Unidirectional interaction between flurothyl seizures and amygdala kindling

BrainResearch, 344(1985) 103-108 Elsevier

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BRE 11027

Unidirectional Interaction between Flurothyl Seizures and Amygdala Kindling REIKO OKADA l, SOLOMON L. MOSHI~ 1.2, KENJI ONO 1and BRUCE J. ALBALA I Departments of l Neurology and 2Pediatrics, Albert Einstein College of Medicine, Bronx, NY 10461 ( U. S. A. ) (Accepted December 27th, 1984) KW words: seizure - - epilepsy - - convulsion - - kindling - - flurothyl - - transfer - - rat

In this report, the interaction between flurothyl convulsions and electrical kindling of the amygdala was investigated. Three flurothyl convulsions decreased the afterdischarge threshold of the amygdala and enhanced the rate of development of electrical kindling without affecting the intensity of postictal refractoriness. On the other hand, 3 generalized kindled convulsions did not alter the flurothyl convulsive threshold. The data suggest that the influence of generalized convulsions on future seizure susceptibility may depend on the agents used to induce the convulsions.

INTRODUCTION R e p e a t e d very low intensity electrical stimulations of various sites of the brain, especially of the limbic system, progressively induce m o r e severe seizures culminating in a generalized convulsion (electrical kindling) s. The r e p e a t e d administration of a variety of convulsants such as cocaine 16,23, lidocaine a7, carbacho126 and p e n t y l e n e t e t r a z o l ( P T Z ) 5,v,1° in subconvulsive doses also lead to the gradual d e v e l o p m e n t of generalized convulsions (pharmacological kindling). The induction of either type of kindling is not limited to the original kindling agent2,3, 4, but results in an increased seizure susceptibility to other kindling agents. This transfer effect may also imply that animals subjected to kindled convulsions, b e c o m e m o r e prone to develop o t h e r types of seizures 15. It is unclear if this change in neuronal communication induced by kindling is the result of: (1) the stimulating agents; (2) the kindling process itself; or (3) the occurrence of generalized convulsions per se. It has been r e p o r t e d that cocaine sensitization does not result in the facilitation of electrical a m y g d a l a kindling 9-20. On the o t h e r hand, r e p e a t e d systemic subconvulsive injections of P T Z facilitate the development of subsequent a m y g d a l a electrical kindling,

irrespective of w h e t h e r or not P T Z - i n d u c e d convulsions occurred 3. H o w e v e r , the P T Z - t r e a t e d rats that experienced generalized seizures have the shortest kindling rates although the difference is not statistically significant. F u r t h e r m o r e , r e p e a t e d convulsions induced by electroconvulsive shock b e c o m e more severe with time 21 or reduce the flurothyl convulsive threshold is. Therefore, the possibility exists that generalized seizures may indeed intensify the epileptic potential of the brain. In this report, we tested this hypothesis by exposing rats to a single or to a short series of generalized convulsions induced by flurothyl (FE) and subsequently d e t e r m i n e d the rate of d e v e l o p m e n t of a m y g d a l a kindling. In addition, we also investigated w h e t h e r a m y g d a l a - k i n d l e d seizures (which have a distinct focus of origin) can alter the threshold of F E - i n d u c e d seizures that p r e s u m a b l y are the result of diffuse neuronal activation. MATERIALS AND METHODS A d u l t S p r a g u e - D a w l e y male rats (275-300 g, M a r l a n d F a r m ) were used. Flurothyl (FE) induced convulsions Flurothyl (bis-2,2,2-trifluroethyl ether, Indoclon,

Correspondence: S. L. Mosh6, Albert Einstein College of Medicine, Department of Neurology, F, G-9, 1300 Morris Park Avenue, Bronx, NY 14061. U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

104 Anaquest, N J) is a volatile convulsant agent. Its administration has been established as an effective technique to measure the seizure threshold of the brain 2& The rats were individually placed in a sealed chamber. Liquid FE was delivered into the chamber through a tube situated above a mesh screen. A piece of filter paper was placed on the screen from which FE vaporized. FE was continuously infused by a Harvard Apparatus pump at the constant rate of 20 ifl/min. The FE convulsive threshold was defined as the latency to the onset of a clonic-tonic generalized convulsion, often followed by loss of posture.

Electrical kindling The rats were implanted stereotaxically with bipolar electrodes (Plastic Products 303/2, Roanoke, VA) in the right amygdala under anesthesia (ketamine 70 mg/kg and Xylazine 10 mg/kg, i.m.) using the following coordinates (from bregma, posterior 1.8, lateral 3.7; depth from skull, 9.1 mm). After a 5-day recovery period, the rats were subjected to the kindling procedure. Each rat was stimulated initially with 50/aA, peak to peak, 60 Hz sinusoidal current for 1 s delivered from an isolated constant current stimulator. The intensity of the current was increased by 50/aA steps every hour until an afterdischarge (AD) occurred. At this point, the current was reduced once by 25/aA. The AD threshold was defined as the lowest current intensity which produced an AD. Subsequently, all the rats were kindled with 400/aA, peak to peak, 60 Hz, stimulations, 1 s in duration, delivered 3-6 times a day. The interstimulation period was at least 1 h. The observed behavioral seizures were classified into 5 stages as described by Racine 19. Stage 1, chewing; stage 2, head nodding; stage 3, unilateral forelimb clonus; stage 4, bilateral symmetrical forelimb clonus with rearing; stage 5, the above and falling. The kindling rate was defined as the number of AD producing stimulations required for the development of the first stage 5 generalized convulsion. After the completion of kindling, the intensity of postictal refractoriness 6,13 was measured in randomly-selected rats (depending on the experimental design) to determine the ability of the rats to suppress recurrent seizures. Eight repetitive stimulations

(400 ~tA, peak to peak, 60 Hz, for 1 s), were delivered at 2 min intervals. The depth EEG was recorded throughout the trial and the behavioral responses were observed in order to determine the seizure and AD indices. Each index was obtained by adding the value of the behavioral seizure stage (1-5) or the duration of the ADs produced by each stimulation and dividing by the number of stimulations (8). A high seizure or AD index or both indicate decreased postictal refractoriness and therefore, the rats are more prone to develop recurrent seizures6,12,13.

Histology and statistics Following the completion of the experiments, the rats were sacrificed. Using standard techniques. 20/am sections were obtained through the neocortex. amygdala, hippocampus, basal ganglia and midbrain in order to verify the electrode placement and to evaluate for any long-term neuronal change. The data were analyzed statistically using the t-test and the one way analysis of variance (F test) with post hoc comparisons, as deemed appropriate.

Experimental protocol Experiment 1. The effect of FE convulsions on kindling The rats were randomly divided to 3 groups. Ten rats were subjects to one FE generalized convulsion. Thirteen rats were subjected to 3 FE generalized convulsions induced at 8-day intervals. Sixteen additional rats, not subjected to any FE convulsions, served as controls. Eight days after the last FE convulsion, the experimental and control rats were kindled.

Experiment 2. The effect of kindled seizures on FE convulsions Eleven rats were kindled and assigned to either a single generalized convulsion group (n = 5) or 3 generalized convulsions group (n = 6). For this experiment, a stage 4 or 5 kindled seizure was considered as a generalized convulsion. After a 6 - 8 day rest period from the last kindled convulsion, the FE convulsive thresholds were determined and compared to the FE threshold obtained from the first FE seizure in Experiment 1 (n = 23).

105 TABLE I

Flurothyl convulsive thresholds prior to kindling Values are the means + S.E.M.

FE convulsive threshold (s)

Single con vulsion (n=lO)

3 convulsions (n = 12) 1st

2nd

3rd

478 ± 15.8"-**

532_+27.8* F(2.36) = 1.95, P > 0.05

531 +__23.5

4 7 0 ± _ ~4 . ,~'**

* Comparison between the single convulsion group and the first seizure of the 3 convulsions group, t-test = 1.48, P > (I. 1. ** Comparison between the single convulsion group and the last seizure in the 3 convulsions group, t-test - (I.23, P > 0.8.

RESU LTS

Experiment i The FE convulsive thresholds for the single and 3 convulsions groups are depicted in Table I. The mean latency in the single convulsion group was 478 + 15.8 (S.E.) s and did not differ statistically from the mean latency to the onset of the first generalized convulsion (5~ -= 532 _+ 27.8 s) in the 3 convulsions group (t-test = 1.48, P > 0.1). Three repeated exposures to FE diminished the convulsive threshold of the third elicited convulsion (X = 470 + 24.3 s) but again the overall difference in the convulsive threshold during the 3 FE exposures was not statistically significant (&.3~ = 1.95, P > 0.05). Furthermore the intensity of the seizures did not appear to change. Thus, 3 FE exposures did not induce a 'kindling-like' effect. Moreover, the convulsive threshold of the third exposure was almost identical to the FE convulsive threshold of the single group (t-test - 0.23, P > 0.8). The effects of FE-induced convulsions on the development of amygdala kindling are shown in Table II. Three previous FE seizures significantly decreased the A D threshold of the amygdala of the 3

convulsions group when compared to both the single convulsion group and/or the control (/~'e,> = 3.49, P < 0.05). However, the durations of the A D s at threshold did not statistically differ among the 3 groups (&.3{, = 2.59, P > 0.05). Along with the decrease in the local threshold, 3 exposures to FE seizures significantly decreased the kindling rate when compared to the kindling rate of the single convulsion and control (F3~e, = 20.2, P < 0.00l) (Fig. 1). Furthermore, the duration of the first generalized seizure was also significantly longer in the 3 convulsions group (/:'>> = 6.34, P < (l.01) when compared to the two other groups. The latter did not differ from each other in any aspect of kindling. The intensity of postictal refractoriness following a kindled convulsion was not affected by previous FEinduced seizures (Table I!I). Neither a single nor 3 FE convulsions significantly altered the seizure or the A D index and thus did not differ from the controls.

Experiment 2 This experiment was designed to investigate the effect of secondarily generalized amygdala kindled sei-

TABLE I1

Susceptibility to kindling jbllowing flurothyl-induced convulsions Values are the means -+ S,E.M. A D , afterdischarge: the term 'kindling rate' is defined as the number of AD :~roducing stimulations required for the development of the first stage 5 generalized convulsion.

(a) Three convulsions ( n = 13) (b) Single convulsion (n= 10) (c) Control ( n = 16)

A D threshold (ILA)

A D duration (s) at threshold

Kindling rate

A D duration (s) at fir.st stage 5

107_+11 *b.~" 175_+21 159_+21 F~ 3~=3.49, P < 0.05

35.7__+6.0 16.2_+2.4 28.8_+6.2 Fc,3~=2.59, P > 0.05

5.9±0.6"**".~ 11.4±0.6 12.6±0.9 FI2,36)=20.2. P < 0.001

96.2±6.9"h,**, 72+8.2 62.1+6.7 /:c 3~,~=6"34, P < (!.()1

Leners depict the individual group differences within the column: * P < (I.05, **P < 0.01, *** P < 0.001.

106

//

any evidence of brain damage other than that associated with the placement of the electrodes.

//

DISCUSSION

g

3t-

\

Z

A Singleconvulsion .

// I 2

I 4

I 6

I 8

Number

Of ADs

I 10

I 12

J 14

Fig. 1. The effect of FE seizures on amygdala kindling. Three FE convulsions markedly facilitate the development of kindling

(P< 0.001).

zures on the F E convulsive thresholds. The mean number of A D s required for the development of a generalized kindled convulsion was = 8.2 _+ 0.8 (S.E.) for the single convulsion group and 10.5 _+ 3.1 for the 3 convulsions group and thus essentially equivalent (t-test = 0.6, P > 0.05):. The 3 convulsions group received 3.1 additional (range 2 - 6 ) electrical stimulations so that each rat had a total of 3 generalized kindled convulsions. The FE convulsive threshold was 486 + 43.5 s for the single convulsion group, 580 + 48.7 s for the 3 convulsions group and 508 + 17.7 s for the control group. The difference was not statistically significant (F2.31 = 1.75, P > 0.05). In addition there was no difference in the intensity of the FE seizures among the 3 groups.

Histology Examination of the brain sections did not reveal T A B L E Ill

Postictal refractoriness Values are the means + S.E.M. The seizure and A D indices are

defined in the text as measurements of the ability of the rats to resist recurrent seizures (postictal refractoriness).

(a) Three convulsions (n=5) (b) Single convulsion (n=5) (c) Control (n=7)

A D index

Seizure index

20+3.2 12+2.6 17_+ 1.7 Fi2.14)=2.37, P > 0.05

1.1 +0.2 1.2+__0.2 113+0.2 F~2,~4)=0.18, P > 0.05

The present study shows that 3 FE seizures increase the local epileptogenicity of the amygdata, facilitate the rate of generalization of amygdalakindled seizures and enhance the duration of a generalized kindled convulsion. A single FE seizure does not induce a similar sequence of events Since repeated (daily) FE exposures can gradually reduce the FE convulsive thresholdl,ls, there is a possibility that the 3 FE seizures produced pharmacological kindling. However, neither the FE convulsive threshold nor the intensity of the FE seizures of the 3 convulsions group were significantly altered as a result of 3 FE exposures occurring in weekly intervals. A similar lack of FE kindling was reported by Adler et al... who did not find any changes in the FE thresholds in rats subjected to 3 convulsions, each separated by 3 weeks. Therefore. our results suggest that repeated generalized FE setzures per se can intensify the epileptic potential of brain in the absence of FE kindling The intensity of postictal refractoriness in the kindled rats previously subjected to FE convulsions does not differ from that of the control group. Fhis indicates that the ability to suppress multiple seizures is not affected by a previous history of convulsions I see also Okada et al.141. Indeed. the development of postictal refractoriness appears to be independent of the enhancement of neuronal excitability associated with kindling and may be an age-related process since its degree is minimal early in life and enhances with maturanon, irrespective of previous convulsions j2. The absence of a facilitating effect of the generalized kindled seizures on the FE convulsive threshold is puzzling and the reason for this unidirectional interaction is not clear. The exact mode and focus of the FE-induced activation is not known, but it has been suggested that FE seizures are the result of diffuse opening up of sodium channels of the neuronal membrane 27. Since our results show that a short series of FE convulsions alter the seizure susceptibility of the limbic system (amygdala), it can be postulated that FE can at least activate limbic sites. On the other hand, if FE seizures were only the result of timbic ac-

107 tivation, electrical kindling, w h i c h p r o d u c e s m u l t i p l e

duces the P T Z c o n v u l s i v e t h r e s h o l d . T h e s e data, in

transfer effects of e n h a n c e d e p i l e p t o g e n i c i t y within

c o m b i n a t i o n with o u r results, suggest that the m e c h a -

the limbic system 8-11,22, s h o u l d h a v e d e c r e a s e d t h e

nism of the c o n v u l s i v e action of P T Z and F E is quite

F E t h r e s h o l d . O u r data did not d e m o n s t r a t e this fa-

d i f f e r e n t and the influence of g e n e r a l i z e d c o n v u l -

cilitation. This suggests that F E seizures m a y re-

sions on future seizure susceptibility m a y d e p e n d on

q u i r e , in a d d i t i o n , the i n v o l v e m e n t of o t h e r site such

the agents used to i n d u c e the convulsions. This hy-

as n e o c o r t e x . I n d e e d , to date, a t r a n s f e r effect f r o m

p o t h e s i s is s u p p o r t e d by the o b s e r v a t i o n s s h o w i n g

a m y g d a l a to n e o c o r t e x has not b e e n r e p o r t e d .

that daily r e p e a t e d F E seizures in rats d e c r e a s e their

P r e v i o u s studies indicate that the p h a r m a c o l o g i c a l effects of a n t i c o n v u l s a n t s on F E seizures r e s e m b l e

t h r e s h o l d to P T Z seizures but increase their resistance to e l e c t r o s h o c k c o n v u l s i o n s I.

their effects on P T Z seizures 25 and it has b e e n sugg e s t e d that these two c o n v u l s i v e agents p r o d u c e convulsions in a similar way~. Y e t , the i n t e r a c t i o n of

ACKNOWLEDGEMENTS

electrical limbic kindling and P T Z seizures is o p p o site to that of electrical kindling and F E seizures. Stri-

This i n v e s t i g a t i o n

was

supported

by N I N C D S

pling and H e n d r i c k s 24 r e p o r t e d that the d e v e l o p m e n t

G r a n t N S 20253. Dr. S. L. M o s h 6 is the r e c i p i e n t of a

of kindling is not facilitated by e i t h e r a single or 3

T e a c h e r I n v e s t i g a t o r D e v e l o p m e n t A w a r d NS 00042 f r o m the N I N C D S .

P T Z seizures and Pinel et al. 15 f o u n d that k i n d l i n g re-

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