Altered glutamate clearance in ascorbate deficient mice increases seizure susceptibility and contributes to cognitive impairment in APP/PSEN1 mice.

Ascorbate (vitamin C) is critical as a first line of defense antioxidant within the brain, and specifically within the synapse. Ascorbate is released by astrocytes during glutamate clearance and disruption of this exchange mechanism may be critical in mediating glutamate toxicity within the synapse. This is likely even more critical in neurodegenerative disorders with associated excitotoxicity and seizures, in particular Alzheimer's disease, in which ascorbate levels are often low. Using Gulo-/- mice that are dependent on dietary ascorbate, we established that low brain ascorbate increased sensitivity to kainic acid as measured via behavioral observations, electroencephalography (EEG) measurements, and altered regulation of several glutamatergic system genes. Kainic acid-induced immobility was improved in wild-type mice following treatment with ceftriaxone, which upregulates glutamate transporter GLT-1. The same effect was not observed in ascorbate-deficient mice in which sufficient ascorbate is not available for release. A single, mild seizure event was sufficient to disrupt performance in the water maze in low-ascorbate mice and in APPSWE/PSEN1dE9 mice. Together, the data support the critical role of brain ascorbate in maintaining protection during glutamatergic hyperexcitation events, including seizures. The study further supports a role for mild, subclinical seizures in cognitive decline in Alzheimer's disease.

Low brain ascorbic acid increases susceptibility to seizures in mouse models of decreased brain ascorbic acid transport and Alzheimer's disease.

Seizures are a known co-occurring symptom of Alzheimer's disease, and they can accelerate cognitive and neuropathological dysfunction. Sub-optimal vitamin C (ascorbic acid) deficiency, that is low levels that do not lead the sufferer to present with clinical signs of scurvy (e.g. lethargy, hemorrhage, hyperkeratosis), are easily obtainable with insufficient dietary intake, and may contribute to the oxidative stress environment of both Alzheimer's disease and epilepsy. The purpose of this study was to test whether mice that have diminished brain ascorbic acid in addition to carrying human Alzheimer's disease mutations in the amyloid precursor protein (APP) and presenilin 1 (PSEN1) genes, had altered electrical activity in the brain (electroencephalography; EEG), and were more susceptible to pharmacologically induced seizures. Brain ascorbic acid was decreased in APP/PSEN1 mice by crossing them with sodium vitamin C transporter 2 (SVCT2) heterozygous knockout mice. These mice have an approximately 30% decrease in brain ascorbic acid due to lower levels of SVCT2 that supplies the brain with ASC. SVCT2+/-APP/PSEN1 mice had decreased ascorbic acid and increased oxidative stress in brain, increased mortality, faster seizure onset latency following treatment with kainic acid (10 mg/kg i.p.), and more ictal events following pentylenetetrazol (50 mg/kg i.p.) treatment. Furthermore, we report the entirely novel phenomenon that ascorbic acid deficiency alone increased the severity of kainic acid- and pentylenetetrazol-induced seizures. These data suggest that avoiding ascorbic acid deficiency may be particularly important in populations at increased risk for epilepsy and seizures, such as Alzheimer's disease.

Intravenous ascorbate improves spatial memory in middle-aged APP/PSEN1 and wild type mice.

The present study investigated the effects of a single intravenous (i.v.) dose of Vitamin C (ascorbate, ASC) on spatial memory in APP/PSEN1 mice, an Alzheimer's disease model. First, we confirmed the uptake time course in ASC-depleted gulo (-/-) mice, which cannot synthesize ASC. Differential tissue uptake was seen based on ASC transporter distribution. Liver (SVCT1 and SVCT2) ASC was elevated at 30, 60 and 120 min post-treatment (125 mg/kg, i.v.), whereas spleen (SVCT2) ASC increased at 60 and 120 min. There was no detectable change in cortical (SVCT2 at choroid plexus, and neurons) ASC within the 2-h interval, although the cortex preferentially retained ASC. APP/PSEN1 and wild type (WT) mice at three ages (3, 9, or 20 months) were treated with ASC (125 mg/kg, i.v.) or saline 45 min before testing on the Modified Y-maze, a two-trial task of spatial memory. Memory declined with age and ASC treatment improved performance in 9-month-old APP/PSEN1 and WT mice. APP/PSEN1 mice displayed no behavioral impairment relative to WT controls. Although dopamine and metabolite DOPAC decreased in the nucleus accumbens with age, and improved spatial memory was correlated with increased dopamine in saline treated mice, acute ASC treatment did not alter monoamine levels in the nucleus accumbens. These data show that the Modified Y-maze is sensitive to age-related deficits, but not additional memory deficits due to amyloid pathology in APP/PSEN1 mice. They also suggest improvements in short-term spatial memory were not due to changes in the neuropathological features of AD or monoamine signaling.

A comparison of low- and high-impact forced exercise: effects of training paradigm on learning and memory.

In this study we compared two types of forced exercise-a low impact paradigm to minimize stress, which included speeds up to 10 m/min and a stressful high impact paradigm, with speeds up to 21 m/min. 150 male C57BL/6J mice were randomly assigned to the low impact, high impact, or sedentary control conditions and were tested on the rotorod and Morris water maze (MWM) as indices of motor learning and spatial memory. We found that 5 weeks of stressful high speed forced exercise led to significant improvement in rotorod performance, as high impact runners outperformed both low impact runners and controls at 15 and 25 rpm speeds. These differences were the result of improved physical fitness due to exercise and likely do not reflect enhanced learning in these mice. In the MWM, 5 weeks of stressful high impact exercise led to significant impairment in spatial memory acquisition compared to low impact runners and controls. Low impact exercise for 10 weeks significantly improved retention of spatial memory compared to high impact exercise. Results suggested that these two paradigms produced different effects of forced exercise on learning and memory. The low impact paradigm led to some improvements, whereas the stressful high impact program caused significant impairment. Comparison of these two paradigms begins to address the window between the beneficial and detrimental effects of forced exercise, and have suggested a boundary of exercise intensity that leads to impairment in learning.

The context preexposure facilitation effect in mice: a dose-response analysis of pretraining scopolamine administration.

The context preexposure facilitation effect (CPFE) is an elaboration of contextual fear conditioning and refers to enhanced contextual conditioning resulting from preexposure to the context prior to a separate, brief context-shock episode. A version of the CPFE developed by Rudy and colleagues in rats has demonstrated greater sensitivity to pre-training hippocampal insult relative to standard contextual fear conditioning preparations. Our aim was to adapt the Rudy CPFE procedures to mice. In Experiment 1 we compared performance of young adult male C57BL6/J mice on two versions of the CPFE. One version - not previously used in mice - adapted methods established by Rudy and colleagues, and the other CPFE task replicated procedures previously established in this mouse strain by Gould and colleagues. In Experiment 2 we compared the effects of pre-training intraperitoneal administration of moderate levels of scopolamine or methylscopolamine on contextual conditioning between mice trained using the Rudy CPFE method and a separate group trained using standard contextual fear procedures. Scopolamine is a muscarinic cholinergic receptor antagonist that impairs hippocampal function. Robust freezing to the conditioning context was observed in mice trained using the Rudy CPFE method (Experiment 1), and greater scopolamine-induced impairments in contextual freezing were observed using this CPFE method relative to mice trained using standard contextual fear procedures (Experiment 2). These findings support use of the Rudy CPFE task as a behavioral assay for hippocampal function in mice.

Age sensitivity of behavioral tests and brain substrates of normal aging in mice.

Knowledge of age sensitivity, the capacity of a behavioral test to reliably detect age-related changes, has utility in the design of experiments to elucidate processes of normal aging. We review the application of these tests in studies of normal aging and compare and contrast the age sensitivity of the Barnes maze, eyeblink classical conditioning, fear conditioning, Morris water maze, and rotorod. These tests have all been implemented to assess normal age-related changes in learning and memory in rodents, which generalize in many cases to age-related changes in learning and memory in all mammals, including humans. Behavioral assessments are a valuable means to measure functional outcomes of neuroscientific studies of aging. Highlighted in this review are the attributes and limitations of these measures in mice in the context of age sensitivity and processes of brain aging. Attributes of these tests include reliability and validity as assessments of learning and memory, well-defined neural substrates, and sensitivity to neural and pharmacological manipulations and disruptions. These tests engage the hippocampus and/or the cerebellum, two structures centrally involved in learning and memory that undergo functional and anatomical changes in normal aging. A test that is less well represented in studies of normal aging, the context pre-exposure facilitation effect (CPFE) in fear conditioning, is described as a method to increase sensitivity of contextual fear conditioning to changes in the hippocampus. Recommendations for increasing the age sensitivity of all measures of normal aging in mice are included, as well as a discussion of the potential of the under-studied CPFE to advance understanding of subtle hippocampus-mediated phenomena.

Differential effects and rates of normal aging in cerebellum and hippocampus.

Cognitive functions show many alternative outcomes and great individual variation during normal aging. We examined learning over the adult life span in CBA mice, along with morphological and electrophysiological substrates. Our aim was to compare cerebellum-dependent delay eyeblink classical conditioning and hippocampus-dependent contextual fear conditioning in the same animals using the same conditioned and unconditioned stimuli for eyeblink and fear conditioning. In a subset of the behaviorally tested mice, we used unbiased stereology to estimate the total number of Purkinje neurons in cerebellar cortex and pyramidal neurons in the hippocampus. Several forms of synaptic plasticity were assessed at different ages in CBA mice: long-term depression (LTD) in both cerebellum and hippocampus and NMDA-mediated long-term potentiation (LTP) and voltage-dependent calcium channel LTP in hippocampus. Forty-four CBA mice tested at one of five ages (4, 8, 12, 18, or 24 months) demonstrated statistically significant age differences in cerebellum-dependent delay eyeblink conditioning, with 24-month mice showing impairment in comparison with younger mice. These same CBA mice showed no significant differences in contextual or cued fear conditioning. Stereology indicated significant loss of Purkinje neurons in the 18- and 24-month groups, whereas pyramidal neuron numbers were stable across age. Slice electrophysiology recorded from an additional 48 CBA mice indicated significant deficits in LTD appearing in cerebellum between 4 and 8 months, whereas 4- to 12-month mice demonstrated similar hippocampal LTD and LTP values. Our results demonstrate that processes of aging impact brain structures and associated behaviors differentially, with cerebellum showing earlier senescence than hippocampus.