Urethane anesthesia blocks the development and expression of kindled seizures

Life Sciences, Vol. 44, pp. 1201-1206

Printed

Pergamon Press

in the U.S.A.

URETHANE ANESTHESIA BLOCKSTHEDEVELOPMENT AND EXPRESSION OF KINDLEDSEIZURES Donald P. Cain, Angela Raithby and Michael E. Corcoran* Departments of Psychology University of Western Ontario London, Ontario, CANADAN6A 5C2 and *University of Victoria Victoria, British Columbia, CANADA V8W 2Y2 (Received in final form February 21, 1989)

Summary The effect of anesthetic and subanesthetic doses of urethane on the development of amygdala kindled seizures and on the expression of previously kindled seizures was studied in hooded rats. An anesthetic dose of urethane (1.5 g/kg) almost completely eliminated evoked afterdischarge and completely eliminated convulsive behavior in both groups. It also eliminated the seizure response to pentylenetetrazol. Subanesthetic doses of urethane (0.25 and 0.5 g/kg) strongly attenuated the expression of previously kindled seizures. These results suggest that urethane may not be an appropriate anesthetic for the study of epileptiform phenomena. Urethane (ethyl urethane; ethyl carbamate) has been used as an anesthetic for over a century, and in recent years has been used frequently to anesthetize rodents and lagomorphs in electrophysiological research. Some of this research has involved models of epilepsy (l-3). In work aimed at finding an anesthetic suitable for use with rodents in acute electrophysiological studies of an epileptiform model of neural plasticity (41, we explored the use kindling, of urethane in both naive and previously kindled rats. We found that urethane consistently blocked both clinical and electrographic seizures evoked by electrical stimulation of the amygdala and by injection of pentylenetetrazol. Methods Twenty-two male hooded rats weighing 250-400 g and housed individually were and received implantation of used. They were anesthetized with pentobarbital one or two bipolar electrodes into the basolateral amygdala using conventional techniques. The electrodes were constructed of twisted Teflon-instereotaxic sulated stainless steel wire 127 urnin diameter and were stereotaxically aimed for the basolateral amygdala. The ends were soldered to small contacts, which into a miniature connector that was attached to the skull with were inserted screws and dental acrylic. One week after surgery the rats were connected to a polygraph and Grass S88 (ADT) . electrical stimulator for determination of the afterdischarge threshold An initial current of 25 ua was applied, which was subsequently raised in small 0024-3205189 $3.00 + .OO Copyright (c) 1989 PerSamon Press plc

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steps until an AD was evoked. All current intensities are expressed as base-topeak values. The ADT was defined as the weakest current that would evoke an AD of 4 set or longer. The current consisted of biphasic square wave pulses, each 1.0 msec in duration, at 60 Hz and a total duration of 1 sec. The rats were then assigned to one of two main groups. The rats in the Urethane Prophylaxis group (n-6) received an anesthetic dose of urethane (SigAll injections were made intraperitoneally. Approximately 30 ma, 1.5 g/kg). min later they were connected to the polygraph and stimulator and received a train of electrical pulses similar to that used during determination of the or behavioral ADT, at an intensity 50 ua greater than the ADT. Electrographic responses were recorded on the polygraph and noted. They were then stimulated at intervals of 1 hr until a total of five stimulations had been applied under urethane. The remaining rats (n=8) were assigned to the Kindled group. Some of them (n=5) received an injection of physiological saline equal in volume to the injection given to the Urethane Prophylaxis group. Beginning 30 min later they were stimulated five times at intervals of 1 hr to provide control data for comparison with the Urethane Prophylaxis group. Beginning 24 hr later they were stimulated once each day until they had exhibited three stage 5 generalized convulsions. The other rats in the Kindled group (n=3) received kindling stimulation once each day until they exhibited three stage 5 convulsions. Approximately two weeks later all rats in the Kindled group received injections of urethane (1.5 g/kg). Beginning 30 min later they received five stimulations at intervals of 1 hr. The first stimulation was applied at an intensity 50 ua greater than the ADT. In order to maximize the possibility of evoking AD under urethane the subsequent stimulations were applied at increasing intensities in the following sequence: 200, 400, 800, 1600 ua. One hr after the last stimulation some of the rats (n=6) received a single injection of pentylenetetrazol and behavioral response was monitored (PTZ, 60 mg/kg) and the electrographic for 60 minutes. This dose of PTZ is sufficient to evoke strong electrographic and clinical seizures in this strain (5). As the results indicated a near-complete blocking of AD by 1.5 g/kg urethane, we wished to have some idea of the effect of smaller doses of urethane in kindled rats. For this purpose we prepared eight additional rats with amygdala electrodes and kindled them as described above. After three stage 5 convulsions had been evoked the ADT was again determined by applying an ascending series of currents beginning at 25 uA. The next day four rats received urethane at 0.25 g/kg and four rats received urethane at 0.5 g/kg. Beginning 30 min latertha ADT was again determined. At the end of testing all rats were deeply anesthetized with pentobarbital and perfused with formalin-saline, and the brains were removed and sectioned on a freezing microtome for verification of the electrode placements. Results The electrode tips of all subjects discussed in or on the border of the basolateral amygdala.

below were confirmed

to lie

Prophylactic treatment with urethane almost completely blocked AD during the five hourly stimulations (Fig. 1). Compared to the controls, which demonstrated a normal progressive increase in AD duration in response to the sequential stimulations, the Urethane Prophylaxis group demonstrated no such increase. None of the Urethane Prophylaxis rats advanced beyond stage 0 (4) by the last stimulation, whereas all of the control rats had reached stage 2 by the last Stimulation. There was no overlap in the AD duration data of the two groups after the administration of urethane.

vol. 44, No. 17, 1989

20 c

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Urethane Anesthesia and Kindled Seizures

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URETHANE CONTROLS

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1

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5

FIG. 1 Afterdischarge (AD) duration in naive rats injected with urethane (Urethane group, 1.5 g/kg) or saline (Controls)and electricallystimulatedat intervals of 1 hr. The values indicatedby ADT were obtained at the AD threshold-determining session, during which no injectionswere given. The values are means f S.E.M.

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FIG. 2 Afterdischargeduration in previously kindled rats before injectionof urethane (the last two stage 5 seizures; first two data points), and after the injection of urethane (last five data points). The first three stimulationswere applied at an intensity 50 UA greater than the AD threshold (ADT + 50), and the remaining stimulationswere applied at the intensitiesshown on the abscissa. The values are means & S. E. M.

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Urethane almost completely blocked evoked AD in the Kindled group during the 2). There was no overfive stimulations of increasing current intensity (Fig. two lap in the AD duration or seizure stage data of any rat during the last stimulations before urethane and the five stimulations under urethane. Stimulation currents as high as 1600 ua were little more effective in evoking AD than None of the Kindled rats exhibited convulsive behavior less intense currents. The injection of above stage 0 in response to any stimulation under urethane. PTZ appeared to increase the rate of respiration and vibrissae twitching, and most rats exhibited spindle activity in the EEG. However, none exhibited epileptiform spiking or convulsive behavior. The effect of smaller doses of urethane on seizures in previously and clinical rats is presented in Table 1. Although electrographic the data activity was not completely blocked by the smaller doses, marked tendency for the smaller doses to elevate the ADT and reduce duration and seizure stage in a dose-dependant manner. Data from the group that received 1.5 g/kg are included in Table 1 for comparison.

kindled seizure show a the AD Kindled

Discussion Urethane exerted a near-canplete blocking effect on the development of kindled seizures in previously unkindled rats and in the expression of seizures The that had been fully kindled before being treated with urethane. in rats Urethane Prophylaxis rats all had electrode placements that supported AD before and which were confirmed to lie in the basothe administration of urethane, kindled lateral amygdala, yet none of them exhibited any tendency to develop seizures after five stimulations at intensities as high as 1600 uA. This stimulation intensity greatly exceeds that normally required for amygdala kindling. This contrasts with the progressive increase in AD duration in response to stimulations that was exhibited by the saline control rats. The sequential induced conclusion that urethane effectively blocked the epileptiform response that urethane completely by kindling stimulation is extended by the fact blocked the electrographic and convulsive response to 60 mg/kg PTZ.

Effects Dose

(g/kg)

N

TABLE1 Of Subanesthetic PreUrethane Afterdischarge

0.25 0.5 1.5

4 4 8

47.5 71.5 88.0

4 4 8

90.0 101.5 68

postUrethane Threshold

Percent Change (uA)

99.0 321.5 1250.0

Afterdischarge 0.25 0.5 1.5

Doses Of Urethane

Duration 16.0 8.7 0.8

+208 +415 +1420 (set) -82 -91 -99

Seizure Stage 0.25 0.5 1.5

4 4 8

5 5 5

1.0 1.25 0

-80 -75 -100

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Doses of urethane well below the anesthetic dose of 1.5 g/kg exerted a marked anticonvulsant effect. Thus, doses of 0.25 and 0.5 g/kg increased the ADT and attenuated the electrographic and clinical seizures in previously These data imply that doses much smaller than the usual aneskindled rats. thetic dose have quite appreciable anticonvulsant effects. of subcortical Although the use of urethane as an anesthetic in studies structures has been advocated (61, the results of the present study indicate that urethane is probably inappropriate in studies of epileptiform activity. Urethane appears to have anticonvulsant effects that are stronger than a number of anesthetics that have been tested in kindled rats, including pentobarbital The dangers of drawing conclusions from data gathered from urethane(7). anesthetized subjects in pharmacological (8,9) and neurophysiological studies (10) have also been emphasized. Urethane is known to greatly alter systems responsible for flash-evoked potentials (10) and EEG rhythms in the neocortex and hippocampus (11). Thus, urethane may be unsuitable as an anesthetic in most studies of normal central nervous system function, with few exceptions (6). One of these exceptions may be the study of neural plasticity using long term potentiation (LTP). The original observations of LTP were made in subjects anesthetized with urethane (121, and many researchers now use urethane anesthesia routinely in LTP research. Preliminary results from our laboratory have shown that rats anesthetized with urethane exhibit strong LTP in the perforant gyrus circuit, path - dentate but do not exhibit AD and kindling shortly thereafter when this is attempted using the same stimulating electrode. Kindling-like increases in the AD duration do not occur even when AD is repeatedly evoked by very high intensity currents in long trains (Cain, Boon & Hargreaves, unpublished observations). Thus, the same dose or urethane (1.5 g/kg) appears to have little or no effect on LTP but completely blocks kindling by raising the AD threshold and greatly attenuating the duration of AD that is evoked. This has possible implications for the question whether kindling and LTP have similar mechanisms (13). Our observations suggest that although LTP may under some circumstances contribute to kindling, the use of urethane during the induction of both models in the same subjects allows a dissociation between the models (14). Urethane appears to be unique in its ability to block amygdala focal AD. Most anticonvulsants, including benzodiazepines and barbiturates, block generalized AD but not limbic focal AD (7,151. Therefore, urethane may be useful in the study of mechanisms of AD generation. 1. 2. 3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13.

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14. D. Cain, Trends Neurosci. 12 6-10 (1988). 15. R. Racine, K. Livingston,r Joaquin, EEG Clin. Neurophysiol.-38 355-365 (1975).