ELSEVIER Molecular Brain Research 26 (1994) 309-319

MOLECULAR BRAIN

RESEARCH

Research Report

Analysis of the hippocampal GABA A receptor system in kindled rats by autoradiographic and in situ hybridization techniques:

contingent tolerance to carbamazepine

Mike Clark *, Gwendolyn S. Massenburg, Susan R.B. Weiss, Robert M. Post Biological Psychiatry Branch, National Institute of Mental Health, Building 10 Room 3N212, Bethesda, MD 20892, USA

Accepted 7 June 1994

Abstract

Tolerance to the anticonvulsant effects of carbamazepine (CBZ) in the amygdala kindling paradigm is a contingent process, since it only develops in rats treated with CBZ before the kindling stimulation and not in those animals treated after the stimulation. The present study was designed to investigate the GABA A receptor system in CBZ contingent tolerance. Receptor autoradiography utilizing various radioligands that bind to different components of the GABA A receptor system and in situ hybridization with oligonucleotides that recognize different subunits of the GABA A receptor were performed. Kindling increased binding to benzodiazepine, picrotoxin, and GABA recognition sites selectively in the dentate gyrus of the hippocam- pus. Kindling also increased levels of mRNA for the 0/4, /31, and /33 subunits but did not change al, a2, or 3'2 subunit levels. Rats tolerant to CBZ showed decreased [3H]muscimol binding, diazepam-insensitive [3H]Ro 15-4513 binding, and decreased 0/4 subunit mRNA content compared to non-tolerant rats, whereas [3H]flunitrazepam binding, [35S]TBPS binding, and the levels of /31, and /33 subunit mRNAs remained elevated. The data suggest an indirect interaction of CBZ with the GABA A receptor system, since CBZ reportedly does not bind to this receptor system.

Keywords: GABAA-benzodiazepine receptor complex; mRNA; Kindled seizure; Hippocampus; Carbamazepine; Receptor autoradiography; Contingent tolerance; In situ hybridization

1. Introduction

Electrical stimulation of various limbic system struc- tures with low intensity current initially produces no behavioral response, but with repeated stimulations a succession of progressively intensifying epileptic events occurs culminating in major motor convulsions. This process of seizure generation was termed kindling and was first described by Goddard et al. [11]. In fully-kin- dled rats, each electrical stimulation reliably elicits a seizure with little variation in duration or intensity of the convulsive episode from one stimulation to the next. Hence, kindling has become a widely used animal model of epilepsy [5,22]. However, the biochemical mechanisms underlying the kindling process are not

* Corresponding author. Fax: (1) (301) 402-0052.

0169-328X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSD1 0169-328X(94)00133-Y

known, although several different receptor systems have been shown to be altered by kindled seizures [5,22].

Much evidence suggests the importance of y- aminobutyric acid (GABA)-mediated neurotransmis- sion in the mechanisms underlying kindling [6,7]. For example, there is a long-term decrease in GABA-medi- ated chloride uptake in brain stem of fully-kindled rats [33] as well as subsensitivity to G A B A in dorsal raphe neurons [12,13] that may be a result of increased G A B A release associated with kindled seizures [9,23]. Addi- tionally, receptor autoradiographic studies have re- vealed increased numbers of both benzodiazepine [24,31] and G A B A [25,31] recognition sites on the GABAA-benzodiazepine receptor-chloride ionophore complex (GABA-BZ receptor complex) in the dentate gyrus of hippocampus in kindled rats.

While carbamazepine (CBZ) is ineffective in the developmental stages of amygdala kindled seizures in rats regardless of dose or route of administration

310 M. Clark et al. / Molecular Brain Research 26 (1994) 309-319

[1,2,35,37], it is a highly effective anticonvulsant against fully-kindled seizures [1,2,14,35,37]. However, develop- ment of tolerance to the anticonvulsant activity of CBZ in the kindling paradigm has been reported to occur rapidly with repeated CBZ administration [14,19,20, 36,37]. Furthermore, this tolerance to CBZ requires the electrical stimulus to be given during periods of CBZ exposure as opposed to drug exposure after the stimulus has occurred [20,36,37]. Therefore, this type of tolerance is a contingent process, since it only devel- ops in rats treated with CBZ before the kindling stimu- lation and is not observed in animals treated with CBZ after the stimulation.

The present study investigated the possibility of changes in the GABA-BZ receptor complex as a po- tential biochemical mechanism of contingent tolerance to CBZ in the amygdala kindling paradigm. Receptor autoradiography was conducted for three distinct re- gions of the GABA-BZ receptor complex, namely, the convulsant (picrotoxin), benzodiazepine, and GABA recognition sites. The results from these autoradio- graphic experiments prompted us to use in situ hy- bridization assays to examine the expression of various GABA A receptor subunit mRNAs in attempt to fur- ther characterize the involvement of GABA-BZ recep- tor complex in contingent tolerance to CBZ.

2. Materials and methods

2.1. Kindling and development of contingent tolerance to CBZ

Male Sprague-Dawley rats (300-350 g) were implanted with bipolar electrodes in the left amygdala and kindled until reliable generalized convulsions (i.e., Stage 5 according to the five-stage rating scale of Racine [29]) were produced with each stimulation as previously reported [37]. Electrode-implanted sham control rats were handled in an identical manner (placed in the kindling apparatus for 3 min with EEG recordings collected), except that no electrical stimulation was delivered. Contingent tolerance to CBZ was pro- duced as previously described [37] with a slight modification in order to match rats for the number of seizures experienced during toler- ance and control conditions. The treatment schedule is presented schematically in Fig. 1. Fully kindled rats were divided into three groups: tolerance induction (CBZ-before), non-tolerant (CBZ-after), and seizure controls (vehicle kindled). One group received CBZ 15 min before each electrical stimulation (CBZ tolerant) until tolerance developed; a second group received CBZ 15 min after each electrical stimulation (CBZ non-tolerant); a third group received vehicle injec- tion either 15 min before or after electrical stimulation (vehicle kindled). The sham animals were divided into two groups and received either CBZ or vehicle either 15 rain before or after place- ment in the kindling apparatus. There were no differences in the before and after treatments within groups for the vehicle-kindled, CBZ-sham, or vehicle-sham groups. Therefore, data from the before- and after-treated animals within these three groups were pooled to give three groups (rather than six) for statistical analysis. This re- sulted in a final of five treatment groups for statistical analysis:

I Surgical Implantation of Electrode

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m Sham Stimulation m I no current passed I I I m no seizures develop m I.. . . . . . i r - - . . . . m

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Electrical Stimulation Order

All groups matched for number of seizures: If a rat in Group 1 did not have a seizure on a given day, then its paired animals in Groups 2 & 3 were given no seizure (i.e., Sham Stimulation) on that day.

Fig. 1. Schematic diagram of the rat treatment schedule.

M. Clark et al. /Molecular Brain Research 26 (1994) 309-319 311

CBZ-before (tolerant) kindled; CBZ-after (non-tolerant) kindled; vehicle-kindled; CBZ-sham; and vehicle-sham (Fig. 1).

Kindled rats receiving CBZ before the stimulation were kindled first. If an animal did not experience a seizure upon stimulation, then its matched CBZ-after and vehicle-kindled animals received sham stimulation on that day in order to match the animals for the number of seizures experienced. Therefore, the kindled rats were matched across groups for the number and pattern of seizures and the number of drug or vehicle injections (Fig. 1). Animals were considered tolerant to CBZ when they did not have an anticonvul- sant response to CBZ for 5 consecutive days. CBZ (15 mg/kg; Sigma Chemical Co., St. Louis, MO) was administered by intraperitoneal injection of a 7 mg/ml solution in vehicle (10% ethanol, 40% proplylene glycol, and 50% saline). Vehicle groups were treated with the same vehicle without CBZ.

2.2. Preparation of brain tissue for receptor autoradiography and in situ hybridization

Animals were sacrificed by decapitation 4 hr after the last kin- dling stimulation, and brains were frozen in isopentane maintained at approximately -45°C, then stored at -70°C until sectioned. Coronal sections (15 g.m) at the level of dorsal hippocampus were cut on a cryostat microtome, thaw-mounted to gelatin-coated glass slides, dried by a fan and stored at -70"C until assayed. Just prior to receptor autoradiographic or in situ hybridization assays, slides were brought to room temperature (23°C) under a stream of 23°C air.

2.3. [ 3H]Muscimol binding

The procedure of Palacios et al. [26] for receptor binding with [3H]muscimol was used with minor modifications. Dried slides were

preincubated in 50 mM Tris-HC1 (pH 7.5; BRL Life Technologies, Inc., Gaithersburg, MD) for 30 min at 23°C and were then incubated in buffer containing 13 nM [3H]muscimol (20.0 Ci/mmol; New England Nuclear, Boston, MA) at 4°C for 45 min. Binding in the presence of 200 g.M GABA (Sigma, St. Louis, MO) was used to define non-specific binding. After the incubation, slides were washed twice for 30 sec (each wash) in buffer at 4°C, rinsed for 10 sec in ice-cold water, and air dried by a fan. Dried slides were apposed to Hyperfilm-3H (Amersham, Arlington Heights, IL) along with tritium standards (Amersham) for 15 days.

2.4. [3H]Flunitrazepam and [3H]Ro 15-4513 binding

[3H]Flunitrazepam and [3H]Ro 15-4513 receptor bindings were according to the methods of Valdes et al. [34] and Sieghart et al. [32], respectively, with minor modifications. Dried slides were preincu- bated in 50 mM Tris-HCl (pH 7.5) for 30 min at 23°C and were then incubated in buffer containing either 1 nM [3H]flunitrazepam (82.5 Ci/mmol; NEN) or 2 nM [3H]Ro 15-4513 (24.1 Ci/mmoi; NEN) for 60 min at 4°C. Nonradioactive diazepam and Ro 15-1788 (10 /zM; generous gifts of Hoffmann-La Roche, Nutley, NJ) were used to define non-specific binding for [3H]flunitrazepam and [3H]Ro 15- 4513, respectively. To determine diazepam-insensitive binding of [3H]Ro 15-4513, 10 /~M diazepam was included in the incubation. After incubation, slides were washed twice for 1 min (each wash) in buffer at 4°C, rinsed for 10 s in ice-cold water, and then air dried. Dried slides were apposed to Hyperfilm-3H along with tritium stand- ards for 7-12 days. Slides showing diazepam-insensitive binding of [3H]Ro 15-4513 were re-exposed for 4 weeks to increase the signal for analyses. Values for diazepam-insensitive [3H]Ro 15-4513 bind- ing were subtracted from [3H]Ro 15-4513 binding in the absence of diazepam to derive the diazepam-sensitive binding for this ligand.

-o

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v

l CBZ-Before (Tolerant)

CBZ-After (Non-tolerant)

Vehicle

mE i CBZ Vehicle

[3ss] TBPS [3H] FLU [3H] Rol 5-451 3

Fig. 2. Examples of autoradiographic film images showing that the kindling-induced increases in radioligand binding to the convulsant and benzodiazepine recognition sites in rat dentate gyrus are not related to contingent tolerance to carbamazepine (CBZ). [35S]TBPS labels the convulsant (i.e., picrotoxin) recognition site which is increased by kindling selectively in the dentate gyrus. The benzodiazepine agonist [3H]flunitrazepam and partial inverse agonist [3H]Ro 15-4513 (in the absence of non-labeled diazepam) label the 'classical' benzodiazepine receptor which is also upregulated by kindling selectively in the dentate gyms. Note that the increases are present to the same extent in all three kindled groups which suggests that contingent tolerance to CBZ does not involve either of these binding sites.

312 M. Clark et al. /Molecular Brain Research 26 (1994) 309-319

[3H]Rol 5-451 3 [ 3H] Muscimol + Diazepam

¢)

t - - ° ~

v

- CBZ-Before (Tolerant)

CBZ-After (Non-tolerant)

. Vehicle

E I CBZ Vehicle

Fig. 3. Examples of autoradiographic film images showing that the kindling-induced increases in radioligand binding to the GABA and diazepam-insensitive receptors could be involved in contingent tolerance to carbamazepine (CBZ). [3H]Muscimol and [3H]Ro 15-4513 (in the presence of 10 gM non-labeled diazepam) label the GABA and atypical (diazepam-insensitive) benzodiazepine recognition sites, respectively. The diazepam-insensitive [3H]Ro 15-4513 binding and the increased [3H]muscimol binding most likely reflect binding to the ot 4 subunit (see Discussion). Note that binding of both radioligands are increased selectively in the dentate gyrus by kindling, and that the CBZ-before (tolerant) group shows a strikingly reduced increase. Compare the binding of [3H]Ro 15-4513 in the presence (Fig. 2) and absence (Fig. 1) of diazepam.

2.5. [35Sit-Butyl bicyclophosphorothionate (TBPS) binding

[35S]TBPS binding was carried out as described by Edgar and Schwartz [10] except that Tris-HCl, pH 7.5, was used as the buffering agent. Briefly, all incubations and washes for [35S]TBPS were carried out at 23°C. Dried slides were preincubated in buffer (50 mM Tris-HCl, pH 7.5; 200 mM NaCI; 1 mM EDTA) for 30 min and, subsequently, incubated in buffer (without EDTA) containing 2 nM [35S]TBPS (134.1 Ci/mmol; NEN) for 3 h. Non-specific binding was defined by including 100 ~M picrotoxin (Sigma) in the assay. After incubation, slides were washed twice for 15 min (each wash) in buffer (without EDTA), rinsed for 10 s in water, and then air dried. Dried slides were apposed to Hyperfilm-MP (Amersham) for 17 h.

2.6. In situ hybridization

To measure the levels of various GABA A receptor subunit mR- NAs (al, a2, a4, /31, /33, ~/2), 45-base antisense oligonucleotides were synthesized and then purified by polyacrylamide gel electrophoresis [30]. The oligonucleotide sequences described by Wisden et al. [41]

were used in the present study. Probes were Y end labeled with [33p]dATP (1614 Ci/mmol; NEN) using terminal deoxynucleotidyl transferase (Stratagene, La Jolla, CA). Sections were fixed in 4% formaldehyde in phosphate buffered saline (PBS; 10 mM NaCI, 1.6 mM NaH2PO 4, 8.4 mM Na 2 HPO 4, pH7.0), rinsed twice in PBS, treated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0), then dehydrated into 100% ethanol and defatted with chloro- form. Sections were then hybridized overnight at 37°C with labeled probes (500,000 dpm/slide) in hybridization buffer containing 4× SSC (1 ×SSC: 150 mM NaCI, 15 mM sodium citrate, pH 7.0), 50% formamide, 1 × Denhardt's solution (Sigma), 250 p~g/ml yeast tRNA, 500 /zg/ml salmon sperm DNA and 10% dextran sulfate. Sections were washed in 1 x SSC (23°C), 2 x SSC/50% formamide (40.5°C), 1 x SSC (23°C), rinsed briefly in water, then dehydrated and exposed to Hyperfilm-MP.

2. 7. Image analysis and statistics

Autoradiograms were digitized and analyzed using the Image software (W. Rasband, NIMH). Measurements were taken in four

Footnote to Table 1 This table presents the analyses of autoradiographic film images for the receptor binding studies. With the exception of [35S]TBPS, all radioligands were tritiated, and the results of triated ligands are presented as fmol/mg wet weight tissue derived from tritium standards. The data for [35S]TBPS binding are presented as arbitrary units based on film density. All values are the mean ± S.E.M. of the number (n) of rats indicated. Supra-DG and infra-DG refer to the dorsal and ventral blades, respectively, of the dentate gyrus. Statistically significant differences between groups for a given hippocampal region are presented as follows: (a) group vs. respective sham group; (b) CBZ-before (tolerant) group vs. both CBZ-after (non-tolerant) and vehicle kindled groups; (c) CBZ-before (tolerant) group vs. vehicle kindled group only.

M. Clark et al. / Molecular Brain Research 26 (1994) 309-319 313

Table 1 Receptor autoradiographic analysis of the GABA-benzodiazepine receptor complex in dorsal hippocampus of kindled rats and carbamazepine tolerant rats

(n = 7) (n = 8) (n = 7) (n = 9) (n = 8) Vehicle CBZ CBZ-before CBZ-after Vehicle Sham Sham Kindled Kindled Kindled

Muscimol Ipsilateral

CA1 325 5:19 327 5:19 278 + 17 311 + 14 339 + 20 CA3 235 ± 15 238 + 13 224 + 12 237 5:12 245 + 14 supra-DG 492 + 32 474 + 27 440 + 24 c 540 + 27 585 _+ 33 infra-DG 527 + 32 515 + 23 478 + 23 b 608 + 28 a 693 + 38 a

Contralateral CA1 288 + 17 299 + 19 247 + 15 280 + 14 300 + 21 CA3 213 + 13 220 + 11 200 + 9 217 5:13 231 5:13 supra-DG 447 5:25 442 5:25 386 5:17 b 494 5:29 520 5:28 infra-DG 493 5:29 468 5:25 442 5:21 b 573 5:31 a 624 5:43 a

TBPS Ipsilateral

CA1 118 5 : 3 113+ 3 115 5 : 2 118 5 : 3 115 5 : 1 CA3 119+ 2 1185: 4 1255: 3 1215: 3 1235: 2 supra-DG 146 5: 2 144 5: 2 177 5: 2a'c 172 5: 3a 167 5: 2a infra-DG 142 5: 3 132 5: 3 174 5: 3a 170 5: 2a 167 5: 3a

Contralateral CA1 116 5 : 3 116 5 : 3 118 5 : 3 120 5 : 4 117 5 : 1 CA3 1195: 1 1 1 5 5 : 2 1 2 4 5 : 2 1 1 8 5 : 2 1 1 6 5 : 3 supra-DG 141 5 : 3 147 5 : 2 175 5 : 3 a 173 5 : 4 a 171 5 : 2 a infra-DG 1405: 3 1 3 9 5 : 2 1 7 5 5 : 4 a 171 5: 4 a 1695: 3 a

Flunitrazepam Ipsilateral

CA1 201 5 : 4 1 8 7 5 : 5 1 9 6 5 : 4 181 5 : 5 1 9 0 5 : 4 CA3 189 5 : 6 189 5:10 179 5 : 4 179 5 : 5 187 5 : 6 supra-DG 257 5 : 5 248 5: 6 297 5: 6a 288 5: 4a 298 5: 5a infra-DG 269 5 : 5 263 5 : 5 308 5 : 5 a 312 5 : 7 a 324 5 : 5 a

Contralateral

CA1 190 5 : 6 190 5 : 4 192 5 : 5 172+ 6 185 5 : 5 CA3 1 8 8 5 : 5 1 8 9 5 : 7 1 8 4 5 : 6 1 7 7 5 : 5 181 5 : 7 supra-DG 255 5 : 4 248 5: 6 284+ 5a 291 5: 4a 291 5: 4a infra-DG 264 5: 2 253 5 : 4 308 5: 5a 312 5: 5a 314 5: 5a

Ro 15-4513 Ipsilateral

CA1 498 5:26 478 5:16 445 + 20 456 5:22 438 5:23 CA3 396 5:28 374 5:19 330 5:13 355 5:14 369 5:13 supra-DG 497 + 24 461 5:23 520 5:23 545 5:16 513 5:31 infra-DG 492 5:26 490 5:15 549 5:27 557 5:25 570 5:32

Contralateral

CA1 475 5:26 500 5:19 439 5:17 434 5:13 431 5:21 CA3 414 5:19 393 5 : 8 377 5:13 375 5:10 378 5:24 supra-DG 480 5:20 474 5:14 510 5:24 551 5:19 520 5:26 infra-DG 485 5:24 488 5:16 567 5:30 535 5:16 562 5:38

Ro 15-4513 + DZP Ipsilateral

CA1 3 8 5 : 2 3 8 5 : 1 43+ 2 4 4 5 : 4 4 2 5 : 2 CA3 3 9 5 : 2 41 5 : 3 3 6 5 : 2 39 5 : 5 39 5 : 3 supra-DG 36 5 : 2 34 5 : 2 67 5: 3a'b 95 5: 7a 85 5: 3a infra-DG 36 5: 3 34 5: 2 81 5: 2a'b 105 5: 7a 105 5: 3a

Contralateral

CA1 24+ 1 22+ 2 3 2 5 : 2 31 5 : 5 3 0 5 : 3 CA3 2 0 5 : 3 1 8 5 : 2 2 0 5 : 3 1 8 5 : 3 2 1 5 : 3 supra-DG 26 5 : 2 24 5 : 2 59 5: 3a'b 80 5: 6a 75 5: 2 a infra-DG 28 5 : 2 21+ 2 73 5: 4a'b 96 5 : 5 a 94 5: 3a

314 M. Clark et al. / Molecular Brain Research 26 (1994) 309-319

regions of the hippocampus (CA1, CA3, dorsal 'blade' of dentate gyrus, and ventral 'blade' of dentate gyrus) and separately on ipsilat- eral (left) and contralateral (right) sides. For receptor autoradiogra- phy, the CA1 and CA3 subfields were measured in the stratum radiatum and oriens layers, respectively; and the molecular layer of the dentate gyrus was analyzed separately in the dorsal and ventral blades. For mRNA analyses, measurements were obtained in the pyramidal cell layers of CAI and CA3; and the granule cell layer of

the dentate gyrus was analyzed separately in the dorsal and ventral blades. For tritiated compounds, film density was converted to and expressed as fmol/mg wet weight tissue based on tritiated standards co-exposed with the tissue sections. However, for [35S]TBPS and [33p]oligonucleotides, values were expressed as arbitrary units based on film density. Data were ~'ested statistically by analysis of variance and Student-Newman-Keuls post-hoc comparisons using the Super- ANOVA software (Abacus Concepts, Calabasas, CA).

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CBZ-After (Non-tolerant)

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Fig. 4. Examples of the effects of kindling and contingent tolerance to carbamazepine (CBZ) on the expression of mRNAs encoding six different subunits of GABA-BZ receptor complex. Sections were hybridized with specific 33p-labeled oligonucleotides and exposed to film. The autoradiograms focus on the dorsal hippocampus. Note that kindling did not alter the expression of mRNA for the al, a2, or 1'2 subunits, while it did increase the levels of mRNA for the ill,/33, and a 4 subunits only in the dentate gyrus. Also note the selective lack of increased a 4 subunit

mRNA in the rats made tolerant to CBZ.

M. Clark et al. / Molecular Brain Research 26 (1994) 309-319 315

Table 2 Expression of mRNA for various subunits of the GABA-benzodi- azepine receptor complex in dorsal hippocampus of kindled rats and carbamazepine tolerant rats

(n=7) (n=8) (n=7) (n=9) (n=8) Vehicle CBZ CBZ-before CBZ-after Vehicle Sham Sham Kindled Kindled Kindled

ot 1

Ipsilateral CA1 33-+3 36_+3 34+3 31+1 37+-3 CA3 32_+2 34+2 28_+2 29_+2 33_+3 supra-DG 37_+4 37_+2 39_+2 41_+3 45_+3 infra-DG 36 -+ 3 34 _+ 2 41 _+ 2 40 _+ 2 44 _+ 3

Contralateral CA1 34_+4 35_+2 32_+2 31_+2 39_+2 CA3 29_+3 30_+3 27_+3 33_+2 29_+2 supra-DG 39_+3 36_+3 42-t-2 405:3 47_+2 infra-DG 35 _+ 3 37 _+ 3 42 _+ 3 37 _+ 2 45 _+ 2

O/2

Ipsilateral CA1 27_+2 25_+2 32+-2 a 31_+2 35_+2 a CA3 49_+2 49_+3 48_+2 49_+3 53_+2 supra-DG. 53_+ 1 55_+ 3 59 + 2 55_+ 4 60_+ 3 infra-DG 59_+1 61_+2 66_+3 60_+2 60_+2

Contralateral CA1 26+_3 26+_2 29_+2 30_+2 34_+2 CA3 45_+1 48_+2 48_+2 48_+1 50+3 supra-DG 56_+ 2 55 _+ 2 63 _+ 4 55 _+ 2 56 _+ 2 infra-DG 59 _+ 3 55 _+ 2 65 _+ 3 59 _+ 2 60 _+ 2

a 4 Ipsilateral

CA1 15_+1 15_+2 15_+2 17_+2 15_+1 CA3 8_+2 14_+2 11_+2 14_+2 11_+2 supra-DG 33 5:1 37_+ 3 48_+ 4 b 69_+ 6 a 73 _+ 4 a infra-DG 36 _+ 4 37 +- 4 53 _+ 5 a,b 76 _+ 3 a 79 _+ 3 a

Contralateral CA1 15_+2 14_+2 13_+1 d 23_+2 19_+3 CA3 9_+2 5_+1 11_+2 10_+2 13_+3 supra-DG 36_+ 3 33 _+ 2 47_+ 3 a,b 68_+ 4 a 69 _+ 3 a infra-DG 39_+2 37_+3 53_+7 a,b 78_+2 a 79_+4 a

Ipsilateral CA1 18_+2 23_+2 26_+3 24_+2 26_+1 CA3 28_+2 27_+1 30_+3 29_+1 32_+3 supra-DG 36_+2 42_+3 51_+4 45_+3 49_+3 infra-DG 37_+2 39_+2 54_+2 a 58-+3 a 57_+2 a

Contralateral CA1 24_+2 23_+1 25_+5 29_+2 26_+2 CA3 28_+2 28_+2 31_+2 29_-t-2 29_+2 supra-DG 38_+2 39_+3 51_+5 46_+3 47_+2 infra-DG 41-+2 41_+2 57_+4 a 56_+3 a 55_+3 a

t~3 Ipsilateral

CA1 30_+2 29_+2 31_+3 29_+2 26_+3 CA3 32_+1 36_+1 33_+2 35_+2 31_+2 supra-DG 48_+ 3 48_+ 2 58_+ 3 a 56_+ 2 62_+ 3 a infra-DG 48_+2 48_+2 67_+4 a 63_+2 a 64_+3 a

Contralateral CA1 27_+3 33_+1 28_+3 26_+2 24_+3 CA3 31_+2 31-+1 34_+1 34_+1 31_+2 supra-DG 50_+1 51_+1 61+3 a 59_+4 56_+2 infra-DG 48_+3 45_+3 68_+4 a 65_+3 a 62_+3 a

Table 2 (continued)

(n=7) (n=8) (n=7) (n=9) (n=8) Vehicle CBZ CBZ-before CBZ-after Vehicle Sham Sham Kindled Kindled Kindled

Y2 Ipsilateral CA1 21_+1 23_+1 24_+2 22_+1 19_+2 CA3 30_+2 25_+2 26_+3 22_+2 26+2 supra-DG 40-+ 1 38_+ 2 43 _+ 2 39_+ 3 39-+ 2 infra-DG 39_+2 38_+2 44_+4 40_+1 40+_2

Contralateral CA1 21_+1 21_+2 23+_2 17-+1 21_+2 CA3 25_+3 25_+1 27-+3 20-+2 20_+2 supra-DG 36_+ 1 34 +_ 2 42_+ 4 35_+ 2 37_+ 3 infra-DG 43_+2 39_+1 44_+3 42_+2 41_+2

This table presents the analyses of autoradiographic film images from the in situ hybridization studies with33p-labeled oligonu- cleotides specific for different subunits. The results are presented as arbitrary units based on film density. All values are the mean -+ S.E.M. of the number (n) of rats indicated. Supra-DG and infra-DG refer to the dorsal and ventral blades, respectively, of the dentate gyrus granule cells. Statistically significant differences between groups for a given hippocampal region are presented as follows: (a) group vs. respective sham group; (b) CBZ-before (tolerant) group vs. both CBZ-after (non-tolerant) and vehicle kindled groups; (d) CBZ-before (tolerant) group vs. CBZ-after (non-tolerant) group only.

3. Results

3.1. Alterations in radioligand binding to different com- ponents o f the G A B A - B Z receptor complex

Fig. 1 shows the animal t r ea tmen t schedule. Exam-

ples of the au to rad iograms f rom radio l igand binding

exper iments are shown in Figs. 2 and 3; and the results

of the image and statistical analyses are p re sen ted in

Tab le 1. No di f ferences in any radiol igand binding in

the h ippocampal CA1 or CA3 regions were found

be tween any of the five t r e a t m e n t groups; and no

d i f ferences be tween CBZ- or veh ic le - t rea ted sham

groups were observed in any h ippocampal region.

However , a l tera t ions in radio l igand binding were ob-

served in the den ta t e gyrus in kindled rats, and the

fol lowing results apply to changes found in the den ta te

gyrus. [3H]Flun i t razepam and [35S]TBPS bindings were

increased bi lateral ly in both the super ior and infer ior

blades in kindled rats. T h e r e were no di f ferences in

these increases be tween the th ree kindled groups

(CBZ-before , CBZ-af te r , vehicle), except that [35S]

TBPS binding in the ipsi lateral super ior b lade of the

C B Z - b e f o r e ( to lerant) k indled group was increased to

a g rea te r extent than that in the same area of the

vehic le k indled group. Diazepam-sens i t ive [3H]Ro 15-

4513 binding showed a t rend toward increased binding

bilaterally, but this effect did not reach statistical sig- nif icance. Inc reased [3H]muscimol binding was ob-

served bi lateral ly in the infer ior b lade of CBZ-a f t e r

(non- to le ran t ) and vehic le k indled rats. Binding of this

radio l igand in C B Z - b e f o r e ( to lerant) k indled rats was

3 16 M. Clark et al./Molecular Brain Research 26 (1994) 309-319

not different from either sham group and was less than both the CBZ-after (non-tolerant) and vehicle kindled groups. Diazepam-insensitive [3H]Ro 15-4513 binding was bilaterally increased throughout the dentate gyrus in all three kindled groups. However, the CBZ-before (tolerant) kindled animals showed a much smaller in- crease than the CBZ-after (non-tolerant) and vehicle kindled rats.

3.2. Alterations in expression of mRNA for different subunits of the GABA-BZ receptor complex

Fig. 1 shows the animal treatment schedule. Exam- ples of the autoradiograms from in situ hybridization experiments are shown in Fig. 4; and the results of the image and statistical analyses are presented in Table 2. No differences between CBZ- or vehicle-treated sham groups were observed for any subunit mRNA in any hippocampal region. The a 2 mRNA level was in- creased only in the ipsilateral CA1 region of CBZ-be- fore (tolerant) and vehicle kindled groups, and the 014 mRNA level in contralateral CA1 of CBZ-before (tolerant) kindled ratswas significantly less than that of the CBZ-after (non-tolerant) group. No other differ- ences in any subunit mRNA in the hippocampal CA1 or CA3 regions were found between any of the five treatment groups. Other than the changes noted above, expression of the 011, 012, and 3'2 subunit mRNAs in any hippocampal region was not different between any of the five treatment groups. However, kindling caused bilateral changes in the levels of 014, i l l , and /3 3 sub- unit mRNAs in the dentate gyrus. The /31, and /3 3 subunits were increased by kindling, with no effect of CBZ treatment, in the inferior blade. There was a trend for these two subunits to be increased in the superior blade, as well, but these elevations were in- consistent in reaching statistical significance. Expres- sion of the 014 subunit mRNA was increased bilaterally in all three kindled groups throughout the dentate gyrus. However, there was strikingly less 014 message induced in the CBZ-before (tolerant) kindled group as compared to both the CBZ-after (non-tolerant) and vehicle kindled groups.

4. Discussion

This study confirmed previous findings from other laboratories that kindling increases binding to the ben- zodiazepine [24,31,34] and GABA [25,31] recognititon sites on the GABA-BZ receptor complex selectively in the dentate gyrus of the hippocampus. The present study extended those findings by showing that kindling also increases [35S]TBPS binding to the convulsant (picrotoxin) recognition site and diazepam-insensitive

[3H]Ro 15-4513 binding. These effects of kindling were also limited to the dentate gyrus. Furthermore, in situ hybridization assays revealed increased levels of mRNA for the a4,/31, and/33 subunits of GABA-BZ receptor complex in kindled rats with no changes in the levels of mRNA for the al, a2, and 3'2 subunits. These kin- dling-induced alterations in GABA-BZ receptor com- plex subunit mRNAs were again limited to the dentate gyrus. However, the most profound findings were noted in rats made contingent tolerant to the anticonvulsant activity of CBZ. Even though the CBZ-before (tolerant) kindled group exhibited the same number of seizures which were of similar duration and intensity as rats in the CBZ-after (non-tolerant) and vehicle kindled groups, the tolerant group had strikingly fewer alter- ations in some of the parameters measured. In con- trast, other parameters revealed no differences among the tolerant, non-tolerant, and vehicle kindled groups. For example, [3H]muscimol binding, diazepam-insensi- tive [3H]Ro 15-4513 binding, and a 4 subunit mRNA content in the dentate gyrus of CBZ-before (tolerant) kindled rats showed either no increase or a greatly reduced increase as compared to the other two kindled groups. The magnitude of increased [3H]flunitrazepam and [35S]TBPS binding and the increased levels of /31 and /33 subunit mRNAs were identical in all three kindled groups.

The validity of the results obtained in this study is supported in several ways. The techniques utilized (re- ceptor autoradiography and in situ hybridization) are well established methods, and the distribution patterns of the film images were consistent with those expected based on examples found in the published literature. Adjacent sections from each rat were used to test the binding of each ligand and for each in situ hybridiza- tion assay; therefore, each animal also served as its own control for investigating differential alterations in binding to the various recognition sites of the GABA- BZ receptor complex and in various receptor subunit mRNAs within a given treatment group, which sub- stantiated the selective changes observed in animals made tolerant to CBZ. Support for the validity of selective differences in the CBZ-before (tolerant) kin- dled rats as compared to CBZ-after (non-tolerant) and vehicle kindled animals is gained from the findings of Wisden et al. [40]. They reported that recombinant receptors expressing the a 4 subunit (in combination with /32 and 3'2 subunits) showed high affinity binding for [3H]muscimol and [3H]Ro 15-4513 but failed to bind benzodiazepine agonists, whereas the a I subunit (in combination with/32 and 3'2 subunits) showed high affinity binding for the benzodiazepines as well. The selective differences in only these three parameters in rats tolerant to CBZ suggest some functional tie be- tween the [3H]muscimol binding site, the diazepam-in- sensitive [3H]Ro 15-4513 binding site, and the a 4 sub-

M. Clark et al. / Molecular Brain Research 26 (1994) 309-319 317

unit mRNA. The 0/4 subunit mRNA may actually encode the GABA-BZ receptor complex protein sub- unit that is responsible for the increased [3H]muscimol and diazepam-insensitive [3H]Ro 15-4513 binding [40].

Increases in [3H]flunitrazepam [24,31,34] and [3H]muscimol [25,31] binding selectively in the dentate gyrus were previously observed in kindled rats. Di- azepam-sensitive binding of the benzodiazepine partial inverse agonist [3H]Ro 15-4513, which is thought to represent binding at the classical benzodiazepine re- ceptor [32], was also tested in the present study. The binding of this ligand followed the same trend as for [3H]flunitrazepam (increased by kindling irrespective of CBZ treatment), but the increase did not reach statistical significance (P values ranging between 0.07 and 0.08) probably due to the larger variance within groups. Nevertheless, it appears that benzodiazepine inverse agonist binding may also be increased by kin- dling. However, only one study [28] has previously examined [35S]TBPS binding in kindled rat brain. They observed decreased [35S]TBPS binding in amygdala, limbic cortex, and thalamus of kindled rats with no changes in the dentate gyrus. However, that study [28] measured [35S]TBPS binding at two different times after the last kindled seizure, at 24 h and 28 days. This was an effort to assess the long term changes that might reflect the 'kindled state'. The present study examined [35S]TBPS binding only at 4 h after the kindled seizure and found [35S]TBPS binding increased selectively in the dentate gyrus. Therefore, it is likely that we measured transient effects resulting from a kindled seizure rather than changes reflecting the 'kin- dled state'. This possiblility for all of the measured parameters is currently under investigation. Four hours after the last seizure was selected on the rationale that changes in mRNA levels would precede alterations in protein synthesis. Since the previously published data from several laboratories reported increased benzodi- azepine and muscimol binding 24 h after the last kindled seizure, a shorter time point (4 h) was selected to increase the likelihood of observing mRNA changes. Furthermore, it was reasonable to expect that changes resulting from the penultimate seizure (28 h) would still be evident, making this study less divergent from previously published results.

At least fifteen different subunits of the GABA-BZ receptor complex have been cloned and consist of 0/1-6, /31-4, '~1-3, ¢~1, and Pl [3]. However, the subunit combination expressing a functional GABA-BZ recep- tor complex in brain is not known, and it is conceivable that several different subunit combinations exist result- ing in various GABA receptors with different func- tions. The results of the present study suggest that kindling-induced increases of [3H]flunitrazepam and [35S]TBPS binding could involve the /31 and /33 sub- units since the changes in these radioligands and sub-

unit mRNAs paralleled each other (i.e., increased in all three kindled groups irrespective of CBZ treatment). Functional significance of this increase may be exem- plified by the lack of cross tolerance to clonazepam [15] (S.R.B. Weiss and R.M. Post, unpublished observa- tions) or diazepam [38] in kindled rats made tolerant to CBZ. On the other hand, the levels of the 0/4 subunit mRNA paralleled the lack of increased [3H]muscimol and diazepam-insensitive [3H]Ro 15-4513 binding ob- served in rats made tolerant to CBZ. Considering the work of Wisden et al. [40] discussed above, our data suggest that the 0/4 subunit may be related to kindling- induced increases in [3H]muscimol and diazepam-in- sensitive [3H]Ro 15-4513 binding in the dentate gyrus and to contingent tolerance to CBZ. It follows that the 0/4 subunit may be involved in contingent tolerance to the anticonvulsant activity of ethanol observed by Kim et al. [16] in amygdala kindled rats, as this subunit was suggested to be involved in the 'ethanol-antagonistic' actions of Ro 15-4513 [40]. This possibility warrants investigation.

The observation of changes in [3H]muscimol binding in CBZ tolerant animals was unexpected, because it was previously reported that CBZ does not inhibit binding of this radioligand in rat brain [21]. The possi- bility of CBZ interacting directly with the GABA-BZ receptor complex does not seem likely but merits futher investigation in light of the current findings. Another possibility is an indirect effect of CBZ on the GABA- BZ receptor complex. CBZ is known to bind [8,21] to the mitochondrial ('peripheral-type') benzodiazepine receptor which is involved in steroidogenesis [17,27]. Furthermore, the anticonvulsant effects of CBZ [39] and contingent tolerance to CBZ [38] were both sug- gested to involve a mitochondrial benzodiazepine re- ceptor mechanism. Cross-tolerance to the anticonvul- sant effects o f PK 11195 (a mitochondrial benzodi- azepine receptor ligand) was observed in animals made tolerant to CBZ in the amygdala kindling paradigm [38]. It is well established that certain steroids interact with the GABA-BZ receptor complex [4,18]. There- fore, contingent tolerance to CBZ could involve modu- lation of GABA-BZ receptor complex, indirectly, through steroids via CBZ binding at mitochondrial benzodiazepine receptors. To our knowledge, direct interaction of neurosteroids specifically with the 0/4 subunit has not been tested.

Whatever the mechanism of tolerance is, it does not appear to be a direct drug effect, because the kindled rats that received CBZ after the electrical stimulation did not develop tolerance. The mechanism is a contin- gent drug effect. Data from the present study suggest the involvement of the GABA-BZ receptor complex, at least the 0/4 subunit, in the anticonvulsant response to CBZ. The possible functional relationship of the bio- chemical changes in the GABA A receptor system to

318 M. Clark et al. / Molecular Brain Research 26 (1994) 309-319

the phenomenon of loss of CBZ efficacy remains to be further explored.

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