We thank the authors of Robinson et al.12 for their contributions to the manuscript from which this protocol is partially derived.

The use of animals are approved by the Oberlin College Institutional Animal Care and Use committee and are in accordance with the Guide for the Care and Use of Laboratory Animals21.
1. Preparation for Viral Infusion
Note: This protocol uses BSL-1 precautions. When employing BSL-2 precautions, a disposable lab coat, gloves, shoe covers, eye protections and a particulate respirator (type N95) are required. All individuals handling BSL-2 compounds must be fit tested for a particulate respirator by a local public health agency. Refer to Lowery &#38; Majewska (2010)22 for additional details on handling and storage of viral vectors.
<ol>
	<li>Upon first use, aliquot and store unused virus in 20 &#181;l microcentrifuge tubes to avoid repeated freezing and thawing.</li>
	<li>Prepare the work surface by removing any unnecessary objects and sterilizing the surface with 70% ethanol. Prepare a 10% bleach solution for decontaminating AAV waste. Place a beaker full of the bleach solution and a sterile 10 &#181;l syringe onto the work surface. Place the microcentrifuge tube with the AAV in a container with crushed ice during set up.</li>
	<li>Place the microcentrifuge tube with the virus into a standard bench vice. Load a 10 &#181;l syringe with at least 4 &#181;l of AAV. Take care not to bend the tip of the syringe against the bottom of the microcentrifuge tube during loading. Ensure that there are no air bubbles in the 4 &#181;l of AAV solution in the syringe.</li>
	<li>Dispose of the empty virus tube in the beaker containing 10% bleach. Store unused portions of the virus as specified by the supplier.</li>
	<li>Add additional 10% bleach to the waste container, and allow waste to sit for 30 min before disposing the decontaminated liquid waste.</li>
	<li>Dispose of all protective equipment and plastic waste in a biohazard container as instructed by institutional guidelines. Decontaminate any equipment or surfaces that came in contact with the virus using 10% bleach.</li>
</ol>
2. Surgery
<ol>
	<li>Prepare the surgical area by placing absorbent bench paper under the stereotaxic apparatus and on an adjacent space designated as a dedicated virus handling area.
	<ol>
		<li>Place a 10% bleach waste container in the dedicated virus handling area near the surgical apparatus.</li>
	</ol>
	</li>
	<li>Induce a steady level of anesthesia and prepare the rat for surgery.
	<ol>
		<li>Place the rat into an induction chamber that contains a mixture of isoflurane gas (range of 1 to 3%) and oxygen (100% at approximately 1 L/min) until there is a loss of consciousness and lack of gross purposeful movement. Maintain isoflurane anesthesia throughout the surgical procedure.</li>
		<li>After 6 deep anesthesia is attained, shave the rat from between the eyes to slightly behind the ears.&#160;</li>
		<li>Place the rat back into the induction chamber for an additional 1-3 min.</li>
		<li>Ensure that the isoflurane anesthesia system is connected to the nose cone of the stereotaxic surgery. Open the stopcock on the isoflurane tubing to begin the flow of isoflurane to the stereotax. Maintain the isoflurane and oxygen at the levels specified above.</li>
		<li>Place the rat in a stereotaxic apparatus by securing the mouth onto the bite bar and securing the ear bars.</li>
		<li>Apply eye lubricant to the eyes and betadine to the surgical site prior to incision.</li>
	</ol>
	</li>
	<li>Make a 2.4 cm midline incision of the skin on the dorsal surface of the skull starting approximately 2 mm caudal to the eyes. Retract the skin to expose the skull. Periodically instill sterile 0.9% sterile saline on the margins of the surgical site to prevent tissues from drying.&#160;</li>
	<li>Clear membranous tissue from the skull until bregma and lambda are clearly visible.</li>
	<li>Ensure that the skull is level by measuring the dorsal-ventral coordinates at bregma and at lambda. Adjust the stereotaxic device accordingly until dorsal-ventral coordinates at bregma and lambda differ by no more than 0.04 mm.</li>
	<li>At this time, reduce the isoflurane gas to between 2 and 2.5%.</li>
	<li>Return the drill to bregma, rezero the coordinates and then move the drill to the desired medial-lateral and anterior-posterior coordinate.</li>
	<li>Drill a hole at the coordinate using a 0.9 mm drill bit as it produces an appropriate size hole for a 28 G infusion syringe.</li>
	<li>Set the infusion pump to deliver virus at a rate of 0.2 &#181;l/min. Place the syringe into the infusion arm of the stereotaxic device and lower the syringe into the desired coordinate. Deliver the desired amount of virus (range 0.1 to 0.8 &#181;l). For example, deliver 0.8 &#181;l of AAV over 4 min. 
	Note: conduct pilot studies to determine the ideal infusion volume and the typical spread of virus in each region of interest.
	<ol>
		<li>After the delivery of virus is complete leave the syringe in place for 10 min to prevent backflow into the needle tract. Slowly raise the syringe at a rate of about 5 mm/min. Repeat until all coordinates have been infused with AAV.</li>
	</ol>
	</li>
	<li>Close the wound using surgical staples and coat the wound with topical antibiotic ointment.</li>
	<li>Follow institutional guidelines for post-operative pain management.</li>
	<li>Place the rat in a cage with bedding, a lid and proper signage and return the rat to the animal facility.
	<ol>
		<li>If working under BSL-2 precautions, collect bedding for the amount of time (typically 48-72 hr) specified by institutional guidelines. Place bedding into a biohazard waste container.</li>
		<li>Dispose of all AAV-contaminated materials as specified by guidelines for their safe disposal.</li>
	</ol>
	</li>
</ol>
3. Behavioral Apparatus
Note: The sensory preconditioning apparatus consists of a standard operant conditioning chamber (12&#8221; L x 9.5&#8221; W x 11.5&#8221; H) with a stainless steel grid floor, 2 Plexiglas sidewalls and 2 metal walls.
<ol>
	<li>Mount a 2.8 W light on one of the aluminum walls to serve as the house light and as the visual stimulus when flashed at 2 Hz. Mount a speaker on the chamber to deliver the auditory stimuli (a 10 sec, 1500 Hz, 78 dB pure tone and a 10 sec 78 dB white noise).
	<ol>
		<li>Use a food hopper to deliver 45 mg food pellets into the food cup. Within the food cup, infrared photocells detect the total time spent in the food cup before, during and after presentations of the stimuli and food rewards.</li>
		<li>House the chambers in sound-attenuating cabinets (22&#8221; W x 22&#8221; H x 16&#8221; D) outfitted with exhaust fans (~68 dB).</li>
		<li>Use a PC computer with Med Associates software to control the operant chamber and acquire the data. During the Conditioning sessions, collect data on the total time spent with the head in the food cup during presentation of the light stimulus (5 sec epoch) and during presentation of the food reward (5 sec epoch). During the Test session, collect data on the total time spent in the food cup in response to presentations of the auditory stimuli (10 sec epoch beginning when the auditory stimuli are terminated).</li>
	</ol>
	</li>
</ol>
4. Overview of the Sensory Preconditioning Paradigm
<ol>
	<li>Following recovery from surgery, gradually food restrict the rats to 85% of their free feeding body weight. Consult veterinary staff for an appropriate food restriction paradigm. 
	Note: The 3-phase sensory preconditioning paradigm is illustrated in Figure 2.</li>
	<li>Preconditioning Sessions (Phase 1; Daily 64 min training sessions conducted across 4 consecutive days):
	<ol>
		<li>Present rats with 12 intermixed trials that consist of delivery of auditory and visual stimuli. Include inter-trial intervals that average 4.5 min (range 4.0 to 5.0 min) after each auditory presentation.</li>
		<li>On 6 of the trials, present a tone (preconditioned stimulus) for 10 sec, followed immediately by a 5 sec presentation of the flashing light stimulus.</li>
		<li>On the other 6 trials, present the white noise (unpaired stimulus) alone for 10 sec.</li>
	</ol>
	</li>
	<li>Conditioning sessions (Phase 2; Daily 64 min training sessions conducted across 5 consecutive days):
	<ol>
		<li>Present rats with 8 trials that consist of delivery of the visual stimulus (the flashing light stimulus) for 5 sec followed immediately by delivery of two 45 mg food pellets. Include inter-trial intervals that average 7 min (range 6.0 to 8.0 min) after each trial. Occasionally rats require more than 5 conditioning sessions to learn the light food association.</li>
	</ol>
	</li>
	<li>Test Session (Phase 3; a single 78 min session):
	<ol>
		<li>Present the rats with 12 intermixed trials that consist of delivery of the auditory stimuli alone. Include inter-trial intervals that average 4.5 min (range 4.0 to 5.0 min) after each auditory presentation.</li>
		<li>On 6 of the trials, present the tone stimulus (the preconditioned stimulus) alone for 10 sec.</li>
		<li>On the other 6 trials, present the white noise (the unpaired stimulus) alone for 10 sec.</li>
	</ol>
	</li>
	<li>Counterbalance the stimuli by completing the study with a second set of rats that receive the white noise as the preconditioned stimulus and the tone as the unpaired stimulus.</li>
</ol>
5. Pharmacological and Behavioral Procedure
<ol>
	<li>Turn on the power to the behavioral apparatus and load the Med-PC code for the appropriate behavioral session. Wire and program the behavioral apparatus such that the fans that are mounted on the sound attenuating cabinets turn on when the power is switched on.</li>
	<li>To prepare a 1 mg/ml solution of clozapine N-oxide (CNO), weigh 5.0 mg of CNO into a 15 ml conical tube and add 5 ml of sterile water or 5 ml of sterile 0.9% saline. Vortex until the solution is clear.
	<ol>
		<li>If CNO does not dissolve into solution or if more concentrated solutions of CNO are desired add a solubilizing agent, such as DMSO23. Specifically, to prepare a 1 mg/ml solution of CNO with 0.5% DMSO, weigh 5.0 mg of CNO and add 25 &#181;l of DMSO to a 15 ml conical tube. Flick gently ensuring that the contents remain at the base of the tube. When the solution becomes clear, add 5 ml of sterile water or 5 ml of sterile 0.9% saline. Further instructions for preparing solutions of CNO are available on the DREADD wiki resource page25.</li>
		<li>Prepare a new CNO solution each day that it is administered.</li>
	</ol>
	</li>
	<li>Inject the CNO intraperitoneally at a dose of 1 mg/kg approximately 30 min prior to behavioral testing. For example, inject a 200 g rat with 0.2 ml of the 1 mg/ml CNO solution. The dose and time course of CNO administration were selected based on previously published reports2,12.
	<ol>
		<li>Inject CNO 30 min prior to each preconditioning session (Phase 1; total of 4 days of injections) but administer the vehicle prior to all subsequent behavioral sessions.</li>
	</ol>
	</li>
	<li>After 30 min, place the rats into the behavioral testing chambers and initiate the computer code for the appropriate behavioral session.</li>
	<li>Upon completion of the behavioral task, immediately remove the rats from the chambers and return the rats to the animal colony.</li>
</ol>
6. Analyses of Behavioral Data
Note: The dependent variable for all behavioral sessions is the amount of time that the rat&#8217;s head is inside the food cup as detected by interruption of the infrared photocells in the food cup. The data (in sec) are collected and recorded by the computer software.
<ol>
	<li>Do not conduct analyses on data generated during the Preconditioning phase.</li>
	<li>For the conditioning sessions: analyze each rats&#8217; ability to learn the light food association by comparing food cup behavior during presentation of the visual stimulus across the 5 behavioral sessions. Specifically, calculate the average time spent in the food cup during the 5 sec light epoch for each of 8 trials/session (i.e., an average of 8 trials that are each 5 sec in duration/rat). Compare average control and experimental values for each of 5 daily sessions (see Figure 3A).
	<ol>
		<li>Analyze the rats&#8217; motivation to obtain food reward by comparing food cup behavior during delivery of the food reward (5 sec epoch) across the 5 behavioral sessions using the same calculation as specified for the 5 sec light epoch above. These values will be consistently higher than those acquired during presentation of the visual stimulus (see Figure 3B).</li>
	</ol>
	</li>
	<li>For the test session: calculate a discrimination ratio that describes the rats&#8217; food cup behavior in response to presentations of each auditory stimulus.
	<ol>
		<li>Specifically, for each rat, divide the average time spent in the food cup following each of 6 presentations of the sensory preconditioned stimulus (i.e., the tone) by the sum of the average time spent in the food cup following each of 6 presentations of the sensory preconditioned stimulus (i.e., the tone stimulus) plus the average of the total time spent in the food cup following each of 6 presentations of the unpaired stimulus (i.e., the white noise stimulus). Each epoch is 10 sec in duration. 
		Note: A discrimination score of 0.5 or greater demonstrates that rats learned the associations inherent in the sensory preconditioning paradigm.</li>
	</ol>
	</li>
</ol>
7. Verification of AAV Placement and Expression
<ol>
	<li>Anesthetize and perfuse the rats transcardially using 4% paraformaldehyde. Due to the use of fluorescent labels, do not exceed a post-fixation time of 2 hr to minimize loss of antigenicity and to preserve the fluorescent signal.</li>
	<li>Dehydrate perfused brains by transferring them into a brain jar containing 30 ml of a 20% sucrose solution in phosphate buffered saline (1x) for a minimum of 2 days or until the brain sinks to the bottom of the brain jar.</li>
	<li>Use a freezing sliding microtome to make coronal brain sections (20-40 &#181;m) throughout the entire rostrocaudal extent of the region of interest.</li>
	<li>Conduct immunohistochemistry directed against the tagged receptor or the reporter protein to ascertain the location and expression of the designer receptor and/or reporter12. An immunohistochemistry staining protocol is provided on the DREADD wiki resource page25.</li>
	<li>Mount the sections onto superfrost-plus slides and coverslip using 100-200 &#181;l of an aqueous mounting medium.</li>
	<li>Perform fluorescence microscopy on brain tissue to verify AAV placement and protein expression12,24.</li>
</ol>

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The authors declare that they have no competing financial interests.

This protocol describes how to apply a pharmacogenetic approach (DREADD) to investigate how a specific brain region contributes to a multi-phase complex learning task. With the ability to temporarily and remotely silence neural activity in discrete brain regions across phases of learning, this combination of approaches provides a platform to investigate a wide range of behaviors, including more nuanced or masked forms of learning. In the example described in this protocol, control rats and rats that express the designer ...

An exciting challenge within the field of behavioral neuroscience is to determine the neural substrates of complex behaviors. A number of techniques such as permanent lesions, temporary brain inactivation via cannulae implants and optogenetics have been employed to identify the contributions of discrete brain regions to subcomponents of complex behaviors. While these approaches inform our understanding of regional specificity during learning, each technique is not without limitations. Specifically, permanent lesions are typically conducted prior to behavioral testing, thus their effects are present throughout the duration of the paradigm. Cannulation studies that involve the presentation of a short-term neural inactivator (e.g., tetrodotoxin) can produce substantial damage to brain tissue and can induce stress in subjects just prior to behavioral testing. Furthermore, inactivation through cannulation is limited to the region of tissue that surrounds the tip of the cannulae. Lastly, while optogenetics offers a range of flexibility for the temporal control of activity in specific brain regions, it is cost prohibitive and technically demanding.
These limitations can be surmounted using a pharmacogenetic approach (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs, DREADDs)1,2. Importantly, while the concept of pharmacogenetics is sophisticated, the execution of the technique is straightforward. Similar to traditional stereotaxic surgical methods that involve infusion of toxin (e.g., NMDA, ibotenic acid) into discrete brain regions, this technique involves infusing an adeno-associated virus (AAV) that contains a DNA fragment for a modified inhibitory G-protein coupled receptor (hM4Di; the designer receptor) into the region of interest of standard laboratory rodents (see Figure 1). The viral vector also contains a fluorescent reporter (mcitrine). Once incorporated into cells, the designer receptor (and reporter protein) are maximally expressed ~3 weeks post-infusion and can be selectively activated for 2-5 hr by systemic administration of the otherwise biologically inert designer drug, clozapine-N-oxide (CNO)1,3. Because the experimenter is endowed with precise, yet remote temporal control over neural activity in specific brain regions, pharmacogenetics combines particularly well with behavioral paradigms that are conducted in multiple phases. In this example, the contribution of the retrosplenial cortex (RSC) to stimulus-stimulus learning is compared to its role in Pavlovian learning, however this combination of approaches is well suited to any number of questions that seek to identify how specific brain regions contribute to complex behavior.
In addition, while not described in the present protocol, viral and transgenic approaches can be used to achieve cell type-specific DREADD expression2. As is inherent in behavioral paradigms that involve pharmacological and/or other types of experimental manipulations, careful consideration of experimental design and subsequent quantitative analysis is required when employing the DREADD approach. Experimenters new to the DREADD approach are referred to a comprehensive review of current DREADD technology2.
Each day, organisms learn about new stimuli and events and their relationships to one another. Even in a familiar environment, such as home, one is quick to detect alterations in the relationships between stimuli because these changes may be predictive of meaningful events. Such stimulus-stimulus (i.e., relational) learning involves the conjoining of multiple stimuli and has traditionally been associated with the hippocampus, which resides centrally within the medial temporal lobe4. However, the hippocampus does not exist nor act in isolation; cortical regions both within and outside of the medial temporal lobe provide critical sensory information to the hippocampal formation5-7. Traditional permanent lesion studies provide compelling evidence for the involvement of a number of cortical regions (e.g., the retrosplenial, postrhinal and entorhinal cortices) in hippocampal-dependent learning but are limited in their ability to discern the role of a particular region during discrete phases of learning8-10.
The present protocol tests the hypothesis that the RSC is necessary for stimulus-stimulus learning by silencing the RSC during a single phase of a 3-phase sensory preconditioning paradigm11,12. Briefly, rats receive infusions of an AAV that contains the designer receptor and ~3 weeks later are administered the designer drug (CNO) 30 min prior to the start of behavioral testing. In the present protocol, experimental rats receive CNO during the first phase of testing (when stimulus-stimulus learning occurs) and they receive vehicle during the next 2 phases of testing. To control for inadvertent effects of CNO on behavior, infuse rats with the designer receptor (hM4Di) and inject with vehicle instead of CNO. To account for general effects of viral infusion and receptor expression, infuse a control virus that does not contain the designer receptor and administer CNO.
A number of different serotypes of AAV are used to deliver genetic material. The current NIH Guidelines for Research Involving Recombinant or Synthetic Molecules maintains that AAV (all serotypes) and recombinant or synthetic AAV constructs, in which the transgene does not encode either a potentially tumorigenic gene product or a toxin molecule and are produced in the absence of a helper virus, require BSL-1 precautions (Appendix B-1. Risk Group 1 (RG1) Agents)13. A number of reviews pertaining to AAV structure, utility and safety are available14,15. Notably, though, due to concerns pertaining to possible reproductive16,17 and potential carcinogenic mechanisms18-20 in rodents, some institutions require the use of BSL-2 precautions when working with AAV. Verify the appropriate BSL prior to use by consulting with oversight committees at individual institutions where the research will be conducted, the Centers for Disease Control and the NIH Guidelines for Research Involving Recombinant DNA Molecules13 when using viral vectors for gene manipulation in the United States. Personal protection, investigator training, vector containment, decontamination, disposal of decontaminated materials, and post-injection animal housing requirements are specified by these guidelines. In addition, consult and follow appropriate Institutional Animal Care and Use committee guidelines or equivalent institutional oversight committee guidelines to ensure the safe handling, administration and disposal of AAV.

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a method for remotely silencing neural activity in rodents during discrete phases of learning

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different forms of learning. Specifically, viral-mediated delivery is used to express a genetically modified inhibitory G-protein coupled receptor (the Designer Receptor) into a discrete brain region in the rodent. Three weeks later, when designer receptor expression levels are high, a pharmacological agent (the Designer Drug) is administered systemically 30 min prior to a specific behavioral session. The drug has affinity for the designer receptor and thus results in inhibition of neurons that express the designer receptor, but is otherwise biologically inert. The brain region remains silenced for 2-5 hr (depending on the dose and route of administration). Upon completion of the behavioral paradigm, brain tissue is assessed for correct placement and receptor expression. This approach is particularly useful for determining the contribution of individual brain regions to specific components of behavior and can be used across any number of behavioral paradigms.

Behavioral Results
Upon completion of the experiment, the effectiveness of the region-specific temporary inactivation should be quantitatively and qualitatively assessed. The present example involves a 3-phase behavioral paradigm (sensory preconditioning), in which CNO was administered to attenuate neural activity in the RSC during the Preconditioning sessions to test the hypothesis that the RSC is necessary for the formation of associations among neutral stimuli12. Importantly, experi...

Watch this Scientific Journal Video about A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning at JoVE.com

A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while rats are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different components of learning.

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and ...

a-method-for-remotely-silencing-neural-activity-rodents-during

Research

JoVE Journal

Behavior

14.6K 조회수.  Oberlin College. This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while rats are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different components of learning.

비디오: A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning

Construction of Microdrive Arrays for Chronic Neural Recordings in Awake Behaving Mice

State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field potentials (LFPs) from populations of neurons and action potentials from individual cells, as the animal engages in experimentally relevant tasks. Chronically implanted microdrives allow for brain recordings to last over periods of several weeks. Miniaturized drives and lightweight components allow for these long-term recordings to occur in small mammals, such as mice. By using tetrodes, which consist of tightly braided bundles of four electrodes in which each wire has a diameter of 12.5 &mu;m, it is possible to isolate physiologically active neurons in superficial brain regions such as the cerebral cortex, dorsal hippocampus, and subiculum, as well as deeper regions such as the striatum and the amygdala. Moreover, this technique insures stable, high-fidelity neural recordings as the animal is challenged with a variety of behavioral tasks. This manuscript describes several techniques that have been optimized to record from the mouse brain. First, we show how to fabricate tetrodes, load them into driveable tubes, and gold-plate their tips in order to reduce their impedance from M&Omega; to K&Omega; range. Second, we show how to construct a custom microdrive assembly for carrying and moving the tetrodes vertically, with the use of inexpensive materials. Third, we show the steps for assembling a commercially available microdrive (Neuralynx VersaDrive) that is designed to carry independently movable tetrodes. Finally, we present representative results of local field potentials and single-unit signals obtained in the dorsal subiculum of mice. These techniques can be easily modified to accommodate different types of electrode arrays and recording schemes in the mouse brain.

The design and assembly of microdrives for in vivo electrophysiological recordings of brain signals from the mouse is described. By attaching microelectrode bundles to sturdy driveable carriers, these techniques allow for long-term and stable neural recordings. The lightweight design allows for unrestricted behavioral performance by the animal following drive implantation.

State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field ...

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.

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 ...

Measuring Neural and Behavioral Activity During Ongoing Computerized Social Interactions: An Examination of Event-Related Brain Potentials

Social exclusion is a complex social phenomenon with powerful negative consequences. Given the impact of social exclusion on mental and emotional health, an understanding of how perceptions of social exclusion develop over the course of a social interaction is important for advancing treatments aimed at lessening the harmful costs of being excluded. To date, most scientific examinations of social exclusion have looked at exclusion after a social interaction has been completed. While this has been very helpful in developing an understanding of what happens to a person following exclusion, it has not helped to clarify the moment-to-moment dynamics of the process of social exclusion. Accordingly, the current protocol was developed to obtain an improved understanding of social exclusion by examining the patterns of event-related brain activation that are present during social interactions. This protocol allows greater precision and sensitivity in detailing the social processes that lead people to feel as though they have been excluded from a social interaction. Importantly, the current protocol can be adapted to include research projects that vary the nature of exclusionary social interactions by altering how frequently participants are included, how long the periods of exclusion will last in each interaction, and when exclusion will take place during the social interactions. Further, the current protocol can be used to examine variables and constructs beyond those related to social exclusion. This capability to address a variety of applications across psychology by obtaining both neural and behavioral data during ongoing social interactions suggests the present protocol could be at the core of a developing area of scientific inquiry related to social interactions.

Research on social exclusion has grown tremendously in recent years. As the field expands, it is imperative to develop sophisticated methodologies allowing for the simultaneous measurement of neural and behavioral outcomes during social exclusion. This protocol utilizes event-related brain potentials to record ongoing neural activity during computerized social interactions.

Social exclusion is a complex social phenomenon with powerful negative consequences. Given the impact of social exclusion on mental and emotional health, ...

The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their behavioral flexibility and required memory duration, the number of goals and pathways, and also the overall task complexity. A confounding feature in many of these tasks is the lack of control over the strategy employed by the rodents to reach the goal, e.g., allocentric (declarative-like) or egocentric (procedural) based strategies. The double-H maze is a novel water-escape memory task that addresses this issue, by allowing the experimenter to direct the type of strategy learned during the training period. The double-H maze is a transparent device, which consists of a central alleyway with three arms protruding on both sides, along with an escape platform submerged at the extremity of one of these arms.
Rats can be trained using an allocentric strategy by alternating the start position in the maze in an unpredictable manner (see protocol 1; &#167;4.7), thus requiring them to learn the location of the platform based on the available allothetic cues. Alternatively, an egocentric learning strategy (protocol 2; &#167;4.8) can be employed by releasing the rats from the same position during each trial, until they learn the procedural pattern required to reach the goal. This task has been proven to allow for the formation of stable memory traces.
Memory can be probed following the training period in a misleading probe trial, in which the starting position for the rats alternates. Following an egocentric learning paradigm, rats typically resort to an allocentric-based strategy, but only when their initial view on the extra-maze cues differs markedly from their original position. This task is ideally suited to explore the effects of drugs/perturbations on allocentric/egocentric memory performance, as well as the interactions between these two memory systems.

The goal of this protocol is to investigate spatial cognition in rodents. The double-H water maze is a novel test, which is particularly useful to elucidate the different components of learning, consolidation and memory, as well as the interplay of memory systems.

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their ...

Quantifying Learning in Young Infants: Tracking Leg Actions During a Discovery-learning Task

Task-specific actions emerge from spontaneous movement during infancy. It has been proposed that task-specific actions emerge through a discovery-learning process. Here a method is described in which 3-4 month old infants learn a task by discovery and their leg movements are captured to quantify the learning process. This discovery-learning task uses an infant activated mobile that rotates and plays music based on specified leg action of infants. Supine infants activate the mobile by moving their feet vertically across a virtual threshold. This paradigm is unique in that as infants independently discover that their leg actions activate the mobile, the infants&#8217; leg movements are tracked using a motion capture system allowing for the quantification of the learning process. Specifically, learning is quantified in terms of the duration of mobile activation, the position variance of the end effectors (feet) that activate the mobile, changes in hip-knee coordination patterns, and changes in hip and knee muscle torque. This information describes infant exploration and exploitation at the interplay of person and environmental constraints that support task-specific action. Subsequent research using this method can investigate how specific impairments of different populations of infants at risk for movement disorders influence the discovery-learning process for task-specific action.

A method is described in which 3-4 month old infants learn a task by discovery and their leg movements are captured to quantify the learning process.

Task-specific actions emerge from spontaneous movement during infancy. It has been proposed that task-specific actions emerge through a discovery-learning ...

Asymmetric Walkway: A Novel Behavioral Assay for Studying Asymmetric Locomotion

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

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

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

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 ...

A Novel Experimental and Analytical Approach to the Multimodal Neural Decoding of Intent During Social Interaction in Freely-behaving Human Infants

Understanding typical and atypical development remains one of the fundamental questions in developmental human neuroscience. Traditionally, experimental paradigms and analysis tools have been limited to constrained laboratory tasks and contexts due to technical limitations imposed by the available set of measuring and analysis techniques and the age of the subjects. These limitations severely limit the study of developmental neural dynamics and associated neural networks engaged in cognition, perception and action in infants performing &#8220;in action and in context&#8221;. This protocol presents a novel approach to study infants and young children as they freely organize their own behavior, and its consequences in a complex, partly unpredictable and highly dynamic environment. The proposed methodology integrates synchronized high-density active scalp electroencephalography (EEG), inertial measurement units (IMUs), video recording and behavioral analysis to capture brain activity and movement non-invasively in freely-behaving infants. This setup allows for the study of neural network dynamics in the developing brain, in action and context, as these networks are recruited during goal-oriented, exploration and social interaction tasks.

This protocol presents a novel methodology for the neural decoding of intent from freely-behaving infants during unscripted social interaction with an actor. Neural activity is acquired using non-invasive high-density active scalp electroencephalography (EEG). Kinematic data is collected with inertial measurement units and supplemented with synchronized video recording.

Understanding typical and atypical development remains one of the fundamental questions in developmental human neuroscience. Traditionally, experimental ...

Feeding Experimentation Device (FED): Construction and Validation of an Open-source Device for Measuring Food Intake in Rodents

Food intake measurements are essential for many research studies. Here, we provide a detailed description of a novel solution for measuring food intake in mice: the Feeding Experimentation Device (FED). FED is an open-source system that was designed to facilitate flexibility in food intake studies. Due to its compact and battery powered design, FED can be placed within standard home cages or other experimental equipment. Food intake measurements can also be synchronized with other equipment in real-time via FED's transistor-transistor logic (TTL) digital output, or in post-acquisition processing as FED timestamps every event with a real-time clock. When in use, a food pellet sits within FED's food well where it is monitored via an infrared beam. When the pellet is removed by the mouse, FED logs the timestamp onto its internal secure digital (SD) card and dispenses another pellet. FED can run for up to 5 days before it is necessary to charge the battery and refill the pellet hopper, minimizing human interference in data collection. Assembly of FED requires minimal engineering background, and off-the-shelf materials and electronics were prioritized in its construction. We also provide scripts for analysis of food intake and meal patterns. Finally, FED is open-source and all design and construction files are online, to facilitate modifications and improvements by other researchers.

Feeding Experimentation Device FED: Construction and Validation of an Open-source Device for Measuring Food Intake in Rodents

Feeding Experimentation Device (FED) is an open-source device for measuring food intake in mice. FED can also synchronize food intake measurements with other techniques via a real-time digital output. Here, we provide a step-by-step tutorial for the construction, validation, and usage of FED.

Food intake measurements are essential for many research studies. Here, we provide a detailed description of a novel solution for measuring food intake in ...

A Computational Method to Quantify Fly Circadian Activity

In most animals and plants, circadian clocks orchestrate behavioral and molecular processes and synchronize them to the daily light-dark cycle. Fundamental mechanisms that underlie this temporal control are widely studied using the fruit fly Drosophila melanogaster as a model organism. In flies, the clock is typically studied by analyzing multiday locomotor recording. Such a recording shows a complex bimodal pattern with two peaks of activity: a morning peak that happens around dawn, and an evening peak that happens around dusk. These two peaks together form a waveform that is very different from sinusoidal oscillations observed in clock genes, suggesting that mechanisms in addition to the clock have profound effects in producing the observed patterns in behavioral data. Here we provide instructions on using a recently developed computational method that mathematically describes temporal patterns in fly activity. The method fits activity data with a model waveform that consists of four exponential terms and nine independent parameters that fully describe the shape and size of the morning and evening peaks of activity. The extracted parameters can help elucidate the kinetic mechanisms of substrates that underlie the commonly observed bimodal activity patterns in fly locomotor rhythms.

A method to quantify the main temporal features seen in fly circadian locomotor rhythms is presented. The quantification is achieved by fitting fly activity with a multi-parametric model waveform. The model parameters describe the shape and size of the morning and evening peaks of daily activity.