192 IgG-saporin lesions to the nucleus basalis magnocellularis (nBM) disrupt acquisition of learning set formation

Brain Research 969 (2003) 147–159 www.elsevier.com / locate / brainres

Research report

192 IgG-saporin lesions to the nucleus basalis magnocellularis (nBM) disrupt acquisition of learning set formation Aileen M. Bailey*, Meghan L. Rudisill, Emily J. Hoof, Michelle L. Loving Department of Psychology, St. Mary’ s College of Maryland, 18952 E. Fisher Road, St. Mary’ s City, MD 20686 -3001, USA Accepted 13 January 2003

Abstract Rats with bilateral 192 IgG-saporin lesions to the nucleus basalis magnocellularis (nBM) were tested on olfactory discrimination learning set (ODLS), olfactory discrimination reversal learning set (DRLS), and open field activity. Control animals demonstrated learning set in both the ODLS and DRLS tasks. The nBM-lesioned animals showed initial acquisition impairment in learning set in the ODLS task but eventually demonstrated learning set in both ODLS and DRLS tasks. There were no group differences in open-field activity. Results suggest that removal of the nBM cholinergic system through 192 IgG-saporin lesions impairs early acquisition of learning set compared to control animals, but does not prevent later use of learning set formation. Implications for the non-cholinergic basal forebrain cells in learning set are discussed.  2003 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behaviour Topic: Learning and memory: systems and functions Keywords: 192 IgG-saporin; Nucleus basalis magnocellularis; Basal forebrain; Learning set formation; Olfactory discrimination

1. Introduction The basal forebrain has been intensely investigated primarily due to its reported deterioration in Alzheimer’s Disease (AD). Research has found that decreases in size and in the density of neurons within the basal forebrain, particularly in the nucleus basalis magnocellularis (nBM), are aspects of the neuropathology of AD [11,32,68,69]. The nBM, one of the nuclei of the basal forebrain, provides the major cholinergic innervation to the frontal, prefrontal, and parietal areas of the cerebral cortex [10,11,27,37,43]. The nBM also sends substantial cholinergic projections to the amygdala [43]. Several researchers have reported that damage to the nBM significantly reduces cortical cholinergic levels [3,14,19,20,24, 30,35,44,54,59] and that decreases in cortical cholinergic levels are associated with cognitive impairments in both humans [11,22] and in nonhuman animals [14, *Corresponding author. Tel.: 11-240-895-4338; fax: 11-240-8954436. E-mail address: [email protected] (A.M. Bailey).

17,19,20,24,34,44,47,54]. Thus, lesions to the nBM in animals have been used in an attempt to understand the cognitive deterioration seen in AD. Bailey and Thomas [1] reported that quisqualic lesions to the nBM produced long lasting impairment in learning set formation (LS). Harlow [25] defined LS as ‘‘learning how to learn efficiently in a situation an animal frequently encounters’’ (p. 51). LS has also been associated with use of the ‘win-stay / lose-shift’ hypothesis [38], that is, development and use of a rule (‘win-stay / lose-shift’) which guides an animal’s responses on subsequent trials and new problems [57]. This hypothesis suggests that when an animal chooses correctly (‘win’) on trial 1 and is rewarded, it learns to ‘stay’ with the object that is associated with that reward on trial 2 and succeeding trials. If the animal chooses incorrectly on trial 1 (‘lose’), optimally it will learn to ‘shift’ to the other object on Trial 2 and thereafter gain the remaining rewards. Evidence of LS is indicated best by improvement in performances on Trial 2 across a number of new problems. Bailey and Thomas [1] found that quisqualic lesions to the nBM impaired learning set in two different paradigms:

0006-8993 / 03 / $ – see front matter  2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02294-7

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olfactory discrimination learning set (ODLS) and olfactory discrimination reversal learning set (DRLS). The ODLS method administers a succession of simple but unique discrimination problems and is the most frequently used procedure for learning set in rats. However, it has been argued that an ‘‘increased efficiency in learning repeated reversals of the same discrimination problem’’ [66, p. 260] can also be used as evidence of LS. If the animal forms a learning set within a discrimination reversal task, they will learn to discriminate problems quicker and optimally within one trial. Despite reporting significant deficits in both tests for LS, Bailey and Thomas [1] were unable to determine if the deficits seen were due to the cholinergic or non-cholinergic cells of the basal forebrain because quisqualic acid produces non-selective neurological damage to the area that it is infused within [18]. Thus, it was our aim here to investigate the involvement of the cholinergic basal forebrain system in learning set formation using the selective cholinergic neurotoxin, 192 Immunoglobulin-saporin. The immunotoxin 192 IgG-saporin is selective to the p75 low-affinity neurotrophin receptor located in the rat cholinergic basal forebrain and leads to neuronal cell death following disruption of ribosome function and protein synthesis [6]. 192 IgG-saporin has been reported to selectively and significantly reduce the number of cholinergic neurons in the basal forebrain [5,6,36,50,70], reduce choline acetyltransferase (ChAT) and / or acetylcholinesterase (AChE) levels in the hippocampus [3,12,13,48,52, 58,63,65], and reduce ChAT and / or AChE levels in the neocortex [7,31,40,42,55,71]. We examined the effects of 192 IgG-saporin nBM lesions on learning set formation to determine if the cholinergic basal forebrain system was critical for learning set performance. Learning set formation was investigated with two procedures: olfactory discrimination learning set (ODLS) and olfactory discrimination reversal learning set (DRLS). We hypothesized that the acquisition of learning set would be impaired in both the ODLS and DRLS task based on the profound impairment in learning set following nBM quisqualic lesions [1] and based on other reports of simple discrimination impairments following IgGsaporin infusions [23,63]. Open-field activity was also measured as an indicator of overall general behavior and activity changes following lesions to the nBM. We did not expect to see any changes in open-field activity following surgery.

2. Materials and methods

2.1. Subjects and maintenance Twenty-three male hooded rats, derived from the Long– Evans strain (Rattus norvegicus) were purchased from Harlan Sprague–Dawley. The rats were 80–90 days old

when purchased, and they were randomly assigned to one of three groups: nBM lesion group (n511), sham lesion group (n56), or nonoperated control group (n56). The rats were housed in individual 25.4 cm (length)320.3 cm (width)318 cm (height) metal cages in a temperature controlled room. Rats were kept on a reverse light / dark cycle where the dark phase was between 9:00 a.m. and 9:00 p.m. local time. All testing was done during the dark phase. All rats were allowed a minimum 3-day adjustment period to the home condition following their arrival from shipment. All rats were gradually introduced to a food deprivation regimen. Each rat was fed a minimum of 20 g of food (Prolab; Brentwood, MO, USA) a day. Each rat was weighed daily and compared to a normal growth weight curve for its species and variety according to data previously collected in this laboratory. If the rat’s weight deviated by more than 10% from the normal weight curve, then the amount of food given was increased to maintain the rat’s weight. The rats were given unlimited access to water throughout the training and testing periods The rats were approximately 115 days old at the beginning of pretraining, and approximately 145 days old at the beginning of olfactory discrimination reversal learning. Maintenance and use of the rats met the policies and procedures recommended by the American Psychological Association’s ethical standard for use of animals in psychological research.

2.2. Stereotaxic surgery All surgeries were carried out under aseptic conditions. The rats undergoing nBM lesions (n511) and sham control lesions (n56) were anesthetized prior to surgery with sodium pentobarbital (60 mg / kg i.p.; Abbot Laboratories, N. Chicago, IL, USA). The rats were additionally pretreated with Diazepam (1.0 mg / kg i.p.; Elkins-Sinn Inc., Cherry Hill, NJ, USA) to minimize the possibility of seizures during surgery, and Rubinol (0.025 mg / kg s.c.; A.H. Robins, Richmond, VA, USA) to facilitate respiratory functioning. Following anesthetization and surgical preparations, the subjects were positioned in a stereotaxic instrument with the incisor bar set at 0.0 mm above the interaural line. The skin and muscle covering the skull were retracted until bregma and the cranium were appropriately exposed. Lesions intended for nBM were based on the following two sets of coordinates: (a) AP50.75 mm posterior to bregma, M / L562.3 mm, and DV527.8 mm below the skull surface at bregma and (b) AP50.75 mm posterior to bregma, M / L563.3 mm, and DV528.1 mm below the skull surface at bregma. When the micropipette was in the correct position, 0.2 ml of 192 IgG-saporin (0.375 mg / ml; CHEMICON International, Temecula, CA, USA) was infused into each the four sites over a period of 2 min, and a further 3 min was allowed for diffusion before the micropipette was retracted. Operated control

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(sham) lesions were subjected to the same procedure, except that 0.2 ml of Dulbecco’s phosphate-buffered saline (Sigma, St Louis, MO, USA) replaced the IgG-saporin. Following infusion of either IgG-saporin or buffered saline, Zephiran-soaked gel-foam (Pharmacia and Upjohn, Kalamazoo, MI, USA) was placed in the trephine holes. The incision area in the skin was cleaned with hydrogen peroxide and Zephiran (Sanufi Pharmaceuticals, Myerstown, PA, USA) and sutured with 3-0 suture material. Nitrofuruzone (Phoenix Pharmaceutical, St. Joseph, MO, USA), an antimicrobial ointment, was applied to the sutured incision areas. Banamine (1.1 mg / kg i.m.; Schering-Plough, Union, NJ, USA), an analgesic, was given immediately following and every 12 h for 48 h after surgery. Body weight and food and water intake were monitored closely following surgery. All surgical animals were given a minimum of 10 postoperative recovery days prior to any behavioral training or testing.

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small cover boards contained a small indentation centered over the food well to hold the ball in place until it was nudged aside by the rat. The discriminanda were odor-bearing ping-pong balls. To prepare the ping-pong balls with the odors, one-quart Mason brand food-storage jars were used to hold the balls and the odoriferous substances. Initially seven drops of an odoriferous substance were placed at the bottom of the jar. A wire screen was placed between the odoriferous substance and the ball to avoid direct contact of the liquid with the ping-pong balls. Two ping-pong balls were kept in each jar and the odoriferous substance was replenished as necessary. Eighteen odoriferous substances were available. Eleven were the following McCormick brand food flavorings: almond, anise, banana, brandy, coconut, lemon, orange, pineapple, rum, strawberry, and vanilla. Five were Durkee brand food flavorings: black walnut, cherry, chocolate, peppermint, and root beer. Two were Kroger brand food flavorings, butter and maple.

2.3. Apparatus and discriminanda 2.4. Behavioral procedures The testing apparatus had two compartments: a holding chamber (HC) for the rat and a stimulus-reinforcement chamber (SRC). The sides and the top of the HC were constructed of wood and painted black. The floor of the HC was constructed of stainless steel rods spaced 1.25 cm apart across the width of the chamber. The inside dimensions of the HC were 31 cm (length)329 cm (width)320 cm (height). The wall facing the SRC had an aperture across its width that could be closed by a guillotine door. The SRC was constructed of wood and painted medium gray. Its inside dimensions were 29 cm (width)320 cm (height)313 cm (length). The SRC was designed to be juxtaposed to the HC. It had no wall on the side closest to the HC; instead, it shared the HC’s wall, which had the aperture described before. The SRC’s wall opposite the side with the aperture had a guillotine door that could be opened to permit the experimenter to set-up the discriminanda on the stimulus tray. The stimulus tray could be reached by the rat through the aperture in the HC. The stimulus tray was constructed of wood and painted medium gray. It had three food wells with diameters of 2.5 cm and depths of 0.5 cm; the centers of the outer food wells were 4 cm from the sides of the tray and the center-to-center distance between the food wells was 8 cm. There was a front wall that mated with the aperture and the wall had three portals, each 435 cm, allowing access to the food wells. The portals were intended to minimize forward rolling of the discriminanda (odor-saturated pingpong balls). Each food well was covered by a small medium-gray board (3.5 cm322.5 cm30.5 cm) that could be moved by sliding back and forth to cover or uncover the food well. The purpose of the cover boards was to enable all food wells to hold food rewards so that the food odors would not provide differential cues, while access to the food rewards could be given when appropriate. These

2.4.1. Pretraining procedures All behavioral training and testing was conducted in a darkened room illuminated only by a red light to enable the experimenter’s vision.

1. For the first 3 days, a rat was placed in the HC with the guillotine door between it and the SRC in the open position. Two food pellets were placed in one of the two, randomly selected, outer food wells, and the rat was allowed to remain in the HC until it consumed both pellets. 2. For the next 3 days, the animal was placed in the HC with the guillotine door closed. The door was raised after 60 s allowing access to the food, and the rat was allowed access until it consumed the food. 3. On the 7th training day, the sliding boards covered about one-third of the food well; on Day 8, about one-half; and on Day 9, about two-thirds. Beginning on Day 10, the boards completely covered the food wells, and after 60 s the boards were moved, and the rat was allowed access to the food. The purpose of using the sliding boards was that later during testing, all food wells would be baited to control for the possible use of the odors emanating from the food pellets. The rat would be given access to the food pellets only when it made a correct choice. 4. On the 11th pretraining day, a ping-pong ball that had not been exposed to the testing odors was introduced. The ping-pong ball was randomly placed over an outer food well such that it covered half of the food well. Thus, the animal had to nudge the ball slightly to displace it and gain access to the food pellets. This procedure was done twice. On the next three trials, the

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ping-pong ball completely covered the food well, and the rat had to nudge the ball out of the indentation holding it in place before the experimenter would slide back the board that covered the food well and expose the reinforcers. The ball’s position over either the right or left food well was randomly determined by the Fellows [21] series. Testing days 12 and 13 followed the same procedure as day 11. 5. On the 14th day, a scented ping-pong ball (randomly selected) was introduced. There were two trials in which the scented ping-pong ball covered half of the food well, and eight further trials where the food well was completely covered by the ping-pong ball. The ball’s position over either the right or left food well was randomly determined by the Fellows [21] series. Testing days 15 and 16 had 10 trials in which a new randomly selected scented ping-pong ball completely covered the food well. 6. Following the 16th training day, the animals were given two different odor saturated balls. One of the two odors was randomly selected to be the correct odor. Placement of the correct odor over either the right or the left food well was determined by the Fellows [21] series. The rats were given 10 trials a day until they made eight out of 10 choices correctly for two successive days (80% criterion). In the event of an error, the trial was re-administered until the correct choice was made or until a total of five such correction trials had been given. After reaching the 80% criterion, the rats were given a new odor-unique discrimination problem. They were again given 10 trials a day until they made eight out of 10 choices correctly on two successive days. The two pretraining problems were used to train the rats a basic odor discrimination task prior to learning set testing.

2.4.2. Oddity and ODLS testing Following pretraining, the rats were tested for oddity concept learning and olfactory discrimination learning set (ODLS) formation, both of which can be assessed at the same time using the same procedure. Specifically, if a rat has learned to respond to the odd odor on the first trial of each new problem, it shows evidence of using the oddity concept. Even if it does not acquire the oddity concept, it might still acquire a learning set by demonstrating chance performance on Trial 1, but above chance performance on Trial 2. Three ping-pong balls were used; two balls had the same odor and the third ball (representing oddity) had a different odor. The two odors were selected randomly from a computer-generated list of numbers, 1 to 18, each of which had been previously assigned to represent one of the 18 odors. The position of the odd ball (the one with the different odor), which was the correct choice, was limited to the left or right food well and its position on a given trial was determined by the Fellows [21] series. The rat

had to nudge the odd ball out of place in order for the experimenter to slide back the board that covered the food well and expose the reinforcers (two regular 45 mg food pellets, Noyes precision pellets, P.J. Noyes, Lancaster, NH, USA). All three food wells were baited to control for possible odor cues from the food reinforcers. In the event of an error, the trial was re-administered until the correct choice was made or until a total of five such correction trials had been given. The subjects were given two odor-unique problems, each for five trials, for a total of 10 trials a day. A total of 50 odor-unique problems was given. The odors used for each of the 50 problems were randomly selected from a random number list except for the restriction that no odor was repeated as odd or non-odd on successive problems. With 18 odors, using two at a time, where either could be the ‘correct / odd’ stimulus enables the construction of 306 odor-unique problems. Note that a given odor might be correct on one problem and incorrect on another, thereby preventing a consistent association of a particular odor with reinforcement.

2.4.3. Olfactory discrimination reversal learning Following oddity and ODLS testing, the rats were given a discrimination reversal (DRLS) task to investigate the transfer of learning set formation to different conditions. Two ping-pong balls, each with a different food odor, were used as discriminanda. The two discriminanda selected were mint and banana, and mint was randomly chosen to be the initially ‘correct’ odor. The position of the two balls was limited to the left and right food wells. The position of the ‘correct’ odor was determined by the Fellows [21] series. The rat had to nudge the correct ball out of place in order for the experimenter to slide back the board that covered the food well and expose the reinforcers. Both of the two outer food wells were baited to control for possible odor cues from the food reinforcers. Ten trials per day were administered. In the event of an error, the trial was re-administered until the correct choice was made or until a total of five such correction trials had been given. The subjects were given one discrimination problem at the rate of 10 trials per day until a criterion of eight correct responses out of 10 were seen on two successive blocks of 10 trials. The subjects were given the first reversal problem beginning the day after reaching criterion; that is, the previously incorrect odor (banana) became the correct choice. A total of 14 such reversals, with the same two odors, were given. 2.4.4. Open field testing The open field was a black, enamel-painted, wooden box measuring 60 cm (length)360 cm (width)328 cm (height). The floor was painted gray and divided into 16 equal squares (15 cm315 cm). Open field activity was examined in a darkened room with only a red light to enable the experimenter’s vision. Rats were placed in the open field

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for 10 min during the third day of general adjustment following their arrival from the supplier and prior to any training or surgery; this was intended to enable general habituation to the open field apparatus. The first open field test was given during the fourth day of adjustment. The second open field test occurred on the 10th postoperative day or on the 10th training day for the nonoperated animals. The third open field test occurred on the day immediately prior to discrimination reversal testing, and the last open field test occurred the day after discrimination reversal testing ceased. During each open field test, an individual rat was placed in the same corner square and the number of squares entered with all four feet was recorded for a 10-min period.

2.5. Histological procedures Upon completion of all behavioral testing, subjects received a lethal dose of sodium pentobarbital (i.p.). Perfusions were carried out by injecting 60 ml of 0.9% physiological saline in the left ventricle which flowed through the circulatory system and exited from a small incision in the right atrium of the heart. An infusion of 150 ml of 4% paraformaldehyde in 0.1 M sodium phosphate buffer and then an infusion of 60 ml of 5% buffered sucrose followed the saline injection. The brains were excised and placed in 10% buffered sucrose for 24 h and were subsequently placed in 25% buffered sucrose solution for 24 h. The brains were frozen and sectioned coronally using a cryostat (IEC Minotome, International Equipment Company, Needham Heights, MA, USA) at 56 mm through the rostrocaudal extent of the nBM region. The sections were transferred to a 0.1 M sodium phosphate buffer solution prior to histological staining. Alternate slices where mounted and stained one with acetylcholinesterase (AChE) histological staining [33] and the other with a stain for Nissl substance (nucleic acids) using cresyl-violet staining methods.

3. Results

3.1. Histological results All stained sections were examined microscopically for both general neurological damage and for AChE content. The cresyl violet-stained sections did not show any obvious changes in the nBM area. There were no signs of overt gliosis in the nBM lesioned animals as is often seen following nonspecific neurotoxic lesions (e.g. ibotenic acid, quisqualic acid). The lesions to the nBM became clear when sections of tissue were examined for AChE content. The control and sham lesioned animals all demonstrated magnocellular multipolar AChE-stained neurons throughout the nBM. These large neurons were missing in the nBM region of the animals that received injections of

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IgG-saporin. AChE was quantified in the frontal cortex, parietal cortex, and nBM region using Scion Image 4.0.2 (Scion, Frederick, MD, USA). There were no significant differences between the control and sham lesioned animals in AChE optical density measurements (all P-values. 0.05) and, thus, they were combined for all histological statistical analyses. Frontal and parietal cortex was examined at 20.30 mm from Bregma. The frontal cortex was measured immediately above the cingulum and the parietal cortex was measured from the same slice but measurements were taken at the lateral extent of the external capsule. Optical density in the frontal cortex was measured in a 13,116 square pixel space for all animals. An independent t-test found a significant difference in optical density in the frontal cortex (t (14)57.92, P,0.001) indicating that the control animals (M5128.78, S.D.524) had significantly more AChE content than the nBM-lesioned animals (M5 45.17, S.D.518; see Table 1). The nBM-lesioned animals showed a 65% decrease in optical density in the frontal cortex compared to the control animals. The parietal cortex was measured in a 14,285 square pixel space for all animals. An independent t-test indicated that the control animals (M596.87, S.D.515.5) had significantly more AChE fibers in the parietal cortex than the nBM-lesioned animals (M537.77, S.D.513.7), t (14)57.76, P,0.001 (Table 1). Thus, the parietal cortex showed a 61% decrease in AChE content in the nBM-lesioned animals compared to control animals. Optical density measurements were also examined in the nBM region. AChE optical density was analyzed 21.30 mm from Bregma in a 2610 square pixel space along the ventral edge of the internal capsule and the ventral medial aspects of the globus pallidus. There was a significant 40% decrease in AChE content in the nBM IgG-saporinlesioned animals (M580.43, S.D.511.15) compared to the control animals (M5133.83, S.D.536.94), t (14)54.34, P50.001. Acetylcholinesterase content was also analyzed in the horizontal limb of the diagonal band (HDB) and in the medial septal area (MS) to examine nBM lesion specificity. There were no significant differences in AChE content in the HDB between the control and nBM-lesioned animals, t (14)51.69, P50.112. Additionally, there were no significant differences in AChE content in the MS between the control and nBM-lesioned animals, t (14)5

Table 1 Acetylcholinesterase density Group

IgG-saporin Control

Region Frontal cortex

Parietal cortex

nBM

45.17618.0* 128.78624

37.77613.7* 96.87615.5

80.43611.15* 133.83636.94

Data are presented as mean (6S.D.) of optical density scans. *P,0.05.

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1.80, P50.09 suggesting an intact septal-hippocampal system. One nBM-lesioned animal was excluded from the following behavioral analyses, as this rat did not have a significant loss of AChE fiber in either the frontal cortex, the parietal cortex, or the nBM and, thus, it was determined that this animal did not have a sufficient lesion to the nBM.

3.2. Behavioral results 3.2.1. Pretraining The nonoperated and sham-operated control rats were combined to form one control group for statistical analyses as there was no statistical difference in their pretraining performance (F (1,10)50.50, P.0.05). The number of trials to criterion was used to examine any group differences in learning the simple olfactory discrimination problems. A two-way mixed analysis of variance (ANOVA; Group3Problem) showed no statistical differences between the control (M565.5, S.D.536.8) and the nBM-lesioned animals (M571, S.D.535.0) in trials to criterion for the pretraining problems, F (1,20),1, indicating that both groups learned the pretraining discrimination problems at the same rate. There was no statistical difference in trials to criterion across the two problems given (F (1,20)51.19, P.0.05), and there was no significant interaction between Group and Problem, F (1,20),1. 3.2.2. Oddity and ODLS testing The nonoperated and sham-operated lesioned animals showed no significant differences in performance in the ODLS test (all P-values.0.05), thus, the groups were combined to create one overall control group for statistical analyses. The overall performance on the five trials for the 50 oddity problems given may be seen in Fig. 1. The binomial approximation was used to determine whether the percentage correct on each of the five trials for each group differed from chance values. It should be noted that chance was viewed in this study as 50%. Because responses could be made to any of the three discriminada, one might view chance as 33%, however, because the odd (correct) discriminandum appeared only in conjunction with the two outer food wells, the more conservative value of 50% was used to indicate chance. Binomial approximation analyses indicated that the control animals performed significantly above chance on Trials 2–5 (all P-values,0.001), whereas the nBM group did not perform significantly above chance until Trial 5 (P,0.01; in all uses of the binomial approximation reported in this article, a 50.01, as a control for familywise error rates and Type I error inflation). A two-way mixed ANOVA (Group3Trial Number, with Trial Number as the within subjects factor) was also used to examine group differences in overall performance on the ODLS task. The ANOVA revealed a significant effect of Trial Number (F (4,80)56.15, P,0.01) indicating that

Fig. 1. Mean (6S.E.M.) percentage of correct responses for the control group and the nucleus basalis magnocellularis (nBM) IgG-saporinlesioned group on each of Trials 1–5 analyzed over the 50 olfactory discrimination learning set problems. The combined non-operated and sham-operated control group performed significantly better than chance on Trials 2–5 (*all P-values,0.01). The nBM-lesioned group performed significantly better than chance only on Trial 5 (*P,0.01).

performance increased with each trial given (Fig. 1). The ANOVA also showed a significant effect of Group (F (1,20)59.2, P,0.01). Post-hoc comparisons indicated that the only significant difference between the groups was on Trial 2 (t (20)52.76, P50.012) with the control animals (M564, S.D.510) performing significantly higher than the nBM-lesioned animals (M553, S.D.57). There was no significant interaction between Trial Number and Group (F (4,80)51.6, P.0.05). As mentioned earlier, an advantage to the ODLS task is that it can be used to measure both oddity concept use and discrimination learning set at the same time. To examine whether or not the animals were using the concept of odd, Trial 1 responses on the 50 discrimination problems were analyzed. If the rats were responding to the concept ‘odd,’ then Trial 1 selection across the 50 problems should have been better than expected by chance [60]. The binomial approximation was used to determine if the rats differed from chance on Trial 1. Neither of the two groups differed significantly from chance values on Trial 1 suggesting that the rats were not using the concept ‘odd’ in this task. The mean percentage correct for the control animals was 50%

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(S.D.59.35; P.0.05) and 52% (S.D.58.91; P.0.05) for the nBM-lesioned group (Fig. 1). To investigate the effect of IgG-saporin-nBM lesions on learning set formation, Trial 2 performances across the 50 problems were examined in successive blocks of 10 problems. The percentage correct on Trial 2 performances for each group of rats and for each block of problems was analyzed using the binomial approximation to determine whether they differed significantly from chance responding. The control animals performed better than chance on Trial 2 by Block 3 (M564.16%, S.D.518.3, P,0.01) and remained above chance over the remaining blocks of oddity problems (Fig. 2). The nBM-lesioned rats did not perform above chance on Trial 2 until Block 5 (M563%, S.D.514.2, P,0.01). However, above chance performance on Block 5 indicates that the nBM-lesioned animals were able to acquire learning set in the ODLS task. A two-way mixed ANOVA (Group3Problem Block, with Problem Block as the within-subjects factor) was

Fig. 2. Mean (6S.E.M.) percentage of correct responses on Trial 2 (deemed most relevant to assess learning set formation) for each group across the five successive 10-trial blocks of oddity problems in the oddity learning set paradigm. The combined non-operated and sham-operated control group performed significantly better than chance on Trial 2 during Blocks 3–5 (*all P-values,0.01). The nucleus basalis magnocellularis (nBM)-lesioned group performed significantly better than chance on Block 5 (*P,0.01).

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computed to assess further difference on Trial 2 performance on the ODLS task. ANOVA analyses indicated a significant effect of Problem Block (F (4,80)55.64, P, 0.001) suggesting an overall increase in Trial 2 percentage correct with increasing problem blocks (Fig. 2). Post-hoc Bonferroni t-tests indicated significant differences between Block 1 and Block 4 (t (21)53.7, P,0.01) and a significant difference between Block 1 and Block 5 (t (21)53.9, P,0.01). There was also a significant effect of Group (F (1,20)57.63, P50.012) with the control animals performing significantly higher (M564%, S.D.517.3) than the nBM saporin-lesioned animals (M553%, S.D.513.4). Planned comparisons indicated that the control animals and the nBM animals were significantly different on Trial 2 percentage correct on Block 3 (t (20)52.25, P50.036) and on Block 4 (t (20)52.05, P50.05). There was no significant interaction between Group and Problem Block (F (4,80),1, see Fig. 2).

3.2.3. Discrimination reversal performance Transfer of learning set formation was also examined in a discrimination reversal (DRLS) paradigm to determine whether the animals could demonstrate the use of learning set on a series of reversal problems. No significant differences were found between the nonoperated and sham-operated control rats in DRLS performance and they were combined to form one control group for statistical comparisons (all P-values.0.05). One nBM-lesioned animal was excluded from statistical analysis on the discrimination reversal tests as this animal inadvertently failed to be given the final reversal problem (reversal [14). Performance was assessed on the DRLS task by examining both trials to criterion and Trial 2 performances for each of the original and 14 reversal problems given. Both groups of rats improved with each successive reversal as measured by the number of trials to criterion (Fig. 3). However, neither group of rats demonstrated the typical increase in trials to criterion on the first reversal problem past the original trial to criterion level [1,15,39]. A two-way mixed ANOVA (Group3Reversal Number, with reversal as the within-subjects factor) was used to analyze the trials to criterion data on the DRLS task. Because of the heterogeneity of variances, a was set to 0.025 to control inflated Type I error rates for the ANOVA analyses. There was a significant effect of Reversal Number indicating a decrease in trials to criterion with each administered reversal problem (F (14,266)515.8, P,0.01; see Fig. 3). There were no group differences in trials to criterion (F (1,19), 1). There was a significant interaction between the group of animals and the number of reversals (F (14,266)53.14, P,0.01) suggesting that the nBM-lesioned animals had a larger decrease in trials to criterion over the first reversal problem than the control animals (Fig. 3). Planned comparisons followed those previously reported by Mackintosh and Holgate [39] and Bailey and Thomas [1]. Comparisons between the trials to criterion for the

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classified as nonperseverative errors. Again, due to the heterogeneity of variance in the data, a was set to 0.025. There were no significant differences between the control (M50, S.D.50) and the nBM-lesioned animals (M53.4, S.D.55.2) in perseverative errors t (19)52.30, P.0.025. Additionally, no significant differences were found in nonperseverative errors, t (19)50.56, P.0.025, between the control (M5127, S.D.572), and nBM-lesioned animals (M5143.8, S.D.558.7). The percentage correct on Trial 2 of the discrimination reversal problems were examined for more precise evidence of learning set formation. Binomial approximations indicated that both the control animals (70.5%, P,0.01) and the nBM-lesioned animals (67.8%, P,0.01) responded significantly better than expected by chance (50%) on Trial 2, suggesting that both groups successfully demonstrated learning set within the DRLS task. An independent t-test indicated that there were no significant differences between the control and nBM-lesioned animals on Trial 2 percentage correct, t (20)50.530, P.0.05.

Fig. 3. Mean (6S.E.M.) trials to criterion for the nucleus basalis magnocellularis (nBM)-lesioned group and the combined non-operated and sham-operated control group on the original discrimination problem and the 14 subsequent olfactory discrimination reversal learning set (DRLS) problems. The control group (P,0.01) and the nBM-lesioned group (P50.01) required significantly fewer trials to criterion on the 14th reversal than they had taken on the original learning. There were no significant differences between the control and nBM-lesioned animals in trials to criterion (P.0.05).

original problem and for the final reversal problem (Reversal 14) revealed a significant decrease in trials to criterion for both the control animals (t (11)53.4, P,0.01) and the nBM-lesioned animals (t (8)53.3, P50.01; see Fig. 3). Additional planned comparisons indicated that there were no significant differences between the control animals (M560, S.D.539.77) and the nBM-lesioned animals (M5 102, S.D.568.6) on trials to criterion for the original discrimination problem (t (20)51.8, P50.08), and no significant differences between the control group (M5 20.83, S.D.52.88) and the nBM-lesioned animals (M520, S.D.50) on the final reversal problem, t (19)50.86, P. 0.05 (Fig. 3). The errors made during the DRLS task were also examined due to previous reports of perseverative errors following nBM lesions [14,51,54]. Errors made while the rats were performing at worse-than-chance levels (two or less correct responses of 10 possible) were defined as perseverative errors (i.e. inability to inhibit responding to the previously correct stimulus). All other errors were

3.2.4. Open-field testing Open field activity was used to examine any differences in general activity levels and emotionality of the rats that might have occurred following lesions to the nBM. There were no differences between the nonoperated control and the sham-operated control animals (F (1,10)51.4, P. 0.05), thus, they were combined to form one control group for statistical analyses. The three postsurgical open-field measurements were averaged to produce one postsurgical measurement for statistical analyses. A two-way ANOVA (Group3Observation Time, with Observation Time as the within-subjects factor) found no statistical differences in activity between the presurgical and postsurgical measurements, F (1,20),1, no group differences between the control animals (M575.93, S.D.523.45) and the nBMlesioned animals (M575.07, S.D.512.93) in activity levels, F (1,20),1, and no significant interaction between Group and Observation Time, F (1,20)51.1, P.0.05. Additionally, as measured by the number of fecal boli, there was no evidence of group differences in emotionality, t (20)51.1, P.0.05.

4. Discussion Lesions to the nBM produced by 192 IgG-saporin significantly impaired acquisition of learning set in the ODLS task. The control animals performed significantly better than chance on Trial 2 by Block 3 (problems 21–30) in the ODLS test indicating the formation of LS. The nBM-lesioned animals performed at chance levels on Trial 2 overall in the ODLS task but performed significantly higher than chance on Trial 2 for problems 41–50 (Block 5). Thus, although the nBM-lesioned animals performed at chance and significantly worse than the control animals

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overall on Trial 2, performance was significantly above chance on Trial 2 for problems 41–50 in the ODLS task, demonstrating use of LS. Additionally, both the control and nBM-lesioned animals demonstrated transfer and use of LS in the DRLS task by performing significantly better than chance on Trial 2 in this paradigm. Thus, the data suggest an initial impairment in the acquisition of LS compared to control animals, but an eventual ability to demonstrate use of LS in both tasks given. The impairment in the acquisition of learning set suggests that the loss of acetylcholine in the nBM and cortex from the IgG-saporin lesion significantly hinders the acquisition of LS but that selective disruption of the basal forebrain acetylcholine system does not prevent LS from occurring. The deficit in LS acquisition reported here does not appear to be due to changes in the olfactory system. The two pretraining discrimination problems given used olfactory discriminada and the nBM-lesioned animals did not show significant differences compared to the control animals as measured by trials to criterion. Such equivalent performance suggests that the nBM-lesioned animals were able to discriminate odors. Additionally, although the nBM lesioned animals did not show above chance performance on Trial 2 until the last 10 problems in the ODLS task, they did show significant intraproblem learning (Fig. 1) in which they performed significantly better than chance on Trial 5 of the new problems indicating that they could learn to discriminate based on olfactory cues. Finally, there was no significant difference in the AChE content between the control and nBM-lesioned animals in the HDB which sends significant cholinergic projections to the olfactory bulb [43]. Thus, a change in olfactory ability does not appear to explain the deficit in learning set acquisition seen in the IgG-saporin nBM-lesioned animals. Deficits in the nBM-lesioned animals in the present experiment also do not appear to be based on a general dysfunction following surgery. The general well-being of the animals was measured by the open-field assessment of general activity and the assessment of emotionality based on the number of fecal boli observed [46]. There were no significant differences between the nBM-lesioned animals or the control animals on either fecal boli or in activity. An inability to perform higher than expected by chance on Trial 2 might arguably be due to a deficit in working memory as opposed to learning set formation. While working memory was not specifically measured here, it seems unlikely to be the source of the impairment in the nBM-lesioned animals. Previous research has indicated that IgG-saporin nBM lesions do not significantly impair working memory [50,61,67]. In addition, nonselective neurotoxic lesions to the nBM have also indicated a lack of working memory deficit [1,16,47,56]. Deficits in working memory that have been reported following both nonselective and selective cholinergic lesions to the nBM [16,44,47,62] tend to dissipate quickly after lesioning (generally within 21 days) and our ODLS testing did not

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begin until roughly 40 days post lesion. Thus, any potential alterations to the working memory system were likely to have dissipated prior to the ODLS testing. Therefore, it appears unlikely that the deficits seen in the ODLS task were due to changes in working memory. Several investigators have suggested that impairments in performance following cholinergic nBM lesions are attentional in nature and do not suggest learning or memory deficits [4,41,42,45,53,64]. Although attentional capacity was not specifically measured in our animals, a deficit in attention does not appear to explain the impairment of learning set acquisition in the ODLS task. The pretraining discrimination problems, the ODLS task, and the DRLS task all require the animals to attend to odor-saturated stimuli. The general presentation of the stimuli in these settings is similar and has similar attentional demands on the animals (i.e. two odors are presented for an unlimited amount of time. While the ODLS task used three pingpong balls, only two odors were presented). However, we found no differences between the control and the nBMlesioned animals in the two olfactory discrimination pretraining problems given and no significant differences between the control and lesioned animals in the DRLS task. Additionally, Turchi and Sarter [62] examined attentional capacity using an olfactory span task. They reported an initial, but transient impairment in olfactory span following IgG-saporin nBM lesions when compared to control animals, however, the nBM-lesioned animals were still able to perform a 10 odor nonmatching-to-sample task [62]. Our animals were only presented with two unique odors which would not appear to tax the olfactory attentional processing capacity of the rats based on the Turchi and Sarter study. Thus, the changes in LS reported here do not appear to be attentional in nature. The impairment of the IgG-saporin nBM-lesioned animals on the ODLS task suggests specific problems in learning set acquisition. The nBM-lesioned animals were not impaired in simple olfactory discrimination as seen by the similar performance between the lesioned animals and the control animals on the two pretraining problems (i.e. both sets of animals were able to make stimulus-reward associations at the same rate when trained to criterion). Our finding is similar to previous reports of intact simple discrimination learning following IgG-saporin injections [8,9,23,63]. The deficit in the ODLS task reported here is one in how quickly the animals can learn which olfactory stimulus is associated with reward in the face of unique novel discriminations each with only five trial presentations. The control animals were able to solve the novel discrimination problems by Trial 2 (i.e. above chance performance), an indication of one-trial learning or learning set formation. However, the nBM IgG-saporin lesioned animals were not initially able to solve the novel problems until Trial 5 (i.e. chance levels on Trials 1–4), indicating an impairment in LS and not in simple discrimination.

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The initial impairment in LS was overcome by the nBM IgG-saporin-lesioned animals during the final block of ODLS problems (problems 41–50) where they performed significantly higher than expected by chance on Trial 2 demonstrating use of LS. Other investigations of IgGsaporin nBM-lesioned animals have reported similar behavioral recovery [9,49] and may suggest a role of the remaining cholinergic cells or other neural areas to compensate for the ACh loss in the nBM [9]. Despite the initial deficit in the ODLS test, the IgGsaporin nBM-lesioned animals were able to eventually demonstrate use of LS. Additional evidence that the IgGsaporin nBM-lesioned animals were able to acquire, use, and transfer learning set stems from behavioral observations on the DRLS task. We examined performance on DRLS following the completion of the ODLS task to examine the transfer of LS to a different task. Performance on a series of discrimination reversal problems can also be examined for learning set formation. Learning set formation can be determined by examining Trial 2 performances and by determining if significant improvement is seen across successive reversals [66]. Both groups showed significant improvement across the 14 reversal problems as indicated by the significant decrease in trials to criterion (Fig. 3). Such improvement over successive reversals alone is arguably sufficient evidence of learning set [66]. However, a more precise measurement of LS in the DRLS task comes from examination of the Trial 2 performances. The nBM-lesioned animals performed similarly to control animals and both groups performed significantly better than chance on Trial 2. Other investigators have previously reported no deficits in other task reversals following IgGsaporin injections to the nBM [23,61]. Although these researchers were not specifically measuring LS, our findings are similar in that no reversal deficits were seen following IgG-saporin lesions to the nBM. It is worth noting that all of our animals performed well on the DRLS task and did not demonstrate the typical rise in trials to criterion on the first reversal [1,15,39]. The ability of our animals to perform so well on the DRLS task may be attributed to the amount of olfactory discrimination training the animals had previously received in the ODLS task. All animals had faced 50 novel odor-unique discrimination problems and were able to solve them by Trial 2 in the ODLS task. Thus, it is not surprising that the animals performed well in the DRLS task and were able to quickly learn the olfactory discrimination reversals. The ability to perform significantly above chance on Trial 2 in the DRLS task demonstrated that the general win-stay / lose-shift rule could be transferred to a different task and that the IgGsaporin lesions did not impair the transfer of LS. An important investigation for the future will be to examine whether IgG-saporin nBM-lesioned animals demonstrate acquisition impairment in the DRLS task when administered first and whether the animals are able to overcome the deficit, and transfer LS to the ODLS task. The results

presented here would predict a transient acquisition impairment in the DRLS task followed by transfer of LS to the ODLS task. The impairment in LS reported here with IgG-saporin lesions to the nBM differ from those reported by Bailey and Thomas [1]. Bailey and Thomas found long-lasting deficits in learning set formation following quisqualic lesions to the nBM, that is, they reported no evidence of learning set formation in either the ODLS or DRLS task. Animals with quisqualic lesions to the nBM never performed significantly different from chance on either the ODLS or DRLS task [1]. Additionally, the quisqualic lesioned animals did not improve significantly with repeated reversal problems in the DRLS task as was seen in the IgG-saporin nBM-lesioned animals. The differences in performance between the quisqualic and IgG-saporin lesioned animals suggests that learning set formation is a cognitive ability that heavily depends on the noncholinergic cells in the basal forebrain, or to a combination of cholinergic and noncholinergic cells in the nBM [18]. However, the cholinergic cells of the nBM alone do not seem to play a critical a role in learning set formation. However, it has been reported that the cholinergic projection to the amygdala from the nBM is spared following IgG-saporin injections, because these cholinergic neurons do not express p75 [5,28,29]. Therefore, it is possible that the cholinergic cells that project to the amygdala are important for LS, and that should be investigated in the future. Other reports of impairment in discrimination reversal tasks following nonselective lesions to the nBM may also indicate a role for the noncholinergic cells of the nBM in learning set formation. Impairments have been seen on spatial discrimination reversal tasks [14,35,51], and in a series of reversals on nonspatial discrimination tasks [1,19,54] following nonselective lesions to the nBM. However, as indicated above, discrimination reversal deficits were not seen here and have not been seen by other researchers following IgG-saporin lesions to the same area [23,61]. Thus, impairment in both spatial and nonspatial reversals following non-selective lesions suggests a possible role for the noncholinergic nBM cells or a combination of cholinergic and noncholinergic cells in the nBM in a general inability to ‘learn to learn’ and benefit across reversed discriminations in a variety of behavioral situations. Learning set formation is considered to be a foundational process for many forms of higher cognitive learning [2,26]. It is likely that this ability to ‘learn to learn’ is well developed in humans and may be impaired as the central nervous system deteriorates with aging or by disease. Although impairment in acquisition of learning set was found, all rats with IgG-saporin nBM lesions were able to acquire learning set suggesting that the loss of the cholinergic cells in the nBM does not block LS completely. The severe impairment in LS reported by Bailey and

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Thomas [1] following quisqualic lesions to the same area suggests a crucial role for the basal forebrain noncholinergic cells in learning set formation. Further investigations are needed to determine which noncholinergic cells of the basal forebrain are most critical for learning set formation.

[15]

[16]

Acknowledgements The authors would like to thank Dr Allen E. Butt for his assistance in histological analysis of the nBM lesions and Dr Roger K. Thomas for editorial comments regarding the manuscript.

References [1] A.M. Bailey, R.K. Thomas, The effects of nucleus basalis magnocellularis lesions in Long-Evans hooded rats on two learning set formation tasks, delayed matching to-sample learning, and open field activity, Behav. Neurosci. 115 (2001) 328–340. [2] G. Bateson, Steps to an Ecology of Mind, Ballantine Books, New York, 1972. [3] M.G. Baxter, D.J. Bucci, L.K. Gorman, R.G. Wiley, M. Gallagher, Selective immunotoxic lesions of the basal forebrain cholinergic cells: Effects on learning and memory in rats, Behav. Neurosci. 10 (1995) 714–722. [4] M.G. Baxter, A.A. Chiba, Cognitive functions of the basal forebrain, Curr. Opin. Neurobiol. 9 (1999) 178–183. [5] J. Berger-Sweeney, S. Heckers, M. Mesulam, R.G. Wiley, D.A. Lappi, M. Sharma, Differential effects on spatial navigation of immunotoxin-induced cholinergic lesions of the medial septal area and nucleus basalis magnocellularis, J. Neurosci. 14 (1994) 4507– 4519. [6] A.A. Book, R.G. Wiley, J.B. Schweitzer, 192 IgG-saporin: I. Specific lethality for cholinergic neurons in the basal forebrain of the rat, J. Neuropathol. Exp. Neurol. 53 (1994) 95–102. [7] P.J. Bushnell, A.A. Chiba, W.M. Oshiro, Effects of unilateral removal of basal forebrain cholinergic neurons on cued target detection in rats, Behav. Brain Res. 90 (1998) 57–71. [8] A.E. Butt, T.D. Bowman, Transverse patterning reveals a dissociation of simple and configural association learning abilities in rats with 192 IgG-saporin lesions of the nucleus basalis magnocellularis, Neurobiol. Learn. Mem. 77 (2002) 211–233. [9] A.E. Butt, M.M. Noble, J.L. Rogers, T.E. Rea, Impairments in negative patterning, but not simple discrimination learning, in rats with 192 IgG-saporin lesions of the nucleus basalis magnocellularis, Behav. Neurosci. 116 (2002) 241–255. [10] D. Collerton, Cholinergic function and intellectual decline in Alzheimer’s disease, Neuroscience 19 (1986) 1–28. [11] J.T. Coyle, D.L. Price, M.R. DeLong, Alzheimer’s disease: A disorder of cortical cholinergic innervation, Science 219 (1983) 1184–1190. [12] W.A. Dornan, A.R. McCampbell, G.P. Tinkler, L.J. Hickman, A.W. Bannon, M.W. Decker, K.L. Gunther, Comparison of site-specific injections into the basal forebrain on water maze and radial arm maze performance in the male rat after immunolesioning with 192 IgG saporin, Behav. Brain Res. 82 (1996) 93–101. [13] K.D. Dougherty, D. Salat, T.J. Walsh, Intraseptal injection of the cholinergic immunotoxin 192-IgG saporin fails to disrupt latent inhibition in a conditioned taste aversion paradigm, Brain Res. 736 (1996) 260–269. [14] B. Dubois, W. Mayo, Y. Agid, M. Le Moal, H. Simon, Profound

[17]

[18]

[19]

[20]

[21] [22]

[23]

[24]

[25] [26]

[27]

[28] [29]

[30]

[31]

[32]

157

disturbances of spontaneous and learned behaviors following lesions of the nucleus basalis magnocellularis in the rat, Brain Res. 338 (1985) 249–258. R.H. Dufort, N. Guttman, G.A. Kimble, One-trial discrimination reversal in the white rat, J. Comp. Physiol. Psychol. 47 (1954) 248–249. S.B. Dunnett, Comparative effects of cholinergic drugs and lesions of nucleus basalis or fimbria-fornix on delayed matching in rats, Psychopharmacology 87 (1985) 357–363. S.B. Dunnett, G. Toniolo, A. Fine, C.N. Ryan, A. Bjorklund, S.D. Iversen, Transplantation of embryonic ventral forebrain neurons to the neocortex of rats with lesions of the nucleus basalis magnocellularis—II. Sensorimotor and learning impairments, Neuroscience 16 (1985) 787–797. S.B. Dunnett, I.Q. Whishaw, G.H. Jones, S.T. Bunch, Behavioural, biochemical and histochemical effects of different neurotoxic amino acids injected into nucleus basalis magnocellularis of rats, Neuroscience 20 (1987) 653–669. J.L. Evenden, H.M. Marston, G.H. Jones, V. Giardini, L. Lenard, B.J. Everitt, T.W. Robbins, Effects of excitotoxic lesions of the substantia innominata, ventral and dorsal globus pallidus on visual discrimination acquisition, performance and reversal in the rat, Behav. Brain Res. 32 (1989) 129–149. B.J. Everitt, T.W. Robbins, J.L. Evenden, H.M. Marston, G.H. Jones, T.E. Sirkia, The effects of excitotoxic lesions of the substantia innominata, ventral and dorsal globus pallidus on the acquisition and retention of a conditional visual discrimination: Implications for cholinergic hypotheses of learning and memory, Neuroscience 22 (1987) 441–469. B.J. Fellows, Chance stimulus sequences for discrimination tasks, Psychol. Bull. 67 (1967) 87–92. H.C. Fibiger, Cholinergic mechanisms in learning, memory, and dementia: a review of recent evidence, Trends Neurosci. 14 (1991) 220–223. A. Fine, C. Hoyle, C.J. Maclean, T.L. Levatte, H.F. Baker, R.M. Ridley, Learning impairments following injection of a selective cholinergic immunotoxin, ME20.4 IgG-saporin, into the basal nucleus of meynert in monkeys, Neuroscience 81 (1997) 331–343. C. Flicker, R.L. Dean, D.L. Watkins, S.K. Fisher, R.T. Bartus, Behavioral and neurochemical effects following neurotoxic lesions of a major cholinergic input to the cerebral cortex in the rat, Pharmacol. Biochem. Behav. 18 (1983) 973–981. H.F. Harlow, The formation of learning sets, Psychol. Rev. 56 (1949) 51–65. H.F. Harlow, Learning set and error factor theory. In: S. Koch (Ed.), Psychology: A Study of a Science. Study 1. Conceptual and Systematic. Vol. 2: General Systematic Formulations, Learning, and Special Processes. McGraw-Hill, New York, 1959, pp. 492–537. S.L. Hartgraves, P.L. Mensah, P.H. Kelly, Regional decreases of cortical choline acetyltransferase after lesions of the septal area and in the area of nucleus basalis magnocellularis, Neuroscience 7 (1982) 2369–2376. S. Heckers, M.M. Mesulam, Two types of cholinergic projections to the rat amygdale, Neuroscience 60 (1994) 383–397. S. Heckers, T. Ohtake, R.G. Wiley, D.A. Lappi, C. Geula, M.M. Mesulam, Complete and selective cholinergic denervation of rat neocortex and hippocampus but not amygdala by an immunotoxin against the p75 NGF receptor, J. Neurosci. 14 (1994) 1271–1289. D.J. Hepler, D.S. Olton, G.L. Wenk, J.T. Coyle, Lesions in nucleus basalis magnocellularis and medial septal area of rats produce qualitatively similar memory impairments, J. Neurosci. 5 (1985) 866–873. L.A. Holley, R.G. Wiley, D.A. Lappi, M. Sarter, Cortical cholinergic deafferentation following the intracortical infusion of 192 IgGsaporin: A quantitative histochemical study, Brain Res. 663 (1994) 277–286. R.W. Jacobs, L.L. Butcher, Pathology of the basal forebrain in

158

[33] [34]

[35]

[36]

[37]

[38]

[39] [40]

[41]

[42]

[43]

[44]

[45]

[46] [47]

[48]

[49]

[50]

[51]

A.M. Bailey et al. / Brain Research 969 (2003) 147–159 Alzheimer’s disease and other dementias, in: A.B. Schiebel, A.F. Wechsler (Eds.), Biological Substrates of Alzheimer’s Disease, Academic Press, New York, 1986, pp. 87–100. M.J. Karnovsky, L.A. Roots, A ‘direct-coloring’ thiocholine method for cholinesterases, J. Histochem. Cytochem. 12 (1964) 219–221. B.J. Knowlton, G.L. Wenk, D.S. Olton, J.T. Coyle, Basal forebrain lesions produce a dissociation of trial-dependent and trial-independent memory performance, Brain Res. 345 (1985) 315–321. P.J. Langlais, D.J. Connor, L. Thal, Comparison of the effects of single and combined neurotoxic lesions of the nucleus basalis magnocellularis and dorsal noradrenergic bundle on learning and memory in the rat, Behav. Brain Res. 54 (1993) 81–90. G. Leanza, O.G. Nilsson, R.G. Wiley, A. Bjorklund, Selective lesioning of the basal forebrain cholinergic system by intraventricular 192 IgG-saporin: Behavioral, biochemical and stereological studies in the rat, Eur. J. Neurosci. 7 (1995) 329–343. J. Lehmann, J.I. Nagy, S. Atmadia, H.C. Fibiger, The nucleus basalis magnocellularis: The origin of a cholinergic projection to neocortex of the rat, Neuroscience 5 (1980) 1161–1174. M. Levine, Hypothesis behavior, in: A.M. Schrier, H.F. Harlow, F. Stollnitz (Eds.), Behavior of Nonhuman Primates, Vol. I, Academic Press, New York, 1965, pp. 97–127. N.J. Mackintosh, V. Holgate, Serial reversal learning and nonreversal shift learning, J. Comp. Physiol. Psychol. 67 (1969) 89–93. M.P. McDonald, G.L. Wenk, J.N. Crawley, Analysis of galanin and the galanin antagonist M40 on delayed non-matching-to-position performance in rats lesioned with the cholinergic immunotoxin 192 IgG-Saporin, Behav. Neurosci. 111 (1997) 552–563. J. McGaughy, J.W. Dalley, C.H. Morrison, B.J. Everitt, T.W. Robbins, Selective behavioral and neurochemical effects of cholinergic lesions produced by intrabasalis infusions of 192 IgGsaporin on attentional performance in a five-choice serial reaction time task, J. Neurosci. 22 (2002) 1905–1913. J. McGaughy, T. Kaiser, M. Sarter, Behavioral vigilance following infusions of 192 IgG-saporin into the basal forebrain: Selectivity of the behavioral impairment and relation to cortical AChE-positive fiber density, Behav. Neurosci. 110 (1996) 247–265. M.M. Mesulam, E.J. Mufson, B.H. Wainer, A.I. Levey, Central cholinergic pathways in the rat: An overview based on an alternative nomenclature (Ch1–Ch6), Neuroscience 10 (1983) 1185–1201. M. Miyamoto, J. Kato, S. Narumi, A. Nagoka, Characteristics of memory impairment following lesioning of the basal forebrain and medial septal nucleus in rats, Brain Res. 419 (1987) 19–31. J.L. Muir, B.J. Everitt, T.W. Robbins, AMPA-induced excitotoxic lesions of the basal forebrain: A significant role for the cortical cholinergic system in attentional function, J. Neurosci. 14 (1994) 2313–2326. N.L. Munn, Handbook of Psychological Research on the Laboratory Rat, Houghton Mifflin, Boston, 1950. C.L. Murray, H.C. Fibiger, Learning and memory deficits after lesions of the nucleus basalis magnocellularis: Reversal by physostigmine, Neuroscience 14 (1985) 1025–1032. O.G. Nilsson, G. Leanza, C. Rosenblad, D.A. Lappi, R.G. Wiley, A. Bjorklund, Spatial learning impairments in rats with selective immunolesion of the forebrain cholinergic system, Neuroreport 3 (1992) 1005–1008. D.S. Olton, G.L. Wenk, Dementia: Animal models of the cognitive impairments produced by degeneration of the basal forebrain cholinergic system. In: H.Y. Meltzer (Ed.), Psychopharmacology: The Third Generation of Progress, Raven Press, New York, 1987, pp. 941–953. T. Perry, H. Hodges, J.A. Gray, Behavioral, histological and immunocytochemical consequences following 192 IgG-saporin immunolesions of the basal forebrain cholinergic system, Brain Res. Bull. 54 (2001) 29–48. A. Peternel, D. Hughey, G. L Wenk, D. Olton, Basal forebrain and

[52]

[53]

[54]

[55]

[56]

[57] [58]

[59]

[60]

[61]

[62]

[63]

[64]

[65]

[66]

[67]

[68]

memory: Neurotoxin lesions impair serial reversals of a spatial discrimination, Psychobiology 16 (1988) 54–58. L. Ricceri, G. Calamandrei, J. Berger-Sweeney, Different effects of postnatal day 1 versus 7 192 Immunoglobulin G-saporin lesions on learning, exploratory behaviors, and neurochemistry in juvenile rats, Behav. Neurosci. 111 (1997) 1292–1302. T.W. Robbins, B.J. Everitt, H.M. Marston, J. Wilkinson, G.H. Jones, K.J. Page, Comparative effects of ibotenic acid- and quisqualic acid-induced lesions of the substantia innomina attentional function in the rat: Further implications for the role of the cholinergic neurons of the nucleus basalis in cognitive processes, Behav. Brain Res. 35 (1989) 221–240. A.C. Roberts, T.W. Robbins, B.J. Everitt, J.L. Muir, A specific form of cognitive rigidity following excitotoxic lesions of the basal forebrain in marmosets, Neuroscience 47 (1992) 251–264. J.K. Robinson, G.L. Wenk, R.G. Wiley, D.A. Lappi, J.N. Crawley, 192 IgG-saporin immunotoxin and ibotenic acid lesions of nucleus basalis and medial septum produce comparable deficits on delayed nonmatching to position in rats, Psychobiology 24 (1996) 179–186. A. Sahgal, A.B. Keith, S. Lloyd, J.M. Kerwin, E.K. Perry, J.A. Edwardson, Memory following cholinergic (NBM) and noradrenergic (DNAB) lesions made singly or in combination: Potentiation of disruption by scopolamine, Pharmacol. Biochem. Behav. 37 (1990) 597–605. A.M. Schrier, C.R. Thompson, Are learning sets learned? A reply, Anim. Learn. Behav. 12 (1984) 109–112. T. Steckler, A.B. Keith, R.G. Wiley, A. Sahgal, Cholinergic lesions by 192 IgG-saporin and short-term recognition memory: Role of the septohippocampal projection, Neuroscience 66 (1995) 101–114. J.D. Stoehr, G.L. Wenk, Effects of age and lesions of the nucleus basalis on contextual fear conditioning, Psychobiology 23 (1995) 173–177. R.K. Thomas, R.B. Lorden, Numerical competence in animals: A conservative view, in: S.T. Boysen, E.J. Capaldi (Eds.), The Development of Numerical Competence: Animal and Human Models, Lawrence Erlbaum, Hillsdale, NJ, 1993. E.M. Torres, T.A. Perry, A. Blokland, L.S. Wilkinson, R.G. Wiley, D.A. Lappi, S.B. Dunnett, Behavioral, histochemical and biochemical consequences of selective immunolesions in discrete regions of the basal forebrain cholinergic system, Neuroscience 63 (1994) 95–122. J. Turchi, M. Sarter, Cortical cholinergic inputs mediate processing capacity: Effects of 192 IgG-saporin-induced lesions on olfactory span performance, Eur. J. Neurosci. 12 (2000) 4505–4514. N. Vnek, L.F. Kromer, R.G. Wiley, L.A. Rothblat, The basal forebrain cholinergic system and object memory in the rat, Brain Res. 710 (1996) 265–270. M.L. Voytko, D.S. Olton, R.T. Richardson, L.K. Gorman, J.R. Tobin, D.L. Price, Basal forebrain lesions in monkeys disrupt attention but not learning and memory, J. Neurosci. 14 (1994) 167–186. T.J. Walsh, R.M. Kelly, K.D. Doughtery, R.W. Stackman, R.G. Wiley, C.L. Kutscher, Behavioral and neurobiological alterations induced by the immunotoxin 192-IgG-saporin: Cholinergic and non-cholinergic effects following i.c.v. injection, Brain Res. 702 (1995) 233–245. J.M. Warren, Primate learning in comparative perspective, in: A.M. Schrier, H.F. Harlow, F. Stollnitz (Eds.), Behaviour of Nonhuman Primates, Vol. 1, Academic Press, New York, 1965, pp. 249–281. G.L. Wenk, J.D. Stoehr, G. Quintana, S. Mobley, R.G. Wiley, Behavioral, biochemical, histological, and electrophysiological effects of 192 IgG-Saporin injections into the basal forebrain of rats, J. Neurosci. 14 (1994) 5986–5995. P.J. Whitehouse, D.L. Price, A.W. Clark, J.T. Coyle, M.R. DeLong, Alzheimer disease: Evidence for selective loss of cholinergic neurons in the nucleus basalis, Ann. Neurol. 10 (1981) 122–126.

A.M. Bailey et al. / Brain Research 969 (2003) 147–159 [69] P.J. Whitehouse, D.L. Price, R.G. Struble, A.W. Clark, J.T. Coyle, M.R. DeLong, Alzheimer’s disease and senile dementia: Loss of neurons in the basal forebrain, Science 215 (1982) 1237–1239. [70] R.G. Wiley, T.N. Oeltmann, D.A. Lappi, Immunolesioning: Selective destruction of neurons using immunotoxin to rat NGF receptor, Brain Res. 562 (1991) 149–153.

159

[71] Z. Zhang, D.A. Lappi, C.C. Wrenn, T.A. Milner, R.G. Wiley, Selective lesion of the cholinergic basal forebrain causes a loss of cortical neuropeptide Y and somatostatin neurons, Brain Res. 800 (1998) 198–206.