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The GABAA receptor complex in experimental absence seizures in rat: An autoradiographic study
Ncuro,wiemc LeHcrs. 140 (1992) 9 12 1992 Elscvier Scientific Publishers Irehmd Ltd. All rights rcserved 0304-3940/92/S 05.00
9
NSL 08650
The GABAA receptor complex in experimental absence seizures in rat" an autoradiographic study O.C. Snead IIF', A. Depaulis", P.K. Banerjee ~<, V. Hechler b and M. Vergnes b "Division ol VcuroD*t~y,(7tildrens Hospital Los Angeles, D¢7~artmenl q/Neurolo£,v, [:niversily q/ Xouther#~ Calf/brnia School ol Medici#l¢', Los A#]I~rCI{',~, (',1 ~ L"X.'t ) aml I'Lahoraloire de" NeurwHo'.violo~ie el Bioloj,,ie des ('omportement,~', CNRS, ('entre de Neurochimie, ,S'tra.vhourg ~k)'ance )
(Received 13 January 1992: Revised vcrsion received 25 February 1992: Accepted 6 March 1992) Key wordy.
7-Ammobutyric acid: Autoradiography: Rat: Absence seizure
Ffic regional distribution of radioactive ligand binding for different receptors of the 7-aminobutyric acidA (GABAO-benzodiazepinc-picrotoxin chloride channel complex was measured on tissue section by autoradiography in brains taken from a genetic strain of Wistar rats with spontaneous absence-like seizures, the genetic absence epilepsy rats from Strasbourg (GAERS), and a control colony. The ligands employed included [~H]musciinol Ik>r high aftinitv GABA agonists sites: [~H]SR 95531 for the low-affinity GABA sites: [3H]flunitrazepam t\~r the benzodiazepine sites: and [~'S]l-but)l bic)clophospfiorotfiionate (TBPS) for the picrotoxin site. There was no significant change between GAERS and control animals in [~tl]flunitrazepam and ["S]TBPS binding. However, there was significantly decreased [~H]muscimol and [~H]SR 95531 binding in the CA2 region of tfic fiippocampus of the GAERS. This was due to a decrease in Bin,,, of both [~H]muscimol and [~H]SR 95531 binding in ifie epileptic strain.
Petil real or generalized absence seizures differ both clinically and experimentally fi'om generalized convulsive or partial seizures in a number of different ways [1]. Absence seizures have characteristic electrographic (EEG) and behavioral changes as well as pharmacologic specificity for anti-absence drugs [12, 25, 28]. One of the defining criteria of experimental absence seizures is that enhancement of brain GABAergic activity exacerbates all experimental forms of absence [5 7, 24, 27, 29] as well as the clinical condition [19, 32]. Because pretreatment with GABA~ agonists is known to result in prolongation of experimental absence seizures [34], we sought to test the hypothesis that GABAergic mechanisms are inwHved in the pathogenesis of absence seizures using a gcnetic animal model developed in our laboratory (Strasbourg), the genetic absence epilepsy rat from Strasbourg (GAERS) [13, 14, 33]. The GAERS meets all criteria for experimental absence seizures [5, 9, 28], including prolongation of seizure by GABAergic agonists [34], and is a valid animal model of absence epilepsy. In these experiments we examined the regional distribution and specilic binding of the macromolecular GABAA receptor complex in thc brains of an epileptic and control strain ('~,rrc,v~om/cm'c. O.C. Snead. Cfiildrens Hospital Los Angeles. 4650 Sunset Boulevard. 1.os Angeles. CA 90027. USA. Fax:(1)(213)6672019.
using autoradiographic techniques. Both the GAERS and control strain have been inbred in our laboratory for more than 10 generations. [3H]Muscimol (20 Ci/mmol), [3H]SR95531 (58 Ci/ mmol), [3H]flunitrazepam (90 Ci/mmol), and [~sS]t-butylbicyclophosphorothionate (TBPS) (61.5 Ci/mmol) were obtained from New England Nuclear/Dupont (Dreieich, FRG). Muscimol, GABA and picrotoxinin were obtained from Sigma (St. Louis, MO, USA). SR 95531 and clonazepam were gifts from Sanofi (Paris, France) and Hoffman-LaRoche (Basel, Switzerland) respectively. All other reagents were from standard commercial sources and were of lhe highest available purity. Sixteen male Wistar rats (350 400 g) were used in the autoradiographic study. Eight animals were chosen from the twelfth generation of the GAERS strain and 8 from a control strain in which no spike wave discharges (SWD) are ever observed [13, 35]. All animals were implanted with epidural electrodes as previously described [3]. Seven days after surgery, the animals underwent EEG recordings in the freely moving state for 60-120 min in order to be certain of the presence or absence of SWD in the epileptic and control animals respectively. Animals were sacrificed by decapitation, the brain rapidly removed and processed as described [30]. The following procedures were performed:
10 TABLE 1 A U T O R A D I O G R A P H I C ANALYSIS O F T H E GABAA R E C E P T O R C O M P L E X IN T H E G E N E T I C A B S E N C E EPILEPSY RAT F R O M S T R A S B O U R G (GAERS) Mean specific binding + S.E.M. (fmot/mg prot). C T X inner and outer, cortical layers IV VI and I-III respectively; Thai., thalamus: Med, medial thalamic nuclei; VL, ventrolateral nucleus of the thalamus; VPL, ventroposterolateral nucleus of the thalamus; VPM, ventroposteromedial nucleus of the thalamus. Subs Nigra, substantia nigra. E refers to the G A E R S while N E refers to control animals. Region
CTX-outer CTX-inner Med thalamus VLThal VPLThal VPM Thai SubsNigra CAI CA2 CA3
[3H]Muscimol
[3H]Flunitrazepam
[3H]SR 95531
[~SS]TBPS
E
NE
E
NE
E
NE
275+_31 146+-21 145 +- 19 118_+ 13 165_+20 191 _+ 20
322+-36 170+_ 18 185 _+ 28 164_+25 206+29 214 +- 15
t10+-9 99+-6 68 +- 4 59_+6 51 +-2
108_+9 94+-2 73 +- 4 50+-5 39±3
171 + 80+105 +44+-
9 8 13 8
200+_ 19 1 0 2 z 18 120 +- 24 83~t24
124+- 18 89+- 11 139+- 17
132+- 18 127+- 18" 144+- 11
86_+8 93+4 89+4 109_+4
77+-4 98_+6 89+-4 109+-4
44+_ 8 217+-22 217+27 222_+30
38+ 6 222~_26 238±33 255+20
E
NE
4.7_+ 1 9 +4 8 +- 0,6 7 ±0.7 7 +4 11 6 5 7
~_: 1 _~0.8 +-0.4 +-0.4
4.4+-0.3 7.8+_0.9 8.9 +- 1 8 ±0.5 7 _+0.6 10 6 4 9
+0.6 _+0.7 +-0.3 +0.4
*P < 0.05 (Mann-Whitney).
[-~H]Muscimol. The method used was a modification of that of Pan et al. [22]. The concentration of [3H]muscitool was 5 nM and that of GABA to account for nonspecific binding was 100 mM. In kinetic studies to determine the Bmax and Kd of [3H]muscimol binding, [3H]muscimol concentrations ranged from 1 to 50 riM. FH]SR 95531. The method used was that of McCabe et al. [16]. The concentration of [3H]SR 95531 was 7 nM and that of GABA to account for non-specific binding was 1 mM. In kinetic studies to determine the Bma x and Kd of [3H]SR 95531 the concentration of [3H]SR 95531 ranged from 1 to 90 nM. FH]Flunitrazepam. The method used was that of McCabe and Wamsley [15] as modified by Olsen et al. [20]. The concentration of [3H]flunitrazepam was ! n M and that ofclonazepam to account for non-specific binding was 1 mM. [3sS] TBPS. The method used was that of Edgar and Schwartz [4]. The concentration of [35S]TBPS was 1 nM and that of picrotoxin to account for non-specific binding was 100/.tM. Autoradiographic images were digitized and analyzed using an MCID computer assisted image analysis system (Imaging Research, Ont., Canada). Five to eight readings were determined and averaged for each anatomic area analyzed. The anatomic regions were identified on Cresyl violet staining and with the assistance of the atlas of Paxinos and Watson [23]. All data was expressed as mean fmol bound/mg protein + standard error (n = 4-8). For each anatomic region, the mean fmol bound/mg pro-
tein was compared between GAERS and their controls using the Mann Whitney test. The Kd and Bm~xwere determined for [3H]muscimol and [3H~R 9553t binding by equilibrium binding analysis of Tobler and Engel [31]. There were 11 13 concentrations of isotope used in each equilibrium binding experiment. The results of this study are shown in Table I. There were no visible images in the non-specific autoradiograms for any of the ligands tested. Particular attention was paid during the analysis to cortical laminae of the frontoparietal cortex and thalamic nuclei m view of the role of thalamocortical pathways in the genesis of SWD in experimental and clinical absence seizures [7, 35]. For this reason the cerebellum was not analyzed for distribution of any ligand. The binding and relative distribution of the high affinity GABAA sites ([3H]muscimot), low affinity GABAA sites ([3H]SR 9553t), benzodiazepine sites ([3H]flunitrazepam) and picrotoxin sites ([3SS]TBPS) were in agreement with that reported in the literature [4, 16. 20. 22]. Statistical analysis revealed significantly reduced [3H]muscimol binding in GAERS compared to their controls in the CA2 region of the hippocampus. Such a difference was not observed in this region with the other ligands. Although not significant, there was an overall trend toward decreased [3H]muscimol and [3H]SR 95531 binding in the GAERS in the cortical and Ihalamic regions whereas no such trend was observed with the other two ligands (Table I). Equilibrium binding analysis of [3H]muscimol and [3H]SR 95531 binding in epileptic and
TABLE I1 R E G I O N A L K I N E T I C ANALYSIS OF [~H]MUSCIMOL A N D [*H]SR 95531 B I N D I N G IN G A E R S The B,,,~,~and K,~ were calculated by the equilibrium analysis of Tobler and Engel [33] as described in the text. VL. ventrolateral nucleus of the thalamus: VPL, ventroposterolateral nucleus of thalamus: CTX, cortex. E refers to the G A E R S and NE to the control animals. Region
CA2 C T X ( I IV) VL VPL
[~H]Muscimol"
[~H]SR 9553F'
K,~
K~
B ......
B ......
E
NE
E
NE
E
NE
E
NE
55.3 54.9 56.1 53.2
60.2 57.4 59.2 56.4
0.72 0.81 0.83 0.79
1.03 1.01 1.05 1.01
41.5 43.0 41.7
40.9 42.5 43.4
95.6 85.7 71.2
110.1 104.3 112.6
' F o r [~H]muscimol binding K d is expressed as nM and B,~,~ as pmol/mg tissue, bFor [~H]SR 95531 binding Kd is expressed as nM and B ....... as fmol/mg tissue.
control rats revealed that the differences in binding were due to a decreased B ...... in hippocampus, cortex and thalamus in the G A E R S (Table II). The observed change in binding of [3H]muscimol to the high affinity site of the GABA A receptor in the CA2 region of hippocampus in the GAERS is of uncertain significance given the fact that this structure is not involved electrographically in the origin or propagation of spike wave discharges in this model of experimental absence seizures [35]. However, abnormalities of other neurotransmitter systems in the hippocampus have been recently reported in another genetic model of absence in rodent, the 'stargazer' mouse [8]. Other authors have examined various components of the GABAA receptor complex in several rodent models of absence seizures. Knight and Bowery [10] reported no statistical difference in the levels of radioligand binding to the high affinity component of the GABAA receptor complex or to the GABAu receptor in the GAERS. Similarly, in an autoradiographic study involving another rat model of spontaneous absence seizures, murase et al. [18] found no significant difference in [3H]muscimol binding between control and epileptic rats. Ortiz et al. [22] have reported increased [~H]flunitrazepam binding in hippocampus, but decreased binding in the rest of the brain in a mouse model of absence. Although our data are not in strong support of a role for the GABA A receptor complex in the pathogenesis of the absence-like seizures in the GAERS, they do not completely disprove the hypothesis since we measured only the regional variation in density of binding to the various GABAx receptor subtypes in these experiments.
More subtle measures of GABA A receptor function such as GABA A receptor-mediated chloride ion flux, the regional anatomic heterogeneity of this parameter [26] as well as allosteric regulation of various components of the macromolecular complex [2, 26], and the relative distribution and binding parameters of all GABAA receptor subtypes particularly in thalamic nuclei and cortical laminae [11, 20] remain to be determined. Allosteric modulation of the GABA~, receptor complex may be a particularly important issue to address in the future since we consistently found no change in binding to the benzodiazepine receptor in the epileptic animals, but observed a decrease in binding to the low affinity GABA receptor (Table I), a site thought to be coupled to the benzodiazepine site [20]. The fact that the G A E R S showed the same distribution of benzodiazepine and low affinity GABA sites as controls, but had a lower density of the low affinity sites (Table II) suggests that there may be an uncoupling of the low affinity GABA site to the benzodiazepine site in the epileptic animals. Alternatively, our data might suggest that the primary cause of the generalized absence seizures in this model is not a change in GABAergic function which remains normal in the GAERS. Rather, preserved GABAergic inhibition in the face of perturbation of some other neurotransmitter system in thalamus and cortex might permit the full expression seizures in this genetic model of absence. A final consideration is that the GABA~ receptor complex may play an excitatory role in generalized absence seizures. The synaptic excitation sustained by the GABAA receptor complex synchronizes the activity of inhibitory interneurons in the hippocampus [17]. The same mechanism could be operative within thalamocortical pathways and modulate the bilaterally synchronous spike wave discharges that characterize this kind of seizure. This work was supported in part by Grant RO1 NS17117 from the National Institutes of Neurologic Disease and Stroke and by Grant FO6TWOI277 from the Fogarty Foundation.
1 Berkovic. S.F., A n d e r m a n n , F.. Andermann, E. and Gloor. R, Concepts of absence epilepsies: discrete syndromes or biologic continuum?, Neurology, 37 (1987)993 1000. 2 Daval, J.L.. Wcrck. M.C., Nehlig, A. and de Vasconcelos, A.P., Quantitative autoradiographic study of the postnatal developmenl of benzodiazepine binding sites and their coupling to G A B A receptors in the rat brain. Int. J. Dev. Neurosci.,9(1991)307 320. 3 Depaulis, A., Bourguignon, J.J., Marescaux, C , Vergnes. M., Schmitt. M., Micheletti, G. and Warter..I.M., Effects of gammahydroxybutyrate and gamma-butyrolactone derivatives on spontaneous generalized non-convulsive seizures in the rat, Neuropharmacology. 27(1988)683 689. 4 Edgar, P.P. and Schwartz, R.D.. Localization and characterization
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11
12 13
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15
16
17
18
19 20
of ~sS-t-butylbicyclophosphorothionate binding in rat brain: an autoradiographic study, J. Neurosci., 10 (1990) 603-612. Fariello, R.A. and Golden, G.T., The THIP-induced model of bilateral synchronous spike and wave in rodents, Neuropharmacology, 26 (1987) 161-165. Gloor, P., Electrophysiology of generalized epilepsy. In P.A. Schwartzkroin and H. Wheal (Eds.), Electrophysiology of Epilepsy, Academic Press, New York, 1984, pp. 107 136. Gloor, E and Fariello, R.G., Generalized epilepsy: some of its cellular mechanisms differ from those of focal epilepsy, Trends Neurosci., 11 (1988) 63 68. Helekar, S.A. and Noebels, J.L., Synchronous hippocampal bursting reveals network excitability defects in an epilepsy gene mutation, Proc. Natl. Acad. Sci. U.S.A., 88 (1991) 4736~740. Jobe, EC., Mishra, P.K., Ludvig, N. and Dailey, J.W., Scope and contribution of genetic models to an understanding of the epilepsies. CRC Critical Reviews in Neurobiology, CRC Press, Boca Raton, 1991, pp. 183-220. Knight, A.R. and Bowery, N.G., GABA receptors in rats with spontaneous generalized non-convulsive epilepsy, J. Neural Transm., in press. Kultas-Ilinsky, K., Fogarty, J.D., Hughes, B. and llinsky, 1.A., Distribution and binding parameters of GABA and benzodiazepine receptors in the cat motor thalamus and adjacent nuclear groups, Brain Res., 459 (1988) 1-16. Lockman, L.A., Absence, myoclonic, and atonic seizures, Pediatr. Clin. North Am., 36 (1989) 331-343. Marescaux, C., Micheletti, G., Vergnes, M., Depaulis, A., Rumbach, L. and Warier, J.M., A model of chronic spontaneous petit real-like seizures in the rat: comparison with pentylenetetrazoleinduced seizures, Epilepsia, 25 (1984) 326-331. Micheletti, G., Vergnes, M., Marescaux, C., Reis, J., Depaulis, A., Rumbach, L. and Warter, J.M., Antiepileptic drug evaluation in a new animal model: spontaneous petit mal epilepsy in the rat, Arzneim.-Forsch./Drug Res., 35 (1985) 483-485. McCabe, R.T. and Wamsley, J.K., Autoradiographic localization ofsubcomponents of the macromolecular GABA receptor complex, Life Sci., 39 (1986) 1937-1945. McCabe, R.T., Wamsley, J.K., Yezuita, J.P. and Olsen, R.W., A novel GABAA antagonist [3H]SR 95531: Microscopic analysis of binding in the rat brain and allosteric modulation by several benzodiazepine and barbiturate receptor ligands, Synapse, 2 (1988) 163 173. Michelson, H.B. and Wong, R.K.S., Excitatory synaptic responses mediated by GABAA receptors in the hippocampus, Science, 253 (1991) 1420.-1423. M urase, K., Nabeshima, T., Kameyama, T., Sasa, M., Takaori, S., Ujibara, H., lshihara, K., Yamada, J. and Serikawa, T., Characteristics of muscarinic, cholinergic, y-aminobutyric acid A and phencyclidine receptors in spontaneously epileptic rats: in vitro quantitative autoradiographic analysis, Neurosci. Lett., 131 (1991 ) 1 -4. Myslobodsky, M., Petit Mal Epilepsy, Academic Press, New York, 1976. Olsen, R.W., McCabe, R.T. and Wamsley, J.K., GABA A receptor subtypes: autoradiographic comparison of GABA, benzodiazepine, and convulsant binding sites in the rat central nervous system, J. Chem. Neuroanat., 3 (1990) 59-76.
21 Ortiz, J.G.. Negron, A.E., Thomas, A.P., Heimer. H., Morreira, J.A., Cordero, M.S., Aranda, J. and Bruno, M.S., GABAe,gic neurotransmission in the C57BL/10 sps/sps mouse mutant: a model of absence seizures, Exp. Neurol., 113 (1991) 338-343. 22 Pan, H.S., Frey, K.A., Young, A.B. and Penney, J.B., Changes in [3H]muscimol binding in substantia nigra, entopeduncular nucleus, globus pallidus, and thalamus after striatal lesions as demonstrated by quantitative receptor autoradiography, J. Neurosci., 3 (1983) 1189-1198.
23 Paxinos, G. and Watson, C.. The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1986. 24 Peeters, B.W.V.M., Van Rijn, C.M.. Vossen, J.M.H. and Coenen, A.M.I_,., Effects of GABAergic agents on spontaneous non-convulsive epilepsy, EEG, and behavior in the WAG/RIJ inbred strain of rats, LifeSci.,45(1989) 1171 1176. 25 Penry, J.K.. Porter, R.J. and Dreifuss, F.E., Simultaneous recording of absence seizures with videotape and electroencephalography. A study of 374 seizures in 48 patients, Brain, 98 (1975) 427 440. 26 Peris, .I., Shawley, A., Dawson, R. and Abendscheim K.H., Regulation of 3~S-TBPS binding by bicuculline is region specific in ral brain, Life Sci., 49 (1991) PL49 PL54. 27 Smith, K.A. and Bierkamper, G.G., Paradoxical role of GABA in a chronic model of petit mal (absence) like epilepsy in the rat. Eur. J. Pharmacol., 176 (1990) 45 55. 28 Snead, O.C., ?'-Hydroxybutyrate model of generalized absence seizures: further characterization and comparison with other absence models, Epilepsia, 29 (1988) 361-368. 29 Snead, O.C., Thc ontogeny of GABAergic enhancement of the yhydroxybutyrate model of generalized absence seizures, Epilepsia, 31 (1990)363 368. 30 Snead, O.C., Hechler, V., Vergnes, M., Marescaux, C. and Maitre, M., Increased ?'-hydroxybutyrate receptors in a genetic model of absence in rat, Epilepsy Res., 7 (1990) 121-128. 31 Tobler, H.J. and Engel, C., Affinity spectra: a novel way for the evaluation of equilibrium binding experiments, Naunyn-Schmiedeberg's Arch. Pharmacol., 322 (1983) 183--192. 32 van der Linden, G.J., Meinardi, H., Meijer, S.W.A., Bossi, L., Gomeni, C.A., A double blind cross over trial with progabide (SL76002) against placebo in patients with secondary generalized epilepsy. In M. Dam, L. Gram and J.K. Penry (Eds.), Advances in Epileptology: Xllth Epilepsy International Symposium, Raven, New York, 1981, pp. 141..144. 33 Vergnes, M., Marescaux, C., Micheletti, G., Reis, J., Depaulis, A., Rumbach, L. and Wafter, J.M., Spontaneous paroxysmal electroclinical patterns in rat: a model of generalized non-convulsive epilepsy, Neurosci. Lett., 33 (1982) 99-101. 34 Vergnes, M., Marescaux, C., Micheletti, G., Depaulis, A., Rumbach, L. and Warter, J.M., Enhancement of spike and wave discharges by GABAmimetic drugs m rats with spontaneous petit reallike epilepsy, Neurosci. Lett., 44 (1984) 91 94. 35 Vergnes, M.. Marescaux, C.. Depaulis, A., Micheletti, G. and Warter, J.M.. Spontaneous spike and wave discharges in thalamus and cortex in a rat model of genetic petit real-like seizures, Exp. Neurol., 96(1987) 127 136.
9
NSL 08650
The GABAA receptor complex in experimental absence seizures in rat" an autoradiographic study O.C. Snead IIF', A. Depaulis", P.K. Banerjee ~<, V. Hechler b and M. Vergnes b "Division ol VcuroD*t~y,(7tildrens Hospital Los Angeles, D¢7~artmenl q/Neurolo£,v, [:niversily q/ Xouther#~ Calf/brnia School ol Medici#l¢', Los A#]I~rCI{',~, (',1 ~ L"X.'t ) aml I'Lahoraloire de" NeurwHo'.violo~ie el Bioloj,,ie des ('omportement,~', CNRS, ('entre de Neurochimie, ,S'tra.vhourg ~k)'ance )
(Received 13 January 1992: Revised vcrsion received 25 February 1992: Accepted 6 March 1992) Key wordy.
7-Ammobutyric acid: Autoradiography: Rat: Absence seizure
Ffic regional distribution of radioactive ligand binding for different receptors of the 7-aminobutyric acidA (GABAO-benzodiazepinc-picrotoxin chloride channel complex was measured on tissue section by autoradiography in brains taken from a genetic strain of Wistar rats with spontaneous absence-like seizures, the genetic absence epilepsy rats from Strasbourg (GAERS), and a control colony. The ligands employed included [~H]musciinol Ik>r high aftinitv GABA agonists sites: [~H]SR 95531 for the low-affinity GABA sites: [3H]flunitrazepam t\~r the benzodiazepine sites: and [~'S]l-but)l bic)clophospfiorotfiionate (TBPS) for the picrotoxin site. There was no significant change between GAERS and control animals in [~tl]flunitrazepam and ["S]TBPS binding. However, there was significantly decreased [~H]muscimol and [~H]SR 95531 binding in the CA2 region of tfic fiippocampus of the GAERS. This was due to a decrease in Bin,,, of both [~H]muscimol and [~H]SR 95531 binding in ifie epileptic strain.
Petil real or generalized absence seizures differ both clinically and experimentally fi'om generalized convulsive or partial seizures in a number of different ways [1]. Absence seizures have characteristic electrographic (EEG) and behavioral changes as well as pharmacologic specificity for anti-absence drugs [12, 25, 28]. One of the defining criteria of experimental absence seizures is that enhancement of brain GABAergic activity exacerbates all experimental forms of absence [5 7, 24, 27, 29] as well as the clinical condition [19, 32]. Because pretreatment with GABA~ agonists is known to result in prolongation of experimental absence seizures [34], we sought to test the hypothesis that GABAergic mechanisms are inwHved in the pathogenesis of absence seizures using a gcnetic animal model developed in our laboratory (Strasbourg), the genetic absence epilepsy rat from Strasbourg (GAERS) [13, 14, 33]. The GAERS meets all criteria for experimental absence seizures [5, 9, 28], including prolongation of seizure by GABAergic agonists [34], and is a valid animal model of absence epilepsy. In these experiments we examined the regional distribution and specilic binding of the macromolecular GABAA receptor complex in thc brains of an epileptic and control strain ('~,rrc,v~om/cm'c. O.C. Snead. Cfiildrens Hospital Los Angeles. 4650 Sunset Boulevard. 1.os Angeles. CA 90027. USA. Fax:(1)(213)6672019.
using autoradiographic techniques. Both the GAERS and control strain have been inbred in our laboratory for more than 10 generations. [3H]Muscimol (20 Ci/mmol), [3H]SR95531 (58 Ci/ mmol), [3H]flunitrazepam (90 Ci/mmol), and [~sS]t-butylbicyclophosphorothionate (TBPS) (61.5 Ci/mmol) were obtained from New England Nuclear/Dupont (Dreieich, FRG). Muscimol, GABA and picrotoxinin were obtained from Sigma (St. Louis, MO, USA). SR 95531 and clonazepam were gifts from Sanofi (Paris, France) and Hoffman-LaRoche (Basel, Switzerland) respectively. All other reagents were from standard commercial sources and were of lhe highest available purity. Sixteen male Wistar rats (350 400 g) were used in the autoradiographic study. Eight animals were chosen from the twelfth generation of the GAERS strain and 8 from a control strain in which no spike wave discharges (SWD) are ever observed [13, 35]. All animals were implanted with epidural electrodes as previously described [3]. Seven days after surgery, the animals underwent EEG recordings in the freely moving state for 60-120 min in order to be certain of the presence or absence of SWD in the epileptic and control animals respectively. Animals were sacrificed by decapitation, the brain rapidly removed and processed as described [30]. The following procedures were performed:
10 TABLE 1 A U T O R A D I O G R A P H I C ANALYSIS O F T H E GABAA R E C E P T O R C O M P L E X IN T H E G E N E T I C A B S E N C E EPILEPSY RAT F R O M S T R A S B O U R G (GAERS) Mean specific binding + S.E.M. (fmot/mg prot). C T X inner and outer, cortical layers IV VI and I-III respectively; Thai., thalamus: Med, medial thalamic nuclei; VL, ventrolateral nucleus of the thalamus; VPL, ventroposterolateral nucleus of the thalamus; VPM, ventroposteromedial nucleus of the thalamus. Subs Nigra, substantia nigra. E refers to the G A E R S while N E refers to control animals. Region
CTX-outer CTX-inner Med thalamus VLThal VPLThal VPM Thai SubsNigra CAI CA2 CA3
[3H]Muscimol
[3H]Flunitrazepam
[3H]SR 95531
[~SS]TBPS
E
NE
E
NE
E
NE
275+_31 146+-21 145 +- 19 118_+ 13 165_+20 191 _+ 20
322+-36 170+_ 18 185 _+ 28 164_+25 206+29 214 +- 15
t10+-9 99+-6 68 +- 4 59_+6 51 +-2
108_+9 94+-2 73 +- 4 50+-5 39±3
171 + 80+105 +44+-
9 8 13 8
200+_ 19 1 0 2 z 18 120 +- 24 83~t24
124+- 18 89+- 11 139+- 17
132+- 18 127+- 18" 144+- 11
86_+8 93+4 89+4 109_+4
77+-4 98_+6 89+-4 109+-4
44+_ 8 217+-22 217+27 222_+30
38+ 6 222~_26 238±33 255+20
E
NE
4.7_+ 1 9 +4 8 +- 0,6 7 ±0.7 7 +4 11 6 5 7
~_: 1 _~0.8 +-0.4 +-0.4
4.4+-0.3 7.8+_0.9 8.9 +- 1 8 ±0.5 7 _+0.6 10 6 4 9
+0.6 _+0.7 +-0.3 +0.4
*P < 0.05 (Mann-Whitney).
[-~H]Muscimol. The method used was a modification of that of Pan et al. [22]. The concentration of [3H]muscitool was 5 nM and that of GABA to account for nonspecific binding was 100 mM. In kinetic studies to determine the Bmax and Kd of [3H]muscimol binding, [3H]muscimol concentrations ranged from 1 to 50 riM. FH]SR 95531. The method used was that of McCabe et al. [16]. The concentration of [3H]SR 95531 was 7 nM and that of GABA to account for non-specific binding was 1 mM. In kinetic studies to determine the Bma x and Kd of [3H]SR 95531 the concentration of [3H]SR 95531 ranged from 1 to 90 nM. FH]Flunitrazepam. The method used was that of McCabe and Wamsley [15] as modified by Olsen et al. [20]. The concentration of [3H]flunitrazepam was ! n M and that ofclonazepam to account for non-specific binding was 1 mM. [3sS] TBPS. The method used was that of Edgar and Schwartz [4]. The concentration of [35S]TBPS was 1 nM and that of picrotoxin to account for non-specific binding was 100/.tM. Autoradiographic images were digitized and analyzed using an MCID computer assisted image analysis system (Imaging Research, Ont., Canada). Five to eight readings were determined and averaged for each anatomic area analyzed. The anatomic regions were identified on Cresyl violet staining and with the assistance of the atlas of Paxinos and Watson [23]. All data was expressed as mean fmol bound/mg protein + standard error (n = 4-8). For each anatomic region, the mean fmol bound/mg pro-
tein was compared between GAERS and their controls using the Mann Whitney test. The Kd and Bm~xwere determined for [3H]muscimol and [3H~R 9553t binding by equilibrium binding analysis of Tobler and Engel [31]. There were 11 13 concentrations of isotope used in each equilibrium binding experiment. The results of this study are shown in Table I. There were no visible images in the non-specific autoradiograms for any of the ligands tested. Particular attention was paid during the analysis to cortical laminae of the frontoparietal cortex and thalamic nuclei m view of the role of thalamocortical pathways in the genesis of SWD in experimental and clinical absence seizures [7, 35]. For this reason the cerebellum was not analyzed for distribution of any ligand. The binding and relative distribution of the high affinity GABAA sites ([3H]muscimot), low affinity GABAA sites ([3H]SR 9553t), benzodiazepine sites ([3H]flunitrazepam) and picrotoxin sites ([3SS]TBPS) were in agreement with that reported in the literature [4, 16. 20. 22]. Statistical analysis revealed significantly reduced [3H]muscimol binding in GAERS compared to their controls in the CA2 region of the hippocampus. Such a difference was not observed in this region with the other ligands. Although not significant, there was an overall trend toward decreased [3H]muscimol and [3H]SR 95531 binding in the GAERS in the cortical and Ihalamic regions whereas no such trend was observed with the other two ligands (Table I). Equilibrium binding analysis of [3H]muscimol and [3H]SR 95531 binding in epileptic and
TABLE I1 R E G I O N A L K I N E T I C ANALYSIS OF [~H]MUSCIMOL A N D [*H]SR 95531 B I N D I N G IN G A E R S The B,,,~,~and K,~ were calculated by the equilibrium analysis of Tobler and Engel [33] as described in the text. VL. ventrolateral nucleus of the thalamus: VPL, ventroposterolateral nucleus of thalamus: CTX, cortex. E refers to the G A E R S and NE to the control animals. Region
CA2 C T X ( I IV) VL VPL
[~H]Muscimol"
[~H]SR 9553F'
K,~
K~
B ......
B ......
E
NE
E
NE
E
NE
E
NE
55.3 54.9 56.1 53.2
60.2 57.4 59.2 56.4
0.72 0.81 0.83 0.79
1.03 1.01 1.05 1.01
41.5 43.0 41.7
40.9 42.5 43.4
95.6 85.7 71.2
110.1 104.3 112.6
' F o r [~H]muscimol binding K d is expressed as nM and B,~,~ as pmol/mg tissue, bFor [~H]SR 95531 binding Kd is expressed as nM and B ....... as fmol/mg tissue.
control rats revealed that the differences in binding were due to a decreased B ...... in hippocampus, cortex and thalamus in the G A E R S (Table II). The observed change in binding of [3H]muscimol to the high affinity site of the GABA A receptor in the CA2 region of hippocampus in the GAERS is of uncertain significance given the fact that this structure is not involved electrographically in the origin or propagation of spike wave discharges in this model of experimental absence seizures [35]. However, abnormalities of other neurotransmitter systems in the hippocampus have been recently reported in another genetic model of absence in rodent, the 'stargazer' mouse [8]. Other authors have examined various components of the GABAA receptor complex in several rodent models of absence seizures. Knight and Bowery [10] reported no statistical difference in the levels of radioligand binding to the high affinity component of the GABAA receptor complex or to the GABAu receptor in the GAERS. Similarly, in an autoradiographic study involving another rat model of spontaneous absence seizures, murase et al. [18] found no significant difference in [3H]muscimol binding between control and epileptic rats. Ortiz et al. [22] have reported increased [~H]flunitrazepam binding in hippocampus, but decreased binding in the rest of the brain in a mouse model of absence. Although our data are not in strong support of a role for the GABA A receptor complex in the pathogenesis of the absence-like seizures in the GAERS, they do not completely disprove the hypothesis since we measured only the regional variation in density of binding to the various GABAx receptor subtypes in these experiments.
More subtle measures of GABA A receptor function such as GABA A receptor-mediated chloride ion flux, the regional anatomic heterogeneity of this parameter [26] as well as allosteric regulation of various components of the macromolecular complex [2, 26], and the relative distribution and binding parameters of all GABAA receptor subtypes particularly in thalamic nuclei and cortical laminae [11, 20] remain to be determined. Allosteric modulation of the GABA~, receptor complex may be a particularly important issue to address in the future since we consistently found no change in binding to the benzodiazepine receptor in the epileptic animals, but observed a decrease in binding to the low affinity GABA receptor (Table I), a site thought to be coupled to the benzodiazepine site [20]. The fact that the G A E R S showed the same distribution of benzodiazepine and low affinity GABA sites as controls, but had a lower density of the low affinity sites (Table II) suggests that there may be an uncoupling of the low affinity GABA site to the benzodiazepine site in the epileptic animals. Alternatively, our data might suggest that the primary cause of the generalized absence seizures in this model is not a change in GABAergic function which remains normal in the GAERS. Rather, preserved GABAergic inhibition in the face of perturbation of some other neurotransmitter system in thalamus and cortex might permit the full expression seizures in this genetic model of absence. A final consideration is that the GABA~ receptor complex may play an excitatory role in generalized absence seizures. The synaptic excitation sustained by the GABAA receptor complex synchronizes the activity of inhibitory interneurons in the hippocampus [17]. The same mechanism could be operative within thalamocortical pathways and modulate the bilaterally synchronous spike wave discharges that characterize this kind of seizure. This work was supported in part by Grant RO1 NS17117 from the National Institutes of Neurologic Disease and Stroke and by Grant FO6TWOI277 from the Fogarty Foundation.
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5
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