Name: reversible cooling-induced deactivations to study cortical contributions to obstacle memory in the walking cat
Uploaded: 2017-12-11T12:30:00
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 wa...

<ol>
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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 ...

The overall goal of the use of cooling-induced deactivations in this study is to assess cortical contributions to obstacle memory in the walking cat. This method can help answer key questions in the field of locomotor control, such as whether the parietal cortex is involved in memory-guided obstacle avoidance. The main advantage of this technique is the ability to reverse the deactivation.This allows each animal to serve as its own control. To begin, construct the apparatus shown here as described in the accompanying text protocol. Next, place the animal onto the testing apparatus while wearing the testing harness, to acclimate the animal to the setup.This leash orientation 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 when working with a moving test subject. Place soft food onto the food platform and then set the animal onto the walkway, allowing it to eat from the platform.Ensure that the animal is comfortable with handling, including instances where the animal must be moved to the start area of the walkway. In order to assess visual obstacle memory, raise the obstacle onto the walkway and place the platform on the far side of the obstacle. Then set the animal at the start area of the walkway and allow the animal to approach the food, stepping over the obstacle with only its forelegs in order to eat from the platform.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. Following a variable delay period, move the food forwards again to encourage the animal to resume walking. Also perform trials where the obstacle is absent in order to prevent habituation to the obstacle and development of a learned avoidance response.Observe hind-leg 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 in comparison to obstacle-absent trials. To assess tactile obstacle memory, begin by ensuring that the obstacle is not raised onto the walkway.Then place the animal at the start of the walkway and allow it to walk towards the food platform placed on the far side of the obstacle slot. As the animal eats, raise the obstacle onto the walkway beneath the food dish, preventing any visual input of the obstacle. Then move the food forward.As the food is moved forwards, the animal should contact the obstacle with their forelegs before stepping over it. Allow the animal to continue eating while straddling the obstacle between their fore and hind legs. During this time, lower the obstacle so that it becomes flush with the walkway to prevent any further visual or tactile inputs.Following a variable delay period, move the food forwards once again to encourage the animal to resume walking. In tactile obstacle-absent trials, have the animal approach and eat from the food platform. However, raise and lower the obstacle before moving the food forward with a variable delay.Perform trials where the obstacle is absent and no foreleg contact occurs, to prevent habituation to the presence of the obstacle or the development of a learned avoidance response. Observe the hind-leg stepping in the obstacle-present and obstacle-absent trials to verify normal locomotor behaviors and intact obstacle memory prior to beginning cortical cooling. To deactivate discrete regions of the brain and to assess their contributions to memory-guided obstacle avoidance, implant CryoLoops bilaterally over areas five and seven according to previously reported surgical procedures as described in the accompanying text protocol.Prior to testing, prepare the cooling circuit by combining 200 milliliters of methanol with 500 cc of dry ice to form an ice bath. Connect the intake tube from the methanol reservoir to the pump. Connect the outflow tube that runs to the ice bath to the other side of the pump.Connect the tubing ends to a dummy CryoLoop and ensure that the ends fit snugly over the inlet and outlet tubes to complete the cooling circuit. Next attach the thermocouple plug to a digital thermometer for continuous temperature monitoring. Ensure that the length of this cable and the tubing are sufficient to reach the head of the animal when connected.Then turn on the piston pump using the switch and verify that all connections are snug and no leaks are present. Ensure that the pump setting, length of tubing inside 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 below 5 degrees Celsius. Once satisfied with the initial setup, switch the pump off and remove the dummy CryoLoop.Place the animal on the testing apparatus and buckle the harness. Then secure the strap and attach the leash. Next, 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, and connect the thermocouple plug to the digital thermometer. Begin the testing session with a visual or tactile obstacle memory trial. After the initial test, follow with additional trials of all four types in a random fashion.Next, switch on the piston pump and wait one to two minutes for the CryoLoop to reach a temperature of three degrees Celsius. Then run a cool block of trials. During the trials, ensure that the temperature of the CryoLoop is maintained around three degrees Celsius.Then switch off the piston pump and wait for the CryoLoop to return to its original temperature. Run a final rewarm block of trials after the piston pump has been switched off. When the behavioral testing is concluded, remove the tubing from inlet and outlet tubes and ensure that the protective caps are replaced.Be conscious of residual methanol that may drip from the tubing ends and may irritate the animal. Remove the leash and harness and then return the animal to the colony. Finally, use a tube cutter to trim the tube ends to prevent leaky connections on the next testing day.When area five of the cat's brain is bilaterally cooled, hind-leg stepping was significantly attenuated in the obstacle-present trials for both the leading and trailing hind legs. The peak step height of the forelegs in the obstacle-present trials and all of the legs in the obstacle-absent trials were not affected by area five deactivation. Similarly, the peak step height for any leg in either obstacle-present or obstacle-absent trials did not differ from the warm condition when area seven was deactivated.In comparison to both warm and area seven cooled conditions, step clearance was reduced in the leading hind-leg step and in the trailing hind-leg step for the area five cooled condition. Additionally, the step trajectory of the trailing hind leg was affected by area five deactivation as the peak occurred before the obstacle, unlike the stepping in both warm and area seven cooled conditions. While attempting this procedure, it's important to remember to monitor the temperature of cooling loops throughout the experiment to ensure proper deactivation.After watching this video you should have a good understanding of how to reversibly deactivate regions of cortex with cooling in a behaving animal.

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