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
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
