Adrenalectomy: Protection from kindled convulsion induced dissociation in rats

Physiology & Behavior, Vol. 20, pp. 469--4"74. Pergamon Press and Brain Research Publ., 1978. Printed in the U~S.A.

Adrenalectomy: Protection from Kindled Convulsion Induced Dissociation in Rats DAN C. MCINTYRE AND PHILLIP D. WANN:

Department o f Psychology, Carleton University, Ottawa, Ontario, Canada (Received August 1977) MCINTYRE, D. C. AND P. D. WANN. Adrenalectomy: protection from kindled c6nvulsion induced dissociation in rats. PHYSIOL. BEHAV. 20(4) 469-474, 1978. - In the first experiment, it was demonstrated that a convulsion provoked from a kindled amygdala focus 10 rain before a trial produced a state of dissociation for a one-trial inhibitory avoidance response. In the second experiment, it was shown that bilateral adrenalectomy alleviated the state-dependent recall deficit in the dissociation paradigm. This abatement of the dissociation was near total when the adrenalectomized animals received their training trial in the normal state and their test trial in the postconvulsive state; on the other hand, a significantly less satisfactory abatement was seen in adrenalectornized animals trained in the postconvulsive state but test in the normal state. The adi~-'nalectomy had no effect on the evolution of the kindling or on the motor convulsion and AD, but did influence the weight g.ain. Kindlingper se seemed to induce an adrenal hYpertrophy effect.

Dissociation

Adrenalectomy

Inhibitory avoidance

Kindledconvulsion

IT IS well documented that several convulsant agents such as ECS, kindling and Metrazol not only provoke amnesia for antecedent events (e.g., [9, I0, 16] ), but also induce a state of dissociation for events experienced following the convulsion (e.g., [ 8, 18, 19]. Recently, Mclntyre [ 11 ] has shown that if bilateral adrenalectomy is performed prior to training in a typical retrograde amnesia paradigm, the amnestic consequences of a kindled convulsion are largely eliminated. The adrenalecforay, however, had no effect on the electrographic seizure or the behavioral convulsion, indicating that some compon e n t of the adrenal response to the seizure was mediating the normally seen amnesia. The purpose of the present experiment was to Rrst, determine whether the kindled convulsion would promote dissociation in the inhibitory avoidance paradigm and second, to assess the effect of bilateral adrenalectomy on the kindled convulsion induction of state-dependent learning and on the rate of development of the kindling itself. The kindled convulsion is generally produced through the daily stimulation of a discrete brain site (the limbic system containing, generally the most sensitive sites) (e.g., [5] ). The initial response to such stimulation is minimal, consisting of a brief arrest of movement and/or ipsilateral eye closure. With daffy repetition, the response evolves to its ~inal form, involving bilateral electrographic afterdischarge (AD) and bilateral clonus of the forelimbs with rearing and falling. The development of a kindled focus results in a permanent disposition to a convulsive response when the focus is activated. The structure selected to serve as the kindled focus was the amygdala, since it has been shown that a kindled

amygdala convulsion produces amnesia for a C E R [10] and inhibitory avoidance response [19], as well as statedependency in a T-maze escape paradigm [ 12]. Experiment l was a demonstration of a kindled convulsion induced dissociation in an inhibitory avoidance paradigm (IA) and in Experiment 2 the influence of bilaterial adrenalectomy on the evolution of the convulsion itselfand the dissociated IA response was assessed. EXPERIMENT

The purpose of this experiment was to assess whether the amygdala kindled convulsion would support state-dependent learning in a one-trial inhibitory avoidance (IA) task, as was previously shown in a T-maze escape paradigm [121. METHOD

Animals There were 48 male Wistar rats used in this experiment. The animals were obtained from Biobreeding Laboratories, Ottawa, and weighed between 2 2 5 - 3 0 0 g at the time of surgery. All animals were housed individually and maintained on feed and water ad lib.

Operative and Histological Techniques All animals were anesthetized with Equi-Thesin (3.1 cc/kg) and were implanted with a bipolar electrode in the amygdala. The electrodes were fashioned from 0.127 mm diameter Diamel-insulated nichrome wire soldered to male connectors and coated with Epoxylite for added insulation.

1This relearch was supported by a NRC grant to the senior author. =Present address: Department of Psychology, Missouri Western State College, St. Joseph, MO.

469

l

470 All electrodes were tested for leaks and short circuits both before implantation and after removal from the brain. The electrodes positioned in the amygdala were implanted using the DeGroot [2] stereotaxic coordinates 0.2 rnm posterior to bregma, 4.5 mm lateral to the midtine and 8.5 mm below the skull. Dental cement, anchored by four stainless steel screws, was used to secure the electrodes to the skull. After completion of testing, the implanted animals were sacrificed with chloroform, and the electrode placements histologically verified. Following perfusion with physiological saline and formol saline, the brains were removed and stored in formol saline for at least three days prior to sectioning. Frozen coronal sections of 40 ~ were made through the electrode tract. The sections were mounted and stained with cresyt violet.

Convulsion Development After one week of postoperative recovery, convulsion development was begun. The kindling procedure was similar to that of Goddard et al. [5]. Each animal designated to undergo kindling was stimulated once every 12 hr until the initial convulsion was elicited. The stimulation was a 60 Hz sine wave delivered at an intensity of 50 uA (peak-to-peak) for a duration of 5 sec. A constant current stimulator was used to deliver the stimulation, and the waveform and intensity were monitored on an oscilloscope. A response was counted as a convulsion only if it continued after the termination of the stimulation. After the first convulsion, each animal was stimulated once per day until five additional convulsions had been elicited.

Inhibitor)' Avoidance Training and Testing Behavioral testing was begun one week after completion of convulsion development. The apparatus used in this experiment consisted of a small wooden box (8 inches long × 6 inches wide × 13 inches high) located in one corner of a 2 ft square grid floor with walls 18 inches high. The small box had a 2 inch semicircular opening covered by a removable guillotine door. The grid floor was connected to a constant current stimulator and scrambler programmed to deliver a 1.0 mA (DC) shock of 5 sec duration. On training and test days, each animal was placed into the start box with its head facing away from the guillotine door. Step-out latencies were recorded as the time elapsing between the raising of the door and the animal placing all four paws on the grid. A 300 sec cut-off criterion was imposed on the latency scores. On the initial trial (training), the door was closed upon completion of the response and the grid electrified. The animals were removed from the apparatus immediately after termination of the footshock and returned to their home cage. Animals trained and/or tested in the postconvulsion state were given the convulsion 10 rain before the trial. The 10 min interval was chosen because pilot data had indicated that normal movement returned by this time following a convulsion, yet brain activity was still frequently abnormal (e.g., inter-ictal spiking and low voltage EEG).

Groups Animals experienced their training and two test trials in either the normal state or 10 min after a convulsion, The first and second test trials proceeded 24 and 48 hr after the

MCINTYRE AND WANN training trial. Shock was not administered after etther test trial. Thus an animal trained in the normal state (N) but tested at 24 and 48 hr in the postconvulsion state t(?) was indicated as belonging to group N/C/C. The eight groups used in this experiment were as follows: N/N/N, C/C/C. N/N/C, C/C/N, N/C/C, C / N / N , N/C/N, and C/N/C'.

Statistical Analysis Step-out [atencies were analyzed with nonparametric techniques [i 7]. Mann-Whitney U Tests were performed on the between group data, while the Wilcoxon MatchedPaired Signed-Ranks Test was used to make within group comparisons..MI tests were two-tailed. RESULTS AND DISCUSSION The average number of stimulations to first convulsion was 27.3, with a clonus onset latency of approximately 1 0 - 1 5 sec and a duration of 2 0 - 3 0 sec. This relatively large number of stimulations to fixst convulsion (27.3) was likely a result of kindling at a regime of 2 stimulations/day, as compared with a 1/day (see Experiment 2) [5]. The median step-out latencies for the training and two test trials are presented in Table 1. There were no reliable differences between groups in initial training latencies. Thus, although the postconvulsion state generally promotes slightly greater activity (at 10 rain) than the normal state [18], this was not manifested in a reliable difference in initial step-out latency. On the 24 hr test, all 4 groups which were examined in the same state as training (either N/N or C/C) indicated excellent retention with no differences between them. The 4 groups which experienced a state change between training and the 24 hr test (N/C and C/N), however, showed little evidence of recall with no differences between them. There was no overlap between the same-state versus changed-state conditions. It is quite clear from these data that amygdala provoked convulsions are quite capable of inducing state-dependent learning for an IA task. These data are very similar to those reported by other using ECS as the convutsant [4, 13, 18]. While demonstrating poorer recall than the groups trained and tested in the same state, the groups tested in the altered state to that of training showed some evidence of retention on the first test trial as compared to their initial training trial step out latency (p<0.05). There were no reliable differences between the altered state groups in this respect. This small increase in latency has been reported also in a retrograde amnesia paradigm [11,19] when using the kindled convulsion from an amygdala focus as the amnesia inducer and does not occur if footshock is not administered following the training trial (unpublished data). It would seem, therefore, that the dissociation was not total. The data from the 48 hr test trial were clearly in the direction predicted by the state dependency hypothesis for all groups, but the small sample size and relatively high within group variability at that point precluded statistical confirmation of the differences (see Table I ). EXPERIMENT 2 It was quite apparent from Experiment I that antmals trained and tested in the same state, either normal or postconvulsive, indicated excellent recall of the training experience. On the other hand, those experiencing a state change between training and testing exhibited comparative-

DISSOCIATION PROTECTION BY ADRENALECTOMY

471

TABLE 1 MEDIAN STEP-OUTLATENCIESDURINGTRAININGAND 24 AND 48 HR TESTING Median step-out latency Training 24-hr test 48-hr test

Group

N

Same-state conditions at 24-hr test

NNN NNC CCC CCN

6 6 6 6

26.5 10.5 27.5 25.5

300.0 300.0 300.0 272.5

300.0 88.5 231.5 138.5

Changed-state conditions at 24-hr test

NCC NCN CNN CNC

6 6 6 6

44.0 32.0 28.0 19.0

93.0 29.5 97.5 94.0

111.0 300.0 79.5 161.0

ly little evidence of recall, and thus good evidence of state-dependency. The purpose of this experiment was to examine the effects of bilateral adrenalectomy on the induction of state-dependent learning in the IA paradigm. F o r this reason, animals were subjected only to change-ofstate conditions between training and testing, either C/N or N/C, since same-state conditions would be expected to permit good recall ':egardl~ss of adrenalectomy. METHOD

A n imals There were 42 male Wistar rats used in this experiment. The animals were purchased from Biobreeding Laboratories, Ottawa, and weighed between 3 0 0 - 3 5 0 g at the time of electrode implanation. All bilateral adrenalectomies were performed by the breeder, using the dorsal approach. These animals were maintained on 0.9% saline ad lib. F o o d and water were otherwise available to all animals ad lib.

Surgical and Histological Procedures Surgical procedures were outlined in Experiment 1, with the exception that here a plastic head cap [14] was used for intracranial stimulation and recording. At the termination of the experiment all animals were subjected to etherization followed, in those animals with electrodes, by perfusion with saline and then Formalin. Prior to the perfusion, the descending aorta was clamped off. The adrenal glands were then excised from the animals possessing them (all were examined). The glands were rinsed in saline, blotted on filter paper, cleansed of extraneous tissue and weighed together. The brains were removed 24 hr after perfusion and stored in Formalin before sectioning (see Experiment 1).

Kindling The procedures for kindling were basically the same as in Experiment 1 except that animals were stimulated once every 24 hr. After the first convulsion was elicited, each animal received 5 additional convulsions at 1/day. The convulsion data of interest were the n u m b e r of trials of amygdala after-discharge (AD) to first convulsion, as well as the duration o f the AD and the latencies and durations of the forelimb clonus. All EEG recordings were taken with a 4 channel Grass Model 7 polygraph. A mechanical relay was used to disconnect the leads from the recorder and ground lead

from the animal during stimulation. These were immediately reconnected after the end of the stimulation.

Groups There were 5 basic groups used in this experiment with the first two groups subdivided into 2 groups each. This subdivision was effected only for the IA task. The groups were as follows: (1) Kindled Intact (KE). Kindled animals with intact adrenals, which received a convulsion either 10 rain before training (C/N group) or testing (N/C group). (2) Kindled Adx (KA). Kindled adrenalectomized animals, which received a convulsion either 10 rain before training (C/N group) or testing (N/C group). (3) Kindled Control (KC). K i n d l e d animals with intact adrenals, which did not receive a convulsion during IA training or testing. (4) Normal Adrenalectomized (NA). Adrenalectomized animals which were handled similarly to the kindled animals but did not receive an intracranial electrode. (5) Normal Control (NC}. Intact animals handled similarly to the others', but were surgically intact. After receiving their 6th convulsion, animals were given one week rest prior to the beginning of IA training.

Data Analysis Step-out latencies were analyzed as in Experiment 1. The convulsion and AD data as well as the adrenal weight data were assessed using analysis of variance and when appropriate, the Newraan-Kuels test. RESULTS

Convulsion and AD The adrenalectomized and intact animals developed convulsions with a mean n u m b e r of 19.4 and 15.8 stimulations to first convulsion; the number of trials with amygdala AD to first convulsion for these two groups was 13.5 and 14.8, respectively. There were no reliable differences between these groups and thus adrenalectomy was without influence on the rate o f kindling. Over the first 6 convulsions, latency to onset progressively decreased ( p < 0 . 0 0 0 1 ) and the duration of clonus increased (p<0.0007). The growth of AD also progressed, not only during the kindling phase, but also during the evocation of the 6 convulsions (p<0.028). There were no reliable differences between any of the kindled groups on any o f these measures.

472

MCINTYRE AND WANN TABLE 2

MEAN CONVULSION LATENCIES, DUATIONS AND AD DURATIONS FOR THE FINAL 3 OUT OF 6 CONVULSIONS DURINGTHE KINDLINGREGIME AND THE INHIBITORYAVOIDANCETRIAL

Group

N

Mean of Final 3 convulsions in series of 6 Lat Dur AD

KC KE KA

6 12 12

4.0 4.4 4.9

22.7 20.9 20.4

40. I 46.8 42.7

One convulsion during IA trial Lat

Dur

AD

-4.2 4.5

-2 7 . 5 67.3 26.6 62.6

TABLE 3 MEAN BODY AND ADRENALWEIGHTSFOR ALL GROUPS

Group

N

Mean Body weight (g) Initial Final

KC KE KA NA NC

6 12 12 6 6

310.1 306.6 328.9 315.2 320.6

477.2 485.1 451.7 4.45.8 480.8

Mean Adrenal weight (nag) Wet Weight Relative Weight 43.7 ~ 4.0 44.9 -- 4.2 --37.5 _*3.3

9.2 ± 1.3 9.6 __*1.4

~'• 300 o C

-

-

200

+

0~

,41,1

100

-+

I

+ NC AC KC

°

l

n ~ c/n KA

dqmq n ~ r./n KE

FIG. 1. Median step-out latencies during training (aottt~l) and retention testing for all groups. For abbreviation see text. the state-dependency which normally follows a kindled amygdala convulsion; this prevention was not complete. however, when the altered state preceded training.

Bode and Adrenal Weights 7.8 _+ 1.1

The convulsion provoked prior to the IA training or test trial was compared to the final 3 of 6 convulsions in the original kindling regime. Although there was an increase in the convulsion duration and AD duration after the one week rest period (p<0.01), the latency to onset remained unchanged and there were no differences between any of the groups on these measures. These data are summarized in Table 2.

Inhibitory Avoidance (IA) The initial step-out latencies for all groups were statistically similar. Therefore, the variables of kindling, adrenalectomy or convulsion provocation 10 min prior to training did not reliably influence the initial step-out response. From the data presented in Fig. I, it may be seen that there were no differences between any' of the control groups (NC, AC or KC) in recall of the IA response, all performed at the ceiling level. On the other hand, the two groups of kindled animals with intact adrenal glands exhibited very poor recall of the training experience, independent of whether the convulsion occurred before training (C/N) or before testing (N/C). The kindled adrenalectomized animals, however, showed significantly greater recall of the training experience than did their intact counterparts (N/C = p < 0 . 0 1 ; C/N = p<0.05). The kindled adrenalectomized animals receiving a convulsion before testing (N/C) were not reliably different from the controls (NC, AC, KC), whereas those receiving their convuJsion before training (C/N) exhibited a significant mter~"erc.-.ce with recall compared to the control animals (F<0.05), 7*'were not reliably different in recall from the other kindled adrenalectomized group (N/C). It is certainly ciear from these data that adrenalectomy prevented the induction of

The adrenal weights of the kindled animals (KE, KC) were compared to the nonkindled controls-(NC). As can be seen in Table 3, kindling had a slight but reliable incremental effect on the adrenal weight, as compared to the controls (p<0.01). This was true for both absolute wet weight and relative weight; the latter is expressed as mg adrenal weight/100 g body weight. The initial body weights of the 5 main groups did not differ; however, the weight increment was sigrtificantiy (+o<0.05) greater in the adrenal-intact groups (KE, KC, NC) as compared to the adrenalectomized animals (KA, NA). Kindling per se did not effect any change in body weight independent of adrenalectomy. Therefore, adrenaleetomy attenuated the body weight increment, and kindling augmented the adrenal weights (see Table 3). GENERAL DISCUSSION In Experiment 1, it was clearly demonstrated that convulsions provoked from an amygdaloid focus 10 min prior to acquisition or retention of an IA response induced a marked period of dissociation for that response. This observation is consistent with previous data [ 12] indicating that amygdaloid convulsions provoked a dissociation for a T-maze escape response when tested immediately following the offset of the forellmb-clonus. Thus a period of potential ~'~ate-dependent memory fixation extends from immediately after the convulsion [12] to at least 10 min after the convulsion. It is not known whether the period immediately after and 10 min after the convulsion are equivalent in ~heir state-dependent property(ies). In the present experiment, when animals did not receive a state change between training and testing, their memory expression was excellent, independent of whether the state was normal or postconvulsive. This apparent equivalence of states is likely a ceficction of the simplicity of the IA paradigm. In the T-maze paradigm, improvement of performance in the aitered state preceded more slowly than in the normal state ~12]. This latter difference, however, may reflect non-

DISSOCIATION PROTECTION BY ADRENALECTOMY associative factors. In general, the dissociative data described in Experiment 1, are very similar to the ECS [ 13,18 ] and Metrazol [ 8 ] provoked state-dependency data. In Experiment 2, only changed state conditions between training and the test trial 24 hr later were examined (C/N and N/C). Here again it was shown that in otherwise intact animals, a kindled convulsion induced a marked period of dissociation for the IA response. On the other hand, bilateral adrenalectomy seriously attenuated this dissociation. When training proceded in the normal state and testing in the postconvulsive state (N/C), the abatement of the state-dependency was total for the adrenalectomized animals compared to controls; in the C/N conditions, however, adrenalectomlaed animals exhibited a significant recall deficit compared to controls, but reliably less interference than that exhibited by the corresponding adrenal intact animals. The reason for this apparent assymetrical attenuation of dissociation in the adrenalectomized animals is not clear. With various pharmacological manipulations and ECS, assymetrical dissociation has been reported [4]. It is, therefore, possible that in the present experiment, the convulsion coupled with the lack of adrenal response at the time of training (a) effects a less satisfactory engram than that achieved in the normal state in adrenalectomized animals or in the postconvulsive state in animals with intact adrenals (C/C conditions in Experiment 1), or (b) precipitates a partial state of dissociation which interferes with subsequent retrieval in the normal state. The data indicate, however, that this postconvuisive state in adrenalectomized animals is only disruptive with respect to training (C/N) and not when antecedent to testing (N/C). In this context, it would be of considerable interest to know what the neurohumoral consequences are of a kindled convulsion in intact versus adrenalectomized animals since it has been suggested that different pharmacological manipulations before training versus before testing protect memory expression in the ECS retrograde amnesia paradigm [1] as well as in the ECS + drug state-dependency paradigm [4]. More explicitly, the manipulated change of a transmitter system prior to training may prove detrimental for recall at testing, but that same manipulation at the time of testing (and not training) has no consequence upon the degree of recall. Similarly, a different pharmacological change prior to testing will effect recall but not if provoked prior to training. The nature of the change produced by adrenalectomy, seen in the protection from the induction of dissociation following a kindled amygdaloid convulsion, is not at all clear. In the context of cyclohexamide induced retrograde

473 amnesia, Nakajima has suggested [ 15 ] that the recall deficit at testing is a function of reduced adrenal corticosteroids at training. This suggestion has been weakened recently somewhat by the data of Dunn and Liebrmann [3], indicating that reduced corticosteroidogenesis at training had no observable effect upon recall 24 hr later. Thus, if the mechanisms of retrograde amnesia and dissociation are in some way related, then perhaps the corticosteroid reduction following adrenalectomy is not involved in the abatement of either phenomenon. There are, of course, other roles the adrenal glands might play in these two phenomena (e.g., posttrial catechoiamine release, [6] ) the memory mechanisms-for which are not at all understood. There were no differences between any of the kindled groups in terms of AD duration either during the kindling regime or during the IA procedures, indicating adrenalectomy was without effect on the evolution and evocation of kindled amygdala convulsions. This observation is consistent with other data [ 11 ] indicating adrenalectomy was without effect on previously kindled amygdala convulsions. A partially discordant or qualifying note by Innes [7] however, has indicated that adrenalectomy retards kindling from the amygdala but only in the dark phase of the diurnal cycle. The animals in the present experiment were kindled, trained and tested during the tight phase (1300-1500 hr); Innes [7] similarly found no effect of adrenalectomy on kindling during the light phase. Also, it should be mentioned that Innes used a strain of rat (CFE, Carworth Farms) which is highly seizure prone by comparison to the Wistar used in the present project. Finally, the adrenalectomized animals showed an attenuated growth rate compared to intact animals, which has been observed previously [7,11]. In addition, kindling produced adrenal hypertrophy as seen in the greater adrenal weights compared to controls. It has been previously noted that this kindling provoked hypertrophy is of the adrenal cortex [7]. In summary, a convulsion from an amygdaloid focus produced a period of dissociation which lastsfor at least I0 min after the convulsion. Similarly, there was no decrement in performance, independent of the training state, provided the test trial proceded in the same state as training. On the other hand, bilateral adrenalectomy totally eliminated the dissociation when the training proceded in the normal state (N/C), but was only marginally effective:in abatement of the dissociation when the training occurred in the postconvulsive state and the testing in the normal state

(C/N).

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