New therapeutic agents developed for treating neurological disorders are often tested successfully on rodents. Testing in an appropriate large animal model where there is longer lifespan and comparable brain size to humans should improve translational success and is frequently expected by regulatory bodies. In this project, we aimed to establish a novel sheep model of Parkinson's disease as a large-brained experimental model for translational research. Our objective was to create a sheep model of Parkinson's disease by unilaterally infusing the neurotoxin 6-hydroxydopamine into the substantia nigra pars compacta. This approach, previously used to induce parkinsonism in rat and non-human primate models, causes dopaminergic imbalance and induces rotational behaviour in quadrupeds challenged with dopaminergic receptor agonists. In the present sheep study, the mixed dopamine receptor agonist apomorphine, 0.25 mg/kg, and dopamine D2 agonist ropinirole, 0.16 mg/kg, were used to induce rotational behaviour and confirm dopamine depletion. Behavioural signs were then measured and characterised in the field using automated movement tracking with simultaneous video recordings. Post-mortem, the extent of the 6-hydroxydopamine lesions was evaluated through tyrosine hydroxylase immunohistochemistry and quantifying levels of catecholamines (dopamine, 3,4-dihydroxyphenylacetic acid and homovanilic acid) quantified using high-performance liquid chromatography. Our new sheep model of Parkinson's disease using 6-hydroxydopamine is safe and offers a number of regulatory, ethical and financial advantages over non-human primate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine models. It provides a platform to evaluate novel antiparkinsonian agents and medical devices in a large brain with the promise of greater success for translation into clinical application.

Epileptogenesis, the gradual process that leads to epilepsy after brain injury or genetic mutations, is a complex network phenomenon, involving a variety of morphological, biochemical and functional brain alterations. Although risk factors for developing epilepsy are known, there is currently no treatment available to prevent epilepsy. We recently proposed a multitargeted, network-based approach to prevent epileptogenesis by rationally combining clinically available drugs and provided first proof-of-concept that this strategy is effective. Here we evaluated eight novel rationally chosen combinations of 14 drugs with mechanisms that target different epileptogenic processes. The combinations consisted of 2-4 different drugs per combination and were administered systemically over 5 days during the latent epileptogenic period in the intrahippocampal kainate mouse model of acquired temporal lobe epilepsy, starting 6 h after kainate. Doses and dosing intervals were based on previous pharmacokinetic and tolerability studies in mice. The incidence and frequency of spontaneous electrographic and electroclinical seizures were recorded by continuous (24/7) video linked EEG monitoring done for seven days at 4 and 12 weeks post-kainate, i.e., long after termination of drug treatment. Compared to vehicle controls, the most effective drug combination consisted of low doses of levetiracetam, atorvastatin and ceftriaxone, which markedly reduced the incidence of electrographic seizures (by 60%; p<0.05) and electroclinical seizures (by 100%; p<0.05) recorded at 12 weeks after kainate. This effect was lost when higher doses of the three drugs were administered, indicating a synergistic drug-drug interaction at the low doses. The potential mechanisms underlying this interaction are discussed. We have discovered a promising novel multitargeted combination treatment for modifying the development of acquired epilepsy.

Agmatine (decarboxylated arginine) exerts numerous central nervous system (CNS) dependent pharmacological effects and may potentially modulate altered neurochemistry seen in neurological disorders. In preclinical studies, injection has been the predominant route of systemic administration. However, a significant translational step would be the use of oral agmatine treatment at therapeutic doses and better understanding of L-arginine metabolic profiles in the CNS post-treatment. The present study systematically investigated the tolerability, safety and brain-plasma neurochemistry following daily oral agmatine sulfate treatment (via gavage) to wild-type (WT) mice up to 900 mg/kg for one week (Experiment 1) or WT and APPswe/PS1ΔE9 transgenic (Tg) mice at 300 mg/kg for fifteen weeks (Experiment 2). Agmatine treatment in both experiments was well tolerated with no marked behavioural impairments, and gross necropsy and organ histology revealed no pathological alterations after 15-week dosing. Moreover, oral treatment increased agmatine levels in the hippocampus and plasma of WT mice (Experiment 1), and in 6 brain regions examined (but not plasma) of WT and Tg mice (Experiment 2), at 30 minutes or 24 hours post-treatment respectively. This study provides fundamental pre-clinical evidence that daily oral delivery of agmatine sulfate to both WT and Tg mice is safe and well tolerated. Exogenous agmatine passes through the blood brain barrier and accumulates in the brain to a greater extent in Tg mice. Furthermore exogenous agmatine has differential actions in the brain and periphery, and its effect on brain putrescine appears to be dependent on the time post-treatment.

While amyloid-beta (Aβ) peptides play a central role in the development of Alzheimer's disease (AD), recent evidence also implicates altered metabolism of L-arginine in the pathogenesis of AD. The present study systematically investigated how behavioural function and the brain and plasma arginine metabolic profiles changed in a chronic Aβ accumulation model using male APPswe/PS1ΔE9 transgenic (Tg) mice at 7 and 13 months of age. As compared to their wild-type (WT) littermates, Tg mice displayed age-related deficits in spatial water maze tasks and alterations in brain arginine metabolism. Interestingly, the plasma arginine metabolic profile was markedly altered in 7-month Tg mice prior to major behavioural impairment. Receiver operating characteristic curve analysis revealed that plasma putrescine and spermine significantly differentiated between Tg and WT mice. These results demonstrate the parallel development of altered brain arginine metabolism and behavioural deficits in Tg mice. The altered plasma arginine metabolic profile that preceded the behavioural and brain profile changes suggests that there may be merit in an arginine-centric set of ante-mortem biomarkers for AD.

L-arginine is a semi-essential amino acid that can be metabolized to form a number of bioactive molecules. Increasing evidence implicates altered L-arginine metabolism in the pathogenesis of Alzheimer's disease. This study measured the plasma levels of L-arginine and its nine downstream metabolites (L-citrulline, L-ornithine, agmatine, spermidine, spermine, glutamine, glutamate and GABA) in 7 and 13 month old male APP SWE PS1 ΔE9 mice (APP/PS1) and their matched wild-type littermates (WT) using high-performance liquid chromatography and liquid chromatography mass spectroscopy. We found that the plasma levels of L-arginine, L-citrulline, L-ornithine, glutamate and GABA increased with age in both the WT and APP/PS1 mice, but with no significant genotype differences. Furthermore, we observed increased agmatine levels with age, but no age-related changes in the polyamines putrescine, spermidine and spermine. There were increased agmatine levels and reduced putrescine and spermine levels in APP/PS1 mice at 7, but not 13, months of age when compared to their age-matched WT mice. No genotype or age effect was found for glutamine or spermidine. These results demonstrate altered plasma levels of L-arginine and its metabolites with age in a metabolite-specific manner, along with altered agmatine, putrescine and spermine levels in APP/PS1 mice mainly at 7 months of age. Future research is required to understand the functional significance and the underlying mechanisms of these changes. Supported by a grant from the Health Research Council of New Zealand.

Alzheimer's disease (AD) is a common neurodegenerative disease among the elderly. Due to complicated idiopathic pathology, AD is best characterized by progressive cognitive decline with memory loss as the earliest prognostic indicator. Mounting evidence indicates that L-arginine metabolism - integral for cell homeostasis, growth and memory - is altered in AD. However L-arginine metabolism has not been investigated in transgenic (Tg) animal models that are used to mimic and understand pathological AD processes. The current study examined the behavioral and L-arginine metabolic profile in 7 and 13 month old male APP SWE PS1 ΔE9 mice using both reference and working memory versions of the Morris water maze task, high-performance liquid chromatography and liquid chromatography mass spectroscopy. We found that 7 month old Tg mice demonstrated mild impairments in spatial working memory, increased agmatine in the parahippocampal cortex, and increased spermine concentrations in the frontal cortex and striatum compared to their wild-type littermates. At 13 months of age, Tg mice were significantly impaired in both versions of the watermaze task and had accompanying alterations in L-arginine, L-ornithine, agmatine, putrescine, spermidine or spermine in at least one of the five brain regions examined. There were no genotype-specific differences in the cerebellum at either age. Nor were there genotype specific differences in glutamate or GABA at either age points in any brain region. These results indicate that L-arginine metabolism is increasingly altered with age in the APP SWE PS1 ΔE9 mice, which reflect and may influence the behavioral impairments observed.

Agmatine, a metabolite of L-arginine, is considered as a novel putative neurotransmitter. It has been detected in axon terminals that synapse with pyramidal cells in the hippocampus, a brain region that is critically involved in spatial learning and memory. However, the role of agmatine in learning and memory is poorly understood. Recently, we demonstrated water maze training-induced increases in tissue levels of agmatine in the CA1 subregion of the hippocampus. This finding has raised an issue whether an endogenous agmatine could directly participate in learning and memory processes as a neurotransmitter. In the present study, quantitative immunogold-labeling and electron-microscopical techniques were used to analyze the levels of agmatine in CA1 stratum radiatum (SR) terminals (n = 600) of male Sprague-Dawley rats that had been trained to find a hidden escape platform in the water maze (WM) task or forced to swim (SW) in the pool with no platform presented. Agmatine levels were significantly increased by ∼85% in the synaptic terminals of SR of trained WM group compared with the SW control group (all P < 0.001). These results, for the first time, demonstrate spatial learning-induced elevation in agmatine levels at synapses in the hippocampus and provide evidence of its participation in learning and memory processing as a novel neurotransmitter.

Amyloid beta fragment 25-35 (Aβ25-35) is the neurotoxic domain of the full-length Aβ1-42 and causes memory impairments in rodents. Recent research suggests that agmatine, decarboxylated arginine, has a neuroprotective role. This study investigated the effects of a single bilateral i.c.v. infusion of aggregated Aβ25-35 (30 nmol) in a battery of behavioural tests conducted during the period 4–6 (Experiment 1) and 4–14 (Experiment 2) weeks post-Aβ25-35 infusion, and evaluated the protective effect of agmatine (40 mg/kg) administered i.p. 30 min prior to Aβ25-35 infusion and once daily for a further nine consecutive days. In Experiment 1, Aβ25-35 rats with saline treatment were not impaired in the elevated plus maze and open field and mildly impaired in the reference memory version of the water maze task, but performed poorly in the working memory version of the water maze task and the object recognition memory task, relative to the control rats that received the i.c.v. infusion of Aβ35-25 (inactive peptide) and saline treatment. By contrast, Aβ25-35 rats with agmatine treatment did not show performance impairments in the working memory version of the water maze task and the object recognition memory task. In Experiment 2, Aβ25-35 rats with saline treatment were significantly impaired in the standard radial arm maze task, but only displayed no or very mild impairments in the delayed non-match to position and reference memory versions of the radial arm maze task, T-maze, object recognition memory task, both the reference and working memory versions of the water maze task, elevated plus maze and open field. By contrast, Aβ25-35 rats with agmatine treatment were not impaired in the standard radial arm maze and performed even better than the controls in the reference memory version of the task. These results demonstrate that agmatine is able to protect against Aβ25-35-induced memory deficits.

Recent evidence suggests that agmatine, the metabolite of arginine by arginine decarboxylase, exists in the mammalian brain and is a novel neurotransmitter. Exogenous agmatine can modulate behaviour function, including learning and memory. The present study investigated the effects of repeated i.c.v. microinfusion of agmatine (once daily) on the reference and working memory versions of the water maze task, as well as the elevated plus maze and open field. Rats with high (100 microg), but not low (10 microg), dose of agmatine displayed reduced exploratory and locomotor activity in the open field relative to the saline controls on day 1 (received three infusions), but not day 12 (received 14 infusions). The three groups performed similarly on both days in the elevated plus maze tested prior to the open field. In the reference memory version of the water maze task, rats with agmatine treatment at both doses performed as well as the saline controls in the cued navigation (day 2), place navigation (days 3-7) and probe test (day 7). In the working memory version of the water maze task (days 8-11), the two agmatine groups generated markedly shorter path length and took significantly less time to reach the platform at the 180 s, but not 30 s, delay as compared to the saline group. These results demonstrate that repeated agmatine treatment produces transient impairments in exploratory and locomotor activity in the open field in a dose-dependent manner. Agmatine significantly facilitates spatial working memory at a longer delay, but not reference memory, suggesting its differential influence on the two types of spatial learning and memory. The underlying mechanisms need to be explored in the future.