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The effects of Type I and Type II corticosteroid receptor agonists on exploratory behavior and spatial memory in the Y-maze
Brain Research 759 Ž1997. 76–83
Research report
The effects of Type I and Type II corticosteroid receptor agonists on exploratory behavior and spatial memory in the Y-maze Cheryl D. Conrad a b
a, )
, Sonia J. Lupien
a,b
, Leila C. Thanasoulis a , Bruce S. McEwen
a
Laboratory of Neuroendocrinology, Box 165, Rockefeller UniÕersity, 1230 York AÕenue, New York, NY 10021, USA Douglas Hospital Research Center-McGill UniÕersity, 6875 BouleÕard Lasalle, Montreal, ´ Que. ´ H4H 1R3, Canada Accepted 4 February 1997
Abstract We investigated the effects of two adrenal steroid agonists on adrenalectomized ŽADX. rats’ performance on the Y-maze. The Y-maze was chosen because memory can be assessed quickly and because it is sensitive to various parameters of exploratory behavior and spatial memory performance. Four days after surgery, ADX rats were injected with aldosterone ŽALDO, a selective Type I receptor agonist., RU362 Ža selective Type II receptor agonist. or sesame vehicle at three different time points Ž120 min prior to Trial 1, immediately after Trial 1 or 120 min after Trial 1.. SHAM-operated rats injected with vehicle were also tested. The results indicate that vehicle-treated ADX rats were impaired on spatial recognition memory compared to SHAM rats. Treatment with ALDO restored spatial recognition memory performance of ADX rats to a level comparable to SHAM-treated rats by acting on acquisition and consolidation, whereas treatment with RU362 did not change the poor spatial recognition memory performance of ADX rats. Discrimination memory was improved only when either agonist was injected prior to the first trial, strongly suggesting a non-selective effect of corticosteroids on discrimination memory such as increasing arousal. A detailed analysis of exploratory behavior showed that both the ALDO- and RU362-treated rats explored the Y-maze more than the ADX and SHAM groups at all periods of the experiment. These results show that the non-specific increase in exploratory behavior induced by replacing corticosteroids targeted at Type I and Type II receptors was used differentially with the ALDO-treated rats learning and consolidating spatial information better than the RU362-treated rats. These data are discussed along with other evidence to suggest that Type II receptors may require the simultaneous occupancy of Type I receptors to affect learning and memory processes. Keywords: Learning; Adrenal steroid; Aldosterone; Type I receptor; Type II receptor; Spatial recognition memory; Discrimination memory
1. Introduction Cognitive deficits following acute administration of corticosteroids or their antagonists have been described in animals and humans w2,3,11,16,23x. These corticosteroid treatment paradigms can be classified as ‘replacement,’ ‘subtractive’ or ‘supplemental’ w14x. The replacement treatment refers to giving corticosteroids to rats whose adrenals have been removed ŽADX., whereas a subtractiÕe treatment involves giving corticosteroid receptor antagonists to intact rats to block specific receptor functions. Abbreviations: ADX, adrenalectomy; ALDO, aldosterone; LTP, long-term potentiation. ) Corresponding author. Fax: q1 Ž212. 327-8634; E-mail: [email protected]
Supplemental treatments involve giving intact animals exogenous corticosteroids, usually glucocorticoids, in order to highly stimulate Type II glucocorticoid receptors. In humans, supplemental effects of corticosteroids on cognition have been manipulated for obvious reasons. Therefore, studies on experimental animals that enable the use of the replacement and subtractive strategies provide an important contribution to our understanding of how the two corticosteroid receptors ŽType I and Type II. may affect behavior. Recently, the subtractive actions of corticosteroids on learning and non-specific memory processes have been investigated in rats w18x and chicks w22x. Oitzl and De Kloet w18x administered Type I and Type II receptor antagonists to intact rats at three time points of testing in the Morris water maze to measure spatial memory acquisi-
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 2 3 6 - 9
C.D. Conrad et al.r Brain Research 759 (1997) 76–83
tion, consolidation and retrieval w18x. The results showed that inactivation of Type I receptors specifically affected the sensory integration of environmental cues Žnon-specific attentional process., while the inactivation of Type II receptors impaired consolidation of acquired information Žspecific memory process.. Similar differential effects of Type I and Type II receptor antagonists have been reported in young chicks using an associational learning paradigm w22x. Taken together, these results suggest that the subtractive actions of corticosteroids have both specific and nonspecific effects on memory via differential activation of Type I and Type II receptors. Whether similar results would be discovered with the replacement paradigm has not been examined. The advantage of investigating the replacement effects of corticosteroids on learning is that the independent contribution of the two corticosteroid receptor subtypes on memory can be separately measured. In the previous studies w18,22x, the inactivation of one receptor subtype by a specific antagonist does not ensure the complete occupancy of the remaining receptor subtype. This is particularly true for Type I receptor antagonists where different stress levels in the animals at the time of testing may lead to large variations in the occupancy of Type II receptors across subjects. Moreover, the experimental paradigms used in the previous studies do not permit a clear differentiation of exploratory vs. spatial memory behaviors, so that the effects of Type I and Type II receptor antagonists on the attentional and mnesic processes were measured and interpreted a posteriori instead of being controlled for a priori. An experimental design in which the replacement effects of corticosteroids on learning can be systematically varied and studied in ADX animals may help overcome some of these potential confounds. In this study, we chose the replacement design in which the activation of each receptor subtype can be manipulated in ADX rats independently of the other receptor. Furthermore, we examined the spatial memory performance of ADX rats injected with either Type I Žaldosterone. or Type II ŽRU362. receptor agonists at three different time points of the experiment; 120 min before the acquisition of the information Žmeasuring the effect of treatment on the acquisition and consolidation process., immediately after the acquisition trial Žmeasuring the effect of treatment on the consolidation process. or 120 min after the acquisition trial Žat time when consolidation has been shown to be completed on this task w5x; measuring the effect of treatment on the recall process.. We used the Y-maze paradigm to measure such behaviors because this task: Ž1. enables memory to be measured quickly before substantial amounts of steroid are metabolized; Ž2. minimizes factors which may influence performance such as motivational or emotional states; and Ž3. has the advantage to measure various parameters of behavior related to spatial exploration Že.g. inspective vs. inquisitive behavior. and spatial memory Že.g. recognition vs. discrimination memory w7x..
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2. Materials and methods 2.1. Subjects Sprague-Dawley male rats were purchased from Charles River Ž200–250 g.. Rats were group housed Ž2–3rcage., maintained on a 12-hr12-h lightrdark cycle Žlights off at 21:00 h. and given rat chow and tap water ad libitum. The rats were allowed to acclimate to the facilities at least 5 days prior to the commencement of the study. 2.2. Y-maze and testing procedure The design and use of the Y-maze has been described previously w5,7x. Briefly, the Y-maze is a two-trial recognition memory test in which performance does not involve the learning of a rule because it taps on an innate tendency of rats to explore new Ži.e. not encountered before. environments. The Y-maze had three identical arms, 50 cm long, 16 cm wide and sides 32 cm high. Visual cues made from colored construction paper and laboratory glassware were placed around the perimeter of the maze and above the top of the black Plexiglas sides. The floor of the maze was covered with soiled animal bedding ŽBeta wood chips.. Rats were tested during the dark phase of their cycle. In Trial 1, one arm was blocked with black Plexiglas and referred to as the ‘novel’ arm in Trial 2. One of the remaining two arms was designated as the ‘start’ arm. The last remaining arm was termed ‘other.’ The blocked, other and start arms were randomized between rats Žbut not within rats. to reduce arm bias effects. At the start of testing, a rat was placed in the start arm and was allowed to explore the start and other arms for 15 min Žacquisition trial.. Entries were manually recorded by an investigator who was not aware of the treatments. Entries into an arm were counted when the rat entered 10 cm of a given arm. At the end of Trial 1, the rat was returned to its home cage and the bedding inside the maze was mixed to reduce the possibility of using odors as a cue. After an intertrial interval ŽITI. of 4 h, the rat was placed in the same start arm as in Trial 1. The previously blocked arm was accessible in Trial 2 and the rat was allowed to investigate all arms for 5 min Žrecall trial.. The dependent variables measured in Trial 2 were: Ž1. first arm entered Žbetween the novel and other arm. which will be referred to as the First Choice; Ž2. the amount of time spent in each arm for each minute ŽDwell.; Ž3. the number of entries made into each arm for each minute ŽEntry.. The First Choice measure reflects the percentage of rats exhibiting recognition of the novel arm Žarm discrimination memory.. The Dwell measure reflects inspective behavior Ži.e. exploratory behavior., while the number of entries reflects inquisitive behavior Ži.e. response to novelty.. Interaction on Dwell andror Entry data between arm choice, treatment and
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period Žtime of injection. reflects spatial recognition memory of the previously unvisited arm w7x.
3. Results 3.1. Preliminary results
2.3. Treatment Aseptic surgical techniques and animal care maintenance were in accordance with the Applicable Portions of the Animal Welfare Act and DHHS ‘Guide for the Care and Use of Laboratory Animals.’ Rats were adrenalectomized or SHAM-operated after being deeply anesthetized with 50 mgrkg sodium pentobarbital i.p. After surgery, the adrenalectomized rats were given a choice of two solutions, both of which contained tap water and 20 m grml corticosterone but one had 3% sodium chloride to help with electrolyte regulation. 48 h prior to Y-maze testing, the corticosterone was removed from the drinking solutions. Y-Maze testing occurred 4 days after surgery and 2 days after the elimination of corticosterone from the drinking solutions. The rats were arbitrarily placed in one of four treatment groups; SHAM Žsesame vehicle.; adrenalectomized ŽADX; sesame vehicle.; adrenalectomizedq aldosterone ŽALDO; 100 m grkg.; adrenalectomizedq RU 28362 ŽRU362; 40 m grkg.. The steroids were injected Žs.c.. at one of the following three time points: 120 min prior to the first Y-maze trial ŽPre., immediately after the first Y-maze trial ŽImm. or 120 min prior to the second Y-maze trial ŽPost.. This led to 12 groups of rats: SHAM-Pre Ž n s 14., SHAM-Imm Ž n s 16., SHAM-Post Ž n s 15., ADX-Pre Ž n s 15., ADX-Imm Ž n s 16., ADX-Post Ž n s 15., ALDOPre Ž n s 14., ALDO-Imm Ž n s 18., ALDO-Post Ž n s 17., RU362-Pre Ž n s 13., RU362-Imm Ž n s 18. and RU362Post Ž n s 14.. The drug doses and 2 h delay between injection and first Y-maze trial was chosen to parallel electrophysiological studies that have demonstrated that these doses and time frame is sufficient to cause specific electrophysiological changes in the hippocampus w19x.
Preliminary analyses performed on Dwell and Entry for Trial 2 showed that differences in rats behaviors were present during Minutes 1 and 2 but were not maintained throughout the 5 min Žno group difference on Period.. Therefore, Dwell and Entries were summed for Minutes 1 and 2 in both experiments. These results are in agreement with Dellu et al. w7x and Conrad et al. w5x. Furthermore, Entry and Dwell for the start and other arms never statistically varied, so the data were averaged for the start and other arm which will be referred to as ‘others.’ Again, these results are in agreement with our earlier findings w5x. The subsequent analyses were performed with Arm ŽNovel, Others. as the only within-subjects factors. 3.2. First Choice (arm discrimination) The number of rats that chose to enter the novel arm immediately after leaving the start arm was analyzed by x 2 analyses ŽTable 1.. Only the rats in the SHAM group consistently entered the novel arm first compared to the other arm at every time period of vehicle injection ŽPre, x 2 s 6.25, P - 0.025; Imm, x 2 s 4.0, P - 0.05; Post, x 2 s 3,27, P - 0.07.. The rats in the ALDO and RU362 groups entered the novel arm first when injected at the Pre time period ŽALDO, x 2 s 9.00, P - 0.005; RU362, x 2 s 8.07, P - 0.005. and Post time period ŽALDO, x 2 s 7.12, P - 0.01; RU362, x 2 s 9.31, P - 0.005. but not the Imm time period. The rats in the ADX group entered the novel arm first when injected with vehicle at the Post time period Ž x 2 s 9.00, P - 0.005. but not at the Pre, nor Imm time periods ŽTable 1.. A log-linear analysis was performed on this data to determine whether a relationship exists between treatment, period and arm for First Choice of entry. A test of
2.4. Statistics Table 1 Discrimination memory ŽFirst Choice.
Dwell and Entries were initially analyzed using a repeated-measures analysis of variance ŽANOVA.. Treatment ŽADX, SHAM, ALDO, RU362. and Period ŽPre, Imm, Post. were the between-subjects variables and Minute Ž1–5. and Arm ŽNovel, Start, Other. were the within-subjects variables. A data transformation using the square-root transformation in addition to a constant variable of 0.5 Žsee Bartlett, 1938. was performed on the Entry data due to the nature of the scale Žnumber of visits in a given time period.. Choice of first arm entered after leaving the start arm was analyzed by two measures, the x 2 test and compared to chance and a log-linear analysis. Data are represented by mean " S.E.M.
Treatment
Pre
Imm
Post
SHAM ADX ALDO RU362
81.3% Ž13r16. a 62.5% Ž10r16. 87.5% Ž14r16. a 86.7% Ž13r15. a
75.0% Ž12r16. a 68.8% Ž11r16. 61.1% Ž11r18. 61.1% Ž11r18.
73.3% Ž11r15. b 87.5% Ž14r16. a 82.4% Ž14r17. a 92.3% Ž12r13. a
Percentage of rats entering the novel arm first after leaving the start arm. First number in parentheses indicates the number of rats that entered the novel arm first and the latter number indicates the total number of rats tested. a P - 0.05, compared to chance Ž50%.. b P s 0.075, compared to chance Ž50%.. Note, there were 7 more rats analyzed for the first choice measure than for the other analyses because these rats made 1 entry after leaving the start arm but did not continue to explore the maze.
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marginal and partial association showed that arm was significant as a main effect Ž x 2 s 51.2, df s 1, P 0.0001. and period with arm was significant as an interaction Ž x 2 s 5.2, df s 2, P s 0.075.. Therefore, a model for a log-linear analysis was performed with a main effect of arm and an interaction between arm with period. The model showed an extremely good fit ŽPearson’s x 2 s 6.88, df s 18, P s 0.991.. First, this model indicates that there is a strong arm component and that rats preferred to enter the novel arm first compared to the other arm, regardless of treatment. Second, marginal table comparisons of this model also indicates that rats injected at the Pre and Post injection times performed similarly but were different from rats injected at the Imm period. Rats injected at the Imm period tended to enter the novel arm first at chance whereas rats injected at the Pre and Post periods entered the novel arm first compared to the other arm. 3.3. Dwell: main effects (inspectiÕe behaÕior) The analysis of Dwell revealed that the SHAM group was less inspective than the ALDO and RU362 groups. The ALDO and RU362 groups spent more time in the arms, as opposed to the center of the maze, when compared to the rats in the SHAM group Ž F3,173 s 3.2, P 0.02.. The behavior of the ADX group fell in between these two groups ŽFig. 1.. All rats spent less time visiting arms on the recall trial when they were injected before the first Y-maze trial ŽPre. than when they were injected later ŽImm or Post, F2,173 s 11.3, P - 0.0001.. 3.4. Dwell: interaction effects (spatial recognition memory) The repeated variable of Arm was significant and interacted significantly with Treatment and Period Žthird-order
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Fig. 2. Spatial recognition memory for dwell. Comparison of the time spent in the novel and other arms during Minutes 1 and 2 for each period ŽPre, Imm, Post.. The SHAM and ALDO groups spent more time in the novel arm compared to the other arm at all periods tested, whereas the ADX and RU362 groups did not spend more time in any arm until the Post period. All groups spent more time in the Novel arm during the Post period which indicates a non-specific effect of injection on recall of spatial recognition memory. ) P - 0.05 compared to the adjacent other arm of the same group. Data are represented as mean"S.E.M.
interaction, F6,173 s 2.1, P - 0.05.. A posteriori analysis breaking this interaction by Treatment revealed that rats in both the SHAM and ALDO groups spent more time in the novel arm, regardless of whether they were injected at Pre, Imm or Post Žsignificant main effect of Arm in both treatments; SHAM, F1,42 s 9.5, P - 0.004; ALDO, F1,46 s 13.6, P - 0.001, with no significant interaction between Period and Arm for either treatment; Fig. 2.. A different pattern of results was noted for the rats in the ADX and RU362 groups. In these groups, the rats did not spend more time in any given arm when they were injected at the Pre or Imm time points. However, when they were injected at the Post time point, the rats spent more time in the Novel arm compared to the Other arms Žsignificant Period by Arm interaction for both treatments, F2,43 s 13.6, P 0.006 for ADX and F2,42 s 15.6, P - 0.0001 for RU362.. 3.5. Entries: main effects (inquisitiÕe behaÕior)
Fig. 1. Inspective behavior. The total amount of time spent in the arms of the Y-maze Žas opposed to the center of the Y-maze. during Minutes 1 and 2. The ALDO and RU362 groups spent more time in the arms compared to the SHAM group indicating that the agonist treatment increased curiosity. P - 0.05 compared to SHAM. Data are represented as mean"S.E.M.
The analysis of Entry showed that the rats in all four groups were more inquisitive by entering more arms, when injected at the Pre time point than when injected at the Imm or Post time points Ž F2,174 s 16.4, P - 0.001.. No significant main effect of Treatment was observed but a significant effect of Arm was noted, showing that all rats tended to enter the Novel arm significantly more often that the Other arms Ž F1,174 s 185, P - 0.0001. and this effect was 4 = stronger for the Post time point of injection compared to the Pre time point Žinteraction between Period and Arm, F2,174 s 13.5, P - 0.001..
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Fig. 3. Spatial recognition memory for entry. Comparison of the number of entries made into the arms during Minutes 1 and 2. The data were transformed as described in Materials and methods. The rats entered the novel arm more often than the other arms at all injection time points but this effect was 4.5= stronger for the Post injection time period compared to the Pre injection time period Ž c s more entries into novel arm during Post injection compared to Pre injection, regardless of treatment.. Data are represented as mean"S.E.M.
Ž r s 0.23; ns. and the other arms Ž r s 0.25, ns. did not reach significance showing that a speed-accuracy trade-off could not explain the absence of similarity between Dwell and Entry data. Another explanation for this result could be that the scale for Entry data was too narrow to allow variability between rats’ performance under these testing circumstances Žrats never entered more than 4 = in a given arm during 1 min.. In order to determine whether the data on Dwell correlated with data on Entry for arm preference, a delta measure for arm preference Žscore on novel arm minus score on other arms. was calculated for both Dwell and Entry and correlated with each other. The correlation coefficient for arm preference between the Dwell and Entry data was significant Ž r s 0.55; R 2 s 0.30, P 0.001. and showed that the rats’ preference for a given arm as measured by Dwell was correlated to the rats’ preference for a given arm as measured by Entry. This result increased our confidence that both Dwell and Entry were measures of spatial recognition memory, suggesting that the narrowness of the scale for Entry data may explain the absence of a third-order interaction for the measure of Entry.
3.6. Entries: interaction effects (spatial recognition memory)
4. Discussion
A significant interaction between Period and Arm Ž F2,174 s 13.5, P - 0.001. showed that, although rats preferred the novel arm at each period, this effect was 4.5 = stronger when rats were injected at the Post time point Ž F s 30.1 and F s 32.4 for the Pre and Imm groups respectively and F s 143.2 for the Post group.. No thirdorder interaction was observed between Treatment, Period and Arm ŽFig. 3..
A novel finding in this study is that corticosteroid effects on memory and exploratory behavior, as assessed by the replacement paradigm, do not affect all aspects of Y-maze behavior in the same way. That is, ALDO and RU362 have selective effects on certain components of Y-maze behavior such as spatial recognition memory but not on other aspects of Y-maze behavior such as discrimination, inspective and inquisitive abilities. For spatial recognition memory, ALDO improved the performance of ADX rats to the SHAM level whereas RU362 did not improve the performance of ADX rats. With regard to the other behaviors measured, inspective behavior appeared to be sensitive to either agonist treatment whereas inquisitive ability and discrimination memory was not affected. Therefore, spatial recognition memory on the Y-maze can be selectiÕely improved in ADX rats by activating the Type I Žbut not Type II. receptors.
3.7. Dwell Õs. Entries (comparison of spatial recognition memory measures) Although treatment differences on spatial recognition memory were observed for Dwell data, no such treatment effect was obtained for Entry data. The main effect of treatment observed for Dwell and Entry suggests the existence of a speed-accuracy trade-off which could explain this discrepancy. A speed-accuracy trade-off is defined as the negative correlation between a measure of speed and a measure of performance w1,4,20x. The main effect of treatment observed for Dwell and Entry showed that rats in the ALDO and RU362 groups visited as many arms but stayed in them longer when injected at the Imm and Post periods than when injected at the Pre period. In order to see if such a speed-accuracy trade-off could explain the absence of similarity between the Dwell and Entry data, a Spearman’s correlation analysis was performed between Dwell and Entry data for both the novel and other arms. The correlation between Dwell and Entry data for the novel arm
4.1. Summary of results
4.2. Exploratory behaÕior and spatial recognition memory The results obtained from the two measures of exploratory behavior Žinspective and inquisitive. for overall performance do not completely parallel each other. The absence of a main effect of group observed for Entry showed that all rats were inquisitive or reacted similarly to novelty and, hence, eliminates the potential confound that differences between treatments were because one group was more or less anxious than another. However, a main effect of treatment for Dwell Žinspective behavior. was
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observed with the RU362 and ALDO groups in that they spent more time in the arms whereas the SHAM and ADX groups spent more time in the center of the Y-maze. This indicates a non-specific effect of corticosteroid receptor agonists that acts primarily by increasing exploratory behavior Žand, by extension, arousal and curiosity. level. The Dwell data obtained on spatial recognition memory behavior extend the exploratory behavior results and show that the increase in exploratory behavior induced by treatment of Type I or Type II corticosteroid receptor agonist was used differently by the ALDO and RU362 group to acquire, consolidate and recall the spatial information. When injected before the acquisition trial ŽPre. or immediately after ŽImm., the ALDO and RU362 behaved differently on the Y-maze, with the ALDO rats performing as well as the controls by spending more time in the novel arm, whereas the RU362 rats performed as poorly as the ADX rats. This result shows that the increase in inspective behavior observed for the ALDO and RU362 rats was used proficiently by the ALDO group to acquire andror consolidate spatial information, whereas the increase in exploration of the RU362 group did not result in improved spatial recognition memory. With regard to the Post period of injection, the ALDO and RU362 groups performed similarly to the SHAM group. Although this result may suggest at first sight that treatment with Type I or Type II corticosteroid receptor agonists do not affect the recall process, the results of the ADX group, which also showed a good spatial memory performance when injected with sesame oil before the recall trial, eliminates such an interpretation. The ADX group did not show a preference for the novel arm when injected before or immediately after the acquisition trial but ADX rats preferred the novel arm when injected before the retrieval period. Since all rats presented this effect when injected at the Post period and the only difference between this time period and the others is the novelty or arousing effect of the injection Žsesame oil or steroid., we have to suggest that the injection by itself induced an increase in arousal which had a positive effect on the recall of information. One way to confirm this in future studies would be to submit the rats to an arousing stimulus Že.g. tail pinch. at a similar time and then to test the effects of this arousal on recall performance. Furthermore, these data indicate that the procedure of adrenalectomy does not completely eliminate spatial recognition memory, as ADX rats can perform in the Y-maze task as well as controls if the conditions allow it Ži.e. by increasing the level of arousal near the time of spatial recognition memory retrieval.. 4.3. Arm discrimination memory The SHAM group was able to recognize the novel arm at every time period, thus, confirming the validity of this measure for recognition memory. However, the ALDO and
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RU362 groups performed identically by entering the novel arm first when they were injected before the acquisition trial ŽPre. or just before recall ŽPost. but they did not enter the novel arm significantly above chance when injected immediately after the acquisition trial ŽImm.. The ADX rats performed the worst by entering the novel arm only when injected before recall ŽPost. but not at the other test periods. Because the performance of the agonist-treated rats was not different than the ADX rats when injected after the acquisition trial for both consolidation ŽImm. and recall ŽPost., one can conclude that the steroids were not specifically improving discriminative memory but were acting indirectly such as changing arousal. The log-linear analysis model supports this hypothesis by indicating that the rats who entered the novel arm on their First Choice were those who received an injection within 2 h of being placed on the Y-maze ŽPre and Post groups. but not when injections were 4 h prior to the rats being placed on the Y-maze ŽImm group.. Furthermore, these data show that discriminative memory as assessed by the Y-maze cannot be differentially modified by activating one specific corticosteroid receptor subtype even through non-specific arousal effects. 4.4. Spatial recognition memory Õs. discrimination memory It may seem strange that the arm discrimination data ŽFirst Choice measure., which is one gauge of memory, lead to different conclusions than the spatial recognition memory data Žinteraction on Dwell and Entry data.. However, the measure of First Choice seems to be testing discrimination memory which is not dependent upon an intact hippocampus whereas the measures of Dwell and Entry appear to be testing a form of memory called spatial recognition memory which is dependent upon an intact hippocampus Žfor a review, see w9x.. The First Choice measure of the Y-maze closely resembles the spontaneously alternated choice observed after one or two forced-choices in the T-maze recognition task. This kind of discriminative memory is generally not impaired in hippocampally damaged animals Žalthough the results of the literature on sensory discrimination are still mixed w10,17x.. Under such conditions that strongly hinder comparisons between cues and eliminates differential choice responses, rats with hippocampal damage perform similar to that of normal rats Žfor a review, see w9x.. In the Y-maze, the rats are required to make a similar comparison in that, when they leave the start arm, they would make a choice between entering a familiar or unfamiliar arm. Under these conditions, Type I or Type II receptor activation had no selective effect on discrimination memory. In contrast, the Dwell and Entry measures of the Y-maze are similar to place learning of the Morris water maze in which the ability to construct, maintain and make use of representational maps are necessary for rats to determine
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their location. This kind of spatial memory is impaired in hippocampal damaged rats. The hippocampus contains one of the highest levels of corticosteroid receptors in the brain w15,21x and is involved in spatial recognition memory w5x, thus, making the hippocampus a likely mediator of these adrenal steroid effects. The results of the present study support the concept that the modulatory actions of corticosteroid agonists are sensitive to spatial, hippocampal-relevant memory but not to discriminative, hippocampal-irrelevant, memory. 4.5. Role of Type I and Type II receptors in spatial memory acquisition, consolidation and retrieÕal These findings are consistent with the hypothesis that Type I receptor activation is involved in sensory integration of spatial environmental cues w18x. Furthermore, Type II receptor activation did not improve spatial memory which is consistent with the hypothesis that Type II receptor activation is associated with the consolidation process of spatial memory formation w18,22x and how these two receptors may interact with each other to affect cognition is discussed in the next section. The negative effects of corticosteroids on cognition may be caused by the inactivation of an attentional process implicated in the acquisition and learning of information. The deleterious effects of corticosteroids on memory could then be the extension of this impaired attentional process, preventing a successful acquisition of the information and, by extrapolation, a successful consolidation and recall of it. The latter suggestion also goes along with results in human subjects showing that endogenous w13x as well as exogenous w12x increases in cortisol levels, although impairing memory, also impairs the process of selective attention. Selective attention is the capacity to discriminate between relevant and irrelevant stimuli and an impairment of this nature will easily prevent the successful acquisition of useful information and may explain how corticosteroids can cause memory impairments w14x. 4.6. Type I and Type II receptors and memory: an oÕerÕiew This study strengthens the hypothesis that corticosteroids and cognitive abilities have an inverted-U relationship by acting via Type I and Type II receptors. Serum corticosterone levels have been shown to have an invertedU relationship with cognition Žfor a review, see w14x. and hippocampal excitability w8x. At very low or high serum corticosterone levels, memory performance is impaired whereas moderate serum corticosterone levels correlate with normal memory performance. Furthermore, the two corticosteroid receptors are hypothesized to be contributing to this non-linear relationship as the two receptors have different affinities for corticosterone, with the Type I receptor exhibiting the higher affinity for corticosterone and the Type II receptor exhibiting the lower affinity.
Therefore, under non-stressful conditions, when corticosteroid levels are low and memory is unimpaired, the Type I receptors are highly occupied whereas the Type II receptors are moderately occupied. But, during stress, when corticosteroid levels are high and memory is impaired, the Type II receptors are highly occupied which suggest a detrimental effect of Type II receptor activation on memory. Our results are consistent with the notion that the inverted-U relationship between serum corticosterone and memory may be selective for spatial recognition memory and, hence, be dependent upon cognitive demands in which the hippocampus plays a critical role. In addition, the results of the present study using the replacement paradigm and the results of the subtractiÕe approach Žsee Introduction. indicate a more complicated role of the two receptors and the non-linear relationship they have with spatial recognition memory. In several studies, we have observed that the Type II receptors demonstrate the capacity to impair spatial recognition memory only when the Type I receptors are occupied. For example, we have found with the replacement paradigm that ADX rats treated with RU362 Žhence, Type II but not Type I receptors were occupied. did not show a change in spatial recognition memory performance compared to vehicle-treated ADX rats. In another study using the subtractiÕe approach, however, intact rats injected with a Type II receptor antagonist Žand, hence, the Type II receptors were unoccupied while the Type I receptors were occupied. were spatial recognition memory impaired w6x Žsee also w18,22x. Furthermore, we found that, even when rats were given high doses of corticosterone Žand, hence, both receptors were highly occupied., spatial recognition memory was impaired w6x. Therefore, the predominate effect of the high occupancy of the Type II receptors was to impair spatial recognition memory when the Type I receptors were occupied but not when the Type I receptors were unoccupied. These results strongly suggest that the Type I receptors must be occupied and functioning in order for the Type II receptors to produce a biphasic enhancement at moderate occupancy levels and then a suppression of spatial recognition memory at high levels of occupancy. A caveat is that ADX rats in our replacement paradigm were already spatial recognition memory impaired and, hence, if RU362 was causing spatial recognition memory impairment, then we might not be able to detect this difference due to a ‘bottom effect’ of our paradigm. Consequently, future studies should be planned to investigate the possible interaction between the two receptors at different occupancy levels and their effects on spatial recognition memory in several kinds of spatial tasks.
Acknowledgements Preliminary results were presented at the 1997 American Physiological Society Conference. Maurice Aubourg’s
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and Nicholas Hasting’s assistance with the surgeries and Dr. Monnie McGee Harper’s statistical advice is gratefully acknowledged. This research was supported by NIH PostDoctoral Grant MH10804 Žto C.D.C.., Fonds de la Recherche en Sante´ du Quebec ´ Scientist Award Žto S.J.L.., and NS Grant 07080 Žto B.S.M... References w1x J.S. Andrews, J.H. Jansen, S. Linders, A. Princen, C.L. Brockkamp, Performance of four strains in the autoshaping, two-object discrimination and swim-tests of learning and memory, Physiol. Behav. 57 Ž1995. 785–790. w2x B.E. Beckwith, T.V. Petros, C. Scaglione, J. Nelson, Dose-dependent effects of hydrocortisone on memory in human males, Physiol. Behav. 36 Ž1986. 283–286. w3x B. Bohus, K. Lissak, Adrenocortical hormones and avoidance behaviour of rats, Int. J. Neuropharmacol. 7 Ž1968. 301–306. w4x J.R. Busemeyer, J.T. Townsend, Decision field theory: a dynamiccognitive approach to decision making on uncertain environment, Psychol. Rev. 100 Ž1993. 432–459. w5x C.D. Conrad, L.A.M. Galea, Y. Kuroda, B.S. McEwen, Chronic stress impairs rats spatial memory on the Y-maze and this effect is blocked by tianeptine pre-treatment, Behav. Neurosci. 110 Ž1996. 1321–1334. w6x C.D. Conrad, S.J. Lupien, B.S. McEwen, Glucocorticoid ŽGC., but not mineralocorticoid ŽMC., receptor agonist and antagonist block spatial memory performance on the Y-maze, Soc. Neurosci. Abstr. 22 Ž1996. 2061. w7x F. Dellu, W. Mayo, J. Cherkaoui, M. LeMoal, H. Simon, A two-trial memory task with automated recording: study in young and aged rats, Brain Res. 588 Ž1992. 132–139. w8x D.M. Diamond, M.C. Bennett, M. Fleshner, G.M. Rose, Inverted-U relationship between the level of peripheral corticosterone and the magnitude of hippocampal primed burst potentiation, Hippocampus 2 Ž1992. 421–430. w9x H. Eichenbaum, T. Otto, N.J. Cohen, Two functional components of the hippocampal memory system, Behav. Brain Sci. 17 Ž1994. 449–518. w10x J.A. Gray, N. McNaughton, Comparison between behavioral effects
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of septal and hippocampal lesions: a review, Neurosci. Biobehav. Rev. 7 Ž1983. 119–188. C. Kirschbaum, O.T. Wolf, M. May, W. Wippich, D.H. Hellhammer, Stress and drug-induced elevation of cortisol levels impair explicit memory in healthy adults, Life Sci. 58 Ž1996. 1475–1483. S. Lupien, C. Gillin, D. Frakes, S. Soefje, R.L. Hauger, M.J. Meaney and P.V. Nair, Delayed but not immediate effects of a 100 minutes hydrocortisone infusion on declarative memory performance in young normal adults, Int. Soc. Psychoneuroendocrinol. Abstr., 25 Ž1995.. S. Lupien, A.R. Lecours, I. Lussier, G. Schwartz, N.P.V. Nair, M.J. Meaney, Basal cortisol levels and cognitive deficits in human aging, J. Neurosci. 14 Ž1994. 2893–2903. S.J. Lupien and B.S. McEwen, The acute effects of corticosteroids on cognition: integration of animal and human model studies, Brain Res. Rev., in press. B.S. McEwen, J.M. Weiss, L.S. Schwartz, Selective retention of corticosterone by limbic structure in rat brain, Nature 220 Ž1968. 911–912. J.W. Newcomer, S. Craft, T. Hershey, K. Askins, M.E. Bardgett, Glucocorticoid-induced impairment in declarative memory performance in adult humans, J. Neurosci. 14 Ž1994. 2047–2053. J. O’Keefe and L. Nadel, The Hippocampus as a Cognitive Map, Clarendon, Oxford, UK, 1978. M.S. Oitzl, E.R. De Kloet, Selective corticosteroid antagonists modulate specific aspects of spatial orientation learning, Behav. Neurosci. 106 Ž1992. 62–71. C. Pavlides, A. Kimura, A.M. Magarinos, ˜ B.S. McEwen, Hippocampal homosynaptic long-term depressionrdepotentiation induced by adrenal steroids, Neuroscience 68 Ž1995. 379–385. W. Read, V.I. Nenov, E. Halgren, Role of inhibition in memory retrieval by hippocampal area CA3, Neurosci. Biobehav. Rev. 18 Ž1994. 55–68. J.M.H.M. Reul, E.R. De Kloet, Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation, Endocrinology 116 Ž1985. 2505–2511. C. Sandi, S.P.R. Rose, Corticosterone enhances long-term retention in one-day-old chicks trained in a weak passive avoidance learning paradigm, Brain Res. 647 Ž1994. 106–112. O.M. Wolkowitz, V.I. Reus, H. Weingartner, K. Thompson, A. Breier, A. Doran, D. Rubinow, D. Pickar, Cognitive effects of corticosteroids, Am. J. Psychiatry 147 Ž1990. 1297–1303.
Research report
The effects of Type I and Type II corticosteroid receptor agonists on exploratory behavior and spatial memory in the Y-maze Cheryl D. Conrad a b
a, )
, Sonia J. Lupien
a,b
, Leila C. Thanasoulis a , Bruce S. McEwen
a
Laboratory of Neuroendocrinology, Box 165, Rockefeller UniÕersity, 1230 York AÕenue, New York, NY 10021, USA Douglas Hospital Research Center-McGill UniÕersity, 6875 BouleÕard Lasalle, Montreal, ´ Que. ´ H4H 1R3, Canada Accepted 4 February 1997
Abstract We investigated the effects of two adrenal steroid agonists on adrenalectomized ŽADX. rats’ performance on the Y-maze. The Y-maze was chosen because memory can be assessed quickly and because it is sensitive to various parameters of exploratory behavior and spatial memory performance. Four days after surgery, ADX rats were injected with aldosterone ŽALDO, a selective Type I receptor agonist., RU362 Ža selective Type II receptor agonist. or sesame vehicle at three different time points Ž120 min prior to Trial 1, immediately after Trial 1 or 120 min after Trial 1.. SHAM-operated rats injected with vehicle were also tested. The results indicate that vehicle-treated ADX rats were impaired on spatial recognition memory compared to SHAM rats. Treatment with ALDO restored spatial recognition memory performance of ADX rats to a level comparable to SHAM-treated rats by acting on acquisition and consolidation, whereas treatment with RU362 did not change the poor spatial recognition memory performance of ADX rats. Discrimination memory was improved only when either agonist was injected prior to the first trial, strongly suggesting a non-selective effect of corticosteroids on discrimination memory such as increasing arousal. A detailed analysis of exploratory behavior showed that both the ALDO- and RU362-treated rats explored the Y-maze more than the ADX and SHAM groups at all periods of the experiment. These results show that the non-specific increase in exploratory behavior induced by replacing corticosteroids targeted at Type I and Type II receptors was used differentially with the ALDO-treated rats learning and consolidating spatial information better than the RU362-treated rats. These data are discussed along with other evidence to suggest that Type II receptors may require the simultaneous occupancy of Type I receptors to affect learning and memory processes. Keywords: Learning; Adrenal steroid; Aldosterone; Type I receptor; Type II receptor; Spatial recognition memory; Discrimination memory
1. Introduction Cognitive deficits following acute administration of corticosteroids or their antagonists have been described in animals and humans w2,3,11,16,23x. These corticosteroid treatment paradigms can be classified as ‘replacement,’ ‘subtractive’ or ‘supplemental’ w14x. The replacement treatment refers to giving corticosteroids to rats whose adrenals have been removed ŽADX., whereas a subtractiÕe treatment involves giving corticosteroid receptor antagonists to intact rats to block specific receptor functions. Abbreviations: ADX, adrenalectomy; ALDO, aldosterone; LTP, long-term potentiation. ) Corresponding author. Fax: q1 Ž212. 327-8634; E-mail: [email protected]
Supplemental treatments involve giving intact animals exogenous corticosteroids, usually glucocorticoids, in order to highly stimulate Type II glucocorticoid receptors. In humans, supplemental effects of corticosteroids on cognition have been manipulated for obvious reasons. Therefore, studies on experimental animals that enable the use of the replacement and subtractive strategies provide an important contribution to our understanding of how the two corticosteroid receptors ŽType I and Type II. may affect behavior. Recently, the subtractive actions of corticosteroids on learning and non-specific memory processes have been investigated in rats w18x and chicks w22x. Oitzl and De Kloet w18x administered Type I and Type II receptor antagonists to intact rats at three time points of testing in the Morris water maze to measure spatial memory acquisi-
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 2 3 6 - 9
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tion, consolidation and retrieval w18x. The results showed that inactivation of Type I receptors specifically affected the sensory integration of environmental cues Žnon-specific attentional process., while the inactivation of Type II receptors impaired consolidation of acquired information Žspecific memory process.. Similar differential effects of Type I and Type II receptor antagonists have been reported in young chicks using an associational learning paradigm w22x. Taken together, these results suggest that the subtractive actions of corticosteroids have both specific and nonspecific effects on memory via differential activation of Type I and Type II receptors. Whether similar results would be discovered with the replacement paradigm has not been examined. The advantage of investigating the replacement effects of corticosteroids on learning is that the independent contribution of the two corticosteroid receptor subtypes on memory can be separately measured. In the previous studies w18,22x, the inactivation of one receptor subtype by a specific antagonist does not ensure the complete occupancy of the remaining receptor subtype. This is particularly true for Type I receptor antagonists where different stress levels in the animals at the time of testing may lead to large variations in the occupancy of Type II receptors across subjects. Moreover, the experimental paradigms used in the previous studies do not permit a clear differentiation of exploratory vs. spatial memory behaviors, so that the effects of Type I and Type II receptor antagonists on the attentional and mnesic processes were measured and interpreted a posteriori instead of being controlled for a priori. An experimental design in which the replacement effects of corticosteroids on learning can be systematically varied and studied in ADX animals may help overcome some of these potential confounds. In this study, we chose the replacement design in which the activation of each receptor subtype can be manipulated in ADX rats independently of the other receptor. Furthermore, we examined the spatial memory performance of ADX rats injected with either Type I Žaldosterone. or Type II ŽRU362. receptor agonists at three different time points of the experiment; 120 min before the acquisition of the information Žmeasuring the effect of treatment on the acquisition and consolidation process., immediately after the acquisition trial Žmeasuring the effect of treatment on the consolidation process. or 120 min after the acquisition trial Žat time when consolidation has been shown to be completed on this task w5x; measuring the effect of treatment on the recall process.. We used the Y-maze paradigm to measure such behaviors because this task: Ž1. enables memory to be measured quickly before substantial amounts of steroid are metabolized; Ž2. minimizes factors which may influence performance such as motivational or emotional states; and Ž3. has the advantage to measure various parameters of behavior related to spatial exploration Že.g. inspective vs. inquisitive behavior. and spatial memory Že.g. recognition vs. discrimination memory w7x..
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2. Materials and methods 2.1. Subjects Sprague-Dawley male rats were purchased from Charles River Ž200–250 g.. Rats were group housed Ž2–3rcage., maintained on a 12-hr12-h lightrdark cycle Žlights off at 21:00 h. and given rat chow and tap water ad libitum. The rats were allowed to acclimate to the facilities at least 5 days prior to the commencement of the study. 2.2. Y-maze and testing procedure The design and use of the Y-maze has been described previously w5,7x. Briefly, the Y-maze is a two-trial recognition memory test in which performance does not involve the learning of a rule because it taps on an innate tendency of rats to explore new Ži.e. not encountered before. environments. The Y-maze had three identical arms, 50 cm long, 16 cm wide and sides 32 cm high. Visual cues made from colored construction paper and laboratory glassware were placed around the perimeter of the maze and above the top of the black Plexiglas sides. The floor of the maze was covered with soiled animal bedding ŽBeta wood chips.. Rats were tested during the dark phase of their cycle. In Trial 1, one arm was blocked with black Plexiglas and referred to as the ‘novel’ arm in Trial 2. One of the remaining two arms was designated as the ‘start’ arm. The last remaining arm was termed ‘other.’ The blocked, other and start arms were randomized between rats Žbut not within rats. to reduce arm bias effects. At the start of testing, a rat was placed in the start arm and was allowed to explore the start and other arms for 15 min Žacquisition trial.. Entries were manually recorded by an investigator who was not aware of the treatments. Entries into an arm were counted when the rat entered 10 cm of a given arm. At the end of Trial 1, the rat was returned to its home cage and the bedding inside the maze was mixed to reduce the possibility of using odors as a cue. After an intertrial interval ŽITI. of 4 h, the rat was placed in the same start arm as in Trial 1. The previously blocked arm was accessible in Trial 2 and the rat was allowed to investigate all arms for 5 min Žrecall trial.. The dependent variables measured in Trial 2 were: Ž1. first arm entered Žbetween the novel and other arm. which will be referred to as the First Choice; Ž2. the amount of time spent in each arm for each minute ŽDwell.; Ž3. the number of entries made into each arm for each minute ŽEntry.. The First Choice measure reflects the percentage of rats exhibiting recognition of the novel arm Žarm discrimination memory.. The Dwell measure reflects inspective behavior Ži.e. exploratory behavior., while the number of entries reflects inquisitive behavior Ži.e. response to novelty.. Interaction on Dwell andror Entry data between arm choice, treatment and
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period Žtime of injection. reflects spatial recognition memory of the previously unvisited arm w7x.
3. Results 3.1. Preliminary results
2.3. Treatment Aseptic surgical techniques and animal care maintenance were in accordance with the Applicable Portions of the Animal Welfare Act and DHHS ‘Guide for the Care and Use of Laboratory Animals.’ Rats were adrenalectomized or SHAM-operated after being deeply anesthetized with 50 mgrkg sodium pentobarbital i.p. After surgery, the adrenalectomized rats were given a choice of two solutions, both of which contained tap water and 20 m grml corticosterone but one had 3% sodium chloride to help with electrolyte regulation. 48 h prior to Y-maze testing, the corticosterone was removed from the drinking solutions. Y-Maze testing occurred 4 days after surgery and 2 days after the elimination of corticosterone from the drinking solutions. The rats were arbitrarily placed in one of four treatment groups; SHAM Žsesame vehicle.; adrenalectomized ŽADX; sesame vehicle.; adrenalectomizedq aldosterone ŽALDO; 100 m grkg.; adrenalectomizedq RU 28362 ŽRU362; 40 m grkg.. The steroids were injected Žs.c.. at one of the following three time points: 120 min prior to the first Y-maze trial ŽPre., immediately after the first Y-maze trial ŽImm. or 120 min prior to the second Y-maze trial ŽPost.. This led to 12 groups of rats: SHAM-Pre Ž n s 14., SHAM-Imm Ž n s 16., SHAM-Post Ž n s 15., ADX-Pre Ž n s 15., ADX-Imm Ž n s 16., ADX-Post Ž n s 15., ALDOPre Ž n s 14., ALDO-Imm Ž n s 18., ALDO-Post Ž n s 17., RU362-Pre Ž n s 13., RU362-Imm Ž n s 18. and RU362Post Ž n s 14.. The drug doses and 2 h delay between injection and first Y-maze trial was chosen to parallel electrophysiological studies that have demonstrated that these doses and time frame is sufficient to cause specific electrophysiological changes in the hippocampus w19x.
Preliminary analyses performed on Dwell and Entry for Trial 2 showed that differences in rats behaviors were present during Minutes 1 and 2 but were not maintained throughout the 5 min Žno group difference on Period.. Therefore, Dwell and Entries were summed for Minutes 1 and 2 in both experiments. These results are in agreement with Dellu et al. w7x and Conrad et al. w5x. Furthermore, Entry and Dwell for the start and other arms never statistically varied, so the data were averaged for the start and other arm which will be referred to as ‘others.’ Again, these results are in agreement with our earlier findings w5x. The subsequent analyses were performed with Arm ŽNovel, Others. as the only within-subjects factors. 3.2. First Choice (arm discrimination) The number of rats that chose to enter the novel arm immediately after leaving the start arm was analyzed by x 2 analyses ŽTable 1.. Only the rats in the SHAM group consistently entered the novel arm first compared to the other arm at every time period of vehicle injection ŽPre, x 2 s 6.25, P - 0.025; Imm, x 2 s 4.0, P - 0.05; Post, x 2 s 3,27, P - 0.07.. The rats in the ALDO and RU362 groups entered the novel arm first when injected at the Pre time period ŽALDO, x 2 s 9.00, P - 0.005; RU362, x 2 s 8.07, P - 0.005. and Post time period ŽALDO, x 2 s 7.12, P - 0.01; RU362, x 2 s 9.31, P - 0.005. but not the Imm time period. The rats in the ADX group entered the novel arm first when injected with vehicle at the Post time period Ž x 2 s 9.00, P - 0.005. but not at the Pre, nor Imm time periods ŽTable 1.. A log-linear analysis was performed on this data to determine whether a relationship exists between treatment, period and arm for First Choice of entry. A test of
2.4. Statistics Table 1 Discrimination memory ŽFirst Choice.
Dwell and Entries were initially analyzed using a repeated-measures analysis of variance ŽANOVA.. Treatment ŽADX, SHAM, ALDO, RU362. and Period ŽPre, Imm, Post. were the between-subjects variables and Minute Ž1–5. and Arm ŽNovel, Start, Other. were the within-subjects variables. A data transformation using the square-root transformation in addition to a constant variable of 0.5 Žsee Bartlett, 1938. was performed on the Entry data due to the nature of the scale Žnumber of visits in a given time period.. Choice of first arm entered after leaving the start arm was analyzed by two measures, the x 2 test and compared to chance and a log-linear analysis. Data are represented by mean " S.E.M.
Treatment
Pre
Imm
Post
SHAM ADX ALDO RU362
81.3% Ž13r16. a 62.5% Ž10r16. 87.5% Ž14r16. a 86.7% Ž13r15. a
75.0% Ž12r16. a 68.8% Ž11r16. 61.1% Ž11r18. 61.1% Ž11r18.
73.3% Ž11r15. b 87.5% Ž14r16. a 82.4% Ž14r17. a 92.3% Ž12r13. a
Percentage of rats entering the novel arm first after leaving the start arm. First number in parentheses indicates the number of rats that entered the novel arm first and the latter number indicates the total number of rats tested. a P - 0.05, compared to chance Ž50%.. b P s 0.075, compared to chance Ž50%.. Note, there were 7 more rats analyzed for the first choice measure than for the other analyses because these rats made 1 entry after leaving the start arm but did not continue to explore the maze.
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marginal and partial association showed that arm was significant as a main effect Ž x 2 s 51.2, df s 1, P 0.0001. and period with arm was significant as an interaction Ž x 2 s 5.2, df s 2, P s 0.075.. Therefore, a model for a log-linear analysis was performed with a main effect of arm and an interaction between arm with period. The model showed an extremely good fit ŽPearson’s x 2 s 6.88, df s 18, P s 0.991.. First, this model indicates that there is a strong arm component and that rats preferred to enter the novel arm first compared to the other arm, regardless of treatment. Second, marginal table comparisons of this model also indicates that rats injected at the Pre and Post injection times performed similarly but were different from rats injected at the Imm period. Rats injected at the Imm period tended to enter the novel arm first at chance whereas rats injected at the Pre and Post periods entered the novel arm first compared to the other arm. 3.3. Dwell: main effects (inspectiÕe behaÕior) The analysis of Dwell revealed that the SHAM group was less inspective than the ALDO and RU362 groups. The ALDO and RU362 groups spent more time in the arms, as opposed to the center of the maze, when compared to the rats in the SHAM group Ž F3,173 s 3.2, P 0.02.. The behavior of the ADX group fell in between these two groups ŽFig. 1.. All rats spent less time visiting arms on the recall trial when they were injected before the first Y-maze trial ŽPre. than when they were injected later ŽImm or Post, F2,173 s 11.3, P - 0.0001.. 3.4. Dwell: interaction effects (spatial recognition memory) The repeated variable of Arm was significant and interacted significantly with Treatment and Period Žthird-order
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Fig. 2. Spatial recognition memory for dwell. Comparison of the time spent in the novel and other arms during Minutes 1 and 2 for each period ŽPre, Imm, Post.. The SHAM and ALDO groups spent more time in the novel arm compared to the other arm at all periods tested, whereas the ADX and RU362 groups did not spend more time in any arm until the Post period. All groups spent more time in the Novel arm during the Post period which indicates a non-specific effect of injection on recall of spatial recognition memory. ) P - 0.05 compared to the adjacent other arm of the same group. Data are represented as mean"S.E.M.
interaction, F6,173 s 2.1, P - 0.05.. A posteriori analysis breaking this interaction by Treatment revealed that rats in both the SHAM and ALDO groups spent more time in the novel arm, regardless of whether they were injected at Pre, Imm or Post Žsignificant main effect of Arm in both treatments; SHAM, F1,42 s 9.5, P - 0.004; ALDO, F1,46 s 13.6, P - 0.001, with no significant interaction between Period and Arm for either treatment; Fig. 2.. A different pattern of results was noted for the rats in the ADX and RU362 groups. In these groups, the rats did not spend more time in any given arm when they were injected at the Pre or Imm time points. However, when they were injected at the Post time point, the rats spent more time in the Novel arm compared to the Other arms Žsignificant Period by Arm interaction for both treatments, F2,43 s 13.6, P 0.006 for ADX and F2,42 s 15.6, P - 0.0001 for RU362.. 3.5. Entries: main effects (inquisitiÕe behaÕior)
Fig. 1. Inspective behavior. The total amount of time spent in the arms of the Y-maze Žas opposed to the center of the Y-maze. during Minutes 1 and 2. The ALDO and RU362 groups spent more time in the arms compared to the SHAM group indicating that the agonist treatment increased curiosity. P - 0.05 compared to SHAM. Data are represented as mean"S.E.M.
The analysis of Entry showed that the rats in all four groups were more inquisitive by entering more arms, when injected at the Pre time point than when injected at the Imm or Post time points Ž F2,174 s 16.4, P - 0.001.. No significant main effect of Treatment was observed but a significant effect of Arm was noted, showing that all rats tended to enter the Novel arm significantly more often that the Other arms Ž F1,174 s 185, P - 0.0001. and this effect was 4 = stronger for the Post time point of injection compared to the Pre time point Žinteraction between Period and Arm, F2,174 s 13.5, P - 0.001..
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Fig. 3. Spatial recognition memory for entry. Comparison of the number of entries made into the arms during Minutes 1 and 2. The data were transformed as described in Materials and methods. The rats entered the novel arm more often than the other arms at all injection time points but this effect was 4.5= stronger for the Post injection time period compared to the Pre injection time period Ž c s more entries into novel arm during Post injection compared to Pre injection, regardless of treatment.. Data are represented as mean"S.E.M.
Ž r s 0.23; ns. and the other arms Ž r s 0.25, ns. did not reach significance showing that a speed-accuracy trade-off could not explain the absence of similarity between Dwell and Entry data. Another explanation for this result could be that the scale for Entry data was too narrow to allow variability between rats’ performance under these testing circumstances Žrats never entered more than 4 = in a given arm during 1 min.. In order to determine whether the data on Dwell correlated with data on Entry for arm preference, a delta measure for arm preference Žscore on novel arm minus score on other arms. was calculated for both Dwell and Entry and correlated with each other. The correlation coefficient for arm preference between the Dwell and Entry data was significant Ž r s 0.55; R 2 s 0.30, P 0.001. and showed that the rats’ preference for a given arm as measured by Dwell was correlated to the rats’ preference for a given arm as measured by Entry. This result increased our confidence that both Dwell and Entry were measures of spatial recognition memory, suggesting that the narrowness of the scale for Entry data may explain the absence of a third-order interaction for the measure of Entry.
3.6. Entries: interaction effects (spatial recognition memory)
4. Discussion
A significant interaction between Period and Arm Ž F2,174 s 13.5, P - 0.001. showed that, although rats preferred the novel arm at each period, this effect was 4.5 = stronger when rats were injected at the Post time point Ž F s 30.1 and F s 32.4 for the Pre and Imm groups respectively and F s 143.2 for the Post group.. No thirdorder interaction was observed between Treatment, Period and Arm ŽFig. 3..
A novel finding in this study is that corticosteroid effects on memory and exploratory behavior, as assessed by the replacement paradigm, do not affect all aspects of Y-maze behavior in the same way. That is, ALDO and RU362 have selective effects on certain components of Y-maze behavior such as spatial recognition memory but not on other aspects of Y-maze behavior such as discrimination, inspective and inquisitive abilities. For spatial recognition memory, ALDO improved the performance of ADX rats to the SHAM level whereas RU362 did not improve the performance of ADX rats. With regard to the other behaviors measured, inspective behavior appeared to be sensitive to either agonist treatment whereas inquisitive ability and discrimination memory was not affected. Therefore, spatial recognition memory on the Y-maze can be selectiÕely improved in ADX rats by activating the Type I Žbut not Type II. receptors.
3.7. Dwell Õs. Entries (comparison of spatial recognition memory measures) Although treatment differences on spatial recognition memory were observed for Dwell data, no such treatment effect was obtained for Entry data. The main effect of treatment observed for Dwell and Entry suggests the existence of a speed-accuracy trade-off which could explain this discrepancy. A speed-accuracy trade-off is defined as the negative correlation between a measure of speed and a measure of performance w1,4,20x. The main effect of treatment observed for Dwell and Entry showed that rats in the ALDO and RU362 groups visited as many arms but stayed in them longer when injected at the Imm and Post periods than when injected at the Pre period. In order to see if such a speed-accuracy trade-off could explain the absence of similarity between the Dwell and Entry data, a Spearman’s correlation analysis was performed between Dwell and Entry data for both the novel and other arms. The correlation between Dwell and Entry data for the novel arm
4.1. Summary of results
4.2. Exploratory behaÕior and spatial recognition memory The results obtained from the two measures of exploratory behavior Žinspective and inquisitive. for overall performance do not completely parallel each other. The absence of a main effect of group observed for Entry showed that all rats were inquisitive or reacted similarly to novelty and, hence, eliminates the potential confound that differences between treatments were because one group was more or less anxious than another. However, a main effect of treatment for Dwell Žinspective behavior. was
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observed with the RU362 and ALDO groups in that they spent more time in the arms whereas the SHAM and ADX groups spent more time in the center of the Y-maze. This indicates a non-specific effect of corticosteroid receptor agonists that acts primarily by increasing exploratory behavior Žand, by extension, arousal and curiosity. level. The Dwell data obtained on spatial recognition memory behavior extend the exploratory behavior results and show that the increase in exploratory behavior induced by treatment of Type I or Type II corticosteroid receptor agonist was used differently by the ALDO and RU362 group to acquire, consolidate and recall the spatial information. When injected before the acquisition trial ŽPre. or immediately after ŽImm., the ALDO and RU362 behaved differently on the Y-maze, with the ALDO rats performing as well as the controls by spending more time in the novel arm, whereas the RU362 rats performed as poorly as the ADX rats. This result shows that the increase in inspective behavior observed for the ALDO and RU362 rats was used proficiently by the ALDO group to acquire andror consolidate spatial information, whereas the increase in exploration of the RU362 group did not result in improved spatial recognition memory. With regard to the Post period of injection, the ALDO and RU362 groups performed similarly to the SHAM group. Although this result may suggest at first sight that treatment with Type I or Type II corticosteroid receptor agonists do not affect the recall process, the results of the ADX group, which also showed a good spatial memory performance when injected with sesame oil before the recall trial, eliminates such an interpretation. The ADX group did not show a preference for the novel arm when injected before or immediately after the acquisition trial but ADX rats preferred the novel arm when injected before the retrieval period. Since all rats presented this effect when injected at the Post period and the only difference between this time period and the others is the novelty or arousing effect of the injection Žsesame oil or steroid., we have to suggest that the injection by itself induced an increase in arousal which had a positive effect on the recall of information. One way to confirm this in future studies would be to submit the rats to an arousing stimulus Že.g. tail pinch. at a similar time and then to test the effects of this arousal on recall performance. Furthermore, these data indicate that the procedure of adrenalectomy does not completely eliminate spatial recognition memory, as ADX rats can perform in the Y-maze task as well as controls if the conditions allow it Ži.e. by increasing the level of arousal near the time of spatial recognition memory retrieval.. 4.3. Arm discrimination memory The SHAM group was able to recognize the novel arm at every time period, thus, confirming the validity of this measure for recognition memory. However, the ALDO and
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RU362 groups performed identically by entering the novel arm first when they were injected before the acquisition trial ŽPre. or just before recall ŽPost. but they did not enter the novel arm significantly above chance when injected immediately after the acquisition trial ŽImm.. The ADX rats performed the worst by entering the novel arm only when injected before recall ŽPost. but not at the other test periods. Because the performance of the agonist-treated rats was not different than the ADX rats when injected after the acquisition trial for both consolidation ŽImm. and recall ŽPost., one can conclude that the steroids were not specifically improving discriminative memory but were acting indirectly such as changing arousal. The log-linear analysis model supports this hypothesis by indicating that the rats who entered the novel arm on their First Choice were those who received an injection within 2 h of being placed on the Y-maze ŽPre and Post groups. but not when injections were 4 h prior to the rats being placed on the Y-maze ŽImm group.. Furthermore, these data show that discriminative memory as assessed by the Y-maze cannot be differentially modified by activating one specific corticosteroid receptor subtype even through non-specific arousal effects. 4.4. Spatial recognition memory Õs. discrimination memory It may seem strange that the arm discrimination data ŽFirst Choice measure., which is one gauge of memory, lead to different conclusions than the spatial recognition memory data Žinteraction on Dwell and Entry data.. However, the measure of First Choice seems to be testing discrimination memory which is not dependent upon an intact hippocampus whereas the measures of Dwell and Entry appear to be testing a form of memory called spatial recognition memory which is dependent upon an intact hippocampus Žfor a review, see w9x.. The First Choice measure of the Y-maze closely resembles the spontaneously alternated choice observed after one or two forced-choices in the T-maze recognition task. This kind of discriminative memory is generally not impaired in hippocampally damaged animals Žalthough the results of the literature on sensory discrimination are still mixed w10,17x.. Under such conditions that strongly hinder comparisons between cues and eliminates differential choice responses, rats with hippocampal damage perform similar to that of normal rats Žfor a review, see w9x.. In the Y-maze, the rats are required to make a similar comparison in that, when they leave the start arm, they would make a choice between entering a familiar or unfamiliar arm. Under these conditions, Type I or Type II receptor activation had no selective effect on discrimination memory. In contrast, the Dwell and Entry measures of the Y-maze are similar to place learning of the Morris water maze in which the ability to construct, maintain and make use of representational maps are necessary for rats to determine
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their location. This kind of spatial memory is impaired in hippocampal damaged rats. The hippocampus contains one of the highest levels of corticosteroid receptors in the brain w15,21x and is involved in spatial recognition memory w5x, thus, making the hippocampus a likely mediator of these adrenal steroid effects. The results of the present study support the concept that the modulatory actions of corticosteroid agonists are sensitive to spatial, hippocampal-relevant memory but not to discriminative, hippocampal-irrelevant, memory. 4.5. Role of Type I and Type II receptors in spatial memory acquisition, consolidation and retrieÕal These findings are consistent with the hypothesis that Type I receptor activation is involved in sensory integration of spatial environmental cues w18x. Furthermore, Type II receptor activation did not improve spatial memory which is consistent with the hypothesis that Type II receptor activation is associated with the consolidation process of spatial memory formation w18,22x and how these two receptors may interact with each other to affect cognition is discussed in the next section. The negative effects of corticosteroids on cognition may be caused by the inactivation of an attentional process implicated in the acquisition and learning of information. The deleterious effects of corticosteroids on memory could then be the extension of this impaired attentional process, preventing a successful acquisition of the information and, by extrapolation, a successful consolidation and recall of it. The latter suggestion also goes along with results in human subjects showing that endogenous w13x as well as exogenous w12x increases in cortisol levels, although impairing memory, also impairs the process of selective attention. Selective attention is the capacity to discriminate between relevant and irrelevant stimuli and an impairment of this nature will easily prevent the successful acquisition of useful information and may explain how corticosteroids can cause memory impairments w14x. 4.6. Type I and Type II receptors and memory: an oÕerÕiew This study strengthens the hypothesis that corticosteroids and cognitive abilities have an inverted-U relationship by acting via Type I and Type II receptors. Serum corticosterone levels have been shown to have an invertedU relationship with cognition Žfor a review, see w14x. and hippocampal excitability w8x. At very low or high serum corticosterone levels, memory performance is impaired whereas moderate serum corticosterone levels correlate with normal memory performance. Furthermore, the two corticosteroid receptors are hypothesized to be contributing to this non-linear relationship as the two receptors have different affinities for corticosterone, with the Type I receptor exhibiting the higher affinity for corticosterone and the Type II receptor exhibiting the lower affinity.
Therefore, under non-stressful conditions, when corticosteroid levels are low and memory is unimpaired, the Type I receptors are highly occupied whereas the Type II receptors are moderately occupied. But, during stress, when corticosteroid levels are high and memory is impaired, the Type II receptors are highly occupied which suggest a detrimental effect of Type II receptor activation on memory. Our results are consistent with the notion that the inverted-U relationship between serum corticosterone and memory may be selective for spatial recognition memory and, hence, be dependent upon cognitive demands in which the hippocampus plays a critical role. In addition, the results of the present study using the replacement paradigm and the results of the subtractiÕe approach Žsee Introduction. indicate a more complicated role of the two receptors and the non-linear relationship they have with spatial recognition memory. In several studies, we have observed that the Type II receptors demonstrate the capacity to impair spatial recognition memory only when the Type I receptors are occupied. For example, we have found with the replacement paradigm that ADX rats treated with RU362 Žhence, Type II but not Type I receptors were occupied. did not show a change in spatial recognition memory performance compared to vehicle-treated ADX rats. In another study using the subtractiÕe approach, however, intact rats injected with a Type II receptor antagonist Žand, hence, the Type II receptors were unoccupied while the Type I receptors were occupied. were spatial recognition memory impaired w6x Žsee also w18,22x. Furthermore, we found that, even when rats were given high doses of corticosterone Žand, hence, both receptors were highly occupied., spatial recognition memory was impaired w6x. Therefore, the predominate effect of the high occupancy of the Type II receptors was to impair spatial recognition memory when the Type I receptors were occupied but not when the Type I receptors were unoccupied. These results strongly suggest that the Type I receptors must be occupied and functioning in order for the Type II receptors to produce a biphasic enhancement at moderate occupancy levels and then a suppression of spatial recognition memory at high levels of occupancy. A caveat is that ADX rats in our replacement paradigm were already spatial recognition memory impaired and, hence, if RU362 was causing spatial recognition memory impairment, then we might not be able to detect this difference due to a ‘bottom effect’ of our paradigm. Consequently, future studies should be planned to investigate the possible interaction between the two receptors at different occupancy levels and their effects on spatial recognition memory in several kinds of spatial tasks.
Acknowledgements Preliminary results were presented at the 1997 American Physiological Society Conference. Maurice Aubourg’s
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and Nicholas Hasting’s assistance with the surgeries and Dr. Monnie McGee Harper’s statistical advice is gratefully acknowledged. This research was supported by NIH PostDoctoral Grant MH10804 Žto C.D.C.., Fonds de la Recherche en Sante´ du Quebec ´ Scientist Award Žto S.J.L.., and NS Grant 07080 Žto B.S.M... References w1x J.S. Andrews, J.H. Jansen, S. Linders, A. Princen, C.L. Brockkamp, Performance of four strains in the autoshaping, two-object discrimination and swim-tests of learning and memory, Physiol. Behav. 57 Ž1995. 785–790. w2x B.E. Beckwith, T.V. Petros, C. Scaglione, J. Nelson, Dose-dependent effects of hydrocortisone on memory in human males, Physiol. Behav. 36 Ž1986. 283–286. w3x B. Bohus, K. Lissak, Adrenocortical hormones and avoidance behaviour of rats, Int. J. Neuropharmacol. 7 Ž1968. 301–306. w4x J.R. Busemeyer, J.T. Townsend, Decision field theory: a dynamiccognitive approach to decision making on uncertain environment, Psychol. Rev. 100 Ž1993. 432–459. w5x C.D. Conrad, L.A.M. Galea, Y. Kuroda, B.S. McEwen, Chronic stress impairs rats spatial memory on the Y-maze and this effect is blocked by tianeptine pre-treatment, Behav. Neurosci. 110 Ž1996. 1321–1334. w6x C.D. Conrad, S.J. Lupien, B.S. McEwen, Glucocorticoid ŽGC., but not mineralocorticoid ŽMC., receptor agonist and antagonist block spatial memory performance on the Y-maze, Soc. Neurosci. Abstr. 22 Ž1996. 2061. w7x F. Dellu, W. Mayo, J. Cherkaoui, M. LeMoal, H. Simon, A two-trial memory task with automated recording: study in young and aged rats, Brain Res. 588 Ž1992. 132–139. w8x D.M. Diamond, M.C. Bennett, M. Fleshner, G.M. Rose, Inverted-U relationship between the level of peripheral corticosterone and the magnitude of hippocampal primed burst potentiation, Hippocampus 2 Ž1992. 421–430. w9x H. Eichenbaum, T. Otto, N.J. Cohen, Two functional components of the hippocampal memory system, Behav. Brain Sci. 17 Ž1994. 449–518. w10x J.A. Gray, N. McNaughton, Comparison between behavioral effects
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