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;

<table><tbody><tr><td>OmniPlex D Neural Data Acquisition System</td><td>Plexon Inc</td><td></td><td></td></tr><tr><td>DigiAmp Digitizing Amplifier</td><td>Plexon Inc</td><td></td><td></td></tr><tr><td>Frame for Floor Projection Maze</td><td>80/20 Inc</td><td></td><td>15 Series T-slot framing</td></tr><tr><td>Short throw projector</td><td>NEC Display Solutions</td><td>WT610E</td><td></td></tr><tr><td>Da-Lite lace and grommet screen</td><td>Da-Lite Screen Company</td><td>81326C</td><td></td></tr><tr><td>Plexiglas</td><td>Modern Plastics</td><td></td><td></td></tr><tr><td>SuperPort Input card</td><td>Med Associates Inc</td><td>DIG-713A</td><td></td></tr><tr><td>SuperPort Output card</td><td>Med Associates Inc</td><td>DIG-726</td><td></td></tr><tr><td>SmartCtrl Interface module</td><td>Med Associates Inc</td><td>DIG-716B</td><td></td></tr><tr><td>Decoder card</td><td>Med Associates Inc</td><td>DIG-700F</td><td></td></tr><tr><td>PCI card</td><td>Med Associates Inc</td><td>DIG-704PCI-2</td><td></td></tr><tr><td>Programmable audio generator</td><td>Med Associates Inc</td><td>ANL-926</td><td></td></tr><tr><td>Programmable Intracranial Self Stimulation Stimulator</td><td>Med Associates Inc</td><td>PHM-150B</td><td>Operated by a PHM-152COM card</td></tr><tr><td>2 Channel electrode</td><td>Plastics1</td><td>MS303/13/SP</td><td>Cut 15-20 mm below the pedestal</td></tr><tr><td>MED-PC IV Software</td><td>Med Associates Inc</td><td>SOF-735</td><td></td></tr><tr><td>OmniPlex Software</td><td>Plexon Inc</td><td></td><td></td></tr><tr><td>CinePlex Software: Tracking and Basic Behavior Modules</td><td>Plexon Inc</td><td></td><td></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 ...

automated-visual-cognitive-tasks-for-recording-neural-activity-using

Research

JoVE Journal

Behavior

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

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 ...

Using MazeSuite and Functional Near Infrared Spectroscopy to Study Learning in Spatial Navigation

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted virtual 3D environments, track a participants' behavioral performance within the virtual environment and synchronize with external devices for physiological and neuroimaging measures, including electroencephalogram and eye tracking.
Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables continuous, noninvasive, and portable monitoring of changes in cerebral blood oxygenation related to human brain functions2-7. Over the last decade fNIR is used to effectively monitor cognitive tasks such as attention, working memory and problem solving7-11. fNIR can be implemented in the form of a wearable and minimally intrusive device; it has the capacity to monitor brain activity in ecologically valid environments. 
Cognitive functions assessed through task performance involve patterns of brain activation of the prefrontal cortex (PFC) that vary from the initial novel task performance, after practice and during retention12. Using positron emission tomography (PET), Van Horn and colleagues found that regional cerebral blood flow was activated in the right frontal lobe during the encoding (i.e., initial na&iuml;ve performance) of spatial navigation of virtual mazes while there was little to no activation of the frontal regions after practice and during retention tests. Furthermore, the effects of contextual interference, a learning phenomenon related to organization of practice, are evident when individuals acquire multiple tasks under different practice schedules13,14. High contextual interference (random practice schedule) is created when the tasks to be learned are presented in a non-sequential, unpredictable order. Low contextual interference (blocked practice schedule) is created when the tasks to be learned are presented in a predictable order.
Our goal here is twofold: first to illustrate the experimental protocol design process and the use of MazeSuite, and second, to demonstrate the setup and deployment of the fNIR brain activity monitoring system using Cognitive Optical Brain Imaging (COBI) Studio software15. To illustrate our goals, a subsample from a study is reported to show the use of both MazeSuite and COBI Studio in a single experiment. The study involves the assessment of cognitive activity of the PFC during the acquisition and learning of computer maze tasks for blocked and random orders. Two right-handed adults (one male, one female) performed 315 acquisition, 30 retention and 20 transfer trials across four days. Design, implementation, data acquisition and analysis phases of the study were explained with the intention to provide a guideline for future studies.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments. Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables noninvasive and portable monitoring of cerebral blood oxygenation changes. This paper summarizes collective use of MazeSuite and fNIR within a cognitive processing learning paradigm.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted ...

Neuroscience

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

An Automated T-maze Based Apparatus and Protocol for Analyzing Delay- and Effort-based Decision Making in  Free Moving Rodents

Many neurological and psychiatric patients demonstrate difficulties and/or deficits in decision making. Rodent models are helpful to produce a deeper understanding of the neurobiological causes underlying the decision-making problems. A cost-benefit based T-maze task is used for measuring decision making in which rodents choose between a high reward arm (HRA) and a low reward arm (LRA). There are two paradigms of the T-maze decision-making task, one in which the cost is a time delay and the other in which it is physical effort. Both paradigms require a tedious and labor-intensive management of experimental animals, multiple doors, pellet reward, and arm choice recordings. In the current work, we invented an apparatus based on traditional T-maze with full automation for pellet delivery, door management and choice recordings. This automated setup can be used for the evaluation of both delay- and effort-based decision making in rodents. With the protocol described here, our lab investigated the decision-making phenotypes of multiple genetically modified mice. In the representative data, we showed that the mice with ablated medial habenular showed aversions of both delay and effort and tended to choose the immediate and effortless reward. This protocol helps to decrease the variability caused by experimenter intervention and to enhance experiment efficiency. In addition, chronic silicon probe or microelectrode recording, fiber-optic imaging and/or manipulation of neural activity can be easily applied during the decision-making task using the setup described here.

This article introduces an automated T-maze apparatus that we invented, and a protocol based on this apparatus for analyzing delay-based decision making and effort-based decision making in free moving rodents.

Many neurological and psychiatric patients demonstrate difficulties and/or deficits in decision making. Rodent models are helpful to produce a deeper ...

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 ...

Utilizing a Reconfigurable Maze System to Enhance the Reproducibility of Spatial Navigation Tests in Rodents

Several maze shapes are used to test spatial navigation performance and behavioral phenotypes. Traditionally, each experiment requires a unique maze shape, thus requiring several separate mazes in different configurations. The maze geometry cannot be reconfigured in a single environment to accommodate scalability and reproducibility. The reconfigurable maze is a unique approach to address the limitations, allowing quick and flexible configurations of maze pathways in a repeatable manner. It consists of interlocking pathways and includes feeders, treadmills, movable walls, and shut-off sensors. The current protocol describes how the reconfigurable maze can replicate existing mazes, including the T-shaped, plus-shaped, W-shaped, and figure-eight mazes. Initially, the T-shaped maze was constructed inside a single experimental room, followed by modifications. The rapid and scalable protocol outlined herein demonstrates the flexibility of the reconfigurable maze, achieved through the addition of components and behavioral training phases in a stepwise manner. The reconfigurable maze systematically and precisely assesses the performance of multiple aspects of spatial navigation behavior.

The present protocol describes a reconfigurable maze, a unique system for testing spatial navigation and behavioral phenotypes in rodents. The adaptability of this maze system enables the execution of various experiments in a single physical environment. The ease of structural rearrangement generates reliable and reproducible experimental results.

Several maze shapes are used to test spatial navigation performance and behavioral phenotypes. Traditionally, each experiment requires a unique maze ...

Virtual Reality Tools for Assessing Unilateral Spatial Neglect: A Novel Opportunity for Data Collection

Unilateral spatial neglect (USN) is a syndrome characterized by inattention to or inaction in one side of space and affects between 23-46% of acute stroke survivors. The diagnosis and characterization of these symptoms in individual patients can be challenging and often requires skilled clinical staff. Virtual reality (VR) presents an opportunity to develop novel assessment tools for patients with USN.
We aimed to design and build a VR tool to detect and characterize subtle USN symptoms, and to test the tool on subjects treated with inhibitory repetitive transcranial magnetic stimulation (TMS) of cortical regions associated with USN.
We created three experimental conditions by applying TMS to two distinct regions of cortex associated with visuospatial processing- the superior temporal gyrus (STG) and the supramarginal gyrus (SMG) - and applied sham TMS as a control. We then placed subjects in a virtual reality environment in which they were asked to identify the flowers with lateral asymmetries of flowers distributed across bushes in both hemispaces, with dynamic difficulty adjustment based on each subject's performance. 
We found significant differences in average head yaw between subjects stimulated at the STG and those stimulated at the SMG and marginally significant effects in the average visual axis.
VR technology is becoming more accessible, affordable, and robust, presenting an exciting opportunity to create useful and novel game-like tools. In conjunction with TMS, these tools could be used to study specific, isolated, artificial neurological deficits in healthy subjects, informing the creation of VR-based diagnostic tools for patients with deficits due to acquired brain injury. This study is the first to our knowledge in which artificially generated USN symptoms have been evaluated with a VR task.

The goal was to design, build, and pilot a novel virtual reality task to detect and characterize unilateral spatial neglect, a syndrome affecting 23-46% of acute stroke survivors, expanding the role of virtual reality in the study and management of neurologic disease.

Unilateral spatial neglect (USN) is a syndrome characterized by inattention to or inaction in one side of space and affects between 23-46% of acute stroke ...

Medicine

A Visuospatial Planning Task Coupled with Eye-Tracker and Electroencephalogram Systems

The planning process, characterized by the capability to formulate an organized plan to reach a goal, is essential for human goal-directed behavior. Since planning is compromised in several neuropsychiatric disorders, the implementation of proper clinical and experimental tests to examine planning is critical. Due to the nature of the deployment of planning, in which several cognitive domains participate, the assessment of planning and the design of behavioral paradigms coupled with neuroimaging methods are current challenges in cognitive neuroscience. A planning task was evaluated in combination with an electroencephalogram (EEG) system and eye movement recordings in 27 healthy adult participants. Planning can be separated into two stages: a mental planning stage in which a sequence of steps is internally represented and an execution stage in which motor action is used to achieve a previously planned goal. Our protocol included a planning task and a control task. The planning task involved solving 36 maze trials, each representing a zoo map. The task had four periods: i) planning, where the subjects were instructed to plan a path to visit the locations of four animals according to a set of rules; ii) maintenance, where the subjects had to retain the planned path in their working memory; iii) execution, where the subjects used eye movements to trace the previously planned path as indicated by the eye-tracker system; and iv) response, where the subjects reported the order of the visited animals. The control task had a similar structure, but the cognitive planning component was removed by modifying the task goal. The spatial and temporal patterns of the EEG revealed that planning induces a gradual and lasting rise in frontal-midline theta activity (FM&#952;) over time. The source of this activity was identified within the prefrontal cortex by source analyses. Our results suggested that the experimental paradigm combining EEG and eye-tracker systems was optimal for evaluating cognitive planning.

The study of cognitive planning combining EEG and eye-tracking systems provides a multimodal approach to investigate the neural mechanisms that mediate cognitive control and goal-directed behavior in humans. Here, we describe a protocol for investigating the role of brain oscillations and eye movements in planning performance.

The planning process, characterized by the capability to formulate an organized plan to reach a goal, is essential for human goal-directed behavior. Since ...

Spatial Memory Testing Using Mazes

Spatial learning and memory are neurological functions that allow us to remember important details associated with our environment. Scientists test this phenomenon in rodents using different types of mazes like Morris water maze, Radial water maze, and Barnes maze. By investigating spatial memory in rodents, neurobehavioral scientists can gain valuable understanding of how these processes are altered following brain damage in humans.
Here, the neurobiology behind spatial memory is briefly reviewed and some general principles behind maze tests are discussed. Then, the video explains generalized protocols on how to conduct trials with specific types of mazes. Lastly, it will explain how behavioral researchers are using these tools to conduct some specific experiments.

Spatial Memory Tests Using Morris Water, Radial Water &amp; Barnes Mazes

Spatial learning and memory are neurological functions that allow us to remember important details associated with our environment. Scientists test this ...