This work is supported by grants from the National Institutes of Mental Health NRSA F32 MH120887-03 (to G.J.B.) and R01 NS045193 and R01 MH115750 (to S.S-H.W.). We thank Drs. Bas Koekkoek and Henk-Jan Boele for helpful discussions for optimizing the DEC setup and Drs. Yue Wang and Xiaoying Chen for helpful discussions for optimizing the DTSC setup.

The animal protocols described here have been approved by the Animal Care and Use Committees of Princeton University.
1. Setting up the SBC
<ol>
	<li>Connect the camera serial interface (CSI) cable to the Raspberry NoIR V2 camera and the camera port on the SBC.</li>
	<li>Download the operating system for the SBC onto the host computer. Write the operating system image to a micro secure digital (microSD) card. 
	NOTE: Detailed instructions for these procedures for a Raspberry Pi SBC can be found elsewhere23. The system has been tested using the following operating systems: Stretch, Buster, Bullseye.</li>
	<li>To enable secure shell communication, create an extensionless file called &#34;ssh&#34; in the boot partition of the microSD card. Once this is done, eject the microSD card from the host machine and insert it into the SBC microSD card slot. Power the SBC by plugging in its power supply.</li>
	<li>Prepare the SBC to accept a wired connection to the host.
	<ol>
		<li>Attach a monitor with an appropriate cable to the SBC. Open a terminal, type the command ifconfig and record the ethernet IP address of the SBC. 
		NOTE: Raspberry Pi model 3B+ has an HDMI display port, while model 4B has a micro-HDMI port.</li>
		<li>Go to the Interface tab of the Raspberry Pi configuration setting and enable the options for Camera, secure shell network protocol (SSH), and Virtual Network Computing (VNC).</li>
	</ol>
	</li>
	<li>Establish a wired connection between the host computer and the SBC.
	<ol>
		<li>Connect an ethernet cable to the ethernet port on the SBC and a host computer. Attach the other end of these cables to an ethernet switch.</li>
		<li>Use a virtual network computing client such as VNC viewer24 and access the desktop using the SBC IP address and the default authentication (user = &#34;pi&#34;, password = &#34;raspberry&#34;).</li>
	</ol>
	</li>
	<li>Download required software included in the protocol steps. 
	CAUTION: Change the default username and password to prevent unauthorized access to the SBC.
	<ol>
		<li>Enter the following command in the SBC terminal to download the rig software: 
		git clone --depth=1&#160;https://github.com/gerardjb/assocLearnRig</li>
		<li>Enter the following commands to download the necessary python libraries. 
		cd assocLearnRig 
		python3 setup.py</li>
		<li>To allow direct control over the microcontroller, connect to the SBC and download the microcontroller integrated development environment (IDE) following steps 1.6.4-1.6.7.</li>
		<li>Open the web browser on the SBC desktop and navigate to&#160;https://arduino.cc/en/software. Download the latest Linux ARM 32 bit version of the IDE.</li>
		<li>Open a terminal window on the SBC desktop and navigate to the downloads directory by typing cd Downloads/</li>
		<li>To install the IDE, type the following commands in the terminal: 
		tar -xf arduino-&#60;version&#62;-linuxarm.tar.xz 
		sudo mv arduino-&#60;version&#62; /opt 
		sudo /opt/arduino-&#60;version&#62;/install.sh 
		(here &#60;version&#62; is the version of the downloaded IDE)</li>
		<li>Open an instance of the microcontroller IDE on the SBC desktop. Select menu option Tools &#62; Manage Libraries. Install the &#34;Encoder&#34; library from Paul Stoffregen.</li>
	</ol>
	</li>
	<li>Expand SBC onboard memory with a USB thumb drive.
	<ol>
		<li>Insert a thumb drive to a USB port on the SBC. Use a USB 3.0 port if available.</li>
		<li>Type in the terminal ls -l /dev/disk/by-uuid/ to find the thumb drive and its unique reference (UUID). Record the UUID.</li>
		<li>To allow the pi user to write to the USB device, type the following commands one by one into the terminal: 
		sudo mkdir /media/usb 
		sudo chown -R pi:pi /media/usb 
		sudo mount /dev/sda1 /media/usb -o uid=pi,gid=pi 
		NOTE: The thumb drive can be added as a device that will auto-mount when the SBC restarts by adding the following line to the end of the fstab file at /etc/fstab: 
		&#8203;UUID=&#60;UUID from step 1.7.2&#62; /media/usb vfat auto,nofail,noatime,users,rw,uid=pi,gid=pi 0 0</li>
	</ol>
	</li>
</ol>
2. Wiring stimulus hardware and assembling stage
<ol>
	<li>Connect and prepare microcontrollers.
	<ol>
		<li>Connect the SBC to the programming port of the microcontroller (Arduino Due) with a USB2 type A to USB2 micro cable. 
		&#8203;NOTE: Use a high-quality cable such as the product in the Table of Materials to ensure proper operation.</li>
		<li>Locate &#34;dueAssocLearn.ino&#34; in the downloaded project repository. Open the sketch with the microcontroller IDE and upload it to the microcontroller connected to the SBC.</li>
		<li>Download and install the appropriate version of the Arduino IDE on the host computer.</li>
		<li>Connect the host computer to the microcontroller (Arduino Uno) with a USB2 type B to USB2 type A cable.</li>
		<li>Go to the GitHub repository (https://github.com/gerardjb/assocLearnRig) and&#160;download the &#34;DTSC_US.ino&#34; sketch to the host computer.</li>
		<li>On the host computer, run the microcontroller IDE and open the &#34;DTSC_US.ino&#34; sketch, then upload it to the microcontroller.</li>
	</ol>
	</li>
	<li>Attach wires to the microcontrollers, breadboard, LEDs, rotary encoder, stepper motor with driver, and solenoid valve with driver as indicated in the Fritzing diagram in Figure 2.</li>
	<li>Power the stepper motor and solenoid valve.
	<ol>
		<li>Properly wire one channel of a power supply to the +V and GND pins of the stepper motor driver.</li>
		<li>Turn on the power supply and set the attached channel voltage to 25 V. 
		&#8203;NOTE: If the connections between the stepper motor, driver, and power supply are correctly configured, a green indicator LED on the stepper motor driver will turn on.</li>
		<li>Properly wire the positive lead of a power supply to the solenoid valve driver hold voltage pin and the other positive lead to the spike voltage pin.</li>
		<li>Attach the negative leads to a ground shared with the control signal.</li>
		<li>Turn on the power supply and set the channel connected to the hold voltage to about 2.5 V and the channel connected to spike voltage to about 12 V.</li>
	</ol>
	</li>
	<li>Connect an air source regulated to a pressure of ~20 PSI to the solenoid valve using the luer adapter.</li>
	<li>Test that all stimulus components and camera are functioning properly.
	<ol>
		<li>Open a terminal on the SBC and type cd ~/assocLearnRig to navigate to the cloned GitHub repository.</li>
		<li>In the terminal, type python3 assocLearnRig_app.py to start the control graphical user interface.</li>
		<li>Start the camera stream by hitting the Stream button.</li>
		<li>Select the DEC Radio button, upload to the microcontroller, and start a session with default parameters by hitting the Start Session button. 
		&#8203;NOTE: After this step, a printout of the data log should appear in the terminal, the message on the camera stream should disappear, and the LED CS and solenoid valve US should turn on and off at appropriate times during each trial.</li>
		<li>After the session ends, repeat the previous steps with the DTSC Radio button selected. 
		NOTE: Sketches in the GitHub repository (&#34;testStepper.ino&#34;, &#34;testRotary.ino&#34;, and &#34;testSolenoid.ino&#34;) can be used to test individual components if the above steps do not provide satisfactory results.</li>
	</ol>
	</li>
	<li>Make the running wheel.
	<ol>
		<li>Cut a 3&#34; wheel from a foam roller. Drill a 1/4&#34; hole in the exact wheel center so that the wheel will not wobble when turned by the mouse&#39;s locomotion.</li>
		<li>Insert a 1/4&#34; shaft into the wheel and fix it in place using clamping hubs placed on each side of the wheel.</li>
	</ol>
	</li>
	<li>Affix the rotary encoder to a 4.5&#34; aluminum channel using an M3 bolt. Stabilize the aluminum channel on the aluminum breadboard using a right angle bracket with a 1/4&#34; bolt, nut, and washer as shown.</li>
	<li>Attach the wheel and rotary encoder using a shaft-coupling sleeve.</li>
	<li>Stabilize the free side of the wheel shaft with a bearing inserted in a right-angle end clamp installed on a breadboard-mounted optical post. 
	&#8203;NOTE: Ensure the wheel spins freely without wobbling when rotated by hand.</li>
	<li>Position the stimulus hardware, head restraint, infrared light array, and picamera around the assembled wheel.
	<ol>
		<li>Position the head restraints using optical posts and right angle post clamps so that the head posts are 1.5 cm in front of the wheel axle and 2 cm above the wheel surface. (Values are for a 20 g mouse).</li>
		<li>Position the CS LED and solenoid valve outlet used for the DEC US less than 1 cm from the eye used for DEC.</li>
		<li>Mount the stepper motor used for the DTSC US</li>
		<li>Mount the picamera on an optical post ~10 cm from where the animal will be. 
		&#8203;NOTE: The design for the picamera mount can be made on a 3D printer from the file in &#34;RaspPiCamMount1_1.stl&#34; in the GitHub repository.</li>
		<li>Place the infrared light array slightly above and directly facing the position of the face on the same side as the picamera.</li>
		<li>Make a tactile stimulus for DTSC by taping foam to the edge of a piece of acrylic mounted to a 1/4&#34; shaft using a clamping hub. Attach the tactile stimulus to the stepper motor shaft. 
		&#8203;NOTE: The design for the acrylic piece can be laser cut following the pattern in &#34;TactileStimDesign.pdf&#34; in the GitHub repository.</li>
	</ol>
	</li>
</ol>
3. Preparing and running behavior experiments
<ol>
	<li>Implanting mouse headplate.
	<ol>
		<li>Anesthetize a mouse using 2% isoflurane and head fix in a stereotactic frame.</li>
		<li>Apply an ophthalmic ointment to the eyes.</li>
		<li>Shave the scalp using soapy water and a sterile scalpel. Inject lidocaine directly underneath the skin of the incision site and clean the surgical site with povidone.</li>
		<li>Make an incision with a scalpel along the midline of the scalp from the back edge of the eyes to the back edge of the skull, being careful not to press too hard on the skull.</li>
		<li>Spread the incision open and clamp both sides with sterile hemostats to hold it open. Gently remove the periosteum using a cotton swab dipped with ethanol and allow the surface of the exposed skull to dry.</li>
		<li>Position the headplate level on the skull, making sure to position the front of the headplate posterior to the eyes. Use cyanoacrylate glue to attach the headplate to the skull and allow the glue to dry fully.</li>
		<li>Mix the dental cement powder (1 scoop), solvent (2 drops), and catalyst (1 drop) in a mixing dish and apply to all areas of exposed bone. Add layers until the surface is flush with the top edge of the headplate, making sure the headplate is securely attached to the skull.</li>
		<li>Suture the skin closed behind and in front of the headplate if necessary.</li>
		<li>Inject post-operative analgesia such as carprofen&#160;per institutional guidelines while allowing the animal to recover for at least 5 days.</li>
	</ol>
	</li>
	<li>Preparing for behavior sessions.
	<ol>
		<li>Allow the test animals to habituate to the platform by mounting them in the head restraint for 30-min sessions for 5 days preceding experiments. 
		NOTE: By the end of the habituation sessions, animals should run comfortably on the wheel.</li>
		<li>(DEC only) Prior to sessions, ensure that the solenoid valve outlet is centered on the target eye positioned &#62;1 cm away.</li>
		<li>(DEC only) Manually actuate an air puff using the push button. Ensure that the mouse promptly produces a blink without showing overt signs of stress such as adopting a hunched posture or grabbing the affected periocular region with the ipsilateral forepaw.</li>
		<li>(DTSC only) Prior to sessions, ensure that the tactile stimulus is centered on the animal&#39;s nose positioned ~1.5 cm away. 
		NOTE: When a DTSC behavioral session is not running, the stepper motor is automatically inactivated to allow manual repositioning.</li>
		<li>(DTSC only) In the SBC terminal, type python3 assocLearnRig_app.py to start the GUI.</li>
		<li>(DTSC only) Run a test session of three trials with the default parameters by hitting the Start Session button in the GUI.</li>
		<li>(DTSC only) Ensure that the logged data that prints to the terminal show a deflection of greater than 20 but less than 100 steps logged on the rotary encoder following the US on each trial. 
		&#8203;CAUTION: To avoid harm and reduce stress to the animal, start the stimulus farther from the animal and move it closer until the required conditions are met.</li>
	</ol>
	</li>
	<li>Running behavioral sessions with data logging.
	<ol>
		<li>Mount a mouse to the head restraint.</li>
		<li>In the terminal of the SBC, type python3 assocLearnRig_app.py to start the GUI.</li>
		<li>To allow camera recordings during the behavioral trials, hit the Stream button. 
		NOTE: Sessions can be run without a camera. In this case, only data from the rotary encoder and stimulus presentation timestamps are logged.</li>
		<li>Input identifying information for the animal into the Animal ID field and hit the Set button.</li>
		<li>Select either the DEC or DTSC from the radio button under the Session Type heading depending on which behavioral paradigm is desired.</li>
		<li>Input the desired experiment parameters to the fields below the Animal ID field and hit the Upload to Arduino button. 
		&#8203;NOTE: Details of the experiment parameters can be found in the GitHub repository README section.</li>
		<li>Hit the Start Session button to begin the session.</li>
		<li>When a session is initialized, data will begin logging in a new directory created in &#34;/media/usb&#34; in the SBC thumb drive mount point.</li>
	</ol>
	</li>
</ol>
4. Exporting and analyzing data
<ol>
	<li>To export all the recorded sessions to the host computer, open a command prompt and input the command pscp -r pi@Pi_IP_address:/media/usb* host_computer_destination, then authenticate with the SBC password. 
	NOTE: The above command is for a Windows machine. On Mac and Linux machines, use terminal and replace &#34;pscp&#34; with &#34;scp&#34;.</li>
	<li>Install Anaconda25 or another python package manager (PPM) on the host computer.</li>
	<li>Go to the GitHub repository and download &#34;analyzeSession.py&#34;, &#34;summarizeSessions.py&#34;, &#34;session2mp4s.py&#34;, and &#34;requirementsHost.txt&#34;.</li>
	<li>Open a PPM prompt and type conda install --file directory_containing_requirementsHostrequirements Host.txt to ensure that the Python package installation has the required python libraries.</li>
	<li>In the prompt, type cd directory_containing_analyzeData to navigate to the directory containing &#34;analyzeData.py&#34; and &#34;session2mp4s.py&#34;. Run the analysis program by typing python analyzeSession.py 
	NOTE: An error message will be generated if using a Python 2 version as python. To check the version, type python -V in the prompt.</li>
	<li>Select the directory containing the data when prompted. Directories with multiple subdirectories will be analyzed sequentially.</li>
	<li>For DEC sessions, for each session directory analyzed, select a region of interest (ROI) containing the mouse&#39;s eye from a trial average image. 
	NOTE: Final analysis data files and summary graphs will populate to a subdirectory of each analyzed session directory.</li>
	<li>Type python summarizeSessions.py to generate summary data across multiple sessions.</li>
	<li>Type in the prompt python session2mp4s.py to convert imaging data files into viewable .mp4 files.</li>
</ol>

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S., Li, L., Scott, B. W., Frankland, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tactile,+acoustic+and+vestibular+systems+sum+to+elicit+the+startle+reflex.">Tactile, acoustic and vestibular systems sum to elicit the startle reflex.</a> Neuroscience and Biobehavioral Reviews. 26 (1), 1-11 (2002).</li><li>. Raspberry Pi Operating system images Available from: <a target="_blank" href="https://www.raspberrypi.com/software/operationg-systems/">https://www.raspberrypi.com/software/operationg-systems/</a> (2021)</li><li>. VNC Server. VNC&#174; Connect Available from: <a target="_blank" href="https://www.realvnc.com/en/connect/download/vnc/">https://www.realvnc.com/en/connect/download/vnc/</a> (2021)</li><li>. Anaconda: The world's most popular data science platform Available from: <a target="_blank" href="https://xddebuganaconda.xdlab.co/">https://xddebuganaconda.xdlab.co/</a> (2021)</li><li>De Zeeuw, C. I., Ten Brinke, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+learning+and+the+cerebellum.">Motor learning and the cerebellum.</a> Cold Spring Harbor Perspectives in Biology. 7 (9), 021683 (2015).</li><li>Badura, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+cognitive+and+social+development+require+posterior+cerebellar+activity.">Normal cognitive and social development require posterior cerebellar activity.</a> eLife. 7, 36401 (2018).</li><li>Koekkoek, S. K. E., Den Ouden, W. L., Perry, G., Highstein, S. M., De Zeeuw, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+kinetic+and+frequency-domain+properties+of+eyelid+responses+in+mice+with+magnetic+distance+measurement+technique.">Monitoring kinetic and frequency-domain properties of eyelid responses in mice with magnetic distance measurement technique.</a> Journal of Neurophysiology. 88 (4), 2124-2133 (2002).</li><li>Kloth, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+associative+sensory+learning+defects+in+five+mouse+autism+models.">Cerebellar associative sensory learning defects in five mouse autism models.</a> eLife. 4, 06085 (2015).</li><li>Boele, H. -. J., Koekkoek, S. K. E., De Zeeuw, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+and+extracerebellar+involvement+in+mouse+eyeblink+conditioning:+the+ACDC+model.">Cerebellar and extracerebellar involvement in mouse eyeblink conditioning: the ACDC model.</a> Frontiers in Cellular Neuroscience. 3, (2010).</li><li>Lin, C., Disterhoft, J., Weiss, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whisker-signaled+eyeblink+classical+conditioning+in+head-fixed+Mice.">Whisker-signaled eyeblink classical conditioning in head-fixed Mice.</a> Journal of Visualized Experiments: JoVE. (109), e53310 (2016).</li><li>Pereira, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+animal+pose+estimation+using+deep+neural+networks.">Fast animal pose estimation using deep neural networks.</a> Nature Methods. 16 (1), 117-125 (2019).</li><li>Mathis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DeepLabCut:+markerless+pose+estimation+of+user-defined+body+parts+with+deep+learning.">DeepLabCut: markerless pose estimation of user-defined body parts with deep learning.</a> Nature Neuroscience. 21 (9), 1281-1289 (2018).</li></ol>

The authors have no conflicts of interest to disclose.

The platform with associated protocols outlined here can be used to reliably track animal behavior in two sensory associative learning tasks. Each task depends on intact communication through the climbing fiber pathway. In the design described here, we incorporate elements to facilitate learning and recording/perturbation of cerebellar response. These include a wheel to allow for free locomotion11,18 as well as head fixation. The wheel allows mouse subjects to lo...

Pavlovian conditioning of sub-second association between stimuli to elicit a conditioned response has long been used to probe cerebellar-dependent learning. For example, in classical delay eyeblink conditioning (DEC), animals learn to make a well-timed protective blink in response to a neutral conditional stimulus (CS; e.g., a flash of light or auditory tone) when it is paired repeatedly with an unconditional stimulus (US; e.g., a puff of air applied to the cornea) which always elicits a reflex blink, and which comes at or near the end of the CS. The learned response is referred to as a conditioned response (CR), while the reflex response is referred to as the unconditioned response (UR). In rabbits, cerebellum-specific lesions disrupt this form of learning1,2,3,4. Further, Purkinje cell complex spikes, driven by their climbing fiber inputs5, provide a necessary6,7 and sufficient8,9 signal for the acquisition of properly-timed CRs.
More recently, climbing fiber-dependent associative learning paradigms have been developed for head-fixed mice. DEC was the first associative learning paradigm to be adapted to this configuration10,11. DEC in head-fixed mice has been used to identify cerebellar regions11,12,13,14,15,16,17 and circuit elements11,12,13,14,15,18,19 that are required for task acquisition and extinction. This approach has also been used to demonstrate how the cellular-level physiological representation of task parameters evolves with learning13,15,16.
In addition to eyeblink, the delayed startle tactile conditioning (DTSC) paradigm was recently developed as a novel associative learning task for head-fixed mice20. Conceptually similar to DEC, DTSC involves the presentation of a neutral CS with a US, a tap to the face sufficient in intensity to engage a startle reflex21,22 as the UR. In the DTSC paradigm, both the UR and CR are read out as backward locomotion on a wheel. DTSC has now been used to uncover how associative learning alters cerebellar activity and patterns of gene expression20.
In this work, a method was developed for flexibly applying DEC or DTSC in a single platform. The stimulus and platform attributes are schematized in Figure 1. The design incorporates the capacity to track animal behavior with a camera as well as a rotary encoder to track mouse locomotion on a wheel. All aspects of data logging and trial structure are controlled by paired microcontrollers (Arduino) and a single-board computer (SBC; Raspberry Pi). These devices can be accessed through a provided graphical user interface. Here, we present a workflow for setup, experiment preparation and execution, and a customized analysis pipeline for data visualization.

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a flexible platform for monitoring cerebellum-dependent sensory associative learning

Climbing fiber inputs to Purkinje cells provide instructive signals critical for cerebellum-dependent associative learning. Studying these signals in head-fixed mice facilitates the use of imaging, electrophysiological, and optogenetic methods. Here, a low-cost behavioral platform (~$1000) was developed that allows tracking of associative learning in head-fixed mice that locomote freely on a running wheel. The platform incorporates two common associative learning paradigms: eyeblink conditioning and delayed tactile startle conditioning. Behavior is tracked using a camera and the wheel movement by a detector. We describe the components and setup and provide a detailed protocol for training and data analysis. This platform allows the incorporation of optogenetic stimulation and fluorescence imaging. The design allows a single host computer to control multiple platforms for training multiple animals simultaneously.

Workflow for DEC experiments and analysis 
Proper experimental parameter selection is important for successful delay eyeblink conditioning (DEC) training. For the data presented here, the GUI was used to choose a CS duration of 350 ms and a US duration of 50 ms. This pairing results in an inter-stimulus interval of 300 ms: long enough to prevent low-amplitude CR production10 and short enough to avoid getting into the regime of poor learning or trace conditioning, a process th...

Watch this Scientific Journal Video about A Flexible Platform for Monitoring Cerebellum-Dependent Sensory Associative Learning at JoVE.com

A Flexible Platform for Monitoring Cerebellum-Dependent Sensory Associative Learning

We have developed a single platform to track animal behavior during two climbing fiber-dependent associative learning tasks. The low-cost design allows integration with optogenetic or imaging experiments directed towards climbing fiber-associated cerebellar activity.

Climbing fiber inputs to Purkinje cells provide instructive signals critical for cerebellum-dependent associative learning. Studying these signals in ...

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JoVE Journal

Neuroscience

3.3K Views.  Princeton University. We have developed a single platform to track animal behavior during two climbing fiber-dependent associative learning tasks. The low-cost design allows integration with optogenetic or imaging experiments directed towards climbing fiber-associated cerebellar activity.

Video: A Flexible Platform for Monitoring Cerebellum-Dependent Sensory Associative Learning

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

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

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

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

Behavior

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 ...

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a living animal&#8217;s brain. Moreover, the lengthy recovery from anesthesia limits the frequency of repeated recording/imaging episodes in longitudinal studies. Hence, new methods that would allow stable recordings from non-anesthetized behaving mice are expected to advance the fields of cellular and cognitive neurosciences. Existing solutions range from mere physical restraint to more sophisticated approaches, such as linear and spherical treadmills used in combination with computer-generated virtual reality. Here, a novel method is described where a head-fixed mouse can move around an air-lifted mobile homecage and explore its environment under stress-free conditions. This method allows researchers to perform behavioral tests (e.g., learning, habituation or novel object recognition) simultaneously with two-photon microscopic imaging and/or patch-clamp recordings, all combined in a single experiment. This video-article describes the use of the awake animal head fixation device (mobile homecage), demonstrates the procedures of animal habituation, and exemplifies a number of possible applications of the method.

This method creates a tangible, familiar environment for the mouse to navigate and explore during microscopic imaging or single-cell electrophysiological recordings, which require firm fixation of the animal&#8217;s head.

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a ...

Combined Shuttle-Box Training with Electrophysiological Cortex Recording and Stimulation as a Tool to Study Perception and Learning

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the necessary equipment is now available by several commercial companies. This protocol provides a detailed description of a two-way shuttle-box avoidance learning paradigm in rodents (here Mongolian gerbils; Meriones unguiculatus) in combination with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical electrophysiological in vivo recordings. The detailed protocol is applicable to study multiple aspects of learning behavior and perception in different rodent species.
Site-specific ICMS of auditory cortical circuits as conditioned stimuli here is used as a tool to test the perceptual relevance of specific afferent, efferent and intracortical connections. Distinct activation patterns can be evoked by using different stimulation electrode arrays for local, layer-dependent ICMS or distant ICMS sites. Utilizing behavioral signal detection analysis it can be determined which stimulation strategy is most effective for eliciting a behaviorally detectable and salient signal. Further, parallel multichannel-recordings using different electrode designs (surface electrodes, depth electrodes, etc.) allow for investigating neuronal observables over the time course of such learning processes. It will be discussed how changes of the behavioral design can increase the cognitive complexity (e.g. detection, discrimination, reversal learning).

Shuttle-box avoidance learning is well-established in behavioral neuroscience. This protocol describes how shuttle-box learning in rodents can be combined with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical in vivo recordings as a tool to study multiple aspects of learning and perception.

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the ...

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 ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze motor-learning induced plasticity, we trained rats using an accelerated rotor rod task. Rats performed the task 10 times at 30-s intervals for 1 or 2 days. Performance was significantly improved on the training days compared to the first trial. We then prepared acute brain slices of the primary motor cortex (M1) in untrained and trained rats. Current-clamp analysis showed dynamic changes in resting membrane potential, spike threshold, afterhyperpolarization, and membrane resistance in layer II/III pyramidal neurons. Current injection induced many more spikes in 2-day trained rats than in untrained controls.
To analyze contextual-learning induced plasticity, we trained rats using an inhibitory avoidance (IA) task. After experiencing foot-shock in the dark side of a box, the rats learned to avoid it, staying in the lighted side. We prepared acute hippocampal slices from untrained, IA-trained, unpaired, and walk-through rats. Voltage-clamp analysis was used to sequentially record miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) from the same CA1 neuron. We found different mean mEPSC and mIPSC amplitudes in each CA1 neuron, suggesting that each neuron had different postsynaptic strengths at its excitatory and inhibitory synapses. Moreover, compared with untrained controls, IA-trained rats had higher mEPSC and mIPSC amplitudes, with broad diversity. These results suggested that contextual learning creates postsynaptic diversity in both excitatory and inhibitory synapses at each CA1 neuron.
AMPA or GABAA receptors seemed to mediate the postsynaptic currents, since bath treatment with CNQX or bicuculline blocked the mEPSC or mIPSC events, respectively. This technique can be used to study different types of learning in other regions, such as the sensory cortex and amygdala.

The slice patch clamp technique is an effective method for analyzing learning-induced changes in the intrinsic properties and plasticity of excitatory or inhibitory synapses.

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Automated Assessment of Fine Motor Performance in Mice Using the Erasmus Ladder

Monitoring Fine and Associative Motor Learning in Mice Using the Erasmus Ladder

Behavior is shaped by actions, and actions necessitate motor skills such as strength, coordination, and learning. None of the behaviors essential for sustaining life would be possible without the ability to transition from one position to another. Unfortunately, motor skills can be compromised in a wide array of diseases. Therefore, investigating the mechanisms of motor functions at the cellular, molecular, and circuit levels, as well as understanding the symptoms, causes, and progression of motor disorders, is crucial for developing effective treatments. Mouse models are frequently employed for this purpose.
This article describes a protocol that allows the monitoring of various aspects of motor performance and learning in mice using an automated tool called the Erasmus Ladder. The assay involves two phases: an initial phase where mice are trained to navigate a horizontal ladder built of irregular rungs (&#34;fine motor learning&#34;), and a second phase where an obstacle is presented in the path of the moving animal. The perturbation can be unexpected (&#34;challenged motor learning&#34;) or preceded by an auditory tone (&#34;associative motor learning&#34;). The task is easy to conduct and is fully supported by automated software.
This report shows how different readouts from the test, when analyzed with sensitive statistical methods, allow fine monitoring of mouse motor skills using a small cohort of mice. We propose that the method will be highly sensitive to evaluate motor adaptations driven by environmental modifications as well as early-stage subtle motor deficits in mutant mice with compromised motor functions.

Author Spotlight: Unveiling Neural Mechanisms Through Automated Evaluation of Motor Learning and Myelin Plasticity Studies Using the Erasmus Ladder 

This article presents a protocol that allows a non-invasive and automated assessment of fine motor performance, as well as adaptive and associative motor learning upon challenges, using a device called the Erasmus Ladder. Task difficulty can be titrated to detect motor impairment ranging from major to subtle degrees.

Behavior is shaped by actions, and actions necessitate motor skills such as strength, coordination, and learning. None of the behaviors essential for ...

Recording Neuronal Activity During Odor Plume Navigation with Miniscope Imaging

Miniscope Recording Calcium Signals at Hippocampus of Mice Navigating an Odor Plume

Mice navigate an odor plume with a complex spatiotemporal structure in the dark to find the source of odorants. This article describes a protocol to monitor behavior and record Ca2+ transients in dorsal CA1 stratum pyramidale neurons in the hippocampus (dCA1) in mice navigating an odor plume in a 50 cm x 50 cm x 25 cm odor arena. An epifluorescence miniscope focused through a gradient-index (GRIN) lens imaged Ca2+ transients in dCA1 neurons expressing the calcium sensor GCaMP6f in Thy1-GCaMP6f mice. The paper describes the behavioral protocol to train the mice to perform this odor plume navigation task in an automated odor arena. The methods include a step-by-step procedure for the surgery for GRIN lens implantation and baseplate placement for imaging GCaMP6f in CA1. The article provides information on real-time tracking of the mouse position to automate the start of the trials and delivery of a water reward. In addition, the protocol includes information on using an interface board to synchronize metadata describing the automation of the odor navigation task and frame times for the miniscope and a digital camera tracking mouse position. Moreover, the methods delineate the pipeline used to process GCaMP6f fluorescence movies by motion correction using the NorMCorre algorithm followed by identification of regions of interest with EXTRACT. Finally, the paper describes an artificial neural network approach to decode spatial paths from CA1 neural ensemble activity to predict mouse navigation of the odor plume.

Author Spotlight: Understanding Processing of Olfactory and Spatial Information by Brain with Real-Time Behavioral Analysis

This protocol investigates the brain-behavior relationship in hippocampal CA1 in mice navigating an odor plume. We provide a step-by-step protocol, including surgery to access imaging of the hippocampus, behavioral training, miniscope GCaMP6f recording and processing of the brain, and behavioral data to decode the mouse position from ROI neural activity.

Mice navigate an odor plume with a complex spatiotemporal structure in the dark to find the source of odorants. This article describes a protocol to ...