sign in sign up
<<
Home , Bicuculline, Convulsant

Brain Research, 244 (1982) 145-153 145 Elsevier Biomedical Press

GABA Binding and in Spinal Cord and Cortical Membranes from Adult Rat and from Mouse in Cell Culture

ROBERT C. FRERE, ROBERT L. MACDONALD and ANNE B. YOUNG

Neurosciences Program, and ( R.L.M. and A.B.Y.) Department of Neurology, University of Michigan, Ann Arbor, MI 48109 (U.S.A.) (Accepted December 24th, 1981)

Key words: GABA receptors -- bicuculline -- cultured neurons

Sodium-independent [ZH]GABA and [3H]muscimol binding was determined in adult rat cerebral cortical and spinal cord membranes and in membranes from fetal mouse cortical and spinal cord neurons in primary dissociated cell culture. In adult rat cerebral cortical membranes, [aH]GABA bound to two sites (Ka -- 8 nM, Bmax -- 0.62 pmol/mg protein; Ka : 390 nM, Bmax -- 3.9 pmol/mg protein) whereas the GABA agonist, [aH]muscimol, bound only to a high affinity site (Ka 5.6 riM, Bm~x -- 1.9 pmol/mg protein). In adult rat spinal cord, only a low affinity site was seen with [aH]GABA (Ka -- 340 nM, Bm~,, -= 9.8 pmol/mg protein) and only a high affinity site was seen with [3H]muscimol (Ka 5.6 nM, Bm~x - 0.25 pmol/mg protein). The inability to measure a high affinity [3H]GABA binding site in spinal cord probably reflects the high ratio of low to high affinity sites in spinal cord (39:1). In membranes from mouse neurons in cell culture, [ZH]GABA bound to two sites on cortical neurons (Ka =9 riM, Bmax : 0.24pmol/mg protein; Ka -- 510 riM, Bm~x - 1.3 pmol/mg protein) and spinal cord neurons (Ka -= 13 riM, Bmax :- 0.12 pmol/mg protein; Ka " 640 nM, Bm ~x 3.2 pmol/mg protein). Again, the ratio of low to high affinity sites in cultured mouse spinal cord neurons was high (27:1). The effects of the potent GABA antagonist, (+)bicuculline, on both low and high affinity [3H]GABA binding was determined. Bicucul[ine appeared to inhibit binding to both sites competitively but the K~ for inhibiting the high affinity site was 5 ltM and for inhibiting the low affinity site was 115/tM. Bicuculline inhibited [aH]muscimol binding in both brain and spinal cord competitively with K~s of 4/~M and 10 ICM respectively. Bicuculline inhibition of [aH]muscimol binding in cultured neuronal membranes was similar to that in adult rat membranes. The binding of the potent GABA agonist, muscimol, only to the high affinity site in both adult rat and cultured mouse neuronal membranes suggests that the high affinity site is the physiologically relevant postsynaptic GABA . The fact that bicuculline inhibits the high affinity site (but not the low affinity site) in concentrations similar to those needed to block GABA-responses in physiological experiments 2s supports this hypothesis.

I NTRODUCTION been measured directly and binding was displaced by GA BA 7,27. [ZH]dib, ydropicrotoxinin binding has Binding studies have been developed in the past also been demonstrated and and pentyl- decade which have made it possible to measure enetetrazole are capable of displacing the bound synaptic GABA receptors biochemically15,16,a1,55, but bicuculline and GABA are notZ2, 48. 56. [3H]GABA binding shows a pharmacology and These findings suggest that the GABA receptor subcellular distribution similar to that expected for complex may be composed of a number of subunits the postsynaptic GABA receptors. Binding is dis- or independent sites which interact in the intact placed by numerous GABA analogs and agonists. living cell but which can be partially dissociated However, only certain antagonists, exemplified by and/or desensitized biochemically upon membrane the bicuculline, appear to act isolation. directly to displace bound GABA15, 56. Other Early studies of [ZH]GABA binding to neuronal GABA antagonists such as , penicillin and membranes demonstrated only one population of pentylenetetrazole have been found to have no effect binding sites15,29,56; however, treatment of mem- on GABA binding33. [3H]bicuculline binding has branes with Triton X-100 (0.05~ v/v) and/or re-

0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press 146 peated freeze-thaw of the membranes followed by cuculline has been said to be a competitive inhibitor extensive washing yielded, in addition, a second, of GABA responses by some 9,1 a and noncompetitive higher affinity GABA binding sitO 6,5'5. It has been by others 41,42,47 but little detailed biochemical work proposed that tritonization removes an endogenous has been performed on its effects on GABA binding inhibitor18, 20,5° which may normally lower tbe affi- in mammalian preparations. In conjunction with the nity of the GABA receptor. The removal of this following paper 28, we have studied the mechanism inhibitor by extensive washing, mild detergent treat- of action of th.is drug and suggest which GABA ment or phospholipase C increases the binding binding site may have relevance to mediation of' affinity for GABA nearly 100-fold ~6,2z. Additional postsynaptic inhibition. evidence suggests that the may prevent the binding of this inhibitor thus enhancing MATERIALS AND METHODS the affinity of the receptor for GABA 20. The high affinity GABA binding site may be Primary dis'sociated cell culture involved in the development of supersensitivity. Primary dissociated fetal mouse neuronal cell After denervation of substantia nigra by hemitrans- cultures were prepared as described previously37 and ections, GABA receptors were found to become used for binding studies. Spinal cords were dissected supersensitive by behavioral studies 5~ and, further- from 12-14-day murine fetuses and cortices were more, the number of GABA receptors increased ~9, removed from 14-16-day fetuses. Dissected tissue 52. However, only the number of high affinity bind- was mechanically chopped and dissociated by tri- ing sites increased after the nigral deafferentation, turation with a Pasteur pipette then trypsinized and with no change observed in the low affinity sites~8,50. grown in Eagles Minimum Essential Medium sup- The high affinity site can be selectively assayed using plemented with heat inactivated horse serum (10°,~i Triton-treated membranes and [aH]muscimol (a po- v/v), fetal calf serum (10°o v/v), glucose (6 gill and tent GABA agonist) 3,4~. sodium bicarbonate (3.7 g/J). The above characteristics of the high affinity Spinal cord cells were plated at a density of binding site suggest its association with GABA- 1/4-1/2 spinal cord per plate and cortical cells at 1/3 mediated postsynaptic inhibition. Nevertheless, the hemisphere per plate. Cells were grown on collagen- actual functions of the high and low affinity sites and coated culture dishes in a humidified 35 ~'C, 10°~,', which site mediates the physiological postsynaptic CO~/90 % air incubator. Cultures were maintained response remain uncertain. It is difficult to draw for 4-8 weeks and the medium changed semiweekly. conclusions from the current literature because one must compare biochemical data from one prepara- Tissue preparation tion with physiological data from another. To Rats were sacrificed by decapitation and the make such comparisons, we have investigated brains and spinal cords rapidly removed and frozen GABA binding and physiology using mouse neu- at 20 ~'C for 1-30 days. Frozen rat rons in primary dissociated cell culture. Properties and spinal cord were homogenized in 50 vols; ice- of GABAergic synaptic transmission have been in- cold 50 mM Tris-citrate buffer, pH 7.1 at 4 °C with vestigated using chick and mouse neurons in cell a polytron homogenizer (setting 7, for 30 s). Triton culture t°,laAT,45,49. The physiological actions of X-100 was added to make the homogenate 0.05°,~i GABA and bicuculline on neurons in cell culture v/v and the samples incubated for 30 min at 37 :"C have also been studied 2,12,25,z6. and then centrifuged at 48,000g for 10 min. The We have studied the properties of GABA recep- pellet was then resuspended in fresh buffer and resedi- tors in membranes from adult rat spinal cord and mented at 48,000 g for 10 min. This washing proce- cortical neurons and from mouse spinal cord and dure was repeated 3 times. For some experiments, cortical neurons in primary dissociated cell culture. crude synaptic membranes were prepared initially We have examined the effects of the potent convul- from fresh brain and spinal cord and tritonized as sant bicuculline on GABA binding in order to previously described a6. compare biochemical and physiological effects. Bi- For tissue culture, spinal cord and cortical cul- 147 tures were homogenized in 200 vols. ice-cold 50 mM Tris-citrate buffer pH 7.1 at 4 °C and Triton X-100 ok added to achieve a final concentration of 0.025~o TOTAL BINDING SITES = v/v. This homogenate was incubated at 37 °C for 30 .8 pmol/rng protein K0=390 nM ~30 TOTAL BINDING SITES = rain and centrifuged at 48,000 g for 20 min. The =o 5,9 pmol/rng protein pellet was resuspended in fresh cold Tris-citrate buffer and washed twice. • KD= 8 nM Binding studies 1.0 fTOTAL BINDING SITES= The final washed pellets were resuspended in ice- !i cold 50 mM Tris-citrate buffer pH 7.1 and aliquots 0 .O~ .04 .O6 08 O 0.02 O.()4 of the tissue suspension (0.1-0.3 mg protein) were BOUND/FREE ( rnl/mg protein) BOUND/FREE (ml/mg protein) incubated in triplicate at 4 °C for 20 rain in 2 ml Fig. 1. Rosenthal analysis of specific [aH]GABA binding in containing various concentrations of [3H]muscimol rat brain (left) and spinal cord (right). Membrane suspensions or [3H]GABA and in the presence or absence of 0.1 (0.2-0.4 mg protein/ml) were prepared as described in the text and incubated in triplicate with increasing concentrations of mM GABA or (+)bicuculline. For competition [aHIGABA (5-800 nM). Specific binding was obtained by studies, the final incubation medium was 2-4 nM subtraction of the binding in the presence of 0.1 mM GABA [aH]muscimol or [aH]GABA. For saturation stu- from the total binding. Each point represents the mean of triplicate determinations. The experiment has been replicated dies, the concentrations of [3H]muscimol varied 4 times. from 3 to 100 nM and those of [aH]GABA from 5 to 800 nM. The assays were terminated by filtration bral cortex was saturable and Scatchard analysis 3s under vacuum through Whatman GF/B filters fol- of the data was most consistent with two sites (Fig. lowed by three 5 ml washings (each less than 3 s) 1; Table l). When the two sites were separated by with ice-cold 25 mM Tris-citrate buffer pH 7.1 at 4 Rosenthal analysis as, the high affinity site had a Ka of ~'C54. The filters were placed in 6 ml ACS (Amer- 8 nM and a Brnax of 0.62 pmol/mg protein; the low sham) and the amount of bound radioactivity deter- affinity site had a Ka of 390 nM and a Bmax of 0.62 mined by liquid scintillation spectrometry (Beckman pmol/mg protein; the low affinity site had a Ka of LS-8100, 38 7/0 efficiency). 'Nonspecific' binding was 390 nM and a Bma× of 3.9 pmol/mg protein. Further estimated as the amount of radioactivity bound in tritonization and/or washing after freeze-thaw of the presence of 0.1 mM GABA or (+)bicuculline. the membranes did not significantly alter the binding 'Specific' binding was obtained by subtracting non- curve. Likewise, when binding studies were perform- specific binding from total binding. Protein was ed on a crude synaptic membrane fraction by a determined by the method of Lowry et al. ~4. centrifugation assayl~, 56 similar saturation curves were obtained. Materials la spinal cord membranes, only a low affinity site Eagles Minimum Essential Medium, horse serum for [aH]GABA binding was demonstrable with a Ka and calf serum were purchased from Gibco. [aH]- of 340 nM and Bmax of 9.8 pmol/mg protein (Fig. muscimol (19 Ci/mmol) and [3H]GABA (58 l; Table 1). Using crude synaptic membrane frac- Ci/mmol) were obtained from Amersham Corpora- tions and a centrifugation assay, a consistent second tion. (+)Bicuculline, imidazoleacetic acid, 2,4-dia- site was also not demonstrable. Varying amounts of minobutyric acid, fl-, /%guandinoproprionic tritonization from 0.025 to 0.10 0/,', did not unmask a acid, F-amino-fl-hydroxybutyric acid, and high affinity site. L- were all purchased from Sigma Chemicals. Muscimol was purchased from Research [3H]muscimol binding in rat membranes Organics. [aH]muscimol binding in tritonized rat cerebral cortex revealed only one site with a Ka of 5.6 nM RES U LTS and Bmax of 1.9 pmol/mg protein (Fig. 2). In spinal [3H/GABA binding in rat membranes cord, [aH]muscimol binding also had only a high [3H]GABA binding in membranes from rat cere- affinity binding site with a Ka of 5.6 nM and Bmax of 148

TABLE I Summary of binding constants obtainedfrom cortical and spinal cord membranepreparations n.d., not determined; n.f., not found.

High affinity site Low affinity Ka Bm ax K,~ Bm ax (riM) (pmol/mgprotein) (riM) (pmol/mgprotein)

[ZH]GABA binding Rat cerebral cortex 8 0.62 390 3.9 Rat spinal cord n.f. n.f. 340 9.8 Cultured mouse cortex 9 0.24 510 1.3 Cultured mouse spinal cord 13 0.12 640 3.2 [ZH]muscimol binding Rat cerebral cortex 5.6 1.90 n.f. n.f. Rat spinal cord 5.6 0.25 n.fl n.f. Cultured mouse cortex 6.3 0.50 n.f. n.f. Cultured mouse spinal cord n.d. n.d. n.d. n.d.

pmol/mg protein) and a high affinity site (Ka 9 nM and Brnax 0.24 pmol/mg protein) (Fig. 3). As in rat m ~ • o2 membranes. [3H]muscimol binding revealed only o ~1. • TOTAL BINDING SITES= o m TOTAL BINDING SITES ~ the high affinity site (Ka 6.3 nM and Bmax 0.5 pmol/mg protein) (inset, Fig. 3) (Table 1). Unlike rat spinal cord membranes, however. N [ ...... ~ ..... membranes of mouse spinal cord neurons in cell 0 0,I 0.2 0,3 0 O~ 02 03 BOUND/FREE (ml/mg protein) BOUND/FREE (rnl/mg protein) culture displayed both a high affinity site (/Ca 13 nM Fig. 2 Scatcbard analysis of specific [3H]muscimol binding in and Bmax 0.12 pmol/mg protein) and a low affinity rat brain (left) and spinal cord (right) membranes. Conditions were identical to those in Fig. 1 with the exception that site (Ka 640 nM and Bmax 3.2 pmol/mg protein) various concentrations of [3H]muscimol (3-100 nM) were with [3H]GABA similar to those seen in membranes used. Each point represents the mean of triplicate determina- from cortical neurons (Fig. 3L The amount of tions. This experiment was typical of 4 separate experiments. membrane obtained from culture plates was small The lines were derived by linear regression. Insets are the Hill plots of the same data. and it was more difficult to obtain accurate satura- tion studies using membranes from neurons in cel! 0.25 pmol/mg protein. Thus muscimol appeared to label specifically a high affinity binding site in wash- % ed tritonized membranes. Furthermore, the Bmax of the muscimol binding site in spinal cord was 0.25 pmol/mg protein which was only 2.5 ~ of the Bmax of the low affinity [3H]GABA binding site. Hill plots ° .. of the binding curves in brain and spinal cord were linear with unity slope and thus the Hill coefficients . • ~ , rexAL ruNe NG S TE_~ r~ i~OP OO3 Or4 were 1.0 (Fig. 2, insets). Fig. 3. Rosenthal analysis of specific [;3H]GABA binding to [3H]GABA binding and [3H]muscimol binding in membranes from cortical (left) and spinal cord (righ0 cell cultures. Membrane suspensions were prepared as described cultured mouse cortical and spinal cord neurons in text and assayed as in Fig. 1. Inset on the left is the [3H]GABA binding to tritonized membranes of Scatchard analysis of specific [ZH]muscimol binding to cort- mouse cortical neurons in dissociated cell culture ical culture under identical conditions. Each poinl is the mean of triplicate determinations. Lines were derived by was similar to that in rat cerebral cortex membranes linear regression. Each experiment was repeated at least 3 with a low affinity site (Ka 510 nM and Bmax 1.3 times. 149 culture than using adult rat membranes. Our studies Bicuculline displacement of ~3H/GABA and f3H!- nevertheless have always indicated curvilinear Scat- muscimol in rat cerebral cortex and spinal cord chard analyses using membranes from spinal cord in membranes cell culture. At present we have not performed (?)Bicuculline displaced [3H]GABA binding to saturation studies with [3H]muscimol using spinal tritonized rat cerebral cortex and spinal cord mem- cord neurons in cell culture. branes (Fig. 4). In brain, (+)bicuculline had an ICs0 of 5/~M whereas in spinal cord, the IC50 was 40/zM. Competition studies of [3H]muscimol binding in adult This appeared as a major discrepancy between brain rat and cultured mouse neuronal tissues Competition studies of [3H]muscimol binding in I00 spinal cord and cerebral cortex membranes from "~'==~z7~---~i~ • SPINAL CORD "-.~- "-~ [; BRAIN adult rat and in membranes from cultured mouse zo so cortical neurons were similar (Table II). The ICs0s of a variety of GABA agonists were similar to those previously reported15,30, ~1,56 for GABA receptors with muscimol being most potent with an IC50 of 6 w 20 o nM, GABA next most potent followed by imidazol- 0 eacetic acid, F-amino-fl-hydroxybutyric acid and /3- -7 -6 -5 -4 -3 -2 -'1 guanidinoproprionic acid./3-Alanine which was quite L0G BICUCULLINE (M) weak in displacing [3H]muscimol binding. Glycine Fig. 4. Semilog plot of displacement of specific [:~H]GABA and glutamic acid, two other putative amino acid binding by (+)bicuculline in brain and spinal cord mem- , were virtually inactive in com- branes. Membrane suspensions were prepared as described in text and incubated in triplicate with [3H]GABA (2-4 nM) and peting for binding as was the uptake inhibitor, 2,4- various concentrations of (+)bicuculline. Specific binding diaminobutyric acid. (+)Bicuculline potently com- was obtained by subtracting the amount bound in the pre- sence of 0.1 mM GABA from the total binding. Each point is peted for [3H]muscimol binding but picrotoxin was the mean of triplicate determinations and the experiment has inactive as previously reported 3. been repeated 5 times.

TABLE II Competitio# studies for [3Hjmuscimol binding in adult rat cerebral cortical and spinal cord membranes and membranes fi'om fetai mouse cortical neurons grown in PDC In these experiments [3H]muscimol concentration was 2-4 nM and the Ka for [3H]muscimol was 4-8 nM. Thus the Ki values would be 2/3 IC~o in each case. (Ki = ICs0/(1 ÷ [L]/Ka)). The data was obtained from competition studies. Each value is the mean of the ICs0s from 3 separate experiments that varied less than 15 ~.

Compotmd IC5o (ttM) fi~r inhibition of [3H]muschnol binding Adult rat Cultured neurons Spinal cord Brain Cortical neurons

Muscimol 0.006 0.006 0.006 GABA 0.030 0.044 0.025 lmidazoleacetic acid 0.9 0.6 0.6 y-Amino-fl-hydroxybutyricacid 1.1 0.7 0.4 fi-Guanidinoproprionic acid 0.6 0.7 0.4 fi-Alanine 10 10 45 G lycine :-~ 1000 ~1000 1000 2,4-Diaminobutyric acid ~:- 1000 -- 1000 1000 L-Glutamic acid > 1000 :~ 1000 :, 1000 ( F)Bicueulline 16 5 5 Picrotoxin ~> 1000 ,- 1000 > 1000 150

I00 ...-¢~ • SPINAL CORD -o.~"4~ 0 BRAIN i ! zs0 O \\ ¢1 0~ 60 °'NX~"'-ED50-- 16iIOpM "~ F z3 2

Lit !!i /-TOTAL BINDING5IRES, ...\\ -~ \( 110 pmol,/mg profein . EOoo- s ~>B.o]}, ~ 20

" o _~ _g .g _4 -~ _; ..

LOG BICUCULLINE (M) ~ 002 0.04 006 0.08 Fig. 5. Semilog plot of displacement of specific PHlmuscimol BOUND/FREE (ml/rng protein) EfOUNE/" FRFE(mltml] protein/ binding by (÷)bicuculline in brain and spinal cord mem- branes. Conditions were identical to those in Fig. 4 except Fig. 6. Scatchard analyses of specific pH]GABA binding to that pH]muscimol (2-4 riM) was used as the ligand. Each brain (left) and spinal cord (right) membranes alone (O) or in point represents the mean of triplicate determinations. The the presence (O) of (+)bicuculline. In brain, the concentra- EDa0s are the mean ± S.E.M. of the ED.~0s from 3 separate tion of bicueu!line was 10/~M and in spinal cord, it was 50 experiments. yM. Saturation curves were performed as described in Fig. 1. The inset in the left-hand figure represents the Rosenthal analyses of the saturation curves from brain. The experiment has been replicated 3 times. and spinal cord and was present whether assays were performed on washed and tritonized whole cord membranes or on crude synaptic membrane frac- Rosenthal analysis as, bicuculline appeared to inhibit tions. In contrast, when (+)bicuculline displace- high affinity [aH]GABA bindiog competitively (Fig. ment of pH]muscimol binding was investigated, the 6, inset). In spinal cord where only a low affinity site lC~0 in spinal cord (16 yM) was much closer to that was seen with [3H]GABA, bicuculline at 50 yM also in brain (5 yM) (Fig. 5). appeared to be competitive (Fig. 6). At the tow Bicuculline is not a stable compound at neutral affinity sites, the Ks for (~-)bicuculline calculated pH 29, therefore solutions were made up fresh daily frcm the dose ratio method s or from the slope of the in 0.02 N HCI and dilutions added to the reaction Scatchard plot '~2 was 115 #M. In both spinal cord mixture just prior to the assay. A series of bicucul- and rat brain membranes, ( i-)-bicuculline appeared line displacement curves of pH]muscimol binding to inhibit pH]muscimol binding competitively were performed at various times after adding (~)- (Fig. 7) with K~s of 4 yM and 10 yM for brain and bicuculline to the 50 mM Tris-citrate buffer pH 7.1. spinal cord, respectively, calculated from slopes of No change in the IC~0 for (+)bicuculline displace- Scatchard plots. These K~s obtained from saturation ment of pH]muscimol was detectable until between studies agree well with Kts obtained from competi- 1 and 2 h after the addition of the (÷)bicuculline to tion studies. neutral medium. By 4 h, there was an approximately 20% increase in the IC~0. Finally, spinal cord dis- placement curves were always run prior to the cerebral cortex curves on any given day to make certain that the increased ICeD in spinal cord was not t o ,o, ...... secondary to a degradative phenomenon. 0'2 "~ / TOTAL aINDINGS=TES; m~ o TOTAL BINDING SITE$- To investigate the mechanism of bicuculline dis- placement of [aH]GABA binding in brain and spinal J cord, saturation curves were performed on brain o- ~ o,~ o 025 050 and spinal cord membranes in the presence of BOUND/FREE (rolling proTeih} BOUND/'F~EE (ml/'m~lprof6in) various concentrations of bicuculline. In brain, Fig. 7. Scatchard analyses of specific [aH]muscimol binding to where two GABA binding sites were demonstrated, brain (left) and spinal cord (right) membranes alone (0)or in (+)bicuculline at 10 yM affected the high affinity the presence (©) of (+)bicuculline (concentrations indicated in figure). Saturation curves were performed as in Fig. 2. site but not the low affinity site (Fig. 6). Further- Lines were derived by linear regression. The experiment has more, by separating the high and low affinity sites by been replicated 4 times. 151

Ioo-.~~~_.. ~. "SPINAL CORD cerebral cortex. However, in extensive studies, we have only been able to demonstrate one binding site for [aH]GABA in spinal cord. This is a low affinity site. Tritiated muscimol on the other hand labeled a high affinity site in both brain and spinal cord. In ED50-12.5pM 2o- brain, the ratio of low to high affinity [aH]GABA binding sites was 6.3:1 and that cf low affinity GABA binding sites to high affinity [aH]muscimol LOG BICUCULLINE(M) binding sites was 2:1. In contrast, in spinal cord the Fig. 8, Semilog plot of displacement of specific [aH]muscimol ratio of low affinity [aH]GABA binding sites to high binding by (+)bicuculline in membranes from cortical and spinal cord cultures. Membrane suspensions were prepared as affinity [aH]muscimo[ binding sites was 40:[. In described in text and assayed with the same conditions as neurons in cell culture, similar relationships were those in Fig. 5. Each point is the mean of triplicate determi- seen. Tbe ratio of low to high affinity [aH]GABA nations and is typical of each of 3 separate experiments. binding sites in brain was 5.3:1 and in spinal cord 27:1. Thus with both adult and cultured murine ( + ) Bicuculline displacement of [aH]muscimol bind- spinal cord neurons, the low affinity [aH]GABA ing to mouse spinal cord and cortical neurons in cell binding site was present in concentrations 25-40 culture times that of the high affinity binding site. [aH]Mus- (?)Bicuculline displaced [aH]muscimol binding cimol labeled only a high affinity site in both brain in membranes from cortical (ICf0 4.5 #M) and and spinal cord tritonized membranes. Hill coeffi- spinal cord (IC5o 12.5 #M) neurons in cell culture cients of unity were found for the dose-response with potencies similar to those found in adult rat curves for [aH]muscimol binding in brain and spinal cerebral cortex and spinal cord membranes (Fig. 8). cord membranes which suggests that there is one There appeared to be a slight tendency for bicuculline population of mutually independent higb affinity to be less potent in spinal cord than brain. Satura- binding sites. tion curves in the presence of bicuculline have not One implication of the high ratio of low affinity been performed in culture secondary to the difficulty [3H]GABA binding sites to high affinity sites in of these experiments. spinal cord is that even at the low ligand concen- trations (2-5 nM) used in routine binding assays DISCUSSION with [aH]GABA 30-50 ~ of the observed binding is to the low affinity site (assuming reversible ligand- GABA receptors are distributed throughout the receptor interactions where Ka = [Bmax] [L]/B and B central nervous system and much is known about bound ligand, Bmax ~ maximum number of their physiological, pharmacological and bioche- binding sites, L = ligand concentration and Ka == mical properties. In brain, biochemical studies of the dissociation constant8). In order to look reliably GABA receptors have revealed two sodium-inde- for changes in the high affinity site in spinal cord, it pendent binding sites for tritiated GABA in trito- would be preferential to study [aH]muscimol bind- nized or frozen and extensively washed mem- ing. In brain, either ligand is adequate since only branes 16J8,3°,~. The density and regional distri- 5-15 ~ of GABA binding is to the low affinity site at bution of the two sites appear to vary independently 2-5 riM. throughout the CNS 35. The function of the different In studying bicuculline displacement of [aH]- binding sites is unknown. We have studied GABA GABA in spinal cord, the effects of the low affinity receptors in adult rat and cultured mouse spinal site also became apparent. (+)Bicuculline had an cord neurons so that biochemical data could be apparent lC~0 of 40/~M in displacing [aH]GABA (2 compared with neurophysiological data obtained riM) in spinal cord. In experiments in spinal cord from the same preparation28. using 100 nM [aH]GABA when 90% of the bind- As observed by others16, 30, we found two binding ing is to the low affinity site, the [C~0 for (+)bicu- sites for [aH]GABA in tritonized membranes from culline displacement of [aH]GABA binding was 100 152

#M (data not shown). When [ZH]muscimol was the relatively bicuculline insensitive. The pharmacology ligand, (+)bicucuUine has an IC50 of l6/~M, which of the low affinity site in spinal t~ord must be studied is similar to but nevertheless slightly higher than the in detail before it can be related to any specific IC50 for (+)bicuculline displacement of [aH]GABA function. Reports of drug effects on mixed high and or [ZH]muscimol binding in brain membranes. low affinity [ZH]GABA binding have utilized ligand Saturation curves of [3H]GABA binding in the concentrations in which 75 o~ of the binding was to presence and absence of 10 #M (÷)bicucultine in the high affinity site 3°. brain showed competitive inhibition of the high The effect of bicuculline on [~H ]muscimol binding affinity binding site with no significant alteration of in both brain and spinal cord appeared competitive the low affinity site. In spinal cord, 10 #M bicucul- (K,is of 4/~M for brain and 10 ~M for spinal cord). line had little effect on the [SH]GABA binding. The Although bicuculline appears to be competitive with K~ for the high affinity site calculated from the GABA in these binding studies, it should be noted Scatchard analyses (slope in presence of bicuculline that apparent competitive inhibition does not neces- equals (I + [I]/K~)) was 5 #M for the high affinity sarily indicate binding of the inhibitor to the same site in brain. At 50 #M however, bicuculline inhibit- site as that of the ligand6. In this respect, there ed [aH]GABA binding competitively in spinal cord are some inconsistent properties of the inhibition of and the K, for (+)bicuculline at the low affinity site GABA binding by bicuculline. Firstly, there is the was 115 #M as calculated from saturation studies in lack of effect of tritonization and/or fi'eeze-thaw and the presence of (+)bicuculline. Previous investiga- extensive washing on the bicucultine IC50 for dis- tions TM have found little or no regional variation in placing GABA binding despite an almost 10-100- the potency of bicuculline for inhibiting [ZH]GABA fold increase in the affinity of GABA itself for its binding but most of these studies were focusing on receptor after these procedures 1~. Furthermore, va- the high affinity site. rious anions enhance the potency of bicuculline for The high K, for bicuculline inhibition of low inhibiting GABA-binding, but the same anions have affinity [3H]GABA binding is less than the ICs~ for no substantial effects on GABA binding5,t6. These bicuculline inhibition of high affinity GABA-trans- properties suggest that the interaction of bicuculline port (about 400 #M)Za,34. The GABA transport with GABA receptors may be more complicated system is very sensitive to triton and freezing, and is than indicated by these kinetic studies. highly temperature- and sodium-dependent'~6. Our studies indicate that the binding properties of These binding studies were performed on frozen and [:~H]muscimol and [3H]GABA in spinal cord and tritonized membranes in the absence of sodium, and cortical neurons in PDC culture are quite similar it is thus unlikely that this low affinity binding site is to those in adult rat nervous system. Two binding the same as the transport site. A further reason sites for [3H]GABA are found in cortical and against the low affinity site representing binding to spinal cord neurons although the ratio of low to the uptake site is that the number of low affinity sites high affinity sites is very much higher in spinal cord have been found not to change in the projection neurons than in cortical neurons. As in rat nervous areas of GABAergic pathways after lesion of the system, ( t-)bicuculline inhibits [3H]muscimot bind- GABAergic neurons 19,5z. For this same reason, it is ing in cultured forebrain neurons slightly more unlikely that the low affinity site represents binding to potently than in spinal cord neurons. Although we presynaptic autoreceptors on GABAergic neurons. have not performed saturation studies in the pre- However, the low affinity site could represent pre- sence of bicuculline in cultured neurons, the simila- synaptic receptors on other non-GABAergic nerve rities of the receptors in the two systems and the terminals. These presynaptic receptors have been neurophysiological data 2s obtained from neurons in demonstrated experimentally and although there is cell culture suggest that bicuculline also would dis- some controversy in the literature, evidence exists play competitive inhibition in cultured neurons. that these sites are pharmacologically distinct from In other systems, the low affinity receptor site is the postsynaptic GABA receptors 1,4,~9,46. Further- thought to be the physiologically relevant~one ~z,5'~ more, these presynaptic sites have been found to be h~ these studies, the potent GABA agonist, musci- 153 tool, binds preferentially to high affinity GABA ACKNOWLEDGEMENTS receptors and bicuculline inhibits high affinity bind- ing at doses similar to those effective physiological- The authors thank Francis Hospod for expert ly 28. The high Ki for bicuculline blockade of the low technical assistance and Aileen BeMent and Kathy affinity site is inconsistent with current knowledge of Lundquist for secretarial help. These studies were the postsynaptic GABA receptor. Thus, our experi- supported by the United Cerebral Palsy Research ments suggest that the high affinity site represents and Educational Foundation, Inc. and USPHS the postsynaptic GABA receptor. The following Grants NS 15140 (A.B.Y.) and NS 15225 (R.L.M). paper gives further evidence for this conclusion by A.B.Y. is the recipient of a NIH Teacher Investi- presenting the physiological properties of postsy- gator Development Award NS 00420. R.L.M. is the naptic GABA responses and their inhibition by recipient of a NIH Research Career Development bicuculline using mammalian neurons primary dis- Award NS 00408. sociated cell culture 's.

REFERENCES 12 Dichter, M. A., Physiological identification of GABA as the inhibitory transmitter for mammalian cortical neurons 1 Arbilla, S., Kamal, L. and Langer, S. Z., Presynaptic in cell culture, Brain Research, 190 (1980) Ill 121. GABA autoreceptors on GABAergic nerve endings of the 13 Enna, S. J., Collins, J. F. and Snyder, S. H., Stereospeci- rat substantia nigra, Europ. J. Pharmaeol., 57 (1979) ficity and structure-activity requirements of G A BA recep- 211 -217. tor binding in rat brain, Brain Research, 124 (1977) 2 Barker, J. L. and Ransom, B. R., Amino acid pharmaco- 185-190. logy of mammalian central neurones grown in tissue 14 Enna, S. J., Ferkany, J. W. and Krogsgaard-Larsen, P., culture, J. Physiol. (Lond.), 280 (1978) 331-354. Pharmacological characteristics of GABA receptors in 3 Beaumont, K., Chilton, W. S,, Yamamura, H. I. and different brain regions. In P. Krogsgaard-Larsen, J. Enna, S. J., Muscimol binding in rat brain: association Scheel-Kruger and H. Kofod (Eds.), GABA-Neurotrans- with synaptic GABA receptors, Brain Research, 148 mitters, Academic Press, New York, 1978, pp. 191-200. (1978) 153-162. 15 Enna, S. J. and Snyder, S. H., Properties of y-amino- 4 Bowery, N. G., Hill, D. R., Hudson, A. L., Doble, A., butyric acid (GABA) receptor binding in rat brain sy- Middlemiss, D. N., Shaw, J. and Turnbull, M., ( ) naptic membrane fractions, Brain Research, 100 (1975) decreases release in the 81-97. mammalian CNS by action at a novel GABA receptor, 16 Enna, S. J. and Snyder, S. H., Influences of ions, enzymes Nature (Lond.), 283 (1980) 92-94. and detergents on 7-aminobutyric acid-receptor binding in 5 Browner, M., Ferkany, J. W. and Enna, S. J., Biochemical synaptic membranes of rat brain, Molec. Pharmacol., 13 identification of pharmacologically and functionally (1977) 442 453. distinct GABA receptors in rat brain, J. Neurosci., 1 17 Farb, D. H., Berg, D. K. and Fischbach, G. D., Uptake (1981) 514 518. and release of [3H]-7-aminobutyric acid by embryonic 6 Cleland, W. W., The kinetics of enzyme catalyzed reac- spinal cord neurons in dissociated cell culture, J. Cell tions with two or more substrates or products. Ill. Pre- Biol., 80 (1979) 651 661. diction of initial velocity and inhibition patterns by 18 Greenlee, D. V., Van Ness, P. C. and Olsen, R. W., inspection, Biochhn. biophys. Acta, 67 (1963) 188-196. Endogenous inhibitor of GABA binding in mammalian 7 Collins, J. F. and Cryer, G., A study of the GABA brain, L~fe Sei., 22 (1978) 1653-1662. receptor using 3H-bicuculline methobromide. In F. 19 Guidotti, A., Gale, K., Suria, A. and Toffano, G., Bio- Fonnum (Ed.), Amino Acids As Chemical Transmitters, chemical evidence for two classes of GABA receptors in Raven Press, New York, 1978, pp. 49%506. rat brain, Brain Research, 172 (1979) 566- 571. 8 Cuatrecasas, P. and Hollenberg, M. D., Membrane re- 20 Guidotti, A., Toffano, G. and Costa, E., An endogenous ceptors and hormone action, Advane. Protein Chem., 30 protein modulates the affinity of GABA and benzodiaze- (1976) 251-45 I. pine receptors in rat brain, Nature (Lond.), 275 0978) 9 Curtis, D. R., Duggan, A. W., Felix, D. and Johnston, 553-555. G. A, R., GABA, bicuculline and central inhibition, 21 Johnston, G. A. R. and Kennedy, S. M., GABA recep- Nature New Biol., 226 (1970) 1222-1224. tors and phospholipids. In F. Fonnum (Ed.), Amino Acids 10 DeFeudis, F. V., Ossola, L., Schmitt, G. and Mandel, P., as Chemical Transmitters, Plenum Press, New York, 1978, High-affinity binding of [3H]muscimol to subcellular pp. 507-516. particles of a neurone-enriched culture of embryonic rat 22 Lehninger, A. L., Biochemistry, 2rid Edn., Worth Publish- brain, Neurosci. Lett., 14 (1979) 195-199. hers, New York, 1975, pp. 197 ft. I 1 DeFeudis, F. V., Ossola, L., Schmitt, G., Wolff, P. and 23 Lloyd, K. G. and Davidson, L., 13H]GABA binding in Mandel, P., Na+-independent binding of [3H]GABA and brains from Huntington's chorea patients: altered regula- [:~Hlmuscimol to subcellular particles of neural primary tion by phospholipids, Science, 205 (1979) 1147 1149. cultures and whole brain, J. Neurochem., 34 (1980) 24 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and 216-218. 154

Randall, R. J., Protein measurement with Folin 40 Scatchard, G., The attractions of proteins for small mole- reagent, J. biol. Chem., 193 (1951) 265-275. cules and ions, Ann. N.Y. Acad. Sci., 51 (1949) 660-672. 25 Macdonald, R. L. and Barker, J. L., Specific antagonism 41 Schechter, P. J. and Tranier, Y., Effect of elevated brain of GABA-mediated postsynaptic inhibition in cultured GABA concentrations on the actions of bicuculline and mammalian spinal cord neurons: a common mode of picrotoxin in mice, Psychopharmacok~gy, 54 (1977) convulsant action, Neurology, 28 (1978) 325-330. 145-148. 26 Martin, D. L., Carrier-mediated transport and removal of 42 Shank, R. P., Pong, S. F., Freeman, A. R. and Graham, GABA from synaptic regions. In E. Roberts, T. N. Chase L. T., Bicuculline and p~crotoxi~ as antagonists of 7- and D. B. Tower, (Eds.), G,4BA in Nervous System aminobutyric and neuromuscular inhibition in the Function, Raven Press, New York, 1976, pp. 347-386. lobster, Brain Research, 72 (1974) 71-78. 27 M6hler, H. and Okada, T., GABA receptor binding with 43 Sine, S. and Taylor, P., Functional consequences of all(+) bicuculline-methiodide in rat CNS, Nature (Lond.), agonist-mediatd state transitions in the cholinergic recep- 267 (1977) 65-67. tor, J. biol. Chem., 254 (1979) 3315 3325. 28 Nowak, L. M., Young, A. B. and Macdonald, R. L., 44 Snodgrass, S. R., Use of 3H-muscimol for GABA receptor GABA and bicuculline actions on mouse spinal cord and studies, Nature (Lond.), 273 (1978) 392.394. cortical neurons in cell culture, Brain Research, 244 (1982) 45 Snodgrass, S. R., White, W. F., Biales, B. and Dichter, 155-164. M.~ Biochemical correlates of GABA function in rat 29 Olsen, R. W., Ban, M. and Miller, T., Studies on the cortical neurons in culture, BraiJ~ Research, 190 (1980) neuropharmacological activity of bicuculline and related 123 -137. compounds, Brain Research, 102 (1976) 283-299. 46 Stool', J. C., Den Breejen, E. J. S and Mulder, A. H., 30 Olsen, R. W., Bergman, M. O., VanNess, P. C., Lummis. GABA modulates the release of dopamine and acetyL S. C., Watkins, A. E., Napias, C. and Greenlee, D. V., choline from rat caudate nucleus slices, Earop. J. Pharma- 7-Aminobutyric acid receptor binding in mammalian col., 57 (1979) 35--42. brain. Heterogeneity of binding sites, Molee. Pharmacol., 47 Takeuchi, A. and Onodera, K., Effect of bicuculline on 19 (1981) 217-227. the GABA receptor of the crayfish neuromuscular 31 Olsen, R. W., Greenlee, D., VanNess, P. and Ticku, M. junction, Nature New Biol., 236 (1972) 55 56. K., Studies on the gamma-aminobutyric acid receptor/ 48 Ticku, M. K., Ban, M. and Olsen, R. W.. Binding of ionophore proteins in mammalian brain. In F. Fonnum [:~H], a ~,,-aminobutyric acid synaptic (Ed.), Amino Acids As Chemical Transmitters, Plenum antagonist, to rat brain membranes, Molec. PharmacoL. Press, New York, 1978, pp. 467-486. 14 (1978) 391-4-02. 32 Olsen, R. W. and Leeb-Lundberg, R., Convulsant and 49 Ticku, M. K., Huang, A. and Barker, J. L., GABA anticonvulsant drug binding sites related to GABA-regu- receptor binding in cultured mammalian spinal cord neu- lated chloride ion channels. In E. Costa, G. DiChiara and rons, Brain Research, 182 (1980) 201-206. G. L. Gessa (Eds.), GABA and Receptors 50 Toffano. G., Guidotti. A. and Costa. E.. Purification of Advanc. Biochem. Psychopharmacol., 26 (1981) 93-102. an endogenous protein inhibitor of the high affinity bind- 33 Olsen, R. W. Ticku, M. K., Greenlee, D. and VanNess, ing of ),-aminobutyric acid to synaptic membranes of rat P., GABA receptor and ionophore binding sites: inter- brain. Proc. nat. ,4cad. Sci. U.S.A.). 75 (1978~ action with various drugs. In P. Krogsgaard-Larsen, J. 4024-4028. Scheel-Krfiger and H. Kofod, (Eds.), GABA Neurotrans- 51 Waddington, J. L. and Cross. A. J.. Denervation super- mitters, Academic Press, New York, 1978, pp. 165-17& sensitivity in the striatonigral GABA pathway, Nature 34 Olsen, R. W., Ticku, M. K., VanNess, P. C. and Greenlee, (Lond.,. 76 (1978) 618-620. D., Effects of drugs on ~,-aminobutyric acid receptors, 52 Waddington, J. L. and Cross. A. J.. Characterisation of uptake, release and synthesis in vitro, Brain Research, 139 denervalion supersensitivity in the striatonigral GABA (1978) 277-294. pathway of the -lesioned rat and in 35 Placheta, P. and Karobath, M., Regional distribution of Huntington's disease. Brain Res. Bull.. 5 (19801 825-828. Na+-independent GABA and benzodiazepine binding 53 Weber. M., David-Pfeuty, T. and Changeux, J.-P., Regu- sites in rat CNS, Brain Research, 178 (1979) 580-583. lation of binding properties of the nicotinic receptor by 36 Ransom, B. R., Bullock, P. N. and Nelson, P. G., Mouse cholinergic ligands in membrane fragments from Torpedo spinal cord in cell culture. II. Neuronal chemosensitivity marmorata. Proc. nat. Acad. Sci. (U.S.A.). 72 ~1975) and its relationship to synaptic activity, J. NeurophysioL, 3443-3447. 40 (1977) 1163-1177. 54 Williams. M. and Risley, E. A.. Characterization of the 37 Ransom, B. R., Neale, E., Henkart, M., Bullock, P. N. binding of 3H-muscimol. a potent 7-aminobutyric acid and Nelson, P. G., Mouse spinal cord in cell culture. 1. agonist, to rat brain synaptosomal membranes using a Morphology and intrinsic neuronal electrophysiologic filtration assay, J. Neurochem., 32 (1979) 7t3-718. properties, J. Neurophysiol., 40 (1977) 1132-1150. 55 Wong, D. T. and Horng, J. S.. Na~-independent binding 38 Rosenthal, H. E., A graphic method for the determination of GABA to the Triton X-100 treated synaptie membranes and presentation of binding parameters in a complex from cerebellum of rat brain, Life Sci., 20 (1977) 445-452. system, Analyt. Biochem., 20 (1967) 525-532. 56 Zukin, S. R.. Young, A. B. and Snyder, S. H.. Gamma- 39 Sawynok, J. and Labella, F. S., GABA and baclofen anainobutyric acid binding to receptor sites in the rat potentiate the K+-evoked release of methionine-enkep- central nervous system, Proc. nat. Aead. Sci "U.S.A.), 7t halin from rat striatal slices, Europ. J. PharmacoL, 70 (1974) 4802-4807. (1981) 103-110.