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
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
3.1.3. Study of sensitivity Acute treatment with PTZ 50 mg kg resulted in
Kindled rats were treated as a PTZ-sensitive group, and
3
seizures scoring 4–5 points. The density B of [ H]-
control rats as a PTZ-resistant group. A lack of response
max
diazepam binding sites was significantly reduced by 15 on day 7 after the seizures induced either by kindling or by
at 30 min after the termination of seizures and by 13 at acute PTZ 50 mg kg was used as a criterion to group the
3
48 h with no change in the K Table 1. No significant rats according to their sensitivity. No differences in [ H]-
d
changes were found on day 7. diazepam binding between the sensitive and insensitive
rats were found on day 7 after the selection Table 2.
3
3.1.2. [ H]-diazepam binding after the kindling Initially, a subconvulsive dose of PTZ 20 mg kg did
3.2. Ten-months-old rats, 6 months after the termination
3
not induce seizures and [ H]-diazepam binding was un- of kindling
changed Table 1. Daily injections of this dose resulted in the development of kindling Fig. 1 and a significant
3.2.1. Seizures retrieval
3
decrease in the B of [ H]-diazepam binding by 19 at
The kindling response of high sensitivity to low doses of
max
30 min and by 16 at 48 h after the termination of PTZ was preserved through the 6-months rest period after
seizures. The K of the binding was unchanged. On day 7, the kindling treatment, but not completely and with some
d
no significant changes were observed Table 1. These attenuation. A subconvulsive dose of PTZ 20 mg kg
findings in the kindled rats are similar to the results found induced no seizures in the control rats Fig. 2A, but this
after an acute injection of the convulsive dose. 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 48 h
Day 7 Control n56
PTZ n56 Control n56
PTZ n56 Control n56
PTZ n56 Pentylenetetrazole acute administration, 50 mg kg
B 1386697
1123676 1360695
1142679 1328690
1302689
max
fmol mg protein K nM
9.0261.08 8.8561.06
9.1060.82 8.9260.80
9.1560.85 9.0960.78
d
Pentylenetetrazole acute administration, 20 mg kg B
1142697 1127694
– –
– –
max
fmol mg protein K nM
8.2960.84 8.3560.79
– –
– –
d
Kindling, 20 mg kg B
1109689 898663
1156672 971668
1130667 1160678
max
fmol mg protein K nM
8.0360.88 7.9660.80
7.8860.79 7.9460.87
8.1260.74 8.4160.92
d
P ,0.05 versus control.
t
A .S. Bazyan et al. Brain Research 888 2001 212 –220
215 Table 2
before, neither in control with acute seizures nor in kindled
3
[ H]-Diazepam binding by cerebellar cortex membranes of rats with
rats.
different sensibility to pentylenetetrazole day 7 after test
3
Animals B
fmol mg protein K nM
max d
3.2.2. [ H]-diazepam binding at seizures retrieval
3
Insensitive n56 923684
7.7860.81
[ H]-diazepam binding was unaltered in the ten months
Sensitive n56 958637
7.6560.35
old control rats, but an acute PTZ challenge 30 mg kg resulted in seizures scores 2–3 points that were accom-
3
points. At a higher dose of PTZ 30 mg kg seizures were panied by a decrease in both indices of [ H]-diazepam
observed in 56 of the control rats maximal scores of binding: the B
to 66, and the K to 73 Table 3.
max d
2–3 points and in 100 of the kindled rats maximal In the kindled control rats with a history of seizures
3
scores of 3–4 points. These observations suggest that the 4–5 points 6 months before, the B
of [ H]-diazepam
max
high sensitivity of rats to PTZ, induced by kindling, was binding was reduced to 54 without a PTZ challenge.
3
attenuated after 6 months, but at the same time the kindled After a rechallenge with PTZ, the B
of [ H]-diazepam
max
rats were more sensitive than the control rats. binding was found to be enhanced to 78, still being
The pattern of seizures in the control and kindled rats significantly lower than in the control rats, with no change
challenged with 30 mg kg of PTZ was different Fig. 2B. in the K
Table 3. This paradoxical finding can be
d
In the control rats, the first peak of seizures occurred at 5 logically explained as shown in Fig. 3. At the time of
min and the second peak was observed at 20 min after the termination of kindling the BDZ receptor density is
PTZ challenge. About 40 of the kindled rats displayed a reduced to 81 versus the control 4-months-old rats. After
period of low-level seizures scoring 1–3 points, which a rest period of 6 months, there was a decrease in BDZ
lasted for 10 min. The first peak of seizures with maximal receptor density to 54 in the kindled rats without a PTZ
scores from 3 to 4 points was observed at 20 min after the challenge. Acute PTZ administration to the kindled rats
PTZ challenge, and the second peak occurred at 35 min. induced seizures and partially restored the BDZ receptor
About 60 of the kindled rats displayed only the low-level density, just to the level of BDZ density found in the
seizures scoring from 1 to 3 points, which lasted for 45 kindled 4-months-old rats 78, which was established 6
min. This type of latency was not observed 6 months months before. At the same tame, the K of BDZ receptor
d
Fig. 2. Pattern of seizures in 10-months-old control and kindled rats. A. Seizures induced by 20 n 525; n
525 and 30 n 525; n
525 mg kg
cont kind
cont kind
x
pentylenetetrazole, averaged by maximal scores. P ,0.05 versus own control, P ,0.05 versus own control and kindled rats at 20 mg kg PTZ. B. Time
F F
course of pentylenetetrazole-induced seizures at 30 mg kg n 514; n
525. P ,0.05.
cont kind
F
Table 3
3
Effects of acute pentylenetetrazole 30 mg kg administration on [ H]-diazepam binding by cerebellar cortex membranes of 10 months old control and
a
kindled rats Control
Control1PTZ Kindled control
Kindled1PTZ n56
acute seizures, n56 n56
rechallenge, n56
?
B 967679
6416106 518681
752680
max
fmol mg protein K nM
10.361.02 7.5660.99
9.5460.78 10.761.21
d a
?
P ,0.05 versus control; P ,0.01 versus control; P ,0.05 versus kindled control.
t t
t
216 A
Fig. 3. Depression of benzodiazepine receptor density B in 4- and 10-months-old kindled rats. The results are expressed as versus control rats.
max 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: P ,0.05 versus control; P ,0.01 versus control; P ,0.05 versus kindled control.
t t
t
binding was unchanged in the kindled rats, whereas in the role is played by intracellular reactions that follow the
control 10-months-old rats that had seizures after an acute receptor occupation. It is known that the activity of BDZ
PTZ challenge the K was significantly altered Table 3. receptors is dependent on protein kinase C [27,44]. Then, a
d
feedback mechanism is suggested: phosphorylation or rephosphorylation of BDZ receptors by a BDZ receptor-
4. Discussion specific protein kinase C. This reaction can be termed
