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- Alzheimer’s disease (AD) is a neurodegenerative condition that dramatically reduces the overall quality of life of patients. As a result, this condition merits extensive research.
- AD has a rising global prevalence of 24 million (Reitz & Mayeu, 2014)
- AD costs the US alone a staggering $172 billion each year (Reitz & Mayeu, 2014)

- Clinical features include:
- Memory loss
- Impaired attention
- Eventual death

- Pathological hallmarks include:
- Amyloid-beta plaques, which consist of aggregations of misfolded proteins
- Neurofibrillary tangles, composed of hyperphosphorylated Tau
- Astrogliosis, also known as reactive astrocytes, defined as an increased number of astrocytes which are involved in neuroinflammation
- Neuronal cell death, which is a major component of neurodegeneration

- Previous studies have developed models to allow for further investigation of AD. These models, which demonstrate some but not all of the pathological hallmarks, include:
- Rodents with genetic mutations (LaFerla and Green, 2012)
- Human induced pluripotent stem cells (Brennand et al., 2015)
- Three-dimensional human neural stem-cell-derived cultures (Choi et al., 2014)

- Most importantly, these models have all failed at recapitulating neuronal cell death
- This gap in the literature highlights the need for more complex theories and models for AD

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https://www.brightfocus.org/alzheimers/infographic/progression-alzheimers-disease

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Check out this video for a more detailed introduction to AD

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Check out this video for a more detailed introduction to AD

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https://www.youtube.com/watch?v=v5gdH_Hydes

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Alzheimer's disease - plaques, tangles, causes, symptoms & pathology

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- The selected article seeks to investigate whether human neuronal precursors derived from stem cells can be transplanted into an AD mouse model to induce complete AD pathology
- This investigation is important because the ability to fully model AD pathology in human neurons within the context of the diseased brain may enable further investigation of human-specific responses to AD pathology as well as genetic analysis

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Experimental, controlled, non-randomized study to investigate AD in a chimeric system.
They used 3 treatment groups:
- Healthy human neurons transplanted into transgenic AD mice
- Healthy human neurons transplanted into WT mice
- Healthy mouse neurons transplanted into WT mice (control).

- The transgenic AD mice were generated by crossing APP PS1 mice (with mutations in the amyloid precursor protein gene and the presenilin 1 gene) with immunodeficient NOD-SCID mice.
The WT mice were also immunodeficient (NOD-SCID), but were wild-type at the APP and PS1 loci.
Immunodeficiency is important to help reduce rejection from transplantation.

1) The authors differentiated human PSCs into neuronal progenitors
-These cells also carry the GFP protein, driven by the chicken b-actin promoter. This allows for easy identification and observation of the human neurons under the microscope

2) They added BMP and Noggin (factors required for neuronal differentiation) to the culture at day 0 and at day 14 (at which point the cultures form rosettes). The neurons were then stereotactically injected into the frontal cortex of mice. The mice were observed for 8 months post transplantation (MPT) until sacrifice, with 4-5 MPT mice being referred as young mice to view AD in early stages, whereas 8 MPT mice were referred to as old mice for late-AD symptoms.

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Result 1: Successful integration of human neurons into AD mouse brain

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Result 1: Successful integration of human neurons into AD mouse brain

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- The first step of this study was to confirm that the human neurons were successfully integrated into the AD mouse brain
- First, they looked for the expression of specific markers including but not limited to:
HuNuclei –> Labels the human neurons; used to confirm that the human neurons were successfully transplanted into the mouse brain
NeuN & MAP2 –> Indicates that the neural precursors differentiated into mature neurons
- Next, they visualized synapse integration between human and mouse neurons, using electron microscopy & GFP immunogold labeling
- Then, they performed RNA sequencing to analyze gene expression. This confirmed the presence of telencephalic, cortical and glutamatergic genes and suggests that the human neurons underwent telencelaphic/cortical specification.

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Result 2: Presence of amyloid-beta plaques

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Result 2: Presence of amyloid-beta plaques

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- The figure below illustrates the co-localization of human neurons (marked with GFP) and amyloid-beta plaques (marked in red by thioflavin) in the AD mouse (box L & M)
- This suggests that the amyloid-beta plaques have formed within the human graft tissue
- There are no amyloid-beta plaques within the human neurons in the WT mice (box J & K)
- This makes sense because, unlike the AD mice, the WT mice do not have a propensity to form amyloid-beta plaques.

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Result 3: Proof of neurodegeneration by neuronal loss

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Result 3: Proof of neurodegeneration by neuronal loss

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- Neurodegeneration in human neurons was assessed by TOPRO3 and GFP staining to observe neuronal density loss
- TOPRO3 is a nuclear stain; as a result, reductions in TOPRO3 staining indicate reductions in overall neuronal populations
- GFP marks the human graft tissue
- Neuron density was significantly lower within the human graft tissue of AD mice compared to the human graft tissue of WT mice (boxes C & F).
- No change in neuron density was observed when comparing the WT host tissue to the AD host tissue (boxes I & L). That is, neuron loss is species-specific and only observed within the human graft tissue of AD mice.
- The degeneration observed in the human graft tissue is derived via a necrosis-mediated mechanism (as opposed to an apoptotic mechanism)
- Necrosis is characterized by swelling of the cytoplasm, enlarged mitochondria, highly dispersed chromatin and, in some cases, rupture of the nuclear membrane
- Note: neurodegeneration was not observed until 6 months post-transplantation (MPT), coinciding with the appearance of amyloid-beta plaques.  This confirms that neuronal loss was not caused by poor integration during transplantation, but rather by exposure of human graft tissue to AB plaques in the AD mice.

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Result 4: Proof of neuroinflammation by activated microglia & astrocytes

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Result 4: Proof of neuroinflammation by activated microglia & astrocytes

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- Amyloid-beta neuroinflammatory responses were assessed and found to be similar between human grafts and mouse host tissue in AD mice.
- Given that neuroinflammatory responses are characterized by the activation of microglia and astrocytes, the researchers stained the brains for GFAP, an astrocytic marker, and Iba1, a microglial marker
- Clustering of GFAP+ astrocytes and Iba1+ microglia cells was found around amyloid-beta deposits in AD mice
- Activated astrocytes & microglia cells show enlarged/phagocytic phenotype

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- Neurofibrillary tangles (NFTs), which are composed of hyperphosphorylated Tau, were then assessed.
- AT8 antibodies were used to assess hyperphosphorylated Tau
-MC1, a conformational antibody, was used to mark the presence of pathological Tau
- AT8+ staining was found in both human and mouse tissues in AD mice at 8 months post-transplantation
-MC1+ staining was only found in the human graft
- This suggests that the pathological conformations of Tau were only present in human tissue
- Despite the Tau pathology, no definite neurofibrillary tangle formation was observed

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Result 6: Gene expression similar to AD patients 

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Result 6: Gene expression similar to AD patients 

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- Finally, gene expression data from the human neurons transplanted into the AD mouse were compared to that of published human AD datasets
- RNA sequencing analysis found:
- Down-regulation of genes involved in synaptic transmission, gated channel activity, neuron projections, cognition, & learning and memory in the human neurons transplanted into AD mice
- Up-regulation of genes involved in cell death and myelination were also detected
- Overall, this gene expression pattern correlates with the phenotypes we know are associated with AD

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- Study goal: To develop a more accurate model of AD that exhibits the full-spectrum of pathologies observed in affected humans.
- Assessment of the model system: This mouse model successfully recapitulated a broad range of neuropathologies associated with AD. This includes: amyloid-beta plaques, microglia and astrocyte activation, tau hyperphosphorylation and alterations to genes involved in learning, cognition and memory.
- This study was also the first to convincingly show neuronal loss in a model of Alzheimer's Disease!
- Important caveats:
1. Amyloid-beta plaques localized to the human graft tissue were more diffuse and smaller than those clustered within the mouse host tissue;
2. The model failed to recapitulate the characteristic accumulation of neurofibrillary tangles found in AD patients.

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1. First study to convincingly demonstrate neuronal loss in a model of Alzheimer’s disease.
- Why is this important?
- Neuronal loss is an important hallmark of Alzheimer's Disease in humans
- Historically, modelling neuronal loss has proven challenging in both transgenic models of AD (Wirths, 2010) and 3D neural cell culture models (Choi, 2014).

2. The neurons in this study were grown and studied in the brain versus in a dish.
- Why is this important?
- Neurons were studied in a natural environment that mimics what we would see in a living system.
- Neurons are exposed to natural cellular, chemical, and microbiological factors.
- This is a major advantage over recent studies that model AD through 3D neural cell cultures, thereby neglecting the interactions and influences that characterize the animal brain (Choi, 2014).

3. These neurons were not genetically manipulated.
- Why is this important?
- This is a major advantage over the most-used model of AD, the transgenic mouse. The transgenic model, typified by specific mutations involved in the development of early onset AD in humans, only realistically mimics 5% of cases of AD (i.e. familial AD) (Price, 1998)
- This model offers a more realistic picture of late onset AD (95% of cases)

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1. No behavioural outcomes were assessed with this model.
- Why is this important?
- It is unclear whether the neuropathological outcomes in this model translate to the characteristic deficits in cognition, learning and memory that we see in human AD patients.
- Next steps: Assess behavioural outcomes!

2. This study failed to demonstrate neurofibrillary tangles.
- Why is this important?
- This limitation is not common to all models of AD. Indeed, the 3D neural-stem-cell-derived in vitro model of AD, proposed by Choi et al. (2014), demonstrated striking neurofibrillary tangles. Transgenic models of AD also successfully model neurofibrillary tangles (Oddo, 2003).

3. Because of the immunodeficient background of the mice, and their associated increased risk of tumor formation, long-term observations (past 8 MPT) are impossible.
- Why is this important?
- It is possible that neurofibrillary tangles would develop in this model at later stages, but because of the limited lifespan of the mice used in this study, it is impossible to confirm this hypothesis.
- Next steps: Perform the same protocol in a longer-living model and assess the presence of neurofibrillary tangles

4. In this study, human neurons were transplanted into the frontal cortex of mice and the resulting phenotype was assessed. Although the frontal cortex is undoubtedly affected in Alzheimer's disease, the medial temporal lobe is of greater clinical relevance and significance. Indeed, the hippocampus has repeatedly been shown to be one of the regions most affected by AD (Anand, 2012).  Transplanting neurons into the hippocampus and assessing the resulting phenotype in that region would provide a clearer picture of the extent and severity of AD pathologies.
-Next steps: assess the phenotypes associated with neuron transplantation into the medial temporal lobe (specifically, the hippocampus).

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Future experiment 1: Search for NFTs using a longer-living strain

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Future experiment 1: Search for NFTs using a longer-living strain

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- For our first experiment, we propose using a longer-living strain of mice and treating it with immunosuppressives, instead of using an immunodeficient background.
- Using the immunosuppressive, tacrolimus, has been validated in a previous study as an effective immunosuppressant after neural stem cell transplantation into the corpus callosum of AD mice.
- Moreover, there are no major side effects of this immunosuppressant, nor has it been found to conflate the pathologies observed in AD mice.
- Using this protocol will also allow us to determine whether the appearance of neurofibrillary tangles (NFT) is age-dependent and, if so, whether this model will eventually develop this phenotype. Markers for hyperphosphorylated Tau, such as pretangle antibodies AT-8, MC-1, or PG-5 can be used to identify tauopathies, and Gallyas silver stain can be used to identify mature NFT formations.

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1 a) Using the immunosuppressive tacrolimus 

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1 a) Using the immunosuppressive tacrolimus 

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Disadvantages of using an immunodeficient background:

- Although the NOD-SCID background (used by Espuny-Camacho and colleagues) is amenable for higher-levels of transplantations and engraftments, these mice have a propensity to develop tumors.  This limits their lifespan to less than 30 weeks, making this type of mice sub-optimal for long-term studies.

- Using a longer-living strain could allow for the detection of other symptoms of AD - especially, neurofibrillary tangles.  This would further support the model as effective, and capable of expressing the full-spectrum of AD neuropathology.

Do AD neuropathology develop in an age-dependent manner?
- Yes!  Several studies have suggested that AD neuropathology, both in models and humans, develop in an age-dependent manner. In a triple transgenic mouse model of AD, AB deposits and synaptic dysfunction developed long before the presence of neurofibrillary tangles (Oddo, 2003).

- Neurofibrillary tangles are essentially aggregates of hyperphosphorylated tau and so are detected long after the identification of tau abnormalities.
- Espuny-Camacho et al., (2017) observed hyperphosphorylated tau at 8 months post-transplantation; the same age at which mice are sacrificed. Therefore, it is possible that neurofibrillary tangles would develop in the Espuny-Camacho model, if given enough time.

How will we test our hypothesis?

- We hypothesize that exposing a longer-living strain of mice to the same protocol outlined by Espuny-Camacho et al., (2017), will result in the development of neurofibrillary tangles in an age-dependent manner.
- To test this hypothesis, we will treat newborn mice with tacrolimus, an injectable immunosuppressant.
- Tacrolimus has been used in a previous study involving the transplantation of neural stem cells in models of Alzheimer’s Disease (McGinley, 2017). We propose using the same protocol.
- This has been shown to be an effective regimen to prevent rejection of transplanted cells.  Most importantly, the treatment of tacrolimus was not found to conflate any of the symptoms associated with AD in the mouse model.

- We expect that the lengthened lifespan of our mice will enable us to potentially detect NFTs in our optimized AD mouse model

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1 b) Search for neurofibrillary tangles (NFTs)

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1 b) Search for neurofibrillary tangles (NFTs)

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- Previous experiments using genetically modified mice observed pretangle-associated epitopes at 11 months in the hippocampus and cortical neurons (Paulson et al., 2008).
- AT-8 and PG-5 pretangle antibodies have been used to detect the presence of hyperphosphorylated tauopathies (Paulson et al., 2008). Mature NFTs pathology can be visualized via Bielschowsky silver stain. Other literature has pointed to Gallyas silver staining as a more powerful approach to visualize mature NFTs (Paulson et al., 2008; Uchihara, 2007). Current literature also reflects that Gallyas staining is indicative of tau+ structures and used in primary diagnostics of AD (Kovacs, 2014).
- In our future experiments, we would apply similar AT8 and PG5 antibody staining methods, as well as the Gallyas method in human derived transplanted neurons to help visualize NFT formation in the immunosuppressed mouse model.

AT-8
- AT-8 is a monoclonal antibody that recognizes phosphorylated tau protein at specific amino acid residues such as serine and threonine (Goedert et al., 1995). Given its capacity to mark phosphorylated tau, AT-8 can mark both early and mature NFTs.

PG-5
- PG5 is also an antibody used to detect phosphorylated tau (Wells et al., 2015). More specifically, it detects hyperphosphorylation at the serine residue (Ahmed et al. 2014; Wells et al., 2015). With AT-8, these two stains specifically target hyperphosphorylated tau, making this a valid marker of NFTs in our model of AD.

Gallyas silver stain
- Gallyas is a silver stain for mature NFTs that visualizes their physical development; it uses silver ions that specifically attach to their targets (Uchihara, 2007). This method allows for the visualization of abundant deposits without staining normal structures (Uchihara, 2007). In conjunction with PG-5 antibody staining, this would allow for visualization and quantification of pretangle and mature NFT inclusions (Ahmed et al., 2014).

- We expect that the use of these techniques for NFT visualization will enable us to detect NFTs in our optimized AD mouse model

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Ahmed, Z., Cooper, J., Murray, T. K., Garn, K., McNaughton, E., Clarke, H., ... O'Neil, M. J. (2014). A novel in vivo model of tau propagation with rapid and progressive neurofibrillary tangle pathology: the pattern of spread is determined by connectivity, not proximity. Acta Neuropathol, 127(5), 667-683.

Anand, K.S. & Dhikav, V. (2012). Hippocampus in health and disease: An overview. Ann Indian Acad Neurol, 15(4), 239-246.

Barbash, S., Hanin, G., & Soreq, H. (2013). Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome. Journal of Visualized Experiments, (76), 1–5.

Brennand, K. J., Marchetto, M. C., Benvenisty, N., Brüstle, O., Ebert, A., Izpisua Belmonte, J. C., … Jaenisch, R. (2015). Creating patient-specific neural cells for the in vitro study of brain disorders. Stem Cell Reports, 5(6), 933-945.

Choi, S. H., Kim, Y. H. , Hebisch, M., Sliwinski, C., Lee, S., D'Avanzo, C., … Kim, D. Y. (2014). A three-dimensional human neural cell culture model of Alzheimer's disease. Nature, 515(7526), 274-8.

Espuny-Camacho, I., Arranz, A. M., Fiers, M., Snellinx, A., Ando, K., Munck, S., … Brion, J. (2017). Hallmarks of Alzheimer’s disease in stem-cell-derived human neurons transplanted into mouse brain. Neuron, 93, 1066-1081.

Goedert, M., Jakes, R., & Vanmechelen, E. (1995). Monoclonal antibody AT8 recognises tau protein phosphorylated at both serine 202 and threonine 205. Neuroscience Letters, 189(3), 167–170.

Jean, Y. Y., Baleriola, J., Fà, M., Hengst, U., & Troy, C. M. (2015). Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus. Journal of Visualized Experiments, (100), 1–7.

Kovacs, G. G. (2015). Invited review: Neuropathology of tauopathies: Principles and practice. Neuropathology and Applied Neurobiology, 41(1), 3–23.

LaFerla, F. M. & Green, K. N. (2012). Animal models of Alzheimer disease. Cold Spring Harb Perspect Med, 2, (11).

Lauckner, J., Frey, P., & Geula, C. (2003). Comparative distribution of tau phosphorylated at Ser262in pre-tangles and tangles. Neurobiology of Aging, 24(6), 767–776.

McGinley, L. M., Kashlan, O. N., Chen, K. S., Bruno, E. S., Hayes, J. M., Backus, C., … Feldman, E. L. (2017). Human neural stem cell transplantation into the corpus callosum of Alzheimer's mice. Ann Clin Transl Neurol, 4(10), 749–755.

Morris, R. G. M. (1981). Spatial localization does not require the presence of local cues. Learn Motiv, 12, 239–260.

Morris, R. (1984). Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods, 11, 47–60.

Oddo, S., Caccamo, A., Shepherd, J. D., Murphy ,M.P., Golde, T. E., Kayed, R., ... LaFerla, F. M. (2003). Triple-Transfenic Model of Alzheimer's Disease with Plaques and Tangles: Intracellular AB and Synaptic Dysfunction. Neuron, 39(3), 409-421.

Paulson, J. B. (2008). Amyloid Plaque and Neurofibrillary Tangle Pathology in a Regulatable Mouse Model of Alzheimer’s Disease. Am J Pathol, 173(3), 762-72.

Price, D.L., Sisodia, S.S. (1998). Mutant genes in familial Alzheimer's disease and transgenic models. Annu Rev Neurosci, 21, 479-505.

Reitz, C., Mayeu, R. (2014). Alzheimer disease: epidemiology, diagnostic criteria, risk factors and biomarkers. Biochem Pharmacol, 88(4), 640-51.

Radde, R., Bolmont, T., Kaeser, S. A., Coomaraswamy, J., Lindau, D., Stoltze, L., ...  Jucker, M. (2006). Aβ42‐driven cerebral amyloidosis in transgenic mice reveals early and robust pathology. EMBO Rep, 7, 940 LP-946.

Sun, X., He, G., Qing, H., Zhou, W., Dobie, F., Cai, F., … Song, W. (2006). Hypoxia facilitates Alzheimer's disease pathogenesis by up-regulating BACE1 gene expression. Proc Natl Acad Sci U S A, 103(49), 18727-32.

Schuff, N., Woerner, N., Boreta, L., Kornfield, T., Shaw, L. M., Trojanowski, J. Q., … Weiner, M. W. (2009). MRI of hippocampal volume loss in early Alzheimers disease in relation to ApoE genotype and biomarkers. Brain, 132(4), 1067–1077.

Uchihara, T. (2007). Silver diagnosis in neuropathology: principles, practice and revised interpretation. Acta Neuropathol, 113(5), 483-499.

Wells, J.A., O'Callaghan, J.M., Holmes, H.E., Powell, N.M., Johnson, R.A., Siow, B., ... Lythgoe M.F. (2015). In vivo imaging of tau pathology using multi-parametric quantitative MRI. NeuroImage, 111, 369-378.

Wirths, O., Bayer, T.A. (2010). Neuron Loss in Transgenic Mouse Models of Alzheimer's Disease. Int J Alzheimers Dis. 2010, 723782.

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Future experiment 2: Assessment of behavioural outcomes after hippocampal injection

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Future experiment 2: Assessment of behavioural outcomes after hippocampal injection

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- For our second experiment, we propose injecting stem-cell derived human neurons into the hippocampus of experimental mice using stereotaxic surgery.
- Characteristically, the hippocampus is severely impacted by Alzheimer's Disease. As a result, assessing the phenotypes in this brain region will provide insights into the extensive consequences of AD (Anand, 2012).
- After injecting human neurons into the hippocampus of experimental mice, we propose assessing behavioural outcomes with the Morris water maze task, which looks at spatial memory.

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2 a) Stereotactic transplantation into hippocampus

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2 a) Stereotactic transplantation into hippocampus

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-The first step of this experiment is to inject human neurons into the hippocampus of AD and WT mice. Stereotactic surgery allows for the targeting of discrete brain regions by identifying them in spatial relation to established landmarks.

1) The brain is first exposed by removing the skull cap, and the syringe will be set to the coordinates relative to the skull cap sutures bregma and lambda (Barbash et al., 2013; Jean et al., 2015).
-Hippocampal CA1 injections have been shown to require the following coordinate relative to bregma-: anterior/posterior axis -2mm, lateral/medial axis +1.8mm and dorsal/ventral axis -1.5mm (Barbash et al., 2013).

2) The brain is then covered and the wound permitted to heal prior to assessment post-op 4-6 weeks (Barbash et al., 2013).
- We propose targeting the hippocampus because it is the structure most commonly and most extensively impacted by AD (Schuff et al., 2009; Anand et al., 2012).

-Our next step would be to assess the pathological and functional consequences of this intervention on our experimental mice. The Espuny-Camacho protocol will be followed to assess the neuropathology of these mice. We propose extending their study through a functional assay. Specifically, we will assess memory through the Morris Water Maze task.

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2 b) Assessing behaviour with Morris water maze 

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2 b) Assessing behaviour with Morris water maze 

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- The Morris water maze (MWM) is a widely used behavioural test to examine spatial learning and memory in animal models, first developed in 1981 (Morris, 1981).
- The typical procedure involves a pool filled with opaque water and a hidden platform that the animal can use to escape (Morris, 1984). After several trials the animals learn and remember the location of the platform, indicated by the reduced length of time that it takes them to mount (Morris, 1984).

- We propose to use the MWM to confirm that the AD model developed by Espuny-Camacho et al. (2017) displays the behavioural phenotype characteristic of AD.
- We expect that the AD mice will take a longer period of time to mount the hidden platform because of their memory impairments (Sun et al., 2006). Overall, this would indicate that the model developed by Espuny-Camacho et al. (2017) was successful in terms of reproducing behavioural deficits of AD.

https://www.jove.com/video/2920/morris-water-maze-test-for-learning-memory-deficits-alzheimers

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1. Preparation

1. Equipment preparation

1. Obtain a circular pool with a diameter of 150cm and a depth of 50cm (Fig. 1). If using black mice, a white pool should be used; if using white mice, a black pool should be used.

2. Arrange the room such that the animal being tested cannot see the experimenter during testing. This can be accomplished with drapes or room dividers.

3. Place high contrast spatial cues about the room, and/or on the interior of the pool at a location which would be above the water surface.

4. Place a 10cm diameter platform in the pool - white for a white pool, clear plexiglass for a black pool. Fill the pool with water until the platform is 1cm above the water surface. Let the water equilibrate to room temperature (22 °C). Depending on the water temperature this may take one to three days, or alternatively hot water can be added to speed up the equilibration.

2. Software preparation

1. Calibrate the pool in the computer software so the camera can create physical distance information from pixel-based information. Divide the pool into 4 quadrants. Specify the platform zone as a variable zone which can change with each trial. Create 5 platform subzones - one in each quadrant, and one in the center of the pool. Save the calibration and use it for the remaining test days. (See example Fig. 2).

2. Set the maximum trial time as 60 sec. If the mouse finds the platform before this time, program the software to stop the trial when the platform is found.

3. Specify the program to begin tracking automatically, when the experimenter exits the testing area. Utilize any "reflection minimization" options your software package provides.

4. Track path length, escape latency, and time spent in each quadrant.

2. Day 1: Visible Platform

1. Computer Program

1. Load the pool calibration into the tracking software.

2. Create 5 trials, with an inter-trial interval appropriate for your experiment. Program the platform location and starting direction to differ with each trial. See Table 1 for an example protocol.

2. Testing procedure

1. Transfer the mice from their housing facility to the behavior room. Keep the mice in an area where they cannot see the pool or spatial cues. Let them adjust to the new environment for at least 30 minutes before testing.

2. Place a flag on the platform to increase its visibility.

3. To begin testing, lift mouse from the home cage by the base of the tail. Support the mouse as you bring it to the testing area. Lifting the mouse by the base of the tail, gently place the mouse into the water, facing the edge of the pool. Quickly leave the testing area.

4. If the mouse finds the platform before the 60 sec cut-off, allow the mouse to stay on the platform for 5 seconds then return it to its home cage. If the mouse does not find the platform, place the mouse on the platform and allow it to stay there for 20 sec before returning it to its home cage.

5. Repeat for all mice in the trail. Begin each subsequent trial with a different platform location and starting direction, as you have programmed into your software.

6. When testing is complete, return the mice to their housing facility. Mice are dried off and normothermia is assured prior to returning to animal facility.

7. In preparation for the following day, remove the flag from the platform and add additional water to the pool to submerge the platform to 1cm below the surface.

3. Days 2-5: Hidden Platform

1. Computer Program

1. Load the pool calibration into the tracking software.

2. Create 5 trials, with an inter-trial interval appropriate for your experiment. Program the platform location to remain in the same position throughout all trials and days, but have the starting direction differ with each trial, each day.

2. Testing procedure

1. For black mice, add non-toxic, white, powdered tempera paint to the pool and mix thoroughly. Use enough paint such that the submerged platform is not visible from the surface of the water. For white mice, a black pool with clear water and a clear plexiglass platform should be used.

2. Follow steps 2.2.3 to 2.2.6.

4. Day 6: Probe Trial

1. Computer Program

1. Load the pool calibration into the tracking software.

2. Create 1 trial with no platform zone, and one starting direction. The starting direction farthest from the platform quadrant used on days 2-5 is preferred. Set the trail length to 60 sec.

2. Testing procedure

1. Remove the platform from the pool.

2. Follow steps 2.2.3 to 2.2.6.

5. Data analysis

1. For each day and each mouse, average the 5 trials to give a single path length and escape latency for each test subject. Calculate the combined error appropriately. For day 6, simply collect the path length, escape latency, and time spent in the platform quadrant for each mouse.

2. If any differences exist between groups on Day 1, it is likely a problem with vision rather than learning and memory. Only proceed with analysis if no differences are seen on day 1.

3. Compare the learning curves for Days 2-5 using statistics appropriate for your data set. A steeper curve represents faster task acquisition; a shallower curve represents a deficit in task acquisition. The data from day 2 to day 5 are analyzed using ANOWA.

4. For day 6, compare the percent of time spent in the previously learned platform quadrant, using statistics appropriate for your data set. A higher percentage of time spent in the platform quadrant is interpreted as a higher level of memory retention.

6. Representative Results

We have used the Morris Water Maze test to examine hypoxia's effect on AD pathogenesis (7) and valproic acid (VPA)'s pharmaceutical potential for AD treatment (6) in transgenic AD model mice. Figure 3 is the representative result we reported in our study on VPA's effect on memory deficits in the APP23 AD mouse model (6). On day 1 (visible platform trials), there is no difference between the VPA treated and control groups in latency (Fig. 3A) and path length (Fig. 3A) indicating that both of the groups have similar motor and visual capabilities. From this we assume the mice are able to see the flagged-platform and the cues in surrounding environment, and can swim acceptably. For days 2-5 (Day 1 to 4 of hidden platform trials) the example shows a difference in the escape latency (Fig. 3C) and path length (Fig 3D) between the groups, suggesting that VPA treated mice performed significantly better than controls over time. The probe trail results on the last day (Day 6) show that the number of times the mice traveled into the third quadrant, where the hidden platform was previously placed, was significantly greater with VPA treatment compared to control (Fig. 3E). These data indicate that VPA treatment significantly improves the memory deficits seen in APP23 mice.

Figure 1. Equipment setup for the Morris Water Maze visible platform test day. The pool is shielded from the experimenter using room dividers. Spatial cues are located on the walls, and maybe placed on the interior of the pool, above the water surface, if desired. The pool is filled with clear water, with the platform located 1cm above the surface. A flag has been placed on the platform to enhance visibility.

Figure 2. Screen capture from the Any-Maze™ Video Tracking System demonstrating pool calibration. The pool is viewed from above by a black and white analog tracking camera with a RTV24 Digitizer. Several zones are defined within the software and the total pool is divided into 4 quadrants. A fifth, platform zone is entered which can vary across trials, with five possible locations: NW, NE, SW, SE, or Center. A calibration line (ticked line across center) is added to allow the software to convert pixel distances into physical distances.

Figure 3. Representative results for the Morris Water Maze. The 7-month APP23 transgenic mice carrying human Swedish mutant APP gene were tested after one month of daily VPA (n=30 mice) or vehicle solution (n=30 mice) injections. (A) During the first day of visible platform tests, the VPA treated and control APP23 mice exhibited a similar latency to escape onto the visible platform. P>0.05 by student's t-test. (B) The VPA-treated and control APP23 mice had similar swimming distances before escaping onto the visible platform in the visible platform test. P>0.05 by student's t-test. (C) In hidden platform tests, VPA treated APP23 mice showed a shorter latency to escape onto the hidden platform on the 3 rd and 4 th day, P<0.001 by ANOVA. (D) The VPA-treated APP23 mice had a shorter swimming length before escaping onto the hidden platform on the 3 rd and 4 th day, P< 0.01 by ANOVA. (E) In the probe trial on the 6th day, the VPA-treated APP23 mice traveled into the third quadrant, where the hidden platform was previously placed, significantly more times than controls. * P<0.005 by student's t-test. (Adapted and reprinted from The Journal of Experimental Medicine 205, 2781-2789, 2008, Rockefeller University Press, Originally published in J. Exp. Med. doi:10.1084/jem.20081588.) (6).

Table 1. Sample water maze protocol*
* Note how both the platform position and starting direction change on day 1, whereas on days 2-5 the platform position remains constant while the starting direction changes. On day 6, there is no platform and a single trial. The starting direction for day 6 is farthest from the previous platform location (SW) so that the mice must travel some distance before entering the previously learned platform quadrant.

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

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1. Preparation Equipment preparation Obtain a circular pool with a diameter of 150cm and a depth of 50cm (Fig. 1). If using black mice, a white pool should be used; if using white mice, a black pool should be used. Arrange the room such that the animal being tested cannot see the experimenter during testing.

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Meeting the creator himself - Dr. Richard Morris

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Meeting the creator himself - Dr. Richard Morris

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Note: the electrophysiology rig is named after him, while other rigs in the lab are named after neuroscientists like Bliss, Lomo, Kandel, etc.