Modification of amygdaloid kindling by diazepam in juvenile rats

Developmental Brain Research, 51 (1990) 249-252

249

Elsevier BRESD 51013

Modification of amygdaloid kindling by diazepam in juvenile rats Timothy E. Albertson, Larry G. Stark and Robert W. Derlet Departments of Internal Medicine and Pharmacology, School of Medicine, University of California, Davis, CA 95616 (U. S. A.) (Accepted 8 August 1989)

Key words: Amygdaloid kindling; Diazepam; Kindled seizure; Immature rat

The amygdaloid kindling phenomenon has been widely used to evaluate and screen potential anticonvulsant compounds in adult rats. In the current study, weanling rats (ages 23-25 days) were implanted chronically with amygdaloid electrodes. They were treated with dimethylsulfoxide (DMSO), 0.5 mg/kg diazepam or 1.0 mg/kg diazepam before twice daily kindling stimulations to determine the effect of diazepam on the acquisition of the kindled seizure. Additional juvenile rats implanted as weanlings and simulated twice daily without drug pretreatment until fully kindled were used to test for the acute anticonvulsant effects of diazepam (0.25-4.0 mg/kg). Diazepam was demonstrated to have anticonvulsant properties in juvenile rats by both prolonging the time to develop the fully kindled response during acquisition and by reducing the elicited seizure severity and the length of the afterdischarge in the fully kindled juvenile rats. Together, these data point to the extension of the anticonvulsant profile of diazepam to now include juvenile amygdaloid kindling in rats. They further point to the potential ability of screening proposed anticonvulsant drugs for their efficacy against amygdaloid kindling in immature rats.

INTRODUCTION T h e a m y g d a l o i d k i n d l i n g m o d e l of e p i l e p s y has b e e n w i d e l y utilized to s c r e e n and e v a l u a t e n e w a n t i c o n v u l s a n t c o m p o u n d s 1"2'9'13'14'17'18. M o s t of t h e s e studies h a v e d e m o n s t r a t e d the effects of v a r i o u s c o m p o u n d s in e i t h e r the p r o g r e s s i v e a c q u i s i t i o n o f the a m y g d a l o i d k i n d l e d r e s p o n s e o r against t h e fully a m y g d a l o i d k i n d l e d r e s p o n s e in adult rats 9. T h e i m m a t u r e o r d e v e l o p i n g rat brain has b e e n s h o w n to be a u n i q u e m o d e l in which to e v a l u a t e the n e u r o l o g i c a l effects o f v a r i o u s c o m p o u n d s 3'8'1°A2"15"22. S e v e r a l studies h a v e d e m o n s t r a t e d differential anticonv u l s a n t activity of c o m p o u n d s w h e n t e s t e d against adult o r i m m a t u r e rats using e l e c t r o s h o c k seizure t h r e s h o l d and p e n t y l e n e t e t r a z o l m o d e l s 1°'11. F e w studies h a v e e x a m i n e d t h e u s e f u l n e s s o f the a m y g d a l o i d kindling m o d e l of e p i l e p s y to study this a g e - d e p e n d e n t differential anticonv u l s a n t p h e n o m e n o n o r e v e n the p o t e n t i a l of this m o d e l to e x a m i n e a n t i c o n v u l s a n t efficacy in the i m m a t u r e brain. T h e p r e s e n t study e x a m i n e d t h e effects of d i a z e p a m on a m y g d a l o i d k i n d l i n g in i m m a t u r e rats. D i a z e p a m is an a n t i c o n v u l s a n t with k n o w n efficacy m o d i f y i n g fully a m y g d a l o i d k i n d l e d seizures and slowing the acquisition of the k i n d l e d a m y g d a l o i d r e s p o n s e in adult rats 1'2'7'13'14"16-18. This c o m p o u n d is o f p a r t i c u l a r i n t e r e s t since it, a l o n g w i t h o t h e r b e n z o d i a z e p i n e agonist c o m p o u n d s , has b e e n r e p o r t e d to result in a s e i z u r e - l i k e b e h a v i o r a l r e s p o n s e in i m m a t u r e rats 3'1°.

MATERIALS AND METHODS Male Sprague-Dawley weanling rats 21-23 days of age, weighing 40-70 g were the subjects. All subjects were anesthetized with Chloropent ® (3.6 ml/kg) containing per ml 42.5 mg chloral hydrate, 21.1 mg magnesium sulfate, 14.3% ethanol and 8.9 mg pentobarbital. Bipolar electrodes were then stereotaxically implanted into the right amygdala at 1.0 mm posterior to bregma, 4.2 mm lateral to midline and 7.2 mm ventral from the surface of the brain. The level of the incisor bar was set to zero. The amygdala electrode was manufactured from 32-gauge Teflon-coated stainless steel wires which were tightly twisted together and insulated except at the cut tips. Each of the paired wires were crimped to a female Amphenol ® pin. Double tightly twisted 32-gauge Teflon-coated stainless steel wires were used to anchor the electrode assembly to the skull and also to serve as the ground electrode as previously described 4-6'2°. The 3 female-pins (two amygdala and one ground electrodes) were covered by short pieces of Silastic tubing (1.02 mm i.d.). The tubing protruded 1-2 mm above the pins to prevent dental acrylic cement from covering them and to help guide the male pins used to connect to the stimulator/electroencephalogram (EEG). The pin and wire assembly and exposed skull were then completely covered with dental acrylic cement. After recovery from anesthesia, the rats were returned to their mothers. All rats were then permanently weaned and housed separately 24 h later. All subjects were stimulated twice daily in a plexiglass box, 30x30x45 cm in size starting on the 25th day of life 48 h or more after surgery. Daily stimulations occurred at least 4 h apart. The cortical and amygdaloid EEG was recorded on a Grass Model 78D polygraph before stimulation. Electrical stimulation was produced by a Grass S-11 stimulator and delivered to the amygdala through an isolated constant-current output. The stimulus consisted of a 1-s train of 60-Hz biphasic square waves, each 1 ms in duration and 400 ~uA in amplitude. At the termination of the stimulus train, the amygdala and the cortical EEG electrodes were electronically switched to the polygraph. Two measures of seizure severity were employed. The first was

Correspondence: T.E. Albertson, Department of Pharmacology, School of Medicine, University of California, Davis, CA 95616, U.S.A. 0165-3806/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

25(I the duration of the afterdischarge (AD) elicited by the stimulus. The AD was defined to be the period during which two or more spikes of at least twice the maximal prestimulus EEG amplitude occurred in the amygdala at a frequency of 1/s or faster. If additional AD occurred within 1 min of the termination of the primary AD, it was included when determining total AD duration. The second measure employed was an assessment of behavioral seizure severity. A modification of the established criteria for kindled seizures in neonate rats was used to stage or rank the seizures 6"2c'. A score of 0 was assigned for no clinical manifestations; 1 indicated facial movement and chewing; 2 indicated head turning or nodding; 3 indicated unilateral clonus or alternating independent forelimb clonus; 4 indicated bilateral forelimb clonus; and 5 indicated rearing with bilateral forelimb clonus or loss of balance and lateral falling.

Experiment 1 Weanling rats were stimulated twice daily 240 min apart for 12 days until stable stage 5 kindled seizures predominated. Prior to the next day of stimulation, they were pretreated with either 0.5 ml/kg i.p. dimethylsulfoxide (DMSO), or 0.25, 0.5, 1.0, 2.0 or 4.0 mg/kg i.p. diazepam (dissolved to give a dose of 0.5 ml/kg DMSO). Thirty min later, all animals were stimulated and the elicited AD duration was recorded and seizure ranks were assigned. Twenty-four h later, all animals were stimulated without pretreatment to document that the elicited seizure response had returned to baseline values. Forty-eight h later animals were again pretreated with another dose of drug and stimulated 30 min later.

TABLE II

Effects of diazepam on the acquisition of amygdaloid-kindted seizures n, number; Data are means ± S.E.M,. ; DZP, diazepam.

Dose

pt

Stimulations to first rank '5' seizures

Control 0.5 mg/kg DZP 1.0 mg/kg DZP

16 9 5

15.9 ± 1.2 19.8 _+ t.0" 22.0 ± 0.5**

* = P ~<0.05 compared to control; ** = P ~<0.01 compared to control.

RESULTS

Experiment 1 Diazepam

induced

reductions

o f fully

amygdaloid

k i n d l e d s e i z u r e s in j u v e n i l e r a t s as d e m o n s t r a t e d in T a b l e I. S i g n i f i c a n t r e d u c t i o n s in b o t h s e i z u r e r a n k s a n d A D duration occurred with diazepam doses of 0.5-4.0 mg/kg. Obvious

prestimulation

behavioral

toxicity

(sedation)

w a s n o t e d w i t h t h e h i g h e s t (4 m g / k g ) d i a z e p a m

Experiment 2 Additional implanted weanling rats received 0.5 ml/kg i.p. DMSO, 0.5 mg/kg i.p. diazepam or 1.0 mg/kg i.p. diazepam once a day 30 min before the first daily kindling acquisition stimulation. These animals then received a second daily stimulation 270 min after the injection or 240 min after the first stimulation. Drug dosing occurred only during the first 5 days after which animals were stimulated twice daily (240 min apart) for an additional 5 days without pretreatment. The number of stimulations to reach the first stage or rank 5 seizure was recorded along with average AD length and seizure rank for each stimulation. After completion of the experiments, all subjects were sacrificed and the location of the amygdaloid electrode was determined. The rates of kindling acquisition for the treatment groups were compared statistically by one-way ANOVA with Dunnett's test used for comparisons of individual means. The grouped Student's t-test was employed to compare AD durations and the Mann-Whitney U-test was used to compare the non-parametric rank scores. Determination of the 50% effective dose (EDso) for control of AD and suppression of generalized seizures was done using a computerized Litchfield and Wilcoxon method 21.

to b e 0.5 m g / k g o f d i a z e p a m w i t h 0 , 2 - 1 . 4 m g / k g d o s e s

80

/ • DMS0 N,16 70 I- Z~ 0.5 mg/kg DZP N=9

so l- " p
T TT

~ 30 2o

i1;,__ . . . . . . .

,,,,,,,,,,,

i

• o

TABLE I

dose

tested. The EDs0 for suppression of AD was determined

Effects of diazepam on fully amygdaloid-kindled juvenile rats % of control trial AD for same animals. Data are means + S.E.M. n, number; AD, Afterdischarge duration; R, seizure rank; DZP, Diazepam. z 6/6 animals sedated with righting reflex intact prior to stimulation.

Dose

n

A D (s)

AD% 1

Control 0.25 mg/kg DZP 0.50 mg/kg DZP 1.0 mg/kg DZP 2.0 mg/kg D Z P 4.00mg/kg DZP z

31 4 7 7 7 6

61.2 + 4.8 34.8+7.2 27.4 + 9.2* 6.6 + 2.5** 19.5 + 4.7** 7.3 + 1.3"*

72.8+ 45.9 + 17.6 + 33.9 + 12.7 +

*P <~0.05 compared to paired control trial; * *P <~0.01 compared to paired control trial.

-

13.2 15.6" 5.9"* 8.5** 2.4**

R 4.4 4.0 1.6" 0.4* 1.6* 0.0"*

: O0

i~='~l

2

I

4

i

'

6

'

i

8

'

'I'

'

'

I

'

~

'

I

i

I

10 12 14 16 16 20

Stimulation Fig. 1. The effect of diazepam (DZP) pretreatment on kindling acquisition. Mean afterdischarge duration (+ S.E.M.) and seizure ranks are presented for daily 0.5 mg/kg i.p. diazepam or DMSO control pretreatments. The dotted line represents when daily drug pretreatment was terminated. Weanling rats were stimulated twice daily 30 min and 270 min after single daily drug dosing for the first 5 days and 240 min apart for the last 5 days.

251 representing the 95% confidence intervals. For suppression of elicited generalized seizures, the EDs0 was determined to be 0.3 mg/kg of diazepam with 0.1-0.8 mg/kg doses representing the 95% confidence intervals. No spontaneous E E G seizure activity was noted prior to electrical stimulation. Experiment 2

The effect of two doses of diazepam pretreatment on kindling acquisition in weanling to juvenile rats is presented in Table II. Five days of diazepam pretreatment prolonged the number of stimulations needed to reach the first stage 5 seizure compared to control animals. Fig. 1 demonstrates the daily average A D duration and seizure ranks for animals pretreated with D M S O and 0.5 mg/kg diazepam. No spontaneous seizure activity was seen after daily diazepam dosing. DISCUSSION The current study has demonstrated that the benzodiazepine receptor agonist diazepam has anticonvuisant properties that include slowing the acquisition of amygdaloid kindling and reducing the severity and length of fully amygdaloid kindled seizures in weanling and juvenile rats. The effect on kindling acquisition and modification of the fully amygdaloid kindled seizure in these animals is similar to or more efficacious than that seen with mature adult rats a'2"7'13"14'16-18. The EDso for diazepam in these immature fully kindled rats of 0.5 mg/kg for A D duration and 0.3 mg/kg for suppression of generalized seizure ranks compares favorably to the previously published EDs0 determinations in adult fully amygdaloid kindled rats of approximately 0.9-1.2 and 1.9 mg/kg for A D duration 1"7 and suppression of seizure severity scores~Srespect fully. Amygdaloid kindling acquisition studies in adult rats have demonstrated that pretreatment with daily diazepam doses of between 0.5 and 10 mg/kg significantly inhibited the rate of kindling ~3~14'~6'~7'23 in a manner

similar to the results of this study with weanling/juvenile rats, despite the fact that once a day drug dosing was coupled with twice daily stimulations. The twice daily stimulation paradigm was a compromise between the grouped-trial kindling approach utilized by some investigators to study the immature brain 6 and the daily stimulation approach used in most adult kindling studies and by some investigators to study the immature brain z°. There are some obvious limitations to this compromised approach. Daily dosing and single daily stimulations offer blood/brain levels of the test c o m p o u n d that are more constant at the time of stimulation than with a single injection followed by twice daily stimulations. In addition, inhibitory processes set off from the first seizure may interact with the test drug on the second elicited seizure of the day. This theoretical interaction was not seen with diazepam. These potential problems with the experimental design must be weighed against the fact that the immature rat has a soft skull and is also growing, both of which threaten the integrity of the chronic electrode. Despite this compromise of twice daily stimulation, similar inhibitory effects were seen with diazepam pretreatment and amygdaloid kindling in the immature and adult rat. Daily pretreatment with diazepam during kindling acquisition was not associated with any behavioral or E E G evidence of spontaneous seizure activity as previously described in immature rats that were somewhat younger (less than 25 days old) than those studied here 3. This finding supports several recent studies that have examined the behavioral and E E G consequences of benzodiazepine exposures in immature rats 8"~1"19. Together, these data point to the potential usefulness of the dynamic kindling model of epilepsy in addition to the pentylenetetrazol and electroshock models of epilepsy to examine as developing immature rat brain for anticonvulsant drug efficacy. Acknowledgements. We wish to note the excellent technical assistance of W. Walby on this project. This work was supported by a grant from the March of Dimes Foundation.

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