Whisker-signaled Eyeblink Classical Conditioning in Head-fixed Mice

Summary

The preparation presented here for whisker-signaled eyeblink conditioning in head-fixed mice precisely stimulates specific whiskers while allowing mice to ambulate on a cylindrical treadmill. A whisker stimulation conditioned stimulus (CS) paired with a periorbital shock unconditioned stimulus (US) results in reliable associative learning on this apparatus.

Abstract

Eyeblink conditioning is a common paradigm for investigating the neural mechanisms underlying learning and memory. To better utilize the extensive repertoire of scientific techniques available to study learning and memory at the cellular level, it is ideal to have a stable cranial platform. Because mice do not readily tolerate restraint, they are usually trained while moving about freely in a chamber. Conditioned stimulus (CS) and unconditioned stimulus (US) information are delivered and eyeblink responses recorded via a tether connected to the mouse's head. In the head-fixed apparatus presented here, mice are allowed to run as they desire while their heads are secured to facilitate experimentation. Reliable conditioning of the eyeblink response is obtained with this training apparatus, which allows for the delivery of whisker stimulation as the CS, a periorbital electrical shock as the US, and analysis of electromyographic (EMG) activity from the eyelid to detect blink responses.

Introduction

Eyeblink conditioning is a form of Pavlovian conditioning and a model system for investigating the neural mechanisms of associative learning and memory. It has been investigated in various species, including humans, rabbits, cats, rats, and mice. The paradigm involves the presentation of two paired stimuli: a neutral conditioned stimulus (CS; e.g., a tone, a flash of light, or whisker stimulation), and a salient unconditioned stimulus (US; e.g., an air puff to the eye, or periorbital shock). The US elicits an unconditioned, reflexive eyeblink response (i.e., UR). Eventually, after several presentations of the paired CS-US, the subject learns....

Protocol

All procedures involving mice were performed in accordance with protocols approved by Northwestern University's Institutional Animal Care and Use Committee based on guidelines issued by the National Institute of Health.

1. The Cylinder (Figure 2A)

1. Construct the cylinder as described by Chettih et al. and Heiney et al. from a long foam cylinder^14-15. Cut a 10 cm length of cylinder and drill a hole through the center to fit the axle, a metal rod 12.7 mm (0.5 in.) in diameter. Mount the cylinder with its axle onto a metal optical breadboard as described by Heiney et al. or another support (....

Results

8-10 week old male C57Bl6/J mice were trained on trace eyeblink conditioning on the head-fixed cylindrical treadmill apparatus. 8 mice were trained with paired CS-US presentations (conditioned group) and 9 mice were trained with unpaired CS and US presentations (pseudoconditioned group).

Example EMG recordings of a conditioned response from a conditioned mouse are shown in Figures 3 and 4. EMG r.......

Discussion

Classical eyeblink conditioning is a form of associative learning that is a useful tool for understanding the neural substrates underlying learning and memory. Previous methods employed for eyeblink conditioning in rodents such as mice involved a chamber that allowed for the animal to move about freely. A head-fixed preparation for eyeblink conditioning in mice, using the apparatus described by Chettih et al. and Heiney et al. and most recently utilized in light-evoked trace eyeblink conditioning in Sie.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Exervo TeraNova Foam Roller 36" x 6"	Amazon	B002ONUM0E	For cylinder
Plexiglas			Custom-made; 1 cm thick
Metal Rods (12.7 mm diameter)			Custom-made
4-40 machine screw (.25 in. long)	Amazon Supply	B00F33Q8QO	For cylinder
Classic Design Hair Comb	Conair	93505WG-320	For whisker stimulation
2-Layer Rectangular Bending Actuator	Piezo Systems	T220-A4-303X	For whisker stimulation
Solder and Flux Kit	Piezo Systems	MSF-003-NI	For whisker stimulation
Magnetic Base	Thor Labs	MB175	For whisker stimulation
Threaded rod for magnetic base			Custom-made
Strips based on 221 series nylon strip connectors from Electronic Connector Corp.			Custom-made, based on Weiss and Disterhoft, 2008
TO-220 Style Transistor	Amazon Supply	B0002ZPZYO	For connector; for the wings
Relia-Tac Sockets	Electronic Connector Corp.	220-S02	For connector
Relia-Tac Pins	Electronic Connector Corp.	220-P02	For headpiece
0-80 stainless steel machine screw (1 in. long)	Amazon Supply	B000FN68EE	Locking Screw
0-80 stainless steel machine screw hex nut (5/32 in. thick)	Amazon Supply	B000N2TK7Y	Locking Screw Head
Loctite Super Glue-Liquid	Loctite	1365896	Cyanoacrylic glue; for the locking screw
Quick Setting Epoxy	Ace Hardware	18613	For connector and whisker stimulation system
Ethernet Cable Wires			Ethernet cable can be taken apart to use the individual wires for the connector
Polyimide coated stainless steel wires (2 in. long, .005 in. diameter)	PlasticsOne	005sw/2.0 37365 S-S	For headpiece, EMG and shock wires
Stainless steel uncoated wire (.005 in. diameter)	AM Systems	792800	For headpiece, ground wires
Tenma Variable Autotransformer	Tenma	72-110	For the whisker stimulation; rheostat to adjust current to the bending actuator
Amplifier	A-M Systems	1700	Amplifier for filtering and amplifying EMG signals
WPI A385R stimulus isolator	World Precision Instruments	31405	For the electrical shock
Isothesia (Isoflurane)	Henry Schein: Animal Health	50031	For surgery; anesthesia
Buprenex Injectable CIII	Reckett Benckiser Pharmaceuticals Inc	NDC 12496-0757-1	For surgery; analgesic
Akwa Tears: Lubricant Ophthalmic Ointment	Akorn	NDC 17478-062-35	Artificial tear ointment to prevent dry eyes while under anesthesia
Povidine-Iodine Prep Pads	PDI	NDC 10819-3883-1	For surgery; antiseptic
Alcohol Prep Pads			May be purchased from any standard pharmacy
Stainless steel surgical scalpel handles (no.3)	Integra Miltex	4-7.	For surgery
Stainless steel surgical scalpel blades	Integra Miltex	4-310 or 4-315	For surgery; number 10 or 15 scalpel blade
3% Hydrogen Peroxide			May be purchased from any standard pharmacy
Micro Clip	Roboz	RS-5459	For surgery, to hold back skin
00-90 stainless steel machine screw (0.0625 in. long)	Amazon Supply	B002SG89X4	For surgery, to wrap ground wire around
Professional Rotary Tool	Walnut Hollow	29637	Hand drill for surgery, to drill holes in skull
Inverted Cone Burr	Roboz	RS-6282C-34	Inverted cone burr size 34; for surgery, to drill holes in skull
Engraving Cutter Drill Bit	Dremel	106	Engraving cutter; 1.6 mm bit; for surgery, to drill holes in skull
C&B Metabond-Quick! Cement System "B" Quick Base	Parkell	S398	For surgery; adhesive luting cement system; important to prevent headpiece avulsion
C&B Metabond-Quick! Cement System Clear L-Powder	Parkell	S399	For surgery; adhesive luting cement system; important to prevent headpiece avulsion
C&B Metabond-Quick! Cement System "C" Universal TBB Catalyst 0.7 ml	Parkell	S371	For surgery; adhesive luting cement system; important to prevent headpiece avulsion
C&B Metabond-Quick! Cement System Ceramic Mixing Dish with temperature strip	Parkell	S387	For surgery; adhesive luting cement system; important to prevent headpiece avulsion
Swiss Tweezers, style #5	World Precision Instruments	504506	For surgery
Puritan Cotton-Tipped Applicators	VWR International	10806-005	For surgery
Dental Caulk Grip Cement Kit	Dentsply	675570	For surgery; dental cement