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Delayed spatial response alternation: Effects of delay-interval duration and lesions of the medial prefrontal cortex on response accuracy of male and female Wistar rats
Behavioural Brain Research, 18 (1985) 41-49 Elsevier
41
BBR00500
D E L A Y E D S P A T I A L R E S P O N S E A L T E R N A T I O N : E F F E C T S OF D E L A Y - I N T E R V A L DURATION AND LESIONS OF THE MEDIAL PREFRONTAL CORTEX ON RESPONSE ACCURACY OF MALE AND FEMALE WISTAR RATS*
FRANS VAN HAAREN, JAN P.C. DE BRUIN, ROB P.W. HEINSBROEK and NANNE E. VAN DE POLL Netherlands Institute for Brain Research, Amsterdam (The Netherlands) (Received February 16th, 1985) (Revised version received August 6th, 1985) (Accepted August 23rd, 1985) Key words: delayed response alternation - delay interval duration - lesion of medial prefrontal cortex - response accuracy male - female - rat
A delayed spatial response alternation procedure was used to assess behavioural differences between male and female Wistar rats, assumed to involve memory functioning. In Expt. I, subjects were required to alternate responses between two levers in an operant environment. The delay between response opportunities was varied between 1, 3, 7.5 and 15 s in different experimental conditions. Incorrect responses produced a time-out from experimental contingencies for the duration of the currently active delay interval. Response accuracy decreased for males as well as females as the duration of the delay interval was increased. Performance improved as subjects were exposed to the different delay interval durations during consecutive trials. Sex differences in behavioural accuracy were not observed. In Expt. II, some subjects who participated in Expt. I received lesions of the medial prefrontal cortex, while others were control-operated. When re-exposed to the 1 and 7.5 s delay conditions of the first experiment, lesioned subjects at first behaved less accurately than control-operated subjects. Accuracy, however, improved after prolonged exposure to the experimental conditions. Sex differences in behaviour after surgery could not be observed.
EXPERIMENT I Behavioural differences between male and female rats have been observed in a n u m b e r o f different learning tasks (for review see ref. 3). F o r instance, sex differences have been reported when rats were exposed to active 4 and passive avoid a n c e procedures 21-23, to taste aversion procedures 5 or to simple schedules o f reinforcement 2. In recent yea~s, research efforts have more and more focused on procedures which allow investigators to examine m e m o r y aspects o f behaviour, on the assumption that m e m o r y for recent and past events plays an important role in behavioural
adaptation to an ever-changing and increasingly complex environment (e.g. ref. 18). F o r m e m o r y to be inferred from the observable behaviour o f a subject, its behaviour at time X has to be under discriminative control o f stimuli which were present at time X - t. Consequently, the discriminative stimuli which are part o f the repertoirecontrolling environment, are not physically present at the time o f response execution. Despite the development o f a n u m b e r o f experimental procedures which satisfy these requirements (delayedmatching-to-sample, radial m a z e and T - m a z e alternation procedures), sex differences in behaviour have not been extensively investigated.
* Some of the data were presented at the Eighth Annual Meeting of the European Neuroscience Association, September 1984, The Hague, The Netherlands. Correspondence: F. van Haaren, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands.
42 This is even more intriguing since basic differences have been postulated between males and females, even at the human level, in cognitive abilities assumed to play an important role in memory functioning. Sex differences in maze learning have been observed in a few studies (e.g. refs. 1, 7). These experiments showed males to be more accurate than females. It has been suggested that sex differences in spatial-perceptual abilities, well-established in marl 16, might also be involved in the observation of behavioural differences between the sexes in maze learning experiments 3. The present experiment was designed to investigate whether or not male and female Wistar rats behave differently in a delayed spatial response alternation procedure, designed to allow for the possibility that differences in memory capacities are reflected in differential decrements in response accuracy. Spatial response alternation tasks have frequently been used to test the effects of different variables on memory capacities in males 6"I0"15'19. The present procedure was adapted from T-maze spatial response alternation procedures, in order to be able to deal effectively with the oestrusrelated behavioural variability 3 usually observed in females. This behavioural variability was experimentally controlled by exposing both male and female subjects to the different experimental conditions for a prolonged period of time. It has been shown in other experiments 1° that response accuracy of males in an operant spatial response alternation task decreases as the duration of the delay interval between presentation of the discriminative stimulus and the opportunity to behave increases. Therefore, male and female Wistar rats were exposed to an operant spatial response alternation procedure in which the duration of the delay interval was manipulated during different conditions of the first experiment.
Method Subjects. Five male and 5 female Wistar rats were obtained from Animal House TNO (Zeist, The Netherlands), when they were 9 weeks old. Upon arrival at the laboratory, they were individually housed and adapted to a reversed light-dark cycle (lights on from 13.30 to 01.30 h). A 23-h food deprivation schedule 11, resulting in a
deprivation to approximately 85 ~o of free-feeding b.wt., was in effect for the duration of the experiment. Experimentation started two weeks after the subjects arrived at the laboratory. Apparatus. Four standard Grason-Stadler (model 111 l-L) rodent operant conditioning chambers were used. Each chamber was made of plexiglass with an aluminium backwall and measured 28 x 30 × 30.5 cm (inside dimensions). The gridfloor consisted of 23 stainless steel grids, spaced 1.25 cm apart. Two non-retractable rodent levers (1.25 cm thick) protruded 1.60 cm into the chamber from the intelligence panel. They were located symmetrically to the side of the pellet retrieval unit and were spaced 13 cm apart (center to center). The levers required a force of at least 0.25 N to be operated. A green (left lever) and a red (right lever) stimulus light were mounted slightly to the side and above each lever. A houselight was located in the upper lefthand comer of the intelligence panel. Each chamber was enclosed in a Grason-Stadler (model 1101) research chest which masked extraneous sounds. A fan provided fresh air. Programming of experimental contingencies and data acquisition were accomplished by means of Grason-Stadler 1200 series programming equipment, located in the experimental room itself.
Procedure Preliminary training. All subjects were first adapted to the experimental chamber and the sound of the pellet feeder for two sessions; they were observed to retrieve the pellet (45 mg Noyes) from the pellet unit at the end of the second session. In the next condition, one of the lights next to one of the levers was illuminated after the expiration of a variable-time (VT) 30-s intertrial interval (ITI). Which fight above which lever was illuminated alternated daily. A press on the lever above which the light was illuminated produced a pellet. Responses during the ITI had no scheduled consequences. Sessions were run 5 days a week (Monday through Friday) and terminated either after 60 pellets had been delivered, or after 30 min, whichever came fn'st. All subjects responded reliably on both levers after 6 sessions in this procedure.
43
Experimental conditions. Subjects were then exposed to a procedure in which both levers were active and responses on alternating levers were reinforced. The houselight and both stimulus lights (one above each lever) were illuminated at the start of the session. A response on the left lever (correct at first) produced a food pellet, extinguished both stimulus lights and started the delay interval. A response on the right lever (not correct at first) produced a time-out (TO) equal to the duration of the delay interval. During TO, houselight and stimulus lights were extinguished. After the expiration of the delay interval, houselight and stimulus lights were again illuminated. TO kept being presented until a response on the correct lever occurred. Which lever was correct and which lever was not correct alternated after every correct response. Responses during TO and the delay interval had no scheduled consequences. Sessions lasted 30 min, or until 60 pellets had been presented, whichever came first. The duration of the delay interval was varied in different experimental conditions. All subjects were exposed to each experimental condition for 20 sessions, which were run 5 days a week, from Monday through Friday, during the last quarter of the subjects' dark hours. The sequence of experimental conditions is summarized in Table I. Results and Discussion Fig. 1 shows, for males and females, response accuracy (number of correct responses/number of correct + incorrect responses) during the different experimental conditions, averaged over two consecutive sessions. One standard deviation is indicated at each datapoint. The data of the final two sessions are also presented in Fig. 2 to facilitate comparison of steady-state behaviour at the different durations of the delay interval. Response accuracy was analyzed using a 3-factorial analysis of variance on the factors sex, duration of delay interval and trials, the latter two variables being repeated measures. As can be seen in Figs. 1 and 2, response accuracy of both males and females was a function of the duration of the delay interval; response accuracy decreased as the duration of the delay interval was increased, F~.24 = 70.67, P < 0.001. Response accuracy
TABLE I
The sequence of experimental conditions in Exp. I (20 sessions each) I
2
3
4
Male subjects 1 2 3 4 5
3 3 3 15 15
15 15 15 3 3
7.5 7.5 7.5 1 1
1 1 1 7.5 7.5
Female subjects 1 2 3 4 5
3 3 15 15 15
15 15 3 3 3
7.5 7.5 1 1 1
1 1 7.5 7.5 7.5
within the different delay interval conditions increased for both sexes with repeated exposure to the experimental conditions, F9.72 = 44.30, P < 0.001. A significant delay by trial interaction, F27,2~6 = 1.92, P < 0.01 suggested that the improvement in response accuracy may have been delay-dependent, although such interaction is not immediately obvious by visual inspection of the data presented in Fig. 1. Even though the data presented in Figs. 1 and 2 suggested that the response accuracy between the sexes may have differed at the 15-s delay interval duration, neither the main effect of sex nor the interaction of sex and delay interval duration reached statistical significance, F~.s = 0.91, n.s. and F3.24 = 1.91, n.s., respectively. As in other procedures designed to measure the discriminative control of behaviour by stimuli which are no longer physically present at the time of response execution ~°, response accuracy of both male and female subjects decreased with increases in the delay interval in the present experiment. This observation suggests that indeed memory aspects were involved in determining response accuracy of the subjects in the present experimental paradigm. That sex differences in behaviour were not observed, might be attributed to the fact that subjects were exposed to the different experimental conditions for a
44
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Fig. 1. The percentage of correct responses (correct responses/correct + incorrect responses) for groups of males and females during the different experimental conditions. Data are presented as averages of two consecutive sessions. One standard deviation is indicated at each datapoint. prolonged period o f time. If such observation proved to be reliable in future experiments, previously observed sex differences in behaviour in other experimental procedures may have to be
attributed to the confounding presence o f variables, which m a y only exert their influence during initial exposure to short-term experimental contingencies. We have previously alluded to the
45
Method Subjects. Three of the 5 male and female sub-
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existence of such variables in other experimental procedures 22. E X P E R I M E N T II
Spatial response alternation in T-maze tasks has frequently been used to test the behavioural effects of lesions inflicted in the prefrontal cortex (PFC). Lesions of the medial PFC are assumed to specifically impair functions necessary to be able to behave efficiently in this procedure 6"8,|3,17. The delayed response alternation procedure used in Expt. I shares important characteristics with the T-maze alternation procedures: (a) spatially different responses are required on alternating trials and (b) the discriminative stimulus which controls the behaviour on trial X, consists of the behaviour executed on trial X - 1. Therefore, the delayed spatial response alternation procedure of Expt. I seemed fit to investigate the effects of lesions in the medial PFC on response accuracy of the male and female subjects which participated in Expt. I. Their post-lesion behaviour was, thus, examined during both the 1- and 7.5-s delay conditions of Expt. I.
jects which participated in Expt. I were randomly chosen to receive bilateral lesions of the medial PFC. The remaining two subjects in each group served as sham-operated controls. Surgical procedures. All 10 male and female subjects were anaesthetized with Hypnorm (0.02% ; 0.10 ml/100 g). They were then placed in a David Kopf stereotaxic instrument. Lesions were made using a high-frequency lesion generator (Radionics Inc, model RFG-4) with thermistor type electrodes, with an uninsulated tip of 1.5 mm and 0.7 mm in diameter. The electrode was placed at an 11 ° angle using the following coordinates for males and females respectively: anterior ofbregma 4.0 and 3.8 mm, lateral ofmidsagittal suture line 1.5 and 1.5 mm and ventral of the dura 3.0 and 2.8 mm. After positioning of the electrode, a current was passed and a tip temperature of 57 °C was maintained for a period of 2 min. Control-operated subjects received the same surgical treatment, but current was not passed through the electrode. Histology. Subjects were anaesthetized with a high dose ofHypnorm, perfused with 0.9% saline, followed by 4% formaldehyde/0.9~/o saline, after which the brains were removed from the skull. Transversal 60-#m Nissl-stained sections were then prepared to evaluate the extent of brain damage. Apparatus. Same as the one used in Expt. I. Procedure. After completion of Expt. I subjects were allowed free access to food and water for two weeks. Thereafter, they underwent surgical procedures and were allowed to recuperate for 1 week. The 23-h food deprivation schedule was reinstated immediately after surgery. Thus, Expt. II started 3 weeks after the completion of Expt. I. During the first experimental condition all subjects were exposed to the 1-s delay condition of Expt. I for 20 sessions; thereafter the delay interval was increased to 7.5 s for another 20 sessions. Subjects were sacrificed immediately after the final experimental session.
Results and Discussion Fig. 3 shows the extent of the median lesion for
46
11.5
11 .~
.5
.5
0.0 Fig. 3. The extent of the median PFC lesion in males (left) and females (right). The coordinates shown refer to those of Krnig and Klippe113.
both males and females based on sections from KOnig and Klippe113. Lesions extended between 2.5 and 3.0 mm on the anterior-posterior axis. The frontal pole was never damaged and the most posterior boundary of each lesion was well anterior of the decussation of the corpus callosum. As can be seen in Fig. 3, the lesions were rather small and did not include the whole of the medial PFC. Damage was largest in the dorsal division of the anterior cingulate area and the medial prec e n t r a l a r e a 14 and affected the deeper cortical layers in all subjects. The most dorsomedial part of the medial precentral area was not affected in
most rats. Histological examination of controloperated subjects did not reveal any permanent brain damage, although some remnants of electrode tracts could still be discovered. Fig. 4 shows response, accuracy for both lesioned and control-operated subjects during the 1- and 7.5-s delay conditions. Due to the very limited number of subjects constituting groups of different sex, the following comparisons are based on the behaviour of lesioned and control-operated subjects, without taking the sex of the subjects into account. An analysis of variance was performed on the factors
47
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SESSIONS Fig. 4. Post-surgery response accuracy for lesioned and control-operated males and females. Data were averaged over two consecutive sessions.
lesion, delay interval duration and trials, the latter two repeated measures. Post-surgery exposure to the delayed response alternation task revealed a significant difference in response accuracy between groups of lesioned and control-operated subjects, F1.8 = 6.69, P < 0.03. Response accuracy was also dependent upon the duration of the delay interval, F~,8 = 18.24, P < 0.01. Subjects responded more accurately during the 1-s than during the 7.5-s delay interval condition as can be seen in Fig. 4. Response accuracy of lesioned and control-operated subjects improved with prolonged exposure to the different experimental conditions, F9.72 = 32.60, P < 0.001. Inspection of Fig. 4 suggested that response accuracy of lesioned subjects improved more than that of controloperated subjects, an observation confLrrned by a significant lesion by delay interaction, F~,8 = 7.25, P < 0.03. This observation is probably due to the fact that control-operated subjects already re-
sponded at almost pre-surgery levels upon initial exposure to the experimental contingencies. The data presented in Fig. 4 seem to show that lesioned males may have recovered more quickly than lesioned females. As already discussed, however, the limited number of males and females in the different experimental groups, precluded an analysis which might have supported such an assertion. The possibility that such sex differences do exist, however, deserves special attention in future experiments. GENERAL DISCUSSION
The present experiments were designed to investigate whether or not an alternative delayed spatial response alternation procedure would allow for the investigation of sex differences in memory capacities in Wistar rats. Such differences have been suggested to exist at the sub-
48 human and human level 3'16. For this purpose, the duration of a delay interval in an operant delayed spatial response alternation procedure was systematically increased. Expt. I showed that both males and females behaved less accurately as the duration of the delay interval was increased from 1 to 15 s. Performance of both males and females improved as subjects were exposed to the different experimental conditions for a prolonged period of time. The results of this experiment, then, suggest that the present procedure indeed offers the possibility to study behaviour in which memory for recent events plays an important role. The present experiment has thus shown that procedures can be designed in which the behavior of males and females may be validly compared, without interference by variables which preclude such an analysis in other experimental procedures. The interpretation of the results obtained in those procedures is often difficult and sometimes impossible, due to the oestrus-related variability in the behaviour of the female. The data of the present experiment do not support the suggestion that male and female Wistar rats have basically different memory capacities, although the latter hypothesis has been offered on the basis of data obtained in aversively motivated experimental procedures9 and maze learning tasks ~,7. Lesions of the medial PFC have been shown to negatively affect the T-maze alternation behaviour of male rats 8"17. In Expt. II, both male and female rats received lesions of the medial PFC and were re-exposed to the delayed response alternation procedure investigated in Expt. I. Upon exposure to this procedure lesioned males and females behaved less accurately than control-operated subjects during the initial sessions of both the 1- and 7.5-sec delay interval conditions. Continued exposure to the experimental procedure, however, produced an increase in response accuracy for lesioned males as well as females. Behavioural deficiencies after medial (pre-)frontal ablations have now been observed in a number of experimental paradigms, all involving delayed responding following the presentation of a distinct interoor exteroceptive stimulus. Response efficiency has been observed to be impaired in spontaneous
alternation, delayed alternation, delayed response and delayed response alternation procedures (for review see ref. 12). All of these tasks require a response controlled by an intero- or exteroceptive stimulus which occurred before response execution and which may or may not be present again at the time of response execution. Responding also needs to occur at a predefined spatial location. An impairment of the ability to behave in a spatially defined environment or an impairment of memory capacities resulting in an inability to recall which recently presented stimulus is to have control over the behaviour, may thus mediate the behavioural deficits in delayed response alternation procedures 12. Although the observed behavioural deficits in the different experimental procedures are consistent with one another, they do not provide any suggestions as to the neuronal mechanisms which are essential for correct performance in the delayed response alternation procedures. The observation that the duration of the delay interval in Expt. II did influence behavioural accuracy suggests, that memory factors may indeed contribute to the observed behavioural deficiencies. Recovery of delayed T-maze alternation after frontal cortex and septal lesions has been observed in male rats in several experiments6,19,2°. The data of the present experiment confirm these findings in a different experimental procedure and tentatively extend them to include recovery of behavioural function also in female subjects. Of course, in Expt. II lesioned subjects had been exposed to the 1- and 7.5-s delay interval durations in Expt. I. This previous experience may have been a factor promoting post-lesion behavioural recovery. Whether or not this variable actually contributed to the observed effect, will have to be investigated in forthcoming experiments. Previous studies 15'19 have suggested that the ventral pregenual cortex (prelimbic area 14) would be the critical site supporting delayed response alternation behaviour in the rat. The results of the present study do not support this hypothesis since the prelimbic area of the subjects participating in this experiment was not damaged or, at most, only its dorsal part. A comparison of the present results with those of others ~9 suggests that per-
49
haps the anterior cingulate area (especially its deeper layers) is the most likely candidate for such a critical site. REFERENCES 1 Barrett, R.J. and Ray, O.S., Behavior in the open field, Lashley III maze shuttle box and Sidman avoidance as a function of strain, sex and age, Dev. Psychol., 3 (1970) 73-77. 2 Beatty, W.W., Effects of gonadectomy on sex differences in DRL behavior, Physiol. Behav., 10 (1973) 177-178. 3 Beatty, W.W., Gonadal hormones and sex differences in nonreproductive behaviors in rodents: organizational and activational influences, Horm. Behav., 12 (1979) 112-163. 4 Beatty, W.W. and Beatty, P.A., Hormonal determinants of sex differences in avoidance behavior and reactivity to electric shock in the rat, J. Comp. PhysioL Psychol., 73 (1970) 446-455. 5 Chambers, K.C., Hormonal influences on sexual dimorphism in rate of extinction of a conditioned taste aversion in rats, J. Comp. PhysioL PsychoL, 90 (1976) 851-856. 6 Corwin, J., Nonneman, A. and Goodlett, C., Limited sparing of function on spatial delayed alternation after two-stage lesions of prefrontal cortex in the rat, Physiol. Behav., 26 (1981) 763-771. 7 Davenport, J., Hagquist, W. and Rankin, G., The symmetrical maze: an automated closed field test series for rats, Behav. Res. Meth. Instr., 2 (1970) 112-118. 8 Divac, I., Frontal lobe system and spatial reversal in the rat, Neuropsychologia, 9 (1970) 175-183. 9 Drago, F., Bohus, B., Scapagnini, U. and De Wied, D., Sexual dimorphism in passive avoidance behavior of rats: relation to body weight, age, shock intensity and retention interval, Physiol. Behav., 24 (1980) 1164-1167. 10 Heise, G.A., Conner, R. and Martin, R.A., Effects of scopolamine on variable intertrial interval spatial alternation and memory in the rat, Psychopharmacology, 49 (1976) 131-139.
11 Hurwitz, H.M.B. and Davis, H., Depriving rats of food: a reappraisal of two techniques, J. Exp. Anal Behav., 40 (1983) 211-213. 12 Kolb, B., Functions of the frontal cortex of the rat: a comparative review, Brain Res. Rev., 8 (1984) 65-98. 13 K6nig, J.F.R. and Klippel, R.A., The Rat Forebrain: A
Stereotaxic Atlas of the Forebrain and Lower Parts of the Brainstem, Williams and Wilkins, Baltimore, 1963. 14 Krettek, J.E. and Price, J.L., The cortical projections of the mediodorsal nucleus and adjacent thalamic nuclei in the rat, J. Comp. Neurol., 171 (1977) 157-192. 15 Larsen, J.K. and Divac, I., Selective ablations within the prefrontal cortex of the rat and performance of delayed alternation, Physiol. Psychol., 6 (1978) 15-17. 16 Maccoby, E.E. and Jacklin, C.N., The Psychology of Sex Differences, Stanford University Press, Stanford, 1974. 17 Nonneman, A.J. and Corwin, J.V., Differential effects of prefrontal cortex ablation in neonatal,juvenileand young adult rats,J. Comp. Physiol. Psychol., 95 (1981) 588-602. 18 Olton, D.S., Characteristics of spatial memory. In S.H. Hulse, H. Fowler and W.K. Honig (Eds.), Cognitive Processes in Animal Behavior, Lawrence Erlbaum Associates, New York, 1978, pp. 341-371. 19 Thomas, G.J. and Brito, N.O., Recovery of delayed alternation in rats after lesions in medial frontal cortex and septum, J. Comp. Physiol. Psychol., (1980) 808-818. 20 Thomas, G.J. and Spafford, P.S., Deficits for representational memory induced by septal and cortical lesions (singly and combined) in rats, Behav. Neurosci., 98 (1984) 394-404. 21 Van Haaren, F. and Van de Poll, N.E., The effects of a choice alternative on sex differences in passive avoidance behavior, Physiol. Behav., 32 (1984) 211-215. 22 Van Haaren, F. and Van de Poll, N.E., The number of pre-shock trials affects sex differences in passive avoidance behavior, Physiol. Behav., 33 (1984) 269-272. 23 Van Haaren, F. and Van de Poll, N.E., Effects of light intensity on passive avoidance behavior of male and female Wistar rats, Physiol Behav., in press.
41
BBR00500
D E L A Y E D S P A T I A L R E S P O N S E A L T E R N A T I O N : E F F E C T S OF D E L A Y - I N T E R V A L DURATION AND LESIONS OF THE MEDIAL PREFRONTAL CORTEX ON RESPONSE ACCURACY OF MALE AND FEMALE WISTAR RATS*
FRANS VAN HAAREN, JAN P.C. DE BRUIN, ROB P.W. HEINSBROEK and NANNE E. VAN DE POLL Netherlands Institute for Brain Research, Amsterdam (The Netherlands) (Received February 16th, 1985) (Revised version received August 6th, 1985) (Accepted August 23rd, 1985) Key words: delayed response alternation - delay interval duration - lesion of medial prefrontal cortex - response accuracy male - female - rat
A delayed spatial response alternation procedure was used to assess behavioural differences between male and female Wistar rats, assumed to involve memory functioning. In Expt. I, subjects were required to alternate responses between two levers in an operant environment. The delay between response opportunities was varied between 1, 3, 7.5 and 15 s in different experimental conditions. Incorrect responses produced a time-out from experimental contingencies for the duration of the currently active delay interval. Response accuracy decreased for males as well as females as the duration of the delay interval was increased. Performance improved as subjects were exposed to the different delay interval durations during consecutive trials. Sex differences in behavioural accuracy were not observed. In Expt. II, some subjects who participated in Expt. I received lesions of the medial prefrontal cortex, while others were control-operated. When re-exposed to the 1 and 7.5 s delay conditions of the first experiment, lesioned subjects at first behaved less accurately than control-operated subjects. Accuracy, however, improved after prolonged exposure to the experimental conditions. Sex differences in behaviour after surgery could not be observed.
EXPERIMENT I Behavioural differences between male and female rats have been observed in a n u m b e r o f different learning tasks (for review see ref. 3). F o r instance, sex differences have been reported when rats were exposed to active 4 and passive avoid a n c e procedures 21-23, to taste aversion procedures 5 or to simple schedules o f reinforcement 2. In recent yea~s, research efforts have more and more focused on procedures which allow investigators to examine m e m o r y aspects o f behaviour, on the assumption that m e m o r y for recent and past events plays an important role in behavioural
adaptation to an ever-changing and increasingly complex environment (e.g. ref. 18). F o r m e m o r y to be inferred from the observable behaviour o f a subject, its behaviour at time X has to be under discriminative control o f stimuli which were present at time X - t. Consequently, the discriminative stimuli which are part o f the repertoirecontrolling environment, are not physically present at the time o f response execution. Despite the development o f a n u m b e r o f experimental procedures which satisfy these requirements (delayedmatching-to-sample, radial m a z e and T - m a z e alternation procedures), sex differences in behaviour have not been extensively investigated.
* Some of the data were presented at the Eighth Annual Meeting of the European Neuroscience Association, September 1984, The Hague, The Netherlands. Correspondence: F. van Haaren, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands.
42 This is even more intriguing since basic differences have been postulated between males and females, even at the human level, in cognitive abilities assumed to play an important role in memory functioning. Sex differences in maze learning have been observed in a few studies (e.g. refs. 1, 7). These experiments showed males to be more accurate than females. It has been suggested that sex differences in spatial-perceptual abilities, well-established in marl 16, might also be involved in the observation of behavioural differences between the sexes in maze learning experiments 3. The present experiment was designed to investigate whether or not male and female Wistar rats behave differently in a delayed spatial response alternation procedure, designed to allow for the possibility that differences in memory capacities are reflected in differential decrements in response accuracy. Spatial response alternation tasks have frequently been used to test the effects of different variables on memory capacities in males 6"I0"15'19. The present procedure was adapted from T-maze spatial response alternation procedures, in order to be able to deal effectively with the oestrusrelated behavioural variability 3 usually observed in females. This behavioural variability was experimentally controlled by exposing both male and female subjects to the different experimental conditions for a prolonged period of time. It has been shown in other experiments 1° that response accuracy of males in an operant spatial response alternation task decreases as the duration of the delay interval between presentation of the discriminative stimulus and the opportunity to behave increases. Therefore, male and female Wistar rats were exposed to an operant spatial response alternation procedure in which the duration of the delay interval was manipulated during different conditions of the first experiment.
Method Subjects. Five male and 5 female Wistar rats were obtained from Animal House TNO (Zeist, The Netherlands), when they were 9 weeks old. Upon arrival at the laboratory, they were individually housed and adapted to a reversed light-dark cycle (lights on from 13.30 to 01.30 h). A 23-h food deprivation schedule 11, resulting in a
deprivation to approximately 85 ~o of free-feeding b.wt., was in effect for the duration of the experiment. Experimentation started two weeks after the subjects arrived at the laboratory. Apparatus. Four standard Grason-Stadler (model 111 l-L) rodent operant conditioning chambers were used. Each chamber was made of plexiglass with an aluminium backwall and measured 28 x 30 × 30.5 cm (inside dimensions). The gridfloor consisted of 23 stainless steel grids, spaced 1.25 cm apart. Two non-retractable rodent levers (1.25 cm thick) protruded 1.60 cm into the chamber from the intelligence panel. They were located symmetrically to the side of the pellet retrieval unit and were spaced 13 cm apart (center to center). The levers required a force of at least 0.25 N to be operated. A green (left lever) and a red (right lever) stimulus light were mounted slightly to the side and above each lever. A houselight was located in the upper lefthand comer of the intelligence panel. Each chamber was enclosed in a Grason-Stadler (model 1101) research chest which masked extraneous sounds. A fan provided fresh air. Programming of experimental contingencies and data acquisition were accomplished by means of Grason-Stadler 1200 series programming equipment, located in the experimental room itself.
Procedure Preliminary training. All subjects were first adapted to the experimental chamber and the sound of the pellet feeder for two sessions; they were observed to retrieve the pellet (45 mg Noyes) from the pellet unit at the end of the second session. In the next condition, one of the lights next to one of the levers was illuminated after the expiration of a variable-time (VT) 30-s intertrial interval (ITI). Which fight above which lever was illuminated alternated daily. A press on the lever above which the light was illuminated produced a pellet. Responses during the ITI had no scheduled consequences. Sessions were run 5 days a week (Monday through Friday) and terminated either after 60 pellets had been delivered, or after 30 min, whichever came fn'st. All subjects responded reliably on both levers after 6 sessions in this procedure.
43
Experimental conditions. Subjects were then exposed to a procedure in which both levers were active and responses on alternating levers were reinforced. The houselight and both stimulus lights (one above each lever) were illuminated at the start of the session. A response on the left lever (correct at first) produced a food pellet, extinguished both stimulus lights and started the delay interval. A response on the right lever (not correct at first) produced a time-out (TO) equal to the duration of the delay interval. During TO, houselight and stimulus lights were extinguished. After the expiration of the delay interval, houselight and stimulus lights were again illuminated. TO kept being presented until a response on the correct lever occurred. Which lever was correct and which lever was not correct alternated after every correct response. Responses during TO and the delay interval had no scheduled consequences. Sessions lasted 30 min, or until 60 pellets had been presented, whichever came first. The duration of the delay interval was varied in different experimental conditions. All subjects were exposed to each experimental condition for 20 sessions, which were run 5 days a week, from Monday through Friday, during the last quarter of the subjects' dark hours. The sequence of experimental conditions is summarized in Table I. Results and Discussion Fig. 1 shows, for males and females, response accuracy (number of correct responses/number of correct + incorrect responses) during the different experimental conditions, averaged over two consecutive sessions. One standard deviation is indicated at each datapoint. The data of the final two sessions are also presented in Fig. 2 to facilitate comparison of steady-state behaviour at the different durations of the delay interval. Response accuracy was analyzed using a 3-factorial analysis of variance on the factors sex, duration of delay interval and trials, the latter two variables being repeated measures. As can be seen in Figs. 1 and 2, response accuracy of both males and females was a function of the duration of the delay interval; response accuracy decreased as the duration of the delay interval was increased, F~.24 = 70.67, P < 0.001. Response accuracy
TABLE I
The sequence of experimental conditions in Exp. I (20 sessions each) I
2
3
4
Male subjects 1 2 3 4 5
3 3 3 15 15
15 15 15 3 3
7.5 7.5 7.5 1 1
1 1 1 7.5 7.5
Female subjects 1 2 3 4 5
3 3 15 15 15
15 15 3 3 3
7.5 7.5 1 1 1
1 1 7.5 7.5 7.5
within the different delay interval conditions increased for both sexes with repeated exposure to the experimental conditions, F9.72 = 44.30, P < 0.001. A significant delay by trial interaction, F27,2~6 = 1.92, P < 0.01 suggested that the improvement in response accuracy may have been delay-dependent, although such interaction is not immediately obvious by visual inspection of the data presented in Fig. 1. Even though the data presented in Figs. 1 and 2 suggested that the response accuracy between the sexes may have differed at the 15-s delay interval duration, neither the main effect of sex nor the interaction of sex and delay interval duration reached statistical significance, F~.s = 0.91, n.s. and F3.24 = 1.91, n.s., respectively. As in other procedures designed to measure the discriminative control of behaviour by stimuli which are no longer physically present at the time of response execution ~°, response accuracy of both male and female subjects decreased with increases in the delay interval in the present experiment. This observation suggests that indeed memory aspects were involved in determining response accuracy of the subjects in the present experimental paradigm. That sex differences in behaviour were not observed, might be attributed to the fact that subjects were exposed to the different experimental conditions for a
44
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Fig. 1. The percentage of correct responses (correct responses/correct + incorrect responses) for groups of males and females during the different experimental conditions. Data are presented as averages of two consecutive sessions. One standard deviation is indicated at each datapoint. prolonged period o f time. If such observation proved to be reliable in future experiments, previously observed sex differences in behaviour in other experimental procedures may have to be
attributed to the confounding presence o f variables, which m a y only exert their influence during initial exposure to short-term experimental contingencies. We have previously alluded to the
45
Method Subjects. Three of the 5 male and female sub-
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existence of such variables in other experimental procedures 22. E X P E R I M E N T II
Spatial response alternation in T-maze tasks has frequently been used to test the behavioural effects of lesions inflicted in the prefrontal cortex (PFC). Lesions of the medial PFC are assumed to specifically impair functions necessary to be able to behave efficiently in this procedure 6"8,|3,17. The delayed response alternation procedure used in Expt. I shares important characteristics with the T-maze alternation procedures: (a) spatially different responses are required on alternating trials and (b) the discriminative stimulus which controls the behaviour on trial X, consists of the behaviour executed on trial X - 1. Therefore, the delayed spatial response alternation procedure of Expt. I seemed fit to investigate the effects of lesions in the medial PFC on response accuracy of the male and female subjects which participated in Expt. I. Their post-lesion behaviour was, thus, examined during both the 1- and 7.5-s delay conditions of Expt. I.
jects which participated in Expt. I were randomly chosen to receive bilateral lesions of the medial PFC. The remaining two subjects in each group served as sham-operated controls. Surgical procedures. All 10 male and female subjects were anaesthetized with Hypnorm (0.02% ; 0.10 ml/100 g). They were then placed in a David Kopf stereotaxic instrument. Lesions were made using a high-frequency lesion generator (Radionics Inc, model RFG-4) with thermistor type electrodes, with an uninsulated tip of 1.5 mm and 0.7 mm in diameter. The electrode was placed at an 11 ° angle using the following coordinates for males and females respectively: anterior ofbregma 4.0 and 3.8 mm, lateral ofmidsagittal suture line 1.5 and 1.5 mm and ventral of the dura 3.0 and 2.8 mm. After positioning of the electrode, a current was passed and a tip temperature of 57 °C was maintained for a period of 2 min. Control-operated subjects received the same surgical treatment, but current was not passed through the electrode. Histology. Subjects were anaesthetized with a high dose ofHypnorm, perfused with 0.9% saline, followed by 4% formaldehyde/0.9~/o saline, after which the brains were removed from the skull. Transversal 60-#m Nissl-stained sections were then prepared to evaluate the extent of brain damage. Apparatus. Same as the one used in Expt. I. Procedure. After completion of Expt. I subjects were allowed free access to food and water for two weeks. Thereafter, they underwent surgical procedures and were allowed to recuperate for 1 week. The 23-h food deprivation schedule was reinstated immediately after surgery. Thus, Expt. II started 3 weeks after the completion of Expt. I. During the first experimental condition all subjects were exposed to the 1-s delay condition of Expt. I for 20 sessions; thereafter the delay interval was increased to 7.5 s for another 20 sessions. Subjects were sacrificed immediately after the final experimental session.
Results and Discussion Fig. 3 shows the extent of the median lesion for
46
11.5
11 .~
.5
.5
0.0 Fig. 3. The extent of the median PFC lesion in males (left) and females (right). The coordinates shown refer to those of Krnig and Klippe113.
both males and females based on sections from KOnig and Klippe113. Lesions extended between 2.5 and 3.0 mm on the anterior-posterior axis. The frontal pole was never damaged and the most posterior boundary of each lesion was well anterior of the decussation of the corpus callosum. As can be seen in Fig. 3, the lesions were rather small and did not include the whole of the medial PFC. Damage was largest in the dorsal division of the anterior cingulate area and the medial prec e n t r a l a r e a 14 and affected the deeper cortical layers in all subjects. The most dorsomedial part of the medial precentral area was not affected in
most rats. Histological examination of controloperated subjects did not reveal any permanent brain damage, although some remnants of electrode tracts could still be discovered. Fig. 4 shows response, accuracy for both lesioned and control-operated subjects during the 1- and 7.5-s delay conditions. Due to the very limited number of subjects constituting groups of different sex, the following comparisons are based on the behaviour of lesioned and control-operated subjects, without taking the sex of the subjects into account. An analysis of variance was performed on the factors
47
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SESSIONS Fig. 4. Post-surgery response accuracy for lesioned and control-operated males and females. Data were averaged over two consecutive sessions.
lesion, delay interval duration and trials, the latter two repeated measures. Post-surgery exposure to the delayed response alternation task revealed a significant difference in response accuracy between groups of lesioned and control-operated subjects, F1.8 = 6.69, P < 0.03. Response accuracy was also dependent upon the duration of the delay interval, F~,8 = 18.24, P < 0.01. Subjects responded more accurately during the 1-s than during the 7.5-s delay interval condition as can be seen in Fig. 4. Response accuracy of lesioned and control-operated subjects improved with prolonged exposure to the different experimental conditions, F9.72 = 32.60, P < 0.001. Inspection of Fig. 4 suggested that response accuracy of lesioned subjects improved more than that of controloperated subjects, an observation confLrrned by a significant lesion by delay interaction, F~,8 = 7.25, P < 0.03. This observation is probably due to the fact that control-operated subjects already re-
sponded at almost pre-surgery levels upon initial exposure to the experimental contingencies. The data presented in Fig. 4 seem to show that lesioned males may have recovered more quickly than lesioned females. As already discussed, however, the limited number of males and females in the different experimental groups, precluded an analysis which might have supported such an assertion. The possibility that such sex differences do exist, however, deserves special attention in future experiments. GENERAL DISCUSSION
The present experiments were designed to investigate whether or not an alternative delayed spatial response alternation procedure would allow for the investigation of sex differences in memory capacities in Wistar rats. Such differences have been suggested to exist at the sub-
48 human and human level 3'16. For this purpose, the duration of a delay interval in an operant delayed spatial response alternation procedure was systematically increased. Expt. I showed that both males and females behaved less accurately as the duration of the delay interval was increased from 1 to 15 s. Performance of both males and females improved as subjects were exposed to the different experimental conditions for a prolonged period of time. The results of this experiment, then, suggest that the present procedure indeed offers the possibility to study behaviour in which memory for recent events plays an important role. The present experiment has thus shown that procedures can be designed in which the behavior of males and females may be validly compared, without interference by variables which preclude such an analysis in other experimental procedures. The interpretation of the results obtained in those procedures is often difficult and sometimes impossible, due to the oestrus-related variability in the behaviour of the female. The data of the present experiment do not support the suggestion that male and female Wistar rats have basically different memory capacities, although the latter hypothesis has been offered on the basis of data obtained in aversively motivated experimental procedures9 and maze learning tasks ~,7. Lesions of the medial PFC have been shown to negatively affect the T-maze alternation behaviour of male rats 8"17. In Expt. II, both male and female rats received lesions of the medial PFC and were re-exposed to the delayed response alternation procedure investigated in Expt. I. Upon exposure to this procedure lesioned males and females behaved less accurately than control-operated subjects during the initial sessions of both the 1- and 7.5-sec delay interval conditions. Continued exposure to the experimental procedure, however, produced an increase in response accuracy for lesioned males as well as females. Behavioural deficiencies after medial (pre-)frontal ablations have now been observed in a number of experimental paradigms, all involving delayed responding following the presentation of a distinct interoor exteroceptive stimulus. Response efficiency has been observed to be impaired in spontaneous
alternation, delayed alternation, delayed response and delayed response alternation procedures (for review see ref. 12). All of these tasks require a response controlled by an intero- or exteroceptive stimulus which occurred before response execution and which may or may not be present again at the time of response execution. Responding also needs to occur at a predefined spatial location. An impairment of the ability to behave in a spatially defined environment or an impairment of memory capacities resulting in an inability to recall which recently presented stimulus is to have control over the behaviour, may thus mediate the behavioural deficits in delayed response alternation procedures 12. Although the observed behavioural deficits in the different experimental procedures are consistent with one another, they do not provide any suggestions as to the neuronal mechanisms which are essential for correct performance in the delayed response alternation procedures. The observation that the duration of the delay interval in Expt. II did influence behavioural accuracy suggests, that memory factors may indeed contribute to the observed behavioural deficiencies. Recovery of delayed T-maze alternation after frontal cortex and septal lesions has been observed in male rats in several experiments6,19,2°. The data of the present experiment confirm these findings in a different experimental procedure and tentatively extend them to include recovery of behavioural function also in female subjects. Of course, in Expt. II lesioned subjects had been exposed to the 1- and 7.5-s delay interval durations in Expt. I. This previous experience may have been a factor promoting post-lesion behavioural recovery. Whether or not this variable actually contributed to the observed effect, will have to be investigated in forthcoming experiments. Previous studies 15'19 have suggested that the ventral pregenual cortex (prelimbic area 14) would be the critical site supporting delayed response alternation behaviour in the rat. The results of the present study do not support this hypothesis since the prelimbic area of the subjects participating in this experiment was not damaged or, at most, only its dorsal part. A comparison of the present results with those of others ~9 suggests that per-
49
haps the anterior cingulate area (especially its deeper layers) is the most likely candidate for such a critical site. REFERENCES 1 Barrett, R.J. and Ray, O.S., Behavior in the open field, Lashley III maze shuttle box and Sidman avoidance as a function of strain, sex and age, Dev. Psychol., 3 (1970) 73-77. 2 Beatty, W.W., Effects of gonadectomy on sex differences in DRL behavior, Physiol. Behav., 10 (1973) 177-178. 3 Beatty, W.W., Gonadal hormones and sex differences in nonreproductive behaviors in rodents: organizational and activational influences, Horm. Behav., 12 (1979) 112-163. 4 Beatty, W.W. and Beatty, P.A., Hormonal determinants of sex differences in avoidance behavior and reactivity to electric shock in the rat, J. Comp. PhysioL Psychol., 73 (1970) 446-455. 5 Chambers, K.C., Hormonal influences on sexual dimorphism in rate of extinction of a conditioned taste aversion in rats, J. Comp. PhysioL PsychoL, 90 (1976) 851-856. 6 Corwin, J., Nonneman, A. and Goodlett, C., Limited sparing of function on spatial delayed alternation after two-stage lesions of prefrontal cortex in the rat, Physiol. Behav., 26 (1981) 763-771. 7 Davenport, J., Hagquist, W. and Rankin, G., The symmetrical maze: an automated closed field test series for rats, Behav. Res. Meth. Instr., 2 (1970) 112-118. 8 Divac, I., Frontal lobe system and spatial reversal in the rat, Neuropsychologia, 9 (1970) 175-183. 9 Drago, F., Bohus, B., Scapagnini, U. and De Wied, D., Sexual dimorphism in passive avoidance behavior of rats: relation to body weight, age, shock intensity and retention interval, Physiol. Behav., 24 (1980) 1164-1167. 10 Heise, G.A., Conner, R. and Martin, R.A., Effects of scopolamine on variable intertrial interval spatial alternation and memory in the rat, Psychopharmacology, 49 (1976) 131-139.
11 Hurwitz, H.M.B. and Davis, H., Depriving rats of food: a reappraisal of two techniques, J. Exp. Anal Behav., 40 (1983) 211-213. 12 Kolb, B., Functions of the frontal cortex of the rat: a comparative review, Brain Res. Rev., 8 (1984) 65-98. 13 K6nig, J.F.R. and Klippel, R.A., The Rat Forebrain: A
Stereotaxic Atlas of the Forebrain and Lower Parts of the Brainstem, Williams and Wilkins, Baltimore, 1963. 14 Krettek, J.E. and Price, J.L., The cortical projections of the mediodorsal nucleus and adjacent thalamic nuclei in the rat, J. Comp. Neurol., 171 (1977) 157-192. 15 Larsen, J.K. and Divac, I., Selective ablations within the prefrontal cortex of the rat and performance of delayed alternation, Physiol. Psychol., 6 (1978) 15-17. 16 Maccoby, E.E. and Jacklin, C.N., The Psychology of Sex Differences, Stanford University Press, Stanford, 1974. 17 Nonneman, A.J. and Corwin, J.V., Differential effects of prefrontal cortex ablation in neonatal,juvenileand young adult rats,J. Comp. Physiol. Psychol., 95 (1981) 588-602. 18 Olton, D.S., Characteristics of spatial memory. In S.H. Hulse, H. Fowler and W.K. Honig (Eds.), Cognitive Processes in Animal Behavior, Lawrence Erlbaum Associates, New York, 1978, pp. 341-371. 19 Thomas, G.J. and Brito, N.O., Recovery of delayed alternation in rats after lesions in medial frontal cortex and septum, J. Comp. Physiol. Psychol., (1980) 808-818. 20 Thomas, G.J. and Spafford, P.S., Deficits for representational memory induced by septal and cortical lesions (singly and combined) in rats, Behav. Neurosci., 98 (1984) 394-404. 21 Van Haaren, F. and Van de Poll, N.E., The effects of a choice alternative on sex differences in passive avoidance behavior, Physiol. Behav., 32 (1984) 211-215. 22 Van Haaren, F. and Van de Poll, N.E., The number of pre-shock trials affects sex differences in passive avoidance behavior, Physiol. Behav., 33 (1984) 269-272. 23 Van Haaren, F. and Van de Poll, N.E., Effects of light intensity on passive avoidance behavior of male and female Wistar rats, Physiol Behav., in press.