Name: reversible cooling-induced deactivations to study cortical contributions to obstacle memory in the walking cat
Uploaded: 2017-12-11T12:30:00.000+00:00
Duration: 9 min 43 s
Description: Watch this Scientific Journal Video about Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat at JoVE.com

We gratefully acknowledge the support of the Canadian Institutes of Health Research, Natural Science and Engineering Research Council of Canada (NSERC), and the Canada Foundation for Innovation. C.W. was supported by an Alexander Graham Bell Canada Graduate Scholarship (NSERC).

All procedures were conducted in compliance with the National Research Council&#39;s Guide for the Care and Use of Laboratory Animals (eighth edition; 2011) and the Canadian Council on Animal Care&#39;s Guide to the Care and Use of Experimental Animals (1993), and were approved by the University of Western Ontario Animal Use Subcommittee of the University Council on Animal Care.
The following procedure can be applied to experiments studying cortical contributions to locomotor control in the walking cat.
1. Apparatus
<ol>
	<li>Construct the apparatus used to assess obstacle memory. 
	NOTE: The apparatus consists of a 2.43 m long, 29 cm wide walkway enclosed by 18 cm high clear acrylic walls (Figure 1). A narrow slot halfway along the apparatus allows a 25.8 cm wide x 3 mm thick obstacle to be raised onto or removed from the walkway using a lever mounted underneath the walking surface.</li>
	<li>To ensure that attention of the animal is maintained on eating, avoid using the hand to raise or lower the obstacle. Instead, the obstacle can be raised or lowered using the experimenter&#39;s leg to move the lever underneath the walkway, allowing the experimenter to continue feeding the animal.</li>
	<li>Properly maintain the lever system to ensure that the obstacle can be raised or lowered soundlessly.</li>
	<li>Use a small elevated platform (23 cm long x 23 cm wide x 16 cm high) upon which soft food is placed, to guide movements of the animal.</li>
	<li>Record all trials using an ethernet camera (54 frames/s) mounted on a tripod 1.85 m away from the midline of the walkway.</li>
</ol>
2. Training Procedures
NOTE: &#160;For successful data acquisition, a period of training preceding behavioral testing ensures that each animal is properly acclimated to the testing room and apparatus. Repeated exposure to a novel environment will aid in reducing startling or other stressful behaviors.&#160;&#160;Acclimation may vary between animals and may require 1-2 months of training. Initial acclimation sessions may be up to 5 min in length depending on the focus and motivation of the animal to eat. Subsequent sessions should aim to increase the duration of time that the animal is motivated to work (typically around 20-25 min).
<ol>
	<li>Acquire mature ( &#62;6 months of age) domestic short hair cats from a commercial laboratory breeder of any weight or sex. 
	NOTE: Motivation to work for food and a cooperative disposition comprise the selection criteria when considering which animals should be included in the study.</li>
	<li>Acclimate each animal to wearing a harness to which a 1 m long leash is attached. Anchor the leash to a shelf above the walkway over the midpoint of the walkway. 
	NOTE: This allows the animal to walk along the central portion of the apparatus without any tension, thus encouraging the animal to remain within this portion of the apparatus. Establishing such boundaries is helpful for working with a moving test subject.</li>
	<li>Place the animal onto the walkway, allowing it to eat from the platform upon which soft food is placed. 
	NOTE: One aim of this initial training is to ensure that the animal readily follows the food platform when moved forwards, and can walk comfortably with the harness and leash.&#160;The use of soft food as positive reinforcement encourages the animal to remain focused throughout each training or testing session, and promotes a comfortable working environment.</li>
	<li>Ensure that the animal is comfortable with handling, including instances where the animal must be moved to the start area of the walkway.</li>
</ol>
3. Behavioral Training and Testing Protocol
NOTE: The obstacle memory is assessed in two paradigms: a visually-dependent obstacle memory task, and a tactile-dependent obstacle memory task. Both paradigms should be used during initial training and subsequent testing.
<ol>
	<li>Visual obstacle memory
	<ol>
		<li>To assess the visual obstacle memory, raise the obstacle onto the walkway (Figure 2A). Place the platform on the far side of the obstacle. Place the animal in the start area of the walkway.</li>
		<li>Allow the animal to approach the food, stepping over the obstacle with only its forelegs in order to eat from the platform.</li>
		<li>As the animal continues to eat, lower the obstacle such that it becomes flush with the walkway to prevent any further visual or tactile inputs.</li>
		<li>Following a variable delay period, move the food forwards again to encourage the animal to resume walking; this delay can be less than 1 s to upwards of 2 min.</li>
		<li>Importantly, perform trials where the obstacle is absent in order to prevent habituation to the obstacle and development of a learned avoidance response. In such visual obstacle-absent trials, ensure that the obstacle is not raised onto the walkway before placing the animal in the start area of the walkway.</li>
		<li>Observe hindleg stepping in obstacle-present and obstacle-absent trials to verify typical locomotor behaviors and intact visual obstacle memory prior to cooling. Ensure that the animal can clear the obstacle without contact, and that stepping of all four legs is significantly elevated in obstacle-present trials. 
		NOTE: Watching videos of training trials may assist in this verification.</li>
	</ol>
	</li>
	<li>Tactile obstacle memory
	<ol>
		<li>To assess the tactile obstacle memory, ensure that the obstacle is not raised onto the walkway before placing the animal in the start area of the walkway (Figure 2B).</li>
		<li>Allow the animal to walk towards the food platform placed on the far side of the obstacle slot.</li>
		<li>As the animal eats, raise the obstacle onto the walkway beneath the food dish, preventing any visual input of the obstacle.</li>
		<li>As the food is moved forwards, note that the animal should contact the obstacle with their forelegs before stepping over it.</li>
		<li>Allow the animal to continue eating while straddling the obstacle between their fore- and hindlegs. During this time, lower the obstacle so that it becomes flush with the walkway to prevent any further visual or tactile inputs.</li>
		<li>Following a variable delay period, move the food forwards once again to encourage the animal to resume walking.</li>
		<li>Importantly, perform trials where the obstacle is absent and no foreleg contact occurs for preventing habituation to the obstacle and development of a learned avoidance response.
		<ol>
			<li>In these tactile obstacle-absent trials, have the animal approach and eat from the food platform, as described in step 3.2.1. However, raise and lower the obstacle (step 3.2.2) before moving the food forward in step 3.2.3. Ensure that a similar delay period where the animal is allowed to continue eating (step 3.2.4) precedes the final continuation of locomotion (step 3.2.5).</li>
		</ol>
		</li>
		<li>Observe the hindleg stepping in the obstacle-present and obstacle-absent trials to verify normal locomotor behaviors and intact visual obstacle memory prior to cooling.</li>
	</ol>
	</li>
</ol>
4. Video Analyses
NOTE: To assess obstacle memory, analyses during initial training and subsequent testing after cooling loop implantation involve quantifying the peak step height, step clearance, and the horizontal distance between the toe and obstacle at the peak of each step for both visual and tactile paradigms (Figure 2C).
<ol>
	<li>Analyze the videos using custom written scripts.</li>
	<li>For every trial, track each foot by marking the position of the toe closest to the camera throughout each step.</li>
	<li>Measure the peak step height as the perpendicular distance between the toe and the surface of the walkway at the highest point in each step trajectory (Figure 2C).</li>
	<li>In the obstacle-present trials, measure the step clearance as the step height directly above the obstacle slot subtracted by the height of the obstacle.</li>
	<li>Additionally, measure the horizontal distance between the toe and the obstacle at the peak of each step in the obstacle-present trials.</li>
	<li>Confirm that the obstacle memory capabilities are intact prior to the cooling loop implantation by verifying that the peak step height is elevated in the obstacle-present trials in comparison to stepping in the obstacle-absent trials.</li>
</ol>
5. Cooling Loop (Cryoloop) Implantation
<ol>
	<li>Implant cryoloops bilaterally over areas 5 and 7 according to previously reported surgical procedures8 (Figure 3).</li>
	<li>In brief, for each hemisphere, perform a craniotomy and durotomy from Horsley-Clarke coordinates20 A15 to A25 to expose the juncture of the ansate and lateral sulci.</li>
	<li>Position individual cooling loops shaped from 23-gauge stainless steel hypodermic tubing with the loop in direct contact with the cortical surface of parietal area 5 or 7.</li>
	<li>Secure the base of each cryoloop to the skull with dental acrylic anchored to the stainless steel screws.</li>
	<li>Close the craniotomies with additional dental acrylic; draw up the skin margins up to the acrylic edges and suture together.</li>
</ol>
6. Cortical Cooling Protocol
<ol>
	<li>Experimental setup 
	NOTE: Before bringing the animal into the testing room, the cooling circuit is prepared and tested. The cooling circuit consists of a methanol reservoir with an intake tube (3.2 mm O.D., 1.6 mm I.D.), a reciprocating piston pump, and dry ice bath connected via polytetrafluoroethylene tubing (1.6 mm O.D., 0.5 mm I.D.; Figure 4). Additionally, a digital thermometer is required.
	<ol>
		<li>Add 500 cc dry ice to 200 mL of methanol in the ice bath. Fit tubing ends snugly over the inlet and outlet of a dummy cryoloop to complete the cooling circuit.</li>
		<li>Attach the thermocouple plug to a digital thermometer for continuous temperature monitoring using a cable composed of two male thermocouple connectors and a thermocouple wire. Ensure that the length of this cable is sufficient to reach the head of the animal when one end is plugged into the thermometer.</li>
		<li>Turn on the piston pump using the switch. 
		NOTE: Methanol should be drawn up from the reservoir, passed through the pump to the dry ice bath where the flowing methanol in the tubing will be cooled to -75 &#176;C. The chilled methanol will then exit the ice bath and run through the attached cryoloop before returning to the methanol reservoir.</li>
		<li>Ensure that the pump setting, length of tubing within the ice bath, and length of tubing from the ice bath to the dummy loops are optimal such that the dummy cryoloop temperature can reach a steady state around -5.0 &#176;C. 
		NOTE: Such temperatures achieved during this initial setup are often sufficient for achieving test temperatures of 3.0 &#177; 1.0 &#176;C when the same system is used to cool an implanted cryoloop. Difficulty in attaining sufficient cooling can be solved by adjusting the speed of the pump, increasing the length of tubing submerged within the ice bath, and/or minimizing the length of tubing from the ice bath to the cryoloop.</li>
		<li>If necessary, lengthen a section of tubing by threading the end of the tube through a tube end fitting and flange the end of the tube with a flanging tool. Attach tubing of a desired length with a similarly flanged end using a connector.</li>
		<li>Verify that all connections are snug and no leaks are present. Once satisfied with the initial setup, switch the pump off, and remove the dummy cryoloop; the circuit is now prepared for a test animal.</li>
	</ol>
	</li>
	<li>Behavioral testing
	<ol>
		<li>Place the animal on the testing apparatus. Slide the harness over the head and secure the strap snuggly around the animal. Attach the leash.</li>
		<li>Remove the protective cap of the implanted cryoloop to expose the inlet and outlet tubes. Fit tubing ends snugly over the inlet and outlet tubes of the cryoloop. Connect the thermocouple plug to the digital thermometer.</li>
		<li>Begin the testing session with a visual (step 3.1) or tactile (step 3.2) obstacle memory trial. Follow with additional trials of all four types (visual obstacle-present, visual obstacle-absent, tactile obstacle-present, tactile obstacle-absent) in a random fashion. 
		NOTE: A typical testing session consists of a &#39;warm&#39; block of trials, where memory-guided obstacle avoidance is observed in the absence of cooling to establish baseline measures.</li>
		<li>Switch on the piston pump, and wait for the cryoloop to reach a temperature of 3.0 &#177; 1.0 &#176;C (1-2 min). Then, run a &#39;cool&#39; block of trials after the piston pump has been switched on. During this block of trials, if needed, assess contributions of the cooled area to memory-guided. Ensure that the temperature of the cryoloop is maintained at 3.0 &#177; 1.0 &#176;C throughout the entire block. 
		NOTE: All four trial types should be randomly interspersed throughout the block.</li>
		<li>Run a final &#39;rewarm&#39; block of trials after the piston pump has been switched off, and the cryoloop has returned to its original temperature. 
		NOTE: Baseline stepping behavior is re-established during this block. Again, all four trial types should be randomly interspersed throughout the block.</li>
	</ol>
	</li>
	<li>Clean-up
	<ol>
		<li>When the behavioral testing is concluded, remove the tubing from inlet and outlet tubes. Be conscious of residual methanol that may drip from the tubing ends and may irritate the animal.</li>
		<li>Ensure that the protective cap is replaced. Remove the leash and harness before returning the animal to the colony. Trim the tubing ends (3-4 mm) using a tubing cutter to prevent leaky connections on the next testing day.</li>
	</ol>
	</li>
</ol>
7. Verifying the Extent of Cooling
<ol>
	<li>At the end of behavioral testing, confirm that the extent of deactivation is restricted to the region of cortex directly beneath each cryoloop using previously reported techniques8. 
	NOTE: This can be verified with thermocline mapping12 or with a thermal imaging camera13,14,19.</li>
</ol>

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The authors have nothing to disclose.

The described paradigm employs cooling-induced deactivations of discrete cortical areas using the cryoloop in order to study memory-guided obstacle locomotion in the cat. The visual and tactile obstacle memory paradigms are fairly simple for animals to execute as they exploit naturalistic locomotor behaviors that occur with minimal effort when an animal is motivated to follow a moving food source. Thus, the majority of the training period is devoted to acclimating the animal the testing room and cooling equipment. Most a...

On naturalistic, uneven terrain, sensory information about an obstacle, which can be acquired via vision or touch, can rapidly modify locomotion for avoidance. This careful coordination of stepping movements involves multiple cortical regions1,2. For example, areas of motor cortex3,4 and parietal cortex5,6,7 have been implicated during complex locomotor tasks such as obstacle avoidance. In quadrupedal animals, step modulations required for obstacle avoidance must extend to both the forelegs and hindlegs. If forward locomotion is delayed between the foreleg and hindleg obstacle clearance (which may arise as an animal treads carefully through a complex, naturalistic environment stalking prey), information about the obstacle maintained in the memory is used to guide the hindleg stepping over the obstacle once walking resumes.
Experimental techniques aimed to deactivate discrete cortical areas can be used to study cortical contributions to memory-guided obstacle locomotion. Cooling-induced cortical deactivation provides a reversible, reliable, and reproducible method for assessing cortical contributions to an overt behavior8. Cryoloops made from stainless steel tubing are shaped specific to the cortical area of interest, ensuring highly selective and discrete deactivation of loci. Once implanted, chilled methanol pumped through the lumen of a cryoloop cools the region of cortex directly beneath the loop to &#60;20 &#176;C. Below this critical temperature, synaptic transmission is inhibited in the region of the cortex directly beneath the loop. Such deactivation can be reversed simply by ceasing the flow of methanol. This method has been used to study cortical contributions to sensory processing and behaviors9,10,11,12,13,14,15,16,17, as well as the motor control of saccadic eye movements18 and memory-guided obstacle locomotion19.
The purpose of this protocol is to use reversible cooling-induced deactivations to assess the involvement of the parietal cortical areas for locomotor coordination in the cat. Specifically, memory-guided obstacle locomotion was examined with or without active parietal cortex. These methods have been used to successfully demonstrate the role of parietal area 5 in memory-guided obstacle avoidance in the walking cat19.

<table><tbody><tr><td>Camera</td><td>IDS Imaging Development Systems GmbH</td><td>Model: UI-5240CP-C-HQ</td><td></td></tr><tr><td>Intake tubing</td><td>Restek</td><td>25306</td><td>Unflanged end is submerged in the methanol reservoir while the flanged end is connected to the pump</td></tr><tr><td>Pump</td><td>Fluid Metering, Inc.</td><td>Model: QG 150</td><td></td></tr><tr><td>Nalgene Dewar vacuum flask</td><td>Sigma-Aldrich</td><td>F9401</td><td></td></tr><tr><td>Teflon tubing</td><td>Ezkem</td><td>A051754</td><td></td></tr><tr><td>Microprobe thermometer</td><td>Physitemp</td><td>Model: BAT-12</td><td></td></tr><tr><td>Flanged tube end fittings</td><td>Valco Instruments Co. Inc.</td><td>CF-1BK</td><td>Assorted colours available for colour coding. Packages include the same number of washers as fittings</td></tr><tr><td>Washers</td><td>Valco Instruments Co. Inc.</td><td>CF-W1</td><td>Extra washers</td></tr><tr><td>Flanging kit</td><td>Pro Liquid GmbH</td><td>201553</td><td></td></tr><tr><td>Tubing connector</td><td>Restek</td><td>25323</td><td></td></tr><tr><td>Tubing cutter</td><td>Restek</td><td>25069</td><td></td></tr><tr><td>Male thermocouple connector</td><td>Omega</td><td>SMPW-T-M</td><td>Used to make cable connection to thermometer</td></tr><tr><td>Thermocouple wire</td><td>Omega</td><td>PP-T-24S</td><td>Used to make cable connection to thermometer</td></tr><tr><td>MATLAB</td><td>MathWorks</td><td>n/a</td><td></td></tr></tbody></table>

reversible cooling-induced deactivations to study cortical contributions to obstacle memory in the walking cat

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For example, in the cat, visual information about an impending obstacle can modulate stepping for avoidance. Locomotor adaptation can also occur independent of vision, as sudden tactile inputs to the leg by an expected obstacle can modify the stepping of all four legs for avoidance. Such complex locomotor coordination involves supraspinal structures, such as the parietal cortex. This protocol describes the use of reversible, cooling-induced cortical deactivation to assess parietal cortex contributions to memory-guided obstacle locomotion in the cat. Small cooling loops, known as cryoloops, are specially shaped to deactivate discrete regions of interest to assess their contributions to an overt behavior. Such methods have been used to elucidate the role of parietal area 5 in memory-guided obstacle avoidance in the cat.

This protocol has been successfully used to examine parietal cortex contributions to obstacle memory in the walking cat19. In this study, cryoloops were implanted bilaterally over parietal areas 5 and 7 in three adult (&#62;6 months of age) female cats (Figure 5A). Animals were assessed in the tactile obstacle memory paradigm in the absence of cooling (warm, control condition), or when area 5 or 7 was bilaterally deactivated.
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Watch this Scientific Journal Video about Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat at JoVE.com

Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat

Complex locomotion in naturalistic environments requiring careful coordination of the limbs involves regions of the parietal cortex. The following protocol describes the use of reversible cooling-induced deactivation to demonstrate the role of parietal area 5 in memory-guided obstacle avoidance in the walking cat.

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For ...

reversible-cooling-induced-deactivations-to-study-cortical

Research

JoVE Journal

Behavior

6.9K Views.  University of Western Ontario. Complex locomotion in naturalistic environments requiring careful coordination of the limbs involves regions of the parietal cortex. The following protocol describes the use of reversible cooling-induced deactivation to demonstrate the role of parietal area 5 in memory-guided obstacle avoidance in the walking cat.

Video: Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat

Operant Procedures for Assessing Behavioral Flexibility in Rats

Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these processes is the ability to adjust behavior in response to changes in one&#8217;s environment (i.e., behavioral flexibility). These processes are essential to normal human behavior, and may be disrupted in diverse neuropsychiatric conditions, including schizophrenia, alcoholism, depression, stroke, and Alzheimer&#8217;s disease. Understanding of the neurobiology of executive functions has been greatly advanced by the availability of animal tasks for assessing discrete components of behavioral flexibility, particularly strategy shifting and reversal learning. While several types of tasks have been developed, most are non-automated, labor intensive, and allow testing of only one animal at a time. The recent development of automated, operant-based tasks for assessing behavioral flexibility streamlines testing, standardizes stimulus presentation and data recording, and dramatically improves throughput. Here, we describe automated strategy shifting and reversal tasks, using operant chambers controlled by custom written software programs. Using these tasks, we have shown that the medial prefrontal cortex governs strategy shifting but not reversal learning in the rat, similar to the dissociation observed in humans. Moreover, animals with a neonatal hippocampal lesion, a neurodevelopmental model of schizophrenia, are selectively impaired on the strategy shifting task but not the reversal task. The strategy shifting task also allows the identification of separate types of performance errors, each of which is attributable to distinct neural substrates. The availability of these automated tasks, and the evidence supporting the dissociable contributions of separate prefrontal areas, makes them particularly well-suited assays for the investigation of basic neurobiological processes as well as drug discovery and screening in disease models.

The ability to assess executive functions such as behavioral flexibility in rats is useful for investigating the neurobiology of cognition in both intact animals and disease models. Here we describe automated tasks for assessing strategy shifting and reversal learning, which are particularly sensitive to disruptions in prefrontal cortical networks.

Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these ...

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 Cognitive Decrements Caused by Cranial Radiotherapy

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For many patients afflicted with CNS and non-CNS malignancies, radiotherapy and chemotherapy offer the best options for disease control. These treatments however come at a cost, and nearly all cancer survivors (~11 million in the US alone as of 2006) incur some risk for developing cognitive dysfunction, with the most severe cases found in patients subjected to cranial radiotherapy (~200,000/yr) for the control of primary and metastatic brain tumors1. Particularly problematic are pediatric cases, whose long-term survival plagued with marked cognitive decrements results in significant socioeconomic burdens2. To date, there are still no satisfactory solutions to this significant clinical problem.
We have addressed this serious health concern using transplanted stem cells to combat radiation-induced cognitive decline in athymic rats subjected to cranial irradiation3. Details of the stereotaxic irradiation and the in vitro culturing and transplantation of human neural stem cells (hNSCs) can be found in our companion paper (Acharya et al., JoVE reference). Following irradiation and transplantation surgery, rats are then assessed for changes in cognition, grafted cell survival and expression of differentiation-specific markers 1 and 4-months after irradiation. To critically evaluate the success or failure of any potential intervention designed to ameliorate radiation-induced cognitive sequelae, a rigorous series of quantitative cognitive tasks must be performed. To accomplish this, we subject our animals to a suite of cognitive testing paradigms including novel place recognition, water maze, elevated plus maze and fear conditioning, in order to quantify hippocampal and non-hippocampal learning and memory. We have demonstrated the utility of these tests for quantifying specific types of cognitive decrements in irradiated animals, and used them to show that animals engrafted with hNSCs exhibit significant improvements in cognitive function3.
The cognitive benefits derived from engrafted human stem cells suggest that similar strategies may one day provide much needed clinical recourse to cancer survivors suffering from impaired cognition. Accordingly, we have provided written and visual documentation of the critical steps used in our cognitive testing paradigms to facilitate the translation of our promising results into the clinic.

Cognitive impairment resulting from the radiotherapeutic management of brain tumors represents a clinically intractable condition that adversely impacts quality of life. The capability to critically evaluate potential interventions for ameliorating radiation-induced cognitive decrements ultimately depends on the capability to undertake rigorous quantitative assessments of cognition.

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For ...

Medicine

Asymmetric Walkway: A Novel Behavioral Assay for Studying Asymmetric Locomotion

Behavioral assays are commonly used for the assessment of sensorimotor impairment in the central nervous system (CNS). The most sophisticated methods for quantifying locomotor deficits in rodents is to measure minute disturbances of unconstrained gait overground (e.g., manual BBB score or automated CatWalk). However, cortical inputs are not required for the generation of basic locomotion produced by the spinal central pattern generator (CPG). Thus, unconstrained walking tasks test locomotor deficits due to motor cortical impairment only indirectly. In this study, we propose a novel, precise foot-placement locomotor task that evaluates cortical inputs to the spinal CPG. An instrumented peg-way was used to impose symmetrical and asymmetrical locomotor tasks mimicking lateralized movement deficits. We demonstrate that shifts from equidistant inter-stride lengths of 20% produce changes in the forelimb stance phase characteristics during locomotion with preferred stride length. Furthermore, we propose that the asymmetric walkway allows for measurements of behavioral outcomes produced by cortical control signals. These measures are relevant for the assessment of impairment after cortical damage.

Here, we present a protocol to quantify precise stepping in rodents. Cortical and the spinal central pattern generator signals are required for precise foot-placement during obstructed locomotion. We report here the novel constrained walking task that directly examines precise stepping behavior.

Behavioral assays are commonly used for the assessment of sensorimotor impairment in the central nervous system (CNS). The most sophisticated methods for ...

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

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

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

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

Neuroscience

Utilizing the Modified T-Maze to Assess Functional Memory Outcomes After Cardiac Arrest

Background: Evaluating mild to moderate cognitive impairment in a global cerebral ischemia (i.e. cardiac arrest) model can be difficult due to poor locomotion after surgery. For example, rats who undergo surgical procedures and are subjected to the Morris water maze may not be able to swim, thus voiding the experiment.
New Method: We established a modified behavioral spontaneous alternation T-maze test. The major advantage of the modified T-maze protocol is its relatively simple design that is powerful enough to assess functional learning/memory after ischemia. Additionally, the data analysis is simple and straightforward. We used the T-maze to determine the rats&#39; learning/memory deficits both in the presence or absence of mild to moderate (6 min) asphyxial cardiac arrest (ACA). Rats have a natural tendency for exploration and will explore the alternate arms in the T-maze, whereas hippocampal-lesioned rats tend to adopt a side-preference resulting in decreased spontaneous alternation ratios, revealing the hippocampal-related functional learning/memory in the presence or absence of ACA.
Results: ACA groups have higher side-preference ratios and lower alternations as compared to control.
Comparison with Existing Method(s): The Morris water and Barnes maze are more prominent for assessing learning/memory function. However, the Morris water maze is more stressful than other mazes. The Barnes maze is widely used to measure reference (long-term) memory, while ACA-induced neurocognitive deficits are more closely related to working (short-term) memory.
Conclusions: We have developed a simple, yet effective strategy to delineate working (short-term) memory via the T-maze in our global cerebral ischemia model (ACA).

This protocol describes the use of a modified T-maze to evaluate functional learning/memory in asphyxia cardiac arrest-induced cerebral ischemia.

Background: Evaluating mild to moderate cognitive impairment in a global cerebral ischemia (i.e. cardiac arrest) model can be difficult due to poor ...

Assessment of Memory Function in Pilocarpine-induced Epileptic Mice

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal model of epilepsy, we generated pilocarpine-treated chronic epileptic mice. We present a protocol for three different behavioral tests using these epileptic mice: novel object location (NL), novel object recognition (NO), and pattern separation (PS) tests to evaluate learning and memory for places, objects, and contexts, respectively. We explain how to set the behavioral apparatus and provide experimental procedures for the NL, NO, and PS tests following an open field test that measures the animals&#8217; basal locomotor activities. We also describe the technical advantages of the NL, NO, and PS tests with respect to other behavioral tests for assessing memory function in epileptic mice. Finally, we discuss possible causes and solutions for epileptic mice failing to make 30 s of good contact with the objects during the familiarization sessions, which is a critical step for successful memory tests. Thus, this protocol provides detailed information about how to assess epilepsy-associated memory impairments using mice. The NL, NO, and PS tests are simple, efficient assays that are appropriate for the evaluation of different kinds of memory in epileptic mice.

This article presents experimental procedures for assessing memory impairments in pilocarpine-induced epileptic mice. This protocol can be used to study the pathophysiologic mechanisms of epilepsy-associated cognitive decline, which is one of the most common comorbidities in epilepsy.

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal ...

An Inescapable Cat Odor Exposure Protocol for Studying Innate and Contextual Threat Conditioning in Rats

Animals respond to threatening situations by exhibiting a number of defensive behaviors, including avoidance, freezing, and risk assessment. An animal model with an ethological approach offers a deeper insight into the biological mechanisms underlying threat responses. This paper describes a methodology to measure defensive behaviors toward both innate and learned aversive stimuli in rats. Animals were individually exposed to predator odor in an inescapable chamber to elicit a measurable, sustained, defensive state. The experimental design involved placing a rat in a familiar chamber for 10 min followed by exposure to cat odor for another 10 min in the same context. The next day, the rats were re-exposed for 10 min to the same context chamber where cat odor exposure occurred. Sessions were video-recorded and defensive behaviors were assessed on both days. 
The behavioral test was coupled with reversible functional inactivation and c-Fos immunohistochemistry techniques to determine the role of the interoceptive cortex in threat responses. Rats exposed to cat odor on the first day and re-exposed to the context chamber on the second day displayed higher levels of defensive behaviors, and that cat odor elicited a robust increase in the neural activity of the interoceptive cortex. Moreover, muscimol inactivation of the interoceptive cortex reduced the expression of defensive behaviors in response to cat odor and impaired contextual threat memory. These results show that this behavioral assay is a useful tool for studying neural mechanisms of defensive behaviors and may offer insight into mechanisms that mediate fear in humans and its related disorders.

Cat skin/fur odor exposure provides a reliable method to investigate neural circuits and mechanisms of defensive responses in rodents and may offer insight into mechanisms that mediate fear in humans. Here, we describe a protocol for investigating the role of the interoceptive cortex in threat responses in rats.

Animals respond to threatening situations by exhibiting a number of defensive behaviors, including avoidance, freezing, and risk assessment. An animal ...

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

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

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

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

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.

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 ...