The effect of an experimental epileptiform syndrome on memory in rats

Jane Mellanby

We are at present working on the effect of epilepsy on memory in rats. A chronic epi- leptiform syndrome can be reproducibly in- duced in rats by injection of minute amounts (a few mouse LD,5,) of tetanus toxin into the hippocampus (Mellanby et al. 1977). With a suitable dose of toxin, the rats exhibit com- plex partial seizures at a frequency of up to 4 times an hour for 4-5 weeks and then recover. After unilateral injection of toxin, seizure discharges are recorded both in the injected hippocampus and, usually simultane- ously, in the opposite hippocampus. The EEG shows frequent inter.icta1 spikes and also short bursts of spikes lasting 1-6 sec.

It seems likely that the effect is a specific result of the pharmacological action of the toxin, since histological examination of the injection site does not show any scarring, and also because control rats, which are similarly injected with neutralized tetanus toxin, never develop the syndrome. We have found im- pairment, in rats who have ,recovered from this syndrome, of their memories for a task which they had learnt before the injection of the toxin. Furthermore, the recovered ani- mals are also impaired in learning new tasks including delayed alternation in a T-maze and a light-discrimination task in a Y-maze (Mellanby &George 1979). The tetanus toxin- induced syndrome involves general behav- ioural changes including hyperreactivity, spo- radic aggression and abnormally passive re- sponses to an intruder rat. However, the de- ficits in learning and memory are still present at a time when the general behaviour has returned to normal. (See Figure 1 for the

timing of the different aspects of the syn- drome and the training and re-training sche- dule.)

It was of obvious interest to find whether controlling overt seizures in the epileptic rats would at the same time reduce or eliminate the memory deficit. it was found that oral administration of cartmmazepine (20 mg/kg twice daily), which produced blood levels within the human therapeutic range (2--10 pg/ml), substantially reduced the frequency of overt seizures while having little effect on the EEG. However, when it was administered twice daily for- 3 weeks after the toxin-in- jection it did not lessen the subsequent im. pairment of memory for a task learned be- fore the opei-ation. This would suggest that the memory impairment may not be pro. duced by the generalized seizures but by the local disorganization of hippocampal func- tion produced by the tetanus toxin.

Tetanus toxin blocks the release of the in- hibitory transmitters, GABA and glycine, in the central nervous system (Curtis & de Groat 1968, Curtis rt al. 1973, Davies & Tongroach 1977, Collingridge &- Davies 1980) and al- though its effect is not specific to such syn. apses (see Mellanby &. Green 1981), it is likely that the epileptic focus results from the local blocking of inhibition.

Summary The memory impairment which is found in rats which have recovered from tetanus toxin- induced limbic epilepsy seems to be related to the local disorganization of hippocampal

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function produced by the epileptic focus ra- ther than by the generalization of seizures. We draw this conclusion from the finding

that carbamazepinr can markedly reduce the number of overt seizures but does not pre- vent the memory deficit (Figures 1 and 2).

Figure 1 Overall progress ofthe epilepttjorm syndrome Experimental design 1 Memory experiments Dgerent groups o/ rats were trained on a light-discri- mination task in a Y-maze and one week later were in-

jected with tetanus toxin ( 5 mouse LD,d bilaterally into thear ventral hqpocampi (and controls were injected similarly with neutralized toxin) Groups of rats were re- trained at 1, 4, 7 or 9 weeks after the injection ofthe tox- in

Rats were injected with toxin (or neutralized toxin) as above and they were traaned on the light-dtscriminatton task for the f i rs t time 7 weeks after toxin injection

g l

: 0 5

0-

*sc*s 2 Learntng experiments IO" n

6om 50

Figure 2. Effect oftreating seizures with carbamazepine on subsequent memory dejicit. The rats were trained on the light-discrimination task and one week later tetanus toxin (or neutralized toxin in controls) was injected bilaterally into their ventral hip- pocampi For the following3 weeks they were dosed orally twice daily with carbamazepine (20 mglkg) or polyethy- lene glycol (in which the drug was dissolved. 1 mllkg). The rats' memory was tested 7 weeks after the injection ofthe toxin (8 weeks after trainind.

,Errors to criterion at initial training minus errors to criteri- on at retraining x 100

Errors to criterion at initial training plus errors to criterion at re-training

Savings score =

Each vertical column in Figure 2 represents the mean score in the group of 12 rats given the treatment indi- cated The vertical bars show the S. E. M.

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References Collingridge G L & Davies J: Reversible effects of low

doses of tetanus toxin on synaptic inhibition in the substantial nigra and turning behaviour in the rat. Brain Res 185: 455-459, 1980.

Curtis D R & de Groat W C Tetanus toxin and spinal inhibition. Brain Res 10: 208-212, 1968.

Curtis D R, Felix D, Game C J A & McCullogh R M: Tetanus toxin and the synaptic release of GABA. Brain Res 51: 35&362, 1973.

Davies J & Tongroach P Tetanus toxin and synaptir inhibition in the substantia nigra and striatum of the rat. J Physiol 290: 23-36, 1979.

Discussion Dam: You talk about seizures. I would like to hear which kind of seizure; what are the clinical manifestations of the seizures. We are treating lots of seizures in patients, ne- vertheless many of the patients may grow worse and will have many psychological symptoms although we are curing the epli- teptic manifestations which we see.

Mellanby: You want to know what the seizures looked like?

What happens is that if you are simultane- ously recording the hippocampal EEG and watching the animal you first of all have a silent period on the EEG when the back- ground spikes disappear and during which the animal “freezes” and its ears go back; you may see a slight chewing. Then it pro- ceeds to stand on its hind legs and myoclon- ic jerks of one or both forepaws occur. There is no lateralisation with respect to which paw starts first, even when you have only used the unilateral injection. They us- ually stand up on their hind paws for 20 seconds, and then sometimes they go almost tonic and fall over. One gets every gradation down to just one paw doing a little bit of myoclonus. S o that is what I mean by an overt fit; something I can see motor signs of. During the period where they just have hippocampal discharges which are not as- sociated with any motor signs of a fit if they occur when the animal is awake it normally suspends animation for a moment and just

Mellanby J &George G: Tetanus toxin and experimental epilepsy in rats. In: Advances in cytopharmacology vol 3, pp 401-408. Eds: B Crccarrlli & F Clemrnti. Raven Press. Nrw York 1979.

Mellanby J, George G, Robinson A & Thompson P: Epileptiform syndrome in rats produced by injecting tetanus toxin into the hippocampus. J Neurol Neu. rosurg Psychiatry 40: 404-414, 1917.

Mellanby J & Green J: How does tetanus toxin act? Neuroscience. In press 198 1.

remains still, and then walks off again as if it had not noticed.

Weiskrantt. When you say that there was no effect on getting rid of the amnesia induced by the toxin, does that mean that your con- trol group showed no change and that your experimental group also showed no change? Or did both groups change relative to each other? There are four possible outcomes. The reason that it is important is because in looking at the clinical results a question has been raised as to whether the drug treat- ment by itself might have an effect on the memory.

Mellanby: We get some impairment of mem. ory by the carbamazepine alone as compared with giving the vehicle, polyethylene glycol, and the impairment is additive with the ef. fect of tetanus toxin.

Weiskmntz A second question is what was the effect of carbamazepine, if any, on the aggression and the hyper-reactivity?

Mellanby: It had no effect on aggression, but it had a small effect on some of the other behavioural changes which I have not ac- tually described. In these animals, during the phase when they are having overt fits and they are being given carbamazepine, we have also done a test of their reactiveness to a strange rat introduced into their cage - the home intruder test. We were hoping,

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to quantify the aggression in this way. We predicted that since the animals are more aggressive if you put your hand into the cage, they would probably be more aggressive to an intruder rat. In fact we found exactly the opposite, that they were much more passivr in response to an intruder rat than were their controls. They simply sat in the corner and “froze”. The carbamazepine released that effect to some extent, so they were not as passive as the epileptic animals were if they had only been given polyethylene gly- col.

Rolls: I am concerned with ceiling effects and floor effects. I wonder if the seizures were so severe that effectively reducing them by a certain amount still left one in some ceiling.

Mellanby: I think this is perfectly possible. It would be nice to reduce them more. We have done a different experiment in whichwe have used ECT to induce seizures in rats, and looked at its effect on the same task, with the same sort of time intervals. One seizure a day, or one every second day, has no effect on memory. That is giving 10 ECT treat- ments and then looking either 24 hours, one week, three weeks or seven weeks after the last one did not produce any impairment.

Blennow: I would like to hear some more comments on the morphology. You said that you had done pathology on them.

imals; were they injected? And how? I Secondly, I wonder about your control an.

Mellanby: They were injected with exactly the same volume of exactly the same amount of toxin, but it was neutralised with anti- toxin. We are at the moment, with Dr Trevor Hughes at the Radcliffe Infirmary in Oxford, trying to do Golgi preparations of the foci, and all I can say is that so far we have not found any obvious changes. But what I have not done is quantitative work, and we ought to. Most of our morphological work so far has involved just looking to see if you get gliosis and other sorts of scarring, and you

do not. In fact it is extremely difficult to find the site of injection.

Blennow: Did you count the neurons?

Mellanby: No,

Stores: I will go back to the behavioural changes associated with the induced seizures, and particularly the aggression. I wonder if it was possible, from your studies, to discern any sex differences or laterality effects. The reason I ask that is that in our own work on children with epilepsy, behavioural dis- turbance, particularly aggression, and over- activity in the sense of gross motor over- activity, were seen in boys with epilepsy but not in girls with epilepsy; and was seen in those with left temporal lobe pathology and not in those with right temporal lobe pa- thology. Are there any counterparts to those findings in your rats?

Mellanby: Of course with the laterality one has a problem with the rats. I suppose one could determine paw preference first, which is something we have not done, and then inject the toxin unilaterally. Yes, the answer is in female rats we have done one study now and there is much less aggression.

Stores: 1 was actually wondering whether it related to the side of injection, or was it al- ways the case that you did bilateral injec- tions?

Mellanby: No. It does not seem to make any difference whether the toxin iiijection is uni- lateral or bilateral, but we have not done big memory studies with unilateral injec- tions. We use unilateral injections when we want to record the EEG so that we can have bilateral electrodes and see what the injected focus is doing to the noninjected side.

Stores: The differences between the boys and girls in our series were over and above the sex differences you get in normal boys and girls.

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Mellanby: That is why we do females, because of your study. They were very much less ag- gressive in this situation.

Kulzg: When did you start administering car- bamazepine in the last study that you talked about?

Mellanby: We administered it the first day postoperatively, which is roughly when we first see seizures in response to injecting the toxin under anaesthetic. If instead you do a different experiment where you have pre- viously implanted a cannula, you can inject the toxin in a non-anaesthetised rat, and then you can get seizures straight away.

Kulig: How specific do you think these effects on memory are to the hippocampus? I ob-

viously assume that you chose the hippocam- pus because you expected a deficit, in terms of what was known about the hippocampus. The other thing is - have you tried other sites outside of the hippocampus? And the other thing that I was surprised about is that you did not use the delayed alternation task.

Mellanby: We have tried injecting the same dose of toxin into a number of other sites (including motor, parietal and occipital cor- tex and the caudate) and we have not seen any gross behavioural changes or overt sei- zures. We have used delayed alternation for looking at the new learning when the an- imals have recovered from the syndrome, and they are impaired on that, which would suggest hippocampal damage of some sort.