0306-4522/90 $3.00 + 0.00 Pergamon Press plc 0 1990IBRO
Neuroscknce Vol. 38. No. 1, pp.93-102, 1990 Printed in Great Britain
MEMORY DEFICITS FOLLOWING NUCLEUS BASALIS MAGNOCELLULARIS LESIONS MAY BE MEDIATED THROUGH LIMBIC, BUT NOT NEOCORTICAL, TARGETS R. P. KEsNEa,*t K. A. CRUTCHE~*$ and H. OMANA* *Department of Psychology, University of Utah, Salt Lake City, UT 84112, U.S.A. SDepartment of Neurosurgery, University of Cincinnati, Cincinnati, OH, U.S.A. Abstract-In order to test the contribution of the target areas of the nucleus basalis magnocellularis to the mediation of item and order recognition memory for spatial locations, one set of rats received lesions of the dorsolateral frontal cortex, parietal cortex, or basolateral amygdala after training in an order recognition memory task, whereas another set of animals received lesions of the nucleus basalis magnocellularis or basolateral amygdala in an item recognition memory task. Animals with basolateral amygdala lesions displayed a deficit for order recognition memory, but no deficit for item recognition memory, a pattern equivalent to that found for animals with nucleus basalis magnocelluiaris lesions. In contrast, animals with dorsolateral frontal cortex displayed no deficit, and animals with parietal cortex lesions displayed only a partial deficit for order recognition memory, results that differ from those found for animals with nucleus basalis magnocellularis lesions. It appears that the nucleus basalis magnocellularis influences item and order recognition memory for lists of spatial locations primarily through projections to hmbic but not neocortical targets.
The basal forebrain choline@ system has been implicated as critically associated with dementia of the Alzheimer type. Evidence for this assertion includes the observation of significant cell loss in this system, with mild chromatolysis of surviving cells and the presence of neurofibrillary tangles and, occasionally, senile plaques. “~20.us8.6*66 In addition, reduced levels of cortical and limbic cholinergic markers have been documented. in Alzheimer’s brains.3,6,8.‘4,‘6,*g~’ The neocortex, amygdala and hippocampus represent major target areas of basal forebrain choline&c neurons,10*13*14*38 which have been implicated in memory function29**0.4sand represent sites of formation of neurofibrillary tangles and senile plaques in Alzheimer’s disea~.Z.7,9,23.25.52’e61 Even though the data suggest that degeneration of the basal forebrain system contributes to memory dysfunction in Alzheimer’s disease, the progressive nature of the disease, the inability to determine the extent of cell loss until autopsy, and the variability in memory dysfunction have made it difficult to ascertain the correlation between the neuropathology and memory functions. In order to test further the exact role of the cholinergic basal forebrain system in mediating mnemonic symptomatology of Alzheimer’s disease, a number of investigators have developed
animal models, for which the rodent has been used extensively.55 In these models the basal forebrain cholinergic system, or its targets, is damaged and behavioral tests used to probe mnemonic function. The assumption is that anatomically and functionally homologous systems can be identified in rats and in humans. However, it is recognized that even though there might not be strict homologous anatomical systems between animals and humans, it is likely that homologous behaviorial functions will emerge. It has been shown that the nucleus basalis magnocellularis (NBM) in rodents is involved in a variety of learning and memory tasks, including passive and active avoidance, water maze and temporal discrimination learning tasks as well as radial arm maze and delayed-matching to sample memory performance tasks l.‘.S.1931.22.33.35.36.11,62@?or an excellent re,ew see Ref. 46.) In a somewhat more complex memory task (order memory) animals with small NBM lesions show impaired recall for the last events (choice order) of a list of spatial locations, whereas large NBM lesions produce a deficit for all events within the list.27 What is not known, however, is to what extent the targets of the NBM, for example, dorsolateral frontal and parietal cortex and basolateral amygdala, contribute to the mediation of order memory for spatial locations. Thus, one question addressed in this study was to what extent lesions of these NBM target areas result in deficits for order memory for a list of spatial locations.
tTo whom correspondence
should be addressed. Abbreuiutions: AChE, acetylcholinesterase; ChAT, choline acetyltransferase; NBM, nucleus basalis magnocellularis. 93
94
R. P. KE~NEKei ai.
Previous research has location information and spatial location information
indicated that spatial temporal ordering of might be mediated by
shared as well as different neural circuitry.3.” Thus, it was of interest to determine the role of NBM and its cholinergic limbic and neocortical targets in item recognition memory for spatial location. Previous research has indicated that lesions of the NBM do not impair item recognition memory for a hst of spatiat locations, even though damage to one of its targets (e.g. parietal cortex) results in a total deficit for all spatial locations within the list.‘8.3o Thus, a second question addressed in this study was to examine whether lesions of the basolateral amygdala (a hmbic target of the NBM) impair item recognition memory for a list of spatial locations. For comparative purposes the data on the effects of NBM lesions on item recognition memory are also presented. EXPERIMENTAL
PROCEDURES
SU&ClS
Forty-seven male Long-Evans rats weighing between 300 and 400 g were used. Each animal was individually housed and maintained at approximately 8540% of its free-feeding weight. Water was continuously available. The animal cages were kept in a laboratory with a 12 h light/dark cycle. Apparurus
An eight-arm radial maze was used. It was painted white, was 94 cm high, and was kept in a well lit room having no windows. one door, and 10 calendar pictures which were placed around the room on the wails. The maze consisted of an octagonal centra1 platform, 4Ocm across, and eight arms which radiated from it like spokes of a wheel. The arms were 71 x 9cm and were attached to the central platform with metal braces. Each arm had i/8” clear Plexiglas sides, which rose 5cm above the surface of the arm and extended from the distal end of the arm to 2.5 cm from the central platform. A food well. 2.5 cm in diameter, was drilled I .5cm deep at the distal end of each arm. A i/8” Plexiglass guillotine door was located at the juncture between the plataform and the arm. This door could be raised from below the maze surface. Each door was IOcm wide and, when in the raised position, extended 23 cm above the surface of the platform. By means of a series of pulleys and strings, each door could be raised and lowered from an adjacent lab room. Strips of Plexiglas (6 x 18 cm) were installed on the central platform in the gaps between the doors in order to prohibit the rats from climbing between the doors. These strips. together with the doors, effectively formed a cyhndrlcai Plexiglas chamber on the central platform into which the animals could be placed through the open top. Finallv. a niece of lineoieum (46 x 150 cm) could be wrapped around the central chamber in such a way that, when in place, the animal could see only the ceiling of the lab. Preo~ruiii~e iruining: general proredure
initially ail animals were trained using the Oiton procedure. In this procedure one piece of Froot Loop cereal was placed at the end of each arm and not replaced during the test session. Errorless performance for an animal was to retrieve each piece of cereal by visiting each arm only once during a test session. Entering a previousiy-visited arm constituted an error. Animals were trained until they performed without errors on three of four consecutive
trials. After reaching this criterion, the Plexiglas doors were introduced so that access to the maze arms was controlled by the experimenter. The reinforcements consisted of one fourth of a Froot Loop cereal placed in the wells at the ends of different arms depending on the specific task to be tested. Order memory preoperative training
After pretraining, 25 animals were started on the serial order probe training procedure. Each trial consisted of a study phase and a test phase. During the study phase a sequence of eight arms was presented. one arm at a time. The study phase sequences differed from trial to trial. As soon as the animal returned to the central chamber from the last arm of the study phase sequence. the linoleum cover was put into place around the Plexiglas chamber. The purpose of this cover was to demarcate the end of the study phase and the beginning of the test phase, as well as to prohibit the rat from seeing the actions of the ex~~menter as he or she baited the correct test arm. Two of the doors previously visited were then opened simultaneously. The selection of the two test doors depended upon which serial position was being tested in that trial and upon the rule that the two doors be either adjacent to each other or separated by only one door. In half of the trials the correct response was to the right and in the other half of the trials the correct response was to the left. The correct response was to the arm that had been visited earlier in the sequence during the study phase of the trial. Approximately 10 s after opening the two test doors the linoleum cover was removed and the subject was allowed to respond by entering one of the two open doors. If the animal made a correct response, it collected one Froot Loop from the well. If the animal made an error, it received no ~info~ment and was not allowed to enter the correct door. Only one test was given for each study phase. Each animal received, on a random basrs. a choice (one test only) of either the first and second (l-2). fourth and fifth (45), or seventh and eighth (7-X) arms in the sequence. The animals were initially given eight trials per serial position for a total of 24 trials. One trial was given per day, 5 days a week. If, after these 24 trials. the animal had not performed at 75% or better in tests of both the l-2 (primacy) and 7-8 (recency) serial position (criterion). then additional blocks of six trials (two trials per serial position) were given until the criterion was met for the most recent block of 24 trials Surgery. After reaching criterion all 25 animals were anesthetized with sodium pentobarbrtal (nembutal, 40 mg/kg, i.p.f and given atropine sulfate (0.2 mg, i.p.) as a prophylactic prior to surgery. They were divided into four groups. The first group (N = 7) received bilateral electrolytic lesions of the basoiateral amygdala. Lesions were produced by passing a 2.0mA anodal current for 20s through a stainless steel electrode (0.25 mm in diameter) insulated with the exception of 0.5 mm at the tip. The lesion coordinates with the rat’s head level were 2.3 mm posterior to bregma, 4.5 mm faterai to midline, and 7.8 mm below the dura. The second group (N = 7) received bilateral parietal cortex lesions by means of aspiration and were intended to destroy parietal areas 5, 7. 39 and 40. The cortex within the following coordinates was aspirated: in the anferiorposterior plane, the lesion included approximately the region from 0.5 mm anterior to bregma to 3.5 mm posterior to bregma: in the m~ial-lateral plane. the lesion began 2 mm lateral to midline and eontinued laterally towards the rhinal suicus. The depth was 2 mm, and attempts were made not to damage the corpus caliosum. The third group (N = 5) received bilateral dorsoiaterai frontal cortex lesions by means of aspiration. The cortex within the following coordinates was aspirated: in the anterior-posterior plane, the lesion included approximately the region from bregma to 3.5 mm anterior to bregma, and
95
Memory deficits following NBM lesions in the medial-lateral plane, the lesion included the area 2-5 mm lateral from midline. The depth was 2 mm. The last group (N = 6) served as sham operated controls. PW operPtive re.rfing. One week following recovery from surgery all animals were given 24 tests (one per day) with eight tests at each order choice (l-2, 4-5, 7-8) position. Item memory preoperative training After pretraining, 22 animals began the item recognition probe training procedure. As in the order memory task, training trials consisted of a study phase and a test phase except that the animal was presented with a sequence of five arms. Each five-arm sequence was selected with the stipulations that no three adjacent arms could be presented in one sequence and that each sequence differed from trial to trial. Once the animal had returned to the central platform from the fifth arm of the study phase, the door was closed and the linoleum cover was lowered around the central structure. Two doors were then immediately opened, one to an arm the animal had visited during the study phase and one to an arm the animal had not. The selection of the two doors was based upon three criteria: first, which serial position was being tested on that particular trial; second, that the selected arms were either adjacent or separated by only one arm; third, that the correct response was 50% of the time on the right or on the left. Approximately 10s after the two test phase doors were opened, the linoleum cover was raised and the animal was allowed to respond by entering one of the two doors. The rule to be learned leading to an additional reinforcement (one piece of Froot Loop cereal) was to choose the arm that had been previously visited during the study phase of the trial (win-stay rule). If the animal chose the arm that was not in the study phase sequence, it was removed from the maxe and placed back into its cage without reinforcement. Each animai received one to three trials per day. On days when more than one trial was piven, each trial was separated by at least 1h. Each animal received 40 tests with eight tests for each of the serial positions. If, after these 40 trials, the animal had not performed 75% or better on the first and last serial position, then additional blocks of 10 trials (two trials per serial position) were given until the criterion was met for the most recent block of 40 trials. Surgery. After reaching criterion all 22 animals were anesthetized with sodium pentobarbital (nembutai, 40 mg/ kg, i.p.) and given atropine sulfate (0.2mg, i.p.) as a prophylactic prior to surgery. The animals were then divided into three groups. The first group (N = 6) received bilateral electrolytic lesions of the basolateral amygdala using the same procedure as described for the order memory experiment. The second group (lv = 9) received ibotenic acid lesions of the nucleus basalis magnocellularis. Ibotenic acid (8 fig/l .O~1 dissolved in 1~1 of Tris buffer) was injected bilaterally using a 10 p 1syringe. One microliter was injected for a duration of 2-3 min. The coordinates with the rat’s head level were 0.7 mm posterior to bregma, 3.2 mm lateral and 6.8 mm below the dura. The third group (IV = 7) served as a control. Four animals received vehicle injections into the nucleus basalis magnocellularis, while three animals received sham operations. Postoperative testing. One week following recovery from surgery, the animals were given 40 tests (one per day) with eight tests at each serial position. Histology At the end of the experiment, all but the ibotenic acid- and saline-injected animals were given a lethal injection of sodium pentobarbital, Riven 0.05 cm’ heparin intracardiaily, and perfused intra~rdially with 10% formaiin solution. The brains were removed from the skulls and stored in 10% formalin for 1 week. They were then photographed, sliced at 50 firn with a freezing microtome and every third section NSC W-D
was mounted, stained with Cresyl Violet, and examined for histological verification of the lesion placement. The ibotenic acid- and saline-injected animals were killed by decapitation and the brains rapidly removed and frozen on dry-ice. Fresh frozen transverse sections (16pm) were cut with a cryostat and stained for Nissl substance or acetylcholinesterase (AChE) histochemistry to determine the extent and location of nucleus basaiis magnocellularis lesions. In addition, two series of sections through the rostrocaudal extent of frontal and parietal neocortex were stained for Nissl substance or AChE histochemistry as described previously. iJ Briefly, the sections used for assessing choiinergic denervation were incubated overnight in a solution containing cupric sulfate, glycine, and acetylthiocholine iodide.” Promethazine was added to inhibit nonspecific esterase activity. The sections were developed with ammonium sulfide and counterstained with Thionin. Some sections were reacted according to the modification of Storm-Mathisen and Blackstads’ in order to intensify the reaction product for dark-field visualization and photomicroscopy. The latter procedure is more sensitive but gives results of lower resolution when compared with the Naik modification.” Histcchemical analysis of the amount of AChE depletion in the neocortex was carried out without knowledge of the behavioral results. In order to obtain a relative indication of the amount of cholinergic denervation following NMB lesions, the AChE-stained tissue sections were examined with both dark-field and bright-field illumination from the frontal pole to the posterior limit of the parietal cortex. Extensive cortical denervation was defined in terms of the complete loss of AChE staining bilaterally within the dorsolateral cortex bordered medially by the cingulate region and ventrolatemlly by the rhinai sulcus (parietal and frontal cortex). Thus, partial denervation was present in thosecasesexhibiting less AChE loss within the dorsolaterai cortical areas when compared with those with extensive depletion. RESULTS Histology The dorsolateral frontal lesions were consistent across subjects. A photomicrograph of a representative lesion is presented in Fig. 1. Note that there is no damage to the medial prefrontal cortex. The parietal cortex lesions were also consistent and included areas 5 and 7. A photomicrograph of a representative lesion is shown in Fig. 2A, B. The corpus callosum was damaged in all animals. Retrograde cell degeneration was present in the lateral posterior, posterior and ventral posterior nuclei of the thalamus, indicating destruction of the intended parietal areas. The amygdala lesions were somewhat variable but included bilaterally the basolateral and central amygdala in all animals. In some animals there was unilateral damage to the cortico-medial amygdala. A representative lesion is presented in Fig. 3. Of the nine NBM lesioned animals, five animals had extensive bilateral AChE depletion of parietal and dorsolateral frontal cortex with minimal AChE depletion of medial prefrontal and orbito-frontal cortex. An example of an NBM lesion that produced extensive bilateral depletion of parietal and parts of frontal cortex, is shown in Fig. 4. Examples of bilateral depletion of AChE in parietal and parts of the frontal cortex have been published previously.27
R. P. KESNERet al
96
Fig. I. Photograph
of a representative dorsolateral frontal cortex lesion.
The remaining four animals had unilateral or smaller bilateral NBM damage with corresponding depletion of AChE in parietal and dorsolateral frontal cortex. Based on the extent (extensive vs partial) of AChE depletion in neocortex the animals were divided into two groups. Behavioral performance-order
memory
Postoperative behavioral performance (mean per cent correct) is shown in Fig. 5 for each group as a function of serial order position (choice order). The graph shows that control subjects and animals with dorsolateral frontal cortex lesions display a serial position curve with good memory for the
first (I-2) and last (7-8) choice order. In contrast, animals with parietal cortex lesions are impaired in remembering the first (l-2) choice order, whereas animals with basolateral amygdala lesions are impaired for all choice orders within the list. A twoway analysis of variance with repeated measures on three choice orders revealed a signScant choice order effect (F = 18.8, df 2.42, P < 0.001) and sig nificant interaction between lesion groups and choice order (F = 3.1, df 6.42, P < 0.05). St&sequent Newman-Keuls tests indicated that far the 1-2 choice order the animals with dorsolateral frontal cortex lesions and controls displayed signScantly better memory than animals with either parietal cortex or
Memory deficits following NBM lesions
Fig. 2. Photographs
of a representative
parietal and posterior parietal association cortex lesion.
97
98
R. P.
KEENER
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Fig. 3. Diagram of a representative
amygdala lesion. Brain sections were taken from Paxinos and Watson.*
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Fig. 4. Diagram of a representative nucleus basalis magnoceliularis lesion that produced extensive a~tylcholin~tc~~ depletion of parietal and dorsolateral frontal cortex. Brain sections were taken from Paxinos and Watson.Lb
99
Memory deficits following NBM lesions
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Fig. 5. Mean percentage responses as a function of choice orders after amygdala, AMY; dorsolateral frontal cortex, DFC; parietal cortex, PC; or control, CONT lesions.
basolateral amygdala lesions (P < 0.05). For the control animals and animals with dorsolateral frontal cortex lesions performance on the l-2 and 7-8 choice order was significantly better than performance for 4-5 choice order (P < 0.05). For the animals with parietal cortex lesions performance for the 7-8 choice order was significantly better than performance for l-2 and 4-5 choice orders (P < 0.05). There were no significant differences between choice orders for animals with basolateral amygdala lesions. Behavioral performance-item
memory
Postoperative behavioral performance (mean per cent correct) is shown in Fig. 6 for the control, NBM, and amygdala lesioned groups as a function of serial position. The graph shows that control subjects and animals with small NBM lesions display excellent performance with somewhat better retention for the first and last items within the list. Animals with basolateral amygdala lesions and large NBM lesions also displayed good performance with slightly poorer retention for the last item within the list. A two-way analysis of variance for groups with repeated measures on serial position revealed non-significant effects for groups and serial position. DlSCUSSlON
The results of the present experiment indicate that, in contrast to NBM lesions, lesions of the dorsolateral frontal cortex do not impair order memory performance. This dissociation implies that the loss of NBM input to dorsolateral frontal cortex does not underlie the deficit in order memory for spatial
SERIALPOSITION
Fig. 6. Mean percentage correct responses as a function of serial position of items (spatial locations) after amygdala, AMY; small or large nucleus basalis magnocellularis, NBM or control, CONT lesions.
location information. In other studies ibotenic acid injections into the NBM produced greater deficits than quisqualic acid in the acquisition of a Morris water maze task, retention of passive avoidance learning and performance of an alternation task in a T-maze, even though quisqualic acid produced similar, or significantly greater, depletion of choline acetyltransferase (ChAT) levels of frontal cortex.‘9.63 Furthermore, there has been a general failure to find significant correlations between decreased frontal ChAT activity and behavioral deficits following NBM lesions.5 There are a number of possible interpretations for the observed dissociations between NBM and dorsolateral frontal cortex. First, the ascending cholinergic input from the NBM might not be. critical for memory performance within a large number of tasks. Instead, NBM lesions might have damaged other non-cholinergic neurotransmitter systems. One such system could be a neocortical descending projection to NBM mediated by glutamate. Damage to the NBM could have produced retrograde degeneration of neocortical neurons that produce glutamate. For order memory this possibility can be ruled out, because lesions of the dorsolateral frontal cortex do not produce a performance deficit. Second, because dorsolateral frontal cortex appears to play an important role in tasks requiring motor learning and time estimation,38.M it is likely that this order memory task for a list of spatial locations and many other tasks as well does not engage the dorsolateral frontal cortex. Thus, one might not expect to find a significant choline@ contribution to the dorsolateral frontal cortex in memory tasks that do not utilize this region.
100
R. P.
KESNER ef ~1.
Finally, there is a possibility that denervation of the target might lead to a greater deficit than total destruction of the target. Some support for this idea comes from the observations of Solomon et a1.,56 who have shown that in rabbits, systemic injections of scopolamine disrupt a classically conditioned nicitating membrane response, whereas removal of the hippocampus or scopolamine injections in rabbits with hippocampus lesions has no disruptive effect. The results of the present experiment also indicate that there are different effects of NBM compared with parietal cortex lesions. Lesions of the parietaf cortex disrupt item recognition for spatial locations,‘” whereas even large lesions of the NBM which result in cortical cholinergic denervation of parietal cortex do not produce any deficits. Similarly, for order memory, lesions of the parietal cortex produce a deficit pattern that is different from that seen following NBM lesions. This dissociation also implies that for item and order recognition memory the ascending chohnergic system does not play a critical role. Parallel deficits have been observed following parietal cortex and NBM lesions in performance of a reference memory task in an eight arm maze and in the acquisition of a water maze or a cheese board (a dry maze analog of the water maze) task.‘9.26.2R.3L It is not clear how these tasks differ from the item and order recognition memory tasks in terms of the utilization of spatial information. In contrast to the dissociations found between NBM and neocortex, the results of the present experiment indicate that there are parallel patterns of results in the item and order memory tasks with basolateral amygdala and NBM lesions, namely an extensive deficit for order memory but no deficit for item memory. Thus, it appears that the NBM might mediate item and order memory through its connections with the basolateral amygdaia. Since reciprocal connections may exist between NBM and basolateral amygdala, one cannot determine in which direction the influence is mediated.‘0~‘2~‘7~37~53 The disruptive effects of NBM lesions on active and passive avoidance learning are certainly comparable to what has been described for basolateral amygdala lesions.” Unfortunately, no one has reported correlations between ChAT levels in amygdala and behavioral deficits observed following NBM lesions. So, it is not possible to determine whether the disruptive effects of NBM lesions are primarily through a chohnergic
projection to the basolateral amygdala. At present, it is not possible to discern whether NBM or basolateral amygdala lesions alter memory for the order of spatial location information. because of a direct influence on the mediation of perceptual or motor processes or memory storage. Elsewhere, Kesner and DiMattia29 have suggested that the amygdala complex is directly involved in maintaining temporary representations of affect-faden as triggered by reinforcement and temporal information, but not spatial location information. Thus, one would predict that amygdala lesions would not after memory for item (spatial iocation and reinforcement) information, but would after memory for order (temporal representation of spatiat location and reinforcement) information. It is then likely that N3M represents a cholinergic modulatory influence on amygdata function. Other components of the basal forebrain cholinergic system also play an important role in memory. For example, the medial septum and the hippocampal formation clearly mediate item and order memory in a parallel fashion,26 suggesting an important influence on memory of the medial septum component of the choline@ basai forebrain system. It is of interest to note that even though the medial septum and the hippocampal formation are directly involved in processing of both item and order spatial location information, the NBM and amygdala are involved primarily in coding order, but not item, spatial location formation. Thus, it appears that there is a dissociation of function between hippocampus and amygdala as well as medial septum and NBM. Finally, it should be noted that the horizontal nucleus of the diagonal band has a cholinergic projection primarily to medial prefrontal and entorhinat cortices. These latter neocortical areas are known to be involved in memory. Thus, it is possible that the horizontal nucleus of the diagonal band plays a role in memory via its neocortical ascending cholinergic contribution. In summary, the NBM appears to influence order but not item memory performance for lists of spatial location, perhaps through cholinergic projections to limbic but not neocortical targets. AcknoH,ledgemenrs-Support for this research was provided bv NIH Grant NS19406-03 and NIH Grant ROINS267?1-04 (RPK), and National Institute on Agin Grant No. AG-07691 (KAC).
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(1987) Behavioral, biochemical and histochemical effects of different neurotoxic amino acids injected into nucleus basalis magnoceliularis of rats. Neuroscience 28, 653669. 20. Ezrin-Waters C. and Resch L. (1986) The nucleus basalis of Meynert. Can. J, Neurot. Sci. 13, 8-14. 21. Flicker C., Dean R. L., Watkins D. L., Fisher S. K. and Bartus R. T. (1983) Behavioral and neur~hemi~al effects following neurotoxic lesions of a major cholinergic input to the cerebral cortex in the rat. Phurmac. Biochem. Bebav. 18, 973-981, 22. Hepler D. J., Wenk G. L., Cribbs B. L., Olton D. S. and Coyle J. T. (1985) Memory impairments following basal forebrain lesions. Bruin Res. 364, 8-14. 23. Hyman B. T., Van Hoesen G. W., Damasio A. R. and Barnes C. L. (1984) Alzheimer’s disease: cell-specific pathology isolates the hip-pal formation. Science 225, 1168-l 170. 24. Johnston M. V., McKinney M. and Coyle J. T. (1981) Neocortical choline&c innervation: a descriotion of extrinsic . and intrinsic components in the rat. Eip! Brain Res. 43, 159-172. 25. Kemper T. L. (1983) Organization of the neuropathology of the amygdala in Alzheimer’s disease. 3ioL Asp. Afrheimer’s Disease Banbury Rep. 15, 31-35. 26. Kesner R. P. (1988) Neuronal organization of memory functions in animals and humans. In Systems with rParning and Memory Abiliries (eds Delacour J. and Levy J. C. S.), pp. 127-151. Elsevier, Amsterdam. 27. Kesner R. P., Crutcher K. A. and Measom M. 0. (1986) Medial septai and nucleus basalis magnocellularis lesions produce order memory deficits in rats which mimic s~ptomatolo~ of Aizheimer’s disease. Neurobiol. Aging 7, 287-295. 2%. Kesner R. P., DiMattia %. V. and Crutcher K. A. (1987) Evidence for neocortical involvement in reference memory. Behav. neural Biol. 47, 40-53. 29. Kesner R. P. and DiMattia B. V. (1987) Neurobiology of an attribute model of memory. In Progress in Psychobiology and Physiological Psychology (eds Epstein A. N. and Morrison A.), Vol. 12, pp. 207-277. Academic Press, New York. 30. Kesner R. P., Adelstein T. B. and Crutcher K. A. (1989) Equivalent spatial location memory deficits in rats with medial septum or hippocampal formation lesions and patients with dementia of the Alzheimer’s type. Brain Cognition 9, 289-300. 31. Kesner R. P. and Berman R. F. (1990) The effects of nucleus basalis magnocellularis lesions on the acquisition of a cheese board (cognitive mapping) task (submitted). 32. Kesner R. P. and Gray M. L. (1989) Dissociation of item and order memory foilowing parietal cortex lesions in the rat. Behav. Neurosci. 103, 907-910. 33. Kessler J., Markowitsch H. J. and Sigg G. (1986) Memory related role of the posterior cholinergic system. Inr. J. Neurosci. 30, 101-l 19. 34. Koelle G. B. (1950) The histochemical differentiation of types of cholinesterases and their localization in tissues of the cat. J. Phurmac. exp. Ther. 100, 158-179. 3.5. Lerer B., Warner J., Friedman E., Vincent G. and Gamzu E. (1985) Cortical cholinergic impairment and behavioral deficits produced by kainic acid lesions of rat magnocellular basal forebrain. Behav. Neurasci. 99, 661677. 36. LoConte G., Bartolini L.. Casamente F., Marconcini-Pepeu 1. and Pepeu G. (1982) Lesions of choline@ forebrain nuclei: changes in avoidance behavior and scopolamine actions. Pharnuzc. Biochem. Behav. 17, 933-937. 37. McGeer P. L., McGeer E. G. and Nagai T. (1983) GABAergic and cholinergic indices in various regions of rat brain after intracerebral injections of folic acid. Bruin Res, 260, 107-l 16. 38. Meclr W. H.. Church R. M. and Olton D. S. (1984) Hippocampus, time and memory. Bahay. Neurosci. 98, 3-22.
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39. Mesuiam E., Mufson J. B., Wainer H. and Levey A. I. (1983) Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Chl-Ch6). Neuroscience 10, 1185-1201. 40. Mishkin M. (1982) A memory system in the monkey. Phil. Trans. R. Sot. Lend. Ser. B. 298, 85-95. 41. Miyamoto M., Shintani M., Nagaoka A. and Nagawa Y. (1985) Lesioning of the rat basal forebrain leads to memory impairments in passive and active avoidance tasks. Bruin Res. 32&l,97-104. 42. Nagai T., Kimura H., Maeda T., McGeer P. L.. Peng F. and McGeer E. G. (1982) Cholinergic projections from the basal forebrain of rat to the amygdaia. J. Neurosci. 2, 513-520. 43. Naik N. T. (1963) Technical variations in Koeiie’s histochemicai method for demonstrating choiinesterase activity. Q. JI microsc. Sci. 104, 89-100. 44. Nakano I. and Hirano A. (1982) Loss of large neurons of the medial septal nucleus in an autopsy case of Aizheimer’s disease. J. Neuropath. exp. Neurol. 41, 341. 45. Ohon D. S. (1983) Memory functions and the hipp~ampus. fn N~~o~io~og~ of&e ~ippocamp~ (ed. Seifert W.). pp. 335-373. Academic Press, New York. 46. Oiton D. S. and Wenk G. L. (1987) Dementia: animal models of the cognitive impairments produced by degeneration of the basal forebrain choiinergic system. In Psychophurmaco/og_v: The Third Generution of Progress (ed. Meltzer H. Y.), pp. 941-953. Raven Press, New York. 47. Paxinos G. and Watson C. (1982) The Rat Brain in Stereotaxic Coordinales. Academic Press, Sydney. 48. Perry E. K., Perry R. H.. Gibson P., Blessed G. and Tomiinson B. E. (1977) A choiinergic connection between normal aging and senile dementia in the human hippocampus. Neurosci. Lett. 32, 643-646. 49. Ferry E. K., Blessed G. and Tomiinson 8. E. (1978) Changes in brain choiinesterases in senile dementia of Alzheimer type. Nearopafh. uppl. Neurobiof. 4, 273-277. 50. Perry E. K., Tomiinson B. E.. Blessed G., Bergmann K., Gibson P. H. and Perry R. H. (1978) Correlation ofchoiinergic abnormalities with senile plaques and mental test scores in senile dementia. Br. med. J. 2, 1457-1459. 51. Pope A. H., Hess H. and Lewin E. (1965) Microchemical pathology of the cerebral cortex in pre-senile dementias. Trans. Am. rteurol. Ass. 89, 15-16.
52. Price D. L. (1986) New perspectives on Alzheimer’s disease. A. Rer. Neurosci. 9, 489-512. basal nucleus. .I. corny. 53. Saper C. B. (1984) Organization of cerebral cortical afferent systems in the rat. I. Ma~~ilular Neurot. 222, 3 13-342. 54. Sarter M. and Markowitsch H. J. (1985) involvement of the amygdaia in learning and memory: a critical review. with emphasis on anatomical relations. Behau. Neurosci. 99, 342-380. 55. Smith G. (1988) Animal models of Aizheimer’s disease: experimental choline@ denervation. Brain Res. Rev. 13, 103-l 18.
56. Solomon P. R., Solomon S. D., Vander Schaaf E. and Perry H. E. (1983) Altered activity in the hippocampus is more detrimental to classical conditioning than removing the structure. Science 220, 331-332. J. and Blackstad T. W. (1964) Choline&erase in the hipp~mpal region. Dist~bution and relation 57. Stop-Mathisen to architectonics and afferent systems. Acta unaf. 36, 2162.53. 58. Tagliavini F. and Pilieri G. (1983) Basal nucleus of Meynert. J. Neural Sci. 62, 243-260. 59. Tomiinson B. E. (1977) Morphological changes and dementia in old age. In Aging and Dementia (eds Smith W. L. and Kinsboume M.), pp. 25-56. Spectrum Publications, New York. 60. Tomiinson B. E. and Kitchener D. (1972) Granuiovacuoiar degeneration of hippocampai pyramidal ceils. J. Pathol. 106, 165. 61. Van Hoesen G. W., Hyman B. T. and Damasio A. R. (1986) Ceil-specific pathology in neural systems of the temporal lobe in Alzheimer’s disease. In Dynamics c$ Cbo~j~e~gic Farnetions fed. Hanin I.), pp. 321-335. Pienum Press, Now York. 62. Wenk G. L. and Oiton D. S. (1987) Basal forebrain choiinergic neurons and Alzheimer’s disease. In Animal Model3 qj’ Dementia: A Synaptic Neurochemical Perspective (ed. Coyie J. T.), pp. 81-101. Alan Liss, New York. 63. Wenk G. L., Markowska A. L. and Oiton D. S. (1989) Basal forebrain lesions and memory: alterations in neurotensin, not acetyichoiine, may cause amnesia. Behaa. Nercrosci. 101, 765-769. 64. Whishaw f. Q., O’Connor W. T. and Dunnett S. B. (1986) The contributions of motor cortex, caudate-putamen and ni~ost~atal dopamine to skilled forelimb use in the rat. Brain IO!& 805-843. for 65. Whitehouse P. f., Price D. L., Clark A. W., Coyie J. ‘I’. and DeLong M. R. (1981) Aizheimer disease-evidence selective loss of choiinergic neurons in the nucleus basaiis. Ann. Neurol. 10, 122-126. 66. Whitehouse P. J., Price D. L., Strubie R. G.. Coyie J. T. and DeLong M. R. (1982) Aizheimer’s disease and senile dementia-loss of neurons in the basal forebrain. Science 215, 1237-1239. 61. Woolf N. J. and Butcher L. L. (1982) Cholinergic projections to the basolaterai amygdaia: a combined Evans Blue and acetylcholinesterase analysis. Brain Res. BUN. 8. 751-763. (Accepted
13 April 1990)
Neuroscknce Vol. 38. No. 1, pp.93-102, 1990 Printed in Great Britain
MEMORY DEFICITS FOLLOWING NUCLEUS BASALIS MAGNOCELLULARIS LESIONS MAY BE MEDIATED THROUGH LIMBIC, BUT NOT NEOCORTICAL, TARGETS R. P. KEsNEa,*t K. A. CRUTCHE~*$ and H. OMANA* *Department of Psychology, University of Utah, Salt Lake City, UT 84112, U.S.A. SDepartment of Neurosurgery, University of Cincinnati, Cincinnati, OH, U.S.A. Abstract-In order to test the contribution of the target areas of the nucleus basalis magnocellularis to the mediation of item and order recognition memory for spatial locations, one set of rats received lesions of the dorsolateral frontal cortex, parietal cortex, or basolateral amygdala after training in an order recognition memory task, whereas another set of animals received lesions of the nucleus basalis magnocellularis or basolateral amygdala in an item recognition memory task. Animals with basolateral amygdala lesions displayed a deficit for order recognition memory, but no deficit for item recognition memory, a pattern equivalent to that found for animals with nucleus basalis magnocelluiaris lesions. In contrast, animals with dorsolateral frontal cortex displayed no deficit, and animals with parietal cortex lesions displayed only a partial deficit for order recognition memory, results that differ from those found for animals with nucleus basalis magnocellularis lesions. It appears that the nucleus basalis magnocellularis influences item and order recognition memory for lists of spatial locations primarily through projections to hmbic but not neocortical targets.
The basal forebrain choline@ system has been implicated as critically associated with dementia of the Alzheimer type. Evidence for this assertion includes the observation of significant cell loss in this system, with mild chromatolysis of surviving cells and the presence of neurofibrillary tangles and, occasionally, senile plaques. “~20.us8.6*66 In addition, reduced levels of cortical and limbic cholinergic markers have been documented. in Alzheimer’s brains.3,6,8.‘4,‘6,*g~’ The neocortex, amygdala and hippocampus represent major target areas of basal forebrain choline&c neurons,10*13*14*38 which have been implicated in memory function29**0.4sand represent sites of formation of neurofibrillary tangles and senile plaques in Alzheimer’s disea~.Z.7,9,23.25.52’e61 Even though the data suggest that degeneration of the basal forebrain system contributes to memory dysfunction in Alzheimer’s disease, the progressive nature of the disease, the inability to determine the extent of cell loss until autopsy, and the variability in memory dysfunction have made it difficult to ascertain the correlation between the neuropathology and memory functions. In order to test further the exact role of the cholinergic basal forebrain system in mediating mnemonic symptomatology of Alzheimer’s disease, a number of investigators have developed
animal models, for which the rodent has been used extensively.55 In these models the basal forebrain cholinergic system, or its targets, is damaged and behavioral tests used to probe mnemonic function. The assumption is that anatomically and functionally homologous systems can be identified in rats and in humans. However, it is recognized that even though there might not be strict homologous anatomical systems between animals and humans, it is likely that homologous behaviorial functions will emerge. It has been shown that the nucleus basalis magnocellularis (NBM) in rodents is involved in a variety of learning and memory tasks, including passive and active avoidance, water maze and temporal discrimination learning tasks as well as radial arm maze and delayed-matching to sample memory performance tasks l.‘.S.1931.22.33.35.36.11,62@?or an excellent re,ew see Ref. 46.) In a somewhat more complex memory task (order memory) animals with small NBM lesions show impaired recall for the last events (choice order) of a list of spatial locations, whereas large NBM lesions produce a deficit for all events within the list.27 What is not known, however, is to what extent the targets of the NBM, for example, dorsolateral frontal and parietal cortex and basolateral amygdala, contribute to the mediation of order memory for spatial locations. Thus, one question addressed in this study was to what extent lesions of these NBM target areas result in deficits for order memory for a list of spatial locations.
tTo whom correspondence
should be addressed. Abbreuiutions: AChE, acetylcholinesterase; ChAT, choline acetyltransferase; NBM, nucleus basalis magnocellularis. 93
94
R. P. KE~NEKei ai.
Previous research has location information and spatial location information
indicated that spatial temporal ordering of might be mediated by
shared as well as different neural circuitry.3.” Thus, it was of interest to determine the role of NBM and its cholinergic limbic and neocortical targets in item recognition memory for spatial location. Previous research has indicated that lesions of the NBM do not impair item recognition memory for a hst of spatiat locations, even though damage to one of its targets (e.g. parietal cortex) results in a total deficit for all spatial locations within the list.‘8.3o Thus, a second question addressed in this study was to examine whether lesions of the basolateral amygdala (a hmbic target of the NBM) impair item recognition memory for a list of spatial locations. For comparative purposes the data on the effects of NBM lesions on item recognition memory are also presented. EXPERIMENTAL
PROCEDURES
SU&ClS
Forty-seven male Long-Evans rats weighing between 300 and 400 g were used. Each animal was individually housed and maintained at approximately 8540% of its free-feeding weight. Water was continuously available. The animal cages were kept in a laboratory with a 12 h light/dark cycle. Apparurus
An eight-arm radial maze was used. It was painted white, was 94 cm high, and was kept in a well lit room having no windows. one door, and 10 calendar pictures which were placed around the room on the wails. The maze consisted of an octagonal centra1 platform, 4Ocm across, and eight arms which radiated from it like spokes of a wheel. The arms were 71 x 9cm and were attached to the central platform with metal braces. Each arm had i/8” clear Plexiglas sides, which rose 5cm above the surface of the arm and extended from the distal end of the arm to 2.5 cm from the central platform. A food well. 2.5 cm in diameter, was drilled I .5cm deep at the distal end of each arm. A i/8” Plexiglass guillotine door was located at the juncture between the plataform and the arm. This door could be raised from below the maze surface. Each door was IOcm wide and, when in the raised position, extended 23 cm above the surface of the platform. By means of a series of pulleys and strings, each door could be raised and lowered from an adjacent lab room. Strips of Plexiglas (6 x 18 cm) were installed on the central platform in the gaps between the doors in order to prohibit the rats from climbing between the doors. These strips. together with the doors, effectively formed a cyhndrlcai Plexiglas chamber on the central platform into which the animals could be placed through the open top. Finallv. a niece of lineoieum (46 x 150 cm) could be wrapped around the central chamber in such a way that, when in place, the animal could see only the ceiling of the lab. Preo~ruiii~e iruining: general proredure
initially ail animals were trained using the Oiton procedure. In this procedure one piece of Froot Loop cereal was placed at the end of each arm and not replaced during the test session. Errorless performance for an animal was to retrieve each piece of cereal by visiting each arm only once during a test session. Entering a previousiy-visited arm constituted an error. Animals were trained until they performed without errors on three of four consecutive
trials. After reaching this criterion, the Plexiglas doors were introduced so that access to the maze arms was controlled by the experimenter. The reinforcements consisted of one fourth of a Froot Loop cereal placed in the wells at the ends of different arms depending on the specific task to be tested. Order memory preoperative training
After pretraining, 25 animals were started on the serial order probe training procedure. Each trial consisted of a study phase and a test phase. During the study phase a sequence of eight arms was presented. one arm at a time. The study phase sequences differed from trial to trial. As soon as the animal returned to the central chamber from the last arm of the study phase sequence. the linoleum cover was put into place around the Plexiglas chamber. The purpose of this cover was to demarcate the end of the study phase and the beginning of the test phase, as well as to prohibit the rat from seeing the actions of the ex~~menter as he or she baited the correct test arm. Two of the doors previously visited were then opened simultaneously. The selection of the two test doors depended upon which serial position was being tested in that trial and upon the rule that the two doors be either adjacent to each other or separated by only one door. In half of the trials the correct response was to the right and in the other half of the trials the correct response was to the left. The correct response was to the arm that had been visited earlier in the sequence during the study phase of the trial. Approximately 10 s after opening the two test doors the linoleum cover was removed and the subject was allowed to respond by entering one of the two open doors. If the animal made a correct response, it collected one Froot Loop from the well. If the animal made an error, it received no ~info~ment and was not allowed to enter the correct door. Only one test was given for each study phase. Each animal received, on a random basrs. a choice (one test only) of either the first and second (l-2). fourth and fifth (45), or seventh and eighth (7-X) arms in the sequence. The animals were initially given eight trials per serial position for a total of 24 trials. One trial was given per day, 5 days a week. If, after these 24 trials. the animal had not performed at 75% or better in tests of both the l-2 (primacy) and 7-8 (recency) serial position (criterion). then additional blocks of six trials (two trials per serial position) were given until the criterion was met for the most recent block of 24 trials Surgery. After reaching criterion all 25 animals were anesthetized with sodium pentobarbrtal (nembutal, 40 mg/kg, i.p.f and given atropine sulfate (0.2 mg, i.p.) as a prophylactic prior to surgery. They were divided into four groups. The first group (N = 7) received bilateral electrolytic lesions of the basoiateral amygdala. Lesions were produced by passing a 2.0mA anodal current for 20s through a stainless steel electrode (0.25 mm in diameter) insulated with the exception of 0.5 mm at the tip. The lesion coordinates with the rat’s head level were 2.3 mm posterior to bregma, 4.5 mm faterai to midline, and 7.8 mm below the dura. The second group (N = 7) received bilateral parietal cortex lesions by means of aspiration and were intended to destroy parietal areas 5, 7. 39 and 40. The cortex within the following coordinates was aspirated: in the anferiorposterior plane, the lesion included approximately the region from 0.5 mm anterior to bregma to 3.5 mm posterior to bregma: in the m~ial-lateral plane. the lesion began 2 mm lateral to midline and eontinued laterally towards the rhinal suicus. The depth was 2 mm, and attempts were made not to damage the corpus caliosum. The third group (N = 5) received bilateral dorsoiaterai frontal cortex lesions by means of aspiration. The cortex within the following coordinates was aspirated: in the anterior-posterior plane, the lesion included approximately the region from bregma to 3.5 mm anterior to bregma, and
95
Memory deficits following NBM lesions in the medial-lateral plane, the lesion included the area 2-5 mm lateral from midline. The depth was 2 mm. The last group (N = 6) served as sham operated controls. PW operPtive re.rfing. One week following recovery from surgery all animals were given 24 tests (one per day) with eight tests at each order choice (l-2, 4-5, 7-8) position. Item memory preoperative training After pretraining, 22 animals began the item recognition probe training procedure. As in the order memory task, training trials consisted of a study phase and a test phase except that the animal was presented with a sequence of five arms. Each five-arm sequence was selected with the stipulations that no three adjacent arms could be presented in one sequence and that each sequence differed from trial to trial. Once the animal had returned to the central platform from the fifth arm of the study phase, the door was closed and the linoleum cover was lowered around the central structure. Two doors were then immediately opened, one to an arm the animal had visited during the study phase and one to an arm the animal had not. The selection of the two doors was based upon three criteria: first, which serial position was being tested on that particular trial; second, that the selected arms were either adjacent or separated by only one arm; third, that the correct response was 50% of the time on the right or on the left. Approximately 10s after the two test phase doors were opened, the linoleum cover was raised and the animal was allowed to respond by entering one of the two doors. The rule to be learned leading to an additional reinforcement (one piece of Froot Loop cereal) was to choose the arm that had been previously visited during the study phase of the trial (win-stay rule). If the animal chose the arm that was not in the study phase sequence, it was removed from the maxe and placed back into its cage without reinforcement. Each animai received one to three trials per day. On days when more than one trial was piven, each trial was separated by at least 1h. Each animal received 40 tests with eight tests for each of the serial positions. If, after these 40 trials, the animal had not performed 75% or better on the first and last serial position, then additional blocks of 10 trials (two trials per serial position) were given until the criterion was met for the most recent block of 40 trials. Surgery. After reaching criterion all 22 animals were anesthetized with sodium pentobarbital (nembutai, 40 mg/ kg, i.p.) and given atropine sulfate (0.2mg, i.p.) as a prophylactic prior to surgery. The animals were then divided into three groups. The first group (N = 6) received bilateral electrolytic lesions of the basolateral amygdala using the same procedure as described for the order memory experiment. The second group (lv = 9) received ibotenic acid lesions of the nucleus basalis magnocellularis. Ibotenic acid (8 fig/l .O~1 dissolved in 1~1 of Tris buffer) was injected bilaterally using a 10 p 1syringe. One microliter was injected for a duration of 2-3 min. The coordinates with the rat’s head level were 0.7 mm posterior to bregma, 3.2 mm lateral and 6.8 mm below the dura. The third group (IV = 7) served as a control. Four animals received vehicle injections into the nucleus basalis magnocellularis, while three animals received sham operations. Postoperative testing. One week following recovery from surgery, the animals were given 40 tests (one per day) with eight tests at each serial position. Histology At the end of the experiment, all but the ibotenic acid- and saline-injected animals were given a lethal injection of sodium pentobarbital, Riven 0.05 cm’ heparin intracardiaily, and perfused intra~rdially with 10% formaiin solution. The brains were removed from the skulls and stored in 10% formalin for 1 week. They were then photographed, sliced at 50 firn with a freezing microtome and every third section NSC W-D
was mounted, stained with Cresyl Violet, and examined for histological verification of the lesion placement. The ibotenic acid- and saline-injected animals were killed by decapitation and the brains rapidly removed and frozen on dry-ice. Fresh frozen transverse sections (16pm) were cut with a cryostat and stained for Nissl substance or acetylcholinesterase (AChE) histochemistry to determine the extent and location of nucleus basaiis magnocellularis lesions. In addition, two series of sections through the rostrocaudal extent of frontal and parietal neocortex were stained for Nissl substance or AChE histochemistry as described previously. iJ Briefly, the sections used for assessing choiinergic denervation were incubated overnight in a solution containing cupric sulfate, glycine, and acetylthiocholine iodide.” Promethazine was added to inhibit nonspecific esterase activity. The sections were developed with ammonium sulfide and counterstained with Thionin. Some sections were reacted according to the modification of Storm-Mathisen and Blackstads’ in order to intensify the reaction product for dark-field visualization and photomicroscopy. The latter procedure is more sensitive but gives results of lower resolution when compared with the Naik modification.” Histcchemical analysis of the amount of AChE depletion in the neocortex was carried out without knowledge of the behavioral results. In order to obtain a relative indication of the amount of cholinergic denervation following NMB lesions, the AChE-stained tissue sections were examined with both dark-field and bright-field illumination from the frontal pole to the posterior limit of the parietal cortex. Extensive cortical denervation was defined in terms of the complete loss of AChE staining bilaterally within the dorsolateral cortex bordered medially by the cingulate region and ventrolatemlly by the rhinai sulcus (parietal and frontal cortex). Thus, partial denervation was present in thosecasesexhibiting less AChE loss within the dorsolaterai cortical areas when compared with those with extensive depletion. RESULTS Histology The dorsolateral frontal lesions were consistent across subjects. A photomicrograph of a representative lesion is presented in Fig. 1. Note that there is no damage to the medial prefrontal cortex. The parietal cortex lesions were also consistent and included areas 5 and 7. A photomicrograph of a representative lesion is shown in Fig. 2A, B. The corpus callosum was damaged in all animals. Retrograde cell degeneration was present in the lateral posterior, posterior and ventral posterior nuclei of the thalamus, indicating destruction of the intended parietal areas. The amygdala lesions were somewhat variable but included bilaterally the basolateral and central amygdala in all animals. In some animals there was unilateral damage to the cortico-medial amygdala. A representative lesion is presented in Fig. 3. Of the nine NBM lesioned animals, five animals had extensive bilateral AChE depletion of parietal and dorsolateral frontal cortex with minimal AChE depletion of medial prefrontal and orbito-frontal cortex. An example of an NBM lesion that produced extensive bilateral depletion of parietal and parts of frontal cortex, is shown in Fig. 4. Examples of bilateral depletion of AChE in parietal and parts of the frontal cortex have been published previously.27
R. P. KESNERet al
96
Fig. I. Photograph
of a representative dorsolateral frontal cortex lesion.
The remaining four animals had unilateral or smaller bilateral NBM damage with corresponding depletion of AChE in parietal and dorsolateral frontal cortex. Based on the extent (extensive vs partial) of AChE depletion in neocortex the animals were divided into two groups. Behavioral performance-order
memory
Postoperative behavioral performance (mean per cent correct) is shown in Fig. 5 for each group as a function of serial order position (choice order). The graph shows that control subjects and animals with dorsolateral frontal cortex lesions display a serial position curve with good memory for the
first (I-2) and last (7-8) choice order. In contrast, animals with parietal cortex lesions are impaired in remembering the first (l-2) choice order, whereas animals with basolateral amygdala lesions are impaired for all choice orders within the list. A twoway analysis of variance with repeated measures on three choice orders revealed a signScant choice order effect (F = 18.8, df 2.42, P < 0.001) and sig nificant interaction between lesion groups and choice order (F = 3.1, df 6.42, P < 0.05). St&sequent Newman-Keuls tests indicated that far the 1-2 choice order the animals with dorsolateral frontal cortex lesions and controls displayed signScantly better memory than animals with either parietal cortex or
Memory deficits following NBM lesions
Fig. 2. Photographs
of a representative
parietal and posterior parietal association cortex lesion.
97
98
R. P.
KEENER
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Fig. 3. Diagram of a representative
amygdala lesion. Brain sections were taken from Paxinos and Watson.*
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Fig. 4. Diagram of a representative nucleus basalis magnoceliularis lesion that produced extensive a~tylcholin~tc~~ depletion of parietal and dorsolateral frontal cortex. Brain sections were taken from Paxinos and Watson.Lb
99
Memory deficits following NBM lesions
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Fig. 5. Mean percentage responses as a function of choice orders after amygdala, AMY; dorsolateral frontal cortex, DFC; parietal cortex, PC; or control, CONT lesions.
basolateral amygdala lesions (P < 0.05). For the control animals and animals with dorsolateral frontal cortex lesions performance on the l-2 and 7-8 choice order was significantly better than performance for 4-5 choice order (P < 0.05). For the animals with parietal cortex lesions performance for the 7-8 choice order was significantly better than performance for l-2 and 4-5 choice orders (P < 0.05). There were no significant differences between choice orders for animals with basolateral amygdala lesions. Behavioral performance-item
memory
Postoperative behavioral performance (mean per cent correct) is shown in Fig. 6 for the control, NBM, and amygdala lesioned groups as a function of serial position. The graph shows that control subjects and animals with small NBM lesions display excellent performance with somewhat better retention for the first and last items within the list. Animals with basolateral amygdala lesions and large NBM lesions also displayed good performance with slightly poorer retention for the last item within the list. A two-way analysis of variance for groups with repeated measures on serial position revealed non-significant effects for groups and serial position. DlSCUSSlON
The results of the present experiment indicate that, in contrast to NBM lesions, lesions of the dorsolateral frontal cortex do not impair order memory performance. This dissociation implies that the loss of NBM input to dorsolateral frontal cortex does not underlie the deficit in order memory for spatial
SERIALPOSITION
Fig. 6. Mean percentage correct responses as a function of serial position of items (spatial locations) after amygdala, AMY; small or large nucleus basalis magnocellularis, NBM or control, CONT lesions.
location information. In other studies ibotenic acid injections into the NBM produced greater deficits than quisqualic acid in the acquisition of a Morris water maze task, retention of passive avoidance learning and performance of an alternation task in a T-maze, even though quisqualic acid produced similar, or significantly greater, depletion of choline acetyltransferase (ChAT) levels of frontal cortex.‘9.63 Furthermore, there has been a general failure to find significant correlations between decreased frontal ChAT activity and behavioral deficits following NBM lesions.5 There are a number of possible interpretations for the observed dissociations between NBM and dorsolateral frontal cortex. First, the ascending cholinergic input from the NBM might not be. critical for memory performance within a large number of tasks. Instead, NBM lesions might have damaged other non-cholinergic neurotransmitter systems. One such system could be a neocortical descending projection to NBM mediated by glutamate. Damage to the NBM could have produced retrograde degeneration of neocortical neurons that produce glutamate. For order memory this possibility can be ruled out, because lesions of the dorsolateral frontal cortex do not produce a performance deficit. Second, because dorsolateral frontal cortex appears to play an important role in tasks requiring motor learning and time estimation,38.M it is likely that this order memory task for a list of spatial locations and many other tasks as well does not engage the dorsolateral frontal cortex. Thus, one might not expect to find a significant choline@ contribution to the dorsolateral frontal cortex in memory tasks that do not utilize this region.
100
R. P.
KESNER ef ~1.
Finally, there is a possibility that denervation of the target might lead to a greater deficit than total destruction of the target. Some support for this idea comes from the observations of Solomon et a1.,56 who have shown that in rabbits, systemic injections of scopolamine disrupt a classically conditioned nicitating membrane response, whereas removal of the hippocampus or scopolamine injections in rabbits with hippocampus lesions has no disruptive effect. The results of the present experiment also indicate that there are different effects of NBM compared with parietal cortex lesions. Lesions of the parietaf cortex disrupt item recognition for spatial locations,‘” whereas even large lesions of the NBM which result in cortical cholinergic denervation of parietal cortex do not produce any deficits. Similarly, for order memory, lesions of the parietal cortex produce a deficit pattern that is different from that seen following NBM lesions. This dissociation also implies that for item and order recognition memory the ascending chohnergic system does not play a critical role. Parallel deficits have been observed following parietal cortex and NBM lesions in performance of a reference memory task in an eight arm maze and in the acquisition of a water maze or a cheese board (a dry maze analog of the water maze) task.‘9.26.2R.3L It is not clear how these tasks differ from the item and order recognition memory tasks in terms of the utilization of spatial information. In contrast to the dissociations found between NBM and neocortex, the results of the present experiment indicate that there are parallel patterns of results in the item and order memory tasks with basolateral amygdala and NBM lesions, namely an extensive deficit for order memory but no deficit for item memory. Thus, it appears that the NBM might mediate item and order memory through its connections with the basolateral amygdaia. Since reciprocal connections may exist between NBM and basolateral amygdala, one cannot determine in which direction the influence is mediated.‘0~‘2~‘7~37~53 The disruptive effects of NBM lesions on active and passive avoidance learning are certainly comparable to what has been described for basolateral amygdala lesions.” Unfortunately, no one has reported correlations between ChAT levels in amygdala and behavioral deficits observed following NBM lesions. So, it is not possible to determine whether the disruptive effects of NBM lesions are primarily through a chohnergic
projection to the basolateral amygdala. At present, it is not possible to discern whether NBM or basolateral amygdala lesions alter memory for the order of spatial location information. because of a direct influence on the mediation of perceptual or motor processes or memory storage. Elsewhere, Kesner and DiMattia29 have suggested that the amygdala complex is directly involved in maintaining temporary representations of affect-faden as triggered by reinforcement and temporal information, but not spatial location information. Thus, one would predict that amygdala lesions would not after memory for item (spatial iocation and reinforcement) information, but would after memory for order (temporal representation of spatiat location and reinforcement) information. It is then likely that N3M represents a cholinergic modulatory influence on amygdata function. Other components of the basal forebrain cholinergic system also play an important role in memory. For example, the medial septum and the hippocampal formation clearly mediate item and order memory in a parallel fashion,26 suggesting an important influence on memory of the medial septum component of the choline@ basai forebrain system. It is of interest to note that even though the medial septum and the hippocampal formation are directly involved in processing of both item and order spatial location information, the NBM and amygdala are involved primarily in coding order, but not item, spatial location formation. Thus, it appears that there is a dissociation of function between hippocampus and amygdala as well as medial septum and NBM. Finally, it should be noted that the horizontal nucleus of the diagonal band has a cholinergic projection primarily to medial prefrontal and entorhinat cortices. These latter neocortical areas are known to be involved in memory. Thus, it is possible that the horizontal nucleus of the diagonal band plays a role in memory via its neocortical ascending cholinergic contribution. In summary, the NBM appears to influence order but not item memory performance for lists of spatial location, perhaps through cholinergic projections to limbic but not neocortical targets. AcknoH,ledgemenrs-Support for this research was provided bv NIH Grant NS19406-03 and NIH Grant ROINS267?1-04 (RPK), and National Institute on Agin Grant No. AG-07691 (KAC).
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13 April 1990)
