Jun 23, 2026
Pavlovian protocol - Fushiki et al 2024
- Akira Fushiki1,2,3
- 1Allen Institute;
- 2Zuckerman Mind Brain Behavior Institute, Columbia University;
- 3Aligning Science Across Parkinsonʼs ASAP Collaborative Research Network
Protocol Citation: Akira Fushiki 2026. Pavlovian protocol - Fushiki et al 2024 . protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmdk79v3p/v1
Manuscript citation:
A Vulnerable Subtype of Dopaminergic Neurons Drives Early Motor Deficits in Parkinsonʼs Disease Akira Fushiki, David Ng, Zachary R. Lewis, Archana Yadav, Tatiana Saraiva, Luke A. Hammond, Christoph Wirblich, Bosiljka Tasic, Vilas Menon, Joaquim Alves da Silva, Rui M. Costa bioRxiv 2024.12.20.629776; doi: https://doi.org/10.1101/2024.12.20.629776
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 23, 2026
Last Modified: June 23, 2026
Protocol Integer ID: 319712
Keywords: ASAPCRN, fushiki et al 2024 pavlovian protocol, pavlovian protocol, pavlovian conditioning protocols for mouse behavioral study, pavlovian conditioning protocol, mouse behavioral study, protocol
Funders Acknowledgements:
Aligning Science Across Parkinsonʼs
Grant ID: ASAP-020551
Abstract
Pavlovian Conditioning Protocols for Mouse Behavioral Studies Derived from Fushiki et al 2024.
Materials
- PyControl system, including a Breakout Board v1.2, Audio Board, Audio Player, Speaker, LED Light, Lickometer v1.0, Port Adapter, Nose-Poke Port, BNC Cables, and Ethernet Cables.
- Custom-built behavioral chamber (17 × 14 × 20 cm; L × W × H) housed within a sound-attenuating enclosure.
- Bonsai (open-source visual programming software).
- 10% sucrose solution (50 g sucrose dissolved in 500 mL water).
- Solenoid valve (LFVA1220310H; The Lee Company, Essex, CT, USA; 12 VDC, 1–30 PSIG), mounted outside the sound-attenuating chamber to minimize noise.
- Top-view camera: 2.0-megapixel USB camera (ELP-USBHD01M-L21, China; DC 5 V; Full HD 1080p).
- Side-view camera: FLIR Point Grey Flea3 camera (FLIR Systems, USA; 1280 × 1024 pixels) equipped with a CS-mount lens.
- Grounding cables used to reduce electrical noise in the lickometer system.
- Collection tube (e.g., 1.5 mL microcentrifuge tube) for measuring reward volume.
- Infrared illumination for monitoring within the sound-attenuating chamber.
Before start
Read through protocol before starting.
System Check and Sucrose–Solenoid Calibration
The behavioral task is controlled using the Python-based PyControl framework, synchronized with Bonsai for video acquisition and event alignment. Rewards consist of 10% sucrose solution (50 g sucrose dissolved in 500 mL water). Reward volume is determined by the solenoid valve open time, typically 120–150 ms, corresponding to approximately 6–8 µL. Because solenoid output can vary across sessions, the calibration is strongly recommended prior to behavioral training.
Power on the PyControl system and verify that all task modules initialize correctly.
Launch Bonsai and confirm that synchronization signals are being received and recorded properly.
Inspect all hardware connections, including the solenoid valve, reward spout, infrared beam sensors, cameras, and associated wiring, to ensure proper alignment and functionality.
Verify that the infrared illumination within the sound-attenuating chamber provides adequate lighting for reliable video acquisition and tracking.
Prepare a fresh 10% sucrose solution or ensure that a previously prepared solution remains clear and free of contamination.
Fill the reward reservoir with the sucrose solution and remove any air bubbles from the tubing connected to the solenoid valve, if present.
Place a clean collection tube (e.g., a 1.5 mL microcentrifuge tube) beneath the reward spout.
Using the PyControl manual control interface, trigger the solenoid valve for a fixed duration (e.g., 130 ms) over 20 consecutive pulses.
Measure the total volume of liquid collected in the tube (e.g., approximately 140 µL).
Calculate the reward volume delivered per pulse using the following equation: Reward volume per pulse = Total collected volume ÷ Number of pulses (Example: 140 µL ÷ 20 pulses = 7 µL per pulse.)
Adjust the solenoid valve open time as necessary to achieve the desired reward volume (typically 6–8 µL per pulse).
Verify that reward delivery remains consistent across at least five additional test pulses.
Confirm that the solenoid valve click is clearly audible outside the sound-attenuating chamber while remaining minimally detectable within the chamber.
Record the calibration parameters and measured reward volume for the session.
Magazine Training
Magazine training was used to habituate food-restricted mice (maintained at approximately 85% of their baseline body weight) to the behavioral chamber and to familiarize them with the location of the reward spout prior to Pavlovian conditioning. This training consisted of a single session conducted over one day. During the session, no conditioned stimuli were presented, and sucrose rewards were delivered at pseudorandom inter-trial intervals (ITIs) ranging from 50 to 70 s.
Confirm that sucrose reward delivery has been calibrated and verify that all system components are functioning properly.
Place the mouse into the behavioral chamber and allow it to acclimate briefly, if required.
Initiate video acquisition and confirm synchronized recording from both the top-view and side-view cameras.
Deliver 10% sucrose rewards at pseudorandom inter-trial intervals ranging from 50 to 70 s.
Allow the mouse to freely explore the behavioral chamber and locate the reward spout.
Continue the session for the designated duration (typically 30–60 min) or until reliable reward retrieval behavior is established, with a maximum of 30 reward deliveries.
Monitor approach and licking behaviors throughout the session to verify successful acquisition of the reward location.
Stop video acquisition and verify that all recordings have been saved successfully.
Remove the mouse from the behavioral chamber and return it to its home cage.
Clean the behavioral chamber and reward spout thoroughly to remove residual sucrose and minimize odor cues for subsequent sessions.
Pavlovian Conditioning
Pavlovian conditioning commenced one day after magazine training and was conducted over five consecutive daily sessions. During training, mice learned to discriminate between a reward-predictive conditioned stimulus (CS+) and a non-rewarded conditioned stimulus (CS−). The CS+ consisted of a 10-s LED light presentation during which a sucrose reward was delivered at cue onset, whereas the CS− consisted of a 10-s auditory tone (4 kHz, 75 dB) that was not paired with reward delivery. Trials were separated by a pseudorandomly varied inter-trial interval (ITI) ranging from 120 to 150 s. One training session was conducted per day, and each session terminated when the mouse had either received 30 sucrose rewards or completed 60 min of training, whichever occurred first.
Verify solenoid calibration and confirm proper synchronization among all system components.
Place the mouse in the behavioral chamber and initiate synchronized video recording from both the top-view and side-view cameras.
Present CS+ and CS− trials in a pseudorandom order with equal probability (50% each).
Pair each CS+ presentation with delivery of a single sucrose reward (~6–8 µL) at cue onset.
Present CS− trials without reward delivery.
Maintain pseudorandom ITIs of 120–150 s between consecutive trials.
Terminate the session after delivery of 30 CS+ rewards or upon reaching a maximum session duration of 60 min, whichever occurs first.
During each conditioned stimulus (CS) presentation, the following behavioral measures were monitored and recorded:
- Lick rate during the cue period
- Latency to the first lick following CS+ onset
- Behavioral activity and locomotion captured using synchronized top-view and side-view video recordings
These behavioral metrics were used to assess associative learning and track the acquisition of cue–reward relationships across the 5-day conditioning period.
Stop video recording and verify that all data have been saved successfully.
Remove the mouse from the behavioral chamber and return it to its home cage.
Clean the behavioral chamber and reward spout thoroughly to remove residual sucrose and prevent carryover of olfactory cues.
Archive all session logs, calibration records, and video files for subsequent analysis and data management.
Protocol references
Akam T et al., 2022; https://elifesciences.org/articles/67846