Morris Water Maze Test for Learning and Memory Deficits in Alzheimer's Disease Model Mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory ¹. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions ^2-4. In addition, the MWM testing environment reduces odor trail interference ⁵. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer’s Disease ^{6, 7}. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer’s disease transgenic model mice.     

水迷宫实验中三种品系小鼠学习记忆能力的比较

目的探讨三种品系小鼠在Morris水迷宫实验中的学习记忆能力差异,为与改善学习记忆能力相关新药研究的实验动物选择提供参考。方法选用C57BL/6小鼠、昆明种小鼠和ICR小鼠,进行定位航行实验(5d)、空间搜索实验(1d)和工作记忆实验(4d),考察其学习记忆能力。结果定位航行实验从第3天起C57BL/6小鼠的潜伏期明显小于昆明种小鼠和ICR小鼠(P0.01);空间搜索实验C57BL/6小鼠穿过原平台位置的次数、在原平台象限游程比率和时间比率均明显多于昆明种小鼠和ICR小鼠(P0.05);动物每天工作记忆实验的潜伏期随测试次数增加而缩短,C57BL/6小鼠缩短的较昆明种小鼠和ICR小鼠稍多。结论C57BL/6小鼠的空间参考记忆能力与工作记忆能力均优于昆明种小鼠和ICR小鼠,且非空间因素对其学习记忆能力的评价干扰较小,可作为优先选择的水迷宫实验动物。     

构树黄酮对小鼠学习记忆的影响(英文)

Morris水迷宫在啮齿目动物的空间学习与记忆的研究中被广泛使用。研究表明摄食抗氧化剂能够增强空间学习与记忆能力。本文目的在于研究构树黄酮对昆明小鼠的空间学习与记忆能力的影响。用构树黄酮固体脂质纳米粒对小鼠灌胃4周,然后进行Morris水迷宫测试。与对照组相比,实验组小鼠的各项指标均有显著改善。这表明构树黄酮能显著增强小鼠的空间学习与记忆能力。同时研究还表明构树黄酮对小鼠的生长发育没有影响。     

The Barnes Maze Task Reveals Specific Impairment of Spatial Learning Strategy in the Intrahippocampal Kainic Acid Model for Temporal Lobe Epilepsy

Neurochemical Research - Temporal lobe epilepsy (TLE) is an acquired form of focal epilepsy, in which patients not only suffer from unprovoked, devastating seizures, but also from severe...     

Intraperitoneal injection of saline modulates hippocampal brain receptor complex levels but does not impair performance in the Morris Water Maze

The involvement of the hippocampus in pain has been demonstrated but key players, i.e. the major brain receptors have not been shown to be modulated by pain. It was therefore the aim of the study to show the concerted action and pattern of brain receptor complex levels in a non-invasive model of moderate pain. C57BL/6J mice were divided into four groups of 14 animals each: trained injected, trained non-injected, yoked injected and yoked non-injected. Animals were tested in the open field and the elevated plus maze for behavioural evaluation and cognitive functions were tested using the Morris Water Maze. Hippocampi were taken 6 h following sacrification. Membrane proteins were prepared by ultracentrifugation and run on blue native gels to keep the native state, blotted to membranes and western blotting was carried out using the primary antibodies against serotonin receptor 5HT1A, muscarinic acetylcholine receptor M1 (mAChR-M1), nicotinic acetylcholine receptor alpha7 (nAChR-alpha7), glutamate (AMPA) receptor (GluR1) and neurokinin receptor 1 (NK-1). There was no difference between performance in behaviour or in the MWM between groups. Brain receptor level changes involved all receptors given above. Pain affected mAChR-M1, GluR1 and NK-1 complex levels when yoked-injected were compared with yoked non-injected animals. Memory mechanisms affected mAChR-M1 complex levels when trained non-injected animals were compared with yoked non-injected controls. Taken together, the neurochemical basis for testing receptor agonists/antagonists on the role of pain and the hippocampus was generated that may be useful for interpretations of the role of this complex area in moderate pain.     

Flavor Preference Learning Increases Olfactory and Gustatory Convergence onto Single Neurons in the Basolateral Amygdala but Not in the Insular Cortex in Rats

The basolateral amygdala (BLA) and the insular cortex (IC) represent two major areas for odor-taste associations, i.e. flavor integration. This learning may require the development of convergent odor and taste neuronal activation allowing the memory representation of such association. Yet identification of neurons that respond to such coincident input and the effect of flavor experience on odor-taste convergence remain unclear. In the present study we used the compartmental analysis of temporal activity using fluorescence in situ hybridization for Arc (catFISH) to visualize odor-taste convergence onto single neurons in the BLA and in the IC to assess the number of cells that were co-activated by both stimuli after odor-taste association. We used a sucrose conditioned odor preference as a flavor experience in rats, in which 9 odor-sucrose pairings induce a reliable odor-taste association. The results show that flavor experience induced a four-fold increase in the percentage of cells activated by both taste and odor stimulations in the BLA, but not in the IC. Because conditioned odor preference did not modify the number of cells responding selectively to one stimulus, this greater odor-taste convergence into individual BLA neurons suggests the recruitment of a neuronal population that can be activated by both odor and taste only after the association. We conclude that the development of convergent activation in amygdala neurons after odor-taste associative learning may provide a cellular basis of flavor memory.     

A new semisynthetic derivative of sauroine induces LTP in hippocampal slices and improves learning performance in the Morris Water Maze

Two semisynthetic acetyl derivatives of the alkaloid sauroine from Huperzia saururus, monoacetyl sauroine, and diacetyl sauroine (DAS) were obtained and their chemical structures were analyzed by NMR. While monoacetyl sauroine is the typical product of acetylation, DAS is an unexpected derivative related to the keto‐enol formation of sauroine. Recordings of field excitatory post‐synaptic potentials from the CA1 region of rat hippocampal slices showed that only DAS acutely applied induced chemical long‐term potentiation (LTP) in a dose‐dependent manner with an EC₅₀ of 1.15 ± 0.09 μM. This effect was blocked by 10 μM D(‐)‐2‐amino‐5‐phosphonopentanoic acid (AP5), suggesting dependence on the NMDA receptor. DAS significantly increased NMDA receptor‐dependent excitatory post‐synaptic currents without affecting α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptor‐dependent currents. Repetitive administration of DAS improved visuo‐spatial learning in the Morris Water Maze. In slices from rats tested in the Morris Water Maze, LTP resulting from electrical synaptic stimulation was 2.5 times larger than in controls. Concentration of DAS measured in the brain after repetitive administration was 29.5 μM. We conclude that slices perfused with DAS display a robust NMDA receptor‐dependent chemical LTP. During chronic treatment, DAS enhances learning abilities through a metaplastic mechanism as revealed by the augmentation of LTP in slices. DAS, therefore, may be a promising compound as a nootropic therapeutic drug.

     

Training in the Morris Water Maze of Female and Male Rats Exposed to Hypoxia at Various Periods of Prenatal Development

Capability for learning was studied in the offspring of rats exposed to hyporbaric hypoxia on the days 11–13, 14–16 or 18–20 of pregnancy. Training in the Morris water maze has been shown to lead to consequences of effect of prenatal hypoxia evident in males, but not in females. The most pronounced changes are found at training in the male rats whose mothers were exposed to hypoxia on the days 14–16 of pregnancy. The revealed differences in the character of learning depend on experimental conditions. Under “severe” stress conditions (at the water temperature of 16–17°C), prenatal hypoxia leads to an improvement of learning parameters as compared with control, while under more favorable conditions (at the water temperature of 23–24°C), to their deterioration.     

Male Age and Experience Increases Mating Success but Not Female Fitness in the Mexican Fruit Fly

Male age and sexual experience are important condition parameters that can influence mate choice and female fitness, yet they are seldom studied simultaneously. Here, we investigated the effect of male age and previous sexual experience on mating success and female fitness in the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae). Males were either young or old, sexually experienced or naïve. Older and sexually experienced males obtained more copulations. However, females did not receive benefits in terms of lifetime fecundity, fertility, or longevity from mating with these males. Results suggest that males become more competitive as they age and gain sexual experience and may be able to maintain a high quality ejaculate compared to young males. Older experienced males may be manipulating females into preferentially mating with them at no benefit to females. Alternatively, females may prefer mating with older more experienced males and possibly receive other indirect benefits, but this remains to be tested.     

CorA Affects Tolerance of Escherichia coli and Salmonella enterica Serovar Typhimurium to the Lactoperoxidase Enzyme System but Not to Other Forms of Oxidative Stress

The enzyme lactoperoxidase is part of the innate immune system in vertebrates and owes its antimicrobial activity to the formation of oxidative reaction products from various substrates. In a previous study, we have reported that, with thiocyanate as a substrate, the lactoperoxidase system elicits a distinct stress response in Escherichia coli MG1655. This response is different from but partly overlapping with the stress responses to hydrogen peroxide and to superoxide. In the current work, we constructed knockouts in 10 lactoperoxidase system-inducible genes to investigate their role in the tolerance of E. coli MG1655 to this antimicrobial system. Five mutations resulted in a slightly increased sensitivity, but one mutation (corA) caused hypersensitivity to the lactoperoxidase system. This hypersensitive phenotype was specific to the lactoperoxidase system, since neither the sensitivity to hydrogen peroxide nor to the superoxide generator plumbagin was affected in the corA mutant. Salmonella enterica serovar Typhimurium corA had a similar phenotype. Although corA encodes an Mg²⁺ transporter and at least three other inducible open reading frames belonged to the Mg²⁺ regulon, repression of the Mg stimulon by Mg²⁺ did not change the lactoperoxidase sensitivity of either the wild-type or corA mutant. Prior exposure to 0.3 mM Ni²⁺, which is also transported by CorA, strongly sensitized MG1655 but not the corA mutant to the lactoperoxidase system. Furthermore, this Ni²⁺-dependent sensitization was suppressed by the CorA-specific inhibitor Co(III) hexaammine. These results indicate that CorA affects the lactoperoxidase sensitivity of E. coli by modulating the cytoplasmic concentrations of transition metals that enhance the toxicity of the lactoperoxidase system.     

Mental Rotational Ability Is Correlated with Spatial but Not Verbal Working Memory Performance and P300 Amplitude in Males

This study investigated how both sex and individual differences in a mental rotation test (MRT) influence performance on working memory (WM). To identify the neural substrate supporting these differences, brain electrical activity was measured using the event-related potential technique. No significant sex differences were observed in a test of verbal WM, however males were significantly faster than females to respond to probe stimuli in a test of spatial WM. This difference was no longer significant after controlling for differences in MRT score, suggesting that rotational ability mediates performance in the spatial memory task for both sexes. A posterior P300 was observed in both tasks as participants encoded information into memory, however the amplitude of the P300 correlated with RT in the spatial task but not in the verbal task. Individual differences in the MRT also correlated with RT and with the amplitude of the P300, but again only in the spatial task. After splitting the analysis by sex, partial correlations controlling for MRT revealed that for males, individual differences in rotational ability completely mediated the correlation between the P300 and RT in the spatial task. This mediating effect was not observed for the female participants. The results therefore suggest a relatively stronger association in males between innate mental rotational ability, spatial memory performance, and brain electrophysiological processes supporting spatial memory.     

Vaccination Method Affects Immune Response and Bacterial Growth but Not Protection in the Salmonella Typhimurium Animal Model of Typhoid

Understanding immune responses elicited by vaccines, together with immune responses required for protection, is fundamental to designing effective vaccines and immunisation programs. This study examines the effects of the route of administration of a live attenuated vaccine on its interactions with, and stimulation of, the murine immune system as well as its ability to increase survival and provide protection from colonisation by a virulent challenge strain. We assess the effect of administration method using the murine model for typhoid, where animals are infected with S. Typhimurium. Mice were vaccinated either intravenously or orally with the same live attenuated S. Typhimurium strain and data were collected on vaccine strain growth, shedding and stimulation of antibodies and cytokines. Following vaccination, mice were challenged with a virulent strain of S. Typhimurium and the protection conferred by the different vaccination routes was measured in terms of challenge suppression and animal survival. The main difference in immune stimulation found in this study was the development of a secretory IgA response in orally-vaccinated mice, which was absent in IV vaccinated mice. While both strains showed similar protection in terms of challenge suppression in systemic organs (spleen and liver) as well as survival, they differed in terms of challenge suppression of virulent pathogens in gut-associated organs. This difference in gut colonisation presents important questions around the ability of vaccines to prevent shedding and transmission. These findings demonstrate that while protection conferred by two vaccines can appear to be the same, the mechanisms controlling the protection can differ and have important implications for infection dynamics within a population.     

Substitution of 2-Aminoadenine and 5-Methylcytosine for Adenine and Cytosine in Hybridization Probes Increases the Sensitivity of DNA Fingerprinting

An oligonucleotide (m⁵C-am²A-m⁵C)₅ containing 2′-amino-deoxyadenosine (am²A) and 5-methyldeoxycytidine (m⁵C) residues has been synthesized and compared with unsubstituted pentadecadeoxyribonucleotide (CAC)₅ as a hybridization probe for DNA fingerprinting. It was shown that considerably higher sensitivity can be achieved with the modified analog.     

Maternal Exercise during Pregnancy Increases BDNF Levels and Cell Numbers in the Hippocampal Formation but Not in the Cerebral Cortex of Adult Rat Offspring

Clinical evidence has shown that physical exercise during pregnancy may alter brain development and improve cognitive function of offspring. However, the mechanisms through which maternal exercise might promote such effects are not well understood. The present study examined levels of brain-derived neurotrophic factor (BDNF) and absolute cell numbers in the hippocampal formation and cerebral cortex of rat pups born from mothers exercised during pregnancy. Additionally, we evaluated the cognitive abilities of adult offspring in different behavioral paradigms (exploratory activity and habituation in open field tests, spatial memory in a water maze test, and aversive memory in a step-down inhibitory avoidance task). Results showed that maternal exercise during pregnancy increased BDNF levels and absolute numbers of neuronal and non-neuronal cells in the hippocampal formation of offspring. No differences in BDNF levels or cell numbers were detected in the cerebral cortex. It was also observed that offspring from exercised mothers exhibited better cognitive performance in nonassociative (habituation) and associative (spatial learning) mnemonic tasks than did offspring from sedentary mothers. Our findings indicate that maternal exercise during pregnancy enhances offspring cognitive function (habituation behavior and spatial learning) and increases BDNF levels and cell numbers in the hippocampal formation of offspring.     

The Expression of BAFF, APRIL and TWEAK Is Altered in Eczema Skin but Not in the Circulation of Atopic and Seborrheic Eczema Patients

The TNF family cytokines BAFF (B-cell activating factor of the TNF family) and APRIL (a proliferation-inducing ligand) are crucial survival factors for B-cell development and activation. B-cell directed treatments have been shown to improve atopic eczema (AE), suggesting the involvement of these cytokines in the pathogenesis of AE. We therefore analyzed the expression of these TNF cytokines in AE, seborrheic eczema (SE) and healthy controls (HC). The serum/plasma concentration of BAFF, APRIL and a close TNF member TWEAK (TNF-like weak inducer of apoptosis) was measured by ELISA. The expression of these cytokines and their receptors in skin was analyzed by quantitative RT-PCR and immunofluorescence. Unlike other inflammatory diseases including autoimmune diseases and asthma, the circulating levels of BAFF, APRIL and TWEAK were not elevated in AE or SE patients compared with HCs and did not correlate with the disease severity or systemic IgE levels in AE patients. Interestingly, we found that the expression of these cytokines and their receptors was altered in positive atopy patch test reactions in AE patients (APT-AE) and in lesional skin of AE and SE patients. The expression of APRIL was decreased and the expression of BAFF was increased in eczema skin of AE and SE, which could contribute to a reduced negative regulatory input on B-cells. This was found to be more pronounced in APT-AE, the initiating acute stage of AE, which may result in dysregulation of over-activated B-cells. Furthermore, the expression levels of TWEAK and its receptor positively correlated to each other in SE lesions, but inversely correlated in AE lesions. These results shed light on potential pathogenic roles of these TNF factors in AE and SE, and pinpoint a potential of tailored treatments towards these factors in AE and SE.     

BDNF Level in the Rat Prefrontal Cortex Increases Following Chronic but Not Acute Treatment with Duloxetine, a Dual Acting Inhibitor of Noradrenaline and Serotonin Re-uptake

Aims Brain-Derived Neurotrophic Factor (BDNF) has a central role in neuronal survival, differentiation, and plasticity. The brain level of BDNF is changed by several mood stabilizers and antidepressant drugs acting on neurotransmitters such as noradrenaline and serotonin. We investigated the effects of acute and chronic treatment with Duloxetine, a new drug blocking the re-uptake of serotonin and noradrenaline (SNRI), on BDNF level in the prefrontal cortex, cerebrospinal fluid, plasma, and serum. Methods Wistar male rats were treated with acute (single treatment) and chronic oral administration (14 days) of different concentrations of Duloxetine (10, 30, and 100 mg/kg/day). At the end of the treatment periods, samples of blood, CSF and the prefrontal cortex were collected. BDNF levels were measured by ELISA. Levels of mature and precursor form of BDNF were measured by Western blot analysis. Results Animals treated with the Duloxetine at all concentrations and examined after 1 and 24 h (single treatment) did not reveal a significant change in the total BDNF level. In animals treated for 14 days with Duloxetine at 30 and 100 mg/kg, the total BDNF level increased significantly in the prefrontal cortex and CSF, but not in the plasma and serum. Using a specific antibody and Western blot we showed that the mature, but not the precursor, form of BDNF was significantly increased in the prefrontal cortex of rats treated for 14 days with Duloxetine at 30 mg/kg/day. Conclusions Our results show a major finding that repeated, but not single, Duloxetine treatment increases the level of BDNF in the prefrontal cortex. Claudio Mannari and Nicola Origlia are contributed equally to this work.