This work was supported by NSF IOS 1146334, NSF EFRI 0937848, DARPA N66001-10-C-2010, and NSF IOS 0522220 to RDB. We thank Stacie Hyatt at Plexon&#160;Inc&#160;for assistance in preparing this manuscript.

All procedures were in accordance with Brown University Institutional Animal Care and Use Committee guidelines.
1. Systems Overview
A video tracking system interacts with a behavioral control system to monitor the rat's progress in a given task, evaluate target behaviors, control stimulus presentation and deliver rewards based on the rat's progress. An vivo electrophysiological recording system collects neural data for event related analyses. (Figure 1A).
<ol>
	<li>The Floor Projection Maze
	<ol>
		<li>The Floor Projection Maze2 is an open field without walls (147.3 cm x 111.8 cm) with a clear colorless Plexiglas floor (0.6 cm thick). The underside of the floor is a unity gain Dual Vision Fabric screen that is stretched over a second rectangle of Plexiglas pieces (147.3 cm x 111.8 cm x 1.25 cm) for rear projection using a short throw projector.</li>
		<li>Connect the projector to the video card in the behavioral system PC. Hold the Plexiglas, screen fabric, and projector using an anodized aluminum frame.</li>
		<li>Install an overhead anodized frame to mount an overhead camera and a pulley system to interface the ICS and headstage tethers to the rat. 
		Note: Connect the frame holding the Floor Projection Maze and overhead frame to ensure electrical continuity and ground to the preamplifier.</li>
	</ol>
	</li>
	<li>Video tracking
	<ol>
		<li>Record, track and analyze videos in real time with a single overhead camera described in step 1.2.2.</li>
		<li>Mount an overhead camera (standard VGA, 80 fps) onto an overhead anodized aluminum frame to monitor the rat</li>
		<li>Use either light emitting diodes (LEDs) attached to the rat's headstage to monitor the position of the rat, or track the centroid of the contour of the rat with an automated tracking system.</li>
		<li>Use the CinePlex Basic Behavior module to analyze the position data online and also save the data file for further offline analysis. 
		Note: We use the CinePlex Behavioral Research System 3.4.1 for recording, tracking and analyzing the rat's position. The CinePlex Tracking module is used for tracking the rat. For effective tracking of the animal's progress, use the CinePlex Basic Behavior module to create zones in two-dimensional space at relevant positions of the maze that have significance in the task. Zones can be combined into sequences, and CinePlex logical events (such as entries into zones and fulfillment of the sequences) are assigned such that if the rat satisfies the criteria for the event, that event becomes true. Assign events with digital outputs and interface via a superport input card to the behavioral system.</li>
	</ol>
	</li>
	<li>A fully automated behavioral control system
	<ol>
		<li>Behavioral test arena: Use an appropriate shaped test arena for the behavioral task on the Floor Projection Maze. Construct the arena using matte white acrylic and place it directly on the Floor Projection Maze. 
		Note: Dimensions can be customized for the behavioral task used. Walls are typically 45-50 cm in height. Here we use double-sided test arenas (bowtie maze12) to maximize the number of trials performed in a session (Figure 1B for dimensions of the bowtie maze). The test arena has four designated areas defined in the tracking software: East Image area, West Image Area, an East Trial Ready Area, and a West Trial Ready Area (Figure 1C).</li>
		<li>Behavioral system: A behavioral interface system controlled by MED-PC IV software runs programs written in-house in MEDstate Notation (see Appendix). Use the events from CinePlex behavioral system to track a rat's progress during the task, control image presentation, deliver audio cues and deliver reward.
		<ol>
			<li>Interface the superport output card to Omniplex digital input to acquire time stamped behavioral event occurrences in the PlexControl software environment for offline analyses. Issue auditory cues using a programmable audio generator. 
			Note: Behavioral events from the apparatus presented here can operate third party equipment (e.g. laser for optogenetic stimulation) via a data transfer board if required.</li>
			<li>House the superport output card, superport input card and programmable audio generator in a tabletop interface cabinet. Interface the cards with a DIG-704PCI-2 PCI card installed in a personal computer (PC) using a DIG-700F Decoder card installed in the tabletop cabinet.</li>
			<li>Deliver bipolar square wave ICS to the rat using a programmable intracranial self stimulation stimulator. Connect the stimulator unit to the PC via a PHM152 COM card. Recommended ICS parameters: Pulse 1 and 2 duration: 500 &#181;sec; Delay between Pulse 1 and 2: 500 &#181;sec; Frequency: 100 Hz. 
			Note: ICS delivery is automated during the task but can be manually delivered using a programmable button box interfaced with a SmartCtrl module.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Neural data acquisition system
	<ol>
		<li>Ensure the Floor Projection Maze frame and ICS system is grounded to the preamplifier of the Omniplex Neural Data Acquisition System to minimize electrical noise in the neural recordings.</li>
		<li>Simultaneously collect neural data, positional coordinates from CinePlex and behavioral event flags from the behavioral system in the data file for offline analyses.</li>
	</ol>
	</li>
</ol>
2. Animal Preparation
<ol>
	<li>Animals
	<ol>
		<li>Obtain na&#239;ve 22 day old male Long-Evans rats.</li>
		<li>Pair house the rats and allow them to acclimatize to the vivarium for one week.</li>
		<li>Handle rats daily for ~5 min.</li>
		<li>Once rats reach 250-275 g, begin food schedules maintaining their body weights at 85-90% of their free feeding weight. Target weights are increased by 10 g/month until rats reach 350 g.</li>
		<li>Single house the rats and continue the food schedule for at least a week before commencing surgery to implant stimulation and recording electrodes.</li>
	</ol>
	</li>
	<li>Surgical implantation 
	Carry out surgical procedures under standard aseptic conditions and according to institutional and regulatory guidelines.
	<ol>
		<li>Anesthetize rats with isoflurane.</li>
		<li>Make an incision in the scalp to reveal the skull for cleaning and identification of bregma and lambda.</li>
		<li>Make craniotomies at the relevant coordinates.</li>
		<li>Affix skull anchor screws.</li>
		<li>Lower the tip of an ICS electrode into the MFB using the following coordinates: antero-posterior, -2.7 mm from bregma; lateral, &#177;1.8 mm; dorsoventral, -8.5 mm from skull surface. A second ICS electrode can be implanted into the opposite hemisphere as a backup in case of electrode malfunction.</li>
		<li>Secure the ICS electrodes to the skull and anchor screws with bone cement. Do not cement the pedestal (plastic housing on the stimulating electrode) of the electrodes.</li>
		<li>Lower the recording electrodes into the site of interest and secure the device with bone cement.</li>
		<li>Position the ICS pedestal away from the recording device and secure the ICS electrodes and recording devices to the anchor screws with bone cement.</li>
		<li>Allow at least seven days recovery before commencing habituation to the shaping protocol.</li>
	</ol>
	</li>
</ol>
3. Behavioral Shaping: General Shaping Consists of Three Stages: Early, Intermediate, and Late Shaping (Figure 2).
Note: The aim of Early and Intermediate shaping is to train the rat to maintain a stationary 'ready position' in the Ready Area for each trial, and perform numerous trials in a session. Semi-automate behavioral shaping so training can be adapted to a rat's individual learning rates. Once a rat is maintaining a successful 'ready position', proceed to task specific shaping (Late shaping) with the aim of transferring the rat onto a fully automated protocol for precise and unbiased behavioral control (Figure 2).
<ol>
	<li>Early shaping (Figure 2A)
	<ol>
		<li>Day 1: Habituate the rat to the behavioral room for 10 min with the equipment switched on then return to the colony.</li>
		<li>Day 2: Repeat step 1, then habituate the animal to the testing arena for 10 min.</li>
		<li>Day 3: Connect the ICS and headstage tethers to the rat, and habituate the rat to the testing arena for 10 min.</li>
		<li>Day 4: Determine the lowest ICS amplitude to establish a place preference using an informal place preference conditioning protocol. Titrate Pulse 1 and 2 amplitudes. Typical amplitude values are 20-80 &#181;A. </li>
		<li>From day 5: Deliver ICS reward to train the rats to associate the Ready Area and east and west image presentations with ICS reward. Continue until the rat is alternating between East and West Image Areas.</li>
	</ol>
	</li>
	<li>Intermediate shaping (Figure 2B).
	<ol>
		<li>Introduce white noise (50 dB) to signal the start of a trial. Turn off the white noise when the rat enters the Ready Area.</li>
		<li>Automate ICS delivery for entry into the Ready Area, and for completing successful 'ready positions'. Adjust the probability of ICS delivery at entry and successful 'ready position' to reinforce behavior. 
		Note: Adjust the reward probabilities manually to suit individual rat's performance during shaping. Decrease reward probabilities for entry into the Ready Area and maintaining successful ready positions. Recommended final probability of reward for fulfilling successful 'ready positions is 5-10%. Rats are rewarded for all correct choices.</li>
		<li>Start with short 'ready position' durations (e.g. 200 msec). Gradually increase the 'ready position' durations in 100 msec steps.</li>
		<li>Turn on the white noise if the rat prematurely breaks the 'ready position' so the rat has to restart the trial.</li>
		<li>Move to Late shaping when rat is holding in the 'ready position' for up to 1,200 msec.</li>
	</ol>
	</li>
	<li>Late shaping (Figure 2C). 
	Note: Training in Late shaping is specific for the task in which the rat will perform. Automate training in this shaping step for precise and unbiased control of all task parameters but maintain flexibility to manually deliver ICS rewards. Training protocols for two tasks are described.
	<ol>
		<li>Visual biconditional discrimination (vBCD) task: Use distinct visual stimuli to train the rats on a simple shape and luminance discriminence.
		<ol>
			<li>Begin the trial by turning on the white noise.</li>
			<li>Randomly impose 'ready position' latencies of 700 &#8211; 1,200 msec.</li>
			<li>Deliver ICS manually if required to reinforce successful 'ready positions'.</li>
			<li>Present a pair of images in the image presentation area. Pseudorandomly present the correct image on the left or right side of the Image Area.</li>
			<li>Deliver ICS for a correct response and clear the floor. A correct response is registered when the rat enters the zone in which the correct image is located. On the first day of training only, issue a 75 dB burst of white noise as a deterrent for an incorrect response.</li>
			<li>Issue a correction trial after an incorrect trial. Correction trials have identical parameters (left or right side and 'ready position' latency) as the previous incorrect trial.</li>
			<li>Once the rats are able to successfully perform the simple discrimination, introduce the different floor patterns to train the biconditional discrimination rule.</li>
			<li>Present a new pair of images and two distinct floor patterns to serve as the context. The correct image is conditional upon the floor pattern; e.g. the black star is correct when the floor is grey and the white circle is correct when the floor is striped (Figure 3C).</li>
			<li>Pseudorandomly assign the floor pattern and the position (left or right) of the correct image for each trial ensuring that trials are counterbalanced.</li>
			<li>Implement correction trials after an incorrect trial in which the trial parameters are identical to the preceding trial.</li>
		</ol>
		</li>
		<li>Visuospatial attention (VSA) task: Present illuminated white circles at defined spatial locations in the arena to train the rats to approach the spatial location of the target circle. 
		Note: For this task the arena has grey circles at different spatial locations in the East and West Image Areas. After a successful 'ready position', the target circle will illuminate (turn white), and the rat must approach that target location (Figure 3B).
		<ol>
			<li>Randomly impose 'ready position' latencies of 1,000-1,600 msec on a trial by trial basis.</li>
			<li>Deliver ICS manually if required to reinforce successful 'ready positions'.</li>
			<li>After completion of 'ready position', randomly illuminate one of the grey circles.</li>
			<li>Deliver ICS when the rat enters the zone of the illuminated circle.</li>
			<li>Turn off illumination and commence the next trial on the opposite (East or West) side.</li>
			<li>At the start of Late shaping, illuminate the circle until the rat approaches it or until the trial ends (5 sec after illuminating the circle). Score each trial as either correct or an omission trial. For omission trials, illuminate the whole floor (white floor presented) and no reward will be available until the next trial.</li>
			<li>When the rat's performance reaches 80% correct, decrease the amount of time the circle remains illuminated to 1 sec. The rat has 5 sec to make a choice.</li>
			<li>Do not reward for incorrect trials. Start the next trial.</li>
			<li>If an omission trial, illuminate the floor and start a new trial.</li>
			<li>At 80% correct further decrease the time the target circle is illuminated to 500 msec.</li>
			<li>Choose target locations randomly before each trial. The rat will continue to have 5 sec to make a choice before the entire floor illuminates to signal the end of that trial.</li>
			<li>Do not reward for incorrect or omission trials. The rat must initiate a new trial on the opposite side of the arena.</li>
			<li>Note: Additional circles may be added in each Image Area to further increase task difficulty.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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Submission fee for this video-article is sponsored by Plexon Inc.

We formally describe protocols to train rats on fully automated complex visual cognitive tasks on the Floor Projection Maze. The Floor Projection Maze has been used successfully to establish neural correlates of object-location conjunctions and error signals in postrhinal cortex during performance on a 2-choice visual discrimination task1.
The behavioral shaping protocol is designed to be flexible so that it can be adapted to suit an individual rat&#8217;s learning rate.&#160;Custom code written for the behavioral system allows the experimenter to control the probability of automated ICS delivery at critical steps during training; e.g. training the &#8216;ready position&#8217;. The code should also allow manual delivery of ICS using a programmable button box during. The aim of Early shaping is to establish effective ICS stimulation for reward delivery to reinforce behavior. When possible we recommend implanting stimulating electrodes bilaterally into MFB in case of ineffective stimulation in one hemisphere. Our experience, however, has been that nearly all implanted ICS electrodes have been functional to deliver a rewarding stimulus. The Intermediate shaping stage is critical for training the rat to maintain a &#8216;ready position&#8217; in the Ready Area. In the &#8216;ready position&#8217;, the rat should be stationary with its nose pointing towards the Image Areas where the choice images will be presented. The &#8216;ready position&#8217; controls when the images appear, the direction from which the rat approaches the images and the distance from which the rat views the images. We recommend tracking LEDs mounted on the rat&#8217;s headstage to monitor the direction in which the rat&#8217;s head is pointing for better control of behavior and more precise control of target image presentation. Manual delivery of ICS can be issued to train rats to maintain the appropriate &#8216;ready position&#8217;. When the rat is maintaining satisfactory &#8216;ready positions&#8217; in each trial, training in Late shaping is task specific. In Late shaping rats are trained to learn specific rules to perform a given task.
We use ICS as our method of reward delivery as this method provides instant feedback to the rat resulting in faster learning during behavioral shaping, and more completed trials in a session when collecting neural data in the automated task. ICS delivery, however, results in electrical artifacts in the neural recordings, and can be problematic for analyzing continuous LFP or multiunit data. The interval around the electrical artifact can be removed offline from the neural recording data file prior to continuous data analysis. Stereotypical artifacts from ICS in high pass filtered spike data can be easily removed offline without affecting single spike analyses. Future optimization of protocols for the Floor Projection Maze includes using optogenetic tools to deliver internal reward to minimize and eliminate artifacts associated with electrical stimulation.

The Floor Projection Maze is optimized for the visual capabilities of rats and is thus better suited for visually guided behavioral tasks. It should be noted that direct comparisons cannot be made between experimental paradigms in the Floor Projection Maze and operant chambers because of different experimental protocols employed and different definitions of criterion for successful acquisition of a given task. We have previously shown, however, that rats acquired a simple 2-choice visual discrimination task in ~50 trials in the Floor Projection Maze2 compared with &#62;300 trials in other studies where images were presented vertically in operant chambers3,10.&#160;The VSA task was modeled after the classical 5-choice serial reaction task13 (5-CSRT) designed to assess visual attentional processes. Typically the 5-CSRT is carried out in an operant chamber and requires the animal to make a nose poke in the appropriate nose-poke hole indicated by a light flash; here the VSA task is carried out in an open arena where animals have to remain stationary at a specific location in the arena, direct their attention to the target locations and wait for the light stimulus to appear. In our experience, rats required ~3,200 trials to reach criterion (75% on two consecutive days) in the 5-CSRT14 compared with ~800 trials in the VSA task in the Floor Projection Maze.

Another benefit of behavioral tasks performed on the Floor Projection Maze is that tasks are carried out in a large open arena, which exploits the natural tendency of the rat to explore its environment, as opposed to tasks that require the animal to fix its gaze to a stationary target. A major advantage of utilizing freely moving rats in an open arena &#160;in the Floor Projection Maze apparatus is that &#160;both allocentric and egocentric spatial information can be collected with visual information. Another advantage of our paradigm is that tasks need not be confined to presentation of static images. Position and features of visual stimuli that are dynamic can be used in future tasks. Using tracking capabilities of the CinePlex Behavioral Research System, tasks can be developed for a more detailed functional understanding of a given brain structure. Combining simultaneous electrophysiological recordings with visually guided cognitive tasks, fundamental questions of how brain activity is related to cognitive processes such as decision-making and visual discrimination can be explored.
Using visually guided tasks, findings from rat studies can be better translated to humans with the ultimate goal of providing therapies for human cognitive diseases.

Visual tasks are commonly used in human and monkey studies to examine mechanisms underlying learning and memory. Rodent models, however, are more readily accessible to researchers for larger scale, better controlled studies, and have the added advantage of permitting more invasive electrophysiological techniques. In combination with other approaches, such as genetic manipulations, electrophysiological recordings in freely moving rats provide a useful model for precisely targeting mechanisms and circuits underlying cognitive processes. Primate visual tasks require the subjects to direct the gaze to visual stimuli on a monitor, whereas rat tasks require the subjects to interact with an environment. The Floor Projection Maze utilizes the natural tendency of rats to actively explore the environment while simultaneously attending to visual stimuli.
Cognitive tasks using touchscreen apparatuses designed specifically for rodents have allowed better translation of findings from rodent models to humans3. Touchscreen tasks are typically carried out in a chamber with two dimensional visual stimuli presented vertically onto walls3-7. These touchscreen tasks require the subject to rear towards the target visual stimulus and break an infrared photobeam or press on a pressure pad to register its response. Anatomical and behavioral evidence, however, suggest that rats process visual information in the lower visual hemifield more effectively for guiding behavior8-10. Our lab has developed cognitive tasks utilizing the Floor Projection Maze2 in which two-dimensional visual stimuli are back projected onto the floor of the test arena. In the Floor Projection Maze rats can be tracked when performing tasks in a large open arena compared with touchscreen apparatus. Thus, spatial information in the recorded neural activity can be obtained in addition to neural correlates of visual information and decision-making.

We deliver intracranial stimulation (ICS) to the medial forebrain bundle (MFB) as a reward11. This method of reward delivery has advantages over food and drink rewards. Food and drink rewards can lead to satiation, even in food-deprived rats, limiting the number of trials an animal will perform and potentially slowing the training process. ICS delivers instant reward providing immediate feedback on task performance. The immediate reward results in faster shaping and acquisition and substantially reduces the duration of training protocols. Moreover, a larger number of trials can be completed in a session, increasing the amount of data collected and resulting in a more reliable sample of task-related behavior.

Using the Floor Projection Maze, we will describe a general protocol to shape the behavior of rats to perform complex cognitive tasks. This general protocol provides a framework for training rats across a variety of tasks currently employed for recording neural correlates of attention and visual discrimination1. Thus, the Floor Projection Maze is optimized for visual abilities of rats and permits stronger comparisons with visual tasks in humans and nonhuman primates.&#160;

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		<col />
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">OmniPlex D Neural Data Acquisition System</td>
			<td style="width:127px;">Plexon Inc</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
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			<td height="21" style="height:21px;width:391px;">DigiAmp Digitizing Amplifier</td>
			<td style="width:127px;">Plexon Inc</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:391px;">Frame for Floor Projection Maze</td>
			<td style="width:127px;">80/20 Inc</td>
			<td style="width:119px;"></td>
			<td>15 Series T-slot framing</td>
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			<td height="40" style="height:40px;width:391px;">Short throw projector</td>
			<td style="width:127px;">NEC Display Solutions</td>
			<td style="width:119px;">WT610E</td>
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			<td height="40" style="height:40px;width:391px;">Da-Lite lace and grommet screen</td>
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			<td height="20" style="height:20px;width:391px;">PlexiGlass</td>
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			<td height="40" style="height:40px;width:391px;">SuperPort Input card</td>
			<td style="width:127px;">Med Associates Inc</td>
			<td style="width:119px;">DIG-713A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:391px;">SuperPort Output card</td>
			<td style="width:127px;">Med Associates Inc</td>
			<td style="width:119px;">DIG-726</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:391px;">SmartCtrl Interface module</td>
			<td style="width:127px;">Med Associates Inc</td>
			<td style="width:119px;">DIG-716B</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:391px;">Decoder card</td>
			<td style="width:127px;">Med Associates Inc</td>
			<td style="width:119px;">DIG-700F</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:391px;">PCI card</td>
			<td style="width:127px;">Med Associates Inc</td>
			<td style="width:119px;">DIG-704PCI-2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:391px;"></td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:391px;">Programmable audio generator</td>
			<td style="width:127px;">Med Associates Inc</td>
			<td style="width:119px;">ANL-926</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:391px;">Programmable Intracranial Self Stimulation Stimulator</td>
			<td style="width:127px;">Med Associates Inc</td>
			<td style="width:119px;">PHM-150B</td>
			<td style="width:167px;">Operated by a PHM-152COM card</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:391px;">2 Channel electrode</td>
			<td style="width:127px;">Plastics1</td>
			<td style="width:119px;">MS303/13/SP</td>
			<td style="width:167px;">Cut 15 &#8211; 20 mm below the pedestal</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:803px;">Software</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:391px;">MED-PC IV Software</td>
			<td style="width:127px;">Med Associates Inc</td>
			<td style="width:119px;">SOF-735</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:391px;">OmniPlex Software</td>
			<td style="width:127px;">Plexon Inc</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CinePlex Software: Tracking and Basic Behavior Modules</td>
			<td style="width:127px;">Plexon Inc</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

automated visual cognitive tasks for recording neural activity using a floor projection maze

Neuropsychological tasks used in primates to investigate mechanisms of learning and memory are typically visually guided cognitive tasks. We have developed visual cognitive tasks for rats using the Floor Projection Maze1,2&#160;that are optimized for visual abilities of rats permitting stronger comparisons of experimental findings with other species.
In order to investigate neural correlates of learning and memory, we have integrated electrophysiological recordings into fully automated cognitive tasks on the Floor Projection Maze1,2. Behavioral software interfaced with an animal tracking system allows monitoring of the animal's behavior with precise control of image presentation and reward contingencies for better trained animals. Integration with an in vivo electrophysiological recording system enables examination of behavioral correlates of neural activity at selected epochs of a given cognitive task.
We describe protocols for a model system that combines automated visual presentation of information to rodents and intracranial reward with electrophysiological approaches. Our model system offers a sophisticated set of tools as a framework for other cognitive tasks to better isolate and identify specific mechanisms contributing to particular cognitive processes.

The goal of the general shaping steps is to acclimate the rat to the testing arena, train the rat to remain in a stationary &#39;ready position&#39; for the presentation of the target visual stimuli, and approach the location of the correct visual stimulus. After habituation to the testing room, arena, and tethers, Early shaping typically requires 100-150 trials until the rats are alternating between the East and West sides of the arena. During Early shaping the rats typically run through the Ready Area in center of the arena and spend most of the time exploring the perimeter of the arena (Figure 4A). Rats at the Intermediate shaping stage gradually learn to maintain a stationary 'ready position' in the Ready Area requiring 600-700 trials. At this point, animals' paths are stereotyped loops from the Ready Area to the Image Area with less time spent exploring the perimeter of the maze. However, rats are not maintaining a stationary &#39;ready position&#39;, indicated by the speed at which rats traverse the center Ready Area (Figure 4B).
By the end of Intermediate shaping, rats maintain a stationary 'ready position' in the Ready Area before approaching the target visual stimulus. The rat then initiates the next trial on the opposite side of the arena (Figure 4C).
The integration of the tracking software and the Neural Data Acquisition System with a behavioral control system allows for event related analyses of neural data. Drivable electrode arrays can be strategically placed to record single-unit and local field potential activity. Recordings were carried out in postrhinal cortex when rats performed the vBCD task. Perievent histograms and raster plots show that cells in postrhinal cortex respond to the onset of the target visual stimulus and to the onset of the floor pattern (Figure 5A). In posterior parietal cortex of rats performing the VSA task cells respond to the image presentation, and when the choice was made by entering the zone that defined the target image (Figure 5B). Local field potential activity in posterior parietal cortex during performance in the VSA task shows strong power in the theta range (~8 Hz) when rats are in the 'ready position' before the image presentation (Figure 5C).
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51316/51316fig1highres.jpg" src="/files/ftp_upload/51316/51316fig1.jpg" width="400" /> 
Figure 1. Experimental setup. A. Schematic of the behavioral and control rooms. The Floor Projection Maze is housed in the behavioral room. Rats are monitored via an overhead camera. The control room houses the equipment to control the task and to collect neural data. B. Dimensions of the bowtie maze. C. Screen capture of CinePlex Studio. Zones are defined by the user. Logical events are sent as CinePlex digital outputs to the behavioral control system (Med Associates) to monitor the progress of the rat. <a href="https://www.jove.com/files/ftp_upload/51316/51316fig1highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51316/51316fig2highres.jpg" src="/files/ftp_upload/51316/51316fig2.jpg" /> 
Figure 2. Schematic of the shaping steps. A. In Early shaping the rat receives ICS for entering the Ready Area, and entering East and West Image Areas. The aim is to train the rat to associate these areas with ICS reward. B. Intermediate shaping focuses on training the rats to maintain a stationary 'ready position' in the Ready Area. The images are presented only after maintaining a successful 'ready position'. Rats continue to receive ICS for approaching the image in the Image area. C. At Late shaping, the rat is successfully maintaining a stationary 'ready position'. Training is task specific, and the rat is trained to learn specific rules to perform a given task. Lightning indicates ICS delivery. <a href="https://www.jove.com/files/ftp_upload/51316/51316fig2highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51316/51316fig3highres.jpg" src="/files/ftp_upload/51316/51316fig3.jpg" /> 
Figure 3. A. Schematic of the vBCD task. When the floor is grey, the black star is rewarded; when the floor is striped, the white circle is rewarded. B. Schematic of the VSA task. Grey circles indicate the target locations in the Image areas. The target stimulus is a brief (500 msec) illumination (white) of one of the circles. The rats are rewarded for approaching the correct target location. <a href="https://www.jove.com/files/ftp_upload/51316/51316fig3highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51316/51316fig4highres.jpg" src="/files/ftp_upload/51316/51316fig4.jpg" /> 
Figure 4. Example paths during shaping. Position data from two minute segments of a rat performing at different shaping steps. Speed of the animal is represented by the heat map. Cold colors represent slowest speeds and hot colors represent fastest speeds. A. During Early shaping rats explore and rear up on the outer walls of the East and West image areas and do not stop in the Ready Area. B. A stereotyped path starts to emerge in Intermediate shaping. Paths form a loop towards the Image areas back to the Ready Area. Rats begin to successfully maintain a 'ready position' in the Ready Area. C. When rats progress onto Late shaping, rats maintain stationary 'ready positions' and the paths of the rats are more stereotyped. <a href="https://www.jove.com/files/ftp_upload/51316/51316fig4highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/51316/51316fig5highres.jpg" src="/files/ftp_upload/51316/51316fig5.jpg" /> 
Figure 5. Examples of perievent related analyses. A. Cells recorded from the postrhinal cortex during the vBCD task with different responses to the presentation of the target image (left), and the presentation of the floor (right). B. Cells recorded from the posterior parietal cortex during the VSA task with different responses to the illumination of the circle (left). Increased firing after choosing the spatial location in which the target circle illuminated (right). C. Spectrogram showing strong theta power in the posterior parietal cortex during the 'ready position'. The vertical red line indicates the end of successful 'ready position' and the presentation of the images. The vertical blue line indicates the presentation of Floor 1 at trial start in the vBCD task. The vertical green line indicates that a choice was registered in the VSA task. <a href="https://www.jove.com/files/ftp_upload/51316/51316fig5highres.jpg" target="_blank">Click here to view larger image.</a>

Watch this Scientific Journal Video about Automated Visual Cognitive Tasks for Recording Neural Activity Using a Floor Projection Maze at JoVE.com

Automated Visual Cognitive Tasks for Recording Neural Activity Using a Floor Projection Maze

We describe protocols for training rats for chronic electrophysiological recordings in fully automated cognitive tasks on a Floor Projection Maze.

Neuropsychological tasks used in primates to investigate mechanisms of learning and memory are typically visually guided cognitive tasks. We have ...

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13.0K Views.  Brown University. We describe protocols for training rats for chronic electrophysiological recordings in fully automated cognitive tasks on a Floor Projection Maze.

Video: Automated Visual Cognitive Tasks for Recording Neural Activity Using a Floor Projection Maze

Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the &#34;cloud&#34; and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in&#160;cabinet; two such cabinets (16 environments) may be controlled by one computer.

Fully automated system for measuring physiologically meaningful properties of the mechanisms mediating spatial localization, temporal localization, duration, rate&#160;and probability estimation, risk assessment, impulsivity, and the accuracy and precision of memory, in order to assess the effects of genetic and pharmacological manipulations on foundational mechanisms of cognition in mice.

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and ...

A Fully Automated Rodent Conditioning Protocol for Sensorimotor Integration and Cognitive Control Experiments

Rodents have been traditionally used as a standard animal model in laboratory experiments involving a myriad of sensory, cognitive, and motor tasks. Higher cognitive functions that require precise control over sensorimotor responses such as decision-making and attentional modulation, however, are typically assessed in nonhuman primates. Despite the richness of primate behavior that allows multiple variants of these functions to be studied, the rodent model remains an attractive, cost-effective alternative to primate models. Furthermore, the ability to fully automate operant conditioning in rodents adds unique advantages over the labor intensive training of nonhuman primates while studying a broad range of these complex functions.
Here, we introduce a protocol for operantly conditioning rats on performing working memory tasks. During critical epochs of the task, the protocol ensures that the animal's overt movement is minimized by requiring the animal to 'fixate' until a Go cue is delivered, akin to nonhuman primate experimental design. A simple two alternative forced choice task is implemented to demonstrate the performance. We discuss the application of this paradigm to other tasks.

A fully automated protocol for rodent operant conditioning is proposed. The protocol relies on precise temporal control of behavioral events to investigate the extent to which this control influences neural activity underlying sensorimotor integration and cognitive control experiments.

Rodents have been traditionally used as a standard animal model in laboratory experiments involving a myriad of sensory, cognitive, and motor tasks. ...

Uncovering Beat Deafness: Detecting Rhythm Disorders with Synchronized Finger Tapping and Perceptual Timing Tasks

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here with the goal of uncovering rhythm disorders, such as beat deafness. Beat deafness is characterized by poor performance in perceiving durations in auditory rhythmic patterns or poor synchronization of movement with auditory rhythms (e.g., with musical beats). These tasks include the synchronization of finger tapping to the beat of simple and complex auditory stimuli and the detection of rhythmic irregularities (anisochrony detection task) embedded in the same stimuli. These tests, which are easy to administer, include an assessment of both perceptual and sensorimotor timing abilities under different conditions (e.g., beat rates and types of auditory material) and are based on the same auditory stimuli, ranging from a simple metronome to a complex musical excerpt. The analysis of synchronized tapping data is performed with circular statistics, which provide reliable measures of synchronization accuracy (e.g., the difference between the timing of the taps and the timing of the pacing stimuli) and consistency. Circular statistics on tapping data are particularly well-suited for detecting individual differences in the general population. Synchronized tapping and anisochrony detection are sensitive measures for identifying profiles of rhythm disorders and have been used with success to uncover cases of poor synchronization with spared perceptual timing. This systematic assessment of perceptual and sensorimotor timing can be extended to populations of patients with brain damage, neurodegenerative diseases (e.g., Parkinson&#8217;s disease), and developmental disorders (e.g., Attention Deficit Hyperactivity Disorder).

Behavioral tasks that allow for the assessment of perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) are presented. Synchronization of finger tapping to the beat of an auditory stimuli and detecting rhythmic irregularities provides a means of uncovering rhythm disorders.

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here ...

Practical Methodology of Cognitive Tasks Within a Navigational Assessment

This paper describes an approach for measuring navigation accuracy relative to cognitive skills. The methodology behind the assessment will thus be clearly outlined in a step-by-step manner. Navigational skills are important when trying to find symbols within a speech-generating device (SGD) that has a dynamic screen and taxonomical organization. The following skills have been found to impact children&#8217;s ability to find symbols when navigating within the levels of an SGD: sustained attention, categorization, cognitive flexibility, and fluid reasoning1,2. According to past studies, working memory was not correlated with navigation1,2.
The materials needed for this method include a computerized tablet, an augmentative and alternative communication application, a booklet of symbols, and the Leiter International Performance Scale-Revised (Leiter-R)3. This method has been used in two previous studies. Robillard, Mayer-Crittenden, Roy-Charland, Minor-Corriveau and B&#233;langer1 assessed typically developing children, while Rondeau, Robillard and Roy-Charland2 assessed children and adolescents with a diagnosis of Autism Spectrum Disorder. The direct observation of this method will facilitate the replication of this study for researchers. It will also help clinicians that work with children who have complex communication needs to determine the children&#8217;s ability to navigate an SGD with taxonomical categorization.

Children who have complex communication needs can benefit from the use of a speech-generating device (SGD). Cognitive skills were identified as having an impact on the ability to navigate between levels of SGDs. This protocol describes the steps needed for Speech-language Pathologists to assess cognitive skills and navigational abilities.

This paper describes an approach for measuring navigation accuracy relative to cognitive skills. The methodology behind the assessment will thus be ...

Using the Activity-based Anorexia Rodent Model to Study the Neurobiological Basis of Anorexia Nervosa

Anorexia nervosa (AN) is a psychiatric illness characterized by excessively restricted caloric intake and abnormally high levels of physical activity. A challenging illness to treat, due to the lack of understanding of the underlying neurobiology, AN has the highest mortality rate among psychiatric illnesses. To address this need, neuroscientists are using an animal model to study how neural circuits may contribute toward vulnerability to AN and may be affected by AN. Activity-based anorexia (ABA) is a bio-behavioral phenomenon described in rodents that models the key symptoms of anorexia nervosa. When rodents with free access to voluntary exercise on a running wheel experience food restriction, they become hyperactive &#8211; running more than animals with free access to food. Here, we describe the procedures by which ABA is induced in adolescent female C57BL/6 mice. On postnatal day 36 (P36), the animal is housed with access to voluntary exercise on a running wheel. After 4 days of acclimation to the running wheel, on P40, all food is removed from the cage. For the next 3 days, food is returned to the cage (allowing animals free food access) for 2 hr daily. After the fourth day of food restriction, free access to food is returned and the running wheel is removed from the cage to allow the animals to recover. Continuous multi-day analysis of running wheel activity shows that mice become hyperactive within 24 hr following the onset of food restriction. The mice run even during the limited time during which they have access to food. Additionally, the circadian pattern of wheel running becomes disrupted by the experience of food restriction. We have been able to correlate neurobiological changes with various aspects of the animals&#8217; wheel running behavior to implicate particular brain regions and neurochemical changes with resilience and vulnerability to food-restriction induced hyperactivity.

Here we present a protocol to induce activity-based anorexia (ABA) in female adolescent mice. ABA is a condition of hyperactivity evoked by imposing food restriction on rodents with access to a running wheel. This phenomenon is being used as a model to study the underlying neurobiology of anorexia nervosa.

Anorexia nervosa (AN) is a psychiatric illness characterized by excessively restricted caloric intake and abnormally high levels of physical activity. A ...

Using Fiberless, Wearable fNIRS to Monitor Brain Activity in Real-world Cognitive Tasks

Functional Near Infrared Spectroscopy (fNIRS) is a neuroimaging technique that uses near-infrared light to monitor brain activity. Based on neurovascular coupling, fNIRS is able to measure the haemoglobin concentration changes secondary to neuronal activity. Compared to other neuroimaging techniques, fNIRS represents a good compromise in terms of spatial and temporal resolution. Moreover, it is portable, lightweight, less sensitive to motion artifacts and does not impose significant physical restraints. It is therefore appropriate to monitor a wide range of cognitive tasks (e.g., auditory, gait analysis, social interaction) and different age populations (e.g., new-borns, adults, elderly people). The recent development of fiberless fNIRS devices has opened the way to new applications in neuroscience research. This represents a unique opportunity to study functional activity during real-world tests, which can be more sensitive and accurate in assessing cognitive function and dysfunction than lab-based tests. This study explored the use of fiberless fNIRS to monitor brain activity during a real-world prospective memory task. This protocol is performed outside the lab and brain haemoglobin concentration changes are continuously measured over the prefrontal cortex while the subject walks around in order to accomplish several different tasks.

Monitoring brain activity outside the lab without physical constraints presents methodological challenges. A fiberless, wearable functional Near Infrared Spectroscopy (fNIRS) system was used to measure brain activity during an ecological prospective memory task. It was demonstrated that this system could be used to monitor brain activity during non-lab based experiments.

Functional Near Infrared Spectroscopy (fNIRS) is a neuroimaging technique that uses near-infrared light to monitor brain activity. Based on neurovascular ...

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer scale. However, current methods mostly rely on tethered cable recordings or only use unidirectional systems, allowing either recording or stimulation of neural activity, but not at the same time or same target. Here, a new wireless, bidirectional device for simultaneous multichannel recording and stimulation of neural activity in freely behaving rats is described. The system operates through a single portable head stage that both transmits recorded activity and can be targeted in real-time for brain stimulation using a telemetry-based multichannel software. The head stage is equipped with a preamplifier and a rechargeable battery, allowing stable long-term recordings or stimulation for up to 1 h. Importantly, the head stage is compact, weighs 12 g (including battery) and thus has minimal impact on the animal&#180;s behavioral repertoire, making the method applicable to a broad set of behavioral tasks. Moreover, the method has the major advantage that the effect of brain stimulation on neural activity and behavior can be measured simultaneously, providing a tool to assess the causal relationships between specific brain activation patterns and behavior. This feature makes the method particularly valuable for the field of deep brain stimulation, allowing precise assessment, monitoring, and adjustment of stimulation parameters during long-term behavioral experiments. The applicability of the system has been validated using the inferior colliculus as a model structure.

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

A wireless, bidirectional system for multi-channel neural recordings and stimulation in freely behaving rats is introduced. The system is light and compact, thus having minimal impact on the animal&#180;s behavioral repertoire. Moreover, this bidirectional system provides a sophisticated tool to assess causal relationships between brain activation patterns and behavior.

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer ...

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.

Automated, Long-term Behavioral Assay for Cognitive Functions in Multiple Genetic Models of Alzheimer&#039;s Disease, Using IntelliCage

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.

Multiple factors&#8212;such as aging and genes&#8212;are frequently associated with cognitive decline. Genetically modified mouse models of cognitive ...

Integrating Visual Psychophysical Assays within a Y-Maze to Isolate the Role that Visual Features Play in Navigational Decisions

Collective animal behavior arises from individual motivations and social interactions that are critical for individual fitness. Fish have long inspired investigations into collective motion, specifically, their ability to integrate environmental and social information across ecological contexts. This demonstration illustrates techniques used for quantifying behavioral responses of fish, in this case, Golden Shiner (Notemigonus crysoleucas), to visual stimuli using computer visualization and digital image analysis. Recent advancements in computer visualization allow for empirical testing in the lab where visual features can be controlled and finely manipulated to isolate the mechanisms of social interactions. The purpose of this method is to isolate visual features that can influence the directional decisions of the individual, whether solitary or with groups. This protocol provides specifics on the physical Y-maze domain, recording equipment, settings and calibrations of the projector and animation, experimental steps and data analyses. These techniques demonstrate that computer animation can elicit biologically-meaningful responses. Moreover, the techniques are easily adaptable to test alternative hypotheses, domains, and species for a broad range of experimental applications. The use of virtual stimuli allows for the reduction and replacement of the number of live animals required, and consequently reduces laboratory overhead.
This demonstration tests the hypothesis that small relative differences in the movement speeds (2 body lengths per second) of virtual conspecifics will improve the speed and accuracy with which shiners follow the directional cues provided by the virtual silhouettes. Results show that shiners directional decisions are significantly affected by increases in the speed of the visual cues, even in the presence of background noise (67% image coherency). In the absence of any motion cues, subjects chose their directions at random. The relationship between decision speed and cue speed was variable and increases in cue speed had a modestly disproportionate influence on directional accuracy.

Here, we present a protocol to demonstrate a behavioral assay that quantifies how alternative visual features, such as motion cues, influence directional decisions in fish. Representative data are presented on the speed and accuracy where Golden Shiner (Notemigonus crysoleucas) follow virtual fish movements.

Collective animal behavior arises from individual motivations and social interactions that are critical for individual fitness. Fish have long inspired ...

Behavioral Assessment of Visual Function via Optomotor Response and Cognitive Function via Y-Maze in Diabetic Rats

The optomotor response and the Y-maze are behavioral tests useful for assessing visual and cognitive function, respectively. The optomotor response is a valuable tool to track changes in spatial frequency (SF) and contrast sensitivity (CS) thresholds over time in a number of retinal disease models, including diabetic retinopathy. Similarly, the Y-maze can be used to monitor spatial cognition (as measured by spontaneous alternation) and exploratory behavior (as measured by a number of entries) in a number of disease models that affect the central nervous system. Advantages of the optomotor response and the Y-maze include sensitivity, speed of testing, the use of innate responses (training is not needed), and the ability to be performed on awake (non-anesthetized) animals. Here, protocols are described for both the optomotor response and the Y-maze and examples of their use shown in models of Type I and Type II diabetes. Methods include preparation of rodents and equipment, performance of the optomotor response and the Y-maze, and post-test data analysis.

Neural degeneration in both eyes and brain as a result of diabetes can be observed through behavioral tests carried out on rodents. The Y-maze, a measure of spatial cognition, and the optomotor response, a measure of visual function, both provide insight into potential diagnoses and treatments.

The optomotor response and the Y-maze are behavioral tests useful for assessing visual and cognitive function, respectively. The optomotor response is a ...