 1990, fO(6): 1719-1727 Physiological Regulation of the Picrotoxin Receptor by r=Butyrolactones and y-Thiobutyrolactones in Cultured Hippocampal Neurons Katherine D. Holland, James A. Ferrendelli, Douglas F. Covey, and Steven M. Rothman Departments of Pharmacology, Anatomy and Neurobiology, and Neurology and Neurological Surgery, Washington University School of Medicine, St. Louis, Missouri 63110 We examined the effects of alkyl-substituted y-butyrolac- and TBLs with alkyl groups in the P-position are convulsants tones (GBLs), and y-thiobutyrolactones (TBLs) on GABA cur- (Klunk et al., 1982a; Levine et al., 1986). GBLs and TBLs rents in cultured, voltage-clamped rat hippocampal neurons. without /3-alkyl substituents are anticonvulsant if the com- Convulsant GBLs and TBLs reversibly diminished GABA re- pounds have alkyl substituentsin the (Y-and/or y-position (Klunk sponses in a concentration-dependent manner. @-Ethyl+- et al., 1982b; Levine et al., 1986). A similar pattern of activity methyl GBL (&EMGBL) completely abolished GABA re- is observed with alkyl-substituted succinimides (Klunk et al., sponses at 3 rnh! (I&,, 390 PM), while TBL and @-ethyl-&methyl 1982~).Early structure-activity studiesand computer-modeling TBL (&EMTBL)-induced inhibition of GABA currents was in- studies of GBLs, TBLs, and succinimidessuggested that these complete, saturating at about 50% of control at 300 @I and compounds may sharea common mechanismof action (Klunk 10 rnh! for &EMTBL and TBL, respectively. B-EMGBL and et al., 1982c, 1983). @-EMTBL both increased the rate of decay of inhibitory post- The site of action of these compounds has recently been ex- synaptic currents (IPSCs) and 8-EMGBL also decreased IPSC amined. Convulsant and anticonvulsant GBLs, TBLs, and suc- peak amplitude. In contrast, the anticonvulsant a-ethyl-a- cinimides all competitively displace%-t-butylbicyclophospho- methyl TBL (a-EMTBL) potentiated GABA currents at all GABA rothionate (TBPS) from the picrotoxin receptor of the GABA, concentrations tested; maximal potentiation was 190% of receptor complex (Weissman et al., 1984; Levine et al., 1985; control at 1 mM a-EMTBL (EC,, 102 PM). Another anticon- Holland et al., unpublished observations). In addition, these vulsant, a-ethyl-a-methyl GBL (a-EMGBL), potentiated re- compounds had little effect on the binding of 3H-flunitrazepam sponses to low (0.5 PM) but not high (2 10 PM) GABA. It also to the benzodiazepine receptor or 3H-muscimol to the GABA blocked the inhibitory effects of picrotoxin and @-EMGBL and recognition site (Holland et al., unpublishedobservations). These the facilitative effect of a-EMTBL on responses to 30 PM data indicated that the site of action of alkyl-substituted GBLs, GABA. a-EMGBL did not interfere with other agents which TBLs, and succinimides is distinct from the GABA, benzo- augment GABA currents. Both a-EMTBL and a-EMGBL de- diazepine, and barbiturate recognition sitesand suggestthat one creased the rate of IPSC decay without altering IPSC peak of the sites of action of these compounds is the picrotoxin re- amplitude. None of these compounds had any direct mem- ceptor. brane effects. Picrotoxin, a convulsant, antagonizes GABA-mediated in- We propose that 8-alkyl GBLs diminish GABA currents, creasesin neuronal chloride conductance. The convulsant tetra- and therefore, we hypothesize that these compounds are methylsuccinimide (TMSM) and a convulsant GBL also had a picrotoxin receptor agonists. &Alkyl TBLs partially diminish similar effect (Barnes and Dichter, 1984; Clifford et al., 1989). GABA currents and may be partial agonists. a-Alkyl TBLs In contrast, resultsof studieson the effects of an anticonvulsant potentiate GABA currents and may be inverse agonists. Fi- succinimide, ethosuximide (ESM), and an anticonvulsant TBL, nally, a-EMGBL potentiates responses to low but not high a-ethyl-a-methyl TBL ((Y-EMTBL), on GABA-mediated inhib- GABA concentrations and may represent a mixed inverse itory processesare contradictory. Barnes and Dichter (1984) agonist, antagonist at the picrotoxin receptor. The present have shown that ESM antagonizesGABA responsesin cultured results demonstrate that the picrotoxin receptor is capable rat cortical neurons. In contrast, Baker and his colleagues(1988) of modulating GABA responses in opposing ways. have shown that c~-EMTBL potentiates GABA responsesin cul- tured chick spinal cord neurons.The concentration ofa-EMTBL Alkyl-substituted y-butyrolactones (GBLs) and r-thiobutyro- usedby Baker in these experiments was well below that needed lactones (TBLs) have convulsant or anticonvulsant activity de- to displace35S-t-butylbicyclophosphorothionate from the picro- pending on the location of the alkyl substituents. Those GBLs toxin receptor found in rat brain (Holland et al., unpublished observations). Thus, it is not clear if the observed modulation Received Aug. 18, 1989; revised Nov. 22, 1989; accepted Dec. 11, 1989. of the picrotoxin receptor by a-EMTBL in chick spinal cord This work was supported by NIH grants GM 07805, NS 14834, and NS 19988. neurons explains the behavioral actions of these compounds. We would like to thank Nancy Lancaster for preparation of the cell cultures. To determine if the opposing behavioral effects of GBLs and Correspondence should be addressed to Dr. James A. Ferrendelli, Department TBLs are mediated by opposing actions at the picrotoxin re- of Neurology and Neurological Surgery, Box 8 11 I, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 631 10. ceptor, we have compared the effects of alkyl-substituted GBLs, Copyright 0 1990 Society for Neuroscience 0270-6474/90/06 I7 19-09$02.00/O TBLs, and succinimideson GABA currents in cultured rat hip- 1720 Holland et al. * Regulatton of Picrotoxin Receptor Butyrolactone pocampal neurons using whole-cell patch voltage-clamp tech- response after drug application was < 90% of the GABA response before niques. In addition, the effects of alkyl-substituted GBLs and drug. Data from 19 of 3 11 cells studied were discarded on this basis. TBLs on inhibitory postsynaptic currents (IPSCs) were exam- The peak current response of the drug/GABA combination was ex- pressed as a percentage of that neuron’s response to GABA alone. Thus, ined. The compounds chosen for this study (Fig. 1), a-ethyl-cu- each cell served as its own control. Each drug concentration was tested methyl GBL ((u-EMGBL), P-ethyl+methyl GBL (P-EMGBL), in at least 2 different cultures. The data were digitized at 0.36 KHz and c~-EMTBL, P-ethyl-P-methyl TBL @-EMTBL), ESM, and TMSM, stored on disk for off-line analysis. Half-maximal effective concentra- were selected because they represent the best-characterized and tions and curve-fitting of sigmoidal dose-response curves were accom- plished using probit analysis (Finney, 197 1). most potent compounds oftheir respective classes. Several other Pairs of neurons with monosynaptic inhibitory connections were also a-substituted GBLs and TBLs were also studied, but in less studied to examine the effects of various butyrolactones on GABAergic detail, for comparison to a-EMGBL and LU-EMTBL. synapses. We recorded from neighboring neurons with 2 whole-cell patch electrodes which were connected to our voltage-clamp circuit and Materials and Methods a standard bridge amplifier. Our extracellular solution lacked tetrodo- toxin, and we used an intracellular solution containing 138 mM K is- Materials. The GBLs and TBLs were synthesized according to previ- ethionate (Kodak), 2 mM KCI, 10 mM NaHEPES (pH 7.3). 2 mM MgATP, ouslv described methods (Khmk et al.. 1982a. b: Levine et al., 1986). 4 mM glucose, and 1.1 mM EGTA. Under these conditions, excitatory Ethdsuximide was providkd by Parke:Davis (M&is Plains, NJ), anh postsynaptic currents (EPSCs) are inward, while IPSCs are outward at TMSM was obtained from ICN Pharmaceuticals (Plainview, NY). Un- a holding potential of -60 mV. Intracellular stimulation of one cell less noted, all other reagents were purchased from Sigma Chemical Co. through the bridge circuit elicited an outward current in the other cell (St. Louis, MO). in about 50% of the successful double recordings. Pairs of cells were Tissue culture. Hippocampal cells were cultured from 1-d-old female considered monosynaptically connected if the onset of the postsynaptic Sprague-Dawley rat pups by a method similar to that used by Huettner current was 55 msec after the peak of the presynaptic action potential and Baughman (1986) for cortical neurons. Hippocampi were dissected and if all of the presynaptic action potentials resulted in postsynaptic under sterile conditions, cut into pieces (< 1 mm), and placed in an responses. The postsynaptic current was inhibitory if stimulation of the enzyme solution containing 1 mg/ml papain (Worthington Biochemical presynaptic cell produced an outward current which was diminished by Corp.) and 0.2 mg/ml BSA in Leibovitz’s L- 15 equilibrated with 95% bicuculline and was insensitive to the application of CNQX, an antag- 0,/5% CO>. After a 20 min incubation, the tissue was resuspended in onistat glutamatereceptors (Honor6 et al., 1988;Yamada et al., 1989). approximately 2 ml of plating media (MEM with no added glutamine Drugs were dissolved in extracellular recording solution and were ap- and 10% Nu Serum; Collaborative Research). The hippocampi were plied by bath perfusion of the culture dish (volume 0.5 ml) at a rate of then gently triturated with a fire-polished Pasteur pipette in 2-3 ml of 1.5 ml/min for 2 min. In some experiments, 30 fin GABA was also plating solution. The freshly dissociated cells were harvested by cen- applied near the postsynaptic neuron from a GABA-containing pipette trifugation (600 x g for 5 min) through 5 ml of plating media containing with 10 psi pressure pulses for 100 msec. The data were digitized at 0.5 10 mg’ml BSA and 10 mg’ml trypsin inhibitor. The cells (2.5 x 105) kHz and stored on disk for off-line analysis.