Name: motor and hippocampal dependent spatial learning and reference memory assessment in a transgenic rat model of alzheimer's disease with stroke
Uploaded: 2016-03-22T13:00:00
Description: Watch this Scientific Journal Video about Motor and Hippocampal Dependent Spatial Learning and Reference Memory Assessment in a Transgenic Rat Model of Alzheimer's Disease with Stroke at JoVE.com

This work was made possible with funding from the Canadian Institutes of Health Research (CIHR) and the authors would like to thank CIHR for their funding support.

The appropriate institutional animal ethics committee should approve all of the behavioral procedures prior to starting experimentation. All animal work described here was approved by Western University Animal Use Subcommittee and follows the Canadian Council on Animal Care guidelines.&#160;These animal experiments were performed during the light phase.
1. Cylinder Task for Gross Forelimb Motor Assessment
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	<li>Equipment Setup
	<ol>
		<li>Acquire a Plexiglas cylinder with a perforated lid that will accommoda...

<ol>
	<li>Auld, D., Kornecook, T., Bastianetto, S., Quirion, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease+and+the+basal+forebrain+cholinergic+system:+relations+to+&#946;-amyloid+peptides,+cognition,+and+treatment+strategies.">Alzheimer's disease and the basal forebrain cholinergic system: relations to &#946;-amyloid peptides, cognition, and treatment strategies.</a> Prog Neurobiol. 68 (3), 209245 (2002).</li><li>Huang, Y., Mucke, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer+Mechanisms+and+Therapeutic+Strategies.">Alzheimer Mechanisms and Therapeutic Strategies.</a> Cell. 148 (6), (2012).</li><li>Querfurth, H., LaFerla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease.">Alzheimer's disease.</a> New Engl J Med. 362 (4), 329-344 (2010).</li><li>Karran, E., Mercken, M., Strooper, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+cascade+hypothesis+for+Alzheimer's+disease:+an+appraisal+for+the+development+of+therapeutics.">The amyloid cascade hypothesis for Alzheimer's disease: an appraisal for the development of therapeutics.</a> Nat Rev Drug Discov. 10 (9), 698-712 (2011).</li><li>Akiyama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+Alzheimer's+disease.">Inflammation and Alzheimer's disease.</a> Neurobiol Aging. 21 (3), (2000).</li><li>Butterfield, D., Drake, J., Pocernich, C., Castegna, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+oxidative+damage+in+Alzheimer's+disease+brain:+central+role+for+amyloid+&#946;-peptide.">Evidence of oxidative damage in Alzheimer's disease brain: central role for amyloid &#946;-peptide.</a> Trends Mol Med. 7 (12), 548554 (2001).</li><li>Kalaria, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+cerebral+ischemia+in+Alzheimer's+disease.">The role of cerebral ischemia in Alzheimer's disease.</a> Neurobiol Aging. 21 (2), 321-330 (2000).</li><li>Selkoe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease:+genes,+proteins,+and+therapy.">Alzheimer's disease: genes, proteins, and therapy.</a> Physiol Rev. 81 (2), 741-766 (2001).</li><li>Snowdon, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+infarction+and+the+clinical+expression+of+alzheimer+disease:+The+nun+study.">Brain infarction and the clinical expression of alzheimer disease: The nun study.</a> JAMA. 277 (10), 813 (1997).</li><li>Clarke, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+APP+provides+neuroprotection+in+the+absence+of+functional+benefit+following+middle+cerebral+artery+occlusion+in+rats.">Overexpression of APP provides neuroprotection in the absence of functional benefit following middle cerebral artery occlusion in rats.</a> Eur J Neurosci. 26 (7), 1845-1852 (2007).</li><li>Whitehead, S., Hachinski, V., Cechetto, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+Between+a+Rat+Model+of+Cerebral+Ischemia+and+&#946;-Amyloid+Toxicity+Inflammatory+Responses.">Interaction Between a Rat Model of Cerebral Ischemia and &#946;-Amyloid Toxicity Inflammatory Responses.</a> Stroke. 36 (1), 107-112 (2005).</li><li>Agca, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+transgenic+rats+producing+human+&#946;-amyloid+precursor+protein+as+a+model+for+Alzheimer's+disease:+Transgene+and+endogenous+APP+genes+are+regulated+tissue-specifically.">Development of transgenic rats producing human &#946;-amyloid precursor protein as a model for Alzheimer's disease: Transgene and endogenous APP genes are regulated tissue-specifically.</a> BMC Neuroscience. 9 (1), 28 (2008).</li><li>Bayona, N. A., Gelb, A. W., Jiang, Z., Wilson, J. X., Urquhart, B. L., Cechetto, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propofol+neuroprotection+in+cerebral+ischemia+and+its+effects+on+low-molecular-weight+antioxidants+and+skilled+motor+tasks.">Propofol neuroprotection in cerebral ischemia and its effects on low-molecular-weight antioxidants and skilled motor tasks.</a> Anesthesiology. 100 (5), 1151-1159 (2004).</li><li>Langdon, K., Clarke, J., Corbett, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+exposure+to+high+fat+diet+is+bad+for+your+brain:+exacerbation+of+focal+ischemic+brain+injury.">Long-term exposure to high fat diet is bad for your brain: exacerbation of focal ischemic brain injury.</a> Neuroscience. 182, 82-87 (2011).</li><li>Brownlow, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+rescue+of+memory+deficits+induced+by+calorie+restriction+in+a+mouse+model+of+tau+deposition.">Partial rescue of memory deficits induced by calorie restriction in a mouse model of tau deposition.</a> Behav Brain Res. 271, 7988 (2014).</li><li>Halagappa, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermittent+fasting+and+caloric+restriction+ameliorate+age-related+behavioral+deficits+in+the+triple-transgenic+mouse+model+of+Alzheimer's+disease.">Intermittent fasting and caloric restriction ameliorate age-related behavioral deficits in the triple-transgenic mouse model of Alzheimer's disease.</a> Neurobiol Dis. 26 (1), (2007).</li><li>Maalouf, M., Rho, J., Mattson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuroprotective+properties+of+calorie+restriction,+the+ketogenic+diet,+and+ketone+bodies.">The neuroprotective properties of calorie restriction, the ketogenic diet, and ketone bodies.</a> Behav Brain Res. 59 (2), (2008).</li><li>Tillerson, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forced+limb-use+effects+on+the+behavioral+and+neurochemical+effects+of+6-hydroxydopamine.">Forced limb-use effects on the behavioral and neurochemical effects of 6-hydroxydopamine.</a> Neuroscience. 21 (12), 4427-4435 (2001).</li><li>Sierksma, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+spatial+memory+function+in+APPswe/PS1dE9+mice+after+chronic+inhibition+of+phosphodiesterase+type+4D.">Improvement of spatial memory function in APPswe/PS1dE9 mice after chronic inhibition of phosphodiesterase type 4D.</a> Neuropharmacology. 77, (2013).</li><li>D'Hooge, R., De Deyn, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+the+Morris+water+maze+in+the+study+of+learning+and+memory.">Applications of the Morris water maze in the study of learning and memory.</a> Behav Brain Res. 36 (1), 60-90 (2001).</li><li>Vorhees, C., Williams, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morris+water+maze:+procedures+for+assessing+spatial+and+related+forms+of+learning+and+memory.">Morris water maze: procedures for assessing spatial and related forms of learning and memory.</a> Nat Protoc. 1 (2), 848-858 (2006).</li></ol>

The combination of stroke and Alzheimer&#39;s disease results in very distinct behavioral pathologies that can affect both motor and cognitive function depending on the severity of each condition. Thus, it is necessary to make use of a variety of behavioral tasks to determine the individual contributions of these conditions, as well as give some insight into the combined and potentially interactive effects in the co-morbid condition. The data presented demonstrate three cost-efficient, time-efficient and sensitive behavi...

Alzheimer&#39;s Disease (AD) is the most prevalent form of dementia in the elderly population and a debilitating neurodegenerative disease. Histopathologically, AD presents itself as amyloid plaques, neurofibrillary tangles and neuronal loss. Amyloid plaques consist primarily of beta-amyloid peptide (A&#946;) that has been produced through an altered proteolytic cleavage of amyloid precursor protein (APP) by &#946;-secretase and &#947;-secretase enzymes1,2,3. The cleavage product, A&#946;, deposits in the brain creating pathological amyloid plaques and has toxic effects on the brain that can lead to the characteristic learning impairments and memory loss. All of these steps together are referred to as the &#34;amyloid cascade hypothesis&#34;3,4. While this hypothesis is important when investigating AD, other cellular changes have been found to precede these plaque formations that strays from the original amyloid cascade pathway. These other cellular changes are thought to contribute to early memory loss, learning impairments and other cognitive dysfunctions involved in AD prior to plaque formation3,5,6.
With AD becoming increasingly prevalent, risk factors for developing AD are becoming an extremely important focus of research. Although age is the principal risk factor for sporadic forms of AD, other risk factors have been identified, including stroke7,8. Stroke is not only a risk factor, but it can also exacerbate already present dementias. For example, clinically, the progression of AD has been shown to be worse in patients who had previously experienced stroke9. Moreover, increased APP expression and A&#946; accumulation has been found in experimental animal models of A&#946; toxicity combined with induced stroke10,11. Since there is this important interaction between stroke and AD, it is essential that these two pathologies be further researched together in co-morbid models to better understand pathophysiology and behaviors implicated in both conditions.
To investigate co-morbid conditions, an appropriate model had to be developed in which a stroke could interact with A&#946; to produce AD-like pathology. For the first time, an APP21 transgenic rat that has a mutated human APP gene incorporated into its DNA was used to achieve an appropriate model of AD. The mutations are the Swedish double missense and Indiana single missense mutations, which have both been implicated in familial forms of AD4,8,12. In the absence of an additional insult, this rat model ages without developing the characteristic A&#946; plaques or neurofibrillary tangles12. Therefore, in an effort to induce AD-like behavioral pathology, a small stroke is introduced into the right striatum to mimic the small subcortical strokes often present in dementia patients9. The stroke in the APP21 transgenic rat embodies the co-morbid condition and allows investigation of various types of behavioral changes implicated in both disease conditions. In particular, this induction of AD-like pathology and cognitive deficits in the adult rat allows us investigate the earliest molecular and cognitive changes preceding AD.
Since the goal is to determine the first signs of behavioral changes and since both stroke and AD have very distinct behavioral pathologies, when studying the co-morbid model, behavior tasks need to assess a variety of behavioral phenotypes. There are a battery of relatively sensitive tests that can be done to analyze motor and cognitive behavior in rodent models that involve a variety of paradigms and equipment. To specifically analyze forelimb and hindlimb motor function, the cylinder task and beam-walk task have been implemented to detect motor deficits and monitor locomotion in this model. Other sensitive tasks designed to specifically assess fine forelimb motor skill (i.e. the staircase task and single pellet reaching task) require food deprivation11,13,14. To avoid any of the known effects of food deprivation on disease pathologies15,16,17, these tests have been deemed unsuitable for this study. The cylinder task assesses the spontaneous use of the rat&#39;s forelimbs during rearing in a novel environment and can detect asymmetry between forelimbs in rats with unilateral stroke10,18. A major benefit to this task is that the apparatus can be utilized for other behavior tasks, such as the Porsolt forced swim task19. Contrary to the cylinder task, the beam-walk task also allows analysis of hindlimb and forelimb motor control, in addition to locmotion10,14. Beam walking includes a locomotor component, a balance component and skilled foot placement. Both of these tests are cost-efficient, straightforward, and time-efficient and elucidate the effects of stroke and AD on differences in limb functioning.
Aside from changes in motor function, AD involves memory deficits that can present in early stages of the disease progression. When addressing AD-like pathologies in a rodent model, it is crucial that hippocampal dependent learning and memory is assessed because the hippocampus is an important brain structure largely affected in AD2. The hippocampus is an essential brain region for spatial learning and memory and its function can be tested using various maze paradigms in rodents. One of the mostly widely used maze tasks for rodent models of different diseases is the Morris water maze20. The Morris water maze utilizes spatial cues to assist the rat in locating a stationary hidden platform and tests spatial reference memory when the platform is removed. A valuable advantage of the water maze setup is that it is highly adaptable depending on the proposed research question20.
For the first time, the techniques described have been used to assess motor and cognitive function in a novel co-morbid rat model of stroke and AD. involving small strokes in an APP21 transgenic rat model.&#160;Co-morbidity was achieved by inducing vasoconstriction of the blood vessels in the striatum to produce a small stroke in APP21 transgenic rats. This stroke model has been well established as a co-morbid condition in an alternative rat model of AD11. Advancement into this novel APP21 transgenic rat model was intended to produce a more translationally valuable model. While the behavioural tasks are described using a co-morbid stroke and AD rat model, these tasks can be further applied to other models of stroke or models of other neurological diseases (i.e. Parkinson&#8217;s disease). The general methodology described will be widely applicable to these other disease states, but behavior timelines and paradigms may require alteration based on the proposed research question and model. In addition to being adaptable, the tasks described are effective in demonstrating minor deficits, while also being cost and time-efficient.&#160;

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			<td height="125" style="height:125px;width:216px;">Cylinder</td>
			<td style="width:115px;">Western University</td>
			<td style="width:167px;">Plexiglas Cylinder 
			Cylinder Diameter: 23 cm 
			Cylinder Height: 40 cm 
			Platform Height: 30 cm 
			Mirror Length: 35 cm 
			Mirror Width: 26.5 cm</td>
			<td style="width:320px;">This was made specifically by Western University Machine Services for our lab. Please contact your own chosen manufacturer to design this product.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Handheld Video Camera</td>
			<td style="width:115px;">JVC</td>
			<td style="width:167px;">GZ-E200</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Video Camera Tripod</td>
			<td style="width:115px;">Slik</td>
			<td style="width:167px;">F163</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AM/FM Clock Radio</td>
			<td style="width:115px;">Sylvania</td>
			<td style="width:167px;">SCR1388</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">White Board</td>
			<td style="width:115px;">Walmart</td>
			<td style="width:167px;">Width: 71.12 cm 
			Height: 55.88 cm</td>
			<td style="width:320px;">These can be purchased at any store (i.e. Walmart, University bookstores)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dry-erase Marker</td>
			<td style="width:115px;">Expo</td>
			<td style="width:167px;">Expo Dry-erase Original Marker</td>
			<td style="width:320px;">These can be purchased at any store (i.e. Walmart, University bookstores)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Wooden Beam</td>
			<td style="width:115px;">Rona</td>
			<td style="width:167px;">Plywood 
			Width: 2 cm 
			Length: 100cm</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">VWR General Purpose Laboratory Tape</td>
			<td style="width:115px;">VWR Intl.</td>
			<td style="width:167px;">89097-920</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">iMovie &#39;11</td>
			<td style="width:115px;">Apple Inc.</td>
			<td style="width:167px;">Version 9.0.9 (1795)</td>
			<td style="width:320px;">This is the version used in this manuscript, but any other iMovie version or video editing software could be used.</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Morris Water Maze Pool with Platform</td>
			<td style="width:115px;">Stoelting Co.</td>
			<td style="width:167px;">60136/60035</td>
			<td style="width:320px;">These are not the exact products used in the video, but these are essentially identical.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Platform Cue</td>
			<td style="width:115px;">-</td>
			<td style="width:167px;">-</td>
			<td style="width:320px;">The platform cue used was created using a small metal stand and white spherical foam ball. These can likely be purchased at any store with home improvement materials (i.e. Walmart, Rona etc.)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mainstays 71&#34; Floor Lamp</td>
			<td style="width:115px;">Walmart</td>
			<td>HW-F0377SLV</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">ANY-Maze Behavioural Tracking Software</td>
			<td style="width:115px;">Stoelting Co.</td>
			<td style="width:167px;">60000</td>
			<td style="width:320px;">There are ANY-maze&#174; bundles that include the camera with or without a computer and accessories.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Compact Video Camera</td>
			<td style="width:115px;">Logitech</td>
			<td style="width:167px;">V-U0023</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Laptop</td>
			<td style="width:115px;">Hewlett-Packard</td>
			<td style="width:167px;">HP&#174; Pavilion dv6 Notebook PC</td>
			<td style="width:320px;">Laptop Specifics: AMD A6-3420M APU with Radeon&#8482; HD Graphics 1.50 GHz, 6.00 GB RAM, 64-bit operating system.</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:216px;">Americana Non-toxic Acrylic Paint</td>
			<td style="width:115px;">DecoArt</td>
			<td>DAO67-9</td>
			<td style="width:320px;">This can be ordered on the DecoArt site or purchased in store at DecoArt&#174; retailers.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Poster Board</td>
			<td style="width:115px;">Walmart</td>
			<td>PA-1961</td>
			<td style="width:320px;"></td>
		</tr>
	</tbody>
</table>

motor and hippocampal dependent spatial learning and reference memory assessment in a transgenic rat model of alzheimer's disease with stroke

Alzheimer&#39;s disease (AD) is a debilitating neurodegenerative disease that results in neurodegeneration and memory loss. While age is a major risk factor for AD, stroke has also been implicated as a risk factor and an exacerbating factor. The co-morbidity of stroke and AD results in worsened stroke-related motor control and AD-related cognitive deficits when compared to each condition alone. To model the combined condition of stroke and AD, a novel transgenic rat model of AD, with a mutated form of amyloid precursor protein (a key protein involved in the development of AD) incorporated into its DNA, is given a small unilateral striatal stroke.
For a model with the combination of both stroke and AD, behavioral tests that assess stroke-related motor control, locomotion and AD-related cognitive function must be implemented. The cylinder task involves a cost-efficient, multipurpose apparatus that assesses spontaneous forelimb motor use. In this task, a rat is placed in a cylindrical apparatus, where the rat will spontaneously rear and contact the wall of the cylinder with its forelimbs. These contacts are considered forelimb motor use and quantified during video analysis after testing. Another cost-efficient motor task implemented is the beam-walk task, which assesses forelimb control, hindlimb control and locomotion. This task involves a rat walking across a wooden beam allowing for the assessment of limb motor control through analysis of forelimb slips, hindlimb slips and falls. Assessment of learning and memory is completed with Morris water maze for this behavioral paradigm. The protocol starts with spatial learning, whereby the rat locates a stationary hidden platform. After spatial learning, the platform is removed and both short-term and long-term spatial reference memory is assessed. All three of these tasks are sensitive to behavioral differences and completed within 28 days for this model, making this paradigm time-efficient and cost-efficient.

The behavioral tasks described were used to demonstrate the effects of stroke in an APP21 transgenic rat model of Alzheimer&#39;s disease. The combination of stroke and the APP21 transgene is expected to result in greater motor deficit in the affected limbs, as well as increased memory deficits.
The cylinder task assessed gross forelimb motor function and is represented as the use of the affected forelimb. Furthermore, the beam-...

Watch this Scientific Journal Video about Motor and Hippocampal Dependent Spatial Learning and Reference Memory Assessment in a Transgenic Rat Model of Alzheimer's Disease with Stroke at JoVE.com

Motor and Hippocampal Dependent Spatial Learning and Reference Memory Assessment in a Transgenic Rat Model of Alzheimer's Disease with Stroke

To investigate the co-morbid Alzheimer&#39;s disease (AD) and stroke condition in a novel model, three behavior tasks are described that assess both motor control and cognitive behaviors. These tasks include the beam-walk task, cylinder task and Morris water maze.

Alzheimer&#39;s disease (AD) is a debilitating neurodegenerative disease that results in neurodegeneration and memory loss. While age is a major risk ...

The overall goal of these behavioral tests is the assess motor function, as well as spacial learning and memory performance in a novel comorbid rat model of Alzheimer's disease and stroke. This method can help answer key questions in the dementia field, such as how stroke and Alzheimer's disease pathology interact, producing a phenotype of potential enhanced motor deficit and accelerated cognitive decline. The main advantage of these techniques is that they test for a range of brain functions while being easy to conduct, cost efficient, and suitable for repeated testing at later time points.The implications of this technique extend toward prevention of dementia after stroke because novel preventative compounds can be tested for their potential to ameliorate the comorid behavioral phenotype. To begin, place a mirror at a 45 degree angle beneath a previous mounted cylinder apparatus. Then affix a video camera to a tripod and position it at a distance of approximately 50 centimeters from the apparatus.Check that the entire diameter of the cylindar in the mirror can be visualized on the camera. Next, choose an acclimatized rat and on a white board write down the animal's number and additional trial information. Place this board in front of the mirror and then press record on the video camera.Continue by picking up the rat by the base of the tail and placing it in the cylinder. Then secure the lid of the apparatus and remove the white board so that it no longer blocks the mirror. Once the white board has been removed, proceed to film the animal for five minutes, a period that constitutes a single trial.At the end of this time, stop the recording, return the rat to it's home cage and clean the cylinder with paper towels and water. Then repeat the previously described steps twice more to achieve a total of three trials per rat. Once data has been collected for all rats, import the camera files into a video editing program and proceed to quantify the number of four limb contacts as described in the text protocol.First, position two shelving units approximately 100 centimeters apart from one another in front of a black wall. Then, use tape to anchor the ends of a previously acquired wooden beam to the shelves so that there is approximately one meter of unsupported wood between them. Next place a video camera on a stand and position it so that the camera captures the entire length of the beam.Proceed to choose a rat that has undergone a beam walking training session and record it's number and additional trial information on a white board. When finished, tape the white board to the wall behind the beam. Continue by placing the animal's home cage and an environmental enrichment tube which serve as a motivating queue which the rat can immediately enter after crossing plank, at one end of the beam.Then press record on the video camera. Immediately afterwards, pick up the rat by the base of the tail and place it on the opposite end of the beam without the cage and tube. Record the animal until it sucessfully traverses the full length of the elevated wood, which marks the end of the trial.If the rat pauses midway across the beam, gently touch it's tail to promote movement. Once the animal completes the task, move it's home cage to the other end of the beam. The change the trial number on the white board and repeat the procedure as previously described.When all animal's trials have been recorded, import the camera files into a video editing program and analyze the relevant step, limb slips, and fall data as describe in the text protocol. Start by securing a video camera above the center of an empty, circular pool. Then, designate the four quadrants of the tank by placing tape along the rim of the pool and align them properly with the outline of the tank in the tracking software program.Next, fill the tank with water to a depth of 36 centimeters and then add black, non-toxic paint until the liquid if opaque in order to ensure the platform is not visible below the surface of the water and to better visualize white rats. Once the pool has been prepared, surround it with blank wall surfaces. Then prepare spatial cues by cutting four shapes from different colors of poster board.Afterwards, attach one shape to each of the walls associated with the north, south, east, and west pool locations. To perform a spatial learning experiment, continue by placing a circular platform in the southwest quadrant of the pool. In the accompanying computer program, check that this structure is aligned with a designated platform area in the quadrant.Proceed by grasping a rat by the base of it's tail and gently placing it at a designated start site in the water along the pool wall. Once the animal is in the tank, quickly move our of it's sight and signal another experimenter to start the tracking software. Allow the rat to swim until it locates and climbs onto the platform.At which point, the other researcher should stop the trial on the computer. Let the animal remain on the platform for 30 seconds. If the rat fails to find the platform within the allotted trial time, here 90 seconds, guide the animal to the structure and let it stay there for 30 seconds.Afterwards, lift the rat by the base of the tail and remove it from the maze. Placing it on a towel draped over an arm. Repeat this procedure three additional times per animal with an inter-trial interval of 20 minutes, so that each rat completes of total of four trials.Once the spatial learning experiment has been completed, remove the platform from the pool to commence the probe trials. In the software, check that the previous platform position, in the southwest quadrant remains defined. Then introduce a rat into the pool.Placing it in the water along the wall of the tank. As before, immediately move out of the animal's sight and instruct another experimenter to start the tracking software. At the end of the 30 second probe trial, grasp the animal by the base of the tail, remove it from the tank and then place it one a towel covered arm.Repeat this procedure one week later. Once spatial learning and probe trials have been performed for all animals, analyze the data as described in the text protocol. Four limb use data from the cylinder apparatus was collected from APP21 transgenic rats three days before and seven and 20 days after stroke induction.When standardized and compared to the value representing equal four limb use, the red line here, these results indicate that comorbid rats do not statistically demonstrate a four limb deficit. Similar results were obtained for wild type animals with or without stroke and APP21 transgenic rats alone. However, comorbid animals showed differences in locomotion associated motor function.Beam task data collected for these rats seven days prior to and 21 days after stroke induction reveals a significant increase in the steps needed to traverse the plank, which was not observed in other groups. Morris water maze results also reveal memory deficits in comorbid animals, as evidenced by this graph showing the percentage change in latency, the time animals take to reach the quadrant of the pool where the platform once was between the first and second probe trials. Here APP21 rats with stroke take significantly longer in the second trial to enter the target quadrant.A difference that was not demonstrated by the transgenic rats alone or wild type animals with stroke. This indicates that comorbid animals have a long term memory deficit. While attempting this procedure, it's important to keep all variables as consistent as possible throughout and across testing sessions and create a stress free environment for the animals.After watching this video, you should have a good understanding of how to assess motor and cognitive performance in rat models of post-stroke dementia.

Research

JoVE Journal

Medicine

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Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

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