This work was supported by the Canadian Institutes of Health Research (CIHR), the Townsend Family, and Jack Brown and Family Alzheimer&#x2019;s Research Foundation (to W. S.). W. S. is the holder of the Canada Research Chair in Alzheimer's Disease. P.L. was supported by a NSERC Alexander Graham Bell Canada Graduate Scholarship Doctoral Research Award and a Michael Smith Foundation for Health Research Senior Graduate Studentship.

1. Preparation
<ol>
<li>Equipment preparation<ol>
 <li>Obtain a circular pool with a diameter of 150cm and a depth of 50cm (Fig. 1). If using black mice, a white pool should be used; if using white mice, a black pool should be used.</li>
 <li>Arrange the room such that the animal being tested cannot see the experimenter during testing. This can be accomplished with drapes or room dividers.</li>
 <li>Place high contrast spatial cues about the room, and/or on the interior of the pool at a location which would be above the water surface.</li>
 <li>Place a 10cm diameter platform in the pool &#x2013; white for a white pool, clear plexiglass for a black pool. Fill the pool with water until the platform is 1cm above the water surface. Let the water equilibrate to room temperature (22 &deg;C). Depending on the water temperature this may take one to three days, or alternatively hot water can be added to speed up the equilibration.</li></ol></li>

 <li>Software preparation<ol>
 <li>Calibrate the pool in the computer software so the camera can create physical distance information from pixel-based information. Divide the pool into 4 quadrants. Specify the platform zone as a variable zone which can change with each trial. Create 5 platform subzones &#x2013; one in each quadrant, and one in the center of the pool. Save the calibration and use it for the remaining test days. (See example Fig. 2).</li>
 <li>Set the maximum trial time as 60 sec. If the mouse finds the platform before this time, program the software to stop the trial when the platform is found.</li>
 <li>Specify the program to begin tracking automatically, when the experimenter exits the testing area. Utilize any &#x201C;reflection minimization&#x201D; options your software package provides.</li>
 <li>Track path length, escape latency, and time spent in each quadrant.</li></ol></li>

</ol>
2. Day 1: Visible Platform
<ol>
 <li>Computer Program<ol>
 <li>Load the pool calibration into the tracking software.</li>
 <li>Create 5 trials, with an inter-trial interval appropriate for your experiment. Program the platform location and starting direction to differ with each trial. See Table 1 for an example protocol.</li></ol></li>
 <li>Testing procedure<ol>
 <li>Transfer the mice from their housing facility to the behavior room. Keep the mice in an area where they cannot see the pool or spatial cues. Let them adjust to the new environment for at least 30 minutes before testing.</li>
 <li>Place a flag on the platform to increase its visibility.</li>
 <li>To begin testing, lift mouse from the home cage by the base of the tail. Support the mouse as you bring it to the testing area. Lifting the mouse by the base of the tail, gently place the mouse into the water, facing the edge of the pool. Quickly leave the testing area.</li>
 <li>If the mouse finds the platform before the 60 sec cut-off, allow the mouse to stay on the platform for 5 seconds then return it to its home cage. If the mouse does not find the platform, place the mouse on the platform and allow it to stay there for 20 sec before returning it to its home cage.</li> 
 <li>Repeat for all mice in the trail. Begin each subsequent trial with a different platform location and starting direction, as you have programmed into your software.</li>
 <li>When testing is complete, return the mice to their housing facility. Mice are dried off and normothermia is assured prior to returning to animal facility.</li>
 <li>In preparation for the following day, remove the flag from the platform and add additional water to the pool to submerge the platform to 1cm below the surface. </li></ol></li>

</ol>
3. Days 2-5: Hidden Platform
<ol>
 <li>Computer Program<ol>
 <li>Load the pool calibration into the tracking software.</li>
 <li>Create 5 trials, with an inter-trial interval appropriate for your experiment. Program the platform location to remain in the same position throughout all trials and days, but have the starting direction differ with each trial, each day.</li></ol></li>
 <li>Testing procedure<ol>
 <li>For black mice, add non-toxic, white, powdered tempera paint to the pool and mix thoroughly. Use enough paint such that the submerged platform is not visible from the surface of the water. For white mice, a black pool with clear water and a clear plexiglass platform should be used.</li>
 <li>Follow steps 2.2.3 to 2.2.6.</li></ol></li>
</ol>
4. Day 6: Probe Trial
<ol>
 <li>Computer Program<ol>
 <li>Load the pool calibration into the tracking software.</li>
 <li>Create 1 trial with no platform zone, and one starting direction. The starting direction farthest from the platform quadrant used on days 2-5 is preferred. Set the trail length to 60 sec.</li></ol></li>
 <li>Testing procedure<ol>
 <li>Remove the platform from the pool.</li>
 <li>Follow steps 2.2.3 to 2.2.6.</li>
 </ol></li>
</ol>
5. Data analysis
<ol>
<li>For each day and each mouse, average the 5 trials to give a single path length and escape latency for each test subject. Calculate the combined error appropriately. For day 6, simply collect the path length, escape latency, and time spent in the platform quadrant for each mouse.</li>
<li>If any differences exist between groups on Day 1, it is likely a problem with vision rather than learning and memory. Only proceed with analysis if no differences are seen on day 1.</li>
<li>Compare the learning curves for Days 2-5 using statistics appropriate for your data set. A steeper curve represents faster task acquisition; a shallower curve represents a deficit in task acquisition. The data from day 2 to day 5 are analyzed using ANOWA.</li>
<li>For day 6, compare the percent of time spent in the previously learned platform quadrant, using statistics appropriate for your data set. A higher percentage of time spent in the platform quadrant is interpreted as a higher level of memory retention.</li>
</ol>
6. Representative Results
We have used the Morris Water Maze test to examine hypoxia&#x2019;s effect on AD pathogenesis (7) and valproic acid (VPA)&#x2019;s pharmaceutical potential for AD treatment (6) in transgenic AD model mice. Figure 3 is the representative result we reported in our study on VPA&#x2019;s effect on memory deficits in the APP23 AD mouse model (6). On day 1 (visible platform trials), there is no difference between the VPA treated and control groups in latency (Fig. 3A) and path length (Fig. 3A) indicating that both of the groups have similar motor and visual capabilities. From this we assume the mice are able to see the flagged-platform and the cues in surrounding environment, and can swim acceptably. For days 2-5 (Day 1 to 4 of hidden platform trials) the example shows a difference in the escape latency (Fig. 3C) and path length (Fig 3D) between the groups, suggesting that VPA treated mice performed significantly better than controls over time. The probe trail results on the last day (Day 6) show that the number of times the mice traveled into the third quadrant, where the hidden platform was previously placed, was significantly greater with VPA treatment compared to control (Fig. 3E). These data indicate that VPA treatment significantly improves the memory deficits seen in APP23 mice.
<img src="/files/ftp_upload/2920/2920fig1.jpg" alt="Figure 1"/> 
Figure 1. Equipment setup for the Morris Water Maze visible platform test day. The pool is shielded from the experimenter using room dividers. Spatial cues are located on the walls, and maybe placed on the interior of the pool, above the water surface, if desired. The pool is filled with clear water, with the platform located 1cm above the surface. A flag has been placed on the platform to enhance visibility. 
<img src="/files/ftp_upload/2920/2920fig2.jpg" alt="Figure 2"/> 
Figure 2. Screen capture from the Any-Maze&trade; Video Tracking System demonstrating pool calibration. The pool is viewed from above by a black and white analog tracking camera with a RTV24 Digitizer. Several zones are defined within the software and the total pool is divided into 4 quadrants. A fifth, platform zone is entered which can vary across trials, with five possible locations: NW, NE, SW, SE, or Center. A calibration line (ticked line across center) is added to allow the software to convert pixel distances into physical distances. 
<img src="/files/ftp_upload/2920/2920fig3.jpg" alt="Figure 3"/> 
Figure 3. Representative results for the Morris Water Maze. The 7-month APP23 transgenic mice carrying human Swedish mutant APP gene were tested after one month of daily VPA (n=30 mice) or vehicle solution (n=30 mice) injections. (A) During the first day of visible platform tests, the VPA treated and control APP23 mice exhibited a similar latency to escape onto the visible platform. P&gt;0.05 by student&#x2019;s t-test. (B) The VPA-treated and control APP23 mice had similar swimming distances before escaping onto the visible platform in the visible platform test. P&gt;0.05 by student&#x2019;s t-test. (C) In hidden platform tests, VPA treated APP23 mice showed a shorter latency to escape onto the hidden platform on the 3rd and 4th day, P&lt;0.001 by ANOVA. (D) The VPA-treated APP23 mice had a shorter swimming length before escaping onto the hidden platform on the 3rd and 4th day, P&lt; 0.01 by ANOVA. (E) In the probe trial on the 6th day, the VPA-treated APP23 mice traveled into the third quadrant, where the hidden platform was previously placed, significantly more times than controls. * P&lt;0.005 by student&#x2019;s t-test. (Adapted and reprinted from The Journal of Experimental Medicine 205, 2781-2789, 2008, Rockefeller University Press, Originally published in J. Exp. Med. doi:10.1084/jem.20081588.) (6).



<table border="1">
 <tbody>
 <tr>
 <td>&nbsp;</td>
 <td colspan="2">Day 1</td> 
 <td>Day 2</td>
 <td>Day 3</td>
 <td>Day 4</td>
 <td>Day 5</td>
 <td>Day 6</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Platform location</td>
 <td>Starting Direction</td>
 <td colspan="4">Platform Location: SW
Starting Location as follows:
</td>
 <td>No platform.</td>
 </tr>
 <tr>
 <td>Trial 1</td>
 <td>SW</td>
 <td>S</td>
 <td>W</td>
 <td>N</td>
 <td>N</td>
 <td>E</td>
 <td>N</td>
 </tr>
 <tr>
 <td>Trial 2</td>
 <td>NW</td>
 <td>N</td>
 <td>S</td>
 <td>W</td>
 <td>E</td>
 <td>S</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trial 3</td>
 <td>NE</td>
 <td>S</td>
 <td>N</td>
 <td>E</td>
 <td>W</td>
 <td>W</td>
 <td>&nbsp;</td> 
 </tr>
 <tr>
 <td>Trial 4</td>
 <td>Center</td>
 <td>E</td>
 <td>E</td>
 <td>W</td>
 <td>S</td>
 <td>E</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trial 5</td>
 <td>SE</td>
 <td>W</td>
 <td>S</td>
 <td>S</td>
 <td>N</td>
 <td>N</td> 
 <td>&nbsp;</td> 
 </tr>
 </tbody>
</table>
Table 1. Sample water maze protocol* 
* Note how both the platform position and starting direction change on day 1, whereas on days 2-5 the platform position remains constant while the starting direction changes. On day 6, there is no platform and a single trial. The starting direction for day 6 is farthest from the previous platform location (SW) so that the mice must travel some distance before entering the previously learned platform quadrant.

<ol>
	<li>Morris, R. G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+localization+does+not+require+the+presence+of+local+cues.">Spatial localization does not require the presence of local cues.</a> Learning and Motivation. 12, 239-260 (1981).</li><li>O'Keefe, 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+review+of+the+hippocampal+place+cells.">A review of the hippocampal place cells.</a> Prog Neurobiol. 13, 419-439 (1979).</li><li>Scoville, W. B., Milner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+recent+memory+after+bilateral+hippocampal+lesions.">Loss of recent memory after bilateral hippocampal lesions.</a> J Neurol Neurosurg Psychiatry. 20, 11-21 (1957).</li><li>Eichenbaum, H., Stewart, C., Morris, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+representation+in+place+learning.">Hippocampal representation in place learning.</a> J Neurosci. 10, 3531-3542 (1990).</li><li>Block, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+ischemia+and+behavioural+deficits.">Global ischemia and behavioural deficits.</a> Progress in Neurobiology. 58, 279-295 (1999).</li><li>Qing, H., He, G., Ly, P. T., Fox, C. J., Staufenbiel, M., Cai, F., Zhang, Z., Wei, S., Sun, X., Chen, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valproic+acid+inhibits+Abeta+production,+neuritic+plaque+formation,+and+behavioral+deficits+in+Alzheimer's+disease+mouse+models.">Valproic acid inhibits Abeta production, neuritic plaque formation, and behavioral deficits in Alzheimer's disease mouse models.</a> J Exp Med. 205, 2781-2789 (2008).</li><li>Sun, X., He, G., Qing, H., Zhou, W., Dobie, F., Cai, F., Staufenbiel, M., Huang, L. E., Song, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+facilitates+Alzheimer's+disease+pathogenesis+by+up-regulating+BACE1+gene+expression.">Hypoxia facilitates Alzheimer's disease pathogenesis by up-regulating BACE1 gene expression.</a> Proc Natl Acad Sci U S A. , 18727-18732 (2006).</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> Brain Research Reviews. 36, 60-90 (2001).</li><li>Brandeis, R., Brandys, Y., Yehuda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+the+Morris+Water+Maze+in+the+study+of+memory+and+learning.">The use of the Morris Water Maze in the study of memory and learning.</a> Int J Neurosci. 48, 29-69 (1989).</li><li>Lipp, H. P., Wolfer, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+modified+mice+and+cognition.">Genetically modified mice and cognition.</a> Curr Opin Neurobiol. 8, 272-280 (1998).</li><li>Sandi, C. <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+and+mechanisms+of+action+of+glucocorticoid+involvement+in+memory+storage.">The role and mechanisms of action of glucocorticoid involvement in memory storage.</a> Neural Plast. 6, 41-52 (1998).</li></ol>

All experiments were conducted in accordance with The University of British Columbia Animal Care and Use committee and CIHR guidelines.

Age, sex, species, and strain differences influence MWM performance (8). Studies indicate that aged mice have poor performance in the MWM, while male rodents perform better than females; additionally, floating is more pronounced in mice than rats (9, 10). Therefore, these elements should be equated across all tests. Evidence also suggests that stressed animals perform more poorly in the MWM (11), thus environmental factors which may cause stress, such as temperature, light, and noise, should be monitored and kept constant over the task.

<table border="1">
	<tbody>
 	<tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 	<td>AnyMaze Video Tracking System</td>
 <td>Stoelting Company</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Tempera Paint</td>
 <td>Reeves &amp; Poole Groups</td>
 <td>White, powdered</td>
 </tr>
 </tbody>
</table>

morris water maze test for learning and memory deficits in alzheimer&#39;s disease model mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4. In addition, the MWM testing environment reduces odor trail interference 5. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer&#x2019;s Disease 6, 7. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer&#x2019;s disease transgenic model mice.

Watch this Scientific Journal Video about Morris Water Maze Test for Learning and Memory Deficits in Alzheimer's Disease Model Mice at JoVE.com

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

The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#39;s Disease Model Mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including ...

morris-water-maze-test-for-learning-memory-deficits-alzheimer-s

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126.7K Views.  University of British Columbia. The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.

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

Low-stress Route Learning Using the Lashley III Maze in Mice

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other aversive stimuli to motivate task acquisition. As animals age, sensory modalities deteriorate. For example, many strains of mice develop hearing deficits or cataracts. Changes in the sensory systems required to guide mice during task acquisition present potential confounds in interpreting learning changes in aging animals. Moreover, the use of aversive stimuli to motivate animals to learn tasks is potentially confounding when comparing mice with differential sensitivities to stress. To minimize these types of confounding effects, we have implemented a modified version of the Lashley III maze. This maze relies on route learning, whereby mice learn to navigate a maze via repeated exposure under low stress conditions, e.g. dark phase, no food/water deprivation, until they navigate a path from the start location to a pseudo-home cage with 0 or 1 error(s) on two consecutive trials. We classify this as a low-stress behavior test because it does not rely on aversive stimuli to encourage exploration of the maze and learning of the task. The apparatus consists of a modular start box, a 4-arm maze body, and a goal box. At the end of the goal box is a pseudo-home cage that contains bedding similar to that found in the animal&#x2019;s home cage and is specific to each animal for the duration of maze testing. It has been demonstrated previously that this pseudo-home cage provides sufficient reward to motivate mice to learn to navigate the maze1. Here, we present the visualization of the Lashley III maze procedure in the context of evaluating age-related differences in learning and memory in mice along with a comparison of learning behavior in two different background strains of mice. We hope that other investigators interested in evaluating the effects of aging or stress vulnerability in mice will consider this maze an attractive alternative to behavioral tests that involve more stressful learning tasks and/or visual cues.

The Lashley III maze is a route-learning task that does not rely on aversive stimuli or visual cues. It is thus a highly attractive option for evaluating learning and memory, especially in aging mice or otherwise where stress is a consideration.

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other ...

Design and Construction of a Cost Effective Headstage for Simultaneous Neural Stimulation and Recording in the Water Maze

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial products are often expensive, not easily customized, and not submersible. Here we describe a method to design and build a customized, integrated circuit headstage for simultaneous 4-channel neural recording and 2-channel simulation in awake, behaving animals. The headstage is designed using a free, commercially available CAD-type design package, and can be modified easily to accommodate different scales (e.g. to add channels). A customized printed circuit board is built using surface mount resistors, capacitors and operational amplifiers to construct the unity gain source follower circuit. The headstage is made water-proof with a combination of epoxy, parafilm and a synthetic rubber putty. We have successfully used this device to record local field potentials and stimulate different brain regions simultaneously via independent channels in rats swimming in a water maze. The total cost is &lt; $30/unit and can be manufactured readily.

We present a low-cost method to design and construct a light headstage pre-amplifier system with simultaneous neural recording and stimulation capability. This device can be waterproofed for use in swimming animals.

Headstage preamplifiers and source followers are commonly used to study neural activity in behavioral neurophysiology experiments. Available commercial ...

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal care. Such a preference memory can last from hours, to days, even life-long, depending on training parameters. Early odor preference learning provides us with a model in which the critical changes for a natural form of learning occur in the olfactory circuitry. An additional feature that makes it a powerful tool for the analysis of memory processes is that early odor preference learning can be lateralized via single naris occlusion within the critical period. This is due to the lack of mature anterior commissural connections of the olfactory hemispheres at this early age. This work outlines behavioral protocols for lateralized odor learning using nose plugs. Acute, reversible naris occlusion minimizes tissue and neuronal damages associated with long-term occlusion and more aggressive methods such as cauterization. The lateralized odor learning model permits within-animal comparison, therefore greatly reducing variance compared to between-animal designs. This method has been used successfully to probe the circuit changes in the olfactory system produced by training. Future directions include exploring molecular underpinnings of odor memory using this lateralized learning model; and correlating physiological change with memory strength and durations.

This protocol introduces lateralized early odor preference learning in rats using acute single naris occlusion. Lateralized learning permits the examination of behavioral outcomes and underpinning biological mechanisms within the same animals, reducing variance induced by between-animal designs. This protocol can be used to investigate molecular mechanisms underpinning early odor learning.

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal ...

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, ...

Assessing Spatial Learning and Memory in Small Squamate Reptiles

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, interest in the cognitive processes of nonmodel species, typically within an ecological context, has also become an emerging field of study. In particular, interest in the cognitive processes in reptiles is growing although experimental studies on reptilian cognition are sparse. The few reptilian studies that have experimentally tested for spatial learning and memory have used rodent paradigms modified for use in reptiles. However, ecologically important aspects of the physiology and behavior of this taxonomic group must be taken into account when testing for spatially based cognition. Here, we describe modifications of the dry land Barnes maze and associated testing protocol that can improve performance when probing for spatial learning and memory ability in small squamate reptiles. The described paradigm and procedures were successfully used with male side-blotched lizards (Uta stansburiana), demonstrating that spatial learning and memory can be assessed in this taxonomic group with an ecologically relevant apparatus and protocol.

This paper describes a modification of the Barnes maze, a standard rodent paradigm used to assess spatial memory and learning, for use in small squamate reptiles.

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, ...

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

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In vivo Optogenetic Stimulation of the Rodent Central Nervous System

A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation

Intracerebroventricular Injection of Amyloid-β Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Assessing Spatial Learning and Memory in Small Squamate Reptiles