Physiology&Behavior.Vol. 52, pp. 971-977, 1992
0031-9384/92$5.00 + .00 Copyright© 1992PergamonPressLtd.
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Behavioral Response of Rats With Cortical Lesions to Cholinomimetics L. G A R O F A L O , * P. J. E L L I O T T t A N D A. C. C U E L L O *l
*Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada, H3G-1 Y6 and i'GLAXO Neuropharmacology Research Group, Ware, Hertz, UK Received 16 D e c e m b e r 1991 GAROFALO, L., P. J. ELLIOTT AND A. C. CUELLO. Behavioralresponse of rats with corticallesions to cholinomimetics. PHYSIOL BEHAV 52(5) 971-977, 1992.--This study examined the performance of cortically lesioned rats and their response to cholinomimetic agents in passive avoidance and water maze tasks. Lesions encompassing mainly the frontal and parietal cortices produce a deficit in a 5-day passive avoidance retention test. This deficit was attenuated by the intraperitoneal (IP) administration of muscarinic agonists or an anticholinesterase. In the Morris water maze task, lesioned vehicle-treated animals showed greater escape latencytimes when compared to their sham counterparts. Cholinomimetics,injecteddaily during acquisition, improved mean escape latency time on days 3 and 4 of testing. The performance of the various groups in a cued version of the water task did not differ. This work demonstrates that performance deficits arising from neocortical loss can be attenuated by cholinergic drugs. Anticholinesterase Passive avoidance
Oxotremorine Pilocarpine Morriswater maze
Arecoline
THE majority of literature presently available on learning and memory processes in animals and humans suggests that central cholinergic systems play an important role. In both rodents and primates, administration of anticholinergic drugs have been shown to induce behavioral deficits in a variety of memorybased tasks (4,51). Decreased memory performance in neurological disorders, such as Alzheimer's disease, is generally associated with deficits in forebrain cholinergic markers and loss or shrinkage of cholinergic neurons in the nucleus basalis of Meynert (42,45,53). In the rat, the counterpart of this nucleus is the nucleus basalis magnocellularis (NBM) (34,41). Various lesioning techniques have shown that damage to this nucleus results in a depletion of cholinergic markers in the NBM and in the neocortex, its area of projection (15,18,20). In addition, unilateral or bilateral injury to the NBM also elicit deficits in memory-based paradigms ( 12,15,19,20,22,31,32,35,38,43). Alternatively, bilateral lesions of the neocortex can also affect a rat's performance in certain behavioral tasks (10,30). Many studies have now shown that administration of cholinergic agents can successfully attenuate behavioral deficits arising from anterograde lesioning of the NBM (12-15,20,22,25,31,32,35,38). In previous studies we have shown that retrograde degeneration of the rat NBM, induced by unilateral lesions of the frontal and parietal cortices, in addition to causing biochemical deficits in cholinergic markers in this area, affect retention and reacquisition of learned Morris water maze and passive avoidance tasks (17).
Physostigmine Cholinergic
Muscarinic
Here we assess, using such rats lacking a large portion of their NBM target site, the ability of cholinomimetic drugs to ameliorate performance deficits in the above tasks. METHODS
Animals Male Wistar rats (325-350 g; Charles River, St. Constant, Quebec) with free access to food and water, on a 12-h light/dark cycle were used in this study. Half the animals had unilateral, devascularizing lesions of the left neocortex while the remaining subjects were sham operated.
Surgical Procedure Rats, under Equithesin anaesthesia (3 ml/kg), were placed in a stereotaxic apparatus and the pia-arachnoid vasculature to the cortex was disrupted unilaterally as previously described (17,49). This procedure results in a gradual cortical atrophy and a loss of the frontal 1 and 3, parietal 1 and portions of the frontal 2, parietal 2 and occipital neocortical areas (see Fig. 1). In addition, a maximal decrease in choline acetyltransferase activity (CHAT) is observed in the NBM 30 days postlesion (9,17,50), at which time subjects with such cortical damage have been found to have performance deficits in memory-based tasks (17). Sham operations consisted of removing an equivalent amount of skull
' Requests for reprints should be addressed to Dr. A. C. CueUo, Department of Pharmacology and Therapeutics, McGill University, 3655 Drummond Street, Montreal, Quebec, Canada H3G-IY6.
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GAROFALO, ELLIOTT AND CUELLO
FIG. 1. Representative fresh coronal br~in slices showing the anterior to posterior extent of the unilateral devascularizing cortical lesion from a subject sacrificed following behavioral studies (51 days postlesion). Coronal brain sections (300-350 um thick) were obtained using a Mcllwain tissue chopper. The extent of the lesion was examined under a Wild (Heerbrugg, Switzerland) microscope and was recorded for every subject. The lesion resulted in a complete atrophy of the frontal 1 and 3, parietal 1 and portions of the frontal 2, parietal 2 and occipital neocortical regions.
bone overlying the neocortex, ensuring that the dura beneath was not damaged.
Behavioral Testing Behavioral testing was initiated 30 days following the surgical procedures.
Ladder- Walking Paradigm Animals were tested for motor coordination in a ladderwalking paradigm. The number of times the rat's paw slipped offthe rungs of a horizontal ladder (100 × 8 cm; rung diameter 4 m m spaced 35 m m apart; suspended 50 cm above a table) were recorded as foot faults. Each rat was allowed five trials in this task.
Passive A voidance Step-through passive avoidance testing was studied in a box (84 × 30 X 30 cm) divided into two equal halves. One com-
partment was painted black and the other white. The dark side was covered with a black top, while the white side was illuminated with two 60 W bulbs. The floor of both sides comprised of steel bars (8 m m in diameter, spaced 10 m m apart); those in the black chamber were connected, via a scrambling device (Lafayette #824000; Lafayette Inst., Co., Lafayette, IN) to a shock generator (Lafayette #58020). Testing consisted of two 3-min habituation trials in the box on consecutive days. On the third day the rats were placed in the white side of the apparatus and the time taken to enter the dark side was recorded. Once inside the dark side the guillotine door was closed and 15 s later a 0.45 m A X 1 s scrambled footshock was applied to the floor bars. Following each shock the subjects (n = 7-8/group) were left in the dark side for a further 15 s and, subsequently, were injected intraperitoneally (1P) with either: vehicle (phosphate buffered saline; PBS), arecoline ( 1.0 mg/kg/ml), pilocarpine (4.0 mg/kg/ml), oxotremorine (0.1 mg/kg/ml), or physostigmine (0.03 mg/kg/ml). Drug dosages were estimated based on previous studies employing these compounds (25,26,29). All the rats were retested
CHOLINOMIMETICS AND BEHAVIOR
973 after which, upon failure to locate the platform, the subject was placed on it for 30 s. The training paradigm consisted of one block of four trials, spaced 15 min apart, per day for 5 consecutive days. Start positions within each block varied between trials. At the end of each block of test trials, sham and cortically lesioned animals were injected IP (n = 7-8/group) with either saline, physostigmine (0.03 mg/kg/ml) or pilocarpine (4 mg/kg/ml). Animals were retested in the water maze 2 weeks later to assess retention (one block of four trials). The day following the retention test these rats were studied in a cued version of the water maze task. In this study, the platform was moved to a new location in the pool and was elevated 3 cm above the surface of the water, thus making it visible. The testing paradigm used was identical to that described above.
Control Lesioned
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FIG. 2. Effect of the cortical lesion on ChAT activity in the ipsilateral and contralateralNBM of adult rats 30 days postlesion.Rats were lesioned unilaterally by cortical devascularization and sacrificed after 30 days. Sham operated rats (control) had an equivalent amount of skull bone removed but were unlesioned. ChAT activity in the NBM and protein content were determined according to the methods of Fonnum (21) and Bradford (7), respectively.*p < 0.05 with respect to control rats, ANOVA post hoc Newman-Keuls' test.
Drugs Oxotremorine, physostigmine, arecoline and pilocarpine were all purchased from Sigma Chemical Co., St. Louis, MO. All drugs were dissolved in PBS on the day of testing prior to their administration.
Biochemical Assays A separate group of rats, similarly cortically lesioned to those prepared for behavioral studies, were sacrificed 30 days postlesion by decapitation. The NBM was microdissected as previously described (50) and the tissue was hand homogenized in 10 volumes of 10 m M Sodium EDTA buffer pH 7.4 containing 0.5% Triton X-100. ChAT activity was determined according to the method of Fonnum (21). Protein content was measured using the method of Bradford (7).
5 days later to investigate retention of the response. When tested, the subjects were not under the influence of any drug-induced effects such as altered pain thresholds, locomotor activity, or environmental perception. White noise was present during both the training and the retention test which occurred between 0900 and 1700 h.
Morris Water Maze
Statistics
Another group of lesioned and sham-operated animals were tested in the Morris water maze task essentially as previously described (17). The apparatus consisted of a white circular plastic pool (140 cm in diameter; 42 cm high) which was filled to a depth of 30 cm with water (18 + 2°C) made opaque with nontoxic powder tempera blue paint (Weber Costello, Mississauga, Ontario). A circular transparent platform (10 cm in diameter) covered with a plastic mesh (Type $74616-F; Sargent-Welch) was placed in one part of the pool such that it was covered with 1 cm of water and was not visible. To assess acquisition of the Morris water maze task, each subject was placed in the water with its face positioned toward the side of the pool. Time taken to find the platform and swimming profile were recorded by an observer blind to the drug treatments. Upon locating the platform, subjects were allowed 30 s to acquire spatial information from external cues in the room. A cutoffpoint of 180 s was used
All results were analyzed using analysis of variance and a post hoc Newman-Keuls test. RESULTS This study shows that unilateral devascularizing cortical lesions significantly decrease ChAT activity in the ipsilateral NBM (Fig. 2) and increase the incidence of multiple footfaults in a ladder-walking task (Table 1). Decreases in rat body weight were noted on postlesion day 10; however, once behavioral studies were initiated (i.e., 30 days postlesion) lesioned rats had recovered normal body weight. The above observations confirm previous work (17). The results of the passive avoidance testing are shown in Fig. 3. Thirty days postoperatively all rats showed similar habituation profiles, entering the dark side of the box within 20 s. In the passive avoidance retention test, sham-operated animals
TABLE 1 NUMBEROF FOOTFAULTSIN LADDER-WALKINGTASK Frontlimb Group
Ipsilateral
Sham (n = 40) Lesioned (n = 40)
16 12
Hindlimb
Contralateral 7 74*
lpsilateral 19 13
Contralateral 21 85*
Thirty days postsurgery sham and lesioned animals were allowed five trials in a ladder walking task. Numbers represent the total number of footfaults observed per group. * p < 0.05 ANOVA, post hoc Newman-Keuls test with respect to sham operated rats.
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though, no significant, F(3, 43) = 2.35, p = 0.1, group effect on the mean escape latency time is noted (Fig. 5), analysis of the swim profiles showed that lesioned vehicle-treated rats used a less direct route to find the platform (Fig. 6B). This was also true during the acquisition phase of this paradigm (Fig. 6A). Figure 4B illustrates the mean escape latency times for all the groups after the platform was moved to a different location in the maze and was made visible. All the subjects showed similar rapid acquisition profiles although in the first trial of the test block sham-operated and drug-treated lesioned groups, in con-
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FIG. 3. Effects of the cortical lesion and cholinomimetics on passive avoidance retention. Veh: vehicle (phosphate buffered saline); Phys: physostigmine; Pilo: pilocarpine; Arec: arecoline; Oxo: oxotremorine. Please see the method section for details. *p < 0.05 with respect to sham operated rats, ANOVA post hoc Newman-Keuls' test. n = 8/group.
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showed no deficit and their performance was not affected by the various drug treatments [ANOVA, F(4, 35) = 0.545, p = 0.71]. Thus, results from vehicle and drug-treated sham-operated animals were pooled. In contrast to sham-operated animals, rats with lesions affecting mainly the frontal and parietal cortices showed a retention deficit in this task. Analysis of variance demonstrated that there was a significant group effect, F(5, 74) = 3.835, p = 0.01. Comparisons of the group means, using a post hoc N e w m a n - K e u l s test, indicated that the IP administration of cholinomimetic drugs to lesioned animals significantly (p < 0.05) attenuated this impairment. No differences were noted in the ability of the different cholinergic agonists (arecoline, oxotremorine, pilocarpine) or the anticholinesterase (physostigmine) to ameliorate the 5-day retention of the passive avoidance task. The mean latency to find the platform for all groups in the Morris water maze task is shown in Fig. 4A. Sham-operated animals rapidly acquired the task as was previously reported (17). Similarly to the passive avoidance results, the behavior of drug-treated sham-operated animals did not differ from their saline injected counterparts. Analysis of variance revealed no differences among the sham-operated groups, F(2, 105) = 1.5, p = 0.22, but did show that escape latency times decreased significantly over trial days, F(4, 105) = 175.5, p = 0.001. The interaction between groups and trial day was not significant, F(8, 105) = I. 15, p = 0.34. Data from vehicle and drug treated sham subjects were, therefore, pooled for each test day. Unilaterally decorticated rats showed a mild impairment in the acquisition of this spatial task. These rats were able to locate the platform; however, their escape latency times were greater than those of sham operated animals. Analysis of variance showed significant effect of groups, F(3, 215) = 2.54, p = 0.05, trial day, F(4, 215) = 133.18, p = 0.00 l, and group by trial day interaction, F( 12, 215) = 3.09, p = 0.001. N e w m a n - K e u l s post hoc tests on the group means revealed that daily IP injections of cholinomimetic drugs, administered subsequent to termination of a block of four test trials, significantly (p < 0.05) decreases the mean escape latency times of lesioned animals on days 3 and 4 of testing (Fig. 4a). Rats were then retested 2 weeks following acquisition of the Morris water maze task to assess retention. Al-
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Doy FIG. 4. Mean escape latency, on each test day, from the Morris water maze with a (A) hidden platform and (B) with the platform now visible but in a new location. Sham operated: O O; lesion + vehicle: • • ; lesion + physostigmine: A A; lesion + pilocarpine: • • . *p < 0.05 with respect to sham operated rats, ANOVA post hoc Newman-Keuls' test. Sham operated, n = 24 animals; lesioned, n = 8 animals/group.
CHOLINOMIMETICS AND BEHAVIOR
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FIG. 5. Mean escape latency to find the hidden platform in the Morris water maze 2 weeks after acquisition of the task. Sham operated, n = 24 animals; lesioned, n = 8 animals/group.
trast to vehicle-treated rats, swam more readily directly towards the platform's old location before heading to the visible one. Following all behavioral testing, the rats were sacrificed and a gross morphological examination of all the brains was undertaken. Lesions were found to be consistent across all groups. Anterior to posterior coronal sections of each brain confirmed that the lesion did not encroach subcortical brain areas (see Fig. 1). However, in some animals, the corpus callosum was at times thinner or absent in more posterior sections.
the NBM (44) rather than due to deafferentation of the cortex. In particular, work using two different neurotoxins (40), suggests that the NBM-cortical cholinergic pathway may have little influence on rodent mnemonic processes as assessed in passive avoidance and Morris water maze tasks. Rather, it has been proposed that NBM lesion-induced deficits occur due to disturbances in motivational behavior arising from disruption of NBM projections to the amygdala (16,40) or have been attributed to alterations in neurochemicals other than acetylcholine (i.e., neurotensin; GABA) (16,52). In animals of the present study, a large portion of the neocortex was damaged unilaterally by a devascularizing lesion. In addition to reliably producing a 40-50% decrease in ChAT activity [present study, (9,17,50)] and cholinergic cell shrinkage (9,49) in the ipsilateral NBM, such a lesion also results in cortical loss and diminished cholinergic innervation of the ipsilateral remaining cortex adjacent to the lesion site. In particular, a significant decrease in the number of synaptic contacts, axonal varicosities and size of cholinergic boutons can be noted within this remaining cortical area (24). The results obtained in the passive avoidance task with these animals are in line with previous studies which show that disruption of fronto-parietal cortical function adversely affects rodent performance in inhibitory
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DISCUSSION
Unilateral devascularizing cortical lesions affecting mainly the frontal and parietal rat cortex cause temporary weight loss, multiple footfaults, and performance deficits in two tasks (passive avoidance and Morris water maze) frequently used to assess learning and memory. The lesions, although extensive, are not overly debilitating to the animals, since they are able, after an initial weight loss, to recover normal feeding and grooming activities. Once behavioral studies were initiated, no gross physiological abnormalities distinguished lesioned subjects from shamoperated rats. The present study sought to determine whether performance deficits previously noted (17) in rats with this particular type of cortical lesion could be attenuated by cholinomimetic drugs. A decline in cholinergic function of the basal forebrain has been thought to underlie the memory dysfunction occurring in the aged and in Alzheimer's disease (5). In animal models which attempt to mimic this deficit, such as the rat with anterograde damage to the NBM, the beneficial effects of cholinomimetic agents have been well documented. In particular, the anticholinesterase, physostigmine, has been shown to improve the performance of rats with bilateral lesions to the NBM in passive avoidance (27) and delayed matching tasks (13), in a reinforced alternation T-maze (36) and to ameliorate escape latency times in the Morris water maze (11). Oxotremorine (1,2,26,29), arecoline (26,29,33,39,46-48) and pilocarpine (29), all muscarinic agonists, have also been shown to improve performance in learning and memory tasks. However, recent data indicate that behavioral impairments following NBM lesions may arise from nonspecific damage to areas in the vicinity of
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FIG. 6. Typical swimming patterns of the subjects during the first trial (A) of each test day and (B) during the first trial 2 weeks after acquisition of the task. Sham operated, n = 24 animals; lesioned, n = 8 animals/ group.
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GAROFALO, ELLIOTT A N D CUELLO
tasks (6,23). The attenuation of the passive avoidance retention deficit by cholinergic drugs in such cortically lesioned rats could be attributed to either activation of the remaining basalo-cortical cholinergic pathway or stimulation of other cholinergic pathways. Indirect support to suggest that the remaining NBM-to-cortex pathway may be involved is provided by studies involving NBM lesioned rats. It has been shown that lesions of the rat frontal cortex, which alone apparently do not affect retention of a passive avoidance response, can abolish the physostigmine-induced recovery of this performance in NBM-lesioned animals (27). Furthermore, rats with unilateral NBM lesions, which also show impairments in a passive avoidance task (19,32), improve their behavior if fetal basal forebrain grafts are transplanted into the cortex (19). However, as previously indicated, alterations in passive avoidance responses by NBM-lesioned rats are also attributed to disturbances in motivation or fear rather than to deficits in learning and memory. In our lesion model, although subcortical brain areas appear undisturbed by the lesion, the amygdala and, in particular, the striatum and thalamus, are affected as exemplified by the presence of gliosis in these subeortical areas (28). Since the amygdala in particular appears to play a role in fear, it is possible that alterations in fear, rather than memory, account for our observations. Although indices of fear have not been examined in detail for these animals, it was noted that lesioned vehicle- or drug-treated rats of the present study did not appear to respond differently to handling than did shamoperated animals. However, in earlier work involving similarly lesioned rats, it was observed that vehicle-treated animals did tend to defecate less on the white side of the box (17). Such an occurrence could be indicative of reduced fear. Furthermore, since the rat cortex is known to receive catecholaminergic (36) and serotoninergic (37) inputs from the locus coeruleus and raphe nucleus, respectively, the contribution of disturbances in these pathways and neurochemicals to the observed behavioral deficits requires assessment. In particular, whether such noncholinergic agents also produce similar behavioral responses in these animals should be tested in future studies. The importance of an intact cortex for spatial localization in the rat has previously been reported. Initial studies by Kolb and collegues (30) showed that bilateral damage to the frontal or cingulate cortex produce deficits in spatial tasks, whereas bilateral parietal lesions produce only mild impairments. DiMattia and
Kesner (10) using rats with more extensive damage to the parietal area have shown that this cortical region plays an important role in spatial memory. The present study demonstrates that impairments in spatial memory, albeit mild, also occur following unilateral devascularizing cortical lesions. However, although lesioned vehicle-treated rats can learn to locate the hidden platform in the water maze and show no significant deficits in mean escape latency times when retested 2 weeks after acquisition, these rats fail to use a direct route to do so. Similar results were obtained with rats subjected to suction ablation cortical lesions (10,30). Thus, rodents lacking frontal/parietal cortices appear to be impaired in their ability to adopt a spatial mapping strategy to solve the task. This impairment seems to be attenuated by cholinomimetic drugs. However, as discussed earlier, with behavioral studies involving brain lesions it is difficult to fully attribute observed performance deficits and their reversal by drugs to changes in learning and memory. In a previous study (17), motor and sensory tests revealed that other than an increase in overnight locomotor activity and deficits in the ability to walk along a horizontal ladder, the latter reconfirmed in the present study, cortically devascularized animals showed no impairments in auditory, olfactory, noxious, and muscle-strength testing paradigms. However, unilateral cortical lesions have previously been reported to induce contralateral sensory neglect (8), and disruption of the NBM-to-cortex pathway has been linked to deficits in visual attentional function (40). The rats in the present study do circle the perimeter of the pool, in a direction ipsilateral to the lesion, when first exposed to the task to a greater degree than do sham-operated animals (see Fig. 6A). However, when tested in the water maze containing a visible platform lesion vehicletreated rats performed similarly to sham-operated rats and quickly located the platform. Although this provides some indirect evidence which could argue against motivation, altered swim speeds and/or motor problems affecting performance in the maze, more detailed studies are required to absolutely ascertain their lack of involvement. ACKNOWLEDGEMENTS We thank Mr. A. Forster for the photographs and Ms. Laura Fernandes for the ChAT assay. This work was supported by the Canadian Medical Research Council and the Centers for Excellence for Neural Repair and Functional Recovery. L.G. received an FRSQ studentship.
REFERENCES 1. Baratti, C. M.; Huygens, P.; Mino, J.; Merlo, A.; Gardella, J. Memory facilitation with post-trial oxotremorine and physostigmine in mice. Psychopharmacology (Berlin) 64:85-88; 1979. 2. Baratti, C. M.; lntroini, I. B.; Huygens, P.; Gusovsky, F. Possible cholinergic-dopaminergic link in memory facilitation induced by oxotremorine in mice. Psychopharmacology (Berlin) 80:161-165; 1983. 3. Bartus, R. T. Four stimulants of the central nervous system: Effects on short term memory in young versus aged monkeys. J. Am. Geriatr. Soc. 27:289-297; 1979. 4. Bartus, R. T.; Johnson, H. R. Short-term memory in the rhesus monkey: Disruption from the anticholinergic scopolamine. Pharmacol. Biochem. Behav. 5:39--46; 1976. 5. Bartus, R. T.; Dean, R. L.; Beer, B.; Lippa, A. S. The cholinergic hypothesis of geriatric memory dysfunction. Science 217:408-417; 1982. 6. Bermudez-Rattoni, F.; Introni-Collison, I. B.; McGaugh, J. L. Reversible activation of the insular cortex by tetrodotoxin produces retrograde amnesia for inhibitory avoidance and spatial learning. Proc. Natl. Acad. Sci. USA 88:5379-5382; 1991. 7. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of dye binding. Anal. Biochem. 72:248-254; 1976.
8. Cowey, A.; Bozek, T. Contralateral 'neglect' after unilateral dorsomedial prefrontal lesions in rats. Brain Res. 72:53-64; 1974. 9. Cuello, A. C.; Garofalo, L.; Kenigsberg, R. L.; Maysinger, D. Gangliosides potentiate in vivo and in vitro effects of nerve growth factor on central cholinergic neurons. Proc. Natl. Acad. Sci. USA 86:20562060; 1989. 10. DiMattia, B. D.; Kesner, R. P. Spatial and cognitive maps: Differential role of parietal cortex and hippocampal formation. Behav. Neurosci. 102(4):471-480; 1988. 11. Dokla, C. P. J.; Thai, L. J. Effect of cholinesterase inhibitors on Morris water maze task behavior following lesions of the nucleus basalis magnocellularis. Behav. Neurosci. 102(6):861-871; 1988. 12. Dubois, B.; Mayo, W.; Agid, Y.; Le Moal, M.; Simon, H. Profound disturbances of spontaneous and learned behaviours following lesions of the nucleus basalis magnocellularis in the rat. Brain Res. 338: 249-258; 1985. 13. Dunnett, S. B. Comparative effects of cholinergic drugs and lesions on nucleus basalis or fimbria-fornix on delayed matching in rats. Psychopharmacology (Berlin) 87:357-363; 1985. 14. Dunnett, S. B.; Toniolo, G.; Fine, A.; Ryan, C. N.; Bjorklund, A.; lversen, S. D. Transplantation of embryonic ventral forebrain neurons to the neocortex of rats with lesions of nucleus basalis mag-
CHOLINOMIMETICS AND BEHAVIOR
15.
16.
17.
18.
19.
20.
21. 22. 23.
24. 25. 26. 27. 28. 29. 30. 31.
32.
33.
nocellularis-ll. Sensorimotor and learning impairments. Neuroscience 16:787-797; 1985. Dunnett, S. B.; Whishaw, I. Q.; Jones, G. H.; Bunch, S. T. Behavioral, biochemical and histochemical effects of different neurotoxic amino acids injected into nucleus basalis magnocellularis of rats. Neuroscience 20:653-669; 1987. Dunnett, S. B.; Everitt, B. J.; Robbins, T. W. The basal forebraincortical cholinergic system: Interpreting the functional consequences of excitotoxic lesions. Trends Neurosci. 14( 11 ):494-501; 1991. Elliott, P. J.; Garofalo, L.; Cuello, A. C. Limited neocortical devascularizing lesions causing deficits in memory retention and choline acetyltransferase activity--Effects of the monosialoganglioside GMI. Neuroscience 31:63-76; 1989. Fine, A.; Dunnett, S. B.; Bjorklund, A.; Clarke, D.; Iversen, S. D. Transplantation of embryonic ventral forebrain neurons to the neocortex of rats with lesions of nucleus basalis magnocellularis--I. Biochemical and anatomical observations. Neuroscience 16:769786; 1985. Fine, A.; Dunnett, S. B.; Bjorklund, A.; lversen, S. D. Cholinergic ventral forebrain grafts into the neocortex improve passive avoidance memory in a rat model of Alzheimer's disease. Proc. Natl. Acad. Sci. USA 82:5227-5230; 1985. Flicker, C.; Dean, R. L.; Watkins, D. L.; Fisher, S. K.; Bartus, R. T. Behavioral and neurochemical effects following neurotoxic lesions of a major cholinergic input to the cerebral cortex in the rat. Pharmacol. Biochem. Behav. 19:309-312; 1983. Fonnum, F. A rapid radiochemical method for the determination of choline acetyltransferase. J. Neurochem. 24:407-409; 1975. Friedman, E.; Lerer, B.; Kuster, J. Loss of eholinergic neurons in the rat neocortex produces deficits in passive avoidance learning. Pharmacol. Biochem. Behav. 19:309-312; 1983. Fukuchi, I.; Susumu, K.; Nakahiro, M.; Uchida, S.; Ishida, R.; Yoshida, S. Blockade of cholinergic receptors by an irreversible antagonist, Propylbenzylcholine mustard (PrBCM), in the rat cerebral cortex causes deficits in passive avoidance learning. Brain Res. 400: 53-61; 1987. Garofalo, L.; Ribeiro-da-Silva, A.; Cuello, A. C. Nerve growth factor induced synaptogenesis and hypertrophy of cortical cholinergic terminals. Proc. Natl. Acad. Sci. USA 89:2639-2643; 1992. Haroutunian, V.; Kanof, P.; Davis, K. L. Pharmacological alleviation of cholinergic lesion induced memory deficits in rats. Life Sci. 37: 945-952; 1985. Haroutunian, V.; Barnes, E.; Davis, K. L. Cholinergic modulation of memory in rats. Psychopharmacology (Berlin) 87:266-271; 1985. Haroutunian, V.; Mantin R.; Kanof, P. D. Frontal cortex as the site of action of physostigmine in nbM-lesioned rats. Physiol. Behav. 47:203-206; 1990. Herrera, D. G.; Cuello, A. C. GFAP immunoreactivity following cortical devascularization. Soc. Neurosci. Abstr. 16:779; 1990. Jung, M.; Costa, L.; Sherman, G. T.; Kelly, P. H. Discriminative stimulus properties of muscarinic agonists. Psychopharmacology (Berlin) 93:139-145; 1987. Kolb, B.; Sutherland, R. J.; Whishaw, I. Q. A comparison of the contributions of the frontal and parietal association cortex to spatial localization in rats. Behav. Neurosci. 970):13-27; 1983. Lerer, B.; Warner, J.; Friedman, E.; Vincent, G.; Gamzu, E. Cortical cholinergic impairment and behavioral deficits produced by kainic acid lesions of the rat magnocellular basal forebrain. Behav. Neurosci. 99:661-677; 1985. LoConte, G.; Bartolini, L.; Casamenti, F.; Marconcini-Pepeu, I.; Pepeu, G. Lesions ofcholinergic forebrain nuclei: Changes in avoidance behavior and scopolamine actions. Pharmacol. Biochem. Behav. 17:933-937; 1982. Meltzer, L. T.; Rosecrans, J. A. Discriminitive stimulus properties of arecoline: A new approach for studying central muscarinic receptors. Psychopharmacology (Berlin) 75:383-387; 1981.
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34. Mesulam, M. M.; Mufson, E. J.; Wainer, B. H.; Levy, A. I. Central cholinergic pathways in the rat: An overview based on alternative nomenclature (Ch l-Ch6). Neuroscience l 0:1185-1201; 1983. 35. Miyamoto, M.; Shinatani, M.; Nagaoka, A.; Nagawa, Y. Lesioning of the rat basal forebrain leads to memory impairments in passive avoidance and active avoidance tasks. Brain Res. 328:97-104; 1985. 36. Moore, R. Y.; Bloom, F. E. Central catecholamine neuron systems: Anatomy and physiology of the norepinephrine and epinephrine systems. Annu. Rev. Neurosci. 2:113-168; 1979. 37. Moore, R. Y.; Halaus, A. I.; Jones, B. C. Serotonin neurons of the midbrain raphe ascending projection. J. Comp. Neurol. 180:417438; 1978. 38. Murray, C. L.; Fibiger, H. C. Learning and memory deficits after lesions of the nucleus basalis magnoceUularis: Reversal by physostigmine. Neuroscience 14:1025-1032; 1985. 39. Overton, D. A. Comparison of the degree of discriminability of various drugs using the T-maze discrimination paradigm. Psychopharmacology (Berlin) 76:385-395; 1982. 40. Page, K. J.; Everitt, B. J.; Robbins, T. W.; Marston, H. M.; Wilkinson, L. S. Dissociable effects on spatial maze and passive avoidance acquisition and retention following AMPA- and ibotenic acid-induced excitotoxic lesions of the basal forebrain in rat: Differential dependence on cholinergic neuronal loss. Neuroscience 43:457-472; 1991. 41. Pepeu, G.; Casamenti, F.; Pedata, F.; Cosi, C.; Pepeu, I. M. Are the neurochemical and behavioral changes induced by lesions of the nucleus basalis in the rat a model Alzheimer's disease? Prog. Neuropsychopharmacol. Biol. Psychiatry 10:541-551; 1986. 42. Perry, E. K.; Perry, R. H.; Blessed, G.; Tomlinson, B. E. Necropsy evidence of central cholinergic deficits in senile dementia. Lancet i: 189; 1977. 43. Riekkinen, P., Jr.; Sirvo, J.; Hannila, T.; Miettinen, R.; Riekkinen, P. Effect of quisqualic acid nucleus basalis lesioning on cortical EEG, passive avoidance and water maze performance. Brain Res. Bull. 24:839-842; 1990. 44. Robbins, T. W.; Everitt, B. J.; Ryan, C. N.; Marston, H. M.; Jones, G. H.; Page, K. J. Comparative effects of quisqualic and ibotenic acid-induced lesions of the substantia innominata and globus pallidus on the acquisition of a conditional visual discrimination: Differential effects on cholinergic mechanisms. Neuroscience 28(2):337-352; 1989. 45. Rossor, M. N.; Svendsen, C.; Hunt, S. F.; Mountjoy, C. G.; Roth, M.; lversen, L. L. The substantia innominata in Alzheimer's disease: A histochemical and biochemical study of cholinergic marker enzymes. Neurosci. Lett. 28:217-222; 1982. 46. Schechter, M. D.; Rosecrans, J. A. Atropine antagonism ofarecolinecued behavior in the rat. Life Sci. 11(11):519-523; 1972. 47. Schechter, M. D.; Rosecrans, J. A. Effect of mecamylamine on discrimination between nicotine- and arecoline-produced cues. Eur. J. Pharmacol. 17:179-182; 1972. 48. Sitaram, N.; Weingarter, H.; Gillin, J. C. Human serial learning enhancement with arecoline (sic) and impairment with scoploamine. Science 201:274-276; 1978. 49. Sofroniew, M. V.; Pearson, R. C. A.; Eckenstein, F.; Cuello, A. C.; Powell, T. P. S. Retrograde changes in cholinergic neurones in the basal forebrain of the rat following cortical damage. Brain Res. 289: 370-374; 1983. 50. Stephens, P. H.; Cuello, A. C.; Sofroniew, M. V.; Pearson, R. C. A.; Tagari, P. C. Effect of unilateral decortication on choline acetyltransferase activity in the nucleus basalis and other areas of the rat brain. J. Neurochem. 45:1021-1026; 1985. 51. Watts, J.; Stevens, R.; Robinson, C. Effects of scopolamine on radial maze performance in rats. Physiol. Behav. 26:845-851; 1981. 52. Wenk, G. L.; Markowska, A. L.; Olton, D. S. Basal forebrain lesions and memory: Alterations in neurotensin, not acetylcholine, may cause amnesia. Behav. Neurosci. 101:765-769; 1989. 53. Whitehouse, P. J.; Price, D. L.; Strubble, R. G.; Clark, A. W.; Coyle, J. T.; DeLong, M. R. Alzheimer's disease and senile dementia loss of neurones in the basal forebrain. Science 215:1237-1239; 1982.
0031-9384/92$5.00 + .00 Copyright© 1992PergamonPressLtd.
Printedin the USA.
Behavioral Response of Rats With Cortical Lesions to Cholinomimetics L. G A R O F A L O , * P. J. E L L I O T T t A N D A. C. C U E L L O *l
*Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada, H3G-1 Y6 and i'GLAXO Neuropharmacology Research Group, Ware, Hertz, UK Received 16 D e c e m b e r 1991 GAROFALO, L., P. J. ELLIOTT AND A. C. CUELLO. Behavioralresponse of rats with corticallesions to cholinomimetics. PHYSIOL BEHAV 52(5) 971-977, 1992.--This study examined the performance of cortically lesioned rats and their response to cholinomimetic agents in passive avoidance and water maze tasks. Lesions encompassing mainly the frontal and parietal cortices produce a deficit in a 5-day passive avoidance retention test. This deficit was attenuated by the intraperitoneal (IP) administration of muscarinic agonists or an anticholinesterase. In the Morris water maze task, lesioned vehicle-treated animals showed greater escape latencytimes when compared to their sham counterparts. Cholinomimetics,injecteddaily during acquisition, improved mean escape latency time on days 3 and 4 of testing. The performance of the various groups in a cued version of the water task did not differ. This work demonstrates that performance deficits arising from neocortical loss can be attenuated by cholinergic drugs. Anticholinesterase Passive avoidance
Oxotremorine Pilocarpine Morriswater maze
Arecoline
THE majority of literature presently available on learning and memory processes in animals and humans suggests that central cholinergic systems play an important role. In both rodents and primates, administration of anticholinergic drugs have been shown to induce behavioral deficits in a variety of memorybased tasks (4,51). Decreased memory performance in neurological disorders, such as Alzheimer's disease, is generally associated with deficits in forebrain cholinergic markers and loss or shrinkage of cholinergic neurons in the nucleus basalis of Meynert (42,45,53). In the rat, the counterpart of this nucleus is the nucleus basalis magnocellularis (NBM) (34,41). Various lesioning techniques have shown that damage to this nucleus results in a depletion of cholinergic markers in the NBM and in the neocortex, its area of projection (15,18,20). In addition, unilateral or bilateral injury to the NBM also elicit deficits in memory-based paradigms ( 12,15,19,20,22,31,32,35,38,43). Alternatively, bilateral lesions of the neocortex can also affect a rat's performance in certain behavioral tasks (10,30). Many studies have now shown that administration of cholinergic agents can successfully attenuate behavioral deficits arising from anterograde lesioning of the NBM (12-15,20,22,25,31,32,35,38). In previous studies we have shown that retrograde degeneration of the rat NBM, induced by unilateral lesions of the frontal and parietal cortices, in addition to causing biochemical deficits in cholinergic markers in this area, affect retention and reacquisition of learned Morris water maze and passive avoidance tasks (17).
Physostigmine Cholinergic
Muscarinic
Here we assess, using such rats lacking a large portion of their NBM target site, the ability of cholinomimetic drugs to ameliorate performance deficits in the above tasks. METHODS
Animals Male Wistar rats (325-350 g; Charles River, St. Constant, Quebec) with free access to food and water, on a 12-h light/dark cycle were used in this study. Half the animals had unilateral, devascularizing lesions of the left neocortex while the remaining subjects were sham operated.
Surgical Procedure Rats, under Equithesin anaesthesia (3 ml/kg), were placed in a stereotaxic apparatus and the pia-arachnoid vasculature to the cortex was disrupted unilaterally as previously described (17,49). This procedure results in a gradual cortical atrophy and a loss of the frontal 1 and 3, parietal 1 and portions of the frontal 2, parietal 2 and occipital neocortical areas (see Fig. 1). In addition, a maximal decrease in choline acetyltransferase activity (CHAT) is observed in the NBM 30 days postlesion (9,17,50), at which time subjects with such cortical damage have been found to have performance deficits in memory-based tasks (17). Sham operations consisted of removing an equivalent amount of skull
' Requests for reprints should be addressed to Dr. A. C. CueUo, Department of Pharmacology and Therapeutics, McGill University, 3655 Drummond Street, Montreal, Quebec, Canada H3G-IY6.
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GAROFALO, ELLIOTT AND CUELLO
FIG. 1. Representative fresh coronal br~in slices showing the anterior to posterior extent of the unilateral devascularizing cortical lesion from a subject sacrificed following behavioral studies (51 days postlesion). Coronal brain sections (300-350 um thick) were obtained using a Mcllwain tissue chopper. The extent of the lesion was examined under a Wild (Heerbrugg, Switzerland) microscope and was recorded for every subject. The lesion resulted in a complete atrophy of the frontal 1 and 3, parietal 1 and portions of the frontal 2, parietal 2 and occipital neocortical regions.
bone overlying the neocortex, ensuring that the dura beneath was not damaged.
Behavioral Testing Behavioral testing was initiated 30 days following the surgical procedures.
Ladder- Walking Paradigm Animals were tested for motor coordination in a ladderwalking paradigm. The number of times the rat's paw slipped offthe rungs of a horizontal ladder (100 × 8 cm; rung diameter 4 m m spaced 35 m m apart; suspended 50 cm above a table) were recorded as foot faults. Each rat was allowed five trials in this task.
Passive A voidance Step-through passive avoidance testing was studied in a box (84 × 30 X 30 cm) divided into two equal halves. One com-
partment was painted black and the other white. The dark side was covered with a black top, while the white side was illuminated with two 60 W bulbs. The floor of both sides comprised of steel bars (8 m m in diameter, spaced 10 m m apart); those in the black chamber were connected, via a scrambling device (Lafayette #824000; Lafayette Inst., Co., Lafayette, IN) to a shock generator (Lafayette #58020). Testing consisted of two 3-min habituation trials in the box on consecutive days. On the third day the rats were placed in the white side of the apparatus and the time taken to enter the dark side was recorded. Once inside the dark side the guillotine door was closed and 15 s later a 0.45 m A X 1 s scrambled footshock was applied to the floor bars. Following each shock the subjects (n = 7-8/group) were left in the dark side for a further 15 s and, subsequently, were injected intraperitoneally (1P) with either: vehicle (phosphate buffered saline; PBS), arecoline ( 1.0 mg/kg/ml), pilocarpine (4.0 mg/kg/ml), oxotremorine (0.1 mg/kg/ml), or physostigmine (0.03 mg/kg/ml). Drug dosages were estimated based on previous studies employing these compounds (25,26,29). All the rats were retested
CHOLINOMIMETICS AND BEHAVIOR
973 after which, upon failure to locate the platform, the subject was placed on it for 30 s. The training paradigm consisted of one block of four trials, spaced 15 min apart, per day for 5 consecutive days. Start positions within each block varied between trials. At the end of each block of test trials, sham and cortically lesioned animals were injected IP (n = 7-8/group) with either saline, physostigmine (0.03 mg/kg/ml) or pilocarpine (4 mg/kg/ml). Animals were retested in the water maze 2 weeks later to assess retention (one block of four trials). The day following the retention test these rats were studied in a cued version of the water maze task. In this study, the platform was moved to a new location in the pool and was elevated 3 cm above the surface of the water, thus making it visible. The testing paradigm used was identical to that described above.
Control Lesioned
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Ipsilaterol NBM
FIG. 2. Effect of the cortical lesion on ChAT activity in the ipsilateral and contralateralNBM of adult rats 30 days postlesion.Rats were lesioned unilaterally by cortical devascularization and sacrificed after 30 days. Sham operated rats (control) had an equivalent amount of skull bone removed but were unlesioned. ChAT activity in the NBM and protein content were determined according to the methods of Fonnum (21) and Bradford (7), respectively.*p < 0.05 with respect to control rats, ANOVA post hoc Newman-Keuls' test.
Drugs Oxotremorine, physostigmine, arecoline and pilocarpine were all purchased from Sigma Chemical Co., St. Louis, MO. All drugs were dissolved in PBS on the day of testing prior to their administration.
Biochemical Assays A separate group of rats, similarly cortically lesioned to those prepared for behavioral studies, were sacrificed 30 days postlesion by decapitation. The NBM was microdissected as previously described (50) and the tissue was hand homogenized in 10 volumes of 10 m M Sodium EDTA buffer pH 7.4 containing 0.5% Triton X-100. ChAT activity was determined according to the method of Fonnum (21). Protein content was measured using the method of Bradford (7).
5 days later to investigate retention of the response. When tested, the subjects were not under the influence of any drug-induced effects such as altered pain thresholds, locomotor activity, or environmental perception. White noise was present during both the training and the retention test which occurred between 0900 and 1700 h.
Morris Water Maze
Statistics
Another group of lesioned and sham-operated animals were tested in the Morris water maze task essentially as previously described (17). The apparatus consisted of a white circular plastic pool (140 cm in diameter; 42 cm high) which was filled to a depth of 30 cm with water (18 + 2°C) made opaque with nontoxic powder tempera blue paint (Weber Costello, Mississauga, Ontario). A circular transparent platform (10 cm in diameter) covered with a plastic mesh (Type $74616-F; Sargent-Welch) was placed in one part of the pool such that it was covered with 1 cm of water and was not visible. To assess acquisition of the Morris water maze task, each subject was placed in the water with its face positioned toward the side of the pool. Time taken to find the platform and swimming profile were recorded by an observer blind to the drug treatments. Upon locating the platform, subjects were allowed 30 s to acquire spatial information from external cues in the room. A cutoffpoint of 180 s was used
All results were analyzed using analysis of variance and a post hoc Newman-Keuls test. RESULTS This study shows that unilateral devascularizing cortical lesions significantly decrease ChAT activity in the ipsilateral NBM (Fig. 2) and increase the incidence of multiple footfaults in a ladder-walking task (Table 1). Decreases in rat body weight were noted on postlesion day 10; however, once behavioral studies were initiated (i.e., 30 days postlesion) lesioned rats had recovered normal body weight. The above observations confirm previous work (17). The results of the passive avoidance testing are shown in Fig. 3. Thirty days postoperatively all rats showed similar habituation profiles, entering the dark side of the box within 20 s. In the passive avoidance retention test, sham-operated animals
TABLE 1 NUMBEROF FOOTFAULTSIN LADDER-WALKINGTASK Frontlimb Group
Ipsilateral
Sham (n = 40) Lesioned (n = 40)
16 12
Hindlimb
Contralateral 7 74*
lpsilateral 19 13
Contralateral 21 85*
Thirty days postsurgery sham and lesioned animals were allowed five trials in a ladder walking task. Numbers represent the total number of footfaults observed per group. * p < 0.05 ANOVA, post hoc Newman-Keuls test with respect to sham operated rats.
974
GAROFALO, ELLIOTT AND CUELLO r - - i Sham Lesioned
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though, no significant, F(3, 43) = 2.35, p = 0.1, group effect on the mean escape latency time is noted (Fig. 5), analysis of the swim profiles showed that lesioned vehicle-treated rats used a less direct route to find the platform (Fig. 6B). This was also true during the acquisition phase of this paradigm (Fig. 6A). Figure 4B illustrates the mean escape latency times for all the groups after the platform was moved to a different location in the maze and was made visible. All the subjects showed similar rapid acquisition profiles although in the first trial of the test block sham-operated and drug-treated lesioned groups, in con-
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FIG. 3. Effects of the cortical lesion and cholinomimetics on passive avoidance retention. Veh: vehicle (phosphate buffered saline); Phys: physostigmine; Pilo: pilocarpine; Arec: arecoline; Oxo: oxotremorine. Please see the method section for details. *p < 0.05 with respect to sham operated rats, ANOVA post hoc Newman-Keuls' test. n = 8/group.
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showed no deficit and their performance was not affected by the various drug treatments [ANOVA, F(4, 35) = 0.545, p = 0.71]. Thus, results from vehicle and drug-treated sham-operated animals were pooled. In contrast to sham-operated animals, rats with lesions affecting mainly the frontal and parietal cortices showed a retention deficit in this task. Analysis of variance demonstrated that there was a significant group effect, F(5, 74) = 3.835, p = 0.01. Comparisons of the group means, using a post hoc N e w m a n - K e u l s test, indicated that the IP administration of cholinomimetic drugs to lesioned animals significantly (p < 0.05) attenuated this impairment. No differences were noted in the ability of the different cholinergic agonists (arecoline, oxotremorine, pilocarpine) or the anticholinesterase (physostigmine) to ameliorate the 5-day retention of the passive avoidance task. The mean latency to find the platform for all groups in the Morris water maze task is shown in Fig. 4A. Sham-operated animals rapidly acquired the task as was previously reported (17). Similarly to the passive avoidance results, the behavior of drug-treated sham-operated animals did not differ from their saline injected counterparts. Analysis of variance revealed no differences among the sham-operated groups, F(2, 105) = 1.5, p = 0.22, but did show that escape latency times decreased significantly over trial days, F(4, 105) = 175.5, p = 0.001. The interaction between groups and trial day was not significant, F(8, 105) = I. 15, p = 0.34. Data from vehicle and drug treated sham subjects were, therefore, pooled for each test day. Unilaterally decorticated rats showed a mild impairment in the acquisition of this spatial task. These rats were able to locate the platform; however, their escape latency times were greater than those of sham operated animals. Analysis of variance showed significant effect of groups, F(3, 215) = 2.54, p = 0.05, trial day, F(4, 215) = 133.18, p = 0.00 l, and group by trial day interaction, F( 12, 215) = 3.09, p = 0.001. N e w m a n - K e u l s post hoc tests on the group means revealed that daily IP injections of cholinomimetic drugs, administered subsequent to termination of a block of four test trials, significantly (p < 0.05) decreases the mean escape latency times of lesioned animals on days 3 and 4 of testing (Fig. 4a). Rats were then retested 2 weeks following acquisition of the Morris water maze task to assess retention. Al-
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Doy FIG. 4. Mean escape latency, on each test day, from the Morris water maze with a (A) hidden platform and (B) with the platform now visible but in a new location. Sham operated: O O; lesion + vehicle: • • ; lesion + physostigmine: A A; lesion + pilocarpine: • • . *p < 0.05 with respect to sham operated rats, ANOVA post hoc Newman-Keuls' test. Sham operated, n = 24 animals; lesioned, n = 8 animals/group.
CHOLINOMIMETICS AND BEHAVIOR
975
'25 20
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ID Sham + vehicle
Lesion + Physo
+ Pilo
FIG. 5. Mean escape latency to find the hidden platform in the Morris water maze 2 weeks after acquisition of the task. Sham operated, n = 24 animals; lesioned, n = 8 animals/group.
trast to vehicle-treated rats, swam more readily directly towards the platform's old location before heading to the visible one. Following all behavioral testing, the rats were sacrificed and a gross morphological examination of all the brains was undertaken. Lesions were found to be consistent across all groups. Anterior to posterior coronal sections of each brain confirmed that the lesion did not encroach subcortical brain areas (see Fig. 1). However, in some animals, the corpus callosum was at times thinner or absent in more posterior sections.
the NBM (44) rather than due to deafferentation of the cortex. In particular, work using two different neurotoxins (40), suggests that the NBM-cortical cholinergic pathway may have little influence on rodent mnemonic processes as assessed in passive avoidance and Morris water maze tasks. Rather, it has been proposed that NBM lesion-induced deficits occur due to disturbances in motivational behavior arising from disruption of NBM projections to the amygdala (16,40) or have been attributed to alterations in neurochemicals other than acetylcholine (i.e., neurotensin; GABA) (16,52). In animals of the present study, a large portion of the neocortex was damaged unilaterally by a devascularizing lesion. In addition to reliably producing a 40-50% decrease in ChAT activity [present study, (9,17,50)] and cholinergic cell shrinkage (9,49) in the ipsilateral NBM, such a lesion also results in cortical loss and diminished cholinergic innervation of the ipsilateral remaining cortex adjacent to the lesion site. In particular, a significant decrease in the number of synaptic contacts, axonal varicosities and size of cholinergic boutons can be noted within this remaining cortical area (24). The results obtained in the passive avoidance task with these animals are in line with previous studies which show that disruption of fronto-parietal cortical function adversely affects rodent performance in inhibitory
A Day:
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DISCUSSION
Unilateral devascularizing cortical lesions affecting mainly the frontal and parietal rat cortex cause temporary weight loss, multiple footfaults, and performance deficits in two tasks (passive avoidance and Morris water maze) frequently used to assess learning and memory. The lesions, although extensive, are not overly debilitating to the animals, since they are able, after an initial weight loss, to recover normal feeding and grooming activities. Once behavioral studies were initiated, no gross physiological abnormalities distinguished lesioned subjects from shamoperated rats. The present study sought to determine whether performance deficits previously noted (17) in rats with this particular type of cortical lesion could be attenuated by cholinomimetic drugs. A decline in cholinergic function of the basal forebrain has been thought to underlie the memory dysfunction occurring in the aged and in Alzheimer's disease (5). In animal models which attempt to mimic this deficit, such as the rat with anterograde damage to the NBM, the beneficial effects of cholinomimetic agents have been well documented. In particular, the anticholinesterase, physostigmine, has been shown to improve the performance of rats with bilateral lesions to the NBM in passive avoidance (27) and delayed matching tasks (13), in a reinforced alternation T-maze (36) and to ameliorate escape latency times in the Morris water maze (11). Oxotremorine (1,2,26,29), arecoline (26,29,33,39,46-48) and pilocarpine (29), all muscarinic agonists, have also been shown to improve performance in learning and memory tasks. However, recent data indicate that behavioral impairments following NBM lesions may arise from nonspecific damage to areas in the vicinity of
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FIG. 6. Typical swimming patterns of the subjects during the first trial (A) of each test day and (B) during the first trial 2 weeks after acquisition of the task. Sham operated, n = 24 animals; lesioned, n = 8 animals/ group.
976
GAROFALO, ELLIOTT A N D CUELLO
tasks (6,23). The attenuation of the passive avoidance retention deficit by cholinergic drugs in such cortically lesioned rats could be attributed to either activation of the remaining basalo-cortical cholinergic pathway or stimulation of other cholinergic pathways. Indirect support to suggest that the remaining NBM-to-cortex pathway may be involved is provided by studies involving NBM lesioned rats. It has been shown that lesions of the rat frontal cortex, which alone apparently do not affect retention of a passive avoidance response, can abolish the physostigmine-induced recovery of this performance in NBM-lesioned animals (27). Furthermore, rats with unilateral NBM lesions, which also show impairments in a passive avoidance task (19,32), improve their behavior if fetal basal forebrain grafts are transplanted into the cortex (19). However, as previously indicated, alterations in passive avoidance responses by NBM-lesioned rats are also attributed to disturbances in motivation or fear rather than to deficits in learning and memory. In our lesion model, although subcortical brain areas appear undisturbed by the lesion, the amygdala and, in particular, the striatum and thalamus, are affected as exemplified by the presence of gliosis in these subeortical areas (28). Since the amygdala in particular appears to play a role in fear, it is possible that alterations in fear, rather than memory, account for our observations. Although indices of fear have not been examined in detail for these animals, it was noted that lesioned vehicle- or drug-treated rats of the present study did not appear to respond differently to handling than did shamoperated animals. However, in earlier work involving similarly lesioned rats, it was observed that vehicle-treated animals did tend to defecate less on the white side of the box (17). Such an occurrence could be indicative of reduced fear. Furthermore, since the rat cortex is known to receive catecholaminergic (36) and serotoninergic (37) inputs from the locus coeruleus and raphe nucleus, respectively, the contribution of disturbances in these pathways and neurochemicals to the observed behavioral deficits requires assessment. In particular, whether such noncholinergic agents also produce similar behavioral responses in these animals should be tested in future studies. The importance of an intact cortex for spatial localization in the rat has previously been reported. Initial studies by Kolb and collegues (30) showed that bilateral damage to the frontal or cingulate cortex produce deficits in spatial tasks, whereas bilateral parietal lesions produce only mild impairments. DiMattia and
Kesner (10) using rats with more extensive damage to the parietal area have shown that this cortical region plays an important role in spatial memory. The present study demonstrates that impairments in spatial memory, albeit mild, also occur following unilateral devascularizing cortical lesions. However, although lesioned vehicle-treated rats can learn to locate the hidden platform in the water maze and show no significant deficits in mean escape latency times when retested 2 weeks after acquisition, these rats fail to use a direct route to do so. Similar results were obtained with rats subjected to suction ablation cortical lesions (10,30). Thus, rodents lacking frontal/parietal cortices appear to be impaired in their ability to adopt a spatial mapping strategy to solve the task. This impairment seems to be attenuated by cholinomimetic drugs. However, as discussed earlier, with behavioral studies involving brain lesions it is difficult to fully attribute observed performance deficits and their reversal by drugs to changes in learning and memory. In a previous study (17), motor and sensory tests revealed that other than an increase in overnight locomotor activity and deficits in the ability to walk along a horizontal ladder, the latter reconfirmed in the present study, cortically devascularized animals showed no impairments in auditory, olfactory, noxious, and muscle-strength testing paradigms. However, unilateral cortical lesions have previously been reported to induce contralateral sensory neglect (8), and disruption of the NBM-to-cortex pathway has been linked to deficits in visual attentional function (40). The rats in the present study do circle the perimeter of the pool, in a direction ipsilateral to the lesion, when first exposed to the task to a greater degree than do sham-operated animals (see Fig. 6A). However, when tested in the water maze containing a visible platform lesion vehicletreated rats performed similarly to sham-operated rats and quickly located the platform. Although this provides some indirect evidence which could argue against motivation, altered swim speeds and/or motor problems affecting performance in the maze, more detailed studies are required to absolutely ascertain their lack of involvement. ACKNOWLEDGEMENTS We thank Mr. A. Forster for the photographs and Ms. Laura Fernandes for the ChAT assay. This work was supported by the Canadian Medical Research Council and the Centers for Excellence for Neural Repair and Functional Recovery. L.G. received an FRSQ studentship.
REFERENCES 1. Baratti, C. M.; Huygens, P.; Mino, J.; Merlo, A.; Gardella, J. Memory facilitation with post-trial oxotremorine and physostigmine in mice. Psychopharmacology (Berlin) 64:85-88; 1979. 2. Baratti, C. M.; lntroini, I. B.; Huygens, P.; Gusovsky, F. Possible cholinergic-dopaminergic link in memory facilitation induced by oxotremorine in mice. Psychopharmacology (Berlin) 80:161-165; 1983. 3. Bartus, R. T. Four stimulants of the central nervous system: Effects on short term memory in young versus aged monkeys. J. Am. Geriatr. Soc. 27:289-297; 1979. 4. Bartus, R. T.; Johnson, H. R. Short-term memory in the rhesus monkey: Disruption from the anticholinergic scopolamine. Pharmacol. Biochem. Behav. 5:39--46; 1976. 5. Bartus, R. T.; Dean, R. L.; Beer, B.; Lippa, A. S. The cholinergic hypothesis of geriatric memory dysfunction. Science 217:408-417; 1982. 6. Bermudez-Rattoni, F.; Introni-Collison, I. B.; McGaugh, J. L. Reversible activation of the insular cortex by tetrodotoxin produces retrograde amnesia for inhibitory avoidance and spatial learning. Proc. Natl. Acad. Sci. USA 88:5379-5382; 1991. 7. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of dye binding. Anal. Biochem. 72:248-254; 1976.
8. Cowey, A.; Bozek, T. Contralateral 'neglect' after unilateral dorsomedial prefrontal lesions in rats. Brain Res. 72:53-64; 1974. 9. Cuello, A. C.; Garofalo, L.; Kenigsberg, R. L.; Maysinger, D. Gangliosides potentiate in vivo and in vitro effects of nerve growth factor on central cholinergic neurons. Proc. Natl. Acad. Sci. USA 86:20562060; 1989. 10. DiMattia, B. D.; Kesner, R. P. Spatial and cognitive maps: Differential role of parietal cortex and hippocampal formation. Behav. Neurosci. 102(4):471-480; 1988. 11. Dokla, C. P. J.; Thai, L. J. Effect of cholinesterase inhibitors on Morris water maze task behavior following lesions of the nucleus basalis magnocellularis. Behav. Neurosci. 102(6):861-871; 1988. 12. Dubois, B.; Mayo, W.; Agid, Y.; Le Moal, M.; Simon, H. Profound disturbances of spontaneous and learned behaviours following lesions of the nucleus basalis magnocellularis in the rat. Brain Res. 338: 249-258; 1985. 13. Dunnett, S. B. Comparative effects of cholinergic drugs and lesions on nucleus basalis or fimbria-fornix on delayed matching in rats. Psychopharmacology (Berlin) 87:357-363; 1985. 14. Dunnett, S. B.; Toniolo, G.; Fine, A.; Ryan, C. N.; Bjorklund, A.; lversen, S. D. Transplantation of embryonic ventral forebrain neurons to the neocortex of rats with lesions of nucleus basalis mag-
CHOLINOMIMETICS AND BEHAVIOR
15.
16.
17.
18.
19.
20.
21. 22. 23.
24. 25. 26. 27. 28. 29. 30. 31.
32.
33.
nocellularis-ll. Sensorimotor and learning impairments. Neuroscience 16:787-797; 1985. Dunnett, S. B.; Whishaw, I. Q.; Jones, G. H.; Bunch, S. T. Behavioral, biochemical and histochemical effects of different neurotoxic amino acids injected into nucleus basalis magnocellularis of rats. Neuroscience 20:653-669; 1987. Dunnett, S. B.; Everitt, B. J.; Robbins, T. W. The basal forebraincortical cholinergic system: Interpreting the functional consequences of excitotoxic lesions. Trends Neurosci. 14( 11 ):494-501; 1991. Elliott, P. J.; Garofalo, L.; Cuello, A. C. Limited neocortical devascularizing lesions causing deficits in memory retention and choline acetyltransferase activity--Effects of the monosialoganglioside GMI. Neuroscience 31:63-76; 1989. Fine, A.; Dunnett, S. B.; Bjorklund, A.; Clarke, D.; Iversen, S. D. Transplantation of embryonic ventral forebrain neurons to the neocortex of rats with lesions of nucleus basalis magnocellularis--I. Biochemical and anatomical observations. Neuroscience 16:769786; 1985. Fine, A.; Dunnett, S. B.; Bjorklund, A.; lversen, S. D. Cholinergic ventral forebrain grafts into the neocortex improve passive avoidance memory in a rat model of Alzheimer's disease. Proc. Natl. Acad. Sci. USA 82:5227-5230; 1985. Flicker, C.; Dean, R. L.; Watkins, D. L.; Fisher, S. K.; Bartus, R. T. Behavioral and neurochemical effects following neurotoxic lesions of a major cholinergic input to the cerebral cortex in the rat. Pharmacol. Biochem. Behav. 19:309-312; 1983. Fonnum, F. A rapid radiochemical method for the determination of choline acetyltransferase. J. Neurochem. 24:407-409; 1975. Friedman, E.; Lerer, B.; Kuster, J. Loss of eholinergic neurons in the rat neocortex produces deficits in passive avoidance learning. Pharmacol. Biochem. Behav. 19:309-312; 1983. Fukuchi, I.; Susumu, K.; Nakahiro, M.; Uchida, S.; Ishida, R.; Yoshida, S. Blockade of cholinergic receptors by an irreversible antagonist, Propylbenzylcholine mustard (PrBCM), in the rat cerebral cortex causes deficits in passive avoidance learning. Brain Res. 400: 53-61; 1987. Garofalo, L.; Ribeiro-da-Silva, A.; Cuello, A. C. Nerve growth factor induced synaptogenesis and hypertrophy of cortical cholinergic terminals. Proc. Natl. Acad. Sci. USA 89:2639-2643; 1992. Haroutunian, V.; Kanof, P.; Davis, K. L. Pharmacological alleviation of cholinergic lesion induced memory deficits in rats. Life Sci. 37: 945-952; 1985. Haroutunian, V.; Barnes, E.; Davis, K. L. Cholinergic modulation of memory in rats. Psychopharmacology (Berlin) 87:266-271; 1985. Haroutunian, V.; Mantin R.; Kanof, P. D. Frontal cortex as the site of action of physostigmine in nbM-lesioned rats. Physiol. Behav. 47:203-206; 1990. Herrera, D. G.; Cuello, A. C. GFAP immunoreactivity following cortical devascularization. Soc. Neurosci. Abstr. 16:779; 1990. Jung, M.; Costa, L.; Sherman, G. T.; Kelly, P. H. Discriminative stimulus properties of muscarinic agonists. Psychopharmacology (Berlin) 93:139-145; 1987. Kolb, B.; Sutherland, R. J.; Whishaw, I. Q. A comparison of the contributions of the frontal and parietal association cortex to spatial localization in rats. Behav. Neurosci. 970):13-27; 1983. Lerer, B.; Warner, J.; Friedman, E.; Vincent, G.; Gamzu, E. Cortical cholinergic impairment and behavioral deficits produced by kainic acid lesions of the rat magnocellular basal forebrain. Behav. Neurosci. 99:661-677; 1985. LoConte, G.; Bartolini, L.; Casamenti, F.; Marconcini-Pepeu, I.; Pepeu, G. Lesions ofcholinergic forebrain nuclei: Changes in avoidance behavior and scopolamine actions. Pharmacol. Biochem. Behav. 17:933-937; 1982. Meltzer, L. T.; Rosecrans, J. A. Discriminitive stimulus properties of arecoline: A new approach for studying central muscarinic receptors. Psychopharmacology (Berlin) 75:383-387; 1981.
977
34. Mesulam, M. M.; Mufson, E. J.; Wainer, B. H.; Levy, A. I. Central cholinergic pathways in the rat: An overview based on alternative nomenclature (Ch l-Ch6). Neuroscience l 0:1185-1201; 1983. 35. Miyamoto, M.; Shinatani, M.; Nagaoka, A.; Nagawa, Y. Lesioning of the rat basal forebrain leads to memory impairments in passive avoidance and active avoidance tasks. Brain Res. 328:97-104; 1985. 36. Moore, R. Y.; Bloom, F. E. Central catecholamine neuron systems: Anatomy and physiology of the norepinephrine and epinephrine systems. Annu. Rev. Neurosci. 2:113-168; 1979. 37. Moore, R. Y.; Halaus, A. I.; Jones, B. C. Serotonin neurons of the midbrain raphe ascending projection. J. Comp. Neurol. 180:417438; 1978. 38. Murray, C. L.; Fibiger, H. C. Learning and memory deficits after lesions of the nucleus basalis magnoceUularis: Reversal by physostigmine. Neuroscience 14:1025-1032; 1985. 39. Overton, D. A. Comparison of the degree of discriminability of various drugs using the T-maze discrimination paradigm. Psychopharmacology (Berlin) 76:385-395; 1982. 40. Page, K. J.; Everitt, B. J.; Robbins, T. W.; Marston, H. M.; Wilkinson, L. S. Dissociable effects on spatial maze and passive avoidance acquisition and retention following AMPA- and ibotenic acid-induced excitotoxic lesions of the basal forebrain in rat: Differential dependence on cholinergic neuronal loss. Neuroscience 43:457-472; 1991. 41. Pepeu, G.; Casamenti, F.; Pedata, F.; Cosi, C.; Pepeu, I. M. Are the neurochemical and behavioral changes induced by lesions of the nucleus basalis in the rat a model Alzheimer's disease? Prog. Neuropsychopharmacol. Biol. Psychiatry 10:541-551; 1986. 42. Perry, E. K.; Perry, R. H.; Blessed, G.; Tomlinson, B. E. Necropsy evidence of central cholinergic deficits in senile dementia. Lancet i: 189; 1977. 43. Riekkinen, P., Jr.; Sirvo, J.; Hannila, T.; Miettinen, R.; Riekkinen, P. Effect of quisqualic acid nucleus basalis lesioning on cortical EEG, passive avoidance and water maze performance. Brain Res. Bull. 24:839-842; 1990. 44. Robbins, T. W.; Everitt, B. J.; Ryan, C. N.; Marston, H. M.; Jones, G. H.; Page, K. J. Comparative effects of quisqualic and ibotenic acid-induced lesions of the substantia innominata and globus pallidus on the acquisition of a conditional visual discrimination: Differential effects on cholinergic mechanisms. Neuroscience 28(2):337-352; 1989. 45. Rossor, M. N.; Svendsen, C.; Hunt, S. F.; Mountjoy, C. G.; Roth, M.; lversen, L. L. The substantia innominata in Alzheimer's disease: A histochemical and biochemical study of cholinergic marker enzymes. Neurosci. Lett. 28:217-222; 1982. 46. Schechter, M. D.; Rosecrans, J. A. Atropine antagonism ofarecolinecued behavior in the rat. Life Sci. 11(11):519-523; 1972. 47. Schechter, M. D.; Rosecrans, J. A. Effect of mecamylamine on discrimination between nicotine- and arecoline-produced cues. Eur. J. Pharmacol. 17:179-182; 1972. 48. Sitaram, N.; Weingarter, H.; Gillin, J. C. Human serial learning enhancement with arecoline (sic) and impairment with scoploamine. Science 201:274-276; 1978. 49. Sofroniew, M. V.; Pearson, R. C. A.; Eckenstein, F.; Cuello, A. C.; Powell, T. P. S. Retrograde changes in cholinergic neurones in the basal forebrain of the rat following cortical damage. Brain Res. 289: 370-374; 1983. 50. Stephens, P. H.; Cuello, A. C.; Sofroniew, M. V.; Pearson, R. C. A.; Tagari, P. C. Effect of unilateral decortication on choline acetyltransferase activity in the nucleus basalis and other areas of the rat brain. J. Neurochem. 45:1021-1026; 1985. 51. Watts, J.; Stevens, R.; Robinson, C. Effects of scopolamine on radial maze performance in rats. Physiol. Behav. 26:845-851; 1981. 52. Wenk, G. L.; Markowska, A. L.; Olton, D. S. Basal forebrain lesions and memory: Alterations in neurotensin, not acetylcholine, may cause amnesia. Behav. Neurosci. 101:765-769; 1989. 53. Whitehouse, P. J.; Price, D. L.; Strubble, R. G.; Clark, A. W.; Coyle, J. T.; DeLong, M. R. Alzheimer's disease and senile dementia loss of neurones in the basal forebrain. Science 215:1237-1239; 1982.
