This work was funded by a Wellcome Trust/Royal Society Henry Dale Fellowship (098400/Z/12/Z), a Medical Research Council (MRC) grant MC-UP-1201/1, a Wellcome Trust and Gatsby Charitable Foundation SWC Fellowship (to T.B.), MRC PhD Studentship (to D.E. and R.V) and a Boehringer Ingelheim Fonds PhD fellowship (to R.V). We thank Kostas Betsios for programming the data acquisition software, the LMB Mechanical and Electrical Workshops for building the experimental arena.

All experiments were performed under the UK Animals (Scientific Procedures) Act of 1986 (PPL 70/7652) following local ethical approval.
1. Setting up the Behavioral Apparatus
NOTE: The main component of the behavioral apparatus is a variant of the Barnes maze13 consisting of a white acrylic circular platform (92 cm in diameter) with 20 equidistant, 5 cm diameter, circular holes, located radially, 5 cm from the edge of the platform. 19 of these are to be closed with black plastic plug (5 mm deep), while the remaining hole leads to a black Perspex shelter (dimensions 15 x 5.8 x 4.7 cm) that mice can easily enter and exit. The central area of the arena consists of a fixed circular platform (22 cm in diameter) and the periphery is mounted on a frame that allows rotation over 360 degrees through a computer-controlled stepper motor. The platform is elevated 45 cm from the floor to prevent the mice from climbing down. See schematic drawing in Figure 1.
<ol>
	<li>Attach visual cues to the periphery of the platform (2D or 3D symbols with different shapes, colors and patterns), at least 2 cm away from the edge to prevent mice from reaching them. 
	NOTE: Examples of visual cues are plain blue squares of 262 cm2, or circles (diameter of 15.5 cm) filled with diagonal purple stripes.</li>
	<li>Add ~2 g of bedding from the mouse&#39;s home-cage to the inside of the shelter to serve as a local olfactory cue. Optionally, position a light emitting diode (LED) at the entrance of the shelter to provide a local visual landmark cue.</li>
	<li>To isolate the mouse from external, distal visual cues, assemble an octagonal or cylindrical wall, extending 40 cm above and 15 cm under the platform surface, around the arena, positioned 10 cm away from the edge of the platform.</li>
	<li>Position an ultrasound speaker 50 cm above the center of the platform, to deliver aversive auditory stimuli.</li>
	<li>Place a translucent screen 64 cm above the arena to back-project overhead aversive visual stimuli using a projector.</li>
	<li>The light provided by the projector will illuminate the behavioral apparatus; calibrate it so that there is 5-10 lx at the center of the platform surface. Place infrared lights and an infrared camera above the platform, with a long-pass filter (&#62;700 nm) to prevent projector flicker in the acquired video.</li>
	<li>Track the position of the mouse online, using either commercially available software or a suitable algorithm. For tracking black mice on a white arena, a simple thresholding operation followed by calculation of the mask centroid is appropriate.</li>
	<li>Ideally, place the behavioral apparatus inside a sound- and light-proof box, to prevent external cues from affecting the experiment.</li>
</ol>
2. Delivering Innately Aversive Stimuli
NOTE: The stimuli described below can be generated with a wide range of different software packages, including LabVIEW and Matlab.
<ol>
	<li>Visual stimulus
	<ol>
		<li>Project the stimuli on to the screen positioned 64 cm above the arena. The stimulus consists of a linearly expanding dark circle on a grey background22 (Weber contrast = -0.98, luminance = 7.95 cd/m2). 
		NOTE: The standard circle should ideally be centered directly above the position of the mouse, subtending a visual angle of 2.6&#176; at onset and expands linearly at 224&#176;/s over 200 ms to 47.4&#176;, at which it remains for 250 ms before offset.</li>
		<li>When delivering the visual stimulus, center it above the mouse. This can be achieved either by waiting until the mouse passes through a pre-defined region in the arena, or by tracking the mouse position online and using the tracking coordinates to define the center of the circle. 
		NOTE: Although we suggest projecting the visual stimulus directly above the mouse, defensive behaviors can also be elicited if the same stimulus is projected ahead of the mouse (while still parallel to the floor). In our experience, this is the case at least until 30 cm ahead of the mouse&#39;s position.</li>
	</ol>
	</li>
	<li>Auditory stimulus
	<ol>
		<li>Trigger a stimulus consisting of a train of three frequency-modulated up-sweeps from 17 to 20 kHz23 over 3 s, lasting 9 s in total. The sound pressure level (SPL), measured at the platform level, should be in a range between 73 and 78 dB.</li>
		<li>Connect a soundcard and an amplifier to the ultrasound speaker positioned above the arena to deliver the auditory stimulus.</li>
	</ol>
	</li>
</ol>
3. Animals:
<ol>
	<li>Use male or female C57BL/6J mice, between 6 and 24 weeks old. Maintain the animals on a 12 h light cycle with free access to chow and water.</li>
	<li>Ensure that the mice are single housed at least 72 h before the behavioral assay and test&#160;them during the light-phase of the light cycle. 
	NOTE: We find that the baseline exploratory behavior of single-housed mice is more comparable across animals than across group-housed mice. In addition, animals with surgical implants for neural activity recording or manipulation often need to be single housed, and it can thus be beneficial to have control datasets in the same housing conditions.</li>
</ol>
4. Standard Behavioral Assay:
<ol>
	<li>Meticulously clean the surface of the platform with 70% ethanol or acetic acid to remove unwanted olfactory cues. Rinse the shelter with water and wash it with 70% ethanol, after which it should be rinsed with water again to lessen the ethanol odor.</li>
	<li>Dry both the platform and the shelter thoroughly, as the mouse may avoid entering the shelter if it is wet.</li>
	<li>Randomize the location of the shelter in the platform for each trial by rotating the platform before the experiment. The shelter location will be anchored in the arena but will be rotated in relation to the behavioral apparatus enclosure.</li>
	<li>Bring the mouse to the experiment room in its home-cage and place the cage on top of the testing platform for a 10 min acclimatization period.</li>
	<li>Remove the mouse from its home-cage and avoid retrieving by the tail: either cup it or let it climb onto an enrichment item that can be lifted. Picking mice by the tail has been show to increase anxiety levels&#160;and thus may affect behavioral responses to threat24.</li>
	<li>Gently place the mouse in the center of the arena and start the video-acquisition.</li>
	<li>Allow for a 7 min habituation period, during which no aversive stimulus is presented. Ensure that the mouse has enough time to find and enter the shelter at least once. Consider it as an entry if all four limbs are inside the shelter. 7 min should be enough for the majority of the mice to find and enter the shelter, but in case this does not happen, extend the habituation period by additional periods of 5 min until at least one entrance has been verified.</li>
	<li>Trigger the delivery of the aversive stimulus (auditory or visual) either manually by observing the live video feed, or automatically by tracking the position of the animal online and pre-defining a region of interest in the arena for triggering stimulus-delivery once the animal reaches it. The visual stimulus is usually delivered directly above the animal using the online video tracking coordinates.</li>
	<li>To elicit more than one escape response in the same session, let the mouse voluntarily leave the shelter. If the mouse did not successfully escape to the shelter upon the last aversive stimulus, wait at least 60 s before presenting another stimulus.</li>
	<li>After the assay is finished, return the animal to its home-cage. In case further testing of the same mouse is required, wait at least 48 h before retesting.</li>
	<li>Clean the platform and the shelter as described in step 4.1 and 4.2 before testing the next animal.</li>
</ol>
NOTE: Steps 5, 6 and 7 are independent variations of the standard behavioral assay and each can be performed individually.
5. &#39;Navigation Strategy&#39; Behavioral Assay:
NOTE: The goal of this assay is to determine what cues mice use to guide escape behavior. In this assay, the movable outer portion of the platform is rotated while the mouse is on the fixed arena center, which radially displaces the shelter, the olfactory cues located inside the shelter and the proximal visual cues attached to the platform. If the mouse is following any of the cues that have been rotated in order to navigate to the shelter, it will correctly escape to the new location of the shelter, but if it uses any other cues that have remained in place, it will navigate to the previous shelter location.
<ol>
	<li>Perform steps 4.1 to 4.8 of the standard behavioral assay described above, eliciting at least one flight response to be used as baseline.</li>
	<li>Let the mouse actively leave the shelter and wait for it to navigate to the central part of the platform. Position a small dish with bedding in the center to encourage exploration and keep the animal in the central platform long enough to allow the rotation of the outer portion of the platform. Add the dish and bedding while the mouse is inside the shelter and after the successful escape that precedes the rotation of the platform. If the mouse fails to enter the shelter during the escape response, wait for it to voluntarily enter the shelter and then add the dish and bedding.</li>
	<li>Use the stepper motor to rotate the platform at least 36 degrees (2 holes) and then immediately use a sensory stimulus to elicit an escape response. It is possible that the rotation triggers a startle and even initiates an escape response. 
	NOTE: From our experience, ~55% of the mice will run outside of the central (non-rotating) platform while the external portion is still rotating, and these trials should be excluded from the analysis.</li>
	<li>Clean the platform and shelter, and return the mouse to its home-cage as described in step 4.10 and 4.11.</li>
</ol>
6. &#39;Dynamic Environment&#39; Behavioral Assay:
NOTE: This assay is designed to evaluate how mice adapt their defensive behaviors to sudden changes in the environment. In these experiments, after eliciting at least one escape response, the location of the shelter is changed and the mouse is allowed to visit it once before a subsequent escape response is triggered. If the mouse perfectly updates the new location of the shelter and computes the absence of the shelter in its previous location, it should escape to the new location. Otherwise, either it has not memorized the new location of the shelter or it has memorized both locations but prefers to escape to the old one.
<ol>
	<li>Perform this assay after steps 4.1 to 4.8 described above, eliciting at least one flight response as a control.</li>
	<li>Once the mouse voluntarily leaves the shelter, remove the shelter and re-position it in a new hole at the opposite end of the platform.</li>
	<li>Allow the mouse to explore the environment until it enters the shelter at its new location.</li>
	<li>When the mouse voluntarily comes out of the shelter, present it with an aversive stimulus and observe its response.</li>
	<li>Use the auditory stimulus for this experiment because of its longer duration, as under prolonged threat, animals may perform additional escape responses to other locations if they fail to find the shelter at first.</li>
	<li>Clean the platform and shelter and return the mouse to its home-cage as described in steps 4.10 and 4.11.</li>
</ol>
7. &#39;No Shelter&#39; Behavioral Assay:
NOTE: The aim of this assay is to understand how the absence of a shelter in the environment affects the expression of defensive behaviors.
<ol>
	<li>Plug all 20 holes. In this experiment, there is no shelter available in the arena.</li>
	<li>After cleaning and drying the platform (steps 4.1 and 4.2), carry out steps 4.4-4.6.</li>
	<li>Allow a 7 min habituation period.</li>
	<li>Present the visual stimulus and observe the defensive response. The lack of a shelter promotes freezing behavior, usually lasting for the duration of the visual stimulus. Use a slowly expanding spot (e.g.&#160;a circle that subtends a visual angle of 2.6&#176; at onset and expands linearly at 11.2&#176;/s over 4 s to 47.4&#176;, at which it remains for 1 s before offset) to facilitate the observation of freezing responses.</li>
	<li>If more than one response per mouse is required, use an inter-stimulus interval of at least 60 s.</li>
	<li>After obtaining the required number of responses in the absence of the shelter, introduce the shelter under one of the holes and let the mouse explore the environment for at least 5 min. 5 min will be enough time for most mice to find and enter the newly introduced shelter, but in case this does not happen, extend this period by additional 5 min blocks until at least one entry is verified.</li>
	<li>Repeat steps 4.8 and 4.9, and observe escape responses performed in the presence of a shelter in the environment.</li>
	<li>Clean the platform and shelter and return the mouse to its home-cage as described in steps 4.10 and 4.11.</li>
</ol>
8. Data Analysis:
NOTE: Each of the following data analysis steps can be performed independently.
<ol>
	<li>Escape responses can be easily identified as a sudden acceleration following the presentation of an aversive stimulus. Quantify the latency to escape as the time between stimulus presentation and either the beginning of a head rotation movement directed at the target of the escape or the beginning of the acceleration movement, whichever comes first.</li>
	<li>Freezing responses can be characterized by total immobility of the animal except for breathing movements25, lasting at least 0.5 s7. Measure the latency to freeze as the time between the presentation of the aversive stimulus and the beginning of the immobility period.</li>
	<li>For each escape response, quantify the linearity of the flight path by calculating the ratio of the distance between the mouse and the shelter and the actual displacement during the flight.</li>
	<li>For each escape response, calculate the accuracy by measuring the distance between the hole where the animal directed its flight to and the hole leading to the shelter. Since there are 20 holes, each hole off target represents a decrement of 10% in accuracy (e.g., 2 holes off target can be expressed as 80% accuracy)</li>
	<li>For the &#39;navigation strategy&#39; behavioral assay, quantify the accuracy of the response relative to the shelter location before the platform rotation. An average accuracy that is not significantly different from the pre-rotation accuracy indicates that the animal is not using any of the sensory cues that were rotated in order to navigate to the escape target.</li>
	<li>For the &#39;dynamic environment&#39; behavioral assay, classify each escape response elicited as being directed towards the new or the old shelter location, or to neither of them. Evaluate how the pattern evolves along the iterations of the elicited defensive behaviors.</li>
	<li>For the &#39;no shelter&#39; behavioral assay, compare the mouse&#39;s speed following the presentation of aversive visual stimulus in the presence and absence of a shelter and calculate the latency to the initiation of the defensive response. Additionally, calculate the probability of triggering a freezing response in response to the stimulus for each situation.</li>
</ol>

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Most of the data included in the results section is a subset of the data presented in Vale et. al 2017.

The assays we describe here are technically straightforward to perform, and with the exception of the platform rotation experiments, can be readily implemented in a standard Barnes maze. Nevertheless, a few complications may arise: on the one hand, the mouse may seem afraid of entering the shelter, which may be due to&#160;the shelter not being sufficiently cleaned or dried. On the other hand, the mouse may stay inside the shelter for very long periods of time. It is critical for these experiments that animals are never removed from the shelter manually, as this can disrupt path integration as well as distress the animals and change their response to threat. We suggest terminating the experiment after 90 min and retesting after 48 h if necessary. Another relevant practical consideration is that when the mouse investigates the edge of the platform, it may not detect visual stimuli if its head is angled over the edge, and thus we recommend withholding the stimuli in such situations. Additionally, mice may jump down off the platform on rare occasions. We suggest ending the experiment and retesting after 48 h. Mice that actively leave the platform once are likely to do it again in the future and may have to be excluded from the study. Finally, from our experience, most mice will respond to the visual stimulus (34/36 mice). However, if the mice do not show any type of response to the visual stimulus (either a startle, a flight or a freezing response), they should be excluded from the study.
An important point to consider is that the control of sensory cues is typically a problem in spatial memory assays, as often it is difficult to eliminate all possible sources of contaminating cues. Our experimental setup reduces external visual cues by surrounding the maze with a wall, and it is placed inside a sound-dampening cabinet for sound isolation. To minimize olfactory cues, we recommend thoroughly cleaning the set-up with 70% ethanol or acetic acid between experiments.
Our behavioral assay adds to previous methods for studying spatial navigation26 by using instinctive defensive behaviors to probe spatial memory and focusing on ethological scenarios. A critical difference between the procedures described here and the standard BM assay is the lack of training sessions. In our assays, the habituation period ensures that the mouse visits the shelter at least once and often many more times, which we have previously shown to be sufficient to memorize the shelter location2. We believe that an important reason for the high success rate and accuracy in finding the shelter despite the lack of training is never passively displacing the animal during the experiment, thus making path integration a viable navigation strategy. We note however that our assay deals with memory formed and assessed during a single session, and that we have not tested for long-term memory of shelter location, which is usually the objective of standard BM experiments. Finally, by using discrete innately aversive stimuli rather than the commonly used fan or buzz for the duration of the session, our assay provides very good experimental control over two behaviors that might use different navigation strategies: foraging and shelter-directed escape. We believe that the use of these assays in combination with modern neuroscience techniques for recording and manipulating neural activity can provide important insights into how neural circuits compute behavior.

Instinctive defensive behaviors are largely considered to be hardwired stimulus-responses, such as the C-start movement in fish and amphibians, which moves the animal away from a threat source1. However, defensive behaviors can be more adaptive if they flexibly take into account&#160;information learned about the current environment. One example of such flexibility is the switch from escape to freezing shown by rodents when faced with threat, depending on prior knowledge about the presence or absence of a shelter in the environment2,3. Other examples of flexibility in innate behaviors include adapting the flight initiation threshold or escape speed depending on the distance between a prey and its shelter4, distance to threat5,6, and previous experience11,12, as well selecting different defensive strategies depending on the sensory properties of the aversive stimulus7, or even suppressing defensive behavior in face of competing motivations such as hunger8,9,10. The dependency of action selection on acquired knowledge about the spatial features of the environment makes instinctive defensive behaviors in mice a powerful model for studying goal selection, spatial memory and navigation. Here, we describe the adaptation of a commonly used behavioral task, the Barnes maze (BM) spatial memory assay13, to determine the influence of the spatial environment on defensive action selection in mice, and their navigation strategies when escaping towards a shelter.
The standard Barnes maze used to study learning and spatial memory in mice consists of a circular ~90 cm diameter platform with 20 equally spaced holes, of which 19 are closed, and one leads to an underground shelter&#160;which mice seek in order to avoid the open-field environment of the platform. Often, weak aversive stimuli (buzzer, bright light, fan) are used continuously throughout the assay to make the environment aversive and thus promote entry into the shelter14. In the most commonly used assay15,16, the animal has one habituation trial where it is guided to the shelter manually by the experimenter immediately after being positioned on the platform. This is followed by a 4-day acquisition period, where each day the mouse is allowed to navigate freely in the maze for 3 min, after which it is again manually guided to the shelter if it does not reach it during the exploration period. The last stage of the assay is one probe trial on the 5th day (although a long-term seven-day memory trial is also commonly performed) when the animal explores the maze with all holes closed. Learning and long-term memory are quantified by the time taken to find the shelter and pokes in the wrong holes during the acquisition period, and by the time spent near the closed shelter hole in the probe trial. Typical results show a decrease in the number of errors and latency to reach the shelter during acquisition, and an above-chance proportion of time spent in the quadrant containing the closed target hole in the probe trial15.
While several variants of the BM assay have been previously described17,18,19, the paradigm we describe here has three fundamental changes from the standard assay. First, the animal is left to explore the maze and find the shelter on its own, and testing is done in the same session, shortly after the shelter has been found, during periods where the animal is engaged in exploratory behavior. Although this setting does not test long-term memory of the shelter location, it is designed to be a naturalistic scenario that mimics the exploration of novel territory, under the threat of predation. In addition, it allows testing of how animals adapt to acute changes in the environment, such as sudden changes in landscape. Second, a key aspect of our assay is that the experimenter never forces the animal in or out of the shelter, which can disorient the animal and exclude path integration20 as a viable navigation strategy21. Path integration is a navigation strategy that uses self-motion cues, such as proprioceptive and vestibular cues, arising from the integration of motor outflow, to update the current position of the animal and navigate towards a goal, which is not possible if the animal is passively moved by the experimenter. Third, we use innately aversive visual22 and auditory23 stimuli to elicit escape to shelter, which is easily distinguished from ongoing foraging behavior and allows the assessment and quantification of specific navigation strategies used during defense from imminent threats. We propose that this assay will be useful for dissecting the role of spatial memory in the selection and implementation of defensive behaviors, and more generally to the broader study of goal-directed navigation and short-term spatial memory. The protocols described here were introduced by Vale et al. in 2017, to which we refer the readers for more in depth discussion of the rationale for experiments and results.

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a behavioral assay for investigating the role of spatial memory during instinctive defense in mice

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often stereotyped actions elicited in response to innately aversive sensory stimuli, but their success requires enough flexibility for adapting to different spatial environments, which can change rapidly. Here, we describe a behavioral assay to evaluate the influence of learned spatial knowledge on defensive behaviors in mice. We have adapted the widely used Barnes maze spatial memory assay to investigate how mice navigate to a shelter during escape responses to innately aversive sensory stimuli in a novel environment, and how they adapt to acute changes in the environment. This new assay is an ethological paradigm that does not require training and exploits the natural exploration patterns and navigation strategies in mice. We propose that the set of protocols described here are a powerful means of studying goal-directed behaviors and stimulus-triggered navigation, which should be of interest to both the fields of instinctive behaviors and spatial memory.

Mice exposed to auditory or visual stimuli initiated fast escape responses with short latencies between the onset of stimulation and initiation of flight. The mean latency to escape from the visual stimulus was 202 &#177; 16 ms (n = 51 responses from 26 animals) and significantly longer for auditory stimulation: 510 &#177; 61 ms (n = 36 responses from 15 animals, p&#60;0.0001 t-test between visual and auditory stimulation; Figure 2A) (step 8.1). Escape responses were accurately directed to the shelter (mean accuracy for visual stimulus: 97.2 &#177; 1.4% and 95.0 &#177; 1.4% for auditory stimulus, not significantly different between the two types of stimuli, p = 0.1655 t-test; Figure 2B) (step 8.4), and flight trajectories were close to a straight line (mean ratio of displacement to distance to shelter for visual stimulus 107.3 &#177; 1.3% and 113.9 &#177; 1.5% for auditory stimulus; Figure 2C) (step 8.3). Observing a short latency from the stimulus is important to distinguish stimulus-evoked behavior from homing runs, and the high linearity of the flight trajectory demonstrates that the escape response has the goal of reaching the shelter, and not simply moving away from the stimulus.
Stimulus presentation after the rotation of the outside part of the maze while the mouse was stationary in the center platform (range = 36&#176;-90&#176;, mean 56&#176;) successfully elicited escape responses (step 5). All mice fled towards the previous location of the shelter, with no decrease in accuracy or linearity of flight trajectory in comparison to the trials before rotation. The mean accuracy, measured in reference to the original shelter position, was 96.3 &#177; 1.3% before rotation (n = 25 responses from 8 mice) and even slightly higher after rotation (100 &#177; 0%, p = 0.009 t-test between pre and post rotation). The mean linearity in relation to the original shelter location was 109.4 &#177; 5.0% pre-rotation (25 responses from 8 mice) and 109.1 &#177; 2.1%, post-rotation (n = 8 responses from 8 mice), p = 0.957 t-test between pre and post-rotation, Figure 3A and B). These results demonstrate that proximal visual cues are not used to guide the flight to the shelter (steps 8.3-8.5).
Changing the location of the shelter after eliciting one successful escape response (step 6) resulted in mice visiting the new shelter location shortly after the change (mean time to visit the new shelter = 33.1 s, range = 4-82 s). Subsequent sequential trials of sound stimulation produced a gradual increase in the probability of escaping to the new location (44.4% on the first trial), reaching 100% across all animals by the fifth trial (n = 9 animals, Figure 3C). These experiments probe the dynamics of updating the shelter location memory to adapt to changes in the environment (step 8.6).
After a 7 min acclimatization period to the maze with no shelter (step 7), the presentation of a 5 s long visual stimulus produced freezing behavior instead of escape (freezing probability = 95.2%, mean freezing time = 7.9 &#177; 2.7 s, n =7 animals). Subsequent introduction of a shelter in the same session and the presentation of the long visual stimulus elicited escape responses reliably, showing that&#160;mice can flexibly switch defensive strategies depending on the presence of refuge (Figure 3D) (steps 8.1 and 8.7).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56988/56988fig1.jpg" /> 
Figure 1: Schematic of the behavioral arena. Drawing of the main components of the behavioral arena viewed from the top (right) and from the side (left). A large fraction of the platform can be rotated, including the shelter and the attached visual cues. Dark circles in the top view are the holes to which the shelter can be attached to, which are all identical (an example shelter location is indicated). The entire arena is enclosed in a sound-dampening cabinet. <a href="/files/ftp_upload/56988/56988fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56988/56988fig2.jpg" /> 
Figure 2: Escape responses are fast and accurate goal-directed behaviors. (A) Latency to escape onset from the onset of visual or auditory stimulation. (B) Accuracy of navigation to the shelter during escape, measured such that the value is 100% if the mouse runs directly to the shelter, and 10% less for each hole away from the shelter (e.g., 80% if the hole reached first was two holes away from the correct one). (C) Displacement of the mouse during escape plotted against the linear distance between the animal&#39;s initial position and the target location, showing that flight is close to linear. Box and whiskers show data from 10-90 percentile and the remaining data points are shown as circles. Red color plots are data from auditory stimulation (n = 36 responses from 15 animals) and black are from visual stimulation (n = 51 responses from 26 animals). <a href="/files/ftp_upload/56988/56988fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56988/56988fig3.jpg" /> 
Figure 3: Mice do not use proximal visual cues or shelter-related cues to guide escape responses. (A) Displacement plot as in Figure 2C, for the responses pre- and post-rotation of the platform. (B) Accuracy of navigation during escape in pre- and post-rotation conditions. Post-rotation accuracy is measured in reference to the initial location of the shelter, and shows that the mice navigate back to the original shelter location instead the new position. (C) Fraction of escape responses directed towards the new shelter location plotted against stimulation number. After the 5th presentation of the stimulus, all animals escape directly towards the new shelter location (n = 9 animals). (D) Mean speed across the animals in response to visual stimulation with a shelter available (red) or absent (blue), showing escape and freezing responses, respectively. Speed has been normalized to baseline and aligned to reaction time (defined as either the beginning of a flight or freezing response, dashed line). Shaded areas show SEM. (n = 7 animals). For 2A and 2B, the box and whiskers show data from 10-90 percentile and the remaining data points are shown as circles. Red color plots are data from auditory stimulation before platform rotation (n = 25 responses from 8 animals) and blue are from responses after rotating the platform (n = 8 responses from 8 animals). <a href="/files/ftp_upload/56988/56988fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice at JoVE.com

A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

Evolution has selected a repertoire of defensive behaviors that are essential for survival across all animal species. These behaviors are often ...

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9.4K Views.  UCL Sainsbury Wellcome Centre for Neural Circuits and Behaviour. This protocol describes a behavioral assay, based on the Barnes maze test, to study how instinctive defensive actions are modified by the knowledge of spatial environment.

Video: A Behavioral Assay for Investigating the Role of Spatial Memory During Instinctive Defense in Mice

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.
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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.

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Assessing Spatial Memory Impairment in a Mouse Model of Traumatic Brain Injury Using a Radial Water Tread Maze

Despite the recent increase in use of mouse models in scientific research, researchers continue to use cognitive tasks that were originally designed and validated for rat use. The Radial Water Tread (RWT) maze test of spatial memory (designed specifically for mice and requiring no swimming) has been shown previously to successfully distinguish between controlled cortical impact-induced TBI mice and sham controls. Here, a detailed protocol for this task is presented. The RWT maze capitalizes on the natural tendency of mice to avoid open areas in favor of hugging the sides of an apparatus (thigmotaxis). The walls of the maze are lined with nine escape holes placed above the floor of the apparatus, and mice are trained to use visual cues to locate the escape hole that leads out of the maze. The maze is filled with an inch of cold water, sufficient to motivate escape but not deep enough to require that the mouse swim. The acquisition period takes only four training days, with a test of memory retention on day five and a long-term memory test on day 12. The results reported here suggest that the RWT maze is a feasible alternative to rat-validated, swimming-based cognitive tests in the assessment of spatial memory deficits in mouse models of TBI.

Here we present a protocol for a mouse-specific test of cognition that does not require swimming. This test can be used to successfully distinguish controlled cortical impact-induced traumatic brain injury mice from sham controls.

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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. ...

Automated, Long-term Behavioral Assay for Cognitive Functions in Multiple Genetic Models of Alzheimer's Disease, Using IntelliCage

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive decline, such as Alzheimer&#39;s disease (AD), have become a promising tool to elucidate the underlying mechanisms and promote the therapeutic advances. An important step is the validation and characterization of expected behavioral abnormality in the models, in the case of AD, cognitive decline. The long-term behavioral investigations of laboratory animals to study the effect of aging demand substantial efforts from researchers. The IntelliCage system is a high-throughput and cost-effective test battery for mice that eliminates the need for daily human handling. Here, we describe how the system is utilized in the long-term phenotyping of a genetic Alzheimer&#39;s disease model, specifically focusing on the cognitive functions. The experiment employs repeated battery of tests that assess spatial learning and executive functions. This cost-effective age-dependent phenotyping allows us to identify the transient and/or permanent effects of genes on various cognitive aspects.

This paper describes a protocol for cognitive assessments for genetic models of the Alzheimer&#39;s disease using the IntelliCage system, which is a high throughput automated behavioral monitoring system with operant conditioning.

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Novel Object Recognition and Object Location Behavioral Testing in Mice on a Budget

Ethologically relevant behavioral testing is a critical component of any study that uses mouse models to study the cognitive effects of various physiological or pathological changes. The object location task (OLT) and the novel object recognition task (NORT) are two effective behavioral tasks commonly used to reveal the function and relative health of specific brain regions involved in memory. While both of these tests exploit the inherent preference of mice for the novelty to reveal memory for previously encountered objects, the OLT primarily evaluates spatial learning, which relies heavily on hippocampal activity. The NORT, in contrast, evaluates non-spatial learning of object identity, which relies on multiple brain regions. Both tasks require an open-field-testing arena, objects with equivalent intrinsic value to mice, appropriate environmental cues, and video recording equipment and the software. Commercially available systems, while convenient, can be costly. This manuscript details a simple, cost-effective method for building the arenas and setting up the equipment necessary to perform the OLT and NORT. Furthermore, the manuscript describes an efficient testing protocol that incorporates both OLT and NORT and provides typical methods for data acquisition and analysis, as well as representative results. Successful completion of these tests can provide valuable insight into the memory function of various mouse model systems and appraise the underlying neural regions that support these functions.

Here we provide a protocol which includes comprehensive instructions for the economical establishment of murine object location and novel object recognition behavioral testing, including the design, cost, and construction of required equipment as well as execution of behavioral testing, data collection, and analysis.

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A Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and dysfunctions in mood and cognition are common and often shared symptoms in neurodegenerative diseases. Shown here is a behavioral test battery for motor, mood, and cognition, which can be repeatedly tested in a longitudinal study. This battery assesses the overall behavioral phenotype in mice by examining each domain of behavior with at least two independent well-accepted tests (i.e., open-field test and rotarod test for motor function, social interaction test, elevated plus maze test, and forced swim test for emotional function, and Morris water maze test and novel object recognition test for cognitive function). Therefore, this sensitive and comprehensive test battery is a powerful tool for the study of behavioral alternation in neurodegeneration.

A comprehensive behavioral test battery of motor skills, mood&#8212;including social interaction, depression, and anxiety&#8212;and cognition is designed for the repeated assessment of neurodegeneration-related behavioral changes in mice.

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Nest Building Behavior as an Early Indicator of Behavioral Deficits in Mice

Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents synthetic and/or natural materials (such as twine, tissue, cotton, paper, and hay) as a gauge of their overall well-being and as an ancillary assessment to predict the possible decline in cognition. Typically, changes in nesting behaviors, such as failure to create a nest, indicate a change in health or welfare. In addition, nesting behavior is sensitive to many environmental and physiological challenges, as well as many genetic mutations underlying pathological disease states. The following protocol describes a nesting behavior paradigm that explores the usage of four types of nesting material. In addition, the protocol utilizes intraclass correlations to demonstrate that inter-rater reliability is higher when nests are constructed out of shredded paper compared to other common nesting materials such as cotton squares, paper twists, and soft cob bedding. The chosen methodology and statistical considerations (i.e., intraclass correlation) for this assay may be of interest for those conducting experiments assessing the quality of living of mice.&#160;

Here, we present a protocol to quantify nest building behavior in mice, which is known to be impaired in several neurological disorders and diseases. This protocol examines the utility of four materials and offers the opportunity to quantify the rater agreement in scoring, improving the validity and reliability of the assay.

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Studying Active Defensive Behaviors in Mice with Modified Fear Conditioning

Modified Fear Conditioning for Inducing Flight Behaviors in Mice

The appropriate manifestation of defensive behavior in a threatening situation is critical for survival. The prevailing theory suggests that an active defensive behavior, such as jumping or rapid darting, is expressed under high threat imminence or actual threat, whereas passive defensive behavior, such as freezing, is expressed when the threat is predicted, but the threat imminence is relatively low. In classical fear conditioning, subjects typically exhibit freezing as a conditioned defensive response, with little expression of active defensive behavior in most cases. Here, we introduce a modified fear conditioning procedure for mice to observe the transition from freezing to flight and vice versa, involving five repetitive pairings of conditioned stimuli (CS; continuous tone, 8 kHz, 95 dB SPL (sound pressure levels)) and unconditioned stimuli (US; foot shock, 0.9 mA, 1.0 s) over two days. This modified fear conditioning procedure requires a relatively large number of conditioning sessions and conditioning days but does not necessitate a high-intensity foot shock for modest expression of flight behavior. Using the same context for conditioning and salient CS presentations is essential to elicit flight behaviors. This modified fear conditioning procedure is a reliable method for observing active defensive behaviors in mice, providing an opportunity to elucidate the fine mechanisms and characteristics of such behaviors in a fearful context.

Author Spotlight: Exploring Neural Correlates of Defensive Behaviors in Fear Learning and Extinction

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Understanding Spatial Learning in Mice Using the Active Place Avoidance Test

The Active Place Avoidance (APA) Test, an Effective, Versatile and Repeatable Spatial Learning Task for Mice

Hippocampus-dependent spatial learning in rodents has been tested using a variety of methods. These include the Morris water maze (MWM), Y-maze, and novel object location (NOL) tasks. More recently, the active place avoidance (APA) task has been developed as an alternative to these more traditional approaches. In the APA task, mice must use spatial cues placed around a rotating arena to avoid a stationary shock zone. Due to the multiple parameters that can be adjusted, the APA task has been demonstrated to be a very versatile approach. It lends itself to being used longitudinally and repeatedly for the same cohort of mice. Here, we provide a detailed protocol to successfully conduct the APA task. We also highlight alternative APA approaches that can be used to examine different components of spatial learning. We describe the data collection and analysis processes. Critical steps during the APA task are discussed to increase the likelihood of successfully conducting the test. The APA task has several advantages over more traditional spatial navigation tests. It is appropriate to use with aged mice or those with disease phenotypes such as Alzheimer's disease. The complexity of the task can be easily altered, allowing a wide range of mouse strains to be tested. Further, the APA task is suitable for testing animals that have undergone surgery or experimental interventions that may have affected motor or neural function, such as stroke or traumatic brain injury.

Author Spotlight: Investigating the Impact of Aging on Hippocampal-Dependent Spatial Learning

Here, we present a protocol for the active place avoidance test, a hippocampus-dependent spatial learning paradigm designed for rodents. Altering key parameters allows for re-testing of animals before and after treatments or over time.

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Spatial Memory Testing Using Mazes

Spatial learning and memory are neurological functions that allow us to remember important details associated with our environment. Scientists test this phenomenon in rodents using different types of mazes like Morris water maze, Radial water maze, and Barnes maze. By investigating spatial memory in rodents, neurobehavioral scientists can gain valuable understanding of how these processes are altered following brain damage in humans.
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Spatial Memory Tests Using Morris Water, Radial Water &amp; Barnes Mazes

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