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Effects of tetrahydroaminoacridine (THA) on functional recovery after sequential lesion of the entorhinal cortex
57
Brain Research, 557 (1991) 57-63 Elsevier Science Publishers B.V. ADONIS 000689939116886T BRES 16886
Effects of tetrahydroaminoacridine (THA) on functional recovery after sequential lesion of the entorhinal cortex J.P. Kesslak, A. Korotzer, A. Song, K. Kamali and C.W. Cotman Department of Psychobiology, University of California, lrvine, lrvine, CA 92717 (U.S.A.) (Accepted 26 March 1991)
Key words: Tetrahydroaminoacridine; Functional recovery; Entorhinal cortex; Serial lesion
Unilateral lesions of rat entorhinai cortex produce a transitory performance deficit on spatial learning tasks, such as reinforced alternation in a T-maze. Tetrahydroaminoacridine (THA), a cholinesterase inhibitor, was administered to determine its effects on behavioral recovery using a reinforced alternation task in a T-maze. Rate of recovery after unilateral entorhinal lesion was not affected by a low dose of THA (0.05 mg/kg), while a higher dose (5.0 mg/kg) impaired recovery. Behavioral recovery was subsequently evaluated in the same rats following lesions to the contralateral entorhinal cortex. Serial bilateral lesions of the entorhinal cortex are known to produce a prolonged performance deficit on the alternation task. The 0.05 mg/kg THA group exhibited an intermediate rate of recovery, between the undamaged control group and bilateral lesion-saline injected groups. The group receiving 5.0 mg/kg of THA after bilateral lesion did not differ from the bilateral lesiun-saline group. The failure of THA to significantly improve functional recovery in rats with lesions of the entorhinal cortex indicates that the compound may have limited applicability in treating human neurodegenerative disorders such as Alzheimer's disease.
INTRODUCTION D a m a g e to rat entorhinal cortex results in learning and m e m o r y i m p a i r m e n t s on a variety of tasks 16'23'25. The deficits o b s e r v e d vary according to the extent of hippoc a m p a l d e n e r v a t i o n and the type of behavioral task. Transient deficits on spatial alternation performance in response to unilateral entorhinal cortex lesions have been r e p o r t e d for the T-maze 15'21'23, Y-maze 2° and H e b b Williams m a z e ~6. M o r e persistent deficits occur on spatial alternation tasks after bilateral entorhinal cortex damage5A2,15,20,22,23,26. T h e rate of behavioral recovery after lesions of the entorhinal cortex can be accelerated by various treatments. A d m i n i s t r a t i o n of gangliosides n'18"19 or nerve growth factor ( N G F 24) increases the rate of recovery on spatial alternation tasks after entorhinal lesions. T h e r e is also evidence that administration of the anticholinesterase t e t r a h y d r o a m i n o a c r i d i n e ( T H A ) m a y improve performance after neural loss, particulary after lesion of the nucleus basalis in rats 4A4. Chronic administration of T H A can i m p r o v e m e m o r y in n o r m a l 7 and age-related m e m o r y i m p a i r e d mice 6. Preliminary clinical results have n o t e d behavioral i m p r o v e m e n t after administration of T H A to patients with A l z h e i m e r ' s Disease ( A D ) 12'27'28, but not in all cases 9. T h e entorhinal cortex experiences
severe neural loss in A D s'l°. This finding is particularly noteworthy since septal cholinergic fibers sprout into the o u t e r molecular layer of the h i p p o c a m p u s after entorhinal lesions in rats 25 and in A D 2'9'10. In the present set of experiments, we have examined the effect of T H A on recovery of p e r f o r m a n c e on a spatial alternation task after unilateral and serial bilateral lesions of the entorhinal cortex in rats. The serial lesion m o d e l was selected to evaluate the effects of progressive neural loss, which also occurs in the course of A D . In the rat, transitory behavioral deficits are seen after m i n o r d a m a g e to the entorhinal cortex (i.e. unilateral lesion), whereas m o r e p r o l o n g e d and severe deficits are p r o d u c e d by increased cell loss (i.e. bilateral lesion) 25. T H A affected recovery after unilateral lesions in a dosed e p e n d e n t m a n n e r , with the smaller dose having no effect and the larger dose having d e t r i m e n t a l effects. A d m i n i s t r a t i o n of T H A after serial bilateral entorhinal lesions did not significantly i m p r o v e p e r f o r m a n c e on the alternation task. MATERIALS AND METHODS
Subjects Male Sprague-Dawley rats (200-225 g), obtained from Charles River, were housed two per cage in a 12/12 h light/dark vivarium. Rats were given ad lib access to Purina Lab Chow. During maze
Correspondence: P. Kesslak, Department of Psychobiology, University of California, Irvine, Irvine, CA 92717, U.S.A.
58 acclimation, pretraining and postsurgery testing the rats were placed on water deprivation. Animals were allowed to drink for 10 min each day beginning at least 30 min after completing the behavioral testing. Body weight was recorded each day of testing. Testing was done during the light phase of the vivarium cycle. Fifty-four rats were initially pretrained (described below) for 5 consecutive days. After pretraining, rats were assigned to experimental groups equated for performance on the alternation task. There were 4 experimental conditions: undamaged receiving saline (CNT; n = 19), unilateral entorhinal lesion receiving saline (SAL; n = 11), unilateral entorhinal lesions receiving 5.0 mg/kg THA (THA5; n = 11), and unilateral entorhinal lesions receiving 0.05 mg/kg THA (THA05; n = 13). All injections were i.p. and administered approximately 30 min before testing. Testing on the spatial task began 2 days after surgery. Following the 15 days of reinforced alternation testing after unilateral entorhinal cortex lesions or sham lesion, the intact contralateral entorhinal cortex was lesioned. The second surgical treatment and alternation testing was continued on 35 male Sprague-Dawley rats. Rats had ad lib access to water for 2 days before the second surgery. After surgery, the rats were given two days for recovery, placed on water deprivation on day three and behavioral testing initiated. Ten rats from the group CNT were randomly chosen to continue in the experiment (n = 10). The contralateral (intact) entorhinal cortex was lesioned in groups that had previously received unilateral lesions (SAL, n = 9; THA5, n = 8; THA05, n = 8). Rats not used in this experiment were randomly chosen for histology to verify lesion placement. The serial bilateral lesion was expected to produce a long-term deficit on the alternation task.
Surgery Rats were anesthetized with Nembutal (54 mg/kg) and placed into a stereotaxic instrument. An incision was made in the scalp exposing the top of the skull and a hole drilled to expose the entorhinal cortex. The head was elevated 10° and the stereotaxic arm set at a 10° angle away from the midline in the coronal plane. There were a total of 9 lesion sites with coordinates from lambda at A/P _+ 0.0, M/L + 3.3, D/V -2.0, 4.0, 6.0; A/P + 0.0, M/L + 4.1, D/V -2.0, 4.0, 6.0; and A/P + 1.0, M/L + 5.1, D/V -2.0, 4.0, 6.0. At each site a no. 2 stainless steel insect pin, insulated except 0.5 mm at the tip, was lowered and an electrolytic lesion made by passing 1 mA current for 45 s. After retracting the electrode, gelfoam was placed over the exposed brain and the scalp sutured. Rats that received unilateral lesions were tested on the alternation task for 15 days. Three days after the alternation test, rats received either lesions of the contralateral entorhinal cortex or sham operations. Rats in the control groups received similar surgical preparation but the electrode was not lowered into the brain. Fifteen days of reinforced alternation was initiated 3 days after the second surgery.
Reinforced-alternation task and group formation Maze acclimation. Each rat was handled for 4 days prior to T-maze acclimation. On the last day of handling the rats were placed on water deprivation and given 10 min access per day. Maze acclimation was conducted for 3 days as follows. On day 1 each rat was placed into the start section of the T-maze and allowed to move freely in the maze for 3 rain, without being removed from the maze. A plastic dish containing 1.5 ml of water was placed at the end of each goal arm and replaced after the rat exited each goal arm. Day 2 was similar to day 1 but after entering the goal arm the rat was allowed to drink, removed from the goal arm, the water replaced and the rat placed in the start arm. Day 3 was similar to day 2 but the gates to the goal arms were closed after the rat entered the reward area. Reinforced alternation. Rats were tested at the same time each day. Each rat was weighed and injected (1 ml/kg) with either 0.9% phosphate buffered saline or THA (Sigma; 5.0 or 0.05 mg/kg) dissolved in phosphate-buffered saline at least 20-30 min before testing. During the 5 days of pretraining all rats received saline
injections. Solutions were prepared daily and coded so that the experimenters had no knowledge of the drugs being injected. The reinforced-alternation task was initiated by placing the rat in the start section of the T-maze, facing the back wall. The animal was then allowed to traverse the maze and enter one of the goal arms. On the first trial of each day the animal was rewarded for entering either arm of the maze. Thereafter, reward was given only if the rat entered the arm opposite the one previously rewarded. After a correct choice the rat received 1.5 rnl of water and allowed 10 s to drink. Testing began 3 days after surgery and lasted for 15 consecutive days. Each rat was tested on 10 consecutive alternation trials per day. If a rat failed to make an alternation choice after 1 min the trial was considered an error. The number of correct choices per day was analyzed with Analysis of Variance and post-hoc tests to determine between group differences.
Histology Rats were sacrificed for verification of lesion placement and size at the completion of behavioral testing. The brains were extracted, placed in dry ice and 25 pm horizontal sections cut using a cryostat (Hacker Instruments). Every 7th and 8th section was taken through the entire brain and stained with Cresyl violet and for acetylcholinesterase (ACHE) respectively.
RESULTS
Histology Brains were examined using light microscopy to determine the extent of the lesion and the presence of cholinergic sprouting in the hippocampus verified from Cresyl violet and AChE stained sections. Examination of the sections indicated that the lesions eliminated the majority of the entorhinal cortex and angular bundle (Fig. 1). The medial and lateral perforant paths were lesioned in all cases, and in a few cases the maximal extent of the lesion impinged on the subiculum with minor involvement of the hippocampus. Loss of the hippocampal afferents from the angular bundle resulted in a dense increase of AChE-positive fibers in the outer molecular layer of the dentate gyrus (Fig. 1). The apparent expansion of the cholinergic fibers into the outer molecular layer verified the loss of entorhinal innervation. There were no apparent differences between brains from saline- and THA-treated animals.
Reinforced-alternation performance Groups receiving unilateral entorhinal lesions without drug treatment showed a transitory deficit on the reinforced-alternation task (Fig. 2). Reinforced alternation performance was close to chance levels (50%) during the first 3 days and recovered to approximately 85% correct by day 15. T H A affected performance on the reinforced alternation task in a dose-dependent manner (Fig. 2). The lower dose of 0.05 mg/kg T H A increased performance slightly above the lesion control group, but this was not significant. Treatment with 5 mg/kg T H A appeared to inhibit recovery and decrease the number of correct responses during the first 6 days of testing after
59 the lesion. P e r f o r m a n c e on the reinforced alternation task was analyzed using a r e p e a t e d measures Analysis of Variance ( A N O V A ) . T h e percent correct alternations for
each day were g r o u p e d into blocks of 3 days as the r e p e a t e d m e a s u r e (i.e. blocks of days 1 - 3 , 4 - 6 , etc.). T h e r e was a significant effect for groups (F3,5o = 8.87, P
Fig. 1. Cresyi violet sections of control animals showed an intact entorhinal cortex (A) (Magnification x20) with an even staining for acetyl cholinesterase in the inner and outer molecular layers of the dentate gyrus (B) (Magnification x150). Lesion of the entorhinal cortex (C) produced a dense increase of cholinergic fibers in the outer molecular layer of the dentate gyrus (D).
60 < 0.001), blocks of days (F4,50 = 41.42, P < 0.001) and group x days interaction (Fz2,z0o = 2.75, P < 0.003). Specific b e t w e e n - g r o u p differences for each block of days were examined post-hoc using a Fisher P L S D ( P < 0.05). C o m p a r e d to CNT, the S A L group had significantly fewer correct alternations during the first 3 blocks of testing (days 1-9). Daily administration of T H A in the T H A 5 group did not facilitate behavioral recovery; in fact they exhibited the poorest performance, scoring significantly worse than C N T on all but the last block of trials. The T H A 0 5 group p e r f o r m e d significantly below the C N T group in the first two blocks of trials and did not differ from C N T for the remaining 12 days of testing. All groups showed significant i m p r o v e m e n t in alternation p e r f o r m a n c e from the first to last blocks of days ( P <
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Following the second surgical treatment, the groups which did not receive entorhinal lesions scored approximately 90% correct for the duration of the reinforcedalternation testing. Serial bilateral lesions appeared to produce a long lasting deficit on the alternation task that improved only slightly during the 15 days of testing (Fig. 3). Analysis of the results indicated a significant effect for groups (F3,31 = 8.37, P < 0.001), blocks of days F3,31 = 9.50, P < 0.001) and group x days interaction (F~2,~24 = 2.45, P < 0.005). Specific between group differences for each block of days were examined post-hoc using a Fisher
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Days of testing Fig. 3. Bilateral lesions of entorhinal cortex produced sustained deficits on the reinforced alternation task for the groups injected with saline (SAL) and 5 mg/kg THA (THA5). Chronic administration of 0.05 mg/kg THA (THA05) produced an intermediate level of behavioral recovery where the group did not differ from either CNT or SAL groups (* differs from control group, P < 0.05).
PLSD (P < 0.05). The SAL group had significantly fewer correct trials than the CNT group across all blocks of trials. The scores of the treatment group with 5 mg/kg T H A (THA5) did not differ from that of the SAL group and had significantly lower scores than the CNT group on all blocks of trials. The group receiving 0.05 mg/kg T H A
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Fig. 4. Rats had a consistent gain in body weight across days of testing after unilateral and bilateral lesions of the entorhinal cortex.
61 (THA05) showed an intermediate level of performance on the alternation task. Group THA05 scored significantly lower than CNT on days 1-6 and 9-12. However, group THA05 did not perform better than group SAL during any of the testing. There appeared to be an overall trend for the bilateral lesioned groups to increase in number of correct alternations with extended training. However, the improved performance of groups SAL and THA5 did not reach the same level of performance as did the undamaged CNT group.
Body weight Body weight for the groups did not differ during any part of the study. During the first 15 days of testing (unilateral lesion) there was a consistent increase in body weight across the days of testing (Fig. 4). The CNT group consistently weighed more than the other groups but this difference was not significant (ANOVA; F3,50 = 0.348, P > 0.05). During the second testing period (bilateral lesion) there was a consistent increase in weight across days of testing (Fig. 4), with no differences between groups (ANOVA; F3,31 = 0.19, P > 0.05). Lesions of the entorhinal cortex and injections of T H A did not affect the weights of the rats relative to undamaged control rats. It should be noted, however, that all groups were in a water deprived condition that could minimize potential differences by limiting overall consumption. However, lesioned rats in non-deprived conditions tend to eat and drink more than undamaged rats but do not show any weight differences 13. DISCUSSION As previously reported, unilateral and bilateral entorhinal lesions impaired performance on a spatial alternation task 15'18'19'21'23. Rats with unilateral lesions exhibited transitory deficits on the alternation task and recovered to undamaged control level by 15 days of testing. T H A did not enhance recovery after unilateral entorhinal lesions, and may be detrimental in high doses. The inability to perform the alternation task after serial entorhinal lesions was similar to that observed after single stage bilateral entorhinal lesions. There was no significant recovery after the 15 days of testing. Delaying the second lesion until after recovery from the unilateral lesion did not appear to affect (i.e. increase) the rate of recovery. Attempts to improve performance on the alternation task by administration of THA after bilateral lesion were not beneficial. Rats treated with a low dose of T H A (0.05 mg/kg) showed a trend for improvement, relative to the lesioned groups receiving either saline or a high dose of T H A (5.0 mg/kg). However, none of the bilateral lesion groups attained the high level of perfor-
mance maintained by the undamaged control and recovered unilateral lesion groups. Following unilateral entorhinal lesion, the gradual recovery to control levels may be due to neural sprouting and/or compensation by other circuits. The contribution of sprouting to recovery after unilateral entorhinal lesions should be considered with regard to the integrity of the total circuit. Minimal processing of information occurs within the deafferented hippocampus, since the majority of the circuit from cortical areas (e.g. entorhinal cortexperforant path) is no longer available. Hippocampal connections on the damaged side are only supplied via the fimbria-fornix and hippocampal commissure. The intact unilateral entorhinal-hippocampal complex remains fully functional and may significantly contribute to behavioral recovery, with sprouting in the deafferented hippocampus providing a minor but perhaps not insignificant role. Increased input through sprouted connections from the intact entorhinal cortex may contribute to this processing of information. An alternative mechanism for recovery is that other structures or pathways substitute for the neural loss by assuming some of the functions. It is apparent, from the results of this study, that at least a partially intact entorhinal-hippocampal circuit is necessary to perform the alternation task, since the elimination of both entorhinal cortices produces a long-term impairment. Since cholinergic axons also sprout within the hippocampus in response to entorhinal damage 2, and cholinergic neurons are involved in learning and memory (for review see ref. 17), it was thought that the anticholinesterase activity of T H A would augment cholinergic mechanisms active during the recovery phase. However, in the present study, T H A did not appear to enhance recovery and may have been detrimental at high doses. Administration of T H A in doses of 1.25, 2.5 and 5 mg/kg in rats with lesions of the nucleus basalis improves performance in a water maze when the distance swum to locate a platform was measured 14. The high doses of T H A (2.5 and 5 mg/kg) did not enhance performance for latency to find the platform and significantly slowed swim speed. A dose of 5 mg/kg T H A has also been shown to enhance performance on a passive avoidance task after lesion of the nucleus basalis 4. However, lesion of the nucleus basalis does not impair passive avoidance learning 4'14 and the enhanced performance may be due to impaired motor function. Thus, T H A may have beneficial effects; however, they are dose-dependent and higher doses may actually impair performance. The impaired performance of rats recovering from neural damage is significant with respect to the effects the drug may have when applied to human neurodegenerative disorders. The neuropathology of AD produces neural loss in
62 entorhinal cortex similar to the deafferentation that occurs in lesioned rats. As a consequence of this neural
intervention to compensate for loss of neurons in the entorhinal cortex of the rat. A n i m a l studies that involve
loss, similar behavioral impairments may be expected. In
damage to the nucleus basalis and septal areas may be more appropriate for testing the efficacy of T H A in
the early stages of A D , after minimal neural loss, it is possible to improve performance on learning tasks with training (for review see ref. 1), a p h e n o m e n o n similar to the extended training required after unilateral entorhinai
treating A D , since it is from these areas that most cholinergic neurons are lost in A D 3. However, therapeu-
inability to store or recall information as neural damage increases, as in the later stages of A D and after bilateral
tic interventions must at some point directly address the loss of entorhinal n e u r o n s which invariably occurs in A D neuropathology 29. Serial entorhinal lesions provide a viable model for the study of progressive entorhinal
entorhinal lesions. Although A D is associated with neural loss in many regions, the use of localized lesions in animal models has the advantage of simplifying the
damage. Alterations in behavior and neural plasticity, in response to the rate and extent of cell loss, need to be considered to supplement our understanding of the
system to examine the
behavioral consequences of
mechanisms for behavioral recovery. It is possible to
specific lesions. The use of partial lesions in future studies will provide a means to determine if there is a 'threshold',
examine these complex issues using well defined systems, such as the e n t o r h i n a l - h i p p o c a m p a l circuit. Finally, the
or critical n u m b e r of neurons, associated with severe
serial entorhinal paradigm provides one possible starting point for testing potential therapeutic interventions for A D , such as T H A , and devising better treatments for
lesions in rats. Both h u m a n s and rats show a significant
long-term memory impairment. Progressive neurotoxin lesions may offer a viable method for simulating the partial neural loss for given brain regions accompanied by sprouting and other plasticity mechanisms proximal and distal to the primary injury. In summary, T H A does not appear to be an effective REFERENCES 1 Brown, R.G. and Marsden, C.D., Subcortical dementia: the neurophysiological evidence, Neuroscience, 25 (1988) 363-387. 2 Cotman, C.W. and Anderson, K.J., Synaptic plasticity and functional stabilization in the hippocampal formation: possible role in Alzheimer's disease. In S. Waxman (Ed.), Adv. Neurol.: Functional Recovery in Neurological Disease, Raven, New York, 47 (1988) pp. 313-335. 3 Coyle, J.T., Price, D.L. and DeLong, M.R., Alzheimer's Disease: a disorder of cortical cholinergic innervation, Science, 219 (1983) 1184-1190. 4 Dokla, C.P.J., Parker, S.C. and Thai, L.J., Tetrahydroaminoacridine facilitates passive avoidance learning in rats with nucleus basalis magnocellularis lesions, Neuropharmacology, 28 (1989) 1279-1282. 5 Engelhardt, E and Steward, O., Entorhinal cortical lesions in rats and runway alternation performance. Changes in patterns of response initiation, Behav. Neural Biol., 29 (1980) 91-104. 6 Fitten, L.J., Flood, J.F., Baxter, C.E, Tachiki, K.H. and Perryman, K., Long term oral administration of memoryenhancing doses of Tacrine in mice: a study of potential toxicity and side effects, J. Gerontol., 42 (1987) 681-685. 7 Flood, J.F., Smith, G.E. and Cherkin, A., Memory enhancement: supra-additive effect of subcutaneous cholinergic drug combinations in mice, Psychopharmacology, 86 (1985) 61-67. 8 Geddes, J.W., Monaghan, D.T., Cotman, C.W., Lott, I.T., Kim, R.C. and Chui, H.C., Plasticity of hippocampal circuitry in Alzheimer's Disease, Science, 230 (1985) 1179-1181. 9 Gauthier, S., Bouchard, R., Lamontagne, A., Baily, P., Bergman, H., Ratner, J. et al.,Tetrahydroaminoacridine-lecithin combination treatment in patients with intermediate-stage Alzheimer's disease. Results of a Canadian double-blind, crossover, multicenter study, N. Engl. J. Med., 322 (1990) 1272-1276. 10 Hyman, B.T., Van Hoesen, G.W., Damasio, A.R. and Barnes, C.L., Alzheimer's disease: cell-specific pathology isolates the hippocampal formation, Science, 225 (1984) 1168-1170. 11 Karpiak, S.E., Ganglioside treatment improves recovery of
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effects of entorhinal cortex lesions in the albino rat, J. Comp. Physiol. Psych., 85 (1973) 70-81. Scheff, S.W. and Cotman, C.W., Recovery of spontaneous alternation following lesions of the entorhinal cortex in adult rats: possible correlation to axon sprouting, Behav. Biol., 21 (1977) 286-293. Stein, D.G. and Will, B.E., Nerve growth factor produces a temporary facilitation of recovery from entorhinal cortex lesions, Brain Research, 261 (1983) 127-131. Steward, O., Assessing the functional significance of lesioninduced neuronal plasticity, Int. Rev. Neurobiol., 23 (1982) 197-254. Steward, O., Loesche, J. and Horton, W.C., Behavioral correlates of denervation and reinnervation of the hippocampal formation of the rat: open-field activity and cue utilization
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Brain Research, 557 (1991) 57-63 Elsevier Science Publishers B.V. ADONIS 000689939116886T BRES 16886
Effects of tetrahydroaminoacridine (THA) on functional recovery after sequential lesion of the entorhinal cortex J.P. Kesslak, A. Korotzer, A. Song, K. Kamali and C.W. Cotman Department of Psychobiology, University of California, lrvine, lrvine, CA 92717 (U.S.A.) (Accepted 26 March 1991)
Key words: Tetrahydroaminoacridine; Functional recovery; Entorhinal cortex; Serial lesion
Unilateral lesions of rat entorhinai cortex produce a transitory performance deficit on spatial learning tasks, such as reinforced alternation in a T-maze. Tetrahydroaminoacridine (THA), a cholinesterase inhibitor, was administered to determine its effects on behavioral recovery using a reinforced alternation task in a T-maze. Rate of recovery after unilateral entorhinal lesion was not affected by a low dose of THA (0.05 mg/kg), while a higher dose (5.0 mg/kg) impaired recovery. Behavioral recovery was subsequently evaluated in the same rats following lesions to the contralateral entorhinal cortex. Serial bilateral lesions of the entorhinal cortex are known to produce a prolonged performance deficit on the alternation task. The 0.05 mg/kg THA group exhibited an intermediate rate of recovery, between the undamaged control group and bilateral lesion-saline injected groups. The group receiving 5.0 mg/kg of THA after bilateral lesion did not differ from the bilateral lesiun-saline group. The failure of THA to significantly improve functional recovery in rats with lesions of the entorhinal cortex indicates that the compound may have limited applicability in treating human neurodegenerative disorders such as Alzheimer's disease.
INTRODUCTION D a m a g e to rat entorhinal cortex results in learning and m e m o r y i m p a i r m e n t s on a variety of tasks 16'23'25. The deficits o b s e r v e d vary according to the extent of hippoc a m p a l d e n e r v a t i o n and the type of behavioral task. Transient deficits on spatial alternation performance in response to unilateral entorhinal cortex lesions have been r e p o r t e d for the T-maze 15'21'23, Y-maze 2° and H e b b Williams m a z e ~6. M o r e persistent deficits occur on spatial alternation tasks after bilateral entorhinal cortex damage5A2,15,20,22,23,26. T h e rate of behavioral recovery after lesions of the entorhinal cortex can be accelerated by various treatments. A d m i n i s t r a t i o n of gangliosides n'18"19 or nerve growth factor ( N G F 24) increases the rate of recovery on spatial alternation tasks after entorhinal lesions. T h e r e is also evidence that administration of the anticholinesterase t e t r a h y d r o a m i n o a c r i d i n e ( T H A ) m a y improve performance after neural loss, particulary after lesion of the nucleus basalis in rats 4A4. Chronic administration of T H A can i m p r o v e m e m o r y in n o r m a l 7 and age-related m e m o r y i m p a i r e d mice 6. Preliminary clinical results have n o t e d behavioral i m p r o v e m e n t after administration of T H A to patients with A l z h e i m e r ' s Disease ( A D ) 12'27'28, but not in all cases 9. T h e entorhinal cortex experiences
severe neural loss in A D s'l°. This finding is particularly noteworthy since septal cholinergic fibers sprout into the o u t e r molecular layer of the h i p p o c a m p u s after entorhinal lesions in rats 25 and in A D 2'9'10. In the present set of experiments, we have examined the effect of T H A on recovery of p e r f o r m a n c e on a spatial alternation task after unilateral and serial bilateral lesions of the entorhinal cortex in rats. The serial lesion m o d e l was selected to evaluate the effects of progressive neural loss, which also occurs in the course of A D . In the rat, transitory behavioral deficits are seen after m i n o r d a m a g e to the entorhinal cortex (i.e. unilateral lesion), whereas m o r e p r o l o n g e d and severe deficits are p r o d u c e d by increased cell loss (i.e. bilateral lesion) 25. T H A affected recovery after unilateral lesions in a dosed e p e n d e n t m a n n e r , with the smaller dose having no effect and the larger dose having d e t r i m e n t a l effects. A d m i n i s t r a t i o n of T H A after serial bilateral entorhinal lesions did not significantly i m p r o v e p e r f o r m a n c e on the alternation task. MATERIALS AND METHODS
Subjects Male Sprague-Dawley rats (200-225 g), obtained from Charles River, were housed two per cage in a 12/12 h light/dark vivarium. Rats were given ad lib access to Purina Lab Chow. During maze
Correspondence: P. Kesslak, Department of Psychobiology, University of California, Irvine, Irvine, CA 92717, U.S.A.
58 acclimation, pretraining and postsurgery testing the rats were placed on water deprivation. Animals were allowed to drink for 10 min each day beginning at least 30 min after completing the behavioral testing. Body weight was recorded each day of testing. Testing was done during the light phase of the vivarium cycle. Fifty-four rats were initially pretrained (described below) for 5 consecutive days. After pretraining, rats were assigned to experimental groups equated for performance on the alternation task. There were 4 experimental conditions: undamaged receiving saline (CNT; n = 19), unilateral entorhinal lesion receiving saline (SAL; n = 11), unilateral entorhinal lesions receiving 5.0 mg/kg THA (THA5; n = 11), and unilateral entorhinal lesions receiving 0.05 mg/kg THA (THA05; n = 13). All injections were i.p. and administered approximately 30 min before testing. Testing on the spatial task began 2 days after surgery. Following the 15 days of reinforced alternation testing after unilateral entorhinal cortex lesions or sham lesion, the intact contralateral entorhinal cortex was lesioned. The second surgical treatment and alternation testing was continued on 35 male Sprague-Dawley rats. Rats had ad lib access to water for 2 days before the second surgery. After surgery, the rats were given two days for recovery, placed on water deprivation on day three and behavioral testing initiated. Ten rats from the group CNT were randomly chosen to continue in the experiment (n = 10). The contralateral (intact) entorhinal cortex was lesioned in groups that had previously received unilateral lesions (SAL, n = 9; THA5, n = 8; THA05, n = 8). Rats not used in this experiment were randomly chosen for histology to verify lesion placement. The serial bilateral lesion was expected to produce a long-term deficit on the alternation task.
Surgery Rats were anesthetized with Nembutal (54 mg/kg) and placed into a stereotaxic instrument. An incision was made in the scalp exposing the top of the skull and a hole drilled to expose the entorhinal cortex. The head was elevated 10° and the stereotaxic arm set at a 10° angle away from the midline in the coronal plane. There were a total of 9 lesion sites with coordinates from lambda at A/P _+ 0.0, M/L + 3.3, D/V -2.0, 4.0, 6.0; A/P + 0.0, M/L + 4.1, D/V -2.0, 4.0, 6.0; and A/P + 1.0, M/L + 5.1, D/V -2.0, 4.0, 6.0. At each site a no. 2 stainless steel insect pin, insulated except 0.5 mm at the tip, was lowered and an electrolytic lesion made by passing 1 mA current for 45 s. After retracting the electrode, gelfoam was placed over the exposed brain and the scalp sutured. Rats that received unilateral lesions were tested on the alternation task for 15 days. Three days after the alternation test, rats received either lesions of the contralateral entorhinal cortex or sham operations. Rats in the control groups received similar surgical preparation but the electrode was not lowered into the brain. Fifteen days of reinforced alternation was initiated 3 days after the second surgery.
Reinforced-alternation task and group formation Maze acclimation. Each rat was handled for 4 days prior to T-maze acclimation. On the last day of handling the rats were placed on water deprivation and given 10 min access per day. Maze acclimation was conducted for 3 days as follows. On day 1 each rat was placed into the start section of the T-maze and allowed to move freely in the maze for 3 rain, without being removed from the maze. A plastic dish containing 1.5 ml of water was placed at the end of each goal arm and replaced after the rat exited each goal arm. Day 2 was similar to day 1 but after entering the goal arm the rat was allowed to drink, removed from the goal arm, the water replaced and the rat placed in the start arm. Day 3 was similar to day 2 but the gates to the goal arms were closed after the rat entered the reward area. Reinforced alternation. Rats were tested at the same time each day. Each rat was weighed and injected (1 ml/kg) with either 0.9% phosphate buffered saline or THA (Sigma; 5.0 or 0.05 mg/kg) dissolved in phosphate-buffered saline at least 20-30 min before testing. During the 5 days of pretraining all rats received saline
injections. Solutions were prepared daily and coded so that the experimenters had no knowledge of the drugs being injected. The reinforced-alternation task was initiated by placing the rat in the start section of the T-maze, facing the back wall. The animal was then allowed to traverse the maze and enter one of the goal arms. On the first trial of each day the animal was rewarded for entering either arm of the maze. Thereafter, reward was given only if the rat entered the arm opposite the one previously rewarded. After a correct choice the rat received 1.5 rnl of water and allowed 10 s to drink. Testing began 3 days after surgery and lasted for 15 consecutive days. Each rat was tested on 10 consecutive alternation trials per day. If a rat failed to make an alternation choice after 1 min the trial was considered an error. The number of correct choices per day was analyzed with Analysis of Variance and post-hoc tests to determine between group differences.
Histology Rats were sacrificed for verification of lesion placement and size at the completion of behavioral testing. The brains were extracted, placed in dry ice and 25 pm horizontal sections cut using a cryostat (Hacker Instruments). Every 7th and 8th section was taken through the entire brain and stained with Cresyl violet and for acetylcholinesterase (ACHE) respectively.
RESULTS
Histology Brains were examined using light microscopy to determine the extent of the lesion and the presence of cholinergic sprouting in the hippocampus verified from Cresyl violet and AChE stained sections. Examination of the sections indicated that the lesions eliminated the majority of the entorhinal cortex and angular bundle (Fig. 1). The medial and lateral perforant paths were lesioned in all cases, and in a few cases the maximal extent of the lesion impinged on the subiculum with minor involvement of the hippocampus. Loss of the hippocampal afferents from the angular bundle resulted in a dense increase of AChE-positive fibers in the outer molecular layer of the dentate gyrus (Fig. 1). The apparent expansion of the cholinergic fibers into the outer molecular layer verified the loss of entorhinal innervation. There were no apparent differences between brains from saline- and THA-treated animals.
Reinforced-alternation performance Groups receiving unilateral entorhinal lesions without drug treatment showed a transitory deficit on the reinforced-alternation task (Fig. 2). Reinforced alternation performance was close to chance levels (50%) during the first 3 days and recovered to approximately 85% correct by day 15. T H A affected performance on the reinforced alternation task in a dose-dependent manner (Fig. 2). The lower dose of 0.05 mg/kg T H A increased performance slightly above the lesion control group, but this was not significant. Treatment with 5 mg/kg T H A appeared to inhibit recovery and decrease the number of correct responses during the first 6 days of testing after
59 the lesion. P e r f o r m a n c e on the reinforced alternation task was analyzed using a r e p e a t e d measures Analysis of Variance ( A N O V A ) . T h e percent correct alternations for
each day were g r o u p e d into blocks of 3 days as the r e p e a t e d m e a s u r e (i.e. blocks of days 1 - 3 , 4 - 6 , etc.). T h e r e was a significant effect for groups (F3,5o = 8.87, P
Fig. 1. Cresyi violet sections of control animals showed an intact entorhinal cortex (A) (Magnification x20) with an even staining for acetyl cholinesterase in the inner and outer molecular layers of the dentate gyrus (B) (Magnification x150). Lesion of the entorhinal cortex (C) produced a dense increase of cholinergic fibers in the outer molecular layer of the dentate gyrus (D).
60 < 0.001), blocks of days (F4,50 = 41.42, P < 0.001) and group x days interaction (Fz2,z0o = 2.75, P < 0.003). Specific b e t w e e n - g r o u p differences for each block of days were examined post-hoc using a Fisher P L S D ( P < 0.05). C o m p a r e d to CNT, the S A L group had significantly fewer correct alternations during the first 3 blocks of testing (days 1-9). Daily administration of T H A in the T H A 5 group did not facilitate behavioral recovery; in fact they exhibited the poorest performance, scoring significantly worse than C N T on all but the last block of trials. The T H A 0 5 group p e r f o r m e d significantly below the C N T group in the first two blocks of trials and did not differ from C N T for the remaining 12 days of testing. All groups showed significant i m p r o v e m e n t in alternation p e r f o r m a n c e from the first to last blocks of days ( P <
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PLSD (P < 0.05). The SAL group had significantly fewer correct trials than the CNT group across all blocks of trials. The scores of the treatment group with 5 mg/kg T H A (THA5) did not differ from that of the SAL group and had significantly lower scores than the CNT group on all blocks of trials. The group receiving 0.05 mg/kg T H A
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61 (THA05) showed an intermediate level of performance on the alternation task. Group THA05 scored significantly lower than CNT on days 1-6 and 9-12. However, group THA05 did not perform better than group SAL during any of the testing. There appeared to be an overall trend for the bilateral lesioned groups to increase in number of correct alternations with extended training. However, the improved performance of groups SAL and THA5 did not reach the same level of performance as did the undamaged CNT group.
Body weight Body weight for the groups did not differ during any part of the study. During the first 15 days of testing (unilateral lesion) there was a consistent increase in body weight across the days of testing (Fig. 4). The CNT group consistently weighed more than the other groups but this difference was not significant (ANOVA; F3,50 = 0.348, P > 0.05). During the second testing period (bilateral lesion) there was a consistent increase in weight across days of testing (Fig. 4), with no differences between groups (ANOVA; F3,31 = 0.19, P > 0.05). Lesions of the entorhinal cortex and injections of T H A did not affect the weights of the rats relative to undamaged control rats. It should be noted, however, that all groups were in a water deprived condition that could minimize potential differences by limiting overall consumption. However, lesioned rats in non-deprived conditions tend to eat and drink more than undamaged rats but do not show any weight differences 13. DISCUSSION As previously reported, unilateral and bilateral entorhinal lesions impaired performance on a spatial alternation task 15'18'19'21'23. Rats with unilateral lesions exhibited transitory deficits on the alternation task and recovered to undamaged control level by 15 days of testing. T H A did not enhance recovery after unilateral entorhinal lesions, and may be detrimental in high doses. The inability to perform the alternation task after serial entorhinal lesions was similar to that observed after single stage bilateral entorhinal lesions. There was no significant recovery after the 15 days of testing. Delaying the second lesion until after recovery from the unilateral lesion did not appear to affect (i.e. increase) the rate of recovery. Attempts to improve performance on the alternation task by administration of THA after bilateral lesion were not beneficial. Rats treated with a low dose of T H A (0.05 mg/kg) showed a trend for improvement, relative to the lesioned groups receiving either saline or a high dose of T H A (5.0 mg/kg). However, none of the bilateral lesion groups attained the high level of perfor-
mance maintained by the undamaged control and recovered unilateral lesion groups. Following unilateral entorhinal lesion, the gradual recovery to control levels may be due to neural sprouting and/or compensation by other circuits. The contribution of sprouting to recovery after unilateral entorhinal lesions should be considered with regard to the integrity of the total circuit. Minimal processing of information occurs within the deafferented hippocampus, since the majority of the circuit from cortical areas (e.g. entorhinal cortexperforant path) is no longer available. Hippocampal connections on the damaged side are only supplied via the fimbria-fornix and hippocampal commissure. The intact unilateral entorhinal-hippocampal complex remains fully functional and may significantly contribute to behavioral recovery, with sprouting in the deafferented hippocampus providing a minor but perhaps not insignificant role. Increased input through sprouted connections from the intact entorhinal cortex may contribute to this processing of information. An alternative mechanism for recovery is that other structures or pathways substitute for the neural loss by assuming some of the functions. It is apparent, from the results of this study, that at least a partially intact entorhinal-hippocampal circuit is necessary to perform the alternation task, since the elimination of both entorhinal cortices produces a long-term impairment. Since cholinergic axons also sprout within the hippocampus in response to entorhinal damage 2, and cholinergic neurons are involved in learning and memory (for review see ref. 17), it was thought that the anticholinesterase activity of T H A would augment cholinergic mechanisms active during the recovery phase. However, in the present study, T H A did not appear to enhance recovery and may have been detrimental at high doses. Administration of T H A in doses of 1.25, 2.5 and 5 mg/kg in rats with lesions of the nucleus basalis improves performance in a water maze when the distance swum to locate a platform was measured 14. The high doses of T H A (2.5 and 5 mg/kg) did not enhance performance for latency to find the platform and significantly slowed swim speed. A dose of 5 mg/kg T H A has also been shown to enhance performance on a passive avoidance task after lesion of the nucleus basalis 4. However, lesion of the nucleus basalis does not impair passive avoidance learning 4'14 and the enhanced performance may be due to impaired motor function. Thus, T H A may have beneficial effects; however, they are dose-dependent and higher doses may actually impair performance. The impaired performance of rats recovering from neural damage is significant with respect to the effects the drug may have when applied to human neurodegenerative disorders. The neuropathology of AD produces neural loss in
62 entorhinal cortex similar to the deafferentation that occurs in lesioned rats. As a consequence of this neural
intervention to compensate for loss of neurons in the entorhinal cortex of the rat. A n i m a l studies that involve
loss, similar behavioral impairments may be expected. In
damage to the nucleus basalis and septal areas may be more appropriate for testing the efficacy of T H A in
the early stages of A D , after minimal neural loss, it is possible to improve performance on learning tasks with training (for review see ref. 1), a p h e n o m e n o n similar to the extended training required after unilateral entorhinai
treating A D , since it is from these areas that most cholinergic neurons are lost in A D 3. However, therapeu-
inability to store or recall information as neural damage increases, as in the later stages of A D and after bilateral
tic interventions must at some point directly address the loss of entorhinal n e u r o n s which invariably occurs in A D neuropathology 29. Serial entorhinal lesions provide a viable model for the study of progressive entorhinal
entorhinal lesions. Although A D is associated with neural loss in many regions, the use of localized lesions in animal models has the advantage of simplifying the
damage. Alterations in behavior and neural plasticity, in response to the rate and extent of cell loss, need to be considered to supplement our understanding of the
system to examine the
behavioral consequences of
mechanisms for behavioral recovery. It is possible to
specific lesions. The use of partial lesions in future studies will provide a means to determine if there is a 'threshold',
examine these complex issues using well defined systems, such as the e n t o r h i n a l - h i p p o c a m p a l circuit. Finally, the
or critical n u m b e r of neurons, associated with severe
serial entorhinal paradigm provides one possible starting point for testing potential therapeutic interventions for A D , such as T H A , and devising better treatments for
lesions in rats. Both h u m a n s and rats show a significant
long-term memory impairment. Progressive neurotoxin lesions may offer a viable method for simulating the partial neural loss for given brain regions accompanied by sprouting and other plasticity mechanisms proximal and distal to the primary injury. In summary, T H A does not appear to be an effective REFERENCES 1 Brown, R.G. and Marsden, C.D., Subcortical dementia: the neurophysiological evidence, Neuroscience, 25 (1988) 363-387. 2 Cotman, C.W. and Anderson, K.J., Synaptic plasticity and functional stabilization in the hippocampal formation: possible role in Alzheimer's disease. In S. Waxman (Ed.), Adv. Neurol.: Functional Recovery in Neurological Disease, Raven, New York, 47 (1988) pp. 313-335. 3 Coyle, J.T., Price, D.L. and DeLong, M.R., Alzheimer's Disease: a disorder of cortical cholinergic innervation, Science, 219 (1983) 1184-1190. 4 Dokla, C.P.J., Parker, S.C. and Thai, L.J., Tetrahydroaminoacridine facilitates passive avoidance learning in rats with nucleus basalis magnocellularis lesions, Neuropharmacology, 28 (1989) 1279-1282. 5 Engelhardt, E and Steward, O., Entorhinal cortical lesions in rats and runway alternation performance. Changes in patterns of response initiation, Behav. Neural Biol., 29 (1980) 91-104. 6 Fitten, L.J., Flood, J.F., Baxter, C.E, Tachiki, K.H. and Perryman, K., Long term oral administration of memoryenhancing doses of Tacrine in mice: a study of potential toxicity and side effects, J. Gerontol., 42 (1987) 681-685. 7 Flood, J.F., Smith, G.E. and Cherkin, A., Memory enhancement: supra-additive effect of subcutaneous cholinergic drug combinations in mice, Psychopharmacology, 86 (1985) 61-67. 8 Geddes, J.W., Monaghan, D.T., Cotman, C.W., Lott, I.T., Kim, R.C. and Chui, H.C., Plasticity of hippocampal circuitry in Alzheimer's Disease, Science, 230 (1985) 1179-1181. 9 Gauthier, S., Bouchard, R., Lamontagne, A., Baily, P., Bergman, H., Ratner, J. et al.,Tetrahydroaminoacridine-lecithin combination treatment in patients with intermediate-stage Alzheimer's disease. Results of a Canadian double-blind, crossover, multicenter study, N. Engl. J. Med., 322 (1990) 1272-1276. 10 Hyman, B.T., Van Hoesen, G.W., Damasio, A.R. and Barnes, C.L., Alzheimer's disease: cell-specific pathology isolates the hippocampal formation, Science, 225 (1984) 1168-1170. 11 Karpiak, S.E., Ganglioside treatment improves recovery of
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