ACTA NEUROBIOL. EXP. 1978, 38: 305-321

FACILITATORY AND INHIBITORY EFFECTS OF STRONG ELECTRICAL HIPPOCAMPAL STIMULATION ON PERFORMANCE

OF TWOAWAY AVOIDANCE RESPONSE IN CATS

Slawomir GRALEWICZ, Krystyna GRALEWICZ and Elibieta SZCZEPANIAK

Department of Animal Physiology, Institute of Physiology and Cytology, University of E6dt, Poland

Abstract. The influence of a strong, seizure inducing electrical stimulation of the hippocampus on the performance of the active avoidance responses in a shuttle-box was studied. The stimulation (a 10 s train of rectangular, negative pulses, 50/s 200 PA) was applied at different stages of training 2 min before experimental sessions. The stimulation evoked a facilitatory or inhibitory effect on the avoidance performance. The kind of effect 'depended on the level of stabilization of the avoidance response. At an early stage of training facilitatory effect dominated, an inhibitory effect prevailed after stabilization of the avoidance response. Intraperitoneal administration of atropine sulphate decreased the inhibitory effect of stimulation. The results confirm the supposition that hippocampal stimulation influences the performance of conditioned active avoidance responses in a similar way as electroconvulsive shock does.

INTRODUCTION

The literature data concerning the influence of hippocampal seizure discharges on the performance of well established conditioned responses are not equivocal. In most cases an impairment or complete inhibition of response was observed (2, 6, 32, 33, 39, 40), though in some instances the animals could perform quite well in qpite of the abnormal activity in the hippocampus (2, 10, 32). Apart from these direct effects, hippocampal seizures may also produce a temporary inhibition or decrease in the performance of conditioned responses (6, 11, 31, 41) or short-lasting amnesia of recent events in patients (4, 7, 9) in the period

2 - Acta Neurobiologiae Experimentalis

after seizures. In some studies, however, reapperance of conditioned responses was observed immediately after the cessation of seizure activity (11, 33, 39).

It is well known that hippocampal stimulation can reproduce most of the effects on memory observed after electroconvulsive shock (ECS) application (3, 17, 23-25, 29, 43). Thus this structure seems to participate in the production of ECS effects or even the phenomena observed after ECS are mainly dependent on an interference in hippocampal functions (20, 26). Some data show that ECS applied before experimental sessions can facilitate the performance of partially forgotten or undertrained conditioned active avoidance responses but inhibits them when they are well established (8, 41). Thus, the level or response stabilization (or forgetting) appears to be an important factor in determining the effect of ECS. Footing on the similarities between the effects of ECS and hippocampal stimulation it may be assumed that different levels of response stabilization might be responsible for the inconsistency of results evoked by hippocampal seizure stimulation. The aim of the present experiment was to find out whether hippocampal stimulation applied before training sessions could modify the performance of an active' avoidance response in the same way as it was observed in the case of ECS application (8), i.e., to facilitate performance when the response was undertrained and inhibit it in later phases of training.

According to previous observations (18) normal cats trained in a shuttle-box attain 40-60°/o level of AAR performance after six to seven sessions of training consisting of ten trials each. After 14 sessions the level of AAR is, as a rule, 90-100°/o. It seemed therefore, that after six sessions of training the response could be regarded as undertrained, after 14 sessions as well trained, and when the criterion had been achieved (ten successive sessions with 90-100~/0 of AAR as minimum) as overtrained. Application of a strong electrical stimulation of the hippocampus before these sessions was expected to evoke different effects on AAR.

EXPERIMENT I

Methods

Subjects. Sixteen male cats, 2-4 years old and weighing 2.5-3.0 kg were used. The animals were randomly divided into two groups. The cats of Group I (n = 9) were stimulated during the experiment. Group I1 (n = 7) served as operated controls.

Surgery and histology. Stainless steel, teflon coated, unipolar electrodes of 120 pm in diam and 0.5 mm bare tip length, were implanted

bilaterally (one electrode into each hemisphere) into the posterior part of the hippocampus in all cats. The stereotaxic coordinates were as follows: A2, L10, H 4 . according to Jasper and Ajmone-Marsan's stereotaxic atlas (22). A detailed description of the surgical technique, as well as the histological procedure used, was published previously (16, 17). Representative sections with the electrode track visible were redrawn with the use of a photographic enlarger to illustrate the electrode placements within the brain.

The experiment was conducted in the same shuttle-box and with the use of the same stimulation equipment as in our previous studies (17, 18): The light of 60 W bulbs located on both sides of the box under the grid floor served as a CS. The US was an electroshock applied to the animals paws through the grid floor.

Training. All cats learned to avoid the electroshock by crossing a bar dividing the shuttle-box into two identical parts in response to the CS. The isolated time of CS was 10 s and the intertrial interval varied between 20 and 40 s (mean 30 s). A detailed description of the train& procedure was published previously (17). After 6 days of training one stimulation session (S1 session) and one control session (Ci session) were performed (sessions 7 and 8 respectively). On the following days training was continued. The procedure was repeated at sessions 14 and 15 (S2 and C, sessions) and after the cats had attained a criterion (S3 and C3 sessions). Ten successive sessions with a 90°/o level of performance was regarded as the criterion. Training sessions consisted of ten trials each. Stimulation and control sessions were composed of 12 trials. The first two trials (antiextinction trials) were performed with the use of US. In Group I S sessions they were followed by a single 10 s train of electrical stimulation of the hippocampus with the following parameters: 200 PA, 50/s, rectangular, negative pulses of 1.0 ms duration. According to our previous observations (18), such a stimulation usually evokes hippocampal seizure dhcharges (Fig. 4A). Two minutes after the end of hippocampal stimulation ten trials were performed without the use of US. The procedure in C sessions was similar, but hippocampal stimulation was not applied. In the cats of Group 11, S qessions and C sessions were performed in the same way. The first two trials were followed by a 2 min pause after which ten trials with no US were performed. The percentage of AAR, the number of intertrial responses (ITR) and the latency of AAR were measured throughout the experiment.

Results

Effects of hippocampal stimulation on AAR performance. Figure 1 shows the results in a graphical form. The training sessions immediately

preceding S sessions are denoted by the letter "T". An analysis cf variances (Lindquist, mixed design, type VI (30)) was used for statistical evaluation of the data. The effects of the following factors were analyzed: A - succession of the experimental sessions, B - treatment and C - experimental groups. The analysis has shown a significant effect of factor A (F2,26 = 6.104, P < 0.001), factor B (F2,26 = 38.737, P < 0.001), factor C (F1,13 = 9.137, P < 0.01) and a significant B-C interaction (F2,26 = 26.903, P < 0.001). Duncan's test (37) applied subsequently has revealed that the level of AAR performance observed in Group I during S sessions differed significantly from that observed in T and C sessions in the same group as well as in T, S and C sessions in Group I1 (P < 0.01 in all comparisons). It was caused by a marked decrease in performance after hippocampal stimulation in all S sessions. Although the detailed analysis failed to discover any statistical differences between successive S sessions in Group I there was no uniformity in the effect of hippocampal stimulation on AAR performance. In fact, inhibition of AAR in S1 was observed in seven out of nine cats. In the remaining two (cat 196 and 283) the percentage of AAR after stimulation was higher in comparison with T1 and C1 session. It is worth noting that these two cats showed the lowest level of AAR at this stage of training. In Sz and SS, an inhibitory effect of stimulation was observed in all cats.

- ; 2 3 4 5 6 7 8 9 W l l 1 2 1 3 l L l 5

t 1 sesslons

T, 5 , cr 1 1 1 t t t 5 52 C? 5 5 3 CI

Fig. 1. Experiment I. Changes in the level of AAR performance after hippocampal stimulation a t different stages of stabilization of the conditioned response. T, ses- sions of training preceding sessions with hippocampal stimulation; S, sessions following hippocampal stimulation; C, control sessions. Solid line, stimulated group;

interrupter line, operated control group (see text for further explanations).

An analogical analysis of the ITR number and the latency of AAR has revealed no statistical differences in the ITR number. An effect of factor A was found, with regard to the AAR latency, (F2:24 = 3.911, P < 0.05) confirming the normal change (shortening) in the speed of crossing as the training progressed.

E f f e c t of hippocampal stimulation o n animal behavior. The symptoms observed during and after stimulation were the same as those described previously (17). A characteristic series of meows appeared each time 8-20 s after stimulation leaving no doubt that hippocampal seizures were evoked. In some cats a period of increased exploratory activity followed the vocalization and lasted 1-2 min.

The results of the above experiment have shown that strong stimulation of the hippocampus, applied before the choosen experimental sessions, in most cases exerts an inhibitory influence on AAR. Since the expected facilitation 01 AAR performance appeared after the first stimulation in only two cats showing the lowest level of avoidance at this time, it seemed likely that the first stimulation was applied after too many sessions of training. Therefore, we decided to repeat this experiment but with the first stimulation applied after three training sessions.

EXPERIMENT I1

Methods

Fourteen male cats were used. Eight animals (Group Ia) were stimulated and the remaining six (Group Iia) were operated controls. Experimental conditions were the same as in Experiment I with the exception that the first stimulation was applied before the fourth session.

Results

The results are presented in Figure 2. Statistical evaluations were performed in the same way as in Expermient I. An analysis of variances revealed a significant effect of factor A (F2,24 = 111.039, P < 0.001) and significant A-C (F2,24 = 20.602, P < 0.001), A-B (F4,4* = 5.246, P < 0.005) and A-B-C (F4,48 = 3.492, P < 0.05) interactions. Further analysis with the use of Duncan's test has shown that in cats of Group Ia the level of AAR performance in S1 session was significantly higher than that observed in T1 and C1 in the same group, as well as in S1 in Group IIa (P < 0.01 in all'comparisons). The opposite effect of stimulation appeared at the end of the experiment: the level of AAR performance in S3 was

significantly lower in comparison with Tg in the same group and with SB in Group IIa (P < 0.05 in both comparisons). No other differences have been found.

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Fig. 2. Experiment 11. All designations as on Fig. 1.

Similarly as in the Experiment I, there was no uniformity in the effects of hippocampal stimulation. A facilitation of AAR performance in SI session appeared in seven out of eight cats. Surprisingly enough the expected inversion of the facilitatory effect (inhibition of AAR) was observed only in two cats (cats 270 and 301)'in S2 and in three (cats 270, 301, 302) in S3. In the remaining animals which showed facilitation in the 4th session, the stimulation failed to evoke inhibitory effects in further S sessions in spite of a high level of AAR performance attained during training. There were no differences in behavior during and after stimulation, between the cats from this experiment and from the preceding one, which might account for the differences in performance after stimulation.

These results have shown that both facilitatory and inhibitory effects on AAR may be observed after hippocampal stimulation. The comparison of the results of the first stimulation from Experiment I and Experiment I1 shows that the relationship between the effect of stimulation and the stage of training is similar to that observed after ECS application (8). The effect of the second stimulation however, does not seem to be determined exclusively by the attained level of AAR. The lack of differences in the immediate effects of stimulation on behavior between the stimulated groups from both experiments suggests that the absence of inhibition observed in some cats from Experiment I1 might Be

somehow related to the occurrence of facilitation after the first stimulation.

Some authors have shown that injections of muscarinic cholinolytics nlay prevent the appearance of the inhibitory effects of ECS on learned habits (1, 42). The aim of the next experiments was to find out whether the same could be observed in the case of th'e inhibitory effect on AAR evoked by hippocampal stimulation. The well known muscarinic blocker - atropine sulphate - was used in the following experiment.

EXPERIMENT I11

Preliminary investigations. In order to find out the optimal dose of atropine, preliminary investigations were carried out on two cats with electrodes implanted bilaterally into the posterior, dorsal and ventral parts of the hippocampus and above the temporo-parietal cortex. EEG recods were made before and after intraperitoneal injections of several doses of atropine sulphate (BDH, London): 0.2, 0.5 and 1.0 mglkg. On the basis of this experiment the 0.5 mglkg dose had been selected for further studies. It evoked typical EEG changes (disappearance of theta rhythm and appearance of slow irregular activity in hippocampal and cortical leads) developing within 40-60 min after the injection, and an increase in locomotor activity. It did not affect the duration of hippocampal seizure discharges evoked by the stimulation but the following period of suppressed EEG activity was markedly shortened (Fig. 4B).

Methods

Six cats in which the hippocampal stimulation evoked strong inhibition of AAR performance had been selected out of Group I and Group Ia from the preceding experiments. Twenty four additional sessions were performed with these cats in the following succession: (i) one control session (C), (ii) one session preceded by intraperitoneal injection of O.gO/o NaCl solution (NaCl), (iii) one session preceded by NaCl injection and hippocampal stimulation (NaC1 + hipp. stim), (iv) one control session, (v) one session preceded by atropine sulphate injection (At) and one session preceded by injection of atropine and hippocampal stimulation (At f hipp. stim). This procedure was repeated 4 times. Intraperitoneal injections of atropine and NaCl were performed 45-50 min before placing the animals in the shuttle-box. The dose of atropine was 0.5 mglkg and the volume of injected solutions was 3 ml in all cases. Hippocampal stimulation with the use of the same parameters as before was performed immediately after placing the animals in the shuttle-box. In sessions in which the stimlllation was not applied the

first trial was performed 2 min after stimulation or after a 2 min stay in the experimental situation. The US was not used in this experiment.

Results

An analysis of variances (Lindquist, treatment X days X subject design) has revealed that only the effect of treatment was significant (F5,25 = 96.887, P < 0.001). A detailed analysis with the use of Duncan's test has shown that the NaCl and At injections alone did not influence the AAR performance. Application of hippocampal stimulation after NaCl injections caused almost complete inhibition of AAR (P < 0.01 in all comparisons). Atropine injected before hippocampal stimulation caused a significant, but not complete reduction of the inhibitory effect (Fig. 3);

CRIW s ITR 00, 10, I),

Fig. 3. Effect of atropine sulphate (At.) on inhibitory effect of hippocampal stimula- tion on AAR performance. A, not injected control; B, NaCI; C, NaCl + hipp. stim.; D, At.; E, At. + hipp. stim. White bars, conditioned responses; dashed bars,

latency of AAR; black bars, intertrial responses.

the level of AAR performance in . At + hipp. stim. sessions was significantly higher than that in the NaCl + hipp. stim. sessions (P < 0.01) but lower than in the remaining sessions in which the hippocampal stimulation was not applied (P < 0.01 in all comparisons). The ITR number in At sessions was significantly higher than in C, NaCl (P < 0.05) and NaCl 4- hipp. stim. (P < 0.01) sessions. In At + + hipp. stim. sessions the ITR number was significantly higher only in comparison with NaCl + hipp. stim. sessions (P < 0.05). No other differences were discovered.

Stimulation of the hippocampus performed after the NaCl injections evoked symptoms which did not differ from those observed in the preceding experiment, although their intensity was slighty increased as a consequence of repeated stimulations (Fig. 4). Injections of atropine

did not affect the immediate effects of hippocampal stimulation. However, the atropine induced increase in locomotor activity was slightly reduced after stimulation.

Anatomical investigations. The results of the histological analysis are presented in Fig. 5. All electrodes were located in the posterior part of th hippocampus. The placements of electrodes were not always symmetrical and in some cases they were located more posteriorly to the aimed area. However, the differences in effects evoked by hippocampal stimulation within groups were not correlated with the different location of electrodes.

DISCUSSION

The experiments showed that a strong hippocampal stimulation applied once before an experimental session might facilitate or inhibit the AAR performance in a shuttle-box in cats. Although no electroencephalographic control of stimulation was performed in the present studies, the symptoms (stupor followed by a series of very characteristic meows) clearly showed that hippocampal afterdischarges were evoked each time by the stimulation (2, 11, 18, 33). As some authors have suggested the postseizure depression of hippocampal activity (32) or a temporary disruption of other extrahippocampal systems (25) may be the factors responsible for the inhibitory effects. I t is known that apart from temporary changes in hippocampal excitability (14) hippocampal seizures may evoke a reorganization of neuronal activity within diencephalic and basal forebrain areas (15, 36) as well as some changes in the activity of cortical regions (34) lasting for some time after seizures. On the other hand, it has been found that the reactivity of brain stem reticular formation, hypothalamus and septum to electrical stimulation does not decrease after seizures (11). Moreover, the experiments performed by Whishaw and Deatherage (42) as well as in our laboratory (18) have shown that animals can learn and perform immediately after seizures though the performance might be blocked when the response has been learned already. Such effects suggest that motor, cognitive and emotional functions are preserved after hippocampal stimulation. Therefore, the observed changes in performance may be better explained as state dependent phenomena.

In the relevant literature most of the authors reported inhibitory effects, or no changes, in one way (6) as well as in two-way (2, 31, 33) active avoidance situations. The present experiments have shcwn that facilitation of AAR may also occur as a consequence of seizure inducing hippocampal stimulation. The results suggest that the kind of effect

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depends on the stage of training and the level of AAR attained what may account for, at least partially, the differentiation of effects of hippocampal seizures known from literature. Certain results suggesting similar relations have also been obtained in our 'previous studies (18). Thus, the effect of hippocampal seizures on shuttle avoidance in cats has proved to be identical as that observed in rats after ECS application in one-way avoidance situations (8, 42).

Similar, bidirectional effects on the. performance of conditioned avoidance as well as alimentary responses have been obtained by Deutsch after intrahippocampal injections of an anticholinesterase (DFP) in rats (12). According to this author, the process of memorization consists of a gradual increase in the sensitivity of postsynaptic membranes in a set of cholinergic synapses up to a certain optimum. The reverse takes place during forgetting. As he assumes, the factors which are able to increase the amount pf free acetylcholine in the brain (intraperitoneal or intracranial injections of cholinergic agents, ECS) may facilitate performance at an early stage of memorization owing to a facilitation of impulse transmission, or inhibit it when an optimal level of synaptic conductivity has been attained owing to a sustained depolarization caused by an excess of the neurotransmitter (see 12, 13). Since our data fit well with the above pattern of changes in performance '(at least when the effect of the first stimulation is considered) they might be interpreted as a result of an activation (when facilitation of AAR occurred) or deactivation (when an inhibition was observed) of synapses responsible for the memory of AAR. Footing on the above it may be assumed that, apart from the changes appearing on the synapses involved originally in the process of learning, hippocampal stimulation might lead to an increase in excitability of other synapses, normally inexcitable, to a degree which made their participation in the condition- ing possible. Depen,ding on the kind of effect of the first stimulation the activity of these synapses might be assuciated with AAR or its absence. Thus, their activation, when the stimulation was applied for the second time, .might be the factor responsible for the maintenance of a high level of AAR performance (observed in some cats in Experiment 11) or a deepening of the inhibitory effect (Experiment I).

The fact that atropine administration prior to stimulation could lead to a diminution of the inhibitory effects might support the assumption that the stimulation, similarly as ECS (8, 42) and intrahippocampal injections of anticholinesterases (12) influenced performance through an activation of the cholinergic system. All of the data, however do not justify the conclusion that the memory synapses are cholinergic. It is very unlikely that the cholinergic or cholinolytic substances injected

into the hippocampus by Deutsch and his associates (see 12) acted directly on memory synapses; there is no evidence that this structure is the site of memory. Therefore, the changes in performance occurring after such injections should be considered exclusively as an effect of some indirectly evoked changes in the activity of other extrahippocampal areas resulting from an activation or deactivation of hippocarnpal cholinoceptive neurons. Recently, Jaffard et al. (21) have shown that electrical stimulation of the hippocampus may lead to marked changes in the activity of the hippocampal cholinergic system suggesting lts activation during stimulation. It is likely that similar effects may be evoked by ECS what might account for the similarity of changes in performance evoked by these three kinds of interventions. The ability of cholinolytics to reverse the effects evoked by ECS (1, 8, 42), cholinergic (12) or electrical stimulation of the hippocampus might result from a depressing effect of these drugs on the activity of hippocampal cholinoceptive neurons; as some authors have suggested the hippocampus is probably one of the central sites of their action (28, 38). Therefore the results cannot be regarded as evidence that the memory synapse is cholinergic. As to the present experiments there is also a possibility that the diminution of inhibitory effects by atropine was caused by the well known facilitatory effect of cholinolytics on the shuttle-avoidance behavior (5, 27, 35) or, what is less likely, by the photophobia which develops after injections of cholinolytics (35). The fact that the reduction of the inhibitory effect evoked by hippocampal stimulation was rarely complete and sometimes barely noticable might support this supposition. Thus, the possibility cannot be excluded that the inhibitory effects of hippocampal stimulation were only masked by the facilitatory effects of atropine. It is worth mentioning here that muscarinic blockers alone injected intraperitoneally (35) or into the hypothalamus (19) may evoke similar changes in performance of the ~hu t t l e avoidance response as those observed after seizure inducing hippocampal stimulation i.e., they may facilitate avoidance when jts level is low and inhibit this response when its level is high.

The similarity of the results to those obtained with the use oi: cholinolytics in similar situations allows one to interpret them as "desinhibition" phenomena. On the other hand, their similarity to the data obtained in experiments with the use of ECS or intrahippocampal injections of cholinergic substances, where the effects of cholinolytics were opposite, makes it possible to interpret them as a result of an indirect activation or deactivation of memory synapses in the way sug- gested by Deutsch hypothesis. We hope that experiments performed in other experimental situations will help us to explain this problem.

This investigation was supported by Project 10.4.1.01 of the Polish Academy of Sciences.

8

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I

Accepted 5 J u l y 1978

S. GRALEWICZ, K. GRALEWICZ and E. SZCZEPANIAK, Institute of Physiology and Cyto- logy, University of E o d t , Rewolucji 1905 r. no. 66, 90-222 L6d2, Poland.

3 - Acta Neurobiologiae Experimentalis