Abstract
Declined learning and memory ability is common in aged individuals. Such age-related cognitive decline is associated with the dysfunction of the prefrontal cortex, hippocampus and its adjacent entorhinal, perirhinal and parahippocampal (postrhinal in rodents) cortices. Although the exact cause of aging is currently unclear, nitric oxide (NO), a bioactive molecule generated by NO synthase (NOS) from L-arginine, has been strongly implicated in the aging process. Agmatine is decarboxylated arginine, and aging appears to affect its tissue concentrations in memory-related brain structures dramatically. Agmatine is considered a novel putative neurotransmitter, interacts with several receptor sub-types, and regulates the production of NO and other metabolites of L-arginine. Recent research has demonstrated that endogenous agmatine may directly participate in the processes of learning and memory, and that exogenous agmatine modulates behavioral function (including learning and memory) and has anti-inflammatory and neuroprotective effects. This thesis further investigated the functional role of endogenous agmatine in learning and memory, and explored the therapeutic potential of exogenous agmatine for age-related cognitive decline through multiple approaches.
Experiment 1 aimed to investigate the effects of aging on agmatine and other metabolites of L-arginine in memory-related structures at the tissue and synaptic levels. This study systematically measured the levels of L-arginine and its down-stream metabolites in the CA1, CA2/3 and dentate gyrus sub-region of the hippocampus and the prefrontal, entorhinal, perirhinal and postrhinal cortices in 4 (young) and 24 (aged) months old male Sprague-Dawley (SD) rats at the tissue (crude homogenates) and synaptoneurosome (a subcellular preparation enriched for synaptic material) levels. As brain water content is decreased in aged brain, neurochemical levels in tissue homogenates normalized by wet tissue weight and protein level were compared. There were significant age-related differences in L-arginine, L-citrulline, L-ornithine, agmatine, putrescine, spermidine, spermine and glutamate, but not g-aminobutyric acid (GABA), in a region-specific manner. The overall pattern of age-related changes in amino acids (L-arginine, L-citrulline, L-ornithine, glutamate and GABA) was largely similar between homogenates and synaptoneurosomes, whereas the pattern for the amines (agmatine, putrescine, spermidine and spermine) was quite different. Furthermore, the pattern of age-related changes in neurochemical levels in tissue homogenates normalized by wet tissue weight and protein level was very similar for all 9 neurochemicals measured. These findings suggest that there are differential effects of aging on L-arginine metabolism at the tissue and synaptoneurosome levels and that the way of data normalization (tissue weight vs protein level) has no or very minor effect on the 9 neurochemicals measured.
Experiment 2 aimed to investigate the effects of aging on the basal levels of extracellular agmatine and extracellular pharmacokinetics of agmatine in the dorsal hippocampus. The optimal in vivo microdialysis conditions were established through the in vitro approaches, which were then used to obtain the basal levels of agmatine and pharmacokinetic profiles of agmatine in 4 (young) and 24 (aged), months old male SD rats. Aged rats had significantly lower basal level of extracellular agmatine in the dorsal hippocampus relative to young rats. For both young and aged rats, the intraperitoneal (i.p.) injections of agmatine sulfate (40 mg/kg) resulted in sharp increases in extracellular levels of agmatine within 5 min post-injection, which returned to baseline levels about 3-4 h post-injection, with significantly higher peak concentration (Cmax) and area under the curve (AUC) value and longer half-life in aged rats. Repeated injections of agmatine sulfate led to increased Cmax and AUC in young, but not aged, rats and elevated baseline levels in both age groups. These findings suggest that agmatine administered systemically can readily cross the blood-brain barrier. However, aging alters the basal level of extracellular agmatine and the extracellular pharmacokinetic profile of agmatine in the hippocampus.
Experiments 3 and 4 aimed to explore the therapeutic potential of agmatine by investigating whether agmatine supplementation could improve behavioral function and attenuate age-related changes in NOS activity and protein expression in memory-related structures in aged male SD rats. Aged rats treated with saline displayed significantly reduced exploratory activity (open field) and impaired spatial reference and working memory (water maze) and object recognition memory relative to young rats. Agmatine (40 mg/kg, i.p.) improved animals’ performance in the working memory version (Expt. 3) or reversal test (Expt. 4) of the water maze and object recognition memory tests (both), but not the open field and spatial reference memory version of the water maze, in aged rats regardless of the treatment regimes [short (17 days) vs prolonged (42 days) treatments]. Agmatine treatment significantly suppressed age-related elevation in total NOS activity (both) and restored endothelial NOS protein to the normal level (Expt. 3). Experiment 4 further demonstrated altered haematology and blood chemistry in aged rats. Prolonged agmatine treatment reduced body weight, abdominal fat and blood albumin level, and increased kidney weight, neutrophils and platelet levels in aged rats. These findings demonstrate that exogenous agmatine selectively improves behavioral function in aged rats, suggesting the therapeutic potential of exogenous agmatine for age-related cognitive decline. However, the toxicity of agmatine sulfate, in aged individuals in particular, needs to be fully assessed in the future.
Experiment 5 used the in vivo microdialysis technique coupled with highly sensitive liquid chromatography/mass spectrometry assay to investigate the dynamic changes of extracellular agmatine in the dorsal hippocampus before, during and after training young male SD rats to find a fixed hidden platform in the water maze. Water maze training resulted in approximately 2-6 folds rapid and sharp increases in extracellular agmatine on the first and forth days of testing, whereas swimming per se had no effect. Learning-induced net agmatine release was dramatically decreased across the 3 blocks of training on day 1, but was significantly increased in the final training block containing a probe trial on day 4. The baseline level of extracellular agmatine was significantly increased after 30 trials of training. These findings provide further evidence for the participation of endogenous agmatine in the processes of spatial learning and memory. Endogenous agmatine in the dorsal hippocampus appears to be involved in the encoding and retrieval phases of memory processing.