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 developed visual cognitive tasks for rats using the Floor Projection Maze1,2 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.
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.
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).
2. Animal Preparation
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).
The goal of the general shaping steps is to acclimate the rat to the testing arena, train the rat to remain in a stationary 'ready position' 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 'ready position', 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).
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. Click here to view larger image.
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. Click here to view larger image.
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. Click here to view larger image.
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. Click here to view larger image.
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. Click here to view larger image.
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’s learning rate. 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 ‘ready position’. 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 ‘ready position’ in the Ready Area. In the ‘ready position’, the rat should be stationary with its nose pointing towards the Image Areas where the choice images will be presented. The ‘ready position’ 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’s headstage to monitor the direction in which the rat’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 ‘ready position’. When the rat is maintaining satisfactory ‘ready positions’ 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 >300 trials in other studies where images were presented vertically in operant chambers3,10. 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 in the Floor Projection Maze apparatus is that 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.
The authors have nothing to disclose.
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 Inc for assistance in preparing this manuscript.
OmniPlex D Neural Data Acquisition System | Plexon Inc | ||
DigiAmp Digitizing Amplifier | Plexon Inc | ||
Frame for Floor Projection Maze | 80/20 Inc | 15 Series T-slot framing | |
Short throw projector | NEC Display Solutions | WT610E | |
Da-Lite lace and grommet screen | Da-Lite Screen Company | 81326C | |
PlexiGlass | Modern Plastics | ||
SuperPort Input card | Med Associates Inc | DIG-713A | |
SuperPort Output card | Med Associates Inc | DIG-726 | |
SmartCtrl Interface module | Med Associates Inc | DIG-716B | |
Decoder card | Med Associates Inc | DIG-700F | |
PCI card | Med Associates Inc | DIG-704PCI-2 | |
Programmable audio generator | Med Associates Inc | ANL-926 | |
Programmable Intracranial Self Stimulation Stimulator | Med Associates Inc | PHM-150B | Operated by a PHM-152COM card |
2 Channel electrode | Plastics1 | MS303/13/SP | Cut 15 – 20 mm below the pedestal |
Software | |||
MED-PC IV Software | Med Associates Inc | SOF-735 | |
OmniPlex Software | Plexon Inc | ||
CinePlex Software: Tracking and Basic Behavior Modules | Plexon Inc |