Brain Research, 244 (1982) 155-164 155 Elsevier Biomedical Press

GABA and Bicuculline Actions on Mouse Spinal Cord and Cortical Neurons in Cell Culture

LINDA M. NOWAK, ANNE B. YOUNG and ROBERT L. MACDONALD Neurasciences Program and ( A.B. Y. and R.L.M.) Department of Neurology, University of Michigan, Ann Arbor, M148109 (U.S.A.) (Accepted December 24th, 1981)

Key words: GABA -- bicuculline -- spinal cord --cortex -- cell culture

The neutral amino acid 7-aminobutyric acid (GABA) produced membrane hyperpolarization and increased membrane chloride ion conductance of spinal cord (SC) and cortical (CTX) neurons in cell culture. GABA dose response curves were obtained for SC neurons by pressure applying known concentrations of GABA from micropipettes with large tips (miniperfusion pipettes). GABA response threshold was about 2 #M and large responses were elicited at GABA concentrations greater than 10/tM. Bicuculline (B1CUC) (0.1-10/~M) reversibly antagonized GABA responses on both SC and CTX neurons with a half maximal inhibitory concentration of about 1 ttM. BICUC antagonism of GABA responses was competitive (Lineweaver-Burke analysis). These results are compared with data on GABA and BICUC displacement of [aH]GABA binding to membranes of SC and CTX neurons in cell culture. It is suggested that high affinity GABA receptors are likely to be relevant for postsynaptic GABA responses while low affinity GABA receptors may be presynaptic.

INTRODUCTION administered systemically or applied topically to cortex2~,aa, 46. Bicuculline has been shown to antago- The neutral amino acid 7-aminobutyric acid nize GABA responses in central nervous system 6,7, (GABA) has a widespread distribution in the central in spinal cord 5, on dorsal root ganglion neurons 10, nervous system9, 24,3s and is a neurotransmitter me- 14,16,31, on invertebrate neurons z4 and on muscle 39. diating both pre 2'5- and postsynaptic inhibition 23. 4a,42. In most preparations where specificity could be GABA interacts with receptors on neuronal mem- studied, bicuculline antagonized only GABA re- branes to increase chloride ion conductance and sponses and did not alter responses to other amino thus to displace membrane potential toward the acids including glycine, fi-alanine or glutamate6, 7, chloride equilibrium potential8,ao, 31. Using [aH]- '~6 Bicuculline antagonized GABA responses at GABA, GABA has been demonstrated to bind to concentrations that did not alter either resting mem- both high and low affinity receptors on brain mem- brane potential or membrane conductance 19. These branesaa,18, 4s. The relationship between GABA- findings have suggested that bicuculline interacts binding and GABA-physiology, however, is uncer- specifically with GABA receptors. tain. For example, are low or high affinity GABA On spinal cord2, 36 and cortical neurons '1 in pri- receptors the physiologically relevant receptors for mary dissociated cell culture, GABA also produces GA BA-mediated postsynaptic inhibition, and which inhibition by increasing chloride ion conductance receptors are relevant for GABA-mediated pre- and producing membrane hyperpolarization. In ad- synaptic inhibition ? dition, specific antagonism of GABA responses is Bicuculline is a phthalide-isoquinoline alkaloid produced by bicucullineHAg, 26. In a companion which antagonizes GABA-mediated inhibition 5 7,,)~ paper, we have demonstrated that saturable, so- by binding to ~9 and displacing GABA from 12,33,49 dium-independent binding of [ZH]GABA and [ZH]- GABA receptors and produces convulsions when muscimol (a GABA agonist) is present in mere-

0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press 156 branes from mouse neurons in cell culture with Louis, MO) to neurons during intracellular record- binding characteristics similar to those of mem- ing was performed in 10 mM Mg ~/DPBS medium branes from adult rat brainlL Thus, spinal cord and which suppressed spontaneous synaptic activity. cortical neurons in cell culture provide preparations GABA (0.5 M; pH 1.3 or 3.3.) was applied ion- in which neurochemical and electrophysiological tophoretically from individual micropipettes data can be directly compared. (50-100 Mf~) using a constant current stimulator. In this paper we present the dose-dependency of Brief (50-250 ms) rectangular current pulses (0 to GABA responses and of antagonism of GABA 34 nA) were used to apply GABA within 2 [~m of responses by bicuculline. We will compare these the neuronal soma membrane preceding, during, results to data presented in a companion paper ~r' and after mini-perfusion of the cell with bicuculline using [aH]GABA binding and its antagonism by or control medium, lntracellular recordings were bicuculline to membranes from spinal cord and made with KCl-containing micropipettes resulting cortical neurons in cell culture. in an increase in intracellutar chloride ions and a change of the reversal potential for GABA from MATERIALS AND METHODS 60 mV to about --20 mV'-k Injection of steady constant current through the KC1 recording micro- Primary dissociated cell culture pipette was used to hyperpolarize the cell membrane Dissociated neuronal cell cultures were prepared to .....70 to --90 mV and thus GABA responses were from spinal cords with attached dorsal root ganglia depolarizing. and cortices dissected from 12 to 14-day-old and i 5 to 16-day-old mouse embryos respectively as describ- Miniperfusion ed previously 37. The cultures were then maintained Miniperfusion was used to locally superfuse for 4-12 weeks prior to electrophysiological experi- neurons with solutions of known concentration. ments. Miniperfusion was achieved by delivering a pressure pulse to the open shank end (0.9 mm lumen dia- Intracellular recording Visually guided penetration of spinal cord and lneter) of a fluid-filled glass micropipette whose tip was large (2--10 #m diameter). A voltage-activated. cortical neurons was accomplished on the modified 3-way pressure valve was controlled by an electronic stage of an inverted phase-contrast microscope with timer. Air pressure to the valve (I-3.5 Ibs/in.") was controlled temperature (35-37 °C). Spinal cord neu- governed by a pressure regulator. When the valve rons were distinguished from dorsal root ganglion was closed, the miniperfusion pipette was at atmos- neurons by appearance and identification was con- pheric pressure. Pipettes were checked at the be- firmed electrophysiologically. Intracellular record- ginning of experiments by bringing the pipette tip ings were made with high impedance (spinal cord up against a small neuronal soma m the culture plate neurons, 25~0 Mf~; cortical neurons, 55-70 M~) on the recording stage of the microscope and ob- glass micropipettes filled with 4 M potassium acetate serving cell movement during 1--2 s pressure pulses. (KAc) or 3 M potassium chloride (KCI). Simultane- During intracellular recording, miniperfusion pi- ous current injection and membrane potential mea- pettes were positioned 15-100 #m from the soma surements with single recording microelectrodes depending upon the size of their tips. A membrane were permitted by using a conventional bridge circuit hyperpolarization, the amplitude of which was pres- and data were recorded on a 6-channel Brush poly- sure-dependent, occurred during fluid ejection with graph or on film by a Grass camera. Recordings were both KAc and KC1 recordings for both drug and made from neurons bathed in Dulbecco's phosphate control solutions. Although this hyperpolarization buffered saline (DPBS; Gibco, Grand Island, NY) could be eliminated by decreasing the line pressure. with 10 mM Mg 2+ and 1 ~ glucose. The pH was or by moving the pipette further from the soma. a 7.2-7.4. small hyperpolarizing artifact was retained often as lontophoresis assurance of miniperfusion. Iontophoretic application of GABA (Sigma, St. Miniperfusion of bicuculline was combined with 157 iontophoresis of GABA. The tip of a GABA ion- responses were reduced to a stable level (defined as 3 tophoretic pipette was positioned adjacent to a consecutive equal responses). The superfusion me- spinal cord neuron after a stable intracellular re- dium was then returned to control. With this techni- cording was obtained. GABA was applied at inter- que, up to 10 changes with 5 different bicuculline vals of 2-5 s and iontophoretic current was adjusted concentrations could be applied to a single cell. to obtain GABA responses of about 10-20 inV. The Reduction of GABA responses by bicuculline was tip of a bicuculline-containing miniperfusion pipette accepted for analysis only if the response returned to was then positioned 10-50 #m from the tip of the control levels after the bicuculline was replaced by iontophoretic pipette. If the GABA response was control medium. reduced when the bicuculline-containing pipette was positioned (an indication of a 'leaking' pipette), the Analysis of dose-response data bicuculline-containing pipette was replaced. Care GABA dose-response curves were obtained by was taken not to direct the miniperfusion pipettes varying the amplitudes of constant duration iontop- directly at the tip of the amino acid iontophoretic horetic pulses of GABA. Log-log plots of such dose pipettes so the depolarizations produced by these response data were also made so that the limiting substances were not attenuated by bulk flow from log log slope at low iontophoretic currents could be the perfusing pipettes. Control medium or two measured. The effect of bicuculline on GABA dose- different concentrations of drug were used in each dependency was determined by obtaining control miniperfusion experiment and dose-dependency was dose-response data and then superfusing the dish taken as further evidence against a bulk flow effect. with a bicuculline-containing solution. During super- Bicuculline concentration was undoubtedly reduced fusion, GABA was applied using a constant ionto- by dilution in the recording medium surrounding phoretic current. When the GABA response reached the cell during miniperfusion, and therefore the a steady reduced value due to bicuculline addition, concentration in the pipette was taken as the upper another GABA dose-response curve was obtained. limit of the dose actually at the cell membrane. Long The bicuculline was then replaced by control medium duration (5 15 s) pressure pulses were used. and dose-response data were again obtained. Data Bicuculline was dissolved in 0.02 N HC1 at the were only accepted if the GABA responses recovered beginning of each experiment and dilutions were to control values following washout of bicuculline. made with the DPBS control medium described. Lineweaver-Burke plots of the dose-response data The pH of more concentrated bicuculline solutions were then made to determine the nature of the anta- was adjusted with NaOH when necessary, and all gonism of GABA responses by bicuculline. bicuculline solutions were stored at 8 °C in light The dose-dependency of the bicuculline antago- proof containers prior to use. All data were obtained nism of GABA responses was studied by obtaining a within 2 h of preparation of bicuculline-containing control GABA response of 10-20 mV amplitude and solutions at neutral pH since bicuculline is unstable then superfusing the culture with several different at neutral pH3L concentrations of bicuculline. Bicuculline was gene- rally added from lowest to highest concentration. Superfusion Data were accepted only if the GABA responses Bicuculline was also applied by superfusion of the returned to control values following superfusion culture plate. After a stable intracellular recording with control medium. was obtained, the tip of a GABA iontophoretic pipette was positioned adjacent to the spinal cord RESULTS neuron and current was adjusted to achieve a re- sponse of about 10-20 inV. Superfusion of control GABA hyperpolarized spinal cord and cortical neu- medium at 0.3-0.5 cc/min was then started. If no rons and increased membrane chloride ion conduc- alteration in GABA response occurred, the culture tance was superfused by a bicuculline-containing solution When GABA was applied by miniperfusion to and superfusion was continued until the GABA spinal cord (Fig. 1A1) and cortical neurons, mem- 158 brane hyperpolarization and an increase in conduc- tance were produced. Short periods of GABA ap- plication (1-2 s) produced responses 10-15 s in dura- 251 tion. If the recording micropipette contained 3 M 20 KC1 rather than 4 M KAc, chloride ions entered the uJ ! 2'1 / neuron changing the equilibrium potential for chlo- Q_ ride ion from about --65 mV to ---20 mV 2, and the GABA responses became depolarizing (Fig. I A2). rr I0 GABA was also applied to spinal cord and cortical neurons using iontophoresis (Fig. 1B) with 200 ms t t 2 545 7 IO pulses producing 400-500 ms depolarizing responses GABA (#M) 0 " (3 M KCl-filled recording micropipettes). -6 -5 -4 The dose-dependency of GABA responses was LOG GABA (M) studied in spinal cord neurons using miniperfusion Fig. 2. GABA responses were dose-dependent. Miniperfusion of GABA and recording with 3 M KCl-containing of SC neurons (n = 13 cells) with 1-10 ffM GABA evoked micropipettes. In each experiment, 3 miniperfusion dosedependent GABA responses with threshold between I and 2 ffM. Responses at each concentration are averages of pipettes were used, each containing a different con- responses on 3-5 cells (6-13 trials): Bars indicate -k the centration of GABA. The cell was impaled by the standard error of the mean (S.E.M.). Log GABA response vs recording micropipette and hyperpolarized to be- log GABA concentration is plotted in inset. See text for further details. tween --70 and --80 mV. The tip of a single mini- perfusion pipette was then brought up to within one cell body (20-40 #m) of the neuron, and GABA was applied for 1 s. The amplitude of the GABA responses could be obtained at l0 ffM. Log-log plot responses recorded using 1-10 ffM GABA were aver- of the dose-response data (inset Fig. 2) was linear at aged and plotted (Fig. 2). Threshold for GABA low GABA concentrations (< 4 #M) but deviated responses was about 2 #M and large (> 20 mV) toward the abscissa at higher concentrations. The slope of the linear portion of the tog-log plot was MINIPERFUSION IONTOPHORESIS 2.8. A I KAc B~ _ GABA SC sc t, Bieuculline antagonized GABA responses Bicuculline rapidly and reversibly reduced the amplitude of iontophoretic GABA responses on spinal cord and cortical neurons (Fig. 3). In spinal lOmV G'ABA 5MM cord neurons, bicuculline was applied locally by 5SEC I SEC miniperfusion as well as by bath superfusion, while A 2 KCI B2 SC CT) in cortical neurons only miniperfusion was used. Results obtained using mini-perfusion of bicuculline were similar to those obtained with superfusion of ~ABA 5.uM bicuculline if small tipped (10-20 #m) miniperfusion Fig, 1.7-Aminobutyric acid (GABA) responses of spinal cord pipettes were used. If larger tips were used. small (SC) and cortical (CTX) neurons. A: miniperfusion of SC (10-20 %) artifactual reductions of GABA-responses neurons with 5/zM GABA increased membrane conductance and evoked hyperpolarizing responses during recordings with were produced. (Ax) 4 M KAc-filled and depolarizing responses with (A2) 3 M Bicuculline had no effect on either resting mem- KCl-filled intracellular micropipettes. Resting membrane po- brane potential or membrane resistance at the con- tentials (RMPs) were --44 mV and --46 mV respectively. B: iontophoretic application of GABA evoked depolarizing re- centrations used in this study (<~ 10 uM). However. sponses from SC (B1) and CTX (Be) neurons during record- at higher concentrations (> 10 #M), bicueulline ings with KCl-filled micropipettes. MPs were --90 mV. produced membrane depolarization with an increase GABA was applied with 200 ms pulses of 3.8 nA (Bx) and 9.0 nA (B~). in membrane resistance (not illustrated) 19. 159

,dGAB A SC 500nM BICUC POST responses was about 200 nM and 100 % antagonism - GABS, / L31CUC .,~ t,~_ was produced by 10 #M. The ICs0 for bicuculline ISEC action was about 1 FM. Although we employed CTX I CONTROL I.uM BICUC POST II°mV fewer bicuculline concentrations with cortical neu- -L_L rons (filled circles), a similar dose-dependency was ~c present. Log-log plots of the data (Fig. 5, inset) were Fig. 3. Bicuculline (BICUC) decreased GABA-responses. The linear at low bicuculline concentrations (< 0.8 #M) scheme on the left shows the experimental paradigm. The but deviated toward the abscissa at higher concen- data traces at the right show that iontophoresis of GABA trations. The slope of the linear portion of the evoked depolarizing responses from SC (upper traces) and CTX (lower traces) neurons and that application of 500 nM log-log dose-response curves was 1.0. and 1 #M BICUC decreased the GABA responses. GABA was delivered by 200 ms constant current pulses of 4 nA to SC Antagonism of GABA responses by bicuculline was cells and 34 nA to CTX cells. Membrane potential was competitive increased to --94 mV (SC) and --72 mV (CTX) by passing hyperpolarizing current through the 3 M KCl-filled recording Dose-response curves were obtained for GABA pipette. applied by iontophoresis. Such curves were non- hyperbolic, having low response amplitude over the Bicuculline reduced the amplitude of GABA re- lower GABA iontophoretic charges (current × du- sponses but did not alter the time course of the ration) and steeply increasing response amplitudes responses. The time courses of GABA responses at higher iontophoretic charges (Fig. 6). Maximal produced by iontophoresis under control conditions GABA responses were not achieved and thus re- and following superperfusion of 0.5, 1 and 2 #M sponses were only obtained up to 15-20 mV. Log-log bicuculline (Fig. 4) are plotted semilogarithmically. plots of GABA dose-response data were linear over In all cases the GABA responses decayed exponen- low iontophoretic charges but responses deviated tially with the same time constant (the decay curves toward the current axis at higher charges (Fig. 6, are parallel despite each GABA response having a different amplitude). .~GABA Antagonism of GABA responses by bicuculline SC ' P 7" ~2,-. °[k~-v' was dose-dependent in both spinal cord and cortical CONTROL 500nM luM 2uM 500msec neurons (Fig. 5). In spinal cord neurons (filled I00- triangles), the threshold for antagonism of GABA 80- • SPINAL CORD

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co 5 ~"'~ -7 -6 5 4 LOG BICUCULLINE (M) -.--.. Fig. 5. BICUC inhibition of GABA responses was dose- dependent. Superfusion and/or miniperfusion of SC and CTX neurons with BICUC reduced the amplitude of GABA re- 50 I00 150 200 250 300 550 400 450 sponses evoked by iontophoresis. The percent inhibition of TIME FROM PEAK (msec) GABA responses obtained from 2-13 SC cells and 3-7 CTX Fig. 4. BICUC reduced GABA response amplitude without cells at each BICUC concentration tested is plotted with changing response kinetics. GABA response amplitude (mV) BICUC concentration (25 nM-10 #M) on the abscissa. Bars from the peak response was plotted as a function of time (ms) indicate ± S.E.M. Log % decrease of GABA responses vs during superfusion of 0, 0.5, 1.0 and 2.0/~M BICUC. GABA log BICUC concentration is plotted in inset. GABA was responses were evoked by 50 ms, 10 nA current pulses. applied by constant current pulses of fixed duration (2.4 nA, Membrane potential was increased to --78 mV by passing 125 ms for data traces above) during application of control current through the 3 M KCl-filled intracellular recording and BICUC-containing solutions. Data above were obtained pipette. Specimen records are presented above the graph. by applying 0, 0.5, 1.0, and 2.0 #M BICUC. 160

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-40 20 ! ,0 40 60 80 I00 12(3 140 160 ; [I/GABA (nC)]2 ~ ~ ~i • -o :4 4~ o~ 5i • Fig. 7. BICUC antagonism of GABA responses was compe- titive. The GABA dose-response data in Fig. 6 is replotted as 0305 0,1 i0 GABA(nC) reciprocal of the response amplitude vs the square of the reciprocal of the GABA charge. The apparent Ka was shifted without changing the apparent V,n~x indicating competitive 0 OI 02 05 04 0.5 antagonism. GABA (nC) Fig. 6. BICUC altered GABA dose-response curves. GABA function on the abscissa to reflect the actual dose-response curves were obtained from single SC neurons in control, and in 1 #M BICUC-containing solutions. Me- log-log slope of the GABA dose-response curve dium was changed by superfusion during recording, GABA used in the experiment, bicuculline still altered only responses evoked by comparable current pulses were smaller the abscissa intercept. Therefore bicuculline ap- in 1 /~M BICUC than in control medium (see data traces) resulting in a shift to the right of the GABA dose-response peared to antagonize GABA responses competi: curve. Log GABA response vs log GABA charge is plotted in tively. the inset. RMP was --57 mV and membrane potential was hyperpolarized to --80 mV by passing current through the 3 DISCUSSION M KCl-filled intracellular recording micropipette.

GABA responses on spinal cord and cortical neurons inset). The average slope ± S.E.M. of the log-log in cell culture plots was 1.34 ~: 0.06 (n ~ 5). We have confirmed that GABA produces mem- After a dose-response curve was obtained on an brane hyperpolarization of spinal cord 2m6 and corti- individual neuron, bicuculline was superfused into cal t~ neurons in primary dissociated cell culture by the culture dish, and the dose-response curve was increasing membrane chloride ion conductance. The repeated. In the example shown, 1 #M bicuculline dose-dependency of GABA responses evoked on shifted the GABA dose-response curve to higher spinal cord neurons in cell culture was similar to GABA charges (Fig. 6) without changing the log- that reported for cortical neurons in cell culture tl log slope (Fig. 6, inset). Lineweaver-Burke plots with a threshold of about 1-2 #M. Sodium-inde- made using reciprocals of response and iontopho- pendent [3H ]GABA binding demonstrated high and retic charge were non-linear and did not intersect low affinity binding sites on membranes from Tri- the ordinate at positive values. However, Line- ton-treated cortical and spinal cord neurons in celt weaver-Burke plots made using a power function (n culture ]5. The potent GABA agonist, [~H]musci- -- 2) on the abscissa were linear and intersected the mol, bound only to high affinity binding sites using ordinate at positive values (Fig. 7). We used the Triton-treated membranes suggesting that high affi- square of the GABA charge since the log-log slope nity binding sites are the relevant sites for post- of iontophoretic GABA dose-response data was synaptic GABA responses. The high affinity GABA about 2.0. Bicuculline altered the intercept on the binding site had a Ka (9 nM) lower than the abscissa without changing the intercept on the ordi- threshold concentration of GABA for evoking nate. Furthermore, if we used a non-integer power GABA responses (2/~M) (Fig. 8) ~5. The difference 161

binding ligand and receptors are in contact for ~ BOUND GABA / o 250 -25 minutes. In the physiological experiments, ligand GABA R ESPOI~ and receptors are in contact only for seconds. When -~. 200 -20~ % GABA was applied to spinal cord neurons for E 150 .15 longer durations (seconds to minutes), responses to z U3 W short duration GABA applications (50 Ins) became m I00 "10 a: < rounded and diminished in amplitude (Macdonald <[m / and Barker, unpublished observation). Further- © 50 / _ more, log-log slopes of GABA dose-response cur- 0 .~,, ves are reduced to unity. Thus, binding and physio- -9 -8 -~ -6 LOG GABA (M) logical experiments may not yield equivalent data. Fig. 8. GABA binding occurred at lower agonist concentra- tions than GABA responses. Sodium-independent high affini- Bicuculline antagonism of GABA responses ty [3H]GABA binding to Triton-treated membranes from Bicuculline antagonized GABA responses evoked spinal cord neurons in cell culture had a lower Ka (9 nM) than the GABA concentration required to evoke threshold GABA- on cortical and spinal cord neurons at similar con- responses (2 #M). For details of binding see Fig. 3 of centrations (0.1-10 #M). Bicuculline displacement preceding paper 15 and Fig. 2 of this paper for details of of [SH]muscimol from high affinity GABA binding GABA dose-response curve. sites was produced over a wider concentration range (0.1-1000 ffM) but which overlapped the concentra- in potency for muscimol and GABA in binding tion range effective in antagonizing GABA respon- studies and for GABA responses may be due to the ses 15. With spinal cord neurons in cell culture, removal of an inhibitory protein (or other endoge- bicuculline antagonized GABA responses with an nous modulator) by Triton-treatment 1s,44. 1C50 of about 1.0 ffM and displaced specific [3H]- When a log-log plot of averaged GABA dose-re- muscimol binding with an IC50 of about 10 ffM (Fig. sponse data was obtained, the curve was linear at 9) aS. With cortical neurons in cell culture, bicucul- low GABA concentrations (1-4 ffM) but deviated line also antagonized GABA responses with an ICso toward the abscissa at higher concentrations (> 5 of about 1 ffM and displaced specific [3H]muscimol ffM). The limiting slope of the log-log dose-re- binding with an lC~0 of about 5 ,uM (Fig. 10) 15. sponse curves is an estimate of the Hill number for the agonlst receptor interaction 47. The log-log slope of the average dose-response curve was greater than SPINAL CORD one (2.8) suggesting that more than one GABA • DECREASERESPONSE GABA molecule is required at each GABA receptor to open G 3H-MUSCIMOL the GABA-coupled chloride ion channel 1,2. The slope of the log log plot of GABA dose-response ~so data obtained for cortical neurons in cell culture was also greater than one (about two) 11 suggesting that GABA receptors on cortical and spinal cord neurons are organized similarly. -8 -7 -6 -5 -4 -3 -2 In contrast, saturation studies of [3H]muscimol LOG BICUCULLINE(M) binding in Tritonized spinal cord and cortical mem- branes were hyperbolic and had Hill numbers of Fig. 9. Spinal cord GABA responses were antagonized at one 15. Triton treatment of neuronal membranes for BICUC concentrations which displaced [SH]muscimol from spinal cord membranes. BICUC displaced [3H]muscimolbe- binding studies may remove an endogenous inhibi- tween 1 nM and I mM BICUC with 50 % displacement at about tor and alter the kinetics of GABA and muscimol 10 ffM BICUC. GABA response amplitude (10-20 mV) was binding to GABA receptors. In addition, receptor reduced by 0.l-10 ffM BICUC with 50% reduction of GABA desensitization may occur during membrane prepa- responses at 1 #M BICUC. For details of displacement experi- ment see Fig. 5 of preceding paper15. BICUC antagonism of ration or during binding experiments where the SC GABA responses is from Fig. 5 of this paper. 162

culture could be produced by either competing with CORTEX IO0 GABA for its receptor (competitive inhibition) or by • DECREASE GABA RESPONSE ~] 3H-MUSCIMOL decreasing chloride channel conductance or open 80 DISPLACEMENT time (noncompetitive inhibition). Since bicuculline ~o reduced the amplitude of GABA responses without ~o altering the time course of GABA responses in the present study, it is likely that bicuculline either 20" competes with GABA for binding to its receptor or

O decreases channel conductance. To differentiate be- LOG BICUCULLINE (M) tween these two alternatives, we performed Line- weaver-Burke analysis of GABA dose-response Fig. 10. Cortical GABA responses were antagonized at curves with and without bicuculline. Bicuculline BICUC concentrations that displaced [SH]muscimol from cortical membranes. BICUC displaced [sI-I]muscimol be- altered the apparent Ka without affecting the appa- tween 1 nM and 1 mM BICUC with 50% displacement at rent Bronx suggesting a competitive interaction be- about 5/~M BICUC. GABA response amplitude (10-20 mV) tween bicuculline and GABA. Similarly, using was reduced by 0.1-10/~M BICUC with 50% reduction of GABA responses at 1 /~M BICUC. For details of displace- Scatchard analysis of [~H]muscimol binding to rat ment experiments see Fig. 5 of preceding paper 15. Details of spinal cord and brain membranes, we have shown reduction of cortical GABA responses are presented in Fig. 5 that bicuculline antagonism of binding was compe- of this paper. titivelL Thus both electrophysiological and neuro- chemical data suggest a competitive interaction be- Thus about 10 #M bicuculline will produce a total tween bicuculline and GABA for binding to high antagonism of 10-20 mV GABA responses which affinity GABA binding sites. The log-log plot of the corresponds to about 50 % and 60 % inhibition of dose-response curve for antagonism of GABA [3H]muscimol binding to GABA receptors on spinal responses by bicuculline was linear at low bicu- cord and cortical neurons respectively. The disparity culline concentrations (< 1 #M) and had a slope of between ICs0s of binding and GABA response anta- 1.0. The low inhibitory Hill constant suggests that gonism probably reflects a difference in the K~ of only one molecule of bicuculline is required to block bicuculline in physiological and binding experiment- each GABA receptor. al conditions. In contrast to its inhibition of high affinity [3H]GABA and [ZH]muscimol binding, bi- Multiple GABA-receptors cuculline displaced low affinity [3H]GABA binding In addition to binding to high affinity GABA only with high concentrations (K~ = 115 #M) tS. The receptors mediating postsynaptic inhibition, GABA necessity for such high concentrations of bicuculline has been demonstrated to enhance the specific bind- for blockade of low affinity binding sites suggests ing of [3H]benzodiazepines2s,43,a~. However, there that the low affinity site is not responsible for post- are somewhat different structural requirements for synaptic GABA responses. Furthermore, since bi- enhancing [3H]diazepam binding and antagonizing cuculline antagonism of high affinity GABA binding [3H]GABA binding suggesting that there are two and GABA responses occurs at lower and similar distinct types of GABA receptors 22, Nonetheless, concentrations, it is likely that the high affinity since benzodiazepine receptors and GABA recep- GABA binding site is the site relevant for physio- tors copurify following solubilization 17, it is likely logical postsynaptic GABA responses. that the high affinity GABA receptor and benzo- On most vertebrate central and peripheral neu- diazepine receptor are part of the same macromo- rons, bicuculline appears to be a competitive inhibi- lecular complex. tor of GABA responses 3,5, 25,27,40 while on cat dorsal What physiological action is produced by acti- root ganglion neurons t6 and invertebrate muscle 41, vating low affinity GABA receptors ? In addition to bicuculline antagonism is noncompetitive or 'mixed' producing postsynaptic inhibition, GABA decreases noncompetitive. The antagonism of GABA respon- release of neurotransmitter from central and perip- ses by bicuculline on spinal cord neurons in cell heral nervous system neurons 4. The pharmacology 163 of presynaptic GABA receptors, however, is quite ACKNOWLEDGEMENTS different from postsynaptic receptors. Presynaptic GABA receptors are insensitive to blockade by We thank Frank Wilk and Robert Frere for bicuculline, poorly bound by muscimol and potently technical assistance and Kathy Lundquist for excel- activated by fl-para-chlorophenyl GABA (bac- lent secretarial assistance. This research was sup- lofen) 4. In contrast, postsynaptic GABA receptors ported by NIH Research Grant NS 15225 to R.L.M. are sensitive to bicuculline, potently activated by and NS 15140 to A.B.Y.L.M.N. was supported in muscimol and insensitive to baclofen. Low affÉnity part by NIMH Training Grant 14279 to the Neuro- GABA binding sites are not bound by muscimol and sciences Program. A.B.Y. was supported in part by are much less sensitive to bicuculline suggesting that NIH Teacher Investigator Development Award NS low affinity GA BA binding sites may be presynaptic 00420. R.L.M. was supported in part by NIH GABA receptors. Research Career Development Award NS 00408.

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