Materials and methods control and kindled group.

A .S. Bazyan et al. Brain Research 888 2001 212 –220 213 mechanisms in kindling seizures retrieval after very long 2.2.1. Three to four-months-old rats times of acute administration of subconvulsive doses of The first series comprised the rats, in which a convulsive convulsants. dose of PTZ 50 mg kg resulted in seizure scores of 4–5 3 Previously, we have found [29] that changes in [ H]- points. They were decapitated 60 min 30 min after the diazepam binding are maximal in the cerebellar cortex, termination of seizures or 48 h later and on day 7 after the rather than in the neocortex, of the kindled rats. It has been injection n536, including control rats. shown that 81 of the total BDZ receptors in cerebellar The second series comprised the rats, in which a cortex contain high-affinity sites for PTZ [6]. In the subconvulsive dose of PTZ 20 mg kg elicited kindled forebrain, only 34 of the total BDZ receptor population seizures scoring 4–5 points, followed by decapitation 60 bear high-affinity sites for PTZ. At the same time, the min 30 min after the termination of seizures or 48 h later cerebellar cortex actualizes movement control. The cere- and on day 7 after the last injection n548, including bellar cortex exerts its influence by inhibiting and dis- control rats. inhibiting motor control action, in the premotor networks The third series comprised the rats endogenously sensi- of the brainstem, sensomotor cortex, and spinal cord [19– tive and insensitive to the convulsant. One group of 22,24]. On the other hand, the cerebellum and its associ- animals n525 was treated with 25 mg kg PTZ and the ated brainstem circuitry does appear to be essential neces- second group with 30 mg kg n525. Then, the rats were sary and sufficient for learning and memory of the selected according to their sensitivity: the first subgroup conditioned response [32,33,62–64,68]. This indicates that n56 with seizures 2 points provoked by 25 mg kg long-term reactions are inherent to the neurons of cere- PTZ; and the second subgroup n56 with no seizures bellum; for instance, in long-term depression [23]. provoked by 30 mg kg PTZ. Both groups were decapitated The goal of the present investigation was to study on day 7 after the injection. 3 long-term characteristics of [ H]-diazepam binding in cerebellar cortex after an acute injection of PTZ in 2.2.2. After 6 months about 10-months-old rats convulsive doses, after kindling development; peculiarities For the period of 6 months both the control and kindled 3 of the retrieval of kindled seizures and of [ H]-diazepam rats were kept in the breeding facility. Their weights at the binding 6 months after the termination of kindling, follow- second stage of experiments reached 360–450 g. ing a challenge with a subconvulsive dose of PTZ. 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

2. Materials and methods control and kindled group.

3 For the study of [ H]-diazepam binding four groups of Male Wistar rats 150–200 g, about 3 months old were animals were used n56 in each group: 1 control rats, used, a total of 266 animals. Five series of experiments no PTZ challenge; 2 acute seizures control rats, 30 min were conducted in two stages. Pentylenetetrazole Sigma, after the termination of acute seizures 2–3 points induced USA or saline control rats were injected intraperitoneal- by a PTZ 30 mg kg challenge; 3 kindled control rats, ly. The seizures were scored by the following scale: 1 a no PTZ challenge, but with a history of seizures 4–5 twitch of the head; 2 occasional clonic seizures; 3 a points 6 months ago; 4 kindling1PTZ challenge, 30 min series of clonic seizures, or clonus of the forepaws; 4 after the termination of seizures 3–4 points induced by a clonic–tonic seizures while standing on the hind paws PTZ 30 mg kg challenge. kangaroo posture; 5 clonic–tonic seizures and falling 3 on one side. 2.3. [ H]-diazepam binding 2.1. Kindling procedure Cerebellar cortex was removed and used to isolate a crude fraction of synaptic membranes. The cerebellar A subconvulsive dose of PTZ 20 mg kg was injected cortex was homogenized and centrifuged for 10 min at in rats n5100 once daily for 24 days. The daily 10003g in the medium: 0.32 M sucrose, 1 mM EDTA and injections gradually increased the sensitivity to the convul- 5 mM Tris–HCl, pH 7.4 at 48C. The supernatant was sant. The dose of PTZ 20 mg kg, which was subconvul- centrifuged for 20 min at 20,0003g. The pellet P was 2 sive in itself, evoked seizures that intensified and finalized frozen at 2408C. After washing by centrifugation, the in a clonic–tonic paroxysm. pellet was resuspended in 4 ml of 50 mM Tris–HCl buffer, pH 7.4. A portion of the suspension of synaptic mem- 2.2. Preselection of animals branes 400 ml was added to 50 ml of an aqueous 3 [ H]-diazepam solution 68 Ci mmol, Amersham, UK The rats were assigned to the different experimental with final concentrations of 0.55; 1.1; 2.19; 4.38; 8.75; groups specified below at the conclusion of kindling, when 17.5; 35 nM. Non-specific binding was assessed in parallel they were about 3–4 months old. samples containing excess unlabeled diazepam. All sam- 214 A ples were run in duplicate. The samples were incubated for 40 min at 48C and filtered through a GF B filter What- man, UK, followed by four successive washes with ice- cold 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 B and the max dissociation constant K were calculated from the Scat- d chard 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