Reactive Oxygen Species in the Regulation of Synaptic Plasticity and Memory

1. Complexes I and III

Complex I, also known as NADH ubiquinone oxidoreductase, is a trans-mitochondrial membrane complex that oxidizes a previously reduced NADH using coenzyme Q10 as the electron acceptor (398). Complex I is the major site of entry of reducing equivalents, followed closely by complex II (398). It is coupled to proton pumping, specifically pumping four protons across the inner mitochondrial membrane, from the matrix to the intermembrane space, thus contributing to the proton gradient that will later fuel the ATP synthase (239). Complex I is the major site of the premature leak of electrons to oxygen, thereby creating the free radical superoxide (469).

Complex III contains two pools of ubiquinone: Q_i faces the mitochondrial matrix and Q₀ faces the intermembrane space (93, 394). Complex III catalyzes the reduction of ubiquinone in a stepwise fashion. First, two electrons are removed from ubiquinone (Q₀) and are passed on to two molecules of cytochrome c to eventually reach complex IV. In a second step, two other electrons are passed on to the Q_i site to reduce quinone to quinol. In total, this sequential reaction releases four protons (398). Complex III participates in the generation of the proton gradient across the mitochondrial membrane by an asymmetric absorption/release of protons rather than pumping protons through the membrane. In terms of ROS production, complex III can leak electrons and participate in superoxide formation (394). It is believed that if the electron transfer from ubiquinone to cytochrome c is delayed for any reason, ubiquinone tends to auto-oxidize and release an electron that is free to attack oxygen for superoxide formation (146).

Animal models carrying mutations in the various components of the electron transport chain, as well as pharmacologic inhibitors, have proven very useful in coupling effects with sources of ROS. We focus the remainder of this section on discussing complexes I and III as potential sources of superoxide involved in modulating learning and memory.

2. Mitochondrial superoxide in learning and memory

Several studies have suggested that ROS of mitochondrial origin might impact signaling during synaptic plasticity and memory, although the studies are at best suggestive. The exact source of ROS during synaptic plasticity has yet to be determined. For example, treatment of rat isolated mitochondria with elevated levels of sodium and Ca²⁺ (thus mimicking the events happening during depolarization of neurons and synaptic transmission of neuronal signals) leads to increased production of superoxide (116). In addition, application of glutamate to cultured forebrain neurons activates NMDA receptors and induces an increase in ROS production that can be blocked by the NMDA receptor channel blocker MK801 (362). NMDA receptor-dependent superoxide production also occluded further superoxide production after uncoupling of mitochondrial proton transport with the inhibitor p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (51). Although the authors of the latter study concluded that their data provided evidence for mitochondrial superoxide production after NMDA-receptor activation, one should not rule out the possibility that p-trifluoromethoxy carbonyl cyanide phenyl hydrazone could be uncoupling proton transport through NADPH oxidase as well (4). Other investigators (113) have used specific inhibitors of complexes I (rotenone) and III (antimycin A) to demonstrate that NMDA receptor-dependent ROS production in cortical neurons originates in the mitochondrial electron transport chain. Although these studies suggest a link between mitochondrial superoxide and synaptic transmission that is essential for plasticity, they have not directly investigated the involvement of mitochondrial ROS in synaptic plasticity.

Perhaps the most direct evidence linking mitochondrial superoxide to synaptic plasticity and memory comes from studies demonstrating that large stimulus-induced increases in cytosolic Ca²⁺ result in production of superoxide by mitochondria (181, 183). This superoxide acts as a modulator of two kinases, CaMKII and PKA, both known to be involved in the induction of synaptic plasticity. On another hand, in the course of investigating the function of mitochondria in CD8⁺ T cell functioning, it was demonstrated that incubation with rotenone, a widely used inhibitor of complex I, leads to decreased production of H₂O₂, Ca²⁺ influx, and ERK phosphorylation (457), another kinase known to be involved in synaptic plasticity (194, 210).

A counter argument to the suggestion that mitochondrial ROS may be involved in synaptic plasticity comes from behavioral and electrophysiological studies in transgenic mice overexpressing SOD-2. These mice have lower levels of mitochondrial superoxide; however, the mice do not exhibit deficits in either LTP or in learning and memory (188), suggesting that mitochondrial superoxide does not contribute in a major way to synaptic plasticity and memory formation under normal physiological conditions. In contrast, mitochondrial superoxide plays a major role in memory dysfunction associated with neuropathological conditions such as aging and AD (115, 277), both discussed later in the review.

Thus, the evidence at this time suggests that there is minimal involvement of mitochondrial ROS in synaptic plasticity and memory, and that other sources of ROS are likely to be involved in these processes.

1. Structure and regulation of the NADPH oxidase

NADPH oxidase first was identified as a membrane-bound enzymatic complex of the phagosome (24, 240) (Fig. 7). It is made up of six subunits: One Rho-GTPase (usually Rac 1 or Rac 2) and five “phox” subunits (phox stands for phagocytic oxidase): gp91^phox (aka NADPH oxidase 2 [Nox2]), p22^phox, p40^phox, p47^phox, and p67^phox. In its latent status, gp91^phox and p22^phox are membrane-bound, whereas the four remaining subunits are cytosolic (24, 240). Upon stimulation, the cytosolic subunits translocate to the membrane to form a complex with the membrane-bound components (240, 404). The assembly of all the subunits yields an active enzyme that catalyzes the oxidation of NADPH into NADP⁺, releasing an electron in the process (240, 404). The electron is coupled to oxygen to generate superoxide. gp91^phox is the catalytic core of the enzyme, responsible for the transmission of an electron to oxygen (240, 404). However, the presence and correct positioning of all subunits is needed for this transfer to occur. p67^phox and Rac act as the activators of gp91^phox, whereas p47^phox acts as the organizer, ensuring that all subunits are properly aligned for optimal function (240, 404). Thus, the regulation of NADPH oxidase occurs via complex interactions between the various subunits, and proper activation and alignment of all subunits is required for full function of the enzyme.

NADPH oxidase also is regulated by multiple signaling pathways. Many of these regulatory pathways also are important for synaptic plasticity and memory (83, 98). Most notably, Ca²⁺ influx, particularly store-operated Ca²⁺ entry, has been shown to play a critical role in the activation of NADPH oxidase and the subsequent production of superoxide in neutrophils (62). Given the important role of Ca²⁺ in synaptic plasticity and memory, Ca²⁺-dependent NADPH activation and superoxide production could very likely be occuring at synaptic terminals, especially since NADPH oxidase recently was found to be localized to neuronal structures in multiple brain areas, including the hippocampus (216, 373, 415). Such regulation and localization suggest a direct role of NADPH oxidase in synaptic plasticity and memory and will be discussed in further depth in the following two sections.

2. NADPH oxidase in the brain

NADPH oxidase is classically linked to respiratory bursts of superoxide serving as host defense against bacteria in polymorphonuclear neutrophils (24, 240). This function and localization of NADPH oxidase have been studied extensively since it was first described over 40 years ago. However, recent findings indicate that this classical view of NADPH oxidase being exclusively an immunological enzyme as being outdated. Instead, in the past decade substantial evidence has accumulated indicating that NADPH oxidase has nonphagocytic functions. These extra-phagocytic functions of NADPH oxidase include regulating cellular growth and death, regulating endothelial function, and mediating intracellular signaling (154, 356).

The first nonphagocytic NADPH oxidase was discovered in 1999 in vascular smooth muscle cells and was named Nox1. This discovery was followed during the next 10 years by a large body of research investigating the existence and function of nonphagocytic Nox (402). We now know of the existence of at least seven different Nox isoforms (Nox 1–5 and Duox 1 and 2). Although structurally very similar and related, each of these isoforms is expressed in different locations and serves a different cellular function. Of particular interest to this review is the discovery that NADPH oxidase can be localized to multiple brain structures, most notably the hippocampus and amygdala (216, 373), which have primary roles in the formation and maintenance of memory.

NADPH oxidase in neurons was first demonstrated by Tammariello and colleagues, who showed that Nox2 subunits were expressed in rat isolated sympathetic neurons in culture. Expression of Nox2 was shown to be necessary for a proper apoptotic response after insult by nerve growth factor withdrawal (409). Shortly after this study, two immunohistochemical studies in mouse and rat tissue extensively characterized the distribution of various NADPH oxidase subunits in the central nervous system (216, 373). Using a variety of antibodies to stain mouse brain sections, widespread distribution of p40^phox, p47^phox, p67^phox, p22^phox, and gp91^phox was observed in neurons in all regions of the neuraxis. Particularly prominent regions included the cortex, striatum, thalamus, hippocampus, and amygdala, suggesting that NADPH oxidase may play a role in both normal neuronal function as well as neurodegeneration in the brain. Similar experiments in rat brain tissue yielded results showing strong p47^phox and Nox2 immunoreactivities in the cortex and hippocampus (373). In contrast, no particular localization of NADPH oxidase subunits was observed in either the thalamus or the amygdala, but some immunoreactivity was observed in cerebellar neurons. Nox4 and Nox5 were also found localized to the brain, particularly to the cortex, cerebellum, and hippocampal pyramidal cells (424). Nox3 was found highly expressed in the inner ear (27), and finally, a fully functional NADPH oxidase was observed in the lens epithelium (354). Besides their localization to the brain, little is known about the regulation of these different Nox isoforms and whether they are involved in ROS signaling in the brain.

Tejada-Simon and colleagues further characterized the mouse hippocampal NADPH oxidase and found that all of its subunits (including Nox2) are present in cell bodies and dendrites of hippocampal neurons. They also found that these subunits were enriched in synaptoneurosome preparations and that p67^phox colocalized with synaptophysin, suggesting that NADPH oxidase is localized at synaptic sites (415). Finally, the authors also showed that hippocampal NADPH oxidase is fully functional, producing superoxide in response to stimulation with phorbol esters (415).

Taken together, these studies clearly demonstrate localization of a functional NADPH oxidase in hippocampal neurons, suggesting that this enzyme might be the source of superoxide required for LTP and memory formation, a subject addressed in the following section.

3. NADPH oxidase in synaptic plasticity

We have argued that given its localization to key brain structures and to its functionality, NADPH oxidase is a prominent candidate as a source for ROS production during synaptic plasticity and memory formation. Another important feature is that NADPH can produce large amounts of superoxide in a very well-controlled manner (349). Thus, ROS production by NADPH oxidase is a punctuate event that can be turned on or off rapidly and specifically in response to particular extracellular stimuli and subsequent signaling events. Signaling events of particular interest to this review include the NMDA receptor-dependent activation of ERK. This type of signaling has been extensively shown to be involved in various forms of synaptic plasticity and memory (340, 405). Inhibiting NADPH oxidase with diphenylene iodonium (DPI) resulted in the inhibition of NMDA receptor-dependent ERK activation, suggesting a direct role for NADPH oxidase in this type of signaling that is associated with synaptic plasticity and memory (225). This observation was supported by studies in transgenic mice lacking the p47^phox subunit. These mice do not have a functional NADPH oxidase and do not exhibit NMDA receptor-dependent ERK activation (225). The p47^phox knockout mice, along with mice lacking the gp91^phox subunit of NADPH oxidase are typically used as models of chronic granulomatous disease, a genetic condition characterized by phagocytic dysfunction (199, 336). In addition, they can be used to study the effect of selective inhibition of NADPH oxidase activity on LTP induction and memory formation. Using these NADPH oxidase mutant mice, it was demonstrated that ablation of NADPH oxidase activity leads to obstruction of the early-phase LTP (223). Nox2 knockout mice also exhibited deficits in posttetanic potentiation, which is a form of short-term synaptic plasticity that is independent of NMDA receptors (223). These studies are unique in that they demonstrate a direct role for NADPH oxidase in LTP induction. This suggests that ROS involved in synaptic plasticity and memory (as will be described later in section IV b) could be generated by the large enzymatic NADPH oxidase complex.

1. Parkinson's disease

PD is a neurodegenerative disease of the basal ganglia, characterized by dopaminergic neuronal loss, mainly in the substantia nigra, and results in bradykinesia, tremor, and postural instability. Although the mechanisms of cell death in PD are not yet fully understood, oxidative stress is known to play an important role. In fact, the oxidation of dopamine during PD generates toxic semiquinones and the accelerated metabolism of dopamine by MAO-B induced the production of excessive H₂O₂ and hydroxyl radicals (313). Further evidence for the role of oxidative stress in PD comes from studies showing that the two main misfolded proteins of PD, parkin and α-synuclein, are a source of oxidative and nitrative stress (59). PD was found to be associated with increased oxidative damage to DNA (38), lipid peroxidation (107, 108, 458), and protein carbonylation (10, 150). Other studies showed increased brain levels of iron (105, 151) and reduced levels of ferritin (106), which creates a source of free radicals that potentiate the oxidative damage, especially in the substantia nigra. Although there is a large body of literature discussing the beneficial effect of antioxidant therapy for PD, no specific reports directly discuss the involvement of oxidative stress in PD-related learning and memory deficits. In a recent study investigating the effect of deprenyl and tocopherol on the PD-related cognitive dysfunction, the authors reported an improvement in the cognitive functions of the group treated with antioxidants. However, the authors do mention that this could be due to recruitment bias and/or limitations of the mini mental state examination (MMSE) test used to assess mental function (423).

2. Diabetes

Growing data report learning and memory problems (135, 161, 441) as well as LTP (21, 202) deficits associated with diabetes mellitus. Expression of neuronal NOS mRNA and protein in the hippocampus of diabetic rats is decreased (357), suggesting a critical role for NO in synaptic plasticity associated with diabetes. Supporting studies, using the active avoidance learning test in rats, demonstrated the involvement of NO imbalance in the occurrence of diabetes-induced learning deficits (234). Multiple studies using a variety of antioxidants to treat and/or prevent the cognitive dysfunction associated with diabetes also demonstrated an involvement of ROS in these events. For example, melatonin and vitamin E have been shown to improve diabetes-induced spatial memory impairments (421). Lycopene also has been shown to decrease markers of oxidative stress associated with diabetes and to attenuate spatial memory deficits (235). Thus, ROS likely play a role in cognitive dysfunction associated with diabetes.

3. Homocysteinuria

Homocysteinuria is a metabolic disorder characterized by deficiency of the enzyme cystathione β-synthase activity leading to accumulation of homocysteine. Homocysteine causes free radical formation and severe neurological dysfunction, which is thought to be due to the increased oxidative stress (263). Administration of homocysteine to adult rats caused severe memory impairments in the step-down inhibitory avoidance test that was prevented by pretreatment with vitamins C and E, suggesting that ROS play a critical role in the onset of memory dysfunction during homocysteinuria (358). Using the same model system it was shown that homocysteinuria caused severe learning and memory deficits in the passive avoidance and Morris water maze tests, and that these deficits were reversed by chronic melatonin treatment, which also significantly reduced the levels of lipid peroxidation (35). Taken together, these findings suggest that homocysteinuria causes cognitive deficits via increased oxidative stress.

1. Superoxide dismutases

SODs are a class of metalloenzymes involved in catalyzing the dismutation of the superoxide radical into oxygen and H₂O₂ and, as such, constitute a very important element of the antioxidant defenses of organisms (136) (Fig. 9A). In mammals, including humans, there are three isoforms of the enzyme that are encoded by three different genes. The SOD isoforms differ with regard to their localization as well as the metal bound to them. SOD-1 is a cytoplasmic enzyme with copper and zinc acting as cofactors, and therefore also is known as Cu/Zn-SOD. SOD-2 is a mitochondrial enzyme with manganese at its core and hence, is also referred to as Mn-SOD. SOD-3 also is a Cu/Zn enzyme, but is localized extracellularly and is often called EC-SOD. The three SOD isoforms have some structural differences, with SOD-1 being functional as a dimer, whereas SOD-2 and EC-SOD are tetramers. The physiological importance of all three SODs is illustrated by the multiple pathologies exhibited in mutant mice lacking each isoform. SOD-1 knockout mice develop hepatocellular carcinoma, age-related mass loss, increased formation of cataracts, and a reduced life span (119, 298). SOD-2 knockout mice survive only a few days postbirth and then die of severe oxidative stress (251). EC-SOD knockout mice do not exhibit an obvious major phenotype, but do develop hypersensitivity to free radical-induced injury (371). The physiological importance of the SODs is also illustrated by studies demonstrating that upregulation of SODs serve to attenuate neuropathological conditions such as ischemic injuries (212) and AD (115, 277).

The use of SOD transgenic mice and/or pharmacological mimetics has been instrumental in determining the functions of these enzymes and the role of superoxide in synaptic plasticity and memory. In line with the dual effect of superoxide and H₂O₂ as crucial signaling molecules in healthy organisms, but involved in oxidative stress in the aging and/or diseased brain, the effects of overexpressing SODs were found to be dependent on the age of the animals.

2. Catalase

Catalase is an antioxidant enzyme found in nearly all living organisms. Its main function is to catalyze the decomposition of H₂O₂ to water and oxygen (82) (Fig. 9B). Catalase is an iron enzyme; it contains four porphyrin heme groups that allow it to interact with H₂O₂. Studies investigating the role of catalase in the mechanisms underlying memory function have done so in relation to H₂O₂ metabolism. For example, LTP in hippocampal slices from adult or aged wild-type mice was examined in the presence or absence of an enzymatic treatment of either catalase or SOD-1. In line with Kamsler and Segal's model of H₂O₂ modulation of synaptic plasticity (205), increased SOD-1 had no effect on LTP from young adult mice, but made LTP impairments worse in old mice (436). It was also found that catalase inhibited LTP in slices from the young adult mice, but reversed age-related LTP deficits in older mice, indicating that in wild-type mice, H₂O₂ plays a critical role in synaptic plasticity at young ages, but becomes detrimental during aging (436). This study supported earlier findings that demonstrated a dual role for H₂O₂ in the regulation of LTP that is mediated by the protein phosphatase calcineurin. H₂O₂ was found to be necessary in young animals, but became detrimental in old animals (204). Several studies investigated the contribution of brain catalase to ethanol-induced learning reinforcement. It was found that catalase is involved in the acquisition of ethanol-induced conditioned place preference (133), as well as learning in the social recognition test (270). Additional supporting evidence of the involvement of catalase in ethanol-induced behavioral reinforcements came from studies of conditioned taste aversion combined with a catalase inhibitor (aminotriazole). These effects are thought to be mediated by acetaldehyde, which is an ethanol metabolite directly produced in the brain by the catalase system (20, 308, 346). In studies directly relevant to aging and neurodegeneration, it was shown that aging-related cognitive dysfunction can be reversed by treatment with synthetic SOD/catalase mimetics, further supporting the idea that free radicals are involved in learning and memory (90, 255).

3. GPx, glutathione reductase, and related enzymes

GPx is a general nomenclature attributed to a group of antioxidant enzymes dedicated mainly to reducing H₂O₂ to water and lipid hydroperoxides to their corresponding alcohol. GPx reduces H₂O₂ to water by oxidizing GSH. Then, the oxidized glutathione disulfide (GSSG) is regenerated back to its reduced form by the action of GR (Fig. 10). GR is constitutively active and inducible upon oxidative stress; therefore, GSH is found almost always in its reduced form, ready to counteract the deleterious oxidant effects (26).

Up to eight isoforms of GPx have been identified in humans thus far, with specific antioxidant mechanisms attributed to each. For example, the cytoplasmic homotetrameric GPx1, the most abundant of these peroxidases, has been shown to preferentially quench H₂O₂, whereas the ubiquitous but less abundant monomeric GPx4 plays an important role in detoxification of membrane lipids. With the exception of increased cataract occurrence, GPx1 knockout mice are phenotypically normal, indicating that this enzyme is not crucial for survival. However, genetic deletion of GPx4 is embryonic lethal, illustrating the different function of the different GPx isoforms (297). GPx levels and/or activity are used widely as an index of oxidative stress and assessment of therapeutic efficacy. In studies of oxidative stress-induced synaptic plasticity and memory impairments, impaired cognition is typically found to be correlated with reduced levels of GPx that are brought back to normal upon certain antioxidant treatments [examples in refs. (86, 172, 301)]. This is in agreement with the evidence discussed in this review, implicating ROS dysfunction with cognitive diseases.

Additional studies using GPx transgenic mice have addressed more directly the role of this antioxidant enzyme in oxidative stress-induced cognitive deficits. For example, moderate increases in GPx1 were shown to reverse hypoxia-induced functional damage to hippocampal slices. Control slices subjected to hypoxia suffered an irreversible loss of EPSPs with an inability to generate LTP. GPx overexpression resulted in a significant recovery of synaptic transmission and LTP (141). In addition, GPx overexpression has been shown to be beneficial to TBI-induced cognitive deficits in the absence of any effect on the lesion anatomy and brain volume. Further, GPx protected hippocampal neurons via its antioxidant effect on early oxidative stress (419). The effect of GPx and GR as antioxidants cannot be dissociated from the main precursor for the system, namely GSH (discussed in details later in this review). In fact, boosting GSH levels can result in increasing the activity of both GPx and GR, as well as other antioxidants (300). In addition, the effectiveness of these enzymes in neuroprotection and improvement of cognitive function depends on the availability of both GSH and GSSG as reducing equivalents (110, 283). In certain instances, increased activity of GPx has been shown to correlate positively with memory impairments, which appears counterintuitive (66). However, the levels of GPx are a better indicator of oxidative stress-induced impaired memory function, which also resulted in the activation of antioxidant defenses, including the increased activity of GPx.

A group of enzymes functionally related to the GSH system are thiol-disulfide oxidoreductases that are primarily involved in the catalysis of thiol-disulfide interchange reactions. This group includes thioltransferase (glutaredoxin [GRX]), thioredoxin (TRX), and protein disulfide isomerase (437). Whereas TRX and protein disulfide isomerase have broad substrate specificities (289), GRX specifically reduces GSH-containing mixed disulfides with greater efficiency than TRX (153, 289). The TRX system contains, aside from TRX, an NADPH-dependent TRX reductase, which reduces the oxidized form of TRX. The GRX system uses GSH as a reducing agent, which then enters the GSH system and is regenerated with GR [reviewed in ref. (201)]. Two gene isoforms of each GRX and TRX have been identified in mammals and a summary of their function can be found in the following review (179). Both GRX and TRX are endogenous antioxidants that contribute to protection against oxidative stress, specifically the formation of intra- and intermolecular disulfide bonds in proteins. As such, both of these systems may modulate memory formation. However, studies specifically investigating the direct involvement of GRX and TRX in synaptic plasticity and memory are limited. TRX upregulation, along with increased ERK-mediated Nrf2 phosphorylation and a reduction in free radical levels, contributes to the beneficial effects of acetyl-L-carnitine on hypoxia-induced spatial memory impairment (29). Additionally, GRX1 and TRX1 are deregulated in AD and contribute to Aβ-induced neurotoxicity (9). However, these studies did not specifically address the involvement of GRX1 and TRX1 in memory dysfunction associated with AD. GRX also was shown to be essential for maintaining the integrity of complex I of the mitochondrial respiratory chain after toxic insult by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a neurotoxin that produces PD-like symptoms through inhibition of complex I (214). This finding suggests that GRX also may be involved in PD pathology; however, its involvement in learning and memory can only be inferred.

Another group of related enzymes, peroxiredoxins (PRXs), is involved in the decomposition of H₂O₂, organic hydroperoxides, and peroxinitrite (79). PRXs are related to TRX because distinct isoforms of TRX are cofactors for PRX. PRX has been shown to contribute its antioxidative capability in the realm of aging- and AD-related cognitive dysfunction. Specifically, PRX II-deficient mice were shown to have increased mitochondrial ROS generation and pronounced age-related LTP impairments compared to their wild-type littermates (218). These mice also exhibited an impairment in the activation of synaptic plasticity-related signaling pathways involving CREB, CaMKII, and ERK, indicating that PRX II is necessary for maintaining redox homeostasis (218). Dietary vitamin E alleviated a number of PRX II deficiency-related impairments, including a restoration of the cognitive decline in aged PRX II-deficient mice (218), suggesting that PRX II helps maintain hippocampal synaptic plasticity against age-related oxidative damage. In a differential proteomic analysis, PRX 1 from aged rat temporal cortex tissue is carbonylated, suggesting that this enzyme contributes to the decline in cognitive function that occurs during aging (431). PRX II also has been shown to be upregulated in response to increase Aβ levels during AD, with the purpose of protecting neurons against Aβ-induced toxicity (456). Conversely, reducing the levels of Aβ in the brains of senescence accelerated mice resulted in reduced PRX II levels (338). Although these findings do not directly link PRX II to AD-related cognitive dysfunction, they do support the idea that PRX II plays a crucial antioxidant role during aging and AD.

This section reviewed evidence for the involvement of the glutathione system, including GSH, GPx, GR, and related enzymes, in reducing oxidative stress and improving cognitive dysfunction, providing more evidence that oxidative stress is associated with neurotoxicity and memory impairment.

4. Metallothionein

Metallothionein (MT) is a family of cycteine-rich low-molecular-weight proteins that are ubiquitous in eukaryotes. Their primary function is to bind essential physiological (copper, zinc, and selenium) and nonessential xenobiotic (cadmium, mercury, silver, and arsenic) heavy metals via their cysteine residues. A significant literature is available (167, 177, 200, 243, 310, 325, 368) describing the physical structure, biochemistry, and molecular biology of MT, as well as their role in protecting cells and organisms from environmental toxicant damage (metal toxicity), but also from oxidative stress. Specific evidence documenting the involvement of MT in synaptic plasticity and memory is also available (248, 459); however, it is not clear whether this involvement is related to their antioxidative capabilities and therefore will not be discussed here.

1. Ascorbate (vitamin C)

Ascorbate (vitamin C) is a potent antioxidant that can neutralize free radicals by donating a hydrogen atom. Vitamin C itself becomes a free radical in the process. However, this does not pose a major concern, as the vitamin C radical is very stable (nonreactive) due to its resonance structure (Fig. 11). Moreover, the reduced form of vitamin C can be readily regenerated by the action of NADH and NADPH-reductases. Excess vitamin C radical becomes a problem in the presence of metal ions such as iron (Fe) or copper (Cu), where it can turn into a powerful pro-oxidant. Normally ferrous iron (II) (Fe²⁺) is oxidized by H₂O₂ to produce ferric iron (III) (Fe³⁺), along with the highly reactive hydroxyl radical and a hydroxyl anion. This is known as the Fenton reaction (Fig. 12). After the Fenton reaction, Fe³⁺ can be reduced back to Fe²⁺ by the action of H₂O₂ to produce a peroxyl radical and a proton in the process. The presence of ascorbate can accelerate the latter free radical producing reaction by acting as an electron acceptor for Fe³⁺, instead of H₂O₂ (Fig. 11). Organisms have developed defenses against this process, such as having iron bound to the transport proteins ferritin and transferritin. However, the exogenous introduction of metal ions from environmental pollutants can cause failure in this defense mechanism.

Vitamin C is an essential dietary supplement because it is required for collagen synthesis and primates have lost the ability to synthesize it. However, its antioxidant properties make it an increasingly desired dietary addition. These antioxidant properties have been explored both in vitro and in vivo and their effect on protein oxidation, lipid peroxidation, and DNA damage have been well characterized. In the interest of this review, we will focus on the role vitamin C plays in the learning and memory processes.

The vast majority of studies investigating the role of vitamin C in learning and memory have done so in the setting of memory dysfunction. In other words, these studies investigate the usefulness and effectiveness of vitamin C in reversing memory deficits induced by various oxidative stress-related conditions, ranging from chemical induction to disease states. Very few studies have addressed the role of vitamin C in normal learning and memory. One study from the early 1980s compared short-term and immediate memory performance of 20 individuals at times when their plasma level of vitamin C was high (owing to supplementation) versus times with low plasma levels of vitamin C (5). This study found no differences in the memory performance, nor did it find any differences in alertness, concentration, or mood. The purpose of this study primarily was to determine the effect of vitamin C on alertness, and therefore the memory test used was a simple procedure consisting of repeating the last five digits of a series of numbers played through headphones. This task was designed to test immediate short-term memory, stemming from concentration as opposed to actual learning (5). Two more recent studies addressed the antioxidant role of vitamin C in learning and memory (374, 422). It was demonstrated that although acute injections of ascorbate had no effect on the learning ability of rats measured with passive avoidance test, short- and long-term supplementation with vitamin C facilitated acquisition and retrieval of learning and memory in rats (374). Tveden-Nyborg et al. went a step further and investigated the effect of chronic vitamin C deficiency on spatial memory as measured by the Morris water maze and also counted hippocampal neurons to assess whether ascorbate had an effect on neurodegeneration (422). In this study, two groups of newborn guinea pigs were randomly assigned to either a vitamin C-sufficient diet or a deficient diet (minimally adequate to prevent scurvy) for 2 months. The objective of the study was to assess the effect of vitamin C deficiency on the neonatal brain, because it is more susceptible to oxidative damage than the adult mature brain. The results showed a significant reduction in spatial memory, compounded by a lower number of neurons in all regions of the hippocampus (422). Collectively, these animal studies indicate that ascorbate, at least partially, contributes to protection of neurons from free-radical-induced memory impairments. Clinical trials investigating the benefits of vitamin C on cognition have yielded more complicated results and are discussed in section V under physiological aging and AD.

2. Tocopherol (vitamin E)

Vitamin E is a generic term referring to a group of tocopherols and tocotrienols, of which α-tocopherol is the best studied. Vitamin E is a fat-soluble vitamin known for its role as an antioxidant, involved in stopping ROS from forming in membranes undergoing lipid peroxidation chain reactions. The role of vitamin E as a systemic antioxidant has been studied extensively, with conflicting results as to its beneficial effects. Similar to ascorbate, the involvement of vitamin E in learning and memory has been for the most part investigated under therapeutics for either ROS-induced disease states or physiological aging (discussed above in section V). Very few studies have addressed the role of vitamin E in normal memory function. In the early 1970s, it was reported that there was no significant effect of vitamin E deficiency on the learning ability of rats, although they needed more trials to acquire a conditioned response (238). The most striking effect of vitamin E deficiency in these experiments was the impaired retention of a one-trial experience, a task dependent on reference memory (238). In similar experiments, the effect of vitamin E deficiency on reference and working memory were subtle at best (367). Studies examining the effect of long-term vitamin E deficiency and vitamin E supplementation in rats found that it had no effect on the acquisition and maintenance of memory tasks, but did impact their learning (195). Collectively, the results of these studies have not conclusively demonstrated whether vitamin E acts as an antioxidant in learning and memory, which may seem surprising given that memory processes depend on the redox status of neurons. In fact, two more recent related studies (449, 450) show a direct role of vitamin E in the modulation of LTP, which is the substrate of learning and memory formation (117). In these studies (449, 450), vitamin E deficiency resulted in LTP impairments and that vitamin E induced a slowly developing long-lasting increase in EPSPs that was independent of NMDA receptor activation. The same effect was not observed with ascorbate. Although these studies clearly argue for a role for vitamin E in LTP (and thus memory formation), the ascorbate study suggests that this effect may not be mediated by ROS (449, 450). Collectively these studies demonstrate that the involvement of vitamin E as an antioxidant in the learning and memory process is not clear and is at best inferred.

3. Glutathione

Glutathione (GSH, l-γ-glutamyl-l-cysteinylglycine) is the predominant cytoplasmic antioxidant molecule in cells. GSH serves to provide reducing equivalents for the maintenance of proper oxidant/antioxidant homeostasis and is required for viability and proper functioning of cells. GSH is a tripeptide synthesized from the amino acids glutamate, glycine, and cysteine (19) (Fig. 11). Given that glycine and glutamate are plentiful in cells, it is cysteine availability that controls the production rate of GSH. This is supported by studies in transgenic flies showing that neuronal overexpression of the rate-limiting enzyme for GSH synthesis increased lifespan by up to 50% (316). The main roles of GSH are to scavenge peroxynitrite and hydroxyl radicals, as well as to convert H₂O₂ to water. A GSH radical is formed in the process; however, it is readily neutralized by combination with another GSH radical to produce GSSG. GSSG can be converted back to GSH by NADPH-dependent GSH reductase (26). Each antioxidant is converted into a radical upon scavenging another radical. This is a concept that was described above for both for vitamin C and E, and it is also true of GSH. All of these antioxidants can recharge each other as such: GSH regenerates vitamin C, which regenerates vitamin E, creating a chain of antioxidant molecule dependency.

With regard to learning and memory, the role of GSH has mainly been investigated after chemical depletion and its effect discussed in the context of schizophrenia, where a deficiency in GSH is common and is associated with cognitive decline. It was demonstrated that low levels of GSH negatively impact LTP and paired-pulse facilitation, indicating that low GSH content can impair short- and long-term forms of synaptic plasticity (11). The effect of GSH depletion in the presence of dopamine, which is a potent pro-oxidant, was studied by inducing GSH depletion in rats by injecting buthionine sulfoximide (BSO) either before or after dopamine administration, and then assessing the spatial memory of rats using the Morris water maze. These investigators found that GSH depletion before the oxidant insult introduced by dopamine led to significant impairments in memory, indicating that GSH is a necessary component of the antioxidant machinery dedicated to maintain a healthy balance of free radicals necessary for proper memory function (389). A specific role for GSH in the acquisition, but not retention, of spatial memory in maze tasks was demonstrated by inducing GSH depletion in rats with systemic diethylmaleate injections and then examining metabolism and cognitive performance in the passive avoidance and Morris water maze tests. These studies showed that GSH depletion resulted in reduced levels of GSH and GPx activity in the hippocampus. It also resulted in reduced acquisition of spatial memory when the drug was administered before training, but did not affect retention of already acquired memory when the drug was administered after memory acquisition (94). More complex assessments of spatial and working memory were performed after transitory GSH depletion by BSO injection in either young normal or mutant rats (osteogenic disorder Shionogi [ODS]—incapable of synthesizing ascorbate). Ascorbate is known to compensate for GSH depletion, and therefore the ODS group served to dissect the direct effect of GSH on cognitive abilities. BSO-ODS rats exhibited impaired spatial memory whether they were tested at a young age, immediately after GSH restoration to physiological levels, or at an older age, long after treatment cessation. In contrast, BSO-normal rats did not exhibit memory impairments at an early age, possibly due to ascorbate compensation, but they did exhibit selective spatial memory impairments at an advanced age when the task required integration of multimodal cues, such as visual and olfactory cues. Although the results of this study were exclusively discussed in the context of schizophrenia, they clearly show that GSH depletion causes complex behavioral impairments associated with increased brain oxidative stress (70). In a more recent study, GSH depletion was induced with 2-cyclohexene-1-one both in rats and mice and then their memory was assessed in the Y-maze test. In both species, GSH depletion caused a significant impairment in short-term spatial recognition memory, suggesting that GSH is necessary for proper spatial memory function, possibly explaining its involvement in psychiatric diseases such as schizophrenia (104). Collectively, these studies outline an important role for GSH in spatial memory formation. On a related note, administration of reduced GSH into the antennal lobes of honeybees prevented oxidative stress-induced impairments in olfactory learning and memory (125), indicating that GSH plays a critical role in learning not only in mammals, but also in invertebrates.

4. Coenzyme Q

Coenzyme Q, also known as coenzyme Q10, ubiquinone, ubidecarenone, or simply Q, is a benzoquinone found in most eukaryotic cells, primarily in the mitochondria. It participates in the electron transport chain in its capacity of electron acceptor/donor and essentially functions as an electron transferring molecule (426) (Fig. 4). Electron transferring ability means that coenzyme Q can act as an antioxidant. Because of this antioxidant ability, coenzyme Q is widely used as a dietary supplement taken to enhance bioenergetics and ameliorate all side effects of increased oxidative stress during aging and a multitude of pathological conditions. Coenzyme Q has been explored as a therapeutic avenue for heart failure (327), mitochondrial dysfunction (160), migraine headaches (47), and neurodegenerative disorders associated with cognitive dysfunction (39). Idebenone, a benzoquinone commercially marketed as a synthetic analog of ubiquinone, also has been extensively studied for its antioxidant properties. We review here the role of both in learning and memory mechanisms.

A general statement that seems to be true of the involvement of quinone in learning and memory is that these compounds do not really affect cognitive abilities under physiological conditions, but rather improve cognitive dysfunction induced by a variety of pathological conditions. One study of the late 1980s directly linked quinones to synaptic plasticity by determining a role for idebenone in enhancing LTP in hippocampal slices (196). Two different concentrations of ubiquinone were tested on several physiological functions that are known to be impaired during aging, including cognitive dysfunction. These investigators supplemented their mice with coenzyme Q at a young age and then assessed their spatial learning and memory at several age points. Although the amount of ubiquinones was increased in the cerebral cortex of treated mice, the low dose treatment did not alter ubiquinone levels. Surprisingly, at higher doses, age-related decreases in learning and memory were made worse, indicating that low coenzyme Q levels had no discernable impact on cognitive dysfunction, whereas higher levels impaired cognitive function (403). These results suggest that under physiological conditions ubiquinone supplementation is not beneficial, but rather can be detrimental to memory. In a similar study investigating the role of coenzyme Q, vitamin E, or both on brain function of aged mice, it was observed that only mice treated with both coenzyme Q and vitamin E exhibited memory improvements (281). Similar experiments with higher doses of coenzyme Q were conducted, and unlike the previous study, there was no adverse effect on memory. On the other hand, the investigators also did not observe any memory improvements. They concluded that coenzyme Q is only beneficial for memory when paired with vitamin E, perhaps because they work in concert, possibly via mutual regeneration of antioxidant function (281). Similar to previous studies, rats fed a ubiquinone-rich diet did not exhibit improvement in the early stages of the Morris water maze test, but did exhibit improvements in the later stages of the test (312). After hyperoxia to induce oxidative stress, rats fed coenzyme Q showed improved memory function over control rats. Repeating the same studies in vitamin E-deficient rats or rats supplemented with vitamin E showed the same pattern of improvement and indicated no synergistic effect of coenzyme Q with vitamin E (312), in contrast to previous studies that demonstrated that coenzyme Q can regenerate the antioxidant powers of vitamin E (281).

Two other conditions in which ubiquinones proved to be beneficial for learning and memory were after ischemia and scopolamine-induced amnesia, which was studied in the context of cholinergic dysfunction during AD. Idebenone ameliorated memory impairment induced by cerebral vascular disturbance in rats. More specifically, the authors demonstrated that idebenone treatment after the acquisition trial of passive avoidance learning caused shortening in the latencies of the retention test trial, that is, idebenone reversed the retrograde amnesia caused by cerebral ischemia (453). In a different study, in embolized rats, idebenone improved learning and memory abilities in the radial arm test (227), as well as passive avoidance test (226), indicating that quinones have a positive effect on learning impairments caused by brain hypofunction.

Perhaps the largest body of literature investigating the effect of quinones in the mechanisms governing memory function is in studies related to AD (AD). Idebenone has long been investigated as a possible therapy for AD with mild, but generally positive, results, suggesting that quinones are involved in preserving the oxidant balance necessary for proper cognitive function. Several groups have studied scopolamine-induced amnesia, which induces deficits in the cholinergic system as seen in AD patients. Idebenone treatment improved scopolamine-induced spatial memory deficits (256) and working memory deficits (452). Using a delayed alternation task in rats, it was shown that idebenone treatment improved short-term memory dysfunction induced by scopolamine, but it did not alter memory in control rats (454). These observations were extended to a serotonin-deficient rat model, where idebenone improved the retardation of discrimination learning induced by central serotonergic dysfunction (455). Because nerve growth factor (NGF) is necessary for the maintenance of cholinergic neurons, its depletion mimics cholinergic deficiency as observed in AD. Unfortunately, NGF cannot be used directly as a therapy because of its inability to cross the blood brain barrier. However, idebenone administration stimulated NGF synthesis in vivo, resulting in an amelioration of the behavioral deficits induced by lesions in the basal forebrain cholinergic system. In these studies, idebenone was beneficial as an NGF stimulator rather than as an antioxidant (307).

An interesting question in the AD field is whether oxidative stress contributes to learning and memory deficits caused by Aβ42. Rats given continuous intracerebroventricular infusion of Aβ42 were treated with either idebenone or vitamin E and then their memory was assessed in the Y-maze, the Morris water maze, and the passive avoidance test. It was observed that both idebenone and vitamin E improved the performance of the Aβ42-treated rats in the Y-maze and Morris water maze, but not in the passive avoidance test (451). These results suggest that quinones might be effective antioxidants to improve learning and memory deficits associated with AD. Finally, streptozotocin (STZ)-treated rats display markers of oxidative damage (increases in thiobarbituric reactive substance, GSH, protein carbonyls, and the activities of the GSH peroxidase and reductase enzymes) and a decline in the level of ATP in the hippocampus and cerebral cortex. Supplementing STZ-infused rats with coenzyme Q reversed all the aforementioned markers, indicating that ubiquinone has neuroprotective effects on cognitive impairments and oxidative damage associated with STZ toxicity (197). In a multicenter, randomized, double-blind, placebo-controlled study in AD patients, treatment with idebenone was effective in delaying memory impairments associated with AD, with no significant side effects (42). In a more recent study, treatment of AD patients with idebenone in the predementia stage resulted in short- and long-term memory improvements in 37% of the patients (427). On a concluding note, although the precise mechanism of action of ubiquinone remains unknown and it does not appear to alter learning and memory under normal physiological conditions (147), it appears to play a central role as an antioxidant molecule involved in restoring the redox balance necessary for improvement of cognitive dysfunction associated with a multitude of pathological conditions, especially AD.

5. Carotenoids

Carotenoids are phytochemicals produced in plants, with β-carotene and lycopene being the most investigated (321). Many epidemiologic studies have made the association of high dietary carotenoid intake with a lower incidence of chronic disease, but the biological mechanisms underlying such protection are not understood. There are several theories that have been put forward to explain the protective effect of carotenoids (321): (a) they can be converted to retinoids and thus acquire pro-vitamin A activity, (b) they can act as potent antioxidants, (c) they can modulate the function of lipooxygenases, and (d) they can modulate expression of genes involved in cell–cell interactions. In the interest of this review, we will focus on the role of carotenoids as antioxidants with an emphasis on their involvement in the regulation of learning and memory. The antioxidant properties of carotenoids lie in their ability to physically quench singlet oxygen and to trap peroxyl radicals. Singlet oxygen is not a free radical but it has electrons in an excited state that may adversely affect molecules containing double bonds. Carotenoids are the most effective singlet oxygen quenchers (109), which results in the formation of excited carotenoids. The excited carotenoids have the capacity to rapidly dissipate newly acquired energy through a series of rotational and vibrational interactions with the solvent, resulting in regeneration of the unexcited carotenoid that can be reused for further cycles of singlet oxygen quenching (67). Several carotenoids, with β-carotene in particular, have the capability of scavenging peroxyl radicals by the formation of a transitory carotenoid adduct radical (67). This radical is highly stable and undergoes decay to a nonradical species, thereby terminating the actions of harmful peroxyl radicals. This latter process destroys carotenoids (442). The antioxidant properties of carotenoids vary depending on the system being used to study them as well as the concentration used. For example, carotenoids can protect cells against oxidant-induced lipid peroxidation in a pro-vitamin A-independent fashion (275). In transformed thymocytes, carotenoids act as potent antioxidants when oxygen tension is low, but turn into pro-oxidants when oxygen tension is high (324). In addition, supraphysiological concentrations of carotenoids may result in pro-oxidative effects (145). These pro-oxidative effects can be prevented by the coadministration of other antioxidants such as vitamin E (323). Many clinical studies have been undertaken to assess the antioxidant ability of carotenoids and produced conflicting results. It was demonstrated that a diet deficient in carotenoids, but otherwise adequate for all other nutrients, resulted in diminished markers of antioxidant capacity in the blood (315). Conversely, supplementing the diet with β-carotene has been shown to increase plasma levels of antioxidant molecules (288). In one study using the oxygen radical absorbance capacity test, no positive role for carotenoids on plasma levels of antioxidants was demonstrated (76). Another report supported this finding by showing that there was no effect of carotenoids on the total antioxidant capacity of the plasma (58). The differences in results between these studies could be due to either different types of supplementation or different age groups and genders of the study individuals. Humans have the capacity to absorb intact carotenoids from the intestines; however, such absorption may differ depending on the type of supplementation offered (within the food as opposed to capsules). Certain forms may be more readily bioavailable than others. For example, lycopene has been shown to be better extracted from heated tomatoes as opposed to raw ones (395). Thus, special care should be put into selecting the right concentrations and form of carotenoids for each particular study setting.

Carotenoids have been investigated as an antioxidant in various pathological conditions associated with learning and memory dysfunction. One carotenoid, retinoic acid (an essential growth factor derived from vitamin A), has been extensively studied for its modulation of the signaling events involved in learning and memory. In fact, retinoic acid activates gene transcription through nuclear receptors, thereby influencing LTP and neurogenesis, particularly in the hippocampus (279, 293). This suggests a role for retinoic acid in learning and memory processes, but it appears to be distinct from the ability of carotenoids to act as antioxidants. Because this review is dedicated to the redox regulation of memory mechanisms, we will limit the discussion in this section to studies specifically addressing the antioxidant function of carotenoids.

A number of epidemiological studies have been undertaken to assess the association between carotenoid levels and either age- or disease-related declines in cognitive function. With the exception of one study, which delineated a pro-oxidant effect of vitamin A in the substantia nigra and striatum at clinical doses (103), all of the other studies showed either neutral or positive correlations with cognitive dysfunction. Engelhart et al. examined whether high plasma levels of vitamins A and E together were associated with lower prevalence of cognitive decline. They performed a cross-sectional study within another AD study and found that if a univariate model was used, high plasma vitamin A and E could be correlated to lower cognitive decline. However, when adjustments were made with respect to age, gender, and total cholesterol, the correlation weakened, and it was concluded that there was no association between plasma levels of vitamin A and E and cognitive decline (122). The same question was examined in a cohort of elderly woman and no correlation between plasma carotenoids and tocopherols and cognitive deficits was observed (206). The same group explored the effect of β-carotene supplementation on cognitive function in men and found that short-term supplementation (mean duration of 1 year) did not affect cognition, whereas long-term supplementation (mean duration of 18 years) provided cognitive benefits (156). The latter study illustrates that careful consideration should be given to every study parameter (such as treatment duration) before conclusions can be drawn.

Five additional studies have described beneficial effects of carotenoids on cognitive deficits. The interaction between carotenoids and the AD susceptibility gene apolipoprotein E was investigated and it was found that among high-functioning older persons, β-carotene supplementation offered protection from cognitive decline, even in persons with greater susceptibility evidenced by the presence of the APO-E4 allele (191). The relationship between plasma carotenoid levels and the MMSE was examined and it was found that lower levels of carotenoids correlated with cognitive impairments (7). As part of the cache county study, high antioxidant intake from food combined with supplementation with vitamin E, vitamin C, and carotene may delay cognitive decline in the elderly (438). Although this study did not dissect a specific role for carotenoids in this protection, it did suggest that carotene is potentially a beneficial antioxidant involved in protection of cognitive abilities. In addition, a significant association between higher carotenoid levels and docosahexanoic acid with higher MMSE scores has been reported, supporting a protective role for both nutrients in aging and AD-related cognitive impairments (433). Finally, the involvement of lycopene in diabetes-associated cognitive decline in rats was examined, and this particular carotenoid has a significant therapeutic potential in diabetes-induced learning and memory impairments (235).

A large body of research has centered on the interesting and widely beneficial effects of a naturally occurring carotenoid chemical called crocin. Crocin is found in the flower Crocus sativus L., more commonly known as saffron. Crocin is the ingredient primarily responsible for the color of saffron. It is a diester formed from a disaccharide (gentiobiose) and the dicarboxylic acid crocetin. Aside from being a potent antioxidant (311, 465), it also has been reported to have anticarcinogenic (1, 88), antidepressant (8), and aphrodisiac properties (184). Given its potent antioxidant capacity, a role for crocin in modulating learning and memory has been proposed. Recent behavioral and electrophysiological studies have shown a beneficial effect of crocin on ethanol-induced LTP deficits and learning and memory impairments. In several separate studies, Saito and associates have demonstrated that crocin can prevent ethanol-induced impairment of hippocampal synaptic plasticity, both in vitro in rat slices (401) and in vivo in anesthetized rats (400). They further investigated the underlying mechanism of these effects and found that crocin specifically antagonized the inhibitory effect of ethanol on NMDA receptor-mediated responses in hippocampal neurons (2). Using the same ethanol-induced impairment model, they also demonstrated that although crocin had no effects on memory registration, consolidation, or retrieval in normal mice, it did improve the ethanol-induced impairments of memory function in all of its constituents (461). In a more recent study (333), crocin counteracted delay-dependent recognition memory deficits in the normal rat, suggesting that this carotenoid can modulate storage and/or retrieval of information. It also attenuated scopolamine-induced spatial memory deficits in the radial arm maze. This study not only demonstrated the beneficial effect of crocin on learning and memory impairments induced by ethanol, but it also suggested that crocin is a compound that can modulate the mechanisms underlying normal recognition and spatial memory (333).

In conclusion, although carotenoids have well-documented antioxidant effects, there continues to be a great deal of uncertainty as to their beneficial role in the mechanisms underlying learning and memory deficits. Depending on the conditions of their use (administration, absorption, duration of administration, etc.), carotenoids appear to offer protection against learning and memory deficits associated with physiological aging as well as a multitude of pathological conditions. The mechanism responsible for the beneficial effects of carotenoids on cognitive function remains to be elucidated.

6. Melatonin

Melatonin is primarily a pineal hormone derived from its precursor serotonin. Its main function is in sleep cycle regulation. Melatonin also is a very powerful scavenger of hydroxyl radicals, making it a potent antioxidant molecule (168). Unlike vitamin C and GSH, which are only effective in aqueous phase, and vitamin E, which is only effective in lipid phase, melatonin can be functional in both phases (412). Melatonin also easily crosses cell membranes and the blood–brain barrier (360), making it a powerful candidate for protecting nuclear and mitochondrial DNA and the central nervous system from damage induced by oxidative stress (359). Many biological effects of melatonin are produced through activation of melatonin receptors (61), so it is important to distinguish its action as an antioxidant on learning and memory.

The discovery of melatonin as an antioxidant was made in 1993 (412). Melatonin is a direct scavenger of hydroxyl and peroxyl radicals. It can also neutralize superoxide, singlet oxygen, H₂O₂, and hypochlorous acid (335). Melatonin also inhibits peroxynitrite formation by inhibiting NOS in brain tissue (245). Melatonin differs from other antioxidants in two important aspects. First, melatonin does not undergo redox cycling; that is, once oxidized, it cannot be regenerated to its reduced form. This happens because melatonin forms stable intermediates upon reacting with free radicals, which makes it a terminal antioxidant (410). Second, the antioxidant action of melatonin involves the donation of two electrons, not one electron, thereby ensuring that melatonin does not become a free radical in the process (410). Melatonin is a much more potent antioxidant than many traditionally used antioxidants. For example, it is twice as effective as vitamin E in protecting cell membranes from lipid peroxidation (330), five times more potent than GSH in scavenging hydroxyl radicals (331), and 60 times more effective than vitamins C and E in providing DNA protection (345). Besides its own ability to scavenge a variety of free radicals, several of its metabolites are themselves potent antioxidants (361, 411). Melatonin can also boost expression and/or activity of other antioxidants, most notably GSH peroxidase (up to eightfold increase), SOD, and catalase (233, 276).

Because the brain has lower GSH concentrations than other organs (164), melatonin plays a particularly important antioxidant role in the central nervous system. In fact, melatonin receptors have been shown to play an important role in the mechanisms of learning and memory in mice (242), and melatonin has been shown to alter the cellular processes associated with memory, such as LTP.

7. Lipoic acid

Lipoic acid is an organosulfur compound that was first postulated to be an effective antioxidant because it prevented symptoms of vitamin E deficiency (334). The mechanism of action for lipoic acid to act as an antioxidant is not well understood. It has been shown that dihydrolipoic acid (the reduced form of lipoic acid) can regenerate oxidized antioxidants, such as GSH, vitamin C, and vitamin E (50, 320), thereby permitting them to enter the circulation to quench more free radicals. Lipoic acid also has been shown to possess the ability to scavenge ROS in vitro; however, there is no clear evidence that this actually occurs in vivo. Recent findings suggest that lipoic acid can boost mitochondrial membrane potential and oxygen consumption, while decreasing mitochondrial production of oxidants (280). This occurs by lipoic acid amplifying key antioxidant protective mechanisms via PPARγ coactivator-1α (PGC-1α) mediation (280).

Lipoic acid has been studied extensively as a therapeutic antioxidant to protect against the damaging effects of oxidative stress on synaptic plasticity and memory. Studies have shown that lipoic acid significantly reversed oxidative stress-induced deficits in LTP (282, 429) and learning and memory in a variety of pathological conditions, such as aging (126, 254, 291, 339), radiation poisoning (268, 269), AD (348, 377, 380), and diabetes (49). In an attempt to identify the underlying mechanism of lipoic acid protection of cognitive functions, α-lipoic acid enhances Ca²⁺ entry through presynaptic N- and P/Q-type channels to cause an increase in evoked glutamate release from rat cerebrocortical synaptosomes (432). This facilitation of glutamate release by α-lipoic acid was found to be due, at least in part, to activation of PKA and PKC (432). Collectively, these studies support the hypothesis that ROS are involved in the modulation of learning and memory processes.

1. Selenium

Although it is toxic in large doses (462), selenium is an essential micronutrient for animals. In humans, selenium is a trace element nutrient that functions as cofactor for multiple enzymes, most notably GSH peroxidases and certain forms of TRX reductase found in animals and some plants (178, 365). This confers to selenium an indirect role as an antioxidant and neuroprotectant mineral. In fact, selenium has been shown to protect the brain from oxidative damage in various models of neurodegeneration. For example, rats treated with intracerebroventricular STZ are a model for oxidative damage and sporadic AD, and display severe deficits in the passive avoidance and Morris water maze tests that is accompanied by high levels of lipid peroxidation, protein carbonylation, reduced GSH, GPx, GR, and ATP in the hippocampus and cortex. Selenium, in line with its powerful antioxidant function, prevented all of the alterations induced by STZ, including the cognitive deficits and the oxidative damage (198). Selenium also has been shown to be neuroprotective and beneficial for memory deficits induced by lead poisoning (237) and psychosis, without affecting baseline behavior (260). The beneficial effects of selenium also were explored in fluoride toxicity (462) and ischemia models (158), where it was shown to enhance ROS scavenging and improve short-term memory impairments. In addition, organic selenium compounds enhanced long-term memory in a novel object recognition test (364). However, memory in the active avoidance shuttle box test was normal in rats that were selenium and vitamin E deficient (32). In a more recent study, using the same model it was shown that the absence of selenium can cause changes in synaptic function in area CA3 of the hippocampus, and inferred that it must play a role in learning and memory (435). These studies suggest that the effects of selenium as an antioxidant beneficial to learning and memory is dependent on the task, as well as the concentration used. Consistent with this idea, it was demonstrated that at higher concentration, selenium could cause toxicity (462).

2. Zinc

After iron, zinc is the second most abundant metal in humans. Zinc is an essential micronutrient for a wide variety of biological functions and has been shown to be a component of more than 1200 enzymes, either acting as a cofactor or as a structural element (137). It is actually the only metal found in all enzyme classes. In the brain, zinc is packaged into vesicles of glutamatergic neurons (53) and plays a critical role in brain excitability (165) as well as synaptic plasticity and learning (303). Similar to selenium, zinc has antioxidant functions, but not without controversy (290). Selenium is an integral component of GSH peroxidases and therefore acts as a general antioxidant, whereas zinc is an antioxidant only at specific sites (45). Although zinc is an integral component of the antioxidant enzyme SOD-1, it plays a structural role as opposed to being a cofactor. In support of this notion, zinc deficiency does not affect SOD-1 activity. On the contrary, SOD-1 activity is depressed with high zinc intake (45). The antioxidant properties of zinc stem mainly from competition with iron and copper for binding to certain proteins, thereby displacing redox-active metals. Zinc also can bind the sulfhydryl groups in proteins, thereby protecting them from oxidation (45). There is a large body of literature discussing the involvement of zinc in synaptic plasticity and memory (44, 303), but this is beyond the scope of this review. To our knowledge, none of these reports have discussed the role of zinc in learning and memory in the context of its antioxidant abilities.

3. Manganese

Manganese is an essential trace element (120) acting as a cofactor for multiple classes of enzymes, including, but not limited to, oxidoreductases, transferases, and hydrolases, most of which are involved in redox homeostasis. The best known manganese-containing enzymes are SOD-2 and arginase (406). As discussed earlier, SOD-2 is the main superoxide scavenger of mitochondria and as such is one of the most characterized antioxidant enzymes. The involvement of manganese in oxidative stress-induced synaptic plasticity and memory stems mainly from its association with SOD-2, which was described earlier in the review.