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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 uncondi....

Protocol

The animal protocols described here have been approved by the Animal Care and Use Committees of Princeton University.

1. Setting up the SBC

  1. Connect the camera serial interface (CSI) cable to the Raspberry NoIR V2 camera and the camera port on the SBC.
  2. 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 Rasp.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
"B" Quick Base For C&B METABOND - 10 mL bottleParkellS398Dental cement solvent
"C" Universal TBB Catalyst - 0.7 mLParkellS371Catalyst
#8 WashersThorlabsW8S038Washers
0.250" (1/4") x 8.00" Stainless Steel Precision ShaftingServocity6341721/4" shaft
0.250” (0.770") Clamping HubServocity545588Clamping hub
1/4" to 6 mm Set Screw Shaft Coupler- 5 packActobotics625106Shaft-coupling sleeve
1/4"-20 Cap Screws, 3/4" LongThorlabsSH25S0751/4" bolt
100 pcs 5 mm 395–400 nm UV Ultraviolet LED Light Emitting Diode Clear Round Lens 29 mm Long Lead (DC 3V) LEDs Lights +100 pcs ResistorsEDGELEC‎ED_YT05_U_100PcsCS LEDs
2 m Micro HDMI to DVI-D Cable - M/M - 2 m Micro HDMI to DVI Cable - 19 pin HDMI (D) Male to DVI-D Male - 1920 x 1200 VideoStar-tech‎HDDDVIMM2MRaspberry Pi4B to monitor cable
256 GB Ultra Fit USB 3.1 Flash DriveSanDisk‎SDCZ430-256G-G46USB thumb drive
3.3 V–5 V 4 Channels Logic Level Converter Bi-Directional Shifter ModuleAmazonB00ZC6B8VMLogic level shifter
32 GB 95 MB/s (U1) microSDHC EVO Select Memory CardSamsung‎MB-ME32GA/AMmicroSD card
4.50" Aluminum ChannelServocity5854444.5" aluminum channel
48-LED CCTV Ir Infrared Night Vision IlluminatorTowallmarkSODIALInfrared light array
4PCS Breadboards Kit Include 2PCS 830 Point 2PCS 400 Point Solderless Breadboards for Proto Shield Distribution Connecting BlocksREXQualisB07DL13RZHBreadboard
5 Port Gigabit Unmanaged Ethernet Network SwitchTP-Link‎TL-SG105Ethernet switch
5 V 2.5 A Raspberry Pi 3 B+ Power Supply/AdapterCanakit‎DCAR-RSP-2A5Power supply for Raspberry Pi 3B+
5-0 ETHILON BLACK 1 x 18" C-3Ethicon668GSutures
6 mm Shaft Encoder 2000 PPR Pushpull Line Driver Universal Output Line Driver Output 5-26 V dc SupplyCalt B01EWER68IRotary encoder
Ø1/2" Optical Post, SS, 8-32 Setscrew, 1/4"-20 Tap, L = 1", 5 PackThorlabsTR1-P5Optical posts
Ø1/2" Optical Post, SS, 8-32 Setscrew, 1/4"-20 Tap, L = 2", 5 PackThorlabsTR2-P5Optical posts
Ø1/2" Optical Post, SS, 8-32 Setscrew, 1/4"-20 Tap, L = 4", 5 PackThorlabsTR4-P5Optical posts
Ø1/2" Optical Post, SS, 8-32 Setscrew, 1/4"-20 Tap, L = 6", 5 PackThorlabsTR6-P5Optical posts
Ø1/2" Post Holder, Spring-Loaded Hex-Locking Thumbscrew, L = 2"ThorlabsPH2Optical post holder
Adapter-062-M X LUER LOCK-FThe Lee Co.TMRA3201950ZSolenoid valve luer adapter
Aeromat Foam Roller Size: 36" LengthAeromatB002H3CMUEFoam roller
Aluminum Breadboard 10" x 12" x 1/2", 1/4"-20 TapsThorlabsMB1012Aluminum breadboard
Amazon Basics HDMI to DVI Adapter Cable, Black, 6 Feet, 1-PackAmazonHL-007347Raspberry Pi3B+ to monitor cable
Arduino  Uno R3ArduinoA000066Arduino Uno (microcontroller board)
Arduino DueArduino‎A000062Arduino Due (microcontroller board)
Bench Power Supply, Single, Adjustable, 3 Output, 0 V, 24 V, 0 A, 2 ATenma72-8335APower supply
Clear Scratch- and UV-Resistant Cast Acrylic Sheet, 12" x 24" x 1/8"McMaster Carr8560K257Acrylic sheet
CNC Stepper Motor Driver 1.0–4.2 A 20–50 V DC 1/128 Micro-Step Resolutions for Nema 17 and 23 Stepper MotorStepper OnlineB06Y5VPSFNStepper motor driver
Compact Compressed Air Regulator, Inline Relieving, Brass Housing, 1/4 NPTMcMaster Carr6763K13Air source regulator
Cotton SwabPuritan806-WCCotton swab
Dell 1908FP 19" Flat Panel Monitor - 1908FPCDell1908FPCComputer monitor
Flex Cable for Raspberry Pi CameraAdafruit2144camera serial interface cable
High Torque Nema 17 Bipolar Stepper Motor 92 oz·in/65 N·cm 2.1 A Extruder MotorStepper Online17HS24-2104SStepper motor
IsofluraneHenry Schein66794001725Isoflurane
Krazy Maximum Bond Permanent Glue, 0.18 oz.Krazy GlueKG483Cyanoacrylate glue
Lidocaine HClVetOne510212Lidocaine
Low-Strength Steel Hex Nut, Grade 2, Zinc-Plated, 1/4"-20 Thread SizeMcMaster Carr90473A029Nuts
M3 x 50 mm Partially Threaded Hex Key Socket Cap Head Screws 10 pcsUxcellA16040100ux1380M3 bolt
NEMA 17 Stepper Motor MountACTOBOTICS555152Stepper motor mount
Official Raspberry Pi Power Supply 5.1 V 3 A with USB C - 1.5 m longAdafruit4298Power supply for Raspberry Pi 4B
Optixcare Dog & Cat Eye Lube Lubricating Gel, 0.70-oz tubeOptixcare142422Opthalimic ointment
Precision Stainless Steel Ball Bearing, Shielded, Trade No. R188-2Z, 13000 rpm Maximum SpeedMcMaster-Carr3759T57Bearing
Premium Female/Female Jumper Wires - 40 x 6"Adafruit266Wires
Premium Female/Male 'Extension' Jumper Wires - 40 x 6" (150 mm)Adafruit826Wires
Premium Male/Male Jumper Wires - 40 x 6"Adafruit758Wires
Radiopaque L-Powder for C&B METABOND - 5 gParkellS396Dental cement powder
Raspberry Pi (3B+ or 4B)Adafruit3775 or 4295Raspberry Pi
Raspberry Pi NoIR Camera Module V2 - 8MP 1080P30Raspberry Pi FoundationRPI3-NOIR-V2Raspberry NoIR V2 camera
Right-Angle Bracket, 1/4" (M6) Counterbored Slot, 8-32 TapsThorlabsAB90ERight-angle bracket
Right-Angle Clamp for Ø1/2" Posts, 3/16" HexThorlabsRA90Right-angle optical post clamp
Right-Angle End Clamp for Ø1/2" Posts, 1/4"-20 Stud and 3/16" HexThorlabsRA180Right-angle end clamp
RJ45 Cat-6 Ethernet Patch Internet CableAmazon‎CAT6-7FT-5P-BLUEEthernet cable
Rotating Clamp for Ø1/2" Posts, 360° Continuously Adjustable, 3/16" HexThorlabsSWCRotating optical post clamps
Spike & Hold Driver-0.1 TO 5 MSThe Lee Co.IECX0501350ASolenoid valve driver
Swivel Base AdapterThorlabsUPHAPost holder adapter
USB 2.0 A-Male to Micro B Cable, 6 feetAmazon‎7T9MV4USB2 type A to USB2 micro cable
USB 2.0 Printer Cable - A-Male to B-Male, 6 Feet (1.8 m)AmazonB072L34SZSUSB2 type B to USB2 type A cable
VHS-M/SP-12 VThe Lee Co.INKX0514900ASolenoid valve
Zinc-Plated Steel 1/4" washer, OD 1.000"McMaster Carr91090A108Washers

References

  1. McCormick, D. A., Lavond, D. G., Clark, G. A., Kettner, R. E., Rising, C. E., Thompson, R. F. The engram found? Role of the cerebellum in classical conditioning of nictitating membrane and eyelid responses. Bulletin of the Psychonomic Society. 18 (3), 103-105 (1981).
  2. McCormick, D. A., Clark, G. A., Lavond, D. G., Thompson, R. F.

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