The authors have no acknowledgements.

The methods detailed in this publication were reviewed by the institutional animal care and use committee (IACUC) at Hilltop Laboratory Animals to ensure proper care, use, and humane treatment of animals in compliance with applicable federal, state, and local laws and regulations, such as the federal Animal Welfare Regulations, or AWRs (CFR 1985), and Public Health Service Policy on Humane Care and Use of Laboratory Animals, or PHS Policy (PHS 1996).
1. General Guidelines for all Behavioral Assessment
<ol>
	<li>Before any animal handling, cover existing cage cards with a new cage card that indicates only the unique, blinded animal identifier. 
	NOTE: Investigators that handle mice during routine compound/placebo dosing are not permitted to handle mice for behavioral assessment.</li>
	<li>Dim or turn off the overhead lights and adjust the lighting such that illumination at the floor of the arena or maze is 30-35 lux.</li>
	<li>For studies that span several weeks, record body weights weekly as an indirect measure of overall health. 
	NOTE: Additional cage-side checks for coat quality, posture, gait and spontaneous locomotion can be included if more robust health checks are warranted.</li>
</ol>
2. Habituating Mice to Handling by Investigators
<ol>
	<li>Two days prior to any behavioral test, habituate the mice to handling. Remove the cage from the cage rack and place on a level surface.</li>
	<li>Remove the lid from the cage. Handle the mouse exactly as it would be handled during performance of the upcoming behavioral test. Place the mouse into a cupped hand above the home cage.</li>
	<li>Measure the latency to jump from the investigator&#39;s hand back into the home cage. Hold mice for a maximum of 5 s. 
	NOTE: Mice that exhibit latencies of &#8805;2 s are deemed &#34;habituated&#34;. Mice that exhibit latencies &#60;2 s during the first trial undergo 2 additional habituation sessions that day.</li>
	<li>Have the mice undergo 2 consecutive days of handling habituation. Note any mouse that is not habituated by the end of the 2nd day.</li>
</ol>
3. Assessing Spatial Working Memory by Measuring Spontaneous Alternation in a Y-maze8
<ol>
	<li>Prior to initial use, thoroughly clean the Y-maze with an unscented bleach germicidal wipe, 70% EtOH, followed by dH2O9. Clearly designate the arms of the maze as &#39;A&#39;, &#39;B&#39; &#38; &#39;C&#39; or other comparable unique identifiers.</li>
	<li>Prior to the start of a testing session, set the data acquisition system or video cameras and set up proper tracking of mice in the maze. Calibrate the distance in the maze using captured video images of a ruler or other object of known length. 
	NOTE: The behavioral methods in this procedure will work with a variety of data acquisition systems and the authors assume anyone performing this procedure is proficient in the use of their chosen data acquisition system. Power analyses indicate that sample sizes of 10-15 mice per group are required for a &#946; &#8804;0.2.</li>
	<li>Remove the cage from the rack and gently place it on a table in close proximity to the Y-maze. Remove the mouse from its home cage and gently place it into one arm of the Y-maze, facing the center. Have the investigator step far enough away from the maze so that the mouse cannot see the investigator.</li>
	<li>Activate the data/video acquisition system immediately after placement of the mouse into the maze.</li>
	<li>Press play and record the spontaneous behavior for each mouse for a period of 10 min. Once a session is complete, gently place the mouse back into its home cage and return the cage to the rack.</li>
	<li>Clean the maze thoroughly between each session with an unscented bleach germicidal wipe, 70% EtOH, followed by dH2O. Repeat from step 3.4 to assess all mice.</li>
	<li>Once all mice have completed exploration of the Y-maze, analyze the data from the acquisition system or manually score the videos of the sessions. An arm entry occurs when all 4 paws of the mouse cross the threshold of the central zone and into the arm and the animal&#39;s snout is oriented toward the end of the arm. 
	NOTE: Endpoints that will be analyzed include: total distance traveled in the maze, total distance traveled within each arm (including the central zone), total time spent in each arm (including the central zone), total number of arm entries, number of entries made into each arm, and a sequential list of arms entered to assess number of alternations made.</li>
	<li>A spontaneous alternation occurs when a mouse enters a different arm of the maze in each of 3 consecutive arm entries. Spontaneous alternation % is then calculated with the following formula. 
	<img alt="Equation 1" src="/files/ftp_upload/55523/55523eq1.jpg" /> 
	NOTE: For example, if the order of arm entry was: ABCCBABCABC, investigator would score a total of 6 spontaneous alternations (in order: ABC, CBA, ABC, BCA, CAB, ABC). With a total of 11 arm entries, the spontaneous alternation % would be 67%.</li>
	<li>Make the following quality control checks to ensure the data represent an unbiased assessment of spontaneous alternation.
	<ol>
		<li>Perform a Pearson&#39;s correlation of spontaneous alternation % to both total distance traveled and number of arm entries made. 
		NOTE: If there is a significant correlation of spontaneous alternation % to either parameter, then data should be further scrutinized due to potential influence of hyperdynamic locomotion on the apparent cognitive endpoint10.</li>
		<li>Analyze number of entries made into each arm with a 1-way ANOVA test. 
		NOTE: If this analysis is significant then this would indicate presence of cues in the environment that attracted mice to a particular region of the maze.</li>
	</ol>
	</li>
</ol>
4. Assessment of Intermediate-term Recognition Memory by Measuring Novel Object Recognition11,12,13
<ol>
	<li>For each phase of this test, thoroughly clean the open field arena with an unscented bleach germicidal wipe, 70% EtOH followed by dH2O prior to initial use.</li>
	<li>One day prior to object exposure, habituate the mice to the open field arena.
	<ol>
		<li>Prior to the start of the habituation session, set up the data acquisition system or video cameras and confirm proper tracking of mice in the maze. Calibrate the distance in the arena using captured video images of a ruler or other object of known length. Mark the corners of the arena in the software to permit scoring of positional biases. 
		NOTE: The behavioral methods in this procedure will work with a variety of data acquisition systems and the authors assume anyone performing this procedure is proficient in the use of their chosen data acquisition system. Power analyses indicate that sample sizes of 15-20 mice per group are required for a &#946; &#8804;0.2.</li>
		<li>Remove the cage from the rack and gently place on a table in close proximity to the arena.</li>
		<li>Remove the mouse from the home cage and gently place the mouse in the center of the arena. Turn on the tracking software and/or video recording system immediately after placing the mouse into the arena.</li>
		<li>Allow mice to freely explore the arena for 30 min. 
		NOTE: During this period, investigators will not disturb the mice.</li>
		<li>After the habituation session, place mice back into their home cage and clean the arena thoroughly with an unscented bleach germicidal wipe, 70% EtOH followed by dH2O.</li>
		<li>Repeat from step 4.2.2 until all mice have been habituated to the arena.</li>
		<li>After all mice have been habituated to the arena, analyze the video. 
		NOTE: Endpoints to analyze include total distance traveled in the arena and time spent near each corner. If relevant to the mouse model, stereotyped behaviors are included in these analyses (i.e., myoclonic corner jumping, circling, etc.). Mice exhibiting biases in time spent in particular regions of the arena are excluded from further experimentation as this will influence object exploration. 
		NOTE: The first phase of novel object recognition involves familiarizing mice to an object. Herein this portion of the novel object recognition procedure will be referred to as the Sample phase.</li>
		<li>Prior to the start of a sample phase session, place objects into the arena and fix them to the floor with a mounting putty so that animals cannot move the objects. Align two identical objects to a particular wall with enough distance between the walls and objects so that the mice can freely explore the objects from all angles.</li>
		<li>Set up the data acquisition system or video cameras and confirm proper tracking of mice and objects in the maze. Calibrate the distances in the arena using captured video images of a ruler or other object of known length.</li>
		<li>Mark the corners of the arena in the software to permit scoring of positional biases. Mark objects in software and track their exploratory behavior separately for each object (i.e., &#34;Object A&#34; and &#34;Object B&#34;).</li>
		<li>Remove the cage from the rack and gently place it on a table in close proximity to the arena.</li>
		<li>Remove the mouse from the home cage and gently place it into the center of the arena, facing the objects.</li>
		<li>Allow the mouse to freely explore the objects for 15 min. During this period do not disturb the mice.</li>
		<li>At the end of the session, gently place the mouse back into its home cage. Clean the arena and objects with 70% EtOH and dH2O. Place these objects back into the arena.</li>
		<li>Repeat step 4.2.11 until all mice are familiarized to an object.</li>
		<li>Once all mice have been familiarized to an object, analyze the videos. 
		NOTE: Object explorations are counted once the following criteria have been met: the mouse is oriented toward the object, the snout is within 2 cm of the object, the midpoint of the animal&#39;s body is beyond 2 cm from the object, and the previous criteria have been fulfilled for at least 1 s. Additionally, if an animal has satisfied the exploration criteria but exhibits immobility for &#62; 10 s then the exploratory bout is deemed finished.</li>
		<li>Calculate an object bias score for each mouse as follows. 
		<img alt="Equation 2" src="/files/ftp_upload/55523/55523eq2.jpg" /> 
		NOTE: Mice exhibiting an object bias score below 20% or above 80% are excluded from further experimentation.</li>
	</ol>
	</li>
	<li>The final phase of novel object recognition involves assessing exploratory behavior directed toward both a novel and familiar object in the environment, referred to here as the Test phase. This phase is performed 2-3 h after completion of sample phase.
	<ol>
		<li>Prior to the start of a test phase session, place objects into the arena and fix them to the floor so that the animals cannot move the objects.
		<ol>
			<li>Place the objects in the same position in the arena relative to the sample phase13.</li>
			<li>Balance the relative position of novel and familiar objects across genotypes and treatment groups.</li>
			<li>Ensure there is enough distance between the walls and objects so that the mice can freely explore the objects from all angles.</li>
		</ol>
		</li>
		<li>Setup the data acquisition system and/or video cameras. Confirm proper tracking of mice and objects in the maze. Calibrate distances in the arena using captured video images of a ruler or other object of known length.</li>
		<li>Mark corners of the arena in the software to permit scoring of positional biases. Mark objects in software and track exploratory behavior for each object individually (i.e., &#34;Novel&#34; and &#34;Familiar&#34;).</li>
		<li>Remove the cage from the rack and gently place it on a table in close proximity to the arena.</li>
		<li>Gently place animals into the center of the arena, facing the objects. Record mice freely exploring objects for 10 min.</li>
		<li>At the end of the test session, remove mice from the arena and place mice back into their home cage. Thoroughly clean arena and objects with an unscented bleach germicidal wipe, 70% EtOH and dH2O after each session.</li>
		<li>Repeat from step 4.4.3 until all animals have been assessed.</li>
		<li>Once object exploration is measured for all mice, videos are analyzed. 
		NOTE: Object explorations are counted once the following criteria have been met: the mouse is oriented toward the object, the snout is within 2 cm of the object, the midpoint of the animal&#39;s body is beyond 2 cm from the object, and the previous criteria have been fulfilled for at least 1 s. Additionally, if an animal has satisfied the exploration criteria but exhibits immobility for &#62; 10 s then the exploratory bout is deemed finished.</li>
		<li>Assess novel object recognition by comparing time spent exploring the novel to familiar object. Three methods are commonly reported in the literature.
		<ol>
			<li>Analyze raw time spent exploring both novel and familiar objects using a repeated measure test. This method is best used when genotype and/or treatment do not affect total exploration time.</li>
			<li>Calculate novelty preference, using the equation: 
			<img alt="Equation 3" src="/files/ftp_upload/55523/55523eq3.jpg" /> 
			NOTE: This provides the percentage of time spent exploring the novel object relative to the total time exploring objects. Values range from 0% (no exploration of novel object) to 100% (exploration only of the novel object), with a value of 50% indicating equal time spent exploring novel and familiar objects.</li>
			<li>Calculate discrimination index11, using the equation: 
			<img alt="Equation 4" src="/files/ftp_upload/55523/55523eq4.jpg" /> 
			NOTE: This yields the difference in time spent exploring the novel and familiar objects relative to the total time spent exploring objects. Values range from -1 (exploration only of the familiar object) to +1 (exploration only of the novel object, with a value of 0 indicating equal time spent exploring novel and familiar objects.</li>
		</ol>
		</li>
		<li>Remove animals that do not participate in the test session due to hyperdynamic locomotion or other stereotypies, from consideration11. 
		NOTE: Criteria used for removal must be objective and determined a priori for the mouse model (i.e., &#60;5th percentile for total exploration time and either &#62;100 average turn angle during test session or &#62;50th percentile time exhibiting myoclonic corner jumping).</li>
	</ol>
	</li>
</ol>
5. Assessment of Corticospinal Function in Mice with Limb Clasping14
<ol>
	<li>Video document the entire session. Record the video using a portable hand-held device (i.e., smartphone or equivalent). 
	NOTE: Power analyses indicate that sample sizes of 10-15 mice per group are required for a &#946; &#8804;0.2.</li>
	<li>Remove the home cage from the rack and place it on a table. Document the animal ID in the video prior to the next step.</li>
	<li>Gently remove the mouse from its cage and suspend by the tail for 5-10 s. The video must record the animal&#39;s hind and forepaws while suspended.</li>
	<li>After capturing at least 5 s of video, place the mouse back into its home cage, and return the cage to the rack.</li>
	<li>Clean the table. Repeat from step 5.2 until all mice have been recorded.</li>
	<li>Score limb clasping from videos of mice suspended by their tail on a scale from 0-4 (see Table 1 for description of scoring). Inspect videos of suspended mice and then assign a score based on the following criteria.
	<ol>
		<li>No limb clasping. Normal escape extension. One hind limb exhibits incomplete splay and loss of mobility. Toes exhibit normal splay.</li>
		<li>Both hind limbs exhibit incomplete splay and loss of mobility. Toes exhibit normal splay.</li>
		<li>Both hind limbs exhibit clasping with curled toes and immobility.</li>
		<li>Forelimbs and hind limbs exhibit clasping and are crossed, curled toes and immobility.</li>
	</ol>
	</li>
	<li>All mice are scored by 2 independent investigators. Any mouse where the 2 scores differ by more than 1 point is rescored once again.
	<ol>
		<li>Scores that differ are averaged.</li>
	</ol>
	</li>
</ol>
<img alt="Table 1" src="/files/ftp_upload/55523/55523table1.jpg" /> 
Table 1: Description of Limb Clasping Scores.

<ol>
	<li>Elder, G. A., Gama Sosa, M. A., De Gasperi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mouse+models+of+Alzheimer's+disease.">Transgenic mouse models of Alzheimer's disease.</a> Mt Sinai J Med. 77 (1), 69-81 (2010).</li><li>Onos, K. D., Sukoff Rizzo, S. J., Howell, G. R., Sasner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+more+predictive+genetic+mouse+models+of+Alzheimer's+disease.">Toward more predictive genetic mouse models of Alzheimer's disease.</a> Brain Res Bull. 122, 1-11 (2016).</li><li>Webster, S. J., Bachstetter, A. D., Nelson, P. T., Schmitt, F. A., Van Eldik, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+mice+to+model+Alzheimer's+dementia:+an+overview+of+the+clinical+disease+and+the+preclinical+behavioral+changes+in+10+mouse+models.">Using mice to model Alzheimer's dementia: an overview of the clinical disease and the preclinical behavioral changes in 10 mouse models.</a> Front Genet. 5, 88 (2014).</li><li>Rodgers, S. P., Born, H. A., Das, P., Jankowsky, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+APP+expression+during+postnatal+development+causes+persistent+locomotor+hyperactivity+in+the+adult.">Transgenic APP expression during postnatal development causes persistent locomotor hyperactivity in the adult.</a> Mol Neurodegener. 7, 28 (2012).</li><li>Hsiao, K., 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=Correlative+memory+deficits,+Abeta+elevation,+and+amyloid+plaques+in+transgenic+mice.">Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice.</a> Science. 274 (5284), 99-102 (1996).</li><li>Santacruz, K., 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=Tau+suppression+in+a+neurodegenerative+mouse+model+improves+memory+function.">Tau suppression in a neurodegenerative mouse model improves memory function.</a> Science. 309 (5733), 476-481 (2005).</li><li>Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> What's wrong with my mouse? : behavioral phenotyping of transgenic and knockout mice. 2nd edn. , (2007).</li><li>Hughes, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+value+of+spontaneous+alternation+behavior+(SAB)+as+a+test+of+retention+in+pharmacological+investigations+of+memory.">The value of spontaneous alternation behavior (SAB) as a test of retention in pharmacological investigations of memory.</a> Neurosci Biobehav Rev. 28 (5), 497-505 (2004).</li><li>Rutala, W. A., Weber, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guideline+for+disinfection+and+sterilization+in+healthcare+facilities.">Guideline for disinfection and sterilization in healthcare facilities.</a> Centers for Disease Control. , (2008).</li><li>Wes, P. D., 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=Tau+overexpression+impacts+a+neuroinflammation+gene+expression+network+perturbed+in+Alzheimer's+disease.">Tau overexpression impacts a neuroinflammation gene expression network perturbed in Alzheimer's disease.</a> PLoS One. 9 (8), 106050 (2014).</li><li>Ennaceur, A., Delacour, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+one-trial+test+for+neurobiological+studies+of+memory+in+rats.+1:+Behavioral+data.">A new one-trial test for neurobiological studies of memory in rats. 1: Behavioral data.</a> Behav Brain Res. 31 (1), 47-59 (1988).</li><li>Taglialatela, G., Hogan, D., Zhang, W. R., Dineley, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermediate-+and+long-term+recognition+memory+deficits+in+Tg2576+mice+are+reversed+with+acute+calcineurin+inhibition.">Intermediate- and long-term recognition memory deficits in Tg2576 mice are reversed with acute calcineurin inhibition.</a> Behav Brain Res. 200 (1), 95-99 (2009).</li><li>DeVito, L. M., Eichenbaum, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+contributions+of+the+hippocampus+and+medial+prefrontal+cortex+to+the+"what-where-when"+components+of+episodic-like+memory+in+mice.">Distinct contributions of the hippocampus and medial prefrontal cortex to the "what-where-when" components of episodic-like memory in mice.</a> Behav Brain Res. 215 (2), 318-325 (2010).</li><li>Lalonde, R., Strazielle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+regions+and+genes+affecting+limb-clasping+responses.">Brain regions and genes affecting limb-clasping responses.</a> Brain Res Rev. 67 (1-2), 252-259 (2011).</li><li>King, D. L., Arendash, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+characterization+of+the+Tg2576+transgenic+model+of+Alzheimer's+disease+through+19+months.">Behavioral characterization of the Tg2576 transgenic model of Alzheimer's disease through 19 months.</a> Physiol Behav. 75 (5), 627-642 (2002).</li><li>Lalonde, R., Lewis, T. L., Strazielle, C., Kim, H., Fukuchi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+expressing+the+betaAPP695SWE+mutation:+effects+on+exploratory+activity,+anxiety,+and+motor+coordination.">Transgenic mice expressing the betaAPP695SWE mutation: effects on exploratory activity, anxiety, and motor coordination.</a> Brain Res. 977 (1), 38-45 (2003).</li><li>Lewis, 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=Neurofibrillary+tangles,+amyotrophy+and+progressive+motor+disturbance+in+mice+expressing+mutant+(P301L)+tau+protein.">Neurofibrillary tangles, amyotrophy and progressive motor disturbance in mice expressing mutant (P301L) tau protein.</a> Nat Genet. 25 (4), 402-405 (2000).</li><li>Spittaels, K., 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=Prominent+axonopathy+in+the+brain+and+spinal+cord+of+transgenic+mice+overexpressing+four-repeat+human+tau+protein.">Prominent axonopathy in the brain and spinal cord of transgenic mice overexpressing four-repeat human tau protein.</a> Am J Pathol. 155 (6), 2153-2165 (1999).</li><li>Terwel, D., 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=Changed+conformation+of+mutant+Tau-P301L+underlies+the+moribund+tauopathy,+absent+in+progressive,+nonlethal+axonopathy+of+Tau-4R/2N+transgenic+mice.">Changed conformation of mutant Tau-P301L underlies the moribund tauopathy, absent in progressive, nonlethal axonopathy of Tau-4R/2N transgenic mice.</a> J Biol Chem. 280 (5), 3963-3973 (2005).</li><li>Merrow, M., Spoelstra, K., Roenneberg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circadian+cycle:+daily+rhythms+from+behaviour+to+genes.">The circadian cycle: daily rhythms from behaviour to genes.</a> EMBO Rep. 6 (10), 930-935 (2005).</li><li>Smarr, B. L., Jennings, K. J., Driscoll, J. R., Kriegsfeld, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time+to+remember:+the+role+of+circadian+clocks+in+learning+and+memory.">A time to remember: the role of circadian clocks in learning and memory.</a> Behav Neurosci. 128 (3), 283-303 (2014).</li><li>Kandel, E. 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+molecular+biology+of+memory+storage:+a+dialogue+between+genes+and+synapses.">The molecular biology of memory storage: a dialogue between genes and synapses.</a> Science. 294 (5544), 1030-1038 (2001).</li><li>Stough, S., Shobe, J. L., Carew, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermediate-term+processes+in+memory+formation.">Intermediate-term processes in memory formation.</a> Curr Opin Neurobiol. 16 (6), 672-678 (2006).</li><li>Stevens, L. M., Brown, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+and+working+memory+deficits+in+the+3xTg-AD+mouse+between+2+and+15-months+of+age:+a+cross-sectional+study.">Reference and working memory deficits in the 3xTg-AD mouse between 2 and 15-months of age: a cross-sectional study.</a> Behav Brain Res. 278, 496-505 (2015).</li><li>Yue, X., 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+estrogen+deficiency+accelerates+Abeta+plaque+formation+in+an+Alzheimer's+disease+animal+model.">Brain estrogen deficiency accelerates Abeta plaque formation in an Alzheimer's disease animal model.</a> Proc Natl Acad Sci U S A. 102 (52), 19198-19203 (2005).</li><li>McAllister, 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=Genetic+targeting+aromatase+in+male+amyloid+precursor+protein+transgenic+mice+down-regulates+beta-secretase+(BACE1)+and+prevents+Alzheimer-like+pathology+and+cognitive+impairment.">Genetic targeting aromatase in male amyloid precursor protein transgenic mice down-regulates beta-secretase (BACE1) and prevents Alzheimer-like pathology and cognitive impairment.</a> J Neurosci. 30 (21), 7326-7334 (2010).</li><li>Blaney, C. E., Gunn, R. K., Stover, K. R., Brown, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+genotype+influences+behavioral+development+of+3xTg-AD+mouse+pups.">Maternal genotype influences behavioral development of 3xTg-AD mouse pups.</a> Behav Brain Res. , 40-48 (2013).</li></ol>

J.M. Levenson is employed by Proclara Biosciences, Inc. C. Miedel, J. Patton, A. Miedel, and E. Miedel are employed by Hilltop Laboratory Animals.

Significance of the Technique with Respect to Existing Methods 
This procedure has been designed to screen for in vivo activity of compounds in transgenic mouse models of &#946;-amyloidosis and tauopathy. The staged approach employed here ensures detection of efficacious compounds in cognitive domains relevant to AD3. Moreover, the approach detailed here uses behavioral tests that have clearly defined endpoints, easily implementable quality control checks, can be run in a moderate throughput format, and require little intervention from the investigator. These characteristics result in assays that exhibit good reproducibility within animals and across cohorts, which results in low intra- and inter-assay variance and effect sizes (2 &#8804;f &#8804;6) that are robust enough to support behavioral profiling in a drug discovery environment.
Critical Steps within the Protocol 
Many mouse models in use for AD drug discovery exhibit behaviors consistent with heightened anxiety and aggression. This makes handling habituation essential for performing any of the behavioral tests described here. As these tests rely on unmotivated behaviors, rough handling by the investigator due to a hyperactive and anxious or aggressive mouse can significantly influence performance. Heightened anxiety could result in failure to perform the task, reducing the overall power of the test. Moreover, light levels in the arena are essential for facilitating the spontaneous locomotion needed for each test. Bright light tends to increase anxiety and suppress locomotion in rodents, therefore care should be taken to adjust ambient light levels to 30-35 lux in the arena.
Another critical aspect of the procedure is minimization of strong environmental cues that would interfere with an animal&#39;s ability to perform the tasks. Cleaning of the arena and objects in between runs is essential as mice are attracted to novel scents in the environment. Failure to thoroughly clean the arena and objects could result in skewing spontaneous activity of the mouse and masking true cognitive performance. Investigators should also minimize use of personal hygiene products and colognes/perfumes when performing these procedures. Lastly, rodents exhibit robust diurnal and circadian changes in many overt behaviors20 including learning and memory21. Therefore, to minimize variance due to diurnal rhythms in basal behaviors and cognitive performance, all tests should be done at the same time of day across cohorts and studies.
Further, specifically with regard to novel object recognition, the delay interval between sample and test phase, and the selection and placement of objects in the environment are critical parameters. Memory exists in 3 distinct forms: short term memory (STM), intermediate term memory (ITM) and long term memory (LTM)22,23. Changing the interval between sample and test phases from minutes (STM) to hours (ITM) or days (LTM) will change the type of memory tested by the procedure12. Moreover, prior to running the novel object recognition test, many objects should be screened in a test cohort of mice for potential biases in exploration. An object that is excessively attractive or repulsive to the test cohort cannot be used when assessing novel object recognition. Ideally all objects that will be employed in the test, when placed into an arena, will elicit equal exploration times from a na&#239;ve cohort of mice. Inadequate testing and optimization of objects can significantly reduce the power of novel object recognition.
Modifications and Troubleshooting 
There are several factors that could increase the apparent variability in the cognitive tests described here. Many mouse models of AD exhibit hyperdynamic locomotion3 which can mask or alter behaviors measured as the cognitive endpoint. Moreover, there is growing evidence that sex24,25,26 and even maternal genotype27 can influence development and progression of neuropathology and cognitive phenotypes in AD mouse models. Unexpected variability or failure to implement a behavioral task could be due to any of these factors. When first implementing a particular behavioral test, results should always be stratified by sex, age and if applicable, maternal genotype. Furthermore, the quality checks outlined in this procedure should always be performed to ensure that hyperactivity or other stereotyped behaviors are not interfering with quantification of cognitive endpoints.
Environment can also influence the spontaneous exploratory behavior of rodents. Scents or sounds that are undetectable to researchers could attract or repel mice, skewing results of cognitive tests that rely on spontaneous behavior. When initially establishing Y-maze or novel object recognition, performance of the control measures to ensure that there are no positional biases in exploration of objects and/or the environment is essential. If positional biases are observed then investigators must thoroughly scrutinize the environment and potentially adjust lighting, arena placement, location of testing room relative to other rooms in the facility (i.e., not near a high traffick area or heavy equipment) and arena cleaning procedures.
Habituation to the testing environment is key to achieving optimal performance in the novel object recognition test. For example, low total exploration times may be due to inadequate habituation. As an alternative to the procedures outlined here for handling (Section 2) and arena (Section 4.2) habituation, habituation to handling and the test environment can be performed as 3, 5 min sessions per day for 2 consecutive days.
Limitations of the Technique 
As with any procedure, these behavioral tests have limitations. These procedures have been employed because they test function of various cortical regions and hippocampus. If the mouse model does not exhibit functional deficits in brain regions probed by these tests, then these techniques will not be useful. Moreover, we have chosen cognitive tests that probe short-term memory. If the mechanism of action of the compound under preclinical assessment is not expected to affect short-term memory then these procedures should be modified accordingly (i.e., increasing the sample-test phase interval to test long-term memory). Lastly, these tests use unmotivated behaviors. Therefore, if a mouse model is excessively hyperactive or displays other stereotyped behaviors that prevent exploration of the environment then these procedures might not be optimal. As an alternative, one could use fear conditioning for Tg2576 or other &#946;-amyloidosis mouse models, or the spatial water maze for rTg4510 or other mouse models of tauopathy3.
Future Applications 
Once these procedures have been successfully adopted in the lab, several modifications or extensions can be made to assess additional cognitive and functional motor measures. For example, changing the novel object recognition task to determine if a mouse can recognize a change in placement of an object13. Alternatively, instead of using objects, one could use other mice and implement a test of social recognition. With respect to limb clasping and motor function, one could supplement that test with the wire hang and/or grip strength tests. The tests detailed in this method form a solid base to screen for compounds that have in vivo efficacy in translational mouse models for AD, and can be adapted or modified in many ways to best interrogate a particular mouse model or meet the needs of a unique drug discovery program.

Alzheimer&#39;s disease (AD) is a progressive neurodegenerative disorder resulting in debilitating cognitive decline that affects approximately 44 million people worldwide. Currently there are no available treatments for AD that are disease-modifying, emphasizing the urgent need for preclinical discovery of novel therapeutic strategies for this disease. A number of different transgenic mouse models have been created that recapitulate various aspects of AD1,2, including deficits in cognitive domains disrupted in patients3. These mouse models represent a useful tool for facilitating efficient screening in vivo.
When assessing a compound for potential in vivo efficacy, a staged approach must be taken that screens for efficacy in appropriate cognitive domains and also monitors behaviors that could influence the specific endpoints used to assess cognition. Many transgenic mouse models of AD exhibit hyperactivity and other behaviors that may interfere with a particular cognitive test, and prohibit its use in drug screening4. Moreover, for an approach to be implemented in a drug screening environment, the particular tests used should sustain at least a moderate throughput, have clearly defined endpoints, and a procedure that requires minimal intervention by the investigators. Using these criteria, behavioral screens can be implemented that exhibit the reproducibility, low intra- and inter-assay variance and effect sizes needed for compound screening. Detailed here are the methods we have employed to screen for compounds effective in mitigating the cognitive and motor phenotypes present in transgenic mouse models of &#946;-amyloidosis and tauopathy5,6. The methods described are adapted from commonly used behavioral paradigms reported in the literature7, with specific optimizations and quality control checks so that they may be used in transgenic mouse models relevant to AD. This protocol can be used with a variety of data acquisition and analysis systems and assumes that the investigator has a working knowledge of the associated software.

<table><tbody><tr><td>Topscan Lite-High Throughput</td><td>Cleversys</td><td></td><td>Automated behavioral analysis. Includes cameras and video acquisition system, laptop.</td></tr><tr><td>ObjectScan</td><td>Cleversys</td><td></td><td>Software module for accurate object exploration quantification</td></tr><tr><td>Open field for mouse</td><td>Cleversys</td><td>CSI-OF-M</td><td>Arena for novel object recognition</td></tr><tr><td>Y-maze for mouse</td><td>Custom</td><td></td><td>Arms: 30 cm long, 10 cm wide, 20 cm high walls, placed 120 deg apart.</td></tr><tr><td>Camera mount for open field</td><td>Custom</td><td>Custom</td><td>76 cm tall, 115 cm wide, cameras mounted @ 30 cm in from either side.&amp;nbsp; Two mounts, each covers two boxes.</td></tr><tr><td>Camera mount for Y-maze</td><td>Custom</td><td>Custom</td><td>76 cm tall, 115 cm wide, cameras mounted @ 30cm in from either side.&amp;nbsp; One mount covers two mazes.</td></tr><tr><td>Marbles</td><td>Inperial Toy</td><td>8565</td><td>Standard (15.5 mm Dia) glass marbles.</td></tr><tr><td>Dice</td><td>Cardinal Industries</td><td>770</td><td>Standard (0.650 inch) white dice with black dots.</td></tr><tr><td>LOCTITE Fun-Tak</td><td>Henkel</td><td>B018A3AG0W</td><td>Standard blue sticky tak</td></tr><tr><td>EtOH</td><td>Nexeo Solutions</td><td>82452</td><td>100% Ethanol Diluted to 70% using distilled Water</td></tr><tr><td>dH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Tulpenhocken Spring Water Co.</td><td>-</td><td>PA D.E.P. #31, NJ D.O.H. #0049, NYSHD Cert. #320</td></tr><tr><td>Paper towels</td><td>Procter &amp;amp; Gamble</td><td>B019DM86LA</td><td>Bounty, White</td></tr><tr><td>Handheld video camera</td><td>Apple, Inc.</td><td>MKV92LL/A</td><td>Acquisition of Limb clasping video, Iphone 6S Plus (or functional equivalent).</td></tr><tr><td>Gloves</td><td>SafePOINT, L.L.C.</td><td>GL640-2</td><td>Standard, Powder free Latex Gloves, Medium</td></tr><tr><td>Light meter</td><td>Dr. Meter</td><td>LX1330B</td><td>Lighting @ the bottom of Open Field= 35 LUX, Lighting @ bottom of Y-Maze= 32 LUX</td></tr><tr><td>Bleach germicidal wipes</td><td>Clorox</td><td></td><td>Sterilization of equipment during &amp;amp; after use</td></tr></tbody></table>

assessment of spontaneous alternation, novel object recognition and limb clasping in transgenic mouse models of amyloid-&#946; and tau neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Aged Tg2576 mice exhibit robust deficits in spontaneous alternations made within a Y-maze15,16, a phenotype that can be replicated using the methods detailed here (Figure 1A). While a trend for increased arm entries is observed in these mice (Figure 1B), the hyperactivity observed in this line of mice did not affect the spontaneous alternation rate (Figure 1C). In contrast, aged rTg4510 mice appear to exhibit increased spontaneous alternation when placed into a Y-maze (Figure 1D). This is due to extreme hyperactivity (Figure 1E) and stereotypy10, which significantly interfere with measurement of spontaneous alternation (Figure 1F). When initially assessing mice in this task, it is critical to ensure that arm entries and/or distance traveled is not significantly correlated with the spontaneous alternation rate.
Prior to assessment of novel object recognition, mice are habituated to the arena where the test will be performed. During the habituation, hyperactivity (Figure 2A) and other stereotyped behaviors relevant to the mouse model can be assessed. During the sample phase, it is critical to measure exploration of each object separately so that mice that exhibit significant biases in exploratory behavior can be excluded from further assessment (Figure 2B, open circles). Novel object recognition is assessed by comparing exploration of a familiar and novel object, and is commonly analyzed in three different ways. If total exploratory time is comparable across genotypes and/or treatment groups, then raw time exploring each object and an appropriate repeated measures tests can be used to determine if there were differences in novel object recognition (Figure 2C). If a particular mouse strain exhibits differences in total exploratory time, novel object recognition can be assessed using either novelty preference (Figure 2D) or discrimination index (Figure 2E).
Limb clasping is a functional motor test that quantifies deficits in corticospinal function. Limb clasping, which is not a cognitive measure, is observed in several transgenic tau mouse models6,17,18,19 and recapitulates some of the functional motor deficits observed in late-stage AD patients. Suspension of mice by the tail elicits an escape response (Figure 3A, &#34;0&#34;). Deficits in the ability to splay the hind limbs and extend the toes are scored based on their severity on a scale from 0-4 (Figure 3A). Using the procedure outlined herein, one can observe significant limb clasping in rTg4510 mice (Figure 3B).
<img alt="Figure 1" src="/files/ftp_upload/55523/55523fig1.jpg" /> 
Figure 1: Spontaneous Alternation in the Y-maze. (A) When placed into a Y-maze, mice adopt a lose-shift search strategy that results in a pattern of exploration whereby each arm is explored just once for every 3 arm entries. Aged Tg2576 mice exhibit a significant deficit in spontaneous alternation. Using the procedures outlined in this method, a significant restoration of spontaneous alternation was observed after treatment with a proprietary compound. Data was analyzed using 1-way ANOVA and post-hoc comparisons to Tg-PBS were performed using Dunnett&#39;s test. **p &#60;0.01. Error bars indicate SEM. (B) Number of arm entries was not significantly different across any of the groups monitored in this experiment. Data was analyzed using 1-way ANOVA test. Error bars indicate SEM. (C) There was no correlation between spontaneous alternation and the number of arm entries made, indicating that any differences in spontaneous locomotor activity did not impact quantification of spontaneous alternation. Correlation test was performed using Pearson&#39;s correlation analysis. (D) When placed into a Y-maze, rTg4510 mice (6 month) appear to exhibit significantly more spontaneous alternation relative to littermate WT mice. Data was analyzed by 1-way ANOVA and post-hoc comparisons to Tg-PBS were performed using Dunnett&#39;s test. **p &#60;0.01. Error bars indicate SEM. (E) rTg4510 mice made significantly more arm entries due to their extreme hyperdynamic locomotion. Data was analyzed by 1-way ANOVA and post-hoc comparisons to Tg-PBS were performed using Dunnett&#39;s test. ***p &#60;0.001. Error bars indicate SEM. (F) Spontaneous alternation behavior significantly correlated with arm entries, indicating the hyperdynamic locomotor phenotype obscured true spontaneous alternation. Correlation test was performed using Pearson&#39;s correlation analysis (r = 0.7, p &#60;0.0001). <a href="http://cloudflare.jove.com/files/ftp_upload/55523/55523fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55523/55523fig2.jpg" /> 
Figure 2: Novel Object Recognition. (A) Arena habituation permits measurement of spontaneous locomotion and other stereotyped behaviors relevant to a particular mouse model. Here, aged Tg2576 mice (22 month) exhibit significantly more spontaneous locomotion relative to littermate WT mice. Data was analyzed by 1-way ANOVA and post-hoc comparisons to Tg-Veh were performed using Dunnett&#39;s test. **p &#60;0.01. Error bars indicate SEM. (B) During sample phase, exploration of two identical objects was tracked separately. Mice that exhibit large biases toward exploration of one of the two objects (open circles) were excluded from test phase. (C-E) Novel object recognition was assessed by measuring the exploration of a novel and familiar object. Novel object recognition was assessed using (C) raw exploration time, (D) novelty preference or (E) discrimination index. Data in panel C were analyzed using a 2-way ANOVA with repeated measures and pairwise comparisons were made using Sidak&#39;s test. Data in panels D-E were analyzed with a 1-way ANOVA and post-hoc comparisons to Tg-Veh were made using Dunnett&#39;s test. *p &#60;0.05, **p &#60;0.01. Error bars indicate SEM. <a href="http://cloudflare.jove.com/files/ftp_upload/55523/55523fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55523/55523fig3.jpg" /> 
Figure 3: Limb Clasping. (A) Representative images of mice exhibiting various degrees of limb clasping as described in Table 1. (B) rTg4510 mice (6 month) exhibit significant limb clasping relative to littermate WT mice as scored using these methods. Data were analyzed using a 1-way ANOVA and post-hoc comparisons to Tg-PBS were performed using Dunnett&#39;s test. ***p &#60;0.001, ****p &#60;0.0001. Error bars indicate SEM. <a href="http://cloudflare.jove.com/files/ftp_upload/55523/55523fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-Î² and Tau Neuropathology at JoVE.co

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

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Research

JoVE Journal

Medicine

26.5K Views.  Hilltop Laboratory Animals. Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of β-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Video: Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-β and Tau Neuropathology

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&#8217;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&#8217;s disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.

This manuscript describes a Morris water maze (MWM) protocol tailored for use with a commonly used mouse model of Alzheimer's disease. The MWM is widely used in transgenic mouse models. Implementation of a procedure sensitive to the background strain of the mouse model is essential for detecting group differences.

 The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, ...

Behavior

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid &beta; protein (A&beta;)1, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by &beta;-secretase and &gamma;-secretase. The amyloid hypothesis entails that A&gamma;-containing plaques as the underlying toxic mechanism in AD pathology2. The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of A&gamma; neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.

One of the pathological characteristics of AD is the formation of Amyloid &#x03B2; protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method.

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

Neuroscience

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid peptide (A&#946;) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of A&#946; (A&#946;42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in A&#946; or tau levels, making it possible to detect subtle alterations over time. In combination with A&#946; and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF A&#946;42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF A&#946; or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why A&#946; or tau levels in CSF change as the disease progresses in human patients. Here, we demonstrate a refined cisterna magna puncture technique for serial CSF sampling from the mouse.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

Biology

Assessment of Social Transmission of Food Preferences Behaviors

Olfactory recognition deficits are suggested to be able to serve as clinical marker to differentiate Alzheimer&#39;s disease (AD) subjects from healthy aging groups. For example, olfactory dysfunction in AD can present as impairment in olfactory recognition, emerging during early stages of the disease and worsening while the disease progresses. The social transmission of food preferences (STFP) task is based on a rudimentary form of communication between rodents concerning distant foods dependent on the transmission of olfactory cues. Healthy wild-type mice would prefer to eat a novel, flavored food that was previously cued by a conspecific, and this food preference would be hampered in transgenic AD mice, such as the APP/PS1 model. Indeed, a strong preference for the cued food in C57Bl6/J mice of 3 months of age was found, and this was reduced in 3 months old transgenic APP/PS1 mice. In summary, STFP task could be a powerful measure to be integrated in present subclinical detection assays of AD.

This paper presents a protocol for the investigation of social transmission of food preference in mice. The advantages and possible applications for this procedure, for instance, in detecting early changes in AD mouse models, are highlighted. To conclude, interpretation of the results in light of critical details are discussed.

Olfactory recognition deficits are suggested to be able to serve as clinical marker to differentiate Alzheimer&#39;s disease (AD) subjects from healthy ...

Olfactory Assays for Mouse Models of Neurodegenerative Disease

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process and may be a useful behavioral marker for early detection. Earlier detection in neurodegenerative disease is a major goal in the field because this is when neuroprotective therapies have the best potential to be effective. Therefore, in preclinical studies testing novel neuroprotective strategies in rodent models of neurodegenerative disease, olfactory assessment could be highly useful in determining therapeutic potential of compounds and translation to the clinic. In the present study we describe a battery of olfactory assays that are useful in measuring olfactory function in mice. The tests presented in this study were chosen because they measure olfaction abilities in mice related to food odors, social odors, and non-social odors. These tests have proven useful in characterizing novel genetic mouse models of Parkinson&#8217;s disease as well as in testing potential disease-modifying therapies.

Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

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.

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 ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Here, we present two novel methodologies, psPACT and mPACT, for achieving maximal optical transparency and subsequent microscopic analysis of tissue vasculature in the intact rodent whole CNS.

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-β and Tau Neuropathology

Summary

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Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice