Original Contributions
Spatial Learning and Memory Deficits Induced by Dopamine Administration with Decreased Glutathione

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Abstract

Administration of buthionine sulfoximine (BSO) selectively inhibits glutathione (GSH) biosynthesis and induces a GSH deficiency. Decreased GSH levels in the brain may result in less oxidative stress (OS) protection, because GSH contributes substantially to intracellular antioxidant defense. Under these conditions, administration of the pro-oxidant, dopamine (DA), which rapidly oxidizes to form reactive oxygen species, may increase OS. To test the cognitive behavioral consequences of decreased GSH, BSO (3.2 mg in 30 μl, intracerebroventricularly) was administered to male Fischer 344 rats every other day for 4 days. In addition, DA (15 μl of 500 μM) was administered every day [either 1h after BSO (BSO + DA group) or 1h before BSO (DA + BSO group), when given on the same day as BSO] and spatial learning and memory assessed (Morris water maze, six trials/day). BSO + DA rats, but not DA + BSO rats, demonstrated cognitive impairment compared to a vehicle group, as evidenced by increased latencies to find the hidden platform, particularly on the first trial each day. Also, the BSO + DA group utilized non-spatial strategies during the probe trials (swim with no platform): i.e., less time spent in the platform quadrant, fewer crossings and longer latencies to the previous platform location, and more time spent around the edge of the pool rather than in the platform zone. Therefore, the cognitive behavioral consequences of decreasing GSH brain levels with BSO in conjunction with DA administration depends on the order of administration. These findings are similar to those seen previously on rod and plank walking performance, as well as to those seen in aged rats, suggesting that the oxidation of DA coupled with a reduced capacity to respond to oxidative stress may be responsible for the induction of age-related cognitive deficits.

Introduction

Oxidative stress (OS) is thought to be a contributing factor to the decrements in cognitive and/or motor performance seen in aging. The “free radical hypothesis of aging” maintains that, with age, the generation and accumulation of reactive oxygen species (such as superoxide and hydroxyl radicals) result in oxidative damage to critical biological molecules, which coupled with the insufficiency of endogenous antioxidant defense mechanisms, contributes to the detrimental effects of aging.1, 2There is evidence to suggest that several indices of antioxidant protection may be reduced in aging. For example, a glutathione (GSH) deficiency has been observed in all tissues in senescent animals,[3]including the brain;[4]a decrease in glutathione reductase and an increase in oxidized glutathione (GSSG) levels have been seen in the brains of 24-month old mice;[5]and reduced glutamine synthetase (an enzyme very sensitive to oxidative damage) was observed in aging.[6]The brain may be particularly vulnerable to the deleterious effects of oxidative damage because it is relatively deficient in free radical protective antioxidant compounds, utilizes high amounts of oxygen, and contains high concentrations of iron and easily peroxidizable fatty acids.7, 8

Glutathione is an endogenous protective agent that plays a critical role in intracellular antioxidant defense. GSH is present in millimolar concentrations in most tissues, including the brain.[9]GSH acts to scavenge reactive oxygen species and is capable of neutralizing free radicals generated by OS.[10]Depletion of GSH in the brain may result in free-radical induced neuronal damage by causing an excess of H2O2 and alkyl peroxides, which can cause oxidative damage to DNA, proteins, and other macromolecules.[1]Because brain GSH may be decreased in aging,4, 5endogenous protection against free-radicals may also be decreased with age.

Buthionine sulfoximine (BSO) is a compound that is known to selectively inhibit γ-glutamylcysteine synthetase, a key enzyme in GSH biosynthesis, and thus lower GSH concentration;[11]therefore, treatment with BSO may lower the protective antioxidant capabilities of the organism to make it more susceptible to neuronal damage by OS, similar to that seen in aging. In addition, administration of the oxidative stressor, dopamine (DA), when GSH levels are compromised, has been found to increase OS, because DA rapidly oxidizes to form reactive oxygen species.12, 13Conditions that increase concentration and/or turnover of DA should increase the potential for the formation of reactive metabolites, especially under conditions in which the ratio of available DA to antioxidant capacity is high.[14]

While Hastings and colleagues12, 13, 15have characterized the reactive oxygen species formed by DA oxidation, there is little information on how these compounds may alter behavior under decreased GSH conditions, such as those seen in aging. One of these is deficits in cognitive function. Memory alterations appear to occur primarily in secondary memory systems, possibly due to the decline in the functioning of the central cholinergic system, and are reflected in the storage of newly acquired information.16, 17Both aged rats and mice show decrements in spatial memory performance, i.e., the ability to acquire a cognitive representation of location in space and the ability to effectively navigate the environment (for reviews see 18, 19, 20, 21). The Morris water maze (MWM) is a known, conventional test of spatial learning and memory; behavioral performance of aged rats is impaired in this paradigm, reflecting a deficit in the ability to utilize spatial information.19, 20, 21

The present study was carried out to examine cognitive behavioral performance using the MWM in animals treated with BSO and DA. We hypothesized that injections of BSO plus DA would be a simple and effective method to lower protection, produce OS, and alter MWM performance in rats.

Section snippets

Animals

Twenty-nine male Fischer 344 rats (Harlan Sprague Dawley, Indianapolis, IN), 6–7 months of age, weighing between 300–400 g, were used in this study. The rats were maintained on a 12 h light/dark cycle and individually housed in hanging wire mesh cages, except during testing when they were housed in plexiglass cages with wood shavings as bedding.

Drug Treatment

Since BSO does not readily cross the blood-brain barrier in adult animals when administered systemically,10, 22, 23it was administered

Behavioral results for the BSO + DA, DA + BSO, and Vehicle Groups

BSO given prior to DA administration selectively impaired cognitive behavior; however, in the reverse condition (DA + BSO), no decrements in performance were observed relative to vehicle administration. There was significant improvement over time in water maze acquisition (learning) performance (i.e., latency to find the platform) on days 1–3 [F(15,210) = 3.20, p < .01]; this result was due to improvements in the DA + BSO and vehicle groups only (p ≤ .05). However, there were no differences in

Discussion

This study showed that depletion of GSH with BSO followed by DA treatment selectively produced deficits in spatial learning and memory behavior; however, in the reverse condition (DA + BSO), no decrements in performance were observed relative to vehicle administration. Additionally, reduction of OS protection via depletion of GSH did not in itself affect cognitive behavior, since treatment with BSO alone did not have an adverse effect on performance, even though GSH was decreased similarly in

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