Long-term reduction of benzodiazepine receptor density in the rat cerebellum by acute seizures and kindling and its recovery 6 months later by a pentylenetetrazole challenge

Brain Research 888 (2001) 212–220 www.elsevier.com / locate / bres

Research report

Long-term reduction of benzodiazepine receptor density in the rat cerebellum by acute seizures and kindling and its recovery 6 months later by a pentylenetetrazole challenge A.S. Bazyan a , *, V.V. Zhulin a , M.N. Karpova b , N.Y. Klishina b , R.N. Glebov b a

Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, Butlerov St. 5 A, GSP-7, Moscow 117865, Russia b Institute of General Pathology and Pathophysiology, Russian Academy of Medical Sciences, Moscow, Russia Accepted 26 September 2000

Abstract Seizures induced by an acute pentylenetetrazole (50 mg / kg) injection were accompanied by a long-term (at 1–48 h, but not on day 7) decrease in the density (Bmax ) of [ 3 H]-diazepam binding to benzodiazepine receptors in rat cerebellar cortex with no change in affinity (Kd ). Kindling for 24 days by daily administrations of pentylenetetrazole (20 mg / kg) led to the same decrease in benzodiazepine receptor density (at 1–48 h, but not on day 7) as that observed after a single dose of pentylenetetrazole (50 mg / kg). This suggests a common mechanism for both acute and kindling-induced seizures, dependent on the long-term receptor changes. The increased susceptibility to seizures persisted for 6 months after the termination of kindling, with BDZ receptor density in cerebellar cortex reduced almost by half. In age-matched controls, an acute dose of PTZ (30 mg / kg) induced seizures and decrease in both Bmax and Kd of [ 3 H]-diazepam binding. In kindled rats, at 6 months post-kindling, the same dose of PTZ (30 mg / kg) restored the benzodiazepine receptor density to the level found 6 months before, at the time of termination of kindling. Also, the severity of seizures was enhanced in the kindled rats. The results are discussed in terms of a balance of inhibitory and excitatory processes, in which the reduced BDZ receptor density at 6 months post-kindling may represent a compensatory reaction to outbalance some alterations in excitatory systems that have been reported to be induced by kindling.  2001 Elsevier Science B.V. All rights reserved. Keywords: Kindling; Cerebellum; Pentylenetetrazole; Benzodiazepine; GABA

1. Introduction It is known that GABAA , benzodiazepine (BDZ), picrotoxin-barbiturate (PCT-BB) receptors and Cl 2 channels are assembled in a single supramolecular complex. Both BDZ and PCT-BB receptors act as allosteric sites for the GABAA receptor and vice versa. Pentylenetetrazole (PTZ) interacts with the PCT-BB receptor of the GABAA / BDZ / PCT-BB receptor complex, inhibiting the GABAA and BDZ receptors, closing the Cl 2 channels and provoking seizures in convulsive doses [5,11,12,25,35,41,46,47, 50,55,65,69]. In subconvulsive doses, chronic administration of PTZ induces kindling phenomena and modifies the GABAA / BDZ / PCT-BB receptor complex, suppressing GABAA , *Corresponding author. Tel.: 17-95-334-7789; fax: 17-95-338-8500. E-mail address: [email protected] (A.S. Bazyan).

BDZ receptors and GABA-stimulated 36 Cl 2 influx into cortical membrane preparations [29,40,49–52,55]. BDZ agonists manifest a marked anticonvulsive effect on clonic–tonic seizures in PTZ-kindled rats, with a marked dose-effect dependence [3,5,13,34,53,54]. Chronic subthreshold stimulation of some brain structures, or chronic subconvulsive doses of convulsants result in kindling which is maintained for a long time [18,29,42,67]. It is known that electrical kindling develops with the active involvement of long-term potentiationinduced NMDA receptors [10,26,37,38,48,61]. While the long-term suppression of the GABAA / BDZ / PCT-BB receptor activity by kindling has been described in a number of studies, little is known about the initial long-term responses. Is the long-term response attributable to kindling development, or initial attribute of GABAA / BDZ / PCT-BB receptors complex, as for NMDA receptors? Similarly, little is known of the GABAA / BDZ / PCT-BB

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )03045-6

A.S. Bazyan et al. / Brain Research 888 (2001) 212 – 220

mechanisms in kindling seizures retrieval after very long times of acute administration of subconvulsive doses of convulsants. Previously, we have found [29] that changes in [ 3 H]diazepam binding are maximal in the cerebellar cortex, rather than in the neocortex, of the kindled rats. It has been shown that 81% of the total BDZ receptors in cerebellar cortex contain high-affinity sites for PTZ [6]. In the forebrain, only 34% of the total BDZ receptor population bear high-affinity sites for PTZ. At the same time, the cerebellar cortex actualizes movement control. The cerebellar cortex exerts its influence by inhibiting and disinhibiting motor control action, in the premotor networks of the brainstem, sensomotor cortex, and spinal cord [19– 22,24]. On the other hand, the cerebellum and its associated brainstem circuitry does appear to be essential (necessary and sufficient) for learning and memory of the conditioned response [32,33,62–64,68]. This indicates that long-term reactions are inherent to the neurons of cerebellum; for instance, in long-term depression [23]. The goal of the present investigation was to study long-term characteristics of [ 3 H]-diazepam binding in cerebellar cortex after an acute injection of PTZ in convulsive doses, after kindling development; peculiarities of the retrieval of kindled seizures and of [ 3 H]-diazepam binding 6 months after the termination of kindling, following a challenge with a subconvulsive dose of PTZ.

2. Materials and methods Male Wistar rats (150–200 g, about 3 months old) were used, a total of 266 animals. Five series of experiments were conducted in two stages. Pentylenetetrazole (Sigma, USA) or saline (control rats) were injected intraperitoneally. The seizures were scored by the following scale: (1) a twitch of the head; (2) occasional clonic seizures; (3) a series of clonic seizures, or clonus of the forepaws; (4) clonic–tonic seizures while standing on the hind paws (kangaroo posture); (5) clonic–tonic seizures and falling on one side.

2.1. Kindling procedure A subconvulsive dose of PTZ (20 mg / kg) was injected in rats (n5100) once daily for 24 days. The daily injections gradually increased the sensitivity to the convulsant. The dose of PTZ (20 mg / kg), which was subconvulsive in itself, evoked seizures that intensified and finalized in a clonic–tonic paroxysm.

2.2. Preselection of animals The rats were assigned to the different experimental groups specified below at the conclusion of kindling, when they were about 3–4 months old.

213

2.2.1. Three to four-months-old rats The first series comprised the rats, in which a convulsive dose of PTZ (50 mg / kg) resulted in seizure scores of 4–5 points. They were decapitated 60 min (30 min after the termination of seizures) or 48 h later and on day 7 after the injection (n536, including control rats). The second series comprised the rats, in which a subconvulsive dose of PTZ (20 mg / kg) elicited kindled seizures scoring 4–5 points, followed by decapitation 60 min (30 min after the termination of seizures) or 48 h later and on day 7 after the last injection (n548, including control rats). The third series comprised the rats endogenously sensitive and insensitive to the convulsant. One group of animals (n525) was treated with 25 mg / kg PTZ and the second group with 30 mg / kg (n525). Then, the rats were selected according to their sensitivity: the first subgroup (n56) with seizures (2 points) provoked by 25 mg / kg PTZ; and the second subgroup (n56) with no seizures provoked by 30 mg / kg PTZ. Both groups were decapitated on day 7 after the injection. 2.2.2. After 6 months (about 10 -months-old rats) For the period of 6 months both the control and kindled rats were kept in the breeding facility. Their weights at the second stage of experiments reached 360–450 g. At the second stage, the persistence of kindling was studied. Two control groups and two groups of kindled rats were treated with 20 and 30 mg / kg PTZ (n525 in each control and kindled group). For the study of [ 3 H]-diazepam binding four groups of animals were used (n56 in each group): (1) control rats, no PTZ challenge; (2) acute seizures control rats, 30 min after the termination of acute seizures (2–3 points) induced by a PTZ (30 mg / kg) challenge; (3) kindled control rats, no PTZ challenge, but with a history of seizures (4–5 points) 6 months ago; (4) kindling1PTZ challenge, 30 min after the termination of seizures (3–4 points) induced by a PTZ (30 mg / kg) challenge. 2.3. [ 3 H] -diazepam binding Cerebellar cortex was removed and used to isolate a crude fraction of synaptic membranes. The cerebellar cortex was homogenized and centrifuged for 10 min at 10003g in the medium: 0.32 M sucrose, 1 mM EDTA and 5 mM Tris–HCl, pH 7.4 at 48C. The supernatant was centrifuged for 20 min at 20,0003g. The pellet (P2 ) was frozen at 2408C. After washing by centrifugation, the pellet was resuspended in 4 ml of 50 mM Tris–HCl buffer, pH 7.4. A portion of the suspension of synaptic membranes (400 ml) was added to 50 ml of an aqueous [ 3 H]-diazepam solution (68 Ci / mmol, Amersham, UK) with final concentrations of 0.55; 1.1; 2.19; 4.38; 8.75; 17.5; 35 nM. Non-specific binding was assessed in parallel samples containing excess unlabeled diazepam. All sam-

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214

ples were run in duplicate. The samples were incubated for 40 min at 48C and filtered through a GF / B filter (Whatman, UK), followed by four successive washes with icecold Tris-buffer. Radioactivity of the filters was measured in Bray’s scintillation cocktail using a Rackbeta (LKB, Sweden) counter. Protein content was determined by the method of Lowry et al. [39]. The density (Bmax ) and the dissociation constant (Kd ) were calculated from the Scatchard plots (M6S.E.M.). For statistical analysis, Student’s unpaired t-test (Tables), two-way (Fig. 2A) and one-way (Fig. 2B) ANOVA test were used.

3. Results Fig. 1. Time course of kindling by daily injections of a subconvulsive dose of pentylenetetrazole (20 mg / kg) for 24 days (n5100).

3.1. Three to four-months-old rats 3.1.1. Acute PTZ (50 mg /kg) injection Acute treatment with PTZ (50 mg / kg) resulted in seizures scoring 4–5 points. The density (Bmax ) of [ 3 H]diazepam binding sites was significantly reduced by 15% at 30 min after the termination of seizures and by 13% at 48 h with no change in the Kd (Table 1). No significant changes were found on day 7.

3.1.3. Study of sensitivity Kindled rats were treated as a PTZ-sensitive group, and control rats as a PTZ-resistant group. A lack of response on day 7 after the seizures induced either by kindling or by acute PTZ (50 mg / kg) was used as a criterion to group the rats according to their sensitivity. No differences in [ 3 H]diazepam binding between the sensitive and insensitive rats were found on day 7 after the selection (Table 2).

3.1.2. [ 3 H] -diazepam binding after the kindling Initially, a subconvulsive dose of PTZ (20 mg / kg) did not induce seizures and [ 3 H]-diazepam binding was unchanged (Table 1). Daily injections of this dose resulted in the development of kindling (Fig. 1) and a significant decrease in the Bmax of [ 3 H]-diazepam binding by 19% at 30 min and by 16% at 48 h after the termination of seizures. The Kd of the binding was unchanged. On day 7, no significant changes were observed (Table 1). These findings in the kindled rats are similar to the results found after an acute injection of the convulsive dose.

3.2. Ten-months-old rats, 6 months after the termination of kindling 3.2.1. Seizures retrieval The kindling response of high sensitivity to low doses of PTZ was preserved through the 6-months rest period after the kindling treatment, but not completely and with some attenuation. A subconvulsive dose of PTZ (20 mg / kg) induced no seizures in the control rats (Fig. 2A), but this dose elicited seizures in 60% of the kindled rats (1–2

Table 1 [ 3 H]-Diazepam binding by cerebellar cortex membranes of 4 months old rats at 30 min, 48 h and 7 days after the termination of seizures at the acute pentylenetetrazole administration and kindling development 30 min Control (n56)

48 h PTZ (n56)

Pentylenetetrazole acute administration, 50 mg / kg Bmax 1386697 1123676* (fmol / mg protein) Kd (nM) 9.0261.08 8.8561.06 Pentylenetetrazole acute administration, 20 mg / kg Bmax 1142697 1127694 (fmol / mg protein) Kd (nM) 8.2960.84 8.3560.79 Kindling, 20 mg / kg Bmax (fmol / mg protein) Kd (nM) *

Pt ,0.05 versus control.

Day 7

Control (n56)

PTZ (n56)

Control (n56)

PTZ (n56)

1360695

1142679*

1328690

1302689

9.1060.82

8.9260.80

9.1560.85

9.0960.78

–

–

–

–

–

–

–

–

1109689

898663*

1156672

971668*

1130667

1160678

8.0360.88

7.9660.80

7.8860.79

7.9460.87

8.1260.74

8.4160.92

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215

Table 2 [ 3 H]-Diazepam binding by cerebellar cortex membranes of rats with different sensibility to pentylenetetrazole (day 7 after test)

before, neither in control with acute seizures nor in kindled rats.

Animals

Bmax (fmol / mg protein)

Kd (nM)

Insensitive (n56) Sensitive (n56)

923684 958637

7.7860.81 7.6560.35

3.2.2. [ 3 H] -diazepam binding at seizures retrieval [ 3 H]-diazepam binding was unaltered in the ten months old control rats, but an acute PTZ challenge (30 mg / kg) resulted in seizures (scores 2–3 points) that were accompanied by a decrease in both indices of [ 3 H]-diazepam binding: the Bmax to 66%, and the Kd to 73% (Table 3). In the kindled control rats with a history of seizures (4–5 points) 6 months before, the Bmax of [ 3 H]-diazepam binding was reduced to 54% without a PTZ challenge. After a rechallenge with PTZ, the Bmax of [ 3 H]-diazepam binding was found to be enhanced to 78%, still being significantly lower than in the control rats, with no change in the Kd (Table 3). This paradoxical finding can be logically explained as shown in Fig. 3. At the time of termination of kindling the BDZ receptor density is reduced to 81% versus the control 4-months-old rats. After a rest period of 6 months, there was a decrease in BDZ receptor density to 54% in the kindled rats without a PTZ challenge. Acute PTZ administration to the kindled rats induced seizures and partially restored the BDZ receptor density, just to the level of BDZ density found in the kindled 4-months-old rats (78%), which was established 6 months before. At the same tame, the Kd of BDZ receptor

points). At a higher dose of PTZ (30 mg / kg) seizures were observed in 56% of the control rats (maximal scores of 2–3 points) and in 100% of the kindled rats (maximal scores of 3–4 points). These observations suggest that the high sensitivity of rats to PTZ, induced by kindling, was attenuated after 6 months, but at the same time the kindled rats were more sensitive than the control rats. The pattern of seizures in the control and kindled rats challenged with 30 mg / kg of PTZ was different (Fig. 2B). In the control rats, the first peak of seizures occurred at 5 min and the second peak was observed at 20 min after the PTZ challenge. About 40% of the kindled rats displayed a period of low-level seizures scoring 1–3 points, which lasted for 10 min. The first peak of seizures with maximal scores from 3 to 4 points was observed at 20 min after the PTZ challenge, and the second peak occurred at 35 min. About 60% of the kindled rats displayed only the low-level seizures scoring from 1 to 3 points, which lasted for 45 min. This type of latency was not observed 6 months

Fig. 2. Pattern of seizures in 10-months-old control and kindled rats. A. Seizures induced by 20 (n cont 525; n kind 525) and 30 (n cont 525; n kind 525) mg / kg pentylenetetrazole, averaged by maximal scores. *PF ,0.05 versus own control, x PF ,0.05 versus own control and kindled rats at 20 mg / kg PTZ. B. Time course of pentylenetetrazole-induced seizures at 30 mg / kg (n cont 514; n kind 525). *PF ,0.05. Table 3 Effects of acute pentylenetetrazole (30 mg / kg) administration on [ 3 H]-diazepam binding by cerebellar cortex membranes of 10 months old control and kindled rats a

Bmax (fmol / mg protein) Kd (nM) a

Control (n56)

Control1PTZ (acute seizures, n56)

Kindled control (n56)

Kindled1PTZ (rechallenge, n56)

967679

6416106*

518681**

752680* ?

7.5660.99*

9.5460.78

10.761.21

10.361.02 ?

*Pt ,0.05 versus control; **Pt ,0.01 versus control; Pt ,0.05 versus kindled control.

216

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Fig. 3. Depression of benzodiazepine receptor density (Bmax ) in 4- and 10-months-old kindled rats. The results are expressed as % versus control rats. [ 3 H]-Diazepam binding was measured using synaptic membranes isolated at the following time points. 4-months-old rats. At 30 min after the last kindled seizures induced by daily pentylenetetrazole (20 mg / kg) injections. 10-months-old rats. Kindled control: at 6 months after kindling with no pentylenetetrazole challenge; kindled1pentylenetetrazole: at 30 min after seizures induced by a pentylenetetrazole challenge (30 mg / kg). Vide statistics in Table 1: *Pt ,0.05 versus control; **Pt ,0.01 versus control; ? Pt ,0.05 versus kindled control.

binding was unchanged in the kindled rats, whereas in the control 10-months-old rats that had seizures after an acute PTZ challenge the Kd was significantly altered (Table 3).

4. Discussion We found that seizures of the same severity, but induced by two different procedures, a single convulsive dose of PTZ (50 mg / kg) and kindling with a subconvulsive dose (20 mg / kg), resulted in similar modifications of BDZ receptors of GABAA supramolecullar receptor complex: a long-term (48 h) decrease in the Bmax , with no change in the Kd , and subsequent normalization on day 7. This result shows that kindling is an elaboration of high sensitivity to subconvulsive doses of PTZ. This result also shows that the characteristics of [ 3 H]-diazepam binding to the BDZ cerebellar receptors of animals with control levels of sensitivity was not different from the characteristics of kindled animals with high level of sensitivity 7 days after the termination of seizures. Normalization of the reaction on day 7 makes it possible to suggest initial binding constants of cerebellum BDZ receptors. A special investigation of the two types of control animals, endogenously sensitive and resistant to PTZ, showed that they also were similar 7 days after the termination of seizures. So, the [ 3 H]-diazepam binding constants of the endogenously sensitive and resistant rats initially were similar. These data allow us to suggest that the change in sensitivity to PTZ of cerebellar BDZ receptors, when PTZ is injected intraperitoneally, may depend not only on the receptor binding constants. It is possible that an important

role is played by intracellular reactions that follow the receptor occupation. It is known that the activity of BDZ receptors is dependent on protein kinase C [27,44]. Then, a feedback mechanism is suggested: phosphorylation or rephosphorylation of BDZ receptors by a BDZ receptorspecific protein kinase C. This reaction can be termed autocontrol feedback reaction. It is possible that autocontrol feedback reaction is modulated by the BDZ receptor and modifies its density. Furthermore, BDZ receptor-dependent protein kinase C mechanism is induced by gene expression [27,44]. It is possible that gene expression can explain the long-term (up to 48 h) decrease of BDZ receptor density in the cerebellum by acute seizures. Extending this logical way, we can suggest that the establishment of kindling is based on the consecutive enhancement of protein kinase C-dependent reaction and modification of BDZ receptors. It is possible that on day 7 after the establishment of kindling the BDZ receptor density was normalized, but intracellular processes still continued. We may suggest, therefore, that the decrease in BDZ receptor density 6 months after kindling is the track of continuing intracellular processes. The high sensitivity induced by kindling was somewhat attenuated after 6 months. This is a typical process for elaborated or learned reactions that attenuate with time without reminder. It is known [59–61] that reminder reconsolidates memory and enhances retrieval. But, at the same time, reminder needs more time for retrieval than it takes immediately after the initial elaboration or learning. We think that the longer latency of seizures retrieval 6 months later (Fig. 2, B) can be explained by PTZ acting as a reminder. In this case, the reminder would switch on

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autocontrol feedback mechanisms which in this situation is manifested as autoenhancement feedback mechanisms, and would retrieve seizures and BDZ receptor constants. But, the theoretical view that seizures are determined by the relationship between excitatory and inhibitory processes is supported by a number of experimental data [7,8,14,18,42,58]. Our primary interest in pursuing this work was initially directed at BDZ receptors, on the assumption that long-term responses are more likely to develop at allosteric modulatory sites of the GABAA receptor complex. This assumption appears to be justified, considering that GABAA , BDZ receptors and Cl 2 channels are allosterically inhibited by PTZ and that 81% of the total BDZ receptors in the cerebellum contain high-affinity sites for PTZ [6]. Accordingly, we feel that the changes in BDZ binding can be interpreted as representative for the whole GABAA supramolecular complex in the cerebellar cortex during seizures and kindling. We found that seizures of the same severity, but induced by two different procedures, a single convulsive dose of PTZ (50 mg / kg) and kindling with a subconvulsive dose (20 mg / kg), resulted in similar modifications of BDZ receptors of GABAA supramolecullar receptor complex: a long-term (48 h) decrease in the Bmax , with no change in the Kd , and subsequent normalization on day 7. It is known [15,16,66] that kindling can lead to a long-term decrease of the GABAA receptor complex activity, due to changes in the synthesis of the respective proteins [30,36]. A series of 40 kindling-induced seizures (by rapid hippocampal stimulation) led to biphasic alterations of GABAA receptor subunit mRNA levels in dentate gyrus with only minor changes in CA 1 –CA 3 [30]. Up to 4 h after the last seizure mRNA expression for alpha 1 subunit was slightly decreased in dentate gyrus, whereas marked reductions were observed for beta 3 and gamma 2 subunits (by 41 and 48%, respectively). Between 12 and 48 h there were major increases of alpha 1 (by 59%) and gamma 2 (by 35%) subunits mRNA levels but no significant changes of beta 3 subunit mRNA expression. The subunits mRNA levels had returned to control values after 5 days [30]. These results are similar to ours: the biphasic changes of GABAA receptor subunits may be related to the onset of normalization of the receptor density. The long-term response induced by acute PTZ treatment is presumably maintained by gene expression. Various types of long-term responses are known to be maintained by gene expression [4,17,56,57]. Therefore, we think that kindling develops because of a long-term response of GABAA / BDZ / PCT-BB receptor complex. It is possible that small (by acute subconvulsive dose PTZ injection and not elicited experimentally) long-term decreases of GABAA receptor complex activity are cumulated to a convulsive threshold in the course of the daily injections and are consolidated by gene expression. The presumed time course of this cumulative process is reflected in Fig. 1.

217

Our results show that kindling is nevertheless preserved at a high sensitivity for as long as 6 months. At the same time, the BDZ receptor response was qualitatively different from the seizures induced by acute PTZ treatment of young and old control animals; in that in young rats only the density of BDZ receptors was reduced, while in older rats both the density and affinity of BDZ receptors was altered. At 7 days after the termination of kindling the BDZ receptor density was normalized, but 6 months later the BDZ receptor density was about two-fold lower. These results correspond with the literature. A normalization of NMDA receptor activity on day 7 was found in amygdala kindling [2], and normalization of GABAA [30] and NMDA [48] receptor subunit mRNA level was observed on day 5 after hippocampal kindling. Some time after the normalization, the receptor response is spontaneously altered again: a decrease in GABAA receptor sensitivity was reported at 3–4 weeks after amygdala kindling [15,16]. An enhanced hippocampal NMDA receptor activity was observed 1 month after kindling [71], and a novel population of NMDA receptors was expressed 28 days after hippocampal kindling [31]. In addition, 6 weeks after kindling an enhancement of the Ca 21 current was observed in the hippocampal CA 1 area of the rat [70]. However, these data are not always confirmed in some reports. No changes of NMDA receptor function were found on day 28 [2] and 3 and 6 weeks [26] after the kindling, as well as of GABAA receptor activity [28] 4 weeks after the kindling. A review of the literature allows us to suggest that the decrease in BDZ binding observed 6 months after the kindling in our study may be unrelated to the mechanism of kindling storage and seizure retrieval. It is possible that the decrease in GABAA / BDZ / PCT-BB receptor complex activity represents a compensatory reaction to a reduced excitatory system activity, since a decrease in NMDA receptor activity has been reported 1 and 1.5 months after kindling [43,45]. We think that only a compensatory reaction of excitatory processes is able to explain the absence of seizures against the background of a one-half decrease of the inhibitory system in our kindled control 10-months-old rats. It is possible that spontaneous changes of the receptor activity can arise after the termination of long-term convulsant treatment or electrostimulation. But the latency of these spontaneous changes in the receptor activity seems to depend on experimental conditions. Our results suggest other mechanisms of seizure retrieval 6 months after the kindling. An acute administration of PTZ (30 mg / kg) to 10-months-old kindled control rats enhances the activity of GABAA / BDZ / PCT-BB receptor complex, increasing it to the level of 4-months-old kindled rats as well as producing seizures. In this case, seizures might be initiated by a simultaneous increase of excitatory system activity, bringing it to the level of 4-months-old kindled rats. We suggest that kindling modifies both

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excitatory and inhibitory systems of the brain, establishing a new balance between them and consolidating it via gene expression (basic mechanism of memory consolidation). At 6 months after the kindling, a subconvulsive PTZ challenge induces seizures by recovering a new consolidated receptor activity. It is a newly consolidated activity, because in 10-months-old intact control rats the seizures induced by a convulsive doze of PTZ (30 mg / kg) are accompanied by a qualitatively different type of BDZ binding response, a decrease in both density and affinity of the BDZ receptor binding. Simultaneous modification of excitatory and inhibitory systems by kindling has been consistently found in a number of studies [7– 9,14,18,42,50,51,58]. When a new consolidated receptor density is attained by acute PTZ administration to 10-months-old kindled rats, it is accompanied by an autoenhancement of seizures (Fig. 2 B). In the first 5 min, no differences in seizure activity are observed between the control and kindled rats. Then, the mechanism of intercellular autoenhancement is switched on and the seizures are intensified. The function of autoenhancement is to accomplish autocontrol or automodification of the GABAA / BDZ / PCT-BB receptor complex activity, initially suppressed by the long-term depression mechanism (Table 1). The alteration of BDZ receptor conformation / density induced by PTZ binding to PCT-BB receptors is maintained for 48 h, when PCT-BB receptors are no longer occupied. These processes can proceed in three stages, the two last stages from it are intercellular: (1) occupation and allosteric depression of the GABAA / BDZ / PCT-BB receptors density; (2) modification of second messengers and protein kinase C-induced phosphorylation / dephosphorylation of the GABAA / BDZ / PCT-BB receptor complex, thus maintaining the depression of GABAA / BDZ / PCT-BB receptor density; (3) modification of gene expression and inhibition of GABAA / BDZ / PCTBB receptor protein synthesis. A newly established level of depression can replace the genetically determined level. Normally, seizures would be observed at stages 2 and 3 of the process. For instance, NMDA receptor activity was found to be normalized at 7–28 days after amygdala and hippocampal kindling [2]. At the same time, metabotropic excitatory amino acid receptor-stimulated polyphosphoinositide hydrolysis was enhanced for a long period of time in amygdala- and hippocampal-kindled rats [1]. We suggest that in our experiments a new consolidated state of receptors is attained independently of the current state of receptors.

Acknowledgements This work was supported by the Russian-Netherlands cooperative grant NWO-RFBR grant 005-RUS99 / 2.

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