The authors would like to thank Christopher Newport University for funding the preparation of this manuscript and The University of Vermont for funding the experiments described in the representative results section.

This protocol is approved by the Institutional Animal Care and Use Committee of Christopher Newport University.
1. Construction of the OFTM Apparatus
<ol>
	<li>Construct the boundary wall (40 cm high and 183 cm diameter) from three pieces of white laminate. Construct the circular maze floor (183 cm diameter) from black laminate and glue it to a plastic table top. Secure the boundary wall to the edge of the table with screws and glue.</li>
	<li>Decorate the interior surface of the boundary wall: Leave one quarter solid white; Paint one quarter solid black; Paint one quarter with black stripes; Paint one quarter with black circles.</li>
	<li>Build four &#8220;food towers&#8221; (8 cm x 8 cm x 20 cm; Figure 1A) cut from a square-shaped white PVC post. Glue a piece of black laminate (8 cm x 8 cm) to the top of each tower. Secure them to the maze floor with crazy glue 54 cm apart from each other.</li>
	<li>Build three &#8220;pre-training towers&#8221; (5 cm, 10 cm &#38; 15 cm high respectively; Figure 1B) from a square-shaped white PVC post. Glue a piece of black laminate (8 cm x 8 cm) to the top of each tower. These towers are taped to the maze floor only during the pre-training phase (described below).</li>
	<li>Glue one black opaque plastic food cup with a screw-top lid (= a clear plastic bead container painted black; 1.75 cm high and 3.75 cm diameter) to the top of each food tower. Drill three small venting holes (3 mm diameter) in each lid. The holes ensure that the rat cannot locate the &#8220;correct&#8221; food tower using olfactory cues. The height of the training towers ensures that the rat cannot locate the &#8220;correct&#8221; tower using the uncapped food cup as a visual cue.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/53227/53227fig1.jpg" /> 
Figure 1. Photographs of the Open-field Tower Maze (OFTM). (A) The rat is able to find and retrieve a food reward from only one reward-baited &#8220;training tower&#8221; during acquisition, as well as all subsequent training and testing. (B) Three &#8220;pre-training towers&#8221; are placed in the center of the maze arena during pre-training. <a href="https://www.jove.com/files/ftp_upload/53227/53227fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. The OFTM Maze Room
<ol>
	<li>Use a lamp with a 60 W light bulb (Lumens: 800) to dimly illuminate the maze room. The lamp is located 3 ft above the floor and 2 ft to the side of the maze, facing toward the ceiling. Play white noise in the background (~70 dB) using a sound machine.</li>
	<li>Mount a digital video camera to the ceiling above the maze to transmit video to a computer. Hang an opaque curtain to separate the maze from the computer monitoring area.</li>
</ol>
3. Rat Handling, Food Restriction &#38; Housing
<ol>
	<li>Have each experimenter handle all rats for seven days (4 min per day) prior to pre-training.</li>
	<li>Place rats on a food restricted diet (1 hr&#160;of free-feeding per day). This diet ensures that rats will be motivated to retrieve rewards during training, while maintaining a healthy weight (~90% of free-feeding weight).</li>
	<li>Double-house rats (if using females) throughout the duration of the experiment. Pair-housed female rats are less anxious than single-housed female rats12.</li>
</ol>
4. Pre-training Procedures
<ol>
	<li>Bait each uncapped food cup on the seven towers with a food reward (e.g., one piece of fruit cereal) inside.</li>
	<li>Place a rat into the maze in a random location until it finds and consumes all seven food rewards before it is removed.
	<ol>
		<li>If the rat does not retrieve all of the food rewards within 10 min, then remove it from the maze and give pre-training procedures again the following day. Note: some rats will take longer than others to initially learn to retrieve the food rewards from the top of the towers.</li>
		<li>If a rat does not retrieve and consume all seven food rewards from the towers within the first two days of pre-training, give it additional 30 min sessions in the OFTM until the food rewards are retrieved. Typically, the rat will find and consume all the food rewards after one or two of these 30 min sessions.</li>
	</ol>
	</li>
</ol>
5. Place-training &#38; Response-training Procedures
<ol>
	<li>Assign each rat two of four start locations. Counterbalance the following: the start location release point within and between each training day; the type of learning (place vs response); the &#8220;correct tower&#8221; for all the place learners; and the turn each response-learner is trained to make (right vs. left).</li>
	<li>At the start of each training day turn on the noise machine and open the video recording program. Bait all four towers and cover three out of the four food cups, leaving the food reward on the &#8220;correct&#8221; tower exposed.</li>
	<li>On each given trial, start video recording (refer to manufacturer&#8217;s user manual for instructions), then place the rat in the maze at its assigned start location. Then, go behind the curtain to observe the rat&#8217;s behavior on a computer monitor. Remove the rat from the maze immediately after it retrieves the reward or after 3 min&#160;have passed and stop video recording.
	<ol>
		<li>For place-learning rats, always use the same &#8220;correct&#8221; food tower, and place the rat in one of two start locations. A place-learner solves the OFTM by finding a specific location using the spatial cues provided in the maze. For example, bait tower D (= the &#8220;correct&#8221; tower) and release the rat from start location &#8216;2&#8217; or &#8216;3&#8217; (solid arrows in Figure 2A, left), or bait tower A and release a rat from start location &#39;1&#39; or &#39;4&#39; (dashed arrows in Figure 2A, left).</li>
		<li>For response-learning rats, switch the &#8220;correct&#8221; food tower depending on the start location. Response-learners solve the OFTM by learning to make a specific turn (e.g., always turn right), regardless of its starting location in the maze. For example, bait tower D and release the rat from start location &#8216;3&#8217;, or bait tower B and release that same rat from a start location &#8216;2&#8217; (solid arrows in Figure 2B, left). For a different rat, bait tower B and release the rat from location &#8216;1&#8217;, or bait tower A and release that rat from location &#8216;4&#8217; (dashed arrows in Figure 2B, left).</li>
	</ol>
	</li>
	<li>Give all rats a total of 48 training trials across 12 days (four trials per day).</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/53227/53227fig2.jpg" /> 
Figure 2. Overhead Schematic Showing Place- and Response-Training &#38; Testing Procedures. This figure shows the start and food reward locations for training place-learners (A, Left) and response learners (B, Left), and also the start and reward locations used during the start location switch test for place-learners (A, Right) and response learners (B, Right). Numbers indicate a rat&#8217;s starting location; gray squares represent food towers; solid and dashed lines point to the baited food tower. Start locations are counterbalanced within each training condition. <a href="https://www.jove.com/files/ftp_upload/53227/53227fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Start Location Switch Testing Procedures
<ol>
	<li>After all the rats perform at their asymptotic level (~80% of correct first choice responding) carry out a &#8220;Start Location Switch Test&#8221; to test the persistence of the learned behavior.</li>
	<li>Counterbalance the novel start locations (i.e., the start locations that have not been previously used for the rat).</li>
	<li>Follow the procedures for each training trial described in protocol section 5 with the new start locations.
	<ol>
		<li>For a place-learning rat that originally was released from location &#8216;2&#8217; or &#8216;3&#8217; and had tower D as its &#8220;correct&#8221; tower, place the rat in either of the new start location &#39;1&#39; or &#39;4&#39; , while still baiting tower D (Figure 2A, right).</li>
		<li>For a response-learning rat that was originally released from location &#8216;2&#8217; or &#8216;3&#8217; and learned to turn right to receive the reward (on towers B and D, respectively), place the rat in either of the new start location &#8216;1&#8217; or &#8216;4&#8217; and bait either tower A or C, respectively (which would still require the rat to turn right in order to find the food reward) Figure 2B, right.</li>
		<li>Administer all rats eight start location switch test trials across two days (four trials per day).</li>
	</ol>
	</li>
</ol>
7. Data Analysis
<ol>
	<li>Have an experimenter who has not trained rats and does not know the treatment of each rat score video-taped trials by counting the number of responses a rat makes to find the &#8220;correct&#8221; tower on each trial (indicated by the rat rearing up over the food cup).</li>
	<li>Calculate the percent of times that a rat finds the reward on its first choice (% of first choice correct responses) within each training day. Construct learning curves using the percent of first choice correct responses across training days. Use the percent of first choice correct response for the statistical analysis.</li>
	<li>Calculate the number of trials it took the rat to reach the a priori set criterion to acquire the task (i.e., first choice correct responding on 8 out of 10 consecutive trials). Exclude rats from analysis that do not reach the a priori criterion.</li>
</ol>

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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=Buspirone+impaired+acquisition+and+retention+in+avoidance+tasks:+involvement+of+the+hippocampus.">Buspirone impaired acquisition and retention in avoidance tasks: involvement of the hippocampus.</a> Chin J Physiol. 41 (1), 33-44 (1998).</li><li>Gisquet-Verrier, P., Alexinsky, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+contextual+change+determine+long-term+forgetting.">Does contextual change determine long-term forgetting.</a> Animal Learning and Behavior. 14 (4), 349-358 (1986).</li><li>Gisquet-Verrier, P., 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=Exposure+to+retrieval+cues+improves+retention+performance+and+induces+changes+in+ACTH+and+corticosterone+release.">Exposure to retrieval cues improves retention performance and induces changes in ACTH and corticosterone release.</a> Psychoneuroendocrinology. 29 (4), 529-556 (2004).</li><li>Packard, M. G., Hirsh, R., White, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+fornix+and+caudate+nucleus+lesions+on+two+radial+maze+tasks:+evidence+for+multiple+memory+systems.">Differential effects of fornix and caudate nucleus lesions on two radial maze tasks: evidence for multiple memory systems.</a> J Neurosci. 9 (5), 1465-1472 (1989).</li><li>Devan, B. D., McDonald, R. J., White, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+medial+and+lateral+caudate-putamen+lesions+on+place-+and+cue-guided+behaviors+in+the+water+maze:+relation+to+thigmotaxis.">Effects of medial and lateral caudate-putamen lesions on place- and cue-guided behaviors in the water maze: relation to thigmotaxis.</a> Behav Brain Res. 100 (1-2), 5-14 (1999).</li></ol>

The authors have nothing to disclose.

Numerous paradigms can be used to investigate learning and memory in rodents, and each has important steps that experimenters should follow. In the OFTM, it is important that maze conditions (e.g., position of maze, intra- and extra-maze cues, dim lighting and background noise level, daily time of training) are kept constant throughout the duration of the experiment. It is also necessary to minimize stress in rat subjects to ensure continuous performance in the OFTM. Stress can be kept to a minimum in female rat...

The open-field tower maze (OFTM) can be used to examine the behavioral and neurobiological mechanisms of spatial learning in rodents. Importantly, the OFTM allows researchers to study both &#8220;place-based&#8221; and &#8220;response-based&#8221; learning types. During place-learning an animal navigates through a maze using the spatial cues in the surrounding environment (e.g., a food reward is located next to a tall beacon). During response-learning the animal makes a specific turn response regardless of its position relative to spatial cues (e.g., make a left or right turn to find a food reward). It has been previously reported that response-learning and place-learning rely on two different memory systems3, 4 and mapping their neural mechanisms has been an ongoing scientific pursuit2, 5-7. The design of the OFTM was adapted from the one previously described8.
Open field paradigms (a component of the OFTM) are commonly used to investigate anxiety in rodents9. During the typical open field anxiety test rats that spend more time in the center of an open field have lower stress and anxiety levels than rats that spend more time near the perimeter. In the OFTM, a rat must enter the center of the maze to find the food reward. Therefore, correct navigation in the OFTM indicates that these rats are non-stressed. One reason why the rats are non-stressed during the training is because unlike standard open-field tests of anxiety training the OFTM requires pre-training phase during which the rat is pre-exposed to the maze over a long period of time. In addition, the training occurs in a dimly lit environment further reducing the anxiety-inducing aspect of an open field which is typically brightly lit.
Another unique feature of the OFTM, compared to some other mazes, is how learning is measured. Experimenters using the Morris Water Maze or Barnes Maze typically measure learning by analyzing the speed and/or path length a rat travels to reach its target location. These dependent measures can be affected by non-learning factors, such as changes in a rat&#8217;s general motor activity. However, the OFTM measures learning using the percent of first-choice correct response a rat makes to find the food reward. This dependent measure is not vulnerable to changes in motor performance and therefore, more accurately assesses learning than measurements of speed and distance. Nevertheless, the experimenter can also measure a rat&#8217;s speed and distance traveled in the OFTM using video tracking software.
Other maze-types, such as the T-maze, Morris Water Maze and Barnes Maze can be used to study place- and response-learning. However, the OFTM is unique in a few notable ways. First, the OFTM is a more sensitive behavioral assay than the T-maze because the OFTM has a chance performance of 25%, whereas the T-maze has a chance performance of 50%. Second, a rat can only turn in one of two ways in the T-maze, but has unrestricted route possibilities when navigating in the OFTM. Third, unlike the Morris Water Maze and Barnes Maze tasks (which have unrestricted route possibilities like the OFTM), experiments using the OFTM do not require rats to navigate through an aversive situation. Rats are motived to navigate in the Morris Water Maze to escape swimming in water and they are motivated to navigate in the Barnes Maze to avoid being in an open, brightly-lit environment. It has been demonstrated that exposure to the Morris Water Maze or Barnes Maze induces the release of stress hormone (i.e., corticosterone) in rodents10. However, rats navigate in the OFTM because they are motivated to find a food reward and training is conducted with dim ambient lighting. Thus, the OFTM is a non-aversive appetitive task. Results from experiments that use a stressful maze paradigm to examine the effects of manipulations such as drug, hormone, exercise, or diet on learning and memory mechanisms can be difficult to interpret because &#8220;stress&#8221;, by itself, may influence cognitive function11.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>OFTM construction supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Safety Glasses, Clear, Uncoated</td>
			<td>Grainger</td>
			<td>4EY97</td>
			<td>Used for general eye protection during maze construction</td>
		</tr>
		<tr>
			<td>Masking Tape, Beige, 18mm x 55m</td>
			<td>Grainger</td>
			<td>15F739</td>
			<td>General supply</td>
		</tr>
		<tr>
			<td>Wiss All-purpose Scissor</td>
			<td>Home Depot&#160;</td>
			<td>37103278647</td>
			<td>General supply</td>
		</tr>
		<tr>
			<td>Lancaster&#160; 6 foot Round Folding table - Heavy Duty white granite plastic - 29&#34;&#160;</td>
			<td>www.webstaurantstore.com</td>
			<td>384YCZ72RND</td>
			<td>Used as base of the maze</td>
		</tr>
		<tr>
			<td>Laminate Countertop sheet in White Matte Finish (48 x 96 x 0.039)</td>
			<td>Home Depot&#160;</td>
			<td>1573603504896</td>
			<td>Used as walls for the maze</td>
		</tr>
		<tr>
			<td>Laminate Countertop sheet in Black Matte Finish (48 x 96 x 0.039)</td>
			<td>Home Depot&#160;</td>
			<td>1595603504896</td>
			<td>Used as floor of the maze</td>
		</tr>
		<tr>
			<td>Adhesive, Spray, 24oz, 17.6oz Net. 3M</td>
			<td>Grainger</td>
			<td>3MA16</td>
			<td>Used to adhere the laminate floor to the table top and pieces of laminate to each other</td>
		</tr>
		<tr>
			<td>16.6 oz #90 hi-strength S</td>
			<td>Lowe&#39;s</td>
			<td>193252</td>
			<td>Used to adhere the laminate floor to the table top</td>
		</tr>
		<tr>
			<td>Superflex RTV, Silicone Adhesive Sealant, Loctile</td>
			<td>VWR</td>
			<td>300040-068</td>
			<td>Used to seal around the baze (between the floor and walls of the maze)</td>
		</tr>
		<tr>
			<td>Industrial Sealant; 10.1oz</td>
			<td>Grainger</td>
			<td>4UH06</td>
			<td>Used to seal around the baze (between the floor and walls of the maze)</td>
		</tr>
		<tr>
			<td>Painter&#39;s Masking Tape; Shurtape; 2in x 60yd</td>
			<td>Grainger</td>
			<td>6FET7</td>
			<td>Used to mark out visual patterns on maze walls</td>
		</tr>
		<tr>
			<td>Mini/Trim paint tray</td>
			<td>ACE Hardware</td>
			<td>1365105</td>
			<td>Used to hold paint&#160;</td>
		</tr>
		<tr>
			<td>Foam mini roller 4&#34; 6pk</td>
			<td>ACE Hardware</td>
			<td>1494871</td>
			<td>Used for painting patterns&#160;</td>
		</tr>
		<tr>
			<td>Val Sample Set Nbase paint</td>
			<td>ACE Hardware</td>
			<td>1505031</td>
			<td>Used as paint for pattern on the walls of the maze</td>
		</tr>
		<tr>
			<td>HP Paint Black</td>
			<td>Walmart</td>
			<td>72450405005</td>
			<td>Used to paint pattern on the walls of the maze</td>
		</tr>
		<tr>
			<td>Brush</td>
			<td>Walmart</td>
			<td>81773801110</td>
			<td>Used to paint pattern on the walls of the maze</td>
		</tr>
		<tr>
			<td>Duct Tape, 2in X 60yd, 10.5mil. Black</td>
			<td>Grainger</td>
			<td>15F767</td>
			<td>Used to wrap the exterior of the maze</td>
		</tr>
		<tr>
			<td>4X4 8ft PT pine Vinyl-CTD Post W/Cap</td>
			<td>Home Depot&#160;</td>
			<td>40933070541</td>
			<td>Used to build towers for the maze</td>
		</tr>
		<tr>
			<td>20 gram Pro Super Glue</td>
			<td>Lowe&#39;s</td>
			<td>125870</td>
			<td>Used to glue tower tops, towers to the floor of the maze and food cups to the tower tops</td>
		</tr>
		<tr>
			<td>Bead Containers 9 - 1/2</td>
			<td>Michaels</td>
			<td>652695766978</td>
			<td>Used as food cups on top of towers</td>
		</tr>
		<tr>
			<td>Spray Paint, Black, 12oz</td>
			<td>Grainger</td>
			<td>6KP22</td>
			<td>Used to paint food cups &#38; covers</td>
		</tr>
		<tr>
			<td>Room prepration</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Conair SU1W Sound Machine; 10 Sounds</td>
			<td>Home Depot&#160;</td>
			<td>204493600</td>
			<td>Used for background noise</td>
		</tr>
		<tr>
			<td>Lamp</td>
			<td>Walmart</td>
			<td>5027698392</td>
			<td>Used for dim lighting of maze room</td>
		</tr>
		<tr>
			<td>ES 14W DayLight Spiral Bulb 4pk</td>
			<td>Home Depot&#160;</td>
			<td>762148208433</td>
			<td>Used for dim lighting of maze room</td>
		</tr>
		<tr>
			<td>Shower Curtain</td>
			<td>Walmart</td>
			<td>3429910901</td>
			<td>Used for separation between the maze and the experimenter</td>
		</tr>
		<tr>
			<td>Curtain Rod</td>
			<td>Bed Bath &#38; Beyond</td>
			<td>76598645104</td>
			<td>Used for separation between the maze and the experimenter</td>
		</tr>
		<tr>
			<td>Froot Loops Cereal</td>
			<td>Walmart</td>
			<td>3800039122</td>
			<td>Used as reward</td>
		</tr>
		<tr>
			<td>Recording equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Media Recorder 3: 4 video</td>
			<td>Noldus</td>
			<td>NMR4-0200</td>
			<td>Used to record training trials</td>
		</tr>
		<tr>
			<td>Monochrome GigE Camera</td>
			<td>Noldus</td>
			<td>XCFW-BG62</td>
			<td>Used to record training trials</td>
		</tr>
		<tr>
			<td>Precision M4800 Laptop</td>
			<td>Dell</td>
			<td>N/A</td>
			<td>Used to run video software</td>
		</tr>
	</tbody>
</table>

place and response learning in the open-field tower maze

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for examining how rats learn to use a place- or response-learning to successfully navigate in an open-field arena. Additionally, this protocol describes how the OFTM differs from other behavioral maze paradigms that are commonly used to study spatial learning in rodents. The OFTM described in this article was adapted from the one previously described by Cole, Clipperton, and Walt (2007). Specifically, the OFTM was created to test spatial learning in rodents without the experimenter having to consider how &#8220;stress&#8221; might play a role as a confounding variable. Experiments have shown that stress-alone can significantly affect cognitive function1. The representative results section contains data from an experiment that used the OFTM to examine the effects of estradiol treatment on place- and response-learning in adult female Sprague Dawley rats2. Future studies will be designed to examine the role of the hippocampus and striatum in place- and response-learning in the OFTM.

The representative results described here have been previously published in the Neurobiology of Learning &#38; Memory, Elsevier2. In this experiment the OFTM was used to test the effects of chronic and cycling estradiol treatments on place- and response-learning in female Sprague-Dawley rats (250 &#8211; 300 g; N = 48). There were two important reasons why the OFTM was used to conduct this experiment. First, estradiol has been shown to induce differential effects on place- and response-learning7. Se...

Watch this Scientific Journal Video about Place and Response Learning in the Open-field Tower Maze at JoVE.com

Place and Response Learning in the Open-field Tower Maze

The Open-field Tower Maze (OFTM) is a maze created to study the behavioral and neural mechanisms of spatial learning (e.g., place- or response-learning) in rats. This maze is especially useful for experimenters who want to use a non-stressful maze paradigm to investigate spatial learning in their research.

This protocol describes how the Open-field Tower Maze (OFTM) paradigm is used to study spatial learning in rodents. This maze is especially useful for ...

place-and-response-learning-in-the-open-field-tower-maze

Research

JoVE Journal

Behavior

10.3K Views.  Christopher Newport University. The Open-field Tower Maze (OFTM) is a maze created to study the behavioral and neural mechanisms of spatial learning (e.g., place- or response-learning) in rats. This maze is especially useful for experimenters who want to use a non-stressful maze paradigm to investigate spatial learning in their research.

Video: Place and Response Learning in the Open-field Tower Maze

Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes of interest allows for enhanced temporally- and regionally-specific control of gene deletion, compared to existing methods. While conditional deletion can also be achieved by mating mice that express Cre recombinase under the control of specific gene promoters with mice carrying a floxed gene, stereotaxic microinjection allows for targeting of discrete brain areas at experimenter-determined time points of interest. In the context of cocaine conditioned place preference, and other cocaine behavioral paradigms such as self-administration or psychomotor sensitization that can involve withdrawal, extinction and/or reinstatement phases, this technique is particularly useful in exploring the unique contribution of target genes to these distinct phases of behavioral models of cocaine-induced plasticity. Specifically, this technique allows for selective ablation of target genes during discrete phases of a behavior to test their contribution to the behavior across time. Ultimately, this understanding allows for more targeted therapeutics that are best able to address the most potent risk factors that present themselves during each phase of addictive behavior.

This article describes how to microinject viral vectors into mouse brain and then test in a conditioned place preference paradigm that includes an acquisition, extinction and reinstatement phase.

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing locomotive impairment in animal models of neuromuscular disease and efficacy of therapeutic drugs that may improve locomotion and/or muscle function. The open field activity measurement provides a different measure than muscle strength, which is commonly assessed by grip strength measurements. It can also show how drugs may affect other body systems as well when used with additional outcome measures. In addition, measures such as total distance traveled mirror the 6 min walk test, a clinical trial outcome measure. However, open field activity monitoring is also associated with significant challenges: Open field activity measurements vary according to animal strain, age, sex, and circadian rhythm. In addition, room temperature, humidity, lighting, noise, and even odor can affect assessment outcomes. Overall, this manuscript provides a well-tested and standardized open field activity SOP for preclinical trials in animal models of neuromuscular diseases. We provide a discussion of important considerations, typical results, data analysis, and detail the strengths and weaknesses of open field testing. In addition, we provide recommendations for optimal study design when using open field activity in a preclinical trial.

Open field activity levels are used to assess locomotive and behavioral activity levels. This protocol provides a well-designed, standardized protocol to use in preclinical trials for neuromuscular disorders.

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing ...

Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of the most commonly used platforms to measure behaviors in animal models. It is a fast and relatively easy test that provides a variety of behavioral information ranging from general ambulatory ability to data regarding the emotionality of the subject animal. As it relates to rodent models, the procedure allows the study of different strains of mice or rats both laboratory bred and wild-captured. The technique also readily lends itself to the investigation of different pharmacological compounds for anxiolytic or anxiogenic effects. Here, a protocol for use of the open field maze to describe mouse behaviors is detailed and a simple analysis of general locomotor ability and anxiety-related emotional behaviors between two strains of C57BL/6 mice is performed. Briefly, using the described protocol we show Wild Type mice exhibited significantly less anxiety related behaviors than did age-matched Knock Out mice while both strains exhibited similar ambulatory ability.

A protocol is provided to use an Open Field Maze to access general locomotor activity, anxiety and emotionality in a laboratory mouse model.

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of ...

The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their behavioral flexibility and required memory duration, the number of goals and pathways, and also the overall task complexity. A confounding feature in many of these tasks is the lack of control over the strategy employed by the rodents to reach the goal, e.g., allocentric (declarative-like) or egocentric (procedural) based strategies. The double-H maze is a novel water-escape memory task that addresses this issue, by allowing the experimenter to direct the type of strategy learned during the training period. The double-H maze is a transparent device, which consists of a central alleyway with three arms protruding on both sides, along with an escape platform submerged at the extremity of one of these arms.
Rats can be trained using an allocentric strategy by alternating the start position in the maze in an unpredictable manner (see protocol 1; &#167;4.7), thus requiring them to learn the location of the platform based on the available allothetic cues. Alternatively, an egocentric learning strategy (protocol 2; &#167;4.8) can be employed by releasing the rats from the same position during each trial, until they learn the procedural pattern required to reach the goal. This task has been proven to allow for the formation of stable memory traces.
Memory can be probed following the training period in a misleading probe trial, in which the starting position for the rats alternates. Following an egocentric learning paradigm, rats typically resort to an allocentric-based strategy, but only when their initial view on the extra-maze cues differs markedly from their original position. This task is ideally suited to explore the effects of drugs/perturbations on allocentric/egocentric memory performance, as well as the interactions between these two memory systems.

The goal of this protocol is to investigate spatial cognition in rodents. The double-H water maze is a novel test, which is particularly useful to elucidate the different components of learning, consolidation and memory, as well as the interplay of memory systems.

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their ...

Quantifying Learning in Young Infants: Tracking Leg Actions During a Discovery-learning Task

Task-specific actions emerge from spontaneous movement during infancy. It has been proposed that task-specific actions emerge through a discovery-learning process. Here a method is described in which 3-4 month old infants learn a task by discovery and their leg movements are captured to quantify the learning process. This discovery-learning task uses an infant activated mobile that rotates and plays music based on specified leg action of infants. Supine infants activate the mobile by moving their feet vertically across a virtual threshold. This paradigm is unique in that as infants independently discover that their leg actions activate the mobile, the infants&#8217; leg movements are tracked using a motion capture system allowing for the quantification of the learning process. Specifically, learning is quantified in terms of the duration of mobile activation, the position variance of the end effectors (feet) that activate the mobile, changes in hip-knee coordination patterns, and changes in hip and knee muscle torque. This information describes infant exploration and exploitation at the interplay of person and environmental constraints that support task-specific action. Subsequent research using this method can investigate how specific impairments of different populations of infants at risk for movement disorders influence the discovery-learning process for task-specific action.

A method is described in which 3-4 month old infants learn a task by discovery and their leg movements are captured to quantify the learning process.

Task-specific actions emerge from spontaneous movement during infancy. It has been proposed that task-specific actions emerge through a discovery-learning ...

Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been maintaining the behavior. There is close correspondence between fear and human anxiety, and therefore studies of extinction learning might provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder, and they might help to develop strategies to treat them. Preclinical research aims to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate the newly formed memory. Vagus nerve stimulation (VNS) is a powerful approach that provides tight temporal and circuit-specific release of neurotransmitters, resulting in modulation of neuronal networks engaged in an ongoing task. VNS enhances memory consolidation in both rats and humans, and pairing VNS with exposure to conditioned cues enhances the consolidation of extinction learning in rats. Here, we provide a detailed protocol for the preparation of custom-made parts and the surgical procedures required for VNS in rats. Using this protocol we show how VNS can facilitate the extinction of conditioned fear responses in an auditory fear conditioning task. In addition, we provide evidence that VNS modulates synaptic plasticity in the pathway between the infralimbic (IL) medial prefrontal cortex and the basolateral complex of the amygdala (BLA), which is involved in the expression and modulation of extinction memory.

Vagus nerve stimulation (VNS) has emerged as a tool to induce targeted synaptic plasticity in the forebrain to modify a range of behaviors. This protocol describes how to implement VNS to facilitate the consolidation of fear extinction memory.

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been ...

Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to drugs of abuse make lasting alterations in the brain function of vulnerable individuals, and these persistent alterations likely serve to maintain the maladaptive drug seeking and taking behaviors characteristic of addiction/dependence2. In rodents, reward experience and contextual associations are frequently measured using the conditioned place preference assay, or CPP, wherein preference for a previously drug-paired context is measured. Behavioral sensitization, on the other hand, is an increase in a drug-induced behavior that develops progressively over repeated exposures. Since sensitized behaviors can often be measured after several months of drug abstinence, depending on the dose and length of initial exposure, they are considered observable correlates of lasting drug-induced plasticity. Researchers have found these assays useful in determining the neurobiological substrates mediating aspects of addiction as well as assessing the potential of different interventions in disrupting these behaviors. This manuscript describes basic, effective protocols for mouse CPP and locomotor behavioral sensitization to cocaine.

This protocol is intended to enable researchers to conduct experiments designed to test these aspects of addiction using the conditioned place preference and locomotor behavioral sensitization assays.

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to ...

The "Motor" in Implicit Motor Sequence Learning: A Foot-stepping Serial Reaction Time Task

This protocol describes a modified serial reaction time (SRT) task used to study implicit motor sequence learning. Unlike the classic SRT task that involves finger-pressing movements while sitting, the modified SRT task requires participants to step with both feet while maintaining a standing posture. This stepping task necessitates whole body actions that impose postural challenges. The foot-stepping task complements the classic SRT task in several ways. The foot-stepping SRT task is a better proxy for the daily activities that require ongoing postural control, and thus may help us better understand sequence learning in real-life situations. In addition, response time serves as an indicator of sequence learning in the classic SRT task, but it is unclear whether response time, reaction time (RT) representing mental process, or movement time (MT) reflecting the movement itself, is a key player in motor sequence learning. The foot-stepping SRT task allows researchers to disentangle response time into RT and MT, which may clarify how motor planning and movement execution are involved in sequence learning. Lastly, postural control and cognition are interactively related, but little is known about how postural control interacts with learning motor sequences. With a motion capture system, the movement of the whole body (e.g., the center of mass (COM)) can be recorded. Such measures allow us to reveal the dynamic processes underlying discrete responses measured by RT and MT, and may aid in elucidating the relationship between postural control and the explicit and implicit processes involved in sequence learning. Details of the experimental set-up, procedure, and data processing are described. The representative data are adopted from one of our previous studies. Results are related to response time, RT, and MT, as well as the relationship between the anticipatory postural response and the explicit processes involved in implicit motor sequence learning.

We introduce the foot-stepping serial reaction time (SRT) task. This modified SRT task, complementing the classic SRT task that involves only finger-pressing movement, better approximates daily sequenced activities and allows researchers to study the dynamic processes underlying discrete response measures and disentangle the explicit process operating in implicit sequence learning.

This protocol describes a modified serial reaction time (SRT) task used to study implicit motor sequence learning. Unlike the classic SRT task that ...

Reinstatement of Drug-seeking in Mice Using the Conditioned Place Preference Paradigm

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to acquire a conditioned place preference in a drug-paired compartment, and after the post-conditioning test, they perform several sessions to extinguish the established preference. The CPP permits the evaluation of the conditioned rewarding effects of drugs related to environmental cues. Then, the extinguished CPP can be robustly reinstated by the non-contingent administration of a priming dose of the drug, and by exposure to stressful stimuli. Both methods will be explained here. When the animal reinitiates the behavioral response, a reinstatement of the conditioned reward is considered to have taken place.
The main advantages of this protocol are that it is non-invasive, inexpensive, and simple with good validity criteria. In addition, it allows the study of different environmental manipulations, such as stress or diet, which can modulate relapse into drug seeking behaviors. However, one limitation is that if the researcher aims to explore the motivation and primary reinforcing effects of the drug, it should be complemented with self-administration procedures, as they involve operant responses of animals.

This protocol describes the Conditioned Place Preference (CPP) as a model of relapse. This procedure permits the measurement of relapse in laboratory animals, considering the impact of drug-associated environmental cues as craving and relapse in abstaining addicts is currently the focus of drug-abuse treatment programs.

The present protocol describes the Conditioned Place Preference (CPP) as a model of relapse in drug addiction. In this model, animals are first trained to ...

Analyzing Spatial Learning and Prosocial Behavior in Mice Using the Barnes Maze and Damsel-in-Distress Paradigms

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. The Barnes maze can also assess other mouse behaviors, such as general motivation to escape from the maze platform and exploratory behavior. The Barnes maze can measure whether a genetic mutation or environmental variable can impact the acquisition and retention of spatial memories, as well as provide information about the search strategy employed by the mice. Here we use the Barnes maze to detect a memory deficit in adult mice following a single developmental ethanol exposure event. The newly described Damsel-in-Distress paradigm exposes a male mouse to a female mouse trapped in a chamber in the open center field of the arena. It provides an opportunity for the mouse to socially respond to the trapped female and exhibit prosocial behavior. The Damsel-in-Distress paradigm can also be used to examine mouse behavior in a novel arena and measure locomotor activity. Both the Barnes Maze and the Damsel-in-Distress protocols require minimal financial investment and most aspects of the tests can be constructed from common lab supplies. These flexible and accessible tools can also be used to detect behavioral changes over the course of development.

This protocol measures spatial learning and memory using the Barnes maze. A novel Damsel-in-Distress paradigm is used to assess locomotor activity and prosocial behavior in mice.

The Barnes maze is a reliable measure of spatial learning and memory that does not require food restriction or exposure to extremely stressful stimuli. ...