BEHAVIORAL AND NEURAL BIOLOGY 58, 72--75 (1992)
BRIEF REPORT Chronic Sodium Azide Treatment Impairs Learning of the Morris Water Maze Task M. CATHERINE BENNETT AND GREGORY M. ROSE1 Medical Research Service, Veterans Administration Medical Center; and Department of Pharmacology (C-236) and Neuroscience Training Program, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, Colorado 80262
terminal enzyme of the electron transport (respiratory) chain, was profoundly decreased in mitochondria isolated from blood platelets of a population of AD patients. Other enzymes of the electron transport chain were not significantly affected. Blood platelets are not known to be a target tissue of AD damage, which suggests that the cytochrome oxidase deficiency reflects a systemic, rather than tissue-specific, defect. In complementary work, a reduction of cytochrome oxidase has been reported in temporal cortex of post-mortem AD brain tissue (Nobrega, DiStefano, & Kish, 1991). In a previous investigation, the chronic and selective inhibition of cytochrome oxidase in rats by sodium azide impaired both hippocampal plasticity and hippocampal-dependent spatial learning in an 8-arm radial maze task (Bennett, Diamond, Stryker, & Parker, Jr., 1992). In the present experiment, we have extended the investigation of the effects of chronic azide t r e a t m e n t on learning and memory using the Morris water maze task (Morris, 1984). Like the radial maze, the Morris water maze is a test of spatial learning; in addition, performance on this task is sensitive to hippocampal damage (Sutherland, Whishaw, & Kolb, 1983). We now report that chronic azide treatment impairs both acquisition and retention of this task. Subjects were adult male S p r a g u e - D a w l e y rats (Harlan Laboratories) which weighed 350-400 g at the time of surgery. The rats were group housed in an AAALAC approved facility under standard laboratory conditions (light:dark, 12:12), with food and water available ad libitum. Under secobarbital anesthesia (50 mg/kg), each rat had an Alzet 2ML4 osmotic minipump implanted subcutaneously. For this implantation, an incision of approximately 1
A reduction in the activity of cytochrome oxidase, a respiratory chain enzyme, has been recently identified in mitochondria from blood platelets and postmortem brain tissue from Atzheimer's disease (AD) patients. We have developed an animal model of this deficit in rats by chronic subcutaneous infusion of sodium azide, a selective inhibitor of cytochrome oxidase, delivered via Alzet 2ML4 osmotic minipumps. In previous work, azide-treated rats were impaired in an appetitively motivated spatial learning task, the radial arm maze. In the present investigation, we tested male Sprague-Dawley rats (350-400 g), which were tonically infused with azide or saline, on an aversively motivated spatial task, the Morris water maze. Azide-treated rats were impaired on both acquisition and retention of this task, without showing evidence of a motor impairment. Thus, the present results are consistent with previous findings showing that chronic azide treatment produces a learning and memory deficit. These findings strengthen the hypothesis that azide treatment in rats produces a useful animal model of some aspects of AD. ©1992 Academic Press, Inc. There is a growing body of evidence that mitochrondrial defects are concomitants of aging (Bandy & Davison, 1990) and numerous neurodegenerative diseases, including Alzheimer's disease (AD) (Blass, Sheu, & Cedarbaum, 1988). Recently, Parker, Fill e y , & Parks (1990) identified a specific site for a mitochondrial defect in AD. These investigators found that the activity of cytochrome oxidase, the 1 This research was supported by grants from the National Institute on Aging (AG10755-01) and the Veterans Administration Medical Research Service. We t h a n k Aaron H u m p h r i e s and Andrew Carrier for t h e i r technical assistance. Correspondence and reprint requests should be addressed to Dr. M. C. Bennett, Dept. of Pharmacology, Box C-236, UCHSC, 4200 East N i n t h Ave., Denver, CO 80262. 72 0163-1047/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
AZIDE TREATMENT IMPAIRS LEARNING TASKS
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FIG. 1. Azide t r e a t m e n t significantly impaired l e a r n i n g of the location of the hidden platform in the Morris water maze (p < .001). Each data point represents the group means ( _+SEM) for four daily trials (saline: n = 11; Azide: n = 11). Performance on the first day of t r a i n i n g was not different between groups.
cm was made in the nape of the neck and the skin was retracted from the muscle and fascia to make a pocket of approximately 1 × 3 cm into which a minipump was inserted. These minipumps have a 2 ml reservoir and provide a constant infusion rate of 2.5 t~l/hr for 28 days. The pumps were filled with either a solution of 160 tLg//zl sodium azide in 0.9% saline (Azide) or the saline vehicle (Control). The Azide pumps delivered a dose of sodium azide equal to 400 ttg/hr. Infusions began 7-21 days prior to the beginning of behavioral testing. Sodium azide crystals of 99% purity were obtained from Aldrich (Milwaukee, WI). Azide (n = 11) and Control (n = 11) rats were trained in a water tank, according to a modification "6 4
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FIG. 2. Azide treatment resulted in poorer performance during a probe t r i a l of retention (p < .02). The bar graph shows the group means for the number of times the r a t swam over the
place where the hidden platform had been located. The 60-s probe trial was given on the 8th day of testing.
73
of the Morris water maze protocol (Morris, 1984). Two Azide rats and three Control rats were eliminated during the testing procedure due to acute infections at the implantation site which were aggravated by the water immersion. Individual rats were given four daily trials of 60 s duration each for 7 consecutive days. A trial consisted of an individual rat being placed into one quadrant of the t a n k filled with water (24-25°C) made opaque by the addition of white Createx, a nontoxic paint. The order of initial quadrant placement was randomized and changed daily, but the same order was used for all subjects on a given day. The experimenter was blind to the t r e a t m e n t of the rats. The task was for the rat to learn the location of a submerged clear platform (hidden from the rat) that occupied a fixed position relative to conspicuous extramaze cues throughout the testing period. The measure of performance was the swim-time (in seconds) to reach the platform. If a rat did not find the hidden platform within 60 s, it was guided to the platform and was given a score of 60 s. All subjects were allowed to remain on the platform for 15 s between trials, regardless of swim-time. On the eighth day of testing, each rat was given a single 60-s probe trial in the water tank with the platform removed. Each rat was placed individually into the water t a n k beginning in the quadrant adjacent to that previously occupied by the platform. The probe test provided a measure of retention, which was determined by counting the number of crossings over the site where the platform had been located. Group performances for the 7 days of training, which reflect the mean swim-times of the daily 4trial blocks, are shown in Fig. 1. The Azide group exhibited overall poorer performance across trials [repeated measures MANOVA main effect of treatment: F(1, 20) = 32.0; p < .001]. First-day performances did not differ significantly between groups (t = 1.00; p > .1, NS). Thus, there was no significant azide-induced deficit on the first day of training. There was a significant effect of time [repeated measures MANOVA main effect of time: F(27,540) = 7.32; p < .001] but no significant interaction [F(27, 540) = 0.95; p > .1, NS]. This analysis reflects the finding that both groups showed significant decreases in swim-times over trials, indicating that both groups exhibited learning during training. The mean number of crossings made by each group over the position that the hidden platform had occupied during the 7 days of training is shown in Fig. 2. The Azide rats made significantly fewer crossings over the position formerly occupied by the
74
BENNETT AND ROSE
platform than did the Control rats (Student's independent t = 2.81, d f = 20; p < .02). This finding suggests that the Azide rats had poorer memory for the former position of the hidden platform than did the Control group. The method of subcutaneous infusion of sodium azide reported in this study results in a 35-39% decrease in cytochrome oxidase activity in mitochondria isolated from soma and synaptic membrane of treated rat brain (Bennett et al., 1992). This level of inhibition has been found previously to be the maximum that could be achieved by chronic systemic azide infusion without also affecting other electron transport enzymes (M. C. Bennett, J. K. Parks, Y. H. Lim, & W. D. Parker, unpublished findings). The activity of cytochrome oxidase found in mitochrondria extracted from AD patients platelets averaged 50% of the age-matched controls (Parker et al., 1990). Thus, the present sodium azide treatment in rats produces a less complete inhibition of cytochrome oxidase than has been reported in AD and accordingly represents a conservative experimental design. In the present investigation, chronic azide treatment impaired both acquisition and retention of an aversively motivated spatial task, the Morris water maze. This finding is consistent with the previous report that chronic azide treatment impaired the acquisition of an appetitively motivated spatial task, the radial arm maze. This result, however, differed from the previous behavioral finding in one respect. In the present investigation, performances of the Control and Azide groups were comparable on the first day of training, then diverged and remained significantly different to the end of the training period. The Azide group was significantly impaired also on a probe trial of retention conducted on the day after training was completed. In the previous study, by contrast, azide treatment impaired only the acquisition of the radial maze task; Azide and Control groups reached asymptote performance levels that did not differ significantly. However, in the present experiment, it is possible that with extended training the performance of the Azide group would have reached the level of the Control group. It is unlikely that azide treatment induced a motor impairment that would account for the performance deficit of the azide-treated group. First day swim-times did not differ significantly between the Azide and the Control groups, suggesting that azide treatment did not impair swimming ability, per se. Moreover, in previous work using the same level of azide exposure as was employed in the present ex-
periment, spontaneous activity in an open field was not affected (Bennett et al., 1992). The finding of an azide-induced learning deficit is consistent with other data that suggest metabolic factors influence cognitive processes. For example, adrenalectomized rats exhibit a transient decrease in basal blood glucose levels (Hall & Gold, 1990). This condition is associated with a memory deficit which can be ameliorated by systemic administration of glucose. A decrease in resting glucose levels may produce bioenergetic consequences similar to those found with electron transport chain inhibition. In related work, perturbations of glucose regulation have been shown to predict memory deficits both in aged rats (Stone, Wenk, Olton, & Gold, 1990) and in elderly humans (Hall, Gonder-Frederick, Chewning, Silveira, & Gold, 1989). Although the mechanism by which an inhibition of bioenergetic metabolism might impair learning and memory is unknown, we have considered several possibilities. First, azide treatment, by its inhibition of cytochrome oxidase, reduces the efficiency of the high energy electron transport system in the mitochrondria. One prediction of decreased energy stores is a generalized decline in cellular function and increased vulnerability of neurons to insult (Armanini, Hutchins, Stein, & Sapolsky, 1990). A second effect of inhibiting the respiratory chain is to force an equilibrium shift from the oxidized NAD + state to the reduced NADH state, the latter species being an electron donor for the respiratory chain. This equilibrium shift could be predicted to have widespread consequences for NAD ÷linked enzymes, resulting in, for example, decreased synthesis of both acetylcholine (ACh) (Gibson & Blass, 1975) and acetyl CoA (Perry, Perry, Tomlinson, Blessed, & Gibson, 1980). A profound reduction in ACh is a well-described concomitant of AD and may partially underlie the learning and memory deficits which are a hallmark of this disease (Whitehouse, Price, Clark, Coyle, & Delong, 1981). A third consequence of inhibiting mitochondrial respiration is the diversion of high energy electrons from the electron transport chain, leading to cytotoxic free radical generation. Oxidant damage is a proposed mechanism of neurodegeneration in AD (Halliwell, 1989). Finally, conditions of hypoxia, ischemia, and hypoglycemia are also associated with increased release of the excitatory amino acids glutamate and aspartate (Pelligrini-Giampietro, Cherici, Alesiani, Carla, & Moroni, 1990). Thus, an excitotoxic component may contribute to an azide-induced behavioral impairment. In future work, we plan to analyze the contri-
AZIDE TREATMENT IMPAIRS LEARNING TASKS b u t i o n of e a c h of t h e s e p u t a t i v e m e c h a n i s m s of azide-induced l e a r n i n g deficits in r a t s , a n d t h e t i m e course w i t h i n w h i c h d a m a g e f r o m e a c h m i g h t occur. I t is l i k e l y t h a t all of t h e a b o v e - m e n t i o n e d m e c h a n i s m s c a n c o n t r i b u t e to a z i d e - i n d u c e d l e a r n i n g deficits, a l t h o u g h w h e t h e r all of t h e m p l a y a role in the behavioral dysfunction seen after a relatively ]brief d i s r u p t i o n of e n e r g y m e t a b o l i s m is not k n o w n . [n t h i s r e g a r d , it w o u l d be of i n t e r e s t to d e t e r m i n e 'whether p r o l o n g e d azide t r e a t m e n t p r o d u c e s beh a v i o r a l deficits t h a t w o r s e n o v e r t i m e . I n s u m m a r y , chronic azide t r e a t m e n t i m p a i r s acquisition a n d r e t e n t i o n p e r f o r m a n c e on t h e M o r r i s 'water m a z e t a s k . T h i s finding is c o n s i s t e n t w i t h o u r p r e v i o u s r e p o r t of a n a z i d e - i n d u c e d deficit on o t h e r b e h a v i o r a l t a s k s ( B e n n e t t et al., 1992). T h e p r e s e n t finding d e m o n s t r a t e s t h a t c y t o c h r o m e oxidase inh i b i t i o n in r o d e n t s m i m i c s s o m e a s p e c t s of t h e l e a r n ing a n d m e m o r y deficit a s s o c i a t e d w i t h AD. W e sugg e s t t h a t a useful a n i m a l m o d e l of a h u m a n d i s e a s e m u s t m e e t two criteria: (1) T h e e x p e r i m e n t a l m a n i p u l a t i o n r e p r o d u c e s a condition w h i c h is associa t e d w i t h t h e n a t u r a l disease; a n d (2) t h e specific m a n i p u l a t i o n of a single clinical c o n c o m i t a n t m i m ics s o m e aspects of t h e p a t h o l o g y of t h e disease. T h e p r e s e n t results, in concert w i t h o u r p r e v i o u s findings, s u p p o r t t h e h y p o t h e s i s t h a t chronic cytoc h r o m e oxidase i n h i b i t i o n b y tonic i n f u s i o n of sod i u m azide p r o d u c e s a p a t h o l o g y in r o d e n t s w h i c h :models s o m e a s p e c t s of AD. REFERENCES Armanini, M. P., Hutchins, C., Stein, B. A., & Sapolsky, R. M. (1990). Glucocorticoid endangerment of hippocampal neurons is NMDA-receptor dependent. Brain Research, 532, 712. ]Bandy, B., & Davison, A. J. (1990). Mitochondrial mutations may increase oxidative stress: Implications for carcinogenesis and aging? Free Radical Biology and Medicine, 8, 523-539. Bennett, M. C., Diamond, D. M., Stryker, S. L., & Parker, W. D., Jr. (1992). Cytochrome oxidase inhibition: A novel animal model of Alzheimer's disease. Journal of Geriatric Psychiatry and Neurology, 5, 93-101.
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Blass, J. P., Sheu, R. K-F, & Cedarbaum, J. M. (1988). Energy metabolism in disorders of the nervous system. Revue Neurologique, 144(10), 543-563. Gibson, G. E., & Blass, J. P. (1975). Decreased synthesis of acetylcholine accompanying impaired oxidation of pyruvic acid in rat brain minces. Biochemistry Journal, 148, 17-23. Hall, J. L., & Gold, P. E. (1990). Adrenalectomy-induced memory deficits: role of plasma glucose levels. Physiology and Behawor, 47, 27-33. Hall, J. L., Gonder-Frederick, C. A., Chewning, W. W., Silveira, J., & Gold, P. E. (1989). Glucose enhancement of performance of memory tests in young and aged humans. Neuropsychologia, 27, 1129-1138. Halliwell, B. (1989). Oxidants in the central nervous system: Some fundamental questions. Is oxidant damage relevant to Parkinson's disease, Alzheimer~s disease, traumatic damage or stroke? Acta Neurologzca Scandanavica Supplement, 126, 23-33. Morris, R. G. M. (1984). Developments of a water-maze procedure for studying spatial learning in the rat. Journal of Neuroscience Methods, 11, 47-60. Nobrega, J. N., DiStefano, L., & Kish, S. J. (1991). Brain cytochrome oxidase activity in Alzheimer's disease. Third IBRO World Congress of Neuroscience, 3, 91. Parker, W. D., Jr., Filley, C. M., & Parks, J. K. (1990). Cytochrome oxidase deficiency in Alzheimer's disease. Neurology, 40, 1302-1303. Pelligrini-Giamipetro, D. E., Cherici, G., Alesiani, M., Carla, V., & Moroni, F. (1990). Excitatory amino acid release and free radical formation may cooperate in the genesis of ischemiainduced neuronal damage. Journal of Neuroscience, 10, 1035-1041. Perry, E. K., Perry, R. H., Tomlinson, B. E., Blessed, G., & Gibson, P. H. (1980). Coenzyme A-acetylating enzymes in Alzheimer's disease: Possible cholinergic "compartment" of pyruvate dehydrogenase. Neurosczence Letters, 18, 105-110. Stone, W. S., Wenk, G. L., Olton, D. S., & Gold, P. E. (1990). Poor blood glucose regulation predicts sleep and memory deficits in normal aged rats. Journal of Gerontology, 45, 169173. Sutherland, R. J., Whishaw, I. Q, & Kolb, B. (1983). A behavioral analysis of spatial localization following electrolytic, kainate- or colchicine-induced damage to the hippocampal formation in the rat. Behavioral Brain Research, 7, 133-152. Whitehouse, P. J., Price, D. L., Clark, A. W., Coyle, J. T., & DeLong, M. R. (1981). Alzheimer's disease: Evidence for selective loss of cholinergic neurons in the nucleus basalis. Annals of Neurology, 10, 122-126.
BRIEF REPORT Chronic Sodium Azide Treatment Impairs Learning of the Morris Water Maze Task M. CATHERINE BENNETT AND GREGORY M. ROSE1 Medical Research Service, Veterans Administration Medical Center; and Department of Pharmacology (C-236) and Neuroscience Training Program, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, Colorado 80262
terminal enzyme of the electron transport (respiratory) chain, was profoundly decreased in mitochondria isolated from blood platelets of a population of AD patients. Other enzymes of the electron transport chain were not significantly affected. Blood platelets are not known to be a target tissue of AD damage, which suggests that the cytochrome oxidase deficiency reflects a systemic, rather than tissue-specific, defect. In complementary work, a reduction of cytochrome oxidase has been reported in temporal cortex of post-mortem AD brain tissue (Nobrega, DiStefano, & Kish, 1991). In a previous investigation, the chronic and selective inhibition of cytochrome oxidase in rats by sodium azide impaired both hippocampal plasticity and hippocampal-dependent spatial learning in an 8-arm radial maze task (Bennett, Diamond, Stryker, & Parker, Jr., 1992). In the present experiment, we have extended the investigation of the effects of chronic azide t r e a t m e n t on learning and memory using the Morris water maze task (Morris, 1984). Like the radial maze, the Morris water maze is a test of spatial learning; in addition, performance on this task is sensitive to hippocampal damage (Sutherland, Whishaw, & Kolb, 1983). We now report that chronic azide treatment impairs both acquisition and retention of this task. Subjects were adult male S p r a g u e - D a w l e y rats (Harlan Laboratories) which weighed 350-400 g at the time of surgery. The rats were group housed in an AAALAC approved facility under standard laboratory conditions (light:dark, 12:12), with food and water available ad libitum. Under secobarbital anesthesia (50 mg/kg), each rat had an Alzet 2ML4 osmotic minipump implanted subcutaneously. For this implantation, an incision of approximately 1
A reduction in the activity of cytochrome oxidase, a respiratory chain enzyme, has been recently identified in mitochondria from blood platelets and postmortem brain tissue from Atzheimer's disease (AD) patients. We have developed an animal model of this deficit in rats by chronic subcutaneous infusion of sodium azide, a selective inhibitor of cytochrome oxidase, delivered via Alzet 2ML4 osmotic minipumps. In previous work, azide-treated rats were impaired in an appetitively motivated spatial learning task, the radial arm maze. In the present investigation, we tested male Sprague-Dawley rats (350-400 g), which were tonically infused with azide or saline, on an aversively motivated spatial task, the Morris water maze. Azide-treated rats were impaired on both acquisition and retention of this task, without showing evidence of a motor impairment. Thus, the present results are consistent with previous findings showing that chronic azide treatment produces a learning and memory deficit. These findings strengthen the hypothesis that azide treatment in rats produces a useful animal model of some aspects of AD. ©1992 Academic Press, Inc. There is a growing body of evidence that mitochrondrial defects are concomitants of aging (Bandy & Davison, 1990) and numerous neurodegenerative diseases, including Alzheimer's disease (AD) (Blass, Sheu, & Cedarbaum, 1988). Recently, Parker, Fill e y , & Parks (1990) identified a specific site for a mitochondrial defect in AD. These investigators found that the activity of cytochrome oxidase, the 1 This research was supported by grants from the National Institute on Aging (AG10755-01) and the Veterans Administration Medical Research Service. We t h a n k Aaron H u m p h r i e s and Andrew Carrier for t h e i r technical assistance. Correspondence and reprint requests should be addressed to Dr. M. C. Bennett, Dept. of Pharmacology, Box C-236, UCHSC, 4200 East N i n t h Ave., Denver, CO 80262. 72 0163-1047/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
AZIDE TREATMENT IMPAIRS LEARNING TASKS
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AZIDE
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TRAINING
FIG. 1. Azide t r e a t m e n t significantly impaired l e a r n i n g of the location of the hidden platform in the Morris water maze (p < .001). Each data point represents the group means ( _+SEM) for four daily trials (saline: n = 11; Azide: n = 11). Performance on the first day of t r a i n i n g was not different between groups.
cm was made in the nape of the neck and the skin was retracted from the muscle and fascia to make a pocket of approximately 1 × 3 cm into which a minipump was inserted. These minipumps have a 2 ml reservoir and provide a constant infusion rate of 2.5 t~l/hr for 28 days. The pumps were filled with either a solution of 160 tLg//zl sodium azide in 0.9% saline (Azide) or the saline vehicle (Control). The Azide pumps delivered a dose of sodium azide equal to 400 ttg/hr. Infusions began 7-21 days prior to the beginning of behavioral testing. Sodium azide crystals of 99% purity were obtained from Aldrich (Milwaukee, WI). Azide (n = 11) and Control (n = 11) rats were trained in a water tank, according to a modification "6 4
0
3 0 ¢0
0
T
2
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0
1
n." W I11
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FIG. 2. Azide treatment resulted in poorer performance during a probe t r i a l of retention (p < .02). The bar graph shows the group means for the number of times the r a t swam over the
place where the hidden platform had been located. The 60-s probe trial was given on the 8th day of testing.
73
of the Morris water maze protocol (Morris, 1984). Two Azide rats and three Control rats were eliminated during the testing procedure due to acute infections at the implantation site which were aggravated by the water immersion. Individual rats were given four daily trials of 60 s duration each for 7 consecutive days. A trial consisted of an individual rat being placed into one quadrant of the t a n k filled with water (24-25°C) made opaque by the addition of white Createx, a nontoxic paint. The order of initial quadrant placement was randomized and changed daily, but the same order was used for all subjects on a given day. The experimenter was blind to the t r e a t m e n t of the rats. The task was for the rat to learn the location of a submerged clear platform (hidden from the rat) that occupied a fixed position relative to conspicuous extramaze cues throughout the testing period. The measure of performance was the swim-time (in seconds) to reach the platform. If a rat did not find the hidden platform within 60 s, it was guided to the platform and was given a score of 60 s. All subjects were allowed to remain on the platform for 15 s between trials, regardless of swim-time. On the eighth day of testing, each rat was given a single 60-s probe trial in the water tank with the platform removed. Each rat was placed individually into the water t a n k beginning in the quadrant adjacent to that previously occupied by the platform. The probe test provided a measure of retention, which was determined by counting the number of crossings over the site where the platform had been located. Group performances for the 7 days of training, which reflect the mean swim-times of the daily 4trial blocks, are shown in Fig. 1. The Azide group exhibited overall poorer performance across trials [repeated measures MANOVA main effect of treatment: F(1, 20) = 32.0; p < .001]. First-day performances did not differ significantly between groups (t = 1.00; p > .1, NS). Thus, there was no significant azide-induced deficit on the first day of training. There was a significant effect of time [repeated measures MANOVA main effect of time: F(27,540) = 7.32; p < .001] but no significant interaction [F(27, 540) = 0.95; p > .1, NS]. This analysis reflects the finding that both groups showed significant decreases in swim-times over trials, indicating that both groups exhibited learning during training. The mean number of crossings made by each group over the position that the hidden platform had occupied during the 7 days of training is shown in Fig. 2. The Azide rats made significantly fewer crossings over the position formerly occupied by the
74
BENNETT AND ROSE
platform than did the Control rats (Student's independent t = 2.81, d f = 20; p < .02). This finding suggests that the Azide rats had poorer memory for the former position of the hidden platform than did the Control group. The method of subcutaneous infusion of sodium azide reported in this study results in a 35-39% decrease in cytochrome oxidase activity in mitochondria isolated from soma and synaptic membrane of treated rat brain (Bennett et al., 1992). This level of inhibition has been found previously to be the maximum that could be achieved by chronic systemic azide infusion without also affecting other electron transport enzymes (M. C. Bennett, J. K. Parks, Y. H. Lim, & W. D. Parker, unpublished findings). The activity of cytochrome oxidase found in mitochrondria extracted from AD patients platelets averaged 50% of the age-matched controls (Parker et al., 1990). Thus, the present sodium azide treatment in rats produces a less complete inhibition of cytochrome oxidase than has been reported in AD and accordingly represents a conservative experimental design. In the present investigation, chronic azide treatment impaired both acquisition and retention of an aversively motivated spatial task, the Morris water maze. This finding is consistent with the previous report that chronic azide treatment impaired the acquisition of an appetitively motivated spatial task, the radial arm maze. This result, however, differed from the previous behavioral finding in one respect. In the present investigation, performances of the Control and Azide groups were comparable on the first day of training, then diverged and remained significantly different to the end of the training period. The Azide group was significantly impaired also on a probe trial of retention conducted on the day after training was completed. In the previous study, by contrast, azide treatment impaired only the acquisition of the radial maze task; Azide and Control groups reached asymptote performance levels that did not differ significantly. However, in the present experiment, it is possible that with extended training the performance of the Azide group would have reached the level of the Control group. It is unlikely that azide treatment induced a motor impairment that would account for the performance deficit of the azide-treated group. First day swim-times did not differ significantly between the Azide and the Control groups, suggesting that azide treatment did not impair swimming ability, per se. Moreover, in previous work using the same level of azide exposure as was employed in the present ex-
periment, spontaneous activity in an open field was not affected (Bennett et al., 1992). The finding of an azide-induced learning deficit is consistent with other data that suggest metabolic factors influence cognitive processes. For example, adrenalectomized rats exhibit a transient decrease in basal blood glucose levels (Hall & Gold, 1990). This condition is associated with a memory deficit which can be ameliorated by systemic administration of glucose. A decrease in resting glucose levels may produce bioenergetic consequences similar to those found with electron transport chain inhibition. In related work, perturbations of glucose regulation have been shown to predict memory deficits both in aged rats (Stone, Wenk, Olton, & Gold, 1990) and in elderly humans (Hall, Gonder-Frederick, Chewning, Silveira, & Gold, 1989). Although the mechanism by which an inhibition of bioenergetic metabolism might impair learning and memory is unknown, we have considered several possibilities. First, azide treatment, by its inhibition of cytochrome oxidase, reduces the efficiency of the high energy electron transport system in the mitochrondria. One prediction of decreased energy stores is a generalized decline in cellular function and increased vulnerability of neurons to insult (Armanini, Hutchins, Stein, & Sapolsky, 1990). A second effect of inhibiting the respiratory chain is to force an equilibrium shift from the oxidized NAD + state to the reduced NADH state, the latter species being an electron donor for the respiratory chain. This equilibrium shift could be predicted to have widespread consequences for NAD ÷linked enzymes, resulting in, for example, decreased synthesis of both acetylcholine (ACh) (Gibson & Blass, 1975) and acetyl CoA (Perry, Perry, Tomlinson, Blessed, & Gibson, 1980). A profound reduction in ACh is a well-described concomitant of AD and may partially underlie the learning and memory deficits which are a hallmark of this disease (Whitehouse, Price, Clark, Coyle, & Delong, 1981). A third consequence of inhibiting mitochondrial respiration is the diversion of high energy electrons from the electron transport chain, leading to cytotoxic free radical generation. Oxidant damage is a proposed mechanism of neurodegeneration in AD (Halliwell, 1989). Finally, conditions of hypoxia, ischemia, and hypoglycemia are also associated with increased release of the excitatory amino acids glutamate and aspartate (Pelligrini-Giampietro, Cherici, Alesiani, Carla, & Moroni, 1990). Thus, an excitotoxic component may contribute to an azide-induced behavioral impairment. In future work, we plan to analyze the contri-
AZIDE TREATMENT IMPAIRS LEARNING TASKS b u t i o n of e a c h of t h e s e p u t a t i v e m e c h a n i s m s of azide-induced l e a r n i n g deficits in r a t s , a n d t h e t i m e course w i t h i n w h i c h d a m a g e f r o m e a c h m i g h t occur. I t is l i k e l y t h a t all of t h e a b o v e - m e n t i o n e d m e c h a n i s m s c a n c o n t r i b u t e to a z i d e - i n d u c e d l e a r n i n g deficits, a l t h o u g h w h e t h e r all of t h e m p l a y a role in the behavioral dysfunction seen after a relatively ]brief d i s r u p t i o n of e n e r g y m e t a b o l i s m is not k n o w n . [n t h i s r e g a r d , it w o u l d be of i n t e r e s t to d e t e r m i n e 'whether p r o l o n g e d azide t r e a t m e n t p r o d u c e s beh a v i o r a l deficits t h a t w o r s e n o v e r t i m e . I n s u m m a r y , chronic azide t r e a t m e n t i m p a i r s acquisition a n d r e t e n t i o n p e r f o r m a n c e on t h e M o r r i s 'water m a z e t a s k . T h i s finding is c o n s i s t e n t w i t h o u r p r e v i o u s r e p o r t of a n a z i d e - i n d u c e d deficit on o t h e r b e h a v i o r a l t a s k s ( B e n n e t t et al., 1992). T h e p r e s e n t finding d e m o n s t r a t e s t h a t c y t o c h r o m e oxidase inh i b i t i o n in r o d e n t s m i m i c s s o m e a s p e c t s of t h e l e a r n ing a n d m e m o r y deficit a s s o c i a t e d w i t h AD. W e sugg e s t t h a t a useful a n i m a l m o d e l of a h u m a n d i s e a s e m u s t m e e t two criteria: (1) T h e e x p e r i m e n t a l m a n i p u l a t i o n r e p r o d u c e s a condition w h i c h is associa t e d w i t h t h e n a t u r a l disease; a n d (2) t h e specific m a n i p u l a t i o n of a single clinical c o n c o m i t a n t m i m ics s o m e aspects of t h e p a t h o l o g y of t h e disease. T h e p r e s e n t results, in concert w i t h o u r p r e v i o u s findings, s u p p o r t t h e h y p o t h e s i s t h a t chronic cytoc h r o m e oxidase i n h i b i t i o n b y tonic i n f u s i o n of sod i u m azide p r o d u c e s a p a t h o l o g y in r o d e n t s w h i c h :models s o m e a s p e c t s of AD. REFERENCES Armanini, M. P., Hutchins, C., Stein, B. A., & Sapolsky, R. M. (1990). Glucocorticoid endangerment of hippocampal neurons is NMDA-receptor dependent. Brain Research, 532, 712. ]Bandy, B., & Davison, A. J. (1990). Mitochondrial mutations may increase oxidative stress: Implications for carcinogenesis and aging? Free Radical Biology and Medicine, 8, 523-539. Bennett, M. C., Diamond, D. M., Stryker, S. L., & Parker, W. D., Jr. (1992). Cytochrome oxidase inhibition: A novel animal model of Alzheimer's disease. Journal of Geriatric Psychiatry and Neurology, 5, 93-101.
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