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.     

The effect of a caudal hippocampus lesion on learning in a Morris water maze in Bank Voles (Clethrionomys glareolus)

The involvement of the caudal hippocampus in spatial learning is presently uncertain, compared to the well established role of the dorsal region. Therefore voles (Clethrionomys glareolus) with large (about 1/3 of the whole hippocampus) caudal cytotoxic lesions were tested in the Morris water maze. A version of the test intended to measure long term spatial memory was used. The lesion inhibited the learning process, as well as reducing the accuracy of platform location memory at early stages of training. The data obtained indicate the involvement of this area in control of spatial learning in rodents.     

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca²⁺), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca²⁺ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca²⁺ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (“slices”) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca²⁺ level. The protocol also allows “in-slice measurement” of absolute Ca²⁺ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca²⁺ signaling as well as the potential involvement of Ca²⁺ in photoreceptor death and retinal degeneration.     

Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment

The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimer’s disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimer’s disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.     

Effects of testosterone on spatial learning and memory in adult male rats

A male advantage over females for spatial tasks has been well documented in both humans and rodents, but it remains unclear how the activational effects of testosterone influence spatial ability in males. In a series of experiments, we tested how injections of testosterone influenced the spatial working and reference memory of castrated male rats. In the eight-arm radial maze, testosterone injections (0.500 mg/rat) reduced the number of working memory errors during the early blocks of testing but had no effect on the number of reference memory errors relative to the castrated control group. In a reference memory version of the Morris water maze, injections of a wide range of testosterone doses (0.0625-1.000 mg/rat) reduced path lengths to the hidden platform, indicative of improved spatial learning. This improved learning was independent of testosterone dose, with all treatment groups showing better performance than the castrated control males. Furthermore, this effect was only observed when rats were given testosterone injections starting 7 days prior to water maze testing and not when injections were given only on the testing days. We also observed that certain doses of testosterone (0.250 and 1.000 mg/rat) increased perseverative behavior in a reversal-learning task. Finally, testosterone did not have a clear effect on spatial working memory in the Morris water maze, although intermediate doses seemed to optimize performance. Overall, the results indicate that testosterone can have positive activational effects on spatial learning and memory, but the duration of testosterone replacement and the nature of the spatial task modify these effects.     

Tool Use Specific Adult Neurogenesis and Synaptogenesis in Rodent (Octodon degus) Hippocampus

We previously demonstrated that degus (Octodon degus), which are a species of small caviomorph rodents, could be trained to use a T-shaped rake as a hand tool to expand accessible spaces. To elucidate the neurobiological underpinnings of this higher brain function, we compared this tool use learning task with a simple spatial (radial maze) memory task and investigated the changes that were induced in the hippocampal neural circuits known to subserve spatial perception and learning. With the exposure to an enriched environment in home cage, adult neurogenesis in the dentate gyrus of the hippocampus was augmented by tool use learning, but not radial maze learning, when compared to control conditions. Furthermore, the proportion of new synapses formed in the CA3 region of the hippocampus, the target area for projections of mossy fiber axons emanating from newborn neurons, was specifically increased by tool use learning. Thus, active tool use behavior by rodents, learned through multiple training sessions, requires the hippocampus to generate more novel neurons and synapses than spatial information processing in radial maze learning.     

Morris water maze: procedures for assessing spatial and related forms of learning and memory

The Morris water maze (MWM) is a test of spatial learning for rodents that relies on distal cues to navigate from start locations around the perimeter of an open swimming arena to locate a submerged escape platform. Spatial learning is assessed across repeated trials and reference memory is determined by preference for the platform area when the platform is absent. Reversal and shift trials enhance the detection of spatial impairments. Trial-dependent, latent and discrimination learning can be assessed using modifications of the basic protocol. Search-to-platform area determines the degree of reliance on spatial versus non-spatial strategies. Cued trials determine whether performance factors that are unrelated to place learning are present. Escape from water is relatively immune from activity or body mass differences, making it ideal for many experimental models. The MWM has proven to be a robust and reliable test that is strongly correlated with hippocampal synaptic plasticity and NMDA receptor function. We present protocols for performing variants of the MWM test, from which results can be obtained from individual animals in as few as 6 days.     

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we describe a pERK immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. Specifically, we used pERK immunostaining to study neuronal activation following Morris water maze (MWM, a classical hippocampal-dependent learning task) in Engrailed-2 knockout (En2^-/-) mice, a model of autism spectrum disorders (ASD). As compared to wild-type (WT) controls, En2^-/- mice showed significant spatial learning deficits in the MWM. After MWM, significant differences in the number of pERK-positive neurons were detected in specific hippocampal subfields of En2^-/- mice, as compared to WT animals. Thus, our protocol can robustly detect differences in pERK-positive neurons associated to hippocampal-dependent learning impairment in a mouse model of ASD. More generally, our protocol can be applied to investigate the profile of hippocampal neuron activation in both genetic or pharmacological mouse models characterized by cognitive deficits.     

Use of spatial information and search strategies in a water maze analog in Drosophila melanogaster

Learning the spatial organization of the environment is crucial to fitness in most animal species. Understanding proximate and ultimate factors underpinning spatial memory is thus a major goal in the study of animal behavior. Despite considerable interest in various aspects of its behavior and biology, the model species Drosophila melanogaster lacks a standardized apparatus to investigate spatial learning and memory. We propose here a novel apparatus, the heat maze, conceptually based on the Morris water maze used in rodents. Using the heat maze, we demonstrate that D. melanogaster flies are able to use either proximal or distal visual cues to increase their performance in navigating to a safe zone. We also show that flies are actively using the orientation of distal visual cues when relevant in targeting the safe zone, i.e., Drosophila display spatial learning. Parameter-based classification of search strategies demonstrated the progressive use of spatially precise search strategies during learning. We discuss the opportunity to unravel the mechanistic and evolutionary bases of spatial learning in Drosophila using the heat maze.     

Behavior strategy learning in rat: effects of lesions of the dorsal striatum or dorsal hippocampus

Depending on task demands, there is a growing body of evidence suggesting that the dorsal striatum plays a critical role in not only learning new response strategies but also in the inhibition of pre-existing strategies when a shift in strategy is required. The present experiment examined the effects of lesions of the dorsal striatum or dorsal hippocampus on acquisition of a response-learning rule and a place-learning rule in a Greek Cross version of the Morris water maze. Specifically, adult Long-Evans rats were prepared with either sham lesions or lesions to one of two subcortical areas of the brain considered necessary for processing nondeclarative or declarative memories, the dorsal striatum or the hippocampus, respectively. An analysis of the trial 2 performance pooled across reversals revealed hippocampus lesions induced accelerated acquisition when a response-learning rule was required. A much smaller enhancement effect was observed in dorsal striatum-lesioned animals in the place-learning paradigm. Dorsal hippocampus- and dorsal striatum-lesioned animals were highly impaired on place learning and response learning, respectively. The present results are congruent with a growing body of literature suggesting that different anatomical substrates are involved in the acquisition and maintenance of different types of information, that these processes can occur simultaneously and in parallel, and that the dorsal striatum is necessary for the mediation of stimulus-response learning, while the hippocampus is necessary to mediate the expression of place learning.     

Barnes Maze Testing Strategies with Small and Large Rodent Models

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g. bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g. distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e. random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g. shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.     

When a Text Is Translated Does the Complexity of Its Vocabulary Change? Translations and Target Readerships

In linguistic studies, the academic level of the vocabulary in a text can be described in terms of statistical physics by using a “temperature” concept related to the text''s word-frequency distribution. We propose a “comparative thermo-linguistic” technique to analyze the vocabulary of a text to determine its academic level and its target readership in any given language. We apply this technique to a large number of books by several authors and examine how the vocabulary of a text changes when it is translated from one language to another. Unlike the uniform results produced using the Zipf law, using our “word energy” distribution technique we find variations in the power-law behavior. We also examine some common features that span across languages and identify some intriguing questions concerning how to determine when a text is suitable for its intended readership.     

Socio-Demographic Predictors and Distribution of Pulmonary Tuberculosis (TB) in Xinjiang,China: A Spatial Analysis

Objectives

Xinjiang is one of the high TB burden provinces of China. A spatial analysis was conducted using geographical information system (GIS) technology to improve the understanding of geographic variation of the pulmonary TB occurrence in Xinjiang, its predictors, and to search for targeted interventions.

Methods

Numbers of reported pulmonary TB cases were collected at county/district level from TB surveillance system database. Population data were extracted from Xinjiang Statistical Yearbook (2006~2014). Spatial autocorrelation (or dependency) was assessed using global Moran’s I statistic. Anselin’s local Moran’s I and local Getis-Ord statistics were used to detect local spatial clusters. Ordinary least squares (OLS) regression, spatial lag model (SLM) and geographically-weighted regression (GWR) models were used to explore the socio-demographic predictors of pulmonary TB incidence from global and local perspectives. SPSS17.0, ArcGIS10.2.2, and GeoDA software were used for data analysis.

Results

Incidence of sputum smear positive (SS+) TB and new SS+TB showed a declining trend from 2005 to 2013. Pulmonary TB incidence showed a declining trend from 2005 to 2010 and a rising trend since 2011 mainly caused by the rising trend of sputum smear negative (SS-) TB incidence (p<0.0001). Spatial autocorrelation analysis showed the presence of positive spatial autocorrelation for pulmonary TB incidence, SS+TB incidence and SS-TB incidence from 2005 to 2013 (P <0.0001). The Anselin’s Local Moran’s I identified the “hotspots” which were consistently located in the southwest regions composed of 20 to 28 districts, and the “coldspots” which were consistently located in the north central regions consisting of 21 to 27 districts. Analysis with the Getis-Ord Gi* statistic expanded the scope of “hotspots” and “coldspots” with different intensity; 30 county/districts clustered as “hotspots”, while 47 county/districts clustered as “coldspots”. OLS regression model included the “proportion of minorities” and the “per capita GDP” as explanatory variables that explained 64% the variation in pulmonary TB incidence (adjR² = 0.64). The SLM model improved the fit of the OLS model with a decrease in AIC value from 883 to 864, suggesting “proportion of minorities” to be the only statistically significant predictor. GWR model also improved the fitness of regression (adj R² = 0.68, AIC = 871), which revealed that “proportion of minorities” was a strong predictor in the south central regions while “per capita GDP” was a strong predictor for the southwest regions.

Conclusion

The SS+TB incidence of Xinjiang had a decreasing trend during 2005–2013, but it still remained higher than the national average in China. Spatial analysis showed significant spatial autocorrelation in pulmonary TB incidence. Cluster analysis detected two clusters—the “hotspots”, which were consistently located in the southwest regions, and the “coldspots”, which were consistently located in the north central regions. The exploration of socio-demographic predictors identified the “proportion of minorities” and the “per capita GDP” as predictors and may help to guide TB control programs and targeting intervention.     

Spiny Mice Modulate Eumelanin to Pheomelanin Ratio to Achieve Cryptic Coloration in “Evolution Canyon,” Israel

Background

Coat coloration in mammals is an explicit adaptation through natural selection. Camouflaging with the environment is the foremost evolutionary drive in explaining overall coloration. Decades of enquiries on this topic have been limited to repetitive coat color measurements to correlate the morphs with background/habitat blending. This led to an overwhelming endorsement of concealing coloration as a local phenotypic adaptation in animals, primarily rodents to evade predators. However, most such studies overlooked how rodents actually achieve such cryptic coloration. Cryptic coloration could be attained only through optimization between the yellow- to brown-colored “pheomelanin” and gray to black-colored “eumelanin” in the hairs. However, no study has explored this conjecture yet. “Evolution Canyon” (EC) in Israel is a natural microscale laboratory where the relationship between organism and environment can be explored. EC is comprised of an “African” slope (AS), which exhibits a yellow-brownish background habitat, and a “European” slope (ES), exhibiting a dark grayish habitat; both slopes harbor spiny mice (Acomys cahirinus). Here, we examine how hair melanin content of spiny mice living in the opposing slopes of EC evolves toward blending with their respective background habitat.

Methodology/Principal Findings

We measured hair-melanin (both eumelanin and pheomelanin) contents of 30 spiny mice from the EC using high-performance liquid chromatography (HPLC) that detects specific degradation products of eumelanin and pheomelanin. The melanin pattern of A. cahirinus approximates the background color of the slope on which they dwell. Pheomelanin is slightly (insignificantly) higher in individuals found on the AS to match the brownish background, whereas individuals of the ES had significantly greater eumelanin content to mimic the dark grayish background. This is further substantiated by a significantly higher eumelanin and pheomelanin ratio on the ES than on the AS.

Conclusion/Significance

It appears that rodents adaptively modulate eumelanin and pheomelanin contents to achieve cryptic coloration in contrasting habitats even at a microscale.     

Rapid Learning of Magnetic Compass Direction by C57BL/6 Mice in a 4-Armed ‘Plus’ Water Maze

Magnetoreception has been demonstrated in all five vertebrate classes. In rodents, nest building experiments have shown the use of magnetic cues by two families of molerats, Siberian hamsters and C57BL/6 mice. However, assays widely used to study rodent spatial cognition (e.g. water maze, radial arm maze) have failed to provide evidence for the use of magnetic cues. Here we show that C57BL/6 mice can learn the magnetic direction of a submerged platform in a 4-armed (plus) water maze. Naïve mice were given two brief training trials. In each trial, a mouse was confined to one arm of the maze with the submerged platform at the outer end in a predetermined alignment relative to magnetic north. Between trials, the training arm and magnetic field were rotated by 180^° so that the mouse had to swim in the same magnetic direction to reach the submerged platform. The directional preference of each mouse was tested once in one of four magnetic field alignments by releasing it at the center of the maze with access to all four arms. Equal numbers of responses were obtained from mice tested in the four symmetrical magnetic field alignments. Findings show that two training trials are sufficient for mice to learn the magnetic direction of the submerged platform in a plus water maze. The success of these experiments may be explained by: (1) absence of alternative directional cues (2), rotation of magnetic field alignment, and (3) electromagnetic shielding to minimize radio frequency interference that has been shown to interfere with magnetic compass orientation of birds. These findings confirm that mice have a well-developed magnetic compass, and give further impetus to the question of whether epigeic rodents (e.g., mice and rats) have a photoreceptor-based magnetic compass similar to that found in amphibians and migratory birds.     

Generation of Murine Cardiac Pacemaker Cell Aggregates Based on ES-Cell-Programming in Combination with Myh6-Promoter-Selection

Treatment of the “sick sinus syndrome” is based on artificial pacemakers. These bear hazards such as battery failure and infections. Moreover, they lack hormone responsiveness and the overall procedure is cost-intensive. “Biological pacemakers” generated from PSCs may become an alternative, yet the typical content of pacemaker cells in Embryoid Bodies (EBs) is extremely low. The described protocol combines “forward programming” of murine PSCs via the sinus node inducer TBX3 with Myh6-promoter based antibiotic selection. This yields cardiomyocyte aggregates consistent of >80% physiologically functional pacemaker cells. These “induced-sinoatrial-bodies” (“iSABs”) are spontaneously contracting at yet unreached frequencies (400-500 bpm) corresponding to nodal cells isolated from mouse hearts and are able to pace murine myocardium ex vivo. Using the described protocol highly pure sinus nodal single cells can be generated which e.g. can be used for in vitro drug testing. Furthermore, the iSABs generated according to this protocol may become a crucial step towards heart tissue engineering.     

Spatial Generalization in Operant Learning: Lessons from Professional Basketball

In operant learning, behaviors are reinforced or inhibited in response to the consequences of similar actions taken in the past. However, because in natural environments the “same” situation never recurs, it is essential for the learner to decide what “similar” is so that he can generalize from experience in one state of the world to future actions in different states of the world. The computational principles underlying this generalization are poorly understood, in particular because natural environments are typically too complex to study quantitatively. In this paper we study the principles underlying generalization in operant learning of professional basketball players. In particular, we utilize detailed information about the spatial organization of shot locations to study how players adapt their attacking strategy in real time according to recent events in the game. To quantify this learning, we study how a make \ miss from one location in the court affects the probabilities of shooting from different locations. We show that generalization is not a spatially-local process, nor is governed by the difficulty of the shot. Rather, to a first approximation, players use a simplified binary representation of the court into 2 pt and 3 pt zones. This result indicates that rather than using low-level features, generalization is determined by high-level cognitive processes that incorporate the abstract rules of the game.     

A Burst-Based “Hebbian” Learning Rule at Retinogeniculate Synapses Links Retinal Waves to Activity-Dependent Refinement

Patterned spontaneous activity in the developing retina is necessary to drive synaptic refinement in the lateral geniculate nucleus (LGN). Using perforated patch recordings from neurons in LGN slices during the period of eye segregation, we examine how such burst-based activity can instruct this refinement. Retinogeniculate synapses have a novel learning rule that depends on the latencies between pre- and postsynaptic bursts on the order of one second: coincident bursts produce long-lasting synaptic enhancement, whereas non-overlapping bursts produce mild synaptic weakening. It is consistent with “Hebbian” development thought to exist at this synapse, and we demonstrate computationally that such a rule can robustly use retinal waves to drive eye segregation and retinotopic refinement. Thus, by measuring plasticity induced by natural activity patterns, synaptic learning rules can be linked directly to their larger role in instructing the patterning of neural connectivity.