Cytochrome Oxidase Inhibition: A Novel Animal Model of Alzheimer’s Disease M. Catherine Bennett, PhD; David M. Diamond, PhD; Stacy L. Stryker, BS; Janice K. Parks, BA; W. Davis Parker Jr, MD
Abstract A profound decrease in activity of the mitochondrial enzyme cytochrome oxidase in blood platelets is a recently identified concomitant of Alzheimer’s disease (AD). We investigated a possible pathogenic link between this finding and the symptoms of AD by mimicking this mitochondrial enzyme deficiency in rats. Rats were infused chronically with a selective inhibitor of cytochrome oxidase, sodium azide, or with saline delivered via subcutaneously implanted osmotic minipumps. The azide treatment impaired both spatial and nonspatial learning. Further, the azide treatment inhibited a lowthreshold form of hippocampal long-term potentiation, primed burst potentiation. The behavioral deficits were not secondary to a sensory or motor impairment. Thus, chronic azide treatment of rats models some characteristics of AD. (I Gerintr Psychid y Neiirol 1992;5:93- 101).
A
lzheimer’s disease (AD) is a progressive neurodegenerative disease marked by a profound impairment of learning and memory and a global cognitive deficit.* There is currently no animal model that exhibits the total pathology associated with AD. Several animal models are being used presently in AD research, each of which mimics specific concomitants of the natural disease. Experimental manipulations to produce these animal models include, for example, lesioning of the central cholinergic system2 and administration of a l ~ r n i n u mAl.~ though no single model produces the full pathology of AD, some of these models have provided useful means for screening therapeutic treatments. Several biochemical markers of mitochondrial dysfunction have been characterized in AD. These correlates of AD include alterations in glucose utilization, alterations in calcium homeostasis, and reReceived April 15, 1991. Received revised July 21, 1991. Accepted for publication August 12, 1991. From the Department of Pharmacology (Drs Bennett and Diamond), and the Departments of Neurology and Pediatrics (Ms Parks and Dr Parker), University of Colorado Health Sciences Center, the Veterans Administration Medical Center (Dr Diamond), and the Department of Biology (Ms Stryker), University of Colorado, Denver, CO. Address correspondence to Dr h1.C. Bennett, Department of Pharmacology, C-236, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denveri CO 80262.
duced mitochondrial activity in skin fibroblasts and in brain.4-9 Recently, Parker et all0 identified a specific site of a mitochondrial enzyme defect in blood platelets of AD patients. These investigators found a profound decrease in the activity of cytochrome oxidase (complex IV), the terminal enzyme in the electron ‘transport (respiratory) chain of mitochondria. Other enzymes of the electron transport chain were spared. Blood platelets are not known to be a target tissue in AD. Therefore, the cytochrome oxidase deficiency in blood platelets may represent a defect that occurs in mitochondria of all cells. The selective nature and widespread occurrence of this mitochondrial enzyme defect identify it as candidate for an early, and possibly pathogenic, defect of AD. One method of addressing the issue of whether cytochrome oxidase deficiency can c m s e the sequelae associated with AD is to examine cytochrome oxidase inhibition in an animal model. In this report, we describe a method for inducing chronic and selective inhibition of cytochrome oxidase in rats via subcutaneous delivery of sodium azide, a selective inhibitor of this enzyme. In the present investigation, we tested the hypothesis that chronic exposure of rats to sodium azide would impair learning and hippocampal plasticity. Hippocampal-dependent learning and hippocampal plasticity were major foci
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of our investigation because the higpocampus is a preferential target of damage in AD. Moreover, the hippocampus has a crucial role in memory formation in humans and other mammal^.'^-^^ Therefore, we studied the effect of azide treatment on the acquisition and performance of behavioral tasks, including a spatial memory task that is known to be hippocampal dependent for Similarly, we investigated the effect of chronic cytochrome oxidase inhibition on hippocampal plasticity. There is considerable evidence that memory formation in humans and other animals involves the capacity to change synaptic strength in the hippocampus."j Long-term potentiation is a long-lasting change in synaptic efficacy that can be induced experimentally in the hippocampus. Long-term potentiation shares many properties with memory and has been used extensively as a physiologic model for memory (see Teyler and Discenna" for review of long-term potentiation). In the present experiment we examined the effect of chronic cytochrome oxidase inhibition on a low-threshold form of long-term potentiation, primed burst potentiation.''-''
Method
Subjects Subjects were adult male Sprague-Dawley rats (Sasco, Omaha, NB) that weighed 350 to 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, except as described for the eight-arm maze protocol. surgery
Under secobarbital anesthesia (40 mg/kg), rats were each implanted subcutaneously with an Alzet 2ML4 osmotic minipump. For each subject, an incision of approximately 1 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 cm by 3 cm into which a minipump was inserted. These minipumps have a 2-mL reservoir and provide a constant infusion rate of 0.5 pg/hr for 28 days. The pumps were filled with either a solution of 160 pg/pL sodium azide in 0.9% saline (azide) or the saline vehicle (control). The azide pumps delivered a dose of sodium azide equal to 400 pg/hr. Sodium azide crystals of 99% purity were obtained from Aldrich, Milwaukee, Wisconsin.
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Behavior
Tzuo-Way Shuttle Box Task.-Azide and control groups were first trained on a shock-motivated, twoway shuttle box task beginning 6 to 9 days after pump implantation. The behavioral apparatus was an automated shuttle box (Omnitech Columbus, Ohio) which consisted of a straight runway divided into two compartments by a partition with a hole in the center. The rats were individually placed in the left compartment of the shuttle box. The position of the animal was detected by photocells. Prior to the first trial only of each day, the rats were given 5 seconds adaptation time. A trial was begun when a light, the conditioned stimulus, was presented in the opposite compartment to the one in which the animal was detected. Five seconds after the conditioned stimulus presentation, the unconditioned stimulus, a ~OO-JLAscrambled shock, was delivered to the dark compartment. The animals' latencies to escape or to avoid (after the first trial) the shock by crossing to the lighted, safe side were recorded automatically for each trial. An avoidance was treated as a 0-second escape latency. Each subject received 15 trials on four consecutive days. Performance was meaured as the latency to cross to the safe side. The entire apparatus was cleaned with 70% ethanovwater between subjects. Operz Field Activity.-The day following the completion of shuttle box training, the azide and control groups were assessed for motor function. Spontaneous activity of the rats was tested in an open field apparatus. Each subject was placed in a Plexiglas box (120 X 120 cm) with a grid of 16 rectangles (30 X 30 cm) marked on the floor. The number of spontaneous line crossings for each subject was recorded for a 5-minute period. The box was cleaned with a 70% ethanolltvater solution between subjects. Test.-Following the open field test, the sensory function of these rats was measured using the flinch-jump test which is a psychometric assessment of threshold responses to footshock. Each rat was given six series (three ascending and three descending) of discrete footshocks (0.5 seconds), ranging from 100 pA to 1000 pA in increments of 100 pA. The intershock interval within a series was 30 seconds, and the interval between series was 2 minutes. The flinch and jump thresholds for each rat were calculated as the amount of current required to elicit those respective responses in 50% of the trials. Fliricli-Jziniy
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Cytochrome Oxidase Inhibition
Eight-Am &did Mnze.-Two additional groups of rats were tested on a spatial task, the eight-arm radial maze. Azide and control groups were implanted with osmotic minipumps 7 days prior to the first day of training. All behavioral experimentation took place between 1 :00 PM and 5: 00 PM using a modification of the method of Barnes et al.’l The testing room was dimly lit with a small source of natural light. There were conspicuous maze-fixed cues in the room and on the walls. The experimenter was blind to the treatment. On the 4th, 5th, and 6th days after pump implantation, each rat was given daily 10-minute acclimation sessions on the eight-arm radial maze in which the rat was free to explore the maze, which was scattered with Fruit Loops cereal broken into quarter pieces. Any rat that did not eat any of the cereal during the acclimation period was not used in the training trials. Twenty-four hours prior to training, the rats were placed on a mild food-deprivation schedule in which each animal was given access to 10 to 12 g of rat chow per day. Rats were weighed daily and no animal was allowed to fall below 80% of its prefasting weight. Beginning on the 8th day after surgery, the rats were placed individually in the radial maze and given a 10-minute trial for each of 12 days. A well in the end of each arm was baited with one quarter of a piece of cereal. A correct choice was one in which the rat entered the baited arm and ate the food. An error was either the entry of the rat into an arm without consuming the bait, or the reentry of a rat into an arm from which it had already eaten the bait. Hippocampal Plasticity A total of six control and six azide rats were used in the electrophysiologic experiment. Two control and two azide rats from the radial maze experiment were used for physiology recordings after being returned to ad libitum access to food for 3 to 5 days. The remaining four rats per group were implanted specifically for the physiology experiment. Recordings from all subjects took place in the 3rd and 4th weeks after pump implantation in order for the duration of azide or saline exposure to be approximately the same for all subjects. The rats, which weighed 350 to 450 g at the time of recording, were removed from the vivarium on the day prior to the recording and individually housed within the laboratory.22 Between 9 : O O AM and 1O:OO Ahi on the day of recording, atropine methyl nitrate was administered (0.2 mgkg, ip) followed by urethane (range, 1.3 to 1.5 g k g , ip). The recording method has been described.= Briefly, the recording. electrode (etched
epoxylite-insulated tungsten; impedance, 0.5 to 1.0 Mohms at 1 kHz) was lowered to the CA1 pyramidal cell layer, which was identified by complex spike activity occurring between 1.8 and 2.3 mm below brain surface. The stimulator electrode (125-pm-diameter stainless steel Teflon-coated wire uninsulated at the tip) was lowered into the left side of the hippocampal commissure (coordinates: AP, -1.8; ML, 1.0). The final coordinates were determined by optimizing the amplitude of the population spike, which is a measure of synchronous firing of CA1 cell activity.24 Population spike responses were displayed on an oscilloscope and stored in an Epson computer for on-line and off-line analysis. Test responses were evoked by constant-current single-pulse stimuli (150-ps duration) presented every 30 seconds for 5 minutes before and 30 minutes following primed burst (PB) stimulation. PB stimulation consisted of a single pulse followed 170 ms later by a burst of four pulses at 200 Hz. PB stimulation current levels were 175% of the current levels used in test pulses. We have reported in previous work that this level of PB stimulation induced PB potentiation in all control subjects.23 Statistical analyses (f-test; P I.05) of the changes in the population spike amplitude were performed for each subject by comparing responses obtained during the 10-minute period immediately prior to PB stimulation to those obtained during the 21- to 30-minute period after PB stimulation. Group comparisons were performed using a repeated measures MANOVA. Assay for Respiratory Chain Enzyme Activity The rats used in the shuttle box task were sacrificed by decapitation approximately 2 weeks after pump implantation and assayed for activities of respiratory chain complexes I through IV. Whole brains except for the cerebellum and hindbrain were used for mitochondrial extraction. Cell body and synaptic membrane mitochondria were isolated by the method of Clark and Nicklas.= Respiratory enzyme complexes were assayed as follows: Complex I (reduced nicotinamide-adenine dinucleotide [NADH]: ubiquinone oxidoreductase) was assayed spectrophotometrically in sonicated mitochondria by following the decrease in the absorption of NADH26 using 60 pmoVL Q1 and 2 mmol/L KCN. Complex IU complex I11 (succinate: cytochrome c oxidoreductase) were assayed as described by Stumpf and Parks.27 For complex I and complex II/III, activity has been expressed as pmlminlmg of mitochondrial protein. Complex IV (cytochrome oxidase) was assayed as a cyanide-sensitive ferrocytochrome . c oxidase in
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cholate-solubilized mitochondriaz6in 20 mmoVL potassium phosphate, pH 7.0, and 25 pmoVL reduced cytochrome c reductase prepared by ascorbate reduction and purified over Sephadex G-50. The oxidation of reduced cytochrome c was followed at 550 nm. Cytochrome oxidase activity has been expressed as a first-order rate constant (rate constant/s/mg mitochondrial protein).
Results
Respiratory Chain Enzyme Activify Respiratory chain enzyme activities were assayed from mitochondria of both cell bodies and synaptic membranes of control and azide rat brains. Chronic azide treatment produced a significant inhibition of cytochrome oxidase in both subcellular compartments: a 35%decrease in the cell body mitochondria and a 39% decrease in the synaptic membrane mitochondria (Figure 1). Based on a previous investigation of dose- and time-response characteristics of respiratory chain enzyme activity to chronic azide treatment, this level of inhibition represents a steady-state degree of inhibition that is reached within 48 hours of pump i m p l a n t a t i ~ n .Azide ~~
treatment did not significantly alter the activity of any other respiratory chain enzyme complex.
Behavior Sluttfle Box Tusk.-Overall, the azide group was significantly impaired with respect to the control group in escape latency (repeated measures MANOVA main effect of treatment: F[1,13] = 8.79; P = 0.01) (Figure 2). For the control group, escape latency for the last day of training was significantIy lower than that of the first training day (two-tailed Student’s t = 2.02; P < .05), indicating an improvement of performance over trials. In contrast, the azide group did not show a significant decrease in escape latency over days (first day versus last day: t = 0.11; P > .1), indicating no improvement in performance. Seiisory and Motor Tests.-The data from the motor and sensory assessments are summarized in Table 1. There was no significant difference in spontaneous activity in an open field between the azide and the control groups (t = 0.28; P > .1). Pain sensitivity of the rats was then assessed using a flinch-jump test. There was no evidence that the two groups differed in their responses to footshock (flinch: t = 0.20; P > .l; jump: t = 0.03, P > .1).The lack of signifi-
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FIGURE 1 Respiratory chain enzyme activity from brains of azidetreated and control rats. Cell body (CB) and synaptic membrane (SM) mitochondrial enzyme activity. Complexes I and IUIII activities are expressed as mean ( 4 SEM) FmoYmidmg of mitochondrial protein; cytochrome oxidase (complex IV) activity is expressed as mean (+ SEM) rate constant/s/mg of mitochondrial protein. * denotes P < .05 on Student t comparisons of azide versus control.
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FIGURE 2 Shuttle box task in seven-azide-treated and eight control rats. Data are expressed in blocks of 15 trials denoting the mean (k SEM) daily performances. The repeated measures MANOVA (derived from the individual scores) revealed significant main effect of treatment (F[1,13] = 9.21; P < .Ol), significant main effect of time (F[11,143] = 2.59; P < .Ol), and a significant treatment by time interaction (F[11,143] = 2.70; P < .05). * denotes trial block significantly different from first trial block (t = 2.02; P < -05).
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Cytochrome Oxidase Inhibition TABLE 1 Motor Activity and Sensitivity to Footshock in Azide-Treated and Control Rats Spontaneous Activity Footshock Sensitivity N 7 8
Azide Control
Line Crossings' 47.6 8.13t 50.0 L 2.81
Flinch' 225 23.lt 230 9.33
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'Mean % SEM. tNot significantly different from control value.
cant differences between the two groups on these measures suggests that the behavioral deficits can not be attributed to a sensory or motor deficit.
Eight-Arm Xrrdial Maze.-The performance of each group on the eight-arm radial maze as measured by the mean daily correct choices is shown in Figure 3. There was a significant azide-induced impairment of performance on this task (repeated measures MANOVA main effect of treatment: F[1,16] = 5.06; P < .05). Although the azide group showed an impairment of acquisition, the two groups reached asymptote performance levels that did not differ significantly. Both groups showed significant improvement in performance over trials, which indicates that both groups showed learning. Overall, there was no statistical difference be-
tween the two groups in the number of errors during acquisition of the spatial task (repeated measures MANOVA: F[1,16] = 2.12; P = .16) (Figure 4). However, the control group, unlike the azide group, exhibited a steady decline in the number of errors over the first 5 days of training. The decrease in the number of errors by the control group reached statistical significance on day 5 (t = 3.31; P < .01, day 1 versus day 5). In contrast, the decrease in the number of errors for the azide group did not reach statistical significance until day 7 (f = 3.98; P < .01, day 1 versus day 7). The lag in acquisition by the azide group provides a second measure of a significant learning impairment in this group. Thus, by both measures of performance, ie, correct choices and errors, azide rats were impaired in spatial learning. In addition, the finding that the two 15
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FIGURE 3 Eight-arm radial maze correct choices for nine azidetreated and nine control rats. Mean (* SEM) number of correct choices per group in daily 10-minute trials. For trials 1 through 12, repeated measures MANOVA main effect of treatment, F(1,16) = 5.04; P < .05; main effect of time, F(11,176) = 32.77; P < .01; treatment by time interaction, F(11,176) = 1.19; P > .1). For trials 1 through 6 (acquisition), repeated measures MANOVA main effect of treatment, F(1,16) = 5.61; P < .05; main effect of time, F(5,80) = 19.52; P < .01; treatment by time interaction, F(5,80) = 0.43; P > . l .
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5
FIGURE 4 Eight-arm radial maze errors for nine azide-treated and nine control rats. Mean (2 SEM) number of errors per group in daily 10-minute trials. Repeated measures MANOVA for trials 1 through 12: main effect of treatment, F(1,16) = 2.12; P > .l; main effect of time, F(11,176) = 17.61; P < .01; treatment by time interaction, F(11,176) = 0.84; P > -1. Compared with the first day of training, the first day in which there was a significant decrease in error number was day 5 for the control group (t = 3.31; P < .01) and day 7 for the azide group (t = 3.98; P < .01). Thus, the decline in errors for the azide group lagged that of the control group by 2 days of training.
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groups reached the same level of performance by both of these measures indicates that the azide rats were neither failing to move about the maze nor were they hypermotor. This result provides additional evidence that the azide-induced impairments on the behavioral tests reflect a learning deficit rather than a motor impairment. Hippocampal Plasticity Azide treatment also significantly impaired the expression of a low-threshold form of long-term potentiation in the hippocampus, PB potentiation. We define PB potentiation as the long-term component of plasticity occurring after the tetanizing stimulus. In the present experiment, it was measured in the 21- to 30-minute interval after the stimulus train was delivered. The measure of the magnitude of the hippocampal response to the test stimuli was the magnitude of the population spike evoked response. The population spike reflects the synchronous firing of the CA1 hippocampal pyramidal cells. Population spike data recorded from the CAI cell layer of the hippocampus of azide and control rats are summarized in Figure 5.
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FIGURE 5 Primed burst potentiation in six azide-treated and six control rats. Data points are mean (? SEM) magnitude of population spike evoked potentials. Patterned stimulus train was delivered at time zero. Baseline current intensity: for the azide group was 69 PA 2 10.3; for the control group, 62 pA rt 10.2 (azide versus control: t = 0.48; P > .1). Baseline magnitude of population spike for the azide group was 1.56 mV 2 0.10; for the control group, 1.65 mV f 0.17 (azide versus control: t = 0.45; P > .1). Repeated measures MANOVA (sampled at 2-minute intervals): main effect of treatment, F(1,lO) = 6.83; P < .05; main effect of time, F(19,190) = 16.51; P < .01; treatment by time interaction, F(19,190) = 1.91; P < .05).
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There was an overall main effect of treatment for the two groups (repeated measures MANOVA: F[1,10] = 6.83; P < .OS), with the azide group showing the lower response magnitude. There was no significant difference between the groups in the short-term component of plasticity, posttetanic potentiation: Evoked responses during the first minute after the tetanizing stimulus did not differ significantly between groups (t = 0.23; P > .1). The difference between the two groups occurred in PB potentiation, the long-term response. Although both groups showed significant PB potentiation, the response of the control group stabilized at approximately 200% over baseline, while the response of the azide group stabilized at approximately 60% over baseline. . There were no significant differences between the azide and control groups either in magnitude of baseline population spikes or in the stimulus current intensities used to evoke the baseline responses (data reported in Figure 5 caption). These results, together with the lack of a difference in posttetanic potentiation magnitudes between the two groups, indicate that the azide-treated rats exhibited normal synaptic transmission and short-term plasticity. Discussion Azide treatment impaired performances on both behavioral tasks tested in the present investigation. One of the tests, the two-way shuttle box task, is relatively insensitive to hippocampal damage. The other task, the radial maze, is a sensitive measure of hippocampal damage.I4 These findings suggest that chronic azide treatment produces a generalized learning deficit. In addition, subjects were examined on measures of sensory and motor function. These tests did not reveal any significant differences between the azide-treated and the control groups. The results of these control studies provide evidence that the impaired performance of the azide group reflects a learning impairment rather than a sensory or motor deficit. We also examined the effect of azide treatment on a form of hippocampal plasticity, PB potentiation. PB potentiation is a form of hippocampal longterm potentiation for which the induction threshold is lower than it is for conventional long-term potentiation. The basis of the reduced threshold for PB potentiation is the use of patterned stimulation that mimics the temporal components of endogenous rhythms of hippocampal activity.*'-'' The results of the electrophysiology experiment paralleled the re-
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Cytochrome Oxidase Inhibition sults of the radial maze experiment: Azide treatment impaired, but did not abolish, both hippocampal PB potentiation and spatial learning. This finding may be related to the relatively short duration of azide exposure prior to testing. Rats treated with azide for 1 to 4 weeks prior to testing may be analogous to patients in the early stages of AD. Although mitochondrial respiration occurs in every cell, there is evidence of differential sensitivity to inhibition of the respiratory chain among neuronal subpopulations. Some cell types of the hippocampus, for example, are more vulnerable to cell death after hypoxia and i~chemia,’~ both of which produce intermediary metabolic consequences similar to respiratory inhibition. There is also evidence to suggest that a specific dysfunction of cytochrome oxidase could cause differential damage in the central nervous system. In recent work, Eells and her coll e a g u e ~have ~ ~ shown that the neurotoxic effect of methanol positioning is secondary to cytochrome oxidase inhibition and that there are regional differences in the vulnerability of the retina to this toxin. Cytochrome oxidase is not distributed uniformly in the nervous system. There is also recent evidence that the heterogeneous pattern of cytochrome oxidase distribution corresponds to the pattern of susceptibility of brain damage in AD. Chandrasekaran et al,3’ from the laboratory of S.I. Rapoport, recently reported evidence that brain regions preferentially afflicted in AD have a higher expression of mRNA for a homologous sequence of the mitochondrial cytochrome oxidase subunit I gene than do brain regions that are relatively spared in AD. The latter two independent lines of evidence30831support the hypothesis that certain brain systems may be differentially susceptible to a defect of cytochrome oxidase. The proposal that a specific mitochondrial enzyme produces a distinctive pattern of brain damage gains support in related observations emerging from the study of Parkinson’s disease. A selective inhibition of complex I of the respiratory chain is a characteristic of the blood platelets in patients with Parkinson’s disease.32 Further, 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP), which produces selective damage to the basal ganglia and Parkinsonlike symptoms is also an inhibitor of complex I.33-36 Consistent with this interpretation, Alpers’ disease and Menkes‘ syndrome, which are both characterized by abnormalities of cytochrome oxidase, have prominent cortical symptom^.^' Despite differences between the clinical manifestations of Alpers’ disease and Menkes’ syndrome compared with AD,
families of inborn (metabolic) errors can produce both clinical and biochemical heterogeneity. Patients with abnormalities of the same gene product can have clinically distinguishable syndrome^.^' We have not yet investigated the mechanism@) for the learning and neurophysiologic impairments produced by depletion of cytochrome oxidase. Dysfunction of cytochrome oxidase can produce numerous changes in cellular metabolism that may contribute to the deficits produced by azide treatment. A discussion of some of these candidate mechanisms follows. First, under normal conditions, electron transport in the mitochondria is coupled to oxidative phosphorylation; thus, a reduction in the efficiency of the electron transport chain also inhibits the phosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP). The inhibition of aerobic respiration also shifts a greater percentage of glucose catabolism, at the stage of pyruvate, to the anaerobic pathway ending in lactate. This metabolic pathway yields less ATP production than does the aerobic Therefore, one effect of respiratory inhibition is a reduction of cellular energy storage that would be predicted to impair cellular function. Second, the inhibition of the mitochondrial respiratory chain also shifts the equilibrium potential from oxidized nicotinamide-adenine dinucleotide (NAD+) to NADH, as NADH is unable to donate its electrons to the chain. The activity of numerous enzymes of intermediary metabolism is linked to the bioavailability of NAD+. Pyruvate dehydrogenase is an NAD+-linked enzyme, and an impairment of pyruvate dehydrogenase decreases acetylcholine synthesis.4o A decline in acetylcholine synthesis and release has been shown to exist under conditions of mild hypoxia and h y p ~ g l y c e m i aand ~ ~ to be associated with aging.42A profound decrease in acetylcholine is a well-described concomitant of AD and may underlie part of the learning and memory deficits of AD.43 Third, the oxidative deamination of glutamate on its entry into the Krebs cycle as a-ketoglutaric acid also requires the presence of the NAD+-linked glutamate dehydrogenase.4 A decrease in NAD+ associated with the inhibition of the electron transport chain might be predicted to increase pools of glutamate. Hypoxia, hypoglycemia, and ischemia have been shown to increase the release of glutamate and aspartate, both excitatory amino acids, and to increase free radical formation.45 These consequences may underlie the delayed loss of neurons ,
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found after ischemia. Further, prolonged exposure to elevated Ievels of glutamate may lead to N-Methyl-D-aspartate-mediated excitotoxicity, which has been proposed to play a role in the pathogenesis of
flecting this aspect of AD should prove to be a valuable tool in screening treatments to mitigate damage resulting from this enzyme defect.
~ ~ - 2 9 ~ 3 6
When the activity of cytochrome oxidase is insufficient to catalyze completely the final reduction step of molecular oxygen to HzO, high-energy electrons are diverted from the electron transport chain. These electrons are associated with active oxygen species, such as the superoxide and hydroxyl radicals, which are c y t o t o x i ~ . ~ 'Free * ~ ~ radical damage has also been proposed to contribute to neurodegeneration in AD.49 The determination of mechanisms by which a cytochrome oxidase deficiency might develop in humans is of considerable clinical significance. The majority of cases of AD is sporadic, ie, appears to be nonfamilial. Three subunits of cytochrome oxidase are encoded in the mitochondria1 genome.50 Mitochondrially encoded defects can appear to be sporadic if the maternal mitochondria are heterop l a ~ m i c . ~ Therefore, '-~~ mitochondrial genetic transmission might account for the non-Mendelian pattern of occurrence of most cases of AD. Alternatively, the present finding that the toxin sodium azide selectively inhibits cytochrome oxidase and produces a cognitive deficit suggests the possibility that similar environmental toxins may be pathogenic. For example, cyanide and carbon monoxide are also selective inhibitors of cytochrome oxidase, and these toxins are components of air pollution and of cigarette smoke. It is likely that a threshold decrease of cytochrome oxidase activity is required before debilitating effects are manifested. Therefore, a toxic load might vary by individual according to genetic or other susceptibility to environmental toxins. In summary, azide-treated rats were significantly impaired on two measures of learning: an aversively-motivated shuttle task and an appetitively-motivated spatial task. The deficit does not appear to be secondary to a sensory or a motor impairment. Furthermore, azide-treated rats did not develop the degree of PB potentiation shown by the control rats. Taken together, these data indicate that chronic azide treatment produces a generalized learning deficit and impairs hippocampal plasticity in rats. These findings are consistent with the hypothesis that a defect of cytochrome oxidase has an etiologic role in AD. Regardless of whether or not the cytochrome oxidase deficiency found in AD is pathogenic, the development of an animal model re-
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Acknowledgments This project was approved by the institutional animal use committee and camed out in accordance with institutional animal care guidelines. This research was supported in part by NIH grants NSOlM7 and NS25382 (W.D.P. Jr) and by a Mental Retardation Research Center grant from NIH. The authors gratefully acknowledge Dr Greg Rose for the generous use of his equipment for this experiment (equipment and supplies purchased from NSF grant BNS8811486 and a grant from the Veterans Administration Medical Research Service) and for his valuable critique of this manuscript. We also thank'Dr Karen Stevens for her extensive data analyses and instruction in SPSS and her helpful comments on this manuscript.
References 1. Corkin 5: Some relationships between global amnesias and the memory impairment in Alzheimer's disease, in Corkin 5, Groivdon JH, Usdin E, Wurtman RJ (eds): Alzheimer's Diseuse: A Rqort of Progress, vol 9. New York, Raven Press, 1982, pp 149-164. 2. Smith G : Animal models of Alzheimer's disease: Fxperimental cholinergic denervation. Brnin Res 1988;472:103-118. 3. McLachlan DRC: Aluminum and Alzheimer's disease. Neitrobiol Agitig 1987;7525-557. 4. Peterson C, Gibson GE, Blass J P Altered calcium uptake in cultured skin fibroblasts from patients with Alzheimer's disease. N Eirgl J Med 1985;312:1063-1065. 5. Peterson C, Goldman JE: Alterations in calcium content in cultured skin fibroblasts from aged and Alzheimer's donors. Proc Nntl Acud Sci U S A 1986;83:2758-2762. 6. Sims NR, Finegan JM, Blass JF:Altered glucose metabolism in fibroblasts from patients with Alzheimer's disease. N Engl J Med 1985;313:638-639. 7. Sims NR, Finegan JM, Blass JF: Altered metabolic properties of cultured skin fibroblasts in Alzheimer's disease. Atiri Neirrol 1987;21:451-457. 8. Sims NR, Finegan JM, Blass JP, et al: Mitochondria1 function in brain tissue in primary degenerative dementia. Bruin Res 1987;436:30-38. 9. Terry RD, Katzman R Senile dementia of the Alzheimer type. Aim Neirrol 1983;14:497-505. 10. Parker WD Jr, Filley CM, Parks J K Cytochrome oxidase deficiency in Alzheimer's disease. Nerrrology 1990;40:13021303. 11. Ball MI, Fisman M, Hachinski V, et al: A new definition of Alzheimer's disease: A hippocampal dementia. Lnricet 1985;1:14-16. 12. Berger TLV, Thompson RF: Identification of pyramidal cells as the critical elements in hippocampal plasticity during learning. Proc Nut1 Acnd Sci USA 1978;75:1572-1576. 13. Scoville WB, Milner B: Loss of recent memory after bilateral hippocampal lesions. Neitrositrg Psychintry 1957;20:11-21. 14. Squire L R Mechanisms of memory. Science 1986;232: 1612-1619. 15. Zola-Morgan 5, Squire LR: Memory impairment in monkeys
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Cytochrome Oxidase Inhibition following lesions limited to the hippocampus. Belznv Neiirosci 1986;100:155- 160. 16. Lynch G, Baudry MP: The biochemistry of memory: A new and specific hypothesis. Scierice 1984;224:1057- 1062. 17. Teyler TJ,Discenna P: Long-term potentiation. Aiiii Rm Neiirosci 1987;10:131-161. 18. Diamond DM, Dunwiddie TV, Rose GM: Characteristics of hippocampal primed burst potentiation in vitro and in the awake rat. ] Neirrosci 1988;8:4079-4088. 19. Rose GM, Dunwiddie -rV: Induction of hippocampal longterm potentiation using physiologically patterned stimulation. Neicrosci Lett 1986;69:244-248. 20. Larson J, Wong D, Lynch G: Patterned stimulation at the theta frequency is optimal for the induction of hippocampal long-term potentiation. Brniri Res 1986;368:347-350. 21. Barnes CA, Nadel L, Honig W K Spatial memory deficit in senescent rats. Cull ] Psychol 1980;34:29-39. 22. Diamond DIM, Bennett MC, Stevens KE, et al: Exposure to a novel environment interferes with the induction of hippocampal primed burst potentiation. Psyclrobiology 1990;18:273-281. 23. Diamond DM, Bennett MC, Engstrom DA, et al: Adrenalectomy reduces the threshold for hippocampal primed burst potentiation in the anesthetized rat. Brniii Res 1989;492:356-360. 24. Anderson P, Bliss TVP, Skrede K K Unit analysis of hippocampal population spikes. Exp Bruin Res 1971;13:208-221. 25. Clark JB, Nicklas WJ: The metabolism of rat brain mitochondria: Preparation and characterization. ] Bid Clfein 1970;245:4724-4731. 26. Whitfield CD, Bostedor R, Goodrum D, et al: Hamster cell mutants unable to grow on galactose and exhibiting an overlapping complementation pattern are defective in the electron transport chain. ] Biol Clierri 1981;2566651-6656. 27. Stumpf DA, Parks J K Human rnitochondrial electron transport chain: Assay of succinate cytochrome c reductase in leucocytes. Bioclrein Med 1981;25:23-238. 28. Bennett MC, Diamond DIM, Stryker SL, Parker WD Jr: Cytochrome oxidase inhibition impairs learning and hippocampal plasticity: Implications for Alzheimer’s disease. Neiirosci Abstr 1990;16:1346. 29. Clark GD: Role of excitatory amino acids in brain injury caused by hypoxia-ischemia, status epilepticus and hypoglycemia. Cliii Periiiutol 1989;16:459-474. 30. Eells JT,Murray TG, Rajani C, Burke JM: Formic acid inhibits cytochrome oxidase activity in retina and cultured retina cells. Neirrosci Absfr 1990;16:611. 31. Chandrasekaran K, Matocha MF, Stoll J, et al: Analysis of mRNA showing region-specific expression in nonhuman primate cortex. Neiirosci Abstr 1990;16314. 32. Parker WD Jr, Boyson SJ, Parks J K Abnormalities of the electron transport chain in idiopathic Parkinson’s disease. A m Neiirol 1990;26719-723. 33. Langston JW, Ballard P, Tetrud JW, Irvin I: Chronic parkinsonism in humans due to a product of meperidine-analog synthesis. Science 1983;219:979-980. 34. Ramsey RR, Salach JI, Singer TP: Uptake of the neurotoxin 1-methyl-4-phenylpyridine (MPP+) by mitochondria and its relation to the inhibition of the rnitochondrial oxidation of NAD+-linked substrates by MPP+. Biochem Biopliys Res Corn111itIi 1986;131:743-748.
35. Ramsey RR, Singer TP: Energy dependent uptake of N-methyl-4-phenylpyridinium,the neurotoxic metabolite of l-methyl4-phenyl-l,2,3,6-tetrahydropyridine, by mitochondria. 1Biol Clierii 1986;261:7585-7587. 36. Vyars I, Heikkila RE, Nicklas WJ: Studies on the neurotoxicity of l-rnethyl-4-phenyl-l,2,5,6-tetrahydropyridine:Inhibition of NAD-linked substrate oxidation by its metabolite N-methyl-4phenylpyridinium. ] Neiirodietii 1986;46:1501-1507. 37. Stumpf DA: Mitochondria1 multisystem disorders: Clinical, biochemical and morphologic features Crrrr Neitrol 1979;2117- 149. . 38. Blass JP, Sheu RK, Cedarbaum JM: Energy metabolism in disorders of the nervous system. Rn, N e d 1988;144:%3-563. 39. Stryker L Chapter 12: Glycolysis, in Biocheinisfry. W.H. Freeman and Company, San Francisco, 1975, p 294. 40. Gibson GE, Blass JP:Decreased synthesis of acetylcholine accompanying impaired oxidation of pyruvic acid in rat brain minces. BioclwiI J 1973;148:17-23. 41. Gibson GE, Blass JP: Impaired synthesis of acetylcholine in brain accompanying mild hypoxia and hypoglycemia. 1Neirrocliein 1976;27:37-42. 42. Gibson GE, Peterson C Aging decreases oxidative metabolism and the release and synthesis of acetylcholine. Neiiroclieni 1981;37:978-984. 43. Whitehouse PJ,Price DL, Clark AW, et al: Alzheimer’s disease: Evidence for selective loss of chohergic neurons in the nucleus basalis. A m Neirrol 1981;10:122-126. 41. Lehninger A L Chapter 16: The tricarboxylic acid cycle and the phosphogluconate pathway, in Biochemistry. Worth Publishers, Inc, New York, 1970; p 439. 45. Pelligrini-Giampietro DE, Cherici G, Alesiani M, Carla V, Moroni F Excitatory amino acid release and free radical formation may cooperate in the genesis of ischemia-induced neuronal damage. J Neitrosci 1990;10:1033-1041. 46. Greenamyre JT, Young AB: Excitatory amino acids and Alzheimer’s disease. Neirrobiol Aging 1990;10:593-602. 47. Graf E, Mahoney JR, Bryant RG, Eaton J W Iron-catalyzed hydroxyl radical formation. ] Biol Cheili 1984;259:3620-3624. . 48. Imlay JA, Linn S: DNA damage and oxygen radical toxicity. Scieiice 1988;240:1302-1309. 49. Halliwell B: Oxidants in the central nervous system: Some fundamental questions. Is oxidant damage relevant to Parkinson‘s disease, Alzheimer’s disease, traumatic damage or stroke? Actn Neirrol Scniid Siippl 1989;126:23-33. 50. Anderson S, Sankier AT, Barrel1 G, et al: Sequence and organization of the human mitochondria1 genome. Nufirre 1981;90:457-465. 51. BoIhuis PA, Bleeker-Wagemakers NJ, Ponne NJ, et al: Rapid shift in genotype of human mitochondria1 DNA in a family with Leber‘s hereditary optic neuropathy. Biocheni Biopliys Res C O ~ I I I 1990;170:994-997. I~I~II 52. Holt IJ, Miller DH, Harding AE: Genetic heterogeneity and mitochondria1 DNA heteroplasmy in Leber‘s heriditary optic neuropathy. J Med Genet 1989;26739-743. 53. Shoffner JM,Lott MT, Lezza AMS, et al: Myoclonic epilepsy and ragged-red fiber disease (MERRF) is associated with a mitochondrial DNA tRNA’YSmutation. Cell 1990;61:931-937.
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Abstract A profound decrease in activity of the mitochondrial enzyme cytochrome oxidase in blood platelets is a recently identified concomitant of Alzheimer’s disease (AD). We investigated a possible pathogenic link between this finding and the symptoms of AD by mimicking this mitochondrial enzyme deficiency in rats. Rats were infused chronically with a selective inhibitor of cytochrome oxidase, sodium azide, or with saline delivered via subcutaneously implanted osmotic minipumps. The azide treatment impaired both spatial and nonspatial learning. Further, the azide treatment inhibited a lowthreshold form of hippocampal long-term potentiation, primed burst potentiation. The behavioral deficits were not secondary to a sensory or motor impairment. Thus, chronic azide treatment of rats models some characteristics of AD. (I Gerintr Psychid y Neiirol 1992;5:93- 101).
A
lzheimer’s disease (AD) is a progressive neurodegenerative disease marked by a profound impairment of learning and memory and a global cognitive deficit.* There is currently no animal model that exhibits the total pathology associated with AD. Several animal models are being used presently in AD research, each of which mimics specific concomitants of the natural disease. Experimental manipulations to produce these animal models include, for example, lesioning of the central cholinergic system2 and administration of a l ~ r n i n u mAl.~ though no single model produces the full pathology of AD, some of these models have provided useful means for screening therapeutic treatments. Several biochemical markers of mitochondrial dysfunction have been characterized in AD. These correlates of AD include alterations in glucose utilization, alterations in calcium homeostasis, and reReceived April 15, 1991. Received revised July 21, 1991. Accepted for publication August 12, 1991. From the Department of Pharmacology (Drs Bennett and Diamond), and the Departments of Neurology and Pediatrics (Ms Parks and Dr Parker), University of Colorado Health Sciences Center, the Veterans Administration Medical Center (Dr Diamond), and the Department of Biology (Ms Stryker), University of Colorado, Denver, CO. Address correspondence to Dr h1.C. Bennett, Department of Pharmacology, C-236, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denveri CO 80262.
duced mitochondrial activity in skin fibroblasts and in brain.4-9 Recently, Parker et all0 identified a specific site of a mitochondrial enzyme defect in blood platelets of AD patients. These investigators found a profound decrease in the activity of cytochrome oxidase (complex IV), the terminal enzyme in the electron ‘transport (respiratory) chain of mitochondria. Other enzymes of the electron transport chain were spared. Blood platelets are not known to be a target tissue in AD. Therefore, the cytochrome oxidase deficiency in blood platelets may represent a defect that occurs in mitochondria of all cells. The selective nature and widespread occurrence of this mitochondrial enzyme defect identify it as candidate for an early, and possibly pathogenic, defect of AD. One method of addressing the issue of whether cytochrome oxidase deficiency can c m s e the sequelae associated with AD is to examine cytochrome oxidase inhibition in an animal model. In this report, we describe a method for inducing chronic and selective inhibition of cytochrome oxidase in rats via subcutaneous delivery of sodium azide, a selective inhibitor of this enzyme. In the present investigation, we tested the hypothesis that chronic exposure of rats to sodium azide would impair learning and hippocampal plasticity. Hippocampal-dependent learning and hippocampal plasticity were major foci
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of our investigation because the higpocampus is a preferential target of damage in AD. Moreover, the hippocampus has a crucial role in memory formation in humans and other mammal^.'^-^^ Therefore, we studied the effect of azide treatment on the acquisition and performance of behavioral tasks, including a spatial memory task that is known to be hippocampal dependent for Similarly, we investigated the effect of chronic cytochrome oxidase inhibition on hippocampal plasticity. There is considerable evidence that memory formation in humans and other animals involves the capacity to change synaptic strength in the hippocampus."j Long-term potentiation is a long-lasting change in synaptic efficacy that can be induced experimentally in the hippocampus. Long-term potentiation shares many properties with memory and has been used extensively as a physiologic model for memory (see Teyler and Discenna" for review of long-term potentiation). In the present experiment we examined the effect of chronic cytochrome oxidase inhibition on a low-threshold form of long-term potentiation, primed burst potentiation.''-''
Method
Subjects Subjects were adult male Sprague-Dawley rats (Sasco, Omaha, NB) that weighed 350 to 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, except as described for the eight-arm maze protocol. surgery
Under secobarbital anesthesia (40 mg/kg), rats were each implanted subcutaneously with an Alzet 2ML4 osmotic minipump. For each subject, an incision of approximately 1 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 cm by 3 cm into which a minipump was inserted. These minipumps have a 2-mL reservoir and provide a constant infusion rate of 0.5 pg/hr for 28 days. The pumps were filled with either a solution of 160 pg/pL sodium azide in 0.9% saline (azide) or the saline vehicle (control). The azide pumps delivered a dose of sodium azide equal to 400 pg/hr. Sodium azide crystals of 99% purity were obtained from Aldrich, Milwaukee, Wisconsin.
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Tzuo-Way Shuttle Box Task.-Azide and control groups were first trained on a shock-motivated, twoway shuttle box task beginning 6 to 9 days after pump implantation. The behavioral apparatus was an automated shuttle box (Omnitech Columbus, Ohio) which consisted of a straight runway divided into two compartments by a partition with a hole in the center. The rats were individually placed in the left compartment of the shuttle box. The position of the animal was detected by photocells. Prior to the first trial only of each day, the rats were given 5 seconds adaptation time. A trial was begun when a light, the conditioned stimulus, was presented in the opposite compartment to the one in which the animal was detected. Five seconds after the conditioned stimulus presentation, the unconditioned stimulus, a ~OO-JLAscrambled shock, was delivered to the dark compartment. The animals' latencies to escape or to avoid (after the first trial) the shock by crossing to the lighted, safe side were recorded automatically for each trial. An avoidance was treated as a 0-second escape latency. Each subject received 15 trials on four consecutive days. Performance was meaured as the latency to cross to the safe side. The entire apparatus was cleaned with 70% ethanovwater between subjects. Operz Field Activity.-The day following the completion of shuttle box training, the azide and control groups were assessed for motor function. Spontaneous activity of the rats was tested in an open field apparatus. Each subject was placed in a Plexiglas box (120 X 120 cm) with a grid of 16 rectangles (30 X 30 cm) marked on the floor. The number of spontaneous line crossings for each subject was recorded for a 5-minute period. The box was cleaned with a 70% ethanolltvater solution between subjects. Test.-Following the open field test, the sensory function of these rats was measured using the flinch-jump test which is a psychometric assessment of threshold responses to footshock. Each rat was given six series (three ascending and three descending) of discrete footshocks (0.5 seconds), ranging from 100 pA to 1000 pA in increments of 100 pA. The intershock interval within a series was 30 seconds, and the interval between series was 2 minutes. The flinch and jump thresholds for each rat were calculated as the amount of current required to elicit those respective responses in 50% of the trials. Fliricli-Jziniy
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Cytochrome Oxidase Inhibition
Eight-Am &did Mnze.-Two additional groups of rats were tested on a spatial task, the eight-arm radial maze. Azide and control groups were implanted with osmotic minipumps 7 days prior to the first day of training. All behavioral experimentation took place between 1 :00 PM and 5: 00 PM using a modification of the method of Barnes et al.’l The testing room was dimly lit with a small source of natural light. There were conspicuous maze-fixed cues in the room and on the walls. The experimenter was blind to the treatment. On the 4th, 5th, and 6th days after pump implantation, each rat was given daily 10-minute acclimation sessions on the eight-arm radial maze in which the rat was free to explore the maze, which was scattered with Fruit Loops cereal broken into quarter pieces. Any rat that did not eat any of the cereal during the acclimation period was not used in the training trials. Twenty-four hours prior to training, the rats were placed on a mild food-deprivation schedule in which each animal was given access to 10 to 12 g of rat chow per day. Rats were weighed daily and no animal was allowed to fall below 80% of its prefasting weight. Beginning on the 8th day after surgery, the rats were placed individually in the radial maze and given a 10-minute trial for each of 12 days. A well in the end of each arm was baited with one quarter of a piece of cereal. A correct choice was one in which the rat entered the baited arm and ate the food. An error was either the entry of the rat into an arm without consuming the bait, or the reentry of a rat into an arm from which it had already eaten the bait. Hippocampal Plasticity A total of six control and six azide rats were used in the electrophysiologic experiment. Two control and two azide rats from the radial maze experiment were used for physiology recordings after being returned to ad libitum access to food for 3 to 5 days. The remaining four rats per group were implanted specifically for the physiology experiment. Recordings from all subjects took place in the 3rd and 4th weeks after pump implantation in order for the duration of azide or saline exposure to be approximately the same for all subjects. The rats, which weighed 350 to 450 g at the time of recording, were removed from the vivarium on the day prior to the recording and individually housed within the laboratory.22 Between 9 : O O AM and 1O:OO Ahi on the day of recording, atropine methyl nitrate was administered (0.2 mgkg, ip) followed by urethane (range, 1.3 to 1.5 g k g , ip). The recording method has been described.= Briefly, the recording. electrode (etched
epoxylite-insulated tungsten; impedance, 0.5 to 1.0 Mohms at 1 kHz) was lowered to the CA1 pyramidal cell layer, which was identified by complex spike activity occurring between 1.8 and 2.3 mm below brain surface. The stimulator electrode (125-pm-diameter stainless steel Teflon-coated wire uninsulated at the tip) was lowered into the left side of the hippocampal commissure (coordinates: AP, -1.8; ML, 1.0). The final coordinates were determined by optimizing the amplitude of the population spike, which is a measure of synchronous firing of CA1 cell activity.24 Population spike responses were displayed on an oscilloscope and stored in an Epson computer for on-line and off-line analysis. Test responses were evoked by constant-current single-pulse stimuli (150-ps duration) presented every 30 seconds for 5 minutes before and 30 minutes following primed burst (PB) stimulation. PB stimulation consisted of a single pulse followed 170 ms later by a burst of four pulses at 200 Hz. PB stimulation current levels were 175% of the current levels used in test pulses. We have reported in previous work that this level of PB stimulation induced PB potentiation in all control subjects.23 Statistical analyses (f-test; P I.05) of the changes in the population spike amplitude were performed for each subject by comparing responses obtained during the 10-minute period immediately prior to PB stimulation to those obtained during the 21- to 30-minute period after PB stimulation. Group comparisons were performed using a repeated measures MANOVA. Assay for Respiratory Chain Enzyme Activity The rats used in the shuttle box task were sacrificed by decapitation approximately 2 weeks after pump implantation and assayed for activities of respiratory chain complexes I through IV. Whole brains except for the cerebellum and hindbrain were used for mitochondrial extraction. Cell body and synaptic membrane mitochondria were isolated by the method of Clark and Nicklas.= Respiratory enzyme complexes were assayed as follows: Complex I (reduced nicotinamide-adenine dinucleotide [NADH]: ubiquinone oxidoreductase) was assayed spectrophotometrically in sonicated mitochondria by following the decrease in the absorption of NADH26 using 60 pmoVL Q1 and 2 mmol/L KCN. Complex IU complex I11 (succinate: cytochrome c oxidoreductase) were assayed as described by Stumpf and Parks.27 For complex I and complex II/III, activity has been expressed as pmlminlmg of mitochondrial protein. Complex IV (cytochrome oxidase) was assayed as a cyanide-sensitive ferrocytochrome . c oxidase in
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cholate-solubilized mitochondriaz6in 20 mmoVL potassium phosphate, pH 7.0, and 25 pmoVL reduced cytochrome c reductase prepared by ascorbate reduction and purified over Sephadex G-50. The oxidation of reduced cytochrome c was followed at 550 nm. Cytochrome oxidase activity has been expressed as a first-order rate constant (rate constant/s/mg mitochondrial protein).
Results
Respiratory Chain Enzyme Activify Respiratory chain enzyme activities were assayed from mitochondria of both cell bodies and synaptic membranes of control and azide rat brains. Chronic azide treatment produced a significant inhibition of cytochrome oxidase in both subcellular compartments: a 35%decrease in the cell body mitochondria and a 39% decrease in the synaptic membrane mitochondria (Figure 1). Based on a previous investigation of dose- and time-response characteristics of respiratory chain enzyme activity to chronic azide treatment, this level of inhibition represents a steady-state degree of inhibition that is reached within 48 hours of pump i m p l a n t a t i ~ n .Azide ~~
treatment did not significantly alter the activity of any other respiratory chain enzyme complex.
Behavior Sluttfle Box Tusk.-Overall, the azide group was significantly impaired with respect to the control group in escape latency (repeated measures MANOVA main effect of treatment: F[1,13] = 8.79; P = 0.01) (Figure 2). For the control group, escape latency for the last day of training was significantIy lower than that of the first training day (two-tailed Student’s t = 2.02; P < .05), indicating an improvement of performance over trials. In contrast, the azide group did not show a significant decrease in escape latency over days (first day versus last day: t = 0.11; P > .1), indicating no improvement in performance. Seiisory and Motor Tests.-The data from the motor and sensory assessments are summarized in Table 1. There was no significant difference in spontaneous activity in an open field between the azide and the control groups (t = 0.28; P > .1). Pain sensitivity of the rats was then assessed using a flinch-jump test. There was no evidence that the two groups differed in their responses to footshock (flinch: t = 0.20; P > .l; jump: t = 0.03, P > .1).The lack of signifi-
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FIGURE 2 Shuttle box task in seven-azide-treated and eight control rats. Data are expressed in blocks of 15 trials denoting the mean (k SEM) daily performances. The repeated measures MANOVA (derived from the individual scores) revealed significant main effect of treatment (F[1,13] = 9.21; P < .Ol), significant main effect of time (F[11,143] = 2.59; P < .Ol), and a significant treatment by time interaction (F[11,143] = 2.70; P < .05). * denotes trial block significantly different from first trial block (t = 2.02; P < -05).
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Cytochrome Oxidase Inhibition TABLE 1 Motor Activity and Sensitivity to Footshock in Azide-Treated and Control Rats Spontaneous Activity Footshock Sensitivity N 7 8
Azide Control
Line Crossings' 47.6 8.13t 50.0 L 2.81
Flinch' 225 23.lt 230 9.33
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'Mean % SEM. tNot significantly different from control value.
cant differences between the two groups on these measures suggests that the behavioral deficits can not be attributed to a sensory or motor deficit.
Eight-Arm Xrrdial Maze.-The performance of each group on the eight-arm radial maze as measured by the mean daily correct choices is shown in Figure 3. There was a significant azide-induced impairment of performance on this task (repeated measures MANOVA main effect of treatment: F[1,16] = 5.06; P < .05). Although the azide group showed an impairment of acquisition, the two groups reached asymptote performance levels that did not differ significantly. Both groups showed significant improvement in performance over trials, which indicates that both groups showed learning. Overall, there was no statistical difference be-
tween the two groups in the number of errors during acquisition of the spatial task (repeated measures MANOVA: F[1,16] = 2.12; P = .16) (Figure 4). However, the control group, unlike the azide group, exhibited a steady decline in the number of errors over the first 5 days of training. The decrease in the number of errors by the control group reached statistical significance on day 5 (t = 3.31; P < .01, day 1 versus day 5). In contrast, the decrease in the number of errors for the azide group did not reach statistical significance until day 7 (f = 3.98; P < .01, day 1 versus day 7). The lag in acquisition by the azide group provides a second measure of a significant learning impairment in this group. Thus, by both measures of performance, ie, correct choices and errors, azide rats were impaired in spatial learning. In addition, the finding that the two 15
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FIGURE 3 Eight-arm radial maze correct choices for nine azidetreated and nine control rats. Mean (* SEM) number of correct choices per group in daily 10-minute trials. For trials 1 through 12, repeated measures MANOVA main effect of treatment, F(1,16) = 5.04; P < .05; main effect of time, F(11,176) = 32.77; P < .01; treatment by time interaction, F(11,176) = 1.19; P > .1). For trials 1 through 6 (acquisition), repeated measures MANOVA main effect of treatment, F(1,16) = 5.61; P < .05; main effect of time, F(5,80) = 19.52; P < .01; treatment by time interaction, F(5,80) = 0.43; P > . l .
1
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FIGURE 4 Eight-arm radial maze errors for nine azide-treated and nine control rats. Mean (2 SEM) number of errors per group in daily 10-minute trials. Repeated measures MANOVA for trials 1 through 12: main effect of treatment, F(1,16) = 2.12; P > .l; main effect of time, F(11,176) = 17.61; P < .01; treatment by time interaction, F(11,176) = 0.84; P > -1. Compared with the first day of training, the first day in which there was a significant decrease in error number was day 5 for the control group (t = 3.31; P < .01) and day 7 for the azide group (t = 3.98; P < .01). Thus, the decline in errors for the azide group lagged that of the control group by 2 days of training.
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groups reached the same level of performance by both of these measures indicates that the azide rats were neither failing to move about the maze nor were they hypermotor. This result provides additional evidence that the azide-induced impairments on the behavioral tests reflect a learning deficit rather than a motor impairment. Hippocampal Plasticity Azide treatment also significantly impaired the expression of a low-threshold form of long-term potentiation in the hippocampus, PB potentiation. We define PB potentiation as the long-term component of plasticity occurring after the tetanizing stimulus. In the present experiment, it was measured in the 21- to 30-minute interval after the stimulus train was delivered. The measure of the magnitude of the hippocampal response to the test stimuli was the magnitude of the population spike evoked response. The population spike reflects the synchronous firing of the CA1 hippocampal pyramidal cells. Population spike data recorded from the CAI cell layer of the hippocampus of azide and control rats are summarized in Figure 5.
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FIGURE 5 Primed burst potentiation in six azide-treated and six control rats. Data points are mean (? SEM) magnitude of population spike evoked potentials. Patterned stimulus train was delivered at time zero. Baseline current intensity: for the azide group was 69 PA 2 10.3; for the control group, 62 pA rt 10.2 (azide versus control: t = 0.48; P > .1). Baseline magnitude of population spike for the azide group was 1.56 mV 2 0.10; for the control group, 1.65 mV f 0.17 (azide versus control: t = 0.45; P > .1). Repeated measures MANOVA (sampled at 2-minute intervals): main effect of treatment, F(1,lO) = 6.83; P < .05; main effect of time, F(19,190) = 16.51; P < .01; treatment by time interaction, F(19,190) = 1.91; P < .05).
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There was an overall main effect of treatment for the two groups (repeated measures MANOVA: F[1,10] = 6.83; P < .OS), with the azide group showing the lower response magnitude. There was no significant difference between the groups in the short-term component of plasticity, posttetanic potentiation: Evoked responses during the first minute after the tetanizing stimulus did not differ significantly between groups (t = 0.23; P > .1). The difference between the two groups occurred in PB potentiation, the long-term response. Although both groups showed significant PB potentiation, the response of the control group stabilized at approximately 200% over baseline, while the response of the azide group stabilized at approximately 60% over baseline. . There were no significant differences between the azide and control groups either in magnitude of baseline population spikes or in the stimulus current intensities used to evoke the baseline responses (data reported in Figure 5 caption). These results, together with the lack of a difference in posttetanic potentiation magnitudes between the two groups, indicate that the azide-treated rats exhibited normal synaptic transmission and short-term plasticity. Discussion Azide treatment impaired performances on both behavioral tasks tested in the present investigation. One of the tests, the two-way shuttle box task, is relatively insensitive to hippocampal damage. The other task, the radial maze, is a sensitive measure of hippocampal damage.I4 These findings suggest that chronic azide treatment produces a generalized learning deficit. In addition, subjects were examined on measures of sensory and motor function. These tests did not reveal any significant differences between the azide-treated and the control groups. The results of these control studies provide evidence that the impaired performance of the azide group reflects a learning impairment rather than a sensory or motor deficit. We also examined the effect of azide treatment on a form of hippocampal plasticity, PB potentiation. PB potentiation is a form of hippocampal longterm potentiation for which the induction threshold is lower than it is for conventional long-term potentiation. The basis of the reduced threshold for PB potentiation is the use of patterned stimulation that mimics the temporal components of endogenous rhythms of hippocampal activity.*'-'' The results of the electrophysiology experiment paralleled the re-
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Cytochrome Oxidase Inhibition sults of the radial maze experiment: Azide treatment impaired, but did not abolish, both hippocampal PB potentiation and spatial learning. This finding may be related to the relatively short duration of azide exposure prior to testing. Rats treated with azide for 1 to 4 weeks prior to testing may be analogous to patients in the early stages of AD. Although mitochondrial respiration occurs in every cell, there is evidence of differential sensitivity to inhibition of the respiratory chain among neuronal subpopulations. Some cell types of the hippocampus, for example, are more vulnerable to cell death after hypoxia and i~chemia,’~ both of which produce intermediary metabolic consequences similar to respiratory inhibition. There is also evidence to suggest that a specific dysfunction of cytochrome oxidase could cause differential damage in the central nervous system. In recent work, Eells and her coll e a g u e ~have ~ ~ shown that the neurotoxic effect of methanol positioning is secondary to cytochrome oxidase inhibition and that there are regional differences in the vulnerability of the retina to this toxin. Cytochrome oxidase is not distributed uniformly in the nervous system. There is also recent evidence that the heterogeneous pattern of cytochrome oxidase distribution corresponds to the pattern of susceptibility of brain damage in AD. Chandrasekaran et al,3’ from the laboratory of S.I. Rapoport, recently reported evidence that brain regions preferentially afflicted in AD have a higher expression of mRNA for a homologous sequence of the mitochondrial cytochrome oxidase subunit I gene than do brain regions that are relatively spared in AD. The latter two independent lines of evidence30831support the hypothesis that certain brain systems may be differentially susceptible to a defect of cytochrome oxidase. The proposal that a specific mitochondrial enzyme produces a distinctive pattern of brain damage gains support in related observations emerging from the study of Parkinson’s disease. A selective inhibition of complex I of the respiratory chain is a characteristic of the blood platelets in patients with Parkinson’s disease.32 Further, 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP), which produces selective damage to the basal ganglia and Parkinsonlike symptoms is also an inhibitor of complex I.33-36 Consistent with this interpretation, Alpers’ disease and Menkes‘ syndrome, which are both characterized by abnormalities of cytochrome oxidase, have prominent cortical symptom^.^' Despite differences between the clinical manifestations of Alpers’ disease and Menkes’ syndrome compared with AD,
families of inborn (metabolic) errors can produce both clinical and biochemical heterogeneity. Patients with abnormalities of the same gene product can have clinically distinguishable syndrome^.^' We have not yet investigated the mechanism@) for the learning and neurophysiologic impairments produced by depletion of cytochrome oxidase. Dysfunction of cytochrome oxidase can produce numerous changes in cellular metabolism that may contribute to the deficits produced by azide treatment. A discussion of some of these candidate mechanisms follows. First, under normal conditions, electron transport in the mitochondria is coupled to oxidative phosphorylation; thus, a reduction in the efficiency of the electron transport chain also inhibits the phosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP). The inhibition of aerobic respiration also shifts a greater percentage of glucose catabolism, at the stage of pyruvate, to the anaerobic pathway ending in lactate. This metabolic pathway yields less ATP production than does the aerobic Therefore, one effect of respiratory inhibition is a reduction of cellular energy storage that would be predicted to impair cellular function. Second, the inhibition of the mitochondrial respiratory chain also shifts the equilibrium potential from oxidized nicotinamide-adenine dinucleotide (NAD+) to NADH, as NADH is unable to donate its electrons to the chain. The activity of numerous enzymes of intermediary metabolism is linked to the bioavailability of NAD+. Pyruvate dehydrogenase is an NAD+-linked enzyme, and an impairment of pyruvate dehydrogenase decreases acetylcholine synthesis.4o A decline in acetylcholine synthesis and release has been shown to exist under conditions of mild hypoxia and h y p ~ g l y c e m i aand ~ ~ to be associated with aging.42A profound decrease in acetylcholine is a well-described concomitant of AD and may underlie part of the learning and memory deficits of AD.43 Third, the oxidative deamination of glutamate on its entry into the Krebs cycle as a-ketoglutaric acid also requires the presence of the NAD+-linked glutamate dehydrogenase.4 A decrease in NAD+ associated with the inhibition of the electron transport chain might be predicted to increase pools of glutamate. Hypoxia, hypoglycemia, and ischemia have been shown to increase the release of glutamate and aspartate, both excitatory amino acids, and to increase free radical formation.45 These consequences may underlie the delayed loss of neurons ,
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found after ischemia. Further, prolonged exposure to elevated Ievels of glutamate may lead to N-Methyl-D-aspartate-mediated excitotoxicity, which has been proposed to play a role in the pathogenesis of
flecting this aspect of AD should prove to be a valuable tool in screening treatments to mitigate damage resulting from this enzyme defect.
~ ~ - 2 9 ~ 3 6
When the activity of cytochrome oxidase is insufficient to catalyze completely the final reduction step of molecular oxygen to HzO, high-energy electrons are diverted from the electron transport chain. These electrons are associated with active oxygen species, such as the superoxide and hydroxyl radicals, which are c y t o t o x i ~ . ~ 'Free * ~ ~ radical damage has also been proposed to contribute to neurodegeneration in AD.49 The determination of mechanisms by which a cytochrome oxidase deficiency might develop in humans is of considerable clinical significance. The majority of cases of AD is sporadic, ie, appears to be nonfamilial. Three subunits of cytochrome oxidase are encoded in the mitochondria1 genome.50 Mitochondrially encoded defects can appear to be sporadic if the maternal mitochondria are heterop l a ~ m i c . ~ Therefore, '-~~ mitochondrial genetic transmission might account for the non-Mendelian pattern of occurrence of most cases of AD. Alternatively, the present finding that the toxin sodium azide selectively inhibits cytochrome oxidase and produces a cognitive deficit suggests the possibility that similar environmental toxins may be pathogenic. For example, cyanide and carbon monoxide are also selective inhibitors of cytochrome oxidase, and these toxins are components of air pollution and of cigarette smoke. It is likely that a threshold decrease of cytochrome oxidase activity is required before debilitating effects are manifested. Therefore, a toxic load might vary by individual according to genetic or other susceptibility to environmental toxins. In summary, azide-treated rats were significantly impaired on two measures of learning: an aversively-motivated shuttle task and an appetitively-motivated spatial task. The deficit does not appear to be secondary to a sensory or a motor impairment. Furthermore, azide-treated rats did not develop the degree of PB potentiation shown by the control rats. Taken together, these data indicate that chronic azide treatment produces a generalized learning deficit and impairs hippocampal plasticity in rats. These findings are consistent with the hypothesis that a defect of cytochrome oxidase has an etiologic role in AD. Regardless of whether or not the cytochrome oxidase deficiency found in AD is pathogenic, the development of an animal model re-
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Acknowledgments This project was approved by the institutional animal use committee and camed out in accordance with institutional animal care guidelines. This research was supported in part by NIH grants NSOlM7 and NS25382 (W.D.P. Jr) and by a Mental Retardation Research Center grant from NIH. The authors gratefully acknowledge Dr Greg Rose for the generous use of his equipment for this experiment (equipment and supplies purchased from NSF grant BNS8811486 and a grant from the Veterans Administration Medical Research Service) and for his valuable critique of this manuscript. We also thank'Dr Karen Stevens for her extensive data analyses and instruction in SPSS and her helpful comments on this manuscript.
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