0361.9230/91

Brain Research Bulletin, Vol. 27, pp. 75-79. c Pergarnon Press plc, 1991. Printed in the U.S.A.

$3.00 + .OO

Intracerebroventricular Administration of K-Agonists Induces Convulsions in Mice M. BANSINATH,

Department

K. RAMABADRAN,’

H. TURNDORF

AND V. K. SHUKLA*

of Anesthesiology, School of Medicine, New York University Medical Center 550 First Avenue, New York, NY 10016 Received 26 November

1990

BANSINATH, M., K. RAMABADRAN, H. TURNDORF AND V. K. SHUKLA. Intracerebroventricular administration of K-agonists induces convulsions in mice. BRAIN RES BULL 27(l) 75-79, 1991. -1ntracerebroventricular (ICV) administration of K-agonists (PD 117302, U-50488H and U-69593) induced convulsions in a dose-related manner in mice. The dose at which 50% of animals convulsed (CD,,) was in nmol ranges for all opioids. Among the opioids used, PD 117302 was the most potent convulsant, ICV administration of either vehicle alone or U-53445E. a non-a-opioid (+) enantiomer of U-50488H did not induce convulsions. The convulsive response of a-agonists was differentially susceptible for antagonism by naloxone and/or MR 2266. Collectively, these findings support the view that convulsions induced by k-agonists in mice involve stereospecific opioid receptor mechanisms. Furthermore, the convulsant effect of a-agonists could not be modified by pretreatment with MK-801, ketamine, muscimol or baclofen. It is concluded that a-opioid but not NMDA or GABA receptor mechanisms are involved in convulsions induced by k-agonists. These results are the first experimental evidence implicating stereospecific k-receptor mechanisms in opioid-induced convulsions in mice.

K-Opioid agonists GABA

Muscimol

PD 117302 Baclofen

U-50488H Convulsions

MK-80 1 NMDA U-69593 U-53445E Stereospecificity Intracerebroventricular (ICV)

standing of the pathophysiological functions and opioid receptor subtypes and their therapeutic implications is highly dependent on additional data derived from using selective opioid ligands (56). Among rodents, rats have frequently been used to assess the effect of opioids on seizure threshold. As mentioned above, the route of administration is an important factor which could qualitatively modify responses to opioids (1). In rats, systemic administration of k-agonists produces anticonvulsant action (14) but evokes generalized seizures when injected ICV (4244). In the same species, intracerebroventricular administration of 6- and e-opioid agonists kindle convulsions (11). Furthermore, a doseand time-dependent anticonvulsant action has been observed following either subcutaneous (SC) or ICV administration of K-agonist, U-50488H in rats (45). Few other investigators have shown that ICV administration of U-50488H does not produce any epileptic discharges, myoclonic contractions or wet dog shakes (15,25). Apparently, the question of K-agonists on ICV administration in rats, inducing pro- or anticonvulsant action still remains open. Upregulation of S-opioid receptors in the brain of seizuresusceptible strain of mouse has been reported (29). In the Swiss Webster strain of mouse, during the course of our continuing investigations aimed to identify the sites at which K-agonists act to induce hypothermia (5,37) and inhibition of gastrointestinal transit (6, 7, 31, 34, 35, 37), we observed that ICV adminis-

THE existing evidence on the role of endogenous opioid mechanisms in the pathogenesis of seizure disorders have recently been reviewed (32). Using many models of epilepsy, a variety of opioids and endogenous opioid peptides have been investigated to define and characterize the role of endogenous opioid systems(s) in the pathophysiology and pharmacotherapy of seizure disorders. Modulation of seizure threshold by opioids and opioid peptides depends on the type of convulsions induced, the route of drug application as well as the species used. After systemic as well as intracerebroventricular (ICV) administration of opioids and opioid peptides, both proconvulsant (10, 16, 18, 43) and anticonvulsant (17, 47, 48, 50) activities have been reported. Currently, despite of many lacunae in understanding their functional significance, the existence of at least three classes of endogenous opioid peptides and receptor subtypes are well established (33). In concert with the concept of multiple opioid receptors, opioids were divided into four classes using threshold for flurothyl-induced seizure in rats (14). According to this classification, p-agonists induced a dose-related anticonvulsant effect along with behavioral depression, while the anticonvulsant effect of u-agonists was associated with behavioral stimulation. The K-agonists did not produce any clear-cut effect, while some other opioids such as meperidine and pentazocine were proconvulsants. However, subsequent reports (2,32) clearly indicate that the distinct role of each opioid receptor subtype in seizure mechanisms is as yet far from clear. Apparently, a full under-

._ ‘Present address: Pharmaceutical Products Research, Department No. 426, AP-9A, Abbott Laboratories, 60064-3500. ‘Visiting Assistant

Professor,

Department

of Biophysics,

Ketamine

Panjab University,

75

Chandigarh

160014, India.

1 Abbott Park Road, Abbott Park, IL

BANSINATH,

76

RAMABADRAN,

TURNDORF

AND SHUKLA

TABLE 1 K-AGONIST

(ICV)-INDUCED

CONVULSIONS*

Dose/Mouse Dw

PD

I 17302

U-69593

U-50488H

I%

nmol

10 20 40 60 80 20 30 40 60 80 20 40 60 80

25 50 101 152 203 56 84 II2 I68 224 42 x5 I28 171

*Linear portion of the dose-effect

Tested

% Convulsed

20

‘5

21 25 25 20 20 20 20 25 20 15 15 15 15

52 64 60 65 25 55 75 60 55

5 II I6 I5 I3 5 II I5 I5 II I 4 7 6

27 -17 -10

data was subjected to probit analysis to calculate the ED,, and potency ratio.

tration of k-agonists induced convulsions. The present study compares the potency, stereospecificity and receptor mechanisms involved in the epileptiform effects of some of the currently available highly selective k-opioid agonists. METHOD

Animals

Male Swiss Webster [(SW)fBR] mice were procured from Taconic Farm. The animals weighing 25-30 g were housed five per cage in a room with controlled temperature (22&2”C), humidity and artificial light (06:30-19:00 h). The animals had free access to food and water and were used after minimum of four days acclimation to housing conditions. All experiments were conducted between 09:00-17:00 h in June-August. Drugs

The following drugs were prepared by dissolving them in sterile water just before use; PD117302( -t- )-trans-N-methylN[2-(pyrrolidinyl)-cyclohexyl]benzo[b]thiphene-4-acetamide (Parke-Davis, Cambridge), U-50488 {3,4-dichloro-N-methyl-N[2-( l-]pyrrolidinyl)cyclohexyl]-Benzacetamide methanesulfonate hydrate, truns-( k)}, U-53445E [non-k (+) enantiomer of U-50488H] (Upjohn Co, Kalamazoo, MI), Dizocilpine (MK801, ( + )-5-methyl-lO,ll-dihydro-5H-dibenzo[u,&yclophepten5,10-iminehydrogen maleate, Merck Sharp & Dohme, West Point, PA), ketamine, naloxone, muscimol and baclofen (Sigma Chemical Co., St. Louis, MO). The K-opioid agonist, U-69593 ({[5R-5u,7a,8B)]-( k)-N-methyl-N-[7-(l-pyrrolidinyl)-loxaspiro[4,5]dec-8yl]-benzene acetamide}, Upjohn Co., Kalamazoo, MI) was dissolved in pyrogen free water acidified with dilute acetic acid. The putative k-antagonist, MR 2266 [( - )-( lR,5R,9R)-5,9-diethyl-2-(3-furyl-methyl)-2’-hy~oxy-6,7benzomorphan] (Boehringer Ingelheim, Federal Republic of Germany) was dissolved in few drops of 0.1 N HCl and made up with distilled water. Experimental

Convulsed

Procedure

All doses of k-agonists were injected ICV in 10 l-t.1volume. Freehand ICV injections into the lateral ventricles of the con-

scious mouse were made using 27-gauge, r/&inch needle attached to a 500~~1 Hamilton syringe assembled on an automatic dispenser (PB 600, Hamilton Company, Reno, NV). The needle was fitted with polyethylene tubing leaving 3 mm of the needle tip exposed. The point of injection was on an imaginary line drawn through the anterior base of the ears and 2 mm from an imaginary midsagittal line (12). In separate groups of mice, the accuracy of the injection technique was histologically verified using India ink injection. After the ICV administration, the animals were individually housed in transparent polycarbonate cages (29 X 19 X 13 cm) and were observed for a minimum of 30 min for signs of seizure activity. In each group, the number of animals showing an episode of clonic jerks persisting for at least five seconds (minimal full seizures) (3) were recorded. In experiments with pretreatment paradigm, the time of ICV challenge of K-agonists was dependent upon the peak effective time for the pretreated drug which was as follows; 15 min for MR 2266 (4,8) and naloxone (36), 30 min for MK-801 and ketamine (37) and 60 min for muscimol and baclofen (24). Doses and routes of administration used are as mentioned in Fig. 1 and Tables 1 and 2. Each mouse was used only once. Data Analysis

Quanta1 dose-response data were analyzed using probit analysis to estimate CD,s, relative potencies and 95% confidence limits by the method of Litchfield and Wilcoxon. Statistical significance was evaluated using probit analysis results, V* test (sample size corrected Pearson chi-square test) (38) and the test of parallelism of regression lines using the computer program of Tallarida and Murray. RESULTS

The dose-response data of the percentage of animals convulsed after ICV injection of k-agonists is shown in Table I. All the K-opioid agonists used, induced minimal full seizure activity. The animals showed one or more of the behavioral signs of convulsions like “popcorn” jumping, myoclonic seizures (isolated jerky limb movement) and all-limb clonic activity (repetitive movements involving all limbs simultaneously, usually

K-AGONISTS INDUCE CONVULSIONS

77

IN MICE

TABLE 3 EFFECT OF OPIOID. NMDA ANTAGONISTS AND GABA AGONISTS ON K-AGONIST (ICV)*-INDUCED CONVULSIONS

A+ /

Convulsed/Tested

0

o PO117302 . U50488H A U69593

25

50

IO0

200

n mol/lO d/mouse FIG. 1. Dose-related convulsive effects of K-agonists after ICV injec-

tion. Each point represents the effect in 15-20 mice. CD,, for each drug mentioned in Table 1. The x2 values for K-agonists and were as follows: 0.4468 (PD 117302), 0.02852 (U 50488H) and 0.00108 (U 69593). The calculated x2 values in all the cases were lower than the table value (3.84, df= 1) suggesting that the percent of animals conis

vulsed was the function of dose of the K-opiate @